Textbook of Pulmonary Vascular Disease

Textbook of Pulmonary Vascular Disease

Jason X.-J. Yuan    Joe G.N. Garcia Charles A. Hales    Stuart Rich Stephen L. Archer    John B. West ●

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Editors

Textbook of Pulmonary Vascular Disease

Editors Jason X.-J. Yuan, MD, PhD Professor of Medicine Institute for Personalized Respiratory Medicine Department of Medicine University of Illinois at Chicago Chicago, IL [email protected] Charles A. Hales, MD Professor of Medicine Massachusetts General Hospital Harvard Medical School Boston, MA [email protected] Stephen L. Archer, MD Harold Hines Jr. Professor of Medicine Chief of Cardiology Department of Medicine University of Chicago Chicago, IL [email protected]

Joe G.N. Garcia, MD Earl Banes Professor of Medicine Vice Chancellor for Research University of Illinois at Chicago Chicago, IL [email protected] Stuart Rich, MD Professor of Medicine Department of Medicine University of Chicago Chicago, IL [email protected] John B. West, MD, PhD, DSc Distinguished Professor of Medicine and Physiology Department of Medicine University of California, San Diego La Jolla, CA [email protected]

ISBN 978-0-387-87428-9 e-ISBN 978-0-387-87429-6 DOI 10.1007/978-0-387-87429-6 Springer New York Dordrecht Heidelberg London Library of Congress Control Number: 2011920684 © Springer Science+Business Media, LLC 2011 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Preface

The major function of the lungs is gas exchange and it does this using a low resistance circulation. The pulmonary circulation (or the pulmonary vasculature) is a unique system that differs dramatically from the systemic circulatory system (e.g., coronary, cerebral, renal arteries) in structure, function, and regulation. A typical example of functional differences between the pulmonary and systemic vasculature is that hypoxia causes pulmonary vasoconstriction but systemic vasodilation. Furthermore, in patients with systemic arterial hypertension (e.g., essential hypertension), pulmonary arterial pressure is normal, while in patients with idiopathic pulmonary arterial hypertension (previously referred to as primary pulmonary hypertension), systemic arterial pressure is usually within the normal range. The divergent vascular responses to hypoxia and the alternative existence of systemic or pulmonary arterial hypertension in patients indicate that the pulmonary vasculature or the pulmonary circulation is unique in terms of its anatomic and histological structure, physiological and pharmacological properties, genetic and epigenetic development as well as cellular and molecular determinants for vasoconstriction, vascular-wall remodeling, and embolus formation. Therefore, the pathogenic mechanisms of pulmonary vascular diseases are rather different from those of systemic circulatory disorders. Development of therapeutic approaches and improvement of clinical management for patients with pulmonary vascular diseases should be directed by understanding the unique physiological and pathological features of the pulmonary vasculature at organ, tissue, cell, and molecular levels. Although many books have addressed clinical aspects of cardiovascular diseases, systemic arterial hypertension and basic science progress about structural and functional studies on systemic arteries (e.g., coronary, cerebral, and other peripheral arteries and microcirculation), very few books have focused on the pulmonary circulation and pulmonary vascular disease. Given the significant differences between the pulmonary and systemic vasculature and between systemic and pulmonary vascular diseases, it is urgent to have a comprehensive reference book specifically designated to describe a) basic structure and function of the pulmonary vasculature or the pulmonary circulation, b) pathophysiology of the pulmonary circulatory system, and c) clinical aspects (diagnosis, treatment, and prevention) of pulmonary vascular diseases. Textbook of Pulmonary Vascular Disease is therefore designed for and is of special interest to a) clinicians (pulmonologists, cardiologists, intensive care physicians, cardiothoracic and vascular surgeons, and emergency physicians), b) physician-scientists and basic-science researchers in the fields of cardiopulmonary and critical care medicine, vascular physiology and pathophysiology, translational medical research, and bioengineering, c) healthcare workers in cardiopulmonary and critical care medicine, and d) clinical and research fellows as well as residents, medical and graduate students. Textbook of Pulmonary Vascular Disease combines basic scientific concepts and knowledge on the pulmonary circulation with clinical diagnosis and treatment on pulmonary vascular diseases. Textbook of Pulmonary Vascular Disease is unique in that no book currently available i) focuses on elucidating the cellular and molecular regulation of normal pulmonary vasculature and the pathogenic mechanisms of pulmonary vascular diseases, ii) includes advanced techniques and technology for basic and clinical research, and iii) includes conventional and molecular approaches currently available for v

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diagnosis and treatment of patients with pulmonary vascular diseases. Another feature of the book is the inclusion of surgical approaches for treatment of pulmonary vascular diseases, which have not been well described in previously published books. Textbook of Pulmonary Vascular Disease is divided into five parts. Part 1 (Structure, Function and Regulation), consisting of nine sections and twenty-nine chapters, is designated for basic knowledge and recent findings related to pulmonary vascular structure, function, and regulation at levels of molecule, cell, tissue, organ and system. Part 2 (Methodological Approaches for Research) is composed of six sections and sixteen chapters that are designed to provide a basic knowledge and spectrum on the techniques and technology that are commonly used to study genetic, molecular, cellular, systemic and pathophysiological aspects of the pulmonary vasculature. Part 3 (Pathology and Pathobiology) includes four sections and nineteen chapters that discuss the potential mechanisms or sequence of events involved in the initiation and progression of abnormalities in the pulmonary vasculature in patients with pulmonary vascular disease. Part 4 (Pulmonary Vascular Diseases) consists of nine sections and thirty-two chapters devoted to describe pathogenesis, epidemiology and pathophysiology of almost all of the pulmonary vascular diseases identified so far by the World Health Organization (WHO). One of the sections is specifically designated to describe pulmonary vascular disease in pediatric patients. Part 5 (Diagnosis and Treatment) includes three sections and twenty chapters designed to illustrate, in details, the diagnostic and therapeutic procedures currently used for patients with pulmonary vascular disease. Textbook of Pulmonary Vascular Disease is written by more than 220 experts in the field including physicians, surgeons, epidemiologists, bioinformaticians, nurses, physician scientists and investigators. All of the contributors are actively involved in clinical, physiological, and pathophysiological studies on the pulmonary circulation and pulmonary vascular diseases. The vast majority of authors are recognized experts in the research area of the topic on which the chapter is based with many contributors also Fellows of the Pulmonary Vascular Research Institute, a not-for-profit, international scientific association focused on the pulmonary circulation and pulmonary vascular disease. Founded in 2007, the Pulmonary Vascular Research Institute (PVRI) has assembled clinical, epidemiological, translational and basic scientists from around the world to perform research and advance education regarding pulmonary vascular disease and right heart failure. There is a focus on performing research in and providing education and treatment to underserved populations of the world. The unique strength of the PVRI is that it brings together a multidisciplinary faculty from around the world within a single focused institute. PVRI members have the expertise to conduct basic, translational, and clinical research at a level that no single academic institution can offer. The publication of an authoritative textbook on pulmonary vascular disease was an initial goal of the Institute. Textbook of Pulmonary Vascular Disease will not only serve as a reference book for physicians, surgeons, private practitioners, translational medical researchers, clinical and research fellows, and medical and graduate students, but also can be used as a guidance manual for technical and marketing personnel in pharmaceutical and biotechnological companies, that are interested in clinical and basic science research in cardiopulmonary diseases, pulmonary vascular diseases, vascular biology, and lung/heart transplantation. We hope that this book will also allow readers to foster new concepts and new collaboration and cooperation among clinicians, physician scientists and investigators so as to further understand the pathogenic mechanisms of pulmonary vascular disease and develop novel therapeutic approaches for the disease. La Jolla, California May 9, 2010

Jason X.-J. Yuan, MD, PhD Joe G.N. (Skip) Garcia, MD Charles A. Hales, MD Stuart Rich, MD Stephen L. Archer, MD John B. West, MD, PhD, DSc

Acknowledgements

The book could not have been completed without the support and encouragement of our families (Ayako Makino, Sue Garcia, Mary Ann Hales, Kathie Doliszny, Andrea Rich, and Penelope West) as well as our colleagues and students at the University of California, San Diego, the University of Chicago, the Massachusetts General Hospital at Harvard Medical School (Boston, MA) and the University of Illinois at Chicago. We are especially grateful to Ms. Melissa Ramondetta at Springer for her encouragement and support in compiling the book, to Dr. Carmelle V. Remillard for her diligence in preparing the figures and editing the text, to Ms. Portia Bridges for tirelessly going through all the details, and to all the contributors for their patience and conscientiousness in writing the manuscripts. In addition, we thank the students, research fellows, and staff of our laboratories who have made considerable contributions to collecting data, selecting representative figures, making schematic diagrams, and reading through the manuscript for grammatical and typographic errors. We sincerely thank Amy Zeifman, Michael Song, Frank Kuhr and Adriana Zimnicka for their diligent work on proofreading and copyediting the galley proofs. Finally, we would like to take this opportunity to thank the publisher, Springer, for its interest in developing such a comprehensive reference book for the field (and readers) of pulmonary circulation and pulmonary vascular disease.

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Contents

Part I  Structure, Function and Regulation The Human Pulmonary Circulation   1  The Human Pulmonary Circulation: Historical Introduction........................... John B. West

3

Structure and Function of the Pulmonary Circulation   2  Microcirculation of the Lung: Functional and Anatomic Aspects..................... Joan Gil

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  3  Pulmonary Vascular Development........................................................................ Rosemary C. Jones and Diane E. Capen

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  4  Pulmonary Vascular Function............................................................................... Robert Naeije and Nico Westerhof

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  5  Pulmonary Vascular Mechanics............................................................................ Alejandro Roldán-Alzate and Naomi C. Chesler

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  6  Modeling of the Pulmonary Vasculature.............................................................. Merryn H. Tawhai and Kelly S. Burrowes

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  7  Metabolic and Clearance Function at the Pulmonary Microvascular Endothelial Surface in Pulmonary Hypertension...................... Stylianos E. Orfanos and David Langleben

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Pulmonary Vasomotor Tone, Vascular Reactivity, and Vascular Permeability   8  The Influence of the Major Vasoactive Mediators Relevant to the Pathogenesis of Pulmonary Hypertension................................. Margaret R. MacLean and Yvonne Dempsie   9  The Normal Fetal and Neonatal Pulmonary Circulation.................................... Steven H. Abman and Robin H. Steinhorn 10  Excitation–Contraction Coupling and Regulation of Pulmonary Vascular Contractility.................................................................... Jeremy P.T. Ward and Greg A. Knock

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11  Endothelial Regulation of Pulmonary Vascular Tone......................................... Stephen Y. Chan and Joseph Loscalzo 12  Acute Lung Injury: The Injured Lung Endothelium, Therapeutic Strategies for Barrier Protection, and Vascular Biomarkers........ Eddie T. Chiang, Ting Wang, and Joe G.N. Garcia

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Signal Transduction 13  Ion Channels and Transporters in the Pulmonary Vasculature: A Focus on Smooth Muscle.................................................................................... Amy L. Firth and Jason X.-J. Yuan 14  Receptor-Mediated Signal Transduction and Cell Signaling.............................. Fiona Murray, Jason X.-J. Yuan, and Paul A. Insel

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15  Role of Calcium as a Second Messenger in Signaling: A Focus on Endothelium........................................................................................ Donna L. Cioffi, Christina J. Barry, and Troy Stevens

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16  Caveolae and Signaling in Pulmonary Vascular Endothelial and Smooth Muscle Cells.................................................................. Geerten P. van Nieuw Amerongen, Richard D. Minshall, and Asrar B. Malik

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Oxygen Sensing and Reactive Oxygen Specials in the Lung 17  The Chemistry of Biological Gases........................................................................ D. Jeannean Carver and Lisa A. Palmer

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18  Role of Oxygen-Derived Species in the Regulation of Pulmonary Vascular Tone.................................................................................. Michael S. Wolin, Mansoor Ahmad, and Sachin A. Gupte

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19  Mitochondrial Reactive Oxygen Species and Redox State in Pulmonary Vascular O2 Sensing........................................................................ Stephen L. Archer and John J. Ryan

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Cell Proliferation and Apoptosis 20  Cellular and Molecular Mechanisms of Pulmonary Vascular Smooth Muscle Cell Proliferation......................................................... Tamara Tajsic and Nicholas W. Morrell

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21  Role of Ca2+ in Vascular Smooth Muscle Gene Expression and Proliferation................................................................................. Karen M. Lounsbury and Patricia C. Rose

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22  Biochemistry and Cellular Mechanisms of Apoptosis in Vascular Smooth Muscle and Endothelial Cells.............................................. Oliver Eickelberg and Fotini M. Kouri

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Coagulation, Thrombosis, and Fibrinolysis 23  The Coagulation Cascade and Its Regulation...................................................... James T.B. Crawley, Jose R. Gonzalez-Porras, and David A. Lane

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24  Platelets in Pulmonary Vascular Physiology and Pathology............................... Michael H. Kroll

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25  Lysis and Organization of Pulmonary Thromboemboli...................................... Timothy A. Morris and Debby Ngo

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Interactions of Pulmonary Vascular Cells with Circulating Blood Cells 26  Interactions of Leukocytes and Coagulation Factors with the Vessel Wall................................................................................... Scott Visovatti, Takashi Ohtsuka, and David J. Pinsky 27  Interaction of the Plasminogen System with the Vessel Wall.............................. Riku Das and Edward F. Plow 28  Endothelial Apoptosis and Repair in Pulmonary Arterial Hypertension............................................................................................. Rohit Moudgil, Manoj M. Lalu, and Duncan J. Stewart

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The Systemic Circulation in the Lung 29  Bronchial Arterial Circulation in the Human...................................................... Gabor Horvath and Adam Wanner

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Part II  Methodological Approaches for Research In vivo and Genetic Animal Models 30  Animal Models of Pulmonary Hypertension........................................................ Jocelyn Dupuis and Norbert Weissmann 31  Transgenic and Gene-Targeted Mouse Models for Pulmonary Hypertension................................................................................. James D. West 32  Animal Models of Increased Lung Vascular Permeability................................. Sara Hanif Mirza, M. Kamran Mirza, and Asrar B. Malik

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Morphological, Functional, and Regulatory Approaches 33  Isolation and Culture of Pulmonary Vascular Smooth Muscle and Endothelial Cells................................................................................. Carmelle V. Remillard, Ayako Makino, and Jason X.-J. Yuan

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34  Conventional Patch Clamp Techniques and High-Throughput Patch Clamp Recordings on a Chip for Measuring Ion Channel Activity................... Carmelle V. Remillard and Jason X.-J. Yuan

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35  Measurement of Pulmonary Vascular Structure and Pulmonary Blood Distribution by Multidetector-Row Computed Tomography and Magnetic Resonance Imaging Techniques........... Sara K. Alford and Eric A. Hoffman

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Molecular Biological Techniques 36  Quantification of DNA, RNA, and Protein Expression....................................... Fiona Murray, Jason X.-J. Yuan, and Paul A. Insel

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37  Gene Cloning, Transfection, and Mutagenesis..................................................... Ellen C. Breen and Jason X.-J. Yuan

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38  Approaches for Manipulation of Gene Expression.............................................. Ying Yu and Jason X.-J. Yuan

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Genomic, Proteomic and Bioinformatical Approaches 39  Bioinformatics, Genomics, and Functional Genomics: Overview...................... Ali Torkamani, Eric J. Topol, and Nicholas J. Schork

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40  Genomic Applications to Study Pulmonary Hypertension................................. Todd M. Bull and Mark W. Geraci

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41  Proteomics and Functional Proteomics................................................................. Dayue Darrel Duan

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Stem Cells 42  Maintenance, Propagation, and Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells.............................. Zoë L. Vomberg, Megan Robinson, Thomas Fellner, and Karl H. Willert

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43  Identification of Adult Stem and Progenitor Cells in the Pulmonary Vasculature...................................................................... Amy L. Firth, Weijuan Yao, and Jason X.-J. Yuan

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44  Differentiation of Embryonic Stem Cells to Vascular Cell Lineages................. Andriana Margariti, Lingfang Zeng, and Qingbo Xu

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Clinical Research Approaches 45  Statistics and Clinical Data Analysis: A Reference Guide.................................. Michael C. Donohue, Tanya Wolfson, and Anthony C. Gamst

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Part III  Pathology and Pathobiology Hypoxia and Hyperoxia in the Development of Pulmonary Hypertension 46  Hypoxic Pulmonary Vasoconstriction................................................................... E. Kenneth Weir, Jesús A. Cabrera, Douglas A. Peterson, Saswati Mahapatra, and Zhigang Hong 47  Pathogenic Roles of Ca2+ and Ion Channels in Hypoxia-Mediated Pulmonary Hypertension....................................................................................... Jian Wang, Dandan Zhang, Carmelle V. Remillard, and Jason X.-J. Yuan 48  Roles of Endothelium-Derived Vasoactive and Mitogenic Factors in the Development of Chronic-Hypoxia-Mediated Pulmonary Hypertension....................................................................................... Rajeev Malhotra and Kenneth D. Bloch

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49  Oxygen-Sensitive Transcription Factors and Hypoxia-Mediated Pulmonary Hypertension....................................................................................... Louise Østergaard, Vinzenz H. Schmid, and Max Gassmann

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50  Developmental Regulation of Pulmonary Vascular Oxygen Sensing................. David N. Cornfield

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51  Pulmonary Vascular Remodeling by High Oxygen............................................. Rosemary C. Jones and Diane E. Capen

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Pulmonary Vasoconstriction and Vascular Remodeling in Pulmonary Hypertension 52  Pulmonary Vascular Remodeling: Cellular and Molecular Mechanisms................................................................................... Kurt R. Stenmark and Maria G. Frid

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53  Carbon Monoxide and Heme Oxygenase in the Regulation of Pulmonary Vascular Function and Structure.................................................. Stella Kourembanas

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54  Shear Stress, Cell Signaling, and Pulmonary Vascular Remodeling................. Shampa Chatterjee and Aron B. Fisher

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55  Pulmonary Hypertension and the Extracellular Matrix..................................... Marlene Rabinovitch

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56  Role of Progenitor Cells in Pulmonary Vascular Remodeling............................ Kurt R. Stenmark, Susan M. Majka, and Maria G. Frid

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57  Receptor Signaling in Pulmonary Arterial Hypertension................................... Patricia A. Thistlethwaite, Robin N. Leathers, Xioadong Li, and Xiaoxue Zhang

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Endothelium and Thrombosis in the Development of Pulmonary Hypertension 58  Role of Endothelium in the Development of Pulmonary Hypertension............. Bryan Ross and Adel Giaid

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59  Coagulation and the Vessel Wall in Pulmonary Embolism................................. Irene M. Lang

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Pulmonary Edema and Acute Lung Injury 60  Alveolar Epithelial Fluid Transport in Lung Injury........................................... Hans G. Folkesson

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61  High-Altitude Pulmonary Edema.......................................................................... Erik R. Swenson

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62  Statins and Acute Lung Injury.............................................................................. Amit K. Mahajan and Jeffery R. Jacobson

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63  Genomics of Acute Lung Injury and Vascular Barrier Dysfunction................. Roberto F. Machado and Joe G.N. Garcia

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64  Ventilator-Induced Mechanical Stress and Lung Vascular Dysfunction........... Konstantin G. Birukov

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Part IV  Pulmonary Vascular Diseases Nomenclature, Classification and Epidemiology of Pulmonary Hypertension 65  Classification of Pulmonary Hypertension: History and Perspectives.............. David B. Badesch

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66  Epidemiology of Pulmonary Arterial Hypertension............................................ Jess Mandel and Darren B. Taichman

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67  Air Pollution and the Pulmonary Vasculature..................................................... Melissa L. Bates and Rebecca Bascom

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Pulmonary Arterial Hypertension 68  Idiopathic Pulmonary Arterial Hypertension...................................................... Jane E. Lewis and Richard N. Channick

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69  Genetics of Familial and Idiopathic Pulmonary Arterial Hypertension........... Eric D. Austin, James E. Loyd, and John A. PhillipsIII

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70  Pulmonary Arterial Hypertension Related to Scleroderma and Collagen Vascular Diseases............................................................................. 1011 Paul M. Hassoun 71  Pathology and Management of Portopulmonary Hypertension......................... 1023 Michael J. Krowka 72  Pathobiology and Treatment of Pulmonary Hypertension in HIV Disease................................................................................. 1033 Michael H. Ieong and Harrison W. Farber 73  Pulmonary Arterial Hypertension Secondary to Anorexigensand Other Drugs and Toxins........................................................ 1043 Kim Bouillon, Yola Moride, Lamiae Bensouda-Grimaldi, and Lucien Abenhaim 74  Pulmonary Arterial Hypertension Related to Gaucher’s Disease, Sarcoidosis, and Other Disorders........................................................... 1061 Judd W. Landsberg and Jess Mandel Pulmonary Arterial Hypertension in Pediatric Patients 75  Pediatric Pulmonary Hypertension: An Integrated View from Pediatric Subspecialists....................................................................... 1083 Judy L. Aschner, Candice D. Fike, Eric D. Austin, Frederick E. Barr, and J. Donald Moore 76  Persistent Pulmonary Hypertension of the Newborn: Mechanisms and Treatment................................................................................... 1109 Steven H. Abman, Robin H. Steinhorn, and Judy L. Aschner

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77  The Pulmonary Circulation in Congenital Heart Disease................................... 1119 Thomas J. Kulik and Mary P. Mullen 78  Pulmonary Hypertension Secondary to Congenital Systemic-to-Pulmonary (Left-to-Right) Shunts................................................... 1139 Antonio A. Lopes 79  Surgical Evaluation of Congenital-Heart-Disease-Associated Pulmonary Hypertension....................................................................................... 1153 Clive J. Lewis and Andrew A. Klein Pulmonary Venous Hypertension 80  Pulmonary Veno-occlusive Disease........................................................................ 1169 Peter F. Clardy and Jess Mandel 81  Left Ventricular Diastolic Heart Function and Pulmonary Hypertension........ 1183 Stuart Rich and Mardi Gomberg-Maitland Pulmonary Hypertension Associated with Hypoxia and Hypoxemia 82  Pulmonary Hypertension Associated with Chronic Obstructive Pulmonary Diseases........................................................................... 1189 Norbert F. Voelkel, Catherine Grossman, and Herman J. Bogaard 83  Pulmonary Hypertension Associated with Interstitial Lung Disease................. 1197 Mary E. Strek and Julian Solway 84  High-Altitude Pulmonary Hypertension.............................................................. 1211 Fabiola León-Velarde and Francisco C. Villafuerte 85  Pulmonary Hypertension and Congenital Heart Defects at High Altitude......................................................................................... 1223 Alexandra Heath de Freudenthal, Franz-Peter Freudenthal Tichauer, Carmen R. Condori Taboada, and Janne C. Lopes Mendes Pulmonary Hypertension due to Chronic Thrombotic and/or Embolic Disease 86  Pulmonary Embolism and Deep Vein Thrombosis.............................................. 1231 Paul F. Currier and Charles A. Hales 87  Pulmonary Hypertension Due to Pulmonary Embolism and Thromboembolic Obstruction of Proximal and Distal Pulmonary Arteries.............................................................................. 1239 Peter F. Fedullo, Kim M. Kerr, and William R. Auger 88  Risk Factors for Chronic Thromboembolic Pulmonary Hypertension............. 1253 Diana Bonderman and Irene M. Lang 89  Evaluation of Small-Vessel Arteriopathy in Chronic Thromboembolic Pulmonary Hypertension......................................................... 1261 Nick H. Kim

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  90  Hemolytic-Anemia-Associated Pulmonary Hypertension: Sickle-Cell-Disease- and Thalassemia-Associated Pulmonary Hypertension..................................................................................... 1269 Elizabeth S. Klings and Mark T. Gladwin Pulmonary Hypertension due to Disorders Directly Affecting the Pulmonary Vasculature   91  Pulmonary Hypertension Due to Schistosomiasis.............................................. 1283 Ghazwan Butrous and Angela P. Bandiera   92  Pulmonary Hypertension Due to Capillary Hemangiomatosis........................ 1297 Mourad Toporsian, David H. Roberts, and S. Ananth Karumanchi Right Heart Dysfunction and Pulmonary Hypertension   93  Molecular Basis of Right Ventricular Hypertrophy and Failure in Pulmonary Vascular Disease....................................................... 1305 Yuichiro J. Suzuki   94  Right Ventricular Dysfunction in Pulmonary Hypertension............................ 1313 Francois Haddad, Mehdi Skhiri, and Evangelos Michelakis Other Disorders of the Pulmonary Circulation   95  Large Vessel Pulmonary Arteritis....................................................................... 1333 Kim M. Kerr   96  Tumors of the Pulmonary Vascular Bed............................................................. 1343 Eunhee S. Yi   97  Cor Pulmonale....................................................................................................... 1355 Mardi Gomberg-Maitland, Thenappan Thenappan, John J. Ryan, Ankush Goel, Nicole Cipriani,Aliya N. Husain, Amit Patel, Savitri E. Fedson, and Stephen L. Archer   98  Pregnancy and Contraception in Patients with Pulmonary Arterial Hypertension........................................................................................... 1377 Barbara A. Cockrill and Charles A. Hales Part V  Diagnosis and Treatment Diagnostic Approaches for Pulmonary Vascular Diseases   99  Cardiac Catheterization in the Patient with Pulmonary Hypertension.......... 1387 Christopher Barnett and Ori Ben-Yehuda 100  Imaging of Pulmonary Vascular Diseases........................................................... 1403 Michael A. Bettmann 101  Histological and Pathological Diagnosis of Pulmonary Hypertension: Pathological Classification of Pulmonary Vascular Lesions.................................................................................................... 1413 Brian B. Graham, Li Zhang, and Rubin M. Tuder

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102  Echocardiography in Pulmonary Vascular Disease........................................... 1425 Paul R. Forfia Therapeutic Interventions for Pulmonary Vascular Diseases 103  Calcium Channel Blockers in the Treatment of Pulmonary Arterial Hypertension.................................................................. 1447 Stuart Rich and Mardi Gomberg-Maitland 104  Prostacyclin and Prostaglandins.......................................................................... 1451 Horst Olschewski 105  Endothelin Receptor Antagonists for the Treatment of Pulmonary Arterial Hypertension.................................................................. 1465 Victor F. Tapson and Robyn J. Barst 106  Phosphodiesterase Inhibitors in the Treatment of Pulmonary Hypertension................................................................................. 1477 Lan Zhao, Zhenguo Zhai, John Wharton, and Martin R. Wilkins 107  Nitric Oxide for Children..................................................................................... 1487 Judy L. Aschner, Candice D. Fike, Eric Austin, J. Donald Moore, and Frederick E. Barr 108  The Serotonin System as a Therapeutic Target in Pulmonary Hypertension................................................................................. 1501 Serge Adnot 109  Combination Therapy for Pulmonary Arterial Hypertension......................... 1509 Ioana R. Preston and Nicholas S. Hill 110  Thrombolytic and Anticoagulant Therapy for Pulmonary Embolism and Chronic Thromboembolic Pulmonary Hypertension.............. 1521 Paul F. Currier and Charles A. Hales 111  Nursing Care of Patients with Pulmonary Arterial Hypertension................... 1531 Christine Archer-Chicko Interventional and Surgical Approaches to Treatment of Pulmonary Vascular Diseases 112  Atrial Septostomy.................................................................................................. 1559 Julio Sandoval, Jorge Gaspar, and Héctor Peña 113  Evaluation of Patients with Chronic Thromboembolic Pulmonary Hypertension for Pulmonary Endarterectomy.............................. 1567 William R. Auger, Peter F. Fedullo, and Kim M. Kerr 114  Pulmonary Endarterectomy................................................................................ 1575 Michael M. Madani and Stuart W. Jamieson 115  Evaluation of Patients with Pulmonary Hypertension for Lung Transplantation..................................................................................... 1591 Gordon L. Yung

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116  Lung Transplantation for Pulmonary Hypertension......................................... 1597 Ashish S. Shah and John V. Conte Jr. 117  Living-Donor Lobar Lung Transplantation for Pulmonary Arterial Hypertension................................................................ 1601 Hiroshi Date and Takahiro Oto 118  Results of Lung Transplantation......................................................................... 1611 Janet R. Maurer Index................................................................................................................................. 1625

Contents

Contributors

Lucien Abenhaim, MD, PhD (Ch: 73) Professor, London School of Hygiene and Tropical Medicine, London, UK Steven H. Abman, MD (Ch: 9, 76) Professor, Department of Pediatrics, University of Colorado, The Children’s Hospital, Denver CO, USA Serge Adnot, MD, PhD (Ch: 108) Professor, Department of Physiology, Henri Mondor Hospital, Creteil, France Mansoor Ahmad, MD, PhD (Ch: 18) Research Associate, Department of Physiology, New York Medical College, Valhalla, NY, USA Sara K. Alford, MS (Ch: 35) Predoctoral Research Fellow, Department of Radiology, University of Iowa, Iowa City, IA, USA Stephen L. Archer, MD (Ch: 19, 97) Harold Hines Jr. Professor and Chief of Cardiology, Department of Medicine, University of Chicago, Chicago, IL, USA Christine Archer-Chicko, MSN, CRNP-BC (Ch: 111) Program Coordinator, Pulmonary Vascular Disease Program, Penn Presbyterian Medical Center, Philadelphia, PA, USA Judy L. Aschner, MD (Ch: 75, 76, 107) Department of Pediatrics, The Monroe Carell Jr. Children’s Hospital, Vanderbilt University School of Medicine, Nashville, TN, USA William R. Auger, MD (Ch: 87, 113) Division of Pulmonary Critical Care Medicine, University of California, San Diego, CA, USA Eric D. Austin, MD (Ch: 69, 75) Assistant Professor, Department of Pediatrics, The Monroe Carell Jr. Children's Hospital, Vanderbilt University, Nashville, TN, USA David B. Badesch, MD (Ch: 65) Professor, Department of Medicine, University of Colorado, Denver, Aurora, CO, USA

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Angela P. Bandiera, MD, PhD (Ch: 91) Consultant Cardiologist, Department of Cardiology, Health Sciences Centre, PROCAPE – University of Pernambuco and Memorials S. Jose Hospital, Recife, Brazil Christopher Barnett, MD, MPH (Ch: 99) Assistant Professor, Department of Cardiology, University of California, San Francisco, CA, USA Frederick E. Barr, MD (Ch: 75, 107) Professor and Division Chief, Department of Pediatrics, The Monroe Carell Jr. Children’s Hospital, Vanderbilt University, Nashville, USA Christina J. Barry, BS (Ch: 15) Graduate Student, Center for Lung Biology, University of South Alabama, Mobil, AL, USA Robyn J. Barst, MD (Ch: 105) Professor, Columbia University College of Physicians and Surgeons, New York, NY, USA Rebecca Bascom, MD, MPH (Ch: 67) Professor, Department of Pulmonary Allergy and Critical Care Medicine, Milton S. Hershey Medical Center, Hershey, PA, USA Melissa L. Bates, PhD (Ch: 67) Postdoctoral Fellow, Department of Pediatrics, University of Wisconsin, Madison, WI, USA Ori Ben-Yehuda, MD, FACC (Ch: 99) Professor, Department of Medicine, University of California, San Diego, San Diego, CA, USA Lamiae Bensouda-Grimaldi, PharmD (Ch: 73) Doctor, Department Paris Santé Cochin, Inserm/La-ser, Paris, France Michael A. Bettmann, MD (Ch: 100) Professor and Vice Chair for Interventional Services, Department of Radiology, Wake Forest School of Medicine, Winston-Salem, NC, USA Konstantin G. Birukov, MD, PhD (Ch: 64) Assistant Professor, Department of Medicine, University of Chicago, Chicago, IL, USA Kenneth D. Bloch, MD (Ch: 48) William Thomas Green Morton Professor, Departments of Anesthesia and Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA Herman J. Bogaard, MD, PhD (Ch: 82) Assistant Professor, Department of Internal Medicine, Virginia Commonwealth University, Richmond, VA, USA Diana Bonderman, MD (Ch: 88) Assistant Professor, Department of Cardiology, Medical University of Vienna, Vienna, Austria Kim Bouillon, MD, MPH (Ch: 73) Doctor, Paris Santé Cochin, La-ser, Paris, France Ellen C. Breen, PhD (Ch: 37) Associate Research Scientist, Department of Medicine, University of California, San Diego, La Jolla, CA, USA

Contributors

Contributors

Todd M. Bull, MD (Ch: 40) Associate Professor, Department of Medicine, University of Colorado, Denver, Aurora, CO, USA Kelly S. Burrowes, PhD (Ch: 6) Postdoctoral Research Fellow, Oxford University, Oxford, UK Ghazwan Butrous, MB, ChB, PhD (Ch: 91) Professor, Division of Cardiopulmonary Sciences, University of Kent, Canterbury, UK Jesús A. Cabrera, MD, PhD (Ch: 46) Assistant Professor, Department of Surgery, University of Minnesota, Minneapolis, MN, USA Diane E. Capen, BA (Ch: 3, 51) Department of Anesthesia, Critical Care and Pain Management, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA D. Jeannean Carver, MD (Ch: 17) Associate Professor, Department of Pediatrics, University of Virginia, Charlottesville, VA, USA Stephen Y. Chan, MD, PhD (Ch: 11) Instructor, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Richard N. Channick, MD (Ch: 68) Associate Professor and Director, Pulmonary Hypertension Program, Department of Pulmonary and Critical Care Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA Shampa Chatterjee, PhD (Ch: 54) Research Assistant Professor, Institute for Environmental Medicine, University of Pennsylvania, Philadelphia, PA, USA Naomi C. Chesler, PhD (Ch: 5) Associate Professor, Department of Biomedical Engineering, University of Wisconsin, Madison, WI, USA Eddie T. Chiang, MA (Ch: 12) Senior Research Technician, Department of Medicine, University of Illinois at Chicago, Chicago, IL, USA Nicole Cipriani, MD (Ch: 97) Fellow, Department of Pathology, University of Chicago, Chicago IL, USA Peter F. Clardy, MD (Ch: 80) Clinical Instructor, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA Barbara A. Cockrill, MD (Ch: 98) Clinical Director, Pulmonary Vascular Disease Center, Brigham and Women's Hospital, Harvard Medical School, Boston, MA, USA Donna L. Cioffi, PhD (Ch: 15) Assistant Professor, Center for Lung Biology, University of South Alabama, Mobile, AL, USA John V. Conte, Jr. MD (Ch: 116) Professor and Director, Heart Transplantation and Mechanical Circulatory Support, Johns Hopkins University School of Medicine, Baltimore, MD, USA David N. Cornfield, MD (Ch: 50) Department of Pediatrics – Pulmonary Medicine, Lucile Salter Packard Children’s Hospital at Stanford, Stanford, CA, USA James T. B. Crawley, PhD (Ch: 23) Department of Haematology, Imperial College London, Hammersmith Hospital, London, UK

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Paul F. Currier, MD, MPH (Ch: 86, 110) Associate Program Director, Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA Riku Das, PhD (Ch: 27) Postdoctoral Fellow, Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Hiroshi Date, MD (Ch: 117) Professor, Department of Thoracic Surgery, Kyoto University, Kyoto, Japan Yvonne Dempsie, PhD (Ch: 8) Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow, UK Michael C. Donohue, PhD (Ch: 45) Assistant Project Scientist, Division of Biostatistics and Bioinformatics, University of California, San Diego, La Jolla, CA, USA Dayue Darrel Duan, MD, PhD (Ch: 41) Professor, Department of Pharmacology, University of Nevada School of Medicine, Reno, NV, USA Jocelyn Dupuis, MD, PhD (Ch: 30) Associate Professor, Department of Medicine, Montreal Heart Institute, Montreal, Quebec, Canada Oliver Eickelberg, MD (Ch: 22) Professor, Institute of Lung Biology and Disease, Comprehensive Pneumology Center, Neuherberg/Munich, Bavaria, Germany Harrison W. Farber, MD (Ch: 72) Professor, Pulmonary Center, Boston University, Boston, MA, USA Savitri E. Fedson, MD (Ch: 97) Assistant Professor and Medical Director of Cardiac Care Unit, University of Chicago, Chicago, IL, USA Peter F. Fedullo, MD (Ch: 80, 87, 113) Division of Pulmonary Critical Care Medicine, University of California, San Diego, CA, USA Thomas Fellner, PhD (Ch: 42) Assistant Director, Human Embryonic Stem Cell Core Facility, University of California, San Diego, La Jolla, CA, USA Candice D. Fike, MD (Ch: 75, 107) Professor, Department of Pediatrics, The Monroe Carell Jr. Children’s Hospital, Vanderbilt University, Nashville, TN, USA Amy L. Firth, PhD (Ch: 13, 43) Postdoctoral Fellow, Laboratory of Genetics, The Salk Institute for Biological Studies, La Jolla, CA, USA Aron B. Fisher, MD (Ch: 54) Professor and Director, Institute for Environmental Medicine, University of Pennsylvania, Philadelphia, PA, USA Hans G. Folkesson, PhD (Ch: 60) Department of Physiology and Pharmacology, Northeastern Ohio Universities College of Medicine, Rootstown, OH, USA

Contributors

Contributors

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Paul R. Forfia, MD (Ch: 102) Professor and Director, Institute for Environmental Medicine, Department of Medicine/Cardiology, University of Pennsylvania, Philadelphia, PA, USA Franz-Peter Freudenthal Tichauer, MD (Ch: 85) Department of Interventional Cardiology, Kardiozentrum Medical Specialty Center, La Paz, Bolivia Maria G. Frid, PhD (Ch: 52, 56) Instructor, Department of Pediatric Critical Care, University of Colorado, Denver, Aurora, CO, USA Anthony C. Gamst, PhD (Ch: 45) Associate Professor, Division of Biostatistics and Bioinformatics, University of California, San Diego, La Jolla, CA, USA Joe G. N. Garcia, MD (Ch: 63, 12) University of Illinois at Chicago, Chicago, IL, USA Jorge Gaspar, MD (Ch: 112) Chief, Interventional Cardiology Department, Ignacio Chávez National Institute of Cardiology, Mexico City, Mexico Max Gassmann, DVM (Ch: 49) Professor, Vetsuisse Faculty, Institute of Veterinary Physiology and Zurich Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland Mark W. Geraci, MD (Ch: 40) Professor and Division Head, Department of Medicine, University of Colorado, Denver, Aurora, CO, USA Adel Giaid, MD, PhD (Ch: 58) Department of Cardiology, McGill University Health Centre, Montreal, Quebec, Canada Joan Gil, MD (Ch: 2) Professor,Department of Pathology, Mount Sinai School of Medicine, New York, NY, USA Mark T. Gladwin, MD (Ch: 90) Professor and Chief, Division of Pulmonary, Allergy, and Critical Care Medicine, University of Pittsburgh, Pittsburgh, PA, USA Ankush Goel, MD (Ch: 97) Fellow, Department of Medicine, University of Chicago, Chicago, IL, USA Mardi Gomberg-Maitland, MD, MSc (Ch: 97, 103, 81) Associate Professor and Director of Pulmonary Hypertension, Department of Medicine/Cardiology, University of Chicago, Chicago, IL, USA Jose R. Gonzalez-Porras (Ch: 23) Department of Haematology, Imperial College London, Hammersmith Hospital, London, UK Brian B. Graham, MD (Ch: 101) Fellow, Department of Medicine, University of Colorado Hospital, Denver, CO, USA Catherine Grossman, MD (Ch: 82) Assistant Professor, Department of Internal Medicine, Virginia Commonwealth University, Richmond, VA, USA

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Sachin A. Gupte, MD, PhD (Ch: 18) Assistant Professor, Department of Biochemistry and Molecular Biology, University of South Alabama, Mobile, AL, USA Francois Haddad, MD (Ch: 94) Attending Cardiologist, Department of Medicine, Stanford University, Palo Alto, CA, USA Charles A. Hales, MD (Ch: 86, 98, 110) Professor, Pulmonary and Critical Care Unit, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA Sara Hanif Mirza, MD (Ch: 32) Internal Medicine Resident, Department of Medicine, University of Illinois at Chicago, Chicago, IL, USA Paul M. Hassoun, MD (Ch: 70) Professor and Director of Pulmonary Hypertension Program, Department of Medicine, Johns Hopkins University, Baltimore, MD, USA Alexandra Heath de Freudenthal, MD (Ch: 85) Head of Pediatric Cardiology, Kardiozentrum Medical Specialty Center, La Paz, Bolivia Nicholas S. Hill, MD (Ch: 109) Professor and Chief of Pulmonary, Division of Pulmonary, Critical Care and Sleep Medicine, Tufts Medical Center, Boston, MA, USA Eric A. Hoffman, PhD (Ch: 35) Professor, Department of Radiology, University of Iowa, Iowa City, IA, USA Zhigang Hong, MD, PhD (Ch: 46) Assistant Professor, Department of Medicine, University of Minnesota, VA Medical Center, Minneapolis, MN, USA Gabor Horvath, MD, PhD (Ch: 29) Associate Professor, Department of Pulmonology, Semmelweis University, Budapest, Hungary Aliya N. Husain, MD (Ch: 97) Professor, Department of Pathology, University of Chicago, Chicago, IL, USA Michael H. Ieong, MD (Ch: 72) Department of Allergy, Pulmonary, and Critical Care Medicine, Boston University Medical Center, Boston, MA, USA Paul A. Insel, MD (Ch: 14, 36) Professor and Vice Chair, Departments of Pharmacology and Medicine, University of California, San Diego, La Jolla, CA, USA Jeffrey R. Jacobson, MD (Ch: 62) Assistant Professor, University of Illinois at Chicago, Illinois, Chicago Stuart W. Jamieson, MD (Ch: 114) Distinguished Professor and Director of Cardiothoracic Surgery Division, Department of Surgery, University of California, San Diego, San Diego, CA, USA Rosemary C. Jones, PhD (Ch: 3, 51) Department of Anesthesia, Critical Care and Pain Management, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA

Contributors

Contributors

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S. Ananth Karumanchi, MD (Ch: 92) Associate Professor, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA Kim M. Kerr, MD (Ch: 87, 95, 113) Associate Professor, Department of Medicine, University of California, San Diego, La Jolla, CA, USA Nick H. Kim, MD (Ch: 89) Division of Pulmonary and Critical Care Medicine, University of California, San Diego, La Jolla, CA, USA Andrew A. Klein, MBBS (Ch: 79) Consultant Anaesthetist, Transplant Unit, Papworth Hospital NHS Trust, Cambridge, UK Elizabeth S. Klings, MD (Ch: 90) Pulmonary Center, Boston University, Boston, MA, USA Greg A. Knock, PhD (Ch: 10) Division of Asthma, Allergy and Lung Biology, King’s College London, London, UK Stella Kourembanas, MD (Ch: 53) Clement A. Smith Professor and Chief, Harvard Division of Newborn Medicine, Children’s Hospital, Harvard Medical School, Boston, MA, USA Fotini M. Kouri, PhD (Ch: 22) Department of Neurology, Northwestern University, Chicago, IL, USA Michael H. Kroll, MD (Ch: 24) Professor and Chief, Department of Benign Hematology, University of Texas, MD Anderson Cancer Center, Houston, TX, USA Michael J. Krowka, MD (Ch: 71) Professor and Vice-Chair, Division of Pulmonary and Critical Care Medicine, Mayo Clinic, Rochester, MN, USA Thomas J. Kulik, MD (Ch: 77) Senior Associate in Cardiology, Department of Cardiology, Children’s Hospital Boston, Boston, MA, USA Manoj M. Lalu, MD, PhD (Ch: 28) Resident Physician, Department of Anesthesiology, Ottawa Hospital, Ottawa, ON, Canada Judd W. Landsberg, MD (Ch: 74) Assistant Professor and Medical Director of Intensive Care Unit, VA San Diego Healthcare System, University of California, San Diego, La Jolla, CA, USA David A. Lane, PhD (Ch: 23) Professor, Department of Haematology, Imperial College London, Hammersmith Hospital, London, UK Irene M. Lang, MD (Ch: 59, 88) Professor, Department of Cardiology, Medical University of Vienna, Vienna, Austria David Langleben, MD (Ch: 7) Professor and Director, Center for Pulmonary Vascular Disease, Department of Cardiology, Jewish General Hospital, McGill University, Montreal, Quebec, Canada Robin N. Leathers, BS (Ch: 57) Staff Research Associate, Division of Cardiothoracic Surgery, University of California, San Diego, San Diego, CA, USA

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Fabiola León-Velarde, DSc (Ch: 84) Professor, Department of Clinical Biology and Physiology, Universidad Peruana Cayetano Heredia, Lima, Peru Clive J. Lewis, MB, BChir, PhD (Ch: 79) Consultant Cardiologist, Transplant Unit, Papworth Hospital NHS Trust, Cambridge, UK Jane E. Lewis, MD (Ch: 68) Postdoctoral Fellow, Department of Medicine, University of California, San Diego, La Jolla, CA, USA Xioadong Li, MD, PhD (Ch: 57) Project Scientist, Division of Cardiothoracic Surgery, University of California, San Diego, San Diego, CA, USA Antonio A. Lopes, MD (Ch: 78) Professor, Department of Pediatric Cardiology and Adult Congenital Heart Disease, Heart Institute, University of São Paulo School of Medicine, São Paulo, Brazil Joseph Loscalzo, MD, PhD (Ch: 11) Bersey Professor and Chairman, Department of Medicine, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA Karen M. Lounsbury, PhD (Ch: 21) Associate Professor, Department of Pharmacology, University of Vermont, Burlington, VT, USA James E. Loyd, MD (Ch: 69) Rudy W. Jacobson Professor, Department of Medicine, Vanderbilt University, Nashville, TN, USA Roberto F. Machado, MD (Ch: 63) Assistant Professor, Department of Medicine, University of Illinois at Chicago, Chicago, IL, USA Margaret R. MacLean, PhD (Ch: 8) Mandy MacLean College of Medical, Veterinary and Life Sciences, University of Glasgow, UK Michael M. Madani, MD (Ch: 114) Associate Professor, Department of Surgery, University of California, San Diego, San Diego, CA, USA Amit K. Mahajan, MD (Ch: 62) Fellow, Department of Medicine, University of Chicago, Chicago, IL, USA Saswati Mahapatra, MS (Ch: 46) Assistant Scientist, Department of Medicine, University of Minnesota, Minneapolis, MN, USA Susan M. Majka, PhD (Ch: 56) Assistant Professor, Department of Cardiovascular Pulmonary Research, University of Colorado, Denver, Aurora, CO, USA Ayako Makino, PhD (Ch: 33) Assistant Professor, Department of Medicine, University of Illinois at Chicago, Chicago IL, USA Rajeev Malhotra, MD (Ch: 48) Fellow, Massachusetts General Hospital, Harvard Medical School, Division of Cardiology, Boston, MA, USA

Contributors

Contributors

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Asrar B. Malik, PhD (Ch: 16, 32) Distinguished Professor and Head, Department of Pharmacology and Center for Lung and Vascular Biology, University of Illinois, Chicago, IL, USA Jess Mandel, MD (Ch: 66, 74, 80) Associate Professor, Division of Pulmonary and Critical Care Medicine, University of California, San Diego, La Jolla, CA, USA Andriana Margariti, PhD (Ch: 44) Cardiovascular Division, King’s College London, Coldharbour Lane, London, UK Janet R. Maurer, MD, MBA, MBC (Ch: 118) Vice President, Medical Director, Department of Provider Services and Coaching Effectiveness, Health Dialog Service Corporation, Desert Hills, AZ, USA Janne C. Lopes Mendes (Ch: 85) Master of Public Health/Epidemiology, General Medicine, Kardiozentrum Medical Specialty Center, La Paz, Bolivia Evangelos Michelakis, MD (Ch: 94) Professor, Division of Cardiovascular Medicine, University of Alberta, Edmonton, Canada Richard D. Minshall, PhD (Ch: 16) Associate Professor, Departments of Pharmacology and Anesthesiology and Center for Lung and Vascular Biology, University of Illinois at Chicago, Chicago, IL, USA M. Kamran Mirza, MD, PhD (Ch: 32) Postdoctoral Research Associate, Department of Pharmacology, University of Illinois at Chicago, Chicago, IL, USA J. Donald Moore, MD (Ch: 75, 107) Assistant Professor, Division of Cardiology, Vanderbilt Children’s Hospital, Nashville, TN, USA Yola Moride, PhD (Ch: 73) Associate Professor, Faculty of Pharmacy, University of Montreal, Montreal, Quebec, Canada Nicholas W. Morrell, MD (Ch: 20) Professor, Department of Medicine, University of Cambridge, Cambridge, UK Timothy A. Morris, MD (Ch: 25) Professor, Department of Medicine, University of California, San Diego, San Diego, CA, USA Rohit Moudgil, MD, PhD (Ch: 28) Postdoctoral Fellow, Department of Medicine, Ottawa General Hospital, Ottawa, Ontario, Canada Mary P. Mullen, MD, PhD (Ch: 77) Associate in Cardiology,Department of Cardiology, Children’s Hospital Boston, Harvard Medical School, Boston, MA, USA Fiona Murray, PhD (Ch: 14, 36) Postdoctoral Fellow, Departments of Medicine and Pharmacology, University of California, San Diego, La Jolla, CA, USA Robert Naeije, MD, PhD (Ch: 4) Professor, Laboratory of Physiology, Erasme Hospital, Brussels, Belgium

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Debby Ngo, MD (Ch: 25) Postdoctoral Fellow, Department of Medicine, University of California, San Diego, La Jolla, CA, USA Geerten P. van Nieuw Amerongen, PhD (Ch: 16) Assistant Professor, VU University Medical Center, Institute for Cardiovascular Research, Department of Physiology, Amsterdam, The Netherlands Takashi Ohtsuka, MD (Ch: 26) Research Fellow, Department of Internal Medicine, University of Michigan, Ann Arbor, MI, USA Horst Olschewski, MD, PhD (Ch: 104) Professor, Department of Internal Medicine, University Hospital, Giessen, Germany Stylianos E. Orfanos, MD, PhD (Ch: 7) Associate Professor, 2nd Department of Critical Care, University of Athens Medical School and Pulmonary Hypertension Clinic, Attikon Hospital, Haidari Athens, Greece Louise Østergaard, PhD (Ch: 49) Zurich Center for Integrative Human Physiology, Vetsuisse Faculty, Institute of Veterinary Physiology, University of Zurich, Zurich, Switzerland Takahiro Oto, MD, PhD (Ch: 117) Senior Assistant Professor and Head of Lung Transplant Program, Department of Cancer and Thoracic Surgery, Okayama University, Okayama, Japan Lisa A. Palmer, PhD (Ch: 17) Associate Professor, Departments of Pediatrics and Anesthesiology, University of Virginia, Charlottesville, VA, USA Amit Patel, MD (Ch: 97) Assistant Professor and Director of Cardiac Magnetic Resonance, Department of Medicine, University of Chicago, Chicago, IL, USA Héctor Peña, MD (Ch: 112) Staff, Cardiopulomonary Department, Ignacio Chávez National Institute of Cardiology, Mexico City, Mexico Douglas A. Peterson, MD, PhD (Ch: 46) Assistant Professor, Department of Medicine, VA Medical Center, University of Minnesota, Minneapolis, MN, USA John A. Phillips III, MD (Ch: 69) Professor and David T. Karzon Chair; Director, Division of Medical Genetics, Vanderbilt University, Nashville, TN, USA David J. Pinksy, MD (Ch: 26) J. Griswold Ruth, MD & Margery Hopkins Ruth Professor; Chief, Division of Cardiovascular Medicine; Director, Cardiovascular Center, Department of Internal Medicine, Cleveland Clinic, Cleveland, OH, USA Edward F. Plow, PhD (Ch: 27) Professor and Chairman, Department of Molecular Cardiology, Lerner Research Institute, Cleveland Clinic, Cleveland, OH, USA Ioana R. Preston, MD (Ch: 109) Assistant Professor and Co-Director, Division of Pulmonary, Critical Care and Sleep Medicine, Pulmonary Hypertension Center, Tufts Medical Center, Boston, MA, USA

Contributors

Contributors

xxix

Marlene Rabinovitch, MD (Ch: 55) Dwight and Vera Dunlevie Professor, Department of Pediatrics; Research Director, Vera Moulton Wall Center for Pulmonary Vascular Disease, Stanford University, Stanford, CA, USA Carmelle V. Remillard, PhD (Ch: 33, 34, 47) Assistant Project Scientist, Department of Medicine, University of California, San Diego, La Jolla, CA, USA Stuart Rich, MD (Ch: 81, 103) Section of Cardiology, University of Chicago Medical Center, Chicago, IL, USA David H. Roberts, MD (Ch: 92) Clinical Director, Division of Pulmonary, Critical Care and Sleep Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA Megan Robinson, BS (Ch: 42) Staff Research Associate, Human Embryonic Stem Cell Core Facility, University of California, San Diego, La Jolla, CA, USA Alejandro Roldán-Alzate, PhD (Ch: 5) Research Associate, Department of Biomedical Engineering, University of Wisconsin, Madison, WI, USA Patricia C. Rose, PhD (Ch: 21) Postdoctoral Fellow, Department of Pharmacology, University of Vermont, Burlington, VT, USA Bryan Ross, BScH (Ch: 58) Department of Cardiology, McGill University Health Centre, Montreal QC, Canada John J. Ryan, MD (Ch: 19, 97) Cardiology Fellow, Department of Medicine, University of Chicago, Chicago, IL, USA Julio Sandoval, MD (Ch: 112) Professor and Chief, Cardiopulmonary Division, Ignacio Chávez National Institute of Cardiology, Mexico City, Mexico Vinzenz H. Schmid, PhD (Ch: 49) Zurich Center for Integrative Human Physiology, Vetsuisse Faculty, Institute of Veterinary Physiology, University of Zurich, Zurich, Switzerland Nicholas J. Schork, PhD (Ch: 39) Professor and Director of Research, Scripps Genomic Medicine; Director of Biostatistics and Bioinformatics, Scripps Translational Science Institute; Department of Molecular and Experimental Medicine, Scripps Research Institute, La Jolla, CA, USA Ashish S. Shah, MD (Ch: 116) Assistant Professor and Director of Lung Transplantation, Department of Surgery, Johns Hopkins University, Baltimore, MD, USA Mehdi Skhiri, MD (Ch: 94) Fellow Heart Transplant and Heart Failure, Department of Medicine, Stanford University, Palo Alto, CA, USA Julian Solway, MD (Ch: 83) Walter L. Palmer Distinguished Service Professor and Associate Dean for Translation Medicine; Vice Chair for Research, Department of Medicine, University of Chicago, Chicago, IL, USA

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Robin H. Steinhorn, MD (Ch: 9, 76) Professor and Division Head of Neonatology, Department of Pediatrics, Children’s Memorial Hospital, Northwestern University, Chicago, IL, USA Kurt R. Stenmark, MD (Ch: 52, 56) Professor, Department of Pediatric Critical Care, University of Colorado Denver, Aurora, CO, USA Troy Stevens, PhD (Ch: 15) Professor and Director, Center for Lung Biology and Departments of Medicine and Pharmacology, University of South Alabama, Mobile, AL, USA Duncan J. Stewart, MD (Ch: 28) Professor, Ottawa Hospital Research Institute, Ottawa, ON, Canada Mary E. Strek, MD (Ch: 83) Associate Professor, Department of Medicine, University of Chicago, Chicago, IL, USA Yuichiro J. Suzuki, PhD (Ch: 93) Professor, Department of Pharmacology, Georgetown University, Washington, DC, USA Erik R. Swenson, MD (Ch: 61) Professor, Department of Medicine, University of Washington, VA Puget Sound Health Care System, Seattle, WA, USA Carmen R. Condori Taboada, MD (Ch: 85) Master of Public Health/Epidemiology, General Medicine, Kardiozentrum Medical Specialty Center, La Paz, Bolivia Darren B. Taichman, MD, PhD (Ch: 66) Assistant Professor and Associate Director, Pulmonary Vascular Disease Program, University of Pennsylvania, Philadelphia, PA, USA Tamara Tajsic, MD (Ch: 20) Department of Medicine, Addenbrooke’s Hospital, Cambridge, UK Victor F. Tapson, MD (Ch: 105) Professor and Director, Pulmonary Vascular Disease Center, Duke University, Durham, NC, USA Merryn H. Tawhai, PhD (Ch: 6) Associate Professor, Auckland Bioengineering Institute, University of Auckland, Auckland, New Zealand Thenappan Thenappan, MD (Ch: 97) Fellow, Department of Medicine, University of Chicago, Chicago, IL, USA Patricia A. Thistlethwaite, MD, PhD (Ch: 57) Fellow, Division of Cardiothoracic Surgery, University of California, San Diego, San Diego, CA, USA Eric J. Topol, MD (Ch: 39) Professor and Director, Scripps Translational Science Institute; Chief Academic Officer, Scripps Health, The Scripps Research Institute; Senior Consultant, Division of Cardiovascular Diseases, Scripps Clinic, La Jolla, CA, USA Mourad Toporsian, PhD (Ch: 92) Instructor, Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA

Contributors

Contributors

xxxi

Ali Torkamani, PhD (Ch: 39) Research Scientist, Scripps Translational Sciences Institute, The Scripps Research Institute, La Jolla, CA, USA Rubin M. Tuder, MD (Ch: 101) Professor, Department of Medicine, University of Colorado, Denver, CO, USA Francisco C. Villafuerte, DPhil (Ch: 84) Department of Clinical Biology and Physiology, Universidad Peruana Cayetano Heredia, Lima, Perú Scott Visovatti, MD (Ch: 26) Clinical Lecturer, Department of Medicine, University of Michigan, Ann Arbor, MI, USA Norbert F. Voelkel, MD (Ch: 82) Professor and Director, Victoria Johnson Center and Department of Internal Medicine, Virginia Commonwealth University, Richmond, VA, USA Zoë L. Vomberg, BS (Ch: 42) Staff Research Associate, Human Embryonic Stem Cell Core Facility, University of California, San Diego, La Jolla,CA, USA Jian Wang, PhD (Ch: 47) Professor, First Affiliate Hospital, Guangzhou Medical University, Guangzhou, Guangdong, China Ting Wang, PhD (Ch: 12) Postdoctoral Fellow, Department of Medicine, University of Illinois at Chicago, Chicago, IL, USA Adam Wanner, MD (Ch: 29) Joseph Weintraub Professor, Division of Pulmonary and Critical Care Medicine, University of Miami, Miami, FL, USA Jeremy P. T. Ward, PhD (Ch: 10) Professor, Division of Asthma, Allergy and Lung Biology, King’s College London, London, UK E. Kenneth Weir, MD (Ch: 46) Professor, Department of Medicine, VA Medical Center, University of Minnesota, Minneapolis, MN, USA Norbert Weissmann, PhD (Ch: 30) Professor, Excellence Cluster Cardio-Pulmonary System, Justus-Liebig University Giessen, Giessen, Germany James D. West, PhD (Ch: 31) Assistant Professor, Department of Medicine, Vanderbilt University, Nashville, TN, USA John B. West, MD, PhD, DSc (Ch: 1) Distinguished Professor, Department of Physiology, University of California, San Diego, La Jolla, CA, USA Nico Westerhof, PhD (Ch: 4) Professor, Department of Pulmonary Diseases, VU University Medical Center, Amsterdam, The Netherlands John Wharton, PhD (Ch: 106) Department of Experimental Medicine and Toxicology, Imperial College Healthcare NHS Trust, London, UK

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Martin R. Wilkins, MD (Ch: 106) Department of Experimental Medicine, Imperial College London, Hammersmith Campus, London, UK Karl H. Willert, PhD (Ch: 42) Assistant Professor, Human Embryonic StemCell Core Facility, University of California, San Diego, La Jolla, CA, USA Tanya Wolfson, MA (Ch: 45) Principal Statistician, Computational and Applied Statistics Laboratory, San Diego Supercomputer Center, University of California, San Diego, La Jolla, CA, USA Michael S. Wolin, PhD (Ch: 18) Professor, Department of Physiology, New York Medical College, Valhalla, NY, USA Qingbo Xu, MD, PhD (Ch: 44) Professor and BHF John Parker Chair, Cardiovascular Division, King’s College London, London, UK Weijuan Yao, PhD (Ch: 43) Associate Professor, Department of Physiology and Pathophysiology, Peking University, Beijing, China E.S. Yi, MD Professor, Department of Anatomic Pathology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA Ying Yu, MD, PhD (Ch: 38) Postdoctoral Fellow, Department of Medicine, University of California, San Diego, La Jolla, CA, USA Jason X.-J. Yuan, MD, PhD (Ch: 13, 14, 33, 34, 36, 37, 38, 43, 47) Professor, Departments of Medicine and Pharmacology, The Institute for Personalized Respiratory Medicine, University of Illinois at Chicago, Chicago, IL, USA Gordon L. Yung, MB, BS (Ch: 115) Professor and Medical Director of Lung Transplant Program, Department of Medicine, University of California, San Diego, San Diego, CA, USA Lingfang Zeng, PhD (Ch: 44) Lecturer, Cardiovascular Division, King’s College London, London, UK Zhenguo Zhai, MD, PhD (Ch: 106) Associate Professor, Beijing Institute of Respiratory Medicine and Beijing Chaoyang Hospital, Capital Medical University, Beijing, China Dandan Zhang, PhD (Ch: 47) Research Scientist, Respiratory Department, Guangzhou Medical College, Guangzhou, Guangdong, China Li Zhang, MD (Ch: 101) Professional Research Assistant, Department of Medicine, University of Colorado, Denver, CO, USA Xiaoxue Zhang, MD (Ch: 57) Staff Research Associate, Division of Cardiothoracic Surgery, University of California, San Diego, San Diego, CA, USA Lan Zhao, MD, PhD (Ch: 106) Lecturer, Department of Experimental Medicine and Toxicology, Imperial College Healthcare NHS Trust, London, UK

Contributors

Part I

Structure, Function and Regulation

Chapter 1

The Human Pulmonary Circulation: Historical Introduction John B. West

Abstract  The history of our gradual understanding of the pulmonary circulation is a fascinating topic, partly because it involves some of the key figures in the development of cardiopulmonary physiology, but also because various misconceptions continue to surface from time to time and are seen even today. Of course, the story has been told before and this account follows the same sequence as one of earlier assays. Naturally, a history like this covering such a vast area must be both derivative and selective, and I apologize in advance to any reader who feels that his contributions have been overlooked. Keywords  Pulmonary circulation • History

1  Introduction The history of our gradual understanding of the pulmonary circulation is a fascinating topic, partly because it involves some of the key figures in the development of cardiopulmonary physiology, but also because various misconceptions continue to surface from time to time and are seen even today. Of course, the story has been told before and this account follows the same sequence as one of my earlier essays [1]. Naturally, a history like this covering such a vast area must be both derivative and selective, and I apologize in advance to any reader who feels that his contributions have been overlooked.

2  Galen’s Scheme The Greco-Roman physician Galen (ca 130–ca 201) and his school elaborated a very comprehensive scheme for blood flow within the body that included a reference to the ­pulmonary J.B. West () Department of Physiology, University of California, San Diego 9500 Gilman Drive, La Jolla, CA 92093-0623, USA e-mail: [email protected]

c­ irculation. One of the remarkable features of this theory (Fig. 1) was the extraordinary influence it had for the next 1,400 years or so. In fact, when William Harvey was at Cambridge University in the late 1500s, part of the instruction included Galen’s writings [2], and indeed Galen’s teachings on bloodletting, for example, were followed for another 200 years. In Galen’s scheme, food underwent “concoction” in the gut and reached the liver, where the blood was formed and imbued with “natural spirit.” It then flowed to the right ventricle, from where some entered the lungs via the pulmonary artery to nourish them, and the remainder of the blood reached the left ventricle through “invisible pores” in the interventricular septum. The existence of these so-called pores was a puzzle for anatomists for over a 1,000 years, but it was a necessary feature of the Galen scheme because it was not appreciated that a large amount of blood flowed from the lung to the heart. In the left ventricle, the blood was mixed with “pneuma” or “soul” from the air that was inhaled, and the result was the formation of “vital spirit,” which was distributed throughout the body by the arterial blood. Some of this reached the brain, where it received “animal spirit,” which was then distributed throughout the body via the nerves, which were thought to be hollow. The generation of vital spirit in the left ventricle gave rise to fuliginous (sooty) waste products that were thought to travel back to the lung through the pulmonary vein. From there they were exhaled in the expired air. Of course, Galen’s scheme seems very fanciful to us now, but in those early times there was no notion of the continuous circulation of the blood nor the fact that energy-producing metabolism occurred in the peripheral tissues. In fact, the belief that all the metabolic changes occurred in the lungs persisted until the end of the 1700s and was espoused by the great Antoine Lavoisier (1734–1794), not receiving its death knell until almost 100 years later as a result of the work of Eduard Pflüger (1829–1910). One of the reasons why Galen’s scheme lasted so long was that European science fell to a low ebb following the sack of Rome in 401. In these so-called Dark Ages, knowledge was mainly preserved by Arabian scholars and it was from one of these that the first intimations of the next advance in understanding the pulmonary circulation came about.

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_1, © Springer Science+Business Media, LLC 2011

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J.B. West

air, pass through the arteria venosa (pulmonary vein) to reach the left chamber of the heart and there form the vital spirit....” This was a remarkably perceptive statement and in the thirteenth century was way ahead of its time. Ibn alNafis was a polymath who, although born in Damascus, worked mainly in Cairo, and he contributed to a large variety of disciplines, including physiology and medicine, but also Sunni theology and law. His writings on the pulmonary circulation were essentially unknown to the Western world until 1924, when the manuscript was discovered in a Berlin library by a young Egyptian physician [3]. This being so, Michael Servetus (1511–1533) is credited with independently discovering the pulmonary transit when he wrote that blood passed from the right ventricle to the left from the pulmonary artery to the vein, not “as is commonly believed” through the interventricular septum. He also said that the color of the blood changed when it went through the lung. The statement was made in the theological book Christianismi Restitutio (“The Restoration of Christianity”), which was considered to be heretical because of Servetus’s views on the both the Trinity and infant baptism and as a result of which he was burned at the stake in Geneva (Fig. 2). Visitors to the Geneva Medical School should look for the small, insignificant memorial near the school which refers to

Fig. 1  The cardiopulmonary system according to Galen and his school. This had an enormous influence for some 1,400 years. (Reproduced with permission [58])

3  The Transit of Blood Through the Lung As we have seen, Galen’s view was that some blood entered the lung from the right ventricle, but there was no realization that it returned to the heart. In fact, it was believed that the products of combustion in the left ventricle moved retrogradely along the pulmonary veins to the lungs, where they could be exhaled. The first person to suggest the pulmonary transit of blood was the Arab scholar Ibn al-Nafis (1213–1288). He was a physician from Damascus and developed the idea that blood from the pulmonary artery reached the lung, where it mixed in some way with the air and then passed through the pulmonary veins to the heart. Writing in a commentary on the works of Avicenna, a Persian physician, Ibn al-Nafis (ca 980–1037), stated “...the blood from the right chamber of the heart must arrive at the left chamber but there is no direct pathway between them. The thick septum of the heart is not perforated and does not have visible pores as some people thought or invisible pores as Galen thought. The blood from the right chamber must flow through the vena arteriosa (pulmonary artery) to the lungs, spread through its substances, be mingled there with

Fig.  2  Michael Servetus (1511–1553). He described the transit of blood through the pulmonary circulation but was burned at the stake for religious heresy. (From Stirling [59])

1  The Human Pulmonary Circulation: Historical Introduction

the execution on one side but has an apology from the citizens of Geneva on the other. What was thought to be the last copy of Servetus’s heretical book was chained to his leg when he was burned, although in fact three copies survived. A few years later in the sixteenth century, Realdus Columbus (1516–1559) of Cremona, but working in Padua, also described the transit of blood through the lung, although since the circulation of the blood was not understood, there was no notion of the continuous flow at this time. Columbus was working as an assistant to the great sixteenth-century anatomist Andreas Vesalius (1514–1564), whose magnificent publication De Humani Corporis Fabrica (“Mechanisms of the Human Body”), produced when he was only 18, marked a watershed in the progress of science from its dominance by the Galenical school to the new thinking of the Renaissance. Any reader who is not familiar with the splendid woodcuts in this book should get hold of a copy forthwith! Nevertheless, Vesalius continued to be influenced by Galen to a large extent although he certainly questioned the existence of interventricular pores between the right and left ventricles, which was a cardinal feature of the Galenical scheme (Fig. 1). Vesalius looked carefully for the pores and, of course, found none. At about the same time, another Italian physician, Andreas Caesalpinus (1519–1603), who was a professor of medicine at Pisa, also described the pulmonary transit.

4  Discovery of the Circulation of the Blood There was bound to be confusion about the nature of blood flow in the body until its continuous circulation was first described by William Harvey (1578–1657) in his watershed book Exercitatio Anatomica de Motu Cordis et Sanguinis in Animalibus (“An Anatomical Study of the Motion of Heart and Blood in Animals”) published in 1628. In fact, his extant lecture notes show that he actually conceived the idea some 10 years earlier [2]. Unlike the scientists named so far, Harvey was an Englishman, but in fact he had to travel to Italy, where the scientific Renaissance was in full swing, to reach the cutting edge of the new medical science, and he spent two of his most influential years in Padua at the same university where both Columbus and Vesalius taught. Harvey initially went to Cambridge University but this was weak in science at the time, whereas at Padua the faculty included the anatomist Fabricius ab Aquapendente (1537–1619) and also the great Galileo Galilei (1564–1642). There is no evidence that Harvey and Galileo met, but there is a nice symmetry about the fact that just as Galileo made his epochal discovery that the Earth circulated around the Sun, Harvey made the equally important discovery that the blood circulated around the body. Harvey was greatly influenced by the fact that

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Aquapendente had described the valves in the veins and indeed part of Exercitatio Anatomica de Motu Cordis et Sanguinis in Animalibus is devoted to this. It is interesting that in spite of Harvey’s great insights into the circulation of blood around the whole body, he did not initially understand the pulmonary circulation, or indeed the nature of respiration. It is clear that for some time the role of the pulmonary circulation was a puzzle to him, and he wrote, “it is altogether incongruous to suppose that the lungs need for their nourishment so large a supply of food, so pulsatorily delivered” [4]. Remarkably, it was not until near the end of his life that he wrote a letter to his friend Slegel in which he described experiments in which the passage of blood from the pulmonary artery to the left ventricle was conclusively demonstrated [4]. However, Harvey had no way of knowing how the blood moved from the arteries to the veins in the peripheral tissues or from the pulmonary artery to the pulmonary veins. Indeed this was the next major advance. Although the concept of peripheral metabolism remained elusive, Harvey argued that the function of the circulation of the blood was to provide nutrition to the peripheral tissues. For example, in some of his lecture notes that are still extant he stated, “It is certain from the experiment . . . that there is a passage of the blood from the arteries to the veins. And for this reason it is certain that the perpetual movement of the blood in a circle is caused by the heartbeat. Why? Is it for the sake of nutrition, or is it rather for the preservation of the blood and of the limbs by means of the infused heat? And the blood by turns heating the limbs and when it is cold is warmed by the heart” [2].

5  Discovery of the Pulmonary Capillaries The name Marcello Malpighi (1628–1694) is usually associated with Bologna, although he spent periods at other Italian universities (Fig. 3). He was one of the first people to use the newly discovered microscope to make extensive studies on both animal and plant tissues. His most famous observations from our point of view were set out in two letters to his friend Giovanni Borelli (1608–1679), who was professor of mathematics at Pisa, but like so many Renaissance scientists had interests in many fields. Malpighi began very modestly by referring to “a few little observations that might increase the things found out about the lungs” [5]. But he then went on to describe the discovery of both the alveoli and the pulmonary capillaries using his microscope to look at the surface of frog lungs. He described the alveoli in his first letter as “an almost infinite number of orbicular bladders just as we see formed by wax plates in the walls of the honeycomb cells of beehives.” The second letter includes the discovery of the pulmonary capillaries, where Malpighi states that the network

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J.B. West

Fig. 3  Marcello Malpighi (1628–1694). Left: he discovered the pulmonary capillaries. Right: his drawing of the capillaries in a frog lung is shown at the top. (From Stirling [59])

of tiny vessels occupies not only the walls but also the floors of the alveoli, and that as the blood flows through the tortuous vessels it is not “poured into spaces but always works through tubules.” Remarkably, he also reported on the appearance of the red blood cells within the capillaries, although he thought that these were fat globules. These two letters make delightful reading even today. Malpighi’s discoveries of the pulmonary alveoli and capillaries completes the circle of the pulmonary circulation and finally resolves much of the confusion that we saw earlier. There is a nice symmetry in the fact that Malpighi was born in 1628, which was the same year as Exercitatio Anatomica de Motu Cordis et Sanguinis in Animalibus was published.

6  First Measurements of Blood Pressure One of the first measurements of blood pressure was made by Stephen Hales (1677–1761), who inserted a tube into the femoral artery of a horse and was astonished to see that the blood rose to the height of about 9 ft. Although he did not measure the pressure in the pulmonary circulation, he made many other important discoveries in that area. For example, in his book Haemostaticks [6] he calculated that the blood spent a little over 2 s in the pulmonary capillary, which was a remarkably accurate estimate since the presently accepted figure in man under resting conditions is about 0.75  s. He also obtained a value of about 17  mm (converted from his measurement in inches) for the diameter of a pulmonary ­capillary, and again this was a good estimate since we now know the value is about 5–10 mm. He noted that the flow in the pulmonary veins was pulsatile and he was well aware of the enormous area of the blood–gas barrier when he ­calculated

the surface area of the interior of a calf’s lung to be 30 m2. The area of the blood–gas barrier in the human lung is now given as about 50–100 m2. For the diameter of the alveoli in the calf lung, he calculated a figure of about 250 mm, and the current value for the human lung is about 300 mm. All these measurements were made using very primitive techniques, but Hales prided himself on his meticulous numerical approach using careful measurements, which explains his term “staticks” and “statistical” essays. Stephen Hales is one of my heroes partly because his investigative energy is so well documented in his very readable books, and partly because his place of work is still extant. He carried out his scientific work while a full-time minister of the parish of Teddington in west London and visitors to that city would be rewarded by a visit to his church, St. Alban’s, which still has the tower that he built, a nice stained glass window celebrating his ministry, and his grave with its fine commemorative stone which was recently refurbished with partial support from the American Physiological Society. Hales also introduced the U-tube manometer, but this was not for measuring blood pressure, but the pressure caused by the rising of sap in plants. Like many scientists of his day, he was equally interested in plants and animals and one of his bestknown books is Vegetable Staticks [7]. In fact, Hales is equally celebrated by plant and animal physiologists. The French physician Jean Poiseuille (1799–1869) was apparently the first person to accurately measure systemic blood pressure and he did this by means of a mercury U-tube manometer. Poiseuille’s name is best known for his law describing the pressure-flow relations in narrow tubes under laminar flow conditions. He recognized that the resistance to the flow in such a tube depends on the fourth power of the radius, a very important concept in respiratory physiology for both blood and air flow.

1  The Human Pulmonary Circulation: Historical Introduction

Of course, it was one thing to measure the blood pressure in a systemic artery but quite another to measure the pressure in the far less accessible pulmonary circulation. Beutner, a student of the great German physiologist Carl Ludwig (1816– 1895), was the first to record the pressure in the pulmonary arteries and he did this in several animals, including cats, dogs, and rabbits [8]. The figure he reported for the mean pressure was between 14 and 23 mmHg depending on the species, and this is within the range that is accepted today. He therefore recognized that the pressures in the pulmonary circulation were a factor of 4 or 5 less than in the systemic circulation, which was a key advance. We now recognize that whereas the systemic circulation needs a high pressure to perfuse the raised arms, for example, the pressure in the pulmonary circulation under resting conditions needs only to be sufficient to raise blood to the top of the lung. The fact that the pulmonary circulation has such a low pressure has many implications, including the thin walls of the pulmonary blood vessels. The measurements made in the Ludwig laboratory were done by opening the chest of animals and cannulating the pulmonary artery. However, this is a very unphysiological situation because the normal negative intrapleural pressure is abolished and some other means of ventilating the lung has to be used. It was therefore an important advance in 1862 when Jean-Baptiste Chauveau (1827–1917) and Etienne Marey (1830–1904) in France conceived the idea of introducing long thin catheters into blood vessels and they did this for both the jugular vein and the carotid artery [9]. In this way they were able to measure the pressures in the right and left ventricles, respectively, and they thus confirmed the large differences between the pressures in the pulmonary and systemic circulations, and the remarkably low pressures in the former. As mentioned above, opening the chest of an animal causes a very striking change in the physiological conditions, and related to this, there ensued controversy for a long time on the effect of the respiratory movements on pulmonary pressures and flows. We now know that during inspiration, for example, venous return is initially increased, but it then falls off. In fact, various respiratory maneuvers, such as the Valsalva maneuver, can have very complicated effects on pulmonary pressures and flows. Interestingly, as early as the eighteenth century, Albrecht von Haller (1708–1777) showed that pulmonary blood flow decreased when the lungs were ventilated with an increased pressure. He did this by perfusing frog lungs with dye.

7  Measurement of Pulmonary Blood Flow A key advance in our understanding of the pulmonary circulation was the introduction of the Fick principle to measure total pulmonary blood flow. Furthermore, because the whole of the output of the right ventricle goes through the lungs, this value is therefore the same as the cardiac output. Adolf

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Fig.  4  Adolf Fick (1829–1901). His “Fick principle” for measuring pulmonary blood flow has been used extensively for nearly 140 years. (Reproduced with permission [1])

Fick (1829–1901) was a student in the Ludwig laboratory referred to earlier and the first mention of his “Fick principle” was a brief note in the proceedings of a medical ­scientific society in Würzburg, Germany [10]. Fick (Fig.  4) is ­remembered eponymously in another context because he described the law of diffusion of gases in fluids, for example, across the blood–gas barrier, when he was only 26 years old. However, it must be said that this very general law of diffusion was previously known to physical chemists. The Fick principle is basically a statement of the conservation of mass, that is, that the amount of oxygen taken in (or carbon dioxide given out) at the mouth must be equal to the amount taken up (or given out) as the blood moves through the lung. Nevertheless, this simple statement gave a great boost to the increase in knowledge of the pulmonary circulation. Admittedly, actual application of the principle is complicated by the fact that the oxygen concentration (for example) in mixed venous blood is required, and this necessitates placing a catheter in the pulmonary artery. This was done by Grehant and Quinquaud in 1886 to measure the pulmonary blood flow in dogs [11], but it was not until many years later that sampling blood from the pulmonary artery became possible in humans. Until this became possible in man, other techniques for esti­ mating the concentration of gases in the pulmonary artery were

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introduced, and these are referred to as indirect Fick methods. Along these lines, Loewy and von Schrötter [12] pointed out in 1903 that if an airway to part of the lung was blocked, the Po2 and Pco2 of the gas distal to the block would become the same as those of mixed venous blood [12]. Later, rebreathing techniques were introduced to obtain equilibration between the partial pressures of gases in the mixed venous blood and a rebreathing bag, although an objection to these techniques is that the respiratory maneuvers required for rebreathing may alter cardiac output. Another technique was not to wait for full equilibration but to infer the Pco2 of mixed venous blood from an extrapolation procedure [13]. Other methods of determining total pulmonary blood flow have been introduced that do not depend on the Fick principle but depend on the uptake of a foreign gas. For example, Markoff et al. [14] and Krogh and Lindhard [15] used nitrous oxide to measure pulmonary blood flow by measuring the rate at which it was taken up from the alveolar gas by the blood. A variant of this technique was introduced by Grollman [16], who used acetylene, which has a much higher solubility in blood than nitrous oxide and is therefore removed more rapidly. Techniques such as these are particularly valuable in unusual environments where the Fick principle cannot be used, and, for example, the nitrous oxide uptake procedure was successfully employed in Spacelab to measure the cardiac output in orbiting astronauts [17].

8  Cardiac Catheterization In retrospect, it is perhaps remarkable that it took so long to develop the technique of human cardiac catheterization. As we saw earlier, Chauveau and Marey in 1863 passed long catheters

Fig. 5  André Cournand (1895–1988) and Dickinson Richards (1895–1973). Together with Werner Forssmann (1904–1979), they were awarded the Nobel Prize in Medicine and Physiology in 1956 for the introduction of cardiac catheterization. (Reproduced with permission [1])

J.B. West

through the jugular vein and carotid artery, ­respectively, in unanesthetized horses, thus entering the right and left sides of the heart. However, there was a general feeling that a procedure like this was too dangerous to be used in humans. In fact, when Werner Forssmann (1904–1979) introduced a ureteric catheter into his own right atrium, the medical establishments scorned the procedure. Forssmann was a young German physician who had the idea of injecting drugs directly into the cavity of the heart via a catheter through a vein rather than by a needle through the chest wall to treat emergencies [18]. He persuaded a surgical nurse to provide the necessary sterile ureteric catheter 65 cm long, he inserted this into the antecubital vein of his left arm after local anesthesia, and with the catheter dangling free, he walked down a flight of stairs to the X-ray department, where he was able to advance it into his right atrium [19]. When his chief, Professor Sauerbruch, heard about it, his comment was to the effect that they ran a clinic there not a circus! Forssmann subsequently abandoned cardiology and was astonished when he was selected as one of the recipients for the Nobel Prize in Medicine and Physiology in 1956. Twelve years later, André Cournand (1895–1988), Dickinson Richards (1895–1973), and their collaborators at Bellevue Hospital in New York developed cardiac catheterization in man (Fig. 5) Beginning in 1941 they reported the use of modified ureteric catheters introduced via a peripheral vein to measure first the pressure in the right atrium, then the right ventricle, and finally, in 1944 the pulmonary artery [20–24]. These studies laid the basis for modern interventional cardiology and they, together with Forssmann, were awarded the Nobel Prize in 1956. A further important advance was when Dexter and colleagues showed that it was possible to wedge the catheter in a small pulmonary artery and thereby obtain a measurement that was approximately equal to the pulmonary venous pressure [25].

1  The Human Pulmonary Circulation: Historical Introduction

Building on these studies, it soon became possible to insert catheters into the left side of the heart via a peripheral systemic artery. These procedures were combined with angiography using radio-opaque contrast material, and a key advance occurred in 1958 when Mason Sones accidentally injected contrast material into the ostium of the right ­coronary artery of a patient and was astonished to record the first coronary angiogram; the patient recovered successfully [26].

9  Advances During the Last 50 Years There has been an enormous increase in knowledge of various aspects of the pulmonary circulation in the last 50 years and space does not permit description of these in detail. Many of these advances will be alluded to in subsequent chapters in this book. The following is a selection that is bound to reflect the author’s interest.

9.1  D  istinction Between Alveolar and Extra-Alveolar Vessels of the Pulmonary Circulation Macklin [27] made an important advance when he injected a latex suspension into the blood vessels of excised lungs and showed a difference in behavior between the small vessels and the large vessels when the lungs were inflated or deflated. He reported that when the lungs with inflated with positive pressure, the caliber of the larger vessels increased, whereas the capillaries were compressed. He concluded from this that the act of breathing helped to move blood through the lung. A much more extensive study was later carried out by Howell et al. [28] when they filled the pulmonary blood vessels with kerosene. Because of surface tension effects, this liquid does not penetrate into the capillaries and other small vessels, and thus the investigators were able to separate the effects of lung inflation on the two types of vessels. To their surprise, they found that when the lungs were inflated with positive pressure, that is, the alveolar pressure was raised with respect to vascular pressures, the larger, kerosene-containing vessels increased their caliber, whereas, as they had expected, the small vessels, not filled with kerosene, were compressed. As a result, they divided the pulmonary vasculature into “compressed” and “expanded” compartments [29]. An interesting related finding was that although the change in volume of the larger, “expanded” vessels was quite small, they were capable of developing surprisingly large pressures. Somewhat later, Mead and Whittenberger [30] named the smaller and larger vessels “alveolar” and “extraalveolar,” respectively, because the former were clearly

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exposed to alveolar pressure and the latter were not. This nomenclature remains in use and the distinction is very important in understanding changes in pulmonary blood flow under a variety of conditions.

9.2  Starling Resistor Effect The critical importance of alveolar pressure in pulmonary hemodynamics became clearer when it was shown that if alveolar pressure exceeds pulmonary venous pressure, pulmonary blood flow is determined by the difference between pulmonary arterial and alveolar pressure rather than the expected pulmonary arterial pressure and pulmonary venous pressure difference. One of the first clear demonstrations of this was by Banister and Torrance [31] in 1960, although a number of previous investigators had recognized the importance of transmural pressure in determining flow through collapsible tubes [32–34]. One of the most extensive studies was then carried out by Permutt et al. [35], who showed that if the pulmonary arterial pressure was held constant in a lung preparation and pulmonary venous pressure was gradually raised, there was no effect on pulmonary blood flow until the venous pressure exceeded the alveolar pressure. In other words, as stated above, flow is independent of venous pressure as long as this is less than alveolar pressure. The phenomenon has been referred to as the “Starling resistor effect” because the English physiologist Ernest Starling used a collapsible tube to maintain the outflow pressure constant in a heart–lung preparation, but it is also called the “vascular waterfall” or “sluice effect” because in both instances the flow over the waterfall or sluice is independent of the pressure in the water below.

9.3  T  opographical Distribution of Pulmonary Blood Flow As long ago as 1887, the German pathologist Johannes Orth suggested that the apex of the lung might have a reduced blood flow under certain conditions because of the weight of the blood, and he speculated that this could be related to the apical development of pulmonary tuberculosis [36]. Some 60 years later when the first measurements of vascular pressures on the right side of the heart were made in humans, as described in Sect. 8, William Dock argued that the pulmonary arterial pressure was so low that it might not be sufficient to raise blood to the apex of the upright lung [37]. However, the gradation in the topographical distribution of blood flow down the upright human lung was first demonstrated when it was shown that the rate of removal of inhaled oxygen-15-labeled carbon dioxide during a short period of

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breath holding was very slow at the apex of the upright lung and gradually increased toward the base [38]. A little later, a very similar distribution of blood flow was reported using radioactive xenon [39]. It was quickly shown that the topographical distribution was very sensitive to posture, exercise (which increased the pulmonary arterial pressure), and acceleration in a centrifuge [40], so gravity was clearly the cause. Then extensive studies using an isolated lung preparation where careful control over the pulmonary arterial, venous, and alveolar pressures was possible showed how the lung could be divided into various zones depending on the magnitudes of the pulmonary arterial, venous, and alveolar pressures [41]. Lung volume was also shown to have an important role, especially at very low states of lung inflation [42]. Subsequently, it has become clear that the topographical distribution of blood flow plays an important role not only in physiological conditions but also in many pathophysiological conditions.

9.4  N  ongravitational Factors Affecting the Distribution of Pulmonary Blood Flow Gravity has a striking effect on the topographical distribution of blood flow in the lung as discussed in the previous section. Blood flow is much higher at the bottom of the upright human lung than at the apex, and indeed some earlier studies found no blood flow at all at the extreme apex [39]. However, it was never contended that blood flow at one particular level in the lung was completely uniform. For example, an early study by Glazier et al. [43] using rapid freezing to look at the histological characteristics of isolated perfused dog lungs clearly showed considerable inequality of red cell filling in adjacent alveolar walls. This inequality was later analyzed theoretically using networks of pulmonary capillaries and it was shown that if there were preferential channels of flow for some reason such as some capillaries being wider than others, blood flow inequality was bound to develop as the perfusion pressure was gradually increased [44]. More recently, the role of nongravitational factors has been increasingly emphasized. For example, Glenny [45] and Glenny and Robertson [46] injected small microspheres into the pulmonary circulation of animals, inflated and fixed the lungs, diced them into small portions, and showed considerable inequality of blood flow between these pieces. Other workers have shown differences in regional vascular conductance in dog lungs [47], and also stratified inequality of blood flow along terminal lung units [48, 49]. Furthermore, in measurements made on astronauts during sustained microgravity in orbital flight, there was clear ­evidence that the topographical inequality of blood was

J.B. West

reduced, but some inhomogeneity of pulmonary perfusion remained [50].

9.5  L ongitudinal Distribution of Vascular Resistance in the Pulmonary Circulation The distribution of vascular resistance along the pulmonary circulation from the pulmonary arteries to the veins has been the subject of numerous studies and there is still not universal agreement on the results. However, there is strong evidence that the major site of vascular resistance is not in the small pulmonary arteries as is the case in the systemic circulation, where the arterioles are highly muscularized and act as throttles. In fact, as we saw earlier, the pulmonary circulation has a very much lower resistance than its systemic counterpart, and the small pulmonary arteries have relatively little vascular smooth muscle, and as a result, they are easily mistaken for pulmonary veins. Some of the most convincing studies have come from the cannulation of small pulmonary blood vessels in experimental animals [51]. The overall message is that the pressure in the pulmonary capillaries is probably about halfway between pulmonary arterial and venous pressures, and that much of the pressure drop in the pulmonary circulation occurs in the capillary bed. This makes sense because the primary function of the pulmonary circulation is to allow the exchange of gas across the pulmonary capillaries, and it is not necessary to direct blood flow to different parts of the lung as is the case in the systemic circulation, where blood flow has to be diverted, for example, to exercising muscles. The result is that the pressure drop in the pulmonary circulation is much more uniform than it is in the systemic circulation, where much of the high aortic pressure is lost in the highly muscularized arterioles. A topic which has resulted in considerable confusion in the past is to what extent the pulmonary venous pressure rises during exercise. At rest, the pressure at the downstream end of the pulmonary veins must be close to left atrial pressure, which is about 5 mmHg in man. However, there is considerable evidence that the pulmonary venous pressure rises substantially on exercise when the cardiac output is increased. Most of this evidence comes from measurements of pulmonary artery wedge pressure, although some direct measurements of left atrial pressure by means of catheters have been made in experimental animals. In one study on normal humans at high levels of exercise, the pulmonary artery wedge pressure increased from 3.4 to 21.1 mmHg, and this was associated with an increase in pulmonary arterial pressure from 13.2 to 37.2 mmHg [52]. Another study on exercising humans reported similar values [53]. Galloping racehorses develop extremely high pulmonary wedge or left

1  The Human Pulmonary Circulation: Historical Introduction

arterial pressures, up to 50 mmHg or more [54, 55]. These animals have been selectively bred for hundreds of years to develop very high maximal oxygen consumptions and these are associated with very high cardiac outputs that necessitate high filling pressures for the left ventricle.

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uniquely suited to modifying blood-borne ­substances such as angiotensin I, bradykinin, serotonin, norepinephrine, some prostaglandins, and leukotrienes. Indeed a substantial fraction of all the vascular endothelial cells in the body are located in the lung. These metabolic functions of the pulmonary circulation are critically important in a number of diseases as discussed later in the book.

9.6  Hypoxic Pulmonary Vasoconstriction As long ago as 1946, von Euler and Liljestrand [56] ventilated anesthetized cats with 10% oxygen in nitrogen and showed that there was a sudden increase in pulmonary arterial pressure compared with air breathing. When the animals were returned to air breathing, the pressure fell. They argued that the hypoxia had a direct action on part of the pulmonary circulation, and the mechanism of the vasoconstriction has been an intense topic of study ever since. In fact, several of the chapters in this book are related in some way to this. There seems no point in reviewing the research here except to say that the constriction apparently involves voltage-gated potassium channels. Many investigators have pointed out that this mechanism has some value in lung disease, for example, asthma, because it results in some blood flow being shunted away from poorly ventilated lung units. However, the chief evolutionary pressure for hypoxic pulmonary vasoconstriction is likely to be the events that occur at birth. The fetal lung has only about 15% of the cardiac output passing through it, with the rest being diverted through the patent ductus arteriosus. At birth, there is a sudden transition from placental to air breathing, which necessitates a very large increase in blood flow through the lung, and this is brought about by a dramatic fall in pulmonary vascular resistance caused in part by the loss of hypoxic vasoconstriction, and in part by inflation of the lung. The high vascular resistance of the fetal lung is assisted by the fact that the walls of the pulmonary arteries contain large amounts of vascular smooth muscle. After birth, considerable involution of the smooth muscle takes place, and the distribution also becomes patchy [57]. The result is that in the adult, hypoxic pulmonary vasoconstriction is uneven, and this is an important factor in some diseases, such as highaltitude pulmonary edema.

9.7  N  onrespiratory Functions of the Pulmonary Circulation Again this topic is addressed in several subsequent chapters in this book and there is little point in reviewing the history here. Suffice to say that since the lung is the only organ except the heart that receives the whole circulation, it is therefore

References 1. West JB (1996) Respiratory physiology: people and ideas. Oxford University Press, New York 2. Keynes G (1978) The life of William Harvey. Clarendon Press, Oxford, p 15 3. Mohyi (1924) Der Lungenkreislauf nach el-Koraschi. Dissertation, Freiburg 1924 4. Harvey W (1989) The works of William Harvey (trans: Willis R). University of Pennsylvania Press, Philadelphia 5. Young J (1929) Malpighi’s De Pulmonibus. R Soc Med Proc 23:1–11 6. Hales S (1733) Statistical essays containing haemastaticks. Innys & Manby, London 7. Hales S (1727) Vegetable staticks. Innys & Innys, London 8. Beutner A (1852) Über die Strom- und Druckkrätfte des Blutes in der Arterie und Vena pulmonalis. Z Rat Med N F 2:97–126 9. Chauveau JBA, Marey EJ (1862) De la force déployée par la ­contraction des différentes cavités du coeur. Coll R Soc Biol (Paris) 4:151–154 10. Fick A. Ueber die Messung des Blutquantums in den Herzventrikeln. S B Phys Med Ges, Würzburg, July 9, 1870 11. Grehant N, Quinquaud CE (1886) Recherches expérimentales sur la mesure du volume du sang qui traverse le poumon en un temps donne. C R Seances Hebd Soc Biol 5:159 12. Loewy A, von Schrötter H (1903) Ein Verfahren zur Bestimmung der Blutgasspannungen, der Kreislaufgeschwindigkeit und des Herzschlagvolumens am Menschen. Arch Anat Physiol Abt 394 13. Kim TS, Rahn H, Farhil E (1966) Estimation of true venous and arterial Pco2 by gas analysis of a single breath. J Appl Physiol 21: 1338–1344 14. Markoff I, Muller F, Zuntz N (1911) Neue Methode zur Bestimmung der in menschlichen Körper umlaufenden Blutmenge. Z Balneol 4:373–411 15. Krogh A, Lindhard J (1912) Measurements of the blood flow through the lungs of man. Skand Arch Physiol 27:100 16. Grollman A (1932) The cardiac output of man in health and disease. Thomas, Springfield 17. Prisk GK, Guy HJB, Elliott AR, Deutschman RA III, West JB (1993) Pulmonary diffusing capacity, capillary blood volume and cardiac output during sustained microgravity. J Appl Physiol 75:15–26 18. Forssmann W (1929) Die Sondierung des rechten Herzens. Klin Wochenschr 8:2085 19. Forssmann W (1974) Experiments on myself. St. Martin’s Press, New York 20. Cournand AF, Ranges HS (1941) Catheterization of the right auricle in man. Proc Soc Exp Biol Med 46:462–466 21. Cournand AF, Lauson HD, Bloomfield RA, Breed ES, Baldwin E (1944) Recording of right heart pressures in man. Proc Soc Exp Biol Med 55:34–36 22. Cournand AF, Riley RL, Breed ES, Baldwin ED, Richards DW, Lester MS, Jones M (1945) Measurement of cardiac output in man using the technique of catheterization of the right auricle or ventricle. J Clin Invest 24:106–116

12 23. Cournand A, Bloomfield RA, Lauson HD (1945) Double lumen catheter for intravenous and intracardiac blood sampling and pressure recording. Proc Soc Exp Biol Med 60:73–75 24. Richards DW (1945) Cardiac output by the catheterization technique in various clinical conditions. Fed Proc 4:215–220 25. Dexter L, Haynes FW, Burwell CS, Eppinger EC, Sagerson RP, Evans JM (1947) Studies of congenital heart disease. II. The pressure and oxygen content of blood in the right auricle, right ventricle, and pulmonary artery in control patients, with observations on the oxygen saturation and source of pulmonary capillary. J Clin Invest 26:554–560 26. Hurst JW (1985) History of cardiac catheterization. In: King SB III, Douglas JS (eds) Coronary arteriography and angioplasty. McGrawHill, New York, pp 5–6 27. Macklin CC (1946) Evidence of increase in the capacity of the pulmonary arteries and veins of dogs, cats and rabbits during inflation of the freshly excised lung. Rev Can Biol 5:199–233 28. Howell JBL, Permutt S, Proctor DE, Riley RL (1961) Effect of inflation of the lungs on different parts of pulmonary vascular bed. J Appl Physiol 16:71–76 29. Permutt S, Howell JBL, Proctor DF, Riley RL (1961) Effect of lung inflation on static pressure volume characteristics of pulmonary vessels. J Appl Physiol 16:64–70 30. Mead J, Whittenberger JL (1964) Lung inflation and hemodynamics. In: Fenn WO, Rahn H (eds) Handbook of physiology, respiration. American Physiological Society, Washington 31. Banister J, Torrance RW (1960) The effects of the tracheal pressure upon flow: pressure relations in the vascular bed of isolated lungs. Q J Exp Physiol 45:352–367 32. Duomarco JL, Rimini R (1954) Energy and hydraulic gradients along systemic veins. Am J Physiol 178:215–220 33. Rodbard S (1955) Flow through collapsible tubes: augmented flow resistance produced by resistance at the outlet. Circulation 11:280–287 34. Holt JP (1941) The collapse factor in the measurement of venous pressure. Am J Physiol 134:292–299 35. Permutt S, Bromberger-Barnea B, Bane HN (1962) Alveolar pressure, pulmonary venous pressure and the vascular waterfall. Med Thorac 19:239–260 36. Orth J (1887) Atiologisches und Anatomisches über Lungensch­ windsucht. Hirshwald, Berlin 37. Dock W (1947) Reasons for the common anatomic location of pulmonary tuberculosis. Radiology 48:319–322 38. West JB, Dollery CT, Naimark A (1960) Distribution of blood flow and ventilation perfusion ratio in the lung, measured with radioactive CO2. J Appl Physiol 15:405–410 39. Anthonisen NR, Milic-Emili J (1966) Distribution of pulmonary perfusion in erect man. J Appl Physiol 21:760–766 40. Glaister DH (1967) The effect of positive centrifugal acceleration upon the distribution of ventilation and perfusion within the human lung, and its relation to pulmonary arterial and intraoesophageal pressures. Proc R Soc Lond B 168:311–334

J.B. West 41. West JB, Dollery CT, Naimark A (1964) Distribution of blood flow in isolated lung: relation to vascular alveolar pressures. J Appl Physiol 19:713–724 42. Hughes JMB, Glazier JB, Maloney JE, West JB (1968) Effect of lung volume on the distribution of pulmonary blood flow in man. Respir Physiol 4:58–72 43. Glazier JB, Hughes JMB, Maloney JE, West JB (1969) Measurements of capillary dimensions and blood volume in rapidly frozen lungs. J Appl Physiol 26:65–76 44. West JB, Schneider AM, Mitchell MM (1975) Recruitment in networks of pulmonary capillaries. J Appl Physiol 39:976–984 45. Glenny RW (1988) Blood flow distribution in the lung. Chest 114:8s–16s 46. Glenny RW, Robertson HT (1990) Fractal properties of pulmonary blood flow: characterization of spatial heterogeneity. J Appl Physiol 69:532–545 47. Beck KC, Rehder K (1986) Differences in regional vascular conductances in isolated dog lungs. J Appl Physiol 61:530–538 48. Wagner PD, McRae J, Read J (1967) Stratified distribution of blood flow in the secondary lobule of the lung. J Appl Physiol 22: 1115–1123 49. West JB, Maloney JE, Castle BL (1972) Effect of stratified inequality of blood flow on gas exchange in liquid-filled lungs. J Appl Physiol 32:357–361 50. Prisk GK, Guy HJB, Elliott AR, West JB (1994) Inhomogeneity of pulmonary perfusion during sustained microgravity on SLS-1. J Appl Physiol 76:1730–1738 51. Bhattacharya J, Staub NC (1980) Direct measurement of microvascular pressures in the isolated perfused dog lung. Science 210: 327–329 52. Wagner PD, Gale GE, Moon RE, Torre-Bueno JR, Stolp BW, Saltzman HA (1986) Pulmonary gas exchange in humans exercising at sea level and simulated altitude. J Appl Physiol 61:260–270 53. Groves BM, Reeves JT, Sutton JR, Wagner PD, Cymerman A, Malconian MK, Rock PB, Young PM, Houston CS (1987) Operation Everest II: elevated high-altitude pulmonary resistance unresponsive to oxygen. J Appl Physiol 63:531–539 54. Jones JH, Smith BL, Birks EK, Pascoe JR, Hughes TR (1992) Left atrial and pulmonary arterial pressures in exercising horses. FASEB J 6:A2020 55. Manohar M (1993) Pulmonary artery wedge pressure increases with high-intensity exercise in horses. Am J Vet Res 54:142–146 56. von Euler US, Liljestrand G (1946) Observations on the pulmonary arterial blood pressure in the cat. Acta Physiol Scand 12:301–320 57. deMello D, Reid LM (1991) Arteries and veins. In: Crystal RG, West JB (eds) The lung: scientific foundations. Raven, New York, pp 767–777 58. Singer C (1957) A short history of anatomy and physiology from the Greeks to Harvey. Dover, New York 59. Stirling W (1902) Some apostles of physiology. Waterlow, London

Chapter 2

Microcirculation of the Lung: Functional and Anatomic Aspects Joan Gil

Abstract  The fixed pulmonary vascular anatomy differs from the systemic anatomy in the arrangement and shape of the capillary segments, but an even more striking peculiarity of the lung is that it needs to adapt itself to three different pressures, which leads to considerable adaptations and changes in morphology. Because of the pressure changes required by respiratory mechanics, the morphology differs both at the level of the capillaries and at the level of the extraalveolar small arteries and veins as a function of the existing mechanical conditions, adapting them in a way that is best suited to fulfill their respective functions. The configuration of the pulmonary capillary network markedly differs from that in the systemic capillary bed. The gas exchange needs are different in the systemic and in the pulmonary capillaries. In the periphery, the capillaries are longitudinal and their number and density in the tissue reflect local needs; in the lung, their purpose is to be capable of picking up from the outside as much oxygen as possible to fulfill the most extreme conceivable needs for gas exchange (the “diffusing capacity”). This capacity is normally not reached and the capillaries tolerate recruitment, de-recruitment, and changes in configuration that support variable quantitative ­levels of gas exchange. Keywords  Gas exchange • Small pulmonary arterioles • Capillary • Flow kinetics • Microvascular circulation

1  Introduction Long gone are the times when the pulmonary circulation was absurdly referred to as the lesser circulation. No other organ circulation, not even when the systemic circulation is studied as a whole, raise issues as complicated and unexpected as those encountered in the lung. This is not limited to the J. Gil () Department of Pathology, Mount Sinai School of Medicine, New York, NY 10029, USA e-mail: [email protected]

biochemical, cellular, and reactive properties of the vessels, but includes the fixed and functional vascular anatomy [1–6]. The fixed anatomy differs from the systemic anatomy in the arrangement and shape of the capillary segments, but an even more striking peculiarity of the lung is that it needs to adapt itself to three different pressures, which leads to considerable changes in morphology. Because of the pressure changes required by respiratory mechanics, the morphology differs both at the level of the capillaries and at the level of the extraalveolar small arteries and veins as a function of the existing mechanical conditions, adapting them in a way that is best suited to fulfill their respective functions. A pulmonary-specific structure required to understand the process is the connective tissue (“fibrous”) continuum [7] (Fig. 1), which inserts itself proximally in the hilum, which is relatively fixed like a fulcrum. At the distal end, however, the visceral pleura is not mechanically fixed, as it moves in solidarity with the parietal pleura and the chest wall only by virtue of the existing subatmospheric air pressure without physical contacts between both of them. While several elements contribute to the generation of recoil pressure of the lung, only the pleural pressure helps to keep the healthy organ open. The connective tissue continuum connects both visceral pleura and hilum, becomes tensed during inhalation, and distributes subatmospheric pressure throughout all interstitial points of the lung. This has many consequences. Nowhere else in the body are capillaries normally exposed to changes in pericapillary pressures. More important yet, the configuration of the capillary ­network markedly differs from that in the systemic capillary bed. The gas exchange needs are different in the systemic and in the pulmonary capillaries. In the periphery, the capillaries are longitudinal and their number and density in the tissue reflect local needs; in the lung, their purpose is to be capable of picking up from the outside as much oxygen as possible to fulfill the most extreme conceivable needs for gas exchange (the “diffusing capacity”). This capacity is normally not reached and the capillaries tolerate recruitment, derecruitment, and changes in configuration and in velocity of the fluid that support variable quantitative levels of gas exchange.

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_2, © Springer Science+Business Media, LLC 2011

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Fig. 1  The fibrous continuum of the lung. a The fibrous continuum extends from the hilum to the visceral pleura extending into all the alveolar wall. b Hexagonal capillary network in the alveolar septum supported by an incomplete layer of elastic and collagenous fibrils and fibers. Occasional interstitial cell elements are not shown. (Reproduced with permission [7])

The lung exhibits two different circulations [1]: (1) a s­ ystemic component arising mostly from the aorta and its intercostal branches that supplies the bronchial walls and the pleura and provides them with nourishment and oxygen and (2) the narrowly designated pulmonary circulation originating in the right ventricle which exchanges gas (oxygen intake and CO2 elimination) to serve the entire body and exhibits the complex properties described by others in this book. Remarkably, both components distally anastomose at the level of their venules in the wall of the small bronchioles and to a lesser extent in the pleura, which creates a permanent right-to-left shunt of variable flow intensity. Pulmonary shunts are about the most perplexing problems of the pulmonary vascular morphology [8].

2  T  echnical Fixation Problems: Vascular Perfusion The lung does not have a rigid, really fixed shape or size: it is a semiliquid organ. Its shape depends in part on the container. It is not easy to determine its true outside anatomy after removing the lung from the chest and placing it on the dissecting table, but the difficulty doubly applies to the internal configuration, raising a complex methodological issue [3] discussed by many authors, as the original morphology is lost when the subject dies and the chest is opened. What should be done to preserve the real anatomy? Supravital microscopy through a chest wall window is appealing but is limited and precludes study of deeper areas [9]. Many in the past [10–12] tried to visualize the “true” in vivo morphology by applying a rapid freezing technique [13]. Liquid CO2 was the medium of choice. Liquid nitrogen is generally not desirable, as it forms a mantle of boiling fluid and insulating gas around the tissue, resulting in slow cooling and poor fixation. In both

cases, unless the lung has been pretreated with a cryoprotector, which frequently defeats the purpose, the slow penetration of the cold temperature results in an unacceptable number of artifacts, in particular large ice crystals and torn membranes, which cast doubt on the quality of the preservation and the dependability of the observations. Generally, only a thin peripheral layer of tissue will be preserved for interpretation, which renders the possibility of representative sampling impossible and limits all observations to the surface. Chemical fixation [3, 6, 7] is a more desirable approach to the preservation of the lungs for light- or electron-microscopic study because it allows unrestricted sampling of wellpreserved tissue. Buffered aldehyde (formalin for light microscopy, glutaraldehyde for electron microscopy) is rapidly instilled into the trachea or a stem bronchus ­following pneumothorax. The use of a controlled instillation pressure in the fluid, some 20 cm H2O [7, 14], is advisable and results in the adjustment of a reproducible and ­well-defined lung volume, corresponding to the plateau of the pressure–volume curve of the fluid-filled lung. Morphologically, this simple approach yields outstanding results, requires little preparation and modest training, and it is currently the method of choice for most morphology studies, for instance, of lung injury. But airway flooding with fixatives is clearly unsuitable for correlative studies of morphology with pulmonary mechanics or lung circulation [3, 6]. At the very least, the alveolar surface tension is abolished and the pressure–volume curves of air- and water-filled lung are very different, so the morphology must also be suspected of being different. For the purpose of this chapter, we are naturally interested in the air-filled natural condition. As repeatedly shown by us [3, 4, 15], this preservation can be achieved only by inflating the lung with a known air volume after an O2-wash-induced atelectasis, full air inflation, subsequent deflation, and adjustment and stabilization of the desired final pressure. This is followed by flush of the pulmonary

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Fig. 2  a Rabbit, fluid-filled lung fixed by vascular perfusion. Note the empty capillary spaces and the undulated appearance of the surface. b Rabbit, air-filled lung fixed by vascular perfusion under zone II ­conditions. Note the smooth surface and the rectangular internal capillary luminal outlines. (Reproduced with permission [4])

circulation through a catheter inserted in the pulmonary main artery and perfusion of the fixative also at a well-known and controlled pressure. Most workers perfuse with buffered, isotonic glutaraldehyde as the fixative, followed by block immersion of the entire or a large fragment of lung and very careful dicing. We [3, 4, 15] believe that a perfusion with 1% buffered OsO4 yields superior results, but it must be warned that osmium emanates highly toxic, dangerous fumes and handling of large volumes of that substance requires training, exhaust equipment, precautions, and great care. Most of our studies and descriptions rely on findings in lungs fixed by vascular perfusion. Figure 2 shows three alveolar walls of two perfusion-fixed rabbit lungs (the capillaries are empty) photographed at the same magnification. The lung in Fig. 2a has been filled with saline, whereas the lung in Fig. 2b is filled with air under zone II conditions (to be discussed later). The difference is striking.

3  S  ystemic Component of Circulation in the Lung: Bronchial and Pleural Circulation The bronchial arteries arise from the descending thoracic aorta and from the posterior intercostal arteries [16]. They supply with oxygen and nutrition the mediastinal bronchi and the following conducting airways all the way to the ­terminal bronchioles. In the wall of the large bronchi they form two plexuses, one in the incomplete muscle layer and the other in the submucosa. The bronchial veins are much smaller and have a lower flow volume. Some of them are deep veins that arise parallel to the bronchial arteries but run in the opposite direction, increasing in volume only modestly because they drop much of their blood in the abundant anastomoses, with the

pulmonary veins returning it to the left atrium. Other veins are superficial and arise in the pleural surface. The bronchial venous blood is drained in the azygos or hemiazygos veins. The lymphatics are also a vascular component. There are none in the alveolar walls, because the overflow compartment of the capillaries is the alveolar space. The pulmonary lymphatics do not arise from closed sacks, as they do in the systemic beds, but instead arise from connective tissue spaces in the periarterial sleeves, partly lined by boundary cells that appear to be either fibroblasts or endothelial cells [17] (Fig. 3). The periarterial lymphatic vessels are barely visible unless the lung is edematous. Lymph nodes are ­frequently found in the subpleural and peribronchial parenchymal areas.

4  R  ole of the Connective Tissue Continuum of the Lung in Relation to the Capillaries A connective tissue continuum of connective tissue of fibers, ground substance, and a few cells extends from the visceral pleura and septa to the hilum [7]. The continuum is fundamental in pulmonary mechanics [18]. Throughout the body, blood vessels and nerves are embedded in the connective tissue stroma of the organ, a filling and supporting component which might be compared to a soft tissue skeleton. In the alveolar walls, however, the pulmonary stroma exhibits peculiar characteristics. The connective tissue continuum (Fig. 1) mostly ­consists of elastic and collagen fibers and fibrils, generally of type 3, is resistant to traction but is thin and malleable, and fulfills the function of connecting the hilum with the visceral pleura [1, 7].

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Fig.  3  a Rat lung fixed by instillation of fixative fluids into the airways following pneumothorax. Note that blood vessels are filled with blood. A represents air spaces, C is a capillary, AV is an extraalveolar vessel surrounded by its own thin sheath of connective tissue, and B is an interstitial cell, probably a fibroblast of an

endothelial cell giving rise to a lymphatic of an incomplete wall. b Rat lung fixed by vascular perfusion under zone II conditions. Note that the extraalveolar vessel (bottom) contains a lymphatic (ly) and a muscular medium. A represents alveolar spaces. (Reproduced with permission [17])

During inhalation, the subatmospheric pressure ­generated in the pleural space pulls on the visceral pleura and causes an expansion of the lung parenchyma. The pull counteracts the effects of the alveolar surface tension and any connective tissue resistance due to overstretching and results in the generation of an interstitial subatmospheric pressure throughout the organ. The opposite happens during expiration. The extraalveolar arteries and veins, like the alveolar capillaries, are embedded within the continuum, although the arrangement and consequences are markedly different in both of them.

bronchioles, arteries, despite the smooth muscle which serves different purposes, lack surface tension to actively retract but the surrounding connective tissue sheath with its changes of pressure allows them to participate in pulmonary mechanics in association with the respiratory movements. The important characteristic (Figs. 3, 5), however, is that unlike the alveoli arteries and veins are never directly exposed to air pressure. During inflation, the alveolar walls radially inserted in the periphery of the connective tissue sheaths are pulled out of the sheath’s perimeter and cause an enlargement of the vascular diameter and therefore an increase of their volume. During expiration, as the alveolar volume is reduced, the radial alveolar wall anchors are somewhat relaxed and the diameter and the volume of the extraalveolar blood vessels accordingly decrease [12]. As we shall see later, this result is exactly the opposite of what happens to the alveolar ­capillaries, whose volume decreases as the alveolar air pressure compresses them during inspiration. During inspiration, arteries and veins therefore become a blood reservoir.

5  B  lood Vessels Inside the Lung but Outside the Alveolar Parenchyma: Extraalveolar Arteries and the Connective Tissue Continuum We have already described how the pulmonary arteries become surrounded with adipose and connective tissue upon their entering the hilum. Structurally, they continue to differ from systemic arteries of comparable caliper only in the smaller thickness of their medial walls. Because of this they stay functionally and anatomically separated from the alveolar wall (Fig. 3), which is occupied mostly by capillaries (Fig. 4) (blood vessels consisting only of an endothelium without a muscle cell layer). Therefore, a major difference exists between the environment in which arteries and veins function and the situation surrounding alveolar capillaries. Unlike the alveoli and the

6  P  ulmonary Circulation: Conducting Arteries 6.1  Arteries Outside the Lung The main pulmonary artery arises directly from the conus arteriosus of the right ventricle. It and its main branches are of the elastic type. This means that the medium is made up of ­parallel

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Fig. 4  a Dog lung fixed by instillation of fixatives into the airways and photographed with the electron microscope at very low power. b Highpower electron micrograph of two air-wall barriers of lung fixed by instillation of the airways. Note the dark erythrocytes at the bottom, and

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the thin endothelial and ­epithelial layers containing many pinocytotic vesicles. EI, epithelial cell; EN, endothelial cell (Reproduced with permission [7])

Fig. 5  The difference between extraalveolar and alveolar vessels during respiratory movements, deflation a and inflation b

elastic lamellae connected to each other by smooth muscle cells inserted obliquely to the lamellae but parallel to each other. As in the systemic circulation, smaller branches become muscular, with a solid medium made up of smooth muscle and

a well-developed intimal layer. The most peculiar characteristic of pulmonary arteries is the relatively thin medial wall when compared with the luminal diameter, which reflects the small prevailing pulmonary pressure. Once they penetrate into the

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pulmonary parenchyma and the hilum, the pulmonary arteries and veins become surrounded by a loose adipose connective tissue which frequently ­contains lymph nodes. An arterial branch that runs in parallel to the bronchial walls is named an axial artery. Evidently, there are as many generations of axial arteries as there are of conducting airways, on average 18 in the human lung [14]. Most parenchymal ­lymphatics arise in the connective tissue sleeve that surrounds the artery rather than the bronchi. In the bronchiolar peripheral territory, the connective tissue sheaths are frequently separated from the bronchiolar sheaths, but are close to them. Additionally to their dynamic changes of ­volume in ­association with mechanics, the periarterial sheaths are also the site of the ­earliest accumulation of interstitial lung edema [6, 18]. The axial arteries are not the only extraalveolar arteries. Hislop and Reid [19] demonstrated that although they are frequently ignored, numerous supernumerary arteries additionally exist and exceed in number the axial ones. Supernumerary arteries branch out of an axial artery and run into the parenchyma surrounded by a connective ­tissue sheath and enter the alveolar wall and the alveolar capillaries in a way that has never been clarified but probably approaches the scheme shown in Fig. 6a, feeding the ­hexagonal ­network of capillary segments in the area of the corner vessels as demonstrated in Figs. 6b and c and 7, at intervals. In the systemic circulation, arterioles (small arteries where the endothelium is surrounded by a single layer of smooth muscle cells) are located between the last arteries and the capillaries and they are responsible for most of the resistance to blood flow and the drop of pressure. Such ­anatomically distinct arterioles are absent in the lung circulation, which raises the issue of the location of the main site of resistance to flow. In some cases it may well be the capillary corner capillaries (see later), which, if this assumption is ­correct, would represent the universal entry point of the arterial blood into the alveolar capillaries. At any rate, in many cases of pulmonary hypertension small extraalveolar vessels of minimal caliper with an abnormal layer of smooth muscle cells can be recognized. The veins inside of the lung rarely receive much attention, but they are also extraalveolar vessels because they are surrounded by connective tissue. They typically originate in the subpleural areas and are located in the interlobular septa, where they increase in size and advance toward the hilum (Fig. 7). Their wall is still thinner than that of the arteries of the same caliper and is frequently less well organized. Finally, the existence of numerous anastomoses between the pulmonary and the bronchopleural systemic circulation needs to be pointed out. They are located at the venular origins of the bronchiolar and the pleural circulations. The existence and possible reversibility of blood flow through these anastomoses is credited with the relative rarity of lung infarcts when compared with the frequency of embolisms.

J. Gil

Fig. 6  a The hexagonal capillary network of an alveolar wall shown as a continuum being drained and fed at intervals by small arteries and veins. b Flat view of the alveolar wall (comparable to a) where capillaries have been filled with a red stain. c Scanning electron micrograph of alveolar walls showing protruding filled capillaries. P is a pore of Cohn. Compare with a and b. (Reproduced with permission [7])

7  I nside the Alveolar Septa: Static Capillary Alveolar Microcirculation 7.1  The Hexagonal Capillary Network The small systemic arteries branch out into arterioles which split into a brush-like bundle of capillaries. Their blood is immediately collected by a mirrorlike assembly of venous capillaries soon assembled into a venule that leads to a drainage vein. This is not so in the pulmonary circulation.

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Fig. 7  Longitudinal section through an alveolar duct showing the relation of axial and supernumerary arteries to alveolar walls and the difference between primary and secondary alveolar walls. Not shown is the fact that arteries and veins are connected by the corner vessels which act as shunts

The first realistic description of the arrangement of the alveolar capillary segments in the alveolar wall was offered by Weibel [14] in his seminal book Morphometry of the Human Lung. Determined to quantify everything quantifiable, Weibel, needing a realistic model, pointed out the major ­difference between the systemic and the pulmonary capillary networks: the capillaries are in the form of a dense anastomosing mesh of capillary segments, which for modeling purposes approach hexagons (Fig.  6). Weibel [7, 20] later revealed the extraordinary size of the capillary endothelial surface, slightly smaller than the epithelial surface, in an average human lung some 120  m.2 In referring to surface areas, however, it is necessary to consider fractal effects. In other words, the size of an area always depends on the magnification at the time of the measurement

7.2  P  rimary Versus Secondary Alveolar Walls Before discussing the intraseptal capillary morphology, we need to point out another anatomic characteristic. Because more than 90% of the alveolar walls is occupied by pulmonary capillaries, it follows that the architecture, geometry, and relationships of alveolar walls must have a decisive influence on the geometry of alveolar capillary networks. In Fig. 7, note two important details: (1) only a small part of the alveolar capillaries are located close to the bronchioles, where the front of bulk air transport normally ends and where gas exchange should logically reach its maximum and (2) there are two different types of alveolar walls [21] — the primary septum at the bottom of alveoli, which separates alveoli open to different ducts (and sometimes possibly to different bronchioles), and the secondary septum, which

rises from the primary septum to separate two neighboring alveoli belonging to the same duct. At the end of the last duct, whenever the final insertion of the alveolar ducts into the pleura or a septum is near, the secondary walls taper off slowly and disappear, simplifying the ductal morphology and transforming alveolar ducts into cones or cylinders [21]. Considering the capillary flow, on a flat section one could be tempted to view the secondary alveolar wall as collateral of the primary wall: the blood that penetrates it will have to rise and fall back, causing considerable turbulence inside the hexagonal capillary segments. On the other hand, if the flow is viewed from a cross section of the duct, the impression would be that of a complex single layer of capillaries flowing constantly from the bronchiolar end toward the pleura, describing a zigzag motion in the secondary septa and a relatively straight line in the primary septa. Both alternatives are difficult to analyze from the hydrodynamic point of view.

7.3  Corner Capillaries Corner capillaries are those located in the corners of alveolar walls, where three septa meet. The word “corner” in this case is based on two-dimensional flat sections (Fig. 7). In the undisturbed three-dimensional space, they are likely to represent long lines at the junctions between primary and secondary septa. They have been observed by multiple authors when attempting to fix the dry lung at different degrees of inflation with high intraalveolar air pressures [21–28]. As we shall discuss below, high-pressure alveolar inflation (with air pressures above the local pulmonary arterial pressure) ­invariably results in the total collapse of the septal capillaries, which are mounted on the connective tissue continuum (Figs.  8, 9a, b) but lack any rigid supporting protective

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Fig. 8  Rat lung fixed by vascular perfusion under zone II conditions. To the right note septal pleat and open capillaries, to the left note a dilated corner vessel opening into a capillary

structure. At the same time, as discussed already, the extraalveolar arteries and veins dilate because of the radial pull of the periarterial alveolar walls. This results in the alveolar blood being squeezed out of the septa and pushed into storage in the extraalveolar vessels. Yet this situation would appear to be unstable as it might conceivably prevent the blood from returning to the veins and the left atrium. It is therefore ­necessary to have a mechanism capable of maintaining the patency of the lung even when the air pressure collapses most of the alveolar network. This is the function of the corner vessels (Figs. 8, 9a, b). They frequently appear dilated, which enables them to carry an increased flow volume possibly at higher velocity [29] and they are not affected by the high air pressure because of their corner location. The alveolar spaces acquire a more or less rounded structure that leaves the corner capillaries well protected in dead spaces. Corner capillaries are the most striking anatomic characteristic of West’s zone I.

7.4  Corner Pleats Gil and Weibel [4, 5, 15] first described in rat lungs fixed by vascular perfusion of chemical fixatives the existence of a reversible alveolar wall feature named “corner pleat” (Figs. 8, 9c) also located, like the corner vessels, in alveolar junctions. This caused confusion, as it was originally interpreted as a mechanism of pulmonary mechanics permitting the relaxation of alveolar corners tensed during inflation [5] and therefore the reduction in volume of the alveolar space. This interpretation is attractive because there is nothing in the alveolar wall to suggest that it might be particularly extensible and because areas of microatelectasis morphologically look like progressions of these pleats with the same structure. Pleats consist of nicked alveolar walls with capillaries folded over themselves and minute gaps filled

Fig.  9  Rabbit lungs fixed by vascular perfusion a, b under zone I, c under zone II, and d under zone III conditions. (Reproduced with permission [4])

with a surfactant-like fluid. It was later observed that alveolar pleats only appear under what is called zone II in West’s nomenclature, which is when the arterial pressure exceeds the air pressure and flow is driven by the difference between air and venous pressures. In this situation many alveolar

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Fig. 10  Summary of the findings in the three zones

walls are also collapsed and it can be assumed that the alveolar pleats fulfill a function similar to that of the corner vessels. In collapsed lumps it can be observed that pleats serve as needs for microatelectasis. Presumably the presence of surfactant reduces the retraction force arising from such areas.

8  F  unctional Adaptation of Alveolar Capillary Networks to Respiratory Movements In all organs of the body except the lung, vascular anatomy is generally fixed. Even if the number or the diameter of open capillaries varies as a function of the local functional or oxygen requirements, the structural arrangements remain unchanged. The reason for that difference is twofold: first, the very limited connective tissue support available to the alveolar structures, which renders them vulnerable to the effects of pressures, and secondly, the existence, in addition to the arterial pressure Pa and the venous pressure Pv, of a third player, the alveolar air pressure PA. It was West who first pointed out three different pressure combinations (named by him “zones”) that result in major changes of the alveolar and capillary configurations. Neither the connective tissue support nor the thin epithelial and

endothelial cells confer any stiffness. Here is a summary of the conditions and the findings [29] (Figs. 9, 10). In all cases it is understood that the alveolar pressure PA is identical to the pleural subatmospheric pressure: Zone I:  PA > Pa > Pv (Figs. 9a, 10), where the air pressure is higher than the arterial pressure, which itself is higher than the venous pressure. In the systemic circulation the arterial flow is driven by the difference between arterial and venous pressure, but here the driving force for the circulation is the difference between PA and Pa. The air pressure (including that generated by the alveolar surface tension) overwhelms any plastic tissue or intracapillary adaptations related to the perfusate and causes an ironing out or flattening of the entire alveolar surface. The exception is, of, course the alveolar corners, where a hidden space remains between the three rounded-off alveolar spaces, occupied by dilated capillaries named “corner vessels,” which most likely are equivalents of arterioles. These corner vessels are hidden from the alveolar pressure and will remain open and permit flow at any extreme alveolar air pressures (for instance, when using the abdominal press or during forced expiration), therefore ensuring a patent path between the right and left chambers of the heart. These corner vessels have similarities with the corner pleats discussed below. Zone II:  Pa > PA > Pv (Figs. 2b, 9b, c, 10, 11a, b), where the arterial pressure is the highest and the air pressure (including pressure generated by the surface tension) is intermediate

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Fig. 11  a Zone II rat lung showing at the bottom left pleated capillaries. b Zone II lung remarkable for a large, dilated corner vessel (top right)

between the arterial and venous pressures. Here the appearance of the tissue and vessels is the result of the ­balance between the arterial and the air pressure. There are areas where the arterial pressure prevails slightly and the alveolar septa are therefore perfused. Other alveolar septa, however, contain only collapsed capillaries. The alveolar epithelial surface remains smooth and the outline of the open capillaries is rectangular, not round (Fig.  10). This finding may result from the small difference between the values of the arterial and air pressures. Even small variations of intracapillary pressures seem to be able to turn the ­balance toward ­perfusion of the alveolar wall (when Pa prevails) or total collapse (when PA is locally higher than Pa). It is remarkable that an entire alveolar wall will show either empty capillaries or open capillaries but never isolated open capillaries in the middle of the wall. This suggests that folds at the intersection of septa may act as flood gates for an entire side of an alveolar wall. Finally, single, dilated, corner vessels are generally absent, but their place is taken by small corner pleats, infoldings of septal walls containing capillaries that acquire the appearance of a bundle and fill the space between the three rounded alveolar walls. The narrow gaps between folded-over alveolar walls are filled with a fluid thought to be alveolar surfactant. We [14] reported in a reconstruction of sections of rabbit lungs fixed by perfusion with osmium that these corner pleats can be seen connected to both an artery and a vein, making it clear that they represent shunt mechanisms aimed, like the corner vessels, at permitting the passage of blood regardless of high alveolar air pressure.  The morphology of this zone is important because it ­corresponds to the situation prevailing in intubated patients with positive end-expiratory pressure. It is relevant to note that the alveolar wall owing to the alternative stitching of the basement membranes of epithelial and endothelial cells to the epithelium of both sides has little capability for ­distension. The small spaces between capillaries contain only connective tissue and some rare cells, such as pericytes or fibroblasts, which may predispose them to folding. It is impossible to determine if these pleats are related or not to the hexagonal pattern. All the above applies only to

air-filled lungs. If the alveolar spaces are flooded with edema fluid, or surfactant the smoothening of the alveolar epithelial surface by alveolar pressure does not happen regardless of pressure relationships, as the pressure is equally transmitted to all points of the surface as described by the principles of solid mechanics [4]. Zone III:  Pa > Pv > PA (Figs. 9d, 10), where both the arterial and the venous pressure exceed the alveolar air pressure. This is the case when the alveolar morphology approaches the textbook description of the pulmonary circulation and the situation encountered in systemic organs, with the flow driven by the difference between Pa and Pv. Since Pv exceeds PA, the alveolar surface is undulated, no longer ironed out, with each alveolar capillary protruding into the alveolar space. No corner vessels and no alveolar pleats can be recognized and all available capillaries appear completely filled. In summary, the study of the morphological equivalents of the three zones of West is difficult and can be achieved only in lungs fixed by vascular perfusion of the fixative (preferably osmium). It is not known whether pressure alone is responsible for all observations described here or whether other factors, for instance, contractile cells also play a role. The observations appear to have the capability of profoundly affecting the functionality of the circulation, but also appear relevant to pulmonary mechanics. In the perspective of a dense hexagonal network of capillary segments fed and drained at regular intervals with a maximal endothelial surface of some 120  m2, without terminal vessels the corner pleats of zone II and the corner vessels seem to represent the secure and uncollapsible entry and exit points of the arterial and venous pulmonary circulations, whereas in zone III the entire morphological capillary network is available to exercise its gas-exchanging and metabolic functions without interference from the alveolar air pressure. Of interest are also the hydrodynamic properties and singularities of the system as described as it concerns flow velocity, pressure distribution and turbulence, and particularly the interplay with pulmonary mechanics. The capillary pulmonary pressure is believed to be of the order of 10 mmHg

2  Microcirculation of the Lung: Functional and Anatomic Aspects

[13, 30–34]. Years ago, there was a popular discussion between the followers of the tube flow theory under Poiseuillian conditions and the theorists of the sheet flow [9, 11, 13, 33, 34], where alveolar flow would be compared to circulation inside a parking garage with a low ceiling interrupted by posts (the center of the hexagons). The sheet flow theory seems to accommodate certain findings in zone II, whereas the tube flow theory suits bests the mainstream zone III circulation pattern.

9  Gas Exchange Few physiological functions are as dependent on physical properties as gas exchange. The morphometric basis of diffusing capacity has been repeatedly described [7, 20]. A simple way to approach the problem is that a tissue barrier or membrane is interposed between gas mixtures of different concentrations (Fig. 4). This barrier consists of thin layers of alveolar epithelium, interstitium, and capillary endothelium. This always applies, although one should additionally consider the distance to the front of inspired air. We have described three different configurations of the relevant gasexchanging structures and it is reasonable to assume that all three of them support this essential function. The most striking characteristic of zone I is that only corner vessels are open. Lamm [26, 27] showed that zone I lungs are capable of gas exchange. From our point of view, we note that the gas-exchanging membrane that surrounds the corner vessels is locally as thin as in other alveolar capillaries. Corner vessels are shunts that connect the arterial to the venous circulation, bypassing all septal vessels. It stands to reason that if they absorb all the circulation in the corners, additionally to some widening they need to be associated with a higher flow velocity of the blood. When all capillaries are open (Zone III) are would assume that their location is the site of capillary drainage. Similar considerations can be made with regard to zone II, which is particularly important in medicine. As described, the alveolar walls, if dry, that is, without alveolar edema, are smooth. Some alveolar walls will include patches of closed and open capillaries which typically have a quadrangular profile, whereas most of the alveolar wall capillaries are compressed and closed. The patches always extend from ­corner to corner, but we cannot explain the reason for this configuration. These are evidently suitable for gas exchange under all conditions. Regarding the corner pleats which represent a more or less circumferential (in two dimensions) convoluted bundle of capillaries inside folded alveolar walls, we have shown that they represent arteriovenous shunts which evidently allow some blood to escape and penetrate into the alveolar septa [15]. They have at least superficially some similarity with the glomerular capillaries in the kidney

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which produce only minimal resistance to perfusion and are therefore suitable for the function. The considerations presented for the corner vessels apply fully: the cluster of capillaries on several sides offers very thin air–blood barriers suitable for gas exchange. Finally, zone III, where the flow is driven only by the pressure difference between arteries and veins, is the model of the commonly shown pulmonary anatomy: the alveolar walls no longer appear flattened out because of the capillary bulges and all capillaries are open and it is also configuration where the diffusing capacity would reach its maximum. One should add that whenever alveolar edema exists and the air spaces are filled with fluid, the surface tension is ­abolished and the configuration is always that of zone III regardless of the pressures. This is shown in Fig. 12, which shows salinefilled rabbit lungs fixed under pressure conditions identical to those in the air-filled lungs shown in Figs. 3, 5, 8, 9.

Fig. 12  Counterpart to the micrographs of air-filled rabbit lungs shown in Fig. 9, but this time, prior to the perfusion with fixatives, the alveolar spaces of the lungs having been filled with saline, thus simulating the conditions of alveolar edema. (Reproduced with permission [4])

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References 1. Gil J (1990) The normal pulmonary circulation. In: Fishman AP (ed) The pulmonary circulation, normal and abnormal. University of Pennsylvania Press, Philadelphia 2. Gil J (1978) Morphologic aspects of alveolar microcirculation. Fed Proc 37:2462–2465 3. Gil J (1990) Controlled and reproducible fixation of the lung for correlated studies. In: Gil J (ed) Models of lung disease: methods in microscopy. Dekker, New York 4. Gil J, Bachofen H, Gehr P, Weibel ER (1979) Alveolar volume surface area relation in air and saline filled lungs fixed by vascular perfusion. J Appl Physiol 47:990–1001 5. Gil J, Weibel ER (1972) Morphological study of pressure-volume hysteresis in rat lungs fixed by vascular perfusion. Respir Physiol 15:190–213 6. Gil J (1988) The normal lung circulation: state of the art. Chest 93:805–825 7. Weibel ER, Gil J (1977) Structure function relationships of the alveolar level. In: West JB (ed) Engineering aspects of lung. Dekker, New York 8. Lovering AT, Stickland MK, Eldridge MW (2006) Intrapulmonary shunt during normoxic and hypoxic exercise in healthy humans. Adv Exp Med Biol 588:31–45 9. Lamm WJ, Bernard SL, Wagner WW, Glenny RW (2005) Intravital microscopic observations of 15-micron microspheres lodging in the pulmonary circulation. J Appl Physiol 98:2242–2248 10. Fung YC, Sobin SS (1977) Pulmonary alveolar blood flow. In: West JB (ed) Bioengineering aspects of the lung. Dekker, New York, pp 267–359 11. Fung Y, Yen RT (1986) A new theory of pulmonary blood flow in zone 2 condition. J Appl Physiol 60:1638–1650 12. Howell JBL, Permutt S, Proctor DF, Riley RL (1961) Effect of inflation of the lung on different parts of the pulmonary vascular bed. J Appl Physiol 16:71–76 13. Sobin SS, Fung YC (1992) Response to challenge to the Sobin-Fung approach to the study of pulmonary microcirculation. Chest 101: 1135–1143 14. Weibel ER (1963) Morphometry of the human lung. Springer, Berlin 15. Ciurea D, Gil J (1996) Morphometry of capillaries in three zones of rabbit lungs fixed by vascular perfusion. Anat Rec 244:182–192 16. Warwick R, Williams PL (eds) (1975) Gray’s anatomy, 35th edn. Longman, London 17. Gil J, McNiff JM (1981) Interstitial cells at the boundary between alveolar and extraalveolar connective tissue in the lung. J Ultrastr Res 76:149–157 18. Lai-Fook JS (1993) Mechanical factors in lung liquid distribution. Annu Rev Physiol 55:155–179 19. Hislop A, Reid L (1973) Pulmonary arterial development during childhood: branching pattern and structure. Thorax 28:129–135 20. Waehrli P, Burri PH, Gil J, Weibel ER (2007) Ultrastructure and morphometry of the human lung. In: Shields TW (ed) General thoracic surgery. Lee and Febiger, Philadelphia

J. Gil 21. Ciurea D, Gil J (1989) Morphometric study of human alveolar ducts based on serial sections. J Appl Physiol 67:2512–2521 22. Gil J, Ciurea D (2004) The functional structure of the pulmonary circulation. In: Peacock AJ, Rubin LJ (eds) Pulmonary circulation, diseases and their treatment, 2nd edn revised. Arnold, London 23. Guntheroth WG, Luchtel DL, Kawabori J (1992) Functional ­implications of the pulmonary microcirculation. An update. Chest 101:1131–1134 24. Bachofen H, Wagensteen D, Weibel ER (1982) Surfaces and ­volumes of alveolar tissue under zone II and zone II conditions. J Appl Physiol 53:879–885 25. Koyama S, Hildebrandt J (1991) Air interface and elastic recoil affect vascular resistance in three zones of rabbit lungs. J Appl Physiol 70:2422–2431 26. Lamm WJ, Obermiller T, Hlastala MP, Albert RK (1995) Perfusion through vessels open in zone 1 contributes to gas exchange in rabbit lungs in situ. J Appl Physiol 79:1895–1899 27. Lamm WJ, Kirk KR, Hanson WL, Wagner WW Jr, Albert RK (1991) Flow through zone I lungs utilizes alveolar corner vessels. J Appl Physiol 70:1518–1523 28. Conhaim RL, Rodenkirch LA (1998) Functional diameters of alveolar microvessels at high lung volume in zone II. J Appl Physiol 85:47–52 29. Naeije R (2004) Pulmonary vascular function. In: Peacock AJ, Rubin LJ (eds) Pulmonary circulation. Arnold, London 30. Pellett AA, Johnson RW, Morrison GG, Champagne MS, DeBoisblanc BP, Levitzky MG (1999) A comparison of pulmonary arterial occlusion algorithms for estimation of pulmonary capillary pressure. Am J Respir Crit Care Med 160:162–168 31. Ehrhart IC, Granger WM, Hofman WF (1986) Effect of arterial pressure on lung capillary pressure and edema after microembolism. J Appl Physiol 60:133–140 32. Gaar KA Jr, Taylor AE, Owens LJ, Guyton AC (1967) Pulmonary capillary pressure and filtration coefficient in the isolated perfused lung. Am J Physiol 213:910–914 33. Zhuang FY, Fung YC (1983) Yen RT analysis of blood flow in cat’s lung with detailed anatomic and elasticity data. J Appl Physiol 55:1341–1348 34. Sobin SS, Fung YC, Tremer HM, Rosenquist TH (1972) Elasticity of the pulmonary alveolar microvascular sheet in the cat. Circ Res 30:440–450

Additional Reading 35. West J, Dollery C, Naimark A (1964) Distribution of blood flow in isolated lungs; relation to vascular and alveolar pressures. J Appl Physiol 19:713–724 36. Staub NC, Storey WF (1962) Relation between morphological and physiological events in lung studied by rapid freezing. J Appl Physiol 17:381–390

Chapter 3

Pulmonary Vascular Development Rosemary C. Jones and Diane E. Capen

Abstract  The vascular beds of the adult lung, the pulmonary and bronchial circulations, are formed by extensive branching systems of large and small arteries and veins, and capillary networks. The focus of this chapter is the development of the pulmonary circulation in the normal lung as this evolves through the embryo/fetus to birth and the postnatal stage; and its continued development through childhood to the adult. In principle, the central large pulmonary arteries and pulmonary veins have a wall structure that reflects their role as conduits of deoxygenated or oxygenated blood. In addition to this function, the distal vascular loops of small thin-walled pulmonary (precapillary) arteries, capillaries, and (postcapillary) veins, which together form the lung’s microcirculation, serve as part of a gas-exchange surface for blood transiting this complex network. Blood vessels form and assemble networks in a series of elegant and intricate steps. Since the lung’s vasculature cannot develop in isolation from its airways, the mechanisms of morphogenesis discussed include, in brief, ones regulating specification of the lung primordium, and the early formation of the lung bud and airways. Mechanisms of alveologenesis, as these pertain to capillary bed formation, are also considered. Keywords  Lung vasculogenesis • Angiogenesis • Alveolo­ genesis • Pulmonary artery • Capillary • Vein

1 Overview of Lung Vascular Development The vascular beds of the adult lung, the pulmonary and bronchial circulations, are formed by extensive branching systems of large and small arteries and veins, and capillary networks. The focus of this chapter is the development of the pulmonary circulation in the normal lung as this evolves through R.C. Jones (*) Department of Anesthesia, Critical Care and Pain Management, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, USA e-mail: [email protected]

the embryo/fetus to birth and the postnatal stage; and its ­continued development through childhood to the adult (Fig.  1a). In principle, the central large pulmonary arteries and pulmonary veins have a wall structure that reflects their role as conduits of deoxygenated or oxygenated blood. In addition to this function, the distal vascular loops of small thin-walled pulmonary (precapillary) arteries, capillaries, and (postcapillary) veins, which together form the lung’s microcirculation, serve as part of a gas-exchange surface for blood transiting this complex network (Fig. 1b). Blood vessels form and assemble networks in a series of elegant and intricate steps. This account of pulmonary vascular development is based on a series of reviews and original publications by investigators who addressed both the larger picture and specific details of cellular and molecular events. Since the lung’s vasculature cannot develop in isolation from its airways, the mechanisms of morphogenesis discussed include, in brief, ones regulating specification of the lung primordium, and the early formation of the lung bud and airways. Mechanisms of alveologenesis, as these pertain to capillary bed formation, are also considered. The development of bronchial vessels (which supply nutrients to large vascular and airway structures) [1, 2], and of their closely associated lymphatics (which drain lung liquid and plasma proteins into the systemic circulation), is discussed elsewhere [3–8]. These systems must develop appropriately with the pulmonary vascular bed and airways to maintain adequate lung function. Following lung organogenesis, fetal vascular development, growth, and maturation to the time of birth, and in the perinatal period, proceeds in a series of time-related steps: a pseudoglandular, canalicular, and saccular phase followed by one of septation/alveologenesis. Although major changes in the overall template (blueprint) regulate lung vascular development in the fetus and neonate, growth and maturation then continue mainly by expansion in the child and through adolescence, until thoracic growth ceases in the adult [9–16] (Table 1). Because of the focus on the mouse in genetic regulation studies of lung development, the timing for this species is given together with that for the human lung (Table 1). The sequence of vascular patterning is constant, but the ­timing of

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_3, © Springer Science+Business Media, LLC 2011

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Fig. 1  Arteriograms and polymer cast of the pulmonary vascular bed. (a) Pulmonary arteriograms showing the human lung at three ages (prepared after injecting the pulmonary arterial bed with a barium sulfate–gelatin mixture), i.e., a newborn (upper left), an 18-month old infant (lower left), and an adult (right). The pre-acinar artery distribution (complete by 28 weeks in the fetus) is present at each time point shown, and the dense background haze, representing the growth of small intra-acinar arteries, increases with age [11]. (b) Photomicrograph of a lung injected with silicone polymer to show the dense capillary networks surrounding each alveolus in the normal pulmonary circulation; polymer perfused (at 50 mmHg infusion pressure) and fixed (2 cm-thick) tissue slices, from an adult lung following acute myocardial infarction, were cleared by incubation in 50, 75, 85, and 100% glycerol (each for 24 h) and examined with a Wild M8 stereomicroscope. Original magnification: (a) ×0.3 (the three lungs are photographed at the same magnification); (b) ×56. (a From Reid [11] copyright American Thoracic Society. (b) Reprinted from [272] with permission)

each phase varies with species [17–19]. These events are summarized in a broad timetable for airway, alveoli, and blood vessel development [20] (Table 2) and are illustrated in a schematic form [21] (Fig.  2). Airway morphogenesis is increasingly recognized as modulated by pulmonary vascular development [22]. For normal vascular development to proceed, the template at each stage must successfully generate vessels appropriate for size, wall structure, and location in the lung; and for age and stage of thoracic growth. Whether catch-up growth is possible will depend on when the normal development program is interrupted or fails. The lung anlage develops as an outpouch (ventral ­diverticulum) at the caudal end of the laryngotracheal groove from the embryonic foregut, and is vascularized by ingrowth of a vascular plexus from the heart/aortic sac and dorsal aorta.

From its connection to the right ventricle of the heart, the main pulmonary artery splits into a right and a left branch, and thereafter one branch remains in close association with each airway branch as these divide to supply the lung lobes and subunits; the central veins connect to the left side of the heart (Fig. 3a). These vessels form as angioblasts (cells committed to an endothelial lineage) and these develop into endotheliallike cells that assemble into tubes: capillary-like structures give rise to small-vessel networks, which increase in complexity by destabilization of existing structures and loop formation. The pulmonary arteries run centrally through the lung and end by supplying capillary segments in the intra-acinar region and pleura (save at the lung hilum). Blood draining from the capillaries enters the pulmonary veins at the periphery of lung units, the most distal veins lying at the edge of the

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Table  1  Timing of structural events in human lung development: organogenesis, pseudoglandular, canalicular, saccular, and alveolar stages (names describing fetal lung development as suggested by the Commission on Embryology Terminology in 1970). Some overlap in the timing of these structural events is recognized. The timing of stages in the mouse lung is included for comparison: the structural changes will be similar but species-specific   (i) Organogenesis (human ~3–5 weeks, mouse E9.5–E14.2a) Ventral outpouching of endodermal cells from anterior foregut into surrounding mesenchyme Ventral diverticulum forms primitive trachea and primary bud divides into two lung buds (endodermal cells forming an inner layer of epithelium, mesenchymal cells a middle layer, and mesothelial cells a thin outer layer) and ventral trachea separates from dorsal esophagus First of six pairs of aortic arch arteries appear and sixth aortic arch branches to lung buds (origin of main pulmonary arteries) a   (ii) Formation of proximal airways and vessels – pseudoglandular stage (human ~5–16 weeks, mouse E14.2–E16.6 ) Lung buds undergo repetitive dichotomous branching to form main and segmental bronchi, bronchioli, and terminal bronchioli – epithelial cells are undifferentiated (especially in the early phase). No respiration possible – airways are hollow blind-ending tubes The sixth left aortic arch develops into the main pulmonary artery and gives rise to the left and right pulmonary arteries Vascular branching parallels airways In human fetal lung, in the child and adult, and in the lungs of several other species studied, the number of arterial venous branches exceeds that of airways. Vessel branches accompanying airways are termed “conventional” and ones that do not accompany them are termed “supernumerary” (iii) Formation of distal airways and vessels – canalicular stage (human ~16–26 weeks, mouse E16.6–17.4a) Airway branching is complete and epithelial cell differentiation advances – ciliated epithelium lines airways and distal epithelium differentiates to “flattened” respiratory type – starting the formation of respiratory bronchioli and the acinar region Cells expressing epithelial type 2 markers herald the onset of surfactant synthesis Main arterial and venous pathways within a segment and side branches are present but are small relative to lung size Peripheral vascular network has developed (iv) Start of formation of acinus – saccular stage (human ~24 weeks to term, mouse E17.4–E19a) Additional respiratory bronchioli and saccules form and terminal saccules give rise to alveolar ducts and saccules Saccule epithelium attenuates to form the septal wall The number of epithelial type 2 cells increases Fetal pulmonary fluid is abundant in potential air spaces The density of small vessels increases and capillaries reorganize to form an air–blood interface At birth alveolar ducts and saccules, in close apposition to capillary networks, form functional gas-exchange units.   (v) Alveolarization (human from birth to ~8 years, mouse from birth to ~6 weeks) At birth, a critical transition from fetal life (in utero) to air-breathing – thin-walled alveoli and saccules are still immature structures After birth, alveoli and capillary structures continue to form and increase in size and number Initial phase – septation of original saccules (2 weeks in mouse, several months in human) Second phase – formation of additional alveoli (4 weeks in mouse, 7–8 years in human) Growth by expansion continues until thoracic growth is complete in the young adult Data from [19, 48, 75, 203, 227, 249, 270] a Embryonic days Table 2  Laws of lung development Law I – airway: The airways (i.e., bronchi and bronchioli) are present by the 16th week of intrauterine life Law II – alveoli: Alveolar structures appear before birth and increase in number, size, and complexity during growth Law III – vessels: The pre-acinar branches of the pulmonary artery (i.e., those accompanying bronchi or bronchioli), and pre-acinar venous tributaries, appear at the same time as the accompanying airways. Intra-acinar vessels appear as the alveoli grow; muscularization lags behind the appearance of new arteries Data from [271]

acinus: venous tributaries arise within alveolar walls and ducts, bronchial walls, the pleura, and connective tissue sheaths, and drain to axial veins that increase in size between the distal lung and hilum [10]. How the developing vascular units are regulated spatially within the surrounding mesenchyme is not known, although Hislop and Reid [13] have shown familial patterns of branching in the human lung, indicating a genetic component for the central vessels. Much still needs to be

understood of the “patterning rules” [23] that first determine formation of the lung’s vascular and microvascular networks, as well as the less ordered (but still hierarchical) vascular networks that form in the lung in injury or disease [24]. A remarkably simple and elegant process is proposed for the airways, for which developmental patterning data indicate three local “modes” of branching orders [25, 26]. Both regular and irregular dichotomous branching systems arise as vascular networks evolve from the expanding central arterial and venous structures by secondary budding, branching, and arborization, and by outgrowth of smaller vascular offshoots. The main arteries accompany airway branches – which branch first in a repetitive, stereotypic pattern (for 16 generations) and then in a nonstereotypic pattern (to generation 23). As the vascular networks increase in size and complexity with lung growth, they are simultaneously pruned by the regression of unperfused units until appropriate for the stage of development (endothelial cell apoptosis likely being triggered by signals related to change in blood flow patterns).

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Fig. 2  Summary of stages of fetal and postnatal lung development and growth, and their timing. Note the overlap between stages, particularly between the alveolar stage and the stage of microvascular maturation. Open-ended bars indicate uncertainty as to exact timing. (Reproduced from Burri [21] with permission from McGraw-Hill companies [21])

Fig.  3  Development of main pulmonary artery branches, and ­conventional and supernumerary artery branches. (a) Arrangement of the main pulmonary vessels in relation to bronchi: the pulmonary arteries arising from the right ventricle (RV) penetrate into the lung alongside the bronchi (left), whereas the main stems of the pulmonary veins leave each lung and enter the left atrium (LA, right). [273] (b) A broncho-arterial bundle illustrating conventional and supernumerary artery branches. Conventional arteries arise at acute angles to

the main axis and supply the respiratory region at the end of the axial pathway. Supernumerary arteries are short and have differing ­diameters. They arise at right angles to the main axis and supply adjacent air spaces, ultimately providing collateral circulation to a respiratory unit via the backdoor [11, 20, 27]. (a Reproduced from Weibel and Taylor [273] with permission from McGraw-Hill ­companies. (b) Reproduced from Jones and Reid [248] with permission from Elsevier)

Although the central arteries branch along with airways, the lung has more arterial than airway branches; at all levels these extra branches, termed “supernumerary” arteries, outnumber the “conventional” artery branches dividing with the airways [27, 28] (Fig. 3b). Typically, the supernumerary branches run a short course to supply the adjacent alveolar region. They appear to have their own vasoregulatory pathways, vasoconstriction being greater than for conventional arteries and the response to nitric oxide is different [29]. Conventional and supernumerary

veins are also present. Distally, small arteries and veins join an extensive capillary network, individual capillary segments being interconnected and running across several alveolar walls [30, 31]. It is ­proposed that the capillary bed represents two sheets of flat membranes, connected by interstitial tissue posts, which allow blood to flow readily across their surface [32, 33]. The size of the capillary network increases as alveolar structures develop. The total number of alveoli is generally accepted to be about 20 million at birth and to have reached the adult

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number of approximately 300 million by 8 years of age. [12, 34–37] In the adult lung, a total alveolar surface area of approximately 70 m2 is invested with a myriad of small arteries and veins and capillary segments. The capillary networks, for example, are calculated to increase in density four-fold between birth and adulthood, when their surface area is approximately 126 m2 [33, 38–40]. As the template changes, the remodeling of artery and vein walls is a striking feature of normal vascular growth and development [9, 10, 14]. In the fetus, vascular cells derive from the mass of mesenchymal cells surrounding the developing airway buds forming from endodermal cells. These mesenchymal cells give rise to a variety of vascular cell types that include endothelial cells and perivascular cells [smooth muscle cells (SMCs), pericytes, and fibroblasts]. Thereafter, these cell types interact to achieve the structural and functional heterogeneity of lung vessels and capillaries. The convention is to identify (class) lung vessels by their wall structure. When SMCs in vessel (artery or vein) walls are enveloped by numerous elastic laminae, as in the main pulmonary vessels and their large branches, these are termed elastic (Fig. 4a). More peripherally, as the number of laminae and SMCs decrease, vessels are termed transitional, and as the number of laminae decrease until SMCs are present between only an internal and an external lamina, they are termed muscular. Although this principle holds true for most segments, often the double laminae (and on occasion even a single lamina) are incomplete in small muscular veins. Distally in the lung, at the entrance to or within the acinus, the SMC layer thins to a few cells; within the acinus, as these cells change pitch to form a spiral, the

Fig. 4  Characterization of segmental and distal (pre- and post-­capillary) artery profiles. (a) Pulmonary arteriogram (from a lung injected with barium sulfate–gelatin mixture) showing an anterior basal segmental artery in a (39-year old) normal man, the region distal to the arrow representing a secondary lobule (left). Enlarged tracings of the same anterior basal segmental artery (#8 by standard nomenclature, as referenced by Elliott and Reid [27]), illustrating its wall structure, by the distribution along its length of elastic, transitional, and muscular regions (right). (b, 1–3) The wall structure of a distal pulmonary artery segment (i.e., pre-capillary segment within the acinus) showing the arrangement of smooth muscle cells (SMCs). In tissue sections examined by light microscopy (after injection of the pulmonary arterial bed with barium sulfate–gelatin mixture), and in cross-sectional profiles, the muscular artery segment has a complete medial smooth muscle coat, the partially muscular segment a crescent of medial smooth muscle, and the non-muscular segment no medial smooth muscle (see b1). Since the transition from muscular (to partially muscular) to non-muscular does not occur at the same point along each pathway, a particular wall structure cannot be predicted by the diameter or location (branch level) of a segment. Morphometric analysis of a population of artery profiles provides a useful way to establish the normal distribution and assess change in disease. For this, wall structure is noted; artery size

Fig. 4  (continued) (external diameter) is assessed by measuring the distance between the two edges of the external elastic laminae (EEL, see b2); and the medial thickness is measured as the distance between the internal elastic lamina (IEL) and the external one (see b3). Wall thickness is similarly measured but also includes endothelium. From the two values, i.e., external diameter and medial thickness/wall thickness, the percent medial thickness, or the percent wall thickness, is calculated to relate the proportion of the wall consisting of smooth muscle to vessel size. To assess the position of an artery profile in the branching pattern, the accompanying airway is used as a landmark, i.e., bronchiolus or terminal bronchiolus (pre-acinar), respiratory bronchiolus, alveolar duct or alveolar wall (intra-acinar). The distal pulmonary veins, which also have a muscular, partially muscular and non-muscular segment, are similarly measured to analyze their population distribution. By transmission microscopy (after injection of a fixative into the pulmonary circulation), SMCs are identified by their characteristic organelles and location in the walls of muscular and partially muscular arteries (dense shading). In non-muscular arteries, and in the non-muscular region of partially muscular ones, intermediate cells are present in normal lung (stippled shading); these are cells with a phenotype midway between a pericyte and a SMC, i.e., a population of precursor cells that rapidly express a SMC phenotype in response to a growth stimulus triggered by injury and/or change in transmural pressure [42, 149, 150, 174]. Original magnification: (a), left ×1, right ×2/3. (a Modified from Elliott and Reid [27] with permission. (b) Modified from Hislop and Reid [16] and Jones et  al. [272] with permission)

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vessels are termed partially muscular and where the cells disappear from the wall, they are termed non-muscular. The walls of the non-muscular vessels typically consist only of endothelium and a single lamina (Fig. 4b). Additional precursor SMCs are present beneath the endothelium in some of these vessels in the normal lung (Fig. 4b), and these cells play an important role in the development of new SMCs in response to injury and disease (see Chapter 42). Capillary walls consist of endothelial cells and basement membrane, with or without pericytes present as supporting perivascular cells [41]. The alveolar–capillary membrane in its thinnest region (1–2 mm) consists of capillary walls encased by epithelial cell processes, with or without an occasional perivascular cell between; in thicker regions, the interstitium of the membrane contains fibroblasts, myofibroblasts, migratory cells, collagen fibers and fibrils, and elastin [41]. Mainly in regions where several capillary segments converge, small vessels (arteries and veins) are embedded in the intervening matrix [42]. The normal distribution for a vessel population, and any change in the population in development or in disease, is revealed by a morphometric analysis of vessel profiles (i.e., vessel segments viewed in cross section) by their wall structure, external diameter, medial or wall thickness (Fig. 4b), and location (level in the branching pattern). Profiles associated with bronchi, bronchioli, or terminal bronchioli are termed preacinar; ones associated with respiratory bronchioli, alveolar ducts, or within the alveolar wall are termed intra-acinar. The acinus, defined as the respiratory unit supplied by a terminal bronchiolus, includes the respiratory bronchioli, alveolar ducts, and alveolar sacs, or alveoli. In the adult lung, the acinus is approximately 1 cm in diameter and consists of many alveoli. The lobule represents a group of three to five acini at the end of an airway. In the adult, the segment from artery to vein is usually short (less than 1 cm in length) and consists of vessels less than 200 mm in diameter. At all levels within this loop, lumen obliteration, occlusion, or restriction by wall thickening is usually the basis of a maintained rise in pulmonary artery pressure (see Chapter 43). The degree of muscularity of vessel walls is related to vessel size, and is proportional to blood flow and transmural pressure [43], save in the small intra-arteries positioned close to the entrance to the acinus, where wall thickness is normally high for vessel diameter. These are the “resistance” arteries of the lung. This local increase in wall thickness encroaches on the lumen rather than increasing the vessel diameter. Changes to the vascular template at birth reflect the lung’s adaptation from the high pressure/low flow system in utero to a low pressure/high flow system [11]. The relatively sparse number of SMCs present in pulmonary artery walls reflects the low resistance and pressure normally present, and is in contrast to the more muscular walls of systemic arteries where the pressure is normally six times greater. For morphometric analyses, the pulmonary vascular bed is prepared under standard conditions, the arterial or venous

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bed being distended with a (radio-opaque) barium sulfate– gelatin mixture at a hypertensive (100 cm H2O) pressure, and the airways are simultaneously distended with fixative at constant (23 cm H2O) pressure. This permits assessment by angiography and gives satisfactory lung fixation for vessels when quantified by brightfield microscopy [44]. When distending vessels greater than 15 mm in diameter, the barium injection medium normally does not cross the capillaries, thereby distinguishing the arteries from the veins. Pulmonary vessels more than 160 mm in diameter are distinctly outlined in the angiogram [45], and smaller vessels are discernible as a background haze (Fig. 1). Typically, the central region of the normal adult lung is characterized by relatively few, but large, conducting arteries or veins on the angiogram, whereas distally the density reflects the presence of small distributing vessels. Sparse in the fetal lung, these increase in size and number during normal growth (Fig. 1). The following provides an overview of lung vascular morphogenesis and signaling systems that regulate vascular development, maturation, and growth (Section 2). The vascular templates of the normal human lung at different ages are then described (Section 3), followed by an account of aberrant patterns of vascular development that result in an adequately functioning lung or ones in which vascular patterning is irreversibly changed (Section 4).

2 Regulation of Lung Vascular Morphogenesis As the lung anlage develops, its formation and remodeling patterns are regulated by a host of transcription and growth factors, the former integrating genetic instructions with growth factor mediated signaling [46–55]. Other factors that influence lung vascular growth and maturation in important ways include physical factors such as intra-thoracic space, lung liquid volume and pressure, amniotic fluid volume, and hormones released by endocrine glands (pituitary, adrenal, and thyroid) [56]. Extracellular matrix signals (particularly basement membrane components such as laminins, entactin, type IV collagen, perlecan, SPARC, and fibromodulin) interact to regulate development [57–63]. Oxygen signaling pathways also play a role in the formation of the lung and its vasculature [64, 65], a low level of oxygen (hypoxia) enhancing distal airway branching and a high level of oxygen inhibiting it and inducing mesenchymal cell apoptosis [66]. Genetic studies in mice continue to reveal the function of a variety of highly regulated factors such as thyroid transcription factor-1 (also known as Nx.1), Gli family genes, retinoid receptors, Smad, N-myc, hepatic nuclear factor-3b, (HNF-3β), helix forkhead-4 (HFH-4), glucocorticoid receptor, cyclic AMP response element binding protein, and CCAAT/enhancer binding proteins, which control the expression of genes encoding

3  Pulmonary Vascular Development

growth factors (which stimulate growth), and of morphogens (which regulate growth responses by forming gradients) in the developing lung [46, 49, 50, 53, 55]. Several of these have dual roles, regulating cell specification in early lung development and the specialized functions of differentiated airway and vascular cells. Each early growth phase in the lung depends on specific interactions between endodermal and mesenchymal cells mediated by growth factors, and morphogens. These include fibroblast growth factor (FGF), transforming growth factor (TGF)-b, epidermal growth factor, vascular endothelial growth factor (VEGF), placental growth factor, platelet-derived growth factor (PDGF), angiopoietin (ANG), and sonic hedgehog (SHH) [46, 49, 50, 53, 55]. Factors known to regulate pulmonary vascular development in the lung are the focus of the present account. In considering the molecular basis of lung vessel formation, Stenmark and Abman [65] included a comprehensive list of molecules likely involved at different stages. Since the factors regulating cell proliferation, migration, differentiation, and survival are not lung-specific, the exquisite and complex architecture of the lung’s vascular bed is determined by their expression patterns and ­interactions, as demonstrated by investigators in the field [49, 50, 52, 53, 55].

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Genetic modifications in mice illustrate examples of failed lung development [48] (Fig. 5a). Once the lung buds form, the trachea extends. The presence of bronchial mesenchyme is

required for airway development to proceed from the trachea by appropriate branching [67]. For example, lung buds cultured without mesenchyme, or with tracheal mesenchyme, fail to branch; and when cultured with the mesenchyme from stomach, gut, or liver, the buds form gastric glands, villi, and hepatic chords, respectively [67]. The lobes form as the developing structures are subdivided by the visceral pleura (Fig. 5b). Left–right (L–R) lung asymmetry, and antero-posterior and proximal–distal patterning are instructed early. Lefty-1, Lefty2, nodal, and Pitx-2 determine L–R asymmetry: [48, 52, 54, 68] transient expression (E8–E8.5) on the left side of prospective floor plate and lateral plate mesoderm indicates that specification of the branching pattern and lobation occurs 24–36 h before the primordial bud appears. Abrogation of Lefty-1 results in bilateral expression of Lefty-2, nodal, and Pitx2 (indicating that Lefty-1 normally restricts Lefty-2 and nodal expression to the left side and leads to left-sided expression of Pitx2). L–R determining genes are expressed when foregut retinoic acid (RA) synthesis/utilization is high and thus are RA targets. HFH-4−/− mice, in which left/right dynein (a protein critical for nonrandom visceral orientation) is abrogated, demonstrate random visceral L–R laterality and lack cilia. These determinants of symmetry/asymmetry are ­superimposed on branching mechanisms effected by signaling between ­epithelium, mesenchyme, and mesothelium [48, 52, 54, 68]. Early restriction of epithelial progenitor/precursor cells [69] results in non-overlapping cell lineages being established for conducting airways (trachea and bronchi) and distal airways (bronchioli, acini, and alveoli) before the definitive lung buds form (at E9–E9.5). Two days later (at E11.5),

Fig. 5  Early lung formation: unsuccessful development in null mutant phenotypes and a dominant negative misexpression phenotype. (a) The HNF3b−/− phenotype is a severe early embryonic lethal phenotype in which the primitive foregut endoderm fails to close into a tube [274]. In the compound null Gli2−/−/Gli3−/− mutant phenotype the primitive lung anlage completely fails to arise from the primitive foregut endoderm [275]. Gli2−/− null mutation and Gli2 gene dosage reduction result in abnormalities of lung lobation. The fibroblast growth factor (FGF)-10−/− phenotype results in the larynx and trachea forming and separating dorso-ventrally from the esophagus, but the primary bronchial branches completely fail to arise from the trachea [276]. The morphologically similar Shh (Nkx2.1) null mutant phenotypes result in the trachea failing to separate dorso-­ ventrally from the esophagus, forming a tracheoesophageal tube from

the sides of which grossly hypoplastic and dysplastic epithelial bags arise. In the Nkx2.1 null mutant, epithelial differentiation is arrested prior to the expression of peripheral epithelial markers; by contrast, in the Shh null mutant, peripheral lung epithelial markers are expressed [277–279]. Finally, the phenotype in transgenic mice that misexpress a dominant negative, tyrosine kinase deleted FGF receptor under the control of the surfactant protein C promoter/enhancer causes bronchial morphogenesis abrogation distal to the primary bronchi [280]. (b) Successive stages in human lung development, at 5 weeks (left), at 6 weeks (center), and at 8 weeks (right). Note the formation of lung lobes by invagination of the visceral pleura (shown in red). (a From Warburton et al. [48], copyright 2000, with permission from Elsevier [48]. b Reprinted from Sadler [281] with permission)

2.1 Early Pre-specification Patterns in Lung Assembly

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labeled precursors of peripheral lung are restricted to a few bronchial tube cells and to cell clusters in bronchial tips and lateral buds: these cells subsequently undergo expansion to form the alveolar surface. It is proposed that the repetitive branching patterns that characterize the development of preacinar airway generations result from a series of complex interactions between signaling molecules in tissue zones termed “morphogenetic signaling centers”: FGF-10/FGF receptor-2 signaling, for example, appears key and the temporal–spatial patterns of expression for selected regulator molecules (such as SHH, TGF-b, bone morphogenetic protein 4, and mSpry) are consistent with their role as modulators of FGF signaling [50, 70]. It is not established if central vascular branching is achieved in a similar way.

R.C. Jones and D.E. Capen

The lung vasculature forms by integrated mechanisms of sprouting angiogenesis and vasculogenesis (Fig. 6). These structures are remodeled in a process termed intussusception (i.e., of itself) whereby the capillary wall invaginates to form a contact zone between opposing endothelial cells. The formation of an intraluminal connective tissue post at the site of endothelial cell contact splits the capillary structure into two segments. Capillary networks continue to expand by intussusceptive microvascular growth [33] as well as simple expansion (Fig. 7). In addition, endothelial cells may remodel the forming capillary segments by their “guided migration” over existing capillary endothelial cells [71]. Small arteries and veins subsequently evolve from the capillary networks by intussusceptive arborization and branch remodeling [72–74] (Fig. 8). On the basis of the analysis of Mercox casts of mouse lung, and scanning and transmission microscopy, deMello et al. described steps in lung vascular formation [75, 76]. The growth of central arterial and venous trunks and their branches proceeds by angiogenesis (Fig. 6c), offshoots of endothelial sprouts at the growth points of these vessels invading the mass of mesenchymal cells surrounding the airway tubes. The solid

tubelike sprouts first formed organize into open tubules as the endothelial cells polarize around a lumen. These tubelike segments continue to elongate by cell migration and proliferation and branch, or fuse (at their blind end) to another vascular sprout to form a new loop. The central artery branches accompany the intrapulmonary airways by E10 in the mouse; [75] by E11.5 there is significant secondary branching and development of further bronchial structures, and by E12.5 arborization is extensive as the central units expand in diameter and length and offshoots grow by expansion and irregular dichotomous branching [75]. At about this time, the segmental pulmonary arteries connect to the central vessels [75]. The vascular side branching pattern of the pulmonary arterial tree, which accounts for up to 40% of the vascular endothelium in wild-type mice, is reduced in transgenic mice highly expressing matrix Gla protein, an inhibitor of bone morphogenetic protein [77]. Such changes are thought to occur in response to reduced levels of phosphorylated Smad5 or Smad8 in alveolar epithelium, and epithelial expression of the activin-like kinase receptor 1 and VEGF (being critical for vascular formation) may lead to loss of coordination (cross talk) between the developing vascular and airway cells [77]. In a separate but concurrent process, vascular segments in the peripheral (sub-pleural) zone of the developing mouse lung form by vasculogenesis [75], the angioblasts forming nests of cells that assemble into capillary-like structures enclosing hemangioblasts (future blood cells) (Fig.  6a, b). Transmission microscopy studies [75] reveal, as early as when the first airway branches form (at E9), abundant blood-filled channels in the peripheral sub-pleural mesenchyme. These lack connections to the central arteries. The intercellular spaces forming the lumen of the first channels arise by loss of vesicles from the apical membranes of mesenchymal cells. By thinning their processes, and reforming their apical membranes, mesenchymal cells evolve to endothelial-like cells (Fig. 6b). The separately developing central and distal vascular systems fuse in the mid fetal stage to complete the vascular circuit (E13–E14) [75]. The extent of growth in the normal mouse lung at this time (E14 and E15) provides a striking example of change in the vascular template (Fig. 9). Although connections between the central pre- and post-capillary vessels and distal vascular networks are sparse by the end of the pseudoglandular stage (E14.2–E16.6), in the canalicular stage (E16.6–E17.4) the arterial–capillary–venous circuit is well

Fig. 6  (continued) an endothelial lineage, and associate into blood islands; developing contacts, the angioblasts assemble into capillary-like structures that subsequently connect and retain hemangioblasts (hematopoietic precursor cells) within their lumen. (c) Transmission electron micrographs of a mouse lung (E9) showing densely packed mesenchymal cells containing cytoplasmic vesicles surrounding the developing lung bud (top left), and intercellular spaces forming (which appear to result from discharge of intracytoplasmic vesicles, leaving a ruptured cell membrane)

between plump, densely packed mesenchymal cells (top right). Thinning of lung mesenchymal cells adjacent to intercellular spaces, in a later fetus (E10), results in endothelial-like cells (lower left). Hematopoietic precursor cells are present in some luminal spaces (lower right). Original magnification: (c) top left ×2,800; top right ×8,750; lower left and lower right ×5,250. (a, b Reprinted from Schoefl [282] with permission from WileyBlackwell. (c) Redrawn and reproduced from deMello et al. [75] with permission, copyright American Thoracic Society [75])

2.2 Lung Vessel Morphogenesis 2.2.1 Endothelial Tube Formation (Vessels and Capillaries)

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Fig. 6   Capillary assembly by angiogenesis and vasculogenesis. (a) Capillary formation by angiogenesis: endothelial cells and their processes, released from the constraint of a basement membrane, and in response to a signal triggering growth, invade the surrounding tissue (sprout in the direction of the growth signal) to form cell-lined channels (top left and top right). Wall destabilization, with focal degradation of the endothelial basement membrane and surrounding matrix, is followed by the formation of a spur as endothelial pseudopodia extend through the gap. Endothelial cell migration in the direction of the growth spur forms a sprout [282, 283]. The delicate microspikes at the leading edge of migrating endothelial cells forming the tip of the sprout lack basement membrane. Rather than migrating singly, they move as a shifting sheet

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of cells [282]. Typically, proliferating cells lying behind the growing tip increase the length of the sprout [283], although sprouts can develop in the absence of endothelial cell proliferation (as in inflammation): an increasing cell population is needed, however, for sustained growth [283, 284]. Although still connected at their origin to a patent vessel or capillary, sprouts continue to elongate and to branch or fuse to an adjacent sprout at their blind end to form a new loop and establish a contiguous network (lower left and lower right). Development of a slit-like lumen followed by the entry of plasma and blood cells completes the formation of a continuous channel. (b) Capillary formation by vasculogenesis: aggregates of undifferentiated mesenchymal cells [281] differentiate in situ into angioblasts, i.e., precursor cells committed to

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Fig. 7  Capillary remodeling and growth: intussusceptive microvascular growth (IMG), and expansion. (a) Steps of intussusception: the capillary lumen is divided as opposing walls protrude to create a contact zone between endothelial cells. Following central perforation of the cellular bilayer, the fused endothelial cells form a transluminal cuff, which is later invaded (and strengthened) by myofibroblasts or pericytes. (b) Growth of an existing vascular bed occurs by simple expansion, by the addition of wall cells and by increase in lumen diameter and segment length (a Reproduced from Kurz et al. [74] with permission. (b) Reprinted from Schoefl [282] with permission from Wiley-Blackwell)

established and connections are widespread [75]. The report of even earlier connections between the two vascular systems in the mouse lung indicates continuity from the time of inception [78]. Other concepts have been proposed, with the vasculature formed, on the one hand, by distal angiogenesis throughout the mouse lung by peripheral vessels connected to the embryonic circulation [79], and, on the other, by vasculogenesis throughout the lung of the chick embryo [80]. The extent to which the central and distal lung vessels develop by processes of angiogenesis and/or vasculogenesis is still subject to debate. Studies demonstrating that endothelial cell function and molecular phenotypes are vessel-­specific, however – those of large vessels having a distinct and different molecular phenotype from microvascular ones, small-vessel endothelial cells arising from within the mesenchyme and large-vessel endothelium originating from the pulmonary truncus by angiogenesis – support the concept that the lung’s vasculature develops by combined processes [81–83]. Although, in general, it has been accepted that capillaries form by angiogenesis and vasculogenesis before birth, and by angiogenesis after birth, their formation by vasculogenesis may also occur postnatally [84]. Of great current interest is the potential role in the growing (and injured) lung of endothelial

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cells, and endothelial cell precursors, derived from the bone marrow and circulating in blood [85, 86]. Circulating hematopoietic progenitor/precursor cells have been reported to contribute to capillary growth and repair in the developing and adult lung [87, 88], and have been detected in the walls of pulmonary arteries in lung disease [89]. The possible population of blood-borne cells fulfilling this role is likely heterogeneous and a true “vascular” progenitor remains to be defined [85]. Studies describing the molecular basis of endothelial channel formation increasingly provide insights into structural events [90–96]. Growth factors such as the FGF family first induce angioblast and hemangioblast formation in the mesenchyme. The homodimeric glycoprotein VEGF is then central in regulating blood vessel formation, maintaining angioblast differentiation and survival via its receptor tyrosine kinases (RTKs), i.e., VEGF-R1 (Flt-1), VEGF-R2 (Flk-1/KDR), and VEGF-R3 (Flt-4). VEGF-A and VEGF-D are abundant in developing lung, VEGF-A being detected in the lung mesenchyme throughout fetal development and VEGF-D from E13.5 [97]. Alternative splicing produces homodimeric VEGF-A isoforms – VEGF-A120/121, VEGF-A164/165, VEGF-A189, and VEGF-A206. Of these, VEGF121 and VEGF165 act as survival factors for endothelial cells, and before E14 these soluble isoforms predominate in the developing lung [97]. VEGF is induced by SHH and is highly expressed by epithelial cells localizing within the matrix at the leading edge of branching airways [96, 98]. The VEGF signals controlling angiogenesis in the lung thus arise from adjacent epithelial cells, stimulating cognate RTKs on endothelial cells. VEGF-R1 and VEGF-R2, in particular, play critical roles in both angiogenesis and vasculogenesis, being required for endothelial cell proliferation, tubule assembly, and wall stabilization [99–101]. Clusters of VEGF-R2+ (Flk-1+) vascular precursors, localized in the mesenchyme close to developing epithelium from E11 to birth, demonstrate that vascularization is initiated, and endothelial cell lineage established, when the lung anlage first develops. VEGF-R1 expression is required by angioblasts to assemble into vascular channels, including tubule formation and sprouting (Fig. 10); VEGF-R2, expressed by endothelial precursors as they differentiate to endothelial cells, results in proliferation [92, 100, 102–105] (Fig. 10). During the differentiation of cells within the mesenchyme there is an ordered progression of surface markers, with the expression of VEGF-R2 being followed by that of vascular endothelial cadherin, platelet-endothelial capillary adhesion molecule (PECAM-1), and CD34 [106]. Yamamoto et  al. [107] characterized developing endothelial cells from undifferentiated mesenchymal cells by their expression of VEGF-R2/VEGF-R1, and PECAM-1, and the shift in temporal–spatial expression patterns of these molecules as ­capillary structures form in the lung’s pseudoglandular and canalicular phase. Early in development (E9.5–E10.5)

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Fig. 8  Capillary and vessel remodeling and intussusceptive microvascular growth IMG, intussusceptive arborization (IAR), and intussusceptive branch remodeling (IBR). (a) Insertion of transluminal pillars (arrowheads) results in rapid expansion of the capillary plexus by IMG. IAR generates feed vessels from the capillary plexus by vertical pillar formation in rows (a arrows), which demarcate future vessels. (b, c) Narrow tissue septa (arrows) formed by pillar reshaping and pillar fusions segregate the new vascular entity. (d) Formation of horizontal

pillars and folds (arrowheads) separate the newborn feeding vessels from the capillary plexus. (e–g) IBR finally adapts the deepness of the branching angle and the diameters of daughter vessels in the newly formed supplying and draining vessels by insertion of transluminary pillars at branching points (arrows). (f–h) Moreover, IBR leads to vascular pruning by repetitively eccentric formation, augmentation, and fusion of pillars (arrowheads). (Reproduced from Djonov et  al. [72] with permission from Lippincott, Williams & Wilkins)

n­ umerous undifferentiated mesenchymal cells and VEGF-R2+ cells, and a few PECAM-1 expressing cells, surround the developing distal airway bud, whereas VEGF-R1+ cells are virtually absent. Subsequently (E11.5–E13.5), fewer undifferentiated mesenchymal cells and VEGF-R2+ cells are present, PECAM-1+ cells increase in number, and small numbers of VEGF-R1+ cells appear. Later, in the pseudoglandular phase (E14.5–E16.5) an organized layer of undifferentiated mesenchymal cells, and few VEGF-R2+ cells surrounding the airway, are in turn now surrounded by an organized layer of PECAM-1 cells, whereas the numbers of VEGF-R1+ expressing cells increase. Finally, in the canalicular/saccular phase (E17.5), VEGF-R1+ and VEGF-R2+ cells are sparse, and a well-organized layer of PECAM-1+ cells surrounds alveolar epithelial cell tubules. Different ligands determine cell differentiation: for example, whereas VEGF promotes VEGF-R2 expressing cells to differentiate to endothelial cells, PDGF-B promotes their differentiation to SMCs and pericytes [108].

While endothelial cells develop in VEGF−/− mice, the ­vessels formated are abnormal. Loss of VEGF164 and VEGF188 isoforms leads to failure to develop a normal distal vasculature and impairs alveolarization [109] (Fig.  11). VEGF expression in (surfactant protein C–VEGF) transgenic mice increases lung blood vessel growth and distorts branching morphogenesis [110]. VEGF-R1−/− mice have normal progenitor cells and abundant endothelial cells that migrate and proliferate but do not assemble into tubules or form functional vessels, whereas VEGF-R2−/− mice have failed vasculogenesis and blood island formation and do not develop endothelial cells, possibly because hemangioblasts fail to differentiate. VEGF stabilizes developing vessel walls by accelerating the development of supporting perivascular cells [111, 112]. Immature vessels that lack perivascular cells need VEGF to prevent their endothelial cells from detaching and undergoing apoptosis, but during postnatal life endothelial cells become VEGF-independent – at least from paracrine signaling

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Fig. 9  Mercox casts illustrating normal pulmonary vascular growth patterns. (a) Scanning electron micrograph of the vascular bed of a mouse (at E14). Note the presence of occasional small capillary buds and branches (arrowheads), intersecting at right angles with the lumen of larger adjacent capillary segments. (b–d) Scanning electron micrographs of the vascular bed of a mouse (at E15). Extensive connections between the central and peripheral systems are now present, revealing a complex peripheral network. (c–d) At higher magnification

(see area indicated in b, arrow) blind-ending pre-capillary branches are seen to approach the future capillary network at right angles. Residual constrictions suggest sites of coalescence (fusion). It seems likely that the vessel upstream from the arrow is central, whereas the more irregular vessels below the arrow are derived from the peripheral system. Original magnification: (a) ×320; (b) ×69; (c) ×320; (d) ×1,200. (Reprinted from deMello et  al. [75] with ­permission, copyright American Thoracic Society [75])

p­ athways – although autologous VEGF signaling (independent of paracrine VEGF signaling) is likely maintained for endothelial cell homeostasis [113]. Gebb and Shannon [96] have shown that endothelial expression of VEGF-R2 within the mesenchyme requires the presence of epithelium, demonstrating a regulatory loop between the two systems; and pulmonary vascular development has been shown to have a rate-limiting role in epithelial branching [114]. The divergent homeobox gene Hex is expressed in the distal lung mesenchyme where primitive blood islands appear and is a specific and early marker of endothelial cell precursors. Unlike VEGF-R2, it is downregulated as endothelial cell differentiation begins. Hex disruption, unlike the disruption of VEGF-R2, has no effect on the presence of hematopoietic precursor cells or vascular network formation [115, 116].

Tie-1 and Tie-2 are a second family of RTKs expressed by endothelial cells (Fig. 10); ANG1, the major ligand for Tie2, is expressed by mesenchymal cells. As yet, no ligand has been identified for Tie-1, which modulates transcapillary fluid exchange. ANG2 is a negative Tie-2 ligand [91, 92, 117–123]. Tie-2 expression modulates VEGF activity and is required for endothelial sprout formation [119, 120, 124]. Although Tie-2−/− mice have normal numbers of endothelial cells, these assemble into immature channels that lack branching networks and the presence of large and small vessels. ANG1−/− mice have vascular defects similar to those of Tie2−/− mice and die of dilated/impaired vessel branching (at E10.5). ANG1 together with VEGF enhances vascular density, whereas ANG2 and VEGF produce longer sprouts, indicating a role for ANG2 in vessel formation in addition to regression

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Fig.  10  Regulation of vascular morphogenesis, maintenance, and remodeling by receptor tyrosine kinases (RTKs) and their ligands. A model for regulation of vascular endothelium is demonstrated by the prototypical angiogenesis factor vascular endothelial growth factor (VEGF) and the class of angiogenic regulators angiopoietin-1 (ANG1) and angiopoietin-2 (ANG2). All three ligands bind to RTKs that have similar cytoplasmic signaling domains, yet their downstream signals elicit distinct cellular responses. Only VEGF binding to VEGF-R2 (Flk1) sends a classic proliferative signal. When first activated in embryogenesis, this interaction induces the birth and proliferation of endothelial cells. In contrast, VEGF binding to VEGF-R1 (Flt1) elicits endothelial cell–cell interactions and capillary tube formation, a process

that closely follows proliferation and migration of endothelial cells. ANG1 binding to Tie-2 RTK recruits and likely maintains association of peri-endothelial support cells (pericytes, SMCs, myocardiocytes), thus solidifying and stabilizing newly formed blood vessels. ANG2, although highly homologous to ANG1, does not activate Tie-2 RTK; rather, it binds and blocks kinase activation in endothelial cells. The ANG2 negative signal causes vessel structures to become loosened, reducing endothelial cell contacts with matrix and disassociating periendothelial support cells. This loosening appears to render the endothelial cells more accessible and responsive to the angiogenic inducer VEGF (and likely to other inducers). (Reprinted from Hanahan [92] with permission from AAAS [92])

Fig. 11  Mercox casts illustrating abnormal pulmonary vascular growth patterns. (a–f) Scanning electron micrographs show different growth patterns in VEGF 120/120 littermate fetal mice (E17): wild-type (a, d), heterozygous (b, e), homozygous (c, f). In the homozygous fetus, there

are fewer small peripheral vessels and they are of larger caliber than those in heterozygous or wild-type littermates. Magnification: Bar (a–c) 152 mm and (d–f) 50 mm. (Reprinted from Galambos et al. [109] with permission, copyright American Thoracic Society [109])

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[124]. In the absence of VEGF and in the presence of ANG2, vessels destabilize as endothelial cells undergo apoptosis. TGF-b1−/− mice have poor endothelial cell differentiation and hematopoiesis and die at mid-gestation (E10.5) [125, 126]. TGF-b1 binds TbRII and activin-receptor-like kinases, ALK1 and ALK5 – the ALK5 (TbRI) receptor phosphorylating Smad2 and Smad3: these complex with Smad4, which translocates to the nucleus and activates transcription target genes [127]. ALK5 (TbRI)−/− mice form blood islands, and their vessels initially are normal, but they develop defects similar to TGF-b1 and TbRII mutants, their vessels lacking hierarchical organized branching and being fragile because supporting perivascular cells fail to differentiate, and the mice die (at E10.5–E11.5) [128]. ALK1−/− mice have fused, dilated major vessels (at E9.5); they fail to form capillary networks, and die (at E11.5). The vascular malformations in these (and endoglin−/−) mice (increased numbers of dilated vessels and hemorrhage) are characteristic of vascular abnormalities in human hereditary hemangiotalactasia [129]. Hoxa-3−/− mice die shortly after birth with heart defects and enlarged pulmonary veins. The walls of their vessels, which normally consist of endothelial cells, SMCs, and an outer layer of cardiac cells, are altered, suggesting that ablation of this gene leads to impaired venous wall formation and vein enlargement resulting from limited cardiac cell migration. Wnt signals are key regulators of cell proliferation and differentiation, and within this growth factor family, signaling of the gene Wnt7b regulates cell proliferation and growth in lung mesenchyme. Analysis of Wnt7blacZ−/− neonatal mice reveals vessel rupture and hemorrhage, indicating a critical role for Wnt7b in lung mesenchymal growth and vascular development [130]. Hox genes regulate lung airway development and can serve to induce an angiogenic phenotype in endothelial cells

[131]. For example, Hoxd-3 colocalizes with avb3 integrin in vessels formed in response to FGF-2 (increasing components of the angiogenic cascade), Hoxb-3 (a Hoxd-3 paralogue) promotes lung capillary formation (controlling expression of ephrin A1/EphA2, see later), whereas Hoxb-7 expressed in fetal lung upregulates angiogenic stimuli (FGF-2, VEGF, and matrix metalloproteinases). Yet other Hox genes suppress an angiogenic phenotype, perhaps to maintain endothelial cells in a fully differentiated state, as in the mature lung. Hoxd-10 is an example of this, being expressed in the adult but not the developing lung, with its expression greater in quiescent endothelial cells, where it blocks FGF-2 dependent migration. As an antiangiogenic factor (inhibiting vessel growth in corneal and Matrigel models), the cytokine-like molecule endothelial monocyte activating polypeptide II (EMAPII) is proposed to negatively regulate lung vascularization. As the lung evolves from a poor to a richly vascularized tissue (E14– E18.5/E19), messenger RNA/protein expression decreases five-fold. Postnatal EMAPII expression levels generally remain low [132, 133]. EMAPII induces endothelial cell apoptosis in adult lung, and its localization to perivascular regions of large arteries may inhibit their branching while the peripheral vasculature continues to develop [132, 133].

Fig. 12  Distribution and sites of action of ephrin B2 and EphB4, a ligand– receptor pair for endothelial cell specification, in arteries and veins of a primary capillary plexus and in a maturing vascular network. [94] They are presumed to interact between opposing arterial and venous endothelial cells in a cis manner (left). During later maturation and remodeling of the

primary plexus (right), by interdigitation, branching, and differential growth of vascular segments, they remain localized to arterial and venous units (cis interactions) but may also interact at the interdigitating surfaces of large vessels (trans interactions). (Reprinted from Yancopoulos et al. [93], copyright 1998, with permission from Elsevier)

2.2.2 Arterial and Venous Specification and Differentiation Arteriovenous relationships are likely established and ­maintained by the expression of the ligand ephrin B2 and its receptor EphB4 [93–95] (Fig.  12). Functional vessels are first reported in the distal mesenchyme in the earliest phase of lung development in the mouse (E11.5), blood flow being

3  Pulmonary Vascular Development

indicated by the delivery of intravascular fluorescein-labeled lectin; later (by E14.5), such vessels extend to the epithelial interface (in response to VEGF signals from epithelial cells) [134]. Despite the evidence of flow, however, the emerging pulmonary vasculature was found to lack arteriovenous specification as determined by ephrin B2 and EphB4 expression [134]. Additional studies are needed to determine the timing of such specification in line with the development of dual pathways reported in structural studies. Notch receptors and ligands are expressed in and around the developing vasculature [135] and this signaling appears to establish the initial arterial and venous identity/fate upstream of EphB4/ ephrin B2 signaling. Notch1–Notch4 interact with five ligands – Delta-like 1, Delta-like 3, Delta-like 4, Jagged1, and Jagged2. Notch1 and Notch4, Delta-like 4, Jagged1, and Jagged2 are each expressed in arterial endothelial cells [135] but only in low amounts, and if at all in venous ones. Notch3 is expressed in arterial but not venous SMCs, and Jagged1 is expressed in arterial endothelial cells and SMCs. Notch1 and Jagged1 appear in larger vessels in the embryonic lung before being expressed in the smaller developing networks [135]. The forkhead box (Fox)f1 transcription factor, which belongs to a family of transcription factors critical to the development of the lung’s microvasculature, may mediate the effects of Notch signaling [136, 137]. EphB4/ephrin B2 signaling is critical for appropriate vascular development, possibly being required for “like” vessels to fuse. The endothelial cells of developing arteries specifically express ephrin B2, and ones of developing veins specifically express EphB4, before the presence of a functional circulation. Ephrin B2−/− and EphB4−/− mice die (at E9.5 and E11.5) with defects in both arteries and veins. Postnatally, EphB4 acts as a negative regulator of blood vessel remodeling and network formation, switching the vascular program from sprouting angiogenesis to circumferential vessel wall growth [138]. Additionally, ephrin B2 and EphB4 are expressed in mesenchymal cells and play a role in endothelial and mesenchymal cell interactions [95].

2.2.3 Vessel and Capillary Wall Maturation (Stabilization) The endothelial channels forming in the lung are stabilized as perivascular cells and develop from within the mesenchyme [17, 91, 92, 117, 139–143]. Their close apposition to endothelial basement membrane triggers differentiation, likely in response to endothelial expression of TGF-b, which also inhibits endothelial cell movement and proliferation [117]. Within the walls of developing vessels, perivascular cells differentiate to SMCs and adventitial fibroblasts, and within capillaries into pericytes. Pericytes develop particularly in those capillary segments that arise directly from a vessel wall, and around postcapillary venules [144–146]. Around forming endothelial channels, they serve to bridge gaps between adja-

39

cent endothelial cells and between the leading edges of merging endothelial sprouts [147, 148]. In some vessel segments adjacent to capillaries (Fig. 4b) the perivascular cells develop into intermediate cells [42, 145, 149, 150] (Fig. 4b). Although the mesenchyme represents an important source of perivascular precursors [142, 151, 152], perivascular cells are also reported to develop from bronchial SMCs and endothelial cells [153], and to express smooth muscle proteins similar to the cells that are mesenchymal in origin [153]. Furthermore, data on the origin of SMCs indicate that embryonic endothelial cells can become mesenchymal cells expressing smooth muscle proteins, and so are also a potential source of these cells [154– 156]. The proteins forming the cytoskeletal and contractile filament networks of the perivascular cells appear sequentially, a-smooth muscle actin (aSMA) followed by calponin, h-caldesmon, a-tropomyosin, and metavinculin, with the smooth muscle myosin heavy chain (SM-MHC) SM1 isoform appearing during the fetal period and the SM-MHC SM2 isoform appearing after birth [142, 157–164]. The contractile filaments of differentiated SMCs confer tensile strength on the vessel wall and an ability to contract [165, 166]. In all but the smallest venules, SMCs are surrounded by basement membrane and are characterized by extensive filaments, fusiform dense bodies, and attachment plaques. To proliferate or migrate, these cells readily disassemble their filaments networks (dedifferentiate); and they may revert to a contractile phenotype by their reassembly. Distally, at the level of the respiratory bronchiolus and proximal alveolar duct, the SMC processes penetrate their surrounding basal lamina to form lateral (SMC–SMC) contacts and the basal lamina of adjacent endothelial cells to form junctional complexes [150]. As vessels approach capillaries, the numbers of endothelial cell contacts increase, and their processes penetrate their basal lamina and the internal elastic lamina to contact SMCs [150]. More distally, at the level of the alveolar duct and alveolar wall, intermediate cells, which have now lost their basal lamina, contact adjacent endothelial cells by their projections [150]. The outermost layer of vessel walls is formed by circumferentially aligned adventitial fibroblasts. Their number, and that of SMCs, determines oxygen diffusion (which becomes restricted once the perivascular tissue cuff is 100 mm or more thick). Although these fibroblasts typically express vimentin, actin isoforms, and nonmuscle myosin, under certain conditions they express two SMCspecific proteins, aSMA and desmin [167]. They are characterized by arrays of microfilaments (4–6  nm in diameter), intermediate-sized filaments, pinocytotic vesicles, and by abundant extensive rough endoplasmic reticulum. Basement membrane is absent, and collagen fibers and fibrils form along their adluminal and abluminal cell margins. Capillary pericytes share the endothelial basement membrane and normally lack extensive filament networks, dense bodies, or attachment plaques [145–147, 168]. Their processes often extend to contact the endothelial cell and rare gap junctions allow nucleotides

40

to pass between the cells [147, 168]. During endothelial sprout formation, they prevent plasma from escaping into interstitial tissue [147, 168]. Possessing cyclic-GMP-dependent kinase, actin, desmin, vimentin, a-tropomyosin, and myosin, capillary pericytes are reported to contract or relax in response to vasomediators [169]. Their ability to express the proteins typical of contractile muscle cells, such as SM-MHC, aSMA, and desmin, however, varies greatly [170–172]. Evidence that interstitial fibroblasts (cells normally present within the interstitium of the alveolar–capillary membrane) are a further source of perivascular cells, and acquire the phenotype of a pericyte or SMC, comes from studies of mature vascular beds, i.e., dorsal and mesenteric capillaries [145, 147, 173], and from studies of distal vessel remodeling in the adult lung in pulmonary hypertension [42, 174–177]. SMCs separate from adjacent endothelial cells and adventitial fibroblasts within the vessel wall as internal and external elastic laminae form along their inner and their outer margin. The essential regulation of smooth muscle growth by elastin in the normal lung is demonstrated by obstructive intimal hyperplasia in (and the death of) mice that lack the elastin gene [178]. In the developing lung, each vascular cell type (endothelial cell, SMC, and fibroblast) is elastogenic [179], although the relative contribution of each to lamina formation is unknown. The process of lamina assembly, as currently understood, derives from data for other sites [118, 178, 180–189]. Expression levels of tropoelastin (a major component of the forming lamina) fall in artery walls and increase in vein walls in the lung after birth, a reverse of the fetal pattern [179]. The presence of endostatin (an inhibitor of endothelial cell proliferation) within matrix and elastic laminae of large vessels may restrict further vascular sprouting from the wall [190]. Homotypic and heterotypic contacts are important for perivascular cell recruitment [191, 192]. At the molecular level, PDGF and in particular (of the genes identified, e.g., PDGF-A, PDGF-B, PDGF-C, and PDGF-D) PDGF-B signaling via its cognate RTK (PDGF-Rb), have a significant role [151, 152, 191, 193, 194]. Typically perivascular cells, including ones in lung vessels, express PDGF-Rb [191, 192, 195, 196] and arterial and capillary endothelial cells express PDGF-B [191, 192, 195, 196]. This suggests that the endothelial cell targets (recruits) the perivascular cell, driving wall formation. Complex shifts in the gradients formed by changing expression patterns of these molecules, however, likely determine this interaction in the developing lung as in the adult [195, 196]. The lack of pericytes in mice deficient in the PDGF-B gene results in capillary dilatation, microaneurysms, and vascular leak and hemorrhage; the absence of the PDGF-Rb gene in mice elicits a similar response (the mice dying at E17.5–E18.5 and at birth, respectively). PDGF-B promotes mesenchymal cell proliferation, initiates their progression through the cell cycle, and induces chemotaxis [191,

R.C. Jones and D.E. Capen

192]; and SMCs may use PDGF-B to suppress ­differentiation (assembly of contractile filaments and networks), enter the cell cycle, and stimulate self-replication via synthesis of PDGF-A [197, 198]. PDGF-B also enhances wall stabilization by inducing perivascular cell expression of VEGF [199, 200]. PDGF retention during vascular development is essential for normal conduit vessels and capillaries to form [201]. Actin reorganization and membrane ruffling (each essential for cell migration within vessel walls) are induced by PDGF-AB and PDGF-B, and therefore via PDGF-Rb. In addition to PDGF-B/PDGF-Rb signaling, ANG and its RTK (Tie-1) are required for perivascular cells to be recruited (Fig. 10). The cells express ANG1 and ANG2 ligands and endothelial cells express their (Tie-1 and Tie-2) receptors. Endoglin (TGF-b RIII), endothelial differentiation gene (Edg-1)/sphingosine isophosphate, and TGF-b1 signaling are also required for appropriate perivascular cell development. Endoglin−/− mice have arrested remodeling of endothelial networks with poor SMC development, dilatation, and hemorrhage (and die at E11.5), whereas Edg-1−/− mice have deficient SMCs/pericytes (and die at E12.5–E14.5). TGF-b1 signaling triggers perivascular cells to express a SMC phenotype. Prx-1 and Prx-2 also are expressed in developing lung [202], regulating patterning of blood vessels and promoting SMC differentiation, and controlling SMC-specific gene expression by enhancing binding of serum response factor to CarG elements (a DNA motif present in many SMC-specific gene promoters).

2.2.4 Vessel and Capillary Network Formation in Distal Lung The lung’s respiratory surface starts to form just before birth and in the immediate period after. Proliferating and organizing endothelial cells expand its capillary and small-vessel networks significantly at this time [32, 203]. A saccule (a primitive structure or sac) first forms at the end of the airway: this is also referred to as a terminal saccule (terminal sac) or primary septum. It is proposed that it forms (a) as a sleeve of inhibition develops around the airway tube to prevent random branching while promoters (such as FGF-10) enhance branching at the tip – the tube initially expanding and then branching under the direction of two such growth-promoter zones separated by a cleft of inhibition – and/or (b) by increased intraluminal pressure, secondary to fluid secretion into the airway (also regulated by FGF-10), which causes expansion of the terminal bud between inhibitory points (of highly resistant tissue areas) created by myofibroblasts making elastin [204]. The terminal saccules are supplied by small vessels and a double capillary system at birth (Fig.  13); during septation the capillary network remodels into a single system. Septation occurs as secondary crests protrude from the primary septa, and as secondary

3  Pulmonary Vascular Development

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Fig. 13  Fetal and postnatal development of the pulmonary capillary bed. (a) In the pseudoglandular stage the epithelial tubes push into the mesenchyme, which contains a loose capillary network (top left). The amount of intervening mesenchyme is reduced and capillaries arrange around epithelium. At sites of epithelium–capillary contact, portions of the air–blood barrier begin to thin as epithelial type 1 and type 2 cells form (top right), whereas, at the periphery, the still cuboidal epithelium allows for further growth and branching. Perinatally, secondary septa are developing (lower left, arrowheads) from primary septa: at this stage, all septa are of the primitive type, containing a double capillary network and a central layer of connective tissue. In the mature lung (lower right), inter-alveolar septa contain a single capillary network meandering through the interstitium. (b) A secondary septum forming from a primary septum by lifting of one

of the two capillary layers (arrows). Elastin deposition (dark regions) is necessary for the formation of alveoli. (c) Photomicrograph of mouse alveolar–capillary membrane (at post-natal day 3): the primary septal wall illustrated is seen to consist of the cytoplasmic extensions of alveolar epithelial (type 1) cells (arrows) separated from capillary endothelial cell processes and the interstitial layer by a continuous basal lamina. The three capillaries present contain erythrocytes in their lumen (e, cl); they form a double capillary layer, together with their basal lamina, ground substance and a portion of an interstitial cell (g, isc). Magnification × 10,000. (c) Bar 1 mm. (a Reprinted www.annual reviews.org from Burri [203] with permission, copyright 1984 Annual Reviews. (b) Reproduced from Burri [203] with permission from McGraw-Hill companies [21]. (c) Reprinted from Amy et al. [205] with permission from Wiley-Blackwell)

septa form, at these points, the primary septum (terminal saccule) divides into alveoli [21, 205, 206]. The secondary septum forms in regions where the capillaries and primary septum can be folded up: fibroblasts proliferating at the sites of origin of the secondary crest and lengthening the secondary septum as it projects (at a right angle) into the alveolar space. Myofibroblasts assemble in the tips of the secondary crests adjacent to elastic fibers. The septal walls of the newly ­forming alveoli then thin as the intervening tissue regresses [207, 208], with interstitial fibroblasts undergoing apoptosis as the capil-

laries fuse [21]. Further insight into the mechanism of septation is provided by studies showing that PDGF-A/ PDGF-Ra signaling is required [151, 152, 209, 210]. In wild-type mice, the distal mesenchyme generates PDGFRa positive myofibroblasts (considered SMC progenitors) in response to PDGF-A, which develop in a cluster around the terminal ends of epithelial buds; in the canalicular stage, these cells spread to surround the prospective terminal sacs, downregulate PDGF-Ra expression, and upregulate tropoelastin expression to start the process of ­septal formation

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Fig. 14  Model of alveolar SMC development. (a) At the pseudoglandular stage, alveolar SMC progenitors (blue) originate as clustered plateletderived growth factor-receptor a positive (PDGF-Ra+) mesenchymal cells located around the epithelial buds. (b) In conjunction with the canalicular stage, these cells normally spread to acquire positions surrounding the prospective terminal sacs; and (c) during the terminal sac to alveolar stages, the cells downregulate PDGF-Ra expression and upregulate tropoelastin expression (black), leading to septation. (d) In PDGF-A−/− lungs, the distal spreading of PDGF-Ra+ progenitors does not occur; (e) consequently, in the absence of alveolar septal elastin deposition, alveogenesis fails. (Reproduced from Lindahl et al. [151] with permission)

forming until approximately 40 days after birth, supports an additional growth process [206] and this appears linked to the action of RA. A high concentration of cellular retinol binding-protein 1 is present in the lung during the period of septation [216]. Furthermore, retinol administration induces alveolus formation, and retinol-storing cells appear first diffusely through the lung (where alveoli form in the septation period at birth) and later only in the subpleural region (where additional alveoli form after septation) [206, 217]. RA receptor (RAR)-b inhibits septation, and in RAR-b−/− mice, septation starts early and alveoli initially form twice as fast as in wild-type mice: later, septation occurs at the same rate as in wild-type mice, supporting the concept of two mechanisms regulating alveolar formation [206]. RA/RAR interaction(s) may regulate septation via cell proliferation, migration, and temporal differentiation by upregulating RA response elements (which modulate other gene expression patterns) [206]. Both RA and RA response elemens increase alveolarization by stimulating proliferation of lung fibroblast progenitors via a PDGF-mediated autocrine mechanism.

3 Vascular Templates in Normal Human Lung at sites along the saccule wall (Fig. 14). PDGF-A mice have −/−

reduced numbers of PDGF-Ra+ myofibroblast progenitor cells, and cells that fail to spread, proliferate, and synthesize elastin, resulting in impaired septation [151, 152, 209, 210] (Fig.  14). The critical role of tropoelastin/elastin deposition and myofibroblast migration for alveolar formation is clearly demonstrated in the neuraminidase-1 (Neu1) null mouse, which is unable to survive [211]. The expression in the ­mesenchyme of Robo (a receptor involved in repellant signaling in developing neuronal systems) and Slit (its ligand) may regulate mesenchymal cell organization [212, 213], and Robo−/− mice lack septation [204]. From this, an intricate mechanism for septal wall formation is proposed in which myofibroblast migration progresses to the points where septa form (a source of high PDGF-A expression) and elastin synthesis is induced along a “morphogen gradient” of PDGF-A ­signals from epithelial cells: subsequent invagination of the ­saccule wall and formation of the secondary septum occurs as capillaries and myofibroblasts following epithelial cells (expressing Robo) are repulsed into the airway space (from a Slit source) [204]. As the distance of these cells from the repulsive signal increases, formation of the septal wall ceases [204]. An alternative form of alveolar septation to that at the time of birth is indicated by the finding that septation of the number of primary saccules present at birth accountsonly for approximately one third of the number of alveoli present at postnatal day 6 and approximately one quarter by day 14 [206, 214, 215]. This discrepancy, and the presence of alveoli

The main features that define the changing vascular template in the normal human lung are the number, diameter, and wall structure of the preacinar and intra-acinar vessels [9, 10, 13, 14, 16, 20, 27, 78, 153, 203, 218–223].

3.1 Fetal Lung Vascular networks begin to form at the onset of organogenesis in the fourth week of gestation: the central pre- and post-­ capillary vessels arise as outgrowths from the great vessels of the heart, the pre-capillary vessels being derived from the fourth aortic arches, and the post-capillary vessels from the primitive atrium. Distal blood lakes appear, in advance of central vascular branches being established, as early as the sixth week (32–44 days) of gestation in the primitive mesenchyme surrounding the lung bud at the neck, and by the seventh week (50.5 days) they are abundant in the sub-pleural mesenchyme [223]. The adult pattern of central vascular and airway structures, consisting of lobar and segmental branches, is present by the sixth week of gestation (42 days) [9, 10, 15, 218, 224– 227]. At this stage the pulmonary artery accompanies airways only to the third or fourth generation [223]. By the eighth week (about 56.5 days), the branching pulmonary artery, a thickwalled blind-ended tube, lags behind the branching airway by two to three generations. Later, at 12–14 weeks, an extensive

3  Pulmonary Vascular Development

capillary network now surrounds the ­distal airway buds, although they are separated from the buds by subpleural mesenchyme. By the 20th week, the full number of preacinar pulmonary vessels has formed in each segment. By 22–23 weeks, the capillary network closely approaches alveolar epithelium, and the pulmonary artery accompanies each airway branch. In their study, deMello and Reid [223] reported the central vessels of the human lung forming by angiogenesis, and the peripheral vascular network by vasculogenesis (as described in their earlier study of vascular development in mouse lung, see Section 2). The first connection between developing distal and proximal networks appears between peripheral lakes and a thin-walled hilar vein, the venous drainage system of the lung thus being established before the pulmonary artery. Studies of insulin-like growth factor expression (a potent angiogenic factor), demonstrating distinct populations of endothelial cells that arise from diverse genetic lineages in the human lung, support the concept of vascular development occurring by both angiogenesis and vasculogenesis [228]. The additional concept that all pulmonary arteries and veins in the human lung arise by vasculogenesis, however, is further proposed from detailed analyses of the lung’s developing vascular (arterial and venous) beds [153, 222]. Although vessel size, length, and diameter increase in the fetal lung, density does not change [9, 10, 16]. Since vessels at the hilum grow faster than ones at the periphery, the gradient of vessel diameter relative to length increases with fetal age. As each airway develops in the fetal lung so do the accompanying conventional arteries with supernumerary arteries. From 18 weeks of gestation onward, the numbers of airways and conventional artery branches are similar, and numerous supernumerary arteries are present [9, 16] (Fig.  15). The ratio of supernumerary to conventional arteries present along the pathway is the same as in the adult (even before airway branching is complete). Analysis of the branching pattern of a lateral pathway in fetal lung (the seventh lateral branch of the axial pathway illustrated in Fig.  15) shows 11 airway generations accompanied by 11 conventional arteries and 40 supernumerary arteries [9]. Additional measurements along the posterior basal segment of a right lung at 40 weeks of age confirmed the same patterns described for the left lung, indicating a similar arterial branching pattern throughout the fetal lung [9]. Conventional and supernumerary veins first appear at a time similar to that for the arteries, developing in a progressive fashion from the hilum to the lung periphery. From 20 weeks of gestation, the number of preacinar conventional veins is within the adult range for the intrasegmental airway number. Although the number of conventional veins in a segment equals that of the airways and conventional arteries, there are more supernumerary veins than supernumerary arteries [10]. By 28 weeks of gestation, a difference is evident in the arrangement of the supernumerary veins: type 1

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Fig. 15  Distribution of arteries (conventional and supernumerary) by size in fetal lung. Conventional and supernumerary artery branches are shown by diameter, relative to the diameter of the axial arterial pathway from which they arise (see the black line), in a fetus (19 weeks gestation). In this (and subsequent images) each branch is seen as a vertical line whose height represents the diameter. From the lobar hilum to the terminal bronchiolus, the 25 airway generations are accompanied by 21 conventional arteries, and there are 58 supernumerary arteries. For clarity, the first eight conventional and 11 supernumerary arteries are shown separately (top image) from the remaining conventional and supernumerary ones (lower image). The asterisk indicates the point of overlap between the top and lower image. Note the change in scale of the vertical axis. At the hilum, the diameter of the main pathway is 1,300 mm. Conventional arteries have a mean diameter of 282.7 mm, and the supernumerary arteries are smaller, with a mean diameter of 62.6  mm. (Reprinted from Hislop and Reid [9] with permission from WileyBlackwell)

supernumerary veins drain lung tissue immediately surrounding an axial vein and receive only occasional tributaries whereas the larger type 11 supernumerary veins drain the more distal capillary bed and are joined by several postcapillary tributaries [10]. Reconstruction of the same pathway in the lung up to 38 weeks of age shows that the branching patterns are more or less the same to the level of the terminal bronchiolus, and are as present in the adult lung. However, many small arteries (and veins) develop late in fetal life and continue to increase at the time of birth – their density (per unit area of lung) increasing as the respiratory region of the lung develops with the appearance of respiratory bronchioli and saccules (Fig. 16). An elastic wall structure extends to the seventh generation along the axial arterial pathway by 19 weeks of gestation [9]; from here on, an elastic structure is present to the same level

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Fig. 16  The acinus at six stages of development. At all ages, airway generations are drawn the same length so that increase in length represents an increase in generations. A given generation may be traced vertically, permitting its structural remodeling to be followed. The actual increase in size is shown by the length from the terminal bronchiolus to the pleura. Note that the changing length of the terminal bronchiolus (the last airway segment that does not give rise to alveoli) is not drawn to scale but is noted numerically. At 16 weeks gestation, the airways end in a tubule close to the pleura. By 19 weeks gestation, the epithelium in the last generation of the airways has thinned and now forms the first respiratory bronchiolus: branching has led to a second-generation respiratory bronchiolus. By 28 weeks gestation, further branching has given rise to a total of three generations of respiratory bronchioli and one generation of transitional ducts, the latter giving rise to primitive saccules. By birth, the number of generations of saccules has increased to three, the last forming the terminal saccule. No true alveoli are present at this time although a few indentations representing future alveoli appeared just before birth. By 2 months,

alveoli have developed from the walls of the respiratory bronchioli, from ­transitional ducts and from saccules, which transforms these last two structures into alveolar ducts. Alveoli also open into the terminal saccules. By 6 years, several changes have occurred – the remodeling of respiratory bronchioli and alveolar ducts may occur in several ways: along some pathways a bronchiolus may transform into an extra generation of respiratory bronchioli by centripetal alveolarization; distal respiratory bronchioli may transform into alveolar ducts; further branching of another one or two alveolar duct generations may occur; or there may be no change. Certainty for some of the above changes is not possible because of variation between acini and cases. At the distal end, however, the terminal sac has probably transformed into the adult atrium, a short, wide passage lined by alveoli, which has given rise to a number of alveolar sacs, formed by budding rather than branching, and each lined by alveoli. This pattern is similar to that seen in the adult: there is probably little further development save an increase in size. (Reproduced from Hislop and Reid [227] with permission from BJM Publishing Group Ltd)

in both the axial and the conventional arteries. These are considered supportive arteries in the fetal lung because of their high tensile strength and ability to maintain vessel patency. The level at which a transitional structure gives way to a muscular structure (Fig. 4a) is established midway through gestation, and the point where a muscular structure gives way to partially muscular and/or non-muscular structures occurs in vessels with a similar diameter throughout fetal life.

Calculation of the percent wall thickness reveals little change with increase in diameter in larger arteries, and that in smaller arteries the percent wall thickness increases rapidly as the diameter decreases, the most rapid increase occurring in arteries less than 200 mm in diameter (Fig. 17). Phenotypically distinct populations of SMCs in the wall of large developing pulmonary arteries raise the possibility of different lineages:[229]: when present in the fetal lung, the SMCs are

3  Pulmonary Vascular Development

Fig.  17  Percent arterial medial thickness at different developmental stages. The percent medial thickness related to the external diameter of arteries (ranging in size from below 200 mm to more than 600 mm) is illustrated for the fetus, a 3-day old infant, and a 4-month old child to adulthood. In the 3-day old infant, arteries over 200  mm in diameter still show the (thicker) fetal pattern of medial thickness, whereas those less than 200 mm in diameter show the (thinner) childhood and adult state. (Reprinted from Hislop and Reid [16], copyright 1981, with permission from Elsevier)

immature. The pulmonary veins are relatively free of muscle: at 20 weeks of gestation, even the largest veins lack muscle, and although muscle fibers are identifiable within vein walls by 28 weeks, these form a continuous muscle layer only at term [10, 16]. At this time, SMCs extend into veins as small as 80 mm but unlike the arrangement in the arteries they are thin-walled and no elastic laminae are present [10, 16]. In a population count of vessels in the normal human lung at term, most arteries above 150 mm in diameter have a muscular wall, and most ranging from approximately 90 mm to approximately 75 mm in diameter have a partially muscular wall. Arteries with a non-muscular wall overlap in size with partially muscular arteries (Fig. 18); below 37 mm, all arteries are non-muscular. Throughout fetal life the shift from ­muscular arteries to a structurally mixed arterial population occurs in the pre-acinar region (proximal to the terminal bronchiolus). The relationship between vessel size and wall structure is similar at all fetal ages [9]. Although the smallest muscular artery, and the range for partially muscular and non-muscular arteries by size, is the same in the fetal and the adult lung, the wall thickness of an artery (of given diameter) is twice as thick in the fetus as in the adult (Fig. 17) Even at birth, however, little muscle is found in the wall of arteries beyond the terminal bronchiolus (Fig. 19). Thus, the fetal lung has a more muscular pulmonary circulation than the adult lung if judged by wall thickness of an artery of given size, but it is less muscular when judged by the presence of muscle in arterial walls, at any level in the branching pattern within the acinus [9]. A striking finding, within hours of birth, is the increased compliance (by wall thinning) of the smallest muscular resistance arteries [11, 218, 230] – associated with a fall in resistance at this time – as the lung transitions from a low-flow/high pressure system to a high flow/low pressure system.

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Fig.  18  Population distribution (count) of vessels in neonatal lung identified by size and wall structure. The percentage of the total number of vessel profiles with a given structure (muscular, partially muscular, or non-muscular) in the human lung at 40 weeks of age is shown for size ranges between 25 and 200 mm in diameter. Smooth muscle is seen to develop in vessel walls in relation to vessel size. In all artery profiles close to 200 mm in diameter, the wall is muscular; below 200 mm, partially muscular artery profiles appear and increase until, at about 100– 75  mm in diameter, they form 68% of the artery population. Below 37 mm all artery profiles are non-muscular, and these then range in size between 125 and 100 mm. (Reprinted from Hislop and Reid [9] with permission from Wiley-Blackwell)

Fig. 19  Extension of arteries with smooth muscle in their wall with age (in the fetus, child, and adult). In the human fetus no arteries with smooth muscle are seen within the acinus (i.e., distal to the terminal bronchiolus). The arteries with smooth muscle extend gradually into the acinus with age, but even by 11 years they are not present within the alveolar wall: only in the adult (19 years) is smooth muscle present in arteries within the alveolar wall. (Reproduced from Hislop and Reid [14] with permission from BJM Publishing Group Ltd)

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3.2  Neonatal and Child Lung At 36 weeks to term, the preacinar pattern of arteries and veins is complete [9, 10]. The lung can now support air breathing but, in terms of its structure, as Reid elegantly described in her study, it is not the adult lung in miniature since, along with need for central vessel growth, its alveolar region is still being formed [231]. At this stage the alveolar region has four generations of respiratory bronchioli and two alveolar ducts proximal to the saccules [226]. It must evolve from this small simple structure into an efficient gas-exchange surface to match functional needs. It responds by a burst of vascular growth: existing vascular units continue to expand in diameter and length and many new intra-acinar units are added as the gas-exchange surface forms [9, 16–18, 227]. From arteriograms of the human lung (between 35 and 48 weeks of gestational age), and the analysis of tissue sections, Frey et al. [232] determined arterial diameter as a function of generation number, and showed that the branching properties of pulmonary arteries in the pre- and post-natal phases of human lung development are unchanged (the dimensions between mother and daughter branches being relatively constant from generation to generation). This demonstration of relatively constant branching ratios is consistent with the fractal pattern/properties of the pulmonary vascular tree [232]. As intra-acinar arterial structures grow and multiply, alveoli develop. Alveoli increase in number but become smaller

Fig. 20  Distribution of arteries (conventional and supernumerary) by size, relative to the diameter of the axial arterial pathway from which they arise (see the black line) in the infant and child lung. (a) In the pre- and intra-lobular arteries of a 4-month old child, the horizontal axis represents the last 5.1 mm of an axial pathway. The acinus is small with only five conventional and six supernumerary arteries. (b) In the pre- and intra-lobular arteries of a 5-year old child, the horizontal axis represents the last 12.6 mm of an axial pathway. The length of the acinus and lobule has increased with age as have the number of conventional and supernumerary arteries (especially the latter). All vessels have also increased in size with age. (Reproduced from Hislop and Reid [14] with permission from BJM Publishing Group Ltd)

R.C. Jones and D.E. Capen

in size as newly forming alveoli divide existing structures. The greatest increase in alveolar number by multiplication occurs between 4 months and 3 years of age. Later, between 4 and 10 years of age, as multiplication slows, alveoli increase in size. In the first 4 months of postnatal life, the number of arteries per unit area of lung, and the density of capillary networks, increase [203]. Within the acinus, an increase in the diameter of the proximal vessels compared with distal ones reflects the burst of small vessels developing at the periphery. After 18 months, the number of new vessels forming slows along with alveolar growth [9, 16]. Between 4 months and 4 years, the number of intra-acinar arteries (up to about 200 mm in diameter) increases per unit area of lung. Although the ratio of their number to the number of alveoli remains similar throughout childhood, their concentration per unit area of lung falls after 5 years of age as alveoli increase in size and separate adjacent arteries [14, 16]. The veins grow at the same time as airways and arteries and, whereas the preacinar drainage pattern is complete halfway through fetal life, the intraacinar venous pattern develops during childhood. Reconstruction of the venous branching system in the lungs of a 3-year-old and a 10-year-old child identifies distal veins arising from the saccular respiratory region [10]. Arteries and veins develop alongside new airway outgrowths in the postnatal lung, with both conventional and supernumerary vessels continuing to appear (Figs.  20, 21). Conventional arteries accompany new airway branches up to

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Fig. 21  Distribution of veins (conventional and supernumerary) by size, relative to the diameter of the axial vein from which they arise (see the black line) in the lung of a 3-year old child. The drainage pattern at the lung periphery is illustrated. The ­supernumerary veins

18 months of age; supernumerary arteries reach the adult number by 5 years of age, and new ones continue to form up to 8 years [14, 16] (Fig.  20). At all ages the conventional arteries are smaller than the parent artery at their point of origin. The mean diameter of these vessels, and that of the supernumeraries, increases with age. Overall, supernumeraries are smaller in diameter than their adjacent conventional arteries [14]. Conventional and supernumerary venous tributaries are found along the length of the axial vein. Conventional veins run in their own connective tissue sheath, enter the axial vein at an acute angle, are of size similar to that of the axial vein, and lie at some distance from the capillary bed they drain [10]. Some supernumerary veins drain the lung tissue immediately around the axial vein (as in the fetus), having no collagen sheath but passing directly through the main vein sheath to the axial vein; others receive postcapillary tributaries and are surrounded by a collagenous sheath continuous with the sheath of the axial vein [10]. Along the axial pathway, the conventional veins are equivalent in number to airway generations and the arteries accompanying them. Both conventional and supernumerary veins become more frequent toward the periphery (Fig. 21). At birth, as in the fetal lung, the percent wall thickness of arteries is still greater overall than later in childhood [14]. After 4 months of age, the wall thickness of larger vessels falls to match that in the adult lung and increases in smaller ones (less than 200 mm in diameter). During childhood, the number of large arteries with an elastic or transitional wall remains constant. Between 4 and 10 months, as vessels increase in size, muscle development lags. Although the wall structure along any axial artery pathway is established midway through fetal life, the distribution of intra-acinar vessels shifts significantly in childhood, more partially muscular and non-muscular arteries than muscular ones now being present than in the fetal or the adult lung [14] (Fig. 22). By 3 days after birth, the distribution of arteries by size and wall structure is similar to that at birth, partially muscular and non-­muscular

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include two types: type I and type II (the type II veins being larger on average than the type I veins). The actual length of the pathway is approximately 11 mm. (Reproduced from Hislop and Reid [10] with permission from BJM Publishing Group Ltd)

Fig. 22  Population distribution of artery profiles by size and wall structure (muscular, partially muscular, and non-muscular) in (a) the normal newborn lung and (b) the lung of an 11-year old child. Partially and nonmuscular structures are now present in larger arteries. (a Reprinted from Kitagawa et  al. [285] with permission from Wiley-Blackwell. (b) Reproduced from Davies and Reid [12] with permission from BJM Publishing Group Ltd)

arteries being as large as 180 and 150 mm in diameter and ones below 40 mm in diameter are non-muscular; indicating

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that muscle growth keeps pace with increase in artery size at this time. The distribution of muscle is then similar in the lung at 18 months of age and at 3, 4, 5, and 11 years of age [14], the largest partially muscular artery being 300 mm and the largest non-muscular one 245  mm. Muscular arteries smaller than 185 mm in diameter were not found. In another lung, at 10 years of age the distribution pattern was similar to that of the adult, indicating a watershed in wall maturation by this time, alongside some degree of individual variation. Muscle gradually extends into arteries within the acinus (to the level of respiratory bronchioli and alveolar ducts) but not distally into the arteries within alveolar walls (Fig. 19). The presence of a high population of thin-walled intra-acinar arteries may provide children with an advantage, since ones as large as 100 mm (or 200 mm) in diameter (with a capillarylike wall structure) can contribute to oxygen transfer [14]. The wall structure of the veins is more developed in the child lung than in the fetal lung: whereas the walls of small veins still consist only of endothelium, some of the larger veins have SMCs [10].

3.3 Adult Lung In 1965, Elliott and Reid [27] provided an account of the pulmonary artery branching pattern in the normal human adult lung. In the lungs studied, they obtained arteriograms and identified arterial branches in tissue sections. They described the distribution of elastic and muscular arteries, relating these by their wall structure and size to their location within the pulmonary arterial tree, and determined the relationship of the arterial branching pattern to that of the bronchi. From this study it emerged that the pulmonary artery is a more muscular vessel than had been previously supposed and a striking divergence first appeared between the branching patterns of the arterial and bronchial trees on the basis of the presence of extra (supernumerary) branches with no counterpart in the bronchial tree (see Sect. 1) [27]. The pattern was defined on the arteriogram (Fig. 4a), or in serially prepared tissue sections, by counting the number of branches as generations – which may also be determined by order [27, 233, 234]. A useful convention is to consider a segmental airway as the first generation (the trachea and lobar branches are counted separately). Using this approach, the inferior lingular segment has about 28 generations and the apical lower lobes as few as 15. In adolescence, new vessels continue to form from preexisting ones by angiogenesis, in line with lung growth. Both the absolute and the relative number of supernumerary arteries remain more numerous than conventional ones (Fig.  23): 3  times as numerous as conventional artery branches, the supernumerary arteries at all levels along an axial pathway remain smaller in size than

Fig. 23  Distribution of pre-lobular and intra-lobular (conventional and supernumerary) arteries in adult lung. A pulmonary artery from the segmental hilum to the pleura, with conventional and supernumerary side branches included down to the start of the lobule is illustrated, and the conventional arteries thereafter. The arterial pathway shown here is the same as that illustrated in Fig. 4a. The 15 conventional arteries were numerically designated by reference to the bronchial generation. This part of the artery was 5 cm in length. (Reprinted from Elliott and Reid [27], copyright 1965, with permission from Elsevier)

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their adjacent conventional branches. They comprise one quarter to one third of the potential cross-sectional area of the branch vessels. The conventional veins are larger than the supernumerary veins at any level and arise from a region of division of an airway. They drain the alveolar region immediately adjacent to the venous pathway. In the adult lung, all preacinar arteries have a complete muscle coat and several elastic laminae. The elastic wall of the main pulmonary artery at the lung hilum has a medial muscle layer between internal and external elastic laminae, and seven or more central elastic laminae. Subsequently, it then has four to seven additional central laminae in its transitional segment [27]. The elastic arteries of the adult lung are more than 3,200 mm in diameter, transitional ones are 3,200–2,000 mm in diameter. An elastic or transitional wall structure extends approximately halfway along an axial pathway to about the ninth airway generation. Distal to this, in arteries 2,000  mm or less in diameter, the wall then changes to a muscular structure with a medial muscle layer between internal and external laminae with four or fewer central laminae: almost all lateral branches of the distal axial pathways have a muscular wall. SMCs finally extend into the most distal arteries in alveolar walls (Fig.  16). A population distribution shows that all arteries greater than 150  mm in diameter are muscular; that partially muscular ones are most frequent in the 40–80-mm size range, and that non-muscular ones appear just below 130 mm, increasing in frequency and representing most of the vessel population below 50 mm (Fig. 24). Arteries as small as 30 mm in diameter, however, may have a muscular wall, and even smaller ones approaching the capillary bed can have a single SMC in their wall (Fig.  19). The wall is thickest in muscular arteries that are 200–500 mm in diameter and found immediately pre-acinar or proximally within the acinus, i.e., the

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resistance arteries of the adult lung. The structural features of the intra-acinar pulmonary veins are similar to those of the pulmonary arteries – first a non-­muscular segment, then a segment with a spiral of muscle before the muscle coat becomes complete as wall thickness and diameter increase. The large elastic and transitional arteries of the aged lung have fewer elastic fibers than ones in the young adult [219, 220]. This is associated with a series of changes: vessel narrowing, caused by intimal and medial wall cells encroaching on the lumen [219, 220], alveolar wall thinning resulting in large and relatively simple alveolar structures, and an overall fall in lung elastic recoil [219, 235]. The intima of large elastic and transitional arteries may be thickened by acellular deposits, although large muscular arteries appear free of these, and hyaline deposits develop in the walls of small vessels less than 150 mm in diameter [219]. The medial thickness of vessels in all size ranges is greater in the aged lung. Most are twice as thick as in the normal young adult lung, and the increase is nearly threefold in ones approximately 3,000  mm in diameter [219]. The range for wall thickness, narrow in the young adult lung, is wide in the aged lung. The wall thickness of pulmonary arteries is greater than in the young adult lung as is the increase in the wall thickness of resistance arteries. When the number of branches arising from the axial pulmonary artery was counted on arteriograms, and the diameter of successive branches to within 0.5 cm of the pleura and the distance between them were measured, no difference was found between the aged and the normal young lung. The axial pathways appear clearer in the aged lung, however, possibly because of a fall in the number of small blood vessels and capillaries [20].

4 Compromised Lung Vascular Development

Fig. 24  Population distribution of vessels by size and wall structure in the adult lung. In a typical pattern, as vessels increase in size the relative number of muscular artery profiles increases: the relative number of nonmuscular arteries decreases, and most partially muscular ones are found in the mid-size range. (Reprinted with permission from Reid [20])

The template for vascular development, growth, and maturation in the normal lung is altered by failed gene–growth factor interactions and by hormones, vitamins, drugs, toxins, radiation, and other factors. The effect of any given agent, and subsequent viability, will depend on when vascular growth is interrupted, be it during organogenesis and cell specification, during the formation of preacinar or intra-acinar vessels, or as the alveolar region forms. It will further depend on the changes in the cascade of signaling pathways regulating lung tissue organization via cell proliferation, migration, differentiation, and survival. The ability to recover from compromises that permanently alter developmental processes and vascular architecture is minimal such that the earlier the challenge to growth the greater the potential for abnormal development. Dysanaptic (unequal) growth

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describes the pattern in the lung whereby the subsequent growth potential of the central conducting airways and vessels is limited compared with that of the acinar region as, for example, during compensatory growth in the postnatal lung [236–238]. Even when the developmental program is normal in relation to its various stages, restricted fetal growth, or normal fetal growth but premature birth, will result in a small and immature lung in terms of cell specialization and differentiation. This increases the risk of respiratory deficiency and postnatal problems [239]. Important examples of factors associated with a compromised intrauterine environment that restrict fetal development and growth include fetal hypoxemia, reduced substrate supply, and hypercortisolemia [240, 241]. Postnatally, the transition from placental to lung-based respiration at birth, i.e., from a hypoxic environment to a relatively hyperoxic one, exposes the perinatal lung to oxidative stress. Surfactant, enzyme, and nonenzymatic antioxidant syntheses normally match each other to protect the lung during this transition. Factors that especially affect the perinatal lung as it undergoes extensive remodeling during alveolar formation and growth include a high or low environmental oxygen tension, as well as interactions between the oxygen tension and genes regulating growth factors, such as VEGF. Dynamic change in the inspired oxygen levels represents a potential signaling mechanism for the expression, and activation, of redoxsensitive transcription factors, cell fate (apoptosis), and downstream proinflammatory cytokine signaling. Redoxsensitive transcription factors are likely differently regulated by oxygen availability to bind specific DNA consensus sequences and activate expression of genes controlling adaptive vascular homeostasis at the time of birth. Hence, both temporal and spatial variance in oxygen abundance will determine lung cell survival or apoptosis via oxygendependent activation of cell regulators and genes.

4.1 Failure to Develop the Normal Quota of Vascular Units In the following conditions, adaptation to disturbance in the lung’s growth pattern is associated with relatively normal function and good prognosis, although under certain circumstances, as in environmental change or in disease, reduced pulmonary reserve becomes apparent.

4.1.1 Single Lung in Agenesis Interference at the earliest stage of the development program results in a single airway branch that gives rise to a single

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lung which typically grows to fill both thoracic cavities. Quantitative analysis of one such lung in a 3-month old infant confirms that the vascular, mesenchymal, and epithelial components produce twice the normal capillary and alveolar quota [242]. Capillary and alveolar multiplication in response to the increased available thoracic space for the single lung thus results in an alveolar number equivalent to that of two lungs. This pattern is consistent with normal lung function.

4.1.2 Congenital Diaphragmatic Hernia and Lung Repair In congenital diaphragmatic hernia (CDH), reduction of the thoracic space by the upward movement of abdominal contents reduces lung volume and the number of airway branches. This pattern of hypoplastic lung development is based predominantly on the mechanical constraints imposed by lack of space. The gestational age at which the hernia is produced determines the pattern of disturbance in lung and vascular growth [243, 244]. Lung vessels are remodeled to reflect the reduced volume receiving blood supply as well as the reduced number of airways and alveoli. Wall thickening by SMCs occurs in arteries in all size ranges and muscle extends into more peripheral vessels than normal. The prognosis of infants with CDH at birth is strongly influenced by impaired vascular development [245, 246]. After CDH repair, lung volume typically increases to fill the available thoracic space, alveolar size becomes abnormally large, but airway, alveolar, and vascular multiplication do not catch up. A gradient of hypoplasia is typically apparent, the ipsilateral lung (particularly its lower lobe) being most compressed. At least during adolescence, lung function is satisfactory, although physiology studies detect reduced function [247].

4.1.3 Musculoskeletal Disorders In certain musculoskeletal disorders, lung growth is impaired metabolically or mechanically [248]. In some, the metabolic disturbance impairing cartilage and bone development also affects tissues needed for lung growth; in others, a reduced thoracic space restricts lung growth and airway development [249]. For example, in osteogenesis imperfecta, lung volume and airway branching are reduced because of reduced thoracic volume and the arteries are crowded and abnormally large, whereas in camptomelic dwarfism the thorax is small, as is lung volume, but airway and arterial size and branching are more appropriate to volume, suggesting that a metabolic effect is less important [249]. A dissociation between thorax size and lung growth, in the absence of metabolic ­impairment,

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is illustrated by a case of Jeune’s syndrome (asphyxiating thoracic dystrophy), a rare inherited malformation characterized by disturbance in utero of bone formation, in which the thorax fails to grow, but the lungs grow to normal size by displacing the diaphragm [250]. The small arteries of this lung had remodeled structurally at the time of death (from hypoxia), and although the lung was normal in volume it was abnormal in shape as it conformed to the abnormal thoracic contours.

4.2 Severe Impairment of Alveolar Growth and Abnormal Vascular Growth: Alveolar–Capillary Dysplasia Here the blood–gas barrier of the lung fails to form, leading to the development of severe respiratory distress shortly after birth. Parenchymal and vascular abnormalities are the basis as discussed by Galambos and deMello: typically, dysplastic thin-walled vessels, somewhat larger than capillaries, form centrally rather than abutting the edge of the airspace, resulting in absence of the normal interface between cells forming the gas-exchange surface [76, 251]. The arteries upstream have thick walls, similar to ones in persistent pulmonary hypertension of the newborn (PPHN). These changes are associated with veins that are misaligned, developing centrally within the lobule (alongside the bronchoarterial sheath) rather than at its periphery (within the interlobular septa). Their presence occurs not only in alveolar–capillary dysplasia, however, but also in other conditions such as alveolar proteinosis and congenital pulmonary venous obstruction [76, 251]. These changes in neonates are rarely compatible with survival of more than a few months, even with supportive measures. Why normal alveolar epithelial and capillary endothelial cell fusion events fail is not known. Galambos and deMello [76, 251] pointed out that, given the importance of VEGF signaling [109], alteration in this signaling pathway may be a causative factor in the development of alveolar–capillary dysplasia, the similar impairment in endothelial nitric oxide synthase (eNOS)deficient mice, and mice administered eNOS inhibitors, reflecting a link between eNOS and VEGF (nitric oxide synthase being an upstream activator, and a downstream target, of VEGF). Of note, in congenital acinar aplasia, a form of hypoplasia that stems from a failure to form saccular structures and alveoli (and thus not compatible with survival beyond a few days), extensive capillary networks develop in the lung despite the lack of respiratory/alveolar surface [76, 251]. It may be that a selective reduction in TGF-b, as shown in a study of this condition, impairs epithelial–mesenchymal cell interactions (in the pseudoglandular phase) [251].

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4.3 Vascular Restriction and Wall Remodeling in Developing Vascular Units: Persistent Pulmonary Hypertension of the Newborn The hypertensive state of the pulmonary vascular bed in utero diverts blood from the lung to the placenta. At birth, the pulmonary vascular bed adapts and pulmonary pressure and resistance fall rapidly as arteries dilate (and stretch) and vascular compliance increases [11, 230]. In PPHN this adaptation fails, blood is diverted (via the foramen ovale and ductus ­arteriosus) to the aorta, and a high pressure persists. Abnormal vascular development, or the remodeling of vascular structures, before birth is the basis of different forms of PPHN [252, 253], which are characterized by (a) dysplasia – abnormal muscularization of intra-acinar arteries associated with increased adventitial collagen formation, and lumen narrowing in preacinar arteries, (b) hypoplasia – the development of too few vessels, and/or vessels that are too small and with abnormally muscular walls, and (c) maladaptation – the failure of small vessels to adapt perinatally [253]. In the idiopathic form, the airways and alveoli develop normally but large numbers of SMCs occur in small arteries, extending to include ones in the alveolar wall [254, 255]., A possible cause of this has been suggested to be relative hypoxia in utero or premature closure of the ductus; however, this currently remains undetermined. The vascular remodeling present in idiopathic PPHN was also found in the lungs of infants with meconium aspiration syndrome (considered a marker rather than a cause of PPHN), who died in the first days of life [256]. In infants with high flow to the pulmonary circulation prior to birth, as in total anomalous pulmonary venous return, and obstructive lesions to the left side of the heart [aortic atresia, aortic stenosis, coarctation of the aorta associated with ventricular septal defect (VSD), and patent ductus arteriosus], similar vascular changes occur and the structural changes are considered secondary to hemodynamic changes [257–260]. In these infants the vein walls are also abnormally thickened by SMCs. Similarly, high pulmonary blood flow before birth resulting from pulmonary (or systemic) arteriovenous malformations, or fistulae, produces severe patterns of arterial wall muscularization. Hypoplasia represents underdevelopment of the lung and is commonly associated with increased muscularization of pulmonary arteries. In the hypoplastic lung that forms in renal agenesis, the cause is metabolic rather than mechanical (as in CDH, for example): pre-acinar arteries and airways are reduced in number, and the alveolar number and size are also reduced [261]. The arteries, although small for their age, are disproportionately large for the lung volume, and more peripheral arteries than normal are muscularized [261]. In pulmonary hypoplasia resulting from phrenic nerve agenesis and diaphragmatic amyoplasia, excessive muscularization

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occurs with typically no change in alveolar and airway ­development [262]. Although the evidence is indirect, infants with VSD and other congenital heart conditions initially adapt in the perinatal stage, but as lung blood flow increases (with right-to-left shunt), pulmonary hypertension develops. Repair of the heart defect before the age of 2 years results in no residual pulmonary hypertension. In a small group of infants with VSD, however, death occurs in the first year. Their lungs typically have resistance arteries that are small and more thick-walled than in the fetus, indicating impaired or failed adjustment to increased flow in the perinatal period.

4.4 Bronchopulmonary Dysplasia In neonates who encounter significant respiratory distress at the time of birth, distal airways may be disrupted and alveolarization impaired, leading to bronchopulmonary dysplasia [65, 230, 249, 263–266]. The importance of impaired vascular growth is also currently recognized. The earlier the interruption to the existing developmental program, the greater the potential for impaired development or growth. Infants with severe bronchopulmonary dysplasia develop pulmonary hypertension [264] likely based on distal vascular restriction. Restricted vascular and alveolar development, including ­capillaries and small vessels developing in the forming alveolar–capillary membrane of a preterm lung or term lung at birth, may be exacerbated by administration of supplemental/iatrogenic oxygen. Supplemental (high) oxygen may impair ­capillary formation (via reduced VEGF signaling) and so alter normal endothelial–epithelial-driven patterns of alveolar formation (see also Chapter 42) [65]. Future function and vascular growth depend on whether catch-up growth is possible.

4.5 Induced Postnatal Growth How to induce, or promote, catch-up growth in the still developing lung and how to induce regenerative growth in the adult lung are important questions. Optimally, the degree of growth will be sufficient to normalize the lung’s vascular system(s). In the developing lung, the question then becomes whether, after correction, appropriate developmental growth and maturation continue to proceed. In a comprehensive review of the mechanisms and limits of induced postnatal growth published by the American Thoracic Society [86], future directions to address the need for growth induction were reviewed. Potential models for stimulated lung growth were discussed in relation to increased alveolarization and vascularization, including the effects of increased lung

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­ etabolic oxygen demand, mechanical strain, reduced-size m lung transplant, chronic hypoxia or high-altitude exposure, and elastic fiber formation. Induced lung growth in response to mechanical signals (septal strain or strain-induced signal transduction) and nonmechanical signals and mediators (hormones, growth factors, retinoids, and hypoxia) were also considered and the limits imposed by the presence of dysanaptic lung growth carefully set out. The final section of the review emphasized the following key issues pertaining to future directions: (a) the need for studies to define similarities and differences between types of lung growth, since the developing lung differs from the adult (non-growing) lung in mechanical stresses, hormonal/growth factor levels, and cell–cell (epithelial–mesenchymal–endothelial) interactions; (b) the need to develop models of mechanical loading on alveolar capillary growth and remodeling, and to isolate and characterize progenitor/precursor cell subpopulations in the lung; (c) the need to understand issues relating to the regulation of alveolar–capillary architecture, and how septal growth is eventually limited; and (d) the need to understand and develop ways to optimally match alveolar to capillary growth. This review is recommended for a greater understanding of some of the important issues surrounding the question of induced and regenerative lung growth [86]. Based on medical imaging techniques, computational modeling studies of the (adult) human arterial and venous tree, including its supernumerary vessels, are currently of interest as part of a renewed effort to relate vessel branching structure to blood flow distribution [267]. Accurate determination of the spatial relationships between airways, vessels, and supporting tissues in these imaging-derived models allows regional interactions within the lung to be determined [268]. This holds significant potential for differentiating normal and disease-related heterogeneity in regional blood flow [268] and for modeling of a virtual lung [269]. The additional aim of these multiscale modeling studies is to provide an effective bridge between the cellular systems of the lung and whole organ function [269]. This approach may offer additional insight into the vascular template(s) required for lung regenerative studies, and serve as a future determinant of their functional success. Although lung or lobar transplant offers hope for patients with dysfunctional lungs from impaired vascular (and/or airway) growth, the constraints determined by the availability of the organ donor pool often restrict this approach [86]. Alternative approaches focus on methods of lung tissue regeneration, and on correction of vascular growth in the postnatal or adult lung by administration (infusion) of progenitor/precursor cells [85, 86]. Here, outstanding questions focus on (a) the need to generate different vascular cell phenotype(s), including the possibility of cells, e.g., endothelial cells, with a functional memory [81], (b) how to achieve the organization of different cell types into tissue structures appropriate to

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proximal or distal regions of the vascular bed, and (c) the need for an understanding of signaling pathways responsible for the modulation of these cells. Clearly, more than one cell type, and the interaction of several signaling systems, will likely be needed to replicate the exquisite coordination evident in lung vascular development and growth [85, 86] but current small steps in this direction offer the potential for future success.

5 Summary of Normal Pulmonary Vascular Development 1. Transcription and growth factors regulate normal pulmonary vascular development in temporal–spatial patterns to achieve the appropriate distribution of vessels, by size, location, and wall structure, for the age and stage of lung growth. 2. Organogenesis begins as the lung anlage arises from the developing foregut; a single airway tube (trachea) develops and buds into the surrounding mesenchyme to form primitive branching airway structures appropriate to a right and left lung; the lung anlage is vascularized by a vascular plexus derived from the heart/aortic sac and dorsal aorta. 3. Pulmonary arteries and veins continue to develop progressively in the central zone, and form from cells within the mesenchyme surrounding the developing airway buds. 4. Central arteries develop in concert with the airways; the central veins develop (midway) between the forming bronchoarterial structures. 5. Distal vascular structures likely develop into networks independently from the central vessels and connect to them in the mid-fetal stage. 6. The venous system is established (functionally) before the lung’s arterial system. 7. Developing central (conventional) arteries branch with airways, and additional (supernumerary) arterial branches form; similar (conventional and supernumerary) venous branches develop. 8. Vascular (arterial and venous) wall structure develops as perivascular cells in the mesenchyme give rise to (medial) SMCs and (adventitial) fibroblasts that surround the endothelial cell networks. 9. Along vascular pathways (from the lung hilum to periphery) wall thickness relates to vessel diameter, save in small intra-acinar (resistance) arteries where thickness is high: in the newborn lung, these arteries are preacinar. 10. Distal to intra-acinar resistance arteries, precapillary arterial and postcapillary venous units (in the alveolar wall) include a thin-walled muscular segment, a partially muscular segment (with muscle spiral), and a non-­

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muscular segment larger in diameter than a capillary but with a similar wall structure; intermediate cells and pericytes represent precursor SMC types in distal (nonmuscular intra-acinar) wall regions. 11. Pulmonary vascular growth before and after birth occurs by an increase in artery and vein size, the number of units, and wall maturation: after birth, this growth occurs during modification to the lung’s alveolar template, and continues by expansion. 12. The overall vascular arrangement described above is similar in all mammalian lungs but vessel size and wall structure, and the position within the branching pattern, are typical within a species and for a given stage of development. Temporal–spatial stages specific to: Fetal lung (mouse E14.2–E19/human 5–38 weeks) 1. Proximal (airways and) vessels form by dichotomous branching (pseudoglandular stage). 2. Distal (airways and) vessels form and central artery and vein branches multiply (canalicular stage). 3. Start of acinus and respiratory region (blood–gas barrier) formation (saccular stage). Neonatal and child lung (mouse birth to 4 weeks/human birth to 8 years) 4. Septation initiates formation of alveoli (alveolarization stage) and alveoli multiply thereafter. Existing vascular structures expand, and new ones form within the enlarging alveolar zone. Adult lung (mouse from 4 weeks/human from 8 years to approximately 22–25 years) 5. Existing vascular structures expand until thoracic growth ceases. Acknowledgements  The writting of this chapter by RJ and DC was supported by a grant from the National Institutes of Health (NIH HL 089252). The sterling work of the colleagues and investigators cited here is gratefully acknowledged. Section 2 dealing with regulation of the molecular basis of lung development is based on the author’s (RJ) presentation, and printed handout, for a symposium on genes and lung development, at a plenary session of an international meeting of the US/ Canadian Society of Pediatric Pathology, held in 2004 in Vancouver, British Columbia, Canada.

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Chapter 4

Pulmonary Vascular Function Robert Naeije and Nico Westerhof

Abstract  The pulmonary circulation is a low-pressure and high-flow circuit. The low pressure prevents fluid moving out of the pulmonary vessels into the interstitial space, and allows the right ventricle to operate at a low energy cost. The flow is matched to ventilation for pulmonary gas exchange. As a low-pressure system, the pulmonary circulation is very sensitive to mechanical influences, and the thin-walled right ventricle is poorly prepared for rapidly increased loading conditions. The pulmonary circulation is functionally coupled to the right ventricle. Pulmonary pressure–flow relationships are determined by a dynamic interaction between ventricular pump function and mechanical properties of the pulmonary arterial tree. In this respect, it is always important to remember that the pulsatility of the pulmonary circulation is greater than that in the systemic bed. The ratio of pulse pressure over mean pressure in the pulmonary artery is about unity, whereas in the aorta, pulse pressure is about 40% of mean pressure, implying that pulsatile energy to be generated. Keywords  Pulmonary vascular function • Pressure-flow relationship • Blood flow • Hemodynamics

1  Introduction The pulmonary circulation is a low-pressure and high-flow circuit. The low pressure prevents fluid moving out of the pulmonary vessels into the interstitial space, and allows the right ventricle to operate at a low energy cost. The flow is matched to ventilation for pulmonary gas exchange. As a low-pressure system, the pulmonary circulation is very sensitive to mechanical influences, and the thin-walled right ventricle is poorly prepared for rapidly increased loading conditions. The pulmonary circulation is functionally coupled to the right ventricle. Pulmonary pressure–flow relationships are R. Naeije (*) Department of Pathophysiology, Free University of Brussels, Campus Erasme CP 604, 808, route de Lennik, Bldg 4. Rm Es.4.108, 1070 Brussels, Belgium e-mail: [email protected]

determined by a dynamic interaction between ventricular pump function and mechanical properties of the pulmonary arterial tree. In this respect, it is always important to remember that the pulsatility of the pulmonary circulation is greater than that in the systemic bed. The ratio of pulse pressure over mean pressure in the pulmonary artery is about unity [1], whereas in the aorta, pulse pressure is about 40% of mean pressure [2], implying that pulsatile energy to be generated by the right side of the heart is relatively more important.

2  Steady-Flow Pulmonary Hemodynamics 2.1  Pulmonary Vascular Resistance The function of a vascular system is defined by pressure ­difference–flow relationships. In the “steady-flow” hemodynamic approach, pressure and flow waves are summarized by their mean values, and a single-point pressure difference– flow relationship is calculated as a resistance. This provides a single-number description of the resistive properties, or “function,” of the vascular system under consideration. The functional state of the pulmonary circulation can thus be defined by the pulmonary vascular resistance (PVR) calculated as the difference between the pulmonary artery pressure (PAP), taken as an inflow pressure, and the mean left atrial pressure (LAP), taken as the outflow pressure, divided by the mean flow (Q):

PVR = (PAP − LAP) / Q.

(1)

This equation is most often implemented in clinical practice by fluid-filled thermodilution catheters. These catheters are balloon-tipped, allowing for an estimate of LAP by an occluded PAP (PAOP). Sometimes, a measurement of LAP or PAOP cannot be obtained, and the total PVR (TPVR) is calculated as

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_4, © Springer Science+Business Media, LLC 2011

TPVR = PAP / Q.

(2)

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R. Naeije and N. Westerhof

Since LAP is not negligible with respect to PAP, TPVR is larger than PVR and is flow-dependent. Thus, TPVR is not a correct characterization of the resistive properties of the pulmonary circulation. A resistance calculation derives from Ohm’s law first derived for electric circuits. The resistance is determined by vessel and fluid properties. A law that governs laminar flows of Newtonian fluids through nondistensible, circular tubes was originally proposed by the French physician Poiseuille and later put into mathematical form by the German physicist Hagen. The law states that the resistance R to flow of a single tube is equal to the product of the length l of the tube and a viscosity constant h divided by the product of fourth power of the internal radius r and p, and can be calculated as a pressure drop DP to flow Q ratio:

R = 8.1h / πr 4 = ∆P / Q.

(3)

The ratio of the pressure drop and flow of an entire vascular bed accounts for the resistances in series and in parallel of the individual vessels The fact that r in the equation is at the fourth power explains why R is mainly located in the ­smallest arteries and arterioles (the resistance vessels) and is exquisitely sensitive to small changes in the caliber of these small vessels (a 10% change in radius results in almost 50% change in resistance) Accordingly, PVR is a good indicator of the state of constriction or dilatation of pulmonary resistive vessels and is helpful to monitor disease-induced pulmonary vascular remodeling and/or changes in tone. The limits of normal or resting pulmonary vascular pressures and flows as derived from measurements obtained in a total of 55 healthy resting supine young adult healthy volunteers [3–5] are shown in Table 1. In that study population, Q Table 1  Limits of normal pulmonary blood flow and vascular pressures Variables

Mean

Limits of normal flow and pressure

  6.4 4.4–8.4 Q (l/min) Heart rate (bpm) 67 41–93 PAP systolic (mmHg) 19 13–26 PAP, diastolic (mmHg) 10 6–16 PAP, mean (mmHg) 13 7–19 PAOP (mmHg)  9 5–13 PCP (mmHg) 10 8–12 RAP (mmHg)  5 1–9 PVR (dyne s cm−5) 55 11–99 SAP, mean (mmHg) 91 71–110 Limits of normal flow and pressure are from the mean minus twice the standard deviation (SD) to the mean plus twice the standard deviation, n = 55 healthy resting volunteers (n = 14 for the measurement of PCP Pulmonary Capillary Pressure) Data from [3–5] Q cardiac output, PAP pulmonary artery pressure, PAOP occluded pulmonary artery pressure, PCP pulmonary capillary pressure (measured by single occlusion), RAP right atrial pressure, PVR pulmonary vascular resistance, SAP systemic arterial pressure

was lower in women, who are smaller than men, and thus PVR was calculated to be higher. However, there were no gender differences in pulmonary hemodynamics after correction for body dimensions. Earlier studies showed that aging is associated with a slight increase in PAP and a decrease in Q, leading to a doubling of PVR over a five-decade lifespan [6–9].

2.2  Pressure–Flow Relationships The inherent assumption of a PVR calculation is that the PAP–Q relationship is linear and crosses the pressure axis at a value equal to LAP, allowing PVR to be constant whatever the absolute level of pressure or flow. Although the relationship between (PAP − LAP) and Q has indeed been shown to be reasonably well described by a linear approximation over a limited range of physiological flows, the zero crossing assumption may be true only in case of well-oxygenated lungs in supine resting subjects, suggesting complete recruitment and minimal distension. Hypoxia and a number of cardiac and ­respiratory diseases increase both the slope and the extrapolated intercepts of multipoint (PAP − LAP)–Q plots [10]. Although an increase in the slope of a PAP–Q plot is easily understood as being caused by a, generally, decreased radius and thus cross-sectional area of pulmonary resistive vessels, the positive extrapolated pressure intercept has inspired various explanatory models. Permutt et al. ­conceived a vascular waterfall model made of parallel collapsible vessels with a distribution of closing pressures [11]. At low flow, these vessels would be progressively derecruited, accounting for a low-flow PAP–Q curve that is concave with respect to the flow axis, and intercepts the pressure axis at the lowest closing pressure to be overcome to generate a flow. At higher flow, completed vessel recruitment and negligible distension account for a linear PAP–Q curve with an extrapolated pressure intercept representing a weighted mean of closing pressures. In this model, the mean closing pressure is the effective outflow pressure of the pulmonary circulation. If LAP is lower than the mean closing pressure, it is irrelevant to the flow. In that situation, a PVR calculation becomes misleading because of flow dependency: increased or decreased flow necessarily decreases or increases PVR, respectively, without a change in the functional state in the pulmonary circulation taking place. These concepts are illustrated in Fig.  1, which represents pulmonary vessels as collapsible with tone or owing to chamber pressure within a device called a “Starling resistor.” This device was conceived by Starling and his coworkers to control systemic arterial pressure in their heart–lung ­preparation. The “waterfall model” of Permutt et al. is also called the “Starling resistor model.”

4  Pulmonary Vascular Function

PAP

CP

Outflow pressure

PAP-LAP 2 CP

PVR = (PAP–LAP) / Q

LAP

Inflow pressure

1

A: LAP > CP B: CP > LAP 1 → 2: vasoconstriction 1 → 3 : no change

A

A

Q

Q

Fig. 1  Starling resistor model to explain the concept of closing pressure within a circulatory system. Flow (Q) is determined by the gradient between an inflow pressure, or mean pulmonary artery pressure (PAP), and an outflow pressure, which is either the closing pressure (CP) or the left atrial pressure (LAP). When LAP > CP, the (PAP − LAP)/Q relationship crosses the origin (A curve) and pulmonary vascular resistance (PVR) is constant. When CP > LAP, the (PAP − LAP)/Q relationship has a positive pressure intercept (B curve), and PVR decreases curvilinearly with increasing Q. Also shown are possible misleading PVR calculations: PVR, the slope of (PAP − LAP)/Q may remain unchanged in the presence of a vasoconstriction (from datapoint 1 to datapoint 2) or decrease (from datapoint 1 to datapoint 3) with no change in the functional state of the pulmonary circulation (unchanged pressure–flow line). (Reproduced with permission [10])

PAP-PAOP (mmHg)

30

E

D

20

B C

10

A

0

1

Resistances Resistances differ are equal

2

3

Vasoconstriction 2

C

Uncertainty

4

2 Uncertainty

1 Vasodilation

Q

B

PVR

PAP-LAP

1

PVR

B

3

63

5

Flow (l/min) Fig. 2  PAP versus flow at two levels of pulmonary hypertension is correctly described by a linear approximation over a physiological range of flows (3–5  l/min). The linearized pressure–flow relationships (dashed lines) have extrapolated pressure intercepts (open squares) that are positive, suggesting a CP higher than the LAP occluded PAP. However, the pressure–flow relations are better described by a curvilinear fit (fully drawn lines), which takes into account the distensibility of the pulmonary vessels. In the curvilinear relations as well as the linear relations over the physiological range, PVR calculations are misleading: from A to B, PVR does not seem change, and from C to B and from D to E, i.e., running over a single relation, PVR decreases, and in the presence of aggravated pulmonary hypertension as assessed by higher pressures at a given flow range the slope, and thus the resistance, may differ little (arrows)

However, distensible vessel models have been developed which explain the shape of PAP–Q curves by changes in resistance and compliance [12, 13]. In fact, as illustrated in Fig. 2, PAP–Q curves can always be shown to be curvilinear with

Fig.  3  Pressure–flow diagram for the interpretation of pulmonary hemodynamic measurements. The central point C corresponds to initial mean PAP, LAP, and flow (Q) measurements. A decrease in PAP − LAP at increased Q can only be explained by pulmonary vasodilatation. An increase in PAP − LAP at decreased Q can only be explained by pulmonary vasoconstriction. Rectangles of certainty are extended to adjacent triangles because negative slopes or pressure intercepts of (PAO − LAP)/Q lines are impossible. Arrows indicate changes in measured PAP − LAP and Q, 1 vasodilatation, 2 vasoconstriction. (Reproduced with permission [10])

concavity to the flow axis provided a large enough number of PAP–Q coordinates are generated and subjected to an ­adequate fitting procedure. However, a derecruitment can be directly observed at low pressures and flows [14]; therefore, it seems reasonable to assume that both recruitment and distension probably explain most PAP–Q curves [15]. According to this integrated view, at low inflow pressure, many pulmonary vessels are closed as a consequence of their intrinsic tone and surrounding alveolar pressure, and those that are open are relatively narrow. As   inflow pressure increases, previously closed vessels ­progressively open (recruitment), and previously narrow vessels progressively dilate (distension). Both mechanisms explain a progressive decrease in the slope of pulmonary vascular pressure—flow relationships with increasing flow or pressure. Whatever the model, PVR determinations are better replaced by multipoint pressure–flow relationships for the evaluation of the functional state of the pulmonary circulation at variable flow. The problem of in vivo ­pressure–flow relationships in intact animals is to alter flow without affecting vascular tone. Exercise to increase the flow may spuriously increase the slopes of PAP–Q plots, in normal subjects [16] as well as in patients with cardiac or pulmonary diseases, leading to linear fitting of PAP–Q plots with negative extrapolated pressure intercepts [17, 18]. An infusion of low-dose dobutamine to increase flow might be preferable [18], although it is always difficult to exclude a possible flow-induced or b-adrenergic or a-adrenergic receptor mediated change in tone, depending on the dose and the preexisting functional state. Most often only a single PVR determination can be obtained for every given functional state of a pathophysiological condition. It is then advised to reason using a pressure– flow diagram as illustrated in Fig. 3. This diagram shows four possible combinations of flow and pressure changes, with

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certainties about true changes in structure- or tone-determined resistance in only two of them: decreased pressure with increased flow as a true decrease in PVR, and increased pressure with decreased flow as a true increase in PVR, the other combinations remaining in an uncertainty domain.

3  Effects of Exercise Supine exercise is associated with proportional increases in cardiac output and pulmonary vascular pressure gradient, with a slight to moderate decrease in PVR. Upright exercise is associated with a marked initial hyperbolic decrease in PVR. At high levels of exercise, PVR at any given load is independent of body position. The sharp curvilinear decrease in PVR at low levels of upright exercise may be explained by a vascular derecruitment, together with an inherent slight curvilinearity of pressure–flow relationships [19]. High levels of exercise markedly increase pulmonary vascular pressures. In athletes able to increase their cardiac output to 25–35 l/min, PAP may increase to 40–45 mmHg and PAOP increased to 25–35 mmHg [19]. None of the previously reported pulmonary hemodynamic measurements at exercise in normal subjects have included direct measurements of LAP through a left-sided heart catheterization. However, PAOP at exercise is unlikely to overestimate LAP. High levels of cardiac output are associated with a complete recruitment of the pulmonary capillary network, which is the condition for the valid estimation of LAP by a PAOP. On the other hand, the filling pressures of both ventricles, as assessed by directly measured right atrial pressure (RAP) and indirectly measured LAP, rise at exercise in relation to stroke volume and exercise capacity [20]. This suggests that the left ventricle tends to overuse the Frank–Starling mechanism (increase stroke volume by an increased preload, or end-diastolic ventricular volume/pressure) to maximize cardiac output at the highest levels of exercise [19]. As mentioned already, PVR increases with age, so the average slope of PAP–Q plots during exercise is 1 mmHg/l/ min in young adults, but more than doubles, up to 2.5 mmHg/l/ min, in old subjects. Much of the slope of PAP–Q is caused by an increase in PAOP (or LAP) [7, 8, 19]. An earlier and more important increase in LAP in older subjects at exercise could be explained by age-related decreased diastolic compliance of the left ventricle [8]. Although PAP–Q relationships at exercise are generally best described by a linear approximation [19], a sufficient number of measurements at high levels of exercise, above the anaerobic threshold, may disclose an increased slope as the cause of a biphasic “takeoff” pattern on log PAP–log VO2 plots [16]. Because of the tight relationship between Q and VO2, this is to be interpreted as an high level of exerciseinduced pulmonary vasoconstriction, caused by sympathetic

R. Naeije and N. Westerhof

nervous system activation, acidosis, and decreased mixed venous oxygenation. An increased slope of PAP–Q plots above the anaerobic threshold may also be related to an increase in LAP. It is intriguing that the takeoff pattern of PAP–VO2 plots at exercise is not observed in patients with pulmonary vascular disease, who rather show a “plateau” pattern [16]. The reason for the decreased slope of PAP–VO2 relationships at high levels of exercise in patients with pulmonary vascular diseases is not clearly understood.

4  P  assive Regulation of Steady-Flow Pulmonary Hemodynamics 4.1  Left Atrial Pressure At a given Q, an increase in LAP is transmitted upstream to PAP in a less than 1:1 proportion, depending on the state of arterial distension and the presence or absence of a closing pressure higher than LAP [10, 15]. In a fully distended and recruited pulmonary circulation, DPAP/DLAP is close to unity.

4.2  Lung Volume An increase in lung volume above functional residual capacity increases the resistance of alveolar vessels, which are the vessels exposed to alveolar pressure, but decreases the resistance of extra-alveolar vessels, which are the vessels exposed to interstitial pressure. A decrease in lung volume below functional residual capacity has the opposite effects. As a consequence, the lowest resultant PVR is observed at functional residual capacity [21].

4.3  Gravity Pulmonary blood flow increases almost linearly from nondependent to dependent lung regions. This inequality of pulmonary perfusion is best demonstrated in an upright lung [22]. The vertical height of a lung is, on average, about 30 cm. The difference in pressure between the extremities of a vertical column of blood of the same size is 23  mmHg, which is quite large compared with the mean perfusion pressure of the pulmonary circulation. Accordingly, the physiological inequality of the distribution of perfusion of a normal lung can be explained by a gravity-dependent interplay between arterial, venous, and alveolar pressures. At the top of the lung, alveolar pressure (PA) is higher than mean PAP

4  Pulmonary Vascular Function

(MPAP) and pulmonary venous pressure (PVP). In this zone 1, flow may be present only during systole, or not at all. Zone 1 is extended in clinical situations of low flow, such as hypovolemic shock, or increased alveolar pressure such as during ventilation with a positive end-expiratory pressure. Further down the lung there is a zone 2 where PAP > PA > PVP. In zone 2, alveolar pressure is an effective closing pressure, and the driving pressure for flow is the gradient between MPAP and PA. As mentioned earlier, such a flow condition can be likened to a waterfall since PVP, the apparent outflow pressure, is irrelevant to the flow as is the height of a waterfall. In zone 3, PVP is higher than PA, so the driving pressure for flow is PAP − PVP. In the most dependent regions of the upright lung, there is an additional region where flow decreases, defining an additional zone, zone 4 [23]. Zone 4 has been attributed to an increase in the resistance of extra-alveolar vessels, because it expands when lung volume is reduced or in the presence of lung edema. Active tone may be an additional explanation for zone 4 as it is also reduced by the administration of vasodilators. The vertical height of lung tissue in a supine subject is, of course, much reduced compared with that of the subject in the upright position, and accordingly, the lung is then normally almost completely in zone 3, with, however, persistence of a still measurable increase in flow from nondependent to dependent lung regions. Three-dimensional reconstructions using single-photonemission computed tomography have shown that there is also a decrease in blood flow from the center of the lung to the periphery [24]. High-resolution methods and fractal modeling of the pulmonary circulation have actually led to the suggestion that the distribution of pulmonary blood flow would be determined as a consequence of the fractal structure of the pulmonary arterial tree, with only secondary minor gravity-dependent adjustments [25]. Subtle differences in arterial branching ratios may indeed influence the flow distribution, with increased heterogeneity as the scale of the inquiry narrows, corresponding to the “what is the length of the coastline” effect. However, the overwhelming evidence remains in favor of the thesis that gravity is the single most important determinant of pulmonary blood flow distribution [26]. Vascular-geometry-related small unit heterogeneity of pulmonary blood flow distribution has not been shown to be relevant to gas exchange.

5  A  ctive Hypoxic Regulation of Steady-Flow Pulmonary Hemodynamics There is an active intrapulmonary control mechanism able to some extent to correct the passive gravity-dependent distri-

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bution of pulmonary blood flow: a decrease in PO2 increases pulmonary vascular tone. Hypoxic pulmonary vasoconstriction was first reported by von Euler and Liljestrand [27], who proposed a functional interpretation that can still be considered valid. In lung tissue, PO2 is determined by the ratio between O2 carried to the lung by alveolar ventilation (VA) and O2 carried away from the lung by blood flow (Q):

PO 2 = VA / Q.

(4)

This is in contrast with hypoxic vasodilation in systemic tissue, where local PO2 is determined by the ratio flow of O2 carried to the tissues (Q) and local O2 consumption (VO2):

PO2 = Q / VO2 .

(5)

The hypoxic pulmonary pressor response is universal in mammals and in birds, but with considerable interspecies and interindividual variability. The attributes of hypoxic ­pulmonary vasoconstriction can be summarized as follows [28]. The response is turned on in a few seconds, fully developed after 1–3 min, and more or less stable thereafter according to the experimental conditions. It is reversed in less than 1 min. It is observed in lungs devoid of nervous connections, and indeed also in isolated pulmonary arterial smooth muscle cells. Hypoxic pulmonary vasoconstriction is enhanced by acidosis, a decrease in mixed venous PO2, repeated hypoxic exposure (in some experimental models), perinatal hypoxia, decreased lung segment size, cyclooxygenase inhibition, nitric oxide inhibition, and certain drugs or mediators which include almitrine and low-dose serotonin. Hypoxic pulmonary vasoconstriction is inhibited by alkalosis, hypercapnia, an increase in pulmonary vascular or alveolar pressures, vasodilating prostaglandins, nitric oxide, complement activation, low-dose endotoxin, calcium channel blockers, b2 stimulants, nitroprusside, and, paradoxically, peripheral chemoreceptor stimulation. The hypoxic pressor response is biphasic, with a progressive increase as PO2 is progressively decreased to approximately 35–40  mmHg, followed by a decrease (“hypoxic vasodilatation”) in more profound hypoxia. The hypoxia-induced increase in PVR is mainly caused by a constriction of precapillary small arterioles [28]. Small pulmonary veins also constrict in response to hypoxia, but this should not normally contribute to more than 20–30% of the total change in PVR [29]. An exaggerated hypoxic constriction of small pulmonary veins could explain the development of pulmonary edema which is observed in a small proportion, of the order of 1–2%, of subjects rapidly taken to high altitudes [5]. Grant et al. [30] used the equations of the control theory and the linear relationships between lobar blood flow and alveolar PO2 (PAO2) found in the coatimundi, an animal with

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a strong hypoxic pressor response, to calculate the efficiency of hypoxic vasoconstriction as a mechanism to stabilize PAO2. They found a maximum gain due to feedback (Gfb) of 0.9 at a PAO2 between 60 and 80 mmHg, falling rapidly off outside these values. A Gfb of 0.9 represents an active correction of 47% of the decrease in PAO2 that would occur in a passive system without hypoxic vasoconstriction. Mélot et  al. used the same equations and linear relationships between compartmental blood flow and PAO2 derived from data for elimination of inert gases obtained in healthy ­volunteers, and found a maximum Gfb of 0.63 at a PAO2 of 60  mmHg, also falling rapidly off at lower and at higher PAO2 [4]. A Gfb of 0.63 represents an active correction by 39% of a decrease in PAO2 that would occur in a passive system without hypoxic vasoconstriction. These studies suggested that the hypoxic pressor response is an only moderately efficient feedback mechanism, acting essentially at PAO2 values higher than are known to occur in severe lung diseases. However, more recent evaluations of the efficiency of hypoxic pressor response using multicompartment lung models [31] fed by biphasic stimulus–response curves using real data [32] led to the conclusions that hypoxic vasoconstriction is really effective in improving gas exchange in severe respiratory insufficiency. A quantification of the efficiency of hypoxic pulmonary vasoconstriction in terms of correction of arterial hypoxemia in chronic obstructive pulmonary disease (COPD) is presented in Fig.  4 [32]. Patients with COPD are hypoxemic because of increased dispersion of the distributions of perfusion and ventilation, with increased perfusion to lung units with a lower than normal VA/Q. Thus, in these patients, altered pulmonary gas exchange can be quantified by the logarithm of the standard deviation of VA/Q dispersion. On the other hand, the strength of hypoxic vasoconstriction can be expressed as PAP in hypoxia divided by PAP in hyperoxia at constant flow. The magnitude of hypoxic vasoconstriction normally ranges from 1 to 4 in the canine and in the human species. It can be seen that, in COPD, arterial PO2 may increase by up to 20 mmHg through the effects of vigorous hypoxic vasoconstriction. The same analysis was performed in patients with acute respiratory distress syndrome (ARDS), who are hypoxemic mainly because of an increased shunt [32]. Thus, in these patients, altered gas exchange can be quantified by intrapulmonary shunt, expressed as a percentage of cardiac output. In ARDS, arterial PO2 could increase by as much as 20 mmHg owing to vigorous hypoxic vasoconstriction. All these predictions are in keeping with the magnitude of decreases in arterial oxygenation observed in patients with ARDS or COPD due to the administration of vasodilating drugs that inhibit hypoxic pulmonary vasoconstriction [32]. The biochemical mechanism of hypoxic pulmonary vasoconstriction remains incompletely understood [28, 33].

R. Naeije and N. Westerhof

Fig.  4  Effects of hypoxic pulmonary vasoconstriction (HPV) in chronic obstructive pulmonary disease (COPD), a lung disease characterized by VA/Q mismatching. LSD logarithmic standard deviation of the log–normal VA/Q ratio distribution. The fraction of inspired O2 ( FIO2) was set to 0.30; other variables were set to normal values and remained unchanged during calculations (barometric pressure 760 Torr, temperature 37°C, hemoglobin 15 g/dl, base excess 0 mmol/l, P50 26.8 Torr, O2 consumption 300 ml/min, CO2 production 240 ml/ min, cardiac output 6.00 l/min, ventilation 7.20 l/min, shunt 0%, dead space 30%). a HPV significantly improved PAO2 at all LSD values and improved SaO2 when it was most decreased. b At PAO2 of 40  Torr (LSD = 2.2), HPV decreased blood flow by 30% in hypoxic lung units (VA/Q < 0.3, alveolar PO2 < 45 Torr) and increased blood flow by 76% in normoxic lung units (VA/Q > 0.5, alveolar PO2 < 60 Torr). (Reproduced with permission [32])

Current thought is that a decrease in PO2 inhibits smooth muscle cell voltage-gated potassium channels, resulting in membrane depolarization, influx of calcium, and cell shortening. However, the nature of the low PO2 sensing mechanism remains elusive. Mitochondria and nicotinamide adenine dinucleotide phosphate oxidases have been discussed as oxygen sensors. Reactive oxygen species, redox couples, and adenosine monophosphate activated kinases are candidate mediators. The reversal of hypoxic vasoconstriction by profound hypoxia is due to an activation of ­ATP-dependent potassium channels [28].

4  Pulmonary Vascular Function

a

c

b 250 125 0

Q

0

0.5 Time (s)

1.0

30 mPAP

20 10 0 0

0.5 Time (s)

1.0

0.20 PAP

As already mentioned, the study of the pulmonary circulation as a steady-flow system is a simplification, since pulmonary arterial pulse pressure, or the difference between systolic PAP (SPAP) and diastolic PAP (DPAP), is of the order of 40–50% of mean pressure, and instantaneous flow varies from a maximum at midsystole to around zero in diastole. Since pressure and flow depend on the heart and arterial load, analysis of wave shapes alone is of limited use in the characterization of the pulmonary arterial tree. However, calculation of the relation between pulsatile pressure and flow allows for a more detailed characterization of the arterial load than by resistance only. PVR characterizes the small vasculature, i.e., the resistance vessels, only. A complete description of pulmonary arterial function requires consideration of pulsatile pressure–flow relationships, or pulmonary vascular impedance (PVZ). PVZ can be calculated from a spectral analysis of the pulmonary arterial pressure and flow waves [36]. This analysis is possible because the pulmonary circulation behaves similarly to a linear system, i.e., a purely sinusoidal flow oscillation produces a purely sinusoidal pressure oscillation of the same frequency. The sinusoidal pressure and flow waves can be related by the ratio of their amplitudes (modulus) and the difference in their phases (phase angle). Thus, both pressure and flow are decomposed into a series of sine waves with frequencies 1, 2, 3, etc. times the heart rate, each with its amplitude and phase angle. PVZ is now the ratio of the amplitudes of the sine waves of pressure and flow (modulus)

Pulmonary artery pressure (mmHg)

6.1  Pulmonary Vascular Impedance

Pulmonary artery flow (ml/s)

6  Pulsatile-Flow Pulmonary Hemodynamics

and their phase differences, and is graphically represented as a modulus and phase angle, both as a function of frequency. A typical PVZ spectrum is illustrated in Fig. 5. Pulmonary arterial impedance at 0  Hz (ratio of mean pressure and mean flow, PAP/Q = Z0) corresponds to TPVR. Normally, the modulus of the impedance decreases rapidly to a first minimum at 2–3 Hz and then oscillates about a constant value. At low frequencies, the phase angle is negative, indicating that flow leads pressure and at higher frequencies the phase hovers around 0°. The precipitous fall in the modulus and the negative phase of the impedance are a measure of total arterial compliance. At high frequencies the rather constant modulus and negligible phase is the socalled characteristic impedance, Zc, of the proximal pulmonary artery. The notion of impedance can be explained as follows. For mean pressure and flow and for low frequencies, impedance is mainly determined by the small resistance vessels, and at zero frequency is equal to TPVR. For intermediate frequencies, the impedance is strongly affected by the elasticity (compliance) of the large arteries. For high frequencies, the characteristic impedance of the proximal arteries determines the input impedance. Taking these observations together, one can say that the higher the frequency, the closer you “look” into the arterial tree.

TPVR=mPAP/Q Impedance modulus (mmHg/(ml/s)

The normal as well as the abnormal pulmonary vascular tone has been shown to be modulated by a series of endotheliumderived and circulating mediators. Endothelium-derived relaxing factors include nitric oxide, prostacyclin, and the endothelium-derived hyperpolarizing factor. The major endothelium-derived contracting factor is endothelin. These observations have been at the basis of efficient treatments of pulmonary arterial hypertension with prostacyclin derivatives, phosphodiesterase-5 inhibitors to enhance nitric oxide signaling and endothelin receptor blockers [34]. The pulmonary circulation is richly innervated by the autonomic nervous system (adrenergic, cholinergic, and nonadrenergic, noncholinergic) [35]. However, the role played by the autonomic nervous system in the control of pulmonary vascular tone appears to be minor. The autonomic innervation of the pulmonary arterial tree is predominantly proximal, suggesting a more important effect in the modulation of proximal compliance.

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0.15

Zc

Compliance

Q

0.10

Characteristic Impedance, Zc ∆/Zc = Reflections

0.05 ∆ 0

0

5 Frequency, Hz

10

Fig. 5  Pulmonary artery flow (a) and pressure (b) and pulmonary arterial input impedance (c). At 0 Hz the total PVR is obtained (mean pressure over mean flow). Between 0 and 4  Hz the impedance is mainly determined by total arterial compliance. The averaged input impedance for high frequencies, usually taken between 4 and 8 Hz, equals the characteristic impedance. The oscillation of the impedance about the characteristic impedance results from wave reflection

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R. Naeije and N. Westerhof

Characteristic impedance is the input impedance without wave reflections. It is measured as the average modulus at higher frequencies (usually 4–8 Hz). It can also be measured as the slope of the early SPAP–flow relationship (Fig. 5). The oscillations of the input impedance about its mean value result from distinct reflections of waves. Increased magnitude of the oscillations implies increased number of reflections. A shift of the first minimum and maximum to higher frequencies indicates an increased wave velocity or a decreased distance of the dominant reflection site. Characteristic impedance depends on the ratio between inertia and compliance of the pulmonary circulation, and can be approximated by the equation Z c = (inertia / area compliance ) = (ρ / A) / (∆A / ∆P ) 1/ 2



1/ 2

= ( ρ / πr ) / (2 πr∆r / ∆P ) , (6) 2

1/ 2

where r is the density of blood, r the mean internal radius, r/A = r/pr2 the inertia, and DA/DP = pDr2/DP = 2prDr/DP the area compliance of the proximal pulmonary arterial tree. The extent of the oscillations of the input impedance around Zc can be used to estimate the degree of arterial wave reflection [2], with Rc, an index of wave reflection, calculated as

Rc = (1 − ∆ / Z c ) / (1 + ∆ / Z c ).

(7)

There have been reports on PVZ in normal subjects [37] and in patients with pulmonary hypertension secondary to mitral stenosis, congenital cardiac defects, congestive heart failure and COPD, and idiopathic pulmonary arterial hypertension [38]. The general pattern has been that of an upward shift of PVZ spectra, (higher TPVR and higher Zc), with the first minima and maxima of the impedance moduli shifted  to higher frequencies (higher pulse wave velocity, thus decreased arterial compliance). The limited amount of available data has not allowed specific patterns to be identified, and even less so the effects of therapeutic interventions. Semi-invasive approaches for the determination of PVZ with fluid-filled catheters and transthoracic Doppler imaging have been reported in patients with idiopathic pulmonary arterial hypertension [39] and the results agree with those reported using highfidelity technology [40]. Pulmonary arterial input impedance is little affected by normal breathing [37], or by disease processes limited to alveolar or juxta-alveolar vessels [41, 42]. In contrast, proximal pulmonary arterial obstruction markedly affects pressure and flow wave morphology, the PVZ spectrum, and at any given PVR, has more important depressant effect on right ventricular (RV) output [41–43].

6.2  T  he Time Constant of the Pulmonary Circulation As mentioned already, the arterial input impedance is a comprehensive and quantitative description of the pulmonary arterial system. However, its use has not gained acceptance because of perceived mathematical complexity and difficulty to interpret data in the frequency domain. Accordingly, Lankhaar et al., [1, 43, 44], modeled PVZ as being ­determined by a dynamic interaction between PVR, total arterial compliance Ca, and Zc. A practical application of this approach was in the quantification of the effects of therapeutic interventions on small and large arteries. These studies also showed that the product of PVR and Ca, i.e., the time constant of the pulmonary circulation, remained constant at approximately 0.7 s in all circumstances. This is illustrated in Fig. 6, which shows PVR and Ca values from patients with pulmonary vascular diseases of various severities and origins. The hyperbolic relationship between compliance and resistance of the pulmonary arterial circulation explains that mild increases in PVR may already markedly increase RV afterload because of associated decrease in compliance. A pharmacological decrease of only mildly increased PVR may markedly improve RV flow output because of a proportionally larger increase in compliance. A lately discovered characteristic of pulmonary vascular function is the tight correlation between SPAP, DPAP, and MPAP, which persists in pulmonary hypertension of all possible origins [45, 46]. Accordingly, MPAP can be calculated from SPAP using a simple formula,

MPAP = 0.6 × SPAP + 2 (45),

(8)

and inversely

Fig. 6  Hyperbolic relationship between pulmonary arterial resistance and compliance in patients with a normal pulmonary circulation (NONPH), with chronic thromboembolic pulmonary hypertension (CTEPH), and with idiopathic pulmonary arterial hypertension (IPAH). (Reproduced with permission of Oxford University Press [43])

4  Pulmonary Vascular Function

DPAP = 0.71MPAP − 0.66 mmHg

Serotonin Pm

and

Control

(9)

SPAP = 1.50 MPAP + 0.46 mmHg (46).

(10)

40 mmHg



69

Pm Pf

Pf

Pb

Pb



SPAP = (TR 2 + 4) + RAP.

(11)

The tight relationships between PAPs imply that the response of the pulmonary circulation to insults and diseases is more monotonous than previously assumed.

6.3  Pressure and Flow Wave Morphology Pressure and flow can also be analyzed by wave-form analysis [48, 49]. The pressure and flow waves consist of a part that travels from the heart to the periphery (forward or initial pressure and flow waves, Pf and Qf) and waves that return toward the heart (backward or reflected pressure and flow waves, Pb and Qb). The derivation of forward and backward waves requires measurement of pressure and flow as a function of time (measured pressure and flow, Pm and Qm) and determination of the characteristic impedance. The mathematical relations are



(

)

P f = P m + Z c Q m / 2,

(12)

Qf = + P f / Zc ,

(13)

P b = P m − Z cQ m / 2,

(

)

(14)

Qb = −P b / Zc .

(15)

and

It should be realized that the value of the characteristic impedance is the value at the site of the measurement of the pressure and flow, i.e., the proximal pulmonary artery. Reflections and forward and backward waves only pertain to oscillatory parts of the pressure and flow. The analysis does not give information on relations between the mean pressure and flow. Figure 7 shows wave separation in the pulmonary artery of the dog [48]. Increased reflection is obtained by vasoconstriction.

Qf

Qf

100 ml/s

This notion is of practical relevance as noninvasive evaluations of the pulmonary circulation in clinical practice often rely on the measurement of a maximum velocity of tricuspid regurgitation (TR) to calculate a SPAP using the simplified form of the Bernouilli equation and a measurement of RAP [47]:

Qm

Qm Qb

Qb

0.5 s Fig. 7  Pressure and flow in a common pulmonary artery are broken down into their forward and backward components. Thick lines are measured waves, Pm and Qm, thin lines are forward waves Pf and Qf, and dotted lines are backward-running waves. With vasoconstriction, reflection, given by Pb/Pf = Qb/Qf, is increased. (From van den Bos et al. [49], reproduced with permission of Wolters Kluwer)

Wave-form analysis, separation into forward and backward waves, has not been often attempted in the pulmonary circulation. Furuno et al. reported high-fidelity pressure and flow measurements in dogs with pulmonary arterial obstruction either in proximal, or large, vessels by arterial banding (which mimics pulmonary embolism), or in distal, or small, vessels by injection of small glass beads (which mimics ARDS) [41]. They were able to explain the typical aspects of late systolic peaking of pulmonary arterial pressure waves and midsystolic deceleration, or notching seen in embolic pulmonary hypertension by wave reflection, i.e., addition of the reflected pressure wave and subtraction of the reflected flow wave on respective forward waves [41]. Nakayama et al. reported higher ­pulmonary arterial pulse pressures in chronic thromboembolic as compared with idiopathic pulmonary arterial hypertension, with the assumption that this is explained by wave reflection, and proposed this as a help for the differential diagnosis between the two conditions [50]. Castelain et al. could not confirm this finding, even though some effect of wave reflection could be identified in pressure wave ­morphology analysis in chronic thromboembolic pulmonary hypertension patients [51]. The same idea but focused on flow waves was reported by Hardziyenka et  al., who showed a shortened time to notching in patients with proximal obstruction due to thromboembolic pulmonary hypertension [52]. The morphology of PAP and flow waves in normal subjects and in pulmonary hypertension is based on the increase in PVR and the decreases in pulmonary arterial compliance and the change in characteristic impedance. These changes in the arterial system explain the altered relation between pressure and flow but cannot explain the whole change in wave shape. Wave shape is also determined by cardiac pump function. It has been shown experimentally that RV output

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decreases with an increased afterload, i.e., if resistance increases and/or compliance decreases [44]. The same finding was recently reported in patients with pulmonary hypertension associated with systemic sclerosis: lower cardiac output in these patients as compared with patients with the idiopathic form of pulmonary hypertension is likely explained by altered RV pump function [53].

7  Right Ventriculoarterial Coupling

20

PAP

Pes PRV

0

Time (s)

Pmaxl

40

Right Ventricular Pressure (mmHg)

Extrapolation

Pmaxl

40

Sine wave fit

Right Ventr. & Pulm. Art. Pressure (mmHg)

Blood pressure and flow depend on the pump (the right ventricle) and the load on the pump (the arterial system). Therefore, in the study of pulmonary arterial hypertension, knowledge of the arterial system alone is not sufficient. It is of special interest to study the power transfer from the pump to the arterial load, because it has been shown that power transfer and ventricular efficiency are near maximal in normal subjects. A simplified approach to test this was given by Sunagawa et  al. [54]. These authors proposed a graphical analysis based on the ventricular pressure–volume diagram [55, 56] of the right ventricle and characterization of the arterial system by means of its arterial elastance (Fig.  8). The diagram allows for the determination of the maximal ventricular elastance (Emax), which is the best possible loadindependent measurement of contractility, and of arterial elastance Ea » PVR/T (where T is the R–R interval), as a measurement of afterload as it is “seen” by the ventricle. The Emax/Ea ratio is a measure of the coupling of the ventricle and the arterial load. The optimal matching of heart and load, i.e., when power transfer and cardiac efficiency are close to maxi­ mal, is found when the Emax/Ea ratio is about 1.5. An isolated increase in Ea (increased vascular resistance), or a decrease in Emax (decreased cardiac contractility), decreases the Emax/Ea ratio, indicating uncoupling of the ventricle from its arterial system, i.e., lower cardiac efficiency. Everything else being the same, a decrease in Emax/Ea is necessarily accompanied by

a decrease in stroke volume. On the other hand, an isolated increase in preload is associated with an increase in stroke volume with unaltered ventriculoarterial coupling. However, although Ea equals the ratio of end-systolic pressure, Pes, and stroke volume and can be found from standard RV catheterization, Emax requires measurement of at least two end-systolic pressures and volumes, since the end-systolic pressure–volume relation does not go through the origin. The common approach is to reduce RV diastolic filling and measure a series of pressure–volume loops [55, 56], but bedside manipulations of venous return are too invasive to be ethically acceptable. In addition, when applied to intact beings, changes in venous return are associated with reflex sympathetic nervous system activation, which affects the ventricular function that is measured. This problem was first approached by Sunagawa et al. for the left ventricle [57]. This so-called single beat method was recently applied to the coupling of the RV to the pulmonary circulation [58]. As shown in Fig. 8, a “maximum pressure (Pmax), is estimated from a nonlinear extrapolation of the early and late systolic isovolumic, isovolumic,” i.e., the pressure of a nonejecting beat with pressure Pmax, portions of the RV pressure curve. This estimated Pmax has been shown to be tightly correlated with Pmax directly measured during a nonejecting beat [58]. A straight line drawn from Pmax to the RV end-systolic pressure of the ejecting beat versus the volume change (stroke volume) allows for the determination of Emax. A straight line drawn from the end-systolic pressure point to the end-diastolic volume point determines Ea. This method avoids absolute volume measurements and related technical complexities. Emax is now calculated as Emax = (Pmax − Pes)/SV, where SV is the stroke volume. With Ea = Pes/SV, the ratio Emax/Ea = (Pmax − Pes)/Pes = Pmax/Pes − 1. The Emax/Ea ratio determined by this single beat method is normally around 1.5, which is similar to values reported for left ventricular–aortic coupling, and is compatible with an optimal ratio of mechanical work to oxygen consumption [54]. The Emax/Ea ratio is decreased by propranolol and increased by dobutamine, and is maintained in the presence of increased

Systole ESPVR

20

Pes

SV

Ea

Emax 1

0

ESV 75

Diastole EDV 150

Volume (ml)

Fig. 8  Single beat method to measure right ventriculoarterial coupling. A maximum pressure (Pmax) is calculated from nonlinear extrapolation of early and late isovolumic portions of the right ventricular pressure curve. A straight line is drawn from Pmax and the end-diastolic volume (EDV) to Pes and the end-systolic volume, thus Emax = (Pmax − Pes)/SV, where SV is the stroke volume. The arterial elastance Ea = Pes/SV

4  Pulmonary Vascular Function

Ea due to hypoxic pulmonary vasoconstriction. In fact, Emax increases adaptedly to increased Ea in hypoxia, even in the presence of adrenergic blockade, which is compatible with the notion of homeometric adaptation of RV contractility. On the other hand, the approach allows for the demonstration that clinically relevant doses of dobutamine do not affect pulmonary arterial hydraulic load [58]. The single beat approach has been used to show the profound decoupling effects of inhaled anesthetics in the same setting [59], this being due to the devastating effects of both negative inotropy and pulmonary vasoconstriction. The method also showed that prostacyclin, at clinically relevant doses, has no intrinsic positive inotropic effect on the right ventricle [60]. Most recently, Kuehne et  al. used magnetic resonance imaging together with RV pressure measurements to generate pressure–volume loops and Emax and Ea determinations in patients with pulmonary arterial hypertension [61]. As compared with controls, RV Emax was increased from 5.2 ± 0.9 to 9.2 ± 1.2  mmHg/ml/100  g, P < 0.05, but RV Emax/Ea was decreased from 1.9 ± 0.4 to 1.1 ± 0.3, P < 0.05, indicating an increased RV contractility in response to increased afterload that was, however, insufficiently coupled to its hydraulic load, with inefficient mechanical work production.

References 1. Lankhaar JW, Westerhof N, Faes TJ et al (2006) Quantification of right ventricular afterload in patients with and without pulmonary hypertension. Am J Physiol Heart Circ Physiol 291:H1731–H1737 2. Murgo JP, Westerhof N, Giolma JP, Altobelli SA (1980) Aortic input impedance in normal man: relationship to pressure wave forms. Circulation 62:105–116 3. Naeije R, Mélot C, Mols P, Hallemans R (1982) Effects of vasodilators on hypoxic pulmonary vasoconstriction in normal man. Chest 82:404–410 4. Mélot C, Naeije R, Hallemans R, Lejeune P, Mols P (1987) Hypoxic pulmonary vasoconstriction and pulmonary gas exchange in normal man. Respir Physiol 68:11–27 5. Maggiorini M, Mélot C, Pierre S et al (2001) High altitude pulmonary edema is initially caused by an increased capillary pressure. Circulation 103:2078–2083 6. Holmgren A, Jonsson B, Sjostrand T (1960) Circulatory data in normal subjects at rest and during exercise in the recumbent position, with special reference to the stroke volume at different working intensities. Acta Physiol Scand 49:343–363 7. Granath A, Strandell T (1964) Relationships between cardiac output, stroke volume, and intracardiac pressures at rest and during exercise in supine position and some anthropometric data in healthy old men. Acta Med Scand 176:447–466 8. Granath A, Jonsson B, Strandell T (1964) Circulation in healthy old men, studied by right heart catheterization at rest and during exercise in supine and sitting position. Acta Med Scand 76:425–446 9. Bevegaard S, Holmgren A, Jonsson B (1963) Circulatory studies in well trained athletes at rest and during heavy exercise, with special reference to stroke volume and the influence of body position. Acta Physiol Scand 57:26–50 10. Naeije R (2003) Pulmonary vascular resistance: a meaningless variable? Intensive Care Med 29:526–529

71 11. Permutt S, Bromberger-Barnea B, Bane HN (1962) Alveolar pressure, pulmonary venous pressure and the vascular waterfall. Med Thorac 19:239–260 12. Zhuang FY, Fung YC, Yen RT (1983) Analysis of blood flow in cat’s lung with detailed anatomical and elasticity data. J Appl Physiol 55:1341–1348 13. Nelin LD, Krenz GS, Rickaby DA, Linehan JH, Dawson CA (1992) A distensible vessel model applied to hypoxic pulmonary vasoconstriction in the neonatal pig. J Appl Physiol 73:987–994 14. Glazier JB, Hughes JMB, Maloney JE, West JB (1969) Measurements of capillary dimensions and blood volume in rapidly frozen lungs. J Appl Physiol 26:65–76 15. Mélot C, Delcroix M, Lejeune P, Leeman M, Naeije R (1995) Starling resistor versus viscoelastic models for embolic pulmonary hypertension. Am J Physiol 267:H817–H827 16. Tolle JJ, Waxman AB, Van Horn TL, Pappagianopoulos PP, Systrom DM (2008) Exercise-induced pulmonary arterial hypertension. Circulation 118:2183–2189 17. Janicki JS, Weber KT, Likoff MJ, Fishman AP (1985) The pressure– flow response of the pulmonary circulation in patients with heart failure and pulmonary vascular disease. Circulation 72:1270–1278 18. Kafi AS, Mélot C, Vachiéry JL, Brimioulle S, Naeije R (1998) Partitioning of pulmonary vascular resistance in primary pulmonary hypertension. J Am Coll Cardiol 31:1372–1376 19. Reeves JT, Dempsey JA, Grover RF (1989) Pulmonary circulation during exercise. In: Weir EK, Reeves JT (eds) Pulmonary vascular physiology and physiopathology. Dekker, New York, pp 107–133 20. Reeves JT, Groves BM, Cymerman A et al (1990) Operation Everest II: cardiac filling pressures during cycle exercise at sea level. Respir Physiol 80:147–154 21. Howell JBL, Permutt S, Proctor DF, Riley RL (1961) Effect of inflation of the lung on different parts of the pulmonary vascular bed. J Appl Physiol 16:71–76 22. West JB, Dollery CT, Naimark A (1964) Distribution of blood flow in isolated lung: relation to vascular and alveolar pressures. J Appl Physiol 19:713–724 23. Hughes JM, Glazier JB, Maloney JR, West JB (1968) Effect of lung volume on the distribution of pulmonary blood flow in man. Respir Physiol 4:58–72 24. Hakim TS, Lisbona R, Michel RP, Dean GW (1993) Role of vasoconstriction in gravity-nondependent central–peripheral gradient in pulmonary blood flow. J Appl Physiol 63:1114–1121 25. Glenny R (2008) Counterpoint: gravity is not the major factor determining the distribution of blood flow in the healthy human lung. J Appl Physiol 104:1533–1535 26. Hughes M, West JB (2008) Point: counterpoint: gravity is/is not the major factor determining the distribution of blood flow in the human lung. J Appl Physiol 104:1531–1533 27. von Euler US, Liljestrand G (1946) Observations on the pulmonary arterial blood pressure in the cat. Acta Physiol Scand 12:301–320 28. Weir EK, Archer SL (1995) The mechanism of acute hypoxic pulmonary vasoconstriction: the tale of two channels. FASEB J 9:183–189 29. Hillier SC, Graham JA, Hanger CC, Godbey P, Glenny RW, Wagner WW (1997) Hypoxic vasoconstriction in pulmonary arterioles and venules. J Appl Physiol 82:1084–1090 30. Grant BJB, Davies EE, Jones HA, Hughes JMB (1976) Local regulation of pulmonary blood flow and ventilation–perfusion ratios in the coatimundi. J Appl Physiol 40:216–228 31. Marshall BE, Marshall C (1988) A model for hypoxic constriction of the pulmonary circulation. J Appl Physiol 64:68–77 32. Brimioulle S, Lejeune P, Naeije R (1996) Effects of hypoxic ­pulmonary vasoconstriction on gas exchange. J Appl Physiol 81: 1535–1543 33. Sommer N, Dietrich A, Schermuly RT et al (2008) Regulation of hypoxic pulmonary vasoconstriction: basic mechanisms. Eur Respir J 32:1639–1651

72 34. Humbert M, Sitbon O, Simonneau G (2004) Treatment of pulmonary arterial hypertension. N Engl J Med 351:1425–1436 35. Downing SE, Lee JC (1980) Nervous control of the pulmonary circulation. Annu Rev Physiol 42:199–210 36. Westerhof N, Stergiopulos N, Noble MIM (2005) Snapshots of hemodynamics an aid for clinical research and graduate education. Springer, New York 37. Murgo JP, Westerhof N (1984) Input impedance of the pulmonary arterial system in normal man. Effects of respiration and comparison to systemic impedance. Circ Res 54:666–673 3 8. Kussmaul WG, Noordergraaf A, Laskey WK (1992) Right ventricular–pulmonary arterial interactions. Ann Biomed Eng 20:63–80 39. Huez S, Brimioulle S, Naeije R, Vachiery JL (2004) Feasibility of routine pulmonary arterial impedance measurements in pulmonary hypertension. Chest 125:2121–2128 40. Laskey W, Ferrari V, Palevsky H, Kussmaul W (1993) Pulmonary artery hemodynamics in primary pulmonary hypertension. J Am Coll Cardiol 21:406–412 41. Furuno Y, Nagamoto Y, Fujita M, Kaku T, Sakurai S, Kuroiwa A (1991) Reflection as a cause of mid-systolic deceleration of pulmonary flow wave in dogs with acute pulmonary hypertension: comparison of pulmonary artery constriction with pulmonary embolisation. Cardiovasc Res 25:118–124 42. Fitzpatrick JM, Grant BJB (1990) Effects of pulmonary vascular obstruction on right ventricular afterload. Am Rev Respir Dis 141:944–952 43. Lankhaar JW, Westerhof N, Faes TJ et al (2008) Pulmonary vascular resistance and compliance stay inversely related during treatment of pulmonary hypertension. Eur Heart J 29:1688–1695 44. Elzinga G, Westerhof N (1973) Pressure and flow generated by the left ventricle against different impedances. Circ Res 32: 178–186 45. Chemla D, Castelain V, Humbert M et al (2004) New formula for predicting mean pulmonary artery pressure using systolic pulmonary artery pressure. Chest 126:1313–1317 46. Syyed R, Reeves JT, Welsh D, Raeside D, Johnson MK, Peacock AJ (2008) The relationship between the components of pulmonary artery pressure remains constant under all conditions in both health and disease. Chest 133:633–639 47. Yock P, Popp R (1984) Noninvasive estimation of right ventricular systolic pressure by Doppler ultrasound in patients with tricuspid regurgitation. Circulation 70:657–662 48. Westerhof N, Sipkema P, van den Bos GC, Elzinga G (1972) Forward and backward waves in the arterial system. Cardiovasc Res 6:648–656

R. Naeije and N. Westerhof 49. van den Bos GC, Westerhof N, Randall OS (1982) Pulse wave reflection: can it explain the differences between systemic and pulmonary pressure and flow waves? A study in dogs. Circ Res 51:479–485 50. Nakayama Y, Nakanishi N, Hayashi T, Nagaya N, Sakamaki F, Satoh N, Ohya H, Kyotani S (2001) Pulmonary artery reflection for differentially diagnosing primary pulmonary hypertension and chronic pulmonary thromboembolism. J Am Coll Cardiol 38:214–218 51. Castelain V, Hervé P, Lecarpentier Y, Duroux P, Simonneau G, Chemla D (2001) Pulmonary artery pulse pressure and wave reflection in chronic pulmonary thromboembolism and primary pulmonary hypertension. J Am Coll Cardiol 37:1085–1092 52. Hardziyenka M, Reesink HJ, Bouma BJ et al (2007) A novel echocardiographic predictor of in-hospital mortality and mid-term haemodynamic improvement after pulmonary endarterectomy for chronic thrombo-embolic pulmonary hypertension. Eur Heart J 28:842–849 53. Overbeek MJ, Lankhaar JW, Westerhof N et  al (2008) Right ventricular contractility in systemic sclerosis-associated and idiopathic pulmonary arterial hypertension. Eur Respir J 31: 1160–1166 54. Sunagawa K, Maughan WL, Sagawa K (1985) Optimal arterial resistance for the maximal stroke work studied in the isolated canine left ventricle. Circ Res 56:586–595 55. Sagawa K, Maughan L, Suga H, Sunagawa K (1988) Cardiac contraction and the pressure–volume relationship. Oxford University Press, New York 56. Suga H, Sagawa K, Shoukas AA (1973) Load independence of the instantaneous pressure–volume ratio of the canine left ventricle and the effect of epinephrine and heart rate on the ratio. Circ Res 32:314–322 57. Sunagawa K, Yamada A, Senda Y, Kikuchi Y, Nakamura M, Shibahara T (1980) Estimation of the hydromotive source pressure from ejecting beats of the left ventricle. IEEE Trans Biomed Eng 57:299–305 58. Brimioulle S, Wauthy P, Ewalenko P et al (2003) Single-beat estimation of right ventricular end-systolic pressure–volume relationship. Am J Physiol Heart Circ Physiol 284:H1625–H1630 59. Kerbaul F, Rondelet B, Motte S et al (2004) Isoflurane and desflurane impair right ventricular-pulmonary arterial coupling in dogs. Anesthesiology 101:1357–1361 60. Kerbaul F, Brimioulle S, Rondelet B, Dewachter C, Hubloue I, Naeije R (2007) How prostacyclin improves cardiac output in right heart failure in conjunction with pulmonary hypertension. Am J Respir Crit Care Med 175:846–850 61. Kuehne T, Yilmaz S, Steendijk P et al (2004) Magnetic resonance imaging analysis of right ventricular pressure–volume loops: in vivo validation and clinical application in patients with pulmonary hypertension. Circulation 110:2010–2016

Chapter 5

Pulmonary Vascular Mechanics Alejandro Roldán-Alzate and Naomi C. Chesler

Abstract  Biomechanics is concerned with the behavior of living tissues when subjected to the action of forces, and the subsequent effects of these living tissues on their environment. In terms of the pulmonary vasculature, we can consider the vascular tissue and the blood as “physical bodies,” and concern ourselves with the biosolid and biofluid ­mechanics principles, respectively, that govern their behavior. In the cardiovascular system, it is generally the blood flowing through the vasculature that exerts forces (e.g., pressure and shear) on the vascular tissue. When the arterial tissue responds to these forces, it subsequently affects the rate and pattern of blood flowing through it, which is its “environment.” Indeed, the interactions between blood flow and the vascular walls are so interrelated that an entire branch of biomechanics is devoted to them – the area of biofluid–biosolid interactions. The mechanics of the pulmonary vasculature are particularly complex because blood flow can be affected not only by the vascular tissue (and vice versa) but also by the state of the airways, especially in the lung capillaries. We begin this chapter by considering the mechanical behavior of blood vessels independent of airways and introduce some basic concepts important to biomechanics, such as stress and strain. We also briefly review some key concepts in biofluid mechanics (e.g., resistance, Reynolds number, and Womersely number) and the mechanical behavior of blood vessel constituents. The subsequent sections are focused on the mechanics of the pulmonary vasculature specifically. First, a review of the mechanics of large pulmonary arteries is presented followed by comments on the intermediate and small vessels in the intra-alveolar region. Next, the effects of breathing on pulmonary vascular mechanics are addressed and the effects of pulmonary vascular tissue mechanics on pulmonary vascular blood flow dynamics are discussed. Finally, we consider the impact of some disease states on these aspects of pulmonary vascular mechanics and conclude with a summary.

N.C. Chesler (*) Department of Biomedical Engineering, University of Wisconsin, 1550 Engineering Dr., 2145 Engineering Centers Building, Madison, WI 53706-1609, USA e-mail: [email protected]

Keywords  Biomechanics • Morphometry • Pulmonary ­vascular tissue mechanics • Flow dynamics

1 Introduction Mechanics is the branch of physics concerned with the state of rest or motion, including deformation, of physical bodies that are subjected to the action of forces, and the subsequent effects of these bodies on their environment [1]. Biomechanics is concerned with the behavior of living tissues when subjected to the action of forces, and the subsequent effects of these living tissues on their environment. In terms of the pulmonary vasculature, we can consider the vascular tissue and the blood as “physical bodies,” and concern ourselves with the biosolid and biofluid mechanics principles, respectively, that govern their behavior. In the cardiovascular system, it is generally the blood ­flowing through the vasculature that exerts forces (e.g., ­pressure and shear) on the vascular tissue. When the arterial tissue responds to these forces, it subsequently affects the rate and pattern of blood flowing through it, which is its “environment.” Indeed, the interactions between blood flow and the vascular walls are so interrelated that an entire branch of biomechanics is devoted to them – the area of biofluid–biosolid interactions. The mechanics of the pulmonary vasculature are particularly complex because blood flow can be affected not only by the vascular tissue (and vice versa) but also by the state of the airways, especially in the lung capillaries. We begin this chapter by considering the mechanical behavior of blood vessels independent of airways and introduce some basic concepts important to biomechanics, such as stress and strain. We also briefly review some key concepts in biofluid mechanics (e.g., resistance, Reynolds number, and Womersely number) and the mechanical behavior of blood vessel constituents. The subsequent sections are focused on the mechanics of the pulmonary vasculature specifically. First, a review of the mechanics of large pulmonary arteries is presented (Sect. 2) followed by comments on the intermediate and small vessels in the intra-alveolar region (Sect.  3).

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_5, © Springer Science+Business Media, LLC 2011

73

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A. Roldán-Alzate and N.C. Chesler

Next, the effects of breathing on pulmonary vascular ­mechanics are addressed (Sect. 4) and the effects of pulmonary vascular tissue mechanics on pulmonary vascular blood flow dynamics are discussed (Sect. 5). Finally, we consider the impact of some disease states on these aspects of pulmonary vascular mechanics (Sect. 6) and conclude with a summary and suggestions for future research (Sect. 7).

1.1 Mechanical Behavior of Blood Vessels All materials deform when sufficiently large forces are applied to them. An elastic material is one that deforms when a force is applied and returns to its original configuration without energy dissipation when the applied force is released. Consider the specimen shown in Fig. 1a, which could be an artery loaded in the axial (longitudinal) direction. Let the initial uniform cross-sectional area of this cylindrical specimen be A (cm2) and the initial unloaded length l0 (cm). Then, ­consider a longitudinal or axial load imposed by a weight F (dyn) which elongates the specimen by an amount Dl (cm). This change in length divided by the initial length is defined as the axial or longitudinal strain, e (cm/cm):

l - l0 Dl = , e= l0 l0

(1)

where l (cm) is the loaded length. The ratio of the axial load to the cross-sectional area of the specimen is defined as the axial stress, s (dyn/cm2):

s = F / A.

(2)

The mechanical behavior of the specimen is fully described by the stress–strain relationship (Fig. 1b). For a linearly elastic, homogenous (behaves identically at each axial location), and isotropic (behaves identically for any loading direction) material, the slope of the stress–strain relationship is the elastic or Young’s modulus, E (dyn/cm2):

E =

s . e

(3)

This relationship is known as Hooke’s law. When a material does not follow Hooke’s law as shown in equation (3), which is the case for most biological materials, including arteries and veins, the stress–strain relationship can be curvilinear (Fig.  1b). In this case, a tangent elastic modulus can be defined as the slope of the curve at any given stress or strain. The tangent modulus, Etan (dyn/cm2), is then

ds Etan = . de

Fig.  1  The stress–strain behavior of materials. (a) Specimen elongation from initial length l0 to new length l due to an applied force F. (b) Linear (Hookean) and nonlinear stress–strain relationships. (c) Blood vessel with dimensions of inner and outer diameter (Di and De, respectively) and wall thickness (h) and loading conditions of internal pressure (Pi), external pressure (Pe), and consequently transmural pressure Pt = Pi – Pe

is typical of most arteries. This type of behavior is often termed strain stiffening. It should be noted that when the specimen is stretched in the axial direction, or loaded in tension, there is a corresponding reduction in the cross-sectional area [lateral strain el (cm/ cm) < 1]. The ratio of the lateral strain to the longitudinal strain is known as Poisson’s ratio, n (cm/cm), and is given by (5)

In vivo, blood vessels are loaded both longitudinally and circumferentially (Fig. 1c). The net outward pressure on the blood vessel wall, or transmural pressure, Pt (dyn/cm2), can stretch the tissue circumferentially, increasing its circumference, pD (cm), and compress the tissue radially, decreasing its thickness, h (cm). Again, the ratio of the lateral strain (for circumferential loading, a change in wall thickness) divided by the longitudinal strain (the change in mean circumference) is Poisson’s ratio. The equations for radial and circumferential stress in a cylindrical specimen with an internal distending pressure Pi and external pressure Pe are

(

)

(

)

æ Pi Ri2 - Pe Re2 ö æ Ri2 Re2 (Pi - Pe ) ö ÷ - ç sr = ç ÷ çè Re2 - Ri2 ÷ø çè R 2 Re2 - Ri2 ÷ø

(

)

(

)

(6)

and

(4)

For the non-Hookean material shown in Fig. 1b, the tangent elastic modulus increases with increasing strain, which

e v = 1 . e





(

)ö + æ (R R (P - P ))ö , ÷ ç ÷ ) ÷ø çè R (R - R ) ÷ø

æ Pi Ri2 - Pe Re2 sq = ç çè Re2 - Ri2

(

2 i

2

2 e

i

2 e

e

2 i

(7)

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where Ri = Di/2 and Re = De/2. Note that these equations are functions of the radius (R). If the vessel wall is thin enough that stress does not change appreciably from Ri to Re and offers little resistance to bending, then the stress in the radial direction can be neglected and the normal stress in the circumferential direction is the hoop stress: s q = Pt R / h.



(8)

Applying this simplified equation requires the “thin-wall assumption,” which we typically invoke when Ri /h > 10. The corresponding equations for strain in the circumferential direction are straightforward:

eq =

2pR - 2pR0 DR = 2pR0 R0

and

h-h Dh er = 0 = , h0 h0

(9)

where R0 and h0 are measured from an initial configuration assumed to be a zero-stress state. However, even when no distending pressure acts, arteries are not in a zero-stress state; residual stresses exist that alter the distribution of stress in the wall at loaded states. Residual stresses can be determined with an opening angle test in which an unloaded ring segment is cut radially. The artery ring springs open and the cross section can be approximated as a circular sector with an opening angle a. Assuming that a single radial cut releases all residual stresses, which is not always the case, the open configuration can be considered a zero-stress state and used as the reference configuration (R0, h0) in the strain equations above. Furthermore, these equations for strain assume infinitesimal or small deformations, which is not the case for arteries exposed to physiological pressures, especially those in the pulmonary circulation. Given the complexities of properly calculating stress and strain in arteries, and thus determining mechanical properties from the corresponding stress–strain relationships, it is often more convenient to calculate an approximate elastic modulus directly from pressure and diameter. For example, Bergel [3] proposed the following elastic modulus based on small changes in pressure and diameter:

Einc = 1.5 D i2 De

( DP / DDe )

(D

2 e

- Di2

)

.

(10)

Here, one must assume a thick-walled tube made of a linear elastic, isotropic, and incompressible (constantdensity) material. Hudetz [4] modified this expression assuming an orthotropic (properties depend on the direction in which they are measured) cylindrical vessel having

a nonlinear stress–strain relationship, which is more realistic:

Hqq =

2P D2 DPt 2 Di2 De + 2 t e2. 2 2 DDe De - Di De - Di

(11)

For either formulation, if the specimen is being tested outside the body (ex vivo) in a testing rig, the specimen must be long enough that the effects of the sutures or clamps that attach the vessel to the testing rig (end effects) can be ignored. Also, the accuracy with which any of these equations approximates the true elastic modulus decreases with increasing strain. Finally, to compare Einc (or Hqq) of one vessel with another (say, from a hypertensive animal), the values must be computed at the same strain; comparing values computed at the same pressure may give spurious results. It is important to note that any calculation of true elastic modulus characterizes the mechanical behavior in a way that is independent of specimen size. That is, whereas it obviously requires a larger pressure to distend a thicker artery, the stresses required to create the same strains in two different size arteries with the same material properties are identical. Thus, unlike stiffness, which is an extrinsic property dependent on the size of the specimen, elastic modulus is an intrinsic property that is not dependent on size. Nevertheless, it is often convenient to discuss the actual forces required to increase arterial diameters in vivo and there is a long list of extrinsic mechanical parameters that have been used to describe blood vessel behavior both in vivo and ex vivo. We briefly review them here. The pressure–strain modulus is the change in transmural pressure (DPt but hereafter we drop the subscript t since we will only consider the transmural pressure) times the initial radius divided by the change in radius:

Ep =

DP · R . DR

(12)

An increase in Ep (dyn/cm2) with disease, for example, could be caused by an increase in elastic modulus, wall thickening, or an increase in initial radius. Like the common usage of stiffness, a vessel with higher Ep requires a higher transmural pressure to generate the same change in vessel diameter. Comparisons of Ep from one vessel to another should be computed at the same pressure. Another parameter used to describe blood vessel mechanical properties based on pressure–diameter measurements in the physiological range is the stiffness coefficient, b (dimensionless):

æ Pöæ D ö - 1÷ , b = ln ç ÷ ç è Ps ø è Ds ø

(13)

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where Ps is a standard (typically physiological) pressure and Ds is the internal diameter of the vessel at Ps. The volume distensibility coefficient, Dv (cm2/dynes), is the ratio of the change in volume, DV (cm3), induced by a change in pressure divided by the initial volume, V = pR2l (cm3):



DV , Dv = V · DP



(14)

Compliance, C (cm5/dyn), is defined as the change in volume for a given change in pressure, which is obviously closely related to the volume distensibility coefficient: C=

DV = Dv · V . DP

(15)

In the systemic circulation, the elastic modulus of the aorta, which is relatively straight (in some regions), of relatively constant diameter, and has only small branches coming off it, has been indirectly measured using the pulse wave velocity, c0 (cm/s):

co =

E ·h , 2R · r

(16)

where r (g/cm3) is the density of blood. Note, this equation is only strictly true for a linearly elastic, homogenous, and isotropic material of straight, cylindrical cross section through which a long-wavelength wave travels. In other words, this equation can be used to estimate but not measure aortic elastic modulus. Its application to the pulmonary circulation is more problematic.

1.2 Fluid Dynamics Whereas other chapters in this book describe the fluid dynamics in the pulmonary vasculature in great detail, here we introduce a few basic concepts that will be germane to our later discussion of the effects of pulmonary vascular mechanics on the dynamics of blood flow in the pulmonary vasculature. Consider the idealized blood vessel described earlier that is a long, straight cylinder of radius R. We now additionally assume that blood flow is steady, has a parabolic velocity profile, and has constant viscosity, m (g/cm s). The pressure gradient in the direction of flow (say, x) required to drive flow at a rate Q (cm3/s) is given by the Hagen–Poiseuille relationship:

dP 8 mQ = . dx pR 4

(17)

If we integrate this pressure gradient over a length of artery L, then we can find the pressure drop in the direction of flow:

8 mQ DP = L. pR 4

(18)

And from this we can calculate the resistance to flow in a tube of these dimensions: DP 8 mL = . Q pR 4

(19)

The general concept of resistance is applicable not only to straight cylindrical tubes with all the assumptions listed above, but also to vascular networks. Clinically, it is useful to measure the pulmonary vascular resistance (PVR; dyn s/ cm5 or, more commonly, mmHg min/L), which is given by the relationship

= DP , PVR Q

(20)

where DP is the difference between pulmonary arterial and left atrial pressure and Q is the cardiac output. From equation (19) it can be seen that in a single vessel, the resistance to blood flow is inversely proportional to the fourth power of the vessel radius. In a vascular network of arteries, capillaries, and veins, the resistance also depends on the pattern of the network. Consider the arterial cast from a section of human lung shown in Fig. 2a. Here, we see arteries in series with other arteries as well as in parallel. Three main schemes have been proposed to describe this complex branching structure: the Weibel model, the Strahler model, and the diameter-defined Strahler model (Fig. 2b–d). The Weibel model (Fig. 2b) assumes a symmetrical branching structure such that all arteries of the same size are in parallel (and make up a generation of the lung) and arteries of different sizes are in series (largest to smallest) [5]. In this case, computing the overall resistance of the system is straightforward. According to Ohm’s law for n parallel resistances (where n = 2 in Fig.  2b), the equivalent single resistance is Req =

( R1 R2 ... Rn ) . For n resistances in series, the equiv( R1 + R2 + ... Rn )

alent resistance is Req = R1 + R2 + ...Rn . So, for the symmetric model in Fig. 2b, the resistance downstream of each level 3 artery is ½ R4, the resistance downstream of each level 2 artery is R3 + ½ R4 , and the overall (or input) resistance is R1 + ½ R2 + ¼ R3 + 1/8 R4 . The Strahler system does not require a symmetric branching structure, which is more physiological (Fig. 2c). In this scheme, one begins numbering from the smallest arteries and when two vessels of the same order meet, the order number of the parent vessel increases by 1. If a small artery joins a large artery, the order number of the large artery does not change. This model has been applied to human and cat lungs [6–8], but did not yield good estimates of resistance. The

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Fig. 2  (a) Cast model of the arterial tree in a human lung showing the arterial network structure. (b–d) Three proposed models of the pulmonary arterial network: the Weibel model (b), the Strahler model (c), and the diameter-defined Strahler system (d), in which the generation numbers are indicated for each vessel. (e) Mean diameter and length

(upper panels) in each generation (order) of the pulmonary arterial tree, and distribution of total cross-sectional area (lower-left panel) of all segments of each order in human lungs. Note the number of pulmonary arterial branches (lower-right panel) increases exponentially from order 1 to order 15 [5, 6, 10]

diameter-defined Strahler model (Fig.  2d) improved upon this scheme by adding a new rule: that when a vessel of order n with diameter Dn meets another vessel of order n, the parent vessel has order n + 1 if and only if its diameter is larger than

Dn + (Sn + Sn+1)/2, where Sn and Sn+1 are the standard ­deviations of the diameters of order n and n+1 [9]. All three schemes have been applied to describe the pulmonary vascular networks of humans, cats, dogs, and rats [10–13].

78

A. Roldán-Alzate and N.C. Chesler Table 1  Percent pressure drop across a given section of the pulmonary vasculature Pulmonary Pulmonary Pulmonary arteries capillaries veins Pattern 1 [14] 35.90 15.40 48.70 Pattern 2 [14] 29.30 21.90 48.80 Gaar et al. [20] 56.00 NA 44.00 Brody et al. [17] 46.00 34.00 20.00 Hakim et al. [15] 40.20 15.60 44.20 Fike and Kaplowitz [18] 61.64 NA 38.36 Pellet et al. [16] 42.65 10.29 47.06 When measurements are not available from precapillary arterioles to postcapillary venules, the pressure drop across the capillaries is not available (NA).

Fig. 3  Pressure as a function of distance from the pulmonary arterial trunk in symmetrically (1) and nonsymmetrically (2) branching models of a cat lung, where the pressures at the pulmonary arterial trunk (Pa), left atrium (Pv), alveoli (PA), and pleural space (PPL) are 20, 2, 0, and −7 cmH2O, respectively. Each tick mark on the horizontal axis represents the location of a vessel exit at a given order (arteries from order 1 to order 11, veins from order −1 to order −11) [14]

The diameter-defined Strahler system best models the human lung vasculature and calculations based on this model best mimic in  vivo hemodynamics. On the basis of this scheme, investigators have identified 15 orders of pulmonary arteries between the main pulmonary artery and the capillaries, and 15 orders of pulmonary veins between the capillaries and the left atrium in healthy human lungs [8]. As shown in Fig.  2e, the diameter and length of individual pulmonary arterial branches decline exponentially from order 1 to order 15, whereas the number of pulmonary arterial branches increases exponentially. The increase in the cross-sectional area of pulmonary arteries, however, is not exponential (Fig. 2e). On the basis of these data, approximately 25% of the total cross-sectional area in the pulmonary vasculature stems from large vessels (diameter more than 0.6 mm), 44% from medium-sized vessels (0.2–0.6 mm), and 30% from small vessels (diameter less than 0.2 mm). Hemodynamic models based on these anatomic data, which assume that PVR is directly proportional to the length (L) of each vessel and inversely proportional to the fourth power of the radius (r) as in equation (19), suggest that pressure drops in pulmonary arteries, capillaries, and veins are equivalent in healthy lungs (Fig. 3, patterns 1 and 2 in Table 1) [14]. Some direct measurements of the pulmonary circulation confirm this modeling result [15, 16], whereas others demonstrate a larger pressure drop in the arteries relative to the veins [17,  18]. Direct measurements in the systemic vasculature show more consistently that the pressure drop predominantly occurs in arteries (Table 2) [19]. In diseased lungs, the pressure distributions, and consequently the major sites of resistance, are not known, in part because insufficient data exist on the branching

Table 2  Percent pressure drop across a given section of the systemic vasculature Arteries Capillaries Veins Mesentery [19] Skeletal muscle [19]

72.70 64.90

8.20 16.10

19.00 18.80

patterns, morphometry, ­morphology, and elasticity of ­pulmonary blood vessels in diseased states. Studies are required to obtain these important data. To close this section, we introduce two dimensionless numbers that are useful to understanding the characteristics of blood flow in any artery, vein, or vascular network: the Reynolds number ( Â ) and the Womersley number (Wo):



rVD Â= , m wr Wo = R , m

(21)

(22)

where w is the heart rate converted to radians (2p × heart rate/60). Â reflects the relative effects of inertial forces and viscous forces; low- Â flows are viscous-dominated (i.e., laminar), whereas high- Â flows are inertia-dominated (i.e., can become turbulent). Analogously, Wo reflects the ratio of unsteady inertial forces to viscous forces; low-Wo flows are viscous-dominated and quasi-steady, whereas ­high-Wo flows are unsteady. As an example of the relevance of these parameters, equations (17), (18), and (19) do not apply for high- Â , high-Wo flows.

1.3 Mechanical Behavior of Blood Vessel Constituents The arterial wall has three layers and is mainly composed of collagen, elastin, vascular smooth muscle cells (vSMCs), and other ground substances. The arrangement of these components

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Table 3  Elastic properties of structural components of arterial wall Physical characteristics Modulus similar to (MPa) Material Type Collagen Protein Nylon ~10 Elastin Protein Rubber ~3.0 Muscle Cellular Active ~0.1–2.0 Endothelium Cellular Wet Kleenex ~0 Matrix Mucopolysaccharide Marmalade Viscous Reprinted with permission from Hayashi et  al. [22] with permission from Taylor & Francis Group.

varies according to the layer in the arterial wall and the location of the artery within the vascular network. Beginning at the right ventricle and ending at the left atrium, one can classify the pulmonary vessels sequentially according to their position in the lung as presegmental arteries, segmental arteries, intra-acinar arteries, microcirculation (consisting of precapillaries, capillaries, and postcapillaries), and veins. In arteries, the ratio of elastin to collagen falls with increasing distance from the heart, whereas the number of vSMCs per unit volume increases [21]. Elastin fibers are highly extensible. Even at very large deformation, elastin behaves almost as a Hookean material with a constant Young’s modulus. Single collagen fibers are also nearly Hookean but are stiffer than single elastin fibers. In the single-fiber configuration, the elastic moduli of collagen and elastin have been estimated to be Ecollagen » 100 MPa » 1,000 Eelastin » 0.1 MPa [21], whereas in more physiological configurations, Ecollagen » 10 MPa » 3Eelastin » 3 MPa [22] (Table 3). Elastin and collagen are the major supporting proteins for vSMCs, maintain internal tension within the artery, and prevent overextension under physiological loading conditions that might damage vSMCs. Although in isolation collagen and elastin behave like linear elastic materials, together elastin and collagen fibers behave nonlinearly under physiological conditions (Fig. 4). Whole, intact arteries are not only nonlinearly elastic but also have viscous (fluid-like) properties. That is, vascular tissue is viscoelastic. In contrast to purely elastic materials, the stress–strain behavior of viscoelastic materials is time-dependent. For example, consider imposing a step change in stress on an elastic material. The material will deform (strain) in response to that stress and the deformation will be linearly proportional to the stress (by the Young’s modulus for a linearly elastic material). In contrast, imposing a step change in stress on a viscoelastic material leads to a rapid deformation followed by a slower deformation, typically to some asymptotic value. This delayed, slower deformation is commonly known as creep (Fig. 5a). Similarly, imposing a step change in deformation on a linearly elastic material causes a step change in stress inversely proportional to the Young’s modulus. In contrast, imposing a step change in deformation on a viscoelastic

Fig. 4  (a) Stress–strain behavior of individual elastin and collagen fibers as well as in combination. (b) An arterial wall with increasing collagen fiber recruitment with increasing load. (Adapted with permission from Chandran et al. [21] with permission from Taylor & Francis Group)

material causes an initial spike in stress followed by a slow decrease, known as stress relaxation (Fig.  5a). Plotting strain versus stress in a viscoelastic material under physiological conditions in which the stress and strain vary cyclically yields a hysteresis loop (Fig. 5b). The area of the loop is related to the energy lost due to the viscosity of the material. Typically, the behavior of viscoelastic materials depends on the frequency of loading and ­unloading. It is also important to note that just as materials can be linearly or nonlinearly elastic, they can also be linearly or nonlinearly viscoelastic. Arteries are usually nonlinearly viscoelastic with dynamic stress–strain behavior as shown in Fig.  5c. For more detailed information on viscoelastic materials and their behavior, we refer the interested reader to other sources [23, 24]. An important implication of vascular viscoelasticity is that it causes energy dissipation in the arterial wall. The consequences of hemodynamic energy loss for ventricular function are largely unknown.

2 Mechanics of Large Pulmonary Arteries The largest of the presegmental arteries, the pulmonary artery trunk, extends about 4 cm beyond the pulmonic valve before dividing into right and left main (or extralobar, also presegmental) pulmonary arteries that feed the lungs. These large arteries are the most accessible pulmonary vessels and their mechanical characteristics have been relatively well studied. As large

80

A. Roldán-Alzate and N.C. Chesler

Fig. 5  Stress–strain behavior of viscoelastic materials. (a) Step increases in stress and strain lead to creep and stress relaxation, respectively. Cyclical changes in stress or strain result in (b) an elliptical stress–strain loop in a linearly viscoelastic material and (c) a nonelliptical stress–strain loop in a nonlinearly viscoelastic material, like an artery. Viscous energy losses are related to the area of the loops. Dashed lines in (a) and (b) represent purely elastic behavior. (c Adapted from Kobs et al. [37] with permission from the American Physiology Society)

arteries, they are typically considered passive conduits whose main function is to transport blood into the pulmonary circulation. However, it has recently been recognized that these large arteries play a key role in the modulation of pulmonary hemodynamics and right ventricular afterload [25]. Both aging and pulmonary hypertension have been shown to affect large pulmonary artery mechanics. For example, Gozna et  al. [26]. performed an angiographic study in 22 nonhypertensive subjects with ages ranging between 1 and 50 years and found a positive correlation between large ­pulmonary artery stiffness (measured via Ep; see equation 12) and age. Indeed, they found that Ep (dyn/cm2) increased linearly with age (years) in (23): Ep = 105 + 2.6 ´ age.

(23)

Other studies subsequently confirmed this relationship [27– 29]. Using a more traditional mechanics approach, Banks et  al. [30] found that circumferential strain decreased with age in studies on circumferential strips of human large pulmonary arteries obtained after death. If the strain decreases for the same imposed stress, the elastic modulus must increase; therefore, the elastic modulus and stiffness of human large pulmonary arteries increase with age. Early, influential work by Hislop and Reid [31] demonstrated that large pulmonary arteries thicken with chronic hypoxia-induced pulmonary hypertension. This process is now thought to be a homeostatic response such that wall stresses are reduced to normal values in the presence of increased pressure (see equation 8). More recently, the large extrapulmonary arteries have been shown to not only stiffen but also increase in elastic modulus and alter their zero-stress state with hypertension [32–40]. As noted already, the opening angle test is typically used to measure changes in the zero-stress state [32, 35, 40]. In human lungs, the opening angle was found to vary in third-order to ninth-order pulmonary arteries (from 92 to 163°) and veins (from 89 to 128°) [41]. Animal experiments demonstrated that these opening

angles change with exposure to hypoxia-induced hypertension [32], suggesting that arterial wall remodeling requires redistribution of stresses within the arterial wall as well as reduction of stresses through wall thickening. Large pulmonary artery stiffness can be measured either with whole-lung inflation [31, 33] or with isolated-vessel tests [34, 37–39]. Elastic modulus can be measured via isolated-vessel tests or the bubble-inflation technique [36, 38]. The bubble test is a useful technique because, as long as the artery radius is large compared with the bubble radius, the mechanical properties of short, highly curved arteries can be measured in either the circumferential or the axial direction. Disadvantages are that the bubble inflation is nonphysiological and in vivo loading conditions cannot be mimicked. With the isolated-vessel technique [42], inflation is physiological and in vivo loading conditions can be mimicked, but it is not appropriate for very short, highly curved arteries such as the main pulmonary trunk. For a complete review of appropriate techniques for measuring arterial mechanical properties, we refer the interested reader to a report by Hayashi et al. [22]. Clearly, changes in the structural components of large pulmonary arteries – collagen, elastin, and vSMCs – are responsible for these changes in mechanics. In larger mammalian species, medial thickening can be accounted for, in large part, by the proliferation of a distinct population of vSMCs residing within the media [43]. Since arterial thickening serves to decrease wall stress, the existence of such a cell population within the media of large arteries may provide the vascular wall with a mechanism to adapt to increased wall tension. Increased vSMC tone with chronic hypoxia, also termed persistent pulmonary vasoconstriction, has been shown to significantly affect resistance ­arteries [44–47], but the existence of this phenomenon in large, conduit pulmonary arteries is unknown. Elastin also plays a significant role in the remodeling of large pulmonary arteries in response to pulmonary hypertension [33, 40, 48]. Although conventional wisdom has been that elastin loading is only relevant at low pressures, a recent study demonstrated that nearly half of the load applied to

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pulmonary arteries was carried by elastin at normotensive pressures, and that about half of the change in stiffness with hypoxia-induced pulmonary hypertension was due to an increase in stiffness of the elastin [40]. In addition, elastindeficient mice have significant pulmonary hypertension, which may be related to large-artery mechanics [48]. Collagen, the stiffer of the two main extracellular matrix proteins, is well known to contribute to conduit artery stiffness. Upregulation of collagen-related genes is known to occur in response to chronic hypoxia [49–51], leading to collagen protein accumulation in large arteries [37, 39, 51, 52]. We and others demonstrated a significant positive correlation between increased collagen content and increased pulmonary artery stiffness [37, 52]. In addition, phenomenological modeling studies have suggested that collagen cross-linking also plays a role in pulmonary conduit artery stiffening with pulmonary hypertension [53]. It is important to note that many studies on arterial stiffness, especially those using imaging techniques, assume circular arterial cross-sectional area. Experiments in isolated, perfused dog lungs demonstrated that the larger arteries tend to be elliptical in cross section when exposed to low transmural pressures. We can explore the impact of elliptical cross section on arterial distensibility with a simple example. Consider an ellipse with major axis a and minor axis b. The cross-sectional area A and perimeter P are

A = pab

(24)

P = p [2(a 2 + b2 )].

(25)

and

Representative values of eccentricity (b/a) for large arteries in isolated dog lungs have been found to be between 0.7 and 0.8. So, if we let a = 0.5  cm and b = 0.35  cm, we obtain a reasonably sized, reasonably elliptical arterial shape. A small increase in pressure might be enough to shift the artery into a circular cross section with r = 0.43 cm, which is the radius of a circle with the same perimeter as the ellipse (i.e., no circumferential distension occurring). This small change in shape could cause a 6.5% change in calculated distensibility even though we might more accurately refer to the shape change as distortion. This analysis suggests that vessel distortion may be responsible for a small portion of the distensibility found in large ­pulmonary arteries [54].

3 Intermediate-Sized Arteries and the Microcirculation Distal to the large, presegmental extralobar and intralobar arteries are the segmental arteries, which accompany the bronchi to supply approximately 20 bronchopulmonary

­segments (in the human). These segmental vessels are intermediate-sized, as are the first generation of intra-acinar arteries that accompany the terminal bronchioles. Distal to the intra-acinar arteries are the precapillary arterioles and, finally, the microcirculation, which act as the site for gas exchange in the lung. Anatomic studies of the segmental vasculature have shown that the first six generations of the segmental divisions are elastic arteries, the next three are transitional (between elastic and muscular), and the remaining divisions are purely muscular [55]. Functional studies have demonstrated that the elastic modulus of the pulmonary arteries increases with increasing distance from the heart [56], as does the collagen-to-elastin ratio [57]. The effects of aging and disease on the segmental intralobar pulmonary arteries have been well studied, despite the fact that these arteries significantly affect pulmonary ­hemodynamics. Cox, the author of several landmark papers in pulmonary vascular mechanics, found that the incremental modulus did not vary significantly between the presegmental extralobar and intralobar arteries [58]. Caro, another early leader in the field, demonstrated that distensibility decreases with distance from the right ventricle in the rabbit lung vasculature using a radiological technique [59]. However, in a comparative study of several species, Ferenza found that the walls of elastic vessels of rabbits were relatively thinner than those of humans, whereas the walls of the muscular vessels were relatively thicker; furthermore, there was no smooth transition from elastic to muscular vessels as is found in the human lung [60]. Thus, the distinction between large arteries (i.e., conduit arteries whose function is to transport the pressure energy and momentum of blood) and intermediate-sized arteries (whose function is to distribute blood) and small, resistance arteries (whose function is to control blood pressure with vSMC activity) is species-dependent and can be blurry. In large animals and humans, isolated-vessel tests are typically used to study the mechanics of the mid-sized pulmonary arteries. However, instead of elastic modulus or stiffness being reported, distensibility, defined as the fractional diameter change for a given pressure change (a), is typically reported. Al-Tinawi et  al. [61] summarized the existing literature and reported an average value of 2%/mmHg for dog, cat, and human pulmonary arteries over the full range of arterial diameters. In smaller animal models, such as rodents, isolated-vessel tests have been performed in side-branch pulmonary arteries [62]. However, typically in this size range, radiological techniques are used to measure pulmonary arterial distensibility. For example, high-resolution X-ray computed tomography [63] has been used to image the vascular tree within isolated, ventilated rat lungs perfused with a contrast agent to obtain vessel diameters over a range of pressures. From these images, local diameters and pressures were measured to quantify the local distensibility, which was within the range of values for pulmonary arteries of larger animals (2.8%/mmHg) [64].

82

In rats, cats, dogs, and humans, the distensibility appears to be independent of vessel size [64]. That is, unlike elastic modulus (an intrinsic mechanical property), distensibility (an extrinsic mechanical property) does not vary significantly with increasing distance from the heart. From equation (3), the elastic modulus (E) is inversely proportional to the deformation (DD/D0 or DR/R0) and directly proportional to stress (approximately PR/h). In contrast, as can be seen in equation (12), the stiffness of an artery is inversely proportional to the radial deformation and directly proportional to the pressure P (not stress). So, if R/h increases, elastic modulus will increase, whereas stiffness will not change. Since R/h typically increases with increasing distance from the heart (i.e., R and h both decrease but h decreases more as a function of distance) [21, 54], this explains how elastic modulus could increase but distensibility (the inverse of stiffness) would remain constant as a function of the location in the arterial tree. In the microcirculation of the lung, because of the low pulmonary arterial pressures, distribution of blood flow is markedly affected by gravity. That is, when a person is in the standing position, the intravascular pressure in the inferior region (base) of the lung is higher than in the superior (apical) region. In contrast, airway pressures are relatively constant. Thus, different zones of the lung can be defined on the basis of the relative differences in upstream and downstream intravascular pressures through a vessel segment and the external, airway pressure. Note, these lung zones are functional, not anatomical. They may disappear with exercise or shift with changes in posture. In zone 3, typically located at the bottom of the lung, hydrostatic effects lead to high arterial and venous pressures, both of which exceed alveolar pressure. Thus, blood flow through the capillaries is driven by the difference between arterial and venous pressure. In zone 2, typically in the middle of the lung, pulmonary arterial pressure exceeds the alveolar pressure, but pulmonary venous pressure does not. As a consequence, blood flow in this zone is determined by the difference between arterial and alveolar pressure instead of the difference between arterial and venous pressure. Finally, in zone 1, typically located at the top of the lung, pulmonary arterial pressure is lower than alveolar pressure. The resulting negative transmural pressure squeezes the capillaries such that no flow is possible. Experimentally, a Starling resistor, which is a collapsible tube subject to an external pressure, has been used to explore the mechanics of capillaries in the different lung zones [21, 65]. Unlike many other capillary beds, the topology of the pulmonary capillary blood vessels is not treelike; it is sheet-like. To characterize the mechanics of the pulmonary capillary network, Fung [66] modeled the vascular space as a sheet of fluid flowing between two membranes held apart by a number of more or less equally spaced posts. A planar view of the sheet shows these posts as regularly distributed circular

A. Roldán-Alzate and N.C. Chesler

obstructions in the center of regular hexagonal spaces (Fig.  6). The sheet flow model is characterized by three parameters: L, the length of each side of the hexagon; h, the average height of the vascular space; and D, the diameter of the post. Whereas the posts are semirigid structures, the sheets are elastic and can either bulge out or in depending on the vascular to alveolar pressure difference. On the basis of this model, the vascular-space tissue ratio (VSTR) is defined as the ratio of the vascular lumen volume to the circumscribing volume delimited by the top and bottom sheet surfaces. In particular, assuming the hexagonal volume minus the post volume is entirely filled with blood, and that the top and bottom sheet surfaces are relatively flat surfaces separated by an average distance h, the VSTR is gven as follows:

region = Volume of hexagonal

Volume of a post = p





VSTR =

3 3 2 L h, 2

D2 h, 4

/2 L2 h 3 - 1/4 pD 2 h p D2 = 1. 2 2 3 /2 L h 3 6 3 L

(26)

(27)

3

(28)

Thus, VSTR is independent of the height h and is equal to the percentage of the area occupied by blood in the plane cross section. In practice, it is difficult to measure L, D, and h from microscopic samples because of randomness and irregularities in the geometry. However, VSTR can be determined by other means [66]. Experiments in cat lungs have shown that VSTR and sheet area are independent of blood pressure, which suggests that higher pressures increase the mean sheet thickness h by an amount that depends on the elasticity of the posts [67, 68]. Indeed, for certain pressure ranges, h has been measured to be a linear function of pressure:

Fig. 6  Pulmonary capillary sheet model. (a) Plane view. (b) Cross section through X-X of (a). (c) Representation of sheet elasticity based on changes in mean sheet thickness as a function of transmural (or transsheet) pressure. (Adapted with permission from Fung [66])

5  Pulmonary Vascular Mechanics



83

h = h0 (1 + aDP ),

(29)

where h0 and a are constants independent of DP (Fig. 6c). The parameter h0 is the sheet thickness at zero transmural (or transsheet or transmembrane) pressure difference (Pcapillary − Palveoli). The parameter a is the distensibility coefficient, as discussed above. The thickness h is an average sheet thickness; it is the mean value averaged over an area that is large compared with the post, but small relative to the alveoli. When the transmural pressure is sufficiently negative, h approaches zero such that blood cannot flow. When the transmural pressure is high, h approaches a constant value, as shown in Fig. 6c. Thus, this conceptual representation of the intralveolar capillaries not only matches experimental data but also fits with the functional definition of lung zones discussed above.

4 Interactions of the Pulmonary Vasculature with the Airways



The effects of lung volume on the pulmonary vasculature and pulmonary mechanics can be dramatic. From residual capacity to total capacity, the lung as a whole can increase in size by a factor of 3. The length of the extra-alveolar blood vessels increases in proportion to the cube root of the lung volume during inspiration [65] (Fig. 7a). In contrast, alveolar vessels narrow with inspiration (Fig. 7b). In general terms, the pulmonary vascular bed behaves as if it were composed of two compartments that respond oppositely to lung inflation; one that enlarges while the other is compressed, such that the overall volume change in the vascular bed is dependent on the relative magnitude of the two effects. Here it is useful to define the internal and external pressures acting on the extra-alveolar and alveolar vessels. We begin with extra-alveolar vessels (Fig. 7c). The net transmural pressure across the arterial wall in the extra-alveolar region is the vascular pressure minus the perivascular pressure: Pt = Pvasc – Pperivasc. The perivascular pressure depends on the lung volume or phase of breathing. In particular, the perivascular pressure for extra-alveolar vessels is equal to the pressure in the lung parenchyma and can be expressed as the difference between the outward-acting pressure in the alveoli (Palv) and the inward-acting effect of alveolar wall recoil (Precoil(A)) such that Pperivasc = Palv – Precoil(A). Under equilibrium conditions, the net outward vascular transmural pressure must be balanced by an equal net inward pressure, which is generated by the recoil pressure of the elastic elements in the vessel wall ( Precoil(W) ), which include elastin and vSMC tone. This recoil pressure in the vascular walls can be expressed as

(

)

Precoil(W) = Pvasc - Palv - Precoil(A) .

Under the influence of Precoil(A) , alveolar septa are also under tension, which tends to expand the extra-alveolar vessels (much like an increase in vascular pressure) as can be seen in Fig. 7 and equation (30). Note, equation (30) assumes equilibrium conditions. A change in diameter will occur if there is an imbalance between the recoil forces in the vascular wall and the difference between the intravascular pressure and the perivascular pressure. If we assume that the difference between the alveolar pressure and the airway wall recoil pressure is always constant (i.e., the alveoli are in equilibrium independent of external pressure), then it is clear from the boundary conditions at the edge of the lung parenchyma that this constant difference must be the pleural pressure such that Ppleural = Palv - Precoil(A) . Then, the pleural pressure is equivalent to the extra-alveolar perivascular pressure as shown in (31):

(30)

(

)

Precoil(W) = Pvasc - Palv Precoil(A) = Pvasc - Ppleural .

(31)

Fig. 7  Effects of changes in lung volume on (a) extra-alveolar and (b) alveolar vessels. The outer surface of the extra-alveolar arteries is exposed to radial traction from the expanding alveoli that surround them (represented by springs); thus, they widen as the lung expands. In contrast, the capillaries within the alveolar septum narrow as the lung expands and the septum lengthens. (c) Extra-alveolar vessels surrounded by lung parenchyma, consisting of alveoli, are subject to an intravascular pressure, a recoil pressure internal to the arterial wall, and an external, perivascular pressure, which in turn is dependent on the net effects of alveolar pressure and recoil forces in the alveolar walls and pleural pressure. (Adapted with permission [65])

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The main pulmonary and extralobar arteries and equivalent veins have intrathoracic pressure as their perivascular pressure, although this pressure may be modified by changes in posture or shifts in the mediastinal structures such as the heart. In the alveolar compartment, the perivascular pressure is simply the alveolar pressure and, as shown in Fig. 7b, the effect of lung inflation is to reduce the area available for flow. During inspiration, the fall in pleural pressure results in an increase in venous return to the right atrium. As a result, capillary blood flow increases. However, because capillary resistance increases with lung volume, capillary blood flow tends to fall as inhalation proceeds [69]. When considering the forces acting on the lung capillaries, a sheet model is typically invoked, after Fung [66], as discussed in Sect. 3. Here, the airways are relatively flat layers of tissue above and below a sheet-like space for blood flow, which is interrupted and connected at regular intervals by posts of connective tissue. At resting lung volumes, these posts act like coiled springs; at high lung volumes, they become stretched and exert a restoring or recoil pressure. In 1961 Permutt et al. developed a technique for quantifying the effects of lung inflation on the volume and compliance of the extra-alveolar and alveolar compartments of the pulmonary vascular bed. In the extra-alveolar compartment, vascular compliance was not significantly affected by inflation of the lungs [70]. However, in the alveolar compartment, inflation of the lung caused a marked decrease in vascular compliance at low pressures; when vascular pressures were raised above 14.7 mmHg, compliance remained constant. Since intravascular pressures throughout the lung can change with posture and exercise, and extravascular pressures can change with region (extra-alveolar vs. alveolar) and lung volume and phase of breathing, the measured mechanical properties of the pulmonary vasculature depend on all of these factors. Indeed, discrepancies in measured pulmonary vascular mechanics, particularly in animal models, may in part be explained by differences in airway pressure, lung volume, and cardiac output (which affects intravascular pressures) [2, 71]. Furthermore, since pulmonary vascular mechanical properties affect pulmonary vascular blood flow, metrics of blood flow such as PVR also depend on posture, airway pressure, lung volume, and blood flow rate.

A. Roldán-Alzate and N.C. Chesler

(Q), both of which are functions of frequency (Fig.  8). Typically, Fourier series analysis is used to calculate impedance from pressure and flow waves measured at a single location in the circulation in response to the native heart beat. These pressure and flow waves are decomposed into mean values and a series of harmonic (sine) waves at multiples of the heart rate frequency [72]. For example, if the heart rate is 60 bpm (i.e., 1 Hz), the first to tenth harmonics occur at frequencies from 1 to 10 Hz. Alternatively, in an isolated lung setup, one can impose pulsatile flow at a range of frequencies with a mechanical pump as pioneered by Caro and McDonald [73]. In either case, the impedance magnitude Z is the modulus of the pressure divided by the modulus of the flow at a given frequency. The impedance phase Q is the phase delay between pressure and flow waves at a given frequency [56]. Unlike PVR, impedance cannot be expressed as a single number; a graphical display is required to show the impedance magnitude and phase spectra. In healthy humans, impedance falls steeply from a relatively high value at 0 Hz to a first minimum at 2–4 Hz, followed by a second maximum at 6–8 Hz, with smaller fluctuations at higher frequencies [72]. The phase typically begins near zero, goes negative, crosses zero, and then fluctuates around zero [56] (Fig. 8). When the general term “impedance” is applied to a vascular system, it is usually referring to input impedance. However, there are four different impedance relationships which may be physiologically or clinically important:

5 Vascular Impedance As noted already, the simplest biofluid mechanical concept relevant to the pulmonary vasculature is PVR. However, resistance is a measure of opposition to steady or mean blood flow in a vascular network. Since blood flow is pulsatile in nature, a more meaningful measure of opposition to blood flow is the impedance [72]. Impedance has a magnitude (Z) and a phase

Fig.  8  Representative pulmonary input impedance spectrum in the adult human. (Adapted with permission [104])

5  Pulmonary Vascular Mechanics

l­ongitudinal, input, characteristic, and terminal impedance. Longitudinal impedance is a function of the pressure drop across a vascular network and the flow through it. This type of impedance depends only on the local properties of the vasculature between the points where the pressure measurements are taken and is somewhat analogous to PVR. On the other hand, input impedance is a function of the pressure and flow measured at the specific point in the vasculature, which may be regarded as the input to the entire vascular tree beyond that point. This type of impedance is dependent not only on the properties local to where the pressure is measured but also on the properties of the segments distal to it. If the input impedance can be measured in the absence of wave reflections, which is possible only theoretically or in certain special circumstances, then it is called “characteristic impedance.” The input impedance measured at the end of the vascular system is called “terminal impedance.” Because impedance is sensitive to blood flow pulsatility, the normal heart rate frequency is an important parameter. In particular, the normal heart rate determines the way in which we interpret changes in impedance. In general, the low-frequency behavior of the impedance is determined by the resistance vessels, the medium-frequency behavior is determined by intermediate vessels, and the high-frequency behavior is determined by the vessels most proximal to the point of measurement. For example, in the aorta, the input impedance is determined by the peripheral vasculature for frequencies below 0.2  times the heat rate, the intermediate arterial network (i.e., the total arterial compliance) for frequencies between 0.2 and 0.3 times the heart rate, and the aorta for frequencies above 3 times the heart rate [74]. If the aorta is solely responsible for the input impedance, then we can assume that wave reflections from downstream locations have essentially cancelled out. Thus, at high frequencies, the input impedance is a reasonable approximation of the characteristic impedance. In practice, the average of the impedance magnitude at frequencies above the first minimum (fmin) is used to approximate the characteristic impedance. Determining the characteristic impedance is useful not only because it yields biofluid mechanical information, but also because it yields biosolid mechanical information about the large, conduit arteries. In particular, the characteristic impedance is directly proportional to stiffness and inversely proportional to cross-sectional area squared [72]. Similarly, the input impedance magnitude at the first harmonic of the frequency, Z1, which occurs at the normal heart rate, is thought to reflect proximal artery stiffness. Another impedance metric, the index of wave reflection, reflects the mismatch in impedance between more proximal and more distal vessels and also the degree of wave reflections [72]. And, increases in fmin have been suggested to indicate the presence of more proximal wave reflection sites or an increase in wave speed throughout the pulmonary vasculature [75].

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However, in the more recent literature, these ­relationships between impedance metrics and arterial function have been shown to be dependent on disease state and species. For example, in one study on the effects of acute emboli-induced pulmonary hypertension in dogs, a measured decrease in the characteristic impedance was interpreted as being caused by dilation of the proximal pulmonary arteries [76]. However, since arteries become increasingly stiff with increasing strain, dilation should also increase stiffness, which should increase the characteristic impedance. Hypoxic pulmonary vasoconstriction, which also increases pressure and therefore dilates the proximal pulmonary arterial tree, did not decrease the characteristic impedance in this species [77]. Thus, perhaps hypoxia modulated stiffness independent of strain via vSMC activity. In pigs, acute hypoxic pulmonary vasoconstriction actually increased the characteristic impedance for reasons not well understood [77]. Our own studies in mice demonstrated that the characteristic impedance did not change significantly with either pulmonary embolization or chronic hypoxia-induced pulmonary hypertension, but did show a slight decreasing trend in both cases, suggesting increased proximal artery dilation that overwhelms the increase in stiffness [78, 79]. Given the difficulties ­identifying the arterial mechanical changes responsible for altered impedance metrics, the dependencies of measured impedance metrics on arterial function should be determined in the species of interest and ascertained with arterial mechanics measurements, as described in Sect. 2, whenever possible. Alternatively, computer models can be used to gain insight into the changes in pulmonary vascular mechanics responsible for changes in pulmonary vascular impedance. The simplest models are lumped-parameter models with a few elements, such as the three- for four-element Windkessel model [74, 80]. These have the advantage of quantifying PVR and compliance but they cannot capture wave reflections. More accurate anatomical models have been made using a combination of medical images and morphometric data [12], fractal models [81–84], and patient-specific medical images [85]. Once the anatomy has been specified, the computational approach must balance computational speed and accuracy. Fully three-dimensional computational models of pulsatile blood flow in the lung are possible, but the computational costs can be daunting. In recent work, good agreement was found between measured and predicted pulmonary pressure–flow relationships with a combination approach consisting of three-dimensional computational modeling coupled with lumped-parameter (impedance) boundary conditions representing the peripheral arteries, capillaries, and veins [85]. Models also have the advantage that they can be used to test surgical repair strategies, predict changes with growth and development, and investigate spatially specific structure–function relationships.

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Despite the additional information provided by impedance, PVR is still the gold standard for diagnosis and prognosis of patients with pulmonary vascular disease. However, advances in noninvasive imaging techniques have been exploited in several recent studies promoting the use of impedance clinically. For example, Huez et  al. [86] used a combination of rightsided heart catheterization and Doppler echocardiography to compute the pulmonary vascular impedance spectrum and demonstrated that impedance was sensitive to pharmacological interventions (inhaled nitric oxide, epoprostenol, and dobutamine). Hunter et al. [87] used similar techniques in children and found that the sum of the first and second harmonics of the input impedance better predicted patient outcomes than PVR alone. Components of the impedance may also be useful for predicting the efficacy of treatment for pulmonary vascular disease [88].

6 Effect of Disease on Pulmonary Vascular Mechanics Changes in pulmonary solid and biofluid mechanics are known to change in the presence of disease. The most well studied disease, in terms of its effects on large and small artery mechanics and blood flow patterns, is pulmonary hypertension. In the preceding sections, we reviewed most of the available literature on the effects of pulmonary hypertension on large and mid-size arteries, the capillary sheet, and pulmonary vascular impedance. Here, we discuss some pulmonary vascular diseases with less well studied pulmonary vascular mechanical consequences, with the hope that it will inspire future research in this area. Marfan syndrome is a connective tissue disorder with manifestations in multiple organs. Dilatation of the main pulmonary artery is one of the standard criteria for the diagnosis of Marfan syndrome. Magnetic resonance imaging has demonstrated that pulmonary artery dilatation is not as severe as aortic root dilatation, which typically requires surgery, but still occurs [89, 90]. The consequences of Marfan syndrome for changes in large-artery pulmonary mechanics (other than an increase in size), pulmonary hemodynamics, or right ventricular function are not well known. An improved understanding of the impact of Marfan syndrome on pulmonary vascular solid and fluid biomechanical changes could improve diagnoses (via arterial stiffening), prognoses (via pulmonary blood flow dynamics), and evaluation of therapy efficacy. Scleroderma (SSc) is another connective tissue disease that has pulmonary vascular consequences. In particular, a common complication of SSc is pulmonary arterial hypertension (PAH). However, the origin of PAH in SSc remains obscure. In patients with CREST syndrome, the evidence

A. Roldán-Alzate and N.C. Chesler

suggests that fibrotic pulmonary vascular changes lead to arteriolar narrowing and subsequent increases in PVR [91]. An important consequence of SSc-PAH is right ventricular dysfunction. Patients with SSc-PAH have been shown to have decreased right ventricular contractility [92], but whether this is due to the effects of SSc on the myocardium or abnormal pulmonary hemodynamics is not known. For example, a decrease in right ventricular contractility can occur owing to increased characteristic impedance and decreased pulmonary arterial compliance (i.e., large-artery stiffening) [93], which would be expected in the presence of long-standing PAH. An improved understanding of the hemodynamic and arterial mechanical consequences of SSc could help target new therapeutic design and development – either to the heart or to the pulmonary vasculature. Although diabetes has mostly been associated with systemic cardiovascular disease, recent studies suggest a link between diabetes and PAH [94]. In a recent study, Lopez-Lopez et al. [95] examined the effect of diabetes on pulmonary endothelial function and more specifically on the release of nitric oxide in response to acetylcholine. In their study, agonist-induced and basal nitric oxide bioactivity were impaired in diabetic pulmonary arteries. In pulmonary arteries from many animal species as well as humans, nitric oxide is the predominant endogenous vasoactive factor accounting for endothelium-dependent relaxation [96]. Although careful biomechanical studies have not been performed on the pulmonary vasculature in the presence of diabetes, suppression of nitric oxide is likely to cause arteriolar constriction, loss of large-artery dilation, and consequently, increases in mean and systolic pulmonary artery pressures. Studies to test these hypotheses, if performed, could have important clinical implications. Williams syndrome is a genetic disorder that involves connective tissue in the vascular and central nervous systems and is associated with cardiac anomalies. In the pulmonary circulation, this disease is manifest by elastin insufficiency and subsequent pulmonary stenosis [97, 98]. As noted earlier, Shifren et al. identified elastin gene dosage as a susceptibility factor for PAH in genetically engineered mice [48], which appears quite relevant to this syndrome. This last study is a good example of the enormous potential that genetically engineered mouse models represent for improving our understanding of pulmonary vascular disease. However, a few important limitations exist when extrapolating from mouse pulmonary vascular mechanics to human pulmonary vascular mechanics. For example, since the blood density and viscosity are similar between species (m = 3.5 cP and r = 1,060 kg/m3) [99] but the diameters, heart rates, and flow rates are dramatically different, the flow regimes are quite different. If we assume a heart rate for mouse and human of 600 and 60 bpm, respectively, a cardiac output of 10 mL/min and 5 L/min, respectively, and a pulmonary artery trunk diameter of 0.1 and 1.2  cm, respectively, we obtain

5  Pulmonary Vascular Mechanics

 = 32 and Wo = 4.4 for mouse and  = 1,339 and Wo = 17 for human (as defined in Sect.  1.2). These dimensionless numbers are important for determining whether flow is parabolic or blunt, the magnitude of wall shear stresses, and pulsatile pressure–flow rate relationships. Finally, in all of these diseases, mechanical cell–cell and cell–matrix interactions may significantly influence the development and progression of pulmonary vascular disease. The mechanical properties of extracellular substrates are known to affect cellular gene regulation and signaling pathways [100, 101]. As a consequence, accumulation of collagen, loss of elastin, or extracellular matrix protein defects might not only stiffen arteries but also redirect the differentiation of vSMCs into a more proliferative phenotype or heighten reactivity to soluble factors such as transforming growth factor b. We direct interested readers to recent reviews [102, 103].

7 Summary and Future Directions Although one might initially think of pulmonary vascular mechanics as the solid mechanics of the pulmonary vasculature, we have emphasized here that vascular solid mechanics affect blood fluid dynamics and vice versa. Disease processes can affect both pulmonary solid mechanics and fluid dynamics, but it is typically the changes in fluid (i.e., blood flow) dynamics that lead to morbidity and mortality in pulmonary vascular disease (via increased right ventricular afterload). The ways in which we study pulmonary vascular mechanics are critical to our ability to properly interpret results and make important discoveries. For example, in the preceding sections, we discussed the various ways in which researchers have calculated elastic modulus and stiffness of large pulmonary arteries, the importance of airway pressures and lung volume on the solid mechanics of mid-sized arteries and the microcirculation, and the ways in which the arterial solid mechanics throughout the lung affect dynamic pressure–flow relationships. Genetically engineered mouse models are an exciting future direction for understanding pulmonary vascular disease at the gene and protein levels. Despite the limitations noted above, techniques for properly measuring mouse pulmonary arterial mechanics and blood flow dynamics, and extrapolating from those results to expected behavior in healthy and diseased human lungs, are an important future direction of research. Also, incorporating pulmonary vascular mechanical analyses into clinical studies is likely to have significant impact on patient care. It is already clear that pulmonary artery stiffness is important to right ventricular afterload and has the potential to be a useful prognostic indicator. In the future, other measures of pulmonary vascular solid mechanics, fluid mechanics, or biosolid–biofluid mechanics may be discovered to improve diagnosis, prognosis, and

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treatment of pulmonary vascular disease. Relatively unexplored areas include: 1. The effects of pulmonary artery viscoelasticity on blood flow dynamics 2. The impact of wave reflections and wave speed on right ventricular function 3. The mechanics of cell–cell and cell–matrix interactions in connective tissue diseases with pulmonary vascular consequences 4. The development of novel diagnostics based on pulmonary arterial mechanics, pulmonary hemodynamics, and/ or the interactions between the pulmonary vasculature and the right ventricle.

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5  Pulmonary Vascular Mechanics 58. Cox RH (1982) Comparison of mechanical and chemical properties of extra- and intralobar canine pulmonary arteries. Am J Physiol 242:H245–H253 59. Caro CG, Saffman PG (1965) Extensibility of blood vessels in isolated rabbit lungs. J Physiol 178:193–210 60. Ferenza C (1969) Pulmonary arterial design in mammals: morphologic variation and physiologic constancy. Johns Hopkins Med J 125:207 61. Al-Tinawi A, Madden JA, Dawson CA, Linehan JH, Harder DR, Rickaby DA (1991) Distensibility of small arteries of the dog lung. J Appl Physiol 71:1714–1722 62. Madden JA, Keller PA, Effros RM, Seavitte C, Choy JS, Hacker AD (1994) Responses to pressure and vasoactive agents by isolated pulmonary arteries from monocrotaline-treated rats. J Appl Physiol 76:1589–1593 63. Karau KL, Molthen RC, Dhyani A et al (2001) Pulmonary arterial morphometry from microfocal X-ray computed tomography. Am J Physiol Heart Circ Physiol 281:H2747–H2756 64. Clough AV, Krenz GS, Owens M, Al-Tinawi A, Dawson CA, Linehan JH (1991) An algorithm for angiographic estimation of blood vessel diameter. J Appl Physiol 71:2050–2058 65. Hughes J, Morrell N (2001) Pulmonary circulation from basic mechanisms to clinical practice. Imperial College Press, London 66. Fung YC (1996) Biomechanics: circulation, 2nd edn. Springer, New York 67. Fung YC, Sobin SS (1972) Pulmonary alveolar blood flow. Circ Res 30:470–490 68. Fung YC, Sobin SS (1972) Elasticity of the pulmonary alveolar sheet. Circ Res 30:451–469 69. Culver BH, Butler J (1980) Mechanical influences on the pulmonary microcirculation. Annu Rev Physiol 42:187–198 70. Permutt S, Howell JB, Proctor DF, Riley RL (1961) Effect of lung inflation on static pressure-volume characteristics of pulmonary vessels. J Appl Physiol 16:64–70 71. Rabinovitch M, Chesler N, Molthen RC (2007) Point: counterpoint: chronic hypoxia-induced pulmonary hypertension does/does not lead to loss of pulmonary vasculature. J Appl Physiol 103:1449–1451 72. Milnor WR (1989) Hemodynamics, 2nd edn. Williams & Wilkins, Baltimore 73. Caro CG, Mc DD (1961) The relation of pulsatile pressure and flow in the pulmonary vascular bed. J Physiol 157:426–453 74. Westerhof N, Lankhaar JW, Westerhof BE (2009) The arterial Windkessel. Med Biol Eng Comput 47:131–141 75. Laskey WK, Ferrari VA, Palevsky HI, Kussmaul WG (1993) Pulmonary artery hemodynamics in primary pulmonary hypertension. J Am Coll Cardiol 21:406–412 76. Ewalenko P, Brimioulle S, Delcroix M, Lejeune P, Naeije R (1997) Comparison of the effects of isoflurane with those of propofol on pulmonary vascular impedance in experimental embolic pulmonary hypertension. Br J Anaesth 79:625–630 77. Maggiorini M, Brimioulle S, De Canniere D, Delcroix M, Wauthy P, Naeije R (1995) Pulmonary vascular impedance response to hypoxia in dogs and minipigs: effects of inhaled nitric oxide. J Appl Physiol 79:1156–1162 78. Tuchscherer HA, Webster EB, Chesler NC (2006) Pulmonary vascular resistance and impedance in isolated mouse lungs: effects of pulmonary emboli. Ann Biomed Eng 34:660–668 79. Tuchscherer HA, Vanderpool RR, Chesler NC (2007) Pulmonary vascular remodeling in isolated mouse lungs: effects on pulsatile pressure-flow relationships. J Biomech 40:993–1001 80. Grant BJ, Paradowski LJ (1987) Characterization of pulmonary arterial input impedance with lumped parameter models. Am J Physiol 252:H585–H593 81. Glenny RW, Robertson HT (1995) A computer simulation of pulmonary perfusion in three dimensions. J Appl Physiol 79:357–369 82. Krenz GS, Linehan JH, Dawson CA (1992) A fractal continuum model of the pulmonary arterial tree. J Appl Physiol 72:2225–2237

89 83. Olufsen MS (1999) Structured tree outflow condition for blood flow in larger systemic arteries. Am J Physiol 276:H257–H268 84. Steele BN, Olufsen MS, Taylor CA (2007) Fractal network model for simulating abdominal and lower extremity blood flow during resting and exercise conditions. Comput Methods Biomech Biomed Eng 10:39–51 85. Spilker RL, Feinstein JA, Parker DW, Reddy VM, Taylor CA (2007) Morphometry-based impedance boundary conditions for patient-specific modeling of blood flow in pulmonary arteries. Ann Biomed Eng 35:546–559 86. Huez S, Brimioulle S, Naeije R, Vachiery JL (2004) Feasibility of routine pulmonary arterial impedance measurements in pulmonary hypertension. Chest 125:2121–2128 87. Hunter KS, Lee PF, Lanning CJ et al (2008) Pulmonary vascular input impedance is a combined measure of pulmonary vascular resistance and stiffness and predicts clinical outcomes better than pulmonary vascular resistance alone in pediatric patients with pulmonary hypertension. Am Heart J 155:166–174 88. Lankhaar JW, Westerhof N, Faes TJ et al (2008) Pulmonary vascular resistance and compliance stay inversely related during treatment of pulmonary hypertension. Eur Heart J 29:1688–1695 89. Nollen GJ, Groenink M, van der Wall EE, Mulder BJ (2002) Current insights in diagnosis and management of the ­cardiovascular complications of Marfan’s syndrome. Cardiol Young 12:320–327 90. Nollen GJ, van Schijndel KE, Timmermans J et  al (2002) Pulmonary artery root dilatation in Marfan syndrome: quantitative assessment of an unknown criterion. Heart 87:470–471 91. Yousem SA (1990) The pulmonary pathologic manifestations of the CREST syndrome. Hum Pathol 21:467–474 92. Huez S, Roufosse F, Vachiery JL et al (2007) Isolated right ventricular dysfunction in systemic sclerosis: latent pulmonary hypertension? Eur Respir J 30:928–936 93. Vonk Noordegraaf A, Naeije R (2008) Right ventricular function in scleroderma-related pulmonary hypertension. Rheumatology (Oxford) 47(Suppl 5):v42–v43 94. Fouty B (2008) Diabetes and the pulmonary circulation. Am J Physiol Lung Cell Mol Physiol 295:L725–L726 95. Lopez-Lopez JG, Moral-Sanz J, Frazziano G et  al (2008) Diabetes induces pulmonary artery endothelial dysfunction by NADPH oxidase induction. Am J Physiol Lung Cell Mol Physiol 295:L727–L732 96. Blanco S, Penin R, Casas I, Lopez D, Romero R (2002) Effects of antihypertensive drugs in experimental type 2 diabetes-related nephropathy. Kidney Int Suppl 62:S27–S31 97. Reesink HJ, Henneman OD, van Delden OM et  al (2007) Pulmonary arterial stent implantation in an adult with Williams syndrome. Cardiovasc Intervent Radiol 30:782–785 98. Land SD, Shah MD, Berman WF (1994) Pulmonary hypertension associated with portal hypertension in a child with Williams syndrome – a case report. Pediatr Pathol 14:61–68 99. Feintuch A, Ruengsakulrach P, Lin A et al (2007) Hemodynamics in the mouse aortic arch as assessed by MRI, ultrasound, and numerical modeling. Am J Physiol Heart Circ Physiol 292:H884–H892 100. Lo CM, Wang HB, Dembo M, Wang YL (2000) Cell movement is guided by the rigidity of the substrate. Biophys J 79:144–152 101. Peyton SR, Kim PD, Ghajar CM, Seliktar D, Putnam AJ (2008) The effects of matrix stiffness and RhoA on the phenotypic plasticity of smooth muscle cells in a 3-D biosynthetic hydrogel system. Biomaterials 29:2597–2607 102. Vogel V, Sheetz M (2006) Local force and geometry sensing regulate cell functions. Nat Rev Mol Cell Biol 7:265–275 103. Peyton SR, Ghajar CM, Khatiwala CB, Putnam AJ (2007) The emergence of ECM mechanics and cytoskeletal tension as important regulators of cell function. Cell Biochem Biophys 47:300–320 104. Matsuzaki T (1994) Pulmonary vascular input impedance in patients with atrial septal defect. Acta Med Nagasaki 39:107–113

Chapter 6

Modeling of the Pulmonary Vasculature Merryn H. Tawhai and Kelly S. Burrowes

Abstract  Mathematical models in physiology aim to describe an observable structure or function (or how they relate) using mathematical equations. A computational model solves a system of equations to predict an output, usually as some controlling parameters are varied over a physiological range. The motivation for this is not simply to duplicate the real process, but rather to provide insight that could not be obtained solely through observation and measurement. Integrative computational models provide a means to relate reductionist measurements to integrated organ function and clinical measurements. They are complementary to experimental studies, and can be used to study integrated function while controlling complexity and interactions that would normally occur during experimentation and therefore potentially obscure some important function. Perfusion of the pulmonary vasculature is a multiscale phenomenon that involves scale-dependent structure and function, therefore requiring different model assumptions for the microcirculation and the arterial or venous flows. The pulmonary vasculature interacts with the surrounding lung tissue, and vessel dimensions (and therefore resistance to flow) are further dependent on hydrostatic pressure gradients, vasoconstriction and vasodilation, and the topology and material composition of the vascular trees. The regional distribution of blood in the lung is determined by the effect of gravity on hydrostatic pressure gradients and regional tissue expansion (and therefore stretch or compression of capillaries), by pulmonary vascular resistance, and by the physical properties of blood. These factors interact in a dynamically expanding and recoiling organ, with time-varying boundary conditions for blood pressure, and subject to the effect of changes in posture. In this chapter, we focus specifically on models of the pulmonary vasculature that have been used to study structure–function relationships in pulmonary perfusion. Keywords  Mathematical model • Computational modeling • Structure-functional relationship • Integration M.H. Tawhai (*) Auckland Bioengineering Institute, University of Auckland, Auckland 1142, New Zealand e-mail: [email protected]

1  Introduction Mathematical models in physiology aim to describe an observable structure or function (or how they relate) using mathematical equations. A computational model solves a system of equations to predict an output, usually as some controlling parameters are varied over a physiological range. The motivation for this is not simply to duplicate the real process, but rather to provide insight that could not be obtained solely through observation and measurement. A model is only a representation, a simplification, an approximation; however, a useful mathematical model provides mechanistic insight and/or suggests new testable hypotheses. The ability to develop models with an increasing amount of complexity has increased in hand with computing power, but introducing model complexity must be reasonably justified: complexity can obscure function, whereas the simplest models are easy to understand and lend themselves to interrogation by standard mathematical techniques [1]. But in some cases we can only achieve insight through employing a c­ omplex model. Integrative computational models provide a means to relate reductionist measurements to integrated organ function and clinical measurements. They are complementary to experimental studies, and can be used to study integrated function while controlling complexity and interactions that would normally occur during experimentation and therefore potentially obscure some important function. Integrative models at the organ and/or tissue level are often structurebased, with realistic model geometry and constitutive laws based on tissue microstructure (e.g., [2, 3] for the heart). Perfusion of the pulmonary vasculature is a multiscale ­phenomenon that involves scale-dependent structure and function, therefore requiring different model assumptions for the microcirculation (see [4, 5] and Sect.  2) and the arterial or venous flows. The pulmonary vasculature interacts with the surrounding­lung tissue, and vessel dimensions (and therefore resistance to flow) are further dependent on hydrostatic ­pressure gradients, vasoconstriction and vasodilation, and the topology and material composition of the vascular trees. The regional distribution of blood in the lung is determined by the effect of

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_6, © Springer Science+Business Media, LLC 2011

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gravity on hydrostatic pressure gradients and regional tissue expansion (and therefore stretch or compression of capillaries), by pulmonary vascular resistance, and by the physical ­properties of blood. These factors interact in a dynamically expanding and recoiling organ, with time-varying boundary conditions for blood pressure, and subject to the effect of changes in posture. A further factor for consideration is the difference in vascular branching geometry between humans and typical laboratory animals, which complicates interpretation of experimental measurements. The relative contribution of these different ­factors to the regional distribution of blood in the lung remains under strong debate [6, 7]. In the following sections we focus specifically on models of the pulmonary vasculature that have been used to study structure–function relationships in pulmonary perfusion. These are integrative physics-based models (in contrast to systems or electrical analog models, see [8, 9] for examples) that solve equations governing function in a domain that ­represents the geometry of the pulmonary vasculature. Our goal is to explain the role of computational models in understanding regional perfusion and its dependence on vascular geometry, interdependence between the vessels and lung ­tissue, and gravity, and to highlight the areas in which further model development would be of immediate value. Model geometry (here “the pulmonary vasculature”) is an important consideration in models that seek to understand structure–function relationships. The degree of detail in the model geometry should be appropriate for the functional study, and where possible the simplest model should be ­chosen. Governing equations go somewhat hand in hand with geometry, for example, three-dimensional (3D) equations cannot be solved in a one-dimensional (1D) structure, and simulations in a symmetric branching tree must adopt an assumption to account for symmetry. The following sections consider simplifications in both geometry and governing equations.

2 Models of the Pulmonary Microcirculation Owing to the vast number of pulmonary capillaries in the lung (approximately 280 billion [10]), it is computationally impossible to represent the flow through each individual ­capillary segment, and it is highly doubtful that such a vast model would give any physiological insight. Models of ­perfusion of the complex pulmonary capillary plexus have therefore sought to reduce the complexity and size of the physical domain by reasonable assumptions [5], or have assumed a tubular network structure for a very small portion of the alveolar tissue [4, 11, 12]. These two types of model are classified here as “sheet-flow” and “tube-flow.” Capillaries in the systemic circulation tend to be longer than those in the pulmonary circulation, and are surrounded on all sides by tissue; hence, systemic microcirculatory blood

M.H. Tawhai and K.S. Burrowes

flow is typically modeled within systems of discrete tubules (the tube-flow method). In contrast, the pulmonary microcirculation is a very dense meshwork of short capillary ­segments that are exposed on each side to air. As the alveolar tissue expands volumetrically, the capillaries are stretched and their height decreases. From these observations, Fung et al. [5, 13] developed a model of the pulmonary microcirculation as a continuous sheet of blood (the sheet-flow model). More recently, the tube-flow approach has been used to simulate perfusion in the pulmonary microcirculation, by taking into account that the pulmonary capillaries deform differently from the systemic capillaries with stretch. In addition to structural factors that govern resistance to blood flow (the topology of the individual conduit vessels and the resulting network, including vessel diameters, length, number, and connectivity of vessels), the apparent viscosity of blood must also be considered at the microcirculatory level. “Apparent viscosity” refers to the viscosity of a nonNewtonian fluid at a specific shear rate. In vessels of diameter less than approximately 300 mm, blood must be considered as a two-phase non-Newtonian fluid: particles suspended in blood, particularly red blood cells (RBCs), strongly ­influence the apparent viscosity and therefore the fluid dynamics in each segment. Blood has shear-thinning properties: at high shear rates (i.e., in the larger vessels) RBCs are dispersed by fluid forces, whereas under low shear the RBCs form aggregates that increase the apparent viscosity. In vessels smaller than 300 mm in diameter, RBCs are preferentially distributed near the vessel center [14, 15]. This “axial migration” results in a layer of zero hematocrit adjacent to the vessel wall, and therefore reduces the apparent viscosity of the blood. This decrease in apparent viscosity with decreasing vessel diameter is known as the Fahraeus–Lindqvist effect [16]. Because the RBCs tend to transit through the center of the vessel, their average velocity is higher than the plasma near the walls. This causes a reduction in the RBC transit time with respect to the plasma or total blood, and a dynamic reduction in vessel hematocrit relative to the entrance hematocrit for a vessel (the Fahraeus effect [17]). A further factor governing the distribution of blood in small branching vessels is the disproportionate distribution of RBCs and plasma at junctions. This “phase separation” at a junction is proportional to the relative flow rates in the daughter vessels [18].

2.1 The Capillary Network Modeled as a Sheet Fung et al. [5, 13] developed the sheet-flow concept on the basis of morphometric observations of the dense pulmonary capillary plexus. This mathematical description approximates blood flow through the pulmonary capillaries as a

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sheet of fluid flowing around “posts” of connective tissue, bounded on either side by compliant capillary endothelium (summarized in Fig. 1). The geometry of the microvascular sheet is characterized by two independent properties: the ratio of the vascular lumen space to the circumscribing tissue space of the network (the vascular space tissue ratio), and the thickness or height of the microvascular sheet. The membrane and post components are elastic and respond to changes in surrounding pressures (blood and air) and lung volume. Sobin et al. [19] conducted a series of experiments where they varied the blood pressure while holding the transpulmonary pressure (Ptp) constant. They showed that the thickness of the capillary sheet varies linearly with transmural pressure (Ptm) when the blood pressure is greater than the alveolar pressure (PA), whereas the plane dimension and diameter of the posts do not change. The thickness of the sheet tends to a constant above an upper limit of about 15  cmH2O for the human lung, and tends to zero, such that the capillaries are collapsed, when the blood pressure is less than the alveolar pressure according to Eq. (1):

H x = H 0 + a ( Px − PA ),

(1)

where Hx is the thickness of the capillary sheet, H0 is the thickness of the sheet when Px = PA, and a is the compliance constant. Flow through the sheet is a function of the change in sheet height at either end of the network described by the following relationship:

Q = C ( Hart )4 − ( H vein )4  .

(2)

2.2 The Capillary Network Modeled as Discrete Tubules

The constant C is given by (3):

C=

SA , 4 mKfL2 α

of the sheet, f is a dimensionless friction factor, and L is the average blood path length between the inlet and the outlet. Simulations in the sheet-flow model require specification of the geometric boundaries of the sheet, such as the locations of and pressures applied at arterioles and venules. In specifying these boundary conditions, one needs to make approximations whereby the flow over a sheet is averaged. The model can be used to analyze the pressure, velocity, thickness, and streamline distributions in an interalveolar septum. It therefore provides a useful description of the general pattern of blood flow through the alveolar walls, and it further provides a realistic estimate of the flow and resistance of the capillary bed under different combinations of boundary pressures. A major advantage of the sheet-flow model is that it is a convenient simplification of the microcirculation for coupling to models of the arteries and veins. Several studies have included sheet flow in models of the full pulmonary circulation [20–22] to represent the pressure drop across the microcirculatory network. The sheet-flow model enables calculation of flow properties in each alveolar septum, but the effects of individual segment geometry and the two-phase nature of blood are accounted for by an increase in the effective resistance to overall flow, therefore segment-to-segment variability in perfusion of individual capillaries cannot be predicted. The sheet-flow model also cannot predict distributions of cellular transit times from arteriole to venule; only average times can be determined. A tube-flow model must be used if this level of detail is required.

(3)

where S is the ratio of vascular space to tissue space, A is the capillary sheet area, m is the apparent viscosity of blood, K is a dimensionless function of the ratio of the thickness to the width

Fig.  1  The alveolar–capillary sheet model illustrating the vascular space between an arteriole and a venule bounded by alveolar–capillary membranes that are connected by interstitial posts. (Reprinted with permission from Elsevier [75])

Dhadwal et  al. [11] and Huang et  al. [12] introduced the concept of modeling flow in the pulmonary microcirculation as transit through a dense network of tubules with elliptical cross section. In these models, the RBC and plasma distributions are dependent on fluid-dynamic effects specific to the small vessels, and the capillary “height” is determined from empirical equations. These tube-flow models describe the flow of blood within discrete capillary segments, giving a description of pulmonary blood flow on a segment-bysegment basis. The pressure, radius, and flow through a pulmonary capillary segment or network of capillaries must consider basic fluid dynamics that is adapted to account for the non-­ Newtonian characteristics of blood, and the unique distensibility of the pulmonary capillaries. An initial Newtonian flow distribution is calculated using conservation of mass and Poiseuille flow. This is modified to account for non-Newtonian dynamics as a result of RBCs, including the Fahraeus effect (reduction of intravascular hematocrit relative to the inflow

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Fig. 2  Generation of an alveolocapillary model for tube-flow computation. a A model alveolus (edges of alveolar septa are black lines) and surrounding points spaced over the surface of a portion of a unit sphere. b Surface Delaunay triangulation (gray triangles) of the points in a. c

M.H. Tawhai and K.S. Burrowes

The Voronoi dual (black hexagons) of the Delaunay triangulation (gray triangles), where the edges of the Voronoi tessellation form the capillaries in the model. d The capillaries projected onto the surface of the model alveolus

Fig. 3  a Blood flow (mm3/s) in the alveolocapillary model (at mid lung height) showing preferential pathways and unrecruited capillaries. b Distribution of red blood cell transit times (s) in the alveolocapillary model from a, exposed to pressure boundary conditions consistent with the upper, mid, and lower regions of the lung

hematocrit of a vessel), the Fahraeus–Lindqvist effect (dependence of hematocrit on vessel diameter), and the phase-separation effect (the disproportionate distribution of RBCs and plasma at bifurcations). Conservation of RBCs and plasma flow is enforced at each junction and the apparent viscosity of the blood within each blood vessel is then adjusted accordingly. Vessel distensibility is updated for each time-dependent flow distribution, based on the internal blood pressure, alveolar and pleural pressures (Ppl), and response to mechanical and metabolic stimuli. Dhadwal et al. [11] represented the capillary geometry in a single alveolar septum as a network of interconnected capillaries arranged into a 6 × 6-square matrix. They studied the influence of random variability in capillary dimension and compliance on flow patterns and pressures within the simple network, finding that preferential flow pathways could develop owing to geometric variability, and that blocking a segment caused changes in local capillary perfusion. Neither of these observations could have been made using a sheet-flow approach. Huang et  al. [12] used a slightly more extensive geometry than Dhadwal et al. [11], with capillary segments randomly positioned within squares, each square representing an alveolar septum. Blood could flow between adjacent septa, entering via a single corner vessel and leaving via a corner vessel on the opposite side of the network. They studied both RBC and neutrophil  transit through multiple alveolar septa, their results

suggesting that neutrophil blocking of capillary segments could regulate RBC and plasma distribution in the pulmonary microcirculation. Capillary continuity is found at least throughout the acinus, between adjacent acini, and possibly extends throughout the lobule. Burrowes et al. [4] developed a 3D model of the capillary plexus surrounding a single alveolar sac, consisting of 19 alveoli and approximately 20,000 capillary segments. The alveolocapillary model geometry was constructed using a Voronoi mathematical meshing technique to create space-filling geometric structures representative of the alveolar and capillary structures found in vivo. The steps in the model generation are summarized in Fig. 2. The method ensures that a continuous capillary network is generated, with only a single capillary layer between adjacent alveoli. The model contains multiple inlet (arteriole) and outlet (venule) vessels spaced at approximately equal distances. Burrowes et  al. [4] studied regional microcirculatory perfusion in the alveolocapillary model, including distributions of cellular transit times and flow rates, by specifying gravity-dependent Ptp and Ptm boundary conditions. Predictions of RBC and neutrophil transit times positioned in the upper, mid, and lower regions of the upright lung showed physiologically consistent trends of a decreasing average transit time for blood cells and an increased homogeneity of transit time distributions in the direction of gravity (Fig.  3). Preferential pathways were predicted in the

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model, particularly at the septal boundaries where adjacent capillary networks converge, and areas of low flow were observed in regions furthest from the arterioles and venules. The preferential pathways in the model of Dhadwal et al. [11] developed owing to variability in the initial height and compliance of the capillaries, whereas in the alveolocapillary model of Burrowes et  al. [4] preferential pathways developed at the convergence of septal networks. Both factors could contribute to preferential pathways in vivo. The inlet and initial hematocrit in the alveolocapillary model was set to 0.4, but the simulation predicted a range of hematocrit in individual capillary segments from 0 to 0.78 in response to the two-phase fluid dynamics. Burrowes et al. [4] also calculated regional neutrophil blockage, finding a far greater incidence of neutrophil trapping in the nondependent (upright apical) tissue. The model predicted no neutrophil trapping in the dependent tissue, whereas in the upper third of the lung, 87% of white blood cells stopped three or more times during their transit through the microcirculation. This regional response is consistent with experimental measurements [23], and may be important in regional differences in the development of emphysema. Early studies of regional perfusion in the human lung, (see [24, 25] for examples), typically measured a vertical gradient of increasing blood flow in the direction of gravity. The classic zonal model for lung perfusion [24] was developed on the basis of these observations, concluding that hydrostatic pressure differences are the main determinant of blood flow distribution. Gravity has a significant effect on perfusion of the alveolocapillary model, with flow increasing approximately linearly in the direction of gravity [4]. A small region of zone 4 flow is predicted in the most dependent tissue. In this model this is caused by a small decrease in the capillary diameter in the base of the lung, which is enough to counteract the increase in blood pressure. The transit time in the dependent tissue is shorter and more homogeneous than in the nondependent tissue (except for the zone 4 region). Inversion of gravity in the capillary model results in an inversion of the perfusion gradient [26]. The tube-flow models of Dhadwal et al. [11], Huang et al. [12], and Burrowes et al. [4] differ mainly in their representation of the capillary geometry. Exposing the models to pressures that are consistent with regional variations in the lung gives model predictions of regional perfusion that match well with the zonal theory. This is not surprising because the capillary network models incorporate the same factors as were considered in the development of the zonal theory (blood pressures, Ptm, Ptp), and the models also neglect the influence of the arterial and venous trees. To form a complete description of pulmonary perfusion, the microcirculation must be coupled to perfusion of the large vessels. This returns us to the sheet-flow model: whereas the tube-flow models give insight into distributions

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of blood cells and plasma within the alveolar walls, creating a model for the full pulmonary circuit requires a simplified representation of the microcirculation that is conveniently provided by the sheet-flow model.

3 Complexity of Structure in Models of Perfusion of the Pulmonary Arterial Tree We can broadly classify models of the structure of the pulmonary arteries and veins by their level of detail: those that use summary information to simplify the geometry (e.g., fractal, symmetric branching [27], or average flow path [28]), and those that include anatomical detail (e.g., [29–32]). Note that even the most anatomically detailed models still employ simplifications. Model geometry is one assumption that must be established in the context of the model application. The summary models are convenient for their small size, whereas geometrically complex models are used in studies where vascular structure is central to the hypothesis. Models that are used to study structure–function relationships in the lung must be consistent with the measured geometry of the lung vasculature. This applies whether the model adopts major simplifications (e.g., symmetric branching) or attempts to closely represent the vascular structure. Castbased morphology studies of the pulmonary vasculature [10, 33] provide geometric information for vessel length, vessel diameter, and the number of vessels in each branching generation and order, down to the level of the capillary bed. The “accompanying” or “conventional” pulmonary arteries have a one-to-one correspondence with the conducting airway tree, and hence follow the airway branching geometry [10]. The pulmonary veins more loosely follow the other two trees, and bifurcate between neighboring airway and arterial bifurcations. Fung [34] described the relationship between the pulmonary arteries and veins as “islands in an ocean,” where the arteriolar regions represent the islands, and the venous regions represent a continuous ocean that fills the space not occupied by airways or arteries. More recently, the lung vasculature can be measured in  vivo using computed tomography (CT) or magnetic resonance imaging. One challenge is to define robust methods for translating these imaging-based measurements into computational models of the pulmonary vasculature.

3.1 Examples of Summary Models The lung is often described as a fractal structure [35], and having fractal perfusion to the level of the alveolar tissue [36, 37].

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A fractal model assumes self-similarity in either structure or function. In its simplest form, a fractal model of the ­pulmonary vasculature specifies the geometric or functional relationship between a parent and child vessel at a bifurcation (e.g., the Horsfield D (delta) model for geometry [38]), and assumes that this relationship is constant throughout the tree. The division of flow can be a constant, or can include random fluctuations. When the flow division is 0.5, the flow is distributed evenly at all bifurcations; when the flow division departs from 0.5, the flow becomes more asymmetric. A convenience of the simplest fractal models is that solutions can be calculated analytically; however, because the foundation of the model is selfsimilarity, a fractal model cannot differentiate between structures that have very different function such as the supernumerary vessels (SVs) and accompanying vessels (AVs) (described further in Sect. 5). Several studies have coupled summary representations of the pulmonary arteries and veins to the sheet-flow model for the pulmonary microcirculation as a model for the full lung circuit. Zhuang et al. [22] presented the first of these models alongside data on the anatomical structure and elasticity of the cat arteries and veins. They calculated steady-state flow through two simplified model structures that had different branching patterns but were both consistent with the anatomical data. Krishnan et  al. [39] later adapted this model to improve its dynamic behavior. The original model gave a good fit to steady-state measurements, but it was necessary to account for the viscous behavior of the vessel walls and slightly modify the vessel dimensions to match the dynamics of vessel occlusion. Marshall and Marshall [40] studied hypoxic vasoconstriction of the pulmonary circulation in the model of Zhuang et al. [22] by specifying the degree of constriction of arteries in orders 1–6 and calculating pressure– flow curves.

3.2 Subject-Specific Models Computational models can now feasibly be based on imaging for an individual. These “subject-specific” models are important for understanding intersubject and interspecies variability, and they allow direct comparison with regional functional imaging that is not relevant for the summary models. Analogous to models of the airway tree [41, 42], subjectspecific models of the pulmonary vasculature can be detailed renderings of the 3D vessel topology or 1D models (in 3D space) that trace and/or virtualize the centerlines of the entire arterial and venous trees. The advantage of the 3D model is that the functional equations are more accurate for simulating fluid dynamics; the 1D model is less precise but covers a far more extensive domain. The selection of either type of model depends on the goal of the study.

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3D models of the pulmonary vasculature are less prolific than their counterparts in the bronchial airways. Models of 3D air flow include analysis of turbulent structures in idealized single bifurcations [43], several generations of bifurcations [44–46], and more recently include accurate imaging-based airway surface topology [41, 47, 48]. 3D models of fluid flow have been used to study transport in the systemic vasculature, for example, recent work in modeling cerebral aneurysms [49] and coronary perfusion [50]. But because these 3D models are limited to a small computational domain, they are typically designed to study a specific (and localized) phenomenon, such as the effect of local plaque formation or vessel constriction. Similar work in the pulmonary vessels is very limited, perhaps because the important questions in the pulmonary vasculature cannot be answered with limited-domain models. Spilker et al. [32] have used a computational method to predict patient-specific surgical outcomes in an application to treatment of arterial stenoses. Their method uses volumetric imaging to define the centerline and diameter of proximal arteries, and virtualizes the distal arteries by attaching 1D summary models with morphometrically consistent geometry. Perfusion of the structure is simulated using 1D equations for fluid flow in elastic vessels, with inlet pressure and outlet impedance as boundary conditions. The dimensions of the virtualized components are “tuned” so that the whole model has pressure and flow distribution consistent with measurements from the patient. The focus of this work is to understand the impact of surgical intervention, and not to study the function of the lung per se. The summary models for the distal arteries that are attached to the more detailed proximal vessels are sufficient for this type of study. To model the accompanying pulmonary vascular trees from the heart to the level of the terminal bronchiole (in the airway tree), Burrowes et al. [26, 29] adapted a method that had previously been used to model the human and ovine conducting airway tree [42, 51]. First, a finite element volume mesh of the lungs or lung lobes was fitted to surface topology that was segmented from multidetector row CT (MDCT). A volumetric rendering of the imaging and the fitted lobe model is shown in Fig. 4a. An initial volume mesh is constructed for each lobe, using high-order shape functions that mathematically represent the surface of the mesh in terms of nodal locations and derivatives at the nodes [52]. The surface topology is represented by densely sampled discrete surface data points (Fig.  4b). The distance from each data point to the surface of the mesh is calculated, and the summation of the distances (plus penalty factors to control curvature in each coordinate direction) forms an objective function. By successive iterations, the nodal locations and derivatives are calculated to minimize the objective function (fitted mesh in Fig. 4c).

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Fig. 4  Generation of a finite element model of each of the five lobes typical of the human lung (right upper lobe in green, right middle lobe in red, right lower lobe in blue, left upper lobe in yellow, left lower lobe in orange). a Surface rendering of volumetric imaging for a single subject from the Human Lung Atlas. b Data points calculated on the sur-

faces of each of the lobes. c Finite element volume meshes to host vascular tree generation. (a (from Tawha et al. [42] reprinted with permission from the American Physiological Society), c From Burrowes et al. [29], reprinted with permission from the American Physiological Society)

Fig. 5  Multiscale and subject-specific models of the pulmonary vasculature. a Arteries and veins segmented using custom-built software (PASS, the University of Iowa). b A subset of 5,000 arteries (red) and veins (blue) manually differentiated from each other. c The trees

extended to the level of the terminal bronchioles via a volume-filling method. d Supernumerary vessels diverging at close to right angles from the main vessel. (Reproduced with permission from the American Physiological Society [29])

The connectivity of approximately 2,500 of the largest pulmonary arterial and venous blood vessels was defined by tracing the centerlines of vessels reconstructed from ­automated segmentation of the volumetric MDCT (Fig. 5b). The nonsegmented arteries that accompany the airways were modeled using a volume-filling method [42, 51]. The volume-filling method generates a tree structure within a volume of arbitrary shape, by successively branching toward the center of mass of seed points that are contained within the volume. The method incorporates subject-specific anatomical data by incorporating segmented airway or blood vessel centerlines, and the shape of the lung and lobes. The method is illustrated schematically in Fig. 6. The volumefilling method has been shown to generate morphometrically consistent tree structures to represent the conducting airways in both human and sheep [42], and the human pulmonary arteries and veins [26, 29]. An advantage of this

approach is that as image resolution increases or image ­processing algorithms improve and they provide additional anatomical information, this is automatically integrated into the model.

4 Functional Study: Contributions of Gravity and Vascular Structure to Arterial Perfusion Although gravity certainly contributes to the global distribution of blood via capillary perfusion and tissue deformation [53, 54], other factors contribute to the heterogeneity that has been measured within isogravitational planes. For example, Glenny et al. [55] demonstrated a persistent flow gradient in the baboon lung, somewhat independent of body posture, yet

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Fig.  6   The volume-filling method. a The subject volume at total lung capacity (a lung, lobe, or sublobar structure) is filled with a uniformly spaced grid of points with density the same as the number of acini. b The grid points are grouped with the closest peripheral vessel. c The center of mass of each subset of points is calculated, and a plane computed from the center of mass and the direction vector of the associated peripheral vessel is used to divide the subset of points in two.

d Centers of mass of the new point subsets are calculated. e New vessels are made that extend from the current tree periphery toward the center of mass of their respective set of grid points, with length equal to 40% of the distance to the center of mass, and branching paths terminate when the vessel is associated with a single grid point or the vessel length is less than a specified length limit. f The method is repeated from b until each grid point is associated with a single terminal vessel

the original zonal model predicts a reversal of the flow gradient with reversal of posture. Musch et al. [56] demonstrated persistent ventilation and perfusion gradients in humans in both the prone and the supine postures favoring caudal lung regions. They also found that flow heterogeneity was unaffected by posture. A consensus between the zonal model and more recent measurements is complicated by differences in experimental protocols, species differences, and the ­influence of mechanisms that are likely to interact during experiments [7]. A computational study of the relative influence of different mechanisms on pulmonary perfusion would ideally use an integrative model with realistic arterial branching, gravity acting on blood and tissue, models for vasocontrol, and a dynamic model for the capillary bed. This level of model sophistication is still some way off. As a first step toward an integrative model, Burrowes et  al. [30, 31] solved the 1D Navier–Stokes equations in an elastic arterial tree geometry of a human lung to study the influence of normal arterial branching, gravity, and posture on regional perfusion. The Navier–Stokes equations govern conservation of fluid mass and momentum. By assuming that the arteries (or veins) are circular in cross section and that the velocity profile over the cross section of each vessel is known, one can reduce the 3D Navier–Stokes equations describing fluid motion can be reduced to 1D Eq. (4) and Eq. (5) [30]. Eq. (4) and Eq. (5) and an empirically based relationship between Ptm and the vessel radius Eq. (6) are solved numerically within the arterial geometries to predict blood pressure, vessel radius, and blood velocity:

∂V ∂V V 2 ∂R 1  ∂p  + (2a − 1) V + 2(a − 1) +  + ρg cos Θ   ∂t ∂x R ∂x ρ ∂x  va V = −2 , 2 (4) a −1 R



∂R ∂R R ∂V +V + = 0,  ∂t ∂x 2 ∂x  R  b  p( R) = G0   − 1 − Pp1 ,  R0  

(5)

(6)

where t denotes time, Q is the vertical angle between the gravitational vector and the vector of the vessel centerline, g is the acceleration due to gravity [g = 9.81  m s-2 (1G) or 0 m s-2 (0G)], r is blood density, and n is the kinematic viscosity of blood (values of 1.05 × 10-6 kg·mm-3 and 3.2 mm s-1 [2], respectively, were used for all simulations in the published studies by Burrowes et  al.). The parameter a defines the velocity profile. a = 1 corresponds to a flat uniform profile, and the profile approaches parabolic as a is increased, where a value of a = 1.33 corresponds to fully developed laminar flow. Elasticity parameters G0 and b in (6) can be obtained from experimental measurements; [57] values of 6.7 kPa and 1.2, respectively, were used by Burrowes et  al. R0 is the unstrained vessel radius at Ptm = 0 kPa. Boundary conditions were prescribed for pressures at the heart, pressures at the terminal arteries (small arteries at the

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level of the terminal bronchiole), a gravitational acceleration vector, and Ptm. It is important to note that the location of the vessels did not change during the simulations. That is, the tissue deformation associated with a postural change was only accounted for via the direction of the Ppl gradient [31]. The distribution of tissue perfusion in the subject-specific model was compared with a distribution of perfusion in a model that had spatially distributed symmetric branching [30], as illustrated in Fig. 7a. “Tissue perfusion” in this analysis refers to the blood delivered to the terminal arteries in the model, which is equal to the blood flow per acinus. A computational model provides the convenience of data analysis at chosen levels of resolution. In Fig. 7 the perfusion results are presented as averaged values within isogravitational sections of lung. Two levels of resolution are presented: averages for 1-mm-thick sections and for 50-mm-thick sections. The first resolution is approximately consistent with microsphere experiments and CT studies, whereas the second resolution is more appropriate for comparison with external scintillation measurements that integrate flow over large sections of lung. Figure 7 shows blood pressure and tissue perfusion for the upright symmetric and anatomical models in normal gravity (1G). Results for zero gravity (0G) in the anatomical model are shown in Fig. 8. For 0G in the symmetric model (not shown here), perfusion of all terminal arteries is equal. When gravity is added, the flow distribution changes to be visually consistent with the zonal model. That is, the

r­ elationship between tissue perfusion and gravitational height is linear, and flow increases in the direction of gravity. In the symmetric model with spatial distribution, the only factor affecting the distribution of blood is the pressure head established between the model inlet and outlets: all perfusion pathways have the same resistance to flow and therefore do not contribute to regional differences. In contrast, the anatomical model results in Figs.  7 and 8 are a consequence of both gravity and variability in path resistance. Tissue perfusion averaged in 50-mm isogravitational sections in the anatomical model in 0G is relatively constant for regions of lung above the pulmonary artery, but decreases systematically below this level. Introducing gravity decreases perfusion above the heart and increases it below the heart with respect to the 0G simulations. A further feature of the anatomical arterial model is that in both the 0G and 1G cases the model predicts a persistent decrease in flow in the basal region (Figs. 7, 8). This decrease is visually consistent with “zone 4,” but could not have originated through compression of extra-alveolar vessels or perivascular cuffing, which are mechanisms that have previously been proposed [58]. The arterial model has elastic vessels, but assumes a nondeforming lung at total lung capacity. Relative compression of the vessels in the gravitationally dependent region therefore cannot be a mechanism in this model. Because the only difference between the symmetric and anatomical models is in their branching structure, a

Fig. 7  Comparison of blood pressure and flow distributions within upright symmetric and anatomical models of the pulmonary arteries in normal gravity (1G). a Blood pressure (kPa) in the symmetric model. b Blood pressure in the anatomically based model. c Blood pressure against gravitationally dependent height (%) for the two

models (S symmetric, A anatomical). d Flow (mm3/s) against vertical location for the two models. The results are presented as averages within isogravitational sections that are 1 mm thick (illustrated with points) and 50  mm thick. (Reproduced with permission from Elsevier [30])

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Fig. 8  Comparison of blood pressure and flow distributions within the upright anatomical model of the pulmonary arteries in zero gravity (0G) and normal gravity (1G). a Blood pressures (kPa) in 0G. b Blood pressures in 1G. c Blood pressure against gravitationally dependent height

(%) for 0G and 1G. d Flow (mm3/s) against vertical location. The results are presented as averages within isogravitational sections that are 1 mm thick (illustrated with points) and 50 mm thick. (Reproduced with permission from Elsevier [30])

c­ onclusion is that the long perfusion pathways to the basal region were the particular feature of the anatomical model that caused persistent reduction of flow in the base [30]. The anatomical model predicts large heterogeneity in tissue perfusion, either with or without gravity. This is consistent with experimental studies [55, 59–62], whereas the 50-mm-slice averages are visually consistent with the earlier studies of West et al. [25]. The perfusion results could also be presented in the standard way, plotting all data and fitting a straight line. However, heterogeneity in the tissue perfusion obscures any gravitational trend and fitting a single line to the data is not appropriate when a significant zone 4 is present. Burrowes and Tawhai [31] further extended the anatomical model to investigate incomplete reversal of flow upon reversal of body posture, finding that although flow with the body in the prone posture was consistently lower than flow with the body in the supine posture, the tissue perfusion at any location was similar in either posture. The arterial tree has regionally dependent path lengths (i.e.,, longest branching paths to the basal regions, then apical, and shortest mid) and the resistance to flow in these model paths is similar regardless of posture. Therefore, there is a persistent (nongravitational) underlying pattern of flow in the model that is likely to exist to some extent in the real pulmonary arteries. The gravitational perfusion gradient for the symmetric and anatomical models in Fig. 7 quantifies the contribution

of gravity within the arterial tree, but not the contribution of the capillaries, tissue deformation, or vasoactive control. Lack of explicit coupling to the microcirculation is the most obvious limiting factor for comparing the theories of gravitational and structural influences on regional perfusion. Pressures prescribed as boundary conditions at the terminal vessels of Burrowes et  al. [30] and Burrowes and Tawhai [31] varied linearly with gravitational height. Coupling with the pulmonary microcirculation could increase the influence of gravity on the simulation results. Contrast this deficit in the anatomical model with the models of Zhuang et al. [22], Krishnan et al. [39], and Marshall and Marshall [40]: in these models the full pulmonary circuit was considered, therefore the influence of both vessel and capillary resistance was included. But the models of the full circuit did not include spatial–anatomical arterial or venous geometry and therefore cannot predict the effect of vascular geometry. This highlights a limitation of computational models; that assumptions and simplifications are necessary in all models to render them both tractable and interpretable. In the case of the anatomical models, this means starting with detailed arterial geometry and neglecting the full circuit. That vascular geometry plays a role in determining the distribution of regional perfusion has been suggested for some time [55, 61, 63, 64]. The contribution of this model analysis is therefore not to propose a new theory, but to

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p­ rovide a mechanistic insight into experimental observations. Experiments are complicated by the interaction of multiple factors: it is extremely difficult to change a single parameter (e.g., blood pressure) without some degree of flow-on effect in the integrated system (e.g., vascular recruitment, effective tissue stiffness, and blood transit times). The modeling study enables the isolation of the effect of arterial geometry and the effect of gravity on blood flow within the arteries, without the confounding influence of other factors.

5 The Function of the Supernumerary Vasculature: A Role for Integrative Structure–Function Modeling One poorly understood feature of the pulmonary vasculature is the supernumerary arteries and veins. Supernumerary vessels (SVs) are small side branches that arise at a large branching angle from the accompanying vessels (AVs) of the lung to supply the closest respiratory tissue without following a matching portion of the bronchial tree [65]. SVs vastly outnumber the AVs. Because these vessels are not visible on angiograms, it has been suggested that they are unperfused under normal conditions [65]. The few experimental studies that have acknowledged the existence and distinct function of the SVs have concluded that they have unique geometry and control of vasoconstriction that limits normal perfusion but would actively divert flow under elevated pressure [65– 72]. To date there has been no explicit study of SV function in the intact lung, yet SVs undoubtedly provide parallel (and possibly dynamically recruited) pathways for perfusion when an artery is blocked by an embolus or indeed in any condition where pulmonary hypertension is a component. Despite the extensive nature of supernumerary branching in the pulmonary vasculature, and despite strong evidence that the recruitment of SVs differs from that of AVs, understanding of the normal function of SVs is poor and their contribution to pulmonary vascular resistance is unknown. Study of the function of the SVs in the intact organ is complicated by methodological issues. The SVs are very small and therefore difficult to visualize on clinical CT. Maximum intensity projection reconstruction can be used to enhance visualization of lateral vessel branching, but without correlation against the airway tree the lateral vessels cannot be classified as SVs or AVs. The volume of blood carried by individual SVs is likely to be small, so it would be very difficult to quantify their perfusion using imaging measurements. In this situation a computational model is likely to give more insight than current experimental measurements, at least until new experiments can be suggested by the model. We now consider the type of model that is most suitable for studying supernumerary function. Fractal models do not

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differentiate between AVs and SVs; a fractal model must either be all-inclusive or all-exclusive of SVs. In the first case, the model operates under the assumption that SVs are as likely to be perfused as the AVs, and in the second case, the SVs are all unperfused. The current body of literature suggests that the second case is the most likely [65, 72]. The pulmonary vascular trees are often described as having many more vessels than the airway tree, and this is represented in Horsfield’s morphometric data for the Strahler branching ratios of the arterial and venous trees of 3.0 and 3.3, respectively [33, 73], both of which are higher than the branching ratio for the bronchial airway tree (2.8). Therefore, a fractal model or structural model based on Horsfield’s measurements must effectively include the SVs. Models that have equivalent number of vessels as in the symmetric Weibel model, or as in the conducting airway tree, effectively exclude the SVs. Burrowes et  al. [26, 29] modeled the supernumerary arteries and veins by assuming that they would arise from parent branches of diameter less than 1.5 mm [10] at approximately 90°, and branch rapidly until they terminated at the nearest pulmonary acinus. The estimated ratio of SVs to AVs (2.5:1 and 3.0:1 for the arteries and veins, respectively [65]) was used to define the number of SVs stemming from the accompanying branch model. The diameter of each SV was specified using a given fraction of the parent vessel diameter. Lengths were calculated using anatomical length-to-diameter ratios [33, 73] and the Strahler order of these new branches was determined on the basis of their diameter. Another criterion that guided the model derivation was that the resulting tree should have Strahler branching ratios close to 3.0 [73] and 3.3 [33] for the arterial and venous trees, respectively. The only computational studies we are aware of that explicitly consider SV function are those of Onuki and Nitta [74] and Burrowes et al. [29]. In both cases the consideration is minor: Onuki and Nitta [74] noted their concern that conventional models of the pulmonary vasculature ignore the SVs, and performed preliminary calculations of perfusion in a model of the dog lung that included SVs; Burrowes et al. [29] compared steady-state perfusion in models with full SV recruitment and with zero SV recruitment, showing that full SV perfusion would result in a far larger range of terminal vessel perfusion than for the model without SV recruitment. In other work, Burrowes et al. have assumed that none of the SVs are recruited, which could impact on the accuracy of the anatomical model distributions. SV function could be studied to a limited degree using summary models, but because the SVs are dynamically recruited structures and likely to be responsive to transmural pressures, the most insight will be gained in a subject-specific model that has a realistic relationship between the regionally varying tethering forces of the lung tissue and the spatially distributed blood vessels.

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6 Summary Computational models of the pulmonary vasculature come in many forms, but all with the common goal of providing insight into the function of the pulmonary circulation that cannot easily be gained through experimentation. Models of components of the pulmonary vasculature have been developed for at least the past 40 years, yet a comprehensive model that explains all of the basic features of regional pulmonary perfusion is still lacking. Medical imaging provides data from which subject-specific models of the geometry of the lung and its blood vessels can be derived. However, incorporating physiological function into these models is challenging because lung structure and function is multiscale (contrast, e.g., blood flow in the large vessels with two-phase cell and plasma transit in the microcirculation) and involves interdependency between lung tissue, blood vessels at each scale, and active mechanisms that contribute to vascular tone. Future computational models of the pulmonary vasculature will contribute to further understanding of basic lung physiological function (e.g., the role of the supernumerary vasculature), disease progression (e.g., estimating regional shear stress as a potential signal for vascular remodeling), and subject-specific function. Acknowledgements  The authors would like to thank collaborators at the University of Iowa and the Iowa Comprehensive Lung Imaging Center for access to imaging data from the Human Lung Atlas. Work by the authors was supported by the Woolf Fisher Trust (Maurice Paykel Post Doctoral Fellowship), the Engineering and Physical Sciences Research Council (EPSRC), and the National Institutes of Health through grant HL064368.

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M.H. Tawhai and K.S. Burrowes 9. Huang W, Tian Y, Gao J, Yen RT (1998) Comparison of theory and experiment in pulsatile flow in cat lung. Ann Biomed Eng 26:812–820 10. Weibel ER (1963) Morphometry of the human lung. Springer, Berlin 11. Dhadwal A, Wiggs B, Doerschuk C, Kamm R (1997) Effects of anatomic variability on blood flow and pressure gradients in the pulmonary circulation. J Appl Physiol 83:1711–1720 12. Huang Y, Doerschuk CM, Kamm RD (2001) Computational modeling of RBC and neutrophil transit through the pulmonary capillaries. J Appl Physiol 90:545–564 13. Sobin SS, Tremer HM, Fung YC (1970) Morphometric basis of the sheet-flow concept of the alveolar microcirculation in the cat. Circ Res 26:397–414 14. Pries AR, Secomb TW, Gaehtgens P (1996) Biophysical aspects of blood flow in the microvasculature. Cardiovasc Res 32:654–667 15. Secomb TW (1995) Mechanics of blood flow in the microcirculation. In: Ellington CP, Pedley TJ (eds) Biological fluid dynamics. Company of Biologists, Cambridge, pp 305–321 16. Fahraeus R, Lindqvist T (1931) The viscosity of the blood in narrow capillary tubes. J Appl Physiol 96:562–568 17. Fahraeus R (1929) The suspension stability of blood. Physiol Rev 9:241–274 18. Pries AR, Secomb TW, Gaehtgens P, Gross JF (1990) Blood flow in microvascular networks. Experiments and simulation. Circ Res 67: 826–834 19. Sobin SS, Fung YC, Tremer HM, Rosenquist TH (1972) Elasticity of the pulmonary microvascular sheet in the cat. Circ Res 30:440–450 20. Gan R, Yen R (1994) Vascular impedance analysis in dog lung with detailed morphometric and elasticity data. J Appl Physiol 77:706–717 21. Zhou Q, Gao J, Huang W, Yen M (2006) Vascular impedance analysis in human pulmonary circulation. Biomed Sci Instrum 42:470–475 22. Zhuang FY, Fung YC, Yen RT (1983) Analysis of blood flow in cat’s lung with detailed anatomical and elasticity data. J Appl Physiol 55:1341–1348 23. Terashima T, Klut ME, English D, Hards J, Hogg JC, van Eeden SF (1999) Cigarette smoking causes sequestration of polymorphonuclear leukocytes released from the bone marrow in lung microvessels. Am J Cell Mol Biol 20:171–177 24. West JB, Dollery CT (1960) Distribution of blood flow and ventilation-perfusion ratio in the lung, measured with radioactive carbon dioxide. J Appl Physiol 15:405–410 25. West JB, Dollery CT, Naimark A (1964) Distribution of blood flow in isolated lung; relation to vascular and alveolar pressures. J Appl Physiol 19:713–724 26. Burrowes KS (2005) An anatomically-based mathematical model of the human pulmonary circulation. The University of Auckland, Auckland 27. Parker JC, Cave CB, Ardell JL, Hamm CR, Williams SG (1997) Vascular tree structure affects lung blood flow heterogeneity simulated in three dimensions. J Appl Physiol 83:1370–1382 28. Dawson CA, Krenz GS, Karau KL, Haworth ST, Hanger CC, Linehan JH (1999) Structure-function relationships in the pulmonary arterial tree. J Appl Physiol 86:569–583 29. Burrowes KS, Hunter PJ, Tawhai MH (2005) Anatomically-based finite element models of the human pulmonary arterial and venous trees including supernumerary vessels. J Appl Physiol 99:731–738 30. Burrowes KS, Hunter PJ, Tawhai MH (2005) Investigation of the relative effects of vascular branching structure and gravity on pulmonary arterial blood flow heterogeneity via an image-based computational model. Acad Radiol 12:1464–1474 31. Burrowes KS, Tawhai MH (2006) Computational predictions of pulmonary blood flow gradients: gravity versus structure. Respir Physiol Neurobiol 154:515–523 32. Spilker RL, Feinstein JA, Parker DW, Reddy VM, Taylor CA (2007) Morphometry-based impedance boundary conditions for patientspecific modeling of blood flow in pulmonary arteries. Ann Biomed Eng 35:546–559

6  Modeling of the Pulmonary Vasculature 33. Horsfield K, Gordon WI (1981) Morphometry of pulmonary veins in man. Lung 159:211–218 34. Fung YC (1991) Dynamics of blood flow and pressure-flow relationship. In: Crystal RG, West JB (eds) The lung: scientific foundations. Raven, New York, pp 1121–1145 35. Weibel ER (1991) Fractal geometry: a design principle for living organisms. Am J Physiol 261:L361–L369 36. Glenny RW, Bernard SL, Robertson HT (2000) Pulmonary blood remains fractal down to the level of gas exchange. J Appl Physiol 89:742–748 37. Wagner WW Jr, Todoran TM, Tanabe N et  al (1999) Pulmonary capillary perfusion: intra-alveolar fractal patterns and interalveolar independence. J Appl Physiol 86:825–831 38. Horsfield K, Dart G, Olson DE, Filley GF, Cumming G (1971) Models of the human bronchial tree. J Appl Physiol 31:207–217 39. Krishnan A, Linehan JH, Rickaby DA, Dawson CA (1986) Cat lung hemodynamics: comparison of experimental results and model predictions. J Appl Physiol 61:2023–2034 40. Marshall BE, Marshall C (1988) A model for hypoxic constriction of the pulmonary circulation. J Appl Physiol 55:1341–1348 41. Lin C-L, Tawhai MH, McLennan G, Hoffman EA (2007) Characteristics of the turbulent laryngeal jet and its effect on airflow in the human intra-thoracic airways. Respir Physiol Neurobiol 157:295–309 42. Tawhai MH, Hunter PJ, Tschirren J, Reinhardt JM, McLennan G, Hoffman EA (2004) CT-based geometry analysis and finite element models of the human and ovine bronchial tree. J Appl Physiol 97: 2310–2321 43. Balásházy I, Hofmann W, Heistracher T (2003) Local particle deposition patterns may play a key role in the development of lung cancer. J Appl Physiol 94:1719–1725 44. Luo HY, Liu Y, Yang XL (2007) Particle deposition in obstructed airways. J Biomech 40:3096–3104 45. Yang XL, Liu Y, Luo HY (2006) Respiratory flow in obstructed airways. J Biomech 39:2743–2751 46. Zhang Z, Kleinstreuer C (2004) Airflow structures and nano-particle deposition in a human upper airway model. J Comput Phys 198:178–210 47. De Backer JW, Vos WG, Gorlé CD et al (2008) Flow analyses in the lower airways: patient-specific model and boundary conditions. Med Eng Phys 30:872–879 48. de Rochefort L, Vial L, Fodil R et al (2007) In vitro validation of computational fluid dynamic simulation in human proximal airways with hyperpolarized 3He magnetic resonance phase-contrast velocimetry. J Appl Physiol 102:2012–2023 49. Ford MD, Nikolov HN, Milner JS et al (2008) PIV-measured versus CFD-predicted flow dynamics in anatomically realistic cerebral aneurysm models. J Biomech Eng 130:021015 50. Suo J, Oshinski JN, Giddens DP (2008) Blood flow patterns in the proximal human coronary arteries: relationship to atherosclerotic plaque occurrence. Mol Cell Biomech 5:9–18 51. Tawhai MH, Pullan AJ, Hunter PJ (2000) Generation of an anatomically based three-dimensional model of the conducting airways. Ann Biomed Eng 28:793–802 52. Fernandez JW, Mithraratne P, Thrupp SF, Tawhai MH, Hunter PJ (2004) Anatomically based geometric modelling of the musculoskeletal system and other organs. Biomech Model Mechanobiol 2:139–155 53. Hopkins SR, Henderson AC, Levin DL et al (2007) Vertical gradients in regional lung density and perfusion in the supine human lung: the Slinky effect. J Appl Physiol 103:240–248 54. Prisk GK, Yamada K, Henderson AC et al (2007) Pulmonary perfusion in the prone and supine postures in the normal human lung. J Appl Physiol 103:883–894

103 55. Glenny RW, Bernard S, Robertson HT, Hlastala MP (1999) Gravity is an important but secondary determinant of regional pulmonary blood flow in upright primates. J Appl Physiol 86:623–632 56. Musch G, Layfield JDH, Harris RS et al (2002) Topographical distribution of pulmonary perfusion and ventilation, assessed by PET in supine and prone humans. J Appl Physiol 93:1841–1851 57. Krenz GS, Dawson CA (2003) Flow and pressure distributions in vascular networks consisting of distensible vessels. Am J Physiol Heart Circ Physiol 284:H2192–H2203 58. West JB (1999) How it really happened-distribution of pulmonary blood flow. Am J Respir Crit Care Med 160:1802–1803 59. Brudin LH, Rhodes CG, Valind SO, Jones T, Hughes JMB (1994) Interrelationships between regional blood flow, blood volume, and ventilation in supine humans. J Appl Physiol 76:1205–1210 60. Hlastala MP, Glenny RW (1999) Vascular structure determines pulmonary blood flow distribution. News Physiol Sci 14:182–186 61. Treppo S, Mijailovich SM, Venegas JG (1997) Contributions of pulmonary perfusion and ventilation to heterogeneity in VA/Q measured by PET. J Appl Physiol 82:1163–1176 62. Walther SM, Domino KB, Glenny RW, Hlastala MP (1997) Pulmonary blood flow distribution in sheep: effects of anesthesia, mechanical ventilation, and change in posture. Anesthesiology 87:335–342 63. Beck KC, Rehder K (1986) Differences in regional vascular conductances in isolated dog lungs. J Appl Physiol 61:530–538 64. Chon D, Beck KC, Larsen RL, Shikata H, Hoffman EA (2006) Regional pulmonary blood flow in dogs by 4D- x-ray CT. J Appl Physiol 101:1451–1465 65. Elliot FM, Reid L (1965) Some new facts about the pulmonary artery and its branching pattern. Clin Radiol 16:193–198 66. Bunton D, MacDonald A, Brown T, Tracey A, McGrath JC, Shaw AM (2000) 5-Hydroxytryptamine- and U46619-mediated vasoconstriction in bovine pulmonary conventional and supernumerary arteries: effect of endogenous nitric oxide. Clin Sci 98:81–89 67. Bunton DC, Fisher A, Shaw AM, MacDonald A, Montgomory I, McGrath JC (1996) Musculo-elastic structure at origin of pulmonary supernumerary artery resembles a baffle valve 119. Br J Pharmacol 119:323P 68. de Mello D, Reid LM (1991) Arteries and veins. In: Crystal RG, West JB, Barnes PJ, Cherniack NS, Weibel ER (eds) The lung: scientific foundations. Raven, New York, pp 767–778 69. Galiè N, Kim NHS (2006) Pulmonary microvascular disease in chronic thromboembolic pulmonary hypertension. Proc Am Thorac Soc 3:571–576 70. Ogata T, Iijima T (1993) Structure and pathogenesis of plexiform lesion in pulmonary hypertension. Chin Med J 106:45–48 71. Yaginuma G, Mohri H, Takahashi T (1990) Distribution of arterial lesions and collateral pathways in the pulmonary hypertension of congenital heart disease: a computer aided reconstruction study. Thorax 45:586–590 72. Shaw AM, Bunton DC, Fisher A et al (1999) V-shaped cushion at the origin of bovine pulmonary supernumerary arteries: structure and putative function. J Appl Physiol 87:2348–2356 73. Horsfield K (1978) Morphometry of the small pulmonary arteries in man. Circ Res 42:593–597 74. Onuki T, Nitta S (1993) Computer simulation of geometry and hemodynamics of canine pulmonary arteries. Ann Biomed Eng 21:107–115 75. Fung YC (1974) Fluid in the interstitial space of the pulmonary alveolar sheet. Microvasc Res 7:89–113 76. Hoffman EA, Clough AV, Christensen GE et al (2004) The comprehensive imaging-based analysis of the lung: a forum for team science. Acad Radiol 11:1370–1380

Chapter 7

Metabolic and Clearance Function at the Pulmonary Microvascular Endothelial Surface in Pulmonary Hypertension Stylianos E. Orfanos and David Langleben

Abstract  The intimal lining of all blood vessels is composed of a single continuous layer of squamous epithelial cells which are known as endothelial cells (ECs). The vascular endothelium is a “dynamic” highly specialized tissue with multiple physiological, immunological, and metabolic functions. Currently, the endothelium is viewed as a tissue comprising functionally heterogeneous ECs, depending on the organ with which it is associated, and the vascular bed location. ECs may be affected by numerous noxious agents and compounds, leading to diverse “endotheliopathies” present in a wide spectrum of acute and chronic abnormalities, such as sepsis, acute lung injury (ALI), and pulmonary arterial hypertension. In the human lung, ECs occupy a surface area of approximately 130 m2. The strategic location of the lungs, combined with the tremendous surface area of the pulmonary microvascular endothelium, allows for interaction with the entire circulating blood volume before it enters the systemic circulation. Thus, pulmonary endothelial functional and structural integrity are essential for the maintenance of adequate pulmonary and systemic cardiovascular homeostasis. This chapter focuses on the physiology and pathophysiology of the pulmonary endothelial metabolic functions, on methods that measure pulmonary microvascular metabolic activities in vivo, and on the application of such methods in the clinical setting, especially in patients suffering from pulmonary hypertension. Keywords  Endothelial cells • Pulmonary microvascular endothelium • Endothelium-derived factor • Lung metabolism

1 Introduction The intimal lining of all blood vessels is composed of a ­single continuous layer of squamous epithelial cells which are known as endothelial cells (ECs). For years the endothelium S.E. Orfanos (*) 2nd Department of Critical Care, University of Athens Medical School and Pulmonary Hypertension Clinic, Attikon Hospital, Haidari Athens 12462, Greece e-mail: [email protected]

was mostly viewed as a “static” semipermeable layer that separated blood from the interstitium and, in the lungs, blood from air. However, in the 1970s, techniques that allowed EC isolation and culture were developed. This event has since triggered extensive research on endothelial function, providing proof that the aforementioned “static” endothelial profile was false and that the vascular endothelium is instead a “dynamic” highly specialized tissue with multiple physiological, immunological, and metabolic functions [1]. Currently, the endothelium is viewed as a tissue comprising functionally heterogeneous ECs, depending on the organ with which it is associated, and the vascular bed location [2, 3]. ECs may be affected by numerous noxious agents and compounds, leading to diverse “endotheliopathies” present in a wide spectrum of acute and chronic disorders, such as sepsis, acute lung injury (ALI), and pulmonary arterial hypertension (PAH) [4]. The development of severe endotheliopathy will lead to the breakdown of normal cardiovascular homeostasis. In the human lung, ECs occupy a surface area of approximately 130 m2. The strategic location of the lungs, combined with the tremendous surface area of the pulmonary microvascular endothelium, allows for interaction with the entire circulating blood volume before it enters the systemic circulation [5]. Thus, pulmonary endothelial functional and structural integrity are essential for the maintenance of adequate pulmonary and systemic cardiovascular homeostasis. This chapter will focus on the physiology and pathophysiology of the pulmonary endothelial metabolic functions, on methods that measure pulmonary microvascular metabolic activities in vivo, and on the application of such methods in the clinical setting, especially in patients suffering from pulmonary hypertension (PH).

2 Pulmonary Endothelial Metabolic Properties The first insights into pulmonary endothelial metabolic function were provided using isolated perfused lung preparations from various animal models. However, it was the ­development

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of the EC culture techniques that enabled precise analysis of pulmonary endothelial physiological, immunological, synthetic, and substance-clearance functions. Well-studied important pulmonary endothelial activities are given in Table  1. Most of these functions are constitutive, whereas others are inducible, expressed after exposure to diverse EC-activating stimuli. In the healthy state, the major pulmonary endothelial properties are related to (1) the promotion of antiaggregation and hemofluidity, (2) barrier function enforcement, and (3) the synthesis, metabolism, or clearance of vasoactive compounds that possess endocrine and paracrine properties that modulate systemic and pulmonary vascular tone, respectively [5]. The pulmonary microvascular endothelium is the major site of pulmonary endothelial metabolic and clearance function, owing to its high ratio of vascular surface to blood volume, the high expression of several enzymes, and the relatively low pulmonary blood flow as compared with the larger extraalveolar vessels [1, 3, 5, 6]. A variety of biologically active compounds are processed, including (1) compounds metabolized at the endothelial surface without plasma uptake – angiotensin I, bradykinin, and adenosine nucleotides; (2) compounds metabolized inside the EC after uptake from plasma – endothelin, serotonin, norepinephrine, and prostaglandins E and F; (3) compounds synthesized within the EC and released into the blood – prostacyclin, prostaglandins E and F, clotting and fibrinolytic factors, lipoprotein lipase, nitric oxide, endothelin, and adrenomedullin; and (4) compounds unaffected by traver­ sing the lungs – epinephrine, prostaglandin A, angiotensin II, dopamine, vasopressin, oxytocin, histamine, vasoactive intestinal peptide, substance P, and prostacyclin [6, 7].

Table 1  Major pulmonary endothelial functions. (Modified with permission [5]) 1

2 3

4 5 6 7 8 9 10 11 12 13 14 15

Synthesis and release of several vasoactive compounds such as angiotensin II, prostacyclin, thromboxane, nitric oxide, and endothelins; regulation of vascular tone Clearance of bioactive peptides such as endothelin-1 Expression of enzymes such as angiotensin-converting enzyme, endothelin-converting enzyme, nucleotidases, nitric oxide synthase and lipoprotein lipase Expression of receptors and signal transduction molecules Cell-surface redox activity (transplasma membrane electron transport systems) Removal and biotransformation of drugs Regulation of coagulation and thrombolysis; promotion of hemofluidity Participation in immune reactions Binding of immune complexes Interaction with bacteria (phagocytosis) and blood components such as leukocytes and platelets Expression of adhesion molecules Production of growth factors Production of cytokines and chemokines Production of reactive oxygen species Barrier function

3 Enzymes Expressed on the Endothelial Surface: Ectoenzymes Several of the aforementioned pulmonary endothelial metabolic activities are due to enzymes bound on the luminal endothelial surface, with their catalytic sites exposed to the bloodstream. These ectoenzymes interact with blood-borne substrates and inhibitors, and their metabolic activity is highly efficient in that it does not require the time and energy expense that would be needed by interactions of circulating substrates with cytosolic enzymes. One such ectoenzyme is the angiotensin-converting enzyme (ACE) [1, 3]. ACE is a monomeric zinc dipeptidyl carboxypeptidase. It is present in most mammalian tissues, particularly on ECs, on the surface of absorptive epithelia, and in the central nervous system [8]. ACE catalyzes the conversion of angiotensin I to angiotensin II, and the degradation of bradykinin [9]. Angiotensin II is an octapeptide with vasoconstrictive and proinflammatory properties; it acts on vascular smooth muscle cells, interacts with the nervous system, and induces aldosterone release. Bradykinin is a nonapeptide with vasodilatory and proinflammatory properties. ACE is uniformly distributed along the luminal pulmonary endothelial surface area [10]. Several other pulmonary endothelial ectoenzymes are expressed in various species [11]. Among them are 5¢-nucleotidase, responsible for the dephosphorylation of extracellular adenosine 5¢-monophosphate to adenosine, a molecule with vasodilatory and antithrombogenic properties; various nucleotidases; aminopeptidase P, which contributes to the breakdown of bradykinin; lipoprotein lipase, which hydrolyzes triglycerides to fatty acids; and carbonic anhydrase, which acts on carbon dioxide [11]. An additional pulmonary endothelial ectoenzyme of paramount importance for the maintenance of pulmonary circulatory homeostasis is the endothelin-converting enzyme (ECE). ECE is expressed in several isoforms, and is involved in the production of endothelin-1 (ET-1), endothelin-2, and endothelin-3 from their precursor big endothelin [12, 13]. ET-1 is the most potent vasoconstrictor circulating in the body, also possessing proinflammatory and proliferative properties [12, 14]. It appears that the human lung is an important site for both production and clearance of ET-1 via the endothelial ETB receptor [7, 15]. Under normal conditions there is a dynamic balance between ET-1 clearance and release in the pulmonary circulation [7].

4 Pathological Features of Pulmonary Arterial Hypertension The updated classification of PH, a product of the fourth World Symposium of Experts on Pulmonary Hypertension held in 2008 in Dana Point, California, has recently been published  [16].

7  Metabolic and Clearance Function at the Pulmonary Microvascular Endothelial Surface in PH

Despite not being the most common form of PH, the PAH ­category (i.e., group 1) has been the main focus of all recent PH classifications. The pathological features of PAH, common to all its subgroups, consist principally of an excess of vascular cells (endothelial, smooth muscle), occlusion of the microvascular lumen, and formation of plexiform lesions [17]. The ­physical loss of perfusion through small precapillary arteries results in a loss of functional microvascular bed. This may be expected to compromise pulmonary endothelial metabolic and clearance functions. In addition, the endothelial dysfunction in PAH  may  contribute [18]. Interest in studying the effects of the ­above-mentioned abnormalities on the availability and function of ­pulmonary endothelial metabolic activity as well as studying the effects of PAH therapy on the availability and function of pulmonary endothelial metabolic activity has led to the ­development of techniques to accurately measure pulmonary microvascular metabolic function.

5 Assessment of Pulmonary Endothelial Metabolic and Clearance Functions Over the years, pulmonary endothelial function has been estimated in vivo, ex vivo, or in situ, principally by means of two methods. The first is estimation of the transpulmonary gradient of either a substrate or an inhibitor, or of a substance that is cleared by the pulmonary circulation. This is achieved by measuring the inflow and outflow concentrations of the compound under investigation, in mixed venous and aortic blood. The second method involves the application of radioisotopicindicator-dilution-type techniques. Both methods provide information on the metabolism or extraction from the pulmonary circulation of a certain compound; the indicator-dilution technique, however, can differentiate between endothelial dysfunction at the capillary level and loss of functional capillary surface area (FCSA), and can additionally provide information on lung transit time and volume of distribution of a compound, as well as measurements of blood flow [1, 8, 19, 20].

6 Transpulmonary Gradient Assessment The transpulmonary gradient technique, although “crude,” has been used in both animal and human investigations. Pulmonary metabolism studies by means of such techniques were performed in PH patients during the 1970s during early attempts to understand the disease [21]. Focusing on the investigation of pulmonary endothelial integrity in PH patients, Sole et al. investigated the extraction of circulating catecholamines by such patients, demonstrating that normal human lungs clear circulating norepinephrine, a feature lost from the pulmonary circulation of PH patients [22]. The

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authors were among the first to conclude that metabolic ­function of the lungs is compromised in PH. More recently, such studies in the human have provided essential scientific information in relation to ET-1-related pathobiology. Stewart et al. estimated arterial and venous plasma concentrations of ET-1 in patients suffering from PH of diverse origins and provided the first evidence that such patients exhibit higher venous ET-1 concentrations than normal volunteers [23]. Patients suffering from primary PH appeared to have increased pulmonary production of ET-1, implying a pathogenic role for ET-1 in the increased pulmonary vascular resistance [23]. This transpulmonary gradient may be modified by administration of prostacyclin therapy for PAH, suggesting one potential mechanism for the beneficial effects of prostanoids in PAH [24]. More recently, the influence of inhaled iloprost on the transpulmonary gradient of the ET-1 precursor big endothelin has been assessed [25]. A significant transpulmonary gradient for big endothelin was observed (i.e., higher concentration in the systemic circulation), and this was attenuated by iloprost inhalation. Application of the same technique in a patient cohort suffering from acute lung vasculopathy, namely, from ALI or acute respiratory distress syndrome (ARDS), provided ­evidence that these patients exhibited abnormal pulmonary ET-1 metabolism, expressed by an early decrease of the net ­balance between pulmonary ET-1 clearance and release. This ­phenomenon was reversible in patients who subsequently recovered [26]. More recently, angiotensin II formation and ET-1 clearance were assessed in ARDS patients put in supine and prone positions. Despite the acute improvement in patients’ oxygenation observed by positioning changes, short-term pulmonary ET-1 net clearance and angiotensin II net formation did not appear to change [27].

7 Indicator-Dilution Techniques for Pulmonary Metabolism Assessment Since the early 1980s, increasing attention has been focused on the monitoring of pulmonary capillary endothelial ectoenzyme activity in several animal models in vivo and in situ [28]. The pulmonary capillary bed provides an ideal environment for the function of these enzymes because it offers very high enzyme concentrations resulting from the combination of low plasma volume and high surface area (i.e., high enzyme mass available for reaction). The most common assay for measuring the activity of pulmonary endothelium-bound ectoenzymes in vivo is a modification of the indicator-dilution technique, originally introduced for cardiac output estimations. The most extensively studied ectoenzyme in animal models, and the sole ectoenzyme studied thus far in humans, is the pulmonary endotheliumbound ACE. Briefly, trace amounts of a radiolabeled substrate are injected as a rapid bolus into a central vein. Simultaneously, arterial blood is withdrawn by means of a peristaltic pump into

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a fraction collector, for the duration of a single transpulmonary passage. Blood is collected into tubes containing “stop” solution, to prevent further activity of plasma-soluble ACE. Blood samples are then collected and the amount of radioactivity associated with both the substrate that survived the single transpulmonary passage and with the formed product is quantified in each sample [1, 8, 20]. This technique allows estimations of very rapid interactions between substrate and the endotheliumbound enzyme, thus minimizing the contribution of plasmasoluble ACE. In addition, microvessels with a diameter of less than 20 mm (i.e., alveolar capillaries) appear to be responsible for the great majority of the product formed, owing to the very high local enzyme concentration. Thus, in this type of study, measurement of pulmonary ACE activity represents in practical terms pulmonary capillary endothelium-bound (PCEB) ACE activity [8].

7.1 ACE Synthetic Substrates

arterial plasma estimated in disintegrations per minute per ­milliliter, where [So] is the sum of the ­concentrations of 3H-BPAP and 3H-BPhe and [S] is the surviving substrate concentration, i.e., the concentration of 3H-BPAP. 3 H-BPAP utilization may also be calculated as percent transpulmonary metabolism (%M), also corrected for the cis-BPAP nonreactive fraction:

%M = 100 ×

7.2 Calculations of ACE Activity Parameters When trace amounts of 3H-BPAP are injected, substrate hydrolysis (v) proceeds under first-order reaction conditions. BPAP transpulmonary hydrolysis is then measured by applying the integrated Henri–Michaelis–Menten equation, corrected for a 7% nonreactive fraction (nrf = 0.07) of BPAP (cis isomer) [30, 31]

{

(

v = ln (1 − nrf) / ([S] / [Sº ]) − nrf

)} = [ E ] × t × k c

cat

/ K m (1)

with [E], tc, kcat, and Km being the capillary enzyme ­concentration, reaction time (capillary mean transit time), catalytic rate ­constant, and Michaelis–Menten constant, respectively. [So] and [S] are the initial and final substrate concentrations in the ­effluent

o

o

(2)

Both v and %M are reflections of ACE activity per capillary. Although v has the advantage of being directly proportional to all three factors that determine product formation (see equation 7.1), %M is a term that investigators and clinicians are more familiar with. The data obtained may be further analyzed utilizing the integrated Henri–Michaelis–Menten equation as modified by Catravas and White [32]:

In the mid-1970s, synthetic radiolabeled substrates with high specificity for ACE were introduced [29]. Unlike the natural ACE substrates angiotensin I and bradykinin, which are also substrates for other naturally occurring enzymes, these ­synthetic compounds could be quantified easily and rapidly. Furthermore, they were adequately reactive to allow measurable hydrolysis during a single transpulmonary passage in vivo. The first synthetic substrate to be used was benzoylPhe-Ala-Pro (BPAP). BPAP is also the only ACE substrate used thus far in human studies. ACE hydrolyzes BPAP to benzoyl-Phe (BPhe). BPAP is pharmacologically inactive, hemodynamically inactive, water soluble, and readily excreted in urine. Other synthetic ACE substrates include benzoylAla-Gly-Pro, benzoyl-Phe-Gly-Pro, and benzoyl-Phe-HisLeu, all of them having been used in animal and cell studies.

{([S ]− [S])/ ([S ]× (1 − nrf))}

Amax / K m = E × kcat / K m = Fp × v

(3)

Amax = E × kcat = Vmax × Qcap

(4)

where

with E, Fp, Vmax, and Qcap being the total enzyme mass, ­pulmonary plasma flow, maximal velocity, and capillary plasma volume, respectively. Amax/Km reflects ACE activity per vascular bed. Under physiological (normal) conditions, as long as the catalytic properties of the enzyme remain unchanged, Amax/Km is an index of PCEB ACE mass (equation 7.3) and, for ACE that is evenly distributed along the luminal endothelial surface, an index of dynamically perfused (i.e., accessible to substrate) capillary surface area (DPCSA). Under toxic conditions, when alterations of enzyme expression and/or kinetic constants may exist, Amax/Km should be viewed as an index of functional capillary surface area (FCSA) related to the enzyme mass available for reaction (the product of DPCSA and the enzyme mass expressed on the endothelial surface) and the enzyme kinetic constants. In recent human studies, Amax/Km has been replaced by the more “clinician-friendly” term FCSA. A more detailed analysis of the derivation of the equations and the additional parameters that can be estimated by this method have been provided elsewhere [20]. These techniques have been used extensively in various animal models; [5]; in the 1990s, they were introduced in humans [1, 20]. Representative arterial outflow ­concentration curves of total 3H (i.e., initial BPAP concentrations) and the surviving 3H-BPAP, obtained after a single substrate passage through the lungs, as well as the corresponding ­transpulmonary hydrolysis from a human volunteer with healthy lungs are ­presented in Fig. 1.

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suffering from ARDS [34]. No changes were seen in the former group, whereas the latter group exhibited decreases in both compound extractions that the authors attributed to the ARDSrelated endothelial injury. An additional study confirmed that serotonin pulmonary extraction decreases in patients with ARDS and correlates with the severity and the evolution of the syndrome [35]. However the above-mentioned studies failed to predict which patients at risk for ARDS would subsequently develop the syndrome; consequently, the utility of assessing lung serotonin metabolism as a tool of earlier ARDS diagnosis in the critical care setting has not been established [36]. Fig.  1  Effluent arterial concentration curves of total tritium (3H, circles) and of the synthetic angiotensin-converting enzyme (ACE) substrate benzoyl-Phe-Ala-Pro (BPAP) (squares) after injection in the right atrium of a representative human volunteer. BPAP hydrolysis (v; diamonds) was estimated from samples in which total radioactivity disintegrations per minute was more than 10 times the background number of disintegrations per minute. (From Orfanos et  al [20]. Reproduced with permission from Lippincott Williams & Wilkins)

8 Indicator-Dilution Technique for Pulmonary Endothelial Clearance Assessment 8.1 Initial Studies on Endothelial Carrier Proteins Indicator-dilution techniques have been used mainly to study the removal of several biologically active compounds from the pulmonary circulation. These involve carrier-mediated processes that require energy and, consequently, a metabolically healthy pulmonary endothelium. Such compounds included the biogenic amines norepinephrine and serotonin, prostaglandin E1, and propranolol [1]. It should be noted, however, that interpretation of these studies proved to be complex, since they needed to take into consideration endothelial transport properties, possible release of metabolites into the bloodstream, binding to blood elements, as well as correction for changes in blood flow [1, 21]. Despite these complexities, several human studies were performed; they all focused on acute lung abnormalities. Dargent et al. performed simultaneous measurements of serotonin and propranolol pulmonary extraction in patients before and after extracorporeal circulation for coronary bypass surgery [33]. No changes were seen in serotonin extraction; in contrast ­propranolol extraction decreased after surgery, whereas it increased in patients who received positive airway pressure. The authors concluded that propranolol extraction was a more sensitive index of lung functional changes in this patient setting. In a similar respect, Gillis et al. estimated prostaglandin E1 and 5-hydroxytryptamine pulmonary removal in patients undergoing cardiopulmonary bypass surgery and in patients

8.2 Indicator-Dilution Techniques for Assessment of ET-1 Clearance by the Pulmonary Endothelium Assessing the first-passage pulmonary clearance of ET-1 by means of indicator-dilution techniques has been introduced more recently [15, 37]. These techniques offer indices that provide information on the extraction occurring on the endothelial surface, and allow quantification of the functional vascular surface involved in the process. Thus, the kinetic and other related parameters obtained with this method provide information comparable with that obtained from pulmonary endothelial ectoenzyme activity determinations. The patient-related technique is similar to that for the BPAP studies in that it employs a centrally placed thermodilution venous catheter for tracer administration and a systemic arterial cannula for blood collection, as well as the peristaltic pump and fraction collector setup described earlier. The injectate contains radiolabeled ET-1 and Evans blue dye–albumin to serve as a plasmatic reference. Details of the preparation and injection can be found elsewhere [38]. Pulmonary blood flow is computed as

q0

Blood flow =

∫

∞

0

CAlb (t ) dt



(5)

where qo is the total amount of Evans blue dye bound ­albumin injected and the denominator represents the area under the fractional recovery versus time curve for the same tracer. The recirculating tracer is mathematically removed by linear extrapolation of the exponential downslope on a ­semilogarithmic plot. The mean tracer ET-1 extraction is calculated by the following equation: ∞



∫ C Ext = 1 − ∫ C

ET −1

0

∞

0

( t ) dt

Alb (t ) dt



(6)

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where the right-hand term represents the difference in the areas of the fractional recovery versus time curves for the tracer albumin and ET-1. The metabolic capacity of the lungs to clear ET-1 from the circulation can be estimated by computation of the permeability–surface area (PS) product for ET-1 removal. The PS product, equivalent to plasmatic clearance, is used to describe the unidirectional uptake of permeating tracers using the indicator-dilution technique [39, 40], and is an index of functional vascular surface area available for clearance. The PS product is stable over a wide range of pulmonary blood flow, despite the expected variations in mean tracer ET extraction [41]. The PS for ET-1 removal by the pulmonary circulation is computed as

In vivo assays under normal conditions have established the normal range of PCEB ACE activity values in several animal models. Importantly, they have additionally allowed ­estimations

of the perfused pulmonary capillary vascular bed. The correlation between changes in Amax/Km and DPCSA has been demonstrated in rabbits, dogs, guinea pigs, and sheep in vivo, under both anesthesia and exercise, and in isolated lung preparations [8]. This correlation is valid if the PCEB ACE kinetic constants are not affected by pulmonary blood flow increases (equation 7.3). We have provided evidence that this is indeed the case in animal studies, by coinjecting a substrate and an inhibitor at different blood flows and obtaining unchanged catalytic and binding ACE properties [1, 8, 19]. In all these studies, transpulmonary hydrolysis of all substrates used was independent of cardiac output, implying that higher pulmonary blood volumes are accommodated mainly through ­parallel recruitment of capillaries with similar enzyme concentrations and capillary transit times. In studies where pulmonary blood flow was increased beyond full recruitment, no additional changes in Amax/Km were observed, whereas substrate hydrolysis decreased mainly owing to decreased transit times through the fully recruited capillary bed [1, 42]. Twelve years ago we introduced the PCEB ACE activity indicator-dilution technique at the bedside, for human volunteers with no lung disease [20]. All subjects received two rapid bolus injections of BPAP through a pulmonary artery catheter: one into the vascular bed of both lungs; the other into the vascular bed of one lung (the right lung in all but one subject). Similar transpulmonary substrate hydrolysis and substrate percent metabolism were observed in one lung and both lungs (Fig.  2), suggesting homogeneous PCEB ACE concentrations and capillary transit times in both human lungs. As in the animals, BPAP hydrolysis and percent metabolism were independent of cardiac index, consistent with similar capillary transit times (tc) among individuals within

Fig. 2  Transpulmonary percent metabolism and hydrolysis (v) of the synthetic ACE substrate BPAP through both lungs and one lung (left panels), and corresponding Amax/Km given in absolute values and normalized per body surface area (BSA; Amax/Km/BSA; right panels). Data

are given as individual values and corresponding means ± the standard error of the mean (SEM). Double asterisk p < 0.01 versus both lungs. (From Orfanos et al [20]. Reproduced with permission from Lippincott Williams & Wilkins)



PS = Fp × ln(1 − Ext)

(7)

where Fp is the pulmonary plasma flow in milliliters per ­second and Ext is the mean tracer ET-1 extraction. From the clinical standpoint, the PS product for ET-1 might be considered analogous to the FCSA (Amax/Km) surface area estimation for BPAP metabolism, although their relative degrees of abnormality might differ in disease states.

9 Pulmonary Endothelial ACE Activity in the Nondiseased Lung

7  Metabolic and Clearance Function at the Pulmonary Microvascular Endothelial Surface in PH

the normal range of pulmonary blood flow at rest. Amax/Km in the right lung was 54% of the total Amax/Km in both lungs (Fig. 2), suggesting that, as in the animals, Amax/Km is also a reliable and quantifiable index of DPCSA in humans [20]. The validity of Amax/Km as a DPCSA index in humans was further confirmed in a pilot study, where repeated PCEB ACE activity determinations were performed in brain-dead subjects with no ALI, at different cardiac outputs in each subject. Unchanged substrate hydrolysis was observed over a wide range of cardiac output, whereas Amax/Km increased linearly with flow, in a pattern similar to that observed in animal studies [11]. In addition to the aforementioned studies that validated PCEB ACE activity in the nondiseased human lung, important pharmacological information was obtained by quantifying PCEB ACE inhibition in humans [43]. Enzyme activity assessment was performed before and twice after intravenous administration of enalaprilat in normotensive subjects. Treatment at the given dose had no significant effect on systemic arterial pressure, but produced reductions in both PCEB and serum ACE activities. Two hours after enalaprilat administration, PCEB ACE inhibition was maintained, whereas serum ACE inhibition was reduced, denoting a ­preferential PCEB ACE inhibitory effect of enalaprilat, and suggesting that the action of this drug may be primarily related to lung endothelial ACE inhibition.

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have been identified in animal studies where changes in ­alveolar pressure and acid–base imbalance affected PCEB ACE activity through changes in DPCSA [44, 45].

10.1 Pulmonary Endothelial ACE Activity in Acute Lung Abnormalities We have estimated PCEB ACE activity in 33 critically ill, mechanically ventilated patients belonging to high-risk groups for ARDS development and suffering from various degrees of ALI/ARDS with lung injury score (LIS) ranging from 0 to 3.7 [46]. Both BPAP transpulmonary hydrolysis (v) and Amax/Km decreased early during the ALI/ARDS continuum and were inversely related to LIS (i.e., to the severity of lung injury; Fig. 3). In contrast to the observations made in animals and humans with normal/healthy lungs, substrate hydrolysis decreased with increasing cardiac output, suggesting decreasing tc at higher cardiac output, a phenomenon probably reflecting the vascular loss occurring from occluded or obliterated vessels. Although Amax/Km increased with higher cardiac output in healthier survivors, such increases did not occur in the more

10 Pulmonary Endothelial ACE Activity Under Pathological Conditions Pulmonary endothelial injury may begin as a subtle metabolic dysfunction and then progress to overt structural alterations and cell death. Numerous studies performed over the last 25 years in models of acute and chronic lung injury have shown that PCEB ACE activity reduction is among the earliest signs of lung damage, preceding changes in all commonly measured parameters such as acid–base balance, gas exchange, hemodynamic parameters, increased permeability, and morphological changes at the light- and electronmicroscopy level [5, 8]. Assessing PCEB ACE activity in lung injury may help distinguish between abnormalities secondary to endothelial dysfunction per se and decreased pulmonary vascular surface area. If endothelial dysfunction is related to either decreased enzyme mass or kinetic constant alterations, then substrate hydrolysis would be altered (equation 7.1). In such a case, Amax/Km is an index of FCSA, related to both enzyme quantity and functional integrity. If loss of DPCSA occurs with neither endothelial dysfunction nor changes in capillary transit times, substrate hydrolysis would not change, whereas Amax/Km would decrease, since the enzyme mass available for reaction would be decreased (equation 7.3). Such patterns

Fig. 3  Hydrolysis of the synthetic ACE substrate BPAP of each patient (v, upper panel) and corresponding Amax/Km values correlate inversely with lung injury score, denoting that the clinical severity of acute lung injury/acute respiratory distress syndrome is related to the degree of pulmonary capillary endothelium-bound ACE activity depression and the loss of functional capillary surface area (FCSA). Linear curves represent least-squares lines. (From Orfanos et al [46]. Reproduced with permission from Lippincott Williams & Wilkins)

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severely ill, implying that in the latter higher blood volumes should have been accommodated mainly through dysfunctional capillaries, whereas no reserves of FCSA were available to be recruited. In our cohort, high Amax/Km values were associated with better survival, raising the interesting possibility that maintenance of high FCSA might be of value as an outcome predictor in ARDS [46]. PCEB ACE activity was more recently assessed in patients undergoing total knee arthroplasty, a surgical procedure often related to fat embolism syndrome after tourniquet release [47]. In most patients capillary recruitment appeared to occur after tourniquet release, a phenomenon that might represent a mechanism by which the lungs are able to accommodate the burden of emboli at the time of tourniquet release in this patient setting, thus preventing dramatic pulmonary hemodynamic alterations.

10.2 Pulmonary Endothelial ACE Activity in Chronic Lung Abnormalities PCEB ACE activity was assessed in subjects suffering from systemic sclerosis (SSc), an autoimmune disease where endotheliopathy is present, in an effort to investigate if pulmonary endothelial dysfunction is an early feature of the ­disease, preceding PH development and overt interstitial lung disease [48]. Two SSc subsets can be identified: the diffuse and the limited subset. PCEB ACE activity was found to be decreased at an early disease stage (absence of PH or pulmonary interstitial fibrosis) in both subsets, whereas this reduction appeared to be mostly related to the SSc subset (lower in the diffuse SSc subset) and the underlying pulmonary hemodynamic parameters. These results indicate that measuring PCEB ACE activity indices offers a means of detecting early pulmonary endothelial dysfunction that is less invasive than lung biopsy; such a technique might in the future assist the clinician in initiating early treatment and in monitoring its results. PCEB ACE activity was more recently assessed in patients suffering from PH from diverse origins. These studies are presented later in this chapter, along with related investigations of pulmonary ET-1 clearance.

10.3 Endothelial ACE Activity in Human Coronary Circulation Although assessing coronary ACE activity is not part of pulmonary metabolism studies, the study by Staniloae et al. provides the first related information obtained by means of indicator-dilution techniques at the bedside in humans [49]. Ten patients were studied and the enzyme activity indices obtained were compared with the patients’ atherosclerotic

S.E. Orfanos and D. Langleben

burdens. Patients exhibited a wide range of coronary ACE activity, whereas the metabolically active vascular surface area (an index similar to FCSA) correlated with myocardial blood flow but not with measures of atherosclerotic burden. It thus appears that coronary ACE activity may be safely assessed at the bedside in humans, providing a good marker of the perfused myocardial surface area. Further studies are needed to fully assess the limitations and the potential of this technique in clinical practice, but it may prove useful in studying changes in coronary perfusion in PH.

11 Pulmonary Endothelial Clearance of ET-1 In 1996, Dupuis et al., applying indicator-dilution techniques in the dog in vivo, provided proof that the ETB receptor of the pulmonary endothelium is completely and exclusively responsible for ET-1 removal from the pulmonary circulation [15]. In parallel, work by the same group identified human pulmonary circulation as an important site for both clearance and production of ET-1 [7]. Approximately half of the circulating ET-1 is cleared in a single transpulmonary passage, whereas a simultaneous equal production takes place. Disruption of this dynamic equilibrium either by reduced pulmonary clearance or increased ET-1 production might contribute to the increased circulating levels found in various cardiovascular diseases [7]. The aforementioned hypothesis has been tested in patients undergoing coronary artery bypass grafting (CABG) with or without cardiopulmonary bypass (i.e., beating heart procedure) [50]. Systemic ET-1 levels were found to be increased in patients who underwent cardiopulmonary bypass. Importantly, the capacity to clear ET-1 from the pulmonary circulation, as derived from the PS product for the compound, was unaffected by cardiopulmonary bypass before and after CABG; it was also found to be similar and unaffected in the beating heart group. Taken together, the above information implies that cardiopulmonary bypass is associated with increased ET-1 release by the pulmonary circulation.

12 Pulmonary Endothelial ACE Activity and ET-1 Clearance Studies in PH Patients The first description of PCEB ACE activity in patients suffering from PH was provided in our initial proof-of-concept clinical study [20]. However, the small number of patients included and the fact that their PH origins were diverse did not permit definite conclusions to be drawn. A more recent study of the effects of PAH on PCEB ACE activity also examined potential differences between two common PAH

7  Metabolic and Clearance Function at the Pulmonary Microvascular Endothelial Surface in PH

subtypes, namely, PAH related to connective tissue disease (PAH-CTD) and idiopathic PAH (IPAH) [51]. Nineteen patients with PAH-CTD and 25 patients with IPAH, who had not at the time received any PAH-specific treatment, were evaluated by means of indicator-dilution-type techniques. The patients’ enzyme activity parameters were compared with those of 23 control subjects with no past or present lung disease. Substrate percent metabolism and hydrolysis, both indices reflecting enzyme activity per capillary, were reduced in PAH-CTD patients, as compared with both IPAH and control subjects (Fig.  4; upper and middle panels, respectively), whereas no differences were observed between IPAH and control subjects. The FCSA (originally termed Amax/Km) normalized to body surface area (BSA) was significantly reduced in both IPAH and PAH-CTD patients as compared with healthy volunteers (Fig. 4; lower panel). The decreases in substrate percent metabolism and hydrolysis obtained in the PAH-CTD subjects could be related to capillary transit time reductions (i.e., reaction time reductions; (equation 7.1)), secondary to vascular remodeling. However, such a phenomenon should also produce inverse relationships of the above-mentioned indices to cardiac output [1], which was not the case. It thus appears that pulmonary endothelial ACE dysfunction or loss is present in PAH-CTD, raising the intriguing probability that the two PAH subgroups posses different pulmonary endothelial phenotypes. In contrast with the findings discussed above, FCSA was equally reduced in both PAH groups, reflecting the PH-related small vessel loss and remodeling. Additional important information is given by the fact that PAH-CTD patients exhibited a linear relationship between FCSA/BSA and carbon monoxide diffusing capacity (DLCO), providing the first functional evidence that the reduced DLCO values in PAH-CTD are related to the degree of FCSA loss [51]. Applying similar techniques, we additionally studied pulmonary endothelial metabolic function in patients with chronic thromboembolic PH (CTEPH) [52]. CTEPH patients exhibited FCSA reductions, whereas substrate percent metabolism and hydrolysis were preserved. Taken together, these findings imply a reduction of the metabolically functional pulmonary capillary bed rather than reduced ACE activity on the pulmonary EC. This reduction might just be the result of decreased capillary recruitment because of upstream vascular plugging by organized thrombus. In such a case, this abnormality should be corrected after pulmonary thromboendarterectomy, a hypothesis to be tested in future studies [52]. In a study that complements the previous two investigations, the first-pass pulmonary ET-1 clearance was assessed in patients suffering from IPAH, PAH-CTD, and CTEPH [38]. Nineteen patients with IPAH, 15 patients with PAHCTD, and 11 CTEPH patients were studied and compared with patients without PH or lung disease. When ET-1 extraction and the PS product were examined, patients with CTEPH

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Fig.  4  Percent metabolism (upper panel) and hydrolysis (v; middle panel) of the synthetic ACE substrate BPAP, along with the corresponding FCSA (i.e., Amax/Km) normalized to BSA (lower panel) in patients with idiopathic pulmonary arterial hypertension (IPAH), pulmonary arterial hypertension related to connective tissue disease (PAH-CTD), and in normal controls. Symbols represent individual subjects; bars show the means ± SEM for each group. Asterisk p < 0.05 versus the other groups in the upper panel and the middle panel and p < 0.05 versus controls in the lower panel. (From Langleben et al [51]. Reproduced with permission from Lippincott Williams & Wilkins)

were similar to normal patients. However, whereas there was a greater data spread within the IPAH and PAH-CTD groups, the great majority of patients in those groups had normal ­levels of extraction and PS products (Fig. 5). Even in patients where the clearance activities were reduced, they were never

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13 Conclusion Studies of pulmonary endothelial metabolism and clearance have been providing important information on pulmonary vascular physiological and pathobiological features in diverse acute and chronic lung abnormalities. Recent investigations have increased our knowledge of the pathophysiological features of PH and offer promising information for future applications in the choice of treatment and the subsequent monitoring of its effects. For instance, the observation that most PAH patients maintain normal levels of functional ETB receptors whereas others have lower levels [38] leads to the intriguing thought that in the future the choice of ET-receptor antagonist might be related to the pulmonary endothelial phenotype of each patient. Although the indicator-dilution techniques are still arduous procedures, there is evidence that they might be replaced by the much simpler “one sample” technique [55] in an effort to develop clinically easy tests for the diagnosis and treatment of pulmonary vascular disease.

References Fig. 5  Endothelin-1 extraction (upper panel) and permeability surface product (lower panel) in patients with IPAH, PAH-CTD, chronic thromboembolic PH, and in normal controls. (From Langleben et al [38]. Reproduced with permission from Lippincott Williams & Wilkins)

absent. These data establish that despite the presumed endothelial dysfunction in PAH, the endothelial ETB receptor is present and functional in most patients with PAH. Dupuis et al. have additionally quantified ET-1 clearance and production in patients with PH related to left-sided heart disease and in controls [53]. Aortic versus pulmonary arterial ET-1 levels tended to be higher in the PH patients, whereas pulmonary extraction was reduced and correlated inversely with the severity of PH. It thus appears that the PH-associated reduction of pulmonary ET-1 clearance contributes to its increased circulating levels. In a similar respect, Staniloae et al. studied pulmonary ET-1 clearance in congestive heart failure (CHF) [54]. ET-1 pulmonary clearance was reduced and correlated inversely with mean pulmonary arterial and pulmonary wedge pressures. Plasma ET-1 levels depicted a strong correlation with right atrial pressure and N-terminal pro-brain natriuretic peptide levels. This interesting study could not differentiate if the observed reduction of ET-1 clearance in congestive heart failure in relation to PH severity is a marker of the latter or if it contributes to the related pulmonary vascular pathobiological features.

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Chapter 8

The Influence of the Major Vasoactive Mediators Relevant to the Pathogenesis of Pulmonary Hypertension Margaret R. MacLean and Yvonne Dempsie

Abstract  The pulmonary circulation exhibits unique responses to vasoactive factors compared with the systemic circulation owing to the many differences in the structure and function of the systemic and pulmonary circulations. The pulmonary circulation is normally virtually fully vasodilated, whereas the systemic circulation is tonically vasoconstricted. This in itself imparts a unique responses to vasoactive factors to the pulmonary arterial bed as vascular tone per se alters the responses to many vasoactive factors. In addition, in vivo and in vitro studies suggest that the pulmonary arterial circulation is composed of phenotypically distinct heterogeneous populations of smooth muscle cells with unique developmental lineages. They differ in their vasoactivity and ability to proliferate and therefore there are regional differences in the responses to many vasoactive factors between the smaller, distal pulmonary resistance arteries and the larger, proximal elastic pulmonary arteries. This differential responsiveness might be expected as the proximal and distal pulmonary arteries have differential developmental origins and proximal pulmonary arteries develop via angiogenesis, whereas distal vessels form by vasculogenesis. There are differences between the proximal and distal pulmonary arteries, for example, in the distribution of K+ channels, endothelin receptors, prostacyclin synthase, nitric oxide (NO) production, NO synthase expression, phosphodiesterase activity, and store-operated Ca2+ entry. This chapter describes the normal pharmacology of the major endogenous vasoactive factors involved in the pathogenic mechanisms of pulmonary arterial hypertension, pulmonary artery regional diversity, and how this alters following experimental and clinical pulmonary hypertension. Keywords  Pulmonary pharmacology • Agonist and antagonist • Receptor • Second messenger • Signal transduction • Vasodilation • Vasoconstriction

M.R. MacLean (*) Mandy MacLean College of Medical, Veterinary and Life Sciences, University of Glasgow, G12 8QQ, UK e-mail: [email protected]

1 Introduction The pulmonary circulation exhibits unique responses to vasoactive factors compared with the systemic circulation owing to the many differences in the structure and function of the systemic and pulmonary circulations. The pulmonary circulation is normally virtually fully vasodilated, whereas the systemic circulation is tonically vasoconstricted. This in itself imparts a unique responses to vasoactive factors to the pulmonary arterial bed as vascular tone per se alters the responses to many vasoactive factors [1]. In addition, in vivo and in vitro studies suggest that the pulmonary arterial circulation is composed of phenotypically distinct heterogeneous populations of smooth muscle cells with unique developmental lineages [2, 3]. They differ in their vasoactivity and ability to proliferate and therefore there are regional differences in the responses to many vasoactive factors between the smaller, distal pulmonary resistance arteries and the larger, proximal elastic pulmonary arteries (PAs). This differential responsiveness might be expected as the proximal and distal PAs have differential developmental origins and proximal PAs develop via angiogenesis, whereas distal vessels form by vasculogenesis [4]. There are differences between the proximal and distal PAs, for example, in the distribution of potassium (K+) channels, endothelin (ET) receptors, prostacyclin (PGI2) synthase, nitric oxide (NO) production, NO synthase (NOS) expression, phosphodiesterase (PDE) activity, and store-operated Ca2+ entry (see below) [5–15]. These differences may also contribute to the observation that hypoxic pulmonary vasoconstriction (HPV) is greater in distal than in proximal PAs [16]. The differences in major vasoconstrictor (pro-proliferative) and vasodilator (antiproliferative) influences in distal and proximal PAs are summarized in Fig. 1. Although this list is by no means complete, it is clear from Fig. 1 that, compared with proximal PAs, the distal PAs have less capability to produce endogenous vasodilators such as NO and PGI2 and are more prone to HPV via voltage-gated K+ channels (also known as delayed-­rectifier K+ channels) (Kv/KDR) and store-operated Ca2+ channels (SOCC). This must make

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_8, © Springer Science+Business Media, LLC 2011

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Fig. 1  Distribution of vasoactive influences in the proximal and distal pulmonary arteries. Note that distal pulmonary arteries have less capability to produce endogenous vasodilators such as NO and prostacyclin (PGI2) and are more prone to hypoxic pulmonary vasoconstriction via voltage-gated potassium channels (also known as delayedrectifier potassium channels) and store-operated Ca2+ channels. This must facilitate vasoconstriction and remodeling in the face of hypoxia and other insults consistent with pulmonary arterial hypertension (PAH) being considered a disease of the distal pulmonary arteries

Fig.  2  Normal pulmonary vascular tone is low owing to a balance between vasoconstrictor and vasodilator influences. Many of these have proliferative and antiproliferative effects also. In PAH there is an increase in vasoconstrictor/proliferative influences (particularly in the

distal pulmonary arteries, which have less endogenous “protection”; see Fig. 1) and this predisposes pulmonary arteries to vasoconstriction and remodeling. BMPRII bone morphogenetic protein receptor 2, PDE phosphodiesterase, PDGF platelet-derived growth factor

them most vulnerable to vasoconstriction and remodeling in the face of hypoxia and other insults. This is consistent with pulmonary arterial hypertension (PAH) being considered a disease of the distal PAs. The vascular tone of the PAs is regulated largely by the endothelium and the smooth muscle cell phenotype. A spectrum of vasoactive substances is synthesized in the endothelium; of these, NO and PGI2 are vasodilators, whereas serotonin (also known as 5-hydroxytryptamine, or 5-HT) and ET-1 are vasoconstrictors. Recent studies have also suggested that NO and PGI2 may also be antiproliferative [17, 18], whereas serotonin and ET-1 are mitogenic [19, 20]. Pulmonary vascular tone is regulated by the influence of many endogenous vasoactive factors, such as serotonin, ET-1, platelet-derived growth factor (PDGF), and

bone morphogenetic proteins, (BMPs) as well as the activity of K+ channels and calcium. The normally low pulmonary vascular tone relies on the endogenous vasodilators maintaining a vasodilated state. However, increased pulmonary vascular tone can ensue in the face of a pulmonary hypertensive insult owing to the balance of vasodilator influence being overcome by increased activity of the endogenous vasoconstrictors. This is frequently also accompanied by increased activity of proliferative agents contributing to pulmonary vascular remodeling (Fig.  2). This review will describe the normal activity of the major endogenous vasoactive factors involved in the development of PAH, PA regional diversity, and how this alters following experimental and clinical PAH. These factors are summarized in Fig. 3.

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Fig.  3  The major vasoactive factors controlling pulmonary vascular tone and involved in the development of PAH. There is endothelial synthesis and abluminal release of serotonin (5-HT), endothelin-1 (ET-1), PGI2, and NO. A rise in cytosolic Ca2+ concentration in pulmonary arterial smooth muscle cells (PASMCs) is due to the stimulation of receptors for 5-HT, ET-1, PGI2, and PDGF, decreased Kv channel activity, membrane depolarization which opens voltage-dependent Ca2+ channels, and activation of receptor-operated Ca2+ channels. Downstream signaling cascades cause pulmonary vasoconstriction and stimulate PASMC proliferation. The bone morphogenetic protein (BMP)signaling pathway leads to antiproliferative and proapoptotic effects on PASMCs. Dysfunction of BMP signaling due to BMPRII mutation and

BMPRII/BMPRIa downregulation attenuate PASMC apoptosis and promote PASMC proliferation. NO and PGI2 synthesis in pulmonary artery endothelial cells has a relaxing effect on pulmonary arteries via accumulation of cylic GMP and cyclic AMP, respectively. Specific PDE isoforms hydrolyze the PDEs and these offer novel therapeutic targets. Activity and expression of the serotonin transporter serves as an additional pathway to stimulate PASMC growth via the mitogen activated protein kinase (MAPK), reactive oxygen species (ROS), and Rho kinase (ROCK) pathways. These pathways can also be activated by receptors such as the 5-HT1B receptor. DAG diacylglycerol, GPCR G-proteincoupled receptor, IP3 inositol 1,4,5-trisphosphate, PKC protein kinase C, PLC phospholipase C

2 Endothelial-Derived Constricting/Proliferative Factors

circulating cells. Eighty percent of ET-1 is secreted to the abluminal side of the endothelial cells and hence ET-1 is likely to act on PASMCs in a paracrine fashion. In humans, ETs mediate their actions via two G-protein-coupled receptor types that have been cloned and classified as ETA and ETB [23]. ETA receptors are located primarily on vascular smooth muscle cells, and ETB receptors are expressed on both endothelial and vascular smooth muscle cells. Both ETA and ETB receptors can mediate vascular smooth muscle cell proliferation and vasoconstriction. In addition, ETB receptors on endothelial cells mediate release of vasodilator and antiproliferative modulators, including NO and PGI2, offsetting many of the effects of ETA receptor activation. ETB receptors also mediate ET-1 clearance in the lung [24]. Thus, understanding the balance between ET receptor expression and activities is

2.1 Endothelin-1 As described in Chapters 48, 58, and 105, increased levels of plasma ET-1, ET-1 expression, and ET-1 activity have been associated with PAH [21, 22]. In mammals, the ET family comprises three endogenous isoforms, ET-1, ET-2, and ET-3. ET-1 is the principal isoform in the human cardiovascular system and is a potent and long-lasting constrictor of human vessels. The ETs are synthesized in the endothelium and to a lesser degree they are also secreted by other cell types, including PA smooth muscle cells (PASMCs), PA fibroblasts, and

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critical for determining therapeutic targets in diseases. ET receptors continue to be classified according to their rank order of potency for the three endogenous isoforms of ET. All three ligands have equal potency at the ETB ­receptor, whereas ET-1 is the more potent ligand at the ETA receptor [23]. 2.1.1 ET Receptors in the PA ET-1 is a very potent vasoconstrictor of PAs. Constriction in human large-conduit PAs has been shown to be mediated by the ETA receptor [25]. However, in human small pulmonary resistance arteries it was shown that constriction is mediated by both the ETA receptor and the ETB receptor [26]. Responses to ET-1 up to 1 nM are inhibited by the ETB antagonist BQ 788 but not by ETA antagonists. Responses to higher concentrations are inhibited by the ETA antagonists but not BQ788. It was subsequently shown that the ETA/ETB antagonist SB 209670 virtually abolishes the contractile response to ET-1 in human small PAs [27, 28]. This differential distribution was subsequently confirmed in PASMCs isolated from distal and proximal PAs where both receptors promote the proliferation of PASMCs in vitro and may contribute to vascular remodeling in PAH [5]. Indeed bosentan (Tracleer) is an ETA/ETB antagonist and its clinical effects have been attributed to both its ability to inhibit pulmonary cell proliferation and migration and its inhibition of ET-1-induced pulmonary vasoconstriction [29]. In the rat there is a similar heterogeneity in the distribution of ET receptors. ET-1 vasoconstriction in the isolated perfused rat lung is mediated by both ETA and ETB receptors [30]. In the large elastic PA of the rat, it is the ETA receptor that mediates vasoconstriction, whereas responses in the small resistance arteries were more typical of an ETBmediated response [6]. In the rat small resistance arteries the order of potency of agonists was sarafotoxin S6C (an ETB agonist) = ET-3 > ET-1, which suggested an “atypical” ETB-like receptor [27]. In addition, the response to ET-1 is resistant to an ETA antagonist, an ETB antagonist, bosentan, and the combination of an ETB and an ETA antagonist, whereas SB 209670 is able to inhibit responses to ET-1. A similar profile of responses to agonist and antagonists has been demonstrated in human bronchus [31]. There is also a very similar “atypical” ET receptor profile in rabbit small PAs [32] whereas in the rabbit larger PAs, ETA and ETB receptors coexist and function in a synergistic fashion [33]. However, there is no evidence for the gene or protein expression of a third ET receptor and it is thought that these phenomena are likely to be due to differences in the kinetic profiles of ETA and ETB binding to agonists and antagonists. In PASMCs from PAH patients there is an increase in ETA and ETB receptor density in distal PAs [5]. Hypoxia causes a redistribution of PASMC phenotypes and accordingly ET receptor pharmacology of PAs changes after exposure to chronic hypoxia. For example, in the chronic hypoxic rat,

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there is a marked increase in the contractile response to ET-1 in the small PAs and this is mediated by the ETA receptor [27], which is consistent with a redistribution of PASMC phenotype from the larger elastic PAs to the more distal PAs [6]. In addition, in the proximal PAs of the chronic hypoxic rat there is an increase in ETA-mediated vasoconstriction as well as ETB-mediated vasoconstriction [34], consistent with the observed increase in ETA- and ETB-receptor messenger RNA (mRNA) expression [35]. Chapter 105 discusses the pros and cons of using either a selective ETA receptor antagonist or nonselective ETA/ ETB antagonists for the treatment of PAH. This will be dictated by the distribution and expression of the ET receptors, which is likely to vary from patient to patient. It is also confounded by the observation that both receptors can mediate constriction and proliferation and that the ETB receptor is responsible for pulmonary ET-1 removal in vivo and blockage of this is likely to be responsible for the rise in the level of plasma ET-1 observed following ETB blockade [24].

2.2 Serotonin The “serotonin hypothesis of anorexigen-induced pulmonary hypertension” arose when it was hypothesized that elevations in the levels of circulating serotonin, secondary to release from platelets, might be involved in anorexigeninduced PAH. Consistent with this, in the 1990s there were reports of elevated plasma levels in patients with PAH as well as PAH occurring in patients with platelet storage pool disease [36, 37]. There is much evidence, however, that serotonin per se may facilitate familial PAH (FPAH) and idiopathic PAH (IPAH) via alterations in various components of the serotonin system [38, 39]. There are four main components of the serotonin system in the pulmonary arterial bed: the serotonin receptor, the serotonin transporter (SERT), the synthesis of serotonin via tryptophan hydroxylase 1, and downstream signaling pathways that are activated following SERT or serotonin receptor activation.

2.2.1 Serotonin Receptors There are 14 different, structurally distinct serotonin receptors, which are divided up into seven families (5-HT1–7) [40]. It is the 5-HT2A receptor that mediates vasoconstriction in most systemic arteries and, prior to 1993 (see below), it had been assumed that it was 5-HT2A that mediated PA vasoconstriction. The 5-HT2A antagonist ketanserin has proved clinically effective in the treatment of systemic hypertension, especially in the elderly [41]. However, in studies on small cohorts of PAH patients, ketanserin at high doses had only a very small effect on pulmonary vascular

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resistance compared with its effect on systemic vascular resistance and often had no effect on pulmonary pressures [42]. Hence, there is no specificity for the pulmonary circulation and the systemic effects have limited its use in PAH, where it fails to improve pulmonary hemodynamics significantly [41]. However, the effectiveness of ketanserin assumes that the 5-HT2A receptor mediates the pulmonary effects of serotonin in man. In 1993, MacIntyre et al. ­[43] conducted an assessment of the anti-migraine drug sumatriptan (a 5-HT1B/D agonist) on the systemic and pulmonary circulations and the coronary artery vasculature. In this study, sumatriptan had a profound effect on pulmonary pressures and resistance. It was subsequently shown that the 5-HT1B receptor mediates serotonin-induced vasoconstriction in isolated human small and large PAs [44, 45]. An increased expression of the 5-HT1B receptor was subsequently demonstrated in a small cohort of PAH patients [46]. 5-HT1B receptors are negatively coupled to adenylate cyclase via Gi protein and many factors present in PAH (increased vascular tone, decreased NOS) can potentiate responses to 5-HT1B agonists via pharmacological synergy [1]. Subsequent studies in experimental ­animals have shown that, in mice and rats, respectively, if the 5-HT1B receptor is knocked out or the animal is treated with a 5-HT1B antagonist, there is a reduction in the development of hypoxia-induced PAH [47]. In chronic overcirculationinduced PAH in growing piglets, there is also an increased expression of the 5-HT1B receptor [48]. It has also recently been shown that the 5-HT1B receptor can mediate proliferation in human PASMCs [49]. The second messenger complex inositol 1,4,5-trisphosphate (IP3)/diacylglycerol is formed from the hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C after activation by Gq (Fig. 3). Via Gq activation, 5-HT2 receptors may also mediate some vascular effects of serotonin, at least in animal models. In rat PASMCs, for example, the 5-HT2A receptor inhibits native Kv and hKv1.5 currents and this may contribute to vasoconstriction [50]. In rat PA fibroblasts, the 5-HT2A receptor also mediates hypoxia-associated serotinin proliferation [51]. The development of hypoxia-induced PAH in mice is ablated in 5HT2B-receptor knockout mice [46] and this receptor may control serotonin plasma levels in mice [52]. Loss of serotonin 5HT2B receptor function, however, may predispose to fenfluramine-associated IPAH in man [53].

2.2.2 The SERT The SERT is particularly highly expressed in the lung and placenta. A single gene encodes the SERT, which is located on chromosome 17q11.2, and SERT expression and function are controlled by a repetitive element of varying length in the promoter region of the gene. Alleles are commonly

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composed of either 14 (short) or 16 (long) repeated elements. The long (L) allele induces a higher rate of SERT gene transcription than the short (S) allele, and is also associated with increased SERT uptake and mRNA and protein expression and activity. The “LL” variant of the SERT was found to be more prevalent in a small group of IPAH patients than in controls [54]. Studies in larger cohorts of PAH patients, however, found no variation in the prevalence of the LL allele of the SERT gene between controls and patients with IPAH or FPAH [55, 56]. One study suggests, however, that patients with FPAH who have the LL variant of the SERT may, however, present earlier than those without it [56]. Experimentally, there is evidence for a role for the SERT in the development of PAH. Mice deficient for the SERT are less susceptible to hypoxia-induced PAH, and develop less hypoxic-induced pulmonary vascular remodeling and right ventricular hypertrophy than wild-type mice [57]. Mice ubiquitously overexpressing the SERT (SERT+ mice) exhibit elevated pulmonary pressures and exaggerated hypoxia-induced PAH, which is associated with elevated hypoxic-induced pulmonary vascular remodeling and right ventricular hypertrophy [58]. Mice which selectively overexpress the SERT in smooth muscle cells also exhibit a similar phenotype [59]. PASMCs derived from IPAH and secondary PAH patients have increased expression of the SERT compared with those from controls, and the proliferative effects of serotonin are exaggerated and abolished by SERT inhibitors [60]. The SERT has also been shown to be involved in proliferation of human, bovine, and rodent PASMCs and PA fibroblasts [49, 51, 61, 62]. There is also evidence that SERT activity can cooperate with the 5-HT1B receptor in the modulation of both vasoconstriction and proliferation [49, 63]. The downstream transcriptional factor GATA-4 mediates serotonin-induced growth of PASMCs [39]. Upstream, signal transduction initiated by serotonin involves SERTdependent generation of reactive oxygen species (ROS) and activation of the extracellular-regulated kinase (ERK) pathway in PASMCs and PA fibroblasts from many species, including human [49, 61, 64]. The small G protein RhoA and its downstream effector Rho kinase (ROCK) play a central role in multiple cellular functions, including proliferation and migration, smooth muscle contraction, and cytoskeletal rearrangement, and ROCK has been implicated in the development of PAH. In platelets and aortic smooth muscle cells, internalized serotonin is transamidated to small GTPases such as Rho A by transglutaminases, rendering these GTPases constitutively active [65, 66]. Interestingly, in PAs (not systemic arteries) from hypoxic rats, when serotonin was transamidated to RhoA, this effect was blocked by the SERT inhibitor fluoxetine [65]. Both ROS and ROCK are involved in the activation of ERK1/2. Studies in human PASMCs have shown ERK1/2 activation to occur via activation of the 5-HT1B receptor, with

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ROS responsible for nuclear translocation of phosphorylated ERK1/2 [49]. In bovine PASMCs, ROS mediates phosphorylation of ERK1/2, whereas activation of ROCK via the 5-HT1B receptor mediates nuclear translocation of phosphorylated ERK1/2 [62]. In Chinese hamster lung fibroblasts, ROCK has been shown to function downstream of the SERT in allowing efficient 5-HT1B-receptor-stimulated mitogenactivated protein kinase/ERK kinase (MEK) phosphorylation of ERK1/2 [67].

2.2.3 Serotonin Synthesis Tryptophan hydroxylase (Tph) catalyzes the rate-limiting step in the synthesis of serotonin from tryptophan. There are two isoforms of Tph, now classified as Tph1 and Tph2 [68]. Tph2 is present exclusively in the brain but not the periphery. The classical Tph gene, now termed Tph1, is mainly expressed in the gut and mediates the generation of serotonin in the peri­ phery [68]. There are dynamic interactions between the PA endothelium and the underlying PASMCs. With respect to serotonin, recent studies have suggested that angiopoietin 1 can stimulate serotonin synthesis in pulmonary endothelial cells via the TIE2 receptor pathway [69] (Fig. 3). Expression of the Tph1 gene is increased in lungs and pulmonary endothelial cells from patients with IPAH [70]. Hence, endotheliumderived serotonin may then act on the underlying PASMCs in a paracrine fashion. Experimentally, de novo serotonin synthesis is required for the development of PAH. For example, hypoxia-induced PAH is ablated in Tph1−/− mice devoid of peripheral serotonin synthesis [71]. Dexfenfluramine-induced PAH is also ablated in Tph1−/− mice [72]. We have also recently shown that in normoxic mice there is little evidence for expression of Tph1 in PA endothelial cells. However, after 2 weeks of hypoxia, there is substantial Tph1 expression, suggesting that hypoxia induces Tph1 expression and de novo synthesis of serotonin. This is consistent with the observation that serotonin is overproduced in lungs and endothelial cells from patients with IPAH [70]. Thus, endothelium-derived serotonin may act on underlying PASMCs in a paracrine fashion.

3 Endothelium-Derived Vasodilators/ Antiproliferative Factors 3.1 Nitric Oxide NO is a potent vasodilator which is synthesized in the vascular endothelium. NO can be synthesized in response to various stimuli such as acetylcholine, ET-1, and shear stress

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caused by increased blood flow [73]. The synthesis of NO involves a multistep reaction in which l-arginine is converted to l-citrulline and NO requiring two cosubstrates, oxygen and reduced NADPH. This reaction is catalyzed by the enzyme NOS, which can occur in three isoforms. Neuronal NOS (nNOS) and endothelial NOS (eNOS) are constitutively expressed forms of the enzyme which are found in high levels in neuronal bodies or endothelial cells, respectively. The third isoform, inducible NOS (iNOS), is a rapidly activated form of the enzyme which is found in alveloar macrophages and is upregulated in response to inflammation. Of the three isoforms, eNOS is the predominant source of NO in the pulmonary vasculature. Interestingly, expression of eNOS is higher in the proximal PAs than in the distal PAs (Fig. 1), and this may contribute to the greater degree of HPV observed in distal PAs. Although eNOS is constitutively expressed, transcriptional and posttranslational modification of eNOS can occur in response to various agonists and physiological stimuli [74]. NO is highly lipid soluble and so can diffuse readily into the cytoplasm of the PASMC, where it mediates activation of guanylate cyclase and thus catalyzes the formation of cyclic GMP (cGMP) from GTP (Fig. 3). cGMP than activates cGMP-dependent protein kinase (PKG), which can decrease intracellular Ca2+ concentrations and promote relaxation of the PASMC via various mechanisms. PKG can inhibit Ca2+ channels, thereby inhibiting Ca2+ entry into the cell [75], while activating the expulsion of Ca2+ via the Ca2+/ATPase pumps and the Na+/Ca2+ exchanger [76, 77]. PKG can also phosphorylate the IP3 receptor and thus inhibit internal release of Ca2+ from the sarcoplasmic reticulum [78]. In addition to its effects on Ca2+ flux, PKG can also inhibit RhoA, which in turn prevents RhoA-induced inhibition of myosin light chain phosphatase [79]. As well as vasodilator properties, NO is also thought to have antiproliferative [80, 81] and proapoptotic [82, 83] effects on PASMCs from various species, including humans. Evidence suggests that NO can inhibit proliferation through both PKG-dependent and PKG-independent pathways. For example, although some studies have shown NO inhibition of vascular smooth muscle cell growth to be attenuated by PKG inhibition [84, 85], others have pointed to NO-induced inhibition of RhoA as the antiproliferative mechanism [86].

3.1.1 Effects of NO on the Pulmonary Vasculature The eNOS isoform plays a role in maintaining the low tone of the pulmonary circulation. Deletion of the eNOS gene in mice causes a small increase in pulmonary pressures under normoxic conditions [87] and in isolated perfused lungs from eNOS null mice, endothelial-dependent relaxation was

8  The Influence of the Major Vasoactive Mediators Relevant to the Pathogenesis of Pulmonary Hypertension

ablated [88]. Dysregulation of this isoform may also contribute to increased pulmonary pressures under hypoxic conditions as eNOS null mice are hyperresponsive to chronic hypoxia [89] and also exhibit a greater HPV response than nNOS or iNOS null mice [88]. Certainly NO is an effective pulmonary vasodilator when pulmonary tone has been increased by hypoxia or other stimuli. NO decreases acute HPV in isolated rat lungs and human volunteers [90, 91]. In rats the pulmonary vasodilator response to NO increases as pulmonary hypertension progresses in response to chronic hypoxia [92]. NO also decreases thromboxane-induced PAH in sheep and endotoxin-induced PAH in pigs [93, 94]. Delivery of the eNOS gene attenuated monocrotalineinduced PAH in rats [95]. Interestingly, NO may also have a role to play in angiogenesis. Coculture of calf PA endothelial cells with rat smooth muscle cells which had been transfected with eNOS resulted in the formation of capillary-like structures [96]. In addition, administration of fibroblasts transfected with eNOS mediated partial regeneration of the pulmonary circulation, and reduction of PA pressure in rats with monocrotaline-induced PAH [97].

3.1.2 NO as a Treatment for PAH Inhaled NO has now been approved by the FDA for the treatment of PAH. Chronic use of inhaled NO has been shown to improve pulmonary hemodynamics in patients with IPAH [98]. In addition, patients who respond to inhaled NO have a better 5-year survival rate that those patients who do not respond [99]. One of the advantages of using NO is that it is rapidly inactivated by hemoglobin in the blood and so, unlike other vasodilators, is selective for the pulmonary circulation [74]. The use of NO as a therapy for PAH is discussed further in Chapter 107.

3.2 Prostacyclin PGI2 is a member of the eicosonoid family of prostaglandins and leukotrienes. The eicosonoids are synthesized in various tissues via the arachidonic acid cascade. Arachidonic acid is cleaved from membrane phospholipid stores by the enzyme phospholipase A2 and broken down via two pathways, either by the cyclooxygenase enzymes to give rise to prostaglandin H2 (PGH2) or by the lipoxygenase enzymes to give rise to the leukotrienes. PGH2 is then further metabolized by various enzymes to give rise to the prostaglandins. The major metabolite of PGH2 in the endothelium of the PA is PGI2 owing to high levels of the enzyme PGI2 synthase. However, in platelets, the major metabolite of PGH2 is thromboxane A2 (TXA2), owing to the high levels of TXA2 synthase present. PGI2 and

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TXA2 have opposing effects and antagonize one another. PGI2 is a potent vasodilator, prevents the aggregation of platelets, and has antiproliferative and anti-inflammatory properties. TXA2, on the other hand, is a vasoconstrictor, activates platelet aggregation, and mediates proliferation of smooth muscle cells [100, 101]. Interestingly, lower levels of PGI2 synthase are present in the distal than in the proximal PAs (Fig.  1), possibly rendering the distal arteries more prone to vasoconstriction in the face of insult [15].

3.2.1 PGI2 Receptor PGI2 exerts its effects via the IP receptor. IP receptors are found in high concentrations on blood platelets and on vascular smooth muscle. The IP receptor is a member of the G-protein-coupled receptor family and is coupled to Gs. Binding of PGI2 to the IP receptor activates Gs, which in turn activates adenylate cyclase and increases the production of cyclic AMP (cAMP) [100] (Fig.  3). cAMP then activates cAMP-dependent protein kinase (PKA), which can phosphorylate several proteins, leading to relaxation of the cell. Among the actions of PKA are the inhibition of myosin light chain kinase and phosphorylation of the IP3 receptor leading to decreased Ca2+ release from intracellular stores [102]. PKA can also initiate gene transcription through its actions at the transcription factor cAMP-response-element-binding protein (CREB) [103].

3.2.2 PGI2 as a Treatment for PAH PAH patients have been shown to have reduced circulating levels of PGI2 and also reduced expression of PGI2 synthase in the PAs [15, 104]. In 1996, PGI2, or epoprostenol, administered intravenously, became the first drug approved by the FDA for use in PAH patients. Several investigations have shown continuous intravenous administration of epoprostenol to be beneficial in both patients with IPAH [105, 106] and in patients with secondary pulmonary hypertension (SPH) [107, 108]. Epoprostenol infusion has also been shown to improve long-term survival in IPAH patients when compared with historical controls [109]. Indeed chronic intravenous administration of epoprostenol is now the first-line therapy for PAH [110]. However, as PGI2 has a very short half life (2–7 min), it is unstable and must be given by ­continuous intravenous infusion. This has led to various problems, such as lack of pulmonary selectivity and thrombotic or infectious complications [111]. Longeracting PGI2 analogues have now been developed for intravenous, inhaled, and oral use. These include treprostinil, iloprost, and beraprost (discussed in greater detail in Chapter 104).

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3.3 cAMP, cGMP, and PDEs 3.3.1 cAMP and cGMP The second messenger cAMP is synthesized by adenylate cyclase, which is stimulated or inhibited by the G proteins Gs (e.g., PGI2) or Gi (e.g., 5-HT1B), respectively (Fig.  3). cAMP activates PKA, which phosphorylates CREB. Phosphorylated CREB is an activated transcription factor, which initiates transcription of specific genes. cGMP is produced by guanylate cyclase, which is stimulated, for example, by NO. cGMP activates PKG, which in turn phosphorylates downstream signaling elements. Early studies showed that there was a decreased expression of both cAMP and cGMP in PAs from hypoxic rat lungs [112]. As both cAMP and cGMP mediate vasodilation and exert an antiproliferative effect in the pulmonary circulation, this may contribute to the development of hypoxia-induced PAH. The cyclic nucleotide PDEs are the endogenous enzymes that hydrolyze cAMP and cGMP [113] (Fig.  3). There are 11 families of PDEs (PDE1–PDE11) and most of these families have more than one gene product (e.g., PDE4A, PDE4B, PDE4C, PDE4D). In addition, each gene product may have multiple splice variants (e.g., PDE4D1–PDE4D9). In total, there are more than 100 specific human PDEs [113]. Those that have been implicated in the regulation of pulmonary vascular tone include PDE1, PDE3, PDE4, and PDE5. For example, in an early study examining changes in PDE activity in the chronic hypoxic rat lung, increased cAMP PDE activity was observed in first-branch and intrapulmonary vessels and was associated with an increase in cilostimide-inhibited PDE (PDE3) activity. Increased total cGMP PDE in the main PA was associated with increases in Ca2+/calmodulin-stimulated (PDE1) activity and an increase in zaprinast-inhibited (PDE5) activity was observed in first-branch and intrapulmonary arteries. These results suggested that decreases in intracellular cyclic nucleotide levels in PAs from pulmonary hypertensive rats were associated with increased PDE activity and, further, that these changes reflect alterations at the level of specific types of PDE isoforms [12]. Subsequently, real-time PCR and immunoblotting demonstrated that the expression of PDE1A, PDE1C, PDE3B, and PDE5A was enhanced in PASMCs from both IPAH and SPH patients compared with control PASMCs [114]. The use of PDE inhibitors as a therapeutic intervention in PAH is discussed further in Chapter 106.

3.3.2 Phosphodiesterase Type 1 PDE1 is encoded by three separate genes, PDE1A, PDE1B (which appears to be absent in human PASMCs), and PDE1C, which hydrolyzes cAMP and cGMP with different affinities and mediates, at least in part, the decrease in cAMP accumulation in

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response to an increase in intracellular Ca2+ concentration [115]. Both PDE1A and PDE1B preferentially hydrolyze cGMP, whereas PDE1C hydrolyzes cAMP and cGMP with equal affinity [115]. PDE1C is present in proliferating aortic smooth muscle cells but is absent in intact human thoracic aorta, findings that suggest PDE1C contributes to the proliferative phenotype of vascular smooth muscle cells [116]. Both PDE1A and PDE1C have been reported to be upregulated in remodeled PAs from IPAH patients [114, 117].

3.3.3 Phosphodiesterase Type 3 PDE3 has high affinity for cAMP but can also hydrolyze cGMP. It hydrolyzes cAMP, however, at 10 times the rate it hydrolyzes cGMP and therefore cGMP effectively acts as a competitive inhibitor for cAMP and consequently for PDE3 [118]. As a result of its high expression in the lung, PDE3 was identified as a potential therapeutic target in pulmonary disease, and indeed PDE3 inhibitors have subsequently been shown to relax PAs [119]. PDE3A/B gene transcript levels are increased in the rat main, first, intrapulmonary, and resistance PAs by chronic hypoxia [120]. This might have led to PDE3 inhibitors being developed for the treatment of PAH. Indeed, the unequivocal effect of PDE3 inhibitors as positive inotropic agents provided a strong rationale for developing such drugs for the treatment of chronic heart disease. However, chronic treatment with milrinone was associated with an increased risk of mortality and this has consequently curtailed the development of PDE3 as a drug target [121].

3.3.4 Phosphodiesterase Type 4 PDE4 is a cAMP-specific PDE and is the largest PDE subfamily, with over 35 different isoforms identified thus far. PDE4 is the most widely characterized PDE isoenzyme and the molecular structure, compartmentalization, and function have been extensively investigated. Cardiomyocytes and vascular smooth muscle cells selectively vary both the expression and the catalytic activities of PDE4 isoforms to regulate their various functions. Altered regulation of these processes can influence the development, or resolution, of cardiovascular disease [122]. In the early 1970s, rolipram, a cAMP PDE inhibitor, was developed as a potential drug to treat depression as it was demonstrated that elevation of the level of cAMP could enhance noradrenergic neurotransmission in the central nervous system. Although rolipram proved to be an effective antidepressant, side effects of nausea and gastrointestinal disturbance terminated its clinical development and these side effects still limit the therapeutic application of PDE4 inhibitors. Total PDE4 activity does not seem to be affected by hypoxia [12]; however, hypoxia was subsequently shown to increase expression of PDE4A10/11, PDE4B2, and PDE4D5

8  The Influence of the Major Vasoactive Mediators Relevant to the Pathogenesis of Pulmonary Hypertension

and activation of certain of these long PDE4 isoforms through PKA phosphorylation [123]. The failure to see any overall increase in PDE4 activity after hypoxic exposure may be due to ERK-mediated phosphorylation and inhibition of particular PDE4 long isoforms. In addition, hypoxia-induced increase in expression of PDE4 isoforms, which are known to interact with certain signaling scaffold proteins, may result in alterations in compartmentalized cAMP signaling [123]. Future application of PDE4 inhibitors in PAH may involve their coadministration with PGI2 analogues such as cicaprost and iloprost as these inhibit mitogen-induced proliferation of human PASMCs in a cAMP-dependent manner. To this effect, isoform-selective antagonists of PDE1, PDE3, or PDE4 inhibited human PASMC proliferation, an effect that was in synergy with the effect of PGI2 analogues [124]. 3.3.5 Phosphodiesterase Type 5 PDE5 activity has been proposed as a modifying factor in the development of PAH for some years. PDE5 is the main factor regulating cGMP hydrolysis and downstream signaling in human PASMCs and sildenafil exerts an antiproliferative effect in PASMCs, mediated by cGMP and activation of PKGs [125]. PDE5 is, in fact, the major cGMP PDE subtype in the pulmonary circulation and it is also more abundant in the lung than in other tissues [126]. Inhibition of PDE5 therefore offers a relatively pulmonary selective antiproliferative, vasodilator mechanism. Experimentally, in rats, PDE5 was shown to be localized throughout the muscularized PAs, including newly muscularized small PAs induced by hypoxia [127]. In addition, sildenafil was shown to attenuate hypoxia-induced PAH [128]. Sildenafil also improved survival, increased cGMP levels, and reduced PAH and pulmonary vascular remodeling in monocrotaline-induced PAH rats [129]. In a randomized, placebo-controlled, double-blind crossover study, sildenafil was subsequently shown to significantly improved exercise tolerance, cardiac index, and quality-oflife indices in patients with FPAH [130] and in June 2005, ­sildenafil (Revatio®) was licensed by the FDA as a treatment for PAH.

4 Other Vasoactive Influences on PASMCs 4.1 Bone Morphogenetic Protein/ Transforming Growth Factor b The transforming growth factor b (TGF-b) superfamily comprises various growth factors which are involved in a large variety of processes, including cell growth, proliferation, survival, differentiation, migration, and extracellular matrix

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synthesis [131]. These include TGF-b isoforms and BMPs. TGF-b isoforms and BMPs are expressed by various cell types; however, in the vasculature they are expressed in endothelial and smooth muscle cells [131]. In 2000, two independent groups identified mutations in the BMPRII gene in around 70% of patients with FPAH [132, 133]. Subsequently, BMPRII mutations have also been identified in 10–40% of patients with IPAH [134–137], although it is likely that some of these patients will have FPAH rather than a de  novo mutation. In addition, PAH in patients with hereditary hemorrhagic telangiectasia has been linked to mutation in a type I receptor gene, the ALK1 gene [138]. These discoveries have led to much research aimed at elucidating the mechanisms linking interrupted TGF-b/BMP signaling to the development of PAH. Various isoforms of BMP inhibit proliferation and promote apoptosis of PASMCs derived from arteries of healthy donors [139, 140]. However, one report suggests that BMP-4 can have site-specific opposing effects within the pulmonary vasculature, inhibiting proliferation of PASMCs derived from proximal arteries while mediating proliferation in PASMCs derived from distal arteries [141]. Once again, this example of the loss of a protective mechanism in the distal PAs may explain why it is these small arteries, rather than the large proximal PAs, which are most prone to pulmonary vascular remodeling. In human fetal lung fibroblasts, BMP-4 mediates a decrease in proliferation and an increase in differentiation of these cells into a smooth muscle cell phenotype [142]. In PA endothelial cells, BMPs appear to have opposing effects to those mediated in PASMCs and PA fibroblasts; thus, in PA endothelial cells BMPs promote survival and protect against apoptosis [143].

4.1.1 BMPRII/TGF-b Receptors Members of the TGF-b family exert their effects via interaction with a receptor complex, which consists of two distinct transmembrane proteins, known as the type I and type II receptors [144]. Upon binding of the ligand, the type II receptor phosphorylates the associated type I receptor, activating an intracellular kinase domain that phosphorylates serine and threonine residues. This leads to activation of the cytoplasmic signaling proteins associated with the TGF-b receptor superfamily, the Smads (Fig.  3). The Smad isoform activated appears to be entirely determined by the type I receptor. For example, the TGF-b receptor TGFR-II can associate with either of the type 1 receptors, ALK1 or ALK5. ALK1 signals through Smads 1 and 5 and possibly 8, whereas ALK5 signals through Smads 2 and 3. The BMP receptor BMPRII can associate with four different type 1 receptors, ALK1, ALK2, ALK3 (BMPR1A), and ALK6 (BMPR1B). All of these type 1 receptors are thought to signal via Smads 1, 5, and 8. All phosphorylated

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receptor-activated smads (R-smads) must bind to the common partner smad (Co-Smad), Smad-4 for nuclear translocation to occur. Each of the R-smads can then regulate gene transcription either by direct binding to DNA or by interacting with DNA binding proteins.

4.1.2 BMPRII and PAH Haploinsufficiency in the gene encoding the BMPRII receptor is the most common mutation found in patients with FPAH [145]. In normal human lungs, BMPRII staining is strongest on the endothelial layer of the PA and is also present on the PASMCs. In lungs from pulmonary hypertensive patients, BMPRII staining is reduced in the distal section of the lung, especially from patients harboring a BMPRII mutation. BMPRII staining is also reduced in patients with SPH but not to the same extent [146]. In line with these observations, the antiproliferative and proapoptotic effects of BMP ligands observed in PASMCs from normal controls and patients with SPH are either considerably attenuated or completely absent in PASMCs from patients with IPAH or FPAH [140, 141]; therefore, it is likely that loss of BMP function in PASMCs may lead to a proliferative response and thus pulmonary vascular remodeling. This has been verified in PASMCs harboring a mutation in the BMPRII gene, which were shown to be unresponsive to the growth-suppressant effects of BMP-4. Therefore, loss of BMP signaling in PASMCs would lead to proliferation and pulmonary vascular remodeling. Interestingly, PASMCs from IPAH patients with no detectable mutation also exhibited a decreased response to BMP-4, but to a lesser extent than the mutation-positive PASMCs [142]. This suggests that even in IPAH or FPAH patients with no BMPRII mutation there is still a loss of BMP function. Conflicting evidence exists as to the effects of loss of BMP function in PA endothelial cells. One study reported that the antiapoptotic effects of BMPs observed in PA endothelial cells from normal subjects were absent in PA endothelial cells derived from IPAH patients [144]. Therefore, loss of BMP function in PA endothelial cells may increase susceptibility of the endothelium to damage, and thus increase the risk of developing PAH [147]. However, a hyperproliferative phenotype in PA endothelial cells taken from patients with IPAH has also been reported [148]. This hyperproliferative phenotype may lead to the formation of plexiform lesions as seen in PAH patients.

4.1.3 Reduced Disease Penetrance in BMPRII Mutation Carriers Despite the prevalence of the BMPRII mutation throughout FPAH and IPAH patient populations, disease penetrance is

M.R. MacLean and Y. Dempsie

low. It is thought that only 15–20% of BMPRII mutation carriers actually develop PAH. Therefore, a loss of signaling through the BMPRII receptor alone is insufficient to induce PAH, and it is likely that interactions with other genes or environmental stimuli must occur in the development of PAH. One study showed an infusion of serotonin to uncover a PAH phenotype in BMPRII+/− mice [149]. It is possible that a reduced ability to suppress pulmonary vascular proliferation only becomes important when pulmonary vascular proliferation is enhanced. As serotonin mediates proliferation of both PASMCs and PA fibroblasts, it is possible that enhanced activation of the serotonin system may provide such a “second hit” risk factor. Another study compared gene expression in PAH patients harboring BMPRII mutations and BMPRII mutation carriers who did not develop PAH. Interestingly, the gene encoding for the estrogen-metabolizing protein CYPIBI was downregulated in female PAH patients with a BMPRII mutation compared with female BMPRII mutation carriers who were unaffected by the disease [150]. FPAH and IPAH are far more prevalent in females than males (more than 2:1), although the reasons for this are currently unclear. Therefore, links between the TGF-b/BMP signaling system and hormonal regulation are of great interest.

4.2 K+ Channels K+ channels play an important role in the regulation of membrane potential in cells throughout the body, including those of the pulmonary vasculature. The resting potential of the PASMC is around −60 mV, with a negative charge inside the cell and a positive charge outside the cell. K+ channels allow the efflux of K+ ions down an electrochemical gradient, thus maintaining the electrical gradient across the cell membrane. When the efflux of K+ ions is inhibited, the positive charge builds up inside the cell and thus the cell becomes depolarized. This allows the entry of Ca2+ ions and, in the case of the PASMC, contraction and proliferation [151, 152] (Fig. 3). There are four main classes of K+ channels, (1) voltagegated K+ channels (Kv), (2) Ca2+-activated K+ channels (KCa), (3) inwardly rectifying K+ channels (KIR), and (4) two-poredomain K+ channels (K2P) [151, 152]. All four K+ channel types have been implicated in the control of resting membrane potential in PASMCs, although it should be noted that evidence for the involvement of K+ channels appears to be both species-specific and cell-specific and varies between vascular beds. For example, in human distal and rabbit proximal PASMCs, the resting potential is set by the two-poredomain acid-sensitive potassium (TASK)-1 channel [153, 154]. In rat, however, the proximal PASMC K+ current is mediated largely by KCa channels, whereas the distal PASMC

8  The Influence of the Major Vasoactive Mediators Relevant to the Pathogenesis of Pulmonary Hypertension

K+ current is mainly conducted by Kv channels (also known as delayed-rectifier channels) [155] (Fig. 1). This heterogeneity of K+ channels is important as PAH is a disease of the distal PAs.

4.2.1 K+ Channels and HPV PAs exhibit a vasoconstrictor response to hypoxic conditions unlike the systemic circulation, which vasodilates in response to hypoxia [156]. Physiologically in mammals, HPV serves to increase pulmonary vascular resistance in the fetus, diverting the circulation away from the lungs. At birth, the pulmonary circulation dilates upon exposure to atmospheric oxygen and from then on receives 100% of the cardiac output. Disruption of this mechanism results in persistent pulmonary hypertension of the newborn (PPHN), a serious condition affecting one or two infants per thousand live birth. Mortality from PPHN is around 10–20% [157]. After birth, HPV becomes an important negative-feedback mechanism diverting blood away from hypoxic areas of the lung and ensuring ventilation–perfusion matching. However, low oxygen concentrations throughout the lung, as observed in patients with chronic obstructive pulmonary disease or following chronic exposure to high altitude, commonly result in the development of PAH [158]. HPV is evident in PAs after the endothelium has been removed, and therefore is not due to endothelial release of vasoconstrictor substances [159]. Evidence suggests that K+ channels play an important role in mediation of HPV. HPV is strongest in resistance PAs, and resistance PASMCs uniquely express an O2-sensitive component to whole-cell K+ current [155]. Various PASMC K+ channels are sensitive to oxygen [160]. However, it is thought that the KCa channel is important in mediation of HPV in the fetus [161], whereas the TASK-1 and/or Kv channels (particularly Kv1.5) are important in mediation of HPV after birth. TASK-1 channels from human distal PASMCs are sensitive to hypoxia and thus have been postulated to play a role in HPV [154]. However other studies have pointed to an important role for the Kv1.5 channel. Rat small pulmonary resistance arteries, which are particularly responsive to HPV, have increased expression of Kv1.5 compared with the larger-conduit arteries, and both the Kv1.5 and the Kv2.1 channels are inhibited by chronic hypoxia in rat PASMCs [162]. Moreover, HPV in the rat is blunted by previous exposure to chronic hypoxia and this can be reversed by aerosol transfection of human Kv1.5 [163]. As TASK-1 channels are open at all voltages, whereas Kv channels are voltage-dependent, it has been suggested that hypoxic inhibition of the TASK-1 channel may depolarize the PASMC and bring the membrane potential into the range where Kv channels are active [164, 165]. Hypoxic inhibition of the Kv channels would then result in further membrane depolarization, influx of Ca2+ through L-type Ca2+ channels, and PASMC constriction.

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4.2.2 K+ Channels and Proliferation It has long been accepted that K+ channels are involved in the maintenance of pulmonary vascular tone. However, more recent findings show that K+ channels also contribute to proliferation of the PASMCs, and thus may contribute to the pulmonary vascular remodeling observed in PAH. Proliferating PASMCs have a diminished whole-cell K+ current, a more depolarized membrane potential, and an increased cytosolic Ca2+ level compared with growth-arrested PASMCs [166]. It is thought that increased levels of intracellular Ca2+ can mediate cellular proliferation via the activation of transcription factors and thus increased expression of pro-proliferative genes and proteins [152].

4.2.3 K+ Channels and PAH There is much evidence to suggest that K+ channels are involved in the development of PAH. PASMCs from patients with IPAH or FPAH have a more depolarized membrane potential and a diminished Kv current compared with PASMCs from patients with secondary PAH or nonpulmonary hypertensive controls [167]. Moreover, gene transcription of Kv1.5 was found to be attenuated in IPAH or FPAH patients compared with secondary PAH patients or nonpulmonary hypertensive patients [168]. Experimentally, gene transfer of Kv1.5 attenuated chronic hypoxic PAH in rats [163], and dichloroacetate, a metabolic modulator that increases mitochondrial oxidative phosphorylation, reversed monocrotaline-induced PAH in rats by upregulating Kv1.5 channels [169].

4.3 Platelet-Derived Growth Factor PDGF was first identified in the 1970s as a mitogen for both smooth muscle cells [170] and fibroblasts [171]. Although PDGF was first purified from platelets [172], it is now known that PDGF can be synthesized in a variety of cells, including endothelial cells, smooth muscle cells, and fibroblasts [173]. PDGF synthesis can be increased in response to various factors, such as hypoxia. It was first thought there were three varieties of PDGF, made up of dimers of disulfide-linked polypeptide chains A and B (PDGF-AA, PDGF-AB, and PDGF-BB). However, two additional members of the PDGF family, PDGF-CC and PDGF-DD, have recently been described [174– 176]. All five members of the PDGF family act via two receptors, PDGFR-a and PDGFR-b, both of which belong to the tyrosine kinase receptor family (Fig. 3). Ligand binding promotes receptor dimerization, phosphorylation, and signaling. PDGF-AA activates PDGFR-aa selectively, PDGF-AB and PDGF-CC activate PDGFR-aa and PDGFR-ab, PDGF-DD

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activates PDGFR-bb, and PDGF-BB is the universal ligand and can assemble and activate any receptor dimer [173]. Evidence exists in both animal models and humans to suggest that PDGF may be involved in the pathogenesis of PAH. PDGF-A, PDGF-B, PDGFR-a, and PDGFR-b mRNA expression and PDGFR-b protein expression are increased in distal PAs from patients with severe PAH [177]. Lambs with chronic intrauterine PAH develop increased PDGFR-a and PDGFR-b lung protein expression [178]. In the rat chronic hypoxic- and monocrotaline-induced models of PAH, imatinib, a PDGF receptor antagonist, increased survival and reversed the increases in right ventricular pressure, vascular muscularization, and right ventricular hypertrophy to near baseline levels [179]. Imatinib has been shown to inhibit both PDGF-BB induction of PDGF-b phosphorylation and PDGF-BB stimulated proliferation and migration in human PASMCs [177], providing a possible mechanism by which it may protect against PAH.

5 Conclusions Here we have described the major vasoactive mediators and influences relevant to the pathogenesis of PAH. PAH involves complex underlying changes is vasoreactivity. It is likely that many vasoactive factors are altered and involved to a greater or lesser extent. It is also likely that their influence and cross talk vary from one patient to another. What is evident is that the distal PAs are particularly vulnerable to pro-proliferative or promitogenic insults, having less protection from endogenous factors under normal conditions. There is also a complex interaction between the PA endothelial cells, PASMCs, and PA fibroblasts, contributing to the complexity of this disease. Identifying which factors and pathways are key to the processes involved in PAH remains the biggest challenge to developing effective therapeutic strategies.

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Chapter 9

The Normal Fetal and Neonatal Pulmonary Circulation Steven H. Abman and Robin H. Steinhorn

Abstract  In utero, the pulmonary circulation is characterized by high pulmonary vascular resistance (PVR) and low pulmonary blood flow, as the fetal lung receives less than 8% of combined ventricular output. At birth, the pulmonary circulation must suddenly undergo a striking vasodilation to allow for an eightfold to tenfold rise in blood flow for the lung to assume its postnatal role in gas exchange. This acute change in pulmonary vascular tone and reactivity is followed by progressive vascular growth, remodeling, and structural adaptations, as the lung circulation continues to grow and mature over time in response to postnatal stimuli. Normal lung vascular development is not only critical for successful adaptation at birth, but ongoing growth and remodeling remains essential throughout postnatal life. The ability of the lung to successfully achieve normal gas exchange requires the growth and maintenance of an intricate system of airways and vessels, including the establishment of a thin yet vast blood–gas interface. Insights into basic mechanisms that regulate lung growth and maturation continue to provide new understanding of lung diseases and their treatment, in newborns and adults alike. Vascular growth in the lung is not only important regarding the risk for pulmonary hypertension, but normal vascular growth and structure are absolutely necessary for establishing sufficient surface area for gas exchange. Developmental abnormalities of the pulmonary circulation contribute to the pathological and pathophysiological processes of diverse diseases, including persistent pulmonary hypertension of the newborn, lung hypoplasia, congenital diaphragmatic hernia, and congenital heart disease. This chapter provides a brief overview of structural and functional changes in the pulmonary circulation that contribute to PVR during fetal life and mechanisms that contribute to the dramatic changes in vascular tone at birth in the normal newborn.

S.H. Abman (*) Department of Pediatrics, The Children’s Hospital, 1717 E. Arizona Ave., Denver, CO, 80210, USA e-mail: [email protected]

Keywords  Newborn • Lung and vascular development • Pediatric pulmonary hypertension • Adaptation • Congenital heart disease

1 Introduction In utero, the pulmonary circulation is characterized by high pulmonary vascular resistance (PVR) and low pulmonary blood flow, as the fetal lung receives less than 8% of ­combined ventricular output. At birth, the pulmonary circulation must suddenly undergo a striking vasodilation to allow for an eightfold to tenfold rise in blood flow for the lung to assume its postnatal role in gas exchange. This acute change in ­pulmonary vascular tone and reactivity is followed by ­progressive vascular growth, remodeling, and structural adaptations, as the lung circulation continues to grow and mature over time in response to postnatal stimuli. Normal lung vascular development is not only critical for successful adaptation at birth, but ongoing growth and remodeling remains essential throughout postnatal life. The ability of the lung to successfully achieve normal gas exchange requires the growth and maintenance of an intricate system of airways and vessels, including the establishment of a thin yet vast blood–gas interface. Insights into basic mechanisms that regulate lung growth and maturation continue to provide new understanding of lung diseases and their treatment, in newborns and adults alike. Lung branching morphogenesis and epithelial differentiation have been studied extensively in past decades; ­however, studies of mechanisms that regulate development of the ­pulmonary circulation have been relatively limited. Until recently, most of the information regarding pulmonary ­vascular development has been largely descriptive in nature. Vascular growth in the lung is not only important regarding the risk for pulmonary hypertension, but normal vascular growth and structure are absolutely necessary for ­establishing sufficient surface area for gas exchange. Recent observations have challenged older concepts that the development of the blood vessels in the lung passively

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_9, © Springer Science+Business Media, LLC 2011

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follows that of the airways. Increasing evidence suggests that lung blood vessels actively promote normal alveolar growth during development and contribute to the maintenance of alveolar structures throughout postnatal life. Disruption of angiogenesis during lung development can impair alveolarization, and preservation of vascular growth and endothelial survival may promote lung growth and structure of the distal airspace. Understanding how alveoli and the underlying ­capillary network develop and how these mechanisms are disrupted in disease states is critical for developing efficient therapies for lung diseases characterized by impaired ­alveolar structure. Developmental abnormalities of the pulmonary circulation contribute to the functional abnormalities of diverse ­diseases, including persistent pulmonary hypertension of the newborn (PPHN; see Chap. 76), lung hypoplasia, congenital diaphragmatic hernia, and congenital heart disease. This chapter provides a brief overview of structural and functional changes in the pulmonary circulation that contribute to PVR during fetal life and mechanisms that contribute to the dramatic changes in vascular tone at birth in the ­normal newborn.

2 Vascular Growth in the Developing Lung Lung development is classically divided into five overlapping stages on the basis of histological features [1]. Although largely descriptive, changes in lung structure across these stages provide a clear sequence of events that occur in ­stereotypical fashion, and allow for subsequent molecular or interventional studies to subsequently examine specific mechanisms. The first four stages, termed the embryonic, pseudoglandular, canalicular, and saccular stages, occur during gestation. At the end of the saccular stage (at about 36 weeks), lungs have formed alveolar ducts and saccules. Alveolarization, in the final stage of lung development, ­primarily occurs postnatally, during the first 2–3 years of life, and may continue at a slower rate beyond childhood [1, 2]. Distinct anatomic changes in the lung circulation have been well described, although the underlying mechanisms regulating structural changes are less clear (Table 1). Changes

in lung vasculature during each stage and the ­relative roles of vasculogenesis and angiogenesis during each stage are described herein.

2.1 Embryonic Period During the embryonic period (weeks 3–7 in humans), the most proximal part of the pulmonary circulation, the pulmonary trunk, is derived from the truncus arteriosus, which becomes divided into the aorta and pulmonary trunk by 8 weeks of gestation in humans by growth of the spiral aorticopulmonary septum [3]. The pulmonary trunk connects to the pulmonary arch arteries, which are derived from the sixth branchial arch arteries, the most caudal of the branchial arteries, by 7 weeks gestation in humans. Recent work suggests that these pulmonary arch arteries originate from a strand of endothelial precursors that connect the ventral wall of the dorsal aortae to the pulmonary trunk [4, 5]. These strands develop a lumen initially at the sites of connection between the pulmonary trunk and the dorsal aortae. The origin of intrapulmonary arteries has been variously described as endothelial sprouts from either the aortic sac or the dorsal aortae, or as originating from a network of capillaries around the foregut. The latter observation comes closest to the results of more recent studies suggesting that the pulmonary artery and vein develop in situ within the splanchnic mesoderm ­surrounding the foregut via at least two processes that likely occur concurrently: (1) vasculogenesis, in which new blood vessels form in situ from angioblasts, and (2) angiogenesis, which involves sprouting of new vessels from existing vessels.

2.2 Vascular Morphogenesis Blood vessel morphogenesis involves discrete steps that occur in a continuum, which are regulated by specific signaling pathways that involve soluble effectors, cytokines and their receptors, proteases, and extracellular matrix (ECM) components. These various pathways control integral events that contribute to the formation of a functional vasculature,

Table 1  Stages of lung growth: vascular development Stage Events Embryonic <6 weeks Vasculogenesis within immature mesenchyme; pulmonary arteries branch from the sixth aortic arches, veins as outgrowths from the left atrium Pseudoglandular <16 weeks Parallel branching of large pulmonary arteries with central airways; lymphatics appear Canalicular <24 weeks Increased vessel proliferation and organization into capillary network around air spaces Saccular <36 weeks Marked vascular expansion with thinning and condensation of mesenchyme: thin air–blood barrier; double capillary network in septa Alveolar <2–3 years Accelerated vascular growth, fusion of the double capillary network with thinning of septa Postnatal 3 years to adulthood Marked vessel growth and remodeling as the surface area increases more than 20-fold

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including endothelial and mural cell (pericyte and smooth muscle) differentiation, cell proliferation and migration, and the specification of arterial, venous, and lymphatic fates. Disruption of these processes may not only result in neonatal pulmonary hypertension but may also contribute to abnormalities of lung structure. Formation of the pulmonary circulation has been primarily described as dependent on two basic processes: vasculogenesis and angiogenesis. Vasculogenesis is the de novo formation of blood vessels from angioblasts or endothelial precursor cells that migrate and differentiate in response to local cues (such as growth factors and ECM) to form vascular tubes. Angiogenesis is the formation of new blood vessels from preexisting ones. It has generally been accepted that the distal vasculature arises by vasculogenesis, and the proximal vasculature by angiogenesis, but this remains controversial [4, 6–8]. Vasculogenesis results in the blood vessel formation from blood islands present within the mesenchyme of the embryonic lung (embryonic day 9, or E9, in the mouse). Angiogenesis starts around E12, when arteries and veins begin to sprout from the central pulmonary vascular trunks. Around E14, peripheral sinusoids and central vessel sprouts connect and establish a vascular network. This union of peripheral and central vascular structures is accompanied by extensive branching of the arteries, which follow the branching pattern of the airways. The idea that blood vessels can arise de  novo within the  mesoderm surrounding the protruding endoderm was originally suggested by Chang [8]. Recent studies using molecular markers of endothelial progenitor cells as well as  endothelial differentiation marker support this concept [9, 10]. These studies describe mesodermal cells expressing primitive endothelial markers at stages of lung development that precede evidence of any connection of blood flow with the established circulatory system. These studies further establish that lung vascular development begins during the embryonic stages of lung development, continues throughout fetal life, and is dependent on epithelial–mesenchymal cross talk [11, 12]. Molecular evidence is supported by ­studies combining electron microscopy utilizing vascular casts of the developing lung. These findings suggest that the large pulmonary arteries develop by angiogenesis from central vessels, that distal vessels likely form via vasculogenic mechanisms within the mesenchyme, and that a third process, vascular fusion, is necessary to ultimately connect the angiogenic and vasculogenic vessels [11–14]. Studies in human fetal lung suggest that airways act as a template for pulmonary artery development. Endothelial tubes form around the terminal buds of the airways, suggesting an inductive influence of the epithelium. More recently, Parera et al. suggested distal angiogenesis as a new mechanism for lung vascular morphogenesis, on the basis of morphological analysis from the onset of lung development (E9.5) until the pseudoglandular stage (E13.5) in Tie2–LacZ transgenic mice [13]. In their model, capillary networks ­surrounding the

137 Table 2  Major angiogenic signals during lung development Growth factors Adhesion molecules bFGF Integrins VEGF ICAM-1 TGF-b VCAM-1 PDGF-BB PECAM-1 Cadherin Selectins Ligands VEGF receptors 1 and 2 Neuropilin Angiopoietins 1 and 2 TGF-b, BMP receptors Ephrin receptors Notch and Jagged

Extracellular matrix Fibronectin Tenascin-c Laminin

Proteases Matrix metalloproteinases

Transcription factors Homeobox (Hox) genes GATA-5, GATA-6 ETS-1, ETS-2 bFGF basic fibroblast growth factor; VEGF vascular endothelial growth factor, TGF transforming growth factor, PDGF plateletderived growth factor, BMP bone morphogenetic protein, ICAM intracellular cell adhesion molecule, VCAM vascular cell adhesion molecule, PECAM platelet/endothelial cell adhesion molecule

terminal buds exist from the first morphological sign of lung development and then expand by formation of new capillaries from preexisting vessels as the lung bud grows. Multiple stimuli contribute to alveolar and vascular growth and development, and in part, involve “cross talk” of paracrine signals between epithelium and mesenchyme (Table  2). For example, vascular endothelial growth factor (VEGF) is expressed in developing lung epithelium, whereas VEGF receptor-2 (VEGFR-2) is localized to angioblasts within the embryonic mesenchyme [15–19]. Diffusion of VEGF to precursors of vascular endothelial cells within the mesenchyme leads to angiogenesis by stimulating endothelial proliferation, a key step in vessel development. These mechanisms may also apply to human lung vascular development. In their studies of human embryos, deMello and Reid reported evidence to support the idea that both ­vasculogenesis and angiogenesis operate cooperatively to form the pulmonary vessels [4]. Hall et al. also proposed that vasculogenesis is a primary mechanism for intrapulmonary vascular development [5]. These investigators also suggested that the venous circulation develops in a fashion similar to that for arteries and may actually precede development of early arteries. Alternatively, this concept has been challenged by studies suggesting that the lung vasculature is formed by “distal angiogenesis,” a process in which the formation of new ­capillaries from preexisting vessels occurs at the periphery of the lung [13]. In this model, epithelial and endothelial interactions are necessary to induce angiogenesis and to coordinate expansion of a vascular network as branching proceeds.

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2.3 Fetal Stages The pseudoglandular period (weeks 5–16) is primarily ­characterized by extensive branching morphogenesis of the airway, in which epithelial buds undergo multiple dichotomous branchings. Gebb and Shannon and Shannon and Deterding demonstrated that epithelial–mesenchymal interactions play a critical role in this process [11, 14]. Early cross talk determines the sites, frequency, and timing of airway branching, but epithelial–mesenchymal communication further dictates early vascular development within the mesenchyme, which is reflected by the close proximity of vascular growth with airway branching. The formation of these ­conventional arteries appears to be followed by the development of smaller, supernumerary arteries between these regions. At the end of the pseudoglandular period, all preac­ inar bronchi are present and accompanied by pulmonary and bronchial arteries. The canalicular stage (weeks 17–26) is a period of expansive organ growth which occurs by differentiation of epithelium into type II cells (surfactant production) and type I cells (key for air–blood barrier). The canalicular stage encompasses the early development of the pulmonary parenchyma, during which the number of lung capillaries markedly increases within the immature mesenchyme. The capillaries begin to arrange themselves around the air spaces and come into close apposition with the overlying epithelium as the lung appears “canalized.” At sites of apposition, thinning of the epithelium occurs to form the first sites of the air–blood barriers. During the saccular stage (24–36 weeks), clusters of ­thin-walled saccules appear in the distal lung and have formed alveolar ducts, the last generation of airways prior to  the development of alveoli. Capillaries form a bilayer (“double capillary network”) within the relatively broad and cellular intersaccular septa at this stage. The interstitium between air spaces contains the capillary network and ­numerous interstitial cells, but only a thin network of ­collagen fibers. During this stage, elastic fibers are deposited within the interstitium, which lays the foundation for subsequent formation of alveoli.

2.4 Postnatal Period The alveolarization stage is largely a postnatal event, with more than 90% of all alveoli being formed shortly after birth, with the greatest surge in alveolar development between birth and 6 months of age, and continuing during the first 2–3 years in humans. The final step of microvascular maturation occurs during this stage [1]. Formation of alveoli occurs by the outgrowth of secondary septa that subdivide terminal saccules into anatomic alveoli. Under electron microscopy, the secondary septa show a central layer of connective tissue with

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numerous cells and very few collagen fibrils, flanked on both sides by capillaries. These capillaries appear to be interconnected over the edge of the crest, and cross-sectioned fibers of elastin can be detected at the tip of the crest or just below the capillary loop. It appears that these new interalveolar walls are formed by the alternate upfolding of one of the two capillary layers on either side of a primary septum. As a result of this process, primary and secondary septa ­contain double capillary network layers. This contrasts with older infants, children, and adults, in which only a single capillary abuts the air space on each side. During the period of alveolarization, thick interalveolar septa begin to thin and the double capillary layer that is characteristic of this stage begins to fuse into a single layer to assume the adult form. Secondary septa initially form as low ridges that protrude into primitive air spaces to increase their surface area. Between birth and adulthood, the alveolar and capillary ­surface areas expand nearly 20-fold and the capillary volume expands by 35-fold. Further expansion of the capillary network subsequently occurs via two angiogenic mechanisms: sprouting angiogenesis from preexisting vessels and intussusceptive growth [20, 21]. Little is known about intussusceptive microvascular growth in the lung. This novel mode of blood vessel formation and remodeling occurs by internal division of the preexisting capillary plexus (insertion of ­transcapillary tissue pillars) without sprouting, which may underlie alveolar growth and remodeling throughout adult life and thus be amenable to therapeutic modulation for lung regeneration. During this time, the lung is undergoing marked microvascular growth and development as well. In addition, the process of septation involves alternate upfolding and growth of capillary layers within primary septa. This mechanism of alveolarization suggests that the failure of capillary network formation and growth, or disruption of the infolding of the double capillary network, could potentially cause failed alveolarization. Much more needs to be learned about the anatomic events underlying lung vascular development and time-specific mechanisms that regulate growth and function at each stage. Likewise, there is a need for endothelial cell-specific surface markers to identify and characterize angioblasts, endothelial progenitor cells, and mature endothelial cells, as well as genetic tools allowing us to better define endothelial cell fate and lineage relationships in the embryonic and the ­ postnatal lung.

2.5 Role of O2 Tension in Vascular Development O2 tension modulates lung development, as hyperoxia impairs lung growth and hypoxia is known to enhance vasculogenesis and angiogenesis. Since normal fetal O2 tension is about

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25 Torr, it is not surprising that in vitro studies of mammalian development appear optimal at low oxygen tension (3%) and that even short exposures to even room-air conditions can be detrimental to embryonic development owing to relative hyperoxia. Recent work with mammalian fetal lung explants has provided evidence that branching morphogenesis is O2dependent [22]. At lower O2 tension values that mimic fetal life, lung explants show a marked increase in airway branches and better maintenance of the mesenchyme [23]. Increases in branching morphogenesis appear to bev confined to the periphery in lung explant cultures, suggesting that hypoxia dependence is strong within the region of active airway bifurcation and vascular cell proliferation. Thus, O2 tension may modulate both physiologic and structural characteristics of lung vascular and airway development. Studies have begun to elucidate signaling pathways through which hypoxia acts to modulate vascular growth and development. Null mutations of HIF-1a in mice has no effect until E9, an age which coincides with the initiation of lung development and vasculogenesis [23]. Consequently, HIF-1a−/− mice display enlarged vascular structures, have impaired early lung morphogenesis, and die by E10.5. Impaired expression of such HIF-regulated genes, such as VEGF, may account for at least part of these findings, since hypoxia upregulates VEGF and VEGFR-2 and inhibition of VEGF signaling mimics the effects of hyperoxia. Several other O2-sensitive genes appear to be involved in this process, including angiopoietins 1 and 2, platelet-derived growth factor B (PDGF-B), fibroblast growth factor (FGF)-9 and FGF10, C/EBP-b, hepatocyte nuclear factor family, transforming growth factor family genes, and erythropoietin, and other mediators may also play important regulatory roles in vascular growth in this setting. Thus, low O2 tension activates unique cellular signaling pathways that are critical for growth and differentiation of cells within the pulmonary vasculature. Studies in the future need to be directed at utilizing the effects of oxygen to study lung vascular development. Such studies will provide better understanding regarding the response of the premature lung that is removed from its normal “hypoxic” environment and placed into relative or absolute hyperoxia at a vulnerable period of lung development. That this transition can inhibit lung development as well as vascularization often leading to significant cardiopulmonary dysfunction is clear.

2.6 Hemodynamic Forces and Vascular Growth Just as vascular growth and structure in utero influence ­pulmonary hemodynamics, hemodynamic forces, including shear (tangential) and stretch (pressure) stress, are major

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determinants of vascular morphogenesis [24]. Endothelial cells in vitro align in the direction of blood flow, and alter their morphology, mechanical properties, and patterns of gene expression in response to shear stress [25]. The mechanisms in which altered blood flow or pressure lead to diverse cellular responses are not clear. The endothelial cell clearly provides a unique interface between intravascular forces and the vessel wall, and appears to serve as a “mechanotransducer,” triggering changes in membrane function, intercellular junctions, and others. Changes in local hemodynamics markedly alter production of vasoactive mediators, including nitric oxide (from shear stress) and endothelin (ET)-1 and PDGF-B (due to stretch or shear stress), which rapidly change vascular tone. Over time, sustained changes in hemodynamic forces and mediators alter vascular structure, as reflected by increases in pulmonary artery smooth muscle cell hyperplasia when exposed to hypertensive stimuli. Recent studies suggest that chronic changes in pulmonary hemodynamics can influence lung vascular growth and distal airspace structure in late-gestation fetal sheep [26, 27]. Intrauterine pulmonary hypertension, caused by surgical compression of the ductus arteriosus (DA), inhibits angiogenesis, resulting in reduced vascular density, lung weight, and alveolarization in near-term fetal sheep [26]. These effects may be at least partly mediated by impaired VEGF signaling, as chronic hypertension reduces lung VEGF ­protein expression by 75%, and treatment with recombinant human VEGF protein protects the lung from these changes [26, 27]. These findings suggest a striking effect of hemodynamic stress on vascular growth during late gestation, and in light of the reduction in lung size and alveolarization, ­suggest a novel mechanism linking pulmonary hypertension and lung hypoplasia, as observed in neonatal lung diseases such as congenital diaphragmatic hernia, lung hypoplasia, and ­bronchopulmonary dysplasia.

3 Developmental Physiology of the Perinatal Pulmonary Circulation 3.1 Physiology of the Fetal Pulmonary Circulation Along with the remarkable progression of lung vascular growth and structure during development, the fetal pulmonary circulation also undergoes maturational changes in function. PVR is high throughout fetal life, especially in comparison with the low resistance of the systemic circulation. As a result, the fetal lung receives less than 3–8% of combined ventricular output, with most of the right ventricular output crossing the DA to the aorta. Pulmonary artery pressure and blood flow progressively increase with advancing

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gestational age, along with increasing lung vascular growth [28]. Despite this increase in vascular surface area, PVR actually increases with gestational age when corrected for lung or body weight. Thus, pulmonary vascular tone actually increases during late gestation, especially prior to birth. Studies of the human fetus support these physiologic observations from fetal lambs. On the basis of multiple Doppler ultrasound measurements that include assessments of fetal left and distal pulmonary artery velocity waveforms, Rasanen et al. have demonstrated that fetal pulmonary artery impedance progressively decreases during the second trimester and the early part of the third trimester. Pulmonary artery vascular impedance does not decrease further during the latter stage of the third trimester despite ongoing vascular growth [29]. Several mechanisms contribute to high basal PVR in the fetus, including low oxygen tension, low basal production of vasodilator products [such as prostacyclin (PGI2) and NO], increased production of vasoconstrictors (including ET-1 and leukotrienes), and altered smooth muscle cell reactivity (such as enhanced myogenic tone) [30–35] (Table  3). In addition to high PVR, the fetal pulmonary circulation is also characterized by progressive changes in responsiveness to vasoconstrictor and vasodilator stimuli (or vasoreactivity). In the ovine fetus, the pulmonary circulation is initially poorly responsive to vasoactive stimuli during the early canalicular period, and responsiveness to several stimuli increases ­during late gestation. For example, the pulmonary vasoconstrictor response to hypoxia, and the vasodilator response to increased fetal O2 tension and acetylcholine increase with gestation [36, 37]. As observed in the sheep fetus, human studies also demonstrate maturational changes in the fetal pulmonary vascular response to increased PaO2.

Table 3  Factors that maintain high pulmonary vascular resistance in the normal fetus 1.  Mechanical: presence of fetal lung liquid, lack of air–liquid interface 2. Structural: e.g., decreased surface area, vessel wall compliance 3.  Functional   (a)  Decreased dilator activity     •  Nitric oxide – adenosine     •  Prostacyclin – adrenomedullin     •  Atrial natriuretic peptide     •  Endothelium-derived hyperpolarization factor (?)   (b)  Increased constrictor stimuli     •  Endothelin-1     •  Leukotrienes C4, D4     •  Endothelium-derived constricting factors (?)   (c)  Altered smooth muscle cell signaling     •  High myogenic tone/responses     •  High cyclic GMP phosphodiesterase type 5 activity     •  Altered calcium handling     •  Impaired K+ channel function/expression

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Maternal hyperoxia did not increase pulmonary blood flow between 20 and 26 weeks of gestation, but increased PaO2 caused pulmonary vasodilation in the 31–36-week fetus [29]. Interestingly, impaired fetal pulmonary vasoreactivity during brief maternal oxygen administration has been shown to predict the severity of ­pulmonary hypoplasia, pulmonary hypertension, and poor outcomes of newborns with congenital diaphragmatic ­hernia. These findings suggest that in addition to structural maturation and growth of the developing lung circulation, the vessel wall also undergoes functional maturation, leading to enhanced vasoreactivity during fetal life. The mechanisms that contribute to progressive changes in pulmonary vasoreactivity during development are not clear, but are likely due to maturational changes in endothelial cell function, especially with regard to NO production [38]. Lung endothelial NO synthase (eNOS; type III) messenger RNA (mRNA) and protein are present in the early fetus and the content increases with advancing gestation in utero and ­during the early postnatal period in rats and sheep [39–41]. The timing of this increase in lung eNOS content immediately precedes and parallels changes in the capacity to respond to endothelium-dependent vasodilators, as assessed by in vivo and in vitro studies [30, 42]. The timing of this increase in lung eNOS content coincides with the capacity to respond to endothelium-dependent vasodilator stimuli, such as oxygen and acetylcholine [30]. In contrast, fetal pulmonary arteries are already quite responsive to exogenous NO much earlier in gestation [42]. Overall, the ability of exogenous NO to dilate fetal pulmonary arteries is greater at less mature gestational ages than responsiveness to vasodilator stimuli that require the endothelium to release endogenous NO. These findings suggest that the ability of the endothelium to produce or sustain production of NO in response to specific stimuli during maturation lags the capacity of fetal pulmonary smooth muscle to relax to NO. This may account for clinical observations that extremely premature newborns are highly responsive to inhaled NO [42]. Although most studies of the perinatal lung have focused on the role of eNOS in vasoregulation, the other NO synthase (NOS) isoforms, including neuronal NOS (nNOS; type I) and inducible NOS (iNOS; type II), have been identified by immunostaining in the rat, sheep, and human fetal lung [43–47]. Lung nNOS mRNA and protein increase in parallel with eNOS expression during development in the fetal rat. iNOS has also been detected in the ovine fetal lung, and is predominantly expressed in airway epithelium and vascular smooth muscle, with little expression in vascular endothelium. Whether the “nonendothelial” (types I and II) isoforms contribute to the physiologic responses of NO-dependent modulation of fetal pulmonary vascular tone is controversial. Treatment of pregnant rats with an iNOS-selective antagonist caused constriction of the great vessels (main pulmonary artery and thoracic aorta) and

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DA in fetal rats. Selective iNOS and nNOS antagonists increase fetal PVR and inhibit shear stress vasodilation at doses that do not inhibit acetylcholine-induced pulmonary vasodilation. These findings support the speculation that iNOS and nNOS may also modulate pulmonary vascular tone in utero and at birth. NOS expression and activity are affected by multiple ­factors, including oxygen tension, hemodynamic forces, hormonal stimuli (e.g., estradiol), paracrine factors (including VEGF), substrate and cofactor availability, and superoxide production (which inactivates NO) [48–52]. Recent studies have shown that estradiol acutely releases NO and upregulates eNOS expression in fetal pulmonary artery endothelial cells. Although estradiol does not cause acute pulmonary vasodilation in  vivo, prolonged estradiol treatment causes late vasodilation which is sustained despite cessation of estradiol infusion. In contrast, VEGF acutely releases NO and causes pulmonary vasodilation in vivo. Chronic inhibition of VEGF receptors downregulates eNOS and induces pulmonary hypertension in the late-gestation fetus [53]. These findings illustrate that diverse hormonal and paracrine factors can regulate NOS expression and activity during development. Vascular responsiveness to endogenous or exogenous NO is also dependent upon several smooth muscle cell enzymes, including soluble guanylate cyclase, cyclic GMP (cGMP)specific (type 5) phosphodiesterase (PDE5), and cGMP kinase [54–57]. Several studies have shown that soluble ­guanylate cyclase, which produces cGMP in response to NO activation, is active before 0.7 of term gestation in the ovine fetal lung. Similarly, PDE5, which limits cGMP-mediated vasodilation by hydrolysis and inactivation of cGMP, is also normally active in utero. Infusions of selective PDE5 ­antagonists, including zaprinast, dipyridamole, E4021, and sildenafil, cause potent fetal pulmonary vasodilation. In the fetal lung, PDE5 expression has been localized to vascular smooth ­muscle, and PDE5 activity is high in the fetal lung in comparison with the postnatal lung. Thus, PDE5 activity appears to play a critical role in pulmonary vasoregulation during the perinatal period, and must be accounted for in assessing responsiveness to endogenous NO and related vasodilator stimuli. Functionally, the NO–cGMP cascade plays several important physiologic roles in vasoregulation of the fetal pulmonary circulation. These include (1) modulation of basal PVR in the fetus, (2) mediating the vasodilator response to ­specific physiologic and pharmacologic stimuli, and (3) opposing the strong myogenic tone in the normal fetal lung. Past studies in fetal lambs have demonstrated that intrapulmonary infusions of NOS inhibitors increase basal PVR by 35% [48]. Since inhibition of NOS increases basal PVR at least as early as 0.75 of gestation (112 days) in the fetal lamb, endogenous NOS activity appears to contribute to vasoregulation through-

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out late gestation. NOS inhibition also selectively blocks pulmonary vasodilation to stimuli such as acetylcholine, oxygen, and shear stress in the normal fetus. In addition to high PVR and altered vasoreactivity during development, the fetal lung circulation is further characterized by its ability to oppose sustained pulmonary vasodilation during prolonged exposure to vasodilator stimuli. For example, increased PaO2 increases fetal pulmonary blood flow during the first hour of treatment; however, blood flow returns toward baseline values over time despite maintaining high PaO2 [58]. Similar responses are observed during acute hemodynamic stress (shear stress) caused by partial compression of the DA or with infusions of several pharmacologic agents [59, 60]. These findings suggest that unique mechanisms exist in the fetal pulmonary circulation that oppose vasodilation and maintain high PVR in utero. We have speculated that this response reflects the presence of an augmented myogenic response within the fetal pulmonary circulation. Recent studies have suggested that NO release plays an additional role in modulating high intrinsic or myogenic tone in the fetal pulmonary circulation. The myogenic response is commonly defined by the presence of increased vasoconstriction caused by acute elevation of intravascular pressure or “stretch stress” [61–64]. Past in  vitro studies demonstrated the presence of a myogenic response in sheep pulmonary arteries, and that fetal pulmonary arteries have greater myogenic activity than neonatal or adult arteries. More recent studies of intact fetal lambs have demonstrated that high myogenic tone is normally operative in the fetus, and contributes to maintaining high PVR in utero. These studies demonstrated that NOS inhibition not only blocks vasodilation to several physiologic stimuli, but acute inhibition of NO production unmasks a potent myogenic response. Thus, these findings support our hypothesis that NOS inhibition unmasks a potent myogenic response, which maintains high PVR in the normal fetus. Further work suggests that downregulation of NO, as observed in experimental pulmonary hypertension, may further increase myogenic activity, increasing the risk for unopposed vasoconstriction in response to stretch stress at birth. In addition to high PVR and low pulmonary blood flow, the fetal pulmonary circulation is characterized by mechanisms that oppose vasodilation. Past work suggests that high myogenic tone contributes to high PVR and may contribute to autoregulation of blood flow in the fetal lung. Rho kinase (ROCK) can mediate the myogenic response in the adult systemic circulation, but whether high ROCK activity contributes to the myogenic response and modulates timedependent vasodilation in the developing lung circulation was recently studied [65]. Fasudil, a ROCK inhibitor, reduced the myogenic response by 90% in chronically prepared fetal sheep. It appears that high ROCK activity

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opposes pulmonary vasodilation in utero and that the myogenic response maintains high PVR in the normal fetal lung through ROCK activation. Although several vasodilators, including PGI2, are released upon stimulation of the fetal lung (e.g., increased shear stress), basal prostaglandin release appears to play a less important role than NO in fetal pulmonary vasoregulation. For example, cyclooxygenase inhibition has minimal effect on basal PVR and does not increase myogenic tone in the fetal lamb [66, 67]. The physiologic roles of other dilators, including adrenomedullin, adenosine, and endothelium-derived hyperpolarizing factor (EDHF) are not clear [68]. EDHF is a short-lived product of cytochrome P450 activity that is produced by vascular endothelium, and has been found to cause vasodilation through activation of calcium-activated K+ channels in vascular smooth muscle in vitro [69]. K+ channel activation appears to modulate basal PVR and ­vasodilator responses to shear stress and increased oxygen tension in the fetal lung, but whether this is partly related to EDHF activity is unknown [33, 70]. Carbon monoxide (CO) is another gaseous molecule that is produced by heme oxygenase and has been shown to have several vascular effects, including vasodilation in the adult systemic and pulmonary vascular beds [70]. CO may act in part through activation of soluble guanylate cyclase, increasing cGMP content in vascular smooth muscle, and causing vasodilation, as described for NO [71]. Despite several ­studies suggesting an important role in vasoregulation in some models, CO has yet to be shown to play an important physiologic role in the fetal lung. For example, inhaled CO ­treatment of the late-gestation fetal lamb had no affect on PVR, and infusions of a heme oxygenase inhibitor did not alter basal pulmonary vascular tone [72]. Further studies are needed to demonstrate its physiologic importance in the developing lung circulation. Vasoconstrictors have long been considered as potentially maintaining high PVR in utero. Several candidate products, including lipid mediators (thromboxane A2, leukotrienes C4 and D4, and platelet-activating factor) and ET-1, have been extensively studied. Thromboxane A2, a potent pulmonary vasoconstrictor that has been implicated in animal models of group B streptococcal sepsis, does not appear to influence PVR in the normal fetus. In contrast, inhibition of leukotriene production causes fetal pulmonary vasodilation [73]; however, questions have been raised regarding the specificity of the antagonists used in these studies. Similarly, inhibition of platelet-activating factor may influence PVR during the normal transition, but data from recent experimental studies are difficult to interpret owing to extensive nonspecific hemodynamic effects. ET-1, a potent vasoconstrictor and comitogen that is ­­produced by vascular endothelium, has been demonstrated to play a key role in fetal pulmonary vasoregulation [74–79].

S.H. Abman and R.H. Steinhorn

PreproET-1 mRNA (the precursor to ET-1) was identified in fetal rat lung early in gestation, and high circulating ET-1 levels are present in umbilical cord blood. Although ET-1 causes an intense vasoconstrictor response in vitro, its effects in the intact pulmonary circulation are complex. Brief infusions of ET-1 cause transient vasodilation, but PVR progressively increases during prolonged treatment. The biphasic pulmonary vascular effects during pharmacologic infusions of ET-1 are explained by the presence of at least two ­different ET receptors. The ETB receptor, localized to the endothelium in the sheep fetus, mediates the ET-1 vasodilator response through the release of NO. A second receptor, the ETA receptor, is located on vascular smooth muscle, and when activated, causes marked constriction. Although capable of both vasodilator and constrictor responses, ET-1 is more likely to play an important role as a pulmonary vasoconstrictor in the normal fetus. This is suggested in extensive fetal studies that have shown that inhibition of the ETA receptor decreases basal PVR and augments the vasodilator response to shearstress-induced pulmonary vasodilation. Thus, ET-1 is likely to modulate PVR through the ETA and ETB receptors, but its predominant role is as a vasoconstrictor through stimulation of the ETA receptor.

3.2 Control of the DA Since most of the right ventricular output crosses the DA in utero, patency of the DA is absolutely vital for fetal survival and well-being. Premature DA closure in utero causes severe pulmonary hypertension, congestive heart failure, hydrops fetalis, or severe hypoxemia. In contrast, an inability of the DA to close after birth may complicate lung disease in the premature newborn with respiratory distress syndrome or cause high-flow pulmonary vascular injury during postnatal life. In addition, maintaining DA patency can be critical for survival in newborns and infants with ductus – dependent cyanotic congenital heart disease. Finally, insights into the unique nature of regulation of the DA, especially in regard to smooth muscle cell tone, proliferation, and synthetic functions, may provide important lessons in vascular biology. For example, changes in oxygen tension have striking effects on DA smooth muscle that are different from those its neighboring smooth muscle cells in systemic (aortic) and pulmonary circulations. Low O2 tension constricts pulmonary vessels but dilates the DA. The increase in O2 tension contributes to the fall in PVR at birth but paradoxically constricts the DA. Past studies demonstrated the important role of intramural prostaglandin production on DA tone, as evidenced by the potent constrictor effects of cyclooxygenase inhibitors. However, recent studies have shown that regulation of the

9  The Normal Fetal and Neonatal Pulmonary Circulation

DA is complex, and involves multiple signals, such as ET-1, CO, the cytochrome P450 system, and K+-channel activities [80–82]. Recent in vitro data suggest that a cytochrome P450 based monooxygenase reaction transduces the signal for DA closure, and that DA constriction is mediated through increased ET-1 production and stimulation of the ETA receptor. Furthermore, CO can relax the DA, but its effects vary, according to ambient oxygen tension, and may act in part by inhibiting ET-1 production. Other studies have demonstrated that ion channels mediate early changes in DA tone, primarily through voltage-gated K+ channels. Decreased oxygen tension inhibits voltage-gated K+ channels, opening Ca2+ channels, and causing constriction in pulmonary artery smooth muscle cells; in fetal DA, oxygen activates a calciumactivated K+ channel and causes vasodilation. ET may act by inhibiting voltage-activated K+ channels.

3.3 Mechanisms of Pulmonary Vasodilation at Birth Within minutes after delivery, pulmonary artery pressure falls and blood flow increases in response to birth-related stimuli. Mechanisms contributing to the fall in PVR at birth include establishment of an air–liquid interface, rhythmic lung distension, increased oxygen tension, and altered production of vasoactive substances. Physical stimuli, such as increased shear stress, ventilation, and increased oxygen, cause pulmonary vasodilation in part by increasing production of vasodilators such as NO and PGI2. Pretreatment with the arginine analogue, nitro-l-arginine, blocks NOS activity, and attenuates the decline in PVR after delivery of near-term fetal lambs. These findings suggested that about 50% of the rise in pulmonary blood flow at birth may be directly related to the acute release of NO. Specific mechanisms that cause NO release at birth include the marked rise in shear stress, increased oxygen, and ventilation. Increased PaO2 triggers NO release, which augments vasodilation through cGMP kinase mediated stimulation of K+ channels [83–85]. Although eNOS has been presumed to be the major contributor of NO at birth, studies suggest that other isoforms (iNOS and nNOS) may be important sources of NO release in utero and at birth as well. Although early studies were performed in term animals, NO also contributes to the rapid decrease in PVR at birth in premature lambs, at least as early as 112–115 days (0.7 of term). Other vasodilator products, including PGI2, also modulate changes in pulmonary vascular tone at birth. Rhythmic lung distension and shear stress stimulate both PGI2 and NO production in the late-gestation fetus, but increased O2 ­tension triggers NO activity and overcomes the effects of prostaglandin inhibition at birth. In addition, the vasodilator

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effects of exogenous PGI2 are blocked by NOS inhibitors, suggesting that NO modulates PGI2 activity in the perinatal lung [2]. Adenosine release may also contribute to the fall in PVR at birth, but its actions may be partly through enhanced production of NO [86]. Thus, although NO does not account for the entire fall in PVR at birth, NOS activity appears important in achieving postnatal adaptation of the lung circulation. Transgenic eNOS knockout mice successfully make the transition at birth without evidence of PPHN [87, 88]. This finding suggests that eNOS−/− mice may have adaptive mechanisms, such as a compensatory vasodilator mechanisms (such as upregulation of other NOS isoforms or dilator prostaglandins) or less constrictor tone. Interestingly, these mice are more sensitive to the development of pulmonary hypertension at relatively mild decreases in PaO2 and have higher neonatal mortality when exposed to hypoxia after birth [89]. We speculate that isolated eNOS deficiency alone may not be sufficient for the failure of postnatal adaptation, but that decreased ability to produce NO in the setting of a perinatal stress (such as hypoxia, inflammation, hypertension, or upregulation of vasoconstrictors) may cause PPHN. Although these studies were performed in term animals, similar mechanisms also contribute to the rapid decrease in PVR at birth in premature lambs [48, 90]. The pulmonary vasodilator responses to ventilation with hypoxic gas mixtures (or rhythmic distension) of the lung or increased PaO2 are partly due to stimulation of NO release in premature lambs at least as early as 112–115 days (0.7 of term) [48]. Other vasodilator products, including PGI2, also modulate changes in pulmonary vascular tone at birth [32, 33, 36]. Rhythmic lung distension and shear stress stimulate both PGI2 and NO production in the late-gestation fetus; increased O2 tension triggers NO activity but does not appear to alter PGI2 production in vivo [30, 32, 36].

4 Conclusions In summary, lung vascular growth and development is a dynamic process, which includes critical changes throughout development, beginning in the embryonic period and continuing throughout gestation and during postnatal life. Production of proangiogenic growth factors, such as VEGF and NO, maintains pulmonary vascular structure in normal and disease states, perhaps owing to enhanced endothelial cell differentiation, survival, and function. Angiogenesis is closely linked with alveolarization in several models of lung development, and clinical and experimental studies have suggested that disruption of vascular growth and signaling may contribute to impaired alveolar development in the setting of lung hypoplasia. Future work is needed to better

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define basic mechanisms of lung vascular growth and development, which will likely lead to novel therapeutic approaches to diseases associated with impaired vascular growth or pulmonary hypertension.

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9  The Normal Fetal and Neonatal Pulmonary Circulation 40. Halbower AC, Tuder RM, Franklin WA, Pollock JS, Forstermann U, Abman SH (1994) Maturation-related changes in endothelial NO synthase immunolocalization in the developing ovine lung. Am J Physiol 267:L585–L591 41. Parker TA, Le Cras TD, Kinsella JP, Abman SH (2000) Devel­ opmental changes in endothelial NO synthase expression in the ovine fetal lung. Am J Physiol 278:L202–L208 42. Kinsella JP, Ivy DD, Abman SH (1994) Ontogeny of NO activity and response to inhaled NO in the developing ovine pulmonary ­circulation. Am J Physiol 267:H1955–H1961 43. Sherman TS, Chen Z, Yuhanna IS, Lau KS, Margraf LR, Shaul PW (1999) NO synthase isoform expression in the developing lung ­epithelium. Am J Phsyiol 276:L383–L390 44. Rairhig R, Ivy DD, Kinsella JP, Abman SH (1998) Inducible NOS inhibitors increase pulmonary vascular resistance in the late-gestation fetus. J Clin Invest 101:15–21 45. Tzao C, Nickerson PA, Russell JA, Noble B, Steinhorn RM (2000) Heterogeneous distribution of type I NOS in pulmonary vasculature of ovine fetus. Histochem Cell Biol 114:421–430 46. Rairigh RL, Storme L, Parker TA, Le Cras TD, Jakkula M, Abman SH (2000) Role of neuronal nitric oxide synthase in regulation of vascular and ductus arteriosus tone in the ovine fetus. Am J Physiol Lung Cell Mol Physiol 278:L105–L110 47. Rairigh RL, Parker TA, Ivy DD, Kinsella JP, Fan I, Abman SH (2001) Role of inducible nitric oxide synthase in the transition of the pulmonary circulation at birth. Circ Res 88:721–726 48. Abman SH, Kinsella JP, Parker TA, Storme L, Le Cras TD (1999) Physiologic roles of NO in the perinatal pulmonary circulation. In: Weir EK, Archer SL, Reeves JT (eds) Fetal and neonatal pulmonary circulation. Futura, New York, pp 239–260 49. Parker TA, Kinsella JP, Galan HL, Richter G, Abman SH (2000) Prolonged infusions of estradiol dilate the ovine fetal pulmonary circulation. Pediatr Res 47:89–96 50. Parker TA, Afshar S, Kinsella JP, Ivy DD, Shaul PW, Abman SH (2001) Effects of chronic estrogen receptor blockade on the pulmonary circulation in the late gestation ovine fetus. Am J Physiol Heart Circ Physiol 281:H1005–H1014 51. MacRitchie AN, Jun SS, Chen Z et al (1997) Estrogen upregulates endothelial NO synthase gene expression in fetal pulmonary artery endothelium. Circ Res 81:355–362 52. Grover TR, Parker TA, Zenge JP, Markham NE, Abman SH (2003) Intrauterine hypertension decreases lung VEGF expression and VEGF inhibition causes pulmonary hypertension in the ovine fetus. Am J Physiol Lung Cell Mol Physiol 284:L508–L517 53. Hanson KA, Burns F, Rybalkin SD, Miller J, Beavo J, Clarke WR (1995) Developmental changes in lung cGMP phosphodiesterase-5 activity, protein and message. Am J Respir Crit Care Med 158:279–288 54. Cohen AH, Hanson K, Morris K et  al (1996) Inhibition of cGMP-specific phosphodiesterase selectively vasodilates the pulmonary circulation in chronically hypoxic rats. J Clin Invest 97:172–179 55. Tzao C, Nickerson PA, Russell JA, Noble BK, Steinhorn RH (2003) Paracrine role of soluble guanylate cyclase and type III nitric oxide synthase in ovine fetal pulmonary circulation: a double labeling immunohistochemical study. Histochem Cell Biol 119: 125–130 56. Thusu KG, Morin FC 3rd, Russell JA, Steinhorn RH (1995) The cGMP phosphodiesterase inhibitor zaprinast enhances the effect of NO. Am J Respir Crit Care Med 152:1605–1610 57. Ziegler JW, Ivy DD, Fox JJ, Kinsella JP, Clarke WR, Abman SH (1995) Dipyridamole, a cGMP phosphodiesterase inhibitor, causes pulmonary vasodilation in the ovine fetus. Am J Physiol 269:H473–H479 58. Accurso FJ, Alpert B, Wilkening RB, Petersen RG, Meschia G (1986) Time-dependent response of fetal pulmonary blood flow to an increase in fetal oxygen tension. Respir Physiol 63:43–52

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146 82. Reeve HL, Weir EK (1999) Regulation of ion channels in the ductus arteriosus. In: Weir EK, Archer SL, Reeves JT (eds) Fetal and neonatal pulmonary circulation. Futura, New York, pp 319–330 83. Archer SL, Huang JMC, Hampl V, Nelson DP, Shultz PJ, Weir EK (1994) NO and cGMP cause vasorelaxation by activation of a charybdotoxin-sensitive K channel by cGMP-dependent protein kinase. Proc Natl Acad Sci U S A 91:7583–7587 84. Rhodes MT, Porter VA, Saqueton CB, Herron JM, Resnik ER, Cornfield DN (2001) Pulmonary vascular response to normoxia and Kca channel activity is developmentally regulated. Am J Physiol Lung Cell Mol Physiol 280:L1250–L1257 85. Tristani-Firouzi M, Martin EB, Tolarova S, Weir EK, Archer SL, Cornfield DN (1996) Ventilation-induced pulmonary vasodilation at birth is modulated by potassium channel activity. Am J Physiol 271:H2353–H2359 86. Konduri GG, Mital S, Gervasio CT, Rotta AT, Forman K (1997) Purine nucleotides contribute to pulmonary vasodilation caused

S.H. Abman and R.H. Steinhorn by  birth-related stimuli in the ovine fetus. Am J Physiol 272: H2377–H2384 87. Fagan KA, Fouty BW, Tyler RC et al (1999) The pulmonary circulation of mice with either homozygous or heterozygous disruption of endothelial NO synthase is hyper-responsive to chronic hypoxia. J Clin Invest 103:291–299 88. Steudel W, Scherrer-Crosbie M, Bloch KD et al (1998) Sustained pulmonary hypertension and right ventricular hypertrophy after chronic hypoxia in mice with congenital deficiency of NOS III. J Clin Invest 101:2468–2477 89. Balasubramanium V, Maxey AM, Morgan DB, Markham NE, Abman SH (2006) Inhaled NO restores lung structure in eNOSdeficient mice recovering from neonatal hypoxia. Am J Physiol Lung Cell Mol Physiol 291:119–127 90. Kinsella JP, McQueston JA, Rosenberg AA, Abman SH (1992) Hemodynamic effects of exogenous nitric oxide in ovine transitional pulmonary circulation. Am J Physiol 263:H875–H880

Chapter 10

Excitation–Contraction Coupling and Regulation of Pulmonary Vascular Contractility Jeremy P.T. Ward and Greg A. Knock

Abstract  Excitation-contraction coupling in vascular smooth muscle (VSM) encompasses a myriad of cellular pathways that contribute to or modulate the coupling of an initial stimulus to the development of force and cell shortening. These pathways converge into those associated with changes in either cytosolic Ca2+ concentration ([Ca2+]cyt) or the sensitivity of the contractile apparatus (Ca2+ sensitivity) to such changes in [Ca2+]cyt; both may be strongly modulated by alterations in the cytoskeleton, though there is emerging evidence that the latter can independently lead to changes in vascular contractility. Contraction in VSM is classically related to the interaction between actin and myosin. Signaling pathways that initiate or regulate contractile force include those that regulate [Ca2+]cyt, Ca2+ sensitivity (through relative alterations in myosin light chain kinase/myosin light chain phosphatase activity), and F-actin assembly and focal adhesions through which the force is transduced. Although the pulmonary vasculature is very similar to the systemic vasculature in terms of its basic structure and key excitation-contraction coupling mechanisms, its function as a low-pressure, low-resistance circulation which is primarily concerned with gas exchange means that it exhibits subtle and not so subtle differences in response to a variety of stimuli. In particular, the pulmonary circulation is unique in that in the adult it constricts to moderate hypoxia, whereas systemic circulations tend to dilate. The mechanisms underlying hypoxic pulmonary vasoconstriction are complex, but as for many other vasoconstrictor stimuli they ultimately involve both an elevation of [Ca2+]cyt and an increase in Ca2+ sensitivity of the contractile apparatus. This chapter describes our current understanding concerning the excitation-contraction pathways in pulmonary vascular smooth muscle. Keywords  EC coupling • Smooth muscle contraction • Contractility • Calcium and calmodulin • Contractile apparatus • Sensitization and desensitization • Myosin light chain kinase • Myosin light chain phosphatase J.P.T. Ward (*) Division of Asthma, Allergy and Lung Biology, King’s College London, London SE1 9RT, UK e-mail: [email protected]

1 Introduction Excitation–contraction coupling in vascular smooth muscle (VSM) encompasses a myriad of cellular pathways that ­contribute to or modulate the coupling of an initial stimulus to the development of force and cell shortening. These ­pathways converge into those associated with changes in either cytosolic Ca2+ concentration or the sensitivity of the contractile apparatus to such changes in cytosolic Ca2+ ­concentration (Ca2+ sensitivity) [1, 2]; both may be strongly modulated by alterations in the cytoskeleton, though there is emerging evidence that the latter can independently lead to changes in vascular contractility [3]. Contraction in VSM is classically related to the interaction between actin and myosin. Force is generated through the ATP- and Ca2+-dependent ratchet-like movement of ­myosin (an ATPase) along fibers of F-actin, a process called cross-bridge cycling. Assembly of myosin units into thick filaments and myosin ATPase activity is promoted by the presence of F-actin. This activity is greatly enhanced by phosphorylation of the 20-kDa myosin light chain (MLC20) by the Ca2+–calmodulin-activated myosin light chain kinase (MLCK). Conversely, MLC20 is dephosphorylated by ­myosin light chain phosphatase (MLCP) [2] (Fig. 1). The net phosphorylation state of MLC20 is therefore primarily a ­product of the relative activities of MLCK and MLCP, although Ca2+independent diphosphorylation of MLC20 may also be of importance in VSM [4]. Signaling pathways that initiate or regulate contractile force therefore include those that regulate cytosolic Ca2+ concentration, Ca2+ sensitivity (through relative alterations in MLCK/MLCP activity), and F-actin assembly and focal ­adhesions through which the force is transduced. Although the pulmonary vasculature is very similar to the systemic vasculature in terms of its basic structure and key excitation–contraction coupling mechanisms, its function as a low-pressure, low-resistance circulation which is primarily concerned with gas exchange means that it exhibits subtle and not so subtle differences in response to a variety of ­stimuli. In particular, the pulmonary circulation is unique in that in the adult it constricts to moderate hypoxia (hypoxic

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Fig.  1  Basic molecular mechanisms underlying force development in smooth muscle, and their regulation. Force is generated by ATP-dependent interaction between the motor domain of myosin and F-actin, regulated by phosphorylation of 20-kDa myosin light chain (MLC20) on Ser-19 and Thr-18 by myosin light chain kinase (MLCK). A rise in cytosolic Ca2+ concentration activates MLCK via Ca2+–calmodulin. MLC20 is dephosphorylated by myosin light chain phosphatase (MLCP), under the regulation of kinases including Rho kinase and protein kinase C. See the text for details

pulmonary vasoconstriction, HPV), whereas systemic circulations tend to dilate (see Chap. 46). The mechanisms underlying HPV are complex, but as for many other vasoconstrictor stimuli they ultimately involve both an elevation of cytosolic Ca2+ concentration and an increase in Ca2+ sensitivity of the contractile apparatus [5]. This chapter describes our current understanding concerning the excitation–contraction pathways in pulmonary VSM, although for some aspects one must refer to systemic VSM as equivalent studies have yet to be performed in the pulmonary vasculature. Section  2 concerns the ­pathways and mechanisms underlying stimulus-induced Ca2+ mobilization, whereas Sect. 3 deals with regulation of Ca2+ sensitivity.

2 Ca2+ Mobilization Ca2+ mobilization in VSM involves Ca2+ entry via a number of Ca2+-permeable plasmalemmal ion channels, Ca2+ release from intracellular stores, and in some cases the Na+–Ca2+ exchanger (NCX) (Fig. 2). Regulation of cytosolic Ca2+ concentration and thus vascular tone involves considerable interaction between the mechanisms that regulate membrane potential, Ca2+ entry, and Ca2+ release, though the precise details are still poorly understood. Another layer of complexity concerns the presence of multiple intracellular stores [sarcoplasmic reticulum (SR), mitochondria, and lysosomes], and the fact that spatial and temporal relationships may be crucial to determining force development.

2.1 Ca2+ Entry Pathways 2.1.1 Voltage-Dependent Ca2+ Entry Regulation of smooth muscle tone, particularly that of VSM, is strongly dependent on membrane potential, with depolarization initiating activation of voltage-dependent, L-type Ca2+ channels (Cav1.2) and consequent Ca2+ entry [1, 6]. Numerous studies have shown that a wide range of agonists cause elevation of cytosolic Ca2+ concentration in pulmonary artery smooth muscle (PASM) either partially or entirely through depolarization [7–9]. Moreover, the discovery that inhibitors of L-type channels suppress HPV [10] led to the long-standing hypothesis that HPV is primarily caused by redox-mediated inhibition of delayed-rectifier K+ channels, depolarization, and voltage-dependent Ca2+ entry [11]. The regulation of PASM membrane potential is complex. Numerous K+ channels have been implicated, including the delayed rectifiers KV1.5, KV2.1, and KV3.4, members of the TASK twin-pore family, KCNQ, and small, intermediate, and large conductance Ca2+-activated K+ channels (SKCa, IKCa, BKCa) (see Chap. 13 [12–16]). Notably, there is significant heterogeneity in KV channel expression, not only between PASM cells (PASMCs) isolated from pulmonary arteries of different sizes but also within segments of the same artery [17, 18]. In addition to K+-channel-mediated outward currents, inward currents through channels permeable to Cl−, Na+, and other cations lead to a resting membrane potential that is several tens of millivolts more positive than the K+ equilibrium potential [19–21] (Fig.  2). There are therefore a plethora of mechanisms by which agonists and

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Fig. 2  Ca2+ mobilization pathways in vascular smooth muscle and pulmonary artery smooth muscle (PASM). The membrane potential is determined by outward (hyperpolarizing) currents through K+ channels including KV, BKCa, and TASK (1) and inward (depolarizing) currents through Ca2+-dependent Cl− (ClCa) channels and nonselective cation channels [store-operated channels (SOCs) and receptor operated channels (ROCs)] (2). Depolarization activates Ca2+ entry through L-type channels (3), and the subsequent local elevation of Ca2+ concentration may activate ryanodine receptors (RyR) on adjacent sarcoplasmic reticulum (SR) to increase the frequency of sparks (4). RyR may also be activated or potentiated by cyclic ADP ribose. Binding of an agonist to a G-protein-coupled receptor activates phospholipase C (PLC) to generate diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3) (5); DAG activates ROCs, eliciting Na+ and Ca2+ entry and depolarization,

whereas IP3 activates clusters of IP3 receptors (IP3R) on the SR, causing Ca2+ release (6), which may also activate adjacent RyR. Store depletion is sensed by STIM1 within the SR, which translocates to and activates SOCs (7). The elevation in subplasmalemmal Na+ concentration and depolarization resulting from activation of nonselective ROCs or SOCs may be sufficient to drive reverse-mode operation of the Na+–Ca2+ exchanger (NCX), leading to Ca2+ entry (8). Much of the Ca2+ entering the cell and released from stores may be sequestered by the superficial SR through sarcoendoplasmic reticulum Ca2+-ATPase (SERCA) and mitochondria via the Ca2+ uniporter (superficial buffer barrier) (9), so that a relatively small proportion enters the central cytosol. Ca2+ is removed from the cell by the plasmalemmal Ca2+-ATPase and normal operation of the NCX (10). Ca2+ is expelled from the mitochondria by the mitochondrial NCX

changes in the environment can induce depolarization and thus vasoconstriction. Many vasoconstrictor agonists elicit depolarization of PASM. In some cases this has been attributed to inhibition of KV channels, possibly via protein kinase C (PKC) [9, 22, 23]. However, there is strong evidence that activation of Ca2+dependent Cl− (ClCa) channels plays a key role in the depolarization of PASM to agonists such as norepinephrine, endothelin-1, and nucleotides, secondary to Ca2+ release from inositol 1,4,5-trisphosphate (IP3)- and ryanodine-sensitive stores [24–27], though the latter has also been reported to inhibit KV channels directly in PASM [28] (Fig. 2). It is also now evident that activation of Na+ influx via nonselective cation channels, as a result of either Ca2+ store depletion (store-operated channels, SOCs) or activation of a receptor– G protein–second messenger pathway (receptor-operated channels, ROCs), is an important primary cause of depolarization and subsequent voltage-gated Ca2+ entry in PASM [8, 29–31] (Fig. 2) and other smooth muscles [32]. Depolarization of PASM in response to hypoxia has long been ascribed to inhibition of K+ channels, and the best known hypothesis suggests that this is due to oxidation of channel

residues, specifically on KV1.5 [14, 33]. However, a number of other mechanisms by which hypoxia could lead to inhibition of these and other KV channels have also been proposed, including elevation of cytosolic Ca2+ concentration owing to Ca2+ release from stores [28, 34], increased synthesis of products of the cytochrome P450 pathway [35], phosphorylation by PKCx following an increase in the level of ceramide [36], and an increase in the level of endothelin-1 [37]. There is also mounting evidence that hypoxia-induced depolarization of PASM is at least partially due to activation of SOCs [5, 30, 31].

2.1.2 Voltage-Independent Ca2+ Entry A number of voltage-independent pathways can contribute to the stimulus-induced elevation of cytosolic Ca2+ concentration in VSM, and these may be of particular importance for the regulation of pulmonary vascular tone in response to a variety of stimuli, including many agonists, hypoxia, stretch, and potentially temperature [38]. In broad terms, these ­pathways can be separated into those involving ROCs, including classic ligand-gated channels such as the P2X receptor and those

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indirectly activated via G proteins and second messengers [39], and SOCs, which are activated by depletion of Ca2+ stores [40] (Fig. 2). However, the terms “SOC” and “ROC” have been used somewhat indiscriminately, and differentiation between the two pathways is not absolute [41, 42]. Here, the term “ROC” is used solely to denote agonist-activated pathways that are independent of store release. The molecular identity of the channels underlying voltage-independent Ca2+ entry is not clear. Until recently, there was a general consensus that in smooth ­muscle at least both ROCs and SOCs are nonselective and permeable to both Na+ and Ca2+, and are most probably formed from members of the transient receptor potential (TRP) protein superfamily [39, 40, 42, 43] (described in other chapters within this tome). However, recent evidence has raised doubts concerning the identity and selectivity of SOCs (see below). A number of important vasoconstrictor agonists have been shown to activate a ROC-mediated Ca2+ entry pathway in PASM and VSM, including ATP (via P2X receptors), endothelin-1, prostaglandin F2a, U-46619, serotonin, ­arachidonic acid, and sphingosylphosphorylcholine (SPC) [8, 26, 44–46]. A similar Ca2+ entry pathway is also activated by sustained HPV [47]. With the exception of that associated with P2X receptors, the inward currents activated by these agonists tend to exhibit similar pharmacological profiles for lanthanides and other channel blockers, and a greater selectivity for Na+ compared with Ca2+, suggesting that the same channel protein may be involved. In PASM and other tissues, the most likely candidate for ROCs is thought to be TRPC6, which is activated by the phospholipase C (PLC) product diacylglycerol (DAG) [32, 39, 42]. Interestingly, TRPC6 has been implicated in at least the initial phase of HPV in genetically modified mice, and its activation and translocation to the membrane may be influenced by a cytochrome P450 epoxygenase product [48, 49]. Conversely, it has recently been reported that the ROC activated by serotonin and arachidonic acid in PASM may involve TRPV4, also activated via a cytochrome P450 epoxygenase product [50]. Voltage-independent Ca2+ entry through SOCs activated by depletion of intracellular Ca2+ stores, variously termed “capacitative Ca2+ entry” (CCE) and “store-operated Ca2+ entry,” seems to play a particularly prominent role in the regulation of pulmonary vascular tone and its response to agonists compared with other vascular beds [5, 51]. In particular, there is strong evidence that CCE is of central importance to the elevation of PASM cytosolic Ca2+ concentration during HPV [5, 30, 31, 47, 52–54]. A number of lines of evidence suggest that in PASM at least, CCE and the associated SOCs are nonselective [42, 51, 54], and involve TRPC proteins, most likely TRPC1 and TRPC4, although TRPC6 may also contribute to CCE at least under pathological c­ onditions [38, 42, 52, 55]. Recently, however, the role of TRPC proteins in SOCs has been questioned, as it has been shown that the SOC ­correlate Ca2+-release-activated channel (CRAC) originally described in inflammatory cells is in fact a combination of plasmalem-

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mal Orai proteins with the endoplasmic reticulum Ca2+sensing protein stromal-interacting molecule (STIM1), which on store depletion associates with Orai and initiates Ca2+ entry [56]. However, CRAC is universally described as highly selective for Ca2+, unlike SOCs in PASM, although several reports now suggest that STIM1 can interact with TRPC proteins to determine their function as SOCs [57, 58]. Notably, in the pulmonary circulation the amplitude of both store-depletion-induced and hypoxia-induced CCE has been correlated with expression of STIM1, TRPC1, TRPC4, and TRPC6 [52]. On the other hand, there is circumstantial ­evidence for more than one type of SOC in PASM, potentially linked to different stores and/or different signaling pathways [54, 59].

2.1.3 The Na+–Ca2+ Exchanger and Plasmalemmal Ca2+-ATPase Removal of Ca2+ from the cell is handled by the plasmale­ mmal Ca2+-ATPase (PMCA) and the NCX [6]. PMCA is stimulated by Ca2+–calmodulin, and by cyclic-nucleotidedependent phosphorylation by protein kinase A (PKA) and protein kinase G (PKG), providing an important ­mechanism by which cyclic AMP (cAMP)- and cyclic GMP (cGMP)raising agents such as prostacyclin, nitric oxide, and sildenafil can reduce PASM cytosolic Ca2+ concentration and cause relaxation in the pulmonary circulation. NCX normally uses the Na+ electrochemical gradient to pump one Ca2+ ion out of the cell in exchange for three Na+ ions; it is therefore inherently electrogenic and dependent on both the membrane potential and the local subplasmalemmal Na+ concentration. Although NCX is normally associated with removal of cytosolic Ca2+ and relaxation, it can operate in reverse mode when the membrane potential is relatively positive and/or there is an elevation in subplasmalemmal Na+ concentration. NCX is known to be expressed and to play an important physiological role in PASM [60, 61]. A number of studies in  PASM and other smooth muscles have suggested that reverse-mode operation of NCX can contribute to the elevation of cytosolic Ca2+ concentration and store refilling following store depletion and activation of CCE [61, 62] and agonist-mediated activation of TRPC6 [63, 64], as a result of Na+ entry through these nonselective pathways (see above) and a consequent increase in subsarcolemmal Na+ concentration (Fig. 2). Similarly, increased expression of NCX in idiopathic pulmonary hypertension and in proliferating PASMCs may lead to enhanced Ca2+ entry that contributes to the phenotypic changes and pathology [65, 66]. Although it has been suggested that inhibition or reversal of NCX may in part underlie the hypoxia-induced elevation of cytosolic Ca2+ concentration in isolated or cultured PASMCs [60, 65, 67], this was not found to be the case in small intrapulmonary arteries of the rat [68]. This may be related to the finding that

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under normal conditions Ca2+ entry via reverse-mode operation of NCX is buffered by uptake into a superficial buffer barrier consisting of closely approximated SR and mitochondria (see later) [69, 70].

2.2  Intracellular Ca2+ Stores The major intracellular Ca2+ store in VSM is the SR, but other organelles, in particular mitochondria and acid lysosomes, also contain Ca2+ at far higher concentrations than the cytosol, and it can be released by physiological and pathophysiological stimuli (Fig. 2). Ca2+ stores play a critical role in the regulation of VSM reactivity; they provide a source of releasable Ca2+ for promoting contraction and other functions, modulate and coordinate the activity of ­plasmalemmal ion channels and other stores, and maintain a low basal cytosolic Ca2+ concentration by avidly sequestering Ca2+. Depletion of the SR also activates CCE (see earlier), which contributes to store refilling and cytosolic Ca2+ signaling. An important function of peripheral SR and mitochondria is to provide a superficial buffer barrier close to the plasmalemma, which buffers Ca2+ entry and strongly limits its ­pervasion into the deeper cytosol, potentially creating intracellular Ca2+ gradients [71] (Fig.  2); preferentially directed Ca2+ release toward the plasmalemma potentiates Ca2+ removal by PMCA and NCX, and regulates the activity of Ca2+-dependent ion channels [6, 69–71]. The superficial ­buffer barrier with its highly regulated Ca2+ release and uptake mechanisms therefore provides another level of ­precise control over the spatial and temporal characteristics of changes to cytosolic Ca2+ concentration. Such dynamic and localized interactions between SR, mitochondria, plasmalemma, and potentially lysosomes within subcellular microdomains underlie Ca2+ oscillations, sparks, and waves, which may be of much more importance to the ­regulation of VSM tone than the mean global cytosolic Ca2+ ­concentration [1, 72].

2.2.1 The Sarcoplasmic Reticulum The SR is distributed as a tubular network largely localized to peripheral and perinuclear regions of the VSM cell [73, 74] (Fig. 2). It sequesters Ca2+ from the cytosol by means of the high-affinity sarcoendoplasmic reticulum Ca2+-ATPase (SERCA), the main isoform in smooth muscle being SERCA-2b [75]. SERCA is regulated via an inhibitory association with phospholamban, a small protein spanning the SR membrane; phosphorylation of phospholamban by PKA or PKG allows enhanced activity of SERCA and thus Ca2+ uptake from the cytosol, another key mechanism by which cAMP- and cGMP-raising agents cause relaxation of

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pulmonary artery [76]. The SERCA inhibitors thapsigargin and cyclopiazonic acid have been used extensively as experimental tools in the study of SR function. Two key mechanisms regulate Ca2+ release from the SR, IP3 receptors (IP3R), and the Ca2+ release channels otherwise known as ryanodine receptors (RyR), as they selectively bind the plant alkaloid ryanodine. The IP3R is the major Ca2+ release channel in VSM, and contributes to the spatial and temporal regulation of local and global Ca2+ signaling (Fig. 2). There are three isoforms of IP3R (IP3R1, IP3R2, and IP3R3); IP3R1 is the predominant or only isoform expressed in VSM, whereas all three are found in the endothelium [77]. Agonists acting via Gq/G11-protein-coupled or tyrosine kinase coupled receptors activate cleavage of phosphatidylinositol 4,5-bisphosphate by PLC to generate DAG (which activates ROCs and PKC, see earlier), and IP3, which binds to IP3R in the SR, causing Ca2+ release. In PASM, vasoconstrictors such as norepinephrine, ATP, prostaglandin FP-receptor agonists, and in some cases endothelin-1 can elicit Ca2+ release and elevation of cytosolic Ca2+ concentration by this mechanism [8, 27, 78], although notably this does not necessarily lead to vasoconstriction [8]. IP3R function is modulated by Ca2+, with increases in cytosolic Ca2+ concentration up to approximately 300 nM potentiating the action of IP3 on IP3R, whereas higher concentrations cause inhibition; the potentiation by moderate elevations of cytosolic Ca2+ concentration leads to the supposition that IP3R could contribute to Ca2+-induced Ca2+ release (CICR) [6]. IP3R are not distributed homogeneously, but form clusters of approximately 30 on the SR membrane, activation of which causes localized elevations of Ca2+, or “Ca2+ puffs”; summation from several clusters leads to a Ca2+ wave and a general elevation of cytosolic Ca2+ concentration [79]. There are few pharmacological inhibitors of IP3R, including xestospongin C and 2-aminoethoxydiphenylborane (2-APB); however, 2-APB may be a more effective blocker of voltageindependent Ca2+ entry pathways than IP3R [46, 80]. The RyR is an extremely large complex of four approximately 600  kDa protein monomers, and all three isoforms (RyR1, RyR2, RyR3) are expressed in small pulmonary arteries, with lower expression of RyR3 in mesenteric and conduit pulmonary arteries [81, 82]. RyR isoforms are heterogeneously expressed within PASMCs, with RyR1 and RyR2 primarily localizing to the cell periphery, and RyR1 and RyR3 to the perinuclear region [74, 81]. Experimentally, RyR are activated by caffeine and locked into a subconductance open state by low concentrations of ryanodine, whereas high concentrations block them. RyR are activated by increases in cytosolic Ca2+ concentration greater than 1 mM, and in cardiac muscle are classically associated with CICR. CICR has also been described in PASM and other VSM [6, 83, 84]. Although in VSM the global cytosolic Ca2+ concentration is never likely to rise sufficiently for activation of RyR, this can occur in highly localized microdomains

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associated, for example, with clusters of IP3R in the SR and/ or of Ca2+ influx pathways in the plasmalemma, giving rise to Ca2+ sparks, and potentially regenerative Ca2+ waves and oscillations (see below) [1, 6]. Ca2+ sparks are an important mechanism in VSM excitation–contraction coupling that can also occur spontaneously, and cause temporally and spatially localized activation of plasmalemmal Ca2+-dependent ion channels (Fig. 2). Activation of BKCa channels leads to spontaneous transient outward currents, hyperpolarization, and relaxation, whereas activation of ClCa channels leads to spontaneous transient inward currents and depolarization [85] (Fig.  2). Ca2+ sparks are observed in PASM, where importantly they seem to preferentially activate ClCa channels, particularly in the functionally significant small intrapulmonary arteries, with subsequent depolarization and contraction [78, 81, 83, 86, 87]. In many systemic arteries sparks preferentially link to KCa channels and cause relaxation [85]. In PASM the response to endothelin-1 (but not norepinephrine) is reported to involve interaction between IP3R and RyR with generation of Ca2+ sparks and vasoconstriction [78, 81, 87]. A number of other factors are known to stimulate or potentiate RyR activity and the frequency of Ca2+ sparks, including Ca2+ loading of the SR store [1], redox state and reactive oxygen species (ROS) [88], and cyclic adenosine diphosphate ribose (cADPR), an endogenous activator of RyR [89, 90] (Fig. 2). cADPR is synthesized by ADP-ribosyl cyclases, a series of multifunctional enzymes that also synthesize nicotinic acid adenine dinucleotide phosphate (NAADP) [89, 91, 92], and a microsomal ADP-ribosyl cyclase has been isolated in PASM [93]. Via its action on RyR, cADPR may participate in the generation or modulation of CICR, Ca2+ sparks, waves, and oscillations, and it has been implicated in the response to numerous stimuli, including agonists such as endothelin-1 and stretch [89]. Although cADPR is generally believed to activate RyR directly or via an ancillary protein, some studies have suggested that it potentiates RyR activity indirectly by enhancing Ca2+ loading of the store [1]. Changes in redox state, ROS, and cADPR may be of particular importance in PASM and the excitation–contraction coupling pathways that are activated during HPV. Ca2+ release from ryanodine-sensitive stores is a critical and early event in the hypoxia-induced elevation of PASM cytosolic Ca2+ concentration [34, 93–95], though the mechanisms by which this is initiated are controversial. Changes in cytosolic redox state and ROS are thought by many to be central to the response to hypoxia [5, 96, 97], and exogenous ROS have been shown to cause Ca2+ release from ryanodine-sensitive stores in PASM [98–101]. Conversely, hypoxia has been shown to increase synthesis of cADPR in PASM, and it has been proposed that this is the primary effector mechanism for the elevation of cytosolic Ca2+ concentration in HPV [92, 93, 95]. Although the substrate for cADPR synthesis is NAD+, the cytosolic concentration of which might be expected to decline in the more

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reduced cytosolic environment associated with hypoxia, it has been suggested that the concomitant increase in NADH inhibits degradation of cADPR [93]. It has also been suggested that activation of AMP-activated kinase during hypoxia may lead to enhanced cADPR concentrations [102]. Nevertheless, there is also evidence that synthesis of cADPR is promoted by ROS [88, 103]. Further studies are required to clarify the role of cADPR in HPV and its upstream signaling pathways. A recurring question is whether there are functionally and spatially separate IP3R- and RyR-containing Ca2+ stores in smooth muscle [104, 105], and specifically PASM. In colonic smooth muscle it has been suggested that a peripheral SR store has both IP3R and RyR, and is refilled by Ca2+ entry across the plasmalemma, whereas a deeper store contains only RyR and is refilled from the cytosol [105]. In canine PASM but not systemic VSM it has been shown that IP3- and ryanodine-sensitive stores are indeed functionally separate, but that this separation is lost with cell culture [86, 106]. Evidence for such complete separation is lacking from other species, although in rat PASM it has been reported that inhibition of SERCA with cyclopiazonic acid only abolishes the initial transient phase of HPV, whereas ryanodine only suppresses the sustained phase [47, 93]. There is also morphological evidence that the SR is distributed heterogeneously between the periphery and central regions of the PASMC, and that this is associated with differential expression of specific RyR isoforms [74, 81]. It is possible, however, that some apparent functional separation might be attributable to localized signaling domains containing other types of store, including lysosomes or mitochondria.

2.2.2 Lysosomes Recent studies have identified the ADP-ribosyl cyclase ­product NAADP as a potentially important mediator for ­RyR-sensitive Ca2+ signaling in PASM. NAADP is a potent Ca2+ mobilizing agent that targets acidic lysosomal Ca2+ stores rather than the SR, and can promote Ca2+ oscillations via a two-pool mechanism that also involves cADPRsensitive Ca2+ stores [91] (Fig.  3). A series of studies in PASM have shown that NAADP elicits localized bursts of Ca2+ from lysosomal stores in the perinuclear region of the cell, which initiate Ca2+ waves apparently mediated by RyR3, which, unlike RyR1 or RyR2, is highly colocalized with lysosomes in the perinuclear region of the cell [74, 107]. This mechanism has been shown to be involved in the response to endothelin-1 of both PASM and coronary VSM [107, 108], and is implicated in integrin signaling in PASM [109]. The concept of a “trigger zone” for NAADP Ca2+ signaling and initiation of Ca2+ waves involving RyR3 is particularly interesting, as gene deletion of RyR3 in mice is reported to selectively suppress the response of PASM to hypoxia, but not norepinephrine [27].

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Fig. 3  Origin of Ca2+ oscillations and waves (based on the concept of Berridge [1]). Agonist binding to G-protein-coupled receptors activates PLC to generate DAG and IP3, causing depolarization via ROCs and additional Ca2+ entry via L-type channels, and Ca2+ release via IP3R, respectively, with consequent activation of SOCs (1). Ca2+ sequestration by SERCA loads the stores, sensitising IP3R and RyR (2). A sufficient rise in store and cytosolic Ca2+ concentration promotes Ca2+ release mechanisms and allows generation of a propagating Ca2+ wave (3), and

activation of ClCa and/or BKCa/SKCa leads to depolarization/hyperpolarization, respectively (4). Cycling between these mechanisms leads to Ca2+ oscillations, the frequency of which will be dependent on the Ca2+ entry mechanisms initiated by receptor activation (1). In the perinuclear region of the cell, a lysosomal/SR trigger region may also generate Ca2+ waves in PASM [74]. Nicotinic acid adenine dinucleotide phosphate stimulates Ca2+ release from acidic lysosomes (5), which activates RyR3 and RyR1 on closely adjacent SR, leading to propagated Ca2+ waves (6)

2.2.3 Mitochondria

and plasmalemma into microdomains where local elevations in Ca2+ concentration may be much larger than in the cytosol as a whole [1]. Whatever the reason, it has been firmly established that mitochondria can buffer moderate elevations of cytosolic Ca2+ concentration initiated by Ca2+ entry and release in VSM, including PASM [70, 111, 112, 114], and may play a role in regulation of SOCs [115]. Intriguingly, it has recently been demonstrated that mitochondria are located much more closely with the plasmalemma and SR of PASMCs than in systemic VSM cells, and that this has functional consequences for signaling [116].

Apart from generating ATP, mitochondria are believed to act as the oxygen sensor for HPV [33, 96]. However, it is also well known that they play an important role in Ca2+ homeostasis in a wide variety of tissues, not only in terms of their capacity to buffer elevations of cytosolic Ca2+ concentration due to Ca2+ release from the SR or Ca2+ entry, but also by regulating these Ca2+ mobilizing mechanisms [70, 79, 110, 111]. They are known to be key components of VSM Ca2+ signaling microdomains, including the superficial buffer barrier, and probably also oxidant-signaling microdomains [1, 6, 72, 111]. The high membrane potential across the mitochondrial inner membrane means that the electrochemical gradient for Ca2+ is strongly inward, and in PASM and other smooth muscles elevations of Ca2+ due to Ca2+ release from the SR or Ca2+ entry across the plasmalemma can be taken up by the mitochondrial Ca2+ uniporter; Ca2+ can be expelled from the matrix by a NCX [70, 111–113]. The Ca2+ uniporter has a relatively low affinity for Ca2+, more than tenfold lower than that of SERCA, but the role of mitochondria as modulators of normal (rather than pathological) Ca2+ homeostasis may be facilitated by very close approximation of mitochondria, SR,

2.3 Integration of Ca2+ Signaling – Ca2+ Waves and Oscillations The highly organized intracellular structure of VSM cells, with closely approximated SR, mitochondria, and lysosomes and/or plasmalemma in localized signaling microdomains, facilitates a high level of cross talk and the ability to engender regenerating Ca2+ waves and oscillations (Fig.  3). It is now believed that these temporally and spatially defined changes in cytosolic Ca2+ concentration are the main determinants of

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VSM function [1, 117]. Various types of Ca2+ waves and oscillations resulting from a variety of stimuli, including endothelin-1, angiotensin II, histamine, and caffeine, have been observed in isolated PASMCs [24, 25, 74, 107, 118, 119]. More recently, with use of a precision-cut lung-slice model, endothelin-1 has been shown to elicit asynchronous Ca2+ oscillations that were propagated as Ca2+ waves along individual PASMCs and airway smooth muscle cells within small pulmonary arteries and bronchioles in situ; the oscillations were mediated by cycling Ca2+ release from ryanodinesensitive stores, but were also dependent on Ca2+ entry [120]. Importantly, use of this model has revealed that the frequency of Ca2+ oscillations is more important than their amplitude or the mean cytosolic Ca2+ concentration for determining pulmonary artery and bronchiole force development and constriction [120, 121]. In addition, it has also been shown, at least in airway smooth muscle, that cAMP-mediated relaxation is caused by a reduction in the frequency of Ca2+ oscillations due to inhibition of IP3R [122]. It is notable that the Ca2+ waves observed in individual PASMCs are asynchronous with those in adjacent cells [120], suggesting that in contrast to many systemic arteries, intercellular gap junctions between PASMCs play at best a minor role in contraction of pulmonary artery, at least in response to agonists. The role of gap junction signaling between PASMCs, and between PASMCs and endothelial cells ­warrants further investigation. The complexity of the signaling microdomains makes disentangling the mechanisms responsible for generation of Ca2+ oscillations and waves difficult, and they are in any case likely to differ between tissues, and probably stimuli. The lung-slice model suggests that at least for endothelin-1 signaling both Ca2+ release from RyR and Ca2+ entry are required [120], and studies in isolated PASMCs have associated endothelin-1-induced Ca2+ sparks, which one can regard as the forerunners of Ca2+ oscillations, with interactions between IP3R and RyR [78, 81, 87]. Endothelin-1 and angiotensin II-induced Ca2+ oscillations have also been associated with iterative Ca2+ release from IP3R and RyR causing an oscillatory inward Cl− current, and consequent Ca2+ influx through L-type channels [25, 118]. One can come to a model, therefore, where an initial agonist-induced generation of IP3 activates IP3R clusters and Ca2+ release (Ca2+ puffs), which subsequently activates adjacent RyR and Ca2+ sparks (Fig. 3). Store depletion activates Na+/ Ca2+ entry via SOCs and the rise in local Ca2+ concentration via ClCa channels, leading to depolarization and additional Ca2+ entry via L-type channels, contributing to the elevation of Ca2+ concentration and loading the stores, so potentiating the release mechanisms. Sufficient stimulation will therefore lead to regenerative Ca2+ oscillations and propagated Ca2+ waves [1]. It is notable that such integrative models rely on a signaling microenvironment close to the plasmalemma.

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In contrast, the proposed NAADP–lysosomal trigger zone model of Ca2+ waves in PASM is largely confined to the perinuclear region of the cell [74, 92, 107] (Fig. 3). Further studies are required to determine whether there is cross talk between these peripheral and central zones. Finally, although the role of Ca2+ oscillations and waves in the response of PASM to hypoxia has yet to be investigated, one can predict that they are of major importance. As the generation and propagation of Ca2+ waves in PASMCs may involve concerted activation of several Ca2+ release and voltage-dependent and voltage-independent Ca2+ entry mechanisms, it is perhaps not surprising that so many of these have been implicated in HPV.

3 Ca2+ Sensitization Ca2+ sensitization is the process by which a stimulus causes an increase in contractile force without the necessity for an increase in intracellular Ca2+ concentration [2]. There are several pathways by which this can occur and most involve MLCP, which, although Ca2+-independent, is nevertheless subject to regulation. Principal among these regulators is the small monomeric G protein RhoA, the importance of which to smooth muscle constriction is well established [123, 124], and its main effector, the serine/threonine kinase Rho kinase [125, 126]. Basal or resting levels of cytosolic Ca2+ concentration are sufficient to support Rho/Rho kinase mediated Ca2+ sensitization and contractile force. However, Rho kinase activation cannot lead to enhanced MLC20 phosphorylation in the complete absence of Ca2+ or MLCK [2]. MLCP activity may also be inhibited through a PKC-mediated pathway and enhanced through cyclic-nucleotide-activated kinases PKG and PKA (see later). MLC20 may also be phosphorylated by kinases other than MLCK, independently of a change in cytosolic Ca2+ concentration (see later).

3.1 Inhibition of Myosin Phosphatase and Phosphorylation of 20-kDa Myosin Light Chain MLCP is a holoenzyme complex composed of three subunits: the 37-kDa catalytic subunit PP1c, a 110-kDa myosin phosphatase targeting subunit (MYPT-1, also known as MBS) which is critical to the specificity of the holoenzyme for myosin, and a 20-kDa subunit (M20) of unknown function [127]. In smooth muscle, PP1c dephosphorylates MLC20 at Ser-19 and to a lesser extent at Thr-18 [128]. MYPT-1 possesses a specific PP1c binding motif and through a series of ankyrin repeats forms a b-hairpin–helix–loop–helix structure; it acts

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as a docking platform for regulatory proteins such as Rho kinase and telokin (see later) [128]. The tonic smooth muscle splice variant of MYPT-1 also possesses a C-terminal leucine zipper which acts as a binding site for PKG [129]. Phosphatase activity is inhibited by several mechanisms, including phosphorylation of MYPT-1, dissociation of the holoenzyme, and phosphorylation of two other regulatory proteins called PKC-potentiated inhibitory protein (CPI-17) and phosphatase holoenzyme inhibitor-1 (PHI-1) [130, 131] (Fig.  4). PP1c catalytic activity is inhibited by hypoxia in PASMCs [132]. MYPT-1 is phosphorylated at two sites relevant to Ca2+sensitization, namely, Thr-695 (Thr-697 in rat) [133] and Thr-850 (Thr-855 in rat) [134]. Phosphorylation of Thr-695 is sufficient for loss of PP1c catalytic activity [135], whereas phosphorylation at Thr-850, which is located in the myosinbinding domain, results in dissociation of the holoenzyme from myosin [134]. Initially, it was thought that Rho kinase was the principal kinase responsible for phosphorylation of MYPT-1 at the inhibitory Thr-695 site [133]. However, it was subsequently shown that recombinant MYPT-1 is phosphorylated by Rho kinase much more strongly at Thr-850 than at Thr-695 [136], and that despite considerable constitutive phosphorylation of Thr-695, only phosphorylation of Thr-850 is sensitive to inhibition by the Rho kinase inhibitor Y27632 [136–138]. We have subsequently shown that this is also the case in PASM stimulated with either prostaglandin F2a (PGF2a) [139], hypoxia [140], or superoxide [141]. There is, however, often a high resting level of Thr-695 phosphorylation that is insensitive to blockade of Rho kinase [137– 139], suggesting that Thr-695 is phosphorylated by a kinase or kinases other than Rho kinase. Indeed, this threonine residue is a known phosphorylation target for zipper-interacting protein kinase (ZIPK) and integrin-linked kinase (ILK) [142, 143] (Fig. 4). The relevance of this site and the kinase(s) responsible in PASM remains to be determined. The two other regulatory proteins that also inhibit PP1c activity, CPI-17 and PHI-1, are both activated by phosphorylation [131, 144]. In cell-free studies, CPI-17 is phosphorylated by Rho kinase, protein kinase N, ILK, and ZIPK [144–146], but most effectively by PKCd [144]. The relative importance of CPI-17 to agonist-mediated Ca2+ sensitization compared with Rho kinase/MYPT-1 is minimal in some smooth muscle preparations [137, 138]. Similarly, although PKC may ­normally contribute to pressure-induced myogenic tone in pulmonary artery [147], PKC inhibitors are much less effective against endothelin-1-mediated constriction and HPV than Rho kinase blockers [148, 149]. On the other hand, PKCa is activated by hydrogen peroxide (H2O2) in PASMCs, and H2O2 causes Ca2+ sensitization and constriction in pulmonary artery that is sensitive to inhibition of PKC but not Rho kinase [101]. Interestingly, although apparently PKCmediated, this constriction was not associated with MLC20

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Fig.  4  Mechanisms underlying phosphorylation and dephosphorylation of MLC20, and Ca2+ sensitization and desensitization. Pro­contractile conditions promote MLC20 phosphorylation by MLCK, integrin-linked kinase (ILK), zipper-interacting protein kinase (ZIPK), and potentially Rho kinase, and suppression of MLC20 dephosphorylation via inhibition of MLCP, via Rho kinase, CPI-17, ILK, and ZIPK. MLC20 dephosphorylation is promoted by cyclic GMP and protein kinase G (and potentially cyclic AMP/protein kinase) by phosphorylation of MYPT-1 and telokin (see the text for details)

phosphorylation and therefore presumably did not occur via activation of CPI-17 [101]. In addition to MLCK, several other kinases have been demonstrated to diphosphorylate and activate MLC20 at ­Ser-19 and Thr-18 (Fig. 4). In cell-free systems Rho kinase directly phosphorylates MLC20 at these sites, but whether this occurs in vivo is unclear, since access of the kinase to MLC20 may be restricted [150]. Other kinases that also diphosphorylate MLC20 are ILK [4, 142], citron kinase (another Rho effector) [151], and ZIPK [152]. The relevance of these pathways in PASM and how they are regulated is unknown. Myosin ATPase activity is also modulated by the two actin-binding proteins caldesmon and calponin. When bound to actin, these proteins inhibit cross-bridge cycling, and this inhibition is relieved by their phosphorylation. The kinase responsible for phosphorylating caldesmon is thought to be p42/p44 mitogen-activated protein kinase (MAPK)

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[153, 154]; however, blockade of this kinase in pulmonary artery only partially inhibits agonist-induced constriction and has no effect on HPV [155, 156]. Calponin, however, is a phosphorylation target for several kinases, including Rho kinase [157] and PKCa [158]. Indeed, PKC-mediated phosphorylation of calponin may explain MLC-independent H2O2-induced constriction in pulmonary artery [101].

3.2 Activation of Rho Kinase Rho kinase has an N-terminal catalytic domain, a central coiled-coil domain containing a Rho-binding region, and pleckstrin-homology (PH) and cysteine-rich domains at the C-terminus [125]. Rho kinase is autoinhibited in the free state through interaction of the C-terminal PH/cysteine-rich domains with the catalytic domain, and this inhibition is relieved by attachment of Rho to the Rho-binding domain [125]. RhoA binding is associated with partial translocation of Rho kinase to the plasma membrane [125] and enhances Rho kinase activity [159]. Rho kinase is also found at stress fibers in stimulated PASMCs [139], presumably through binding of the PH domain to myosin [160]. Rho kinase also translocates in PASMCs in response to hypoxia and superoxide, but not H2O2 [140, 141]. Although Rho kinase activation by hypoxia in PASMCs is inhibited by C3 exoenzyme, suggesting a dependency of Rho kinase activation on RhoA [161], agonist-induced MLC20 phosphorylation is not always abolished in the absence of RhoA [124], suggesting alternative pathways for the activation of Rho kinase. Indeed, lipids such as arachidonic acid, sphingosine 1-phosphate, SPC, and lysophosphatidic acid (LPA) also bind to and activate Rho kinase in a fashion similar to RhoA, resulting in release of the catalytic domain from autoinhibition [127, 162, 163]. In PASM, MYPT-1 phosphorylation and tension are increased by LPA [132]. However, the relative importance of Rho-mediated and Rho-independent activation of Rho kinase remains to be determined, since the concentrations of lipid mediators required to activate Rho kinase are usually in the supraphysiological range, as is the case with SPC in pulmonary artery [46].

3.3 Activation of Rho The Rho family of small monomeric G proteins, of which RhoA is the principal mediator of constriction in smooth muscle, act as a molecular switch cycling between inactive GDP-bound and active GTP-bound forms. Upon stimulation with agonists [164] or ROS [165], Rho translocates to the plasma membrane, a process requiring prenylation [166]. Exogenous GTP-RhoA causes constriction and MLC20 phosphorylation in permeabilized arteries [167], and RhoA small

J.P.T. Ward and G.A. Knock

interfering RNA (siRNA) knockdown abolishes agonistinduced vasoconstriction [124]. The exchange of GDP for GTP is catalyzed by structurally diverse yet highly G protein specific families of guanine nucleotide exchange factors (GEFs) which enhance the rate of nucleotide dissociation, therefore allowing for the binding of the much more abundant GTP [168]. Conversely, the intrinsic GTPase activity of the G protein is promoted by specific GTPase-activating proteins (GAPs), thus inactivating the G protein [168]. Stability of inactive GDP-RhoA is maintained by GDPdissociation inhibitors (GDIs), which keep Rho in the cytosol by masking the prenylation site and preventing binding of the active G protein to membrane-bound effectors [169]. Of the 70-odd RhoGEFs in the human genome, at least 12 are expressed in the cardiovascular system, including vav2/3, leukemia-associated RhoGEF (LARG), p115 RhoGEF, PDZ-RhoGEF, and most recently smgGDS [126, 170, 171]. The role of RhoGEFs in pulmonary artery and how they are regulated is largely unexplored, except for PDZ-RhoGEF, which is expressed in PASM, and a constitutively active fragment of which directly constricts b-escin-permeabilized pulmonary artery, an effect that is reversed by Rho kinase inhibition, confirming the importance of Rho/RhoGEF in the activation of Rho kinase [170]. The activation pathways for RhoGEFs are complex and not fully understood; however, depending on which RhoGEF is involved, they involve direct interaction with heterotrimeric G protein subunits, tyrosine phosphorylation of the GEF, or a combination of the two. Most RhoGEFs have two conserved domains: the PH domain, which directs intracellular location of the GEF, and the Dbl homology domain, which confers the catalytic activity [168]. Three of these GEFs, p115-RhoGEF, PDZ-RhoGEF, and LARG, also contain a regulator of G-protein-signaling domain. This domain can specifically interact with Ga12/13 or Gaq/11 [172, 173], which are coupled to most classes of vasoconstrictor receptors. Activation of the vav family of RhoGEFs has an absolute requirement for tyrosine phosphorylation for activation, which relieves autoinhibition of the Rho-binding domain [174, 175]. Ga12-mediated activation of LARG and PDZRhoGEF is also associated with tyrosine phosphorylation of  the GEFs by (among others) src-family kinases (srcFK) [172, 176]. Some RhoGAPs and RhoGDIs are also phosphorylation targets for src, RhoGDI being phosphorylated to prevent its binding to Rho, thus p­ reserving Rho activity [177].

3.4 Role of src-Family Kinases in Ca2+ Sensitization Tyrosine phosphorylation has been implicated in smooth muscle constriction and Ca2+ sensitization for some time without specific tyrosine kinases having been identified. For

10  Excitation–Contraction Coupling and Regulation of Pulmonary Vascular Contractility

example, contractile agonists induce tyrosine phosphorylation of multiple protein targets in a number of VSM preparations, including PASM [139, 178, 179], and nonselective tyrosine kinase inhibitors such as genistein and tyrphostin A23 inhibit agonist-induced constriction, RhoA activation, and MLC20 phosphorylation in several a-toxin- or b-escin-permeabilized smooth muscle preparations, including pulmonary artery [149, 178, 180, 181]. In addition, vanadate, an inhibitor of protein tyrosine phosphatases, induces Y27632-sensitive constriction and MLC20 phosphorylation and causes genisteinsensitive translocation of RhoA from the cytosol to the plasmalemma [182]. More recent evidence points toward an early central role for non-receptor tyrosine kinases, particularly srcFK, in Ca2+ sensitization, and that this is upstream of RhoA/Rho kinase activation (Fig.  5). For example, SPC-mediated ­constriction is dependent on Rho kinase mediated phosphorylation of MYPT-1 and these actions are blocked by the srcFK ­inhibitor PP1 [183]. srcFK activity requires dephosphorylation at the C-terminal Tyr-527 followed by subsequent ­autophosphorylation at Tyr-417, thus uncovering the active site [184]. Activation also uncovers the SH2 domain, which allows translocation of the kinase and interaction with other tyrosine phosphorylated proteins [184]. SPC induces ­autophosphorylation and translocation to the plasma ­membrane of the srcFK fyn as well as Rho kinase [183],

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and the Rho kinase translocation is blocked by the src-specific inhibitor PP1 [183]. In PASM, PGF2a causes srcFK autophosphorylation, constriction, translocation of Rho kinase to stress fibers, and Y27632-sensitive phosphorylation of MYPT-1 and MLC20, and these effects are all blocked by the srcFK-specific inhibitors PP2 or SU6656 [139]. Similarly, HPV is also blocked by these inhibitors and hypoxia enhances srcFK autophosphorylation in pulmonary artery, and hypoxia-mediated translocation of Rho kinase and Rho kinase mediated phosphorylation of MYPT-1 and MLC20 in PASMCs are inhibited by siRNA directed against src and fyn or by SU6656 [140]. How srcFK are activated by contractile stimuli remains unclear. It may occur through direct contact with some Ga subunits [185], or lipid mediators [186], or indirectly via activation of protein tyrosine phosphatase-a, which specifically dephosphorylates Tyr-527 of srcFK in response to some stimuli [187]. Alternatively, ROS may, through oxidation of specific cysteine residues, directly inactivate tyrosine phosphatases, thus producing a net amplification of srcFKdependent tyrosine phosphorylation of target proteins [188, 189]. This may be of particular relevance to the vascular response to hypoxia and HPV, as it has been proposed that this is initiated by an increase in generation of mitochondriaderived ROS [96]. Although it has not been directly shown that hypoxia ­activates srcFK via superoxide or H2O2 in PASM, this has been reported in other smooth muscles [190], and it has recently been demonstrated that exogenously generated superoxide causes cytosolic Ca2+ concentration independent constriction, Rho kinase translocation, and Rho kinase dependent MYPT-1 phosphorylation in pulmonary artery, and the constriction is sensitive to srcFK inhibition [141]. Therefore, at least one pathway through which hypoxia may cause constriction in pulmonary artery is through ROS-enhanced srcFK activity, activation of RhoA possibly via src-specific phosphorylation of RhoGEF, and subsequent Rho kinase dependent Ca2+ sensitization [140, 141]. Along with focal adhesion kinase (FAK), src is also involved in activation of p42/p44 MAPK (see earlier), the p38/HSP27 pathway, actin polymerization, and the myogenic response (see Sect. 4).

3.5 Ca2+ Desensitization

Fig. 5  Activation pathways for src, RhoA, and Rho kinase, and phosphorylation of guanine nucleotide exchange factor and GDP-dissociation inhibitor (see the text for details)

Cyclic-nucleotide-mediated relaxation such as that induced by nitric oxide includes a significant component due to Ca2+ desensitization [191]. Pulmonary artery constriction elicited by inhibition of endothelial nitric oxide synthase by Nw-nitrol-arginine methyl ester is inhibited by Rho kinase blockers, agonist-induced constriction of pulmonary artery is blocked by cGMP analogues [163, 192, 193], and cGMP causes PKG-mediated activation of myosin phosphatase and actin

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disassembly [123, 194, 195], suggesting antagonism between Rho/Rho kinase and PKG. There are four potential pathways through which this may occur. Firstly, RhoA is directly phosphorylated by PKG and PKA [123], which enhances its interaction with GDI, thus sequestering it in the cytoplasmic compartment [127], and this has been suggested as an explanation for the effectiveness of phosphodiesterase inhibitors (e.g., sildenafil) against RhoA/Rho kinase mediated constriction in pulmonary hypertension [196]. Secondly, PKG and PKA phosphorylate MYPT-1 at serine residues adjacent to the inhibitory sites of Thr-695 and Thr-850, respectively (Fig. 4). This phosphorylation, although not directly enhancing phosphatase activity, inhibits Rho kinase mediated phosphorylation of the inhibitory sites [143, 163, 197]. Thirdly, PKG can activate the phosphatase through interaction with the leucine zipper binding site of MYPT-1 [198]. Finally, PKG may phosphorylate and activate telokin (Fig. 4). Telokin is an independently expressed C-terminal myosinbinding domain of smooth muscle MLCK, with no ­catalytic activity of its own. Telokin indirectly modulates the level of myosin light chain phosphorylation. This occurs either through activation of MLCP [199, 200] or via competition with MLCK for myosin [201]. Either way, telokin activity is regulated by phosphorylation at three residues (Ser-12, Ser15, Ser-18) by PKA/PKG (Fig. 4), and is fully active when fully phosphorylated [200]. Although telokin is believed to be of most importance in phasic smooth muscle, it may also play a role in tonic smooth muscle [199]. Recently it has been reported that telokin is expressed in PASM of cat, and that its phosphorylation is reduced by hypoxia in small pulmonary arteries [202]. It has therefore been suggested that phosphorylated telokin helps maintain a low resting tone in pulmonary artery, and that relief of this phosphorylation by hypoxia contributes to MLC20 phosphorylation and constriction [202]. Further studies are, however, required to determine whether this mechanism plays a ­significant role in HPV.

4 Actin Polymerization, Focal Adhesions, and Mechanotransduction Growing evidence suggests that smooth muscle constriction is also strongly influenced by factors that control the polymerization of F-actin and the attachment of F-actin fibers to the plasma membrane [3], and the findings of a number of studies in VSM, including PASM, are consistent with this concept [124, 138, 203, 204]. F-actin is anchored to the plasma membrane through a complex of adhesion proteins associated with the intracellular portions of integrins at sites called focal adhesions or membrane-associated dense plaques [3]. Actin polymerization is dynamic and is enhanced by ­contractile agonists [124, 138] and ROS [205], and occurs in

J.P.T. Ward and G.A. Knock

Fig. 6  Interaction between pathways promoting actin polymerization (see the text for details)

response to hypoxia and LPA in PASMCs and pulmonary artery, respectively [132, 204] (Fig.  6). Only a small net increase in the quantity of F-actin may occur in response to contractile stimuli [3, 206], and yet disrupting this process, either by capping of F-actin with cytochalasin [203, 204, 207] or by inhibition of actin nucleation initiation proteins [206] ,greatly inhibits constriction. It has been suggested that this polymerization, centered around focal adhesions, increases the ability of the cells to transmit force throughout the tissue by triggering an increase in the number of ­attachments of existing myosin-associated F-actin filaments to focal adhesions and therefore to the extracellular matrix [3, 206]. This process involves recruitment and activation of integrins, various adaptor proteins, tyrosine kinases (FAK, src, and PYK2), ILK, small monomeric G proteins, and heat-shock proteins [3, 208] (Fig. 5), and occurs ­independently of cytosolic Ca2+ concentration and MLC20 phosphorylation [206, 207]. In some preparations, including PASMCs and pressurized pulmonary artery, actin polymerization seems to be particularly dependent on RhoA and Rho kinase, since siRNA directed against smgGDS RhoGEF, or RhoA [124, 171], or Rho kinase inhibition [132, 204], causes disruption of F-actin and of actomyosin assembly. Rho kinase phosphorylates LIM kinase, which in turn phosphorylates and inhibits cofilin and/or elongation factor-1a, both of which can sever and

10  Excitation–Contraction Coupling and Regulation of Pulmonary Vascular Contractility

depolymerize F-actin [209]. Actin polymerization is also regulated by heat-shock proteins 27 and 20 (HSP27 and HSP20) (Fig. 6). Both proteins encourage the stabilization of F-actin filaments and/or their anchoring within cytoplasmic dense bodies, thus promoting force generation [210, 211]. HSP27 is phosphorylated and activated by MAPK-activated protein kinase-2 via p38 MAPK in response to vasoconstrictors and cellular stresses such as H2O2 [205]. In association with src-dependent phosphorylation of cytoskeletal adaptor proteins [208], this activation encourages its binding to actin [212]. HSP20, on the other hand, is phosphorylated by PKA or PKG in response to vasorelaxants, causing it to dissociate from actin [210]. Both agonist- and hypoxia-induced constrictions in pulmonary artery are associated with phosphorylation of HSP27 and are suppressed by inhibitors of p38 MAPK or HSP27 antibodies [155, 156, 213], and p38 MAPK may contribute to Ca2+ sensitization through antagonism of underlying NO/PKG-mediated Ca2+ desensitization, perhaps at the level of heat-shock protein/actin interaction [156]. p38 MAPK/HSP27-mediated actin polymerization occurs independently of the Rho/Rho kinase pathway [211]. Focal adhesions are also the sites at which mechanical strain or movement of extracellular protein attachments induce Ca2+ influx and constriction in VSM (the myogenic response) [203]. Pressure-induced, integrin-dependent activation of FAK and src, in combination with actin polymerization [203], results in tyrosine phosphorylation of multiple targets, including the adaptor protein paxillin and perhaps the L-type Ca2+ channel itself, and subsequent enhancement of Ca2+ influx [154, 208]. This response may be of particular importance for the generation of de novo myogenic tone in PASM of animals with pulmonary hypertension [147], but integrin stimulation also causes Ca2+ release from ryanodinesensitive stores and lysosomes in PASMCs from healthy ­animals [109].

5 Summary Our understanding of excitation–contraction coupling in smooth muscle has increased substantially in recent years, and it has become clear that it is much more complex than originally presumed. This, of course, applies equally to ­pulmonary VSM, where our knowledge of some important aspects of excitation–contraction coupling is considerably more sparse than in other tissues. It is becoming apparent, however, that despite functional differences, for example, the response to hypoxia, most if not all of the underlying mechanisms are present in PASM. Key functional differences between pulmonary and systemic VSM therefore almost ­certainly reflect quantitative rather qualitative differences in the various procontractile and prorelaxant processes that ultimately

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determine force generation. Dysregulation of the balance between these processes can lead to the altered PASM function in pulmonary hypertension. Areas where our understanding is particularly limited in the pulmonary vasculature, but by comparison with other smooth muscles are likely to prove extremely important, include the role and regulation of Ca2+ oscillations and waves, the pathways regulating Ca2+ sensitization (which seem to be of specific importance in PASM), and the emerging importance of the actin cytoskeleton. It is to these areas that future studies should be directed. Acknowledgements  Work in the authors’ laboratories was supported by the Wellcome Trust and the British Heart Foundation.

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J.P.T. Ward and G.A. Knock 191. Kawada T, Toyosato A, Islam MO, Yoshida Y, Imai S (1997) cGMP-kinase mediates cGMP- and cAMP-induced Ca2+ desensitization of skinned rat artery. Eur J Pharmacol 323: 75–82 192. Nagaoka T, Morio Y, Casanova N et  al (2004) Rho/Rho kinase signaling mediates increased basal pulmonary vascular tone in chronically hypoxic rats. Am J Physiol Lung Cell Mol Physiol 287:L665–L672 193. Badejo AM Jr, Dhaliwal JS, Casey DB, Gallen TB, Greco AJ, Kadowitz PJ (2008) Analysis of pulmonary vasodilator responses to the Rho-kinase inhibitor fasudil in the anesthetized rat. Am J Physiol Lung Cell Mol Physiol 295:L828–L836 194. Wu X, Somlyo AV, Somlyo AP (1996) Cyclic GMP-dependent stimulation reverses G-protein-coupled inhibition of smooth ­muscle myosin light chain phosphate. Biochem Biophys Res Commun 220:658–663 195. Sandu OA, Ito M, Begum N (2001) Selected contribution: insulin utilizes NO/cGMP pathway to activate myosin phosphatase via Rho inhibition in vascular smooth muscle. J Appl Physiol 91: 1475–1482 196. Guilluy C, Sauzeau V, Rolli-Derkinderen M et al (2005) Inhibition of RhoA/Rho kinase pathway is involved in the beneficial effect of  sildenafil on pulmonary hypertension. Br J Pharmacol 146: 1010–1018 197. Nakamura K, Koga Y, Sakai H, Homma K, Ikebe M (2007) cGMPdependent relaxation of smooth muscle is coupled with the change  in the phosphorylation of myosin phosphatase. Circ Res 101:712–722 198. Surks HK, Mochizuki N, Kasai Y et al (1999) Regulation of myosin phosphatase by a specific interaction with cGMP-dependent protein kinase Ia. Science 286:1583–1587 199. Choudhury N, Khromov AS, Somlyo AP, Somlyo AV (2004) Telokin mediates Ca2+-desensitization through activation of ­myosin phosphatase in phasic and tonic smooth muscle. J Muscle Res Cell Motil 25:657–665 200. Khromov AS, Wang H, Choudhury N et al (2006) Smooth muscle of telokin-deficient mice exhibits increased sensitivity to Ca2+ and decreased cGMP-induced relaxation. Proc Natl Acad Sci U S A 103:2440–2445 201. Silver DL, Vorotnikov AV, Watterson DM, Shirinsky VP, Sellers JR (1997) Sites of interaction between kinase-related protein and smooth muscle myosin. J Biol Chem 272:25353–25359 202. Madden JA, Dantuma MW, Sorokina EA, Weihrauch D, Kleinman JG (2008) Telokin expression and the effect of hypoxia on its phosphorylation status in smooth muscle cells from small and large pulmonary arteries. Am J Physiol Lung Cell Mol Physiol 294:L1166–L1173 203. Cipolla MJ, Gokina NI, Osol G (2002) Pressure-induced actin polymerization in vascular smooth muscle as a mechanism underlying myogenic behavior. FASEB J 16:72–76 204. Boer C, van Nieuw Amerongen GP, Groeneveld AB et al (2004) Smooth muscle F-actin disassembly and RhoA/Rho-kinase ­signaling during endotoxin-induced alterations in pulmonary ­arterial compliance. Am J Physiol Lung Cell Mol Physiol 287:L649–L655 205. Huot J, Houle F, Marceau F, Landry J (1997) Oxidative ­stress-induced actin reorganization mediated by the p38 mitogenactivated protein kinase/heat shock protein 27 pathway in vascular endothelial cells. Circ Res 80:383–392 206. Zhang W, Wu Y, Du L, Tang DD, Gunst SJ (2005) Activation of the Arp2/3 complex by N-WASp is required for actin polymerization and contraction in smooth muscle. Am J Physiol Cell Physiol 288:C1145–C1160 207. Saito SY, Hori M, Ozaki H, Karaki H (1996) Cytochalasin D inhibits smooth muscle contraction by directly inhibiting contractile apparatus. J Smooth Muscle Res 32:51–60

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Chapter 11

Endothelial Regulation of Pulmonary Vascular Tone Stephen Y. Chan and Joseph Loscalzo

Abstract  This chapter focuses on the central significance of the pulmonary endothelium in the control of pulmonary vascular tone in pulmonary development, homeostasis, and pathophysiological processes. We review the upstream stimuli known to regulate the expression and activity of endothelium-specific mediators of vascular tone. Subsequently, we focus on the known actions of the endothelium-derived vasoactive effectors most important for the balance of control of vasomotor tone – vasodilators (nitric oxide and prostacyclin) and vasoconstrictors (thromoboxane A2 and endothelin). Additional mediators that may affect pulmonary vasomotor tone and endothelium-derived hyperpolarizing factor related activity are discussed, along with more poorly characterized effectors. Finally, the complex balance of these vasoactive factors and the intersection of their downstream signaling pathways is presented in the context of prototypical in vivo physiological and pathophysiological processes in the pulmonary circulation. Keywords  Endothelium-derived constriction and relaxing factor • Endothlin • Thromboxane • Nitric oxide • Prostacyclin • Vasoconstriction • Vasodilation

1 Introduction Under baseline conditions in the adult, the pulmonary circulation represents a low-pressure and low-resistance circuit that accommodates the entire cardiac output at less than 20% of systemic pressures. The first insights into this feature of the pulmonary vasculature originated from characterization of anatomic differences among pulmonary arteries and ­systemic arteries. Both large pulmonary arteries and the ­pulmonary capillary bed carry a lower number of vascular smooth muscle cells (VSMCs) than their systemic counterparts. The presence of this relatively thin layer of vascular J. Loscalzo (*) Brigham and Women’s Hospital, Harvard Medical School, Boston 0415, MA, USA e-mail: [email protected]

muscle translates to an inability to mount high levels of ­pulmonary pressure and resistance at the baseline. As a result of these anatomic characteristics, the pulmonary circulation, in contrast to the peripheral circulation, is specifically and substantially influenced by a number of passive factors that can alter pulmonary vascular resistance (PVR). These can include changes in cardiac output from the right side of the heart, changes in left atrial pressure, and changes in airway or interstitial parenchymal pressures, gravitational forces, and vascular obstruction or recruitment. These factors can influence PVR, independent of active changes in vascular tone and independent of active regulation of cellular function in the vascular wall itself. Nonetheless, subsequent physiology studies in both humans and animal models have emphasized the ­importance of active changes in pulmonary vascular tone under both physiological and pathophysiological conditions. Much of the active control of vasomotor tone is localized to small ­pulmonary arteries and arterioles (with internal diameters of several hundred micrometers). Vascular endothelial cells act as a central sensor of such physiological stimuli and injurious stimuli (such as hypoxia, shear stress, inflammation, and toxins) to modulate a variety of vascular functions. Correspondingly, stemming from results of classic ex vivo studies of arterial explants, active modulation of vascular tone in both peripheral and pulmonary arteries relies ­predominantly on the function of an intact endothelial monolayer. Our ability over the past few decades to characterize the pulmonary vasculature at both the cellular and the molecular levels has led to a more sophisticated understanding of this dependence. This function primarily involves the tightly regulated production and secretion of specific vasoactive effectors from endothelium. These factors tend to act either in an autocrine fashion to autoregulate endothelial function or in a paracrine fashion to influence neighboring VSMCs. Modulation of these factors is active during normal physiological processes, such as the abrupt and dramatic transition during birth from vasoconstriction to vasodilation in the ­pulmonary vasculature. Additionally, dynamic regulation of these vasoactive factors is especially apparent under pathophysiological conditions, such as ­pulmonary arterial hypertension (PAH), resulting in chronic vasoconstriction and endothelial and vascular smooth

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_11, © Springer Science+Business Media, LLC 2011

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­ uscle proliferation. Unlike the historical theories of passive m tone, it has also become clear that the balance of these ­factors is critical for homeostasis of pulmonary vascular tone in the adult as well. Control of pulmonary vascular tone by endothelial cells at the cellular and molecular levels is extraordinarily complex and is still only understood to an incomplete degree. A significant portion of our knowledge of this process is derived from studies of various endothelium-dependent vasoactive mediators in peripheral vessels with extrapolation to the pulmonary circulation. Some of these data were subsequently verified in the context of the pulmonary vasculature; however, other mechanisms and responses are based on incomplete circumstantial evidence. Currently, it appears that a number of distinct but complex stimuli can alter ­pulmonary vascular tone via control of multiple intersecting signaling pathways in the endothelial cell, ultimately leading to the release of vasoactive effectors (Fig.  1). Of these effectors, those that have predominant roles in control of vascular tone include vasoconstrictors, such as endothelin (ET-1) and thromboxane A2 (TxA2), and vasodilators, such as nitric oxide (NO) and prostacyclin (PGI2). The ­so-called endothelium-derived hyperpolarizing “factors” (EDHFs) may additionally contribute via their action as non-NO, non-prostanoid-dependent vasodilatory agents. Finally, novel factors, such as vasoactive intestinal peptide (VIP) and leukotrienes, have emerged as significant effectors of vascular tone, especially in the context of PAH. This chapter will highlight our current understanding of the complex and incompletely characterized interplay of exogenous upstream stimuli with downstream endothelium-specific vasoactive effectors that ultimately

Fig. 1  The pulmonary vascular endothelium is integral for the control of pulmonary vascular tone. The pulmonary vascular endothelial cell acts as a sensor of physiological and pathophysiological conditions, transmitted primarily in the forms of mechanical, humoral, neural, and hypoxic stimuli. In response, endothelial cells produce and secrete a tightly regulated balance of vasoactive agonists in the vascular milieu. These effectors can then act in an autocrine fashion to autoregulate endothelial function or in a paracrine fashion to mediate contractile function of neighboring vascular smooth muscle cells (VSMCs). In specific cases (i.e., S-nitrosation and nitrite formation from nitric oxide), mechanisms exist to allow for the transport and survival of the agonist signal to anatomically remote regions of the pulmonary vasculature

S.Y. Chan and J. Loscalzo

regulate pulmonary ­vascular tone under both physiological and pathophysiological conditions.

2 Upstream Exogenous Stimuli of Vasoactive Effectors The upstream stimuli that regulate pulmonary vascular tone via endothelium-dependent mechanisms are quite varied. In general, they can be divided into neural, humoral, mechanical, and hypoxic stimuli (Fig.  1). In this section, we will introduce the known pathways by which these stimuli can affect pulmonary vasomotor tone, mainly by the induction of vasoactive factors from the endothelium. Some of these stimuli have been studied to a sufficient degree to describe mechanisms by which they induce the release of specific vasoactive factors. These will be described in later sections devoted to the individual factors. Comprehensive descriptions of all downstream signaling mechanisms from these stimuli are lacking and are the subject of ongoing study.

2.1 Neural Mechanisms The autonomic nervous system can modify pulmonary vascular tone under both physiological and pathophysiological conditions through release of neurotransmitters that activate autonomic receptors. Those receptors that are expressed on pulmonary endothelium include the adrenergic (a2 and b2), muscarinic (cholinergic) (M3), purinergic (P2Y), and tachykinin (NK1) receptors. The adrenergic and cholinergic subtypes represent the best studied autonomic receptors in the pulmonary endothelium. Adrenergic stimulation via neural secretion of the neurotransmitters norepinephrine and serotonin can induce potent pulmonary arterial vasoconstriction primarily by activating a1 receptors and serotonin receptors, respectively, on VSMCs. Notably, a marked potentiation of the contractile response to norepinephrine and serotonin has been observed after removal of pulmonary vascular endothelial cells [1]. Multiple mechanisms may regulate this endothelium-dependent response. First, pulmonary endothelial cells may blunt the contractile response to adrenergic stimulation by scavenging and degrading norepinephrine, serotonin, and ATP. Second, pulmonary endothelial cells may release NO, a vasodilator gas, in response to adrenergic stimulation, as indicated by augmentation of contraction after electrical field stimulation (EFS) of intramural adrenergic nerves in the presence of a nitric oxide synthase (NOS) inhibitor, N6-monomethyl-l-arginine [2]. In isolated dog intrapulmonary arteries and veins, removal of endothelial cells augments EFS-induced norepinephrine release; this response is inhibited by subsequent exposure to basal

11  Endothelial Regulation of Pulmonary Vascular Tone

effluent from endothelium-intact donor arteries [3]. More recently, enhanced release of NO has been described to contribute to the long-term b-adrenergic-induced desensitization of pulmonary vascular smooth muscle [4]. Therefore, although multiple factors contribute directly to vasomotor control, the major effect appears to rely upon the downstream release of vasodilator NO to drive the inhibition of adrenergic contraction. Responses in pulmonary vascular tone to cholinergic (acetylcholine) stimulation may also be primarily mediated by NO. Acetylcholine induces vasodilation of the pulmonary vasculature, likely by inducing endothelial production of NO. Uncertainty still exists regarding the mechanism by which acetylcholine, which is released at the adventitiomedial border by cholinergic nerve endings, can transmit its effects remotely to endothelial cells. Nonetheless, in the ­preconditioned pulmonary vasculature of the cat, vagal stimulation induces a frequency-dependent vasorelaxation, which is inhibited by the NOS inhibitor Nw-nitro-l-arginine methyl ester (l-NAME) and by inhibition of guanylyl cyclase, thereby implicating the importance of the NO signaling cascade. Furthermore, NOS inhibitors decrease the pulmonary vasodilatory response to acetylcholine in both feline and ovine [5, 6], as well as ex vivo blood-perfused rat, pulmonary vascular beds [2]. In contrast, l-NAME has no inhibitory effects on vasorelaxation induced by other drugs with alternative mechanisms of action, including adenosine, isoproterenol, sodium nitroprusside, nicorandil, prostaglandin E1, and 8-bromo-cyclic GMP; however, diffusion through the vascular smooth muscle layer is presumed to be involved. Finally, the endothelium-dependent production of NO app­ ears at least partially responsible for nonadrenergic, non­ cholinergic (i-NANC) vasodilatory pulmonary responses [7]. ATP, the possible i-NANC mediator in the pulmonary circulation, acts as an endothelium-dependent pulmonary vasodilator via stimulation of NO production. As a result, although multiple types of autonomic signals appear to modulate endothelial function to control vasomotor tone, their downstream effects appear to rely upon the common vasodilatory action of NO.

2.2  Humoral Mechanisms In addition to neural input, the actions of additional vasoactive substances generated from nonendothelial sources directly depend upon the function of the pulmonary vascular endothelium to modulate vascular tone. First, pulmonary vascular endothelial cells are known to play important roles in the metabolism and degradation of some of these upstream vasoactive factors. As noted already, pulmonary vascular endothelium endocytoses and degrades vasoactive amines, such as norepinephrine, serotonin, and ATP, which are secreted

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from neural fibers and platelets to induce vasoconstriction. Pulmonary endothelial cells also express angiotensinconverting enzyme, which catalyzes formation of the potent vasoconstrictor angiotensin II (AT-II) from secreted angiotensinogen and degrades bradykinin and tachykinins. The net result induces endothelium-dependent pulmonary arterial vasoconstriction, primarily by activation of AT-II receptors on VSMCs and downstream myofibril contraction. Endothelial cells also express neutral endopeptidase, which degrades enkephalin and other short peptides such as tachykinins. Lack of stimulation of enkephalin and tachykinin receptors on VSMCs leads to vasorelaxation. Therefore, these endothelium-specific metabolic activities help to maintain homeostasis in pulmonary vascular tone. Second, a number of secreted vasoactive substances directly stimulate receptors on the cell surface of pulmonary vascular endothelium to alter vasomotor tone. These include histamine, serotonin, bradykinin, ATP/ADP, substance P, thrombin, and arachidonic acid (AA). Although the signaling pathways involved are complex and typically intersect with each other, some common downstream kinase pathways and second messengers are induced, including the Rho kinase, phosphoinositide 3-kinase (PI3K), and mitogen-activated protein kinase (MAPK) pathways. Consequently, the downstream endothelial responses typically involve production of one or several secreted vasoactive mediators, as described in detail later. More recently, novel humoral mechanisms of endothelial regulation of vascular tone have been described (as reviewed by Villar et al. [8]). These include the actions of lipopolysaccharide, which can bind toll-like receptors on endothelial cells, resulting in vasodilator production. Alternatively, sex hormones such as estrogen may regulate an EDHF pathway (see the description later), resulting in selective vasodilation in females in comparison with males. The impact of lipopolysaccharide and sex hormones awaits further elucidation in the pulmonary circulation.

2.3 Mechanical Factors These flow-dependent changes in endothelial function and vascular tone may also affect pulmonary vascular disease states. Notably, increased flow through the pulmonary circulation has long been associated with development of PAH. Certain types of congenital heart disease with functional systemic-to-pulmonary shunts, such as unrestricted ventricular septal defects (VSD) and large patent ductus ­arteriosus (PDA), invariably lead to pulmonary vascular remodeling and the clinical syndrome of PAH during childhood (Eisenmenger’s syndrome). The presence of atrial ­septal defects (ASD) with systemic-to pulmonary shunts may also lead to PAH over time [12]. Yet, in contrast to the cases of

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unrestricted VSD and PDA, only 10–20% of all ­persons with ASD progress to PAH [13]. This observation may reflect differences in the response of the pulmonary ­vasculature to pressure overload (as seen in shunts with VSD and PDA) as compared with volume overload (as seen in shunts with ASD); however, the underlying mechanisms are unclear. In part, this challenge stems from the difficulty of studying directly the in vivo flow patterns at the anatomic level of the pulmonary arteriole. Recently, ex vivo modeling of pulsatile flow with high levels of shear stress and chronic inhibition of vascular endothelial growth factor (VEGF) has demonstrated apoptosis of pulmonary artery endothelial cells followed by outgrowth and selection for proliferating apoptosis-resistant cells [14]. It remains to be seen if production of NO, PGI2, or other vasoactive factors is dysregulated in these cells. Nonetheless, it is plausible that chronically elevated flow in the pulmonary circulation may allow for selection of endothelial cells with dysregulated growth and augmented vasoconstrictive function.

2.4 Hypoxic Mechanisms Exposure of the pulmonary vasculature to hypoxia induces vasoconstriction acutely and induces vascular remodeling chronically. As an acute response, hypoxic pulmonary vasoconstriction (HPV) appears to rely primarily upon direct activation of calcium-dependent vasoconstriction in pulmonary VSMCs. It is specific for the pulmonary vasculature, and it is important for maximizing the efficiency of matching ventilation and perfusion. Although a complex process involving all vascular cell types, modulation of acute HPV depends critically upon the function of the pulmonary endothelium. Mechanical injury to the pulmonary endothelium can enhance HPV as well as hypoxic contractions, suggesting a vasodilatory role of the endothelium during HPV. Induction of such vasodilation to modulate the contractile response relies on at least two main mechanisms. First, as noted already, the endothelium may increase metabolism of circulating vasoconstrictors, thereby reducing their action during hypoxia. Second, and perhaps more importantly, endothelial cells appear to inhibit directly the contractile response by secreting increased levels of the vasodilators NO and PGI2 [15]. The homeostatic role of NO and PGI2 on modulating acute HPV was first suggested by Brashers et  al. [16] and was later confirmed by several groups [17, 18], who demonstrated a potentiation of hypoxic vasoconstriction by pharmacologic NO inhibitors in ex vivo animal models of pulmonary vascular beds. The dependence of this response on the endothelium has also been demonstrated by ex vivo enhancement of hypoxic contraction after removal of endothelium and by inhibition of NO using a variety of NO inhibitors [19]. Subsequently, PGI2 release was discovered to increase during HPV [20], with inhibition of PGI2 synthesis

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by cyclooxygenase (COX) inhibitors leading to enhancement of pulmonary pressure [20, 21] and reduction of blood flow  [22] during hypoxia in animal models. Together, these experiments appear to identify endothelium-specific NO and PGI2 as functional antagonists to HPV. However, it is unclear as to whether the above-mentioned experiments accurately reflect the physiological setting of HPV in vivo. Specifically, these experiments were conducted either on large-conduit pulmonary arteries or under conditions of severe hypoxia. Furthermore, this in vitro hypoxic contraction has been demonstrated, as well, in systemic arteries, including the aorta [23], coronary arteries [24], and femoral arteries [25]. In contrast, induction of HPV in vivo is typically observed in smaller vessels, in less extreme conditions of hypoxia, and is unique to pulmonary vessels [15]. Chronic hypoxia can also lead to increased pulmonary pressures and pulmonary hypertension; however, this ­context may induce distinct cellular processes through pulmonary vascular remodeling (as reviewed in [26]). Notably, chronic hypoxia leads to decreased production of NO and PGI2 by endothelial cells (as shown by Badesch et al. [27] and Faller [28] among others), accompanied by increases in the production of vasoconstrictive factors such as platelet-activating factor, vasoactive AA metabolites, ET, serotonin, ATP, and platelet-derived growth factor. Significant changes in permeability, coagulant, inflammatory, and protein synthetic capabilities in pulmonary endothelial cells have also been observed in response to hypoxic exposure. These factors can directly or indirectly influence neighboring VSMCs and/or adventitial fibroblasts, thus contributing to chronic abnormalities in vascular tone and structure [26]. Improved ability to define the in  vivo effects of these endothelium-derived effectors on pulmonary vascular tone awaits the development of a tractable animal model for physiological and pathophysiological exposure to hypoxia and subsequent study of the pulmonary vasculature.

3 Endothelium-Specific, Vasoactive Mediators of Vascular Tone Downstream of the physiological and pathophysiological stimuli to the pulmonary circulation, endothelium-specific responses that lead to regulation of pulmonary vascular tone primarily rely upon the balance of specific vasoactive factors that control vasodilation and vasoconstriction. Most of these effectors also play significant roles in modulating vascular tone in the peripheral circulation, upon which most mechanistic studies have been based. In the next section, we will review both the direct evidence of the function of these effectors in the pulmonary vasculature and extrapolations of their mechanisms of action from prior studies in the peripheral vascular context.

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3.1 Nitric Oxide

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3.1.1 Nitric Oxide: Production and Mechanisms of Action

pulmonary tissue from mice deficient in a specific NOS isoform. nNOS and iNOS play minimal roles in the maintenance of the already low pulmonary resting tone; in contrast, eNOS appears to have the predominant role in influencing vasomotor tone under basal conditions. Successful study of the crystal structures of these NOS isoforms has provided an important basis for understanding the multitude of regulatory interactions important for function (as reviewed in [32] and schematically depicted in Fig.  2). Each NOS monomer has three main functional domains: a reductase domain, a calmodulin binding seq­ uence, and an oxygenase domain. Catalytic activity requires homodimerization, which is dependent on heme binding, zinc, and the cofactor tetrahyrobiopterin (BH4). The oxygenase domain is the active site of NO production and is dependent upon binding heme, l-arginine, and BH4. The reductase domain then modulates electron transfer to molecular ­oxygen (O2) bound to heme iron. Without l-arginine or BH4, “uncoupling” of eNOS can ensue. In this setting, molecular O2 is the recipient of electron transfer across the eNOS dimer, leading to the production of superoxide and subsequent reactive ­oxygen species (ROS) rather than NO. A genetic murine model of NO deficiency and pulmonary hypertension via “uncoupling” of eNOS through BH4 deficiency has suggested the in  vivo importance of endothelium-specific NO in  regulating pulmonary vascular tone [33]. Therefore, the presence of BH4 is critical for NO production by stabilizing a favorable spin state of the Fe(II)–O2 heme intermediate. Subsequent two-step oxygenation of the guanidino nitrogen of l-arginine leads to NO and l-citrulline synthesis. After production by eNOS, NO can diffuse to neighboring VSMCs and activate multiple signaling pathways to increase

NO is a free-radical gas that is produced through catalytic oxidation of l-arginine by NOS. Three isoforms of NOS have been described with overlapping expression patterns (as reviewed in [30]). Endothelial NOS (eNOS) is abundantly expressed in vascular endothelial cells and is responsible for the majority of NO production in the pulmonary vasculature. Neuronal NOS (nNOS) and inducible NOS (iNOS) are expressed in bronchial airway epithelium, as well as at low levels in VSMCs. Notably, iNOS can be upregulated in nearly all types of pulmonary cells after exposure to inflammatory stimuli, such as cytokines or endotoxin. Historically, the in  vivo study of NO in the pulmonary vasculature employed the use of chemical inhibitors that nonspecifically modulate all isoforms of NOS. More recently, the mechanistic description of the role of NO synthesis and vasodilatory function in the pulmonary vasculature has been greatly aided by the study of genetically modified murine models deficient in specific NOS isoforms. The physiological importance of each NOS isoform in regulating homeostasis of pulmonary vascular tone was initially evaluated by Fagan et  al. [31]. This study entailed the isolation and perfusion of whole

Fig.  2  Nitric oxide (NO) production by the pulmonary endothelium induces classical [cyclic GMP (cGMP)-dependent] and alternative (cGMP-independent) signaling pathways. NOS nitric oxide synthase, sGC soluble guanylyl cyclase, PDE phosphodiesterase, PKG protein kinase G, SNO-products S-nitrosated products. (Modified from Coggins and Bloch [29]. Reprinted with permission from Lippincott, Williams & Wilkins)

The first seminal advance in the mechanistic description of the endothelial control of vascular tone was the observation in 1980 by Furchgott and Zawadzki [23]: an intact and functional endothelial lining was required for vasorelaxation of ex vivo aortae upon exposure to acetylcholine . Vasorelaxation was mediated by a labile, secreted factor that was named endothelium-dependent relaxing factor (EDRF). Without EDRF, vasoconstriction, rather than vasorelaxation, was induced by acetylcholine stimulation of vascular smooth muscle. Subsequent studies in animal models and human vascular samples have extended the implications of this discovery, demonstrating the in  vivo significance of EDRF in the homeostatic maintenance of basal peripheral tone as well as in response to a variety of physiological stimuli (as reviewed by Coggins and Bloch [29]). The subsequent identification of EDRF as a gaseous factor, NO, has led to an appreciation of its central and sophisticated role in control of pulmonary vascular tone, as well as a number of pleiotropic functions in the vasculature in general. Specifically, the critical role of NO in the pulmonary vasculature in states of homeostasis and vascular abnormality has been established through studies of the regulation of NO production, downstream NO signaling, and animal models of pulmonary injury and pulmonary hypertension, along with specific genetic modifications in the NO pathway.

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pulmonary vasorelaxation (Fig.  2). The classical signaling pathway involves the NO-dependent activation of soluble guanylyl cyclase (sGC). Crystal structures of sGC have described a heterodimeric structure carrying single a and single b subunits with an intervening heme group bound by the apposed N-terminal domains of the subunits (as reviewed in [34]). The predominant sGC isoform in pulmonary VSMCs is a1b1, with a second isoform, a2b1, only minimally present in the general vasculature. Reaction of NO with the ferrousheme group of sGC stimulates a conformational change at the catalytic site leading to a marked increase in production of cyclic GMP (cGMP) from GTP (as reviewed in [35]). cGMP can then activate a variety of signaling pathways via interaction with its effector proteins. These include cGMP-gated ion channels, cGMP-dependent protein kinase (PKG), and cGMP-regulated phosphodiesterases (PDEs). Activation of these pathways can induce vasorelaxation in VSMCs through several mechanisms, including hyperpolarization of the plasma membrane, inhibition of intracellular calcium influx, and decreasing myofilament calcium sensitivity through activation of PKG-mediated phosphorylation and, subsequently, myosin light chain phosphatase (MLCP). Additionally, PKG can phorphorylate and inactivate the GTPase RhoA, preventing downstream activation of Rho kinase and Rho kinase dependent inhibition of MLCP [36]. cGMP is primarily degraded to GMP by PDE enzymes (as reviewed in [37]). Multiple types of PDEs can degrade both cyclic AMP (cAMP) and cGMP. Others, such as PDE5, which is abundantly expressed in the adult pulmonary vasculature [38], can specifically recognize only cGMP. Notably, specific PDE5 inhibitors, such as sildenafil, have been utilized as specific pulmonary vasodilators as a result of these effects. Additional pathways have been characterized that induce vasodilatory responses to alternative modes of endotheliumdependent NO signaling in pulmonary VSMCs. A predominant mechanism relies upon S-nitrosation, an enzymatic process of specific covalent attachment of NO groups to a broad range of proteins via sulfhydryl groups on cysteine amino acids [39]. S-nitrosated proteins may represent stable compounds that allow for transport of NO at greater anatomic distances than would be possible by simple diffusion of the gas molecule itself. In addition to the NO radical gas molecule, other sources of NO can include other S-nitrosated ­proteins, metal–NO complexes, or other NO oxidation products such as nitrite. For example, the oxidation of plasma NO to nitrite is likely catalyzed by ceruloplasmin, which competes with the scavenging function of hemoglobin [40]. Nitrite has a significantly longer half-life than NO in both tissues and plasma, allowing for transport and survival of the NO signal. Nitrite can then release NO under low-pH conditions [41]. As a result of this function, the pulmonary endothelium may serve as a source of active NO for the peripheral circulation. Additionally, blood-borne S-nitrosated

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proteins themselves may exert NO-like vasodilatory effects on the pulmonary vasculature itself (as reviewed in [42]). Future studies of NO signaling in the pulmonary vasculature will likely identify increasingly important roles for S-nitrosation and nitrite in mediating vasodilation. Finally, a model of in vivo “uncoupling” of eNOS through BH4 deficiency has also suggested the importance of NO production by the pulmonary endothelium. A relevant murine model has been described under prolonged hypoxic exposure that carries either a genetic deficiency or a transgenic rescue of guanosine triphosphate cyclohydrolase-1 (GTPCH1), the enzyme that catalyzes the rate-limiting step in BH4 biosynthesis [33]. In the genetically null model under normoxic conditions, levels of pulmonary-specific BH4 correlate inversely with right ventricular systolic pressure (RVSP) and demonstrate decreased pulmonary NOS activity with resulting right ventricular hypertrophy and evidence of pulmonary vascular remodeling. Transgenic rescue of GTPCH-1 allows for restoration of BH4, which correlate with normalization of RVSP and improvement of right ventricular hypertrophy [43]. The hypertensive response in the pulmonary vasculature in response to chronic hypoxia is exaggerated in the null mouse but is repressed in transgenic-overexpressing mice. Taken together, these experiments demonstrate an essential role for eNOS function to produce specific NO for homeostasis of pulmonary tone in both normoxia and hypoxic stress. Currently, it is unclear if BH4 deficiency plays a ­specific causative role in pulmonary hypertension in humans; however, it may represent an attractive pharmaceutical target for amelioration of pulmonary hypertension.

3.1.2 Nitric Oxide: Mechanisms of Regulation At a molecular level, extracellular stimuli induce intracellular regulatory events to modulate expression or activity of eNOS. These effects can be accomplished by transcriptional regulation, posttranscriptional stability of message, posttranslational modifications, protein–protein interactions, and change in intracellular localization (translocation). Initially, it was believed that eNOS is constitutively expressed by the endothelium, whereas iNOS was thought to be inducible and, therefore, the major source of regulation of NO production in the vasculature. This belief was largely supported by the rapidity of the transcriptional response and the short half-life of the iNOS RNA transcript. However, it has become apparent that eNOS transcription can be actively regulated in pulmonary endothelial cells in response to both mechanical and humoral stimuli. A primary example involves the activation of the signaling pathway of Rho/Rho kinase to downregulate eNOS transcription in both the peripheral and the pulmonary vasculature. Rho is a GTP-binding protein that activates its downstream target, Rho kinase, in response

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to activation of a variety of G-protein-coupled receptors (GPCRs) in a number of cell types in the vessel wall, including endothelial cells (as reviewed in [44]). As a result, Rho kinase modulates the reorganization of the actin cytoskeleton by inhibiting MLCP, and in so doing, modulates the downregulation of eNOS. These findings have been verified in the pulmonary vasculature. Recently, elevated Rho kinase activity has been demonstrated in animal models of pulmonary hypertension [45, 46]. Furthermore, intravenously administered fasudil, a selective Rho kinase inhibitor, has induced pulmonary vasodilation and regression of pulmonary hypertension in various animal models (among the earliest reports were [45, 47]) as well as in patients with severe pulmonary hypertension who were otherwise refractory to conventional therapies [48]. Interestingly, in a mouse model of chronic hypoxia, pulmonary hypertension improves after therapy with simvastatin, a 3-hydroxy-3-methylglutaryl (HMG) CoA reductase inhibitor known also to inhibit Rho kinase activity [49]. Taken together, these data suggest that Rho kinase is active in regulating eNOS expression as well as other endothelium-specific effectors; as a result, it may control a master molecular “switch” in the pulmonary artery, initiating an activated state in disease from a quiescent state in health. In addition to transcriptional regulation, eNOS is subject to posttranscriptional control at the level of messenger RNA (mRNA) stability. Interestingly, eNOS mRNA demonstrates significant stability under basal physiological conditions; however, a number of diffusible and pathological perturbations, including exposure to oxidized low-density lipoprotein, thrombin, inflammatory cytokines, and hypoxia, can destabilize eNOS mRNA through unclear mechanisms. An antisense inhibitor RNA, sONE, has been postulated as a key  regulator of the stability of the eNOS transcript [50]; however, again, these findings await validation in the context of the pulmonary endothelium. The subcellular localization of eNOS in caveolae is also important in the regulation of its activity. Caveolae are flaskshaped invaginations found on the surface of the plasma membrane in a variety of vascular cell types, including pulmonary endothelium, VSMCs, and fibroblasts (as reviewed in [51]). Caveolae and their main coat protein caveolin-1 (CAV-1) are potential regulators of signaling function that spatially organize and concentrate signaling molecules on the basis of an enrichment of cholesterol and sphingolipids in their membrane composition. eNOS is specifically ­targeted to cellular membranes by irreversible myristoylation at its N-terminal glycine [52]. Membrane anchoring specifically in caveolae is stabilized by reversible palmitoylation at the N-terminal cysteine residues, allowing for eNOS localization to caveolae during inactive conditions [53]. Owing to this localization in endothelial cells, CAV-1, which lies in close proximity to eNOS in caveolae, directly binds to eNOS at the calmodulin binding site and inhibits its function. Activation

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of eNOS is accompanied by dissociation from CAV-1 and translocation to intracellular sites via binding calmodulin and reversible depalmitoylation. Therefore, both binding of eNOS to CAV-1 and palmitoylation of eNOS appear to provide additional levels of dynamic control of eNOS activity [30]. Correlation of these mechanisms awaits verification in the pulmonary vasculature; however, the actions of caveolae appear to represent a possible upstream regulatory pathway of the development of pulmonary hypertension. CAV-1 is depleted in plexiform lesions and muscularized pulmonary arterioles from patients with PAH [54]. Furthermore, CAV-1null mice develop a dilated cardiomyopathy and pulmonary hypertension [55] with impaired NO and calcium signaling. Although a number of possible pathogenic mechanisms can be envisioned, regulation of eNOS function certainly appears to be a likely causative factor. Definitive proof, however, of the specific pathogenic actions of caveolae and CAV-1 in pulmonary hypertension is lacking. The actions of endogenous NOS inhibitors also afford an additional mechanism to regulate NO production. One of the earliest identified inhibitors was asymmetric dimethylarginine (ADMA) (as reviewed in [56]), which is degraded by the enzyme dimethylarginine dimethylaminohydrolase (DDAH). In a rat model of chronic hypoxia and pulmonary hypertension (10% inspired O2 for 1 week), increased ADMA levels and decreased levels of DDAH correlated with increased pulmonary arterial pressures [57]. Correspondingly, in a murine model heterozygous for a DDAH1-null mutation, decreased DDAH activity and consequent increased ADMA levels were associated with increased RVSP indicative of increased pulmonary vascular pressure along with thickened walls of pulmonary arterioles consistent with vascular remodeling [58]. These results correlated in certain human populations suffering from idiopathic pulmonary hypertension who were also found to have higher plasma ADMA ­levels than control populations [59]. It is predicted that endogenous upregulation of other NOS inhibitors, such as the antisense RNA molecule sONE [50], may also correlate with increased pulmonary arterial pressures; however, confirmatory evidence is still pending. Downstream of expression, regulation of eNOS activity in endothelial cells relies upon the activity of the calmodulin and calcium signaling, as well as upon posttranslational modifications such as palmitoylation, phosphorylation, and S-nitrosation of eNOS itself. As noted above, eNOS activity depends upon calmodulin binding to its active site to allow for electron transfer from the reductase to oxygenase domain. Calmodulin binding to eNOS is inhibited by phosphorylation by protein kinase CK 2 [60]. In addition, regulation of intracellular calcium concentration itself appears to control tightly calmodulin binding, as well as the electron flux at the active site; as a result, a variety of pathways that mobilize calcium rapidly induce eNOS activity [30]. In general, calcium is

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released from sequestered calcium stores upon activation of phospholipase C (PLC). Activated PLC then cleaves phosphatidylinositol 4,5-bisphosphate into diacylglycerol and inositol 1,4,5-trisphosphate (IP3), leading to the activation of protein kinase C (PKC) and IP3 receptors. IP3 receptor activation then initiates a number of complex signaling cascades involving the activation of numerous ion channels and an increase in the level of intracellular calcium for eNOS activation. Conversely, prolonged stimulation of eNOS activity can lead to eNOS depalmitoylation by the enzyme APT-1 [53], which allows for transport of eNOS out of caveolae into the cytoplasm and deactivation of eNOS activity [61]. Dynamic phosphorylation of eNOS affords an additional method of stimulatory and inhibitory control. Activity of PI3K and its downstream effector, the kinase Akt, are principal determinants of phosphorylation of eNOS at Ser-1177 [62] and Ser 635 [63], allowing for activation of eNOS in response to agonists or mechanical stimuli such as shear stress. Ser-617 may represent an additional site of phosphorylation by Akt, and may serve both to sensitize eNOS to calmodulin binding and to increase phosphorylation at additional amino acids [62, 64]. In contrast, phosphorylation of eNOS at Thr-495 [65] and Ser-116 can attenuate eNOS activity by inhibiting binding to calmodulin. In addition to phosphorylation, phosphatase activity can ­regulate eNOS activity. Calcineurinactivated pathways may culminate in dephosphorylation of Ser-116 and/or Thr-495 to increase eNOS activity. Further­ more, despite its additional ability to dephosphorylate Thr-495, protein phosphatase 2A likely functions primarily as an inhibitor of eNOS activity by dephosphorylating Ser-1177. Finally, S-nitrosation of eNOS at cysteine residues (Cys-94 and Cys-99) has recently been appreciated as a mechanism for inhibition of eNOS under basal conditions. The mechanism by which S-nitrosation inhibits activity of eNOS is unclear, but it may rely upon dissociation or dysfunction of eNOS dimerization for electron transfer [66]. As a result, a variety of regulatory checkpoints of eNOS activity exist, thereby creating complex but specific posttranslational control of eNOS in various biological contexts in the pulmonary circulation.

3.1.3 Nitric Oxide: Linking Mechanisms of Regulation to Exogenous Stimuli As noted already, endothelial production of NO in the ­pulmonary vasculature can be induced by multiple types of exogenous stimuli, including neural, humoral, mechanical, and hypoxic signals. In most cases, control of eNOS activity and expression primarily modulates this response. Some of the best understood neural and humoral activators that regulate expression of eNOS and NO in the pulmonary endothelium exert their molecular effect via activation of cell-surface seven transmembrane guanine nucleotide binding proteins, GPCRs. These receptors are linked on their

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cytoplasmic side to heterotrimeric G proteins that consist of a and bg subunits. Each subunit is further subgrouped into classes that comprise at least 20 Ga, 6 Gb, and 12 Gg isoforms, allowing for a number of combinations that each may activate distinct signaling cascades, which can control eNOS expression and activity [30]. One of the best characterized exogenous eNOS agonists is bradykinin, a soluble hormone that activates the bradykinin B2 receptor, a GPCR that is widely expressed in peripheral and pulmonary vasculature [67]. Local concentrations of bradykinin are regulated by kininogenases, which cleave the precursor kininogens, and by kininases, which degrade bradykinin. As noted already, endothelium-specific angiotensin-converting enzymes represent important kininases that control bradykinin levels in the vasculature [68]. Other endogenous GPCR ligands that activate eNOS include acetylcholine (which binds and activates the M2 muscarinic receptors), histamine, adenosine, and ATP/ADP [69]. These ligands all activate GPCRs that consequently activate PLCb and mobilize intracellular calcium stores to promote robust eNOS activation [70]. By highresolution microscopy, it appears that these calcium waves occur preferentially at the cell edges where eNOS and some GPCRs congregate [71]. Alternatively, thrombin can bind GPCRs to induce inhibition of eNOS by phosphorylation at Ser-1177 via inhibition of Akt [72] and by downregulation of eNOS mRNA by Rho/Rho kinase activation [73]. Finally, other GPCR ligands, such as the platelet-derived lipid mediator lysophosphatidic acid, stimulate eNOS through activation of Akt-dependent phosphorylation [74]. The platelet-derived sphingolipid sphingosine 1-phosphate (S1P) can activate GPCR-dependent Akt signaling as well as nonselective cation channels to allow for an influx of ­calcium, both leading to eNOS activation [74, 75]. Other humoral factors that activate non-GPCR cell-­ surface receptors play significant roles in regulating eNOS activity. VEGF is an angiogenic polypeptide growth factor that can induce eNOS activation via binding receptor tyrosine kinases. VEGF recognizes at least four independent receptor subtypes, most notably the KDR receptor on vascular endothelium [76]. VEGF was first suspected to be integral to eNOS regulation when it was shown that endotheliumdependent vasodilation in canine coronary arteries is sensitive to both NOS inhibitors and tyrosine kinase inhibitors [77]. Since then, it has been demonstrated that VEGF can robustly activate eNOS function by multiple, potentially interacting pathways. VEGF can activate eNOS via both Akt-dependent signaling and phosphorylation of Ser-1177 on eNOS itself [78]. Akt activation is likely mediated by both PI3Ka and PI3Kb pathways, but PKC may also ­contribute. VEGF also activates the phosphatase calcineurin, leading to dephosphorylation of Ser-116 on eNOS and ­resulting in stimulation of its activity. Similar to S1P, VEGF can increase intracellular calcium levels and, thereby, eNOS activity by stimulation of the tyrosine kinase c-Src and

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s­ ubsequent stimulation of PLCg [79]. Finally, reversible ­denitrosation of eNOS appears to be stimulated by VEGF [80]. Insulin can also serve as an exogenous stimulus, activating eNOS by insulin receptor tyrosine kinases that act upstream of PI3K, Akt-mediated Ser-1177 phosphorylation, and eNOS denitrosation [80, 81]. Diabetic vasculopathy may also lead to eNOS activation by irreversible glycosylation to  form O-linked N-acetylglucosamine conjugates [82]; however, the contribution of these modifications in the ­pulmonary vasculature is not clear. Finally, estrogen is a sex steroid hormone that binds intracellular estrogen receptors (ERa and ERb) that can also regulate vascular tone via ­modulation of eNOS activity [83–85]. To activate eNOS, estrogen can induce calcium transients and can activate PI3K/Akt and MAPK. Taken together, these multiple receptor-coupled signaling pathways constitute specific and complex means of regulating eNOS by neural and humoral stimulation. It is tempting to speculate that these agonists and downstream signaling pathways act cooperatively, either competitively or synergistically, in  vivo to afford multiple levels of regulation. Shear stress can robustly activate eNOS and vasorelaxation via still unknown pathways. Significant prior work focused mainly on the endothelium of the peripheral vasculature, suggesting that laminar flow induces a vasorelaxed, quiescent state whereas turbulent flow leads to a vasoconstricted, activated state. Part of the downstream response appears to be dependent upon the transcription factor KLF2, which may then activate PI3K/Akt pathways in a calciumindependent manner. Conversely, activation of calciumdependent (KCa) potassium channels and hyperpolarization may be important in the response to shear stress, allowing for the release of NO and other endothelium-specific vasodilators. The mechanisms upstream of these intracellular events are even less clear. Evidence exists to suggest that some GPCRs, such as P2X4 purinoreceptors [86], may sense fluid shear stress. Conversely, some G protein subunits may be directly activated by shear stress, independent of GPCR expression, suggesting their role as mechanoreceptors [87]. Nonetheless, definitive identification is elusive for mechanoreceptors in the pulmonary endothelium that can convert mechanical forces to intracellular signals specific for eNOS activation. Furthermore, it is unclear if the best studied flowdependent phenotypes are recapitulated in the pulmonary vasculature. In part, this stems from the difficulty of directly studying the in vivo flow patterns at the anatomic level of the pulmonary arteriole. A number of animal models have been developed to study “flow-dependent” PAH, notably involving surgical creation of a shunt in rodents and larger mammalian species (as reviewed in [88]). In addition, attempts at computer modeling have shown promise [89, 90]. These may play important roles in the future for delineating better the molecular effects of increased flow on eNOS and NO in the pulmonary vasculature.

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Under physiological and pathophysiological stressors, such as acute or chronic hypoxia, the role of NO signaling is less clear, but it appears to play at least some role in regulating pulmonary vessel tone. For example, molecular O2 is necessary for NOS activity, and a decreased production of NO is observed in hypoxic conditions and during HPV. Accordingly, HPV is augmented in NOS3-null mice [91], suggesting the importance of endothelial NO production in tempering that response. Additional evidence exists regarding the importance of the NO pathway in the adaptation of the pulmonary vasculature to chronic hypoxia leading to pulmonary hypertension. In rodent models of chronic hypoxia (1–3-week exposure times), pulmonary eNOS expression is increased [92]. Furthermore, two groups have reported that prolonged hypoxia leads to increased levels of pulmonary hypertension and vascular remodeling in NOS3-null mice compared with wild-type controls [93, 94]. Taken together, these data also suggest a role for activated eNOS and increased NO signaling during hypoxia and, therefore, they suggest its function in relief of pulmonary hypertension. Because the response to hypoxia in vivo is quite complex, however, seemingly conflicting data have emerged from the use of different animal models regarding the actual role of NO in response to hypoxia. Notably, one study reported that NOS3-null mice are protected from, rather than prone to, pulmonary hypertension and vascular remodeling in response to prolonged hypoxia [95]. Conversely, in a study of ex vivo pulmonary artery explants from rats exposed to hypoxia for 1 week, decreased NO production was reported, which correlated with decreased Ser-1177 phosphorylation and, thus, tighter association of eNOS with CAV-1 but not with activating partners, such as calmodulin and hsp90 [96]. These associations indicate that hypoxic exposure may favor the inactive state of eNOS, thereby causing an absence of NO and resulting vasoconstriction. These apparently paradoxical results likely stem from the activities of multiple intersecting signaling pathways during hypoxic stimulation that affect both the regulation of endothelial production and downstream effects of NO itself on the pulmonary vasculature. As a result, although NO appears to play a significant role in controlling vasomotor tone in response to acute and chronic hypoxia, its specific regulation and downstream consequences in  vivo have yet to be clearly elucidated. Because of the predominant role of NO signaling in ­controlling pulmonary vascular tone, production of NO by eNOS, NO itself, degradation of cGMP, and sGC activity have all been targeted for therapeutic application in a variety of pulmonary vascular diseases. These will be reviewed in detail in other chapters but will be mentioned briefly here as their efficacy reinforces the notion of a primary role for the NO pathway in control of pulmonary vascular tone. First, inhaled NO has been used as therapy for pulmonary hypertension in the setting of persistent pulmonary hypertension of the newborn (PPHN), right ventricular myocardial infarction,

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implantation of a left ventricular assist device, lung transplantation, a variety of cardiac surgeries, and acute chest syndrome and vasoocclusive crises in sickle cell disease (as reviewed in [29]). Second, gene transfer of eNOS to rat lungs via inhaled adenoviral vectors has also produced prolonged improvement of pulmonary vascular tone in animal models of hypoxic pulmonary hypertension [97] and in eNOS-mull mice [98]. Furthermore, infusion of bone-marrow-derived endothelial progenitor cells overexpressing eNOS in animal models has resulted in normalized RVSP, improved microvascular perfusion, and improved survival [99, 100]. Third, effectors that influence the regulation of eNOS have been studied for therapeutic benefit in pulmonary vascular diseases, such as HMG CoA reductase inhibitors [101, 102]. Finally, in VSMCs, inhibitors of PDE5, such as sildenafil, increase vasorelaxation, reduce PVR, and improve exercise tolerance in cases of pulmonary hypertension (as reviewed in [103]). A number of sGC sensitizers and activators are under current study to further improve pulmonary vascular tone. As a result, since the initial identification of the role of NO in control of pulmonary vascular tone, our understanding of its mechanism of action and its vast effects on the pulmonary vasculature have improved considerably. Ongoing research now links NO production in the lung as a source for systemic vascular networks, suggesting that the actions of the pulmonary endothelium not only directly regulate ­pulmonary vasomotor tone but also indirectly regulate ­systemic vascular tone. Certainly, these discoveries raise new questions in the investigation of NO and continue to be useful in the design of new therapeutic strategies for treatment of pulmonary vascular pathophysiological processes.

3.2 Arachidonic Acid Metabolites: Prostacyclin and Thromboxane A2 AA and its various eicosanoid metabolites are well-known modulators of inflammation in multiple cells types; in pulmonary endothelial cells, these represent significant effectors that regulate vasomotor tone. The initial discovery of PGI2, which is produced predominantly by endothelial cells as an endothelial relaxing factor, first underscored the importance of the endothelium and the AA metabolic cascade in regulating vascular tone. Subsequently, TxA2 was identified as a functionally anatagonistic eicosanoid that is predominantly generated by platelets to induce vasoconstriction and platelet activation. These prostaglandins play crucial roles in modulating a number of endothelium-specific functions such as thrombosis, cellular proliferation, migration, viability, permeability, and leukocyte adhesion. More importantly, in the pulmonary circulation, maintenance of the balance of these two eicosanoids has been proposed as a

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central mechanism by which vascular tone is modulated. Conversely, an imbalance appears to be a predominant feature of vasoconstriction under pathophysiological conditions, such as pulmonary hypertension [104]. In this section, we will focus on the contribution of the pulmonary endothelium to eicosanoid generation with an emphasis on the roles of PGI2 and TxA2 in the control of vascular tone. 3.2.1 Arachidonic Acid Metabolites: Production and Mechanisms of Action Production of eicosanoids in the pulmonary endothelium begins with the liberation of AA from membrane-bound phospholipids via the action of phospholipase A2 (PLA2) (as reviewed in [105]). Various isoforms of PLA2 (cytosolic, secretory, and calcium-independent) are regulated by cytoplasmic calcium, cAMP, and/or phosphorylation events; activation of PLA2 is subsequently controlled by transcriptional and posttranscriptional mechanisms, as well as by regulation of subcellular localization (with predominant action occurring at the nuclear envelope). When this happens, AA can be metabolized further or secreted for exchange of free AA between cells; in fact, multiple types of endothelial cells, including aortic, microvascular, and pulmonary cells, express fatty acid binding proteins to facilitate AA uptake. Whether originating from the extracellular space or from membrane phospholipids, AA then undergoes further chemical modification via oxygenation by three predominant routes – catalysis by COXs, lipoxygenases, or cytochrome P450 (CYP) monooxygenases (Fig. 3). COX catalyzes a two-step process, leading to the formation of prostaglandin H2 (PGH2) and its downstream products, collectively known as prostanoids. COX catalyzes the incorporation of two molecules of O2 into AA to form prostaglandin G2 (PGG2), and this is followed by a peroxidase step that allows for a two-electron reduction of PGG2 to PGH2. Depending upon the cell-specific context, isomerization or reduction of PGH2 then leads to production of TxA2, PGI2, or a variety of other prostanoids. Differential regulation and production of these specific prostanoids affect their balance in the pulmonary circulation and, therefore, can profoundly affect vascular tone. Alternatively, lipoxygenase may catalyze the formation of leukotrienes, lipoxins, or hydroxyeicosatetraenoic acids (HETEs); among these, leukotrienes, as described later in the chapter, and HETEs such as 20-HETE [106] can function as direct vasoactive mediators of pulmonary vascular tone. Finally, CYP promotes the production of epoxyeicosatrienoic acids (EETs), which have been shown to function as EDHFs and consequent vasodilators [107], also discussed in a subsequent section. Although prostanoids control a number of endothelial functions, including migration, proliferation, and viability, much of the active control of vasomotor tone occurs at the

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Fig. 3  Production of vasoactive arachidonic acid (AA) metabolites in the pulmonary vascular endothelium. In the pulmonary endothelium, AA can be metabolized into a variety of products to influence a number of vascular functions. This diagram depicts the pathways that lead to the principal metabolites involved in control of pulmonary vascular tone. These metabolic cascades include the production of prostanoids via cyclooxygenase

activity, the production of leukotrienes via lipooxygenase activity, and the production of ­epoxyeicosatetraenoic acids (EET) via cytochrome P450 oxygenase activity. Receptors are noted in parentheses. PG prostaglandin, LT leukotriene, H(P)ETE hydro(pero)xyeicosatetraenoic acid, Tx thromboxane, PPAR-a peroxisome proliferator-activated receptor-a. (Modified and reprinted with permission [107])

level of the vascular smooth muscle. Prostanoids do not function as a circulating hormone but rather act as local paracrine mediators at the site of production. TxA2, prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), prostaglandin F2 (PGF2), and PGI2 are classified as conventional prostanoids that all exert their effects through activation of GPCRs expressed on the plasma membrane [107]. The GPCRs that recognize each prostanoid specifically include five basic types: TP, DP, EP, FP, and IP, respectively [108]. Notably, each prostanoid typically recognizes its single cognate receptor without a significant degree of recognition of the other receptors; however, the downstream signals from a single receptor can be quite varied depending upon the vascular cell type in the pulmonary circulation. For example, the IP receptor, which binds and is activated by PGI2, can be coupled to either Gs or Gq effectors, depending upon the specific cell type. In VSMCs, binding of PGI2 leads to IP receptor binding and Gq activation. In turn, this leads to cAMP-dependent and PLC-induced increases in intracellular calcium leading to activation of myosin light chain kinase (MLCK) and subsequent phosphorylation of myosin and vasodilation. Other IP-dependent downstream signaling cascades induce endothelial proliferation, whereas

activation of IP receptors on platelets inhibits activation and aggregation. An even more complex regulatory cascade is induced by TxA2 upon binding the TP receptor. The TP receptor may be coupled to any one or a combination of Gq, Gs, Gi, and G12/13 units. As a result, the TP receptor may activate cAMP-, calcium-, and Rho-dependent signaling pathways [109] with downstream activation of Rac, protein kinase A (PKA), PKC, p42/p44 MAPK, c-Jun N-terminal kinase (JNK), and Akt. In aggregate, these pathways lead to a dephosphorylation of myosin and vasoconstriction. Alter­ natively, TxA2 can bind platelet receptors, leading to activation and aggregation [110]. PGE2 can similarly induce vasodilation, whereas PGF2 can activate vasoconstriction; PGD2 can activate constriction or dilation depending on the context (as summarized in Table 1). Unlike PGI2 and TxA2, however, these likely play minor roles in controlling vascular tone. Other nonconventional prostanoids such as prostaglandin J2 and prostaglandin A2 can be produced from PGH2 in endothelial cells exposed to inflammatory stimuli [111] and, in turn, can activate a variety of downstream nuclear receptors; however, unlike conventional prostanoids, their function typically centers on regulating vasomotor tone.

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S.Y. Chan and J. Loscalzo Table 11.1  Differential effects of endothelium-derived prostanoids on pulmonary vascular tone Arachidonic acid metabolite Vascular tone Thromboxane A2 Prostaglandin D2 Prostaglandin F2 Prostaglandin E2 Prostacyclin

↑ ↓↑ ↑ ↓ ↓

The role of prostanoids in the intact pulmonary vasculature has been interrogated, but a complete description of their mechanisms of action in vivo is still preliminary. In humans, PGI2 and TxA2 have been identified as important mediators of pulmonary vasomotor tone in physiological processes at birth as well as in pathological conditions such as pulmonary hypertension (as discussed later in this chapter). Recognition of their importance has led to the therapeutic success of prostanoid therapy for patients with severe PAH (as reviewed in [112]). A number of murine models with genetic deletions of COX isoforms, the IP receptor, and the TP receptor have been reported, demonstrating significant effects on vascular development and angiogenesis; however, a description of the direct effects on pulmonary vascular tone is lacking. These models may represent important tools for future study of these prostanoids during development, under basal conditions, and in pathophysiological settings such as pulmonary hypertension. 3.2.2 Arachidonic Acid Metabolites: Mechanisms of Regulation The expression and catalytic activity of COX in the pulmonary endothelium are tightly controlled, and the resulting production of prostanoids, such as TxA2 and PGI2, greatly influences the critical balance of vasoconstriction and vasodilation in the pulmonary circulation. Furthermore, COX has been demonstrated to affect vascular tone profoundly in a variety of physiological and pathophysiological contexts, such as hypertension, preeclampsia, atherosclerosis, diabetes, cerebral blood flow, and tumor angiogenesis. Essentially two isoforms of COX have been identified with approximately 60–65% homology: COX-1 and COX-2 (additionally, a splice variant of COX-1 named COX-3 has been reported). Both isoforms are present in equal proportions in the luminal surface of the endoplasmic reticulum and the inner and outer membranes of the nuclear envelope [113]. In vascular endothelium, COX-1 is constitutively expressed under basal conditions [8]; however, it can also be induced by shear stress and a variety of transcription factors [114]. COX-2 is typically considered an inducible isoform that can be upregulated by a variety of conditions, such as cytokines, cholesterol, lipoproteins, and hypoxia. Both endothelial cells and VSMCs express COX, but endothelial cells produce up to 20 times more than VSMCs [115]. Regulation of expres-

sion and activation of COX, particularly the COX-2 isoform, typically determines prostanoid production with consequent implications for pulmonary vascular tone. Exogenous agonists can bind to endothelial cell surface receptors to activate a variety of kinase signaling cascades to induce COX-2 expression, resulting in production of PGI2 or TxA2 in both the peripheral and the pulmonary circulations. Interleukins, such as IL-1a and IL-1b, can activate extracellular-signal-regulated kinases (ERK), JNK/ stress-activated protein kinase, and p38 MAPK [116] to increase expression of both COX-2 and PLA2, leading to increased PGI2 production [117]. Blood-borne high-density lipoproteins can synergize with these cytokines at the cell surface to upregulate COX-2 in endothelial cells [118]. Activation of antioxidant enzymes such as catalase [119] can also lead to COX-2 transcription via similar pathways. Lysophosphatidylcholine, a component of oxidized lowdensity lipoprotein, can induce p38 MAPK and the transcription factors CREB and ATF-1, leading to COX-2 induction [120]. Alternatively, phorbol 12-myristate 12-acetate (PMA), an activator of PKC, induces expression of COX-2 via ERK1 and ERK2 activation [121]. Downstream of these exogenous stimuli are more central signaling cascades that allow for direct transcriptional control of COX-2; however, because of the relative paucity of studies of these regulatory pathways in the pulmonary vasculature, our mechanistic understanding relies heavily on extrapolated data from studies in the peripheral circulation. For example, the signaling pathways resulting from cytokine-induced inflammation and hypoxic stress typically culminate in the activation of the transcription factor nuclear factor kB (NF-kB), which can directly bind to a response element in the COX-2 promoter to upregulate transcription [122]. In other settings of hypoxia, a member of the highmobility group I(Y) protein family is also activated to facilitate ­transactivation of the promoter of COX-2 [115]. cAMP, which is an intracellular second messenger for downstream PGI2-dependent function, serves to regulate upstream COX ­expression in a more complex fashion. Although increased intracellular AMP levels inhibit COX-1 activity, they conversely induce COX-2 transcription [123]. Alternatively, in the context of PMA stimulation, extracellular cAMP can be upregulated ultimately to inhibit expression of COX-2 in human pulmonary microvascular endothelial cells [124]. The mechanistic explanations for these seemingly paradoxical observations are unclear. A similarly complex, tissuespecific regulatory pathway of COX expression appears to result from the actions of the peroxisome proliferator-­ activated receptor (PPAR) family of transcription factors. In pulmonary endothelium, PPARg is highly upregulated during hypoxic stress [125] and can be activated to modulate diverse cellular programs of inflammation, proliferation, and differentiation [126]. In macrophages, COX ­transcription is

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i­ nhibited by PPARg [127]. Similarly, PPARg agonists inhibit NF-kB activity and consequently inhibit COX-2 induction [128]; however, direct binding of PPARg to the promoter of COX-2 increases its transcription in epithelial cells [129]. The effects of PPAR activation in pulmonary endothelial cells are currently unclear but are likely to have significant consequences for vasomotor tone via these downstream effects on prostanoid production. In addition to upregulation of transcription, COX activity can be independently modulated to alter the balance of PGI2 and TxA2 expression. An unusual feature of COX involves the autoinactivation of enzymatic activity after a finite turnover of catalysis (as reviewed in [130]). An additional factor that allows for independent activity of COX-1 is the negative allosteric regulation of COX-1 at low concentrations of arachidonate leading to increased eicosanoid synthesis [113]. Vascular effectors have also been associated with specifically increasing the activity of COX. These include NO, which can increase COX activity in vascular endothelial cells [131]. This complex mechanism depends upon the level and source of NO and the specific COX isoform; it may involve the production of peroxynitrite (produced from the interaction of NO and superoxide anions), a ROS that can directly activate COX. Interestingly, COX activity itself also carries an ability to co-oxidize substances, such as NADPH, which allow for production of further superoxide species [132]. This suggests a potential positive-regulation loop for the potentiation and activation of COX activity. Furthermore, a difference in the threshold for hydroperoxide activation between COX-1 and COX-2 may allow for independent function of these two enzymes in the same cell type. In contrast to our mechanistic understanding of COX activity in endothelium, the molecular control of the multiple cell-specific synthases and isomerases that direct subsequent steps converting PGH2 to conventional prostanoids such as PGI2 and TxA2 and other nonconventional prostanoids have been less well characterized. In general, the activities of these enzymes appear mostly cell-specific and are constitutively active. Upon COX activation, platelets, mast cells, and monocytes/macrophages synthesize TxA2, PGD2, PGE2, and PGF2 whereas pulmonary endothelial cells release mostly PGI2 [133]. Endothelial production of PGI2 can be stimulated by additional factors, including hypoxia, endotoxin, interleukins, and fluid shear stress. Conversely, proinflammatory cytokines and oxidative stress can promote endothelial release of TxA2, PGD2, PGE2, and PGF2. However, the ­mechanisms beyond COX regulation by which the balance of ­constrictive and dilatory prostanoid effectors is affected are less clear. As a result, the AA cascade serves as a major mechanism for generation of endothelium-specific effectors that play important roles in regulating pulmonary vascular tone. Much of our mechanistic understanding of these factors to date

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relies upon evidence from the peripheral circulation. Their action in the pulmonary circulation awaits more direct study in the pulmonary vasculature.

3.3 Endothelin 3.3.1 Endothelin: Production and Mechanisms of Action In contrast to vasodilators such as NO and PGI2, the production of vasoconstrictive factors by pulmonary endothelial cells allows for further control of tone in the pulmonary circulation. ET is such a potent vasoconstrictive peptide that is secreted by pulmonary endothelium and greatly influences vasomotor tone in physiological and especially in pathophysiological settings. ET was first isolated from conditioned medium of coronary arterial endothelial cells in 1985 and was initially named endothelium-derived contractile factor [134]. In 1988, ET was purified and identified as a novel 21 amino acid peptide. Since then, three endogenous isoforms have been described (ET-1, ET-2, and ET-3); and they are expressed at different levels depending on tissue context [135]. ET-1 is the predominant vasoactive isoform produced in endothelial cells, and it can profoundly alter vasomotor tone in both the peripheral and the pulmonary circulations. Specifically, ET-1 has been discovered to have integral roles in a number of cardiovascular diseases, including systemic hypertension, cerebrovascular spasm, chronic renal failure, atherosclerosis, ischemic heart disease, and chronic heart failure, as well as pulmonary vascular diseases such as pulmonary hypertension (as reviewed in [136]). In endothelial cells, ET-1 is first translated as a precursor, named preproendothelin. After the signal peptide removal, the precursor is processed by a furin-like peptidase to yield a biologically inactive intermediate called big ET. Big ET is further converted into active ET by endothelin-converting enzyme (ECE). Four different isoforms of ECE exist (ECE-1a, ECE-1b, ECE-1c, ECE-1d), derived from differential splicing from one transcript. These isoforms are present in different cellular sites. ECE-1a is particularly highly expressed in endothelial cells, resides in intracellular secretory vesicles, and is constitutively transported to the cell surface. The precursor of ET-1 appears also to be contained in these vesicles for processing by ECE-1a and transport to the plasma membrane for secretion. In contrast, ECE-1b is located in the cytoplasm near the trans-Golgi network; ECE-1c and ECE-1d are located directly at the cell surface and may act as ectoenzymes. Another isoform, ECE-2, was identified in 1995 with a different pH sensitivity for enzymatic activity compared with ECE-1 [137]. The existence of additional isoforms is possible because high levels of fully

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processed ET-1 are still present in the setting of ECE-1 and ECE-2 double-homozygous null mice [138]. Upon processing and secretion, ET-1 binds one of two main ET receptors in the pulmonary vasculature: ETA receptor and ETB receptor. Both receptors belong to a family of heptahelical GPCRs. In the pulmonary vasculature, both cell types are predominantly expressed on VSMCs. The ETA receptor binds ET-1 and ET-2 with more affinity than ET-3; whereas the ETB receptor can bind the three isoforms with equal affinity. Owing to the low homeostatic levels of bloodborne ET-1 in the pulmonary circulation under basal physiological conditions, it was initially hypothesized that ET-1 is secreted by endothelial cells for paracrine action [136]. The vasoconstrictor response of ET-1 primarily relies upon its binding to the ETA receptor subtype on neighboring VSMCs [139]. After binding, activation of the ETA receptor causes a transient, followed by a more sustained increase in the level of intracellular Ca2+ ions [140, 141]. The transient increase originates from an intracellular pool of free Ca2+ through activation of PLC; the sustained increase results from extracellular influx, mediated by activation of nonselective cation channels and a store-operated Ca2+ channels. Activation of the ETB receptor may play less of a role in control of pulmonary vasomotor tone [139], yet still can induce vasoconstriction by VSMCs [142]. In contrast, stimulation of ETB receptors on endothelial cells themselves induces vasodilation via the release of NO, PGI2, and stimulation of ATPgated potassium channels (as shown by Hasunuma et  al. [143] among others). Notably, activation of the ETA receptor also results in a potent mitogenic signal in endothelial cells and smooth ­muscle cells that can affect vasomotor tone via its effects on vascular remodeling. Cellular proliferation ensues from activation of PKC, MAPK, and the early growth response genes c-fos and c-jun [144]. The mitogenic action of ET-1 on pulmonary VSMCs can occur through the ETA receptor and/or the ETB receptor subtype, depending on the anatomic location of cells. The ETA receptor predominantly mediates mitogenesis in the main pulmonary artery, whereas mitogenesis in resistance arteries relies upon contributions from both subtypes. In contrast, some reports have suggested that activation of the ETB receptor specifically may induce an apoptotic pathway through unclear mechanisms.

3.3.2 Endothelin: Mechanisms of Regulation In contrast to our more sophisticated understanding of the regulation of NO and prostanoids, our description of the mechanistic links between exogenous stimuli and ET production is still emerging. although certain neural (i.e., norepinephrine), humoral (i.e., vasopressin, bradykinin, AT-II,

S.Y. Chan and J. Loscalzo

tumor necrosis factor a (TNF-a), and transforming growth factor-b (TGF-b); as reviewed by Touyz and Schiffrin [145]), mechanical [11], and hypoxic stresses [146] can increase expression of ET-1 at the transcriptional level, the complete panel of relevant receptors and downstream molecular pathways that transmit these signals have not been fully characterized. Although induction of ET-1 expression mainly occurs at the transcriptional level, modulation of mRNA stability also has been described [147, 148], indicating that alternative pathways other than direct transcriptional control may be involved in modulating ET expression. The existence of multiple processing ECE isoforms suggests the possibility for multiple levels of posttranslational regulation of ET-1 activity; however, again, direct evidence is lacking. More recently, it has become more evident that ET-1 expression is coordinately regulated with NO production [149]. Inhibition of NOS activity in peripheral porcine aortae increases both basal and thrombin-stimulated ET-1 secretion; conversely, NO donors suppress ET-1 expression [150]. In addition, inhibition of eNOS enhances both basal and hypoxic expression of ET-1, whereas NO donors suppress basal and hypoxia-induced ET-1 [151]. Inhibition of NOS activity also reduces shear-stress-mediated downregulation of ET-1 expression [152]. Conversely, activation of ETB receptors can stimulate eNOS activity, as described previously. ET-1 can also increase expression of eNOS in cultured endothelial cells [153]. Taken together, these data demonstrate the existence of an intricate system of molecular “cross talk” between NO and ET-1 to regulate vascular tone. It remains to be seen how this type of coordinate regulation impacts other vasoactive factors and how these modes of regulation specifically modulate pulmonary vascular tone. The physiological and pathophysiological significance of ET-1 on pulmonary vascular tone is highlighted by the success of chronic ET receptor antagonists in the treatment of pulmonary hypertension. It was initially expected that the advent of genetically engineered animal models would aid in better elucidating the in vivo mechanisms of action of ET-1 on vascular tone. However, these animal models have not provided a clear explanation of these effects. ET-1 homozygous-null mice have an embryonic lethal phenotype [154]. Heterozygous ET-1-deficient mice surprisingly demonstrate elevated systemic blood pressure despite low plasma and ­tissue levels of ET-1 expression [154]. It appears that this hypertension partially stems from high sympathetic tone, and it has been suggested that this may result from reduction in vasorelaxation mediated through ETB receptors. Non­ etheless, these unexpected phenotypes in the vasculature likely reflect notable complexities of function and signaling that remain to be determined. As a result, ET represents an endothelium-specific and highly active vasoconstrictor in the pulmonary vasculature.

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This vasoconstrictor response appears particularly active during times of active physiological or pathophysiological stress. Notably, the complete upstream physiological and pathogenic mechanisms that result in elevated levels of ET-1 and dysregulated signaling await further clarification but likely involve multiple layers of coordinate and feedback regulation.

3.4 Other Vasoactive Mediators 3.4.1 Endothelium-Derived Hyperpolarizing Factors In addition to NO and PGI2, other endothelium-specific factors have been characterized that induce vasorelaxation of pulmonary and peripheral arteries via hyperpolarization of endothelial cells and neighboring VSMCs. Initially, evidence for EDHF activity emerged from studies of eNOS/COX-1 double-null murine models, which are unable to produce NO and prostanoids in the vascular endothelium [155, 156]. In resistance arteries of female mice, endothelium-dependent relaxation is preserved, whereas relaxation in resistance arteries of corresponding males is impaired [157]. This characteristic led to the origin of the term “endothelium-derived hyperpolarizing factor” (EDHF) to describe a putative vasodilatory effector, functioning independently of PGI2 and NO [158]. Since that time, however, it has become apparent that, rather than a single diffusible substance, a number of endothelium-specific vascular mediators carry hyperpolarizing capabilities and can induce vasorelaxation. Therefore, the acronym EDHF may be misleading in light of more ­specific mechanistic data that are emerging [159]. The in  vivo significance of non-prostanoid-, non-NOdependent EDHF activity in control of vascular tone has been demonstrated mostly in resistance arteries in the peripheral circulation; however, some parallel studies have focused on the pulmonary vascular endothelial cells, especially in the setting of study of specific effectors with EDHF activity. In cell culture of endothelial cells, both nonspecific agonists of GPCRs and calcium ionophores, independent of effects from NO or prostanoids, can increase intracellular calcium concentration, leading to endothelial hyperpolarization followed by hyperpolarization of neighboring VSMCs and vasorelaxation [160, 161]. From studies with pharmacologic inhibition, the activation of endothelium-specific SKCa and/or IKCa [162, 163], rather than BKCa [164], calcium-activated potassium channels on the plasma membrane is necessary for these EDHF-type responses. Two mechanisms appear to be active in the subsequent propagation of hyperpolarization into neighboring VSMCs: (1) endothelium-specific hyperpolarized potential transmitted directly to VSMCs via myoendothelial gap junctions and connexin-40 or, alternatively,

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(2) accumulation in the intracellular cleft of potassium ions released from the endothelial cells, leading to direct activation of potassium channels and/or Na+/K+-ATPase on VSMCs and subsequent hyperpolarization [159]. A variety of supportive evidence for both mechanisms exists from pharmacology studies in cell culture and genetic manipulation of mice in vivo; and the involvement of gap junctions and potassium ions can occur simultaneously, sequentially, or synergistically. The contributing roles of each of these mechanisms in the pulmonary vasculature still remain unclear. EETs have been proposed as significant, soluble vascular effectors that may possess significant EDHF activity. EETs and its major metabolites, vic-dihydroxyeicosatrienoic acids, result from the metabolism of AA by CYP monooxygenases [165]. CYP enzymes are multifunctional and are integral to the metabolism of cholesterol, steroids, vitamins, bile acids, and xenobiotics, and in the production of ROS. EET production primarily occurs in the endothelial compartment, as no EET production has been observed in VSMCs [166]. As with other soluble vasoactive effectors, multiple levels of control of EET production exist in the vascular mileu (as reviewed by Fleming [167]). Transcription of CYP with subsequent formation of EETs can be increased by humoral stimulation with exogenous factors such as bradykinin. Exposure to chronic hypoxia can also induce upregulation of CYP with increased EET production. CYP activity is regulated, as well, at the level of posttranslational modification by phosphorylation, subcellular localization, proteosomal degradation, and NO-dependent modification of the heme moiety. Finally, after production by CYP, the levels of free EET products are tightly regulated by sequestration in membrane-bound phospholipids. EETs are lipophilic compounds with over 90% present as circulating species in the plasma as esterified phospholipids in lipoproteins. These lipids are incorporated into cell membrane phospholipids, and these may serve as an intracellular storage form from which EETs may be produced, independent of CYP activation. The exact mechanisms of action of EETs on the pulmonary vascular wall have not been fully described. Specific cell-surface receptors for EETs have yet to be identified [168]. Nonetheless, EETs can induce multiple signaling cascades involving tyrosine kinases, p42/44, and p38 activity. This results in activation of calcium-dependent potassium channels, particularly by modulating GPCR-dependent and PKA-dependent pathways. In addition to membrane hyperpolarization, 5,6-EET, 11,12-EET, and 14,15-EET isoforms may separately allow for calcium entry via activation of calcium-specific plasma membrane channels, such as BKCa [169, 170]. In doing so, membrane hyperpolarization in endothelial cells leads to efflux of potassium ions into the extracellular milieu. This increase in extracellular potassium then acts as a direct paracrine factor to stimulate smooth

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muscle cell hyperpolarization by activating K+ conductances. In contrast, myoendothelial gap junctions do not appear to figure prominently in hyperpolarization by EETs. As a result of regulating membrane hyperpolarization, EETs appear to affect vasodilatory activity, at least as indicated by mechanistic studies in cell culture. In vivo evidence also exists for this function, particularly in the peripheral arterial vasculature. In studies of explanted peripheral arteries, pharmacology studies have suggested that an EDHF contributes to bradykinin dilatation in human forearm [171, 172] and dog coronary [173] arteries. Furthermore, these studies have indicated that EDHF activity results from a CYP metabolite. In systemic hypertensive patients, dilation in response to bradykinin is markedly reduced and unaffected by NOS inhibition, but is inhibited by sulfaphenazole, an inhibitor of EET activity [172]. A more complex role for EETs appears to be in the pulmonary vasculature. In newborn piglet pulmonary resistance arteries, 5,6-EET has been demonstrated to induce vasodilation; but this activity appears to depend upon at least one additional endothelium-specific factor [174]. To complicate this interpretation, 5,6-EET can induce either dilation [175] or constriction [176] of pulmonary arteries, depending upon the anatomic location in the arterial tree and the presence of COX activity [177]. 5,6-EET appears to activate Rho kinase signaling, resulting in phosphorylation of myosin light chain and induction of contractile function [178]. Further mechanistic evidence is necessary to understand better the in  vivo consequences of this balance in vasomotor tone that is dependent on EETs. In addition to EETs, other vascular factors have been ­proposed to function as EDHF-like vasodilators in various contexts. For example, ROS, such as hydrogen peroxide, may function as hyperpolarizing factors. Both endothelial cells and VSMCs produce significant amounts of ROS in response to a variety of hormonal, neural, and mechanical stimuli. In addition, non-NO, non-prostanoid-mediated EDHF responses in certain peripheral vessels can be inhibited or entirely abrogated by catalase; conversely, agonistinduced-EDHF responses in these vessels are associated with increased endothelial production of hydrogen peroxide [179]. However, subsequent studies have suggested that hydrogen peroxide is, at best, a weak hyperpolarizing factor and may not have canonical EDHF activity. It is currently unclear if hydrogen peroxide can directly activate endothelial potassium channels, known to be essential in the mechanism of EDHF responses, as noted above. In addition, these hyperpolarizing responses depend critically on the type of artery and may not be applicable to the pulmonary circulation [180]. Additionally, other diffusible factors, such as C-type natriuretic peptide [181], have been proposed as eff­ ectors of EDHF responses. However, like EETs and hydro­ gen peroxide, their exact roles as endothelium-dependent vasodilators still await characterization, especially in the pulmonary vasculature.

S.Y. Chan and J. Loscalzo

3.4.2 Leukotrienes In addition to the previously described AA metabolites (i.e., prostanoids and EETs), the production of inflammatory leukotriene products from AA by 5-lipoxygenase (5-LO) appears to modulate endothelium-specific pulmonary vasomotor tone (as reviewed in [182]). 5-LO catalyzes a two-step oxygenation reaction, converting AA ultimately to leukotriene A4 (LTA4). In the presence of cell-specific nuclear or cytosolic enzymes, LTA4 may then undergo hydrolysis to generate leukotriene B4 (LTB4) or undergo conjugation with glutathione to produce leukotriene C4 (LTC4). The glutathione moiety can be then be sequentially cleaved to yield leukotriene D4 (LTD4) and leukotriene E4 (LTE4). The 5-LO catalytic pathway is abundantly expressed in macrophages, foam cells, and inflammatory cells (such as dendritic cells, mast cells, and neutrophilic granulocytes) – cell types especially prominent after vascular remodeling in a number of pulmonary vascular diseases in response to hypoxia, abnormal flow, or idiopathic stimuli. 5-LO is tightly regulated by a number of signaling elements. Initially, it is activated by a membrane-bound peptide named 5-LO activating protein (FLAP), followed by calcium-dependent subcellular translocation of 5-LO to the nuclear membrane [183]. 5-LO can also be stimulated by ATP (and to a lesser extent by other free nucleotides). Furthermore, activation may be induced by phosphorylation via the MAPK pathway [184] in response to a number of proinflammatory cytokines, such as TGF-b, or calcium mobilizing agents as well as physiological cellular stress such as osmotic shock, UV exposure, and heat shock. Expression of 5-LO may also be induced by a number of circulating effectors including, 1,25-dihydroxyvitamin D3, and by the proinflammatory cytokines granulocyte–macrophage colonystimulating factor, TNF-a, and IL-3 [185]. Alter­natively, PKA-induced phosphorylation of 5-LO inhibits activation in response to adenosine, PGE2, and b-adrenergic agonists, among others. Similarly, exposure to cytokines such as IL-4 and IL-13 downregulates expression of 5-LO [186]. Notably, pulmonary endothelial cells carry minimal or absent activity of 5-LO; however, these cells and VSMCs carry more abundant quantities of enzymes that act downstream in the synthesis of leukotrienes. As a result, in the presence of excessive adhesion of leukocytes to the endothelium in settings of inflammation, unstable LTA4 is generated, followed by transcellular transfer to endothelial cells for ­further processing. Although LTB4 is generated mainly in neutrophils and macrophages, LTC4, LTD4, and LTE4, the socalled cysteinyl-leukotrienes, are formed in endothelial cells and VSMCs for active function [187, 188]. Once generated, the cysteinyl-leukotrienes exert their effect on vascular function by binding specific seven transmembrane domain GPCRs. The receptors that bind LTB4 (BLT1-R and BLT2-R) are highly expressed in macrophages and leukocytes [189] to allow for activation and chemotaxis of these cell types

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to enhance proinflammatory conditions. Alternatively, the receptors that bind LTC4 and LTD4 (cysLT1-R and cysLT2-R) are more prominently expressed in the vascular wall: endothelial cells themselves express cysLT2-R, whereas VSMCs express cysLT1-R to a much higher degree. As a result of these latter receptors, cysteinyl-leukotrienes can also exert direct vasoactive effects. In pulmonary arteries, LTC4, LTD4, and LTE4 all induce vasoconstriction [190], leading to increased intracellular calcium concentration and activation of MLCK to augment contractile function. This effect appears specific for activation of leukotriene receptors, as dose-dependent increases in contractility via exposure to LTC4 or LTD4 in cultured rat artery smooth muscle cells are attenuated by montelukast, a selective cysLT1-R antagonist [191]. Furthermore, this vasoconstrictive effect is at least partially endothelium-dependent, as such selective cysLT1-R antagonists can inhibit acetylcholine-induced contraction elicited in an endothelium-dependent vessel explant exposed to indomethacin (to inhibit COX activity and subsequent prostanoid production) and precontracted with phenylephrine [191]. In human saphenous veins, LTC4 and LTD4 induce vasodilation at low concentrations but induce vasoconstriction at higher doses. In contrast, coronary arteries appear unresponsive to either leukotriene at basal conditions [192]. Notably, cumulative evidence exists from studies of the peripheral vasculature that abnormal or diseased vasculature may exhibit increased formation and/or sensitivity to various leukotrienes [192]. This sensitization may be especially prominent in the setting of AT-II-dependent arterial tone [188, 193]. It is tempting to extrapolate these interpretations to the pulmonary vasculature; however, because of the known specificity of effects of leukotrienes on select anatomic v­ascular bed, the results of studies in the peripheral vasculature should not be fully extrapolated to the pulmonary circulation without more direct evidence.

3.4.3 Other Gaseous Vasodilators Similar to NO, carbon monoxide (CO) and hydrogen sulfide (H2S) are gaseous vasodilators produced by endothelial cells, deficiencies of which may adversely affect pulmonary vascular tone and lead to the development of pulmonary hypertension. Mice that are null in heme oxygenase-1 (HO-1), a primary enzyme that produces CO in the pulmonary vasculature, exhibit less tolerance for hypoxia, with resulting right ventricular dysfunction [194]. In contrast, overexpression of HO-1 in the lung prevents development of pulmonary hypertension in murine models of chronic hypoxia [195] and in rat models of monocrotaline-induced PAH [196]. H2S also functions as a vasodilator and inhibitor of vessel wall proliferation, which can protect against the development of PAH in rat models [197, 198]. Studies elucidating the precise regulation and mechanisms of action of CO and H2S in basal states and during disease progression are currently ongoing.

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3.4.4 Vasoactive Intestinal Peptide VIP may also play a role in the control of pulmonary vascular tone. VIP is produced by pulmonary endothelial cells and is a pulmonary vasodilator, an inhibitor of proliferation of VSMCs, and an inhibitor of platelet aggregation. Decreased concentrations of VIP have been reported in serum and lung tissue of patients with pulmonary hypertension [199]. VIP-null mice suffer from moderate pulmonary hypertension [200]. Furthermore, both pulmonary arterial pressure and PVR decrease after treatment with VIP [201]. Improvement of hemodynamics and clinical course has also been observed with inhaled VIP in a small number of PAH patients [199]. Key questions regarding the molecular actions of VIP, the mode(s) of regulation of VIP expression, and its putative  causative role in pulmonary hypertension remain unanswered.

4 In Vivo Balance of Endothelium-Specific Vasoactive Effectors Although our understanding of the molecular functions of the previously described effectors has improved substantially over the past few decades, their in vivo effects and complex interactions with one another in the pulmonary vasculature are not entirely clear. Some unifying mechanisms have become more apparent in the context of certain physiological and pathophysiological states, linking the regulation of these effectors into a more cohesive model. The perinatal transition represents a prototypical example of a physiological change in pulmonary vascular tone, whereas PAH represents a well-studied pathophysiological example of dysregulated vasomotor tone. These processes emphasize the in vivo relevance of these endothelium-specific factors on vascular tone. Additionally, they emphasize the in vivo consequences of “cross talk” among these factors to allow for endotheliumdependent regulation.

4.1 Endothelium-Specific Changes in Perinatal Pulmonary Vascular Tone The fetal pulmonary circulation is marked by high PVR, allowing for blood flow to be diverted from the pulmonary vessels to the systemic circulation via the foramen ovale and ductus arteriosus. At birth, an acute decrease in PVR facilitates blood flow to the pulmonary circulation, allowing for gas exchange and then closure of the foramen ovale. This abrupt and dramatic transition of the pulmonary vasculature tone from in utero to postnatal life determines survival and depends profoundly on endothelial action. Although the

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entire process of this physiological adaptation is poorly understood, it likely results from a combination of the development, structure, and function of the endothelial cell along with its response to physiological stimuli via release of effectors of vasodilation. First, the specialized structure of the fetal endothelium is thought to augment mechanically the levels of vascular resistance. By the end of gestation, endothelial cell morphology is significantly different from that seen in the mature pulmonary circulation. In the adult, the endothelium is composed of a thin layer of elongated cells with a broad base adjacent to the basement membrane, a high surface-to-volume ratio, and limited overlap of adjacent lateral cell borders. As a result, vascular permeability is low and pulmonary blood flow is high to accommodate larger circulatory volumes at low pressure. In contrast, in the term fetus, pulmonary endothelial cells are cuboidal, have a narrow base adjacent to the basement membrane, a low surface-to-volume ratio, and significant overlap of adjacent lateral cell borders [202, 203]. As a result, the internal diameter of pulmonary arteries is small, resulting in an elevated resistance to forward blood flow. Correspondingly, electron microscopy has demonstrated that fetal endothelial intercellular junctions are ­fenestrated and are typically associated with bundles of microfilaments, indicative of high vascular permeability and poor barrier function. This permeability may reflect a combination of actions by several factors such as ET-1, VEGF, AT-II, and hypoxic stress, and is necessary for continuous cross talk with fluid-filled alveolae in the absence of pulmonary blood flow. These unique structural specifics of the fetal endothelium appear important to the ability for maintain elevated vascular resistance until the time of adaptation of extrauterine life. At birth, these structural adaptations of the endotheliumv dramatically change, especially in the precapillary vessels. From studies in animal models, in less than 5 min, endothelial cells become thinner as the lumen expands; the cell

s­ urface-to-volume ratio increases, and cell-to-cell overlap decreases. The endothelial replication rate is also reduced, suggesting that cell spreading accounts for increases in endothelial surface area. Historically, these postnatal changes have been attributed to passive reactions secondary to relaxation by neighboring VSMCs. However, recent studies have indicated that the transition of hypoxia to normoxia may ­trigger active events of cytoskeletal actin remodeling and changes in cell–cell junctional composition. Although these cellular processes certainly affect other aspects of endothelial function, such as control of cellular proliferation, migration, and permeability, they also appear to augment the acute changes in vascular resistance. Interrogation of the molecular pathways that lead to these structural changes is an active area of ongoing research; however, these studies have been challenging owing to the absence of easily tractable animal models or cell culture models that accurately recapitulate this complex physiological event. Reflecting their structural adaptation, pulmonary vascular endothelial cells tightly regulate the balance of vasoactive factors that favor either vasoconstriction during gestation or vasodilation at birth (as depicted in Fig. 4). During fetal life, ET-1 contributes most predominantly to high PVR. The release of ET-1 is stimulated by hypoxia, especially relevant in the intrauterine environment [204]. To a smaller degree, fetal levels of AT-II may contribute to increases in production of ET-1 [205]. In the porcine fetal lung, ETA and ETB receptors are expressed in pulmonary VSMCs to allow for intracellular calcium influx and intense vasoconstriction [206]. Correspondingly, the level of circulating ET-1 is high in the normal-term human fetus, and specifically is elevated in lung parenchyma and pulmonary arteries at birth (as observed in porcine models) [207]. At birth, the balance favoring vasoconstriction shifts to that of vasodilation. Historical studies from the 1960s by Dawes [208] implicated several physiological stimuli in this transition to a low PVR, including drainage of fetal lung

Fig.  4  Perinatal changes in pulmonary vascular tone critically depend upon endothelial function to shift the net balance of vasoactive factors. During fetal life, the pulmonary endothelium is exposed to a variety of humoral and environmental stimuli, leading to high levels of expression of endothelin-1 (ET-1) to induce intense vasoconstriction via activation of

ETA and ETB receptors on VSMCs. At the time of birth, in response to distinct exogenous stimuli, the pulmonary endothelium expresses increased levels of ETB receptors and is stimulated to produce NO and prostacyclin (PGI2), leading to rapid vasodilation. AT-II angiotensin-II, VEGF vascular endothelial growth factor. (Modified and reprinted with permission [224])

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fluid, rhythmic distension of the lung, and an increase in O2 tension. Since those initial studies, the overarching mechanism for this response is still unclear: no single factor has yet been identified that is responsible for the initiation of vasodilation and the nature and cellular site of the O2 sensor is unknown. Currently, it is hypothesized that onset of breathing is associated with an increase in pulmonary blood flow, leading to increased shear stress. This mechanical stimulation then may result in an increase in the level of bradykinin and the release of PGI2 and NO from the endothelium to overwhelm the vasoconstrictive effects of ET-1 and lead to a fall in PVR [209]. The in vivo evidence for the role of these factors is varied. In the case of NO, eNOS protein expression and enzyme activity increases in late gestation [206], and the peak of pulmonary eNOS expression in the fetal lamb precedes the development of a physiological vasodilatory response to acetylcholine or increased O2 levels [210]. Correspondingly, pulmonary sGC is abundantly expressed in VSMCs of pulmonary resistance vessels of fetal and perinatal animals, with peak activity in the perinatal period, suggesting a role in response to NO in the acute drop of PVR at birth [211]. Notably, eNOS-null mice are still viable and have normal litter sizes, leading some investigators to conclude that the NO pathway is not essential for initial vasodilation at birth; however, these eNOS-null pups are not entirely normal, as they are frequently cyanotic at birth with a high rate of neonatal loss (40% within 1 h of birth) [212]. Defects in pulmonary angiogenesis have been noted in the setting of both eNOS-null mice and those exposed to the NOS inhibitor l-NAME. These morphologic abnormalities have been compared to those seen in patients with the syndrome of alveolar capillary dysplasia, as seen in human infants with PPHN [213]. Finally, the importance of the NO pathway has been implicated in this transition by study of the failure to adapt properly to extrauterine life, such as in hypoxia-induced neonatal pulmonary hypertension. In animal models of chronic perinatal hypoxic exposure, isolated pulmonary arteries fail to establish normal relaxation in response to acetylcholine [96]. Furthermore, although still abundant, eNOS protein expression is reduced, and the activity of calcium-dependent and calcium-independent eNOS does not increase at birth [206]. Chronic hypoxia also impairs endothelial calcium metabolism and normal coupling between eNOS and CAV-1, leading to further eNOS sequestration and inhibition [96]. Furthermore, expression of DDAH II is reduced, leading to increased levels of the endogenous inhibitor of eNOS, ADMA [214]. Elevated levels of ADMA have been detected in the urine of neonates with pulmonary hypertension [215]. Finally, neonatal pulmonary hypertension appears to be marked by inhibition of l-arginine transport and subsequent inhibition of NO production [216]. In newborn lambs suffering

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from pulmonary hypertension secondary to surgically-created systemic-to-pulmonary shunts and increased shear stress, circulating l-arginine levels are reduced [217]. In addition, in newborn lambs exposed to hypoxia or infusion of the thromboxane analogue U46619, an increase in pulmonary pressure has been attenuated by pretreatment with l-arginine [218]. As a result, although an intact NO pathway alone does not appear to be necessary for vasodilation at birth, its appropriate upregulation still appears to contribute significantly to this complex physiological function. In addition to the NO pathway, the actions of AA metabolites, such as PGI2, may figure strongly in the perinatal transition of the pulmonary vasculature to a vasodilated state. In early fetal life, the levels of PGI2 synthase and PGI2 are low, potentially owing to the inhibitory action of plasma glucocorticoids on endothelial COX-1 gene transcription and expression [219]. PGI2 synthase and PGI2 levels subsequently increase at birth [220], with significantly higher levels of PGI2 synthesis in newborn rather than fetal pulmonary vessels [221]. Historically, this transition has been attributed to a combination of PGI2 release by bradykinin in response to mechanical distention of the lung and of PGI2 release by increased shear stress by resulting (pulsatile) pulmonary blood flow. In vitro studies on ovine fetal pulmonary arterial endothelial cells have also demonstrated that physiological levels of estradiol-17 induce COX-1 gene expression [222] and subsequently PGI2 synthesis [223]. In correlation, fetal plasma estrogen levels increase at birth, as observed in sheep, guinea pigs, and humans, suggesting a regulatory role for estrogen in this process. Similar to the case with NO, it is unclear if PGI2 itself is necessary alone for perinatal vasodilation. Recent hypotheses exist that suggest that NO and PGI2 likely act coordinately on the pulmonary vasculature, necessitating the presence of both factors to augment downstream signaling pathways of vasodilation [224]. Nonetheless, the significance of PGI2 in this transition is highlighted by its reduced expression in newborns with pulmonary hypertension. In addition, historical studies in the 1970s and 1980s reported that chronic COX inhibition in utero results in the anatomic and physiological feature of PPHN [225], therefore implicating AA metabolites and, perhaps, PGI2 in particular in modulating the proper transition to a vasodilated state. These data are further corroborated by more recent evidence of increased levels of LTC4 and LTD4 in neonates with PPHN [226] and increased levels of TxA2 in an ovine model of PPHN [227]. At this time, however, the molecular aspects of the in vivo regulation of AA metabolism in hypoxia or other neonatal pathological contexts have been poorly explored. Finally, in parallel to the modulation of NO and PGI2, transition of the vasoconstrictive activity of ET-1 has also been implicated in regulation of these events. ET-1 levels remain elevated during birth but then decrease at 2 days [207]. Interestingly, during the acute phase of initiation of

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air breathing, ETB receptors are transiently expressed on pulmonary arterial endothelial cells [206, 228]. Binding of ET-1 to these receptors leads to a shift in function from vasoconstriction to vasodilation, as verified in ovine [229] and porcine [206, 228] models. For unclear molecular reasons, this ­specific vasodilatory effect by ET-1 is most prominent in the presence of already existing high vascular tone, such as during childbirth. Conversely, in experimental models of PPHN induced either by hypoxia [207] or fetal ligation of the ductus arteriosus [230] the levels of circulating ET-1 and ETA receptors remain high in the pulmonary vasculature. Elevated ET-1 levels have also been reported in human infants with PPHN [231]. In contrast, expression of endothelial ETB receptors is blunted in hypoxic models. Consequently, isolated pulmonary arteries from hypoxic piglets demonstrate an increase in contractile response to ET-1 and lack of response to ETB antagonists [232]. Taken together, it is clear that endothelial regulation of the balance of vasoactive factors appears crucial for the appropriate physiological transition of pulmonary vascular tone at birth. In addition to NO, AA metabolites, and ET-1, a number of other less understood soluble effectors likely play integral roles in this process in vivo. Key objectives of further investigation include a better mechanistic understanding of the mechanotransduction and hypoxia-mediated signaling pathways that lead to modulation of these vasoactive factors; in addition, a more sophisticated characterization of the intersection and possible coordinate regulation of signaling pathways downstream of these vasoactive factors is still lacking.

4.2 Endothelium-Specific Changes in Pulmonary Arterial Hypertension Similar to its importance in regulating perinatal transition of vascular tone, the pulmonary endothelium also plays a critical role in the development of pathophysiological conditions. PAH is a prototypical and complex pulmonary vascular disease state that causes significant morbidity and mortality and is clinically characterized by an increase in PVR. The histopathological changes are marked by vascular proliferation/ fibrosis, remodeling, and vessel obstruction. Development of PAH involves the complex interaction of multiple vascular effectors, many of which are endothelial-cell-dependent (as depicted in Fig.  5). Subsequent vasoconstriction, thrombosis, and inflammation ensue, leading to vessel wall remodeling and cellular hyperproliferation as the hallmarks of severe disease. These processes are influenced by genetic predisposition as well as diverse endogenous and exogenous stimuli. Despite these complexities, the overall pattern of dysregulation centers upon an imbalance of the aforementioned vascu-

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lar effectors controlling vasodilation and vasoconstriction (as summarized in Table 2). Alteration of the endogenous production of NO has been linked to the progression of PAH. Reduced levels of eNOS have been demonstrated in the pulmonary vasculature of patients with PAH, suggesting a mechanism of dysregulated vasoconstriction [233, 234]. Correspondingly, a murine model that genetically lacks eNOS is more susceptible to developing pulmonary hypertension in response to other endogenous stimuli, as compared with the wild-type control [94]. Furthermore, the impact of NO has been reflected in its therapeutic role in PAH, as inhaled NO (as reviewed in [235]) and the NO-dependent PDE5 inhibitor sildenafil (and as reviewed in [236]). Nonetheless, the exact mechanisms of eNOS and NO dysregulation in PAH have not been fully elucidated. First, increased levels of eNOS have been described specifically in plexiform lesions in idiopathic PAH [237]. Increased NO levels may provoke endothelial cell proliferation in these lesions; however, this link has not been verified. Second, dysregulation of NO depends not only upon NOS activity but also upon still incompletely characterized processes of NO transport in blood [238]. Third, NO levels are intricately associated with oxidative stress, another likely but incompletely described regulator of pulmonary hypertension [239]. Finally, specific polymorphisms of NOS have been associated with pulmonary hypertension [240, 241]; ­however, mechanistic details are lacking. Again, further characterization of the dysregulated action of NO and potential interactions with other pathogenic factors may depend upon the development of a genetically tractable animal model of severe PAH. PGI2 and TxA2 also play crucial roles in vasoconstriction, thrombosis, and, to a certain degree, vessel wall proliferation. Protein levels of PGI2 synthase are decreased in small and medium-sized pulmonary arteries in patients with PAH, particularly in those with the idiopathic form [242]. Biochemical analysis of urine in patients with PAH has shown decreased levels of a breakdown product of PGI2 (6-keto-prostacyclin F2a), accompanied by increased levels of a metabolite of TxA2 (thromboxane B2) [243]. Therefore, it appears that production of these effectors is coordinately regulated, with the imbalance toward TxA2 favored in the development of PAH. The mode of this regulation remains to be characterized, but it may rely upon the enhancement of the free-radical environment in the vasculature. Nonetheless, recognition of this imbalance has led to the success of PGI2 therapy and improvement of hemodynamics, clinical status, and survival in patients with severe PAH (as reviewed in [112]). Dysregulated leukotriene production by 5-LO may also affect the severely vasoconstricted state of PAH. As noted already, 5-LO regulates the synthesis of leukotrienes, which, in turn, can promote cytokine release. 5-LO may represent a

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Fig. 5  Pulmonary arterial hypertension involves the coordinated action of multiple endothelium-derived vascular effectors. The histological progression of the pulmonary vasculature from quiescence to pathogenic activation and vasoconstriction in PAH in large part involves the dysregulation of endothelial function. Initial injury to the endothelium may initiate pathogenic signaling pathways. These pathways activate an imbalance of secreted vascular mediators that drive the vascular responses of vasoconstriction, proliferation, thrombosis, and dysregulation of apoptosis, leading to the formation of a layer of “neointima.”

Table 2  Dysregulation of vascular effectors in pulmonary arterial hypertension (PAH) Vascular effector Nitric oxide Thromboxane A2 Prostacyclin Endothelin-1 Vasoactive intestinal peptide Rho kinase 5-Lipoxygenase

Change in activity in PAH

Effect on vasoconstriction

Decreased (increased in plexiform lesions) Increased Decreased Increased Decreased

Increased

Increased Increased

Increased Increased

Increased Increased Increased Increased

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Pathological phenotypes that may influence disease progression include vessel inflammation, transdifferentiation of endothelial cells to VSMCs and transdifferentiation of fibroblasts and VSMCs to myofibroblasts. Engraftment and differentiation of vascular progenitor cells may contribute as well. (Modified and reprinted with permission from Elsevier [104]. The micrographs of pulmonary arteries are courtesy of http://www.scleroderma.org and the New England Journal of Medicine, copyright 2004, Massachusetts Medical Society. All rights reserved [253])

possible upstream factor involved in inciting the end-stage inflammatory state that predominates end-stage PAH [244]. 5-LO promotes endothelial cell proliferation in cell culture, and elevated levels of 5-LO have been detected in macrophages and pulmonary endothelium derived from patients suffering from idiopathic PAH [245]. In both a monocrotaline-treated rat model [246] and a genetically susceptible bone morphogenetic protein receptor 2 (BMPR2) +/− mouse model [247] of PAH, overexpression of 5-LO via adenoviral delivery worsened pulmonary arterial pressures and vascular remodeling. Furthermore, 5-LO inhibitors have attenuated pulmonary hypertension in both of these models. It is tempting to speculate that 5-LO may possess an upstream regulatory role in PAH progression and may be intricately regulated with the production of other vasoactive AA metabolites, such

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as PGI2 and TxA2. Yet, it is also possible that 5-LO and its enzymatic products and derivatives may merely reflect the severity of the end-stage inflammatory state. Dysregulation of ET-1 has also been implicated in PAH. Plasma levels of ET-1 are increased in animal and human subjects suffering from PAH owing to a variety of causes [231, 248]. Improvement in hemodynamics, clinical status, and survival of PAH patients treated with chronic ET receptor antagonists highlights the significance of these effects (as reviewed in [249]). The complete upstream pathogenic mechanisms that result in elevated levels of ET-1 and dysregulated signaling have not been fully clarified. Furthermore, it remains to be seen if selective ETA receptor antagonists allow for improved vasomotor relaxation, as this is predicted to preserve induction of vasorelaxation by the ETB receptor. Thus, in parallel to the tightly regulated physiological process of vasodilation at birth, a dysregulated balance of these endothelium-derived factors appears to be active in the progression of different forms of PAH. Yet, although our understanding of the specific action of each individual effector has matured, the in vivo vascular responses to their coordinate action remain unclear. In fact, none of these factors have yet been definitively linked to the root pathogenesis of disease. Insight into this topic is offered by the fact that the above-mentioned effectors are likely subject to upstream, overarching regulatory pathways that affect the action of multiple vasoactive molecules [250]. Some examples of potential overarching and overlapping regulatory pathways may include those that function through Rho kinase, voltageactivated potassium channels, and caveolae, among others. Characterization of these regulatory mechanisms is ongoing and may eventually allow for identification of primary molecular triggers of disease and offer novel therapeutic targets for drug development.

5 Conclusion and Future Directions Multiple cell types in the pulmonary vasculature and pulmonary circulation contribute to the control of vasomotor tone. Of these cell types, the endothelium plays a predominant and critical regulatory role in response to neural, humoral, mechanical, and hypoxic stimuli, leading to modulation of a complex array of vasoactive effectors and subsequent activation of distinct and overlapping signaling pathways in the vascular wall. Since the discovery of NO as the first endothelium-specific effector nearly 30 years ago, a more sophisticated understanding has emerged, focusing on the regulation of each individual vasomotor effector in health and disease in response to primary endothelial stimuli. Recently, some unifying mechanisms of disease have become more apparent, linking the coordinate regulation of these effectors into a

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more cohesive model. However, the understanding of these complex cellular processes at the molecular level is incomplete. In part, this stems from the lack of a small animal model with pulmonary vasculature that can be precisely observed and genetically manipulated. This is especially evident in studies of the perinatal changes of pulmonary vascular tone, given the challenges of specific physiological and molecular interrogation of the fetal and neonatal pulmonary systems. Recent advances have hinted at the possibility of more tractable animal models of pulmonary vascular disease, which would be useful for combining genetic study and physiological and pathophysiological exposures. Furthermore, although our attention has centered on these discrete soluble factors for control of pulmonary ­vascular tone, it is becoming increasingly clear that regulation of the differentiation of the pulmonary endothelium itself also contributes to vascular tone. In particular, it has been hypothesized that processes of differentiation and dedifferentiation of endothelial cells and their progenitors may also impact pulmonary vascular tone both by altering secretion of vasoactive factors and by altering the structure and anatomy of the vessel wall itself. Especially in the setting of vascular injury such as PAH or even during fetal development when vascular tone is high, altered regulation of circulating or resident progenitor cells and/or transdifferentiation of endothelial cells to VSMCs may contribute [251, 252] to increased vasoconstriction (Fig. 5). As a result, a number of exciting hypotheses have emerged recently, focusing on endothelial lineage commitment as a novel process for ­ultimately regulating vasomotor tone. Direct evidence in favor of these hypotheses is currently lacking. Finally, our continued incomplete understanding of the regulation of vasomotor tone in general may also stem from the limitations of utilizing a reductionist approach to define a complex and dynamic in vivo process. In the postgenomic era, it is conceivable that complex physiological processes and complex pathological conditions such as PAH will become redefined, on the basis of growing genomic, transcriptional, and proteomic data. A “systems-based” network analysis can be envisioned where specific genetic determinants (such as eNOS and COX-1) may interact with environmental or acquired perturbations (such as hypoxia and shear stress) and “stress response” modifying genes (such as those that modulate angiogenesis or inflammation) to lead to one of several phenotypes (such as vasodilation and vasoconstriction) or pathophenotypes (such as PAH and right ventricular failure) [250]. With the aid of the genomic database, this resulting human disease network would offer several unique opportunities: to provide a mechanistic rather than an observational description of pulmonary vascular tone, to predict risk factors for related pathological states such as PAH, and to identify and quantitate regulatory determinants that otherwise would be ignored. By combining a network-based approach with more tractable animal models

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and innovative ideas regarding the developmental aspects of the endothelium itself, the results would certainly improve our ability to identify common, overarching pathways of regulation and aid in our understanding of the mechanistic links among primary disease triggers, vasoactive effectors, and end-stage phenotype. Such information would be valuable not only for improvement of our understanding of pulmonary vascular tone as a fundamental biological process, but also, perhaps, for identification of therapeutic targets useful for regression of the related pathogenic processes themselves. Acknowledgements  This work was supported in part by NIH grants HL61795, HV28718, and HL81587. We thank Stephanie Tribuna for expert secretarial assistance.

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11  Endothelial Regulation of Pulmonary Vascular Tone 223. Sherman TS, Chambliss KL, Gibson LL, Pace MC, Mendelsohn ME, Pfister SL, Shaul PW (2002) Estrogen acutely activates ­prostacyclin synthesis in ovine fetal pulmonary artery endothelium. Am J Respir Cell Mol Biol 26:610–616 224. Wojciak-Stothard B, Haworth SG (2006) Perinatal changes in pulmonary vascular endothelial function. Pharmacol Ther 109:78–91 225. Levin DL (1980) Effects of inhibition of prostaglandin synthesis on fetal development, oxygenation, and the fetal circulation. Semin Perinatol 4:35–44 226. Stenmark K, James S, Voelkel N, Toews W, Reeves J, Murphy R (1983) Leukotriene C4 and D4 in neonates with hypoxemia and pulmonary hypertension. N Engl J Med 309:77–80 227. Abman SH, Stenmark KR (1992) Changes in lung eicosanoid ­content during normal and abnormal transition in perinatal lambs. Am J Physiol 262:L214–L222 228. Hislop AA, Zhao YD, Springall DR, Polak JM, Haworth SG (1995) Postnatal changes in endothelin-1 binding in porcine ­pulmonary vessels and airways. Am J Respir Cell Mol Biol 12: 557–566 229. Ivy DD, Lee DS, Rairigh RL, Parker TA, Abman SH (2004) Endothelin B receptor blockade attenuates pulmonary vasodilation in oxygen-ventilated fetal lambs. Biol Neonate 86:155–159 230. Black SM, Johengen MJ, Soifer SJ (1998) Coordinated regulation of genes of the nitric oxide and endothelin pathways during the development of pulmonary hypertension in fetal lambs. Pediatr Res 44:821–830 231. Allen S, Chatfield B, Koppenhafer S, Schaffer M, Wolfe R, Abman S (1993) Circulating immunoreactive endothelin-1 in children with pulmonary hypertension. Association with acute hypoxic pulmonary vasoreactivity. Am Rev Respir Dis 148:519–522 232. Schindler M, Hatch D, Hislop A, Haworth S (1997) Enhanced constriction of bronchi and pulmonary arteries to endothelin in pulmonary hypertension. Circulation 96:427 233. McQuillan L, Leung G, Marsden P, Kostyk S, Kourembanas S (1994) Hypoxia inhibits expression of eNOS via transcriptional and posttranscriptional mechanisms. Am J Physiol 267:H1921–H1927 234. Giaid A, Saleh D (1995) Reduced expression of endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension. N Engl J Med 333:214–221 235. Griffiths M, Evans T (2005) Inhaled nitric oxide therapy in adults. N Engl J Med 353:2683–2695 236. Barnett C, Machado R (2006) Sildenafil in the treatment of ­pulmonary hypertension. Vasc Health Risk Manag 2:411–422 237. Mason N, Springall D, Burke M, Pollock J, Mikhail G, Yacoub M, Polak J (1998) High expression of endothelial nitric oxide synthase in plexiform lesions of pulmonary hypertension. J Pathol 185:313–318 238. Gladwin M, Schechter A (2004) NO contest: nitrite versus S-nitroso-hemoglobin. Circ Res 94:851–855 239. Bowers R, Cool C, Murphy R, Tuder R, Hopken M, Flores S, Voelkel N (2004) Oxidative stress in severe pulmonary hypertension. Am J Respir Crit Care Med 169:764–769 240. Yildiz P, Oflaz H, Cine N, Erginel-Unaltuna N, Erzengin F, Yilmaz V (2003) Gene polymorphisms of endothelial nitric oxide synthase

195 enzyme associated with pulmonary hypertension in patients with COPD. Respir Med 97:1282–1288 241. Kawaguchi Y, Tochimoto A, Hara M, Kawamoto M, Sugiura T, Katsumata Y, Okada J, Kondo H, Okubo M, Kamatani N (2006) NOS2 polymorphisms associated with the susceptibility to pulmonary arterial hypertension with systemic sclerosis: contribution to the transcriptional activity. Arthritis Res Ther 8:R104 242. Tuder R, Cool C, Geraci M, Wang J, Abman S, Wright L, Badesch D, Voelkel N (1999) Prostacyclin synthase expression is decreased in lungs from patients with severe pulmonary hypertension. Am J Respir Crit Care Med 159:1925–1932 243. Christman B, McPherson C, Newman J, King G, Bernard G, Groves B, Loyd J (1992) An imbalance between the excretion of thromboxane and prostacyclin metabolites in pulmonary hypertension. N Engl J Med 327:70–75 244. Voelkel N, Tuder R, Wade K, Höper M, Lepley R, Goulet J, Koller B, Fitzpatrick F (1996) Inhibition of 5-lipoxygenase-activating protein (FLAP) reduces pulmonary vascular reactivity and pulmonary hypertension in hypoxic rats. J Clin Invest 97:2491–2498 245. Wright L, Tuder R, Wang J, Cool C, Lepley R, Voelkel N (1998) 5-Lipoxygenase and 5-lipoxygenase activating protein (FLAP) immunoreactivity in lungs from patients with primary pulmonary hypertension. Am J Respir Crit Care Med 157:219–229 246. Jones J, Walker J, Song Y, Weiss N, Cardoso W, Tuder R, Loscalzo J, Zhang Y (2004) Effect of 5-lipoxygenase on the development of pulmonary hypertension in rats. Am J Physiol Heart Circ Physiol 286:H1775–H1784 247. Song Y, Jones J, Beppu H, Keaney JJ, Loscalzo J, Zhang Y (2005) Increased susceptibility to pulmonary hypertension in heterozygous BMPR2-mutant mice. Circulation 112:553–562 248. Giaid A, Yanagisawa M, Langleben D, Michel R, Levy R, Shennib H, Kimura S, Masaki T, Duguid W, Stewart D (1993) Expression of endothelin-1 in the lungs of patients with pulmonary hypertension. N Engl J Med 328:1732–1739 249. Langleben D (2007) Endothelin receptor antagonists in the ­treatment of pulmonary arterial hypertension. Clin Chest Med 28:117–125, viii 250. Loscalzo J, Kohane I, Barabasi A (2007) Human disease classification in the postgenomic era: a complex systems approach to human pathobiology. Mol Syst Biol 3:124 251. Zhu P, Huang L, Ge X, Yan F, Wu R, Ao Q (2006) Transdifferentiation of pulmonary arteriolar endothelial cells into smooth muscle-like cells regulated by myocardin involved in hypoxia-induced pulmonary vascular remodelling. Int J Exp Pathol 87:463–474 252. Sakao S, Taraseviciene-Stewart L, Cool C, Tada Y, Kasahara Y, Kurosu K, Tanabe N, Takiguchi Y, Tatsumi K, Kuriyama T, Voelkel N (2007) VEGF-R blockade causes endothelial cell apoptosis, expansion of surviving CD34+ precursor cells and transdifferentiation to smooth muscle-like and neuronal-like cells. FASEB J 21:3640–3652 253. Humbert M, Sitbon O, Simonneau G (2004) Treatment of ­pulmonary arterial hypertension. N Engl J Med 351(14): 1425–1436

Chapter 12

Acute Lung Injury: The Injured Lung Endothelium, Therapeutic Strategies for Barrier Protection, and Vascular Biomarkers Eddie T. Chiang, Ting Wang, and Joe G.N. Garcia

Abstract  The vascular endothelium can be considered as an organ/tissue which comprises a monolayer of endothelial cells which serve as a semipermeable cellular barrier separating the inner space of blood vessels from its surrounding tissue and to control the exchange of fluids and cells between the two compartments. Since the pulmonary circulation receives the entire cardiac output, the large surface area of the lung microvasculature is well suited for sensing mechanical, chemical, and cellular injury by inhaled or circulating substances. This endothelial barrier is dynamically regulated through exposure to these various stimuli of physiological and pathological origin and serves to regulate multiple key biological processes (including lung fluid balance and solute transport between vascular compartments). For example, an increase in vascular permeability is a necessary feature of the body’s defense mechanism to provide injured tissues with access to leucocytes, resulting in tissue edema due to fluid extravasation. However, during conditions of intense lung inflammation such as observed in acute lung injury or its severer form of acute respiratory distress syndrome, the large surface area becomes a liability and provides the opportunity for profound vascular permeability resulting in massive fluid accumulation in the alveolar space and progressively leading to pulmonary failure. Alterations in vascular permeability occur not only in acute inflammatory lung disorders primarily caused by sepsis, pneumonia, and trauma which result in high rates of patient morbidity and mortality, but are an attractive target for therapeutic intervention in subacute lung inflammatory disorders such as ischemia–reperfusion injury, radiation lung injury, and asthma. Thus, understanding the mechanisms of endothelial barrier dysfunction is vital for the management and treatment of key and enigmatic pulmonary disorders.

Keywords  Lung vascular permeability • Barrier function and dysfunction • Endothelium • Epithelium • Acute lung injury • Respiratory distress syndrome

J.G.N. Garcia (*) Department of Medicine, University of Illinois at Chicago, Chicago, IL 60612, USA e-mail: [email protected]

1 Introduction 1.1 Overview of Lung Endothelial Cell Barrier Regulation in Inflammatory Lung Injury The vascular endothelium can be considered as an organ/ tissue which comprises a monolayer of endothelial cells (ECs) which serve as a semipermeable cellular barrier separating the inner space of blood vessels from its surrounding tissue and to control the exchange of fluids and cells between the two compartments. Since the pulmonary circulation receives the entire cardiac output, the large surface area of the lung microvasculature is well suited for sensing mechanical, chemical, and cellular injury by inhaled or circulating substances. This endothelial barrier is dynamically regulated through exposure to these various stimuli of physiological and pathological origin and serves to regulate multiple key biological processes (including lung fluid balance and solute transport between vascular compartments). For example, an increase in vascular permeability is a necessary feature of the body’s defense mechanism to provide injured tissues with access to leucocytes, resulting in tissue edema due to fluid extravasation. However, during conditions of intense lung inflammation such as observed in acute lung injury (ALI) or its severer form of acute respiratory distress syndrome (ARDS), the large surface area becomes a liability and provides the opportunity for profound vascular permeability resulting in massive fluid accumulation in the alveolar space and progressively leading to pulmonary failure. Alterations in vascular permeability occur not only in acute inflammatory lung disorders primarily caused by sepsis, pneumonia, and trauma which result in high rates of patient morbidity and mortality [1, 2], but are an attractive target for therapeutic intervention in subacute lung inflammatory disorders such as ischemia–reperfusion injury [3], radiation lung injury [4, 5], and asthma [6, 7]. Thus, understanding the mechanisms of endothelial barrier dysfunction is vital for the management and treatment of key and enigmatic pulmonary disorders.

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_12, © Springer Science+Business Media, LLC 2011

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1.2 Transcellular Versus Paracellular Permeability A key concept of the dynamically regulated lung EC barrier is the notion that two general pathways, transcellular and paracellular, that describe the movement and flow of fluid, macromolecules, and leukocytes into the interstitium (and subsequently the alveolar air spaces) produce clinically significant pulmonary edema during inflammatory lung processes (Fig. 1). The transcellular pathway utilizes a tyrosine kinase dependent, gp60-mediated transcytotic albumin route, an active process of albumin transport in which endocytic vessels fuse with the endothelium in response to surface glycoprotein (gp60) receptor ligation [8]. However, there is general consensus that the primary mode of fluid and transendothelial leukocyte trafficking occurs by the paracellular pathway as shown by the elegant electron microscopy studies of Majno and Palade [9, 10], who demonstrated lung EC rounding and paracellular gap formation at sites of active inflammation within the lung vasculature.

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Disruption of the integrity of the EC monolayer is now recognized as a cardinal feature of inflammation, ischemia– reperfusion injury, and angiogenesis and occurs in response to a variety of mechanical stress factors, inflammatory mediators, and activated neutrophil products [reactive oxygen species (ROS), proteases, cationic peptides]. The dramatic cell shape change which results in paracellular gap formation implicates the direct involvement of endothelial structural components composed of cytoskeletal proteins (microfilaments and microtubules). Thus, although once perceived as a passive cellular barrier, ECs are now recognized as a highly dynamic tissue contributing to the multiple dimensions of EC function, including interactions with a number of barrier-regulatory effectors via the endothelial cytoskeleton. The duration and outcome of inflammatory disease processes depends upon the balance between the severity of endothelial injury caused by adhesive biophysical forces, mechanical shear stress (SS), or receptor ligation by specific inflammatory mediators and the efficiency of endogenous repair mechanisms to restore vascular integrity [1, 2]. In this chapter, we will (1) address the role of cytoskeletal rearrangement in mechanistic regulation of pulmonary vascular barrier function and permeability, (2) define current strategies designed to enhance the integrity of the lung vascular endothelium, and (3) identify vascular biomarkers and potential prognostic determinants of acute inflammation.

2 Role of the Cytoskeleton 2.1 Endothelial Cell Cytoskeleton Components: Overview

Fig. 1  Paracellular and transcellular routes of transport. The paracellular pathway involves diffusive transport of molecules or fluid across the vascular barrier between the cell–cell junctions of adjacent endothelial cells. The diagram illustrates polar organization of tight and adherens junctions of endothelial cells and interactions of linking proteins that form junctions. Cell–cell connections include tight junctions composed of transmembrane occluding proteins linked to the actin cytoskeleton by the zona occludens family (ZO-1) and adherens junctions mediated by Ca2+-dependent association of vascular endothelial cadherin proteins linked to the a-catenin, b-catenin, and g-catenin complex which are linked to the actin cytoskeleton. Transcellular pathway involves vesicular (nondiffusive) transport. The diagram illustrates vesicular transport of albumin by either solid-phase or fluid-phase pathways. The endothelial cell surface expresses albumin binding proteins that bind albumin (solid circles). Formation of vesicles contains albumin bound to albumin binding proteins and those free in cytosol. The vesicle membrane fuses with the abluminal cell membrane, and albumin bound to binding protein and free albumin are extruded to the abluminal side

It is now well accepted that dynamic cytoskeletal elements, actin, microtubules, and intermediate filaments (IFs), are key elements of vascular barrier regulation. The vast majority of the studies contributing to this recognition have focused on agonist-mediated signaling to the actomyosin cytoskeleton with subsequent effects on lung vascular barrier-regulatory properties. Historically viewed as separate and distinct cytoskeletal systems, microtubules and actin filaments are now known to interact functionally during dynamic cellular processes. The microtubule scaffolding complex [11, 12], with a central role of tubulin dynamics, actively contributes to cytoskeletal rearrangement and in transducing competing barrier-regulatory forces, often in close collaboration with microfilament elements. Much less is known about IFs, an enigmatic component of the EC cytoskeleton consisting of dimer structured a-helical proteins which combine to form fibrils. IF proteins are expressed in a specific manner, with

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vimentin the primary IF protein found in ECs. The role of IFs in regulating EC barriers represents a fertile area for future investigations as only limited information is available [13, 14]. Nevertheless, cytoskeletal constituents together provide the capacity for dynamic regulation of cell shape and, as a consequence, of moment-to-moment adaptation to an ever-changing vascular environment.

2.2 Actin Microfilaments and Myosin Actin, a globular protein with a centrally located ATP-binding site, is critical to many cellular processes, including cell motility, cell division, cell signaling, and as we and others have shown, EC permeability [15–17]. G-actin reversibly assembles to form polymerized actin fibers called filamentous actin (F-actin) or actin microfilaments (7-nm diameter), conferring strength to structural elements regulating cell shape, particularly when accompanied by phosphorylated myosin. Dynamic remodeling of actin filaments within peripherally distributed cortical bands is essential for maintenance of endothelial integrity and basal barrier function, with inhibition of actin polymerization (cytochalasin D) directly increasing EC permeability [16]. Edemagenic agents initiate dramatic cytoskeletal rearrangement characterized by the loss of peripheral actin filaments with a concomitant increase in organized actin cables that span the cell, known as “stress fibers.” Critically involved in regulating the spatial locale and level of actin cycling (polymerization–depolymerization) are numerous actin-binding proteins which serve as cross-linking/ bundling proteins, polymerization/depolymerization proteins, and capping/severing proteins. One key actin-binding protein and central regulator of the EC contractile apparatus is the Ca2+/calmodulin-dependent nonmuscle isoform of myosin light chain kinase (nmMLCK). Phosphorylation of the substrate myosin light chain (MLC) by nmMLCK is central to paracellular gap formation and increased permeability by many edemagenic agents, including thrombin [18] and vascular endothelial growth factor (VEGF) [19], both in  vitro and in preclinical models of inflammatory lung injury. Studies with nmMLCK knockout mice have revealed protection from sepsis-induced ALI and our laboratory has shown that nmMLCK knockout mice, as well as mice treated with an inhibitory peptide which reduces MLC kinase (MLCK) activity, are protected against ventilator-induced lung injury (VILI) [20]. In addition, we have shown that genetic variants (single-nucleotide polymorphisms) in MYLK, the gene on chromosome 3q21 encoding MLCK, confer significant susceptibility to sepsis, and sepsisand trauma-induced ALI [21], as well as contributing to risk of severe asthma in African Americans, another inflammatory lung disorder [22]. A key regulatory feature of nmMLCK

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is the posttranslational modification (PTM) by increased levels of nmMLCK tyrosine phosphorylation catalyzed by either p60src kinase or c-abl kinase, or by inhibition of tyrosine phosphatases (vanadate). This PTM serves to increase kinase activity and modulates EC barrier responses [15, 23–25]. Diperoxovanadate, a potent tyrosine phosphatase inhibitor, also increased nmMLCK activity, the number of stress fibers, and EC contraction via activation of p60src kinase. The nmMLCK isoform binds cortactin, another actin-binding protein and EC barrier regulator which localizes to numerous cortical structures within cells [25]. The SH3 domain in cortactin binds the proline-rich areas in nmMLCK [18, 26, 27], with this interaction enhancing cortical actin formation and tensile strength. The central region of cortactin binds and cross-links actin filaments, with its C-terminus site for p60src kinase-mediated phosphorylation which reduces cross-linking activity. Tyrosine phosphorylation of cortactin by p60src potentiates and stabilizes actin polymerization, and strengthens cortactin–nmMLCK interactions [28], and is a key step in a sequence of events that produce cytoskeletal changes, reassembly of adherens junctions (AJs), and barrier restoration during lung inflammation.

2.3 Microtubules Microtubules are 25-nm polymers of a-tubulin and b-tubulin that form a lattice network of rigid hollow rods spanning the cell in a polarized fashion from the nucleus to the periphery while undergoing frequent assembly and disassembly [29, 30]. Important functions of microtubules include intracellular transport of vesicles and organelles, as well as signal transduction and cytoskeletal structure. In addition, microtubules act in concert with the actin cytoskeleton to promote EC barrier integrity. Microtubules and actin filaments exhibit complex, but intimate functional interactions during dynamic cellular processes [29–32]. Microtubule disruption with an agent such as nocodazole or vinblastine induces rapid assembly of actin filaments and focal adhesions, isometric cellular contraction that correlates with the level of MLC phosphorylation, increased permeability across EC monolayers, and increased transendothelial leukocyte migration, events that can be reversed or attenuated by microtubule stabilization with paclitaxel [31, 32]. The mechanisms involved in these effects are poorly understood but are likely to be mediated through interaction with actin filaments, suggesting significant microfilament–microtubule cross talk. Disruption of microtubules causes actin cytoskeletal remodeling, cell contraction, and decreased transendothelial resistance through a Rho kinase induced phosphorylation of MYPT1, a MLC phosphatase [31, 33]. Nocadozole causes formation of stress fibers and myofilament assembly accompanied by increases

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in MLC phosphorylation, remodeling of AJs [34, 35], and barrier disruption [31]. Microtubule stabilization with paclitaxel inhibits the formation of stress fibers and preserves cellular shape and intercellular contacts [32]. Although these effects are poorly understood, microfilament–microtubule cross talk represents an intriguing area of EC barrier regulation [32, 36].

2.4 Intermediate Filaments IFs, the third major element involved in EC cytoskeletal structure, were defined on the basis of their 10–12-nm filament structure which distinguished them from 7-nm microfilament and 25-nm microtubules. Despite greater diversity than the highly conserved components of either actin microfilaments or microtubules, IF proteins share a common dimer structure containing two parallel a-helices which combine to form polar fibrils that associate with an array of IF-binding proteins while connecting to the nuclear envelope, peripheral cell junctions, and other cytoskeletal components. IF proteins are expressed in a highly cell specific manner, with vimentin being the primary IF protein found in ECs and other cells of mesenchymal origin. Although these data suggest potential roles for IFs in EC cytoskeletal structure and barrier function, these effects are likely to be subtle and subject to compensation by biological redundancy and the function of IFs in EC barrier regulation is much less understood [15]. Assembly of IFs is a complex process likely highly regulated by signaling cascades associated with cell motility. Vimentin is a dynamic structure undergoing constant assembly/disassembly, as well as anterograde and retrograde movements. Microtubulebased movement of IFs is likely critical for assembly and maintenance of the vimentin IF network [37, 38]. The physical and dynamic properties of the vimentin network in the vascular EC are likely important in regulation of cell shape and resistance to hemodynamic stress that accompanies blood flow and resistance to shear strain, physiological changes regulated by the IF cytoskeleton, and IF-associated proteins which serve as internal scaffolding for ECs, linked to the plasma membrane, and to junctional contacts. Vimentin protein expression is higher in macrovascular EC lining vessels subjected to the highest hemodynamic strain, such as the aorta, compared with microvascular EC lining vessels under less SS. Vimentin knockout mice develop normally without gross blood vessel abnormalities, but with reduced mesenteric artery vessel dilation in response to flow [37, 38]. Downstream responses to flow may be the result of intracellular mechanosignaling events triggered by deformation of the IF cytoskeleton. Changes in unidirectional laminar flow results in rapid adaptation of the EC vimentin network, with directional displacement within minutes of initial exposure. As noted with microfilaments and microtubules, over a period of hours, cytoskeletal

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filaments align themselves in the direction of flow, with significantly larger change in the vimentin distribution around the nucleus compared with displacement occurring in the cytosol closer to the substrate. These observed spatial changes may be a means of distribution of local shear force transmission throughout the cell and therefore convey cell signaling messages via a mechanosignaling pathway. Thus, vimentin IFs are likely critical for maintaining the structural integrity of ECs under SS, and may also be a conduit for signaling cascades triggered by mechanical force, again an exciting area for future examination.

2.5 Adhesive Protein–Cytoskeleton Linkages Dynamic equilibrium exists between EC contractile forces and the adhesive protein–cytoskeleton linkages with cell– cell and cell–matrix interactions necessary for proper barrier function. A major contributor to the intact cellular barrier is the tight apposition of individual ECs with neighboring cells via intercellular junctions which collectively contribute to basal endothelial barrier function. The two primary types of intercellular contacts between ECs are AJs and tight junctions (TJs), both of which link the EC actin cytoskeletons of neighboring cells to each other while providing mechanical stability and mediating signal transduction [15] (Fig. 1). In addition to cell–cell junction stability, cell–matrix interaction also contributes to stability of the barrier function. Specific components of the focal adhesion complex, i.e., the integrin-based linkage between the extracellular matrix (ECM) and the endothelial cytoskeleton, provide strong tethering of the endothelium to the vessel wall and thus enhanced barrier integrity. AJs are composed of cadherins bound together in a homotypic- and Ca2+-dependent fashion to link adjacent ECs [39]. Cadherins interact through their cytoplasmic tail with the catenin family of intracellular proteins (primarily b-catenin), which in turn provide anchorage to the actin cytoskeleton [40]. The primary adhesive protein present in human endothelial AJs, vascular endothelial cadherin (VE-cadherin) [41], is critical to maintenance of EC barrier integrity as demonstrated by increased vascular permeability induced in mice after infusion of VE-cadherin blocking antibody [42]. Similarly, in cultured ECs, VE-cadherin blocking antibody enhanced neutrophil transendothelial migration while producing reorganization of the actin cytoskeleton [43]. Anchorage of VE-cadherin to the actin cytoskeleton is crucial to maintaining barrier integrity since a cytoplasmic-deleted VE-cadherin which cannot anchor to the actin cytoskeleton still forms cadherin–cadherin binding but results in increased vascular permeability [41]. TJs, or zona occludens, are areas that surround the entire apical perimeter of adjacent cells and are formed by the

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fusion of the outer layers of the plasma membranes. These associations are sufficiently tight as to form a virtually impermeable barrier to fluid [44, 45] and are composed of occludins, claudins, and junctional adhesion molecules coupled to cytoplasmic proteins [46] (Fig.  1). Similar to VE-cadherin at AJs, ECs express a cell-type-specific transmembrane adhesion protein, claudin-5, at TJs. The cytoplasmic components of TJs are linked to the EC actin cytoskeleton by the zona occludens family (ZO-1). TJs are particularly abundant and prominent in the brain microvasculature and epithelial cells, where strict control of permeability is needed. In contrast, most microvascular beds of ECs, particularly leaky lung microvasculature, have less defined TJ structures and more prominent AJ structures. Therefore, AJs and not TJs have historically been considered the primary targets involved in junctional protein dissociation resulting in increased paracellular permeability, but there is growing evidence that TJs may play a larger role in the regulation of paracellular permeability in the lung than previously thought [47]. Finally, focal adhesions are intimately involved in lung EC barrier regulation via signaling between the cytoskeleton to the ECM. Focal adhesions are attachments of ECs to the underlying ECM and are mediated by ECM proteins (i.e., collagen, fibronectin, laminin, etc.), integrins, and cytoplasmic adhesion plaques (containing vinculin, talin, and paxillin) [48, 49]. Integrins couple the ECM to the cytoskeleton and transmit signals from the surrounding environment and play a key role in the formation of cell adhesion complexes which attach to the actin cytoskeleton via the cytoskeletal proteins actin, vinculin, talin, and a-actinin. Focal adhesions, primarily through integrins, form a bridge for bidirectional signal transduction between the actin cytoskeleton and the cell–matrix interface. Disruptions of the integrin–ECM connection can increase EC permeability [50, 51] and integrins modulate EC permeability to SS and inflammatory mediators [52]. Integrin– ECM binding stimulates tyrosine phosphorylation of proteins such as paxillin, cortactin, and focal adhesion kinase (FAK), as well as calcium influx [53, 54]. FAK is the principal kinase which catalyzes the downstream reactions of integrin engagement and focal adhesion assembly [52], with FAK activity regulated by tyrosine phosphorylation mediated by the Src family. Activation of FAK through tyrosine phosphorylation produces cell contraction and increased EC barrier permeability. We previously reported that integrin b4 expression is dramatically upregulated upon challenge with the barrier-protective agent simvastatin [55]. Furthermore, the upregulation of integrin b4 attenuates endotoxin- and ventilator-induced expression of inflammatory cytokines interleukin (IL)-6 and IL-8 [56], suggesting a novel mechanism of modulating endothelial barrier function via integrin b4 and focal adhesion signaling.

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3 Mechanisms of Increased Permeability Mediators of Barrier Dysfunction 3.1 Endothelial AJs/TJs: Dissociation and the Disruption of Vascular Integrity Inflammatory mediators increase vascular permeability by disrupting endothelial junctions and focal adhesion complexes as well as inducing cellular contraction to open paracellular gaps [49, 57–59]. As TJs and AJs are ideally situated in a locale between cell–cell junctions, they logically are key participants in the control of vascular paracellular permeability and monolayer integrity. Recent studies in brain ECs have focused on the importance of claudins in TJ formation and maintenance [60, 61]. Mice with claudin-5 gene knocked out did not have a morphologically altered vascular network or TJ structures, but the claudin-5-deficient pups died within 10 h of birth owing to size-selective loosening of the blood– brain barrier against molecules of less than 800 kDa. It appears that moderate redundancy among the claudin isoforms may allow for the formation of the TJ, but not for the complete function of the TJ. Claudin-3 appears to act in concert with claudin-5 to form the tightly organized strand network, but in claudin-5 mutants, claudin-3 can only maintain the barrier against larger molecules [60], suggesting claudin-3 is a structural barrier, whereas claudin-5 is crucial for the dynamic regulation of TJ permeability. Gene inactivation of claudin-1 and occludin also has no effects on vascular morphology or barrier permeability, suggesting a minor role in TJ function in endothelium as compared with claudin-5 and claudin-3 [62]. The family of junctional adhesion molecules (JAM-A, JAM-B, JAM-C) and EC-selective adhesion molecules (ESAM) are transmembrane glycoproteins that associate with TJ strands but are not part of the strands per se [63]. Inactivation of these genes in mice does not cause any defect in the development of the vascular system in the embryo, but in adult mice these molecules play an important role in modulating leukocyte diapedesis through ECs. JAM-Cs however, is unique in that unlike other junctional proteins, it increases endothelial permeability when expressed at the EC surface, suggesting a role in promoting and/or organizing junction formation [64]. This activity is mediated by VE-cadherin activity and actin organization, as well as by kinases and phosphatases that modulate TJ protein phosphorylation and endothelial permeability. Many of the studies on TJ have been using brain ECs, where the adhesion molecules are prominent. Nevertheless, recent studies on lung ECs have demonstrated that despite the less prominent formation of TJs as compared with AJs, TJs may play a critical role in the endothelial barrier dysfunction associated with exposure to particulate matter from air pollution, which has been shown to induce a gradual and prolonged barrier dysfunction in

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cultured lung endothelium [47, 65]. AJs were found unexpectedly to be unaltered but the TJs, specifically ZO-1, were degraded through a calpain-dependent proteasome pathway, a novel mechanism of lung endothelial barrier regulation. In contrast to TJs, the regulation of lung vascular integrity involving AJs has been well characterized. Although VE-cadherin is present in high concentration in all ECs, different types of vessels appear to modify VE-cadherin expression to complement the vascular barrier function of that particular vessel. Four modes of AJ protein regulation of permeability have been described, all involving VE-cadherin: phosphorylation, internalization, cleavage, and expression. Simultaneous coordination of VE-cadherin phosphorylation and internalization appears to be crucial for a rapid response to an increase in permeability [66], whereas VE-cadherin cleavage and expression are progressive alterations. Edemagenic stimuli induce tyrosine phosphorylation of AJ proteins (VE-cadherin, b-catenin, and p120 catenin), which parallels increases in permeability, with the tyrosine kinase Src implicated in the phosphorylation of AJ proteins as it directly associates with the VE-cadherin/catenin complex, and src gene inactivation or treatment with inhibitors blocks VEGF-induced VE-cadherin phosphorylation [67]. Phosphorylation of VE-cadherin is dependent on kinase activation as well as inhibition of associated phosphatases such as the endothelial-specific phosphatase VE-PTP, which also associates with VE-cadherin, and inactivation of the VE-PTP gene leads to a phenotype comparable to that of VE-cadherin null embryos, suggesting that vessels cannot form correctly if VE-cadherin is constantly phosphorylated [68]. Permeability may also be regulated by VE-cadherin internalization. Typically, p120 catenin binds to VE-cadherin and acts as a plasma membrane retention signal to prevent VE-cadherin internalization; however, upon challenge with barrier-disrupting stimuli, activated Src phosphorylates Vav2, a guanine exchange factor (GEF) for Rac, which then phosphorylates VE-cadherin at Ser665, inducing b-arrestin 2 recruitment and promoting clathrin-dependent VE-cadherin internalization [66]. Angiopoietin 1 induces Src trapping by mDia, reducing its activity at AJs, and thus reducing vascular permeability [69]. The third pathway that may induce vascular permeability is VE-cadherin cleavage. VE-cadherin is particularly susceptible to enzymatic proteolysis, specifically elastase and Adam-10, which are released in high amounts by leukocytes, promoting VE-cadherin cleavage, cell extravasation, and vascular leakage. Lastly, permeability control may also be achieved through VE-cadherin gene expression. The VE-cadherin promoter contains several binding sites for transcription factors, TAL-1, ERG, and hypoxia-inducible factors. Therefore, the interendothelial junction is a key site of regulating vascular permeability, with various stimuli targeting either the TJ or the AJ, or both. Furthermore, there are various combinatory modes of regulating the AJ that promote

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dissociation of adhesion proteins in the cell–cell junction. However, additional factors often accompany junctional dissociation, such as disbanding of cortical cytoskeleton and increase in cellular contraction, which augment the barrier dysfunction.

3.2 Regulation of Vascular Permeability by Stress Fiber Formation and Endothelial Cell Contraction The monolayer integrity is regulated by the dynamic equilibrium which exists between contractile forces and tethering forces [18, 70, 71]. Transcellular stress fiber formation and activation of actomyosin interaction, along with the cortical actin ring disassembly, results in contractile tension that induces cell rounding, which contributes to cell–cell gap formation (Fig.  2), with inhibition of this cytoskeletal reorganization attenuating barrier dysfunction [72, 73]. Contraction triggered in ECs is regulated by nmMLCKcatalyzed MLC phosphorylation on Thr18 and Ser19 which increases actomyosin ATPase activity and shifts the equilibrium between the folded and unfolded myosin forms [74], thus providing the assembling and functioning of the contractile apparatus of the cells. The MYLK gene on chromosome 3 in humans encodes three proteins: the nmMLCK isoform, the smooth muscle MLCK isoform (130–150 kDa), and telokin [75–78]. In smooth muscle, nmMLCK is expressed at relatively low level, being present together with a shorter smooth muscle isoform, whereas only nmMLCK can be detected in ECs [78] and exists as a 1,914 amino acid high molecular weight (214-kDa) protein. The nmMLCK shares essentially identical catalytic and CaM regulatory motifs with smooth muscle MLCK, but contains a unique 922 amino acid N-terminal domain comprising potential novel PTM sites [79]. Inflammatory agonists such as VEGF and thrombin produce rapid increases in MLC phosphorylation, reflecting coordinated nmMLCK activity and the small GTPase Rho and its effector, Rho kinase, result in phosphorylation and, thereby, inhibition of the MYPT1 myosin phosphatase, resulting in stabilization and accumulation of phosphorylated MLC. The aggregated result is actomyosin interaction and EC permeability which is significantly attenuated by MLCK or Rho kinase inhibitors [19, 80–82]. Despite the clear contribution of MLCK/Rho kinase driven increases in MLC phosphorylation to tension development and increased vascular permeability, MLCK-independent pathways are also involved in the regulation of cellular contraction. Protein kinase C (PKC)-mediated pathways exert a prominent effect on barrier regulation in a time- and speciesspecific manner without significantly increasing MLC

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Fig.  2  Intracellular signaling pathways evoked by endothelial cell edemagenic agents and contractile agonists, and resulting in paracellular gap formation. In this model, under basal conditions, a balance exists between actomyosin contractile and cellular adhesive forces. When contractile forces predominate, as depicted in the thrombinstimulated model, actin stress fibers form and endothelial cells pull apart to form paracellular gaps, favoring barrier disruption. Thrombin cleavage of the proteinase-activated receptor-1 on the surface of endothelial cells activates both heterotrimeric G proteins (Gq, G12/13) and small GTPases such as Rho. Activated Rho induces Rho kinase, which via phosphorylation of the phosphatase regulatory subunit inhibits the

myosin light chain phosphatase. Rho kinase and myosin light chain kinase (MLCK) activation occurs via independent pathways. Increased level of cytosolic Ca2+ (via inositol trisphosphate production) activates the Ca2+/calmodulin-dependent MLCK, with conformational changes allowing the enzyme to access the preferred substrate myosin light chain. Rho kinase and MLCK activation both culminate in increased myosin light chain phosphorylation, which enables actomyosin contraction, resulting in increased stress fiber formation, cellular contraction, paracellular gap formation, and ultimately endothelial barrier dysfunction. Also depicted are other edemagenic agonists that result in stress-fiberdependent contraction leading to endothelial barrier dysfunction

phosphorylation and without inducing formation of actin stress fibers, but with alterations in other components of the endothelial cytoskeleton [18, 83, 84]. PKC-mediated increases in EC permeability involve phosphorylation of caldesmon, an actin-, myosin-, and calmodulin-binding protein present in smooth muscle actomyosin cross-bridges as a 145-kDa protein and in ECs as a 77-kDa protein [84]. The phosphorylation of caldesmon alters smooth muscle crossbridge activity [85]. Caldesmon-mediated regulation of actomyosin ATPase in smooth muscle is also modified by the actin cross-linking protein filamin and gelsolin [86]. Although filamin participates directly in barrier regulation via CaM kinase II activation [87], its effects on actin cytoskeletal rearrangement are regulated through Rho family GTPases [88, 89], thereby providing another link with a known modulator of EC barrier function. The cytokine tumor necrosis factor a (TNF-a) induces slow-onset barrier disruption in cultured ECs independent of MLCK activity [11]. Finally, p38 kinase activation also has been linked to contractile regulation in smooth muscle [90], EC migration [91, 92], and lipopolysaccharide (LPS)-induced EC permeability [93]. The mechanism through which p38 exerts these effects is unclear but may involve the actin-binding protein hsp27 [94], a known p38 mitogen-activated protein kinase (MAPK) target whose

actin-polymerization-inhibiting activity dramatically decreases after phosphorylation [95, 96] in association with stress fiber development [92, 97].

3.3 Mechanical Stress and Vascular Barrier Function The pulsatile nature of blood pressure and flow exposes blood vessels to constant hemodynamic forces in the form of SS and cyclic stretch (CS). The flow of blood parallel to the vessel surface produces fluid SS from the friction of blood against the vessel wall. In contrast, CS is an important mechanical force generated in the lung circulation either by circulating blood, which results in the rhythmic, pulsatile distension of the arterial wall, or by tidal breathing. The endothelium converts these mechanical stimuli to intracellular signals that effector cellular functions including proliferation, migration, remodeling, apoptosis, and permeability, as well as gene expression. The cytoskeleton is the key structural framework for the ECs to transmit mechanical forces between its luminal, abluminal, and junctional surfaces to its interior, including the cytoplasm, nucleus, and focal adhesion sites. Changes in mechanical stress

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activate multiple sensing mechanisms and signaling networks in ECs, resulting in physiological and pathological functional responses. Blood pressure is the major determinant of vessel stretch, although arterial wall distension normally does not exceed 10–12% stretch. At physiological levels, both SS and CS provide barrier-enhancing and barrier-maintenance stimuli [98, 99]. However, pathological levels of mechanical stress, as induced by mechanical ventilation, may serve as a barrierdisrupting stimulus. Our studies using human ECs stretched at excessive distension of 18% CS demonstrated increased Rho activation and sensitivity to edemagenic agonists, suggesting barrier-disrupting phenotype [99, 100]. Long-term CS increased gene expression and protein content of signaling and contractile proteins including Rho GTPase, MLCs, MLCK, zipper-interacting protein kinase, protease-activated receptor (PAR)-1, caldesmon, and HSP27, suggesting regulation at both the translational and the posttranslational level [99, 101]. Acute lung overdistention caused by mechanical ventilation at high tidal volumes can induce remodeling of ECM constituents such as collagen, elastin, proteoglycans, glycosaminoglycans, and matrix metalloproteinases [102], processes which influence cellular responses to mechanical stress via increased inflammatory cytokine production, macrophage activation, acute inflammation, and barrier dysfunction resulting in pulmonary edema. Reduction in tidal volume causes less ECM disorganization [102] and has improved patient mortality from VILI [103], a topic reviewed in the following.

3.4 Pulmonary Vascular Response to Oxidative Stress Among the organs in the body, the lung exists in a high-oxygen environment and is susceptible to injury by oxidative stress. Cigarette smoking and inhalation of airborne pollutants/toxins/ oxidant gases and particulate matter result in direct lung damage as well as the activation of lung inflammatory responses [104–106]. Long-term exposure of lungs to higher oxygen tension (hyperoxia), as observed with premature infants and critically ill patients on ventilators, causes oxidative stress and lung injury [107]. Thus, increased ROS production has been directly linked to inflammatory lung diseases such as asthma, chronic obstructive pulmonary disease, and ARDS. ROS are essential for normal lung/endothelial function [108], but an imbalance of the redox equilibrium may contribute to pulmonary edema [109, 110]. The imbalance of oxidants produced to oxidants detoxified, i.e., a change in the redox equilibrium appears important in the development of various inflammatory lung diseases, and increased ROS production have been directly linked to oxidation of DNA, proteins, lipids and sugars, remodeling of ECM, alteration of mitochondrial respiration, and apoptosis. Furthermore, increased levels of ROS have been implicated in initiating

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signaling cascades of activation of transcription factors (NF-kB and AP-1), chromatin remodeling, and gene expression of proinflammatory mediators [106, 111]. Also, ROS generated by phagocytes that have been recruited to sites of inflammation and excess generation of ROS by vascular cells are a major cause of edema and lung injury. Generation of ROS and ROS signaling in lung endothelium alter vascular permeability in vivo [112, 113] and in endothelial monolayers [24, 114, 115]. Despite several potential sources of ROS [mitochondrial electron transport chain, cytochrome P-450 enzymes, xanthine oxidase, nitric oxide synthases, myeloperoxidase (MPO) system], the vascular NADPH oxidase family of proteins has been shown to be a major contributor of endothelial ROS in response to hyperoxia [116] since NADPH oxidase mediated superoxide production increases endothelial permeability [117, 118].

3.5 Bioactive Agonists Which Increase Lung Vascular Permeability A variety of agonists, cytokines, growth factors, and mechanical forces alter pulmonary vascular barrier properties and serve to increase vascular permeability [11, 15, 17, 19, 24, 99, 119, 120]. The serine protease thrombin represents an ideal model for the examination of agonist-mediated lung endothelial activation and barrier dysfunction as thrombin evokes numerous EC responses that regulate hemostasis and thrombosis, and is recognized as an important mediator in the pathogenesis of ALI [15]. Thrombin increases EC leakiness to macromolecules by ligating and proteolytically cleaving the extracellular N-terminal domain of the thrombin receptor, a member of the family of PARs [121–123]. The cleaved N-terminus, acting as a tethered ligand, activates the receptor and initiates a number of downstream effects, including cytoskeletal rearrangement (Fig.  2). In vivo studies have detailed events which followed thrombin infusion into the pulmonary artery of the chronically instrumented lung lymph sheep model initiating a cascade of events that culminate in intravascular coagulation, inflammation, and vascular leak [124–126]. Naturally occurring agonists, such as the cytokines TNF-a and IL-1b, have a prominent effect early in ALI, causing microthrombosis, and eliciting a cascade of inflammatory signals which result in capillary endothelial production of P-selectin, an adhesion molecule which enhances leukocyte-EC migration [127–129] and actin reorganization, and paracellular gap formation [130]. TNF-a also increases tyrosine phosphorylation of VE-cadherin, leading to increased paracellular gaps in human lung endothelium [129]. Much less is known about pre-B-cell colony-enhancing factor (PBEF), a relatively unknown cytokine we identified via functional genomic approaches as a novel ALI candidate

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gene [131, 132]. PBEF is also known as visfatin, following its identification as a visceral fat hormone [133], and nicotinamide phosphoribosyltransferase (Nampt), as it serves as the rate-limiting component in the NAD biosynthesis pathway that catalyzes the conversion of nicotinamide and phosphoribosylpyrophosphate into nicotinamide mononucleotide. We demonstrated PBEF as a novel biomarker in sepsis and sepsis-induced ALI with genetic variants conferring ALI susceptibility [131, 132]. Furthermore, PBEF is highly expressed in polymorphonuclear neutrophils (PMNs) of sepsis subjects, with expression upregulated by mechanical force and inflammatory cytokines, and is involved in EC barrier regulation [131, 134, 135]. We explored the mechanistic participation of PBEF in ALI and VILI and demonstrated that recombinant human PBEF is a direct neutrophil chemotactic factor and elicits marked increases in the levels of bronchoalveolar lavage (BAL) PMNs and PMN chemoattractants (KC and MIP-2) after intratracheal injection in mice [136], changes accompanied by modest increases in lung vascular and alveolar permeability. Dramatic increases in BAL PMNs, BAL protein, and cytokine levels (IL-6, TNF-a, KC) were observed in recombinant human PBEF- and VILI-challenged mice [136], whereas heterozygous PBEF+/− mice were significantly protected (reduced BAL protein levels, BAL IL-6 levels, peak inspiratory pressures) when exposed to a model of severe VILI and exhibited significantly reduced expression of VILI-associated gene expression modules. The role of the renin–angiotensin system in pulmonary vascular regulation is now well recognized with angiotensin II, a key component of the renin–angiotensin system, generated primarily by angiotensin-converting enzyme (ACE) from angiotensin I and its effects are mediated through angiotensin type I (AT-1) and angiotensin type II (AT-2) receptors which are expressed in the normal lung. The pulmonary endothelium represents a major site of ACE expression and angiotensin II production, with ACE2, a homologue of ACE, expressed in the lung inactivating angiotensin II, leading to the downstream generation of angiotensin 1-7, which acts through AT-2 receptors to induce vasodilatation. Although components of the renin–angiotensin system have been implicated in a variety of lung diseases, including pulmonary hypertension and fibrotic lung diseases, the system has been strongly linked to the pathophysiology of pulmonary vascular leak syndromes. For example, ACE2 serves as the receptor for the coronavirus, first identified in 2003, responsible for severe acute respiratory syndrome [137, 138], with a mortality rate of more than 50% in the elderly. ACE and AT-2 serve a protective role in ARDS, whereas ACE2, angiotensin II, and AT-1 mediate lung edema and injury associated with ARDS. A role for ACE via angiotensin II and/ or bradykinin in ALI was proposed [139]. Reductions in ACE activity by captopril attenuated the inflammatory response and apoptosis, whereas blocking bradykinin receptors did not attenuate the anti-inflammatory and antiapoptotic effects of

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captopril [140]. Captopril did not attenuate ACE activity or necrosis, indicating that inflammation and apoptosis in VILI is due to ACE-mediated Ang angiotensin II production [141]. New blood vessel formation, or angiogenesis, is defined by the generation of new capillaries by ECs either by sprouting or by splitting from pre-existing vessels. Sprouting angiogenesis involves EC detachment from the basement membrane, migration, and subsequent proliferation, tube formation, and, finally, functional maturation of the new vessel [142]. VEGF is key in vasculogenesis as mice lacking the VEGF receptor Flt-1 fail to develop fully functional blood vessels [143]. Inhibition of VEGF as a promising therapeutic strategy in the management of patients with advanced malignancies [144]. Pulmonary hypertension is a devastating disease with many similarities to neoplastic processes and is characterized by aberrant angiogenesis, with VEGF serving as a target in pulmonary hypertension [145, 146]. VEGF increases EC permeability and was originally named “vascular permeability factor” for its profound effects on vascular barrier function [147]. VEGF levels are highest in the lungs and plasma and VEGF levels are increased in patients with ARDS compared with the other groups [148]. VEGF increases cytosolic calcium levels and levels of MLC phosphorylation at high doses and VEGF inhibition decreases EC permeability [148, 149]. Additional angiogenic factors with barrier-regulatory properties include angiopoietin 1 and angiopoietin 2, which are critical for normal vascular development. The angiopoietin family is compopsed of vascular growth factors which are ligands to the family of tyrosine kinases that are selectively expressed in the vascular endothelium. VEGF induces EC differentiation and migration, whereas angiopoietin 1 stabilizes vascular networks [150–152]. Angiopoietin 1 and angiopoietin 4 modulate EC permeability by altering the state of AJs and specifically inhibit vascular leakage in response to VEGF or other barrier-disruptive agents, as well as promoting vessel maturation. Angiopoietin 2 antagonizes angiopoietin 1 and promotes barrier dysregulation by blocking the ability of angiopoietin 1 to activate its receptor [152].

4 Mechanisms of Increased Barrier Integrity: Therapeutic Strategies Understanding the mechanisms of barrier dysfunction offers the advantages to design therapeutic strategies which target barrier-integrity preservation or reverse established barrier dysfunction by restoring vascular integrity. Prior to the last decade, permeability-reducing strategies primarily consisted of cyclic AMP (cAMP) augmentation, producing only modest barrier enhancement [153–156]. More recently, a number of barrier-promoting agents have been identified which share common signal transduction mechanisms

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which are distinct from cAMP signals and target the endothelial actin cytoskeleton to facilitate barrier-restorative processes. The dynamic process of actin polymerization allows for the rapid reorganization of actin structures, with profound functional consequences for barrier regulation that are highly dependent on the exact spatial location of this actin rearrangement occurring as either barrier-disrupting cytosolic stress fibers or as a barrier-enhancing thickened cortical actin ring. We have demonstrated that the quiescent EC phenotype is characterized by a cortical actin ring and few stress fibers, a structure which favors cell–cell adhesion and cell–matrix tethering. We have conceptualized a paradigm whereby barrier recovery after edemagenic agonists involves development of a cortical actin ring to anchor cellular junctions and a carefully choreographed (but poorly understood) gap-closing process via formation of Rac GTPase-dependent lamellipodial protrusions into the paracellular space between activated ECs (Fig.  3) Within these lamellipodia, signals are transduced to actin-binding proteins (nmMLCK and cortactin) and phosphorylated MLCs in spatial-specific cellular locations. Lamellipodia also require formation of focal adhesions (regulated by the cytoskeleton) critical to the establishment of the linkage of the actin cytoskeleton to target effectors that restore cell–cell adhesion and cell–matrix adhesion. This process is essential to the restoration of endothelial barrier in response to exposure to agonists such as sphingosine 1-phosphate (S1P), hepatocyte growth factor (HGF),

simvastatin, activated protein C (APC), ATP, oxidized phopholipids, and hyaluaron [33, 134, 157–161]. Central to these events is the activation of small GTPases, Rac and cdc42 [162], which follows ligation of barrier-protective receptors and drives cortical actin remodeling and lamellipodia formation (Fig. 3). In addition to lamellipodia, there is increased actin polymerization at the cell periphery (i.e., the cortical actin ring) which occurs with increased force driven by the actin-binding proteins cortactin and nmMLCK, which also translocate to this spatially defined region. Like lamellipodia formation, Rac GTPase-dependent increases in the level of cortical actin follow exposure to multiple barrier-enhancing levels of SS or to potent barrier-enhancing agonists [134, 158, 159], including S1P [157, 158], HGF [33], ATP [159], simvastatin [158], APC [134], prostaglandin E2 [163], and oxidized phospholipid 1-palmitoyl2-arachidonoyl-sn-glycero-3-phosphochlorine (OxPAPC) [160] (Table 1). These observations serve to highlight the importance of the cellular location of cytoskeletal proteins in maintaining or enhancing EC barrier function, with cortactin directly interacting with nmMLCK, an association which is increased by p60src tyrosine phosphorylation of either cortactin or nmMLCK [26]. Rac activation is in conjunction with Akt-mediated phosphorylation events known to be involved in EC proliferation and migration [164] and EC barrier enhancement. Akt-induced phosphorylation of the S1P1 receptor is important in barrier enhancement produced by high molecular weight hyaluronan [161, 165].

Fig. 3  Intracellular signals elicited by barrier-protective agonists with cortical cytoskeletal linkage to target junctional adhesion components. Increased endothelial cell barrier function by barrier-enhancing agonists is depicted. A low concentration (0.5–1  mM) of sphingosine 1-phosphate (S1P), a platelet-derived lipid growth factor, activates a specific G-protein-coupled receptor, leading to profound cytoskeletal rearrangement and increased barrier function in  vitro and in  vivo.

Ligation of S1P1 results in activation of the small GTPase Rac, a signaling cascade that results in cytoskeletal rearrangement and increased cortical actin formation with MLCK colocalization. Phosphorylation of myosin light chain at the periphery mediates increased linkage to the adherens junction, resulting in increased endothelial barrier integrity. Also depicted are other barrier-enhancing agonists that result in cortical actin formation leading to enhanced endothelial barrier function

MLCK inhibitors S1P

Summary/Function

References

Attenuation of the actin–myosin contractile apparatus which augments paracellular permeability [20, 168, 169] [157, 170, 180, 186, 188] S1P induces rapid and potent endothelial barrier enhancement through reduction of the numbers of central actin stress fibers and enhancement of cortical actin formation to stabilize cell–cell junctions. S1P attenuated endotoxin-induced pulmonary edema in mice and canine models of injury [158, 191, 193, 195] Simvastatin RhoA/Rac1 Patients on cholesterol-reducing statin regimens have exhibited improved vascular function. The HMG-CoA reductase inhibitor mitigates VEGF signaling through RhoA inhibition and Rac activation. In vitro, simvastatin pretreatment protects EC from thrombin-induced stress fiber formation and barrier dysfunction ATP Gi/Go protein, but not [159, 196, 198] ATP induces endothelial barrier enhancement through a Rac-dependent cytoskeletal rearrangement with ATP receptor reduction of the numbers of central actin stress fibers with increase cortical actin formation. In vivo, nonhyrolyzable ATP protected mice from endotoxin-induced lung injury [33, 202, 203] HGF c-Met receptor HGF, an angiogenic factor, induces endothelial barrier enhancement via ligation of c-Met receptor, which transactivates CD44 into caveolin-enriched microdomains to activate Rac-dependent cytoskeletal rearrangement. Unlike S1P-mediated pathways, HGF activates PI-3-kinase activity, with important roles for MAPK (extracellular signal-regulated kinase [ERK] and p38) and PKC APC EPCR that transactivates [134, 204, 207] APC binds EPCR and transactivates S1P1 receptor signaling. APC pretreatment prevents and reverses thrombinS1P1 receptor induced barrier dysfunction by increasing Rac-dependent cortical actin formation and MLC phosphorylation. In 2001, the FDA approved the use of recombinant APC for treatment of severe sepsis in adults Oxidized Putative receptor which [160, 209–211, 214] OxPAPC-mediated endothelial barrier enhancement via transactivation of S1P1 receptor to activate Rac and phospholipids transactivates S1P1 Cdc42. OxPAPC accentuates peripheral F-actin in a unique ziplike configuration with novel interaction between focal adhesion and AJ complexes. In vivo, OxPAPC protects rats from mechanical-stress-induced receptor lung injury MNTX mOP-R that inhibits S1P3 [215–217] Pretreatment with mOP-R antagonist protects ECs from thrombin- and LPS-induced barrier dysfunction through receptor an mOP-R-independent antagonism involving inhibition of RhoA-dependent S1P3 receptor. The FDA recently approved MNTX for treatment of postoperative ileus, and it may rapidly translate into a treatment for pulmonary edema [131, 136] Extracellular PBEF PBEF is significantly upregulated in the lung during injury. Extracellular release of PBEF promotes endothelial Anti-PBEF barrier dysfunction and neutrophil extravasation. Anti-PBEF neutralizing antibodies, which target neutralizing extracellular PBEF without altering beneficial intracellular PBEF, protected lungs from ventilator-induced antibody lung injury in mice adherens junction, activated protein C, endothelial cell, endothelial protein C receptor, filamentous actin, hepatocyte growth factor, hydroxy-3-methylglutaryl coenzyme A, lipopolysaccharide, mitogen-activated protein kinase, myosin light chain kinase, methylnaltrexone, mu opioid receptor, phosphatidylinositol 3-kinase, 1-palmitoyl-2-arachidonoyl--glycero-3-phosphochlorin, pre-Bcell colony-enhancing factor, protein kinase C, sphingosine 1-phosphate, vascular endothelial growth factor

Targets

MLCK S1P1 receptor

Therapeutic molecules

Table 1  Potential therapeutic agents to reverse vascular permeability and restore barrier integrity

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4.1 Strategies to Reverse Permeability and Restore Barrier Integrity 4.1.1 MLCK Inhibitors Historically, cyclic nucleotides have represented the sole strategy for retarding the edema phase observed in inflammatory lung syndromes, possibly via cAMP-dependent protein kinases that phosphorylate proteins such as MLCK and inhibit F-actin reorganization [153, 154, 166, 167]. We examined nmMLCK as a molecular target involved in increase of lung epithelial and EC barrier permeability utilizing genetically engineered mice and complementary strategies to reduce nmMLCK activity or expression. Both MLCK inhibition (membrane-permeant oligopeptide, PIK) and silencing of nmMLCK expression in the lung significantly attenuate LPS-induced lung permeability and inflammation. We also targeted pulmonary vessels and utilized ACE antibody-conjugated liposomes with nmMLCK small interfering RNA (siRNA) as cargo in a murine VILI model, again with significant attenuation of VILI. Furthermore, nmMLCK−/− knockout mice were significantly protected when exposed to a model of severe VILI. Thus, the multidimensional cytoskeletal protein nmMLCK represents an attractive target for reducing lung vascular permeability and lung inflammation in the critically ill [20, 168, 169]. 4.1.2 SIP and Closely Related Analogues S1P is a sphingolipid resulting from the phosphorylation of sphingosine, a product of sphingomyelinase catabolism of sphingomyelin, catalyzed by sphingosine kinase [170]. S1P ligates a family of receptors known as S1P receptors (also termed endothelial differentiation gene or Edg receptors) with prominent effects on the vasculature, promoting EC mitogenesis, chemotaxis, and angiogenesis. Our earlier studies were the first to demonstrate that S1P is the most potent EC chemoattractant in serum [171] and to link S1P and its receptor ligation to enhanced vascular barrier regulation and demonstrated that physiological doses of S1P induce EC activation, marked cytoskeletal rearrangement, and stabilization of lung EC barrier function in vitro [157]. This novel function for S1P was of particular relevance to clinical medicine as thrombocytopenia is well known to be associated with increased vascular leak [172] and although the mechanism of this effect was unknown, we demonstrated that activated platelets are an important source of S1P and directly enhance barrier function via S1P1 receptor ligation [173]. Platelets contain significant levels of sphingosine kinase but reduced levels of sphingosine lyase, thereby serving as enriched sources for the barrier-promoting S1P [173]. Ligation by S1P of the barrier-enhancing Gi-protein-coupled S1P1 receptor (also

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known as Edg1) [157, 170, 174, 175] increases Rac GTPase activity [157], cytosolic calcium level [176], and aggregation of key barrier-regulatory signaling components into caveolin-rich lipid rafts, including the Rac GTPase target p21-associated Ser/Thr kinase (PAK) and its downstream target cofilin, an actin-binding protein [177], nmMLCK, cortactin, and c-Abl. PAK and cofilin allow polymerization–depolymerization cycling to occur and thus facilitate rearrangement of actin from primarily transcytoplasmic to primarily cortical in a spatially distinct organization as a cortical actin cellular ring, processes which are integral to EC barrier function [157]. Increases in MLC phosphorylation within a peripheral distribution within the cortical actin ring [157] provide strength to this spatially directed scaffolding force and enhance cell–cell tethering as we described via atomic force microscopy [178]. Immunofluorescence studies demonstrated that overexpressed green fluorescent protein–nmMLCK distributes along cytoplasmic actin fibers, but rapidly translocates to the cortical regions of the cell after S1P treatment, rapidly catalyzing MLC phosphorylation. In addition, confocal microscopy studies showed ECs challenged with S1P demonstrate colocalization of nmMLCK with the key actin-binding and EC barrierregulatory protein cortactin [158]. The interaction of cortactin and nmMLCK decreases cortactin-stimulated actin polymerization [26, 158] and is essential to S1P barrier protection. The p60 src is not involved in this pathway, but other tyrosine kinases such as c-abl are likely involved [158]. S1P-induced cytoskeletal rearrangement produces increased linkage of actin to AJ components, as well as S1P-induced phosphorylation of focal-adhesion-related proteins paxillin and FAK, with translocation of these proteins to the EC periphery, further implicating S1Pinduced cell–cell adhesive changes as part of the mechanism of S1P-induced barrier enhancement [176, 179]. The potential utility of S1P in restoring lung water balance in patients with inflammatory injury was underscored in studies involving small- and large-animal models of ALI in which S1P provided dramatic attenuation of LPS-mediated lung inflammation and permeability [170, 180]. Mice treated with S1P had significantly less histological evidence of inflammatory changes/lung injury, with decreased neutrophil alveolitis on BAL and decreased lung MPO activity [180]. Interestingly, mice treated with S1P after intratracheal administration of LPS also showed an attenuated renal inflammatory response compared with controls, measured by tissue MPO activity and Evans blue dye extravasation as a measure of capillary leak. S1P also protected against intrabronchial LPS-induced ALI and concomitant VILI in a canine model, with decreased shunt fraction, decreased BAL protein, decreased extravascular lung water, and improved oxygenation [181]. Use of a large-animal canine model

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allowed investigation of regional lung changes in ALI and the effect of S1P on these changes. Computed tomography scans of animals subjected to LPS/VILI found that animals treated with S1P had a dramatic improvement in alveolar air content (with decreased edema) in all lung regions [181]. Additional in  vivo studies found that S1P protects against VILI in a murine model as assessed by Evans blue dye extravasation [181]. We have also evaluated a potential role for S1P in ameliorating lung ischemia–reperfusion injury, a common sequela of lung transplantation, which is characterized by alveolar damage, edema, and inflammation in donor lungs and is a significant cause of transplant failure. Utilizing a rat model of ischemia–reperfusion injury (pulmonary artery ligation and reperfusion), we determined that rats pretreated with S1P exhibited reduced lung vascular permeability and inflammation compared with controls [181]. Lung MPO activity, an index of parenchymal leukocyte infiltration, and levels of IL-6, IL-1b, and IL-2 were also attenuated in S1Ptreated animals exposed to ischemia–reperfusion injury [182]. Together, these findings suggest that S1P may serve as an effective permeability-reducing agent in diverse conditions which share an element of lung inflammatory burden. Despite the profound attractiveness of S1P as a therapeutic agent which targets the endothelium in high-permeability states, S1P has several attributes which limit its potential utility as a permeability-reducing strategy. With an affinity for ligation of the S1P3 receptor, intratracheal S1P has been implicated as a cause of pulmonary edema via endothelial/ epithelial barrier disruption [182]. S1P also causes bradycardia via ligation of cardiac S1P3 receptor [183]. These findings generated increased interest in FTY720, a derivative of the natural immunosuppressant myriocin [184], and a recently described immunosuppressive agent that causes peripheral lymphopenia by inhibiting cellular egress from lymphoid tissues. FTY720 is structurally similar (but not identical) to S1P and is phosphorylated by sphingosine kinase to FTY720-phosphate, which is an agonist at S1P receptors [184]. This characteristic prompted investigation of the effect of FTY720 on EC barrier function. FTY720 did not have superior efficacy compared with mycophenolate mofetil in preventing renal transplant rejection [185], but it is in phase III clinical trials as an immunosuppressant in multiple sclerosis patients. The clinical availability of FTY720 makes it attractive as a potential mediator of EC barrier function in patients with ALI. Our in vivo studies demonstrated that intraperitoneally administered FTY720 protected against intratracheally administered LPS in a murine model of ALI, as measured by Evans blue dye extravasation [180]. The mechanism of FTY720-induced EC barrier enhancement diverges from the mechanism described for S1P in several ways, including the delayed kinetics of the rise in total energy requirement (TER) compared with S1P [186]. Decreased expression of the S1P1

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receptor prevented an S1P-induced increase in TER but only partially altered FTY720-induced TER increases. Unlike S1P, FTY720 did not result in threonine phosphorylation of the S1P1 receptor, nor did inhibition of phosphatidylinositol 3-kinase (PI-3-kinase) prevent FTY720-induced EC barrier enhancement [186]. Furthermore, FTY720 did not cause the increased intracellular calcium level, the MLC phosphorylation, or the cytoskeletal rearrangement seen in response to S1P [186]. Downregulation of Rac or cortactin using siRNAs attenuated the barrier-enhancing effect of S1P, but not that of FTY720 [186]. Although FTY720 is an S1P receptor agonist, its mechanism of barrier enhancement is distinct from that of S1P and does not require the S1P1 receptor. We are currently pursuing novel S1P and FTY analogues for use in inflammatory lung injury models [187–189].

4.1.3 Simvastatin Another class of prominent barrier-protective agonists under intense scrutiny is the statin family of compounds known as 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCoA reductase) inhibitors [190]. These drugs inhibit cholesterol synthesis in the liver, are commonly used in clinical practice as lipid-lowering agents, and prevent acute coronary events. A plethora of reports have now demonstrated that the benefits of statin therapy cannot be entirely attributed to decreased serum cholesterol level. We have been interested in the effect of statins on endothelial function in ALI as an ever-growing body of literature demonstrates improved outcomes in patients with sepsis who are treated with statins, with decreased mortality in bacteremic patients admitted to the hospital while on statin therapy [190]. A retrospective study in human patients with multiple organ dysfunction syndrome found that those receiving statins had significantly lower 28-day mortality and hospital mortality compared with matched controls not receiving statin therapy [191]. Animal studies suggest dramatically improved survival in mice treated with simvastatin prior to initiation of sepsis by cecal ligation and puncture compared with mice which were not pretreated with simvastatin [192]. We have pursued the mechanism of statin action on the endothelium and found that simvastatin attenuated thrombininduced stress fiber formation, paracellular gap formation, and barrier dysfunction [193]. Co-incubation with mevalonate (the product of HMG-CoA reductase activity) eliminated the protective effect of simvastatin against thrombin-induced EC permeability, indicating this effect is due to HMG-CoA reductase inhibition and did not involve either intracellular increased cAMP levels or increased levels of endothelial nitric oxide synthase. Statins inhibit geranylgeranylation of small GTPases, essential for GTPase interaction with cell membranes [158], and translocation of the small GTPases

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Rac and Rho to the plasma membrane. EC pretreatment with simvastatin prevented thrombin-induced translocation of Rho to the plasma membrane [193] and simvastatin was found to confer greater protection against thrombin-induced barrier dysfunction than Rho inhibition alone. Rac inhibition may be protective via decreased activation of NADPH oxidase and resultant superoxides that induce barrier dysfunction, and this was also found to be important in simvastatin-induced EC barrier protection [194]. Simvastatin pretreatment resulted in reduced diphosphorylated MLC levels, reduced numbers of stress fibers, increased Rac GTPase activation [158], cortactin translocation to the EC periphery [158], and increased cortical actin and decreased paracellular gap formation after thrombin treatment. Unlike S1P, simvastatin does not cause an increased baseline TER [158]. Simvastatin elicits changes in EC gene expression with downregulation of caldesmon and the thrombin receptor PAR-1, as well as upregulation of integrin b4 (known to function in cell–cell adhesion), Rac 1, and GEFs, which may regulate Rho GTPase activity [158]. The importance of new protein synthesis to the barrier protective effect of simvastatin was established by the elimination of the protective effect by coincubation of ECs with simvastatin and the protein synthesis inhibitor cycloheximide [158]. In vivo data from an intratracheal-LPS murine model of ALI support the in vitro finding that simvastatin is protective of EC barrier function and against markers of inflammatory lung injury compared with controls, with decreased BAL neutrophil count and MPO activity, decreased vascular permeability, and a marked reduction of inflammatory histological changes [195]. Investigation of gene expression in lung tissue of mice pretreated with simvastatin in this LPS-induced model of ALI found that simvastatin caused differential regulation of several families of genes, including inflammatory and immune response genes, as well as NFkB regulation and cell adhesion genes [195]. Simvastatin may prove to be clinically relevant in treating ALI, as ALI typically has a prolonged course, and treatment with simvastatin along the trajectory of the illness may be beneficial. To this end, a blinded, randomized controlled clinical trial of simvastatin in ALI is currently under way.

4.1.4 Adenosine Triphosphate ATP is found in abundance in the EC microenvironment and participates in EC barrier regulation, with constitutive release of ATP across the EC apical membrane in basal conditions [196, 197]. ATP reduced EC albumin permeability in a concentration-dependent manner in ECs from a variety of origins, including porcine aorta and pulmonary artery, bovine aorta, and human umbilical vascular endothelial cells [198]. The mechanism of ATP-induced EC barrier enhancement involves

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Gi/Go proteins [196] but does not involve adenosine receptors [198], increased PKC activity, or increases in cyclic GMP levels [198]. However, ATP-induced decreases in EC permeability were found to involve the phospholipase C signaling pathway [198], as well as alterations in EC MLC phosphorylation [199, 200]. We demonstrated that ATP produces Ca2+- and p42/44 MAPK-independent increases in cell–cell interfaces (VE-cadherin staining) and increased thickness and continuity of zona occludens (ZO-1) in TJs [196], mediated in part via cAMP-independent activation of protein kinase A (PKA). We also noted that ATP produced a biphasic effect on MLC phosphorylation, with an initial increase followed by a decrease in levels of phosphorylated MLC. However, the delayed decrease in the levels of phosphorylated MLC was prevented by phosphatase inhibitors, emphasizing the importance of G-protein-mediated phosphatase activity in the ATP-induced decrease in MLC phosphorylation and ATP-induced barrier enhancement [196]. Similar to S1P (as well as HGF, APC, etc.), ATP-mediated barrier enhancement required Rac-dependent cytoskeletal rearrangement with decreased numbers of central actin stress fibers, increased cortical distribution of actin, peripheral MLC phosphorylation, and cortactin translocation to the cortical actin ring [159]. In addition, a rapid, transient increase in MLC diphosphorylation was observed after ATP stimulation, with phosphorylated MLC localized at the cell periphery, a stark contrast to the central, stress-fiber-associated phosphorylated MLC seen in ECs treated with thrombin [159]. As an extension of these in  vitro studies, the effect of purinergic stimulation was assessed in a murine model of ALI with intratracheally administered LPS. As ATP is rapidly degraded intravascularly, the nonhydrolyzable analogue ATPgS was used for in vivo studies. Mice given ATPgS intravenously concomitant with intratracheal administration of LPS were protected from LPS-induced ALI compared with controls as assessed by neutrophil infiltration and MPO activity [201]. ATPgS also attenuated the lung microvascular permeability elicited by LPS, with decreased BAL protein and decreased Evans blue–albumin extravasation in mice treated with ATPgS compared with controls [201]. ATPgStreated animals were also protected from the LPS-induced decrease in body weight that was seen in control mice [201]. In addition, in vitro studies found that ATPgS alone produced an increased TER in ECs and also showed delayed protection against the reduction in TER caused by LPS [201].

4.1.5 Hepatocyte Growth Factor Alterations in vascular permeability are requisite steps in the angiogenic process [157, 171]. We were the first to report that HGF, a well-known angiogenic factor, like S1P, is a potent EC barrier-protective agonist [33] and acts via

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stabilization of the EC actin cytoskeleton. HGF-mediated EC protection from the barrier-disrupting effect of thrombin [202] evolves via increased Rac activation involving the Racspecific GEF Tiam1 as well as decreased Rho activation with increased PAK1 phosphorylation [202]. HGF signals via a tyrosine kinase receptor, c-met, and serves to recruit CD44v10, a key transactivated receptor for CD44, into caveolin-enriched microdomains (CEMs) or lipid rafts [203]. In experiments using siRNA, both c-met and CD44 were found to be important in HGF-induced increases in EC TER [203]. Furthermore, pretreatment of ECs with the CEMinterfering compound methyl-b-cyclodextran also prevented HGF-induced increases in TER [203]. In addition, Rac activation by HGF was found to require CEM formation, c-met, CD44, Tiam1, and dynamin-2 [203]. In a mouse model of LPS-induced ALI, HGF was protective against markers of lung inflammation, an effect not noted in CD44 knockout mice [203]. The signaling mechanism involved in HGFinduced EC barrier enhancement is complex, with important roles for c-met, CD44, and CEM formation. HGF produced Rac-dependent increases in the levels of cortical actin, cortactin translocation, and cortical levels of phosphorylated MLC [33]. Further mechanistic studies found that HGFinduced EC barrier enhancement critically involves PI-3-kinase activity, distinguishing the mechanism of HGFinduced barrier enhancement from that of S1P [33], with important roles for MAPKs (ERK and p38) and PKC in HGF-induced EC barrier enhancement [33]. Attention to the role of improved cell–cell or cell–matrix adhesion elicited by HGF found that HGF produced increased b-catenin localization to the EC periphery alongside cortical actin and increased association of b-catenin with VE-cadherin [33]. The cell signaling effectors of HGF (PI-3-kinase, ERK, p38, PKC) were found to converge at phosphorylation of glycogen synthase kinase-3b, which regulates the association of b-catenin and cadherin, thereby controlling cell–cell adhesion [33].

4.1.6 Activated Protein C APC is a serine protease that modulates coagulation and inflammation. In 2001, the Food and Drug Administration approved Xigris®, or recombinant human APC (rhAPC), also known as drotrecogin alfa (activated), for treatment of severe sepsis in adults after a randomized trial found a 28-day survival benefit in treated patients [204]. Because severe sepsis involves ALI and systemic increased vascular permeability, the effect of APC on pulmonary EC permeability is intriguing. Interest in the effect of the anticoagulant APC on EC permeability is also related to the well-described role of the procoagulant thrombin in EC barrier disruption. Furthermore, the mechanism of the survival benefit imparted by treatment with rhAPC is unclear, as APC given to human subjects in the setting of

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endotoxin infusion improved hemodynamics but did not have an anti-inflammatory or antithrombotic effect [205], suggesting that a different mechanism may be involved. We demonstrated that APC prevented and was able to reverse thrombin-induced increased permeability [134]. APC also increased MLC phosphorylation and the level of actin at the EC periphery and decreased the number f central stress fibers. The barrier-enhancing effect of APC was found to be mediated by Rac1 activation, similar to the barrier-enhancing effect of S1P, simvastatin, and HGF [134]. The endothelial protein C receptor (EPCR) is critical to APC-induced barrier enhancement and MLC phosphorylation. Furthermore, EPCRmediated transactivation of the S1P1 receptor via PI-3-kinase is essential and involves direct interaction between EPCR and S1P1 receptor [134]. This novel pathway for APC-induced EC barrier enhancement may contribute significantly to the survival benefit offered by rhAPC in patients with severe sepsis. More recent work has focused on APC in animal models of ALI. Using a rat model of intestinal ischemia–reperfusion injuryinduced ALI, investigators found that APC treatment just prior to reperfusion attenuated subsequent pulmonary edema, which was accompanied by fewer neutrophils on histological examination and a marked improvement in the histological appearance compared with animals that did not receive APC [206]. In addition, rats treated with APC prior to intestinal reperfusion had lower serum levels of TNF-a, IL-6, and D-dimer compared with controls [206]. Investigation of APC in a mouse model of VILI found that APC pretreatment was protective against VILI caused by high tidal volume ventilation, with mice pretreated with APC exhibiting significant reductions in BAL protein and Evans blue dye extravasation compared with controls [207].

4.1.7 Oxidized Phospholipids Oxidized phospholipids are derived from oxidized low-density lipoproteins and have been the focus of much investigation in the areas of vascular injury and inflammation [36], with increased levels noted in ALI [104]. Oxidized phospholipids resulting from the oxidation of OxPAPC activate MAPKs ERK, and c-Jun N-terminal kinase, but not p38 or its downstream target, Hsp27 [36], and increased the activity of both PKC and PKA [36] and Src kinases, processes involved in OxPAPC-mediated EC barrier enhancement, whereas Rho, Rho kinase, ERK, p38, and PI-3-kinase were not involved [208]. Furthermore, OxPAPC resulted in phosphorylation of the actin-binding protein cofilin as well as phosphorylation of the focal adhesion proteins FAK and paxillin, indicating that OxPAPC may affect the EC actin cytoskeleton and cell– cell adhesions [36]. OxPAPC protects against EC barrier dysfunction in  vitro [160, 209] after thrombin and LPS stimu­lation [210]. OxPAPC accentuates peripheral F-actin in a unique, ziplike configuration [160, 210, 211] and results in

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continuous focal adhesions with accumulation of b-catenin [210]. The signaling pathways involved in OxPAPC-mediated endothelial barrier protection involve Rac and Cdc42 [160], the Rac effector PAK1 [160], the upstream Rac/Cdc42specific GEFs Tiam1 and bPIX [212], and the actin-binding proteins cortactin and Arp3 [212]. OxPAPC was found to cause a novel interaction between focal adhesion and AJ complexes, a process mediated by association of paxillin and b-catenin and dependent upon Rac and Cdc42 [213]. In vivo studies have shown that intravenous OxPAPC delivery results in significant attenuation of LPS-induced inflammation in a rat model [214] and VILI [209]. OxPAPC protects ECs from mechanical-stress-induced injury via cytoskeletal rearrangements and changes in Rho and Rac activation and remains a potential therapy for the profound pulmonary edema associated with inflammatory states.

4.1.8 Methylnaltrexone Methylnaltrexone (MNTX) is a peripherally restricted mu opioid receptor (mOP-R) antagonist recently approved by the Food and Drug Administration for the treatment of postoperative ileus and also recently found to work synergistically with 5-fluorouracil and bevacizumab to inhibit VEGF-induced pulmonary EC proliferation and migration [215]. Antagonists of mOP-R are of interest as potential EC barrier-enhancing agents because of the barrier-disruptive properties of the mOP-R agonist morphine [216]. Pretreatment of human pulmonary microvascular ECs with 0.1 mM MNTX was found to protect against the decrease in TER caused by the mOP-R agonists morphine and DAMGO and also protected against the barrier-disruptive effects of thrombin and LPS, which act independently of mOP-R [217]. MNTX augments the barrier-enhancing effect of S1P [217]. EC pretreatment with naloxone, a charged mOP-R antagonist, protected against morphine and DAMGO-induced barrier disruption, but was not protective against barrier disruption caused by thrombin or LPS. These data, together with the observation that siRNA targeting mOP-R had a minimal effect on MNTXinduced protection against thrombin and LPS, suggest that the protective effect of MNTX cannot be attributed to mOP-R antagonism alone [217]. Further experiments found that MNTX confers its barrier-protective effect by inhibiting the association of the RhoA-activating GEF p115RhoGEF with the S1P3 receptor and resultant RhoA activation that is caused by barrier-disrupting agents [217]. Complementary in  vivo experiments found that intravenous administration of MNTX after ALI had been established via intratracheal administration of LPS was protective against ALI at 24 h, as assessed by histological examination and BAL protein and TNF-a levels [217].

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4.1.9 PBEF Neutralizing Antibodies As noted already, PBEF is a biomarker in sepsis and sepsisinduced ALI and intratracheal injection of recombinant PBEF into mice results in increased lung inflammation and vascular permeability [136, 218], indicating that extracellular PBEF promotes endothelial barrier dysfunction. Intracellular PBEF may have a contrasting beneficial response in ALI function via effects on cell apoptosis. Neutrophils in sepsis patients increase expression of PBEF, which promotes cell survival through the enzymatic process of NAD biosynthesis via nicotinamide phosphoribosyltransferase (Nampt) activity, a feature cancer cells have utilized to prevent cell death. The Nampt inhibitor FK-866 is currently in trials as a cancer drug to promote apoptosis. Thus, PBEF therapies are complicated, with intracellular PBEF appearing to have beneficial effects in cells by promoting cell survival, whereas extracellular PBEF appears to induce inflammatory response. To specifically target extracellular PBEF that may induce deleterious cellular response, we generated neutralizing antibodies against PBEF to act as a molecular sponge for extracellular PBEF without altering intracellular PBEF function, which may be beneficial for the cell. Using a mouse model of lung injury, we demonstrated that the anti-PBEF neutralizing antibodies significantly protected lungs from VILI by reducing the availability of extracellular PBEF from sensitizing the lung endothelium [136]. The study implicates PBEF as a key inflammatory mediator intimately involved in both the development and the severity of ventilator-induced ALI and demonstrated that anti-PBEF neutralizing antibody has potential clinical utility.

5 Vascular Biomarkers of Acute Inflammation Various molecules participating in the activation of inflammation in ALI serve as indicators for the progression of normal to pathological biological processes, providing important tools to detect disease and support diagnostic and therapeutic decisions. Ideally, vascular biomarkers have strong correlation between the presence/absence of a disease state and clinical outcome and provide predictive points of intervention to slow or reverse the disease. Furthermore, the indication of a specific biomarker may allow for customized therapies that are more effective in different phases of the disease. New research and novel understanding of the molecular mechanisms of ALI have revealed an abundance of exciting new biomarkers with high potential value as prognostic tools (Table 2).

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Table 2  Prognostic biomarkers of acute lung injury and acute respiratory distress syndrome Biomarkers Descriptions Summary/Prognostic Indication S1P S1P3 receptor (tyrosine-nitrated) IL-8/IL-8 receptor Protein C Thrombomodulin

PAI-1 sICAM-1 IL-6 PBEF

Sphingolipid; angiogenic factor Sphingolipid receptor Cytokine; inflammation Procoagulant activity Cofactor in the thrombin-induced activation of protein C in the anticoagulant pathway Inhibitor of plasminogen activator in plasma Marker of EC activation; adhesion molecules Cytokine; inflammation Cytokine; inflammation

Low S1P level is predictive of vascular dysfunction Tyrosine nitration of S1P3 receptor released from cell surface as microparticles is predictive of pathological disease state IL-8 increase is predictive of death Low protein C level is predictive of death Reduced plasma thrombomodulin level is predictive of higher mortality and worse system dysfunction

References [157, 173] [219, 220] [222, 264] [264, 265] [229, 239]

PAI-1 level increase is predictive of death

[240, 242]

ICAM-1 level increase is predictive of death

[249, 264]

IL-6 level increase is predictive of death [255, 256] Secretion of extracellular PBEF upon mechanical stress induces [131, 136] pulmonary edema and neutrophil extravasation in mice ICAM-1 intercellular adhesion molecule-1, interleukin, PAI-1 plasminogen activator inhibitor-1, sICAM-1 soluble intercellular adhesion molecule-1

5.1 Sphingosine 1-Phosphate The importance of sphingolipids to maintain physiological vascular integrity has been well established and thrombocytopenia, a clinical condition in which there is a deficient number of circulating platelets, is associated with increased vascular leak [172] via an unknown mechanism. Activated platelets are an important source of S1P and contain significant levels of sphingosine kinase but reduced levels of sphingosine lyase, thereby serving as enriched sources for the barrier-promoting S1P [173] which directly enhance barrier function via S1P1 ligation [173].

5.2 S1P3 Receptor (Tyrosine-Nitrated) Although the role of S1P at physiological concentration is critical to maintaining normal endothelial barrier function, the differential ligation to S1P receptors has differential responses. In contrast to the ligation to S1P1, the ligation of S1P3 induces endothelial barrier dysfunction via activation of Rho-dependent actin stress fiber and cell–cell gap formation. Recently, we discovered that culture of ECs challenged with barrier-disrupting agents induces tyrosine nitration of S1P3 receptors, which are released into media in microparticles or exosomes [219, 220]. The occurrence of protein tyrosine nitration under disease conditions is now firmly established and represents a shift from the physiological signaling actions of •NO to oxidative and potentially pathogenic pathways. Protein tyrosine nitration is an irreversible PTM mediated by reactive nitrogen species, a process that suggests the regulatory function of proteins that undergo phosphorylation in signal transduction cascades might be seriously compromised by peroxynitrite-promoted nitration. We explored S1P3 as a potential biomarker and observed from immunoblot analysis of serum from mice

exposed to various models of vascular injury that they had significant tyrosine-nitrated S1P3 expression [219, 220]. In addition, we examined serum from patients with sepsis and ALI and discovered tyrosine-nitrated S1P3 receptor was correlated with disease progression [219, 220]. Therefore, our data indicate tyrosine-nitrated S1P3 receptor is released from challenged ECs in microparticles and serves as a novel biomarker for vascular injury in various disease models.

5.3 IL-8/IL-8 Receptor The family of PMN chemotactic cytokines, known as the CXC chemokines have been described and characterized and include IL-8, GRO-a, GRO-b, GRO-g, ENA-78, and granulocyte chemotactic peptide (GCP)-2. These chemokines are all produced by human alveolar macrophages and contain a glutamyl– leucyl–arginine (ELR) motif that is critical to their neutrophil binding and chemotactic functions [221]. IL-8 is present in biologically significant concentrations in BAL fluid from patients with ARDS, tracking PMN concentrations [222]. Although GRO-a and ENA-78 concentrations are higher than IL-8 concentrations, IL-8 is the predominant chemoattractant in ARDS BAL fluid via its high-affinity binding to CXC chemokine receptors, CXCR1 and CXCR2, on human PMNs. Unlike ENA-78, GRO-a, GRO-b, and GRO-g with a high-affinity binding only to CXCR2, IL-8 and GCP-2 can bind to either receptor with high affinity [223]. In the presence of a systemic inflammatory process such as severe sepsis, CXCR2 is tonically downregulated and the function of only CXCR1 receptor predominates [224]. Thus, of the multiple neutrophil chemotactic factors produced in humans, there appears to be a small group that is particularly relevant to patients with ARDS, with IL-8 and its cognate receptor CXCR1 being the dominant receptor–ligand pair. IL-8 also binds to its circulating high-affinity polyclonal

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IgG3 and IgG4 autoantibodies naturally [225], therefore preventing binding to CXC chemokine receptors on PMNs [226]. These autoantibodies are present in lung fluids from patients who are at-risk for ARDS as well as in patients after the onset of ARDS. The ratio of IL-8 autoantibody:cytokine complex was significantly higher at the onset of ARDS than in patients at risk for ARDS. In addition, patients with ARDS with an elevated anti-IL-8-autoantibody:IL-8 complex ratio are more likely to die than patients with lower concentrations of anti-IL-8-autoantibody:IL-8 complex [227]. Thus, the antiIL-8-autoantibody:IL-8 complex ratio in lung fluid samples was more revealing than lung fluid protein concentrations to predict the development of ARDS in patients who were at-risk, and also for predicting mortality in patients with ARDS [228].

5.4 Protein C / Thrombomodulin The protein C pathway is one of the most important regulators of blood coagulation and serves as a critical link between coagulation and inflammation in sepsis and ALI [229–231]. Protein C is a vitamin-K-dependent plasma glycoprotein that is synthesized by the liver and circulates as a two-chain biologically inactive zymogen. It is transformed to its active form, APC, by the thrombomodulin–thrombin complex on the cell surface. APC suppresses further thrombin formation by proteolytically inactivating coagulation factors Va and VIIIa [232]. The membrane-bound EPCR potentiates this activation about 20-fold [233]. Recent evidence suggests that, in addition to its anticoagulant effects, APC also has anti-inflammatory properties. Thus, the protein C pathway is important for the control and modulation of both coagulation and inflammation [230]. APC inhibits the production of TNF-a via NFkB activation in monocytes and ECs [234], and inhibits neutrophil activation and chemotaxis through interaction with a cell-surface receptor similar to the EPCR [235]. Decreased protein C activation on the pulmonary vascular endothelium surface may contribute to the widespread microvascular thrombosis that occurs in the acutely injured lung and may also be proinflammatory and proapoptotic. Administration of APC attenuates experimental sepsis-induced lung injury. In human studies, an infusion of APC 2 h prior to and 6 h after administration of an intravenous injection of LPS prevented LPS-induced increase in tissue factor expression and thrombin formation in plasma after LPS injection, as well as circulating levels of IL-6 or TNF-a, markers of inflammation [236]. Loss of thrombomodulin and EPCR from the cell surface results in a decreased ability to activate protein C, a phenomenon that has been implicated in the pathogenesis of sepsis and lung injury. Release of the protein C pathway components throm-

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bomodulin and EPCR into the plasma has been reported in experimental sepsis models [237]. In clinical studies, plasma protein C levels were reduced in patients with severe sepsis, with 90% of patients meeting the criteria for acquired protein C deficiency. Low levels of protein C were associated with ventilator dependency and a higher prevalence of ARDS and correlated with higher mortality [238]. Another study demonstrated that patients with severe sepsis varied markedly in their ability to generate APC [239]. Modulation of coagulation and inflammation through the activation of protein C is a critical mechanism in the pathogenesis of sepsis and ALI [231]. Protein C levels and thrombomodulin levels are lower early in the course of ALI and reduced plasma protein C and thrombomodulin levels are associated with higher mortality and more nonpulmonary organ system dysfunction, with the combination of low levels of protein C and other predictors such as high levels of plasminogen activator inhibitor-1 (PAI-1) conferring an even higher risk of mortality. The prognostic value of protein C and thrombomodulin was not altered by exclusion of patients with coexisting sepsis [229].

5.5 Plasminogen Activator Inhibitor-1 The balance between activation of coagulation and activation of fibrinolysis is likely an important determinant of the amount and duration of fibrin deposition in the injured lung, and the fibrinolytic system is profoundly altered in patients with ALI/ARDS, both systemically and in the alveolar compartment. Plasminogen activator (PA) and PAI-1 regulate fibrinolysis, the dissolution of fibrin clots, through modulation of the conversion of plasminogen to plasmin, a major fibrinolytic enzyme [240]. Upregulation of PAI-1, the major inhibitor of fibrinolysis, appears to play a primary role in the shift from profibrinolytic to antifibrinolytic phenotypes in a variety of cell types, including ECs, indicating a risk factor for ALI and sepsis. There are two forms of PA, urokinase-type PA (uPA) and tissue-type PA (tPA). uPA is a cell-surface protein that is responsible for activating fibrinolysis at the tissue level, whereas tPA is a soluble protein that activates intravascular fibrinolysis [241]. Two major endogenous PA inhibitors have been identified, PAI-1 and PAI-2, which are produced by platelets, endothelial, mesothelial, and epithelial cells, including those of the lung [241]. PAI-1 is the major PA inhibitor in plasma and extravascular fluids and has been implicated in the fibrinolytic defect associated with ALI [242]. Human lung ECs isolated from patients with ARDS constitutively express greater levels of PAI-1 than controls with lower fibrinolytic potential as measured by the PA to PAI-1 ratio. In limited ALI/ARDS clinical studies, reduced fibrinolytic capacity and an increase in uPA and in PAI-1 activity was noted in ARDS patents, with levels of PAI-1 higher

12  Acute Lung Injury

in both pulmonary edema fluid and plasma of ALI/ARDS patients, and correlated with mortality in patients with ALI/ ARDS [243, 244]. A variety of strategies are being explored to develop inhibitors of PAI-1 that might be of therapeutic use in ALI/ARDS or other diseases associated with high levels of PAI-1 such as cardiovascular disease [240].

5.6 Soluble Intercellular Adhesion Molecule-1 Intercellular adhesion molecule-1 (ICAM-1; CD54) is an adhesion molecule constitutively expressed in the normal lung and is a critical participant in pulmonary innate immunity [245]. Soluble ICAM-1 (sICAM-1) represents a circulating form of ICAM-1 that is constitutively expressed or is inducible on the cell surface of different cell lines [246]. Structurally, ICAM-1 belongs to the immunoglobulin superfamily, serving as a counterreceptor for the leukocyte integrin LFA-1. Interaction between ICAM-1, present on ECs, and LFA-1 facilitates leukocyte adhesion and migration across the endothelium; however, sICAM-1 binding to LFA inhibits lymphocyte attachment to ECs [247]. sICAM-1 is found in BAL fluid and the release of sICAM-1 is induced by several cytokines and various factors, including IL-1, IL-6, TNF-a, interferon-g, and angiotensin II via proteolytic cleavage of ICAM-1 or direct transcription from its messenger RNA [248]. Studies correlating sICAM-1 levels to disease have led to the identification of sICAM-1 as a marker for diseases such as viral infections, autoimmune disease, atherosclerosis, coronary heart disease, cancers, and neurological disorders [249]. Increased BAL sICAM-1 has been described in adults with granulomatous lung diseases such as sarcoidosis, tuberculosis [250], hypersensitivity pneumonitis, and radiation pneumonitis [251], and in children exposed to second-hand smoke [252]. Importantly, the level of sICAM-1 is increased in pediatric ARDS during high-frequency oscillatory ventilation [253] and in ALI patients [254].

5.7 Interleukin-6 IL-6, a well-recognized ALI candidate gene and ALI biomarker [255, 256], and is produced by a wide range of cells, including ECs, in response to stimulation by endotoxin, IL-1b, and TNF-a [257]. IL-6 in the acute-phase response stimulates synthesis of C-reactive protein from hepatocytes in vitro and in vivo [258]. Elevated levels have been described in acute conditions such as burns, major surgery, and sepsis and may predict development of multiple organ failure and the severity of ARDS of different orgins, such as sepsis and

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acute pancreatitis [255]. The elevation of the level of and persistence of circulating IL-6 has been associated with increased mortality in critically ill patients with ARDS, sepsis, and trauma, and IL-6 concentrations have been shown to be elevated in the BAL fluid from patients with established severe ALI [259]. Functional polymorphisms in the promoter region of the IL-6 gene exist (G174C), with the C allele associated with reduced gene promoter activity, lower circulating IL-6 concentrations, and a lower mortality rate in patients with acute respiratory failure admitted to the ICU [260]. In the multispecies ALI studies performed, significant IL-6 gene expression across all species as well as differential region-specific expression in the canine ALI model has been noted. All of these facts suggest that the role of IL-6 in ALI is complex and IL-6 may have a dual role in the temporal response to sepsis and mechanical stress.

5.8 Pre-B-cell Colony-Enhancing Factor The PBEF gene is one of a handful of genes with extremely high level of expression across the range of ALI models used and in human ALI samples. Whereas we were the first to report that PBEF is significantly upregulated in the lung as well as in models of lung injury [131], the published literature on PBEF is quite sparse [261, 262]. This gene encodes for a proinflammatory cytokine, originally described for its role in the maturation of B-cell precursors, with gene expression upregulated in amniotic membranes from patients undergoing premature labor, especially with amniotic infections. PBEF protein levels were significantly increased in both BAL fluid and serum of human, murine, and canine ALI models as well as in cytokine- or CS-activated lung microvascular endothelium [131, 136]. Triple immunohistochemical staining of canine lungs revealed colocalization of increased PBEF expression in lung endothelium, type II alveolar epithelial cells, and infiltrating neutrophils, as well as upregulation of PBEF expression in inflammatory cytokine-stimulated human pulmonary microvascular ECs in  vitro [131]. These results support PBEF as a potential biomarker in ALI and potentially involved in inflammatory lung processes, a notion supported by recent studies in patients with sepsis which convincingly demonstrate that PBEF inhibits neutrophil apoptosis [131, 136]. Common variants in the human PBEF gene are also confirmed to be associated with susceptibility to sepsis-associated ALI [263]. The T allele in the C-1543T single-nucleotide polymorphism in the PBEF promoter region was associated with a nearly twofold decrease in the reporter gene expression. This result is consistent with our observations from animal models of ALI, human patients with ALI, and

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in vitro cell culture experiments, and suggests that higher expression of PBEF is implicated in the pathogenesis of ALI. These results further suggest that genetically determined increased PBEF expression contributes to susceptibility to ALI.

6 Conclusion and Perspectives Despite decades of frustration in the pursuit of potent barrierregulatory therapies, progress has now been made for alleviation of the human suffering associated with uncontrolled lung vascular leakage and alveolar flooding. Novel biologically compatible agents have now been identified which can preserve or restore vascular integrity, leveraging new insights into the mechanisms which govern the integrity of the vascular endothelium, particularly the role of cytoskeletal linkages to junctional proteins. In addition, several endothelial target proteins or protein pathway participants also serve as potentially novel biomarkers in the management of these patients. The newly revised scientific armamentarium offers promise for the future management of pulmonary edema associated with increased vascular leak in the critically ill as well as other lung conditions which exhibit strongly dysregulated barrier function such as radiation pneumonitis, acute chest syndrome in sickle cell patients, and in subacute inflammatory disorders such as asthma. Nearly each barrier-regulatory agent discussed herein has been successfully evaluated in preclinical models of ALI and one agent, FTY720, is in phase III trials, whereas three agents, statins, APC and MNTX,arecurrentlyapprovedbytheFoodandDrugAdministration for other medical conditions. Thus, the prospects for the rapid translation of these lung vascular barrier-protective strategies to clinical practice are high. Additional translational bench-to-bedside genomic and genetic strategy approaches combined with dissection of the basic mechanisms of endothelial structure/function during inflammation will lead to greater specificity in advancing clinical trials of agents for the treatment of inflammatory lung injury in a manner which represents personalized medicine for critically ill individuals.

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221 induced acute lung injury: an experimental study in a rat model. Am J Surg 195:861–873 207. Finigan JH, Boueiz A, Wilkinson E et al (2009) Activated protein C protects against ventilator-induced pulmonary capillary leak. Am J Physiol Lung Cell Mol Physiol 296:L1002–L1011 208. Birukova AA, Chatchavalvanich S, Oskolkova O, Bochkov VN, Birukov KG (2007) Signaling pathways involved in OxPAPC-induced pulmonary endothelial barrier protection. Microvasc Res 73:173–181 209. Nonas S, Birukova AA, Fu P et al (2008) Oxidized phospholipids reduce ventilator-induced vascular leak and inflammation in vivo. Crit Care 12:R27 210. Nonas S, Miller I, Kawkitinarong K et al (2006) Oxidized phospholipids reduce vascular leak and inflammation in rat model of acute lung injury. Am J Respir Crit Care Med 173:1130–1138 211. Nonas SA, Miller IL, Birukova AA, Chatchavalvanich S, Garcia JG, Birukov KG (2006) Protective effects of oxidized phospholipids on ventilator-induced lung injury. Proc Am Thorac Soc 3:A245 212. Birukova AA, Malyukova I, Mikaelyan A, Fu P, Birukov KG (2007) Tiam1 and bPIX mediate Rac-dependent endothelial barrier protective response to oxidized phospholipids. J Cell Physiol 211: 608–617 213. Birukova AA, Malyukova I, Poroyko V, Birukov KG (2007) Paxillin-b-catenin interactions are involved in Rac/Cdc42mediated endothelial barrier-protective response to oxidized phospholipids. Am J Physiol Lung Cell Mol Physiol 293:L199–L211 214. Nonas SA, Miller IL, Kawkitinarong K, Garcia JG, Birukov KG (2005) Protective effects of oxidized phospholipids on endotoxininduced lung injury. Proc Am Thorac Soc 2:A837 215. Singleton PA, Garcia JG, Moss J (2008) Synergistic effects of methylnaltrexone with 5-fluorouracil and bevacizumab on inhibition of vascular endothelial growth factor-induced angiogenesis. Mol Cancer Ther 7:1669–1679 216. Liu HC, Anday JK, House SD, Chang SL (2004) Dual effects of morphine on permeability and apoptosis of vascular endothelial cells: morphine potentiates lipopolysaccharide-induced permeability and apoptosis of vascular endothelial cells. J Neuroimmunol 146:13–21 217. Singleton PA, Moreno-Vinasco L, Sammani S, Wanderling SL, Moss J, Garcia JG (2007) Attenuation of vascular permeability by methylnaltrexone: role of mOP-R and S1P3 transactivation. Am J Respir Cell Mol Biol 37:222–231 218. Liu P, Li H, Cepeda J et al (2009) Critical role of PBEF expression in pulmonary cell inflammation and permeability. Cell Biol Int 33:19–30 219. Zhao J, Singleton PA, Mirzapoiazova T et al (2010) Sphingosine1-phosphate receptor 3 (SIP3) is a novel biomarker for acute lung injury and idiopathic pulmonary fibrosis. AM J Respir Crit Care Med 131:A2674 220. Zhao J, Singleton PA, Mirzapoiazova T et al (2009) Sphingosine1-phosphate receptor 3 (S1P3) is a novel biomarker for acute lung injury. Clin Soc Clin Res; CSCR Abstract 221. Goodman RB, Pugin J, Lee JS, Matthay MA (2003) Cytokinemediated inflammation in acute lung injury. Cytokine Growth Factor Rev 14:523–535 222. Jorens PG, Van Damme J, De Backer W et al (1992) Interleukin 8 (IL-8) in the bronchoalveolar lavage fluid from patients with the adult respiratory distress syndrome (ARDS) and patients at risk for ARDS. Cytokine 4:592–597 223. Van Damme J, Wuyts A, Froyen G et  al (1997) Granulocyte chemotactic protein-2 and related CXC chemokines: from gene regulation to receptor usage. J Leukoc Biol 62:563–569 224. Cummings CJ, Martin TR, Frevert CW et  al (1999) Expression and function of the chemokine receptors CXCR1 and CXCR2 in sepsis. J Immunol 162:2341–2346 225. Sylvester I, Suffredini AF, Boujoukos AJ et al (1993) Neutrophil attractant protein-1 and monocyte chemoattractant protein-1 in human serum. Effects of intravenous lipopolysaccharide on free

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E.T. Chiang et al. 245. Mendez MP, Morris SB, Wilcoxen S, Greeson E, Moore B, Paine R 3rd (2006) Shedding of soluble ICAM-1 into the alveolar space in murine models of acute lung injury. Am J Physiol Lung Cell Mol Physiol 290:L962–L970 246. Witkowska AM, Borawska MH (2004) Soluble intercellular adhesion molecule-1 (sICAM-1): an overview. Eur Cytokine Netw 15:91–98 247. Ronald JA, Ionescu CV, Rogers KA, Sandig M (2001) Differential regulation of transendothelial migration of THP-1 cells by ICAM-1/ LFA-1 and VCAM-1/VLA-4. J Leukoc Biol 70:601–609 248. Mastruzzo C, Crimi N, Vancheri C (2002) Role of oxidative stress in pulmonary fibrosis. Monaldi Arch Chest Dis 57:173–176 249. Constans J, Conri C (2006) Circulating markers of endothelial function in cardiovascular disease. Clin Chim Acta 368:33–47 250. Demir T, Yalcinoz C, Keskinel I, Demiroz F, Yildirim N (2002) sICAM-1 as a serum marker in the diagnosis and follow-up of treatment of pulmonary tuberculosis. Int J Tuberc Lung Dis 6:155–159 251. Baumer I, Zissel G, Schlaak M, Muller-Quernheim J (1998) Soluble intercellular adhesion molecule 1 (sICAM-1) in bronchoalveolar lavage (BAL) cell cultures and in the circulation of patients with tuberculosis, hypersensitivity pneumonitis and sarcoidosis. Eur J Med Res 3:288–294 252. Grigg J, Riedler J, Robertson CF (1999) Bronchoalveolar lavage fluid cellularity and soluble intercellular adhesion molecule-1 in children with colds. Pediatr Pulmonol 28:109–116 253. Samransamruajkit R, Prapphal N, Deelodegenavong J, Poovorawan Y (2005) Plasma soluble intercellular adhesion molecule-1 (sICAM1) in pediatric ARDS during high frequency oscillatory ventilation: a predictor of mortality. Asian Pac J Allergy Immunol 23:181–188 254. Conner ER, Ware LB, Modin G, Matthay MA (1999) Elevated pulmonary edema fluid concentrations of soluble intercellular adhesion molecule-1 in patients with acute lung injury: biological and clinical significance. Chest 116:83S–84S 255. Nonas SA, Finigan JH, Gao L, Garcia JG (2005) Functional genomic insights into acute lung injury: role of ventilators and mechanical stress. Proc Am Thorac Soc 2:188–194 256. Flores C, Ma SF, Maresso K, Wade MS, Villar J, Garcia JG (2008) IL6 gene-wide haplotype is associated with susceptibility to acute lung injury. Transl Res 152:11–17 257. Chung KF (2005) Inflammatory mediators in chronic obstructive pulmonary disease. Curr Drug Targets Inflamm Allergy 4: 619–625 258. Mortensen RF (2001) C-reactive protein, inflammation, and innate immunity. Immunol Res 24:163–176 259. Park WY, Goodman RB, Steinberg KP et al (2001) Cytokine balance in the lungs of patients with acute respiratory distress syndrome. Am J Respir Crit Care Med 164:1896–1903 260. Marshall RP, Webb S, Hill MR, Humphries SE, Laurent GJ (2002) Genetic polymorphisms associated with susceptibility and outcome in ARDS. Chest 121:68S–69S 261. Samal B, Sun Y, Stearns G, Xie C, Suggs S, McNiece I (1994) Cloning and characterization of the cDNA encoding a novel human pre-B-cell colony-enhancing factor. Mol Cell Biol 14: 1431–1437 262. Ognjanovic S, Bao S, Yamamoto SY, Garibay-Tupas J, Samal B, Bryant-Greenwood GD (2001) Genomic organization of the gene coding for human pre-B-cell colony enhancing factor and expression in human fetal membranes. J Mol Endocrinol 26:107–117 263. Kamp R, Sun X, Garcia JG (2008) Making genomics functional: deciphering the genetics of acute lung injury. Proc Am Thorac Soc 5:348–353 264. McClintock D, Zhuo H, Wickersham N, Matthay MA, Ware LB (2008) Biomarkers of inflammation, coagulation and fibrinolysis predict mortality in acute lung injury. Crit Care 12:R41 265. Ware LB, Fang X, Matthay MA (2003) Protein C and thrombomodulin in human acute lung injury. Am J Physiol Lung Cell Mol Physiol 285:L514–L521

Chapter 13

Ion Channels and Transporters in the Pulmonary Vasculature: A Focus on Smooth Muscle Amy L. Firth and Jason X.-J. Yuan

Abstract  Spanning the plasmalemmal membrane of all cells are many ion channels, exchangers, and transporters, all interacting to control vascular tone via the regulation of cytosolic Ca2+ concentration. Among these channels are voltagegated K+, Na+, and Ca2+ channels, voltage-independent Ca2+ and nonselective cation channels, Cl− channels, and ligandgated ion (K+, Ca2+, and Na+) channels. Plasmalemmal Ca2+–Mg2+ -ATPase (or Ca2+ pump) and Na+–K+ -ATPase (or Na+ pump) and an array of exchangers – the Na+–Ca2+ exchanger (NCX), Na+–H+ exchanger (NHE), and Na+–Mg2+ exchanger – all coexist to maintain ionic homeostasis in the cell. Comprehensive studies of ion channel function under both normal and pathophysiological conditions have been made possible owing to the development of cell isolation and electrophysiological techniques. This chapter introduces the ion channels and transporters present in the pulmonary vasculature (focusing on their expression and function) and discusses the potential pathogenic role of ion channels and transporters in the development of pulmonary hypertension. Keywords  Membrane potential • Potassium channel • Calcium channel • Sodium-calcium exchanger • Sodium pump • Calcium pump • Channelopathy

and Na+–K+-ATPase (or Na+ pump) and an array of exchangers – the Na+–Ca2+ exchanger (NCX) [1], Na+–H+ exchanger (NHE) [2], and Na+–Mg2+ exchanger [3] – all coexist to maintain ionic homeostasis in the cell. Comprehensive studies of ion channel function under both normal and pathophysiological conditions have been made possible owing to the development of cell isolation and electrophysiological techniques [4]. Pulmonary artery smooth muscle cells (PASMCs) have been shown to actively maintain an elevated cytosolic K+ concentration (approximately 140 mM) and a low cytosolic Na+ concentration (approximately 10  mM), whereas the resting cytosolic Cl− concentration is approximately 50 mM (Table 1). PASMCs have a unique resting permeability to ions; a higher basal permeability to both Cl− and Na+ relative to K+ helps to explain why the resting membrane potential (Em) of −40 to −60 mV is substantially less negative than predicted by the equilibrium potential for K+ (EK = −83 mV). It is beyond the scope of this chapter to discuss in detail the fundamental biophysical properties of cells; however, it is essential to describe some background to understand key aspects of resting Em. To comprehend the contribution of each ion to Em in PASMCs, it important to appreciate their equilibrium potential, as determined by the Nernst equation:

1 Introduction Spanning the plasmalemmal membrane of all cells are many ion channels, exchangers, and transporters, all interacting to control vascular tone via the regulation of cytosolic Ca2+ concentration. Among these channels are voltage-gated K+, Na+, and Ca2+ channels, voltage-independent Ca2+ and nonselective cation channels, Cl− channels, and ligand-gated ion (K+, Ca2+, and Na+) channels. Plasmalemmal Ca2+–Mg2+-ATPase (or Ca2+ pump) A.L. Firth (*) Laboratory of Genetics, The Salk Institute of Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA, 92037, USA e-mail: afirth @salk.edu

Eion =

RT [X ]o . log zF [X ]i

Eion reflects the equilibrium potential of ion X, R is the gas constant, T is the temperature (in kelvins), z is the valence of the ion, F is the Faraday constant, and [X]o and [X]i are the concentrations of ion X in the extracellular space and in the cytoplasm, respectively. At body temperature (37°C), RT/zF is constant and equal to 60.8 mV. The equation assumes that the membrane is permeable to a given ion for this ion to generate a transmembrane potential. A schematic illustration of the electrochemical gradients for K+, Na+, and Cl− that contribute to Em as determined by data collected from rabbit PASMCs is featured in Fig.  1 [5]. At resting membrane potential a net cytoplasmic loss of K+ and Cl− and a net gain of Na+ occurs. If the cell membrane were solely permeable to

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_13, © Springer Science+Business Media, LLC 2011

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A.L. Firth and J.X.-J. Yuan Table 1  Cytosolic electrolyte concentrations determined by various methods in different types of vascular smooth muscle cells Species SMC type [Na+]cyt (mM) [K+]cyt (mM) [Cl−]cyt (mM) Methoda Rat

Dog Rabbit

Portal vein Aorta A7r5 cells Mesenteric artery Carotid artery Aorta Pulmonary artery

45 74 4.4 11 68 70 15

138 110

86

88 129 134

70 54 51

A A B A A A A

Mean ± SD 41.1 ± 30.5 119.8 ± 20.8 65.2 ± 16.1 Method of determination: atomic absorption spectrophotometry and/or flux measurements using radioisotopes (A); use of the ionsensitive fluorescent indicator SFBI Na+ binding benzofuran isophthalate (B) SMC smooth muscle cell, SD standard deviation a

Fig. 1  Electrical and chemical gradients for K+, Na+, Ca2+, and Cl− in pulmonary artery smooth muscle cells (PASMCs). Resting membrane potential (Em) for a PASMC is −40 to −60 mV. EK, ENa ECa, and ECl represent the reversal potentials for K+, Na+ Ca2+, and Cl−, respectively

K+, then Em would be directly equal to EK. As previously mentioned, Em is actually more depolarized (or less negative than EK), being in the region of −50 mV in PASMCs; therefore it is indicative of other ion conductances contributing. The constant-field Goldman equation quantitatively analyses the contribution of all three of these ions to Em,

Em =

P [K ]o + PNa [Na ]o + PCl [Cl ]i RT log K , zF PK [K ] i + PNa [Na ]i + PCl [Cl]o

where PK, PNa, and PCl are the absolute membrane permeabilities to K+, Na+, and Cl− in centimeters per second and [X]o and [X]i are the extracellular and cytoplasmic concentrations of each ion in millimoles per liter. This chapter will introduce the ion channels and transporters present in the pulmonary vasculature, focusing on their expression and function.

2 Calcium Channels and Transporters Pulmonary arteries function to regulate oxygenation of the blood by changing vessel contractility to control blood flow.

Cytosolic Ca2+ concentration is critical for the regulation of vessel tone. During homeostasis, cytosolic Ca2+ concentration is maintained at approximately 100 nM, creating a gradient greater than 10,000-fold with the extracellular environment (approximately 1.6 mM) [6]. There are several fundamental systems that coordinate to control cytosolic Ca2+ concentration: (1) extracellular Ca2+ entry via voltage-dependent Ca2+ channels (VDCCs), (2) receptor-operated cation channels (ROCs), (3) store-operated channels (SOCs) activated by depletion of the sarcoplasmic reticulum (SR), (4) Ca2+ release and sequestration from and into the SR, (5) Ca2+-ATPase and NCX responsible for plasmalemmal Ca2+ efflux, and (6) mitochondrial Ca2+ release and sequestration.

2.1 Role of Intracellular Ca2+ in PASMC Contraction and Proliferation Intracellular Ca2+ is involved in (or required for) (1) muscle contraction (skeletal, cardiac, and smooth muscle) and cell motility, (2) neurotransmitter release, neuronal excitability, learning and memory, (3) fertilization and development, (4)

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cell proliferation, differentiation, and apoptosis, and (5) gene transcription. Ca2+ signals are precisely regulated to coordinate all of these functions. The contractile characteristics and the mechanisms that cause contraction of vascular smooth muscle (including pulmonary vascular smooth muscle) are very different from those for cardiac muscle and skeletal muscle. PASMCs undergo slow, sustained, and tonic contractions. The major contractile apparatus (or contractile proteins) in PASMCs are actin and myosin, which are not organized into distinct bands as they is in cardiac muscle and skeletal muscle cells, but are well organized for maintaining sustained and tonic contractions and reducing lumen diameter. Contraction in PASMCs can be initiated by two major mechanisms: (1) electromechanical coupling, the mechanism that causes contraction or relaxation through changes in Em, and (2) phamacomechanical coupling, the complex of mechanisms that can cause contraction or relaxation independent of changes in Em. Regardless of the initial mechanisms, an increase in cytosolic Ca2+ concentration is the major trigger for smooth muscle cell contraction and pulmonary vasoconstriction. When cytosolic Ca2+ concentration rises, Ca2+ binds to calmodulin (CaM) and forms a Ca2+/CaM complex, which activates myosin light chain kinase to phosphorylate the myosin light chain. This phosphorylation increases the activity of myosin ATPase, which hydrolyzes ATP, thereby releasing energy. The subsequent cycling of the myosin cross-bridges (actomyosin) produces displacement of the myosin filament in relation to the actin filament causing contraction (Fig. 2). The phosphorylated myosin light chain can be dephosphorylated by myosin light chain phosphatase (MLCP), leading to

smooth muscle cell relaxation. Increased Ca2+/CaM in the cytosol also activates RhoA/Rho kinase, which subsequently phosphorylates and inactivates MLCP. Therefore, activation of the RhoA/Rho kinase pathway can also cause smooth muscle contraction by inactivating MLCP (Fig. 2). In addition to causing PASMC contraction, cytosolic Ca2+ also stimulates cell proliferation by activating Ca2+-sensitive signal transduction proteins (e.g., CaM kinase and mitogen-activated protein kinase) and transcription factors (e.g., nuclear factor of activated T cells, cyclic AMP response element binding protein, c-Fos/c-Jun, and nuclear factor kB) (Fig. 3). Since the nuclear envelope has high permeability to Ca2+, a rise in cytosolic Ca2+ concentration can rapidly increase nuclear Ca2+ concentration and activate Ca2+-sensitive pathways in the nucleus. Maintaining a sufficient level of Ca2+ in the SR (or endoplasmic reticulum, ER) is also critical for cell proliferation; the Ca2+– Mg2+-ATPase (or Ca2+ pump) on the SR/ER membrane (SERCA) is the major active transporter that sequesters cytosolic Ca2+ into the SR/ER and maintains a high Ca2+ concentration (approximately 0.1–0.5 mM) in the SR/ER. The Ca2+ concentration in the SR/ER may regulate cell proliferation by modulating the amplitude and frequency of Ca2+ signals in the cytosol and nucleus. Ca2+ also participates in regulating PASMC proliferation through its intimate involvement with the cell cycle. There are four Ca2+/CaM-sensitive steps in the cell cycle: (1) transition from G0 (resting state) to G1 phase (the beginning of DNA synthesis); (2) from G1 to S phase (an interphase during which replication of the nuclear DNA occurs); (3) from G2 to M phase (mitosis); and (4) through M phase. A rise in cytosolic Ca2+ concentration in the PASMC, which also rapidly increases nuclear Ca2+ concentration and gradually enhances the

Fig.  2  Sequence of events for Ca2+-mediated smooth muscle contraction. When cytosolic Ca2+ concentration rises, Ca2+ binds to calmodulin (CaM), which activates myosin light chain kinase to phosphorylate the myosin light chain. This phosphorylation increases the activity of myosin ATPase, which hydrolyses ATP, thereby releasing energy. The subsequent cycling of the myosin cross-bridges

(phosphorylated actomyosin) produces displacement of the myosin filament in relation to the actin filament, causing contraction. A rise in cytosolic Ca2+ concentration and activation of the small GTPase RhoA also activate RhoA/Rho kinase, which subsequently inactivates myosin light chain phosphatase and causes contraction (or inhibit relaxation)

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concentration of intracellularly stored Ca2+ in the SR/ER, plays an important role in facilitating quiescent cells to get into the cell cycle and propelling cell passage through the cell cycle.

A.L. Firth and J.X.-J. Yuan

Therefore, an increase in cytosolic Ca2+ concentration not only induces pulmonary vasoconstriction by triggering PASMC contraction, but also mediates pulmonary vascular remodeling by stimulating proliferation and migration of PASMCs (and other cell types). Sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling are the two major causes for the elevated pulmonary vascular resistance in patients with pulmonary arterial hypertension (PAH; Fig. 3).

2.2 Regulation of Cytosolic Ca2+ Concentration in PASMCs 2.2.1 Voltage-Dependent Ca2+ Channels

Fig. 3  An increase in cytosolic Ca2+ concentration causes PASMC contraction and proliferation. When cytosolic Ca2+ concentration rises, Ca2+ binds to CaM, which causes PASMC contraction by activating myosin light chain kinase. Cytosolic Ca2+ also activates CaM kinase and mitogen-activated protein kinase, as well as various transcription factors (e.g., nuclear factor of activated T cells, cyclic AMP response element binding protein, AP-1 family members, and nuclear factor kB), to facilitate cell passage through the cell cycle and cause cell proliferation. MLCP myosin light chain phosphatase, PAFB pulmonary artery fibroblasts, PVR pulmonary vascular resistance, PAP pulmonary arterial pressure, SR sarcoplasmic reticulum, ER endoplasmic reticulum

Fig. 4  Structure and function of voltage-dependent calcium channels (VDCCs). (a)A planar representation of four VDCC subunits coassembled to form a channel. (b) A VDCC channel in the plasma membrane with associated subunits. The pore region forming between the S5 and S6 subunits is indicated. (c) Representative current–voltage (I–V) relationship for a VDCC in control (squares), when activated by BAYK8644 (triangles), and when inhibited by dihydropyridines (circles). (c Reproduced from Moosmang et al. [155] with permission from Macmillan Publishers Ltd, copyright 2003)

Changes in the ionic gradients determine the plasmalemmal membrane potential, which is largely regulated by K+ channels (discussed in Sect. 3). Depolarization drives Ca2+ entry in a process known as excitation–contraction coupling. In excitation– contraction coupling, VDCCs are activated when depolarization surpasses a threshold; this varies among vascular beds owing to the complement of ion channels present in the plasmalemmal membrane. Only a few millivolts is necessary to open VDCCs and allow Ca2+ influx, causing contraction [7]. VDCCs are ubiquitously expressed in vascular smooth muscle cells (VSMCs) and comprise four subunits each containing six transmembrane spanning domains (S1–S6) enclosed by N- and C-termini. A variety of subunits can coassemble, giving rise to heterogeneity of VDCCs; subunits include a1, b1–b4, g1–g8, and a2d1–a2d3. The a1 subunit, containing the Ca2+-selective pore (a pore-forming loop between S5 and S6) and voltage sensor (S4), is essential for channel function (Fig. 4). Sites for channel regulation by intracellular second messengers, toxins, and drugs also reside in the a

13  Ion Channels and Transporters in the Pulmonary Vasculature: A Focus on Smooth Muscle Table 2  Expression and function of voltage-dependent Ca2+ channels Subtype Voltage sensitivity a-Subunit gene Associated subunits L

T

Strong depolarization

Weak depolarization

Cav1.1(a1S) Cav1.2(a1C) Cav1.3(a1D) Cav1.4(a1F)

Cav3.1(a1G) Cav3.2(a1H) Cav3.3(a1I)

a2d b g1, g2 a2d

R

Strong depolarization

Cav2.3(a1E)

b1, b3, b4 Possibly g Not known

N P, Q

Strong depolarization Strong depolarization

Cav2.2(a1B) Cav2.1(a1A)

Not known Not known

subunit. The core a subunit is commonly associated with an intracellular b subunit and a disulfide-linked transmembrane a2d subunit. To date, ten encoding genes have been identified; Cav1.1–Cav1.4 (channels contain a1S, a1C, a1D, and a1F), Cav2.1–Cav2.3 (channels contain a1A, a1B, and a1E) and Cav3.1–Cav3.3 (channels contain a1G, a1H, and a1I). The combination of a1 subunits with the accessory subunits forms six functionally distinct subfamilies; the L-, N-, P-, Q-, R-, and T-type channels [8]. Table  2 provides a summary of the expression and pharmacological regulation of VDCCs in the pulmonary vasculature. Of these, the dihydropyridine-sensitive, high-voltage-activated and slowly inactivating L-type and the low-voltage-activated, rapidly inactivating T-type are most extensively studied in VSMCs. L-type (Cav1) channels have been substantially characterized in PASMCs and are important in elevating cytosolic Ca2+ concentration during hypoxia. Furthermore, differences in Ca2+ channel density and oxygen sensitivity have been observed along the pulmonary artery tree in studies using specific VDCC inhibitors and direct measurement of VDCC currents [9]. L-type channels are high-voltage-activated and have a 25-pS single channel conductance (Cav1.2b in the lung) in isotonic Ba2+ solution. A high-affinity binding site for inhibitors such as the dihydropyridines (nifedipine), the phenylalkylamines (verapamil), and the benzothiazepines (diltiazem) resides in the transmembrane a1 subunits close to the pore region [10]. Ca2+ channel blockers, such as nifedipine, are used as vasodilator therapies for pediatric PAH; a more variable response is observed in adults and significant side affects can be experienced. Inhibition of L-type channels in PASMCs attenuates voltage-gated Ca2+ influx and improves pulmonary arterial pressure in more than 40% of infants with PAH [11]. L-type channels are shown to be upregulated in hypoxia-induced pulmonary hypertension and associated with a Ca2+-dependent resistance [12]. The electrophysiological characteristics of VDCCs in PASMCs include an activation threshold between −40 and −50  mV,

227

Activators

Inhibitors

References

BAYK8644

Dihydropoyridines

[134]

Unknown

Phenylalkylamines Benzothiazepines Mibefradil

[8, 17]

Unknown Unknown Unknown

Amiloride Nickel SNX-482 Nickel w-Conotoxin-GVIA w-Agatoxin IVA

[8] [8] [8]

with the maximum inward current amplitude occurring at approximately 0 mV [13]. An example of the electrophysiological properties of the L-type VDCC is shown in Fig. 4. The other common Ca2+ channel in the pulmonary vasculature is the low-voltage-activated T-type channel (Cav3). Its single channel conductance of 8-pS is insensitive to the common L-type channel blockers and the physiological relevance of this channel in the pulmonary vasculature is presently undetermined. R-type (Cav2.3) currents have been shown to be activated by endothelin-1 [14] and cause enhanced cerebral artery constriction during subarachnoid hemorrhage [15, 16]. Recent studies have also suggested that R-type channels are important in the proliferation of human PASMCs [17]. The expression and function of T-type channels, however, remain poorly described in the pulmonary vasculature and their electrophysiological properties are yet to be fully characterized.

2.2.2 ROCs and SOCs Depletion of Ca2+ from the ER or SR is able to trigger an influx of Ca2+ across the plasmalemmal membrane. This phenomenon is known as store-operated Ca2+ entry and occurs via channels commonly termed “store-operated channels” (SOCs). Activation of membrane receptors by ligand binding, diacylglycerol and protein kinase C (PKC) can stimulate ROCs, leading to Ca2+ influx and Na+ efflux. Until recently the precise molecular entity of ROCs and SOCs has remained enigmatic. In the vasculature there is increasing evidence that transient receptor potential (TRP) channels are capable of forming functional ROCs and SOCs. In addition, Ca2+-release-activated Ca2+ channels (CRACs) have been identified as a functional Ca2+ entry pathway in several cell types. Perhaps TRP channels and CRACs are not mutually exclusive; recent studies have provided new evidence demonstrating a coexistence and interaction between CRACs and TRP channels in smooth muscle cells [18].

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TRP Channels Similar to VDCCs, TRP channels are part of the superfamily of six transmembrane spanning cation channels; however, they lack the voltage-sensing moiety. They function as voltageindependent, nonselective cation channels, being variably permeable to Na+, K+, Cs+, Li+, and Mg2+, in addition to the predominantly carried ion Ca2+ [19]. Several subtypes of TRP channels have been identified in the pulmonary vasculature on the basis of their activation stimuli and presence of regulatory domains in the N- and C-termini; these include the classical or canonical TRP (TRPC1–TRPC7), vanilloid-receptor-related TRP (TRPV1–TRPV4), and melastatin-related TRP (TRPM1– TRPM8) channels. Other TRP subfamilies, which tend to be associated with specific genetic disorders, are the polycystins (TRPP), mucolipidins (TRPML), the ankyrins (TRPA) and the mechanoreceptor potential C (TRPN) [20].

In VSMCs more than ten TRP isoforms have been detected; the function and expression of these channels are summarized in Table  3 and the membrane topology and representative currents are depicted in Fig.  5. Distinct domains in the N- and C-termini reflect the capability to form specific protein–protein interactions; some are ubiquitous, others are isoform-specific. All TRP isoforms, for example, have a series of ankyrin repeats; however, the number of repeats may differ among isoforms. Other functionally recognized domains include CaM-binding sites, present in TRPC1 and TRPC4, acting to regulate Ca2+dependent feedback inhibition of store-operated Ca2+ entry via TRPC1 [21]. TRPC1 is ubiquitously expressed in the vasculature and it is proposed to be one of the pore-forming subunits comprising SOCs in VSMCs [22, 23]. Furthermore, TRPC1 is a critical TRP isoform responsible for capacitative-Ca2+-entry-induced pulmonary vasoconstriction and

Table 3  Expression and function of transient receptor potential (TRP) channels Pulmonary expression Modulation Family Subunit Selectivity PCa/PNa

Antagonists

References

TRPC

Gd/La

[25, 27, 135–137]

TRPM

TRPV

TRPA

1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 1 2 3 4 5

Nonselective 2.7 1.6 7 9.5 5 1.9 Nonselective ~0.3 1.6 MVC-selective MVC-selective DVC-selective DVC-selective 3.3 3.8 3 2.6 6 >100

SMC, EC SMC, EC SMC, EC SMC, EC SMC, EC SMC, EC SMC, EC EC SMC SMC, EC SMC, EC – EC SMC, EC SMC, EC SMC, EC SMC, EC SMC SMC, EC –

6 1

>100 0.8

– ND

Store depletion, PLC pathway DAG Store depletion, DAG Store depletion? La3+, calmidazolium Store depletion? La3+, NO DAG, PIP3, Ca2+, 20-HETE Store depletion, DAG Translocation? ADP-ribose/AA/NAD/H2O2/Ca2+ Osmotic cell swelling/store depletion? Ca2+/voltage/PIP2 Ca2+/voltage/PIP2/heat Translocation ATP/H+/phosphorylation/PIP2 Cool/menthol/icilin/pH/PIP2 Heat/vanilloids/anandamide/PIP2/camphor Heat/osmotic cell swelling/exocytosis Warm/menthol/PUFAs Warm/osmotic cell swelling/5¢6¢-EET Low Ca2+ concentration/hyperpolarizaton/ exocytosis Store depletion, exocytosis Cold/icilin/isothiocuanates/cannabinoids/ bradykinin/DAG/PUFAs pH/Ca2+/proteolytic cleavage

BRP2/Gd/La Ca2+/2-APB 2-APB 2-APB/La Ca2+/La [137–139] Clotrimazale Mg2+ AMP/ATP Spermine Mg2+ Mg2+ Capsazepine Capsazepine DAT La Gd/La Ca2+/Cd Ca2+/Cd Amiloride

[137–139]

[137]

Amiloride? [137] 1 MVC-selective ND 2 – 3 – TRPP 2 Nonselective ND Ca2+/TRPP1/EGF/PIP2/actin cytoskeleton Flufenamate/Cd/La? [137] 3 4.3 Ca2+ Gd/La/Ni? 5 – – Question marks represent controversial observations.AA arachidonic acid, CaM calmodulin, DAG diacylglycerol, DAT 1,3-di(arylalkyl)thioureas, DVC divalent cation, EC pulmonary artery endothelial cells, PLC phospholipase C, MVC monovalent cation, ND not determined, PIP2 phosphoinositide 4,5-bisphosphate, PIP3 phosphatidyl 3,4,5-trisphosphate, PUFAs polyunsaturated fatty acids, SMC pulmonary artery smooth muscle cell, 2-APB 2-Aminoethoxydiphenyl Borate, 20-HETE 20-Hydroxy Arachidonic Acid, 5'6' EET 5′6′ Epoxyeicosatrienoic acid, EGF Epidermal Growth Factor, BRP2 TRPML

13  Ion Channels and Transporters in the Pulmonary Vasculature: A Focus on Smooth Muscle

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Fig. 5  Structure and function of transient receptor potential (TRP) channels. (a) Planar views of TRPC1, TRPV1, and TRPM1 channel subunits. (b) Representative traces reflecting changes in cytosolic Ca2+ concentration recorded in smooth muscle cells, capacitative calcium entry subsequent to cyclopiazonic acid (CPA)-dependent depletion of the endoplasmic reticulum/sarcoplasmic reticulum (ER/SR) Ca2+ stores (in the absence of extracellular Ca2+). The lower panel shows representative current traces and I–V curves recorded in human PASMCs

overexpressing TRPC6 before and after CPA exposure. (c) A currently proposed functional interaction between TRP channels, STIM, and Orai. Activation of G-protein-coupled receptors (GPCRs) increases inositol 1,4,5-trisphosphate mediated depletion of Ca2+ from the ER/ SR causing STIM dimerization and redistribution to discrete puncta in the ER/SR membrane in closer proximity to the plasma membrane where interactions with Orai (and TRP) channels can occur increasing Ca2+ influx

PASMC proliferation [24, 25]. In addition to forming homomultimeric channels, TRPC1 is also reported to form heteromultimeric channels with TRPC5, TRPC3, and TRPP2; such interaction may be crucial to the trafficking or translocation of TRPC1 to the plasma membrane [26]. TRPC4 is widely expressed and is known to be present in VSMCs [27]; however, its role is more pertinent in the endothelium, where it regulates vascular permeability and vasorelaxation [28, 29]. TRPC1, TRPC4, and TRPC6 protein expression varies with the location in the pulmonary vascular tree: higher expression was observed in the distal pulmonary artery than in the proximal pulmonary artery; this correlated with changes in cytosolic Ca2+ concentration occurring during hypoxic pulmonary vasoconstriction (HPV) [30]. TRPM2–TRPM4, TRPM7, TRPM8, and TRPV1– TRPV4 have all been detected using reverse transcription PCR in the pulmonary artery [31]. For more detail on TRP channels in the pulmonary vasculature, please refer to Firth et al. or Townsley et al. [32, 33].

Ca2+-Release-Activated Ca2+ Channels Recent evidence suggests a significant role for CRACs in voltage-independent Ca2+ influx into cells and it is postulated that this may contribute to SOCs in PASMCs [18]. Orai-1 is the fundamental CRAC pore-forming subunit in the plasma membrane [34]. ER/SR Ca2+ concentration is sensed by stromal interaction molecule 1 (STIM-1) and upon store depletion it oligomerizes, enabling it to redistribute to discrete sites in the ER/SR membrane in close proximity to the plasma membrane [35]. The precise functional association between STIM-1 and Orai-1 is currently unknown and the mechanism of Ca2+ influx mediated by Orai-1 remains to be fully elucidated.

2.2.3 Plasmalemmal Ca2+-ATPase The PMCA is a plasmalemmal P-type Ca2+-ATPase regulated by CaM which extrudes Ca2+ from cells in VSMCs to

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maintain Ca2+ homeostasis. It is encoded by four genes (PMCA-1, PMCA-2, PMCA-3, and PMCA-4), of which PMCA-4 and, to a lesser extent, PMCA-1 are expressed by vascular smooth muscle [36]. Alternative splicing of these four genes has created 20 isoforms of the PMCA. Each PMCA is a ten transmembrane spanning domain with cytosolic N- and C-termini. The CaM-binding site is located in a key regulatory domain in the C-termini along with PKC and protein kinase A (PKA) phosphorylation sites [37]. Interestingly, its activity has been shown to increase in the presence of oxidants, such as hydrogen peroxide (1  mM), and matrix metalloprotease 2 in PASMCs [38]. The pump has recently been purified from bovine PASMCs, which will open the door for more detailed studies on its ion transport mechanisms [39].

2.2.4 Na+–Ca2+ Exchanger The NCX represents the second system functioning to export Ca2+ from intact cells. The NCX has a much larger capacity than the PMCA and its importance as a primary mechanism for Ca2+ extrusion has been acknowledged by four dedicated international conferences. The expression and function of the NCX have been widely documented in both normal and diseased PASMCs and pulmonary artery endothelial cells [40–42]. The NCX operates in parallel with Ca2+ channels and pumps in the plasma membrane and can move Ca2+ either into or out of cells. Its activity is regulated by the electrochemical gradient across the membrane using Na+ gradients as a source of energy. The NCX is noticeably inhibited in PASMCs exposed to mild hypoxia, decreasing the rate of Ca2+ extrusion from the cytosol [43]. There are three currently identified isoforms of the NCX (NCX1–NCX3) and a further four isoforms for a potassiumdependent NCX (NCKX) [44]. The isoforms have a similar overall structure consisting of an intracellular loop, which connects to a five transmembrane domain at the N-terminus and a six transmembrane domain at the C-terminus. Messenger RNA (mRNA) for the NCX1 and NCKX3 isoforms has been isolated from cultured human PASMCs [45]. This study also reported that the NCX could contribute to Ca2+ entry owing to NCX activity in the reverse mode (inhibited by KB-R9756). At this stage it was predicted that blockade of the reversemode NCX may be a possible therapeutic target in the treatment of pulmonary hypertension [45]. More recent evidence, however, suggests that a contractile response, resembling that observed in HPV induced by the removal of extracellular Na+ in intact pulmonary arteries, was not due to inhibition of the NCX and thus may not be supportive of a direct role for the NCX in HPV [46].

A.L. Firth and J.X.-J. Yuan

2.2.5 Functional Interaction of TRPC Channels and NCX in Caveolae Caveolae are cholesterol- and sphingolipid-rich flask-shaped invaginations of the plasma membrane, whose principal structural component is caveolin. In the vasculature, caveolae appear to be more prominent in endothelial cells than in smooth muscle cells. However, the number of caveolae and the expression level of caveolin are significantly increased in PASMCs in patients with idiopathic PAH (Fig.  6a). The “simplest” physiological role of caveolae and caveolin is to centralize, concentrate, and colocalize cooperative receptors, signaling molecules/proteins, and effectors (e.g., ion channels and transporters) within microdomains. A caveolin-scaffolding domain (CSD) on caveolin itself can associate with membrane proteins. The TRPC1 channel proteins contain the C-terminal CSD-consensus binding sequence that allows for their physical and functional interaction with caveolin-1 (cav-1) in the caveolae of PASMCs (Fig. 6b). Cav-1 also binds to an N-terminal cav-1-binding motif (amino acids 322–349) of TRPC1 (Fig. 1a). Truncation of cav-1 (i.e., removal of CSD, membrane anchoring domain, and palmitoylation sites) significantly decreases store-operated Ca2+ entry through TRPC channels and downregulates TRPC1 and cav-1 expression. The presence of TRPC channels and NCX proteins in these caveolar lipid raft domains seems to constitute an important factor in their functional roles in regulating intracellular Ca2+. In addition to functioning as Ca2+ channels, TRPC channels are also permeable to Na+. TRPC-mediated Na+ influx into the cytosol of PASMCs, such as that provoked by activation of G-protein-coupled receptors (GPCRs) (Fig. 6c), is one of the major mechanisms to increase cytosolic Na+ concentration. A rise in cytosolic Na+ concentration (especially in the submembrane area) functionally converts the forward mode of NCX (inward Na+ and outward Ca2+ transportation) to the reverse model (outward Na+ and inward Ca2+ transportation). The inward Ca2+ transportation through the reverse mode of NCX plays an important role in maintaining a high level of cytosolic Ca2+ and refilling the SR/ER with Ca2+ via SERCA (Fig. 6c). By binding with cav-1 in both N- and C-termini, TRPC can be efficiently regulated upon activation of receptors. As shown in Fig. 6c, accumulated ligand (e.g., growth factors, mitogenic and angiogenic ligands, and vasoactive substances) in caveolae activates the receptors (GPCRs or receptor tyrosine kinases) located in caveolae and increases synthesis of inositol 1,4,5-trisphosphate (IP3) and diacylglycerol. By binding with IP3 receptors (IP3R) on the ER/SR membrane, the second-messenger IP3 induces a transient increase in cytosolic Ca2+ concentration owing to Ca2+ release from the SR/ER to the cytosol. Opening of the Ca2+ release channels or IP3R is an efficient way to deplete Ca2+ from the

13  Ion Channels and Transporters in the Pulmonary Vasculature: A Focus on Smooth Muscle

231

Fig. 6  Functional coupling of receptors, TRPC channels, and Na+–Ca2+ exchangers (NCX) in caveolae in PASMCs. (a) Ultrastructure of plasma membrane of PASMCs isolated from normal subjects and patients with idiopathic pulmonary arterial hypertension (IPAH) was assessed by electron microscopy. IPAH PASMCs have increased flask-like invaginations of the plasma membrane consistent with the morphology of caveolae (indicated by arrows) and as determined by the number of caveoli per membrane length. (b, c) The caveolin-1 binding domains in

TRPC1 (b) and the potential mechanisms (c) that are involved in ligandmediated regulation of cytosolic Ca2+ concentration through GPCRs, TRP channels, and NCX in caveolae. CBM caveolin-1-binding motif, PBD protein 4 binding domain, CSD caveolin-scaffolding domain, PM plasma membrane, ER/SR endoplasmic reticulum and sarcoplasmic reticulum, G, G protein, IP3 inositol 1,4,5-trisphosphate, IP3R inositol 1,4,5-trisphosphate receptor, SERCA the sarcoplasmic reticulum/endoplasmic reticulum Ca2+–Mg2+-ATPase, Cav-1 caveolin-1

intracellular stores (i.e., SR or ER). The store depletion then activates TRPC-encoded SOCs, which are located in caveolae, and promotes Ca2+ influx or causes store-operated Ca2+ entry. Since TRPC channels are also highly permeable to Na+, the local accumulation of intracellular Na+ would then activate the reverse mode of Na+–Ca2+ exchange and further enhance Ca2+ entry (Fig. 6c). The proximity of the receptor, the channel, the transporter, and the caveolin allows the ligand-mediated increase in cytosolic Ca2+ concentration to be efficiently and rapidly regulated for signal transduction and for stimulating functional changes of the cell.

nary vasculature are (1) voltage-gated K+ channels (KV), (2) large-, intermediate-, and small-conductance Ca2+-activated K+ channels (BKCa, IKCa, and SKCa respectively), (3) ATPsensitive K+ channels (KATP), and (4) two-pore-domain K+ channels (K2P). The Na+–K+-ATPase (or Na+ pump) is the chief mechanism for Na+ extrusion, transporting both Na+ and K+ against their concentration gradients.

3 Potassium Channels and Transporters Potassium channels are the key regulators of cellular excitability, being fundamentally involved in the regulation of cellular Em. Efflux of K+ after activation of K+ channels leads to membrane hyperpolarization, inhibition of VDCCs, and vasodilation. A small change in K+ channel activity can have a substantial effect on membrane potential and thus factors targeting K+ channels can modulate vessel tone [47]. The main families of potassium channels identified in the pulmo-

3.1 Voltage-Gated K+ Channels Voltage-gated K+ channels, or KV channels, are the most diverse group of K+ channels ubiquitously expressed in VSMCs. There are currently over 40  KV channel members grouped into 12 subfamilies (the properties are summarized in Table  4). KV channels are tetramers of six transmembrane spanning proteins formed from symmetrically arranged KV a subunits. They align in either in a homomultimeric or in a heteromultimeric combination, surrounding a central pore highly selective to the transportation of K+ across the cell membrane (depicted in Fig. 7a) [48]. The voltage sensor is located in the S4 transmembrane domain and contains a preserved region of positive arginine or lysine amino acid residues (repeats of Arg

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Table 4  Expression and function of K+ channels in the pulmonary vasculature Family Subunits Accessory subunits Activators Inhibitors KV

BKCa

KATP

Antagonists

References

Kv1.1–Kv1.7

Kvb1

Depolarization

Hyperpolarization

4-AP

[69, 130, 131, 140–145]

Kv1.10 Kv2.1

Kvb2

PKA

PKC

Kv3.1b/3.3/3.4

Kvb3

Kv4.1.1/4.2 Kv5.1 Kv6.1–Kv6.3 Kv9.1/9.3 Kv10.1 Kv11.1-1.7 Slo1/KCNMA1

Decreased pHcyt Increased cytosolic Ca2+ concentration Cytochrome c Rho kinase Hypoxia

CytochromeP450 inhibitors (clorimazole) Correolide

b1

PKG/PKA

PKC

b2 b3 b4

NO EETS CO AA PKA/PKG

ROS Decreased pHcyt c-Src

CGRP Decreased ATP Pinacidil Depolarization Increased pHcyt PKA

Ca2+-activated PP2B PKC

Kir6.1

SUR2B

Kir6.2

K2PD

Increased ATP

IBTX, ChTX, paxilline, TEA (>1 mM) PDE inhibitors

[142, 146–149]

Glybenclamide, tolbutamide

[76, 143]

TASK1/2 Not yet identified Hyerpolarization Zn2+, anandamide [86–88, 150] THIK-1 Decreased pHex TRAAK TREK-1/2 TWIK-1/2 KCNQ KCNQ1 Not yet identified Depolarization Hyperpolarization [79, 80, 143] 4-AP aminopyridine, AA arachidonic acid, CO carbon monoxide, CGRP calcitonin gene related peptide, EETS epoxyeicosatrinoic acids, NO nitric oxide, PKA protein kinase A, PKC protein kinase C, PKG protein kinase G, PDE phosphodiesterase, PP2B protein phosphatase 2B, ROS reactive oxygen species, SUR2B sulfonylurea receptor subunit 2B, TEA tetraethylammonium, IBTX Iberiotoxin, CHTX Charybdotoxin

Fig. 7  Structure and function of K+ channels. (a) Planar membrane topologies of single potassium channel subunits for KV, BKCa, and Kir channels, respectively. The pore-forming loop and the voltage sensor in transmembrane unit 4 are indicated. (b) Representative current traces recorded in human pulmonary arterial smooth muscle cells indicating diversity in the Kv currents; from left to right, (1) rapidly activating and slowly inactivating IK(V), (2) rapidly activating and noninactivating IK(V), (3) slowly activating and noninactivating IK(V), and (4) rapidly activating and rapidly inactivating IK(V). (b Reproduced with permission [142])

13  Ion Channels and Transporters in the Pulmonary Vasculature: A Focus on Smooth Muscle

or Lys-Xaa-Xaa(7)) essential for depolarization-dependent opening of the channel [49]. Selectivity to K+ is energetically favored through a flexible pore lined by a backbone of carbonyl groups, a sequence highly conserved within the K+ channel family [50]. Although the cytoplasmic C-terminus differs among KV channel a subunits, the cytoplasmic N-terminus is highly conserved throughout the KV channel family. This 120 amino acid domain contains a tetramerization domain (T1-S1) comprising the molecular determinants for the formation of functional tetrameric channels from specific a-subunit assembly [51]. The tetrameric T1 domain exists as a region distinct from the transmembrane channel and, owing to a structural resemblance, is commonly referred to as a ball and chain structure [52]. The T1 domain is fundamental in influencing the channel conformation, gating, and electrophysiological properties [53]. KV channels are activated by depolarization and are central to the regulation of resting membrane potential in PASMCs [54, 55]. Whole-cell currents carried by KV channels, denoted IKv, are determined by the following equation,

I KV = N × Popen × iK ,

where N denotes the total number of KV channels, Popen is the steady-state open probability of a KV channel, and iK is the current amplitude through a single KV channel. Decreased IKv may occur owing to (1) a decrease in the total channel protein due to transcriptional inhibition of the KV genes via transcriptional silencer elements (e.g., the KV1.5 repressor element in the promoter region of the gene) [56, 57], (2) a decrease in the functional surface expression of KV channels due to inhibition of channel trafficking by the KV channel interacting proteins (e.g., KChIP) [58] or due to the inhibition of a–a subunit assembly via the cytoplasmic N-terminal tetramerization domain (T1) [51], (3) association with the regulatory b subunits either decreasing activation and/or increasing inactivation of KV channels [59, 60], (4) inhibition of channel activity by PKC-mediated phosphorylation of the channel a and b subunits [61], (5) pharmacological blockade of the KV channels by selective [e.g., 4-aminopyridine (4-AP), bepridil, and correolide] [62, 63] and nonselective (e.g., nicotine, endothelin-1, serotonin, fenfluramine, acute hypoxia, and dichloroacetate) inhibitors [64–66], and (6) transcriptional/translational and functional inhibition of KV channels by antiapoptotic proteins (e.g., Bcl-2) [67]. In PASMCs, a heterogeneity of KV channel currents exists [68, 69] (Fig. 7b). Different KV channel currents were recorded by Smirnov et al. in PASMCs isolated from conduit pulmonary arteries. Sixty-seven percent of the cell population comprised a large, rapidly activating IKv inhibited by 4-AP but insensitive to tetraethylammonium (TEA) (termed IKv1) and 33% of the cells expressed a smaller, slower activating current which was more sensitive to TEA than to 4-AP (termed IKv2).

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Resistance pulmonary arteries exhibited a third more uniform current resembling that of IKv1, but distinguished by a higher current density, a significantly larger time constant for activation, and more negative IKv activation and inactivation [69].

3.2 Ca2+-Activated K+ Channels Ca2+-activated K+ (KCa) channels are subcategorized on the basis of their conductance; large (BK), intermediate (IK), and small (SK). Like KV channels, BKCa channels also form tetramers which consist of four a and four b subunits. BKCa channels are ubiquitously expressed in VSMCs and can be activated by changes in both membrane potential and intracellular Ca2+ concentration[70]. The a subunit has seven transmembrane domains and features extracellular N-termini and intracellular C-termini [71]. The channel is regulated by the b subunit, which comprises two membrane-spanning domains separated by an extracellular loop. BKCa channels may act as a negative-feedback mechanism in response to depolarization and thus increased cytosolic Ca2+ concentration during vasoconstriction. Cox and Rusch showed that inhibition of Ca2+ efflux decreased the BKCa current and increased the KV current, indicating that cytosolic Ca2+ levels are critical regulators of these channels [72]. BKCa channels are present in PASMCs; however, their overall contribution to the whole cell current has species diversity and varies in different pulmonary artery tree regions [73].

3.3 ATP-Sensitive K+ Channels and Inwardly Rectifying K+ Channels KATP is another ubiquitous class of K+ channels; these channels show little or no voltage dependence and have low open probability under basal conditions. Structurally, KATP channels consist of an octameric complex of four pore-forming inward rectifier K+ (Kir) channel subunits (Kir 6.1 or 6.2) and four sulfonylurea receptors (SURs) [74]. Coexpression of SUR subunit 2B (SUR2B) with Kir 6.1 or 6.2 produces channels with the characteristics of two distinct KATP families, KNDP (named reflecting a primary role for various nucleotide diphosphates in their activation) and KATP (predominantly sensitive to ATP), respectively [75]. The expression of Kir 6.1 with SUR2B has been detected in human PASMCs [76]. Some evidence suggests they may contribute to resting membrane potential in PASMCs, although the regulatory mechanisms are still to be elucidated [76]. Additionally, there is no proposed role in HPV. Robertson et al. suggested that KATP channels are normally closed in the pulmonary arteries and are not activated by the levels of hypoxia that cause a constriction [77].

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3.4 KCNQ Channels KCNQ channels, previously known as members of the KV7 family of voltage-dependent K+ channels, were first identified in cardiac cells where mutations in a chromosomal locus on the KVLQT1 gene encode for an inherited form of long QT syndrome [78]. Five isoforms (KCNQ1–KCNQ5) have been identified and, of these, only KCNQ1 is known to be expressed in PASMCs [79, 80]. Linopirdine and XE991, KCNQ blockers, were demonstrated to be potent and selective pulmonary artery constrictors and data suggest that KCNQ channels could contribute to the regulation of resting membrane potential in PASMCs [79, 80].

3.5 K2P Channels K2P channels have four transmembrane segments (denoted M1–M4), two pore-forming domains (P1 and P2), a short cytoplasmic N-terminus, and a long cytoplasmic C-terminus. In addition, an extracellular loop exists between the M1 and P1 regions (as shown in Fig. 8) [81]. It is believed that two a subunits of K2P are necessary to form functionally active

Fig.  8  Structure and function of two-pore-domain K+ channels (K2P) channels. (a) Membrane topology of a K2P channel subunit featuring two pore regions, P1 and P2, and four transmembrane spanning domains, M1–M4, and cytoplasmic N- and C- termini. (b) Representative trace and I–V relationship recorded in TASK-expressing COS cells at pH 6.1, 7.4, and 8.4. The arrow indicates the zero-current level. (c) Summarized data indicating the pH dependence of TASK activity in COS cells at −50, 0, and +50 mV. (b, c Reproduced from Duprat et al. [89] with permission from Macmillan Publishers Ltd, copyright 1997)

A.L. Firth and J.X.-J. Yuan

channels. K2P channels are expressed in PASMCs and have a noninactivating current, denoted IKN, characterized by a lack of both voltage and time dependency [82]. To date, 14 K2P channels have been identified and these are subcategorized into five structural groups: 1 . TWIK-1 (KCNK1), TWIK-2 (KCNK6), and KCNK7 2. TASK-1 (KCNK3), TASK-3 (KCNK9), and TASK-5 (KCNK15) 3. TREK-1 (KCNK2), TREK-2 (KCNK10), and TRAAK (KCNK4) 4. TASK-2 (KCNK5), TALK-1 (KCNK16), and TALK-2 (KCNK17) 5. THIK-1 (KCNK13) and THIK-2 (KCNK12) Of these channels, TASK-1, TASK-2, TREK-1, TREK-2, and TWIK-2 have been detected in the pulmonary circulation [83–86]. K2P channels are considered proponents of the regulation of resting membrane potential; TASK-1 (TWIKrelated acid-sensitive K+ channel [59]), in particular, is able to regulate membrane potential by 10 mV in both a hyperpolarizing and a depolarizing direction [87]. In PASMCs from rats and rabbits [88] and, most recently from humans [87], the pharmacological profile of IKN has been shown to match TASK-1, being insensitive to glibenclamide, TEA, and Ca2+ channel blockers and having a high sensitivity to external pH but not cytosolic Ca2+ concentration. TASK-1 currents display outward rectification under physiological conditions conforming to the Goldman–Hodgkin–Katz current equation predicting the I–V curve, thus suggesting that TASK-1 is a pure K+-selective leak channel (as shown in Fig. 8) [89]. Inhibition of this channel by acid (H+) is dependent upon membrane voltage and potassium concentration, indicative of a pore-blocking inhibition mechanism. It has been shown that TASK-1 senses changes in pH within a physiological range via protonation of a histidine residue in the P1 pore-forming domain which leads to blocking of the channel [90]. It is also noteworthy that TASK-1, in addition to its acid sensitivity, exhibits sensitivity to hypoxia and, as such, these channels are proposed to be functionally important in mechanisms underlying HPV [87]. There is currently limited evidence suggesting a direct role of TASK in HPV or PAH; recently; however, it has been shown that endothelin-1, a vasoconstrictive agonist known to be upregulated in PAH, was shown to inhibit TASK-1 in human PASMCs [91].

3.6 Na+–K+-ATPase Na+–K+-ATPase, or Na+–K+ pump, is ubiquitously expressed and was the first enzyme to gain recognition as an ion pump. It enables the electrogenic, active transportation of both

13  Ion Channels and Transporters in the Pulmonary Vasculature: A Focus on Smooth Muscle

Na+ and K+. In each “cycle” the pump extrudes three Na+ and the cell accumulates two K+, the net change in charge hyperpolarizes the cell; the basic function of the Na+–K+ATPase is to maintain low cytosolic Na+ concentration and high cytosolic K+ concentration. The pump is a heterodimer formed from a catalytic a subunit and a glycosylated b subunit [92]. The a subunit is functionally important, conferring the binding sites for the physiological ligands (namely, Na+, K+, Mg2+, and ATP) and for ouabain, an inhibitor which displays tissue-specific affinity for the pump. The b subunit acts as a chaperone that ensures correct insertion of the a subunit in the plasma membrane and modulates the affinity of the enzyme for cations. Several distinct isoforms of the a subunit exist; the a1 isoform is ubiquitously expressed, whereas the a2–a4 isoforms are more limited in their distribution. It has recently been shown that the a2b1 and a1b1 isoforms of Na+–K+-ATPase exist in small membrane microdomains known as caveolae in PASMCs, whereas only the a1b1 isoform is detected in the noncaveolae regions of the plasma membrane [93]. The Na+–K+-ATPase utilizes energy derived from ATP hydrolysis to undergo conformational changes to enable binding and release of Na+ and K+ [94]. The pump may also be regulated by phosphorylation; PKA and PKC have been postulated to phosphorylate the a subunit [95]. Recently, small membrane proteins with a characteristic FXYD motif have been shown to associate with the Na+–K+-ATPase and modulate its properties for the transport of Na+ or K+, possibly implicating a role in pathophysiological states (reviewed by Geering [96]). Regulation of the Na+–K+-ATPase has a crucial role in the

Fig. 9  Structure and function of Na+ channels. (a) Planar schematic diagram showing structural arrangement of Na+ channel a and b1 and b2 subunits. (b) Representative currents showing inward INa in human PASMCs; the traces represent depolarizations, from a holding potential of −70 mV to test potentials between −80 and +80 mV in 20-mV increments. The inset indicates the summarized I–V relationship for INa. (c) Representative INa from PASMCs before, during, and after extracellular application of 1 mM tetrodotoxin. (b, c Reprinted from Platoshyn et al. [100] with kind permission from Springer Science+Business Media)

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regulation of the contractility of the vasculature [97] and, indeed, in the canine pulmonary artery, hypoxia inhibits the Na+–K+ ATPase [98].

4 Sodium Channels and Transporters 4.1 Voltage-Dependent Na+ Channels A rapidly activating and inactivating inward current (Iin) in rabbit PASMCs, dependent upon extracellular Na+, was first described by Okabe et  al. [99]. The current has since been fully characterized in cultured human PASMCs [100]. Although the presence of Na+ channels is certain, their functional role in the pulmonary artery is not clear, especially as PASMCs have a resting membrane potential more positive than that at which the Na+ channels would be inactivated. Furthermore, tetrodotoxin (TTX), a specific Na+ channel inhibitor, has no effect upon membrane potential, cytosolic Ca2+ release, or proliferation in pulmonary arteries and, therefore, Na+ channels are generally regarded as unimportant in the control of the normal function of these vessels [100]. Na+ channels consist of four internally homologous subunits each containing six transmembrane domains; the S5–S6 transmembrane domains fold to create an internal pore (Fig. 9). These subunits form a single principal pore region: the structural basis of this pore determines the selectivity and conductance properties of the channel and was described in detail by

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Table 5  Expression and function of Na+ channels Species/cell

Subunit (gene)

Accessory subunits (gene)

Electrophysiology

Pharmacology

References

Inactivation TTX-sensitive [100] V0.5 = −75.5 ± 3 mV ta = 0.485 ± 0.06 ms (at 0 mV) th = 2.85 ± 0.41 ms (at 0 mV) Cultured human PASMC ND ND Inactivation 30% TTX-sensitive [102] V0.5 = −75.5 ± 0.8 mV K = 10.73 Primary cultured human ND ND Activation 44% TTX-sensitive [151] PASMC V0.5 = −25 ± 4 mV K = 4.2 ± 0.5 Primary cultured rabbit main ND ND Activation TTX-sensitive [99] PASMC V0.5 = −65 mV K = 5.7 ND not determined, V0.5 half-activation/inactivation potential, K slope factor for activation/inactivation, PASMC pulmonary artery smooth muscle cell,t time constant (dependency) of activation/inactivation, TTX tetrodotoxin Cultured human PASMC

SCN2A–SCN4A, SCN7A–SCN9A, SCN11A

SCN1B, SCN2B

Marban et al. [101]. Currently more than ten voltage-dependent Na+ channel genes (SCN1A–SCN11A) have been identified; of these it is predominantly those forming TTX-insensitive channels (Nav1.5) that have been identified at the mRNA expression level in rabbit PASMCs (Table 5). A TTX-sensitive current has, however, been recorded in rabbit PASMCs, characterized by a very rapid inactivation (less than 10 s) [99]. In cultured human PASMCs, mRNA was detected for Na+ channel genes SCN2A, SCN3A, SCN4A, SCN7A, SCN8A, SCN9A, and SCN11A and accessory b subunits SCN1B and SCN2B (Table 5) [100]. The rapid outward current in cultured human PASMCs was sensitive to submicromolar concentrations of TTX [100, 102]. Figure 9 shows representative Na+ currents recorded from cultured human PASMCs at a series of voltages and confirms the TTX sensitivity of these currents.

4.2 Amiloride-Sensitive Epithelial Na+ Channel The amiloride-sensitive epithelial Na+ channel (ENaC) is most frequently associated with defective chloride transport in patients with cystic fibrosis. With regard to the pulmonary circulation, ENaC is implicated in the progression of highaltitude pulmonary edema, characteristically associated with exaggerated hypoxia pulmonary hypertension. During highaltitude pulmonary edema, hypoxia inhibits reabsorption of Na+ and concomitant fluid from the alveolar space by inhibiting ENaC and Na+–K+-ATPase.

4.3 Na+-Dependent Exchangers In addition to the NCX, discussed earlier in this chapter, two other Na+-dependent exchangers are expressed and are func-

tional in the pulmonary circulation; the Na+–Mg2+ exchanger and the Na+–H+ exchanger (NHE). Such extrusion mechanisms are functionally important in the maintenance of ion homeostasis.

4.3.1  Na+–H+ Exchanger The NHE is electrogenic, exchanging Na+ for H+ and is primarily involved in pH homeostasis in many mammalian cell types, acting alongside several other ion transport systems, such as the Na+-independent Cl−–HCO3 and Na+-dependent Cl−–HCO3 transporters. The NHE is activated by an increase in intracellular H+ concentration and extracellular Na+ is exchanged for intracellular H+ in a 1:1 stoichiometry. Excessive stimulation of the exchanger will ultimately increase the concentration of intracellular Na+, which, in turn, can drive the Na+–K+-ATPase, increasing the energy demands of the cell, and/or activate the NCX, driving Ca2+ into the cell. The NHE has recently been studied in PASMCs and significant roles in apoptosis, proliferation [2, 103], and hypoxia-induced pulmonary hypertension are evident [104, 105]. In PASMCs from 3-week-old hypoxic rats, it was found that pHcyt and NHE1 mRNA expression levels were significantly elevated and that inhibition of the NHE by 5-(N,N-dimethyl) amiloride elevated the rate of apoptosis [103]. Additionally, exposure of mouse PASMCs to chronic hypoxia increased pHcyt, NHE activity, and NHE1 expression [105]; such changes have been confirmed to contribute to the development of pulmonary hypertension [104]. Deficiency of NHE1 in mice prevented the development of hypoxia-induced pulmonary hypertension and vascular remodeling [104]. The NHE has additionally been shown to aid in recovery from acid loads during hypoxia [106]. Since the cloning of the first

13  Ion Channels and Transporters in the Pulmonary Vasculature: A Focus on Smooth Muscle

NHE (NHE1) by Pouyssegur’s laboratory in 1989, nine isoforms of the exchanger have been identified (NHE1–NHE9) [107]. NHE9 is more restricted in its distribution, unlike NHE1, which is ubiquitously expressed in the plasma membrane. The structures of the 815 amino acid proteins are similar; NHE2–NHE4 share 45–65% amino acid homology within the transmembrane domains. The exchangers contain ten to twelve helical hydrophobic membrane-spanning domains, an approximately 500 amino acid N-terminus functionally responsible for catalyzing the amiloride-sensitive NHE and an approximately 300 amino acid C-terminus acting as a regulatory domain [108].

4.3.2 Na+–Mg2+ Exchanger The Na+–Mg2+ exchanger has been widely studied in erythrocytes and in VSMCs, with a pertinent role in systemic hypertension having been identified; however, is yet to be cloned and extensively studied in the pulmonary vasculature. The Na+–Mg2+ exchanger presents a predominant mechanism for Mg2+ extrusion from the cell and has been identified in a variety of tissues, including sheep ruminal epithelial cells [109], VSMCs [3], and erythrocytes [110]. Interestingly, activation of the NHE has been shown to modulate Na+–Mg2+ transport in VSMCs and increased activity of NHE could influence the change in Na+dependent regulation of cytosolic Mg2+ concentration observed in spontaneously hypertensive rats [111]. A role for intracellular Mg2+ in the regulation of KV channels in rat PASMCs has recently been shown and presents a potential for the activity of the Na+–Mg2+ exchanger to contribute to the development of HPV and/or PAH [112].

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5 Chloride Channels and Transporters Despite chloride being the most abundant intra- and extracellular ion, until recently there was very little data on the presence and regulation of Cl− channels in the pulmonary vasculature. Activation of Cl− channels results in the movement of Cl− ions along their electrochemical gradient from inside the cell to the extracellular environment, resulting in a net positive current in the cell (i.e., inside the cell becomes more positive and the cell depolarizes). Cl– ions can be actively accumulated in the cell via one of three uptake mechanisms: the Na+–K+–2Cl− cotransporter, the Cl–HCO3 exchanger, or the relatively unknown “pump III” [113]. In the pulmonary circulation, the most extensively studied Cl– channel current is stimulated by an elevation of cytosolic Ca2+ concentration, commonly referred to as the calcium-activated chloride current (IClCa). In addition, voltage-gated and volume-sensitive Cl− channels exist, though they are not as well characterized in the pulmonary vascular system (Table 6). Chloride transportation is involved in cell-volume regulation and is implicated in essential cellular functions such as progression of the cell cycle, cell proliferation, and contraction, thus having a pertinent role in the development of pulmonary edema and potentially pulmonary vascular remodeling.

5.1 Ca2+-Activated Cl− Channels Calcium-activated chloride channels are stimulated by an elevation of cytosolic Ca2+ concentration,due to either intracellular store (SR) release of Ca2+ or influx of Ca2+ ions. In the pulmonary artery of rabbits and rats it has been shown that

Table 6  Expression and function of Cl− channels Ca -activated (ICl(Ca))

cGMP-activated (ICl(cGMP,Ca))

160–600 nM ~74 nM 1–4 pS I−>Br−>Cl− Below 1 mM at resting cytosolic Ca2+ concentration 2–20 mM 15–250 mM Independent

No effect 3–6 mM

2+

EC50 cytosolic Ca2+ EC50 cGMP Conductance Halide permeability Voltage/time dependence

Niflumic acid IC50 DIDS IC50 Zn2+ IC50 TFP IC50 Protein tyrosine kinase inhibition

Br−>I−, Cl − or Cl−>I− Independent

No effect at 100 mM No effect at 200 mM ~2–6 mM

Gene Bestropohin-3 References [152] [152] DIDS 4 4′-diisothiocyanato-stilbene-2,2′-disulfonic acid, TFP trifluoperazine

Ca2+/CaMKII-activated (ICl(CaMKII))

Volume-sensitive (ICl(swell))

~430 nM Not sensitive 25–30 pS I−>Br−»Cl−>>gluconate Voltage-dependent and voltage-independent Ca2+ binding Not sensitive

5.7 mM Tyrphostin B46 (50 mM), genistein (100 mM) [153]

[154]

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both norepinephrine and histamine (GPCR agonists) activate IClCa [114, 115]. Furthermore, activation of the reverse mode of the NCX can also activate IClCa, a potential mechanism in the development of pulmonary hypertension [116]. Many investigators have characterized the biophysical and pharmacological properties of IClCa in human and animal PASMCs. In a study on rat PASMCs, it was shown that depolarization or agonist-mediated elevation of cytosolic Ca2+ concentration elicited a time-dependent outward Cl− current that reversed near to the equilibrium potential for Cl−, approximately 0 mV (Fig. 10a), and that was inhibited by the Cl− channel blocker niflumic acid (10–50 mM) [117]. Cl− channel inhibition also attenuated serotonin-induced membrane depolarization and agonist-induced pulmonary artery contraction [117]. Although the electrophysiological and pharmacological properties of IClCa are relatively well defined (Fig. 10b), the molecular correlate for IClCa in the pulmonary artery is not precisely known. The ClCa channels are heterogeneous with several different characteristics; currently they can be divided into three distinct families: (1) channels activated directly by Ca2+, (2) channels requiring Ca2+/CaM-dependent protein kinase II, and (3) cyclic GMP (cGMP)-dependent channels. A group of proteins, known as CLCA, and controversially associated with ICaCl, are the bestrophin family [118]. Expression of bestrophin proteins (Best-1 to Best-4) in different cell types correlates with the appearance of a Cl− conductance. The cGMP-dependent Ca2+activated Cl− current was recently characterized in several vascular beds, including the pulmonary artery. This current had a high cytosolic calcium sensitivity Ca2+ concentration sensitivity, a linear voltage dependence, and permeability to anions were found [118, 119]. With use of small interfering RNA, Best-3 was shown to be a critical component of the endogenous ICaCl in smooth muscle cells of intact pulmonary artery walls [118]. Although Best-3 may encode a “nonclassical” Ca2+-activated Cl− current owing to the lack of voltage sensitivity, it is the “classical” Ca2+-activated Cl− current that is expressed at its highest level in the pulmonary artery. In a recent breakthrough study, Caputo et  al. provided evidence that the membrane protein TMEM16A is an intrinsic component of the voltage-sensitive Ca2+-dependent Cl− channel; its expression in the pulmonary vasculature remains to be investigated [120]. TMEM16A is an 840 amino acid, eight transmembrane spanning protein with cytoplasmic N- and C-termini; it undergoes alternative splicing, generating a variety of isoforms with significant sequence homology (e.g., TMEM16b, approximately 60% sequence homology). The TMEM16 proteins may represent subsets of chloride channels with a biophysical profile that remains to be characterized. Bovine pulmonary endothelial cells also have ICaCl; elevation of cytosolic Ca2+ concentration activated an outwardly rectifying Cl− current for which the reversal potential was dependent upon extracellular Cl− concentration and the current inactivated rapidly at negative membrane potentials and

A.L. Firth and J.X.-J. Yuan

Fig.  10  Structure and function of Cl− channels. (a) A representative family of traces for a Ca2+-activated Cl− current in PASMCs elicited by a series of test potentials from −60 to +60 mV in 20-mV increments (from a holding potential of −70 mV). The cells are superfused with modified Kreb’s solution including 1.8  mM Ca2+. (b) Representative traces of Ca2+-activated Cl− currents recorded in the presence of either 3 mM ATP (left) or AMP-PNP 5′-adenylyl-beta,gamma-imidodiphosphate (right) at various intracellular Ca2+ concentrations. The voltage protocol is featured below the traces. (a Reproduced with permission [117]. (b) Copyright 2006 Angermann et al. [156])

had a single channel conductance of 7.9 ± 0.7 pS. Furthermore, strong calcium chelation with 1,2-bis(2-aminophenoxy) ethane-N,N,N¢,N¢-tetraacetic acid prevented ATP-induced activation of ICaCl, and 4,4¢-Diisothiocyanatostilbene-2,2¢disulfonic acid (DIDS) and niflumic acid inhibited it in a voltage-dependent manner [121].

13  Ion Channels and Transporters in the Pulmonary Vasculature: A Focus on Smooth Muscle

5.2 Voltage-Gated Cl− Channels The CLCN family of voltage-gated chloride channels has nine identified members. Although a significant similarity in sequence homology exists, there is a distinct diversity in function. CLCN1–CLCN7, CLCNKa, and CLCNKb are the currently identified voltage-sensitive chloride channel genes. CLCN channels characteristically have ten transmembrane spanning regions with intracellular C- and N-termini and it is suggested that functional channels arise from dimerization of genes. The expression of CLCN3 has been shown in the human fetal lung and in smooth muscle cells and endothelial cells of the large and small pulmonary arteries, pulmonary epithelial cells, and bronchial smooth muscle cells [122]. The first functional evidence demonstrated an upregulation of the ClCn-3 gene in both rat pulmonary artery in the monocrotalineinduced model of pulmonary hypertension and PASMCs cultured from canines and incubated with inflammatory mediators. Furthermore, overexpression of CLCN3 improved resistance of these cells to reactive oxygen species [123], potential role in the pathogenesis of pulmonary hypertension.

5.3 Volume-Sensitive Cl− Channels Volume-sensitive chloride channels are ubiquitously expressed and activated in response to cell swelling. Yamazaki et al. demonstrated that functional volume-sensitive chloride channels were present in canine PASMCs [124]. Exposure of cells to hypotonic solutions increased Cl− conductance, a current exhibiting outward rectification in a symmetrical Cl− gradient. This channel was sensitive to inhibition by DIDS, ATP, and tamoxifen. It is currently not known whether this current is carried by a homomeric channel or CLCN3 in heteromeric association [124].

6 Aquaporins Aquaporins are a family of water-transporting membranebound proteins. To date 13 mammalian aquaporin homologues have been detected; several of these are expressed and functional in the human lung. Aquaporins are six transmembrane spanning proteins with five interconnecting loops and intracellular N- and C-termini. Two hydrophobic loops, the cytoplasmic B loop and the extracellular E loop, each containing a signature motif consisting of a tripeptide sequence of asparagine, proline, and alanine, are proposed to from the aqueous pore region of the channel [125]. Aquaporins are important for water permeability across microvessels in intact lungs; more specifically, in knockout mice, aqua-

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porin-1 was demonstrated to provide an important route for osmotic and hydrostatic water movement between the air space, interstitial compartment, and microvessels [126, 127]. Aquaporins are most influential in regulating pulmonary fluid homeostasis and, in a bleomycin-induced pulmonary edema rat model, the expression of aquaporin-1 has been shown to be significantly reduced. It is postulated that alterations in aquaporin expression may lead to pathophysiological conditions [126, 128].

7 Interaction of Ion Channels During Homeostasis and Disease Ion channels form a complex signaling system functioning to maintain a cellular homeostasis: a balance between apoptosis and proliferation, between contraction and relaxation, between cell shrinkage and cell swelling, and control of cellular excitability and resting membrane potential. Alterations in the expression and function of ion channels are a significant factor in the mechanisms leading to the development and persistence of PAH. To avoid repetition with other chapters in this book, the involvement of ion channels in the development and progression of pulmonary vascular disease will only be discussed very briefly here using K+ channels as an example.

7.1 Hypoxic Regulation of KV Channel Expression and Function Potassium channels are key regulators in cell signaling in response to hypoxia and, therefore, the development of acute HPV and chronic hypoxia-induced pulmonary hypertension. Post et al. were the first to observe that a decrease in oxygen tension to hypoxic levels directly inhibited K+ currents in concert with membrane depolarization in isolated canine PASMCs, an effect not present in canine renal arterial smooth muscle cells [129]. Subsequently, Archer et  al. identified KV1.5 and KV2.1 as the potential molecular correlates for the slowly inactivating, 4-AP-sensitive, charybdotoxin-insensitive delayed rectifier currents in resistance artery PASMCs [130]. Furthermore, acute hypoxia has been shown to depolarize PASMCs by inhibiting K+ currents, and extended exposure to hypoxia (more than 72 h) is known to decrease KCNA5 gene expression in these cells [66]. A distinct heterogeneity in KV channel sensitivity to hypoxia exists, and is postulated to be due to differential expression and functional sensitivity of the KV channels to hypoxia [131]. Reviews by Mauban et  al. and Weir and Olschewski convey the key aspects of the role of ion channels in HPV [132, 133].

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7.2 Role of Plasmalemmal K+ Channels in Apoptosis and Proliferation in PASMCs Initiation of apoptosis is recognized by cell shrinkage, a process regulated by a change in the ionic composition of the extracellular and intracellular environments of the cell. Apoptotic volume decrease is an early apoptotic event in which K+ efflux through open plasmalemmal K+ channels creates a positive potential outside the cell; in turn, Cl− leaves the cells along its concentration gradient. An osmolarity imbalance is created and, in an effort to maintain the osmotic balance, water leaves the cell via aquaporins and cells shrink. Several studies have confirmed the highly influential role of K+ channels in the process of apoptosis. In PASMCs, overexpression of KCNA5, encoding KV channel isoform KV1.5, enhanced apoptosis, whereas inhibition of K+ channels by 4-AP reduced staurosporine-induced apoptotic volume decrease and apoptosis [62]. Furthermore, overexpression of antiapoptotic oncogene Bcl-2 causes a downregulation of KV channel a subunit mRNA and decreases KV channel activity [67].

8 Summary Ion channels are fundamental to the maintenance of cellular homeostasis and to the excitability of cells in response to a variety of stimuli. Alterations in the expression and function of ion channels are key features in the development and pathogenesis of pulmonary vascular disease and thus ion channels and consequential signaling cascades are potential sites for therapeutic intervention. Acknowledgements  The authors acknowledge the California Institute of Regenerative Medicine and the NIH for supporting their research.

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Chapter 14

Receptor-Mediated Signal Transduction and Cell Signaling Fiona Murray, Jason X.-J. Yuan, and Paul A. Insel

Abstract  The maintenance of low resistance, pressure, and tone in the pulmonary circulation is dependent on the interaction of circulating and locally produced vasomodulatory regulators; many such vasoactive mediators act via receptormediated signaling pathways. Many types of receptors that regulate pulmonary vascular tone are expressed on the plasma membrane of cells, although vasomotor activity is also influenced by intracellular receptors, such as calcium-release receptors and receptors that regulate transcription (e.g., steroid hormone receptors). The pulmonary vasculature expresses a wide variety of receptor classes and subtypes that facilitate the interaction of cells of the pulmonary circulation with their extracellular environment (hormones, neurotransmitters, and other factors in the extracellular milieu play key roles in modifying blood flow under both physiological and pathophysiological conditions). One can identify such receptors by assessing the binding of radioligands, molecular cloning and expression studies, antisense approaches, and/or by conducting studies with transgenic or knockout animals. Receptormediated signaling in the pulmonary circulation changes with development and disease, is highly species specific, cell-type specific, and often depends on an intact endothelium. Moreover, the accessibility of plasma membrane receptors for neurotransmitters and hormones from the extracellular environment makes them excellent drug targets. The major classes of membrane receptors that regulate pulmonary vascular tone are G-protein-coupled receptors, ligand-gated ion channels, and receptor protein kinases [receptor tyrosine kinase and serine/threonine kinase receptors]. This chapter provides an overview of signaling by cell-surface receptors in the pulmonary circulation and highlights mediators whose activation regulates pulmonary vascular development, tone, and permeability. Keywords  Signal transduction • Intracellular signaling cascades • Molecular pharmacology • G protein-coupled receptor • Tyrosine kinase receptor F. Murray (*) Departments of Medicine and Pharmacology, University of California at San Diego, BSB 3073, 9500 Gilman Drive, La Jolla, CA, 92093-0636, USA e-mail: [email protected]

1  Introduction The maintenance of low resistance, pressure, and tone in the pulmonary circulation is dependent on the interaction of ­circulating and locally produced vasomodulatory regulators; many such vasoactive mediators act via receptor-mediated signaling pathways [1–3]. Many types of receptors that regulate pulmonary vascular tone are expressed on the plasma membrane of cells, although vasomotor activity is also influenced by intracellular receptors, such as calcium-release receptors and receptors that regulate transcription (e.g., steroid hormone receptors). The pulmonary vasculature expresses a wide variety of receptor classes and subtypes that facilitate the interaction of cells of the pulmonary circulation with their extracellular environment (hormones, neurotransmitters, and other factors in the extracellular milieu play key roles in modifying blood flow under both physiological and pathophysiological conditions). One can identify such receptors by assessing the binding of radioligands (i.e., radiolabeled agonists or antagonists), molecular cloning and expression studies, antisense approaches, and/or by conducting studies with transgenic or knockout animals. Receptor-mediated signaling in the pulmonary circulation changes with development and disease, is highly species specific, cell-type specific, and often depends on an intact endothelium [1–4]. Moreover, the accessibility of plasma membrane receptors for neurotransmitters and hormones from the extracellular environment makes them excellent drug targets. The major classes of membrane receptors that regulate ­pulmonary vascular tone are G-protein-coupled receptors (GPCR), ligand-gated ion channels, and receptor protein kinases [receptor tyrosine kinase (RTK) and serine/threonine kinase receptors]. This review provides an overview of signaling by cell-surface receptors in the pulmonary circulation and highlights mediators whose activation regulates pulmonary vascular development, tone, and permeability. Receptor-dependent signaling cascades contribute to the progression of diseases such as pulmonary arterial hypertension (PAH) and pulmonary edema; however, these topics will be discussed in more detail in subsequent chapters and have been reviewed elsewhere [1–3].

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_14, © Springer Science+Business Media, LLC 2011

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2 G-Protein-Coupled Receptors Over 30% of all prescription drugs, in addition to many ­hormones and neurotransmitters, act via GPCRs. GPCRs are a superfamily of receptors that have a similar general structural arrangement: seven transmembrane spanning domains, which are seven a-helices, linked by three alternating intraand extracellular loops. GPCRs are the largest receptor family in the human genome, constituting approximately 3% of the genes [5–7]. Activation of these receptors occurs when ligands bind to the receptors (depending on the GPCR, to extracellular or transmembrane binding pockets), thereby altering receptor confirmation and promoting coupling, in particular by the third intracellular loop and proximal portion of the C-terminal tail, to intracellular heterotrimeric (G) proteins, which comprise a guanosine diphosphate (GDP)-bound Ga subunit and a Gbg complex (Fig. 1). In mammalian cells at least 15 genes encode for the Ga subunits, five for the Gb subunits, and 12 for Gg subunits, but in addition, there are

Fig. 1  G-protein-coupled-receptor (GPCR)-mediated signaling in the pulmonary artery smooth muscle cell (PASMC). GPCR activation promotes the exchange of the G-protein-bound GDP for GTP, thereby inducing a conformational change and dissociation of Ga from the Gbg and thus initiating downstream signaling. The Gbg subunits can directly activate phosphatidylinositol 3-kinase (PI3K) and phospholipase Cb (PLCb). PLCb, activation (by Gbg and Gaq) promotes the hydrolysis of phosphatidylinositol 4,5-bisphosphate and yields the intracellular messengers 1,2-diacylglycerol (DAG) and inositol 1,4,5-trisphosphate. DAG remains membrane-bound and promotes the translocation of protein kinase C from the cytoplasm to the membrane and its subsequent activation. Ga12/13-dependent pathways (guanine nucleotide exchange factors for Rho activate the small G protein RhoA followed by Rho kinase) and Gai-dependent pathways (inhibition of adenylyl cyclase decreases cyclic AMP accumulation) lead to PASMC vasoconstriction and proliferation. In contrast Gas stimulates the production of cyclic AMP via activation of adenylyl cyclase, which in turn decreases PASMC proliferation and enhances vasodilation

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many splice variants of each [8]. Ligand binding triggers the exchange of guanosine triphosphate (GTP) for GDP on the a-subunit, thus facilitating dissociation from the Gbg and usually from the receptor as well. This classic model has recently been challenged, on the basis of evidence suggesting that a conformational change within the G protein may not necessarily induce subunit dissociation and that such dissociation may not be obligatory for the activation of G-proteinregulated effector molecules [9]. Inactivation of Ga, which occurs when GTP is hydrolyzed to GDP, determines the rate and length of the signal derived from GPCR-promoted activation of heterotrimeric G proteins. Intrinsic GTPase activity of the Ga subunits can be promoted by GTPase-activating proteins, such as regulator of G protein signaling (RGS) proteins, or even by effector molecules, such as phospholipase Cb (PLCb); specific RGS proteins terminate the activation of particular G proteins [10]. To date, limited information is available regarding the role of particular RGS proteins in cells from the pulmonary circulation; however, since knockdown of specific RGS family members, such as RGS2, can lead to systemic hypertension by amplifying signaling responses to vasoactive mediators, this may be an interesting topic for future research and may represent a novel way to manipulate GPCR signaling in the pulmonary circulation [11]. G proteins are divided into four main classes according to their a subunit, Gas, Gai, Gaq/11, and Ga12/13, which each lead to the activation/inactivation of signaling pathways that control the production of second messengers and gene expression: Gas stimulates the membrane-associated enzyme adenylyl cyclase (AC) which increases the level of cyclic adenosine 3¢,5¢-monophosphate (cAMP), and regulates Ca2+ channels; Gai inhibits AC activity, decreasing the level of cAMP, and also regulates K+ and Ca2+ channels; Gaq/11 ­stimulates membrane-bound PLCb, which cleaves phosphatidylinositol 4,5-bisphosphate into inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG); Ga12/13 regulates GTPase RhoA, a low molecular weight G protein. Gbg can act as a single entity to activate both concordant and disconcordant signals, for example, by directly regulating Kir3 K+ channels, PLCb, AC, voltage-gated calcium channels, and even other proteins, such as PDZ-domain-containing proteins [5]. The stoichiometric relationship of GPCR signaling pathways, the absolute concentrations and relative proportions of each component, is important for defining the efficiency of each pathway. Data in a number of cell types for the b-adrenergic receptor (AR) pathway indicate that the ratio of receptors (fewer than 10,000 per cell) to G proteins (about 1,000,000 per cell cell) to ACs (about 30,000 per cell) is approximately 1:100:3, suggesting that although receptor activation of Gas is important for amplification of signaling, AC is the critical component that limits maximal b-AR response [12]. Limited data are available for the stoichiometric ratios of other classes of G proteins and their effectors; defining the critical components in Gaq/11, Ga12/13, and for Gbg subunits will be important

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for determining which components determine potency (concentration dependence of agonists) and efficacy (maximal response) of their signaling pathways. Stoichiometry cannot fully explain the efficient activation and amplification of response to GPCR agonists: such observations have provided a rationale for the assessment of compartmentation (i.e., colocalization) of GPCRs and their downstream effectors via “scaffolding” proteins or in specific plasma membrane domains [13–15]. The spatial and temporal organization of GPCR signaling is regulated by scaffolding and GPCR interacting proteins such as b-arrestin, A-kinase anchoring proteins (AKAPs), and PDZ domain proteins that colocalize membrane proteins in specific subcellular domains: PDZ domains primarily bind proteins containing a C-terminal S/TXV(L/I) motif, which is present in some GPCRs such as the b2-AR, in which that domain facilitates binding to a Na+–H+ exchanger regulatory factor [16]. In addition to such adaptor proteins there are membrane microdomains, such as clathrin-coated pits, lipid (membrane) rafts, and caveolae (a subset of lipid rafts that contain the structural protein caveolin) that serve as cellular platforms and bring receptors together with downstream mediators, thereby facilitating receptor-, tissue-, and cellspecific signal transduction. The movement of receptors and their effectors into and out of these complexes contributes to both the qualitative (i.e., as a determinant of “preferred” signaling partners) and quantitative (e.g., rapidity and reversal) regulation of cell signaling. A number of GPCRs localize in membrane rafts/caveolae either before (e.g., b-AR subtypes) or after (e.g., bradykinin receptors) stimulation by agonists [15]. Caveolae are highly expressed in the pulmonary ­circulation – indeed, the pulmonary vascular endothelium is the most enriched source of caveolae in the body and appears to be functionally important and also contributes to disease: moreover, increased expression in PASMC occurs in PAH; the role of microdomains in pulmonary function is discussed in detail in subsequent chapters [15, 17]. Further levels of complexity have recently emerged regarding GPCR signaling. For example, GPCRs were previously thought to couple preferentially to only one G protein; however, coupling to more than one G protein can occur; for example, b2-AR can activate both Gas and Gai [18]. In addition, cross talk between GPCR pathways occurs, such that agonists that activate Gaq can potentiate stimulation of GaS [19]. In addition “ligand-directed” signaling appears to occur, i.e., particular agonists that bind to the same receptor can stimulate distinct G protein signaling cascades [20, 21]. The large number of isoforms of downstream mediators, such as AC and phospholipase C (PLC), or the fact that multiple active receptor conformations exist, may be important for this complex regulation. Assembly of individual GPCRs into homodimers or heterodimeric combinations also can occur, although the precise physiological role of such multimers is not yet well defined [22].

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2.1 GPCRs and the Pulmonary Circulation Individual cells can express up to 100 different GPCRs so it is likely that the full range of GPCRs in the pulmonary circulation have yet to be fully defined. Recent work profiling GPCR expression indicates that the lung expresses previously unappreciated GPCRs, many of which may have physiological and pathological roles [23]. Given this caveat, we speculate that key GPCRs involved in regulation of the pulmonary circulation may not have yet been studied. Endogenous mediators acting via specific GPCRs can have differing effects depending on the predominant receptor subtype in the vessel, the presence or absence of endothelium, and on the initial tone of the pulmonary circulation [1– 4]. For example, angiotensin II (Ang II), endothelin (ET)-1, or 5-hydroxytryptamine (5-HT) antagonists do not vasodilate the normal pulmonary circulation but they attenuate hypoxic pulmonary vasoconstriction, and under normoxic conditions some vasodilators have little effect on pulmonary tone [1, 24, 25]. Together, these data imply that when vascular tone is increased, the lung is more tightly under the control of vasoactive mediators than in the normal pulmonary circulation. Because effects of agonists and antagonists can be influenced by the experimental conditions employed, caution should be taken when interpreting data from isolated pulmonary arteries (PAs) on which tone is added, or even cultured isolated pulmonary cells that are typically grown in enriched nutrients and O2 without the interaction of other cells. Thus, the response of a mediator in vitro does not necessarily define its response in the pulmonary circulation in vivo.

2.1.1 Mechanisms of G-Protein-Mediated Pulmonary Arterial Vasodilation GPCR agonists that vasodilate the pulmonary circulation act via increasing the intracellular concentration of the second messengers cAMP in the case of b2-AR and cyclic guanosine 3¢,5¢-monophosphate (cGMP) [e.g., for agonists that increase the levels of nitric oxide (NO)] in PA smooth muscle cells (PASMCs; Fig. 1). Agonists that activate Gas stimulate the production of cAMP via activation of AC. Nine membranebound isoforms of mammalian ACs have been characterized, each with their own tissue distribution and regulation; AC6, which is Gas-coupled, is highly expressed in both PASMCs and PA endothelial cells (PAECs) [26, 27]. The intracellular level of cAMP is also dependent on its hydrolysis by a family of 11 enzymes, phosphodiesterases (PDEs). The basal activity of PDEs (PDE1, PDE3, and PDE4 appear to control cAMP degradation in normal PASMCs and PAECs) maintains a high level of intracellular cyclic nucleotides and thus a low level of vasomotor tone. cAMP acts via a number of downstream effectors, the most well characterized

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being cAMP-dependent protein kinase (PKA) [28]. PKA phosphorylates serine and threonine residues on targets such as myosin light chain kinase (MLCK), resulting in vasodilation. In addition cAMP can also act via PKA-independent mechanisms, in particular cyclic-nucleotide-gated (CNG) channel, and the more recently identified effector Epac (exchange protein directly activated by cAMP; Epac-1 and Epac-2 are known), a guanine nucleotide exchange factor (GEF) for the a low molecular weight G protein Rap-1, the role of which in the pulmonary circulation is as-yet poorly defined [29]. The activation of the specific cAMP effectors may be GPCR-dependent, for example, in airway smooth muscle cells b2-AR agonists decrease proliferation preferentially via Epac [30]. AKAPs also aid in coordinating the biological effects of cAMP by targeting cAMP effectors to distinct compartments in the cell and bringing them in close proximity to specific intracellular mediators, in particular by facilitating effects of PKA activation [31]. cAMP not only produces relaxation of the PA but also can reduce PASMC proliferation and alter gene expression. For example, cAMP (via PKA) modulates expression of genes by transcriptional activation of CREB, the cAMP response element binding protein. CREB alters gene expression through its interaction with cis-regulatory cAMP-responsive DNA elements, which are located in cAMP/PKA-target genes, such as cyclooxygenase-2, an increase in activity of which persists long after the cAMP has been degraded [32, 33]. In addition, cAMP acts via PKA to protect PAEC barrier function by promoting cell–cell and cell–matrix association through enhanced phosphorylation of particular proteins [34]. GPCR stimulation, for example, via ET-1, on PAECs can also lead to vasodilation through effects on PASMCs: activation of Gaq receptors on PAECs raises the intracellular Ca2+ concentration ,which stimulates the production of NO and prostacyclin (PGI2), which diffuse to PASMCs and raise, respectively, the intracellular concentrations of cGMP and cAMP. NO stimulates soluble guanylyl cyclase, which catalyzes the hydrolysis of GTP to cGMP. NO can also have cGMP-independent effects, such as downregulating the number and binding activity of Ang II receptors, thereby blunting the vasoconstrictor effect of Ang II and activating K+ channels [35, 36]. The intracellular concentration of cGMP can also be increased via atrial natriuretic peptide (ANP) acting on natriuretic peptide receptors (NPR-A and NPR-B) on PASMCs; natriuretic peptide receptors are sometimes termed “enzyme receptors,” because of their intrinsic guanylyl cyclase activity. Interestingly, ANP is released in response to acute hypoxia, presumably as part of a negative-feedback loop that helps keep pulmonary vascular tone low [37]. As with cAMP, cGMP levels are also determined by the activity of PDEs: PDE1 and PDE5 appear to be the main cGMP PDEs in the pulmonary circulation [28]. Increasing the intracellular cGMP concentration in PASMCs activates protein kinase G (PKG), which reduces the intracellular Ca2+ concentration by inhibiting voltage- and receptor-operated calcium

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channels, resulting in the uncoupling of the contractile apparatus by activating myosin light chain phosphatase and can also promote hyperpolarization of the membrane by activating K+ channels [38]. In PAECs cGMP can directly activate CNG ion channels, which mediate membrane depolarization following activation of store-operated calcium entry [39]. Thus, an intact endothelium is essential for low basal tone of the pulmonary circulation, since many of the endogenous mediators that stimulate release of NO and PGI2 from PAECs can lead to vasoconstriction if they directly act on PASMCs.

2.1.2 Mechanisms of G-Protein-Mediated Vasoconstriction and Vascular Cell Proliferation Activation of GPCRs that link to Gai, Gaq/11, and Ga12/13, e.g., by 5-HT on PASMCs, leads to vasoconstriction and in many cases increased cellular (in particular PASMC) proliferation (Fig. 1). Stimulation of Gai decreases the intracellular cAMP concentration by inhibiting the activity of specific ACs; AC2 and AC8 are the main Gai-regulated ACs in the PA [26, 27]. Agonist stimulation of Gaq increases the activity of PLCb, which by elevating IP3 levels increases intracellular Ca2+ concentration by opening receptor-operated Ca2+ channels and inducing Ca2+ mobilization from the sarcoplasmic reticulum; IP3 also opens store-operated Ca2+ channels directly or indirectly by store depletion to further increase intracellular Ca2+ concentration. Intracellular Ca2+ can bind to calmodulin or activate intracellular enzymes, such Ca2+–calmodulindependent protein kinase (CAMKII) and MLCK, leading to phosphorylation of the myosin light chain. That phosphorylation stimulates myosin ATPase and promotes hydrolysis of ATP, thereby generating energy for the cycling of the myosin cross-bridges with the actin filament. In parallel, DAG, which is also derived from the action of PLCb, binds protein kinase C (PKC) and promotes its association with the cell membrane. PKC, several isoforms of which coexist in PASMCs (PKCa, PKCb, PKCd, PKCe, and PKCz), can activate mitogen-activated protein kinases (MAPK), and phosphorylate a number of substrates involved in the contractile process [40]. Gaq can also activate RhoA (a low molecular weight monomeric G protein) and Rho kinase (ROCK), which mediate Ca2+ sensitization and can promote sustained vasoconstriction [41]. Activation of Ga12/13, for example, by thrombin, promotes vasoconstriction of the PA and proliferation of PASMCs, primarily by activating Rho GEFs (p115 RhoGEF, PDZRhoGEF) that are linked to the activation of Rho. Rho, in turn, can activate ROCK, inhibit cadherin-mediated aggregation, activate or inhibit one or more Na+–H+ exchangers, and activate phospholipase Ce (PLCe) [42, 43]. Ga12/13 can also directly activate PLCe, AKAP 110, and heat shock protein 90 [43]. Interaction of Ga12/13 and other G proteins, in particular Gaq, can produce overlapping biological responses such as sustained Ca2+ influx and Rho activation;

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Table 1  G-protein-coupled receptors (GPCR) expressed on pulmonary vascular cells Endogenous ligand GPCR Cell type G protein Angiotensin II Endothelin-1

Norepinephrine/epinephrine

Acetylcholine

Bradykinin Vasopressin Adrenomedullin Vasoactive intestinal polypeptide Calcitonin gene-related peptide Substance P Histamine Urotensin II Adenosine ATP, ADP, UTP, UDP 5-Hydroxytryptamine Prostacyclin Thromboxane A2 Sphingosine Thrombin

AT1 ETA ETB ETB b2-AR a1-AR a2-AR M1 M3 M3 B2 V1 CRLR/RAMP VPAC-2 CGRP CGRP NK1 NK2 H1 UT

PASMC PASMC PASMC PAEC PASMC PASMC PAEC PAEC PAEC PASMC PAEC PAEC PASMC PAEC PASMC PASMC PAEC PAEC PASMC PASMC PASMC

Gaq Gaq Gaq Gaq Gas Gaq/Ga12/13 Gaq Gaq Gaq Gaq Gaq Gaq Gas Gaq Gas Gas Gaq Gaq Gaq Gaq Gaq

A2B P2Y2, P2Y4, and P2Y6 5-HT1B 5-HT2A IP FP and TP S1P1 S1P3 PAR1 PAR1/PAR2

PASMC PASMC PAEC PASMC PASMC PASMC PASMC PAEC PAEC PAEC PASMC

Gas Gaq Gaq Gai Gaq/Ga12/13 Gas Gaq Gai Gai/Gaq/Ga12/13 Gaq/Ga12/13 Gaq/Ga12/13

Response Proliferation/vasoconstriction [160] Proliferation/vasoconstriction Proliferation/vasoconstriction Vasodilation [77–85] Vasodilation [48–56] Vasoconstriction Vasodilation Vasodilation [58–61] Vasodilation Vasoconstriction Vasodilation [161] Vasodilation [162] Vasodilation [163] Vasodilation Vasodilation [164] Vasodilation/antiproliferation [165] Vasodilation [166] Vasoconstriction Vasoconstriction [167] Vasoconstriction large pulmonary artery [168] Vasodilation [63–66] Vasoconstriction [63, 64, 68–73] Vasodilation Both proliferation/vasoconstriction [92–98] Vasodilation/anti-proliferation [104] Proliferation/vasoconstriction [109] PAEC barrier protection PAEC barrier dysfunction [115–118] PAEC barrier dysfunction [111–114] Proliferation/vasoconstriction

AR adrenergic receptor, PAEC pulmonary artery endothelial cell, PASMC pulmonary artery smooth muscle cells

coordinated action of these proteins occurs with pulmonary smooth muscle contraction [44, 45]. Gbg can recruit phosphatidylinositol 3-kinase (PI3K), a potent mitogen, to the cell membrane and activate PI3K by direct interaction with its catalytic subunit (p110, Fig.  1). PI3K occurs in several subtypes and can also be activated by phosphorylation by RTKs, non-receptor protein tyrosine kinases of the src family, and focal adhesion kinase. Several downstream targets of PI3K have been identified, including phosphoinositide-dependent kinase-1, Akt, mammalian target of rapamycin (mTOR), and p70 ribosomal protein S6 kinase (p70S6K), all of which can play a role in cell proliferation [5, 46]. A direct role for PI3K in Ang-II-induced DNA synthesis and proliferation occurs in PA adventitial fibroblasts in response to hypoxia [47]. The main GPCRs in the pulmonary circulation, their preferred G protein, and their functional response on ligand binding are summarized in Table  1; a number of which will be discussed in more detail in the following.

2.1.3 Adrenergic Receptors Sympathetic nerves help maintain basal pulmonary vascular tone via both a-ARs and b-ARs, and their multiple subtypes, although this contribution is lower than the sympathetic tone that exists in numerous peripheral vascular beds. In the normal pulmonary circulation, low tone is maintained predominantly via b-ARs since in response to sympathetic stimulation a-antagonists lead to vasodilation, whereas b-AR antagonists enhance vasoconstriction [48]. In the PA of humans, rats, and mice b1-AR and b2-AR (but not b3-AR) on PASMCs mediate vasodilation, although the contribution of each receptor is species-specific [48–51]. Functional studies have shown b2-AR predominates in human, murine, and rat PA. b2-AR stimulation leads to activation of the Gas–cAMP pathway and PASMC relaxation [50, 51]. Depending on the size of the PA investigated and the initial level of precontraction, b-AR-mediated relaxation can be either dependent on or independent of an intact endothelium [50].

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Sympathetic nerve stimulation regulates vascular tone and maintains an adequate ventilation/perfusion matching under physiological conditions. However, excessive stimulation of a1-ARs produces vasoconstriction and proliferation; the pulmonary circulation expresses both a1-ARs and a2ARs on PASMCs [49, 52, 53]. Agonists for a-AR such as norepinephrine, and phenylephrine promote pulmonary vasoconstriction and proliferation via activating Gaq or Ga12/13, and as outlined earlier, increase intracellular Ca2+ concentration [54]. Furthermore, a1-ARs block membrane K+ ion channels via PKC activation, thereby leading to influx of Ca2+ through voltage-dependent channels secondary to membrane depolarization [55]. In contrast, a2-ARs on the surface of PAECs, via production of NO, can stimulate vasodilation of PAs [56]. Eckhart et  al. (1996), showed that hypoxia increased the de novo synthesis of a1-ARs in smooth muscle cells both in  vivo and in  vitro, shifting the balance further in favor of vasoconstriction and proliferation [57].

2.1.4 Muscarinic Cholinergic Receptors The pulmonary circulation receives parasympathetic input but this parasympathetic influence is less than that derived from the sympathetic system. Five muscarinic cholinergic receptor isoforms (M1–M5) have been identified; M1 and M3 are most important for the regulation of the pulmonary circulation [58]. The effects of acetylcholine (Ach), an endogenous muscarinic receptor agonist, on the pulmonary circulation differ depending on the relative number of receptors on the PAECs and PASMCs, the integrity of the endothelium, and the initial level of pulmonary vascular tone [59–61]. Under basal tone, Ach increases PA pressure, which is enhanced by removal of the endothelium, whereas under increased tone, Ach relaxes the PA [61]. In human PAs Gaqcoupled M1 and M3 receptors located on the endothelium are responsible for Ach-induced relaxation, whereas Achinduced contraction is mediated by Gaq-coupled M3 receptors on PASMCs: M3-receptor activation on PAECs leads to release of both NO and PGI2 in an IP3/Ca2+-dependent manner. Unlike in human PA, M1 receptors mediate pulmonary vasoconstriction in vivo in the rabbit pulmonary circulation, thus highlighting species-specific effects of receptor activation in the pulmonary circulation [62].

2.1.5 Purinergic Receptors: GPCRs and Ligand-Gated Ion Channels Purinergic receptors are comprise adenosine (P1) receptors and the P2 receptors, which respond to nucleotides, including ATP, ADP, UTP, and UDP [63]. The P2 receptors are divided into two families: P2Y, which are GPCRs, and P2X, which

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are ligand-gated ion channels. Pharmacological profiling has subdivided P1 receptors into four subtypes (A1, A2A, A2B, and A3), P2X into seven subtypes (P2X1, P2X2, P2X3, P2X4, P2X5, P2X6, and P2X7), and P2Y into eight subtypes (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12, P2Y13, and P2Y14). Ectonucleotidases in the circulation and on cell surfaces act to rapidly degrade extracellular ATP into ADP, AMP, and adenosine, thereby terminating signaling at P2 receptors but potentially promoting signaling at P1 receptors. Purinergic signaling influences normal vascular tone in both PAECs and PASMCs [63, 64]. Adenosine, the breakdown product of ATP, inhibits PASMC proliferation and induces pulmonary vasodilation in an endothelium-independent manner by raising the intracellular cAMP concentration via binding to Gas-dependent A2 receptors: A2B adenosine receptors mediate P1-mediated vasodilation [64, 65]. Adenosine attenuates the vasoconstrictor responses of 5-HT in intact endothelium and denuded rabbit PAs [66]. However, adenosine can also promote apoptosis of PAECs, which appears to depend on the generation of adenosine from extracellular ATP by ectonucleotideases and on its cellular uptake [67]. UTP, UDP, and ATP act via P2Y receptors but only ATP acts via P2X to modulate vascular tone. The specific P2 receptors in the pulmonary circulation, their distribution, and species specification is not yet entirely clear [63, 64]. In both large and small rat PA, P2 receptor agonists enhance vasoconstriction via P2 receptors on PASMCs at resting tone but lead to relaxation via PAEC P2 receptors when the tone is raised [68, 69]. Molecular and pharmacological approaches have implicated Gaq-coupled P2Y2, P2Y4, and P2Y6 receptors as potentially important regulators of the function of PA [68, 70]. In PASMCs stimulation of P2Y receptors raises the intracellular Ca2+ concentration, leading to contraction, whereas in PAECs an increase in intracellular Ca2+ concentration leads to NO- and PGI2-promoted vasodilation [71, 72]. ATP stimulates proliferation of both PASMCs and PAECs, a response that appears dependent on increased PI3K activity, phosphoERK1/2 (extracellular signal-regulated kinase) phosphoAkt, and p70S6K, all known to be associated with the proliferation [73]. ATP binding to ligand-gated P2X receptors facilitates the nonselective passage of cations across the plasma membrane, resulting in an increase in intracellular Ca2+ concentration and depolarization of the cell membrane [74]. P2X receptors contain two transmembrane-spanning domains with intracellular hydrophobic C- and N-termini and a large extracellular loop with ATP-binding sites. The presence of P2X1, P2X2, P2X3, P2X4, P2X5, and P2X7 has been established in human PA; P2X1 receptors most likely mediate the vasoconstriction of PASMCs [68, 69, 73–75]. Shear stress stimulates PAECs to release ATP, which acts in an autocrine fashion to activate Ca2+ influx via P2X4 receptors; in the absence of P2X4 Ca2+ influx is blunted, less NO is released, and vasodilatation is

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prevented [76]. Extracellular ATP thereby protects PAECs exposed to oxidant stress and enhances vasodilation, thus emphasizing a key role of purinergic receptors in maintaining normal pulmonary vasculature function.

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increased circulating levels of ET-1 are found in the lung of patients with PAH [91].

2.1.7 5-HT Receptors 2.1.6 ET Receptors ET-1 is a 21 amino acid polypeptide produced by PAECs and one of the most potent endogenous vasoconstrictors. ET-1 acts via two ET receptor subtypes; both ETA and ETB are expressed on PASMCs but only ETB is expressed on PAECs [2, 77–79]. ET receptors in PAECs clear ET from the circulation and induce release of endogenous NO and PGI2 via Gaq-dependent pathways; the contribution of ETB receptors on PAECs to pulmonary vascular tone appears low as removal of the endothelium does not significantly enhance ET-1-induced pulmonary vasoconstricton [78–81]. ET-1 acting on PASMCs causes a concentration-dependent contraction in isolated PAs in vitro and increases pulmonary vascular resistance in vivo [78, 81, 82]. The relative expression of each type of ET receptor varies depending on the size of the PA studied, which is likely due to the distribution of phenotypically distinct PASMCs: in human PAs the ETA receptor is predominant in larger vessels, with an increased proportion of ETB receptors in more distal branches [78, 83]. ET receptor distribution differs between species in that ETA and ETB receptors coexist in both large and small rabbit PAs [84]. Hypoxia can lead to the redistribution of the ET receptor subtypes, increasing ETA-mediated vasoconstriction in both large and small rat PAs [85]. McCulloch et al. (1998), demonstrated that the ET-1 response in pulmonary resistance vessels after hypoxic exposure could only be blocked by a mixed ETA/ETB antagonist [78, 85]. Thus, ET receptor subtype distribution differs among branches of the PA and species and can be altered by various stimuli. Both ETA and ETB receptors couple to Gaq-dependent pathways, leading to increased concentrations of intracellular Ca2+, PKC activation, ERK1/2 activation, and induction of c-fos and c-jun [82, 86]. ETA receptors also can enhance intracellular Ca2+ concentration by activating nonselective calcium channels on the surface of smooth muscle cells and depolarize human PASMCs via PKC activation and TASK-1 phosphorylation (two-pore domain K+ channel) [87, 88]. Furthermore, ET-1, via Rho/ROCK activation, has been implicated in agonist-stimulated Ca2+ sensitization of 20-kDa myosin light chain phosphorylation and contraction in smooth muscle [89]. ET-1, via ETA receptors, can also act as a co-mitogen in PASMC and pulmonary fibroblasts, in part as the result of the rapid activation of the prosurvival PI3K/ Akt signaling pathway, a mechanism that appears to depend on p38 MAPK [89, 90]. Since ET-1 is a potent pulmonary vasoconstrictor and mitogen, it is perhaps not surprising that

Another vasoactive mediator that has a key role in pulmonary vasoconstriction and proliferation of PASMCs is 5-HT, also known as serotonin [92]. 5-HT is synthesized from L-tryptophan by tryptophan hydroxylase and is released from PAECs. There are at least 17 different 5-HT receptors, including 5-HT1A-F, 5HT2A-C, 5-HT3, and 5-HT4 [93]. In the pulmonary circulation vasoconstriction by 5-HT is mediated by 5-HT1B, 5-HT2A, and 5-HT2B receptors, with their various contributions depending on the level of pre-existing tone and the species [94, 95]. In the absence of tone, 5-HT2A mediates the vasoconstrictive effect of 5-HT in bovine and rat PAs, whereas in the presence of tone, 5-HT elicits contraction via 5-HT1B receptors [96–98]. However, in small and large human PAs, 5-HT1B receptors promote vasoconstriction, even in the absence of tone; this is important since this role of 5-HT1B receptors contrasts with most of the effects of 5-HT in the systemic arteries, which occur via 5-HT2A [96, 97]. 5-HT acting by 5-HT1B receptors induces contraction via a Gai-dependent mechanism, thereby decreasing the level of cAMP by negatively coupling to AC, whereas 5-HT2A receptors activate Gaq-dependent pathways, increasing intracellular Ca+ concentration and activating PKC: Gaq and Gai pathways are thought to synergize to enhance the contractile effect of 5-HT. The 5-HT transporter (SERT), which is located on PAMSCs, appears key for the mitogenic effects of 5-HT since selective SERT inhibitors prevent the remodeling of the pulmonary vasculature [99]. Intracellular accumulation of 5-HT via SERT appears to induce reactive oxygen species (via activation of monoamine oxidase or NADPH oxidase), which leads to the phosphorylation of ERK1/2 and the activation of transcription factors such as GATA4 [100]. As with ET-1, 5-HT can activate Rho/ROCK, thus providing another mechanism by which increased contraction and proliferation is achieved [101]. Sweeney et al. (1995), demonstrated that increasing the level of cGMP could inhibit the ability of 5-HT to constrict PAs [102]. Such results imply that high circulating levels of cyclic nucleotides in the pulmonary circulation, by attenuating the effect of vasoactive mediators, help in maintaining a low pulmonary vascular tone.

2.1.8 Prostanoid Receptors Arachidonic acid (AA) is metabolized through the cyclooxygenase and lipoxygenase pathways to form prostaglandins, thromboxanes, and leukotrienes, which can promote

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constriction or relaxation in PA [1, 103]. Predominant eicosanoids produced by the pulmonary circulation are PGI2 and prostaglandin E2 (PGE2), which both cause vasodilation, and prostaglandin F2a (PGF2a) and thromboxane A2 (TXA2), which are vasoconstrictors: many eicosanoids are cleared by the lung. Prostanoid receptors are classified into five main types: DP, EP, FP, IP, and TP. PGI2 synthase predominates in PAECs and directs metabolism toward PGI2, which is both a very potent pulmonary vasodilator and antiproliferative [104]. PGI2 binds to Gas-coupled IP receptors on PASMCs, leading to an elevation of the cAMP level and a subsequent decrease in the intracellular Ca2+ level, helping to keep vasomotor tone low. PGI2 and its analogues inhibit DNA synthesis and cell proliferation in distal human PASMCs, whereas proximal human PASMCs are comparatively unresponsive [105]. Such results imply that regional heterogeneity and variation in IP receptor expression occurs in the pulmonary arterial tree. Transgenic mice overexpressing PGI2 synthase do not show PASMC hypertrophy following exposure to hypoxia, and PGI2 has been shown to be increased 2.7-fold after 7 days of hypoxia, suggesting that PGI2 has a protective effect in the pulmonary circulation [104, 106]. In fact, synthetic stable analogues of PGI2, such as iloprost and beraprost, are used in the treatment of PAH [107]. In contrast, the eicosanoids PGF2a and TXA2 are both potent vasoconstrictors that bind to FP and TP receptors, respectively, on PASMCs, leading to activation of Gaqdependent pathways and an influx of Ca2+ and release of Ca2+ from store-operated channels. TXA2, acting via PKCz, inhibits voltage-dependent K+ channels, leading to depolarization and activation of L-type Ca2+ channels, thereby constricting the PA [108, 109]. Since a balance of eicosanoids is key in the control of vascular tone under physiological conditions, altered eicosanoid production, possibly via endothelial dysfunction, could shift the balance to the vasoconstrictors and be detrimental to pulmonary function.

2.1.9 Endothelial Barrier Function: Role of Protease-Activated Receptor-1 and Sphingosine 1-Phosphate An intact endothelium is vital for maintaining a low tone in the pulmonary circulatory bed. In addition to growth factors (which are discussed below), certain GPCR agonists, in particular thrombin and sphingosine 1-phosphate (S1P) play an integral role in the regulation of endothelial cell barrier function; thrombin reduces whereas S1P enhances barrier integrity [110]. Adherens junctions, which are composed of vascular endothelial cadherin (VE-cadherin), a-catenin, b-catenin, and p120 catenin, determine the stability of the connections between endothelial cells. The serine protease thrombin directly acts on PAECs, changing their shape and forming gaps; thrombin acts by cleaving

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the extracellular N-terminal domain of the protease-activated receptor (PAR1-4 have been identified): both PAR1 and PAR2, which activate Gaq- and Ga12/13-dependent pathways, mediate the vascular effects of thrombin [111–114]. Thrombin binding to PAR1 acts via Gaq to activate PLCb, thus increasing intracellular Ca2+ concentration and activating kinases and phosphatases that lead to the dephosphorylation of VE-cadherin and b-catenin and phosphorylation of p120 catenin [112]. Gaq- and Ga12/13-dependent pathways also stimulate RhoA via its interaction with p115RhoGEF, activating ROCK and LIM kinase; both pathways increase the numbers of F-actin stress fibers and actomysin contraction. Cytoskeletal rearrangement leads to disruption of the endothelial barrier, thereby increasing albumin permeability. Inhibition of MLCK and ROCK inhibits thrombin-dependent barrier dysfunction [113]. Intercellular gaps that form as a result of PAEC contraction facilitate the ability of thrombin to access and bind to protease-activated receptors on PASMCs, thereby increasing contraction and proliferation; recent data indicate that PASMCs express high levels of PAR1 and PAR2 [114]. Activation of PAR1 can stimulate S1P1-signalling pathways, thus highlighting another protective mechanism of the pulmonary circulation, in this instance to keep PAEC barrier function intact [111]. In contrast, S1P (primarily released by platelets) acts via S1P receptors on PAECs to enhance barrier function. S1P1 and S1P3 receptors are highly expressed in the pulmonary circulation: S1P1 couples to Gai, whereas S1P3 couples to Gai, Gaq, and Ga12/13 [115]. Reverse transcription PCR profiling has revealed that expression of SIP1 receptors is highest in the lung compared with 40 other tissues of the body [23]. S1P1 receptors promote cytoskeletal rearrangement and adherens junction assembly in a Rac1-dependent manner by increasing the recruitment of PI3K and increasing the concentration of intracellular Ca2+ and thereby enhancing polymerization of F-actin and myosin light chain phosphorylation [116]. Inhibition of Gai, PLC, or IP3 prevents the S1P-activated increase in Ca2+ concentration, Rac activation, adherens junction assembly, and subsequent endothelial barrier enhancement [117]. The levels of S1P to which the PAECs are exposed are crucial: high levels (more than 5 mM) can activate S1P3 receptors and mediate RhoA-dependent barrier disruption [118]. How an increase in intracellular Ca2+ concentration both enhances and disrupts the barrier function of PAECs depending on the agonist and agonist concentration is not well understood; however, compartmentalization of the receptor and its effectors may, at least in part, explain such effects. S1P recruits S1P1 receptors to lipid-rich microdomains in PAECs in a PI3K-dependent manner; altering these domains (by depleting them of cholesterol) inhibits S1P signaling and barrier enhancement [119]. Such findings may help explain why caveolin-1 knockout mice (which have a loss of caveolae) develop PAH in association with a disruption of the endothelial layer [17, 119].

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3 Receptor Tyrosine Kinases RTKs, of which there are 90 in the human genome, are receptors that have intrinsic enzyme activity [120, 121]. RTKs are composed of N-terminal extracellular ligand-binding domains and C-terminal intracellular tyrosine kinase domains, which, upon ligand binding, catalyze the transfer of a phosphate residue from ATP to tyrosine residues in the substrates, thereby altering their activity, subcellular location, and stability. RTK ligands include growth factors such as basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), plateletderived growth factor (PDGF), vascular endothelial growth factor (VEGF), and insulin-like growth factor-1 (IGF-1). Growth factors acting via RTKs trigger the activation of several MAPKs, the best characterized of which include the extracellular-signal-regulated kinase (ERK1/2, also known as p42/p44 MAPK), the c-Jun N-terminal kinase (JNK), p38, and ERK5 [120, 121]. MAPK cascades are involved in cellular proliferation, differentiation, and migration [122]. Activation of MAPK involves three sequentially activated kinases: MAPK kinase kinase (MKKK) phosphorylates and activates MAPK kinase (MKK), which in turn phosphorylates and activates MAPK (Fig. 2). Specific MKKK and MKK upstream mediators induce each MAPK subfamily. MAPK phosphatases (MKPs) dephosphorylate the threonine and tyrosine residues of MAPKs both in vitro and in vivo: MKP-1 and MKP-2 are widely distributed and induced in response to robust growth factor stimulation, suggesting that they negatively feed back on MAPK activity [123]. Most RTKs exist as monomers but form dimers following ligand binding; such dimers initiate the autophosphorylation of the RTK, which facilitates the binding of enzymes (Src, PLCg, Shp-2 and PI3K), adaptor proteins (Grb2 and Shc), or docking proteins (IRS, FRS and Gab/Dok) that contain Srchomology 2 (SH2) or phosphotyrosine binding (PTB) domains [120, 121]. The interaction of RTK with adaptors and docking proteins promotes the recruitment of mediators and activation of numerous signaling cascades. For ERK1/2, RTKs promote the activation of the low molecular weight G protein Ras through the recruitment of Grb2/Sos complexes, which activates Ras, thereby recruiting Raf-1 to the plasma membrane through its interaction with the N-terminal domain of Ras (Fig. 2) [124]. Raf-1 can phosphorylate MEK (MAPK/ERK Kinase), which in turn phosphorylates ERK1/2. Downstream effectors of ERK1/2 include cytoplasmic proteins such as cytosolic phospholipase A2 and transcription factors such as c-Myc and c-Fos, which contribute to alterations in gene expression. RTK activation can also trigger PLC- and PI3K/Akt-dependent signaling. MAPK scaffolds have been identified that include the kinase suppressor of Ras, which bring all the components of the specific MAPK cascade together either at the plasma membrane or at other subcellular compartments, thereby

Fig.  2  Receptor tyrosine kinase (RTK)-mediated signaling in the PASMC. Activation of RTKs leads to signaling by three kinases that are connected in series: a mitogen-activated protein kinase (MAPK) kinase kinase that phosphorylates and activates a MAPK kinase, which in turn activates MAPK. For the extracellular-signal-regulated kinase (ERK) pathway, Ras activation occurs through the recruitment of Grb2/Sos complexes, which leads to the recruitment of Raf-1, which phosphorylates MEK, which in turn phosphorylates ERK1/2. ERK1/2 can activate transcription factors such as Elk-1, c-Myc, and c-Fos, therefore altering gene expression. RTK can also activate the PI3K/Akt pathway in PASMCs, which in turn increases transcription of antiapoptotic and pro-proliferative genes

determining the strength, duration, and location of ERK signaling [120]. Furthermore, as with GPCRs, the localization of RTKs in membrane microdomains, such as rafts/caveolae, facilitates activation: for example, the EGF receptor (EGFR) contains a 60 amino acid region that mediates targeting to the raft/caveolae microdomain [15, 125]. MAPKs are not only activated by growth factors but also by GPCR agonists and cytokines and thus can serve as a point of convergence and cross talk among multiple signaling pathways; receptor cross talk is discussed in more detail in the following.

3.1 RTKs in the Pulmonary Circulation A number of growth factors, including PDGF, bFGF, EGF, and VEGF, have been implicated in the development and maintenance of the pulmonary circulation [126]. Different growth factors can function at different stages of the cell cycle, highlighting the importance of multiple signals operating in concert. Excessive production of growth factors and/ or increased expression of their receptors can also play a role in pulmonary vascular disease, such as PAH [1–3].

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A number of studies have shown that VEGF-A is highly expressed in lung and is important for vascular development and maintenance of PAEC function and survival. VEGF is essential for embryonic lung development: knocking out a single allele is embryonically lethal and inhibition of signaling mediators prevents development in neonatal rats [127]. VEGF, which acts via VEGF receptor-1 (FIT) and VEGF receptor-2 (KDR), increases NO and PGI2 formation from PAECs; VEGF binding leads to c-Src activation, which, via PLCg, DAG, and IP3, leads to PKC activation and increased intracellular Ca2+ concentration, and in parallel Akt activation via PI3K, both of which can activate NO synthase [128, 129]. Interestingly VEFG overexpression protects against monocrotaline-induced PAH, presumably by protecting endothelial integrity [130]. PDGF, bFGF, IGF-1, and EGF, via RTK activation, also contribute to the development of the pulmonary arterial tree, for example, by aiding tube formation [126, 131]. These mitogens also have a key role in pulmonary vascular remodeling in response to hypoxia or vascular injury: inflammatory cells, platelets, and cytokines can all stimulate their release [1–3]. Inhibition of PDGF receptor and EGFR can reverse the progression of pulmonary arterial remodeling [132, 133]. PDGF-mediated PASMC proliferation, via PI3K/Akt signaling, has been shown to be associated with c-Jun/STAT3induced upregulation of transient receptor potential channel 6 expression, an increase in capacitative Ca2+ entry, nuclear export, and proteasomal degradation of CREB [134, 135]. Recently, PDGF was shown to regulate PASMC proliferation and phenotype by inducing miR-221, a noncoding RNA that plays a role in negative posttranscriptional regulation of genes [136]. Furthermore, fibroblast growth factor 2 (FGF2) acting via fibroblast growth factor receptor 1 in PAECs, is linked to the pathogenesis of PAH by altering endothelial function; FGF2 increases the expression of the antiapoptotic proteins XIAP and BCL-X via PKC [137]. It is important for the pulmonary circulation to tightly regulate growth factor expression after development since chronic exposure can lead to the muscularization of the small PAs and increased PA pressure.

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members act via a receptor complex of type I and type II serine/threonine kinase receptors (seven type I and five type II have been identified), which comprise a cysteine-rich extracellular domain, a single transmembrane domain, and a conserved intracellular Ser-Thr kinase domain. Upon ligand binding, the preformed dimers of receptor type II phosphorylate the preformed dimers of type I receptor on serine or threonine residues, which can then recruit and phosphorylate receptor-regulated Smads (R-Smads; Smad1, Smad2, Smad3, Smad5, and Smad8) in a receptor-dependent manner (Fig. 3). These activated R-Smads form complexes with Smad4, which allows translocation to the nucleus and regulation of gene transcription such as of c-myc and cyclin D1, chromatin-remodeling complexes, and histone-modifying enzymes. The activation of R-Smads can be inhibited by inhibitory Smads (Smad6 and Smad7), which lead to degradation or phosphorylation of the complex, thereby attenuating signaling. In addition, nonSmad-dependent signal pathways downstream of TGF-b receptors have been proposed: these include interaction with MAPK pathways; for example, TGF-b receptors associate with TRAF6, which recruits TAK1 to activate JNK/p38

4 Receptor Serine/Threonine Kinases Transforming growth factor (TGF)-b family members are multifunctional cytokines: over 30 members that include TGF-b isoforms, activins, and the protein nodal, bone morphogenetic proteins (BMPs), and growth and differentiation factors are present in mammals [138, 139]. These factors are pleiotropic, playing vital roles during embryogenensis and maintaining normal tissue function by coordinating cell proliferation and differentiation. TGF-b family

Fig.  3  Receptor serine/threonine kinase mediated signaling in the PASMC. The transforming growth factor b (TGF-b)/bone morphogenetic proteins (BMP) signaling pathway is initiated after the formation of a heterotetrameric complex of type I and type II receptors at the cell membrane. TGF-b signaling can activate Smad2/Smad3, whereas BMP signaling induces Smad1/Smad5/Smad8 activation. Activated Smads form complexes with Smad4, which allows translocation to the nucleus and regulation of gene transcription, such as of c-myc and cyclin D1. The activation of receptor-regulated Smads can be inhibited by inhibitory Smads (Smad6 and Smad7) that shut off signaling

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[140]. TGF-b signals in most cells via TGF type II receptor and ALK5, activins via activin receptor types IIA and IIB and ALK4, and BMPs via BMP type II receptor (BMPR-II). Depending on the cell type, the specific ligand, and the environment, binding to these receptors can have different functional outcomes.

4.1 Receptor Serine/Threonine Kinases in the Pulmonary Circulation BMP signaling is essential for development of the lung and maintenance of the pulmonary vasculature since altered expression can lead to death during early embryogenesis [141]. BMP-2, BMP-4, and BMP-7 exert their effects by interacting with BMP receptor IA (ALK3) or BMP receptor IB (ALK6) and their coreceptor, BMPR-II. Ligand binding to BMPR-II, which are expressed in PAECs and PASMCs, results in the formation of a complex with BMP type I receptor that activates Smad1, Smad5, and Smad8, whereas in parallel TGF-b acts via an ALK5/Smad3dependent pathway [142]. The endogenous expression of the BMP inhibitors noggin and Smurf1 blunts BMP signaling and is crucial in pulmonary development by the inhibition of branching caused [143]. The tight regulation of BMP signaling may be key in preventing pulmonary remodeling and keeping tone low since mutations in BMPR-II that lead to reduced BMPR-II expression and Smad1/ Smad5 phosphorylation are associated with the development of PAH [144, 145]. Smad1 is essential for both the BMP-2- and the BMP-4dependent survival of PAECs and growth suppression of PASMCs; however, p38 and ERK1/2 are required for their mitogenic response in PASMCs [142, 145, 146]. BMPs are either proliferative or antiproliferative in PASMCs depending on the branch of the pulmonary arterial tree from which the smoothe muscle cells are isolated [145, 146]. Other effectors of BMP signaling have recently been noted, for example, the antiproliferative effect of BMP-2 on PASMCs has been shown to involve activation of the transcription factor ­peroxisome proliferator-activated receptor g (PPARg), independent of Smad1/Smad5/Smad8 and correlated with reduced nuclear phospho-ERK and apoE RNA expression [147]. BMPR-II can also recruit the canonical Wingless (Wnt) signaling pathway via ERK1/2 to promote PAEC survival, while in parallel BMPR-II can also stimulate the noncanonical Wnt pathway via Smad1 to activate RhoA and Rac1 to induce migration [148]. These data highlight the complexity of the signaling of the pulmonary circulation with respect to the distribution of receptors and effectors in different cells or even the same cell type from different regions of the pulmonary arterial tree.

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5 Receptor Cross Talk and Convergence It is well established that intracellular signaling pathways from different classes of receptors can both positively and negatively modulate each other’s activity and thereby govern their biological response. Such interactions can occur either from the spatial organization of the signaling pathways via multifunctional adaptor proteins or through convergence among receptors: such associations may be important in the homeostasis of the pulmonary circulation. “Cross talk” between signaling pathways initiated by GPCRs and those by RTKs have been widely studied, with GPCRs or components of GPCR-induced pathways being either upstream or downstream of RTKs: for example, ET-1 can activate EGFR in the absence of growth factors [149]. On the basis of as-yet indirect data, it appears that activation of certain G–proteins, for example, Gai, can induce the extracellular activity of a transmembrane metalloproteinase causing ectodomain shedding of a transmembrane RTK ligand precursor, such as heparin-binding EGF, which in turn activates the EGFR [150]. GPCRs can also activate cytoplasmic tyrosine kinases, such as c-Src and Pyk, which phosphorylate RTK, or by formation of multimeric complexes via interaction of components of GPCR-mediated signaling, such as b-arrestin and GRKs (G-protein coupled receptor kinases), can integrate signaling pathways (the latter phenomenon is termed “transactivation”) [151]. For example, association of b-arrestins with GPCRs can promote the recruitment of signaling molecules such as adaptor protein complex 2, clathrin, Src, Hck, and c-Raf-1, several of which are important for ERK activation [151]. Novel convergence mechanisms of GPCRs with other receptors have recently been shown, e.g., a GPCR/RhoA/ Cyr61/integrin pathway contributes to sustained GPCR responses and b-arrestins can interact with components of Wnt pathways (e.g., Dvls and Axins), thereby allowing GPCRs to recruit components of the canonical Wnt pathway [152, 153]. Such receptor cross talk may play a critical role in the normal homeostasis of the pulmonary circulation as well as in the development of disease. For example, Gas activation, via increasing the intracellular concentration of cAMP and PKA activation, antagonizes mitogenic pathways as a consequence of phosphorylation of Raf-1 on Ser-43 and Ser-621, thereby attenuating smooth muscle cell proliferation [154]. cAMP can blunt PI3K-dependent pathway activation as part of its antiproliferative effects [155]. In addition, ANP, which increases cGMP levels, inhibits proliferation of PASMCs, in part, by preventing TGF-b -induced Smad2 and Smad3 nuclear translocation and increases in extracellular matrix expression [156]. Moreover, cGMP via PKG can activate RGS2, thereby attenuating Gaq-coupled vasoconstriction of smooth muscle; such an interaction may help explain some of the beneficial effects of the PDE5 inhibitor sildenafil in treating PAH [11].

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These data suggest that receptor-mediated pathways can also work as “physiological antagonists,” i.e., by having actions that oppose one another in the pulmonary circulation. In contrast, in the setting of disease, receptor-mediated signaling can act synergistically to enhance constriction and proliferation. For example, 5-HT through the activation of ERK1/2 and phosphorylation of the linker region of Smad1 can antagonize the BMP pathway [157]. In addition, decreased levels of cGMP can enhance 5-HT-promoted vasoconstriction of PASMCs [102]. Decreased PKA activity via Gai activation enhances ERK1/2-mediated signaling in PAECs, thereby contributing to cytoskeletal reorganization and barrier dysfunction [158]. In addition, TGF-b1 can downregulate the expression of AC1, AC2, and AC4, increase Gai protein levels, and reduce IP receptor messenger RNA expression, thereby impairing PGI2 signaling in PASMCs [159]. Such effects work together and contribute to vascular remodeling in response to these mediators: these data imply that cross talk of signaling can regulate disease progression via the action of multiple signaling components.

6 Summary The maintenance of low tone in the pulmonary vascular tree is the result of a tight balance of endogenous vasoconstrictors and vasodilators. Receptor-mediated signaling plays a pivotal role in coordinating cellular functions: receptor cross talk can either attenuate or augment changes in the vasculature. Receptormediated downstream pathways in the PA are complex: they are species- and cell-specific and dependent on an intact endothelium and on basal tone. Such complexity implies that studies of receptor function in PASMCs derived from the proximal pulmonary arterial vasculature may not be representative of the full range of effects in vivo. It remains unclear why the response of certain receptor ligands can alter dramatically when tone is increased and why blockade of certain receptors has little or no effect on basal tone. Thus, a comprehensive understanding of the normal regulation of pulmonary function by receptors and signaling pathways is necessary before researchers and clinicians can fully appreciate the complexity (and exploit information) regarding abnormalities that accompany and may lead to disease and that may be therapeutic targets. With increased understanding of receptors and postreceptor components involved in signal transduction mechanisms and the development of molecular tools to interrogate such receptors and signaling components, future studies will likely assess the role of previously unappreciated receptors (especially GPCRs that have not previously been studied) and their effectors in the pulmonary circulation. We hypothesize that newly recognized receptors may prove to be more important than those highlighted above. Recent data have indicated that

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the profiling of GPCR expression can cluster tissues that comprise classical physiological systems (cardiovascular, gastrointestinal, etc.), thus implicating such receptors in homeostatic regulation. Despite the large number of physiological responses that are regulated by cell-surface receptors, especially GPCRs, only a small percentage are currently targeted therapeutically; therefore, identification of “novel” (normally expressed but not previously recognized) receptors and their function in the pulmonary circulation could be promising for the discovery of new targets for drugs for the treatment of pulmonary vascular disease. Acknowledgements  This work was supported by grants from the National Heart, Lung, and Blood Institute of the National Institutes of Health (5K99HL091061-02), the Leukemia & Lymphoma Society (7332-06), and the Ellison Medical Foundation (AG-SS-1662-06).

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F. Murray et al. 152. Bryja V, Gradl D, Schambony A, Arenas E, Schulte G (2007) Beta-arrestin is a necessary component of Wnt/beta-catenin ­signaling in vitro and in vivo. Proc Natl Acad Sci U S A 104:6690–6695 153. Walsh CT, Radeff-Huang J, Matteo R et al (2008) Thrombin receptor and RhoA mediate cell proliferation through integrins and cysteinerich protein 61. FASEB J 22:4011–4021 154. Graves LM, Bornfeldt KE, Raines EW et al (1993) Protein kinase A antagonizes platelet-derived growth factor-induced signaling by mitogen-activated protein kinase in human arterial smooth muscle cells. Proc Natl Acad Sci U S A 90:10300–10304 155. Azam MA, Yoshioka K, Ohkura S et al (2007) Ca2+-independent, inhibitory effects of cyclic adenosine 5’-monophosphate on Ca2+ regulation of phosphoinositide 3-kinase C2alpha, Rho, and myosin phosphatase in vascular smooth muscle. J Pharmacol Exp Ther 320:907–916 156. Li P, Wang D, Lucas J et al (2008) Atrial natriuretic peptide inhibits transforming growth factor beta-induced Smad signaling and myofibroblast transformation in mouse cardiac fibroblasts. Circ Res 102:185–192 157. Long L, MacLean MR, Jeffery TK et al (2006) Serotonin increases susceptibility to pulmonary hypertension in BMPR2-deficient mice. Circ Res 98:818–827 158. Liu F, Verin AD, Borbiev T, Garcia JG (2001) Role of cAMPdependent protein kinase A activity in endothelial cell cytoskeleton rearrangement. Am J Physiol Lung Cell Mol Physiol 280: L1309–L1317 159. El-Haroun H, Clarke DL, Deacon K et al (2008) IL-1beta, BK, and TGF-beta1 attenuate PGI2-mediated cAMP formation in human pulmonary artery smooth muscle cells by multiple mechanisms involving p38 MAP kinase and PKA. Am J Physiol Lung Cell Mol Physiol 294:L553–L562 160. Morrell NW, Upton PD, Kotecha S et  al (1999) Angiotensin II activates MAPK and stimulates growth of human pulmonary artery smooth muscle via AT1 receptors. Am J Physiol 277:L440–L448 161. Taraseviciene-Stewart L, Scerbavicius R, Stewart JM et al (2005) Treatment of severe pulmonary hypertension: a bradykinin receptor 2 agonist B9972 causes reduction of pulmonary artery pressure and right ventricular hypertrophy. Peptides 26:1292–1300 162. Smith AM, Elliot CM, Kiely DG, Channer KS (2006) The role of vasopressin in cardiorespiratory arrest and pulmonary hypertension. QJM 99:127–133 163. Upton PD, Wharton J, Coppock H et  al (2001) Adrenomedullin expression and growth inhibitory effects in distinct pulmonary artery smooth muscle cell subpopulations. Am J Respir Cell Mol Biol 24:170–178 164. Petkov V, Mosgoeller W, Ziesche R et al (2003) Vasoactive intestinal peptide as a new drug for treatment of primary pulmonary hypertension. J Clin Invest 111:1339–1346 165. Chattergoon NN, D’Souza FM, Deng W et  al (2005) Antiproliferative effects of calcitonin gene-related peptide in aortic and pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 288:L202–L211 166. Pedersen KE, Buckner CK, Meeker SN, Undem BJ (2000) Pharmacological examination of the neurokinin-1 receptor mediating relaxation of human intralobar pulmonary artery. J Pharmacol Exp Ther 292:319–325 167. Ortiz JL, Labat C, Norel X, Gorenne I, Verley J, Brink C (1992) Histamine receptors on human isolated pulmonary arterial muscle preparations: effects of endothelial cell removal and nitric oxide inhibitors. J Pharmacol Exp Ther 260:762–767 168. MacLean MR, Alexander D, Stirrat A et  al (2000) Contractile responses to human urotensin-II in rat and human pulmonary arteries: effect of endothelial factors and chronic hypoxia in the rat. Br J Pharmacol 130:201–204

Chapter 15

Role of Calcium as a Second Messenger in Signaling: A Focus on Endothelium Donna L. Cioffi, Christina J. Barry, and Troy Stevens

Abstract  Endothelial cells line blood vessels and lymphatic vessels, and in so doing separate circulating solutes, macromolecules, and cells from the underlying tissues. In this capacity, endothelium fulfills a gatekeeper role, responding to mechanical and chemical signals in the blood and tissues to regulate the directed transport of molecules and cells. The endothelial cell barrier is established by cell–matrix interactions that connect endothelium to the basement membrane, and by cell–cell junctions that connect adjacent endothelial cells together. The strength of both the cell–matrix and the cell–cell junctions is adjusted dynamically, modulated in part by cytosolic calcium concentrations. Whereas basal cytosolic calcium concentrations are maintained at low nanomolar levels, inflammatory first messengers increase cytosolic calcium concentrations. This increase in calcium concentration disrupts cell–matrix and cell–cell adhesion, inducing interendothelial cell gaps that provide a paracellular transport pathway. Calcium channels on the cell membrane allow for calcium entry that mediates this change in cell shape. However, recent advances have identified that endothelial cells express many different calcium channels, with quite distinct cellular functions. This chapter reviews how calcium homeostasis is controlled in tissue compartments, how cells such as endothelial cells compartmentalize calcium, and how endothelial cells utilize calcium signals as a second messenger to regulate their shape. Keywords  Lung microvascular endothelium • Endothelial cells • Signaling cascades • Vascular permeability • Barrier function

1 Introduction Endothelial cells line blood vessels and lymphatic vessels, and in so doing separate circulating solutes, macromolecules, and D.L. Cioffi (*) Center for Lung Biology, University of South Alabama, MSB 2316, 5851 USA Drive N., Mobile, AL 36688, USA e-mail: [email protected]

cells from the underlying tissues [1, 2]. In this capacity, endothelium fulfills a gatekeeper role, responding to mechanical and chemical signals in the blood and tissues to regulate the directed transport of molecules and cells. The endothelial cell barrier is established by cell–matrix interactions that connect endothelium to the basement membrane, and by cell–cell junctions that connect adjacent endothelial cells together. The strength of both the cell–matrix and the cell–cell junctions is adjusted dynamically, modulated in part by cytosolic calcium concentrations. Whereas basal cytosolic calcium concentrations are maintained at low nanomolar levels, inflammatory first messengers increase cytosolic calcium concentrations [3, 4]. This increase in calcium concentration disrupts cell–matrix and cell–cell adhesion, inducing interendothelial cell gaps that provide a paracellular transport pathway. Calcium channels on the cell membrane allow for calcium entry that mediates this change in cell shape. However, recent advances have identified that endothelial cells express many different calcium channels, with quite distinct cellular functions. This chapter reviews how calcium homeostasis is controlled in tissue compartments, how cells such as endothelial cells compartmentalize calcium, and how endothelial cells utilize calcium signals as a second messenger to regulate their shape.

2 The Origins of Calcium Calcium is an alkaline-earth metal that occurs in either an ionized or a combined state. It is the fifth most abundant element in the earth’s crust. In 1808 Sir Humphry Davy first discovered calcium by electrolyzing a mixture of lime and mercuric oxide [5]. A critical role for calcium in mammalian physiological function was established in 1883 when Sydney Ringer demonstrated that isolated frog hearts require calcium in the bathing solution to continue beating [6]. Since its discovery, calcium has been found to play significant roles in numerous biological functions [7], including egg fertilization [8], tissue development [8], conduction of nerve impulses to muscle [9], cell adhesion, metabolism, proliferation, secretion, and learning and memory [7].

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3 Calcium Homeostasis in the Blood We now recognize that calcium is a mineral that is necessary to sustain life. Calcium can be found in many food sources, such as milk, cheese, and broccoli. Once calcium has been consumed, stomach acid converts inorganic calcium into a soluble, ionized state to be absorbed in the small intestine. Calcium absorption by the small intestine consists of a passive paracellular pathway and an active transcellular pathway [10–12]. The paracellular mechanism is a diffusional process that functions throughout the length of the intestine [13]. On the other hand, the transcellular mechanism predominantly occurs in the duodenum and upper jejunum and is regulated by vitamin D, or the hormonal form 1,25-dihydroxyvitamin D3 [14]. The transcellular mechanism is a three-step process. First, calcium is driven by the electrochemical gradient across the brush border through the transient receptor potential vanilloid (TRPV) calcium channels TRPV5 and TRPV6 [15–18]. Calcium then diffuses toward the basolateral membrane, and is extruded into blood via the plasma membrane calcium ATPase 1b (PMCA1b) and sodium/calcium exchanger [19]. Blood calcium concentrations are highly regulated, and are maintained at approximately 10  mg/dL (5  mEq/L, 2.5  mmol/L). Deviations from the normal calcium range result in either hypercalcemia or hypocalcemia, which can lead to disease. To maintain strict blood calcium concentrations, the small intestine, bones, and kidneys supply or remove calcium from the blood (Fig. 1). Dietary calcium is absorbed in the small intestine, which contributes to regulation of blood calcium concentration by adjusting the rate of calcium absorption. For example, when dietary calcium intake is low, calcium absorption by the transcellular process is func-

Fig. 1  Regulation of blood calcium levels. When calcium concentration in the blood is low, the body responds by stimulating secretion of the parathyroid hormone, which in turn results in increased production of vitamin D. Vitamin D enhances intestinal absorption of calcium. The parathyroid gland and vitamin D increase the release of calcium from bone and decrease the renal excretion of calcium

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tionally important [20, 21]. On the other hand, when dietary calcium intake is high, calcium absorption by the transcellular process is reduced and calcium is predominantly absorbed by the paracellular process [21]. Bone serves as a vast calcium reservoir, storing 99% of the body’s calcium [22, 23]. When blood calcium levels are low, osteoclasts release calcium from the bone in an effort to restore blood calcium concentrations [24, 25]. In contrast, when blood calcium concentrations are high, osteoblasts store calcium in bone [24, 25]. The kidney filters calcium [25, 26]. Under normal physiological conditions, 98–99% of filtered calcium is reabsorbed, preserving blood calcium levels [27]. Approximately 60% of the reabsorption occurs in the proximal tubules and the remainder in the ascending limb of the loop of Henle and the distal tubule. However, when blood calcium concentrations are high, the kidney tubular calcium reabsorption decreases, allowing more calcium to be excreted [25, 27]. Thus, the small intestine, bones, and kidneys are critical components of calcium homeostasis and are the main organs that regulate calcium blood concentrations. In addition to the small intestine, bone, and kidney, calcium homeostasis is regulated by hormonal control systems. The parathyroid hormone, vitamin D, and calcitonin are three hormones that control blood calcium concentrations. The parathyroid hormone, also known as parathormone and parathyrin, is a peptide hormone secreted by the parathyroid in response to a low blood calcium level [25, 28]. Parathormone increases the blood calcium level by three main mechanisms. First, it increases the number of osteoclasts that break down bone matrix and release calcium into the blood. Second, parathormone maximizes tubular calcium reabsorption within the kidney. Third, it stimulates kidney vitamin D production to increase calcium absorption in the intestines. Vitamin D increases calcium blood concentrations [29]. Vitamin D is a steroid hormone that promotes intestinal calcium absorption. Vitamin D can be found in food or, under normal sunlight exposure, can be synthesized by the skin. As mentioned previously, parathormone converts vitamin D to its active form and facilitates small intestine calcium absorption. In concert with parathormone, vitamin D releases calcium from bone. Calcitonin is a polypeptide secreted by parafollicular cells of the thyroid; it reduces blood calcium levels [23, 25, 29]. Calcitonin suppresses renal tubular calcium reabsorption, and therefore enhances calcium excretion. Calcitonin also inhibits calcium removal from bone by osteoclasts, promoting bone formation. By inhibiting calcium removal, calcitonin minimizes calcium flux from bone to blood. Parathormone, vitamin D, and calcitonin each contribute to blood calcium homeostasis.

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Calcium functions in biological systems when it is in an uncomplexed form, as well as when it is in complexed forms. In the free form, calcium is an ion with a +2 oxidation state. Whereas low millimolar calcium concentrations are found in blood and interstitium, cells maintain just nanomolar calcium concentrations, establishing an approximately 20,000:1 concentration gradient across the plasma membrane. Although cellular calcium can exist in an uncomplexed state, its lifetime as a free ion is very short, on the order of 3 × 10−5 s, compared with buffered calcium, which exhibits a lifetime of approximately 1  s [30]. The short free calcium lifetime is largely a reflection of the abundance of negatively charged proteins and molecules that exist within the cell. Indeed, there are numerous calcium-binding proteins with binding affinities that cover a wide range. The higher the binding affinity, the more tightly the protein holds on to calcium, whereas a protein with a weak binding affinity will be more apt to release calcium. Calcium-binding proteins play variable roles within the cell, including calcium buffering, transcription, ion channel regulation, cell proliferation, and homeostasis. Although there are thousands of known calcium-binding proteins, they generally fall into one of several categories. First is the wellknown superfamily of EF hand calcium-binding proteins. The EF hand motif is one of the most common domains encoded by the human genome. This is a small domain comprising 29 residues in the form of a helix–loop–helix structure (Fig. 2a) [31]. The loop, which is 12 or 14 amino acids long, is the actual calcium-binding moiety (reviewed in [32, 33]). Most EF hand proteins contain adjacent pairs of the motif that are connected by a variable-length linker [32, 33].

There are more than 120 proteins within the EF hand superfamily [33]. Some of the more widely studied include calmodulin, calcineurin, calreticulin, and the S100 subfamily. Calmodulin is a ubiquitously expressed calcium-binding protein that is conserved throughout evolution. Calcium binding to calmodulin changes the conformation of calmodulin, and enables calmodulin to regulate the function or activity of binding partners [34]. Calmodulin has two globular domains forming a dumbbell shape, each of which has two EF hand motifs [35], and thus can bind a total of four calcium ions (Fig. 2b). Within each motif there are six key amino acids which play an important role in calcium binding [31]. Although the calcium-binding sites exhibit similarity to one other, they nonetheless exhibit quite distinct binding affinities as well as distinct conformational changes induced by calcium binding [31]. Calmodulin exhibits another interesting feature. In addition to binding calcium, it also binds to proteins that bear a calmodulin-binding domain. When calmodulin is not bound to a protein, its binding to calcium is positively cooperative between the two EF hands within each of the globular domains, yet there is no cooperativity between the domains. However, when calmodulin is bound to peptides expressing calmodulin-binding domains, there appears to be cooperativity between all four calcium-binding sites [34]. Annexins comprise another superfamily of calcium-binding proteins. Annexins are perhaps best known for their role in apoptosis. However, with the identification of more than 1,000 proteins within this superfamily, it is believed that the annexins have numerous other functions (reviewed in [36]). The identifying feature of annexins is that they are not only calcium-binding proteins, but that they are also phospholipid-binding proteins (reviewed in [37]). The annexin repeat, which is in all annexins described to date, contains four repeating units, each with approximately 70 amino acids (Fig. 2c). The repeats contain up to five a-helices and usually have a “type 2” calcium-binding

Fig. 2  Calcium-binding proteins. a. The basic calcium-binding structure of the EF hand motif is a helix–loop–helix (reprinted by permission of MacMillan Publishers Ltd from Burgoyne [31] copyright 2007). b Calmodulin exhibits two globular domains each of which has two EF hand motifs which can cooperatively bind two calcium ions (yellow spheres) (reprinted from Valeyev et  al. [35]). c The annexin

core contains four repeats (blue, yellow, green, and red), each of which has five a-helices. Each annexin repeat can bind one calcium ion. The red spheres denote oxygen atoms that are involved in calcium binding. Importantly, annexin association with membrane surfaces is mediated through bound calcium (reprinted by permission of MacMillan Publishers Ltd from Gerke et al. [143] copyright 2005)

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motif [36]. Indeed, with its positive charge, calcium regulates the interaction between annexin and the negatively charged phospholipids within the plasma membrane.

4.2 Intracellular Calcium Compartments Both prokaryotic and eukaryotic cells rely on the precise regulation of free calcium in a temporal and concentrationdependent manner. The cell has multiple ways in which it controls the free calcium concentration, including different cell compartments, transporters, and channels and various calcium handling modes. Different cell compartments often act as calcium “stores” in which they contain a much higher calcium concentration as compared with the cytosol. The cytosol typically has the lowest concentration of free calcium, in the unactivated state, which is on the order of 100 nM for both aortic and pulmonary artery endothelial cells [38, 39]. Importantly, the cytosol is a calcium-storage compartment. Other compartments are formed by organelles that include the endoplasmic/sarcoplasmic reticulum, mitochondria, and even the nucleus. Intracellular vesicles also act as calciumstorage compartments. There are different types of calciumstorage vesicles. Calciosomes are small vacuoles that contain, in addition to calcium, calcium-binding proteins similar to calsequestrin [40]. Calsequestrin is found within the lumen of junctional sarcoplasmic reticulum of cardiac and skeletal muscle, and functions to bind calcium in a high capacity, yet with moderate affinity [41]. Calciosomes were first described by Volpe et al. [42], who postulated that they might be the intracellular target of inositol 1,4,5-trisphosphate (InsP3). In non-muscle-cell calciosomes, the calsequestrin-like protein was later identified as calreticulin [43]. Another type of calcium-storage vesicle is characterized by acidic lumenal pH, similar to the acidocalcisomes of bacteria and unicellular eukaryotes [40]. In mammalian cells, these acidic calciumstorage depots come in multiple forms, such as secretory granules, endosomes, lysosomes, and the trans-Golgi network (reviewed in [44]). For example, human platelet dense granules are acidic compartments which contain calcium and inorganic polyphosphate and pyrophosphate [45]. In conjunction with other similarities with the bacterial acidocalcisomes, it is suggested that these organelles are evolutionarily conserved from bacteria to humans. Polyphosphates, which are polymers of inorganic phosphates linked through high-energy phosphoanhydride bonds, act as strong cation chelators and can thus serve as a calcium-storage polymer (reviewed in [46, 47]). Indeed, compartmental calcium storage is a key regulator of cytosolic calcium concentration. Given the physiological importance of precisely regulating cytosolic calcium concentration, it is not surprising that the cell possesses several different calcium regulatory mechanisms.

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4.3 Calcium Transporters Calcium transporters and ion channels are another way in which the cell regulates its free calcium concentration. Calcium transporters in general move the ion against its concentration gradient, thus requiring energy input in the form of ATP hydrolysis. There are numerous calcium transporters in mammalian cells. On the plasma membrane, an ATP-dependent calcium pump, known as the calcium-ATPase or the plasmalemmal calcium pump, is expressed and is responsible for extruding calcium from the cytosol into the extracellular space. This pump, which acts via active transport, exhibits high calcium affinity (reviewed in [48]). Interestingly the calcium-ATPase has been shown to be localized to caveolae along with InsP3receptor-like protein, thus implicating caveolae in control of cytosolic calcium concentration [49]. In support of this idea, plasma membrane calcium-ATPase function is inhibited and calcium extrusion is reduced in caveolin-1 knockout mice [50]. The sodium/calcium exchanger similarly extrudes calcium from the cell. Although calcium is still moved against its concentration gradient, this transporter does not require ATP hydrolysis. Instead, calcium movement out of the cell is coupled with sodium movement into the cell, down its concentration gradient (reviewed in [51]). Although this antiporter system displays a high calcium transport capacity, it has only a weak calcium affinity (reviewed in [48]). Different cells express different types and amounts of calcium transporters. The sodium/calcium exchanger will compete with other calcium transport pathways, and the importance of the sodium/calcium exchanger depends upon the abundance of other transport pathways. In some tissues, e.g., the liver, its importance is low, whereas in other tissues, e.g., kidney, smooth muscle, brain, and heart, its importance is high (reviewed in [51]). Although calcium storage and calcium extrusion represent the principal ways of lowering cytosolic calcium concentration, the cell also possesses mechanisms whereby it can increase cytosolic calcium concentration. There are two key mechanisms to increase cytosolic calcium concentration, including calcium release from internal stores and calcium entry through channels in the plasma membrane.

4.4 Calcium Release Calcium mobilization from internal stores and external calcium entry are critically dependent upon the action of the calciummobilizing second messenger, InsP3. InsP3 couples external hormone signals to intracellular calcium release signals [52, 53]. InsP3 is a small water-soluble molecule generated at the plasma membrane, from where it detaches and rapidly diffuses through the cytosol. InsP3 then binds to specific receptors on

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intracellular calcium stores, such as the endoplasmic reticulum [54]. Once InsP3 has bound to specific receptors, it induces calcium store release and quickly raises the cytosolic calcium concentration [55, 56]. InsP3 is generated by phospholipase C (PLC) cleavage of lipid phosphatidylinositol 4,5-bisphosphate (PIP2). Two pathways activate PLC and ultimately generate InsP3 [53]. The first pathway is a G-protein-coupled-receptor pathway, specifically the Gq pathway, which activates PLCb1. Activated PLCb1 cleaves PIP2 to generate two products, InsP3 and diacylglycerol. InsP3 diffuses through the cytosol and binds to its receptor on the calcium store. Diacylglycerol, on the other hand, remains embedded in the membrane, and can either be further cleaved to release arachidonic acid or specifically activate the serine/threonine protein kinase referred to as PKC. The second pathway that generates InsP3 is the tyrosine kinase pathway [53, 57]. Tyrosine kinase receptors are unified by the presence of a single membrane-spanning domain and an intracellular tyrosine protein kinase catalytic domain. In general, a ligand binds to the tyrosine kinase receptor, resulting in receptor dimerization. Receptor dimerization activates the kinase activity of both intracellular chains, resulting in cross-phosphorylation of several tyrosine residues. Once the tyrosine kinase receptor has been phosphorylated, the receptor is activated. The activated receptors stimulate PLCg1, which in turn hydrolyzes PIP2 to InsP3 and diacylglycerol. Once InsP3 has been generated, it has a half-life of just a few seconds [58]. The average InsP3 half-life is approximately 9 s [59]. InsP3 is inactivated by either phosphorylation to inositol 1,3,4,5-tetrakisphosphate or dephosphorylation to inositol 1,4-bisphosphate, which is rendered an ineffective ligand at the InsP3 receptor [57]. InsP3 is also subject to rapid metabolism by InsP3 3-kinase isoforms [57, 60] and membrane-associated InsP3 5-phosphatase [61]. Because InsP3 possesses a short half-life, most InsP3 receptors are located close to the plasma membrane. InsP3 is unique to the various inositol phosphates and phospholipids in that it is the only molecule that has a channel as a target molecule [62]. The InsP3 receptor is a tetrameric channel embedded in the membrane of the endoplasmic reticulum and functions as a calcium release channel [63– 65]. The InsP3 receptor is located on the endoplasmic reticulum close to the plasma membrane and PLC complexes. This prime receptor location is well situated to respond to locally generated InsP3. The InsP3 receptor has an N-terminal InsP3binding domain and a C-terminal channel forming a sequence separated by a large intervening regulatory domain. The InsP3 receptor consists of three different isoforms that share a 60–70% amino acid sequence homology. Most cells express more than one InsP3 isoform. Once InsP3 has activated its receptor, calcium is rapidly released from the store into the cytosol [58].

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The InsP3 receptor itself is under the dual control of InsP3 and calcium [53, 66]. Low levels of calcium activate the receptor, whereas a cytosolic calcium concentration above 300 nM inhibits the receptor [67–70]. However, the ability of calcium to promote its own release is regulated by InsP3, such that in the absence of InsP3, calcium has little effect on the InsP3 receptor [53]. As the concentration of InsP3 increases, the receptor’s sensitivity to calcium increases [53]. Whereas InsP3 receptor activation is the principal calcium release mode in endothelial cells, other cells rely on ryanodine receptors and cyclic ADP ribose mechanisms as well. Indeed, the ryanodine receptor is responsible for the calciuminduced calcium release mechanism that is critically important for skeletal muscle contraction [71, 72], and cyclic ADP ribose dependent calcium release may contribute to the second messenger response to hypoxia [73].

4.5 Calcium Entry The activation of Gq-linked agonists or receptor tyrosine kinases, as described earlier, leads to generation of InsP3 and subsequent release of calcium from the endoplasmic reticulum­ through the InsP3 receptor. In many cell types, especially nonexcitable cells such as endothelial cells, depletion of stored calcium, i.e., in endoplasmic reticulum, opens channels in the plasma membrane to allow for calcium influx into cells (Fig. 3). These particular ion channels, which are opened in response to store depletion, are known as storeoperated entry channels (reviewed in [74]). It has been a little over 20 years since Putney first described this store-operated entry channel process [75]. Since his original description of this phenomenon, numerous groups worldwide have committed substantial time and resources to try to answer two critical questions. First, what is the signal(s) that links store depletion to channel activation and, second, what is the molecular identity of the channel(s) itself? Interestingly, 20 years later, the answers to both questions remain unsettled. From the literally thousands of experiments that have been performed trying to identify the signal(s) linking store depletion to channel activation, three principal hypotheses have been formulated (reviewed in [76]). One hypothesis promotes the idea that the InsP3 receptor in the endoplasmic reticulum is directly and physically coupled to the store-operated entry channel in the plasma membrane. In this model it is proposed that when calcium is released from the endoplasmic reticulum through the InsP3 receptor, the proteins that constitute the receptor change conformation. As the InsP3 receptor is believed to be physically tethered to the store-operated entry channel, a change in receptor conformation will induce a subsequent change in store-operated entry channel conformation,

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Fig.  3  Activation of Gq or receptor tyrosine kinases promote storeoperated channel (SOC) entry. Agonists which bind to G-proteincoupled receptors or tyrosine kinase receptors activate phospholipase C (PLC). PLC, in turn, cleaves phosphatidylinositol 4,5-bisphosphate to produce the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). SOC entry involves diffusion of IP3 to the endoplasmic reticulum (ER), where it binds to IP3 receptors. The IP3 receptor is itself a calcium channel which when activated releases calcium from the ER store. Depletion of the calcium store triggers, by an unknown mechanism, opening of SOCs on the plasma membrane. Receptoroperated channels can be activated by a number of second messengers, including DAG, without requiring calcium store depletion. Adapted from Li et al. [145]

and in doing so gate open the channel. A variant of this model proposes a physical link between the InsP3 receptor and the store-operated entry channel, but not via direct interaction. The second hypothesis proposes that the signal linking store depletion to store-operated entry channel activation is a soluble factor that is released from the endoplasmic reticulum. This soluble factor is believed to diffuse through the cytosol and bind to and activate the store-operated entry channel. Although much work has been devoted to this hypothesis, to date, no single protein or molecule has been identified which fulfills the criteria of the soluble factor. The third and final hypothesis is the vesicle-insertion model. Here, the storeoperated entry channels are believed to reside in vesicles that directly underlie the plasma membrane. Upon store depletion, the vesicles are stimulated to move to and insert themselves into the plasma membrane, thus exposing the store-operated entry channels to the extracellular medium. Although the quest to identify store-operated entry channel proteins has been difficult, some progress has been made. The first identified candidate for a store-operated entry channel was the transient receptor potential (TRP) protein found in the Drosophila melanogaster eye [77]. As the Drosophila phototransduction cascade was known to follow the path of activated PLC and generation of InsP3, the TRP became a candidate for a store-operated entry channel. Indeed, when expressed in vitro, the Drosophila TRP protein can be activated by store depletion, i.e., it acts as a store-operated entry channel, whereas the related TRP “leak” (TRPL) protein is constitutively active [78]. Homologues of Drosophila TRP proteins have been identified in mammals, Caenorhabditis elegans, and zebrafish, and now TRP proteins constitute a superfamily (reviewed in [79]). Interestingly, the Drosophila TRP was cloned at roughly the

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same time as Putney described store-operated entry channels (reviewed in [80]. Indeed, this early observation set the stage for experiments over the following 20 years. The mammalian TRPC (canonical) subfamily most closely resembles the Drosophila TRP (reviewed in [81]). They are six transmembrane-spanning-domain proteins with intracellular C- and N-termini. On the basis of homology with voltage-gated potassium channels, the functional TRPC channel likely comprises four subunits that coalesce and form a pore through the transmembrane 5 and 6 regions. Seven members of the TRPC subfamily (TRPC1–TRPC7) have been identified to date. Of these seven, TRPC1, TRPC3, TRPC4, and TRPC5 have been shown to be able to perform in a store-operated capacity [82–92]. Although numerous studies have determined that the TRPC1, TRPC3, TRPC4, and TRPC5 proteins fulfill the criteria for storeoperated entry channels, there is a problem. For as many reports supporting the idea that these TRPCs are storeoperated, there are just as many refuting the idea. Indeed, the concept that TRPC proteins can contribute to storeoperated entry channels has become quite controversial. At present, this issue remains unresolved, although it appears that cell-specific channels can form from variable combinations of TRPC proteins, perhaps contributing to the difficulty in data interpretation. Just recently, another protein, orai1, was introduced as a potential store-operated entry channel. Unlike TRPC proteins, orai1 is a four-transmembrane-spanning-domain protein. Similar to the Drosophila TRP, orai1 was originally identified as a potential store-operated entry channel when a naturally occurring mutation surfaced. It was observed that patients with a particular form of severe combined immunodeficiency syndrome exhibited a single point mutation, R91W, in T cells [93]. Further, these cells lacked the originally described store-operated entry channel current, ICRAC, leading to the hypothesis that orai1 proteins form the ICRAC channel. Considerable excitement was generated within the scientific community when the heterologous expression of orai1 was found to restore ICRAC to cells naturally deficient in this current. However, not all cells expressing orai1 possess an ICRAC [94–97], and recently Liao et al. [98, 99] and Chang et  al. [100] independently recognized that orai1 interacts with TRPC proteins, where it functions to link calcium store depletion to activation of calcium entry through the TRPC channel. It will be interesting to follow the progression of this story over the next few years. Indeed, only when the molecular identity of a store-operated entry channel has been accurately described will we then be able to determine how the channel is gated. Although there is still much uncertainty regarding the identity and regulation of store-operated entry channels, significant progress in the field has been made. Endothelial cells possess a specific store-operated entry channel current, referred to as ISOC, that has been described by Vaca and

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Kunze [78], Fasolato and Nilius [101]; Wu et al. [102, 103]. ISOC is a calcium-selective channel, meaning that it prefers to conduct calcium even in the presence of other ions. This characteristic is reflected in the reversal potential of the current, which is approximately +40 mV. ISOC is a small current, on the order of 1–1.5 pA/pF and is inwardly rectifying. The molecular identity of the ISOC channel that underlies ISOC has been partially unraveled. In the first set of studies, TRPC1 expression was inhibited using small interfering RNA [103]. TRPC1 knockdown decreased store-operated channel (SOC) entry and inhibited ISOC, but did not effect a left-shift in the reversal potential. The observation that there was only a partial inhibition of ISOC, and not a complete abolishment, led to the idea that TRPC1 contributes at least one subunit to the channel, but it is not the critical subunit that somehow senses store depletion. Thus, there must be at least one other type of subunit in the ISOC channel. The second observation, i.e., no shift in the reversal potential, revealed that TRPC1 does not play a role in determining the calcium selectivity of the channel, again suggesting that there is at least one other type of subunit in the channel’s molecular makeup. The second set of studies focused on the contribution of TRPC4 to the ISOC. Here, a TRPC4 knockout mouse was developed [104]. In aortic endothelium, not only was the ISOC reduced to a negligible current, the reversal potential was left-shifted to zero. These results were exciting because they suggested critical roles for TRPC4 in ISOC activation (or sensing store depletion), and in determination of the calcium-selective nature of this channel. If indeed the ISOC is formed by four subunits, then we can be confident of at least two, TRPC1 and TRPC4. Although the identity of the other two subunits is uncertain, on the basis of the molecular identity of other tetrameric ion channels, it is likely that the remaining two subunits exhibit structures homologous to those of TRPC1 and TRPC4. Thus, we can hypothesize that the remaining two subunits are likely to be some combination of TRPC1, TRPC4, and/or other TPRC homologues. Since orai1 has only four transmembrane-­spanning domains, and as such does not exhibit a structure homologous to the structures of the TRPC proteins, we predict that it does not contribute to the pore-forming part of the channel structure. As the molecular identity of ISOC has been partially resolved, so too has the mechanism that underlies channel activation. Early work in platelets and endothelial cells revealed that the cytoskeleton plays a critical role in activation of store-operated entry channels. The cytoskeleton constitutes a molecular framework which provides structural integrity to the cell. It is composed of numerous proteins, including actin filaments, intermediate filaments, and microtubules. In some cell types, including endothelial cells, this spectrin–actin meshwork contributes to the formation of a unique structure directly underlying the plasma membrane, the membrane skeleton. In the well-studied erythrocyte (reviewed in [105, 106]), the membrane skeleton has been

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Fig. 4  The membrane skeleton. Filamentous actin (yellow) is crosslinked by spectrin (brown) to form a scaffolding network that provides membrane support. Protein 4.1 (blue) and ankyrin (green) link transmembrane proteins to the membrane skeleton; additionally protein 4.1 forms a ternary complex with spectrin and actin. (Reprinted with permission from [3])

shown to provide structure and support to the plasma membrane, important for cell shape; additionally, the erythrocyte membrane skeleton appears to limit movement of integral membrane proteins, phospholipids, and cholesterol. Although much less is known about specific membrane skeleton functions in nonerythroid cells, proteins which contribute to erythrocyte membrane skeleton composition have also been identified in nonerythroid cells [107–113]. The proteins spectrin and actin represent principal components of the membrane skeleton (reviewed in [106]), with spectrin cross-linking actin filaments [114] to form a molecular meshwork underlying the plasma membrane (Fig. 4). The membrane skeleton physically interacts with the plasma membrane, providing structural support to the cellular membrane. Protein 4.1 and ankryin are principally responsible for linking the membrane skeleton to the membrane, by simultaneously binding to spectrin in the membrane skeleton and transmembrane proteins in the plasma membrane (reviewed in [115]). Initial studies examining whether an intact actin cytoskeleton was important for activation of SOC entry supported the physical coupling model, in that disruption of filamentous actin prevented activation of SOC entry [116–118]. Importantly, calcium release from the store as well as basal cytosolic calcium levels were not affected by filamentous actin disruption. However, these studies utilized pharmacological agents that grossly stabilized (e.g., with jasplakinolide) or disrupted (e.g., with cytochalasin D) filamentous actin; thus, specific protein–protein interactions within the cytoskeleton important for channel activation could not be determined. Subsequent studies in endothelial cells focused on the role of the membrane skeleton in store-operated entry channel activation, and further sought to determine whether specific protein–protein interactions within the membrane skeleton were important for channel activation. To address these questions, more refined experiments were designed. Antibodies that targeted and disrupted specific protein–protein interactions were microinjected into endothelial cells and SOC entry was measured [102]. An antibody to the protein 4.1–spectrin interaction decreased the overall SOC entry and completely inhibited the ISOC channel, whereas an antibody that disrupted

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the actin–spectrin interaction had no effect. These observations led to the exciting hypothesis that protein 4.1 functionally links the ISOC channel to the membrane skeleton, supporting the conformational coupling model of store-operated entry channel activation. Subsequent sequence analysis of TRPC1 and TRPC4 revealed that TRPC4 contains a conserved protein 4.1 binding domain [119], and protein 4.1 coimmunoprecipitates with TRPC4 [74], indicating that these two proteins physically interact. The protein 4.1 binding domain on TRPC4 is located on the cytosolic C-terminus approximately 50 amino acids downstream of where the last transmembrane domain exits the membrane. As the pore region of the channel is predicted to form by coalescence of the five to six transmembrane domains of the four subunits (reviewed in [120]), this would place the protein 4.1 binding domain in direct proximity to the pore region. If protein 4.1 binds at its binding domain on TRPC4, then it would be in close proximity to the pore region of the channel, and as such, in a manner analogous to the ball-and-chain model of ­voltage-gated channels, it is ­conceivable that protein 4.1 acts as the “ball” and gates the ISOC channel. Indeed, this idea represents a novel function of protein 4.1. Although SOC entry represents a principal mechanism of calcium entry into nonexcitable cells, it is not the only mechanism. For instance, multiple other channels have been identified in pulmonary endothelial cells, including receptoroperated channels, cyclic-nucleotide gated channels, TRPV (vanilloid)-containing channels, and T-type calcium channels. These channels are distinct from store-operated entry channels in that activation is not triggered by store depletion per se. Channels that are formed from TRPV4 subunits can be activated by several different factors, including elevated temperature, mechanical stress, and arachidonic acid metabolites (reviewed in [121]). Cyclic-nucleotide-gated channels are directly activated by cyclic AMP and/or cyclic GMP (reviewed in [122]). Pulmonary artery endothelial cells possess a cyclicnucleotide-gated channel that is a nonselective cation channel which can be activated downstream of SOC entry [123]. Receptor-operated calcium channels were originally described as voltage-insensitive channels that are activated by receptor agonists such as norepinephrine, acetylcholine, and prostaglandins (reviewed in [124]). Subsequently, with the discovery of the TRPC subfamily of proteins, the term “receptor-operated channel” almost became synonymous with TRPC3, TRPC6, and TRPC7. The receptor-operated TRPC channels exhibit similarities to SOCs. Receptors coupled to Gq/11 or receptor tyrosine kinases activate both channel types, through generation of InsP3 and diacylglycerol. Whereas InsP3 diffuses through the cytosol to bind to its receptor on the endoplasmic reticulum, as described previously, diacylglycerol remains associated with the plasma membrane, where it activates receptor-operated channels (Fig. 3). In general, receptor-operated calcium channels exhibit nonselective currents. Given that store- and receptor-operated

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currents are both activated by similar agonists, it can be difficult to differentiate their independent contributions to shifts in cytosolic calcium concentration. Fortunately, studies of SOCs have been facilitated by the discovery of thapsigargin. Thapsigargin is a plant alkaloid that blocks the calcium-ATPase pump on the endoplasmic reticulum [78, 125]. It functions by preventing calcium reuptake into the endoplasmic reticulum, and as the endoplasmic reticulum exhibits a slow, constitutive calcium leak, in the presence of thapsigargin it will soon become depleted of its calcium content. This, in turn, activates SOCs. Use of thapsigargin allows one to follow events triggered solely by store depletion, without the confounding effects of additional signal transduction events that occur with Gq-linked agonists. The receptor-operated community has relied on the use of diacylglycerol analogues, e.g., oleylacylglycerol, to specifically activate receptor-operated channels without concomitantly activating SOCs. To date, in pulmonary endothelial cells, receptor-operated calcium entry has not been as intensely studied as store-operated calcium entry. Another calcium entry pathway that has been described in pulmonary endothelium is the T-type voltage-gated channel. This discovery was a surprise, considering that endothelial cells are considered to be nonexcitable cells that do not express voltage-gated ion channels. However, in the pulmonary vasculature, pulmonary microvascular endothelial cells express the T-type channel, whereas pulmonary artery endothelial cells do not (reviewed in [126]). The T-type channel in microvascular endothelial cells possesses an a1G subunit, and exhibits a window current in the range of voltages between −60 and −30 mV. The window current range of voltages lies between the two peaks of the bimodal resting membrane potential of the microvascular endothelial cells (reviewed in [127]). These peaks are at −60 to −70  mV and −40 to −10  mV. The relationship between the resting membrane potential and window current range of voltages is believed to be physiologically important with respect to activation of the T-type channel. Inflammatory agonists such as thrombin, and other Gq-linked agonists, cause an initial transient hyperpolarization which is then followed by a sustained depolarization. Either hyperpolarization from the −40 to −10 mV peak or depolarization from the −60 to −70 mV peak could transition the membrane potential into the window current range of voltages and thereby activate the T-type channel allowing for calcium entry. As the different calcium entry pathways were described in the pulmonary endothelium, an interesting observation was made. Not all subsets of pulmonary endothelial cells express the same calcium entry pathways. For instance, both the T-type calcium channel and TRPV4 are found in pulmonary microvascular endothelial cells but not pulmonary artery endothelial cells [126, 128, 129]. The TRPC1 and TRPC4 proteins of the ISOC channel are expressed in both pulmonary artery and microvascular cells, and indeed ISOC can be

15  Role of Calcium as a Second Messenger in Signaling: A Focus on Endothelium

activated in both cell types [130]. However, ISOC activation is differentially regulated among cells. Whereas thapsigargin alone can activate ISOC in pulmonary artery endothelial cells, it is insufficient to activate ISOC in microvascular cells.

5 Physiological Functions of Calcium Entry For most of its scientific history, endothelium was recognized as an inert, squamous epithelial layer [1, 2, 131]. More recently, the systematic study of structure and function has begun to resolve the highly dynamic and complex nature of endothelium. From this work has come a realization that considerable heterogeneity exists in endothelium, not only when comparing cells between different organs, but perhaps most provocatively when comparing cells along the vascular tree within an organ [132, 133]. The lung is no exception, as striking endothelial cell heterogeneity is seen along the artery–capillary–vein axis within this organ. The study of calcium channel expression and function has greatly aided our understanding of pulmonary endothelial cell heterogeneity. Activation of store-operated calcium entry channels increases endothelial cell permeability [134–136]. More specifically, ISOC activation appears to provide a calcium source that is uniquely coupled to cytoskeletal reorganization, disruption of cell–cell adhesions, and intercellular gap formation [130, 137]. Use of thapsigargin has been pivotal in resolving these mechanisms both in  vitro and in the intact circulation. In the intact circulation, thapsigargin application increases extra-alveolar permeability, resulting in perivascular cuff formation that impairs lung compliance [138]. Rolipram is a type 4 phosphodiesterase inhibitor that increases the level of cyclic AMP. Rolipram prevents thapsigargin from increasing extra-alveolar permeability, but reveals a thapsigargin-induced capillary leak site [130]. Thus, although the mechanisms responsible for ISOC activation in extra-alveolar and capillary endothelium are different, the result is the same, as calcium permeation through this channel results in gap formation and increased permeability. Whereas thapsigargin typically increases permeability in extra-alveolar vessels, it fails to do so in animals with heart failure. Indeed, Alvarez et al. [139] placed an aortocaval fistula in rats causing development of heart failure. As heart failure developed, the permeability response to thapsigargin was abolished. This functional response to heart failure was accompanied by the downregulation of TRPC1, TRPC3, and TRPC4 in extra-alveolar endothelium. Although the thapsigargin-induced increase in permeability was abolished in these animals, the permeability response to a different calcium agonist, 14,15-epoxyeicosatrienoic acid, was retained, bringing into question how the response to one agonist, thapsigargin, could be abolished whereas the response to a second agonist, 14,15-epoxyeicosatrienoic acid, was not.

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14,15-Epoxyeicosatrienoic acid activates a calcium channel belonging to the TRPV proteins, TRPV4 [140]. Endothelial cell TRPV4 expression in the pulmonary circulation is concentrated in the capillaries, and this expression pattern is not changed in animals with heart failure [129, 141]. Hence, channel activation results in a calcium response that increases capillary permeability, but does not increase extraalveolar permeability [138, 141]. Unlike the activation of SOC entry, TRPV4 activation does not result in endothelial cell gap formation, but causes loss of cell–matrix tethering. The mechanisms allowing for store-operated entry channels to control cell–cell adhesion and the TRPV4 channel to control cell–matrix adhesion are still incompletely understood. Not all calcium channels regulate endothelial cell barrier function. Wu et  al. recently discovered that lung capillary endothelium expresses a T-type calcium channel [142]. Activation of this channel does not increase endothelial cell permeability, but rather is responsible for the P-selectin upregulation that is important for neutrophil trafficking [141]. Thus, while T-type channel activation is not responsible for increasing permeability, it is involved in the endothelial cell response to inflammation. The systematic study of these three calcium channels, the ISOC channel, TRPV4, and the T-type calcium channel, has greatly advanced our understanding of lung endothelial cell heterogeneity and basic cell biology. Whereas ISOC activation induces interendothelial cell gaps in all vascular segments, TRPV4 activation decreases cell–matrix interaction in capillaries. Whereas the TRPV4 channel controls permeability in capillaries, the T-type calcium channel controls P-selectin surface expression in this vascular segment. A major challenge is now to determine how these discrete intracellular calcium pools are coupled to their physiologically relevant effectors with such high fidelity, how these mechanisms are engaged in the course of inflammation, and how such diverse mechanisms are coordinated to elicit an appropriate inflammatory response.

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Chapter 16

Caveolae and Signaling in Pulmonary Vascular Endothelial and Smooth Muscle Cells Geerten P. van Nieuw Amerongen, Richard D. Minshall, and Asrar B. Malik

Abstract  Caveolae exist in most cell types (with certain exceptions, e.g., erythrocytes, lymphocytes, and neurons) and are particularly abundant in endothelial cells (ECs) and smooth muscle cells (SMCs) of blood vessels. It is clear that the major plasmalemma vesicle structure in ECs and SMCs is caveolae as opposed to clathrin-coated vesicles. The number of caveolae is high in continuous endothelium (e.g., 73 caveolae per square micrometer in ECs of intramuscular capillaries) and low in fenestrated or discontinuous endothelium. Caveolae have a lipid composition similar to that of membrane rafts, but in addition, they possess other proteins, including the organellespecific structural protein caveolin and the more recently identified cavin. Caveolae appear to represent a specialized form of membrane raft domain, where caveolin-1, glycosphingolipids, and cholesterol are preferentially concentrated. Instead of assembling these structures in the plasma membrane, cells may in fact build caveolae in the Golgi apparatus and then send them to the plasma membrane proper, where they are incorporated, a process involving Na/K-ATPase. This chapter describes the structure and function of caveolae and caveolin proteins and their interaction with membrane receptors, transporters and signaling proteins that are related to the pulmonary vascular permeability and pulmonary hypertension. Keywords  Caveolae • Caveosome • Caveolin • Clathrin • Lung microvascular endothelial cell • Vascular permeability • Signal transduction

1 Introduction 1.1 Structure and Function of Caveolae Caveolae exist in most cell types (with certain exceptions, e.g., erythrocytes, lymphocytes, and neurons) and are G.P. van Nieuw Amerongen (*) VU University Medical Center, Institute for Cardiovascular Research, Department of Physiology, van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands e-mail: [email protected]

p­ articularly abundant in endothelial cells (ECs) and smooth muscle cells (SMCs) of blood vessels. It is clear that the major plasmalemma vesicle structure in ECs and SMCs is caveolae as opposed to clathrin-coated vesicles (Fig. 1) The number of caveolae is high in continuous endothelium (e.g., 73 caveolae per square micrometer in ECs of intramuscular capillaries [1]) and low in fenestrated or discontinuous endothelium. On the basis of electron microscopy data, caveolae exist luminally, abluminally, and as free cytoplasmic vesicles, with the largest number in perijunctional zones between endothelia [2]. One issue often unappreciated is that caveolae number, quantified by electron microscopy, decreases 10- to 1,000-fold in cultured ECs (0.1–9 per square micrometer of plasma membrane), although ample caveolin-1 protein expression is detected in cultured cells [3]. Caveolae have a lipid composition similar to that of membrane rafts, but in addition, they possess other proteins, including the organelle-specific structural protein caveolin [4, 5] and the more recently identified cavin [6, 7]. Caveolae appear to represent a specialized form of membrane raft domain, where caveolin-1, glycosphingolipids, and cholesterol are preferentially concentrated [4, 8, 9]. Instead of assembling these structures in the plasma membrane, cells may in fact build caveolae in the Golgi apparatus and then send them to the plasma membrane proper, where they are incorporated, a process involving Na/K-ATPase [10]. Caveolae were originally described by Palade during electron microscopy analyses of capillary ECs [11] and somewhat later in gall bladder epithelium [12]. In collaboration with Simionescu [13, 14], Palade demonstrated the participation of caveolae in macromolecular transport across ECs by transcytosis. Recent dynamic intravital fluorescence microscopy has revealed that caveolae operate effectively as pumps, moving antibodies within seconds from blood across the endothelium into lung tissue, even against a concentration gradient [15]. In larger blood vessels, medial SMCs likewise display abundant caveolae [16] (Fig.  2) and various stimuli and ligands have been reported to induce caveolae internalization in SMCs. Recent studies have confirmed that caveolae are directly involved in the internalization of membrane constituents, extracellular ligands and macromolecules, bacterial toxins,

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Fig. 1  (a) Transmission electron microscopy micrograph of small muscularized mouse artery showing predominance of caveolae vesicular structures in a smooth muscle cell (SMC) and an endothelial cell (EC) (scale bar 2 mm). (b) Higher magnification of the EC in (a) showing numerous caveolae along the luminal plasma membrane above the nucleus (scale bar 0.5 mm). (c) A ×10 zoom of EC caveolae open to the lumen (solid arrow) (scale bar 200 nm). (d) Higher magnification of (a) showing SMC caveolae attached to the plasma membrane. (e) A ×10 zoom of SMC caveolae attached to basal membrane (scale bar 200 nm). (Courtesy of Oleg Chaga, Center for Lung and Vascular Biology, University of Illinois at Chicago)

Fig.  2  Fluorescent micrographs of caveolin-1 and 4¢,6-diamidino-2phenylindole nuclear immunostaining of mouse artery. Note abundant green caveolin-1 staining along the plasma membrane of ECs aligned with the direction of blood flow, and relatively weaker caveolin-1 staining in SMCs aligned perpendicular to the flow. (Courtesy of E.C. Eringa, Institute for Cardiovascular Research, VU University Medical Center)

and nonenveloped viruses [17–19]. The internalized substances have been suggested to be delivered, whether directly or indirectly, to various intracellular compartments, such as the Golgi apparatus and the endoplasmic reticulum (ER) [20]. The caveolae-mediated pathway is considered to be distinct from the clathrin-mediated internalization pathway [21, 22]. In contrast to cargo sorting and vesicle formation by clathrin,

COPI, and COPII coats, caveolae contain permanently stabilized membrane-integrated coats that do not undergo cyclic association and dissociation. Efficient sorting is achieved by releasing cargo in response to local cues received in a particular compartment, such as low pH in endosomes [19]. The interaction and association between these two internalization pathways, if any, remains largely unknown. The term “caveosome” has been proposed to identify a caveolin-containing multivesicular structure thought to mediate sorting in a manner akin to, but independent of, endosomes in the clathrin-coated-pit/clathrin-coated-vesicle endocytic pathway [22, 23]. However, recent data indicate that caveosomes correspond to late endosomal compartments, wherefore this term no longer should be used [24]. This review will not deal with lipid transport/cholesterol homeostasis (not so much related to signaling function of caveolae).

1.2 Lessons from Cav-1-/- and Cavin-/- Mice Studies revealed that caveolin-1-null (Cav-1-/-) mice show (1) lack of caveolae, (2) downregulation (degradation) of caveolin-2 but normal expression of caveolin-3, (3) vascular

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dysfunction with reduced endothelium-dependent (NO) relaxation, contractility, and myogenic tone, and (4) lung abnormalities with thickened alveolar septa [25–28]. It was further observed that caveolin-1/caveolin-3 double-knockout mice lack caveolae both in muscle and in nonmuscle cells and develop a severe cardiomyopathy with myocyte hypertrophy, inflammation, and interstitial fibrosis [29]. Although the phenotypes of caveolin-1-deficient animals appear less serious than expected: the findings stress the importance of caveolin proteins and caveolae in the cardiovascular system. The primary observations from Cav-1-/- mice and small interfering RNA (siRNA)-mediated caveolin-1-depleted mice for the purposes of this review are (1) absence of caveolae in capillary endothelium, (2) persistence of caveolae in cardiomyocyte and vascular SMC sarcolemma, and (3) increased thickness of alveolar septa [27] and capillary basement membrane [30]. Cav-1-/- mice exhibit disruption of EC barrier properties and edema formation, particularly in the lung. Studies in Cav-1-/- models do not distinguish between the actions of caveolins within or outside of caveolae. The absence of the caveolin protein not only results in the lack of caveolae as a structure but also a total lack of interaction and modulation of activity of enzymes/molecules (e.g., endothelial nitric oxide synthase, eNOS) to which caveolin binds (whether inside or outside caveolae). Cavin, another caveolae-specific protein localized to the cytosolic face of plasma membrane adipocyte caveolae [31], colocalizes with caveolin-1 [32, 33]. It is highly expressed in adipocytes, lungs, heart, and colon, in lesser amounts in the thymus, spleen, kidney, and testis, and is weakly expressed in liver and brain [32, 33]. Animals deficient in cavin do not have caveolae in any cell type and, interestingly, also lack significant expression of all three caveolin isoforms even though caveolin messenger RNA expression is normal. Cavin-/- mice are viable and of normal weight but have higher circulating triglyceride levels, significantly reduced adipose tissue mass, glucose intolerance, and hyperinsulinemia – characteristics that constitute a lipodystrophic phenotype [6, 7] also observed in Cav1-/- mice [34].

2 Signaling 2.1 Caveolin-1 as a Scaffolding Molecule One of the most important structural components of caveolae is caveolin-1, a protein of 21–24 kDa. Caveolin-1 is a member of the caveolin family, which comprises caveolin-1, caveolin-2, and caveolin-3 [35–37]. (Fig.  3) Caveolin-1 and caveolin-2 are found in most cells, whereas caveolin-3 is

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Fig. 3  Phosphorylation map of the caveolin family of proteins. Caveolins are highly homologous and conserved proteins. All isoforms contain membrane-spanning, oligomerization and caveolin-scaffolding domains. (a) The caveolin-1a isoform consists of 178 amino acids with phosphorylation sites at Tyr14 and Ser80. The caveolin-1b isoform lacks the first 31 amino acids, and thus it does not contain the tyrosine phosphorylation site. Residues 75–158 (part of scaffolding domain, membrane-spanning domain, and most of the C-terminus) are involved in binding dynamin 2. Scaffolding and membrane-spanning domains of caveolin-1 also participate in homo-oligomerization and formation of heteroligomers with caveolin-2. (b) Caveolin-2 is approximately 50% homologous to caveolin-1, and the caveolin-2a isoform contains 162 amino acids with phosphorylation sites at Tyr19, Tyr27, Ser23, and Ser36. The caveolin-2b isoform, produced by alternative splicing of messenger RNA, is truncated by 13 N-terminal amino acids, whereas no information is yet available about the phosphorylation of the caveolin-2g isoform. (c) Caveolin-3 is a muscle-specific isoform, which contains 151 amino acids and is very homologous to caveolin-1. No phosphorylation sites have been demonstrated for caveolin-3 to date. (Modified with permission [37])

muscle-specific (most strongly expressed in skeletal and heart muscle cells). In smooth muscles, even in the same cell, caveolin-1 and caveolin-3 show different distributions, suggesting that the two caveolins form distinct caveolae, and that caveolin-1 and caveolin-3 serve different functions [38]. Two isoforms of caveolin-1 exist in the lung: caveolin-1a is expressed predominantly in ECs and caveolin-1b is expressed in epithelial cells [39]. Deletion of the caveolin-1 gene in mice resulted in cardiac hypertrophy and lung hypercellularity and matrix deposition [27], indicating its importance in cardiac and lung development. Expression of caveolin-1 induces the formation of caveolae-like structures [40, 41] and, hence, it appears to play a pivotal role in the biogenesis of caveolae. Coexpression of caveolin-1 and caveolin-2 facilitates the formation of deep caveolae [38, 41]. Recent studies have begun to identify the functions of caveolin-1. Caveolin-1 assembles as 12–18-mers in a stoichiometric complex with cholesterol [42]. Caveolin-1 appears to regulate caveolar internalization by stabilizing caveolae at the plasma membrane rather than controlling the shape of the membrane invagination [35]. Internalized caveolin-1 also colocalized with endocytosed transferrin in

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Rab5-labeled, Rab4-labeled, or early endosome antigen-1labeled compartments where caveolin-1 was phosphorylated; it then moved to a Rab11-associated compartment. Immunogold electron microscopy also revealed that internalized caveolin-1 colocalized with Rab5 or Rab4 in vesicles larger than caveolae. These results suggest that the internalized caveolae interact with early endosomes [43, 44]. Three palmitoylation sites in the C-terminal region next to the transmembrane domain contribute to anchoring caveolin proteins to the membrane. The N-terminal region of all caveolins contains a conserved FEDVIAEP motif that has been defined as the “caveolin signature” sequence, and has been suggested to be important for the binding of caveolin to cholesterol and glycosphingolipid-rich membrane domains [45, 46]. Caveolin-1, caveolin-2, and caveolin-3 molecules form homoand hetero-oligomers in the membrane and interact directly with cholesterol. They also interact with a multitude of signaling molecules (via a caveolin-binding motif in the latter) identified by proteomic methods [47–50]. As of yet, a definitive list of proteins (which may be cell-type-specific and context-dependent) that interact with caveolin-1 has not been established as outlined by Springer and Horrevoets [51]. The central segment of caveolin proteins contains the scaffolding domain (CSD), which allows oligomerization of caveolin monomers and direct interaction with other proteins, presumably regulating their activity [52]. Because caveolin-1 is a scaffolding protein, it has also been hypothesized to function as a “master regulator” of signaling molecules in caveolae [53]. In the endothelium, caveolin-1 regulates signaling by binding to and inhibiting eNOS. Increased cytosolic Ca2+ concentration or activation of the kinase Akt leads to eNOS activation and its dissociation from caveolin-1 [54]. The CSD (residues 82–101) has been considered to be a single functional domain for some time. In the last few years, however, it has been proposed that the CSD could be functionally divided into two regions, the former (82–95) mediating signaling protein (such as eNOS) inhibition and the latter (95–101) involved in cholesterol and membrane binding. Notably, the association with caveolin seems in most cases (eNOS, Ras, Wnt, and Erk1 and Erk2 cascades) to be inhibitory and to maintain the signaling molecules in an inactive state [53, 55]. Patel et al. [56] proposed therefore “that interaction of signaling proteins with the CSD regulates signal transduction events by sequestering components away from their signal transduction partners, such that in the ‘basal’ state signaling is inhibited, but colocalization in caveolae facilitates the interaction of components upon activation of the signaling pathway.” In addition, caveolin proteins contain several serine and tyrosine residues within their intracellular domains that are substrates for a variety of kinases and become phosphorylated in response to different stimuli [57, 58] (Fig. 3). Caveolin-1 was originally identified as a substrate of v-Src tyrosine kinase and, in fact, is heavily phosphorylated in v-Src-transformed cells [59]. The activation of temperature-sensitive v-Src

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kinase induced rapid tyrosine phosphorylation of caveolin-1 followed by a marked redistribution of phosphorylated caveolin-1 into cytoplasmic vesicular compartments [60]. Tyrosine phosphorylation of caveolin-1 and endocytosis of caveolae are also induced by oxidative stress or phosphatase inhibitors, such as hydrogen peroxide, okadaic acid, sodium orthovanadate, and pervanadate [43, 61–64]. Tyrosine phosphorylation of caveolin-1 was also reported in cells stimulated by insulin, insulin-like growth factor-1, epidermal growth factor, and albumin [62, 63, 65–69]. Although earlier work suggested that phosphorylated caveolin-1 (pY14Cav1), which comprises less than 1% total caveolin-1 under basal conditions, localizes mainly to focal adhesions and is largely absent from caveolae [57, 70], more recent work has indicated that the presence in focal adhesions likely reflects an artifact of the antibody used [71].

2.2 G-Protein-Coupled Receptors and Receptor Tyrosin Kinases in Caveloae Caveolae are rich sites of a large number of G-protein-coupled receptors (GPCRs; including the bradykinin, angiotensin, muscarinic, endothelin, TNFa, and b-adrenergic receptors), heterotrimeric G proteins, G-protein-regulated effectors, and receptor tyrosine kinases [vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), epidermal growth factor] [56]. As implied by the caveolin signaling hypothesis, caveolae bring downstream effectors near to receptors (e.g., GPCRs) to help initiate receptor-, tissue-, and cellspecific signal transduction. These effectors are thought to reside in caveolae owing to direct interaction with caveolins (via the CSD) or by other caveolae-associated proteins. Palmitoylation may aid or enhance caveolar localization of proteins; the reversibility of palmitoylation may help regulate the movement of molecules into and out of caveolae in response to stimulation by agonists. In spite of the localization of certain GPCR and postreceptor signaling components in rafts or caveolae (see below), the precise determinants of this localization are not known. Certain GPCR and postreceptor signaling components show cell-selective patterns of localization in caveolae microdomains, but a generally accepted explanation for such patterns is as of yet not available. Certain non-receptor tyrosine kinases, such as members of the Src family (c-Src, Fyn, Lyn), are enriched in caveolae owing to N-terminal myristoylation and formation of complexes with caveolin [72]; palmitoylation of caveolin-1 at Cys156 is essential for caveolae/c-Src interaction [73]. Caveolin-1, owing to interaction via the CSD, suppresses the activity of c-Src and Fyn [58, 74]. Tyrosine phosphorylation of caveolin-1 (Tyr14) and caveolin-2 (Tyr19) facilitates recruitment of SH2-domain-containing proteins, such as Grb7 and matrix metalloproteinases [57, 59]. Caveolin-1 mediates

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the association of integrins with Src kinases, which can activate extracellular-signal-regulated kinase (ERK) and promote cell cycle progression [75, 76].

2.3 Caveolae and Ca2+ Handling In ECs, a number of molecules responsible for Ca2+ handling have been shown to be compartmentalized in caveolae, including a d-myoinositol 1,4,5-trisphosphate receptor like protein [77], dihydropyridine-sensitive Ca2+ channels [78], a Ca2+-ATPase [77], and Trp1 channels involved in capacitive Ca2+ entry [79]. Some of the proposed endothelium-derived hyperpolarizing factors (EDHFs), such as the epoxyeico­ satrienoic acids (EETs), seem to interact directly or in a membrane-delimited fashion with these Ca2+-regulatory proteins. Saliez et al. [80] demonstrated that expression of caveolin-1 is required for EDHF-related relaxation by modulating membrane location and activity of TRPV4 channels and connexins, which are both implicated at different steps in the EDHF-signaling pathway. Furthermore, caveolin-1 interaction can directly modulate the open probability of some channels such as the “big conductance” Ca2+-dependent potassium channels (BK), a potential end effector of EDHFmediated relaxation. Moreover, it has been demonstrated recently that the gap junction protein connexin (Cx43) and caveolin-1 partially colocalize in cells where they are endogenously expressed, mainly at junctional membranes of contacting cells. Electron microscopy studies also showed that caveolae are in close proximity to the ER [81]. The functional importance of caveolae with regard to Ca2+ release and reuptake was assessed [82]. It was demonstrated that caveolae are the preferred sites of Ca2+ entry when ER Ca2+ stores are depleted. Caveolae are thus the compartment involved in regulating store-operated Ca2+entry [83]. Patel et al. [84] recently showed that increased caveolin-1 expression in SMCs from patients with idiopathic pulmonary arterial hypertension was associated with enhanced Ca2+ entry in response to Ca2+ store depletion, indicating that increased caveolin-1 expression contributes to Ca2+ influx in SMCs. Cav-1 knockout (Cav-1-/-) mouse studies showed that loss of caveolin-1 expression in ECs abrogated Ca2+ entry due to Ca2+ store depletion [85]. Moreover, reexpression of wild-type Cav-1 in Cav-1-/- ECs rescued Ca2+ entry [85], indicating the role of caveolin-1 in regulating Ca2+ entry in ECs. We have shown that CSD peptide markedly reduced ER Ca2+ store depletion as well as store-depletion-activated Ca2+ entry in response to thrombin in ECs [86]. We also observed the interaction of the CSD with TRPC1 C-terminal residues 781-789 [87] and that the CSD interacts with inositol 1,4,5-trisphosphate receptor 3 and TRPC1 to regulate the Ca2+-store-releaseinduced Ca2+ entry in ECs [88].

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2.4 Regulation of eNOS/Akt eNOS-derived NO production is tightly regulated by direct interaction with caveolin-1 and is activated during caveolaemediated endocytosis [54]. Caveolar localization of eNOS in ECs was identified independently by two laboratories [89, 90]. eNOS localization is tightly regulated by a variety of transcriptional, posttranscriptional, and posttranslational mechanisms such as dimerization, phosphorylation, and protein–protein interactions. The activity of eNOS is determined by its localization and by phosphorylation at Ser1177 by several serine/threonine kinases, such as Akt. Several proteins residing in or recruited to caveolae serve as regulators of eNOS. Some of the proteins that modulate eNOS activity in addition to caveolin-1 include heat shock protein (HSP90), cationic amino acid transporter 1 (arginine transporter), Ca2+–calmodulin, ion channels, Ca2+ pumps, soluble guanylate cyclase, bradykinin B2 receptor, and VEGF receptor 2 [82, 91–95]. The localization of signaling molecules within caveolae provides the proximity necessary for a rapid and efficient propagation of signals to downstream targets. A novel mechanism of eNOS activation and NO production in ECs was recently described in which eNOS phosphorylation, dissociation from caveolin-1, and activation was induced by caveolae-mediated endocytosis and activation of caveolae transport machinery [54]. Inhibition of Gbg activation and downstream Src, Akt, and phosphatidylinositol 3-kinase pathways blocked caveolae-mediated endocytosis of albumin as well as NO production induced by the activation of albumin-binding protein gp60 in caveolae [54, 63]. In response to various stimuli such as VEGF and shear stress, eNOS binds to HSP90 which facilitates calmodulininduced displacement of caveolin-1 from eNOS, illustrating that the interrelationship of caveolin-1 and eNOS is a dynamic one [96, 97]. Recently, internalization of eNOS via caveolae has been shown to be a mechanism involved in the regulation of plateletactivating factor (PAF)-induced endothelial permeability, suggesting the novel concept that eNOS internalization into the cytosol is a signaling mechanism that leads to the onset of microvascular hyperpermeability in inflammation [98].

3 Regulation of Vascular Function by Caveolae Acute vascular inflammation [99] and progression of lung fibrosis [100] in mice were prevented by systemic administration of the CSD peptide, presumably by sequestering signaling proteins required for the response. These and many other studies strongly argue that caveolin-1, through its scaffolding domain, regulates EC and vascular SMC signal transduction, and thereby, vascular function.

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3.1 Differences Between EC and SMC Caveolae

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ECs and SMCs. Because SMCs also express caveolin-3 and thus caveolae still form in SMCs of caveolin-1 null mice (whereas they do not in ECs from these mice), it seems likely that caveolae and caveolin-1 may have differing functions in vascular regulation and homeostasis.

EC caveolae are thought to play an important role in transcellular transport and regulate basal endothelial permeability, oncotic pressure, and the delivery of hormones, drugs, and immunoglobulins [17]. In SMCs, caveolae have also been shown to mediate the uptake of solutes, although it seems less likely that they mediate a transport or transcellular permeability function. Rather, there is an emerging role of the caveolins in organizing and modulating SMC functions, including growth and proliferation, contraction, and metabolic support systems that support these functions [101]. In both cases, caveolae, via the scaffolding function of caveolin-1 and protein sequestering function of lipid raft domains, serve to concentrate signaling molecules and thereby control the kinetics of receptor-mediated signaling events, including Ca2+ influx and eNOS activation [62]. It remains unclear as to whether cytoskeleton–caveolae interactions are similar between ECs and SMCs and if this provides equivalent control of vesicular trafficking and domain-specific signaling events. Thus, it remains to be determined whether molecular signaling events specific to ECs and SMCs are regulated similarly by caveolin-1 or whether caveolin-1 controls critical cellular functions such as migration, proliferation, activation or reactivity, endocytosis, and exocytosis (secretion) of peptide hormones and adhesion molecules in a similar fashion in

Although caveolae form in vascular SMCs, they likely do not have a significant transport function, but rather are likely used for internalization of fatty acids and steroid hormones bound to albumin as well as for insulin-dependent signaling and glucose uptake [101]. Signaling pathways mediating the formation and release of caveolae from the plasma membrane are poorly understood [22]. Phosphorylation events are likely important since caveolar fission is increased by phosphatase inhibition and decreased by kinase inhibition [37, 61]. Caveolin-1 is phosphorylated by Src family kinases [102] on Tyr 14 [59], suggesting a relationship between tyrosine kinase activity and release of caveolae from the membrane [37, 63, 65, 74, 103] (Fig.  4). Immunoprecipitation studies showed that the 60-kDa albumin-binding glycoprotein (gp60) associates with caveolin-1 in ECs after gp60 activation [65]. In fact, both proteins are tyrosine-phosphorylated during caveolaemediated endocytosis of albumin [63, 65, 74]. In addition,

Fig.  4  Model of caveolae-mediated endocytosis in endothelial cells. Caveolin-1 plays a central role because it serves a scaffolding function for components of the “caveolar release complex,” Gi and Src, the signaling machinery responsible for endocytosis. Stimulation of G proteincoupled receptors or tyrosine kinase receptors, for example upon engagement and clustering of albumin binding protein gp60 (1, 2), activated Src phosphorylates tyrosine residues on caveolin-1 (Y14) and

caveolin-2 (Y19, Y27) (3) and also the GTPase dynamin-2 (Y231, Y597) which is thought to help recruit and activate dynamin-2 at the plasma membrane (4). The caveolar release complex thus engaged (i.e., phosphorylated caveolin, dynamin-2, and Src) activates vesicle fission. Src-dependent phosphorylation events may be the trigger that activates caveolar fission by decreasing the rigid structure of the caveolar coat and activating dynamin pinchase function. (Modified with permission [37])

3.2 Endocytosis Versus Transcytosis

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inhibition of G protein signaling with pertussis toxin or Gaiantagonist mini-gene peptide, as well as dominant-negative Src, blocked albumin-activated endocytosis in ECs [65]. Because both gp60 and Src activation are required for albumin endocytosis, Minshall et al. [65] addressed the role of Src phosphorylation of caveolin-1 and dynamin-2 in ECs in signaling the fission of caveolae and their release into the cytosol. Endocytosis of fluorescently tagged albumin or cholera toxin subunit B in ECs was blocked by filipin and methylb-cyclodextrin [65, 104], sterol binding agents that disassemble cholesterol-rich caveolae [5, 105]. Coincident with the endocytosis of albumin (within 1  min after gp60 activation), caveolin-1 and dynamin-2 were tyrosine-phosphorylated at residues 14 and 597, respectively [63, 103]. In both cases, pretreatment of cells with Src kinase inhibitor PP1 or PP2 abolished the phosphorylation. The functional importance of these events with respect to caveolae-mediated endocytosis was investigated in pulmonary microvessel ECs stably expressing non-Srcphosphorylatable caveolin-1 or dynamin-2 mutants. Expression of either Y14F caveolin-1 or Y597F dynamin-2 abolished albumin and cholera toxin subunit B endocytosis, indicating Src phosphorylation of these residues is required for signaling caveolar-mediated endocytosis [63, 103]. Furthermore, association between caveolin and dynamin was increased when dynamin was phosphorylated at Tyr597 and reduced by the nonphosphorylatable dynamin mutant [63, 103]. More recent data provide further support for the novel concept that a large component of pulmonary vascular hyperpermeability induced by activation of polymorphonuclear neutrophil (PMNs) adherent to the vessel wall is dependent on signaling via caveolin-1 and increased caveolae-mediated transcytosis. Thus, it is important to consider the role of the transendothelial vesicular permeability pathway that contributes to edema formation in developing therapeutic interventions against PMN-mediated inflammatory diseases such as acute lung injury [72]. Whether this also applies in SMCs is not known, but it at least seems plausible that agonists such as VEGF and insulin or engagement of cell adhesion molecules in SMCs may also regulate caveolae formation and fission. Caveolin-1-independent pinocytosis and transcellular transport has received some attention recently as a small pool of vesicles, speculated to be less than 1% of the caveolar vesicular population in wild-type mouse ECs [28], were observed in caveolin-1 knockout ECs and were shown by transmission electron microscopy to be similar in size to or slightly larger than (100–120-nm diameter) caveolae resembling vesicular-vacuolar organelles [28, 106]. Thus, assembly of these cellular structures does not require the presence of caveolin-1. This finding, reported by the three independent groups that generated the Cav1-/- mice [25, 26, 28], indicates there may be an additional pool of vesicles in ECs that

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is neither clathrin-coated nor caveolin-1-coated. As clathrincoated vesicles do not compensate for the loss of caveolae in Cav1-/- ECs, a proposed raft-mediated mechanism of endocytosis may predominate in the absence of caveolin-1 [107, 108]. The role of this endocytic pathway in transcellular transport, nutrient delivery, and receptor signaling in vascular ECs and SMCs is only now being investigated.

3.3 Paracellular Permeability Several lines of evidence indicate that caveolin-1 not only is essential for caveolae-mediated transcellular permeability, but also affects paracellular permeability. In addition to defective endocytosis of albumin, studies carried out in caveolin-1 knockout mice showed increased junctional (paracellular) permeability of the endothelial barrier [109, 110], providing a compensatory mechanism responsible for survival of the mice. Indeed, ultrastructural studies indicated opening of interendothelial junctions in caveolin-1 null mice [110] and following siRNA-mediated caveolin-1 depletion in  vivo [109]. Remarkably, caveolin downregulation also contributes to leakiness of tumor vessels [111] and structural abnormalities of the tumor vasculature. Furthermore, inhibition of caveolae internalization by blocking caveolar scission with a dominant-negative dynamin mutant (K44Adynamin) inhibited PAF-induced hyperpermeability to fluorescein isothiocyanate–dextran-70 [98] as well as albumin [63]. However, thrombin-induced myosin light chain phosphorylation and stress fiber formation were not altered in caveolin-1-depleted cells, suggesting that caveolin-1 is not indispensable for all forms of vascular permeability [112]. There are several possible mechanisms by which caveolae/caveolin-1 might affect the paracellular or junctional endothelial barrier. Perhaps the most striking observation regarding the phenotype of Cav1-/- mice was the fivefold increase in plasma NO levels [27] thought to mediate opening of the junctional or paracellular pathway. This effect of NO may be due to enhanced phosphorylation of vascular endothelial cadherin (VE-cadherin) or b-catenin in the junctional complex, resulting in NO-mediated disassembly of the adherens junctions. Another possible mechanism of integrating caveolae-mediated transport and endothelial paracellular permeability involves the recently described role of the intersectin family of scaffolding proteins [113–115]. These proteins are present in abundance in ECs, where they regulate the activity of dynamin and mediate Cdc42-dependent actin polymerization. Studies showed that siRNA-induced knockdown of the long form of intersectin-2 resulted in decreased caveolae-mediated internalization but at the same time increased inter-EC dimensions, thereby regulating the paracellular permeability pathway. Alternatively, increased

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VEGF receptor 2 phosphorylation and decreased association with the adherens junction protein VE-cadherin [116] might also contribute to decreased endothelial barrier function in Cav1-/- mouse lungs. No direct evidence has been provided to indicate whether caveolin-1 interacts with the VE-cadherin complex, although it is known that b-catenin can be recruited to caveolae and that caveolin-1 binding to b-catenin blunts Wnt/lef-1 signaling by preventing interactions with glycogen synthase kinase-3b and the nuclear targeting of b-catenin [55]. Depletion of caveolin-1 in mouse brain microvascular ECs also reduces the expression of adherens junction proteins b-catenin and VE-cadherin as well as tight junction proteins ZO1 and Occludin [117, 118]. Additional studies are needed to determine how caveolin-1 regulates endothelial barrier function, how elevated NO levels perturb junctional integrity, and how caveolin-1 keeps ECs in a contact-inhibited state to maintain proper tissue organization in organs. Understanding the mutual interactions between junctional and caveolae-mediated permeability will likely be important in delineating how lung fluid balance is controlled and vascular homeostasis is maintained.

3.4 Mechanosensation and Shear Stress ECs and SMCs concentrate transmembrane receptors, and signaling proteins that transduce chemical and mechanical signals into the cell and mediate, for example, Rho GTPase activation [106, 119, 120]. In vascular SMCs, stretch-induced activation of focal adhesion kinase, Erk1/2, and Rho does not require caveolin-1 [121]. In ECs, Akt activation is insensitive to stretch, but is activated by shear stress in a caveolin-1dependent fashion. These data suggest that endotheliumdependent sensing of shear stress is primarily associated with caveolin-1-dependent Akt phosphorylation, whereas stretch sensing by vascular SMCs involves rapid mitogenactivated protein kinase and slow Rho–cofilin signaling. Exposure of mesangial cells to 10% equal biaxial cyclic mechanical strain caused Rho activation that was dependent on Rho–caveolin-1 interactions and was abrogated by disruption of caveolae using filipin or methyl-b-cyclodextrin treatment [122]. Stretch-induced activation of both RhoA and Rac1 through caveolae was reported in cardiomyocytes [123]. It was suggested that in these cell types, activation of RhoA and Rac1 localized in caveolae was essential for sensing externally applied forces and transducing these signals to the actin cytoskeleton and for Erk1/2 translocation [75]. A role for caveolin-1 or caveolae in shear-mediated EC responses has also been suggested. Caveolae have been proposed to mediate EC responses to flow in vitro [124, 125]. In mice, caveolin-1 expression is required for dilation and Akt signaling in response to acute changes in shear, as well as

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long-term remodeling after carotid artery ligation and resultant reduced blood flow [121, 125]. eNOS activation is enhanced in caveolae isolated from lungs exposed to flow, supporting the concept that mechanosignaling can occur via proteins found in caveolae [124]. Furthermore, Cav1-/- mice in the ApoE-/- background show decreased atherosclerosis [126]. However, caveolin-1 is also a critical negative regulator of eNOS, and Cav1-/- ECs have constitutively active eNOS and elevated NO production [127, 128]. Thus, the effects of caveolin-1 deletion might be explained in part by the dysregulation of eNOS; alternatively, Cav1-/- mice have an elevated high density lipoprotein level, reduced hepatic low density lipoprotein (LDL) production, and decreased LDL transcytosis across ECs [129]. Thus, a role for caveolin-1 in mechanotransduction per se has not been established.

3.5 Vascular Tone In ECs, caveolin-1 acts as a scaffolding protein to cluster lipids and signaling molecules within caveolae and, in some instances, regulates the activity of proteins targeted to caveolae. Specifically, putative mediators of EDHF-mediated relaxation are located in caveolae and/or regulated by the structural protein caveolin-1, such as potassium channels, calcium regulatory proteins, and connexin 43, a molecular component of gap junctions. Comparing relaxation in vessels from caveolin-1 knockout mice and their wild-type littermates, we observed a complete absence of EDHF-mediated vasodilation in isolated mesenteric arteries from caveolin-1 knockout mice. The absence of caveolin-1 is associated with an impairment of calcium homeostasis in ECs, notably, a decreased activity of Ca2+permeable TRPV4 cation channels that participate in NO- and EDHF-mediated relaxation. Moreover, morphological characterization of caveolin-1 knockout and wild-type arteries showed fewer gap junctions in vessels from knockout animals associated with a lower expression of connexins 37, 40, and 43 and altered myoendothelial communication. Finally, we showed that TRPV4 channels and connexins colocalize with caveolin-1 in the caveolar compartment of the plasma membrane. Expression of caveolin-1 is required for EDHF-mediated relaxation by modulating membrane location and activity of TRPV4 channels and connexins, which are both implicated at different steps in the EDHF-signaling pathway [80]. In pulmonary artery SMCs from patients with idiopathic pulmonary arterial hypertension, an increase in caveolin-1 expression is associated with increased formation of caveolae, enhanced capacitive Ca2+ entry, and accelerated SMC proliferation [84]. Furthermore, disruption of caveolae was shown to inhibit pulmonary vasoconstriction [130]. For a review of the role of caveolin-1 in pulmonary arterial hypertension, see Mathew et al. [131] and Chap. 69, where the role of caveolae in pulmonary hypertension is described.

16  Caveolae and Signaling in Pulmonary Vascular Endothelial and Smooth Muscle Cells

3.6 SMC Mitogenic Signaling The role of caveolin and caveolae functions in SMC physiologic and pathophysiologic processes is not yet known. Studies thus far suggest that caveolae are less abundant in synthetic than in contractile SMCs [132] and are less abundant in proliferating than in nonproliferating cells [115, 132]. Furthermore, Peterson et al. [134] used cultured human coronary vessel SMCs to study (1) how PDGF, a potent SMC mitogen and an important factor in atherogenesis [114, 133, 134], affects caveolin-1 expression, and (2) how overexpression of caveolin-1 influences the response to PDGF [133, 135]. The results demonstrate that exposure of serum-starved SMCs to PDGF caused a dose-dependent reduction in caveolin-1 levels as determined by immunoblotting. Electron microscopy analysis further showed that the loss of caveolin-1 was accompanied by a decrease in the number of cell-surface caveolae. In vivo confirmation of these findings was demonstrated in that the decrease in caveolin-1 protein in the PDGF-treated cultures was paralleled by a distinct increase in caveolin-1 messenger RNA content. Hence, caveolin-1 downregulation in response to PDGF could not be ascribed to an inhibition of caveolin-1 gene transcription. On this basis, it was inferred that the PDGF-induced reduction in caveolin-1 was due to enhanced degradation, via either lysosomes or the ubiquitin-proteasome pathway. Overexpression of caveolin-1 in SMCs did not interfere with early events in PDGF signal transduction (Erk1/2 activation), but blocked entrance into the G1 phase of the cell cycle (cyclin D1 synthesis) and progression into S phase (DNA synthesis), thereby shifting the cells into an apoptotic program [136]. Taken together, these findings suggest that stretch-induced growth signaling in vascular SMCs is dependent on cholesterol-rich plasma membrane domains such as caveolae. These findings indicate that the transition of SMCs from a contractile to a synthetic phenotype involves a marked decline in the number of plasma membrane caveolae. In parallel, caveolin is internalized and redistributed to Golgi-associated vesicles in the perinuclear cytoplasm [132].

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Caveolae appear to provide a strategic pathway worthy of targeting. They contain intravenously accessible, tissue-specific proteins that mediate active transport of macromolecules into the tissue, overcoming the restrictive EC barrier. The caveolae gateway may therefore provide a selective, speedy shuttle service for imaging and therapeutic applications [15, 138]. Although mutations in caveolin-3 have been reported to cause autosomal dominant limb-girdle muscular dystrophy and caveolin-1 mutations occurring during the early stages of mammary transformation have been suggested to be critical upstream/initiating events leading to increased ERa levels, no such mutations have been associated with cardiovascular ­disease. Inhibition of caveolar endocytosis may help to limit vascular hyperpermeability associated with inflammation [72, 98]. The study by Saliez et al. [80] emphasizes the role played by caveolin-1 in EDHF-related signaling and raises the possibility of targeting caveolae to improve vascular relaxation in the context of coronary or peripheral ischemic diseases characterized by deficient endothelium-dependent relaxation. Mutations in caveolin-1 have been observed in 16% of human breast cancers [115, 139], and many human tumors have reduced levels of caveolin-1 expression [114]. For some tumors, however, caveolin-1 expression appears to be related to cell survival and growth [140, 141]. Thus, depending on the tumor origin, caveolin-1 can function either as a tumor suppressor or as a tumor promoter. The molecular mechanisms underlying these cell-type-specific growth-regulatory behaviors are currently unknown. Dysregulation of caveolin-3 results in muscular defects, reflecting its restricted tissue distribution to muscle cells. Patients with Duchenne muscular dystrophy show increased levels of caveolin-3 and caveolae at the sarcolemma. The opposite situation occurs in limbgirdle muscular dystrophy, where mutations in caveolin-3 result in severe reduction of caveolin-3 expression and in the number of caveolae in muscle. Alterations in caveolin-3 have also been described for hereditary rippling muscle disease, which is linked to a locus that spans Cav3.

References 4 Concluding Remarks Caveolinopathy needs to be considered as a differential diagnosis in a range of clinical situations, including patients who do not have clinical symptoms. Mutations in the caveolin-3 gene (CAV3) can lead to a broad spectrum of clinical phenotypes. Five different mutations were identified. Phenotypes that have so far been associated with primary caveolin-3 deficiency include limb-girdle muscular dystrophy, rippling muscle disease, distal myopathy, and hyperCKemia [137].

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16  Caveolae and Signaling in Pulmonary Vascular Endothelial and Smooth Muscle Cells 127. Ju H, Zou R, Venema VJ, Venema RC (1997) Direct interaction of endothelial nitric-oxide synthase and caveolin-1 inhibits synthase activity. J Biol Chem 272:18522–18525 128. García-Cardeña G, Martasek P, Masters BS et al (1997) Dissecting the interaction between nitric oxide synthase (NOS) and caveolin. Functional significance of the NOS caveolin binding domain in vivo. J Biol Chem 272:25437–25440 129. Frank PG, Pavlides S, Cheung MW, Daumer K, Lisanti MP (2008) Role of caveolin-1 in the regulation of lipoprotein metabolism. Am J Physiol Cell Physiol 295:C242–C248 130. Schach C, Firth AL, Xu M et al (2008) Regulation of pulmonary vasoconstriction by agonists and caveolae. Exp Lung Res 34:195–208 131. Mathew R, Huang J, Gewitz MH (2007) Pulmonary artery hypertension: caveolin-1 and eNOS interrelationship: a new perspective. Cardiol Rev 15:143–149 132. Thyberg J, Roy J, Tran PK, Blomgren K, Dumitrescu A, Hedin U (1997) Expression of caveolae on the surface of rat arterial smooth muscle cells is dependent on the phenotypic state of the cells. Lab Invest 77:93–101 133. Raines EW, Ross R (1996) Multiple growth factors are associated with lesions of atherosclerosis: specificity or redundancy? Bioessays 18:271–282 134. Peterson TE, Guicciardi ME, Gulati R et  al (2003) Caveolin-1 can regulate vascular smooth muscle cell fate by switching plate-

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Chapter 17

The Chemistry of Biological Gases D. Jeannean Carver and Lisa A. Palmer

Abstract  Today there are at least four biological gases: oxygen (O2), nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S). Except for molecular oxygen, many of these gases were originally known for their detrimental physiological effects. However, the current consensus suggests that these gases play a role in signal transduction, modulating physiological function. The roles of these gases are complex as each is dependent on the rate of production, concentration, chemical reactivity, and availability of target proteins. Moreover, many of these gases share the same molecular targets, and thus are able to modify the responses to each other. This chapter discusses the chemistry of each of these gases, their biological production, potential interactions, and physiological consequences. Keywords  Reactive oxygen species • Radicals • Oxidant • Superoxide • Nitric oxide • Carbon monoxide • Hydrogen sulfide • Nitrosylation

1 Introduction Today there are at least four biological gases: oxygen (O2), nitric oxide (NO), carbon monoxide (CO), and hydrogen sulfide (H2S). Except for molecular oxygen, many of these gases were originally known for their detrimental physiological effects. However, the current consensus suggests that these gases play a role in signal transduction, modulating physiological function. The roles of these gases are complex as each is dependent on the rate of production, concentration, chemical reactivity, and availability of target proteins. Moreover, many of these gases share the same molecular targets, and thus are able to modify the responses to each other. This chapter will discuss the chemistry of each of these

D.J. Carver (*) Department of Pediatrics, University of Virginia, Charlottesville, VA, USA e-mail: [email protected]

gases, their biological production, potential interactions, and physiological consequences.

2 Oxygen and Reactive Oxygen Species Oxygen (O2) makes up 21% of the air we breathe. It is carried by hemoglobin in red blood cells, which deliver oxygen to and remove carbon dioxide from tissues in the body. Changes in oxygen levels can act as a molecular switch; turning on ion channels [1], altering blood flow [2], and modifying the activity or expression of transcription factors [3–5]. With respect to the pulmonary vasculature, inhaled oxygen is a prompt vasodilator and the classic therapy for acute pulmonary hypertension. O2 is also necessary in the production of energy by being the terminal electron acceptor in the mitochondrial transport chain and in the process generating reactive oxygen species. These radical and reactive oxygen species produced from oxygen are, in normal physio­ logical conditions, necessary for normal redox-mediated vascular signaling. In disease states where they are ­overproduced or poorly controlled, however, they are likely counterproductive, with resultant proliferation of vascular smooth muscle and increased vascular tone.

2.1  Reactive Oxygen Species Biologically, molecular oxygen generates cellular respiration through its mitochondrial reduction to water in a series of oneelectron transfers. During this process, reactive oxygen species are also produced, including superoxide radical (O2•−), hydrogen peroxide (H2O2), hydroxyl radical (HO•), and peroxynitrite (in the presence of radical nitric oxide). Radicals are chemical entities with unpaired electrons in an otherwise open-shell configuration, i.e., all valence electrons have not been shared in forming chemical bonds. These electrons are highly “reactive” in that they are quickly engaged in forming new chemical bonds. Most radicals, therefore, have short half-lives (Fig. 1).

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_17, © Springer Science+Business Media, LLC 2011

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Fig.  1  Biosynthesis of oxygen radicals and other reactive oxygen species

D.J. Carver and L.A. Palmer

Fig. 3  NADPH oxidase and the biosynthesis of reactive oxygen species. NADPH oxidase is activated by a number of luminal stressors, with resultant liberation of a free electron necessary for oxygen radical formation

Fig. 2  Enzymes, catalysts, and cofactors in the biosynthesis of reactive oxygen species

Peroxide and hypohalous acids, such as HOCl and HOBr, although not radicals per se, are potent and “reactive” ­oxidizing molecules that serve as intermediate molecules in the conversion of one radical oxygen species to another. The production of oxygen radicals may be catalyzed by a number of tissue- or cell-specific enzymes or by metals, especially iron and copper (Fig. 2).

2.2  Superoxide The major source of superoxide radical is the one-electron reduction of oxygen in the electron transport chain of mitochondria and in the endoplasmic reticulum. Additionally, O2•− is produced in the vascular wall primarily by the constitutively active enzyme NADPH oxidase found in endothelium, vascular smooth muscle cells, and adventitial fibroblasts [6, 7]. Catalysts of vascular superoxide production include NADPHdependent oxidases, xanthine oxidases, lipoxygenases, and mitochondrial oxidases (Fig.  2). Studies have identified NADPH oxidase as the most important source of superoxide anion in the vasculature, playing a significant role in the vascular remodeling and endothelial dysfunction found in cardiovascular diseases [8] (Fig. 3). Likewise, superoxide may be produced during the conversion of hypoxanthine to xanthine by xanthine oxidase, and by nitric oxide synthase (NOS) when l-arginine and tetrahydrobiopterin are limiting [9]. Once formed, superoxide radical is typically converted to significantly more reactive and biologically dangerous oxygen species through (1) the slower metal-catalyzed Haber– Weiss reaction to hydroxyl radical (Fig. 4) or (2) interaction with nitric oxide to peroxynitrite (ONOO−) (Fig. 1).

Fig.  4  The Haber–Weiss reaction. The Haber–Weiss reaction is the iron-catalyzed transformation of superoxide and hydrogen peroxide to the hydroxyl radical. The second step of it is referred to as the Fenton reaction

2.3  Hydrogen Peroxide H2O2 is not a classic radical as it has no unpaired electrons and may have a longer half-life, diffusing easily through cell membranes and to distant locations from its site of origin. It is, however, an important intermediate product in the conversion of the superoxide radical (O2•−) to the more toxic hydroxyl radical (HO•) and is produced in microsomes, mitochondria, and phagocytic cells. H2O2 is produced from superoxide nonenzymatically, or by superoxide dismutase, whereupon it may react with ferrous iron (Fe2+) or copper (Cu+) to produce the hydroxyl radical (Fig. 5). Additionally, H2O2 can be produced by monamine oxidase, galactose oxidase, xanthine oxidase, and amino acid oxidase [10]. In addition to the hydroxyl radical, H2O2 can be oxidized by eosinophil-specific peroxidase and neutrophil-specific peroxidase from halide substrates (Cl− or Br−) to form potent oxidant hypohalous acids (HOX) and other reactive halogenating species. Furthermore, H2O2 can inactivate enzymes through oxidation of essential thiol groups. In opposition to the damaging effects of H2O2, the enzyme catalase neutralizes it to water and diatomic oxygen.

17  The Chemistry of Biological Gases

Fig.  5  The influence of superoxide dismutase activity on the vasculature

Fig. 6  The production of the hydroxyl radical from hypochlorous acid

2.4  Hydroxyl Radical The hydroxyl radical is a more dangerous compound than superoxide because it reacts with an oxidizable compound faster than it can undergo enzymatic detoxification. In fact, most of the damage produced by superoxide and peroxide in  vivo is due to the production of HO•, although its high reactivity limits its actions locally to the site of initiation. The hydroxyl radical may be generated by the Haber–Weiss reaction (Fig. 4) or by the one-electron reduction of hypochlorite by ferrous iron or superoxide (Fig. 6). It can oxidize, disabling proteins, carbohydrates, nucleic acids, and lipids, resulting in injury to the cell membrane, DNA mutation, and protein signaling dysfunction. Fortunately, abundant endogenous antioxidants such as glutathione reduce its toxicities in the normal physiological state.

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mitochondrial electron transport chain) generates a diffusible mediator (i.e., reactive oxygen species) to regulate an effector protein (i.e., voltage-gated potassium and calcium channels). An analogous mechanism for regulating oxygen diffusion and circulation also exists in other specialized mammalian O2-sensitive tissues, such as the carotid body and ductus arteriosus [14]. Reactive oxygen species influence other physiological processes as well, including host immunity, hormone biosynthesis, and cell signaling [15]. The physiologic regulatory effects of reactive oxygen species specific to vascular function include modulation of cell growth, apoptosis, immune cell migration, inflammation, secretion, and extracellular matrix protein production [7]. However, in ­disease states where pro-oxidants may overwhelm ­antioxidant capacity, an imbalance in the redox state may result, whereby reactive oxygen species contribute to vascular dysfunction and remodeling through oxidative damage, or “stress.” This may alter a number of redox-sensitive signaling pathways involved in the functional and structural vascular changes associated with various abnormalities, including hypertension, hyperlipidemia, atherosclerosis, diabetes, chronic renal failure, and pulmonary hypertension [7, 16, 17] (Fig. 7). Several recent studies have provided evidence that reactive oxygen species produced specifically by the activities of NADPH oxidase may contribute significantly to alterations in vascular tone in disease states associated with chronic or intermittent hypoxia-induced pulmonary hypertension, such as bronchopulmonary dysplasia, persistent pulmonary hypertension of the newborn, and obstructive sleep apnea [18–22]. A better understanding of the role played by radical and reactive oxygen species in pulmonary vascular function may provide significant opportunities for the therapy of pulmonary hypertension.

2.5  Physiology Oxygen and its radicals play important roles in the regulation of vascular function, both for normal physiological function but also in disease, regulating endothelial NOS (eNOS) and nitric oxide (NO) [11–13]. Under physiological conditions, low concentrations of intracellular oxygen radicals play an important role in the normal redox signaling of vascular function and integrity. One example is hypoxic pulmonary vasoconstriction (HPV), the homeostatic and widely conserved vasomotor response of resistance pulmonary arteries to alveolar hypoxia. HPV balances ventilation and perfusion, diverting pulmonary blood flow away from unoxygenated lung, and optimizing systemic pO2 by reducing ­intrapulmonary shunt. It is a phenomenon intrinsic to the lung, modulated by endothelium, but mechanistically occurring in the smooth muscle cell. The “redox theory” for the mechanism of HPV proposes the synchronized action of a redox sensor (the proximal

Fig.  7  Reactive oxygen species and vascular cell function. Reactive oxygen species influence vascular cell function through a variety of pathways, including ion channels, transcription factors, metalloproteinases, posttranslational modifications by nitric oxide, and classic kinase signaling cascades

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mediated through interactions with oxygen and reactive oxygen species, transition metals, and thiols.

3 Nitric Oxide and Reactive Nitrogen Species Nitric oxide (NO) is a small diatomic molecule. Historically, it was known for its adverse effects on the environment, including the generation of smog, depletion of ozone, and in the formation of acid rain. In the late 1970s, NO was found to increase the activity of guanylate cyclase and relax smooth muscle [23, 24], and an endothelium-derived factor, named endothelium-derived relaxing factor (EDRF), was found to relax isolated vascular strips [25]. In 1987, EDRF was ­identified as NO [26, 27]. Since that time, NO has been found to be involved in a variety of biological functions, including regulation of vascular tone, platelet function, cellular adhesion, extravasation of leukocytes into tissue, neuronal transmission, immune system function, mitochondrial respiration, and regulation of iron bioavailability [28].

3.1  Nitric Oxide Biosynthesis NO is produced endogenously from l-arginine by the action of NOSs, Eq. (1).

L - Arginine + NADPH + O 2 → L - citrulline + NO + NADP +

(1)

NOSs exist in three isoforms: neuronal (or type I) NOS (nNOS), inducible (or type II) NOS (iNOS), and eNOS (or  type III). Each enzyme is a homodimer requiring three cosubstrates (l-arginine, NADPH, and O2) and five cofactors (FAD, FMN, calmodulin, heme, and tetrahydrobiopterin) for activity. Each NOS is composed of an N-terminal oxygenase domain, a calmodulin binding domain, and a C-terminal reductase domain. The oxygenase domain binds heme, tetrahydrobiopterin, and l-arginine, which forms the active site where NO synthesis takes place. The reductase domain binds FMN, FAD, and NADPH. During NO synthesis, the reductase flavins acquire electrons from NADPH and transfer them to the heme iron, permitting it to bind and activate O2, catalyzing­ the formation of NO [29]. NOS abundance is regulated by transcriptional, translational, posttranslational, and biochemical means [30]. NO production is controlled by (1) the availability of substrate l-arginine via the activity of arginase [31], (2) the availability of cofactors [32], and (3) the ­presence of the endogenous inhibitors such as asymmetric dimethyl l-arginine [33]. The varied functionality of NO is due to its wide range of chemical reactivity and number of potential reactive targets (lipids, proteins, nucleic acids, and sugars). Chemical interactions of NO are determined by the local redox environment which determines its chemical form: free radical (NO.), nitrosyl anion (NO−), and nitrosonium cation (NO+). Chemically, the actions of NO are

3.2  Nitric Oxide Reactions with Oxygen/ Reactive Oxygen Species The effects of NO can be mediated through the formation of reactive nitrogen oxide species from reactions of NO with either O2 or O2−. Reactions with oxygen, as represented by Eqs. (2–4) are slow at physiological concentrations: nanomo­ lar concentrations of NO; micromolar concentration of oxygen.

2NO + O 2 → 2NO 2

(2)

NO 2 + NO → N 2 O3

(3)

N 2 O3 + H 2 O → 2HNO3

(4)

However, under pathological conditions where the production of NO is elevated or in microenvironments (membranes or in the hydrophobic core of a protein), these reactions may be more relevant. NO and O2− rapidly combine to form peroxynitrite (ONOO−, Eq. (5)), which has been proposed to play a central role in physiological processes involving NO and pathological changes caused by NO. H

+

NO + O − → ONOO −  − ONOOH →·NO +·OH (5) 2 2 This reaction occurs close to the diffusion-controlled limit, with an average rate constant of 1010 M−1s−1 [34, 35]. In healthy cells, the levels of NO are regulated by the activity of NOS and the presence of oxyhemoglobin, whereas the levels of O2− are regulated by the activity of superoxide ­dismutase [36]. Thus, peroxynitrite production in biological systems is determined by the rates of production of O2− and NO, their relative concentrations, and their proximity to one another. Peroxynitrite is a transient species with a biological halflife of 10–20 ms [37]. Although peroxynitrite anion is relatively stable, peroxynitrous acid decays rapidly as it homolyzes to form nitrogen dioxide (NO2) and hydroxyl radical, Eq. (5). However, in the absence of a target, the main product from peroxynitrite decay is nitrate. Peroxynitrite reacts directly with certain amino acids, the most ­susceptible being the sulfur-containing amino acids (cysteine and methionine) and the aromatics (tryptophan, tyrosine, phenylalanine, and histidine) [38]. Peroxynitrite can also react with carbon dioxide to form nitrogen dioxide and carbonate radicals [39], Eq. (6) This reaction occurs with a second-order rate constant of 4.6 × 104 M−1s−1 at pH 7.4 and 37°C [40].

17  The Chemistry of Biological Gases

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(6)

ONOO − + CO 2 →·NO 2 + CO3.−

Transition metal centers (heme and non-heme iron, copper, and manganese ions) are also targets of peroxynitrite [38], forming a secondary oxidizing species at the metal center plus nitrogen dioxide, as described by Eq. (7) ONOO

−

+M

n+

→ ONOO

−

−M

n+

→·NO 2 +

−.

O−M

n+

(7)

Hydrogen peroxide does not act directly with nitric oxide. However, interactions can occur between hypervalent metal–oxo complexes following reactions between hemes and peroxides. These hypervalent metal–oxo complexes are integral components to the catalytic cycle of peroxidases [glutathione peroxidase, catalase, heme oxygenase (HO)-1, myloperoxidase] and thus have the potential to alter signaling mediated by NO. NO interactions with hypervalent metal–oxo complexes are discussed next.

3.3  Nitric Oxide Reactions with Metals Nitric oxide can interact with a variety of transition ­metals. Direct interactions of these metal centers can form metal nitrosyl complexes with no net change in redox state, Eq. (8).

Fe (II )(H 2 O )6

2+

+ NO → Fe (II )(H 2 O )5 NO 2 + + H 2 O (8)

These nitrosyl complexes can be formed by the process of nitrosation through NO+ donation or through oxidative nitrosation where the thiol is oxidized to a radical prior to the addition of NO. NO can interact directly with metal–oxygen complexes. One good example of this reaction is the interaction of NO with oxyhemoglobin to form methemoglobin and nitrate Eq. (9). This is considered to be one of the primary ways in which NO radical is consumed as the reaction is rapid (3 × 107 M−1s−1) and oxyhemoglobin is at a high concentration (4–8 mM) [28].

Hb (Fe − O 2 ) + NO → Met Hb (Fe3+ )+ NO3−

(9)

NO can interact with high-valence metal complexes formed from the oxidation of metal species or metal–oxygen complexes by hydrogen peroxide, Eqs. (10, 11). A reduction of the hypervalent complexes to a lower-valence state protects tissues from peroxide-mediated damage.

Fe (II ) + H 2 O 2 → Fe (IV )O + NO → Fe (II ) + NO 2 − (10) Fe (III ) + H 2 O 2 → Fe (V )O + NO → Fe (III ) + NO 2 − (11)

3.4 Nitric Oxide Reactions with Thiols Traditionally, nitric oxide synthase is thought to generate NO radical (NO.). NO. diffuses into target cells and signals through the activation of guanylate cyclase. In 1992, S-nitrosothiols (SNOs) were discovered within a biological system [41]. Since that time, numerous proteins have been found to be S-nitrosylated. These include hemoglobin [42], hypoxia-inducible factor-1a [43–46], SP-1 [47], eNOS [48–58], and cycloxygenase-2 [51], to name a few. In addition, genetic and biochemical data [52–55] suggest a role of SNOs in mediating many of the guanylate cyclase independent actions of NOS. SNOs can be synthesized, transported, and metabolized. Synthesis can occur through a variety of ways. SNOs can be formed by a direct interaction with thiol radicals or thiols to form SNOs or SNO radicals. SNO radicals may be stabilized through the loss of an electron or through protonation [55, 58]. In addition, SNOs can be produced through the oxidation of NO to NO+ followed by a reaction with thiols [55–58]. Oxidation of NO can be catalyzed by oxygen, aromatic residues, and transition metal ion complexes. Both iron (hemoglobin [59]) and copper (ceruloplasmin [57]) catalyze the formation of SNOs. Hemoglobin can be viewed as an S-nitrosothiol synthase [60] where nitrite is the substrate. Under physiological concentrations and time constraints, nitrite in the presence of deoxyhemoglobin forms an intermediate with the properties of Fe3+NO which  is converted to SNO–hemoglobin upon oxygenation. Thus, hemoglobin, converts NO into SNOs through an allosterically modulated heme–NO redox reaction. Ceruloplasmin can also serve as an enzyme to synthesize  S-nitrosoglutathione (GSNO), an endogenously produced SNO. In this reaction, copper serves as the electron acceptor, and NO+ is transferred to the thiolate of glutathione. Transport of GSNO into cells can be mediated by g-glutamyl transpeptidase (gGT), the L-AT transport ­system, or protein disulfide isomerase [61–64]. One enzyme that plays a physiological role in the catabolism of SNOs is S-nitrosoglutathione reductase (GSNO-R) [65, 66]. This ubiquitously expressed enzyme is responsible for  the breakdown of GSNO to oxidized glutathione and ammonia. GSNO-R does not directly affect SNO–proteins; however, these SNO–proteins may be affected indirectly through altered transnitrosation equilibria with GSNO. Besides GSNO-R, other enzyme systems have been ­implicated in the catabolism of SNOs in  vivo. These include thioredoxin/thioredoxin reductase [67], Cu/Zn superoxide dismutase [68–70], and gGT [61–63]. Physiologically, SNOs have been found to play a role in  signaling hemoglobin desaturation [71], signaling hypoxic ventilatory drive [61], and regulating hypoxic gene regulation [72].

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3.5  Tyrosine Nitration Tyrosine nitration is mediated by reactive nitrogen species (ONOO−) and nitrogen dioxide (NO2) formed as secondary products of NO metabolism in the presence of oxidants which include superoxide, hydrogen peroxide, and transition metal centers [73]. In vivo, different nitration pathways can contribute to tyrosine nitration. Most involve free-radical biochemical reactions with carbonate radials and/or oxo– metal complexes oxidizing tyrosine to tyrosyl radicals followed by a diffusion-controlled reaction with .NO2 to yield 3-nitrotyrosine.

3.6 Relationship Between Nitric Oxide and Oxygen The relationship between oxygen and nitric oxide is complex. Nitric oxide affects arteriolar tone and blood flow, thus affecting oxygen delivery to cells and tissues. Yet, nitric oxide release by NOS depends on oxygen concentration. The Km values for oxygen for each of the three NOS isoforms are significantly different: 350  mM for nNOS, 130  mM for iNOS, and 4  mM for eNOS [74]. Release of nitric oxide in the form of S-nitrosothiols from hemoglobin has been connected with oxygen saturation [2] and is thought to optimize ventilation/perfusion within the lung [71]. Nitric oxide can also alter cytochrome c oxidase activity, thus regulating mitochondrial respiration and tissue oxygen consumption. Lastly, nitric oxide can enhance the stabilization of hypoxia-inducible factor-1a [44–46], altering hypoxic signaling and potentially leading to the development of disease such as pulmonary hypertension [72]. Taken together, interactions between nitric oxide and oxygen can alter the bioavailability of both molecules, through a variety of mechanisms.

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transferred to acceptor proteins via transnitrosation reactions from hemoglobin [76, 77]. In the nitrite reductase model, NO signaling is related to nitrite reduction by deoxygenated hemoglobin. The conversion of nitrite to NO is allosterically regulated: increases in oxygen binding increase the rate at which nitrite is converted to NO [78]. Stepwise, nitrite reacts with deoxygenated ­hemogloblin to make methemoglobin and NO. It is proposed that to prevent scavenging of NO by resident oxyhemoglobin within the red blood cell, an intermediate species is formed in the nitrite reductase reaction. This intermediate diffuses out of the red blood cell and decomposes into NO or a chemical species with NO-like bioactivity. One potential candidate for this intermediate is N2O3 [79]. The formation of N2O3 could occur through the formation of an adduct with Fe2+–NO2− character from the interaction of methemoglobin and nitrite. The radical nature of this adduct would mean the adduct would quickly react with NO, forming N2O3, which can homolyze into NO and NO2., affecting vascular smooth muscle tone (Fig. 8). In this second model, red blood cells are a vehicle for O2-regulated delivery of NO signals in the form of S-nitrosothiols (Fig. 9). Hemoglobin binds NO at cysteine b93 and converts it to bioactive SNO as a function of blood O2 saturation [77, 80, 81]. The binding of O2 changes the conformation of hemoglobin from the deoxygenated state to the oxygenated state [77, 81]. This permits the transfer of nitrosonium (NO+) equivalents from the heme (Fe2+) to the thiol (cysteine b93) [77, 82, 83]. Upon deoxygenation, NO+ equivalents are transferred from cysteine b93 to a receptor cysteine in the cytosolic N-terminus of anionexchange protein 1 (AE1) [82, 83] on the red blood cell membrane. The NO+ equivalents located on AE1 are then exported from the red blood cell by a ­mechanism that is not well defined, but may occur directly through cell–cell transfer or indirectly through the formation of a serum intermediate such as S- GSNO or S-nitrosocysteine [83]. Subsequent entry of GSNO into cells can be mediated by gGT or other transporters [64].

3.7  Hemoglobin and Nitric Oxide Bioavailability

4 Carbon Monoxide

Hemoglobin plays a number of roles in regulating NO bioavailability. Initial observations demonstrated that hemoglobin is a NO sink owing to its rapid reactions with both oxygenated and deoxygenated hemoglobin, limiting the presence of NO in the blood. This catabolic reaction is exemplified in (11), where NO reacts with oxyhemoglobin to form methemoglobin and nitrate. However, in recent years, NO bioactivity has been proposed to be generated through interactions with hemoglobin either as a nitrite reductase [75] or

Carbon monoxide (CO) is an endogenously produced gas generated by enzymatic heme metabolism. It is colorless with low water solubility but chemically stable under physiological conditions. The reaction is catalyzed by HO in the reticuloendothelial system of the spleen and liver [84]. The biological effects of CO are dependent on its ability to bind to heme proteins, which are related to the concentration of CO, the concentration of O2, and the availability of reduced transition metals.

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Fig. 8  Nitrite- and hemoglobin-mediated N2O3 export from the red blood cell. Nitrite reacts with deoxyhemoglobin to make methemoglobin and nitric oxide. Most of the nitric oxide binds to the heme moiety of deoxyhemoglobin or forms nitrate and methemoglobin from oxyhemoglobin. Methemoglobin binds nitrite to form an

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adduct with Fe2+–NO2 character (hemoglobin–NO2.). This adduct can ­rapidly react with NO to form N2O3. N2O3 can diffuse out of the red cells, homolyze to NO and NO2., allowing for NO export. (Reprinted with permission from Macmillan Publishers Ltd [79]. Copyright 2007)

4.1  Carbon Monoxide Biosynthesis There are three specific isoenzymes of HO: HO-1, HO-2, and HO-3. HO-1 is the inducible isoform, whereas HO-2 and HO-3 are constitutive isoforms. The distribution of these enzymes is tissue-specific and linked to specific biological actions. Mechanistically, HO forms a complex with ­NADPH-dependent flavoprotein reductase (cytochrome P450 reductase) and biliverdin reductase on the endoplasmic reticulum. HO breaks the a-methylene carbon bond of the porphyrin ring using NADPH and molecular O2, releasing equimolar amounts of biliverdin, iron, and CO (Fig.  10). NADPH and oxygen are required cofactors in this reaction. The end products, biliverdin and bilirubin, are powerful endogenous antioxidants, whereas CO is a signaling molecule.

4.2  Carbon Monoxide Chemistry

Fig. 9  Oxygen-regulated delivery of NO in the form of S-nitrosothiols. S-Nitrosothiol binding, formation and release is allostericallly regulated by red blood cell hemoglobin and oxygen saturation. At high pO2, hemoglobin is in the R-state configuration. In this configuration, the cysteine 93 residue of the b chain is protected in a hydrophobic pocket. With deoxygenation, the conformation of hemoglobin changes to the T state, exposing the cysteine 93 residue of the b chain. In the T state, a subpopulation of hemoglobin reacts with NO., NO2−, or low-mass S-nitrosothiols to produce a nitrosylated heme in the b chain. The change in configuration to the R state, brings the cysteine b93 residue close to the nitrosylated heme. This is followed by a nitrosylation reaction between the NO heme and the cysteine b93 residue. (Reprinted with permission [77])

Carbon monoxide itself is unreactive in biological systems. Transition metals are effective in promoting the reduction of CO [85]. CO binds iron only in its reduced state – Fe(II) – which is in contrast to what is seen with NO. Transition metals are effective in promoting the reduction of CO [85]. CO readily forms metal carbonyls, which are susceptible to attack of the CO oxygen atom by electrophiles. Once formed in the body, metal carbonyls are relatively stable until CO is displaced by molecular oxygen. However, most CO is bound to hemoglobin as carboxyhemoglobin, which is carried to the lungs for excretion.

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Fig. 10  Heme oxygenase mediated formation of carbon monoxide. (Reproduced with permission [84])

4.3  Biological Function of Carbon Monoxide The biochemical effects of CO depend on its ability to bind heme proteins or proteins that contain other transition metals at their active site, thus altering protein function. CO can activate guanylate cyclase, influencing vascular tone, gene regulation, and neurotransmission. CO binds tightly to hemoglobin ferrous hemes, interfering with the oxygencarrying capacity of hemoglobin. This results in a reduction of the oxygen content of arterial blood, hinders the release of oxygen, and ultimately lowers tissue pO2 [86]. CO can also bind to the ferrous heme a3 of cytochrome c oxidase, the terminal electron acceptor of the mitochondrial respiratory chain. This results in a slower rate of cellular respiration, an increase in complex III peroxide leak rates, and a limited turnover of adenosine diphosphate [86]. The increase in peroxide level may affect the stabilization of hypoxia-inducible factor 1, cell proliferation, and mitochondrial biogenesis [86]. Lastly, the ability of CO to bind to iron may limit iron availability for the participation in redox cycling. This attribute may be an important antioxidant mechanism that can limit superoxide-driven Fenton reaction within a cell. This has been proposed to be the mechanism for the protective effects of CO in cerebral malaria [86].

4.4  Carbon Monoxide/Nitric Oxide Interactions There is known interplay between CO and NO. The affinity of NO for Fe(II) is 1,500 times greater than that of CO [87]; however, the dissociation constant for CO is greater than that for NO or for O2 [88]. Both CO and NO bind iron in the heme moiety of heme proteins, share downstream signaling pathways, and have similar regulatory functions. For instance, low concentrations of CO stimulate NO release in blood platelets and vascular cells [89], which is proposed to be due to redistribution of NO in the cells.

5 Hydrogen Sulfide Another naturally occurring and biologically relevant gas is hydrogen sulfide (H2S). Known widely as an environmental pollutant with extreme toxicity at high levels, it has recently gained new notoriety as a naturally produced gas of significant physiological influence, with major effects on vascular tone. When applied exogenously in very small doses, it also has provided remarkable protective effects from the cell level to the whole-animal level.

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Fig. 11  Biosynthesis of hydrogen sulfide. Biosynthesis of hydrogen sulfide requires the activity of cystathionine a-lyase and/or cystathionine b-synthetase on a cysteine substrate

5.1  Hydrogen Sulfide Biosynthesis H2S formation is widespread in mammalian cells and occurs through the activity of two pyridoxal phosphate-dependent enzymes on cysteine and homocysteine substrates (Fig.  11): cystathionine a-lyase – abundant in peripheral tissue – and cystathionine b-synthetase – abundant in brain tissue and liver.

5.2  Hydrogen Sulfide Catabolism H2S is a labile and easily diffusible gas with a half-life of minutes. H2S is rapidly metabolized (Fig. 12) through sequestration by hemoglobin binding, or reacting chemically with oxyradicals, peroxynitrite, hypochlorous acids, and homocysteine, or rapid enzymatic oxidation to thiosulfate and sulfite, and is finally eliminated in the urine mainly as sulfate in free and conjugated forms. All details of its oxidation to thiosulfate are not known, and it takes place with more efficiency in some tissues than in others. Sulfide oxidase, glutathione, and heme-containing compounds have all been proposed as catalysts for this oxidative conversion. It can also undergo methylation to CH3SH, but the importance of this is debated.

5.3  Hydrogen Sulfide Biochemistry H2S, known for its rotten egg odor, is colorless, flammable, and has a structure similar to that of water. O H

H

However, sulfur is not nearly as electronegative as ­oxygen, so hydrogen sulfide is also not nearly as polar as water. Because of this, comparatively weak intermolecular forces exist for H2S as compared with water. H2S is soluble and a weak acid in water or plasma, with a pKa at 37°C of 6.76.

H 2S ↔ HS− + H + Both sodium hydrosulfide (NaHS) and H2S may be dissolved in physiological liquid solution of pH 7.4, forming approximately 18.5% H2S and 81.5% hydrosulfide anion (HS−).

NaHS ↔ HS− + Na + In more acidic conditions, the percentage of H2S is higher.

S H

Fig. 12  Catalysis of hydrogen sulfide. Sulfide is metabolized by hemedependent nonenzymatic and enzymatic reactions, then excreted mostly in the urine

H

NaHS + H + ↔ H 2S + Na +

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The potential for biological activity is also enhanced by its highly lipophilic properties, and ability to freely penetrate cell membranes. High-concentration H2S (millimolar range) exhibits toxicity by its ability to inhibit mitochondrial cytochrome c oxidase, carbonic anhydrase, and monamine oxidase. It is made endogenously, however, in several normal tissues, including plasma and brain, in the 50–100-mM range, where it has many important physiological regulatory properties. Circulating blood levels have been described in the 10–100-mM range [90]. These measurements have been classically spectrophotometric, likely measuring not H2S per se, but total sulfide concentration. The H2S physiological functions described below may occur through a variety of chemical activities of the gas. Sulfides can bind proteins in  vitro, presumably as persulfides, then subsequently be liberated by the chemical reaction of dithiothreitol [91]. This binding may alter protein activity. H2S also reacts with many metals to produce the corresponding metal sulfides. It is known to react with (and be sequestered by) hemoglobin to form sulfhemoglobin [91], having the potential to modulate activities of heme-containing enzymes. H2S, a thiol with strong reducing properties, may also be important as an antioxidant molecule, similar to other small thiols such as cysteine and glutathione. It can interact and neutralize a number of species abundant in tissues, including superoxide radical (O2•−), hydrogen peroxide (H2O2), peroxynitrite (−ONOO), and /or hypochlorite (ClO−). These interactions may result in some of the cytoprotective effects of H2S at physiological concentrations.

2H 2S + 2O 2• − ↔ O 2 + 2OH − + HS − SH ↔ SO32 − + H 2 O 8H 2S + 8H 2 O 2 ↔ S8 + 16H 2 O H 2S + − ONOO ↔ NO 2 − + H 2S = O H2S may also counteract cellular injury caused by hypochlorous acid, an oxidant product of peroxide (H2O2 + Cl → HOCl + OH−).

5.4 Physiological Effects of Hydrogen Sulfide 5.4.1 Blood Pressure Control H2S was shown to affect vascular tone in both aortic rings and the whole animal in a biphasic dose-dependent manner. At higher doses, it activated ATP-sensitive K+ channels to vasodilate, but at low H2S concentrations, NO was scavenged, resulting in vasoconstriction [92]. This endothelial NO-dependent vasoconstrictor effect was also hypothesized by others to be due to eNOS inhibition [93]. However, a very recent study of mutant mice deficient in the enzyme that

­generates H2S from l-cysteine displayed pronounced s­ ystemic hypertension and decreased endothelium-­dependent vasorelaxation. The vasorelaxation thus attributed to H2S was felt to be consistent with that of other endotheliumderived relaxing factors, such as NO, and not to changes in reactive oxygen species or glutathione activity [94]. 5.4.2 Hypoxic Pulmonary Vasoconstriction and Pulmonary Hypertension Elevated serum levels of H2S achieved through daily intraperitoneal NaHS dosing in hypoxic rats attenuated the hypoxic pulmonary vasoconstrictor response and increased the HO-1 level in the pulmonary vascular smooth muscle cells [95]. However, the vascular effect of sulfide is dramatically dependent on the ambient oxygen concentration and pH [96, 97].

5.4.3 Suspended Animation H2S is also a potent reversible inhibitor of complex IV (cytochrome c oxidase) in oxidative phosphorylation, having the powerful potential to control cellular metabolism. Mice treated with 80  ppm H2S had a significant and dramatic reduction in metabolic rate along with induction of a suspended-animation-like state. After a 6-h treatment, VO2 was reduced by 90%, body temperature decreased to 2°C above ambient, and respiratory rate decreased from 120 to ten breaths per minute, with no apparent adverse effects [98]. Investigators have shown that the reduction in metabolic rate caused by H2S was independent of body temperature, and was more likely due to inhibition of the sinus node and therefore heart rate, with an associated increase in vascular resistance [92]. 5.4.4 Neutralization of Reactive Species Multiple studies have demonstrated that H2S neutralizes a variety of reactive species, including oxyradicals, peroxynitrite, hypochlorous acid, and homocysteine. Several reports also noted that micromolar levels of H2S can upregulate endogenous antioxidant systems and enhance known antioxidants, such as glutathione, N-acetylcysteine, and superoxide dismutase [91].

5.4.5 Anti-inflammatory Effects Multiple studies have also demonstrated a number of antiinflammatory effects, such as decreased immune cell ­adhesion and infiltration, decreased edema following injury,

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decreased coronary artery restenosis following balloon angioplasty, fibrosis pathway inhibition, and upregulation of a number of anti-inflammatory and proangiogenic genes, such as HO-1, vascular endothelial growth factor, insulinlike growth factor receptor, and transforming growth factorb receptor pathway genes.

5.4.6 Cytoprotective Effects H2S improved myocardial preconditioning and cell survival of injury due to ischemia/reperfusion, b-agonist therapy, and cardiopulmonary bypass. One report suggests that the beneficial effects of garlic on cardiovascular disease are due to its role in the biological production of H2S leading to vasorelaxation [99].

5.4.7 Hypoxia Tolerance A 20-min pretreatment of mice with the gas resulted in ­survival from near anoxia for up to 3.5 h, presumably due in part to the reduction in metabolic rate [100]. The suggested hypothesis was that prior exposure to H2S reduced oxygen demand, making it possible for mice to survive extreme hypoxia, with cytochrome c oxidase playing a major role as the place where supply and demand meet in aerobic cells. The investigators further speculated that H2S decreases ­temperature through decreased mitochondrial function to generate­ heat in brown fat, and H2S decreases respiratory rate by inhibition of mitochondrial function in the brainstem or lung chemoreceptors.

5.4.8 Hemorrhagic Shock Sulfides improved survival following lethal hemorrhage.

6 Summary Biological gases play important roles in the physiological process and pathological changes of the pulmonary vasculature. The influence of each of the gases is dependent on the relative abundance, location (subcellular distribution, cell type, organ type), and presence of target molecules. Cross talk between these gases regulates metabolism, signaling cascades, as well as gene expression. In health, the interplay between these gases may be important in finetuning the physiological responses to luminal and/or alveolar conditions. In disease, this interdependence may be disrupted.

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299 84. Wu L, Wang R (2005) Carbon monoxide: endogenous production, physiological functions, and pharmacological applications. Pharmacol Rev 57:585–630 85. Shriver DT (1981) Activation of carbon monoxide by carbon and oxygen coordination. In: Ford PC (ed) Catalytic activation of carbon monoxide. American Chemical Society, Washington, pp 1–18 86. Piantadosi CA (2008) Carbon monoxide, reactive oxygen signaling, and oxidative stress. Free Radic Biol Med 45:562–569 87. Keilin D (1966) Inhibition of cell respiration by carbon monoxide. The history of cell respiration and cytochrome. Cambridge University Press, Cambridge, pp 252–268 88. Gibson QH, Olson JS, McKinnie RE, Rohlfs RJ (1986) A kinetic description of ligand binding to sperm whale myoglobin. J Biol Chem 261:10228–10239 89. Thom SR, Xu Y, Ischiorpoulos H (1997) Vascular endothelial cells generate peroxynitrite in response to carbon monoxide exposure. Chem Res Toxicol 10:1023–1031 90. Lefer DJ (2008) Potential importance of alternations in hydrogen sulphide (H2S) bioavailability in diabetes. Br J Pharmacol 155:617–619 91. Szabo C (2007) Hydrogen sulphide and its therapeutic potential. Nat Rev Drug Discov 6:917–935 92. Ali MY, Ping CY, Mok Y-YP et al (2006) Regulation of vascular nitric oxide in vitro and in vivo; a new role for endogenous hydrogen sulphide? Br J Pharmacol 149:625–634 93. Kubo S, Doe I, Kurokawa Y, Nishikawa H, Kawabata A (2007) Direct inhibition of endothelial nitric oxide synthase by hydrogen sulfide: contribution to dual modulation of vascular tension. Toxicology 232:138–146 94. Yang G, Wu L, Jiang B et al (2008) H2S as a physiologic vasorelaxant: hypertension in mice with deletion of cystathionine g lyase. Science 322:587–590 95. Qingyou Z, Junbao D, Weijin Z, Hui Y, Chaoshu T, Chunyu Z (2004) Impact of hydrogen sulfide on carbon monoxide/heme oxygenase pathway in the pathogenesis of hypoxic pulmonary hypertension. Biochem Biophys Res Commun 317:30–37 96. Koenitzer JR, Isbell TS, Patel HD et al (2007) Hydrogen sulfide mediates vasoreactivity in an O2-dependent manner. Am J Physiol Heart Circ Physiol 292:1953–1960 97. Lee SW, Cheng Y, Moore PK, Bian JS (2007) Hydrogen sulfide regulates intracellular pH in vascular smooth muscle cells. Biochem Biophys Res Commun 358:1142–1147 98. Blackstone E, Morrison M, Roth MB (2005) H2S induces a suspended animation-like state in mice. Science 308:518 99. Benavides GA, Squadrito GL, Mills RW et  al (2007) Hydrogen sulfide mediates the vasoreactivity of garlic. Proc Natl Acad Sci U S A 104:17977–17982 100. Blackstone E, Roth MB (2007) Suspended animation-like state protects mice from lethal hypoxia. Shock 27:370–372

Chapter 18

Role of Oxygen-Derived Species in the Regulation of Pulmonary Vascular Tone Michael S. Wolin, Mansoor Ahmad, and Sachin A. Gupte

Abstract  Early studies detected potentially important roles for reactive oxygen-derived species (ROS) in influencing pulmonary vascular tone in models of lung injury and in responses of the pulmonary vasculature to hypoxia. In addition, observations on the inhibitory effect of superoxide on endothelium-dependent relaxation and its association with producing relaxation through stimulating the soluble, or heme-containing, form of guanylate cyclase (sGC) in pulmonary arteries were major contributing factors to Lou Ignarro’s Nobel-prize-associated work in identifying the role of nitric oxide (NO) as an endothelium-derived relaxing factor. Studies on the roles of ROS and their interaction with NO (generating reactive NO-derived species, NOx) evolved into evidence that these species may have major roles in influencing vascular function under various physiologically adaptive and pathophysiological situations to which the pulmonary circulation is exposed. For example, ROS and their interaction with NO appear to be part of the progressive adaptation of the pulmonary circulation to changes in flow, pressure, and inflammation. Although this chapter emphasizes interactions of ROS (and NOx) with signaling systems that acutely control vascular force generation or tone, it is important to note that some of the signaling systems considered are also likely to be major factors in the adaptive remodeling of the pulmonary circulation. Keywords  Oxygen radicals • Guanylate cyclase • Nitric oxide • Pulmonary vasoconstriction • Endotheliumderived factors

1 Introduction: Where Are Reactive Oxygen-Derived Species Important in the Regulation of Pulmonary Vascular Tone? Early studies detected potentially important roles for reactive oxygen-derived species (ROS) in influencing pulmonary vascular tone in models of lung injury [1, 2] and in responses of the pulmonary vasculature to hypoxia [3–8]. In addition, observations on the inhibitory effect of superoxide on endothelium-dependent relaxation and its association with producing relaxation through stimulating the soluble, or heme-containing, form of guanylate cyclase (sGC) in pulmonary arteries were major contributing factors to Lou Ignarro’s Nobel-prize-associated work in identifying the role of nitric oxide (NO) as an endothelium-derived relaxing factor [9]. Studies on the roles of ROS and their interaction with NO (generating reactive NO-derived species, NOx) evolved into evidence that these species may have major roles in influencing vascular function under various physiologically adaptive and pathophysiological situations to which the pulmonary circulation is exposed. For example, ROS and their interaction with NO appear to be part of the progressive adaptation of the pulmonary circulation to changes in flow [10], pressure [11], and inflammation [12]. Although this chapter emphasizes interactions of ROS (and NOx) with signaling systems that acutely control vascular force generation or tone, it is important to note that some of the signaling systems considered are also likely to be major factors in the adaptive remodeling of the pulmonary circulation.

2 Overview of the Role of ROS in Vascular Function M.S. Wolin () Department of Physiology, New York Medical College, Valhalla, NY 10595, USA e-mail: [email protected]

Various stimuli to which the pulmonary vasculature is exposed, such as those highlighted in Fig.  1, may directly alter the activity of oxidases which generate ROS. The species

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3 Regulation of the Sources of ROS Generation Fig. 1  Model showing how exposure of the pulmonary vasculature to various physiological and pathophysiological conditions modulates the production of reactive oxygen species by oxidases that interact with redox-regulated components of signaling mechanisms regulating vascular reactivity and remodeling Table 1  Processes activating oxidases Oxidases Activation mechanisms Nox1 & Nox2

Receptors and cellular stress promote p47phox binding by a PKC-mediated phosphorylation and cytosolic subunit aggregation Receptors and oxidants stimulate rac1 binding by a Src kinase, EGF receptor, PI3-kinase pathway Nox4 Basal activity may participate in oxygen sensing? Nox5 Direct calcium binding plus PKC phosphorylation Increased mitochondrial NADH and electron Mitochondria (electron density (complex I) transport chain) Increased coenzyme Q semiquinone (complex III) Opening mitochondrial K+ channels and mild depolarization Inhibition by severe depolarization/uncoupling May participate in oxygen sensing? Nitric oxide Signaling associated with activation of NO synthase production plus oxidation/loss of tetrahydrobiopterin cofactor Xanthine oxidase Activation of p38 MAP kinase, thiol oxidation, and proteolysis Cytochrome P450 Receptor signaling? PKC protein kinase C, PI3-kinase phosphatidylinositol 3-kinase, EGF epidermal growth factor, MAP mitogen-activated protein kinase

produced influence vascular force through redox signaling mechanisms that influence the release of endotheliumderived vasoactive mediators and/or directly control vasodilator or vasoconstrictor mechanisms within vascular smooth muscle. In some situations ROS can function as tissue hormone-like mediators that regulate vascular force. The activities of oxidases are controlled by very specific signaling mechanisms (Table 1). There is emerging evidence for a high level of organization of signaling mechanisms within vascular cells which appear to be associated with compartmentalization of signaling based on the activities of ROS metabolizing systems and the ability to alter redox systems linked to the metabolism of the species generated. The specific mechanisms regulating the activities of oxidases and their subcellular colocalization with redox-controlled signaling systems allow individual ROS to have either vasodilator or vasoconstrictor activity in the same regions of the circulation in a manner which is determined by the stimuli-promoting responses mediated through ROS.

The lung seems to contain most of the oxidases that have been associated with regulating vascular function (Table 1). Vascular smooth muscle in pulmonary arteries has been shown to contain NAD(P)H oxidases (Nox), including Nox2 and Nox4 [13], and mitochondria [14], which appear to be active generators of ROS under physiological conditions. Mice deficient in the gp91phox subunit of Nox2 show decreased levels of superoxide in pulmonary arterial smooth muscle [15]. Although this defect does not seem to alter the acute response to hypoxia [15], it appears to suppress the progression of changes associated with the development of pulmonary hypertension elicited by chronic hypoxia [16]. There is evidence that Nox are significant sources of ROS in pulmonary vascular endothelium, and that pathophysiological conditions cause ROS generation by endothelial nitric oxide synthase (NOS) [10]. Other systems which potentially generate ROS, including Nox1, xanthine oxidase, cyclooxygenase, and cytochrome P450, are also thought to be associated with the pulmonary vasculature, but less is known regarding their role as a source of vasoactive ROS. Table 1 contains a list of many of the subunit components and signalinglike mechanisms that potentially control ROS production by the vascular-associated oxidases, but only limited studies have been reported on the function of these signaling mechanisms in the pulmonary vasculature. There is substantial evidence that hypoxia can modulate ROS production by Nox [8] and mitochondria [6, 7] in a manner that modulates pulmonary arterial force. Pathophysiological conditions appear to increase ROS generation by Nox and NOS uncoupling in endothelium in a manner which influences the release of endothelium-derived vasoactive mediators [10]. For example, there is substantial evidence that changes in laminar and oscillatory flow through systemic arteries have major effects on vascular function through mechanisms associated with increased Nox activity in the endothelium [17, 18]. In addition, ROS production by NOS and mitochondria may also be a contributing factor in flow-elicited responses [10, 19]. Although there is evidence that some vasoactive stimuli, cytokines, and growth factors can increase the levels of mitochondrial ROS [19], little is known regarding the direct influence these alterations in mitochondrial function have on the control of vascular contracting and relaxing mechanisms. However, changes in expression of subunits influencing the activities of oxidases by flow, pressure, cytokines, and other contributors to vascular pathophysiological function appear to be important factors in the progression of pulmonary vascular diseases which are associated with alterations in vascular reactivity [20].

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3.1 NAD(P)H Oxidases Studies on the oxidases in vascular tissue have detected evidence for the presence of Nox1, Nox2, Nox4, and Nox5 [21, 22]. These oxidases differ in their subunit content and cellular and subcellular localizations, and Nox5 seems to be expressed primarily in humans. Nox1 was initially termed Mox1, and its expression was associated with cellular growth and proliferation. Nox2 was actually the first characterized enzyme in this group as the gp91phox oxidase present in neutrophils and related phagocytic cells. It appears that Nox1 and Nox2 are generally associated with cell membrane structures and they seem to have similar mechanisms for activation. Stimulation of phosphorylation of a cytosolic p47phox subunit by protein kinase C (PKC) seems to be one of the best known mechanisms of initiating activation of these oxidases. Other subunits, including p67phox and p40phox, may bind to the p47phox subunit and these normally cytosolic proteins then bind to the membrane-bound Nox1 or Nox2 subunits which are already associated with a p22phox subunit. Although a Nox01 subunit can replace the phosphorylated p47phox subunit and a NoxA1 subunit can substitute for the p67subunit, little is known about the roles of these alternative subunits in pulmonary vascular regulation. Endothelium appears to contain the p67phox and p40phox subunits; however, there is little evidence for their importance in pulmonary vascular smooth muscle. There is a second mechanism of activating Nox1 and Nox2 which appears to enhance and prolong the initial p47phox binding-associated activation through a subsequent binding of rac1 to the membrane-bound Nox subunits. One pathway controlling rac activation appears to originate from Nox-derived ROS initiating a c-Src/epidermal growth factor receptor kinase signaling mechanism mediated through stimulating phosphatidylinositol 3-kinase [23]. Nox4 appears localized to internal cellular structures, and this oxidase is known to consist of the membrane-bound Nox4 and p22phox subunits. Currently, changes in Nox4 activity seem to be closely associated with the levels of expression of its Nox4 and p22phox subunits, and the enzyme does not appear to be regulated by signaling mechanisms controlling Nox1 and Nox2. Peptide mediators associated with the progression of pulmonary hypertension such as urotensin II have been associated with increased expression of Nox4 and activation of vascular smooth muscle proliferation-associated signaling [24]. Nox5 is different from the other Nox in that it has a calcium binding site that may be regulated through a PKC mechanism, and it lacks a requirement for the p22phox subunit or other cytosolic subunits known to influence the activity of Nox. Although Nox5 appears to be expressed in human vascular tissue, little is known about if it has a role in regulating pulmonary vascular function. Our studies suggest that increasing passive force on pulmonary

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arteries by stretching them causes an acute activation of Nox2 associated with enhancing their reactivity to vasoconstrictors [25] and increasing the expression of Nox4 by organ-culturing pulmonary arteries with transforming growth factor b causes a hypoxia-reversible depression of force mediated by an increased level of hydrogen peroxide [26]. Thus, the activities of Nox2 and Nox4 may have important roles in regulating vasoconstrictor and vasodilator signaling mechanisms in pulmonary arterial smooth muscle.

3.2 Mitochondria Mitochondria are present in essentially all types of cells potentially involved in vascular regulation, and studies suggest that they have important roles in regulating force in pulmonary arterial smooth muscle through their influence on mechanisms associated with both energy metabolism and the generation of ROS [6, 7, 27]. Mitochondrial function is very sensitive to energy-consumption-associated processes, metabolic conditions, hypoxia, and some cellular signaling mechanisms such as changes in intracellular calcium levels. Studies on the properties of mitochondria suggest that complex I and complex III in the electron transport chain appear to have sites which can transfer electrons to oxygen, generating superoxide anion [6– 8]. It appears that high levels of electron density in complex I are associated with increased superoxide generation in the mitochondrial matrix, and rotenone appears to increase the generation of superoxide from this site. The conditions controlling the production of superoxide by complex III are less well understood. It is thought that the one-electron-reduced semiquinone form of coenzyme Q reacts with oxygen to generate superoxide. Superoxide appears to be generated in both the matrix and the intermembrane space when it is produced by the complex III site. Increased electron flow into complex III seems to promote superoxide production, and this has been suggested to occur in vascular tissue when it is exposed to mitochondrial K+ channel opening drugs such as diazoxide or under conditions of a partial depolarization of the mitochondrial membrane potential by H+-transporting agents known as protonophores or mitochondrial uncouplers [28]. Rotenone inhibits electron flow from complex I into complex III in a manner which attenuates superoxide generation at this site, and the interaction of antimycin with complex III increases superoxide generation at the complex III site [7, 8]. There is a possibility that pulmonary vascular mitochondria may have different properties because both rotenone and antimycin appear to inhibit ROS generation [14, 29]. Recent studies have suggested that metabolic conditions increasing the level of mitochondrial NADH may be a factor in increasing mitochondrial superoxide generation in vascular tissue, and exposure to

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conditions which strongly depolarize mitochondria seems to be associated with decreased superoxide production [30]. Owing to the actions of manganese superoxide dismutase (SOD) in the matrix of mitochondria and Cu,Zn-SOD in the region between the inner and outer membranes of mitochondria, hydrogen peroxide may be the main ROS released from this organelle. Hypoxia appears to be a major regulator of mitochondrial ROS production, and there is convincing evidence that hypoxia can both increase [7] and decrease [6] mitochondrial ROS production in pulmonary arterial smooth muscle. On the basis of recent work on systemic arteries [30], the effects of hypoxia under the conditions studied on mitochondrial NADH redox may be a major factor in determining if hypoxia increases or decreases mitochondrial ROS generation.

3.3 Nitric Oxide Synthase The NOS enzyme in endothelium produces NO from l-arginine when a variety of stimuli, such as the shear force of flow, stimulate signaling mechanisms activating electron flow from NADPH into the flavoprotein–heme region of this protein. Although endothelial cell calcium is a key factor is stimulating NOS activity, extensive studies (generally in systemic vascular tissue) have identified several other protein binding interactions, phosphorylation signaling systems, and other posttranslational modifications that have major roles in influencing the function of this NO-producing enzyme [31]. It seems that the availability of tetrahydrobiopterin is one of the most influential factors in maintaining NO biosynthesis once the activity of this enzyme is stimulated. When this cofactor is oxidized or not available, the NOS reaction undergoes what is described as “uncoupling,” and it can become a major source of superoxide production in the endothelium. Although many of the regulatory processes influencing NOS activity, including the availability of l-arginine, seem to influence NOS uncoupling, the significance of these mechanisms in functional vascular tissue is not well understood. A tetrahydrobiopterin-controlled NOS uncoupling seems to be a major factor in the loss of endothelium-derived relaxation by NO seen in multiple vascular diseases [32], and uncoupled NOS has been reported to be a source of endothelium-derived hydrogen peroxide, the levels of which control vascular contractile function. The uncoupling of NOS promotes the formation of reactive NO-derived species which may also contribute to changes in endothelial function. A more dominant source of quantities of NO that promote formation of reactive NO-derived species in amounts that directly influencing vascular function seems to be associated with increased expression of the inducible form of NOS by inflammatory mediators and other stimuli.

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3.4 Other Oxidases There are a variety of other oxidases which potentially influence the pulmonary vasculature, but there is only minimal documentation of their influence on vascular contractile function. Although cytochrome P450 has been demonstrated to be a potential source of endothelium-derived relaxing levels of hydrogen peroxide in systemic arteries [33], the role of this process in pulmonary vascular regulation is not well documented. Lung injury is associated with activation of xanthine oxidase and the gp91phox (Nox2) in phagocytic cells in models where these sources of ROS produce pulmonary edema potentially associated with vasoconstriction [34, 35].

4 Metabolic Redox Processes Controlling the Concentrations of Vasoactive Species and Interactions with Signaling Systems 4.1 Metabolic Control of ROS Levels The activities of oxidases, mitochondria, and NOS discussed in Sect. 3 are the most dominant factors influencing the levels of ROS in the subcellular environments of cells. In addition, the levels of enzymes metabolizing ROS have major roles in determining the local concentrations of individual vasoactive species such as superoxide, peroxide, and peroxynitrite, which have important roles in controlling signaling mechanisms and regulating vascular force and reactivity. (Fig. 2) The concentrations of Mn-SOD in the mitochondrial matrix and Cu,ZnSOD in other intracellular regions probably maintain superoxide concentrations in the picomolar range. The efficient

Fig.  2  Model showing how the competition between superoxide ­dismutase and NO for reaction with superoxide (O2.–) generated by oxidases regulated by signaling mechanisms described in Table 1 goes on to modulate signaling mechanisms controlling vascular force described in the text and Table 2 through subsequent chemical and enzymatic reactions of peroxynitrite (ONOO–) and hydrogen peroxide (H2O2). Some actions of ONOO and its derived nitrogen dioxide (.NO2) include the oxidation and/or nitrosation/nitration of thiols (RSH), including iron (Fe–S) and zinc (Zn–S) binding sites, unsaturated fatty acids resembling arachidonic acid, and heme. The efficient metabolism of peroxide by enzymes including catalase, glutathione peroxidases, peroxiredoxins, and heme peroxidases in the cellular regions exposed to peroxide are thought to cause localized redox effects which influence signaling mechanisms

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intracellular conversion of superoxide into hydrogen peroxide by the SOD enzymes then generates fluxes of peroxide which are continually being metabolized by the peroxide-metabolizing systems present in subcellular regions. Although glutathione (GSH) peroxidases are probably the major consumers of peroxide in most subcellular regions of vascular tissue, other peroxide-consuming enzymes, including catalase and peroxiredoxins, are also likely to be significant consumers of peroxide in the poorly documented regions where these proteins are found. The availability of reducing cofactors, including GSH and thioredoxin for GSH peroxidase and peroxiredoxins, both controls the abilities of these enzymes to consume peroxide and generates oxidized forms of GSH (GSSG) and thioredoxin that are potentially involved in regulating the signaling systems discussed in the next section. Metabolic sources of NADPH generation in subcellular compartments, such as glucose 6-phosphate dehydrogenase (G6PD) in the pentose phosphate pathway of glucose metabolism, seem to be key factors in maintaining the availability GSH and thioredoxin for the metabolism of peroxide. These metabolizing systems are thought to maintain intracellular peroxide concentrations in the low nanomolar range. As superoxide levels increase, superoxide begins to efficiently scavenge NO in the region where it is produced. Since NO reacts with superoxide at a rate which is three- to fourfold faster than the rate superoxide reacts with SOD, NO will become a significant consumer of superoxide when its concentrations approach the local levels of SOD. Under these conditions, the generation of peroxynitrite (ONOO–) becomes a significant factor. Vascular smooth muscle secretes an extracellular Cu,Zn-containing form of SOD, which seems to have a major role in protecting NO from being consumed by superoxide in the extracellular environment of systemic and pulmonary arteries.

4.2 Metabolic Interactions of ROS with Signaling Systems Direct chemical reactions of ROS with components of signaling systems and redox changes resulting from the metabolism by enzymes such as peroxidases seem to contribute vascular regulatory processes. Owing to the low levels of superoxide resulting from its consumption by SOD, there seems to be only minimal evidence for direct interactions of superoxide with components of signaling systems beyond its reaction with NO and conversion to peroxide. The scavenging of NO by superoxide attenuates the direct interactions of NO with sites such as the ferrous heme of sGC. When significant amounts of ONOO are generated in subcellular regions, it appears to become a major factor in biological regulation as a result of its chemical reactivity with thiols, unsaturated fatty acids, and other biological molecules. Although the chemical

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reactions of ONOO are complex, and may involve species such as nitrogen dioxide (.NO2) and bicarbonate radical, its formation is likely to be a major pathway through which superoxide is involved in biological regulation beyond its role in generating peroxide. The consequences of these reactions include thiol oxidations that disrupt iron and zinc binding sites, the formation of protein–protein and protein–GSH (S-glutathionation) containing disulfides, protein tyrosine nitration, heme oxidation, and fatty acid oxidation and/or nitration which potentially have important roles in vascular regulatory mechanisms and the progression of pathophysiological processes. Since the fastest rates of reactions of peroxide with biological molecules seem occur with peroxidases, including the key enzymes which metabolize peroxide, these processes should be the most dominant route for peroxide to influence signaling systems. There are several enzymes with which peroxide reacts that appear to have important roles in biological regulation. A component of the mechanism through which peroxide initiates prostaglandin production by cyclooxygenase is its metabolism by the heme peroxidase produced by reaction of this enzyme, an oxidation process needed for the production of prostaglandin G2 [36]. The peroxide prostaglandin G2 (and/or hydrogen peroxide) then sustains generation of prostaglandin H2 by cyclooxygenase based on the availability of arachidonic acid. Peroxide metabolism by catalase appears to be able to stimulate sGC [4, 37]. The metabolism of peroxide through GSH peroxidases generates GSSG, and the shift in the redox status of GSH to oxidation promotes reversible protein thiol oxidations through glutaredoxin and protein–disulfide isomerase reactions. The metabolism of peroxide by peroxiredoxins seems to be linked to the oxidation of thioredoxin, which also appears to function as a reversible regulator of protein thiol redox. Although these thiol modifying systems seem to be very logical pathways for the redox regulation of signaling, very little is currently known about how these systems ­function, and it is often assumed regulation occurs through the direct reactions of peroxide with thiols on proteins being regulated [38]. The direct rates of reaction of peroxide with thiols on proteins such as protein phosphatases appear to be very slow, and the oxidase generating peroxide would have to colocalize in the immediate vicinity of the thiol being oxidized for regulation to occur through chemical reactions. The redox status of NADPH in subcellular regions is likely to be a major factor in determining the activities of thiol redox-controlled systems because NADPH-dependent enzymes including GSH and thioredoxin reductases maintain the redox status of GSH and thioredoxin. Thus, the balance of interactive of systems controlling redox in subcellular regions is a major factor in determining the behavior of redox-controlled processes. The biological chemical reactions of ONOO and the species it generates such as nitrogen dioxide (.NO2) and bicarbonate radical under physiological conditions seem complex and are

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not well understood [39–41]. However, some of the products of reactions of these reactive NO-derived species (NOx) detected in biological systems appear to influence vascular function in a manner which provides evidence for potentially important regulatory roles. Although thiols seem to function as scavengers of NOx, there is evidence that these reactions result in nitrosation (RSNO) and nitration (RSNO2) of thiols, and oxidation reactions such as disulfide (RSSR) formation which may have roles in regulating signaling systems [39–41]. For example, RSNOx are potent vasodilators, and they may be a tissue source of NO that is released over time. In addition, the reactions of protein thiols with NOx seem to be important contributors to the regulation of systems controlled by thiol redox. It is well established that ONOO results in nitrotyrosine formation, and this has been hypothesized to also be a contributor to biological regulation. For example, prostaglandin I2 synthase appears to be readily inactivated by tyrosine nitration, and this can shift prostaglandin-mediated regulation from vasodilation to vasoconstriction as a result of the loss of prostaglandin I2 and increased availability of prostaglandin H2. Unsaturated fatty acids, such as arachidonic acid, also directly react with NOx to generate nitrated and oxidized fatty acids with potent effects on vascular reactivity [42].

Table 2  Redox mechanisms regulating vascular force Mechanism Signaling systems Autocoids and endotheliumderived vasoactive factors

NO-mediated relaxation Prostaglandins

5 Roles for ROS in Controlling Signaling Pathways Regulating Force in the Pulmonary Vasculature The various ways oxidases are regulated and the availability of multiple signaling mechanisms with which individual ROS can potentially interact in subcellular regions helps provide explanations for why these species can be involved in both vasoconstrictor and vasodilator mechanisms that potentially regulate pulmonary vascular function. This section considers some of the documented interactions of ROS and redox with signaling systems listed in Table 2 that are potentially involved in regulating pulmonary vascular function. It is also important to consider that the level of tone in the pulmonary vasculature may enhance the expression of constrictor mechanisms at low tone and vasodilator mechanisms when pressure is increased by vasoconstriction of the pulmonary circulation. For example, peroxide can stimulate vasoconstriction under basal conditions in the saline-perfused rabbit lung through thromboxane A2 generation [2], and vasodilation is observed when tone is elevated in the rabbit lung preparation with the thromboxane A2/prostaglandin H2 receptor agonist U46619.

Action

Superoxide scavenges NO preventing relaxation Peroxides stimulate vasodilator or vasoconstrictor prostaglandin production ONOO and other oxidants inactivate PGI2 synthase converting vasodilation by PGI2 to vasoconstriction by PGH2 Other endothelium-derived factors Decreased level of NO promotes vasoconstriction through endothelin release Systems regulated by NADPH oxidation promotes the NADPH oxidation is associated with increased SR Ca2+ uptake by the cytosolic NAD(P)H redox coordination of multiple systems SERCA pump, decreased intracellular Ca2+ release, and influx that promote relaxation through lowering intracellular Ca2+ level Relaxation through increased Stimulation of cGMP generation by sGC is activated by NO, peroxide metabolism by catalase, and other cGMP signaling sGC mechanisms sGC stimulation by NO is inhibited by superoxide, and oxidation of its heme and thiol sites Stimulation of PKG cGMP stimulates PKG Peroxides promote a thiol-oxidation-mediated subunit dimerization that directly activates PKG Ion channels K+ channels Oxidant mechanisms open K+ channels, promoting relaxation through hyperpolarization Increased levels of cytosolic Ca2+ Many redox-controlled interactive systems promote or inhibit the release promoting vasocontriction and/or reuptake of intracellular Ca2+ Protein kinases promoting ERK Peroxide and thiol oxidants stimulate vasoconstriction PKC Receptors activate PKC, stimulating oxidant signaling which promotes vasoconstriction Rho Stimulated by Nox2-derived peroxide PGI2 prostaglandin I2, PGH2 prostaglandin H2, SR sarcoplasmic reticulum, SERCA sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, sGC soluble guanylate cyclase, cGMP cyclic GMP-dependent protein kinase, ERK extracellular-regulated kinse, PKC protein kinase C

18  Role of Oxygen-Derived Species in the Regulation of Pulmonary Vascular Tone

5.1 Autocoids and Endothelium-Derived Vasoactive Factors 5.1.1 Prostaglandins One of the most potent effects of exposing microvascular preparations to ROS-generating systems seems to be the responses mediated through peroxide stimulating the generation of vasoactive prostaglandins [1, 43]. As mentioned already, one of the earliest responses reported [1] in the pulmonary circulation of rabbits exposed to ROS was vasoconstriction that appeared to be mediated through increased thromboxane A2 generation. Although the cellular sources of ROS-stimulated prostaglandin generation which influence pulmonary vascular function are not well understood, it has been established that peroxide stimulates phospholipase A2 to release arachidonic acid in pulmonary vascular endothelium [44]. Peroxide stimulation of arachidonic metabolism by cyclooxygenase to generate prostaglandin H2 is a fundamental property of the cyclooxygenase enzyme [36]. In addition, ROS may shift the profile of prostaglandins produced toward vasoconstrictors, especially under conditions where an inhibition of vasodilator biosynthesis by enzymes such as prostaglandin I2 synthase is observed.

5.1.2 Nitric Oxide It is well established that endothelium-derived NO often functions to depress the expression of vasoconstriction in the pulmonary circulation. Attenuation of the influence of NO by its reaction with superoxide is often a major factor in many aspects of physiological and pathophysiological aspects of pulmonary vascular regulation, especially under conditions where the vasculature is exposed to high levels of flow, pressure, and inflammation-related processes. Since NO is transported as a membrane-permeable dissolved gas, it will be removed by a combination of processes including diffusion into the blood of the circulation or the airway region and by reaction with any intracellular and extracellular source of superoxide in the vessel wall such as endothelium, smooth muscle, fibroblast, and inflammatory-cell-associated-oxidases. Increased expression of the inducible-inflammatory form of NOS can also be a factor in depressing pulmonary vascular force through NO or in the generation of ONOO. The complex chemical reactions of ONOO and its interactions with signaling systems make it difficult to predict most aspects of its role in biological regulation. It is likely that ONOO may initially have signaling effects which directly regulate vascular force, but as the vasculature is exposed to ONOO enzymes become inactivated, and ONOO may have effects resembling

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other forms of oxidative stress. Thus, exposure of the pulmonary vasculature to increased levels of ONOO is likely to be a major factor in the progression of many adaptive and pathophysiological processes.

5.1.3 Other Endothelium- and InflammatoryCell-Derived Vasoactive Factors Endothelin is often released from the endothelium under conditions where NO levels are decreased by processes often associated with oxidative stress. Many stressful conditions, and the autocoids and cytokines they generate, have the potential to increase oxidant signaling in the vessel wall through stimulating oxidases, changing the expression of subunits which influence the activities of activities, or altering the expression of ROS-metabolizing enzymes. Most of the studies published in this area focused on examining the progression of pulmonary vasculature adaptation related to disease processes. The adaptation process potentially influences both ROS-dependent and redox-independent mechanisms regulating pulmonary vascular tone and reactivity.

5.2 Redox-Controlled Mechanisms Regulating Vascular Smooth Muscle Force Force generation by vascular smooth muscle is a combination of the activity of signaling mechanisms promoting contraction and relaxation, and the interactions between these mechanisms. The topics will be discussed on the basis of mechanisms that seem to be redox-regulated, without separating them into processes promoting vasodilation and constriction because removal of an existing vasoactive process will elicit the opposite response.

5.2.1 Systems Regulated by Cytosolic NADP(H) Redox Studies on the actions of inhibitors of G6PD have resulted in evidence that glucose metabolism by the pentose phosphate pathway maintains the levels of cytosolic NADPH, and that its oxidation or increasing the ratios of cytosolic NADP/NADPH appears to coordinate multiple processes that lower intracellular calcium levels associated with vascular relaxation [45, 46]. These processes appear to include stimulation of calcium uptake into the sarcoplasmic reticulum by the sarcoplasmic/endoplasmic reticulum Ca2+ATPase (SERCA) pump, and inhibition of intracellular

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calcium release and calcium influx. Owing to the existence of multiple ion transport systems and channels sites that could be regulated by redox and the interactive nature of the systems controlling intracellular calcium levels around the sarcoplasmic reticulum and plasma membrane, the actual sites regulated by NADPH redox remain to be defined. Although K+ channels appear to have only a minor role in the NADPH-oxidation-associated relaxation of coronary arteries by inhibitors of G6PD [45], the Kv channel has a prominent role in the relaxation of rat pulmonary arteries exposed to these inhibitors [47]. Thus, the metabolic control of cytosolic NADPH can be a factor controlling force, and it has been suggested that increased levels of NADPH in pulmonary arteries from processes such as decreased peroxide generation and metabolism under hypoxia can be a factor in regulating pulmonary arterial tone [3, 48]. In addition, it appears that pulmonary arteries maintain higher levels of cytosolic NADPH than coronary arteries as a result of increased expression of G6PD [13]. The increased levels of cytosolic NADPH in pulmonary arteries have been proposed to maintain the production of vasodilator levels of peroxide by Nox. Thus, hypoxia can cause vasoconstriction by depressing the consumption of NADPH needed for both the generation and the metabolism of peroxide. Since NADPH redox potentially has a major influence on other redox systems, such as the processes controlling thiol redox in subcellular regions discussed in Sect. 4, it is likely to be a factor in most of the redox mechanisms regulating vascular smooth muscle force that are considered next.

5.2.2 Cyclic GMP Signaling The cytosolic form of guanylate cyclase, or sGC, is a major site of redox regulation: it promotes vascular relaxation through generating cyclic GMP (cGMP) and stimulating cGMP-dependent protein kinase (PKG) activity. Many processes including multiple systems regulating the level of intracellular calcium and its influence on force generation by vascular smooth muscle seem to be directly controlled by PKG [49]. Since NO directly stimulates sGC by binding its Fe2+ heme, it is a major target for the influence of superoxide and other factors that control the bioavailability of NO in the region of vascular smooth muscle. The heme of sGC seems to be readily oxidized by ROS and ONOO, and this also seems to be a factor in the expression of NO-mediated vasodilation [50]. Thus, the oxidant stress associated with most vascular pathophysiology is a major factor in attenuating NO-mediated vasodilation through its effects on sGC–cGMP signaling. There are other redox factors that potentially contribute to the regulation of sGC–cGMP signaling. Peroxide can stimulate

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relaxation of pulmonary arteries through cGMP signaling [37]. One process may involve the direct stimulation of sGC activity resulting from peroxide metabolism by catalase, and this mechanism is sensitive to inhibition by superoxide [37]. Another process may involve peroxide promoting a disulfideelicited dimerization of the PKG protein subunits which directly stimulated PKG-mediated vasodilation [51]. Cytosolic NADPH is potentially a factor in influencing all of the mechanisms discussed in this section as a result of its role in controlling NO biosynthesis, Nox activity, peroxide metabolism, and protein thiol and heme redox. Peroxide stimulation of sGC appears to be suppressed by NO [52], and it is likely that NADPH redox influences the thiol-oxidation-elicited stimulation of PKG. Thus, the redox regulation of sGC and that of PKG are potentially major factors in the coordination of multiple processes promoting vascular relaxation that are controlled by cGMP-associated signaling. Although there is general agreement on the role of superoxide attenuating NO-mediated relaxation in pulmonary vascular pathophysio­ logical processes, the roles of most other aspects of redox regulation of cGMP signaling in regulation of the pulmonary circulation remain to be better defined.

5.2.3 Ion Channels Interest in the mechanism of hypoxic pulmonary vasoconstriction has resulted in studies and hypotheses on the redox regulation of potassium channels and processes controlling intracellular calcium levels in pulmonary arterial smooth muscle. Although this topic has been debated in many recent reviews [6–8], the sites directly regulated by redox remain to be more precisely defined. This is because most potassium channels and processes controlling intracellular calcium levels have sites such as cysteine thiols which can be regulated by redox. In addition, many of the processes regulating calcium and potassium channels influence each other through redoxindependent interactions. Redox-regulated systems such as cGMP may also control the function of many of the ion channels suggested to be redox-regulated. Some of best documented aspects of redox-regulated ion channels in pulmonary and systemic vascular smooth muscle include potassium channels and the SERCA pump. Weir and Archer have reported the findings of studies that show that plasma membrane K+ channels are opened by oxidant-related conditions that are associated with hyperpolarization [3, 14, 53]. Hyperpolarization promotes vascular relaxation through processes which include the closing of voltage-regulated calcium channels. One of best documented redox-regulated ion transport systems is the SERCA pump. Thiol oxidation of sites on the SERCA pump that are thought to occur under physiological conditions seem to stimulate calcium uptake into the sarcoplasmic reticulum [54]. Filling of the sarcoplasmic

18  Role of Oxygen-Derived Species in the Regulation of Pulmonary Vascular Tone

reticulum with calcium is thought to inhibit the influx of calcium through the plasma membrane through processes associated with the capacitive regulation of calcium. This interactive system may be part of mechanisms which then coordinate the lowering of intracellular calcium level and relaxation. It could also be hypothesized that the regulation of K+ channels and hyperpolarization may be part of this mechanism through effects such as sarcoplasmic reticulum filled with calcium closing calcium-regulated potassium channels and TRP channels associated with calcium influx. Hyperpolarization would then also close voltage-regulated calcium channels. In addition, the SERCA pump is also stimulated by PKG, allowing cGMP signaling to regulate this interactive system by lowering the cytosolic calcium level in a manner similar to oxidants.

5.2.4 Protein Kinases Promoting Vasoconstriction There are several protein kinases which appear to be redoxrelated and generally function to enhance the actions of calcium in promoting contraction through mechanisms such as the inhibition of myosin phosphatase. Some of the best documented systems include the Src kinase, PKC, extracellularregulated kinase (ERK), and Rho kinase systems [25, 55–58]. Although the precise redox-controlled process regulating each of these kinase systems is not well understood, it is likely that a step involving inhibition of protein phosphatases by oxidants is often involved. Since the PKC and Src kinase systems participate in the activation of Nox [23], they may also contribute to feed-forward mechanisms where oxidants increase the production of ROS. The ERK system appears to be activated in pulmonary arteries by stretch through mechanisms that appear to involve increasing the generation of Nox-derived peroxide, Src kinase activation, and a thiol-oxidation-mediated process [25]. Although its activation causes enhancement of the force generation by contractile stimuli, ERK activation does not appear to participate in the bovine pulmonary artery contractile response elicited by an acute exposure to hypoxia that was studied [25]. Oxidants have been shown to activate Rho kinase in vascular tissue [56, 58]. Rho kinase activation seems to be an important contributing factor to changes in pulmonary artery reactivity that occur when pulmonary arteries are exposed to prolonged periods of hypoxia [55]. Sorting out what is occurring with the PKC and Rho kinase systems is complicated because they may also be activated and regulate force by redox-independent mechanisms associated with signaling activated by contractile agents. In addition, cGMP–PKG signaling may inhibit systems such as Rho kinase [49]. Thus, poorly understood aspects of the redox regulation of several different protein kinases may be important factors in the control of pulmonary vascular function.

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6 Coordination, Integration, and Changes in Pathophysiological Aspects of ROS Signaling Smooth muscle of the pulmonary vasculature is probably normally exposed to low levels of NO which is derived from the endothelium and other sources in the lung in amounts that partially stimulate relaxation through cGMP. In addition, if the concept that pulmonary arterial smooth muscle is exposed to vasodilator levels of peroxide derived from sources within such as Nox and mitochondria under basal normoxic conditions is correct, it is also probably exposed to sources of peroxide generation that activate vasodilation through processes which might involve mechanisms such as the stimulation of sGC and thiol redox changes influencing ion channels and perhaps PKG. Since most of the redox-regulated vasoconstrictor mechanisms in the pulmonary circulation seem to involve enhancing the sensitivity of the contractile apparatus to calcium when the vasculature is exposed to vasoconstrictors, these mechanisms may have only minor roles in influencing the pulmonary circulation under the minimal levels of basal tone that appear to exist under normoxic physiological conditions. Pathophysiological conditions influencing the pulmonary circulation generally increase the generation of ROS and activate adaptive responses that are poorly understood. Although stimuli such as changes in the shear of flow, pressure, and inflammation, and exposure to hypoxia, vasoactive mediators, cytokines, and other regulatory factors have been reported to influence the generation of ROS and their interaction with NO, there is generally a lack of a consensus on which oxidases are involved and how they influence redox signaling. Some observations suggest ROS and redox-signaling processes are part of the progressive adaptation of the pulmonary circulation to pathophysiological conditions [11, 16, 59]. In addition to structural changes in the vasculature, there are a diversity of reports of changes in the expression of components of signaling systems associated with ROS and redox regulation. Studies examining vascular reactivity under these conditions have in some cases detected a loss of endothelium-derived relaxation associated with superoxide scavenging NO, changes in the function of relaxing and contractile mechanisms, and the profile of prostaglandins and other eicosanoids which either have been demonstrated or could be hypothesized to be linked to the direct influence of ROS signaling on these processes [1, 11, 12, 55, 59]. For example, an increased role for Rho kinase enhancing vasoconstriction which could originate from changes in many different redoxregulated processes seems to be an important factor in the expression of pulmonary hypertension [56]. Although these processes could be influenced by acute therapies that target ROS, a more important factor may be reversing the adaptive

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effects of stimuli promoting dysfunction. It is interesting to note that the pulmonary circulation generally shows little evidence of contributing to the oxidant-associated pathological process of circulating factors in systemic vascular diseases such as hypertension and diabetes. Although the reasons for this are not understood, some factors such as angiotensin II increasing the expression of endothelial NOS through activation of pulmonary endothelial angiotensin type 2 receptors [60] could be a factor. The many facets of ROS redox-regulated processes originating from the subcellular localization of multiple distinct signaling pathways allow these systems to have many different roles in the sequential progression and expression of pulmonary vascular disease processes which remain to be defined.

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18  Role of Oxygen-Derived Species in the Regulation of Pulmonary Vascular Tone 37. Burke TM, Wolin MS (1987) Hydrogen peroxide elicits pulmonary arterial relaxation and guanylate cyclase activation. Am J Physiol 252:H721–H732 38. Forman HJ, Fukuto JM, Torres M (2004) Redox signaling: thiol chemistry defines which reactive oxygen and nitrogen species can act as second messengers. Am J Physiol Cell Physiol 287:C246–C256 39. Wolin MS (2000) Interactions of oxidants with vascular signaling systems. Arterioscler Thromb Vasc Biol 20:1430–1442 40. Pacher P, Beckman JS, Liaudet L (2007) Nitric oxide and peroxynitrite in health and disease. Physiol Rev 87:315–424 41. Pryor WA, Houk KN, Foote CS et al (2006) Free radical biology and medicine: it’s a gas, man! Am J Physiol Regul Integr Comp Physiol 291:R491–R511 42. Balazy M, Iesaki T, Park JL et  al (2001) Vicinal nitrohydroxyeicosatrienoic acids: vasodilator lipids formed by reaction of nitrogen dioxide with arachidonic acid. J Pharmacol Exp Ther 299:611–619 43. Wolin MS, Rodenburg JM, Messina EJ et al (1987) Oxygen metabolites and vasodilator mechanisms in rat cremasteric arterioles. Am J Physiol 252:H1159–H1163 44. Chakraborti S, Gurtner GH, Michael JR (1989) Oxidant-mediated activation of phospholipase A2 in pulmonary endothelium. Am J Physiol 257:L430–L437 45. Gupte SA, Arshad M, Viola S et al (2003) Pentose phosphate pathway coordinates multiple redox-controlled relaxing mechanisms in bovine coronary arteries. Am J Physiol Heart Circ Physiol 285:H2316–H2326 46. Gupte SA, Wolin MS (2006) Hypoxia promotes relaxation of bovine coronary arteries through lowering cytosolic NADPH. Am J Physiol Heart Circ Physiol 290:H2228–H2238 47. Gupte SA, Li KX, Okada T et al (2002) Inhibitors of pentose phosphate pathway cause vasodilation: involvement of voltage-gated potassium channels. J Pharmacol Exp Ther 301:299–305 48. Wolin MS, Ahmad M, Gao Q et  al (2007) Cytosolic NAD(P)H regulation of redox signaling and vascular oxygen sensing. Antioxid Redox Signal 9:671–678 49. Lincoln TM, Dey N, Sellak H (2001) Invited review: cGMPdependent protein kinase signaling mechanisms in smooth muscle: from the regulation of tone to gene expression. J Appl Physiol 91:1421–1430

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50. Stasch JP, Schmidt PM, Nedvetsky PI et  al (2006) Targeting the heme-oxidized nitric oxide receptor for selective vasodilatation of diseased blood vessels. J Clin Invest 116:2552–2561 51. Burgoyne JR, Madhani M, Cuello F et  al (2007) Cysteine redox sensor in PKGIa enables oxidant-induced activation. Science 317:1393–1397 52. Mohazzab HK, Fayngersh RP, Wolin MS (1996) Nitric oxide inhibits pulmonary artery catalase and H2O2-associated relaxation. Am J Physiol 271:H1900–H1906 53. Olschewski A, Hong Z, Peterson DA et al (2004) Opposite effects of redox status on membrane potential, cytosolic calcium, and tone in pulmonary arteries and ductus arteriosus. Am J Physiol Lung Cell Mol Physiol 286:L15–L22 54. Tong X, Ying J, Pimentel DR et al (2008) High glucose oxidizes SERCA cysteine-674 and prevents inhibition by nitric oxide of smooth muscle cell migration. J Mol Cell Cardiol 44:361–369 55. Jernigan NL, Walker BR, Resta TC (2008) Reactive oxygen species mediate RhoA/Rho kinase-induced Ca2+ sensitization in pulmonary vascular smooth muscle following chronic hypoxia. Am J Physiol Lung Cell Mol Physiol 295:L515–L529 56. Gupte SA, Kaminski PM, George S, et al (2009) Peroxide generation by p47phox-Src activation of Nox2 has a key role in protein kinase C-induced arterial smooth muscle contraction. Am J Physiol Heart Circ Physiol 296:H1048–H1057 57. Thakali K, Davenport L, Fink GD et al (2007) Cyclooxygenase, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase MAPK, Rho kinase, and Src mediate hydrogen peroxide-induced contraction of rat thoracic aorta and vena cava. J Pharmacol Exp Ther 320:236–243 58. Jin L, Ying Z, Webb RC (2004) Activation of Rho/Rho kinase signaling pathway by reactive oxygen species in rat aorta. Am J Physiol Heart Circ Physiol 287:H1495–H1500 59. Jankov RP, Kantores C, Pan J et al (2008) Contribution of xanthine oxidase-derived superoxide to chronic hypoxic pulmonary hypertension in neonatal rats. Am J Physiol Lung Cell Mol Physiol 294:L233–L245 60. Olson SC, Dowds TA, Pino PA et  al (1997) ANG II stimulates endothelial nitric oxide synthase expression in bovine pulmonary artery endothelium. Am J Physiol 273:L315–L321

Chapter 19

Mitochondrial Reactive Oxygen Species and Redox State in Pulmonary Vascular O2 Sensing Stephen L. Archer and John J. Ryan

Abstract  Mammals possess a homeostatic O2-sensing system that comprises the resistance pulmonary arteries, ductus arteriosus, carotid body, neuroepithelial body, systemic arteries, fetal adrenomedullary cell, and fetoplacental arteries. Together these specialized tissues form a homeostatic system that increases the organism’s ability to survive hypoxia, whether encountered during development, at altitude, or during disease. Thus, the homeostatic O2-sensing system optimizes O2 uptake and delivery. One important part of the homeostatic O2-sensing system is hypoxic pulmonary vasoconstriction (HPV), a vasomotor response of resistance pulmonary arteries to alveolar hypoxia, that optimizes ventilation/perfusion matching and optimizes systemic O2 tension. The core mechanisms of hypoxic pulmonary vasoconstriction resides in the smooth muscle cell, although it is modulated by the endothelium. This chapter explores and updates the redox theory for the mechanism of hypoxic pulmonary vasoconstriction. Keywords  Oxygen sensing • Hypoxic pulmonary vasoconstriction • Redox signaling • Reactive Oxygen Species (ROS)

1 Introduction There is now considerable evidence that HPV results from the coordinated action of a redox sensor (possibly the proximal mitochondrial electron transport chain, ETC) which generates a diffusible mediator (a reactive oxygen species, ROS) that regulates effector proteins [voltage-gated K+ (Kv) channels]. The controversy whether HPV results from hypoxic increases in the production of superoxide anion or from hypoxic withdrawal of ROS was discussed in a recent published debate [2, 3]. Although the identity of the sensor (mitochondria vs. NADPH oxidase, NOX) and its function (to increase the level of superoxide or decrease the level of S.L. Archer (*) Department of Medicine, University of Chicago, Chicago, IL 60637, USA e-mail: [email protected]

H2O2) remains controversial, there is better (although imperfect) consensus regarding the downstream effector pathway (K+ channel inhibition, Ca2+ channel activation). It appears that hypoxia ultimately elicits vasoconstriction by inhibiting Kv channels (e.g., Kv1.5 and Kv2.1) in the PASMC which depolarizes the PASMC, thereby activating voltage-gated Ca2+ channels, increasing Ca2+ influx and causing vasoconstriction. HPV is sustained and enhanced by capacitative calcium entry, activation of Rho kinase, which mediates calcium sensitization, and also by a variety of paracrine factors, such as endothelin. A role for cyclic ADP ribose as a redox-regulated mediator of intracellular calcium release has been proposed. Here we discuss the redox theory for HPV. The redox theory for HPV is increasingly recognized to be relevant to a variety of diseases. Disorders of the O2 sensing may contribute to the proliferative, apoptosis-resistant phenotype of PASMCs in pulmonary arterial hypertension and cancer. Prior to discussing identification of a redox sensor for HPV, it is critical to define some of the characteristics of HPV (which must be satisfied by any putative sensor). HPV is a homeostatic vasomotor response of small, muscular “resistance” PAs to alveolar hypoxia. HPV actively diverts perfusion to optimally ventilated lung segments. The central mechanism of HPV involves a redox-based O2 sensor (e.g., mitochondria or NOX), which alters the production of a diffusible redox mediator (e.g., superoxide, H2O2, or redox couples such as reduced/oxidized glutathione of NADH) in direct or inverse proportion to changes in inspired O2 concentration. Our group has reported that hypoxia creates a reduced environment characterized by an increased ratio of reduced glutathione to oxidized glutathione and decreased levels of superoxide and H2O2; other groups, who otherwise subscribe to the redox theory of O2 sensing, find diametrically opposed redox signaling (hypoxia increasing the level of superoxide). In either case, the resulting alteration of the redox environment must inhibit certain Kv channels in PA SMCs (PASMCs). Upstream of this pathway, the magnitude of HPV is impacted by the endothelium (e.g., enhanced by endothelin, inhibited by nitric oxide); downstream it is modulated

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_19, © Springer Science+Business Media, LLC 2011

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Fig. 1  Initial (1986) redox model for hypoxic pulmonary vasoconstriction (HPV). The concept is that redox state, determined by metabolism (in the mitochondria and elsewhere), regulates sulfhydryl balance in the membranes. This controls pulmonary artery smooth muscle cell K+ flux and membrane potential and thereby regulates the L-type Ca2+ channel and thus the cytosolic Ca2+ level. This pathway was also proposed as a means of regulating insulin secretion in pancreatic b cells. (Reproduced with permission [1])

by Rho kinase through sensitization of the contractile apparatus to calcium. This chapter, extracted from two recent reviews [4, 5], presents a critical appraisal of the redox theory for the mechanism of HPV (Figs. 1, 2).

2 Homeostatic Oxygen-Sensing System Before discussing the mechanism of HPV, it is useful to review the homeostatic oxygen-sensing system, to which HPV is a contributor. Mammalian cells depend on appropriate amounts of oxygen for survival. There are a group of specialized tissues that contain these specialized cells which detect and govern responses to changes in oxygen tension all to optimize systemic oxygen delivery. These specialized cells are situated within the carotid body, ductus arteriosus, PAs, systemic arteries, and neuroepithelial cells [6] in the lungs. Fetoplacental arteries [7] and the fetal adrenomedullary cells [8] also contain specialized tissue that senses local oxygen tension [9].

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Fig. 2  Updated redox theory of HPV. In this model the mitochondrial electron transport chain senses hypoxia and decreases reactive oxygen species (ROS) production. A decrease in ROS (superoxide anion and/or H2O2) production inhibits the effector Kv1.5 and Kv2.1 channels. Kv channels depolarize the membrane and activate Ca2+ entry via L-type Ca2+ channels. Other theories, which are not mutually exclusive, include NADPH oxidase as a sensor and source of ROS, cyclic adenosine diphosphate ribose as a redox-activated mediator that causes intracellular Ca2+ release by activating ryanodine receptors [80] in the sarcoplasmic reticulum, and changes in the function of adenosine monophosphate kinase. In addition, activation of Rho kinase may enhance HPV by sensitizing the contractile apparatus to Ca2+

2.1 Carotid Body This organ, derived from neural crest cells, is located at the bifurcation of the carotid arteries. The carotid body plays a key role in oxygen sensing and maintaining oxygen homeostasis by stimulating the brainstem respiratory center at times of hypoxia. It is through this mechanism that a decline in arterial oxygen level results in a compensatory increase in ventilation (and our sense of “huffing and puffing” at altitude). The carotid body has a considerable blood supply from branches of the external carotid body. The carotid body contains glomus cells which determine the levels of hypoxia in the capillaries which flow through the carotid body. In turn, the afferent sensory fibers from the carotid body join the glossopharyngeal nerve, which signals the hypoxia to the respiratory center of the brain. Efferent activity is likely mediated through autonomic neurons. Hypoxia activates neurosecretion by inhibiting potassium channels in the glomus cells [10], a mechanism that is (more or less) conserved in all specialized O2-sensitive tissues.

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2.2 Ductus Arteriosus In utero, the ductus arteriosus allows blood from the PAs to bypass the lungs, travel through the aorta, and undergo oxygenation in the placenta. At birth, when the neonate breathes in air and the lungs expand, there is an increase in arterial oxygen tension. As a response, the PAs dilate and there is a large increase in blood flow into the lungs. Concurrently, the ductus arteriosus responds to this increase in oxygen level in the exact opposite manner by constricting and closing. When the ductus arteriosus contracts, the infant experiences the switch from fetal circulation to neonatal circulation. Blood exiting the right ventricle into the PA now is forced to enter the lung to undergo oxygenation. Closure of the ductus arteriosus happens within hours after birth (by active vasoconstriction) and the lumen is subsequently permanently closed by a process of remodeling (mediated by SMC migration and fibrosis). In the ductus SMCs, inhibition of K+ channels also initiates acute constriction in response to the rise in PO2 that occurs with the first breath [11, 12].

3 The Pulmonary Circulation and HPV The adult pulmonary circulation is a unique, low-resistance circuit designed for gas exchange. It is perfused by a thinwalled, afterload-intolerant right ventricle. The pulmonary circulation accommodates the entire cardiac output at one fifth the pressure and resistance of the systemic vasculature. The large arteries, which serve as conduits, manifest only weak HPV, and may dilate in response to hypoxia, whereas the small intrapulmonary arteries, which control pulmonary vascular resistance (PVR), are the site of maximal HPV [13, 14]. In most forms of pulmonary hypertension (PHT), the burden of disease is also localized to these small arteries. This anatomical (proximal–distal) and functional (conduit– resistance) distinction extends to the cellular level. SMCs from these PA segments have different cellular electrophysiological characteristics, with the strongest being the oxygen-sensing mechanism evident in small muscular PAs that are less than 100 mM in diameter [15]. Indeed, proximal and distal PAs have different embryological origins. Conduit PAs are derived from the sixth aortic arch, whereas the distal PAs are derived from expansion of the capillary plexus, originating by vasculogenesis from the mesenchymal lung bud [16, 17]. HPV is defined as the increase in PVR that occurs when the alveolar PO2 falls below a threshold level. The first observation of HPV was made by Bradford and Dean [18] in 1894. Von Euler and Liljestrand recognized the potential homeostatic role of HPV in optimizing systemic PO2, noting that it increases the blood flow to better aerated lung areas, which

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leads to improved conditions for the utilization of alveolar air [19]. In atelectasis, when the hypoxia is segmental, HPV is restricted to the vascular segments serving the hypoxic lobe, thereby achieving ventilation/perfusion matching and optimizing systemic PO2, without elevating PA pressure (PAP) [20, 21]. During global hypoxia, as occurs at altitude or in hypoventilation syndromes (such as sleep apnea), HPV increases PAP, causing PHT. HPV is found in virtually all mammals, although its incidence is diminished in races or species that are adapted to life at altitude (Tibetan natives [22], yaks). Likewise chronic hypoxia (10% O2 for more than 3 days) impairs HPV in rats, despite enhanced constriction to other stimuli [23].

3.1  Properties of HPV in Humans The nature of HPV in humans helps us define physiologic hypoxia and delineates the fundamental characteristics of HPV. Results from reductionist models should be judged against these studies because some of the basic science of HPV has been conducted under conditions that may not be relevant to HPV (i.e., using anoxic PO2s, cell lines, conduit arteries, etc.). At the summit of Mount Everest (27,559 ft, barometric pressure 272 mmHg), the mean PaO2 in ambient air was a remarkable 24.6 mmHg [24]. This serves as a practical definition of “physiologically tolerable” hypoxia. To go higher (in altitude) or lower (in PO2) is not sustainable even for those acclimated to hypoxia; yet much of the molecular science of HPV is conducted at PO2s below 30 mmHg with no measure of pH or PCO2. Focusing on literature that uses “physiologically tolerable” hypoxia helps clarify the HPV literature. The onset of HPV is rapid (seconds) [25] and the elevation in PVR is sustained [26]. In humans, the maximum elevation of PVR occurs within 15 min and the magnitude of HPV is not potentiated by repeated hypoxic challenges [27]. Moderate hypoxia (12.5% O2, PO2 ~ 50 mmHg) doubles PVR. In healthy volunteers, 8 h of hypoxia increases PVR from 1 to 3 Wood units within 2 h; thereafter the PVR remained at a stable level of elevation. Systemic vascular resistance decreases in parallel, falling from 19 to 14 Wood units and PVR reverts to normoxic levels upon return to breathing 20% O2 [28]. The intrinsic nature of these opposing responses to hypoxia in pulmonary versus systemic circulations is recapitulated in the isolated, serially perfused, lung–kidney model [29]. This reminds us that any proposed sensor–effector combination should have opposing effects in cells from the pulmonary and systemic circulations. In humans, HPV is significantly impaired by hyperventilation and the resulting respiratory alkalosis; however, HPV is not reduced by inhibition of synthesis of eicosanoids or endothelin receptor antagonism (reviewed in [4]), consistent with endothelium independence of the core mechanism of

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HPV. Support for the proposed role of K+ and Ca2+ channels in HPV can also be found in patients with chronic obstructive pulmonary disease (COPD). HPV is attenuated by 53% in COPD patients by nifedipine, an L-type, voltage-gated Ca2+ channel blocker, and this occurs at doses that do not lower systemic vascular resistance [30].

3.2 HPV in Animal Models In contrast to the clarity of the in  vivo and isolated lung research, there is disagreement about the characteristics of HPV in PA rings. Although we find that HPV causes a rapid constriction in resistance PA rings that gradually plateaus and is sustained [31], much as occurs in vivo, several groups have found that hypoxia elicits a biphasic response consisting of an immediate, endothelium-independent constriction which peaks in approximately 10 min (phase I) and a subsequent slowly developing endothelium-dependent contraction peaking at approximately 30–40 min (phase II) [32]. The basis for the discrepancy is uncertain, but likely relates to the duration of the hypoxic challenge (our work focuses on the first 10 min of the response, other groups focus on longer hypoxic exposure), details of tissue selection (resistance versus conduit arteries), and details of tissue handling/ viability (which are often obscure in the methods sections).

4 General Features of HPV Certain features of HPV must be accounted for by any proposed mechanism: 1. HPV begins rapidly, is intrinsic to PAs, and is strongest in resistance PAs. HPV occurs in isolated lungs [33], small PAs [34], and PASMCs [13, 35, 36]. Although most PA segments constrict to hypoxia [37], HPV is strongest in small PAs (greater than the third division, less than 200 mm) [14, 15, 37, 38]. Proximal PAs dilate in response to hypoxia [15, 39]. The restricted occurrence of hypoxic constriction of intrapulmonary PAs (and to a lesser extent veins) reflects the localized abundance of HPV’s molecular apparatus in these segments [40]. 2. HPV is triggered by airway hypoxia. Resistance PAs are surrounded by terminal airways and preferentially respond to moderate alveolar hypoxia, rather than hypoxemia. 3. HPV involves the coordinated inhibition of Kv channels and activation of L-type Ca2+ (CaL) channels. HPV is initiated, at least in part, by the inhibition of a family of O2sensitive Kv channels leading to membrane depolarization, opening of CaL channels, and vasoconstriction [41–43]. Hypoxia dilates systemic arteries, and hypoxic inhibition of whole-cell K+ current (IK) occurs in PASMCs, but not

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in systemic arterial SMCs [41, 43]. The activity of the CaL channel is largely regulated by membrane potential (Em), which is established by K+ channels [20]. However, the CaL channel is also directly O2-sensitive, being activated by hypoxia (independent of Em) [44]. Accordingly, Ca2+ channel blockers (nifedipine) inhibit HPV [33], whereas Kv channel inhibitors (4-aminopyridine, 4-AP [15, 45]), and CaL agonists (BAYK8644) enhance HPV [46, 47]. 4. HPV is reversible. PVR returns to the baseline level rapidly (within 1 min) with discontinuation of hypoxic ventilation. Indeed, perfusion defects that relate to atelectasis from proximal airway obstruction cause chronic HPV that reverses with relief of air obstruction even after years [48]. This reversibility is consistent with the redox theory but is hard to explain if one postulates a major role for Rho kinase activation in HPV.

5 Mechanisms of O2 Sensing in HPV The redox mechanism for HPV was proposed initially in 1986 [1] (Fig. 1), and has been refined subsequently (Fig. 2) (reviewed in [4]). This theory proposes that a drop in alveolar O2 concentration decreases production of a redox mediator virtually instantaneously (less than 30 s). The mediator, in turn, alters the function of one or more effector proteins, which ultimately increases cytosolic Ca2+ concentration, thereby activating the PASMC’s contractile machinery. There are two potential cytochrome-based O2-sensitive sources of ROS which are candidate O2 sensors, the mitochondrial ETC and a family of classical and novel nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOX). The final common pathway of all forms of pulmonary vasoconstriction involves activation of the contractile apparatus. Thus, actin and myosin, and their regulatory systems, such as Rho kinase, are very important determinants of the magnitude of HPV. However, since they appear not to be unique to the pulmonary circulation and act downstream from the “O2-sensing unit,” they are not discussed in this chapter. The role of Rho kinase activation in HPV becomes increasingly important as the duration of hypoxic exposure is sustained. Advocates of the theory that hypoxia increases superoxide production note that superoxide anion causes pulmonary vasoconstriction by activating Rho kinase [49, 50].

5.1 Mitochondria as O2 Sensors The mitochondrion’s role as a primary site for O2 consumption during aerobic metabolism makes it an ideal candidate for early detection of changes in PO2. The evidence suggesting that the mitochondrial ETC could be important in O2 sensing is based in part on the concordant effects of certain ETC inhibitors

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and hypoxia. Inhibitors of the mitochondrial ETC and oxidative phosphorylation cause pulmonary vasoconstriction in isolated blood-perfused lungs [51]. Moreover, inhibitors of complex I (rotenone) and complex III (antimycin A), but not inhibitors of complex IV (cyanide), mimic hypoxia’s hemodynamic effects, causing pulmonary vasoconstriction, dilatation of the human ductus arteriosus [11], and systemic circulation [29]. Rotenone and antimycin not only inhibit subsequent acute HPV but their constrictor potency, like that of hypoxia, is selectively suppressed in chronic hypoxic PHT (CH-PHT), a condition in which constriction to most stimuli (e.g., angiotensin II) is enhanced [52]. The parallels between hypoxia and ETC inhibitors are also evident in nonvascular tissue. ETC inhibitors mimic hypoxia’s effects on the carotid body [53]. The initial phase of the pulmonary vasoconstriction induced by both metabolic inhibitors and hypoxia appears to occur without ATP depletion. ATP is preserved in HPV induced by moderate hypoxia; conversely, anoxia, which does deplete ATP, results in only transient pulmonary vasoconstriction followed by pulmonary vasodilatation [54]. We find that rotenone and antimycin reduce lung and PA ROS production in a manner analogous to that of physiological hypoxia [29, 42, 55]. However, the effects of hypoxia and mitochondrial inhibitors in the pulmonary circulation are controversial and other groups that have similarly concluded that mitochondria are sensors in HPV disagree on the signaling mediator [56, 57]. Notably, Schumacker and Chandel [56, 58] and Ward [50] found that hypoxia increases superoxide production and implicated this as a means by which cytosolic Ca2+ concentration increases in PASMCs, leading to HPV. On a cellular level, the proximal ETC inhibitors also mimic hypoxia. Hypoxia, inhibits K+ current and depolarizes PASMCs, but does not have this effect in systemic arterial SMCs [41, 43]. Like hypoxia, rotenone and antimycin A (and some other ETC inhibitors) inhibit IK in PASMCs [42]. As would be predicted from the redox theory, the ETC inhibitors, which dilate the ductus arteriosus, increase IK in human ductus arteriosus SMCs [11]. In terms of the ROS up versus down controversy, the K+ current in PASMCs is inhibited and the membrane potential is depolarized by reducing agents, not by ROS or oxidants [59]. The PASMC’s O2-sensing mechanism appears to relate to the small proportion of total electron flux involving unpaired electrons and the associated production of intramitochondrial ROS. Teleologically, this uncoupled electron flow provides an early warning of potential risk to the upstream supply of electrons and their originating reducing equivalents which could limit ATP production. Thus, there is some poorly understood but obligatory link between signaling ROS and redox couples. Moreover, the role that compartmentalized redox signaling (cytosolic vs. mitochondrial) plays is unclear. However, new redox probes permit dissection of the various subcellular ­compartments [60, 61].

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The ETC comprises four megacomplexes (each with many subunits) that mediate transfer of electrons down a redox potential gradient through a series of carriers, resulting in final acceptance of the electron by O2, producing ATP, with water as an end product. In the course of electron transport, H+ ions are translocated, creating a proton gradient that generates the mitochondrion’s extremely negative membrane potential (∆ym). This potential energy is used to produce ATP. Complexes I and III produce most of the mitochondrial ROS [62], although there is tissue heterogeneity in which complex predominates. Complex I, which is inhibited by the isoflavinoid rotenone, is an NADH ubiquinone oxidoreductase where NADH is oxidized to NAD+ as it transfers two electrons to ubiquinone. Complex III is a ubiquinol cytochrome c oxidoreductase that transfers electrons from FADH2 to cytochrome b, and subsequently to the final electron acceptor, cytochrome c. Complex IV, composed of cytochromes a and a3, is a cytochrome oxidase that is inhibited by cyanide. Complex IV accepts electrons from reduced cytochrome c and passes them to O2. Inhibition of a complex proximal to the site of ROS production should decrease ROS production (as we observe with rotenone); conversely, distal inhibition would disrupt electron flow, diverting electrons to react with O2 and generate ROS, a phenomenon we have observed with cyanide [42]. This suggests the site of critical ROS generation is proximal to or at complex III. To detoxify the mitochondrial ROS, the mitochondria uniquely express an inducible superoxide dismutase (SOD) isoform (manganese superoxide dismutase, SOD2). SOD2 transforms toxic superoxide radicals to H2O2. We hypothesize that H2O2 is a diffusible redox mediator connecting the sensor (mitochondria) to the effector (K+ channel), linking redox state and vascular tone [29]. In this redox theory the low normoxic pulmonary vascular tone is due to tonic generation of ROS which maintains the O2-sensitive Kv channels in the PASMC oxidized and open (thereby relaxing the PASMC) (Fig.  2). Conversely, HPV results from the withdrawal of these ROS and Kv channel closure. Differences in mitochondrial ETC function and ROS generation appear to account for the observed heterogeneity in SOD2 expression in PA versus renal artery mitochondria, the levels of ROS and SOD2 being far higher in PASMCs [29]. In general, SOD2 levels are inversely proportional to mitochondrial ROS production. Whereas the redox theory views SOD2 as a H2O2 generator, others view it as an antioxidant-eliminating superoxide (it is in reality both).

5.2 NOX or NOX Isoforms as O2 Sensors Another potential O2-sensor is NAD(P)H oxidase or one of the NOX isoforms. Lung levels of NADPH increase with hypoxia [63]. This flavocytochrome contains two membrane-bound

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subunits (p22phox and gp91phox) and two cytosolic proteins (p47phox and p67phox). NOX (or a variant that preferentially uses NADH as a substrate) produces ROS in proportion to the PO2 and has been suggested as an O2 sensor [64]. Production of ROS by NOX isoforms has been demonstrated in cells derived from several O2-sensitive tissues, including the neuroepithelial body, PASMCs, and carotid body. Much of the evidence favoring NOX as an O2 sensor derives from experiments using diphenylene iodonium (DPI). DPI inhibits NOX and, in several respects, mimics hypoxia. After causing slight vasoconstriction, DPI decreases subsequent pressor responses to hypoxia (reviewed in [4]). Moreover, it reduces normoxic ROS production in the PA and inhibits PASMC IK. Unfortunately, DPI nonspecifically inhibits flavoproteincontaining enzymes, including nitric oxide synthase and complex I of the mitochondrial ETC (reviewed in [4]). Mice deficient in the 91phox-containing NOX (Nox-2) manifest dramatically lower normoxic lung ROS production than backcrossed control mice [55]. However, HPV and the O2-sensitive portion of PASMC IK are preserved [55]. Moreover, rotenone constriction is preserved or enhanced, consistent with the mitochondrial O2-sensor hypothesis [55]. Preserved O2 sensing has also been reported in the type 1 cell of the carotid body from these mice [65]. Overall, these findings argue against the classical NOX system as an O2 sensor in HPV, although they do not exclude a role for other novel oxidases. Moreover, since the NOX is simply another cytochrome-based electron shuttle, it may combine with a mitochondrial signal to regulate K+ channels and vascular tone.

6 Diversity in Redox Signaling Because PAs constrict and systemic arteries relax in response to hypoxia, it appears probable that the redox signals generated in response to hypoxia differ between these vascular beds. ROS levels are higher in normal PAs than in systemic arteries, such as the renal artery, and only the PAs have a ROS signal which is hypoxia-inhibited [29]. The relatively depolarized mitochondria in PASMCs (relative to renal artery SMCs) may account for the higher ROS levels and the hypoxia-sensitive changes in ROS production in PASMCs. PAs also differ from coronary arteries (both endotheliumdenuded arteries) in that the activity of glucose 6-phosphate dehydrogenase, the rate-limiting enzyme generating cytosolic NADPH, is 1.5-fold higher in bovine PAs than in coronary arteries. This may explain why bovine PAs had 4.2-fold higher levels of NADPH and greater superoxide levels, despite similar NOX2 and NOX4 expression [66]. Elevated levels of cytosolic NADPH in PASMCs may drive NOX activity [66]. Thus, differences in mitochondrial function and the pentose phosphate pathway may account for the

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unique occurrence of a hypoxia-induced redox shift in the pulmonary circulation.

7 Effector Mechanisms of HPV: K+ Channels K+ channels are proteins consisting of four transmembranebound a-subunits and four regulatory b-subunits. The ionic pore, which determines the channel’s intrinsic conductance and ionic specificity, is created by the formation of tetramers of a-subunits. The Kv channels also have, in their S4 region, a voltage sensor. b-subunits associate with many K+ channels and alter their expression and kinetics. There are several types of K+ channel a-subunits, including Kv, inward rectifier (Kir), and two-pore (TASK) channels. Inhibitors of Kv channels in general (e.g., 4-AP) [41, 45, 67] and inhibitors of Kv1.x channels (e.g., correolide) cause pulmonary vasoconstriction [40, 45]. Additionally, preconstriction of resistance PAs with the Kv blocker 4-AP eliminates subsequent HPV [40]. Hypoxia inhibits IK and depolarizes EM in canine PASMCs, but not in renal, arterial SMCs [41, 43] and is now widely accepted. In the last decade, significant advances have been made in the molecular identification of the relevant O2-sensitive K+ channels. How do K+ channels control pulmonary vascular tone? In PASMCs, as in most vascular SMCs, closure of Kv channels decreases the tonic efflux of K+ that occurs down the intracellular/extracellular concentration gradient (145 mM/5 mM). Channel closure renders the cell interior relatively more positive (depolarized) and when Em exceeds approximately −30 mV, the open probability of L-type voltage-gated Ca2+ channels increases, and intracellular Ca2+ concentrations rise. K+ channel inhibition increases both cytosolic Ca2+ and K+ concentrations, which not only promotes contraction, but also induces a phenotype that favors proliferation and suppression of apoptosis. Thus, regulation of K+ channel activity, and the subsequent regulation of Ca2+ concentration, may be important for maintaining both low PVR and the pulmonary circulation’s thin-walled vascular morphology. Eleven Kv channel families have been identified. A variety of putative O2-sensitive channels exist in the PASMC (Kv1.2, Kv1.5, Kv2.1, Kv3.1b, and Kv9.3) (reviewed in [4]). Certain K+ channels are specially suited to O2 sensing, by virtue of possessing key cysteine and methionine groups. Reduction or oxidation of sulfhydryl residues in these channels by a redox mediator, such as ROS, can cause conformational changes in the channel, thereby altering pore function and channel gating. Only certain Kv channels appear to be effectors of HPV (e.g., Kv1.5 and Kv2.1) [4]. In PASMCs, oxidants increase IK (e.g., H2O2, diamide, and oxidized glutathione); in contrast, reducing agents (e.g., reduced glutathione) and agents that facilitate electron shuttling (e.g., duroquinone) inhibit

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IK (reviewed in [4]). Moreover, oxidants (e.g., diamide) dilate the pulmonary circulation (mimicking O2), whereas reducing agents mimic hypoxia. Hypoxia and redox agents may alter the function of K+ channels directly, through modulation of levels of electron donors (NADH) or by modulating ROS production. The role of Kv1.5 and Kv2.1 in HPV can best be seen in two models in which HPV is selectively suppressed, the CH-PHT model and the Kv1.5 knockout mouse. Suppression of acute HPV and the O2-sensitive portion of IK in CH-PHT results, in part, from loss of Kv1.5 expression [52, 68]. Kv1.5 adenoviral gene transfer restores Kv expression, O2-sensitive IK, and HPV in rats with CH-PHT [69]. In mice with targeted Kv1.5 deletion, HPV is also impaired and the size of the O2sensitive IK in PASMCs is reduced [31]. Similar effects on vascular tone of correolide, a selective Kv1.x channel inhibitor [40], and hypoxia suggest that a Kv1.x channel contributes to the mechanism of HPV [40]. In patch–clamp studies, intracellular dialysis of PASMCs with anti-Kv1.5 antibodies (immunoelectropharmacology) depolarizes resistance, but not conduit, PASMCs. Indeed, the combination of anti-Kv1.5 and anti-Kv2.1 causes a large depolarization, and hypoxia has no additional effects [40, 70]. Kv1.5 channels are preferentially expressed in resistance PASMCs (within the vascular segment with the greatest HPV) [40]. Heterologously expressed human Kv1.5, cloned from normal PA, generates an outward Kv current that is active near the resting Em of –65 mV in Chinese hamster ovary cells and which is inhibited by hypoxia [40]. Thus, the consequence of enriched Kv1.5 expression in resistance PAs is relative hyperpolarization of resistance versus conduit PASMCs (about -60mV vs. about −35 mV) and a unique O2 sensitivity that underlies the localized enhancement of HPV in resistance PAs.

8 Controversies Regarding the Role of ROS in HPV Even among groups that agree that there is a redox basis for HPV and that the mitochondria are the source of signaling ROS, there remains controversy as to the nature of the effects of hypoxia on ROS generation (decreased versus increased).

8.1 Hypoxia Decreases ROS Generation During alveolar hypoxia, we and others have found that ROS production falls in proportion to the amount of inspired O2 [42, 71, 72]. Higher normoxic levels of ROS cause physiological oxidation of K+ channels in PASMCs. In support of this

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view, we measured PA ROS production using three different detection methods (lucigenin-enhanced chemiluminescence, Amplex Red H2O2 assay, and 2¢,7¢-dichlorodihydrofluorescein diacetate, DCF). In endothelium-denuded, resistance PA rings, a concordant decrease in ROS levels during hypoxia or exposure to ETC inhibitors was detected by all three methods [29]. Likewise, Paky et  al. found that hypoxia reduces lung ROS production, measured using lucigenin [73]. They noted that the SOD-mimetic tiron or antimycin A decreased lung ROS production. Conversely, ROS production in rat lungs or isolated lung mitochondria increases in direct proportion to PO2. Wolin’s group also suggested that it is the withdrawal of normoxic ROS (H2O2) that elicits HPV, although they attributed the source of the ROS to NADH oxidase [74]. In summary, we find that regardless of the technique of ROS measurement, both in isolated lungs and in isolated PA rings, hypoxia consistently and rapidly decreases ROS production prior to the onset of HPV (reviewed in [4]). This fall in ROS production is not an artifact of vasoconstriction as the vasoconstrictors angiotensin II and KCl have no effect [42, 55].

8.2 Hypoxia Increases ROS Generation In contrast, Marshall et al. found an increase in ROS production during hypoxia in isolated PA myocytes, although they attributed this to NOX [75]. In experiments using both isolated perfused lungs and cultured PASMCs, the Schumacker group showed that hypoxia increased intracellular DCF fluorescence in cultured PASMCs and this was blocked by the complex III inhibitor myxothiazol [56]. Many of their measurements of HPV were conducted in isolated PASMCs (passage number unspecified but more than 6 days in culture) using a surrogate for PVR (SMC shortening) [56]. Since cultured cells rapidly lose ion channels and also have diminished O2 sensitivity, at least in the ductus arteriosus [11], we believe that conclusions about O2 sensing should be derived from experiments in freshly isolated tissue (when possible). Nonetheless, using sophisticated techniques (including redox-sensitive green fluorescent proteins that permit compartment-specific ROS measurement), these investigators continue to find that hypoxia increases ROS [58]. Schumacker recently noted increased ROS production with hypoxia in the cytosol of PASMCs, although he did note a simultaneous reduction in ROS production in the mitochondria (personal communication). The relative importance of the various redox pools to oxygen sensing remains uncertain. Although agreeing that PASMC mitochondria function as O2− sensors, the Schumacker group concluded that increased ROS production triggers HPV. Supporting their conclusions, they observed that a variety of antioxidants (pyrrolidinedithiocarbamate, ebselen, and diethyldithiocarbamate)

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abolished HPV [56]. They measured ROS in cultured PASMCs using DCF. DCF is a suboptimal ROS probe, as discussed in a recent review on controversies in HPV [4]. However, it is unlikely that the use of cultured cells and the use of DCF are the only explanations for the divergent findings among groups because Liu et al. recently reported that hypoxia increases ROS production (measured by DCF fluorescence), and “tended” to increase ROS production (using lucigenin-enhanced chemiluminescence), and variably increased ROS production (measured by electron paramagnetic resonance spectra) [76]. Like the Schumacker group, they found that SOD and catalase reduced HPV in porcine PAs. The latter finding contradicts our previous report that liposomal delivery of SOD plus catalase enhances HPV [77]. Knock et al. concluded that hypoxia increases superoxide generation, although this is inferred from the effects of LY83583, a putative generator of superoxide anion on vascular tone and calcium in small PAs [50]. In that work, ROS were not directly measured and LY83583 did not itself cause relaxation of systemic arterial rings (i.e., did not mimic this aspect of hypoxia). Moreover, 6-anilino-5,8-quinolinequinone (LY83583) is not a specific generator of ROS; it is also an inhibitor of guanylate cyclase [78, 79].

8.3 Points of Agreement and Likely Causes for Disagreements For a review of the controversies of ROS signaling in HPV, the interested reader should consult other sources [2, 3]. Both camps have concluded that ROS produced by the proximal ETC signals the hemodynamic response to hypoxia and ETC inhibitors. Moreover, the isolated lung data from the Schumacker group agree with our observations (i.e., rotenone increases normoxic pressure and inhibits subsequent HPV, whereas cyanide does not) [56, 57]. The Schumacker group found that the proximal ETC inhibitors rotenone (50 ng/mL) and myxothiazole (50 ng/mL) inhibit HPV, whereas antimycin A (1 ng/mL) does not. However they acknowledge that antimycin A at 10  ng/mL does cause pulmonary vasoconstriction and inhibits HPV [56], consistent with our findings [42]. They discount the effects of antimycin A because it also inhibits constriction to U46619 [56]. Interestingly, myxothiazole does not cause pulmonary vasoconstriction, which they interpret as indicating that it targets the relevant ROS generator mediating HPV. Thus, their conclusions about the site of ROS generation are based on the assumption that the relevant “sensor” complex(es) when blocked will selectively inhibit HPV but will not mimic hypoxia by increasing PAP. In contrast, we assume that blocking the sensor should elicit both vasoconstriction and inhibition of subsequent HPV. Since

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these metabolic inhibitors eventually lower ATP levels, perhaps over the 30 min for which the Schumacker group observed contraction in their cultured cell model, the confounding effects of ATP depletion may have occurred. The resulting KATP channel activation could promote relaxation and might explain the observed reduction in U46619 contraction. In our studies, where ROS and PVR were measured simultaneously, the changes in PVR and ROS occurred within seconds/minutes of administration of hypoxia or ETC inhibitors and ROS levels fell before constriction commenced (as expected if the ROS are indeed a signal). It is likely that the discord in the literature, generated by high-quality investigative teams, relates to divergence in the techniques employed to measure ROS, differences in the timing of the measurement, variations in incubation conditions of the ROS assay (pH, PO2, and PCO2), and differences in the precise tissue preparations studied. We advocate using multiple independent measurement techniques (luminol, lucigenin, L-012, Amplex Red) to measure ROS in freshly isolated, resistance PA rings [29] and isolated perfused lungs [4]. In conclusion, the data indicating HPV is sensed by mitochondria, the sensor is in the proximal ETC, the mediator is a ROS, and the mechanism resides in the PASMC are more concordant than discordant. The redox model (Fig. 2) offers a comprehensive explanation for HPV and is relevant to studies of ROS in other organs. Although there are undoubtedly deficiencies in the detail of the model and areas of controversy remain, this redox hypothesis is testable and we encourage its widespread evaluation with a greater focus on the physiologic attributes of HPV, as observed in vivo.

References 1. Archer S, Will J, Weir E (1986) Redox status in the control of pulmonary vascular tone. Herz 11:127–141 2. Weir EK, Archer SL (2006) Counterpoint: hypoxic pulmonary vasoconstriction is not mediated by increased production of reactive oxygen species. J Appl Physiol 101:995–998 3. Ward JPT (2006) Point: Hypoxic pulmonary vasoconstriction is mediated by increased production of reactive oxygen species. J Appl Physiol 101:993–995 4. Moudgil R, Michelakis ED, Archer SL (2005) Hypoxic pulmonary vasoconstriction. J Appl Physiol 98:390–403 5. Archer SL, Michelakis ED, Thébaud B et al (2006) A central role for oxygen-sensitive K+ channels and mitochondria in the specialized oxygen-sensing system. Novartis Found Symp 272:157–171 6. Youngson C, Nurse C, Yeger H, Cutz E (1993) Oxygen sensing in airway chemoreceptors. Nature 365:153–155 7. Hampl V, Bibova J, Stranak Z et al (2002) Hypoxic fetoplacental vasoconstriction in humans is mediated by potassium channel inhibition. Am J Physiol Heart Circ Physiol 283:H2440–H2449 8. Rychkov GY, Adams MB, McMillen IC, Roberts ML (1998) Oxygen-sensing mechanisms are present in the chromaffin cells of the sheep adrenal medulla before birth. J Physiol 509:887–893

19  Mitochondrial Reactive Oxygen Species and Redox State in Pulmonary Vascular O2 Sensing 9. Weir EK, López-Barneo J, Buckler KJ, Archer SL (2005) Acute oxygen-sensing mechanisms. N Engl J Med 353:2042–2055 10. López-Barneo J, López-López J, Ureña J, González C (1988) Chemotransduction in the carotid body: K+ current modulated by PO2 in type I chemoreceptor cells. Science 241:580–582 11. Michelakis ED, Rebeyka I, Wu X et  al (2002) O2 sensing in the human ductus arteriosus: regulation of voltage-gated K+ channels in smooth muscle cells by a mitochondrial redox sensor. Circ Res 91:478–486 12. Tristani-Firouzi M, Reeve HL, Tolarova S, Weir EK, Archer SL (1996) Oxygen-induced constriction of rabbit ductus arteriosus occurs via inhibition of a 4-aminopyridine-, voltage-sensitive potassium channel. J Clin Invest 98:1959–1965 13. Madden JA, Dawson CA, Harder DR (1985) Hypoxia-induced activation in small isolated pulmonary arteries from the cat. J Appl Physiol 59:113–118 14. Shirai M, Ninomiya I, Sada K (1991) Constrictor response of small pulmonary arteries to acute pulmonary hypertension during left atrial pressure elevation. Jpn J Physiol 41:129–142 15. Archer SL, Huang JM, Reeve HL et al (1996) Differential distribution of electrophysiologically distinct myocytes in conduit and resistance arteries determines their response to nitric oxide and hypoxia. Circ Res 78:431–442 16. Hall SM, Hislop AA, Pierce CM, Haworth SG (2000) Prenatal origins of human intrapulmonary arteries: formation and smooth muscle maturation. Am J Respir Cell Mol Biol 23:194–203 17. Hall SM, Hislop AA, Haworth SG (2002) Origin, differentiation, and maturation of human pulmonary veins. Am J Respir Cell Mol Biol 26:333–340 18. Bradford J, Dean H (1894) The pulmonary circulation. J Physiol 16:34–96 19. von Euler U, Liljestrand G (1946) Observations on the pulmonary arterial blood pressure in the cat. Acta Physiol Scand 12: 301–320 20. Weir EK, Archer SL (1995) The mechanism of acute hypoxic pulmonary vasoconstriction: the tale of two channels. FASEB J 9:183–189 21. Archer S, Michelakis E (2002) The mechanism(s) of hypoxic pulmonary vasoconstriction: potassium channels, redox O2 sensors, and controversies. News Physiol Sci 17:131–137 22. Groves BM, Droma T, Sutton JR et al (1993) Minimal hypoxic pulmonary hypertension in normal Tibetans at 3,658 m. J Appl Physiol 74:312–318 23. McMurtry IF, Petrun MD, Reeves JT (1978) Lungs from chronically hypoxic rats have decreased pressor response to acute hypoxia. Am J Physiol 235:H104–H109 24. Grocott MP, Martin DS, Levett DZ, McMorrow R, Windsor J, Montgomery HE (2009) Arterial blood gases and oxygen content in climbers on Mount Everest. N Engl J Med 360:140–149 25. Jensen KS, Micco AJ, Czartolomna J, Latham L, Voelkel NF (1992) Rapid onset of hypoxic vasoconstriction in isolated lungs. J Appl Physiol 72:2018–2023 26. Carlsson AJ, Bindslev L, Santesson J, Gottlieb I, Hedenstierna G (1985) Hypoxic pulmonary vasoconstriction in the human lung: the effect of prolonged unilateral hypoxic challenge during anaesthesia. Acta Anaesthesiol Scand 29:346–351 27. Bindslev L, Jolin A, Hedenstierna G, Baehrendtz S, Santesson J (1985) Hypoxic pulmonary vasoconstriction in the human lung: effect of repeated hypoxic challenges during anesthesia. Anesthesiology 62:621–625 28. Dorrington KL, Clar C, Young JD, Jonas M, Tansley JG, Robbins PA (1997) Time course of the human pulmonary vascular response to 8 hours of isocapnic hypoxia. Am J Physiol 273:H1126–H1134 29. Michelakis E, Hampl V, Nsair A et al (2002) Diversity in mitochondrial function explains differences in vascular oxygen sensing. Circ Res 90:1307–1315

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322 52. Reeve HL, Michelakis E, Nelson DP, Weir EK, Archer SL (2001) Alterations in a redox oxygen sensing mechanism in chronic hypoxia. J Appl Physiol 90:2249–2256 53. Duchen MR, Biscoe TJ (1992) Relative mitochondrial membrane potential and [Ca2+]i in type I cells isolated from the rabbit carotid body. J Physiol 450:33–61 54. Buescher P, Perse D, Pillai R, Litt M, Mitchell M, Sylvester JT (1991) Energy state and vasomotor tone in hypoxic pig lungs. J Appl Physiol 70:1874–1881 55. Archer SL, Reeve HL, Michelakis E et al (1999) O2 sensing is preserved in mice lacking the gp91 phox subunit of NADPH oxidase. Proc Natl Acad Sci USA 96:7944–7949 56. Waypa GB, Chandel NS, Schumacker PT (2001) Model for hypoxic pulmonary vasoconstriction involving mitochondrial oxygen ­sensing. Circ Res 88:1259–1266 57. Waypa GB, Marks JD, Mack MM, Boriboun C, Mungai PT, Schumacker PT (2002) Mitochondrial reactive oxygen species trigger calcium increases during hypoxia in pulmonary arterial myocytes. Circ Res 91:719–726 58. Waypa GB, Guzy R, Mungai PT et al (2006) Increases in mitochondrial reactive oxygen species trigger hypoxia-induced calcium responses in pulmonary artery smooth muscle cells. Circ Res 99:970–978 59. Reeve HL, Weir EK, Nelson DP, Peterson DA, Archer SL (1995) Opposing effects of oxidants and antioxidants on K+ channel activity and tone in rat vascular tissue. Exp Physiol 80:825–834 60. Dooley CT, Dore TM, Hanson GT, Jackson WC, Remington SJ, Tsien RY (2004) Imaging dynamic redox changes in mammalian cells with green fluorescent protein indicators. J Biol Chem 279: 22284–22293 61. Hanson GT, Aggeler R, Oglesbee D et al (2004) Investigating mitochondrial redox potential with redox-sensitive green fluorescent protein indicators. J Biol Chem 279:13044–13053 62. Duchen MR (1999) Contributions of mitochondria to animal physiology: from homeostatic sensor to calcium signalling and cell death. J Physiol 516:1–17 63. Gupte SA, Okada T, McMurtry IF, Oka M (2006) Role of pentose phosphate pathway-derived NADPH in hypoxic pulmonary vasoconstriction. Pulm Pharmacol Ther 19:303–309 64. Mohazzab-H K, Wolin M (1994) Properties of a superoxide aniongenerating microsomal NADH oxidoreductase, a potential pulmonary artery PO2 sensor. Am J Physiol 267:L823–L831 65. Roy A, Rozanov C, Mokashi A et al (2000) Mice lacking in gp91 phox subunit of NAD(P)H oxidase showed glomus cell [Ca2+]i and respiratory responses to hypoxia. Brain Res 872:188–193 66. Gupte SA, Kaminski PM, Floyd B et al (2004) Cytosolic NADPH may regulate differences in basal Nox oxidase-derived superoxide generation in bovine coronary and pulmonary arteries. Am J Physiol Heart Circ Physiol 288:H13–H21

S.L. Archer and J.J. Ryan 67. Yuan X-J (1995) Voltage gated K+ currents regulate resting membrane potential and [Ca2+]i in pulmonary artery myocytes. Circ Res 77:370–378 68. Smirnov SV, Robertson TP, Ward JPT, Aaronson PI (1994) Chronic hypoxia is associated with reduced delayed rectifier K+ current in rat pulmonary artery muscle cells. Am J Physiol 266:H365–H370 69. Pozeg ZI, Michelakis ED, McMurtry MS et al (2003) In vivo gene transfer of the O2-sensitive potassium channel Kv1.5 reduces pulmonary hypertension and restores hypoxic pulmonary vasoconstriction in chronically hypoxic rats. Circulation 107:2037–2044 70. Archer SL, Souil E, Dinh-Xuan AT et al (1998) Molecular identification of the role of voltage-gated K+ channels, Kv1.5 and Kv2.1, in hypoxic pulmonary vasoconstriction and control of resting membrane potential in rat pulmonary artery myocytes. J Clin Invest 101:2319–2330 71. Paky A, Farrukh I, Michael J, Gurtner G (1987) Basal superoxide production in the isolated perfused rabbit lung. Fed Proc 46:995 72. Archer SL, Nelson DP, Weir EK (1989) Simultaneous measurement of O2 radicals and pulmonary vascular reactivity in rat lung. J Appl Physiol 67:1903–1911 73. Paky A, Michael JR, Burke-Wolin T, Wolin MS, Gurtner GH (1993) Endogenous production of superoxide by rabbit lungs: effects of hypoxia or metabolic inhibitors. J Appl Physiol 74:2868–2874 74. Mohazzab KM, Fayngersh RP, Kaminski PM, Wolin MS (1995) Potential role of NADH oxidoreductase-derived reactive O2 species in calf pulmonary arterial PO2-elicited responses. Am J Physiol 269:L637–L644 75. Marshall C, Mamary AJ, Verhoeven AJ, Marshall BE (1996) Pulmonary artery NADPH-oxidase is activated in hypoxic pulmonary vasoconstriction. Am J Respir Cell Mol Biol 15:633–644 76. Liu JQ, Sham JS, Shimoda LA, Kuppusamy P, Sylvester JT (2003) Hypoxic constriction and reactive oxygen species in porcine distal pulmonary arteries. Am J Physiol Lung Cell Mol Physiol 285: L322–L333 77. Archer SL, Peterson D, Nelson DP et al (1989) Oxygen radicals and antioxidant enzymes alter pulmonary vascular reactivity in the rat lung. J Appl Physiol 66:102–111 78. Lodygin D, Menssen A, Hermeking H (2002) Induction of the Cdk inhibitor p21 by LY83583 inhibits tumor cell proliferation in a p53independent manner. J Clin Invest 110:1717–1727 79. Schmidt MJ, Sawyer BD, Truex LL, Marshall WS, Fleisch JH (1985) LY83583: an agent that lowers intracellular levels of cyclic guanosine 3',5'-monophosphate. J Pharmacol Exp Ther 232: 764–769 80. Wilson HL, Dipp M, Thomas JM, Lad C, Galione A, Evans AM (2001) ADP-ribosyl cyclase and cyclic ADP-ribose hydrolase act as a redox sensor. a primary role for cyclic ADP-ribose in hypoxic pulmonary vasoconstriction. J Biol Chem 276:11180–11188

Chapter 20

Cellular and Molecular Mechanisms of Pulmonary Vascular Smooth Muscle Cell Proliferation Tamara Tajsic and Nicholas W. Morrell

Abstract  Pulmonary arterial hypertension is the end result of a complex series of events termed “vascular remodeling,” which includes proliferation of resident smooth muscle cells in the walls of small muscular pulmonary arteries. This process leads to increased thickness of the smooth muscle component of the vessel wall, which then contributes to reduced lumen diameter and increased contractility. Since smooth muscle cell proliferation contributes significantly to the process of vascular remodeling, it is important to appreciate the main factors driving this process in the hypertensive lung. This chapter will review the basic mechanisms of vascular smooth muscle cell proliferation, focusing on work in pulmonary vascular cells. Keywords  Cell proliferation and growth • DNA synthesis • Growth factor • Signal transduction • Pulmonary vascular remodeling

1 Introduction Pulmonary arterial hypertension (PAH) is the end result of a complex series of events termed “vascular remodeling,” which includes proliferation of resident smooth muscle cells (SMCs) in the walls of small muscular pulmonary arteries. This process leads to increased thickness of the smooth muscle component of the vessel wall, which then contributes to reduced lumen diameter and increased contractility. Since SMC proliferation contributes significantly to the process of vascular remodeling, it is important to appreciate the main factors driving this process in the hypertensive lung. This chapter will review the basic mechanisms of vascular SMC (VSMC) proliferation, focusing on work in pulmonary vascular cells.

T. Tajsic (*) Department of Medicine, Addenbrooke’s Hospital, Hills Road, Box 157, Cambridge, CB4 1LL, UK e-mail: [email protected]

2 The Cell Cycle Cell proliferation involves an ordered sequence of events in which a cell duplicates its content and then divides into two daughter cells each containing chromosomes identical to those of the parental cell. This cycle of duplication and division is known as the cell cycle. Control of the cell cycle is critical for normal development of multicellular organisms and regulation of cell number. In eukaryotes, the cell cycle is composed of four discrete, temporally regulated phases: G1, S, G2, and M phase. The M phase consists of the division of the nucleus, called mitosis, and the division of the cytoplasm, referred to as cytokinesis. The period between two consecutive M phases is referred to as interphase and includes the other three phases: the S phase, during which the cell replicates its DNA content, and two gap periods, G1, between the end of the M phase and beginning of the S phase, and G2, between the end of the S phase and the beginning of the next M phase. During these two gap phases the cell grows, monitors intracellular conditions, and integrates signals from the environment (other cells) to assess if there is a need for the cell to proliferate and if so whether the cell is adequately prepared to commit itself to the demands of DNA replication and cell division. The M phase takes up a relatively small portion of the cell-cycle time as it takes approximately 1 h for most human cells to divide, whereas the interphase can last from several hours up to a whole lifetime in different cell types. The cell cycle is a highly complex, ordered system of biochemical switches. The transition through the cell cycle is regulated by a series of heterodimeric protein kinases that are conserved from yeasts to mammals. The regulatory subunits, cyclins, control cell-cycle events, and their concentration changes as cells progress through the cell cycle. Cyclindependent kinases (Cdks), the catalytic subunits of the protein kinases, have no kinase activity unless they are associated with a cyclin. Each Cdk associates with specific cyclins and the associated cyclin determines which proteins are phosphorylated by a particular cyclin–Cdk complex. Different cyclin– Cdk complexes trigger different steps of the cell cycle. The concentration of each cyclin rises gradually and then falls

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sharply at a specific time in the cell cycle as a result of ubiquitination and proteosome degradation. The proliferation of human cells is controlled by a complex network of signaling pathways that integrate extracellular signals about environmental cues, the identity, and density of neighboring cells. If the growth factors are present through G1, the increase in concentration helps activate the appropriate Cdk, whereas the rapid fall in cyclin concentration returns the Cdk to its inactive state. The proliferation of human cells is principally regulated by a variety of extracellular growth factors. If the growth factors are present through G1, representing the need of the organism for the specific cell type/cell to proliferate, this will stimulate G1 phase cyclin synthesis and transition through the G1 restriction point. Principally, G1 cyclins, cyclins D1,D2, and D3, bind to Cdk4 and Cdk6 and drive progression through the G1 phase. Then, the concentration of the G1/S cyclin, cyclin E, increases and forms a complex with, and thereby activates, Cdk2. This leads to activation of the proteins that regulate initiation and completion of DNA replication (Cdc6, etc.) and their subsequent binding to the origin-recognition complexes: specific nucleotide sequences scattered at various locations along each chromosome. The prereplication complex is thus formed and the cell is now ready to replicate its DNA. The rise in S phase cyclin, cyclin A, concentration, and its binding to Cdk2 triggers the replication process. Additionally, retinoblastoma protein, normally serving as a molecular brake, binding to gene regulatory proteins and preventing them from driving transcription of genes necessary for cell proliferation, is phosphorylated by G1/S and S cyclin–Cdk complexes, altering its conformation and releasing the bound proteins which are now free to activate the genes necessary for proliferation. The cyclin A–Cdk2 complex also prevents re-replication, thus preventing the risk of gene amplification. This complex leads to phosphorylation of the prereplication complex regulatory

Fig. 1  The cell cycle

T. Tajsic and N.W. Morrell

p­ roteins, which then dissociate from the origin-recognition complexes and are subsequently degraded, ensuring that the replication is not reinitiated in the same cell cycle. Once DNA has been faithfully and accurately replicated, the cell can engage in mitosis. In this step, cyclin B (the M phase cyclin) levels rise, leading to the formation of a complex with Cdk1, which then phosphorylates proteins involved in chromosome condensation, nuclear envelope breakdown, mitotic spindle formation, and mitosis (division of the nucleus) occurs, commonly followed by cytokinesis (division of the cytoplasm). At the end of mitosis, all Cdk activity is reduced to the baseline level, and the M cyclin is degraded. This results in the cell exiting mitosis and re-entering the G1 phase. In the mature adult most human cells are highly or terminally differentiated, and they withdraw from the cell cycle during G1 phase, entering the G0 phase. For most cells there is no need for further replication and the concentration of growth factors is not high enough to drive them through the restriction point in G1. In these cells the molecular brakes are applied and they enter the resting state of G0. In some tissues cells can be stimulated to re-enter the cell cycle and replicate when a need arises or in pathological conditions. The biological processes of molecular brakes are not entirely understood, but the cell-cycle inhibitors (CKIs) seem to play an important role. They block the assembly or activity of certain cyclin–Cdk complexes, leading to cell-cycle arrest. In mammalian cells, two families of CKIs have been identified. The Ink4 family members (p15, p16, p18, p19) specifically bind to and inhibit Cdk4 and Cdk6, thus inhibiting transition through the G1 checkpoint. The members of the Cip/Kip family of CKIs (p21, p27, p57) can bind to Cdk2, Cdk4, and Cdk6, and their complexes with cyclins A, E, and D, and can arrest the cell cycle at the G1 or G2 checkpoint [1–3] (Fig. 1).

20  Cellular and Molecular Mechanisms of Pulmonary Vascular Smooth Muscle Cell Proliferation

Pulmonary artery SMCs (PASMCs) in normal, adult lung blood vessels proliferate at an extremely low rate, and are thus mostly quiescent. In common with other VSMCs, PAMSCs are not terminally differentiated and are able to modulate their phenotype, profoundly and reversibly, and exit their quiescent state in response to changing local environmental conditions that normally regulate phenotype.

3 The Pulmonary Vasculature It has become apparent that PASMCs are not uniform in phenotype throughout the pulmonary circulation, but are heterogeneous both at a single anatomical site and along the vascular bed. Studies of the bovine main pulmonary artery revealed the presence of phenotypically distinct SMC subpopulations throughout the media. These specific populations where differently oriented, as seen by light microscopy, and they exhibited a differential expression of messenger RNA for procollagen and tropoelastin [4]. When conventional histochemical staining techniques and light microscopy are used, the media of the human pulmonary arteries appears homogeneous, but when a panel of antibodies to contractile and cytoskeletal proteins is used, several phenotypes can be discerned, which arise early during development of the arterial media [5]. The subpopulations from bovine arteries exhibit very stable and specific functional capabilities both in vivo and in vitro. They are distinct with regard to their state of differentiation, expression of smooth muscle markers, proliferative response to growth factors, and to hypoxia [4–6]. When isolated in tissue culture, these subpopulations display markedly different rates of proliferation, and matrix protein expression. Thus, in any segment of the vessel wall there may be distinct SMC subpopulations that may play diverse roles in vascular homeostatis and can respond differently to injury and to changes in the environmental cues [4–11]. Furthermore, heterogeneity in SMCs is also observed in cells isolated from different anatomical locations in the lung, i.e., proximal versus peripheral arteries. Human PASMCs from peripheral pulmonary circulation (arteries 1–2 mm in diameter) proliferate more rapidly than cells isolated from the main pulmonary artery and they exhibit different responses to prostacyclin analogs and bone morphogenetic protein (BMP) 4 [12, 13]. There is a possibility that these distinct subpopulations have different embryonic origins, but no fate-mapping analysis of their origin has been undertaken thus far.

4 Factors Involved in PASMC Proliferation As previously noted, in a normal mature blood vessel, VSMCs are contractile and exhibit extremely low rates of proliferation, although growth factors may be present. Furthermore,

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exposure of intact blood vessels to exogenous growth factors, both in vivo and in vitro, does not lead to rapid proliferation [14]. However, VSMCs are stimulated to proliferate mostly in pathological conditions such as a response to injury, or changes in the environment, suggesting that factors exist in the normal vessel wall, which inhibit stimulation of VSMC proliferation by growth factors. Inhibitory signals that prevent VSMC proliferation are thought to be cell–matrix interaction, soluble mediators (such as growth factors), as well as cell– cell adhesion [14–16]. VSMCs in the tunica media of blood vessels are embedded in extracellular matrix (ECM) comprising elastin, collagen (types I, III, IV, VI, VIII, and XIII), proteoglycans, hyaluronan and adhesive glycoproteins (fibronectin and laminin). ECM components are produced by vascular cells. When normal medial VSMCs are placed in culture, within a few days they lose their contractility and myofilaments and develop an extensive rough endoplasmic reticulum and a large Golgi complex, shifting from the normally contractile and quiescent phenotype into a synthetic and proliferative phenotype [17]. Interestingly, laminin, a constituent of normal media, has been shown to inhibit this shift, whereas fibronectin promotes the shift to the “synthetic” phenotype [18]. Culture of VSMCs on polymerized collagen type I or collagen type IV inhibits VSMC proliferation and promotes the quiescent, contractile phenotype [14, 19]. Mutation of one allele of elastin, previously thought to play a purely structural role, is sufficient to induce subendothelial proliferation of VSMCs [20]. Heparan sulfate, produced by VSMCs, has long been recognized as potent inhibitor of smootrh muscle cell proliferation [21]. In addition to direct effects, a number of growth factors bind to the heparin sulfate rich ECM [22], and thus can serve as a local site of storage. Heparin is also critical for oligomerization of fibroblast growth factor (FGF) and subsequent stimulation of endothelial cells and VSMCs [23]. Another group of glycoproteins, termed matricellular or matrix-associated proteins, are a class of secreted proteins that interact with other ECM components, multiple specific cell-surface proteins, and growth factors to modulate cell– matrix interactions. Four members of the group, osteopontin, SPARC, thrombospondin, and tenascin, have been shown to exert common “antiadhesive” functions involved in cell migration and proliferation [24]. In pulmonary arteries, tenascin C localizes to sites of active vascular remodeling, where it colocalizes with proliferating cells and epidermal growth factor (EGF) [25]. Tenascin C augments the proliferative response to growth factors by a mechanism that involves binding of its cell-surface integrin (avb3), leading to a rearrangement of the cytoskeleton and clustering and priming of growth factor receptors (e.g., EGF receptors) [26]. Matrix metalloproteinases (MMPs) are a family of zinccontaining enzymes thought to be responsible for the turnover of ECM components at physiological pH. Increased MMP activity has been demonstrated after injury of systemic blood

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vessels, and therefore it was proposed that MMPs may modulate VSMC proliferation as well as migration [27]. Three synthetic inhibitors of MMPs [28, 29] as well as the adenoviral overexpression of tissue inhibitors of metalloproteinases [30] reduced MMP-induced SMC proliferation. Mechanisms proposed to explain this phenomenon focus on MMPs affecting growth-factor-mediated cell proliferation by regulating their bioavailability and/or activity through cleavage of either matrix or nonmatrix (growth-factor-bound) proteins with the consequent release of active growth factors such as FGF2 [16, 31].

4.1 Growth Factors 4.1.1 Receptor Tyrosine Kinases Receptor tyrosine kinases (RTKs) play an important role in the control of most fundamental cellular processes, including cell proliferation. They are membrane-spanning cell-surface receptors with intrinsic tyrosine kinase activity and they serve as receptors for various growth factors, hormones, and cytokines. All RTKss activate Ras through the exchange of GTP for GDP by the guanine nucleotide exchange factor Sos. The adaptor protein Grb2 forms a complex with Sos, resulting in recruitment of the complex to an activated RTK, thus translocating Sos to the plasma membrane, enabling it to be close to Ras and perform the guanine nucleotide exchange and activate Ras [32]. Once activated, Ras reacts with several effector proteins such as Raf and phosphatidylinositol 3-kinase. Activated Raf stimulates the mitogen-activated protein kinase (MAPK) kinase cascade, eventually phosphorylating extracellular-regulated kinase (ERK), which then translocates to the nucleus, where it phosphorylates and activates various transcription factors [33, 34]. This highly conserved signaling cascade plays an important role in regulating cell cycle, cell proliferation, and other important cellular processes. Phosphatidylinositol 3-kinase activation leads to stimulation of various intracellular responses, a particularly important one being cell survival by phosphorylating the proapoptotic protein BAD, which then blocks its complex with Bcl-2 and Bcl-xl and prevents apoptotic cell death [35]. Several RTK ligands such as platelet-derived growth factor (PDGF), FGF2, insulin-like growth factor (IGF), and EGF had been shown to stimulate PASMC proliferation. The PDGF family consists of PDGF-A, PDGF-B, PDGF-C, and PDGF-D, which are inactive as monomers, but form active homo- and heterodimers (PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, PDGF-DD). Most often studied is the PDGF-BB homodimer, which potently stimulates PASMC proliferation [36, 37]. This effect is inhibited by PDGF RTK inhibitors, including imatinib [37]. PDGF is a more potent PASMC mitogen than FGF2, IGF, and EGF.

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4.1.2 Transforming Growth Factor b Superfamily Transforming growth factor b (TGFb) ligands are a large group of cytokines that regulate many cellular functions, including cell growth, adhesion, migration, cell-fate determination, differentiation, and apoptosis. They are also among the key cytokines that control vascular function [38]. Malfunctions in TGFb signaling cause serious human diseases such as cancer, fibrosis, wound-healing disorders [39], and several hereditary conditions such as familial PAH [40], hereditary hemorrhagic telangiectasia [41, 42], and fibrodysplasia ossificans progressiva [43]. The TGFb superfamily of ligands comprises some 30 genes in mammals, including three TGFb isoforms (TGFb1, TGFb2, and TGFb3), and more than 20 BMPs [43]. TGFb superfamily receptors are transmembrane serine/threonine kinases, functionally divided into two classes: type II and type I receptors. There are five type II (TGFbRII, ActRII, ActRIIB, BMPRII, AMHRII) and seven type I (activinreceptor-like kinases, ALK1–ALK7) receptors. Access of ligands to the signaling type I and type II receptors is regulated by soluble ligand binding proteins and by accessory type III receptors. Examples of the latter class that have been most intensively studied are endoglin and b-glycan [44, 45]. These two accessory receptor are highly structurally related transmembrane proteins that facilitate TGFb ligand binding to TGFbRII [46–49]. Endoglin can exert this function only when bound to TGFbRII. However, it is able to bind directly to BMPs [50–52]. Binding of the ligand dimer causes bridging of the preformed dimers of type II receptors and dimers of type I receptors to form heterotetrameric, active receptor complexes. This enables the constitutively active type II receptor to phosphorylate, and thereby activate, the type I receptor in a juxtamembrane region. Once activated, the type I receptor is able to recruit and subsequently phosphorylate receptorregulated Smads (R-Smads) on two serine residues at their extreme C-termini. ALK4, ALK5, and ALK7 recruit, and phosphorylate Smad2 and Smad3, whereas ALK1–ALK3 and ALK6 recruit and phosphorylate Smad1, Smad5, and Smad8. This C-terminal phosphorylation allows the R-Smads to form complexes with the common Smad, Smad4, and, also, to form homomeric complexes [53–56]. These complexes accumulate in the nucleus and regulate gene transcription usually in association with other transcription factors [53–55]. TGFb signaling is negatively regulated by the inhibitory Smads (I-Smads), Smad6 and Smad7. I-Smads are transcriptionally induced in response to TGFb and BMPs in a Smad-dependent manner and are thought to function in signal termination [53–56]. In VSMCs, most TGFb effects appear to be mediated via the TGFbRII/ALK5 complex. Subsequent downstream signaling is complex, not only involving Smad2/Smad3 but also

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Fig. 2  Effects of transforming growth factor b superfamily ligands on pulmonary artery smooth muscle cells (PASMCs)

kinases such as p38 MAPK, p42/p44, and c-Jun N-terminal kinase as well as downstream transcription factors [57, 58]. TGFb1 can attenuate VSMC activation by opposing the effects of mitogenic growth factors, proinflammatory cytokines, and genes that affect vascular remodeling. This antimitogenic effect on VSMCs was found to be ALK5-mediated via signaling pathways dependent on both Smad3 and p38 MAPK [59] (Fig. 2) VSMCs are also influenced by BMPs: BMP2, BMP4, BMP6, and BMP7. BMPs interact with three type 2 receptors, BMPRII, ActRII, and ActRIIB, to activate different type I receptors ALK2, ALK3, and ALK6 [57]. The effect of BMPs depends on the source of VSMCs studied as well as the local environment. The serum-stimulated proliferation of cells harvested from the main or lobar pulmonary arteries tends to be inhibited by BMP2, BMP4, and BMP7. With use of a dominant negative Smad1 construct, the growth inhibitory effects of BMPs have been shown to be Smad1-dependent [12]. Another study demonstrated that BMP2 prevents PDGF-induced VSMC proliferation via induction of the peroxisome-proliferator-activated receptor g/apoE axis [60, 61]. In contrast, in PASMCs isolated from pulmonary arteries of 1–2-mm diameter, BMP2 and BMP4 stimulate proliferation [12, 60, 61]. This pro-proliferative effect of BMPs in peripheral cells is dependent on the activation of ERK1/2 and p38 MAPK. Both Smad and MAPK pathways are activated to a similar extent in cells from both locations, but the integration of these signals by the cell seems to differ [62]. (Fig. 2)

4.2 Calcium and Smooth Muscle Proliferation Contraction is, probably, the most important property of the smooth muscle. It is triggered by a rise in the free cytosolic Ca2+ concentration, because a Ca2+ binding protein,

calmodulin, binds Ca2+ as its cytosolic concentration increases. This complex then activates myosin light chain kinase, which, in turn, phosphorylates the myosin light chain. The phosphorylated myosin light chain stimulates the activity of myosin ATPase, hydrolyzing ATP to release energy for the subsequent cycling of the myosin crossbridges with the actin filament. The formation of these cross-bridges underlies SMC contraction [63]. Cytosolic Ca2+ concentration in PASMCs can be increased by (a) Ca2+ influx through voltage-dependent Ca2+ channels (VDCCs), receptor-operated Ca2+ channels, and store-operated Ca2+ channels, and (b) Ca2+ release from intracellular stores, the most important one being the sarcoplasmic reticulum (SR), via Ca2+-release channels [e.g., inositol 1,4,5- trisphosphate (IP3) receptors and ryanodine receptors]. In contrast, cytosolic Ca2+ concentration in PASMCs can be decreased by (a) Ca2+ expulsion by the Ca2+–Mg2+-ATPase (Ca2+ pump) and by the forward mode of the Na+/Ca2+ exchanger in the plasma membrane, and (b) Ca2+ sequestration by the Ca2+– Mg2+-ATPase in the SR (SERCA) [64–66]. Resting membrane potential in VSMCs normally ranges between −70 and 50 mV [67]. Decreased expression and/or function of K+ channels leads to sustained membrane depolarization and contributes to sustained elevation of cytosolic Ca2+ concentration by (a) activating VDCCs and (b) facilitating the production of IP3, which stimulates the release of SR Ca2+ into the cytoplasm [68]. Free cytosolic Ca2+ concentration is an important determinant not only for VSMC contraction but, also, for its proliferation. Elevated cytosolic Ca2+ concentration leads to a rapid increase in the nuclear concentration of Ca2+, which pushes the cells from quiescence into the cell cycle, where they undergo mitosis, thus promoting their proliferation [69]. Transition through all four phases of the cell cycle appears to be sensitive to Ca2+/calmodulin complex activation. Some of the signaling molecules involved in mitogenic pathways appear to be Ca2+-dependent. MAPK II, an enzyme involved

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in the phosphorylation cascade that transduces the mitogenic signals of various growth factors activated by a rise in cytosolic Ca2+ concentration [70]. It has also been shown that resting cytosolic Ca2+ concentration is significantly elevated in proliferating PASMCs compared with that in growth-arrested cells [71, 72]. Interestingly, sustained elevation in cytosolic Ca2+ concentration due to Ca2+ influx has been shown to upregulate the expression of activating protein-1 (AP-1) family proteins c-Fos and c-Jun [73]. AP-1 is a family of transcription factors composed of Jun (v-Jun, c-Jun, JunB, and JunD), Fos (v-Fos, c-Fos, FosB, Fra1, and Fra2), or activating transcription factor subunits [74]. The target genes for AP-1 are often involved in regulation of cell proliferation, migration, and apoptosis, for example, endothelin (ET)-1, PDGF, and others [75]. Hence, the upregulation of the AP-1 family members c-Fos and c-Jun by the elevated cytosolic Ca2+ concentration could stimulate production of mitogens such as PDGF or ET, which, in turn, stimulate VSMC proliferation [76]. It has also become apparent that the Ca2+ stored in the SR plays an important role in the initiation of DNA synthesis and cell proliferation. Depleting the IP3-sensitive Ca2+ stores with the Ca2+–Mg2+-ATPase inhibitors leads to cell-cycle arrest, whereas Ca2+ rescue via SERCA pumps allows the resumption of the S phase of the cell cycle [77]. Therefore, increased cytosolic and increased SR Ca2+ concentration are both required for PASMC mitosis and ­proliferation. (Fig. 3)

Fig. 3  Increased cytosolic Ca2+ concentration exerts mitogenic effects on PASMCs through direct stimulation of the cell-cycle transition and through increased expression of the activating protein-1 transcription factor family members c-Fos and c-Jun which stimulate production of the powerful mitogens platelet-derived growth factor and endothelin-1

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4.3 Vasoactive Substances Serotonin (5-hydroxytryptamine, 5-HT) is a monoamine neurotransmitter whose platelet and plasma levels are increased in PAH patients [78, 79]. It is produced by the enterochromaffin cells in the gut, serotoninergic neurons in the central nervous system, and pulmonary neuroendocrine cells and neuroepithelial bodies [80]. Apart from vasoconstriction, 5-HT induces hypertrophy and hyperplasia of human PASMCs [81–85] and also acts as a comitogen since in combination with other growth factors, such as PDGF, FGF, and EGF, the proliferative response is greater than with each mitogen alone. Unlike the vasoconstrictive effects of 5-HT that are believed to be receptor-mediated, proliferative effects require 5-HT internalization via the 5-HT transporter (5-HTT) [82, 83]. Interestingly, 5-HTT is abundantly expressed in the lung, where it is predominantly located in the PASMCs [86]. In PASMCs, mitogenic and comitogenic effects of 5-HT are dose-dependently inhibited by selective 5-HTT inhibitors, but not by 5-HT2A or 5-HT1B/1D receptor antagonists [87]. Once internalized, 5-HT can exert its mitogenic and comitogenic effects on PASMCs, but the underlying intracellular mechanisms are still elusive. It is believed to work through phosphorylation of GTPase-activating protein and through stimulation of MAPK kinase and ERK1/2, which could account for its mitogenic function [88–90].

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Endothelin (ET) is a potent vasoconstrictor produced by endothelial cells under pathological conditions. There are three ET isoforms, ET-1, ET-2, and ET-3, ET-1 being the predominant one. ET-1 is initially produced as a 38 amino acid peptide and then processed into a 21 amino acid peptide by the action of ET-converting enzyme. ET can be secreted by PASMCs, airway epithelium, macrophages, and pulmonary artery endothelial cells [91, 92]. ET-1 exerts its cellular functions through two receptor subtypes: ETA and ETB [93]. ET-1 is a potent mitogen for PASMCs. Inhibition of endogenous ET-1 release or its action attenuated sum-stimulated proliferation of PASMCs [93, 94]. Angiotensin II is part of the renin–angiotensin system and is another vasoactive factor that was shown to exert mitogenic effects on PASMCs [95]. Angiotensin II is an active form processed from its precursor angiotensin I by the action of the angiotensin-converting enzyme which is found predominantly in the capillaries of the lung. Thromboxane A2 (TXA2), a metabolite of arachidonic acid, is also a vasoconstrictor and a PASMC mitogen [76, 95]. It is produced in platelets, lungs, and several other tissues. Human TXA2 receptor (TP), a typical G-protein-coupled receptor (GPCR) with seven transmembrane domains, has two splice variants, TPa, and TPb, that vary in their C-terminal regions. TPs communicate mainly with G12/13 and Gq, resulting in phospholipase C activation and RhoGEF activation. Mitogenic effects of TXA2 are believed to be ERK1/2-mediated [96, 97]. Nitric oxide, prostacyclin, and vasoactive intestinal peptide. Nitric oxide (NO) is generated from l-arginine and the reaction is catalyzed by NO synthase (NOS), which has three distinct isoforms [98] NOS-III (i.e., endothelial NOS), which is a Ca2+-dependent constitutive isoform, is expressed basally in vascular endothelial cells, whereas NOS-I (i.e., neuronal NOS) is expressed in neuronal cells. NO generated by NOS-I and NOS-III is responsible for maintaining normal vascular tone and neuronal signal transduction. NOS-II (i.e., inducible NOS), which is expressed in a variety of cells within the body, is a Ca2+-independent isoform, and is induced in response to certain cytokines and endotoxins. NO induces cyclic GMP (cGMP)-mediated vasodilatation and inhibition of PASMC proliferation. Prostacyclin is an important endogenous vasoactive factor that acts through adenylate cyclase and cyclic AMP to induce vascular dilatation and antimitogenic effects on PASMCs. Vasoactive intestinal peptide is a neuropeptide that functions primarily as a neurotransmitter and is, also, a potent vasodilator and PASMC-proliferation inhibitor. It acts through two receptor subtypes (VPAC-1 and VPAC-2) which are coupled to adenylate cyclase and cGMP systems [76, 95, 99].

4.4 Hypoxia Chronic hypoxia leads to the development of PAH, characterized by medial thickening [100, 101]. Hypoxia has

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d­ istinct effects on PASMCs in different parts of the pulmonary vascular bed, as the cellular composition of the arteries changes along the longitudinal axis of the pulmonary circulation. Data from small animals (rats and mice) suggested that this medial thickening is largely attributable to PASMC hypertrophy and ECM deposition in proximal pulmonary arteries [102–104]. However, data available from proximal arteries of large mammals, including humans, revealed that hypoxia stimulates proliferation of some of the distinct PASMC subpopulations within the media [105, 106]. As described previously, the tunica media of pulmonary and systemic arteries comprises several distinct SMC populations that exhibit different ion-channel and receptor expression and could be derived from different lineages [4–11, 105, 106]. The PASMC subpopulation that exhibits the proliferative response to chronic hypoxia is characterized by a less differentiated or an “undifferentiated” phenotype, as assessed by expression of smooth muscle markers compared with other PASMCs in the vessel media. These data were consistent with those of in  vitro studies showing that less differentiated SMCs exhibit a proliferative response to chronic hypoxia, whereas the SMCs with a more differentiated phenotype show a growth-inhibitory response to hypoxia [6, 7, 9]. The mechanisms underlying this proliferative response of the specific proximal PASMC subpopulation have not been elucidated. It has been demonstrated that the proliferation-prone PASMCs show augmented responses to GPCR agonists [6, 7, 9] and to the stimulation of the protein kinase C pathway compared with other PASMC subpopulations [107]. Hypoxia-induced GPCR activation, leading to ERK1/2 activation, has been implicated in the hypoxia-induced proliferation [108]. PASMCs isolated from more peripheral (distal) arteries (1–2  mm in diameter) show a proliferative response to hypoxia. When exposed to chronic hypoxia, distal arteries show profound medial thickening caused by PASMC hyperplasia [100, 104]. However, some researchers have questioned whether the cells that are proliferating in the media of distal arteries in response to hypoxia are actually SMCs or myofibroblasts [103]. The mechanisms underlying hypoxia-induced proliferation of PASMCs could be either direct and/or through hypoxia-induced release of vasoactive factors by endothelial cells and/or platelets. Hypoxia causes intrinsic changes in potassium and calcium homeostasis of PASMCs leading to decreases in KV channel currents, membrane depolarization, elevated intracellular Ca2+ concentration causing vasoconstriction, and PASMC proliferation [72, 109–111]. It seems that Ca2+ influx channels rather than VDCCs mediate these effects [103, 112]. In addition, hypoxia reduces KV channel activity and reduces messenger RNA and protein expression of several pore-forming a-subunits of the K+ channels in rat PASMCs (KV1.1, KV1.5, KV1.6, KV2.1, and KV4.3) [72, 109–111, 113].

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Hypoxia has been shown to reduce the production of prostacyclin and NO [114–118] and to increase the production of ET, 5-HT, and PDGF by endothelial cells [119, 120] and this imbalance may promote mitogenesis of PASMCs. A number of studies have addressed whether hypoxic exposure of cultured PASMCs leads to cell proliferation. Some studies concluded that hypoxia inhibits PASMC proliferation, or is not a direct stimulus [108, 120, 121]. Other studies concluded that hypoxia exerts mitogenic actions on PASMCs [6, 122–129]. The different findings of these studies may arise from (a) a different origin of the cells used for experiments (e.g., proximal versus peripheral arteries), (b) different subpopulations of SMCs isolated, (c) severity of hypoxia – more moderate hypoxia (1–5%) more often had a mitogenic effect than severer hypoxia or anoxia, and (d) the concentration of serum in the culture media – serum concentrations of more than 2% seem to favor the proliferative response to hypoxia [104]. It has also been shown that moderate (5% O2) hypoxia enhances PDGF-AB-, FGF2-, and EGF-driven PASMC proliferation and that this effect is hypoxia-inducible factor (HIF)-1a-dependent and HIF-1bdependent [130].

4.5 Mechanical Stress and PASMC Proliferation VSMCs in the blood vessel media are exposed to circumferential, radial, and axial stress resulting from pulsatile blood flow. This stress is counterbalanced by the vessel wall tone induced by VSMC contraction, elastin, and collagen [131]. Exposure of the vessel wall to stretch leads to deformation of the ECM of the tunica media, which indirectly stimulates cell membrane receptors of the VSMCs embedded within. VSMC membrane cytoskeleton and contractile apparatus are functionally associated; hence, they are all prone to distortion by stretch [131]. Most of the data available on effects of mechanical stretch on VSMCs come from studies on systemic VSMC. Several studies using various levels of stretch (from 5 to 24%) found that it inhibited VSMC proliferation as assessed by thymidine incorporation [132], 5-bromodeoxyuridine incorporation [133], or an increase in p21 expression leading to G1 cell cycle arrest [134]. In contrast to these studies, most other studies have reported an induction of VSMC proliferation by various levels of stretch [135, 136]. The stretch-induced proliferation was initially attributed to the autocrine action of PDGF [136], or stretch-induced phosphorylation of the PDGF receptor a [137]. One of the studies demonstrated the induction of ERK phosphorylation by 20% stretch, leading to an increase in DNA synthesis [138]. More recent studies have clarified the role of focal adhesions and the focal adhesion kinase (FAK) in stretch-induced VSMC

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proliferation in  vitro. Focal adhesions are regions of tight, intimate contact of the cells with the underlying ECM that provide a structural link between the actin cytoskeleton and the ECM which enables the cells to sense the mechanical changes in ECM and transduce signals related to cell growth and proliferation [139]. Focal adhesion assembly is regulated by GTP-binding protein Rho. FAK is a cytoplasmic soluble tyrosine kinase that acts as a biomechanical sensor and a signaling switch stimulated by stretch in  vitro [140–142]. When VSMCs are subjected to sustained stretch, activation of the Rho-GTPases stimulates cell contractility, which, in turn, leads to the bundling of actin filaments and the aggregation of the ECM receptors, integrins. Clustering of the integrins recruits FAK to focal adhesions, where they bind to talin [139, 143]. FAK is then autophosphorylated at Tyr397, activating an attachment site for c-Src [144, 145]. The FAK/cSrc complex then stimulates MAPKs, such as eERK1/2 and p38 MAPK [146]. ERK1/2 drives proliferation of VSMCs exposed to stretch, whereas p38 MAPK increases apoptosis [147]. In the presence of stretch, ERK1/2 activation in VSMC requires an intact actin cytoskeleton as well as a functioning Rho/Rho kinase pathway. Disruption of the actin cytoskeleton using cytochalasin-D, as well as the use of RhoA and Rho kinase inhibitors, attenuates ERK1/2 activity despite VSMC stimulation using stretch [148]. Several studies showed an increase in DNA synthesis in PASMCs of arteries exposed to static force [149]. One of these studies suggested that mechanical stretch leads to production of VEGF and FGF by PASMCs, with FGF acting as a mitogen [150].

4.6 Inflammation and PASMC Proliferation An inflammatory milieu (i.e., the release of circulating cytokines, chemokines, and growth factors by inflammatory cells) can contribute to PASMC proliferation. Inflammation is thought to contribute to the pathobiological processes in PAH. Fractalkine is a chemokine produced by circulating CD4+ and CD8+ lymphocytes that promotes the chemokine (C-X3-C motif) receptor 1 (CX3CR1)-expressing leukocyte recruitment [151]. Both human and rat PASMCs were shown to express CX3CR1 (the receptor for fractalkine). Fractalkine induced proliferation in cultured rat PASMCs, suggesting that it may act as a growth factor for PASMCs. The level of fractalkine was also shown to be increased in the plasma of PAH patients [152]. Another cytokine, synthesized by vascular cells, is CC ligand 2, formerly called monocyte chemoattractant protein 1. This cytokine is a potent mediator of monocyte/macrophage activation and migration but has also been shown to stimulate PASMC proliferation [153].

20  Cellular and Molecular Mechanisms of Pulmonary Vascular Smooth Muscle Cell Proliferation

Fig. 4  Balance of vasoactive factors. Increase in availability/concentration of the pro-proliferative factors such as 5-hydroxytryptamine, endothelin-1, angiotensin II, and thromboxane A2 with decreased levels of antiproliferative factors such as nitric oxide, prostacyclin, and vasoactive intestinal peptide could contribute to proliferation of PASMCs

5 Conclusions This chapter has reviewed the complex processes known to contribute to SMC proliferation (Fig. 4), one of the key factors in the pathobiological processes in PAH. Although the contributions of numerous pathways have been discussed, there are major connections between pathways and common mechanisms that are crucial to the process of proliferation. In addition, ongoing research needs to determine the hierarchy of these processes. Such knowledge will assist in the development of novel therapies to treat PAH.

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Chapter 21

Role of Ca2+ in Vascular Smooth Muscle Gene Expression and Proliferation Karen M. Lounsbury and Patricia C. Rose

Abstract  The importance of Ca2+ in pulmonary smooth muscle physiological function, including events such as contraction, gene transcription, and cell proliferation, is well established. Yet, there is much to learn regarding the complex interplay between Ca2+ signaling, gene expression, and the development of lung diseases such as pulmonary arterial hypertension. Under normal physiological conditions, vascular smooth muscle cells in adult blood vessels show low rates of proliferation and synthetic activity. Mature smooth muscle cells, unlike cardiac muscle cells, are not terminally differentiated, and thus have the unique ability to dedifferentiate and alter their phenotype in response to changes in the extracellular environment. Phenotypical switching is an important survival mechanism in response to injury, and is necessary during vascular development. However, abnormal signal transduction results in the development of a proliferative synthetic phenotype rather than one that is quiescent and contractile, leading to pathologic vascular remodeling. Ca2+ is known to be a key transcriptional regulator of both the onset and the maintenance of many genes associated with vascular remodeling. This chapter focuses on events that underlie changes in gene expression and proliferation that are mediated by Ca2+ signaling in pulmonary smooth muscle. In addition, the role of Ca2+ in the development of lung disease as it relates to cell signaling is discussed in the chapter. Keywords  Calcium signaling • Smooth muscle contraction • Cell proliferation • Calcium-sensitive proteins • Pulmonary vasoconstriction • Vascular remodeling

1 Introduction The importance of Ca2+ in pulmonary smooth muscle ­physiological function, including events such as contraction, gene transcription, and cell proliferation, is well established. K.M. Lounsbury (*) Department of Pharmacology, University of Vermont, Given Building, 49 Beaumont Avenue, Burlington, VT 05452, USA e-mail: [email protected]

Yet, there is much to learn regarding the complex interplay between Ca2+ signaling, gene expression, and the development of lung diseases such as pulmonary arterial hypertension (PAH). Under normal physiological conditions, vascular smooth muscle cells (VSMCs) in adult blood vessels show low rates of proliferation and synthetic activity [1]. Mature smooth muscle cells, unlike cardiac muscle cells, are not terminally differentiated, and thus have the unique ability to dedifferentiate and alter their phenotype in response to changes in the extracellular environment [1, 2]. Phenotypical switching is an important survival mechanism in response to injury, and is necessary during vascular development. However, abnormal signal transduction results in the development of a proliferative synthetic phenotype rather than one that is quiescent and contractile, leading to pathologic vascular remodeling [3]. Ca2+ is known to be a key transcriptional regulator of both the onset and the maintenance of many genes associated with vascular remodeling. This chapter will focus on events that underlie changes in gene expression and proliferation that are mediated by Ca2+ signaling in pulmonary smooth muscle. In addition, the role of Ca2+ in the development of lung disease as it relates to cell signaling will be discussed.

2 Sources of Ca2+ in Pulmonary Artery Smooth Muscle Cells The classic pathway for modulation of Ca2+ in arterial smooth muscle relies on membrane depolarization to stimulate the opening of L-type voltage-dependent Ca2+ channels on the plasma membrane. This influx promotes activation of multiple Ca2+-regulated enzymes, including calmodulin (CaM)dependent protein kinases and phosphatases, Raf kinase, phosphoinositol 3-kinase, and phospholipase C (PLC). The Ca2+ signal is amplified by PLC-mediated production of inositol 1,4,5-trisphosphate (IP3) that activates Ca2+ release from the sarcoplasmic reticulum (SR). If the Ca2+ stores are substantially depleted, store-operated Ca2+ channels on the plasma membrane are opened to restore SR Ca2+ levels and

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Fig.  1  Changes in Ca2+ signal regulation during vascular smooth muscle cell (VSMC) phenotype switching. Maintenance of the differentiated phenotype is promoted by L-type voltage-dependent Ca2+ channel (VDCC) function and large-conductance Ca2+-activated K+ channel (BKCa) activation by Ca2+ sparks released from ryanodine receptors (RyRs). The proliferative phenotype is associated with downregulation of VDCC, BKCa, RyR, and SERCA2a. The proliferative phenotype is associated with an upregulation of transient receptor

potential channels (TRPCs)/Orai channels, inositol 1,4,5-trisphosphate receptors, and SERCA2b, which leads to store-operated Ca2+ entry as the primary Ca2+ signal. GFR growth factor receptors, Gq-R Gq-coupled receptor, PLC phospholipase C, SOS son-of-sevenless, Raf Ras-activated kinase, MEK mitogen-activated protein kinase/ extracellular-signal-regulated kinase kinase, ERK extracellular-signalregulated kinase, CaMK Ca2+/CaM-dependent protein kinase, ROK Rho kinase, CaN calcineurin

further amplify the Ca2+ signal. The Ca2+ signal is attenuated by hyperpolarizing K+ channel activity, Ca2+-binding proteins (buffering), and Ca2+ pumps on the SR and plasma membranes. The amplitude and duration of the Ca2+ signal is important for both muscle contraction and gene transcription. Dysfunctional regulation of any of these steps could disrupt Ca2+ homeostasis, thus leading to vasoconstriction and vascular remodeling. The following section details the individual Ca2+ regulatory components and their role in pulmonary artery smooth muscle cell (PASMC) gene expression and proliferation, with an overall summary depicted in Fig. 1.

(SMMHC) [5]. This loss is accompanied by an increase in expression of T-type Ca2+ channels that can be activated at the more negative membrane potentials observed in dedifferentiated VSMCs. Although not directly studied in PASMCs, the alteration in expression levels of voltage-gated Ca2+ channels could contribute to a change in the Ca2+ homeostasis that promotes cell growth signaling.

2.1 Voltage-Dependent Ca2+ Channels PASMCs, like most smooth muscle cells, express predominantly L-type voltage-dependent Ca2+ channels, which are activated by high voltage and characterized by large singlechannel conductance and slow voltage-dependent inactivation [4]. Influx of Ca2+ through these channels is the primary mechanism of excitation–contraction and excitation–transcription coupling through Ca2+/CaM signaling, resulting in cell contraction and maintenance of the differentiated phenotype. When the signal is prolonged owing to loss of Ca2+ buffering/extrusion or when combined with effectors that prolong the signal, Ca2+ influx through L-type Ca2+ channels can contribute to activation of growth factor signaling that leads to cell proliferation and vascular remodeling. Loss of functional L-type Ca2+ channels in arterial smooth muscle has been associated with cell dedifferentiation and loss of expression of differentiation markers such as smooth muscle a-actin and smooth muscle myosin heavy chain

2.2 IP3 and Ryanodine Receptors VSMCs, like all eukaryotic cells, use agonist signaling through G-protein-coupled receptors to transduce intracellular signals. Signaling through Gq-coupled receptors activates PLC, which hydrolyzes phosphatidylinositol 4,5-bisphosphate to form the second messengers IP3 and diacylglycerol. IP3 selectively activates IP3 receptor ion channels on the SR membrane, initiating a wave of Ca2+ release from the SR into the cytoplasm. These cytoplasmic Ca2+ signals have been termed “Ca2+ waves” and have been shown to be necessary for many cell functions, including cell migration and proliferation. All muscle cells and many other cell types also express ryanodine receptors (RyRs) on the SR membrane that are activated by a rise in cytoplasmic Ca2+ level. Distinct release events from RyRs have been termed “Ca2+ sparks,” and their signaling function is cell-type specific. In cardiac myocytes, Ca2+ sparks are stimulated by L-type Ca2+ channel activation and serve to augment the Ca2+ influx signal in excitation– contraction coupling and promote cell contraction [6]. In most types of smooth muscle cells (freshly isolated or intact), Ca2+ sparks oppose contraction by directly coupling to Ca2+-activated

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K+ channels [7]. In isolated PASMCs, multiple subtypes of RyRs are expressed, but, unlike in other smooth muscle cells, RyR activity is activated by IP3-receptor-mediated Ca2+ release and coupled with membrane depolarization rather than hyperpolarization [8, 9]. This coupling is similar to what is observed in nonmuscle cells, but the significance with regard to pulmonary artery function is not known. It is not completely clear whether the SR pools of Ca2+ used by IP3 receptors and RyRs are distinct or conjoined. In a study of PASMCs, depleting the RyR pool using caffeine and ryanodine did not alter IP3-mediated Ca2+ transients, suggesting that the two stores are distinct [10]. However, similar studies performed in renal artery VSMCs suggested that the two types of receptors used the same SR Ca2+ pool. The spatial organization of these receptors is further complicated by the finding that culturing PASMCs results in a combined pool utilized by both RyRs and IP3 receptors [11]. It is possible that these changes in SR pool regulation may involve store-operated Ca2+ mechanisms that make it more difficult to detect distinct pools of Ca2+. Alternatively, the pools of Ca2+ may be dynamic in nature and their compartmentalization may change with cell conditions. The VSMC proliferative phenotype has been correlated with a loss of RyR expression and an increase in IP3 receptor activity. Sustained Ca2+ signals necessary to promote proliferation in cultured VSMCs have been attributed to IP3 receptor activity, and IP3 receptor antagonists block VSMC proliferation [12]. RyRs are downregulated when VSMCs are cultured, and their suppression is accompanied by an increased expression of IP3 receptors [13]. Furthermore, in an animal model of atherosclerosis, resting arterial Ca2+ levels and IP3-mediated Ca2+ release are both increased, correlating sustained elevations in the levels of store-released Ca2+ with a proliferative arterial disease state [14]. In summary, channels in the SR membrane are clearly important in smooth muscle phenotype, including PASMCs, and the growth phenotype is characterized by a downregulation of RyRs and an upregulation of IP3 receptors and IP3mediated Ca2+ signals (Fig. 1).

2.3 Store-Operated Ca2+ Entry It has long been known that release of intracellular stores of Ca2+ upon stimulation by numerous ligands is necessary for sustained Ca2+ level increases and important in the regulation of gene expression in many cell types, including smooth muscle. It was also found that release of Ca2+ from IP3sensitive stores activated influx of extracellular Ca2+, but the molecular identity of this store-operated Ca2+ entry (SOCE) channel proved difficult to ascertain. The majority of evidence supported the hypothesis that the calcium-release-

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activated current (Icrac) was due to activation of the nonselective ion channel family of transient receptor potential channels (TRPCs). This hypothesis was strongly supported by the observation that Icrac and SOCE were lost in TRPC4 and TRPC1 knockout mice, respectively [15, 16]. TRPC alterations are also linked to vascular diseases, where spontaneously hypertensive rats have been shown to express elevated levels of arterial TRPC3 when compared with normal control rats, and inhibition of TRPC1 in coronary arteries was found to reduce neointimal growth after balloon injury [17, 18]. It was later discovered that the SR membrane protein stromal interaction molecule 1 (STIM1) and the ion channel Orai1 are the only components necessary to make a functional SOCE unit. The importance of this process in vascular smooth muscle proliferation was demonstrated by the finding that suppression of STIM1 using RNA interference reduces VSMC proliferation in rat carotid arteries following balloon injury [19]. The two seemingly separate hypotheses have been reconciled by the finding that overexpression of TRPC proteins increases Orai1 activity and SOCE and that there is a functional interaction between Orai1, TRPCs, and STIM1 [20]. Thus, although TRPC proteins are not likely the channels that move Ca2+ in SOCE, they are clearly directly critical to SOCE regulation and VSMC function. In PASMCs, there are several lines of evidence that upregulation of TRPCs is linked to increased SOCE, cell proliferation, and PAH [21]. Pulmonary arteries express the TRPC isoforms 1, 3, and 6, with the most evidence for TRPC1 having store-operated function [22]. During proliferation, PASMCs exhibit an increase in TRPC1 and TRPC6 levels following serum or platelet-derived growth factor (PDGF) stimulation, respectively [23, 24]. SOCE has been shown to be important for normal physiological functions related to both contraction and proliferation in PASMCs, however TRPC3 and TRPC6 are dysfunctionally upregulated in arteries from patients with PAH. Furthermore, silencing of TRPC6 using small interfering RNA inhibits proliferation in PASMCs from patients with PAH [25]. Overall, the evidence thus far suggests that PAH is accompanied by an increase in TRPC expression. This induction enhances store-operated Ca2+ signaling through the Orai1 channel and results in sustained cytoplasmic Ca2+ transients that are sufficient to alter gene expression leading to a proliferative phenotype (Fig. 1).

2.4 Ca2+ Pumps Restoration of resting Ca2+ levels is achieved by Ca2+–Mg2+ATPases (PMCA pumps) and Na2+/Ca2+ exchangers on the plasma membrane, as well as sarcoendoplasmic reticulum Ca2+–Mg2+-ATPases (SERCA pumps) on the SR membrane.

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The maintenance of expression and function of these pumps is essential for maintenance of Ca2+ homeostasis to keep cytoplasmic Ca2+ levels low and to refurbish both ryanodineand IP3-sensitive Ca2+ stores in the SR. The PMCA pumps and Na+/Ca2+ exchanger are essential for movement of Ca2+ from the cytoplasm up its concentration gradient to the extracellular space. There are four isoforms of PMCA, with PMCA1 and PMCA4 expressed in most cell types, including VSMCs, and having distinct functions with respect to proliferation. Overexpression of PMCA1 inhibits proliferation of cultured serum-stimulated VSMCs, whereas PMCA4 is upregulated during proliferation. In addition, PMCA1 expression is negatively regulated by c-Myb, which is selectively expressed during G1/S phase of the cell cycle, whereas PMCA4 upregulation is thought to be protective against apoptosis [26]. Thus, PMCA1 is essential for maintaining homeostasis in differentiated smooth muscle, whereas PMCA4 is induced and required for responses to phenotype change or cellular stress [27]. It has been proposed that these diverse functions are related to the decreased sensitivity of PMCA4 to Ca2+ and CaM levels, which would only be obtained during conditions of agonist stimulation, cell proliferation, or stress. In the case of the Na+/Ca2+ exchanger, the proliferative state is associated with a change in both its expression and its function. In cultured PASMCs, the Na+/Ca2+ exchanger is expressed at high levels and functions in reverse mode, promoting sustained Ca2+ influx and cell proliferation. In addition, upregulation of the Na+/Ca2+ exchanger contributes to the enhanced Ca2+ entry in PASMCs from patients with idiopathic PAH (IPAH) [28]. The SR Ca2+ store replenishment is known to be important for growth signals in VSMCs, and selective subtype expression of SERCA pumps has been shown to determine the VSMC proliferative state. Culturing of mesenteric arterial myocytes results in downregulation of SERCA2a expression, upregulation of SERCA2b expression, and an increase in the amplitude of store-operated Ca2+ transients [13]. Overexpression of SERCA2a inhibits proliferation of cultured VSMCs, and in PASMCs, inhibiting the SERCA pumps causes depletion of the SR store that correlates with cell cycle arrest [29]. Thus, although function of SERCA is important in maintaining the differentiated state, selective overexpression of the SERCA2b isoform results in a proliferative phenotype (Fig. 1).

2.5 K+ Channels as Regulators of Ca2+ Because many of the Ca2+ cascades are dependent on voltage-gated Ca2+ influx, K+ channels that regulate the resting membrane potential of the VSMCs are important indirect regulators of Ca2+ influx. Ca2+-activated K+ channels have been shown to be altered in the development of arterial rest-

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enosis, with large-conductance Ca2+-activated K+ (BKCa) channels being replaced by intermediate-conductance Ca2+activated K+ (KCa3.1) channels. A link to their role in phenotypic switching was made by the finding that inhibition of KCa3.1 activity by in vivo local administration of a selective blocker significantly reduces the development of angioplastyinduced restenosis in a porcine carotid artery model [30]. Although the mechanisms involved in phenotype modulation are not yet known, KCa3.1 function results in voltage-independent membrane hyperpolarization which is likely important in enhancing Ca2+ signaling through TRPCs and induction of mitogenic pathways. Kv channels have also been widely studied for their important role in Ca2+ homeostasis [31, 32]. Because the membrane potential set point is dominated by membrane K+ permeability, a loss of Kv channel activity will cause the resting membrane potential to be more depolarized, which would facilitate activation of L-type Ca2+ channels and prolong the Ca2+ signal. The increased level of Ca2+ leads to both pulmonary vasoconstriction and the promotion of PASMC proliferation and remodeling through Ca2+-regulated gene transcription. In proof, the expression and function of Kv channels (Kv1.2, Kv1.5) is reduced in PASMCs from IPAH patients when compared with patients with other forms of hypertension or cardiopulmonary disease [33, 34]. On the basis of these lines of evidence, the proliferative phenotype is exemplified by a reduction in BKCa and Kv channel expression and an increase in intermediate-conductance KCa3.1 channel expression which could have effects on gene expression through changes in membrane potential set point and store-operated Ca2+ signaling (Fig. 1).

3 Ca2+ -Regulated Transcription Factors The importance of Ca2+ in cellular and physiological events such as muscle contraction, proliferation, and transcription is well established. Thus, dysregulated Ca2+ signaling in VSMCs as a consequence of altered cytoplasmic Ca2+ homeostasis can activate numerous Ca2+-sensitive transcription factors that could contribute to disease [35]. Several Ca2+-regulated transcription factors whose activity has been associated with altered Ca2+ signaling in diseases associated with pulmonary arteries will be discussed.

3.1 Nuclear Factor of Activated T Cells Nuclear factor of activated T cells (NFAT) was first characterized as a Ca2+-regulated transcription factor necessary for the regulation of genes that encode cytokines and their receptors in activated T cells [36, 37]. Although NFAT expression

21  Role of Ca2+ in Vascular Smooth Muscle Gene Expression and Proliferation

is critical for the activated T-cell phenotype, it also functions in nonimmune cells such as skeletal, cardiac, and smooth muscle cells [36, 37]. The NFAT family of transcription factors consists of four Ca2+-dependent isoforms, NFATc1, NFATc2, NFATc3, and NFATc4, and one Ca2+-independent isoform, NFAT5 [36–38]. Ca2+-dependent nuclear localization of NFAT defines its level of activation. NFAT is constitutively expressed, but its nuclear localization signal is concealed when it is in its phosphorylated form [38]. Elevation of cytoplasmic Ca2+ level activates the Ca2+/CaM-dependent phosphatase calcineurin, which dephosphorylates NFAT, exposing its nuclear localization signal. Upon nuclear translocation, NFAT binds and activates promoters of genes involved in processes ranging from the immune response and inflammation to smooth muscle cell differentiation marker gene expression [36, 38]. Because of its sensitivity to Ca2+/calcineurin signaling, NFAT can respond quickly to changes in cytoplasmic Ca2+ levels and is affected by the frequency of Ca2+ oscillations [39, 40]. NFAT signaling has been substantiated in both cultured and native arterial smooth muscle cells [41, 42]. In cultured VSMCs, NFAT is required for proliferation and migration in response to thrombin or PDGF through a pathway that includes upregulation of interleukin-6 [43, 44]. In intact cerebral arteries, the vasoconstrictor UTP has been shown to stimulate NFATc3 nuclear accumulation in VSMCs. This signaling requires both L-type Ca2+ channel function and release of Ca2+ from intracellular stores, suggesting that membrane depolarization alone is not sufficient to result in nuclear localization of NFATc3 [42, 45]. Because NFAT nuclear export is stimulated by serine/threonine kinases such as c-Jun amino-terminal kinase (JNK), it is likely that both an increase in Ca2+ level and a decrease in JNK activity are required for optimal NFAT induction. Indeed, in JNK knockout animals, a rise in intracellular Ca2+ levels is sufficient to elicit NFAT nuclear import [46]. Thus, although Ca2+ level elevation is necessary, it may not be the rate-limiting step in NFAT nuclear accumulation in VSMCs. Several isoforms of NFAT have been evaluated in VSMCs and correlated with both maintenance of differentiation and disease, depending on the NFAT isoform. In both aortic VSMCs and PASMCs, the nuclear translocation of NFATc1 correlates with expression of the differentiation marker smooth-musclespecific SMMHC promoter, and blocking NFAT signaling promotes a proliferative phenotype [47–49]. These data suggest that NFATc1 signaling is required for PASMC differentiation. In contrast to this study, NFATc2 activity is upregulated in PASMCs from patients with IPAH when compared with normal subjects [50]. Increased NFATc2 activity in IPAH is associated with a more depolarized membrane potential as a consequence of NFATc2-mediated downregulation of Kv channel expression, increased intracellular K+ levels and entry of Ca2+ through L-type Ca2+ channels [33, 50]. The resulting increased level of intracellular Ca2+ in PASMCs facilitates a

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forward-feeding loop of NFATc2 activity, which may ultimately lead to PASMC hyperplasia. Therapeutic targeting of NFATc2 has thus been proposed for the treatment of IPAH. The NFATc3 isoform has been shown to be necessary for vascular development, regulation of VSMC contraction, and maintenance of a differentiated smooth muscle phenotype [51, 52]. However, abnormal Ca2+ signaling can lead to events that trigger changes in the vasculature through altered NFATc3 regulation. Similar to IPAH, in hypoxia-induced PAH, Kv channel expression is reduced, which results in a depolarized membrane potential and Ca2+ influx through L-type Ca2+ channels [53]. Hypoxia also stimulates NFAT activity and enhanced nuclear translocation of NFATc3 [54]. NFATc3 knockout animals or animals treated with the calcineurin antagonist cyclosporine A do not exhibit hypoxia induction of smooth muscle a-actin or an increase in arterial wall thickness, suggesting that NFATc3 is a key regulator of arterial remodeling during hypoxia-induced PAH [54]. Taken together, these results implicate that selective NFAT isoform expression can direct Ca2+ signals to different outcomes. In the development of PAH, upregulation of NFATc2/NFATc3 and downregulation of NFATc1 are observed, and study of how these changes are associated with expression of different Ca2+ regulatory pathways may lead to treatment strategies that take advantage of these subtype selectivities (Fig. 2).

3.2 Serum Response Factor Serum response factor (SRF) is a member of the MADS box family of transcription factors, and binds to serum response elements (SREs) within the promoters of many genes related to VSMC proliferation and differentiation [55]. As its name implies, SRF was discovered as a growth factor signaling molecule, and was shown to bind SREs within the promoter of immediate early genes such as c-fos and egr-1 [56]. The SRE is also referred to as a CArG box element on the basis of its consensus sequence, CC(A/T)6GG. A connection between SRF and the Ras/mitogen activated protein kinase (MAPK) growth factor signaling pathway was found to be through MAPK-mediated phosphorylation/activation of the Elk-1 transcription factor, which then forms a complex with SRF on the SRE [57]. SRF was also found to be necessary for the induction of muscle-specific genes during early development and differentiation as well as downregulation of these genes in response to injury or inflammation [58, 59]. SRF binding sites have been identified on smooth-muscle-specific genes, including caldesmon, calponin, SM22, smooth muscle a-actin, SMMHC, and smoothelin-A [60–62]. The question becomes: How does a single transcription factor, binding to a consensus DNA response element, participate in two seemingly opposite gene expression profiles and phenotypic outcomes?

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Fig.  2  Transcriptional control of VSMC phenotype switching. Expression of myocardin (Mycd) directs serum response factor (SRF) to gene targets such as smooth muscle a actin and smooth muscle myosin heavy chain and requires L-type VDCC and Rho kinase (ROK) activities. Downregulation of Mycd and repressor element 1-silencing transcription factor leads to a loss of RyR and Kv channels in conjunction with upregulation of Elk-1-directed SRF targets, including TRPCs, and

KCa3.1, resulting in overactive store-operated Ca2+ entry and a proliferative phenotype. GFR growth factor receptors, Gq-R Gq-coupled receptor, PLC phospholipase C, SOS son-of-sevenless, Raf Ras-activated kinase, MEK mitogen-activated protein kinase/extracellular-signal-regulated kinase kinase, ERK extracellular-signal-regulated kinase, CaMK Ca2+/ CaM-dependent protein kinase, CaN calcineurin, CREB Ca2+/cyclic AMP response element binding protein, OPN osteopontin

Signal discrimination in the context of growth versus muscle differentiation is actively under investigation, and current evidence supports a model that includes gene specificity through CArG element degeneracy and due to selective expression of SRF coactivators that target SRF to select gene targets. The presence of a single conserved C or G nucleotide substitution in the A/T-rich central domain of the CArG element is found only in the promoters of smooth-muscle-specific genes, and when these elements are replaced by the c-fos CArG element, the differentiation genes are induced instead of downregulated in response to injury [63]. This mechanism of CArG degeneracy explains the ability of SRF to activate transcription of growth-specific promoters and suggests that under these conditions, the CArG elements controlling smooth-muscle-specific genes are inactive. The hypothesis that transcriptional coactivators might also play a role in SRF specificity was based on the observation that the level of SRF protein was not altered in response to vascular injury, nor did it change during the transcription of smooth-muscle-specific genes [64]. A bioinformatics screen of unknown cardiac and smooth-muscle-specific proteins identified myocardin, which became the first member of a family of myogenic transcriptional coactivators [65]. Myocardin forms a ternary complex with SRF bound to CArG elements on cardiac and smooth-muscle-specific gene promoters [64]. The selectivity of SRF gene targets has also been shown to be Ca2+-sensitive. Ca2+ influx through L-type Ca2+ channels has been shown to increase smooth muscle a-actin, SMMHC,

myocardin, and c-fos messenger RNA (mRNA) levels as well as the level of activity on their specific promoter elements [66]. Again, the question arises: How could the upregulation of genes that are so disparate in their function be simultaneously regulated by Ca2+? Although not completely clear, data suggest that in contrast to c-fos, the regulation of smoothmuscle-specific genes requires both Ca2+ influx through L-type Ca2+ channels and activation of the Rho-dependent protein kinase signaling pathway. Alternatively, regulation of growth-related genes can be induced by Ca2+ signals through L-type Ca2+ channels but also by ligands that activate IP3mediated release of Ca2+ from the SR Ca2+ store [66, 67]. The dependence of cofactor expression on Ca2+ signaling that is modulated by secondary signaling pathways allows for tight regulation of gene expression patterns in VSMCs. Taken together, these studies have revealed the existence of an intimate balance wherein vascular injury causes induction of MAPKs and Elk-1 binding to SRF on growth-related CArG elements, while simultaneously causing repression of myocardin expression to prevent activation of SRF on muscle-specific promoters (Fig.  2). These pathways have not been specifically studied in PASMCs, nor have they been examined in models of PAH or human pulmonary artery. Because these pathways are strongly affected by alterations in expression of Ca2+-regulatory channels and pumps known to be altered in PAH, it is likely that SRF selectivity is an important untested component of pulmonary artery remodeling in response to PAH.

21  Role of Ca2+ in Vascular Smooth Muscle Gene Expression and Proliferation

3.3 Ca2+/Cyclic AMP Response Element Binding Protein Initially identified in neurons, the Ca2+/cyclic AMP (cAMP) response element (CRE) binding protein (CREB) regulates transcription of hundreds of genes in all eukaryotic cells through its interactions with Ca2+/CREs on the promoter of gene targets [68]. CREB is activated through phosphorylation of serine 133 in its kinase-inducible domain, which promotes its binding to CREB-binding protein (CBP300) and other transcriptional coactivators necessary for the activation of transcription [69]. Many kinases are capable of phosphorylating CREB, including cAMP-dependent protein kinase, Ca2+/ CaM-dependent protein kinase II, and p90RSK [70, 71]. Inactivation of CREB through phosphatase activity can also be regulated by Ca2+ through Ca2+/CaM activation of calcineurin which indirectly leads to dephosphorylation and inactivation of CREB [38]. CREB and NFAT are inversely regulated by calcineurin; thus, its activity may define the dominant Ca2+-mediated transcriptional activity. In cultured VSMCs, CREB phosphorylation is induced by multiple Ca2+ signals, including L-type Ca2+ channel activation, agonist-mediated IP3 production, and blocking SERCA pumps to stimulate SOCE [42, 72]. Immediate early genes such as c-fos as well as cell-cycle-specific CRE-containing genes, including cyclins A and E, are upregulated following increases in the Ca2+ level [42, 73]. PDGF-stimulated expression of the migratory protein osteopontin has also been shown to be mediated by CREB-dependent binding to a functional CRE site within the osteopontin promoter [74]. A microarray analysis comparing transcription patterns in human VSMCs following stimulation of different sources of Ca2+ showed regulation of overlapping, yet distinct sets of genes, with SOCE activation of CRE-containing genes that reflected a growth phenotype [75]. In human PASMCs, the mitogenic effects of ATP have been partly attributed to CREB-dependent upregulation of TRPC4 expression and activity that results in an increase in SOCE and basal levels of cytoplasmic Ca2+ [76]. Thus, in VSMCs from different arterial beds, CREB activity correlates with a proliferative and migratory phenotype. Evidence for altered CREB function in intact tissue and arterial diseases has also been reported. Cerebral arteries from hypertensive animals exhibit elevated levels of VSMC Ca2+, phospho-CREB, and c-fos mRNA [77]. Interestingly, these effects are reversed by inhibition of L-type Ca2+ channels after arterial dissection, suggesting that the CREB activation may be a result of a defect in the “off” mechanism. The proliferative response to angiotensin II-induced hypertension or oxidative endothelial injury has also been correlated with increased levels of phospho-CREB and a proliferative response [78]. Furthermore, expression of dominant negative CREB constructs in an angioplasty model reduces neointimal formation and promotes apoptosis [79]. Transient

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in vivo ischemia also results in CREB phosphorylation and an upregulation of CRE-mediated BCL-2 expression [80]. Together, these results suggest a role for CREB in both survival and proliferation of VSMCs following injury. An exception to the correlation between CREB activity and the proliferative phenotype is a group of studies showing that hypoxia results in a loss of CREB [81]. This mechanism for CREB reduction was found to include long-term PDGF signaling through phosphoinositol 3-kinase, nuclear export of CREB, and proteasomal degradation [82]. The differences in these findings when compared with the evidence linking CREB to proliferation can be explained by the timing of CREB activation as it relates to gene regulation and effects on arterial phenotype. It is also possible that although CREB levels are reduced, phospho-CREB activity is still elevated.

3.4 Activating Protein-1 The activating protein-1 (AP-1) family of transcription factors is composed of multiple family members subclassed as Jun (v-Jun, c-Jun, JunB, and JunD), Fos (v-Fos, c-Fos, Fra1, Fra2, and FosB) or activating transcription factor (ATF2, ATF3/LRF1, B-ATF), which combine as homo- or heterodimers and bind to AP-1 binding sites (TGAG/CTCA) within the promoters of multiple genes. These transcription factors are the gene products of classical immediate early genes that are themselves regulated by CRE and AP-1 elements. AP-1 family members are upregulated within minutes of growth factor signaling through Ca2+ transients and/or Ras/extracellular-signal-regulated kinase (ERK) pathway activation and become a self-generating logarithmic on switch through generation of their own transcription. The kinase cascade is stopped by phosphatase activity, and AP-1 family members are rapidly removed by ubiquitin-mediated proteasomal degradation [83]. Although AP-1 sites are found in both growth and tissue-specific gene promoters, elevated levels of AP-1 family members have been directly associated with proliferative disorders, including vascular diseases and cancer (reviewed in [84, 85]). Ca2+ regulation of AP-1 signaling and cell proliferation through agonist and IP3-mediated SOCE has been demonstrated in several arterial cell models. Induction of the proliferation-associated protein osteopontin by UTP, angiotensin II, or PDGF is mediated by an AP-1 site bound by c-Fos and c-Jun in aortic VSMCs [86]. Stimulation of SOCE by depleting SR Ca2+ stores induces ERK activation and c-fos transcription in both cultured and intact cerebral VSMCs, and these activities are dependent on Ca2+ entry [72, 75]. ERK activity is elevated in cerebral arteries from hypertensive animals, and also in carotid arteries following balloon injury [87]. Arterial injury is also associated with increased levels of

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c-fos mRNA and AP-1 binding activity [88]. As with cancer, it is likely that the elevated ERK and AP-1 activity is due to defects in the off switch for the pathway, and thus investigations of Ras inactivation and phosphatase activity will be important for understanding the abnormality and developing therapies. AP-1 signaling in the pulmonary vasculature has both direct and indirect links to Ca2+ signaling, PASMC growth, and vascular remodeling. In PASMCs, upregulation of TRPC6 expression and cell proliferation in response to PDGF stimulation requires induction of Jun and STAT3 transcription factors. The increase in SOCE due to TRPC6 upregulation was found to be necessary for the growth trigger [24, 35]. The response of the pulmonary artery endothelial cells to hypoxia includes Ca2+ influx through SOCE and an increase in AP-1 binding activity. This AP-1 response leads to expression of mitogens by endothelial cells that ultimately influence PASMC growth [89]. These findings indicate a strong relationship between AP-1 signaling and Ca2+-mediated vascular remodeling that could have important implications in the development of pulmonary hypertension (Fig. 2). Pharmacologic agents that block PDGF receptors or MAPKs are currently used in the clinics for treatment of cancer, and these agents may also be important therapeutic agents in the future treatment of pulmonary vascular diseases.

4 Genes Altered in PAH Correlate with Altered Ca2+ Signaling and Cell Proliferation Microarray analysis is a powerful and efficient method to examine genes altered in specific diseases because of its ability to simultaneously measure the expression of thousands of genes from a single cell type or from an intact model with multiple cell types. The limitation is primarily at the analysis level owing to the multiple statistical comparison difficulties, and it is therefore important to validate selected results by other methods. This section will outline the results of four microarray studies performed on PASMCs that link PAH with alterations in ion channel homeostasis and regulation of cell proliferation/survival in human tissue [90, 91] and a mouse model of hypoxia-induced PAH [92, 93]. A microarray analysis performed by the Voelkel group used lung tissue obtained from patients with severe forms of PAH, including sporadic primary pulmonary hypertension (SPPH) and familial primary pulmonary hypertension (FPPH), a ­disease linked to germline mutations of the bone morphogenic protein (BMP) receptor II [94–96]. The lung tissue was compared with normal lung tissue from patients with other diagnoses for

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changes in gene expression [90]. Several genes were found to be significantly changed, with notable increases in Ca2+-regulated survival factors such as BCL-2 and FYN as well as ion channels, including the inward rectifier K+ channel (GIRK) and the voltage-gated Na+ channel (SCN1B) (Table 1). Downregulation was observed for the IP3 receptor type 3 Ca2+ channel (ITPR3), the voltage-gated K+ channel (KCNA), and caveolin 1 (CAV1) (Table 2). Cluster analysis unexpectedly found that the overall gene expression pattern from the FPPH lungs was more closely related to the normal lung profile than that of primary pulmonary hypertension (PPH) lungs. The limitations of this study were the small number of patient samples and the use of whole lung, which dilutes the pulmonary artery signal, but overall the changes support the hypothesis that severe PAH is associated with an environment that has a depolarized membrane potential and activation of survival signals. A study by the Yuan group compared patterns of gene expression in PASMCs derived from patients with IPAH and PASMCs from normal patients [91]. Because BMP-2 signaling is lost in severe forms of PAH, the authors also tested the effects of BMP-2 treatment on gene expression in PASMCs from control and IPAH sources. This study had the advantage that the tissue source was specific to pulmonary artery; however, the cells were exposed to culture conditions, and the control cells were not isolated, but commercially available cultured PASMCs. The results of the study indicated that BMP-2 has opposing effects on PASMCs from the lungs of subjects with IPAH when compared with normal subjects. BMP-2 was shown to cause a reduction in BCL-2 expression and induction of apoptosis in cultured PASMCs [89]. The upregulation of growth factors, ion channels, and signaling molecules important in Ca2+ signaling pathways, along with the downregulation of voltage-gated K+ channels and the gene that encodes a Ca2+-ATPase pump, strongly supports a role for Ca2+mediated regulation of proliferation and remodeling in pulmonary artery hypertensive disease (Tables 1, 2). There is also the suggestion that Ca2+-regulated transcription factors may be important not only in proliferation but also in the upregulation of antiapoptotic genes, i.e., BCL-2 and IP3 receptor type 3 [90, 91]. Additionally BCL-2 is upregulated in the lungs of subjects with FPPH and SPPH, suggesting a resistance to apoptosis in primary pulmonary hypertension [90]. Finally, there is a strong correlation between the Fantozzi and Geraci studies that suggests that mutations in BMP receptor II leads to altered signaling by BMP-2 and the upregulation of genes in PASMCs that favor the proliferative, antiapoptotic phenotype observed in PAH. Although data obtained from microarray analyses done on lung homogenates reveal important clues that explain some of the phenotypical changes observed in PAH, intrapulmonary arteries accounts for less than 10% of lung tissue. Thus, the

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Table 1  Representation of genes upregulated in different types of pulmonary arterial hypertension Function Gene Hypertension Signaling proteins/ transcription factors

BCL-2 (B-cell lymphoma 2) CALM1 (calmodulin 1) CAV2 (caveolin 2) CBP (CREB-binding protein) CREB (cyclic AMP response element binding protein-1) c-Fos EGR1 FYN (SRC-related oncogene) SRF (serum response factor) ATF3 (activating transcription factor) EDN-1 (endothelin-1) GIRK (inward rectifier K + channel) SCN1B (voltage-gated Na+-channel type Ib polypeptide) 5HT-2B (serotonin receptor subtype 2B) IP3 receptor 1 (inositol 1,4,5-trisphosphate receptor) EGF (GRB2) PDGF-b

Lung model

Reference

SPPH, FPPH IPAH IPAH IPAH IPAH IPAH IPAH SPPH, FPPH HIPH HIPH HIPH SPPH, FPPH SPPH, FPPH IPAH IPAH IPAH HIPH, IPAH

Intact [90] Cultured, BMP-2 [91] Cultured, BMP-2 [91] Cultured, BMP-2 [91] Cultured, BMP-2 [91] Cultured, BMP-2 [91] Cultured, BMP-2 [91] Intact [90] Mouse, hypoxia [92] Mouse, hypoxia [93] Mouse, hypoxia [93] Membrane receptors/ion Intact [90] channels Intact [90] Cultured, BMP-2 [91] Cultured, BMP-2 [91] Growth factors Cultured, BMP-2 [91] Mouse, hypoxia or [91, 92] cultured, BMP-2 TGF-b HIPH Mouse, hypoxia [92] Ca2+ binding/Ca2+ FKBP1a (FK506 binding protein 1A) HIPH Mouse, hypoxia [92] signaling S100A4 HIPH Mouse, hypoxia [92] BMP-2 bone morphogenetic protein-2, FPPH familial form of primary pulmonary hypertension, HIPH hypoxia-induced pulmonary hypertension, IPAH idiopathic pulmonary arterial hypertension, SPPH sporadic primary pulmonary hypertension, CREB cyclic AMP response element binding protein, EGF epidermal growth factor, PDGF-b platelet derived growth factor b, TGF-b transforming growth factor b

Table 2  Representation of genes downregulated in different types of pulmonary arterial hypertension Function Gene Hypertension Signaling proteins/ transcription factors Membrane receptors/ion channels

CAV1 (caveolin 1)

SPPH, FPPH

AGTR2 (angiotensin II type-2 receptor) ITPR3 (inositol 1,4,5-trisphosphate receptor type 3)

IPAH IPAH/SPPH, FPPH

Lung model

Reference

Intact

[90]

Cultured, BMP-2 [91] Mouse, hypoxia or [90, 91] cultured, BMP-2 SPPH, FPPH NT [90] KCNA (voltage-gated, shaker-related K+ channel) Enzymes ATP2A3 (Ca2+-transporting ATPase) IPAH BMP-2 [91] BMP-2 bone morphogenetic protein 2, FPPH familial primary pulmonary hypertension, IPAH idiopathic pulmonary arterial hypertension, NT no treatments, SPPH sporadic primary pulmonary hypertension

data obtained from whole-lung studies may not fully represent altered gene expression in intact PASMCs. Investigators from the Fink group thus used an animal model of hypoxiainduced PAH, followed by dissection of the PASMCs using laser scanning microscopy for microarray analysis [92]. This model has the advantage of specifically studying PASMCs and an increased number of samples and time points, but it has the disadvantage that it is not completely representative of the development of human PAH. The results of the study showed that multiple growth factors, including PDGF-b, as well as the transcription factor SRF are significantly upregulated following chronic hypoxia [92]. The McLoughlin group also used microarray analysis in a hypoxia-induced PAH mouse model, but they analyzed mRNA levels in whole lung and focused on expression patterns of overrepresented transcription-factor-binding sites

after different times of hypoxia exposure [93]. A cluster analysis of genes altered by hypoxia revealed a significant increase in the expression of genes that are regulated by CREB and AP-1 family members (Table 1), and this pattern correlated well with protein levels of phospho-CREB. Surprisingly, myocardin upregulation was also identified in this cluster, suggesting that the loss of myocardin may occur later in the development of PAH. The disadvantage of examining whole lung was countered by the advantage of multiple time points and further comparisons with transcription factor activity in human lung microvascular endothelial cells. ATF3, endothelin-1, and follistatin were the only genes validated, but other CREB targets related to hypertrophy, including eukaryotic translation initiation factors, would be interesting to follow in the future. Overall, this model supports the notion that the development of PAH is associated with

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induction of Ca2+-regulated transcription factors known to regulate VSMC growth. In conclusion, the microarray analyses presented here displayed patterns of gene expression in four different PAH models that favor a proliferative phenotype and are related to remodeling as a consequence of proliferation. The microarray study in cultured PASMCs from PAH patients shows an upregulation of genes that relate to cell growth and proliferation, and, taken together with the altered ion channel expression from the intact lung study, it is tempting to conclude that in PAH, dysregulated gene expression supports an environment that facilitates the activation of Ca2+-regulated transcription factors. It is also interesting to note that the genes altered in the hypoxia mouse models represent changes that would be expected as part of the immediate early gene response (i.e., growth factor alterations), whereas the analysis of human disease represents later effects following the immediate early gene response (i.e., ion channel alterations). A study of longer time points in the animal model and a study of healthy patients with the FPPH genotype may lead to better insights related to the role of Ca2+ signaling in the onset and progression of PAH.

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Chapter 22

Biochemistry and Cellular Mechanisms of Apoptosis in Vascular Smooth Muscle and Endothelial Cells Oliver Eickelberg and Fotini M. Kouri

Abstract  Cell death plays a pivotal role in physiological development and tissue homeostasis. There are different ways for cellular death, arising from differential conditions and producing differential effects. The fact that perturbations in the death pathways can lead to diseases such as cancer, Alzheimer’s, atherosclerosis, and pulmonary vascular diseases reveals the essentiality of cell death for tissue homeostasis. This chapter briefly discusses the historical aspects and focus on the mechanisms of programmed cell death, or apoptosis, in pulmonary vascular smooth muscle and endothelial cells, which constitute the pulmonary vessel wall, in the context of health and disease. Keywords  Apoptosis • Programmed cell death • Caspase • Apoptotic volume decrease • Pulmonary vascular remodeling • Cytochrome c • Mitochondria

1 Apoptosis, Retrospectively The statement “life would be impossible without death” could not be more appropriate to describe the pivotal role of cell death in physiological development and tissue homeostasis. There are different ways for cellular death, arising from differential conditions and producing differential effects. The fact that perturbations in the death pathways can lead to diseases such as cancer, Alzheimer’s, atherosclerosis, and pulmonary vascular diseases reveals the essentiality of cell death for tissue homeostasis [1]. This chapter will briefly discuss the historical aspects and focus on the mechanisms of programmed cell death in pulmonary vascular smooth muscle and endothelial cells, which constitute the pulmonary vessel wall, in the context of health and disease.

O. Eickelberg (*) Institute of Lung Biology and Disease (iLBD), Helmholtz Zentrum München, Ingolstädter Landstraße 1, 85764, Neuherberg/Munich, Germany e-mail: [email protected]

Apoptosis, or programmed cell death, is a common biological process. It is a kind of cellular suicide occurring when the organ or tissue demands it, leading to cell elimination. In concert with cell division, it is necessary for the regulation of cell number in a multicellular organism. Apoptosis is recognized to be a key mechanism for many physiological processes, especially during development, and its impairment contributes to disease. “Apoptosis,” derived from the Greek words apo, meaning “from,” and ptosis, meaning “falling,” means the “dropping or falling off.” The actual process was first described by the German scientist C. Vogt in 1842, but it was not until the end of the 1960s that programmed cell death was demonstrated by J.F.X. Kerr, while studying liver tissue by electron microscopy [2]. Furthermore, in 1972, Kerr et al. [3] characterized this novel phenomenon of programmed cell death, or apoptosis, which was distinct from the already known cell necrosis. The paper at that time received severe criticism regarding the term chosen to characterize this mode of cell death, since the word “apoptosis” was originally used to describe the falling of leaves. There were several constraints on whether this term was etymologically correct and should be used for this purpose. In addition, the fact that apoptosis was suggested to be a truly controlled process was hardly accepted. The apoptotic event was opposing the already known type of cell death called necrosis, which resulted from a tissue injury and led to plasma membrane breakage and release of intracellular components into the extracellular space, creating an inflammatory response. According to Kerr et al., there was much more to apoptosis than simply another way of cell death. With Kerr’s discovery of apoptosis, a novel field of research was born. Therefore, it did not take long for a vast number of studies to focus on the complexity of apoptotic cell death. To date, it is well established that the morphological signature of apoptosis includes cell shrinkage or apoptotic volume decrease (AVD), chromatin condensation, nuclear fragmentation, and blebbing of the plasma membrane [4]. The hallmark of an apoptotic cell is its DNA fragmentation, which originally was thought to take place at the linker region between the nucleosomes, creating 180 base pair fragments that have

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the characteristic morphology of a “DNA ladder” under gel electrophoresis [5]. In-depth analysis demonstrated that although it may vary among cell types, this is a two-phase process. The first phase includes DNA cleavage into around 50 kilobase pair fragments that undergo further cleavage to 180 base pair fragments in the second phase [6]. The final event of apoptosis is the formation of apoptotic bodies, which are cellular fragments, including parts of the nuclear lamina and the cytoskeleton, surrounded by the cell’s plasma membrane. An early critical feature of the apoptotic cell is that the phosphatidylserine in the plasma membrane, which normally faces the internal part of the cell, now is in contact with the extracellular space, allowing an easier detection of early apoptotic cells. The resulting apoptotic bodies are ingested rapidly either by neighboring cells, something common during embryogenesis, or by components of the innate immune system, such as macrophages and dendritic cells, using mechanisms similar to those used to recognize and remove pathogens, in the absence of an inflammatory response [7–9]. Most of the basic knowledge on apoptosis comes from extensive studies in the field of embryogenesis and development. There is a plethora of developmental processes where apoptosis participates. For example, programmed cell death is vital for correct development of the immune and nervous system. Apoptosis is responsible for 50% of neuronal cell inactivation during development, and one billion immune cells commit suicide on a daily basis in the body [10–12]. Mammary gland development is another example of the significance of apoptosis, where removal of detached epithelial cells from their surrounding extracellular matrix is crucial for the correct development of the gland. During the development of the nematode Caenorhabditis elegans as well, many cells undergo apoptosis frequently. Therefore, C. elegans was used as a genetic model to obtain more insight into the apoptotic machinery, which is finely tuned and highly conserved across species, from nematodes to humans [13].

2 Apoptotic Pathways The apoptotic cell-signaling pathway is responsible for converting a death stimulus, which can originate from the intracellular space (intrinsic pathway) or extracellular space (extrinsic pathway), into cellular effects. Apoptosis is divided into an initiation phase, involving the transmission of a specific death signal, and an execution phase, involving destruction of cellular compartments and structures and final cell death. After extensive studies, it is now known that apoptosis can have two different, well-conserved initiation phases, depending on where the death signal comes from. Each initiation phase leads to the activation of a different transduction pathway, however both lead to death. There is the

O. Eickelberg and F.M. Kouri

intrinsic pathway, which utilizes mitochondria and the endoplasmic reticulum (ER), and the extrinsic pathway, which utilizes specific transmembrane receptors.

2.1 Intrinsic Pathway The intrinsic pathway is activated by a variety of signals, such as lack of growth factors, hypoxia, toxins, reactive oxygen species (ROS), and DNA damage. Although the exact mechanism downstream of these stimuli is not totally clear, the end effect is the activation of the intrinsic apoptotic pathway and eventually cell death. The central organelle for activation of the intrinsic pathway is the mitochondrion [14]. The loss of stability of the outer mitochondrial membrane leads to the release of proapoptotic factors, such as cytochrome c, which are confined within the inner and outer mitochondrial membranes under physiological conditions. Cytochrome c, except for its role in apoptosis, is also involved in the electron transport chain that leads to ATP synthesis. The electron transport chain consists of several protein complexes (I–V) found on the inner mitochondrial membrane. During the transportation of electrons from one complex to the next, protons are produced and pumped from the mitochondrial matrix to the intermitochondrial space. This causes the formation of an electrochemical membrane potential, known as the mitochondrial transmembrane potential (DYm). Upon release of cytochrome c, DYm is lost. In response to an apoptotic stimulus, due to opening of the permeability transition pore, a nonspecific channel on the outer mitochondrial membrane, there is a mitochondrial permeability transition. The permeability transition pore is composed of three components, the voltage-dependent anion channel (VDAC), the adenine nucleotide transporter, and the cyclophilin D. Much attention has been given to the VDAC because of its diverse functions. The VDAC protein family is composed of the VDAC1, VDAC2, and VDAC3 isoforms, which are located on the outer mitochondrial membrane. The presence of VDAC on the outer mitochondrial membrane is critical, since it regulates the energy synthesis via controlling the metabolite transport across the membrane. Downregulation of this protein can lead to growth arrest, due to reduction in the energy synthesis [15]. Recent studies have implicated the VDAC in the regulation of the apoptotic intrinsic pathway as well. It has been shown that a decrease in the expression levels of VDAC1, by small interfering RNA, is able to attenuate the proapoptotic effect of endostatin on endothelial cells [16,17]. Furthermore, VDAC1 expression levels are increased upon treatment of endothelial cells with endostatin and subsequent induction of apoptosis. In another study, depletion of VDAC1, again by small interfering RNA, reduced the cytochrome c release and

22  Biochemistry and Cellular Mechanisms of Apoptosis in Vascular Smooth Muscle and Endothelial Cells

further apoptosis in response to cisplatin [18]. On the other hand, overexpression of VDAC1 was able to induce apoptosis in a variety of cell types. In particular, in endothelial cells, VDAC1 overexpression induced the activation of caspase-9, increased ROS levels, and the endostatin-induced apoptotic effect [16,19]. Several potential mechanisms have been suggested for the mode of action of the VDAC. According to one, the VDAC undergoes oligomerization and allows the release of cytochrome c. This hypothesis was further substantiated by in  vitro experiments [20]. The VDAC was shown to assemble into dimers, trimers, and tetramers [21]. The oligomerization of the VDAC was also shown by atomic force microscopy and NMR spectroscopy [22,23]. Evidence also suggests that the VDAC also has binding sites for members of the Bcl-2 family, Ca2+, as well as hexokinase, which is a glycolytic enzyme. Members of the Bcl-2 superfamily, such as the proapoptotic molecules Bax and Bak, can either interact with the VDAC and lead to the opening of the channel and further apoptosis, or oligomerize by themselves, forming a porelike structure, and translocate to the mitochondria, where they lead to cytochrome c release within minutes after the apoptotic stimulus [24,25]. Although it has been suggested that Bax and Bak form a pore, the actual mechanism remains elusive [26]. It has been shown that the regulation of VDAC activity and permeabilization of the mitochondrial outer membrane by Ca2+ occurs in a VDAC-dependent manner [27]. It is very interesting that the glycolytic enzyme hexokinase by binding to the VDAC is able to block apoptotic cell death [19]. Further evidence suggests that this could be the pathway utilized by cancer cells to escape from cell death and proliferate indefinitely [28]. After cytochrome c has been released from mitochondria, the next pivotal step of the intrinsic pathway is the formation of a receptor known as the apoptosome. The main structural unit of the apoptosome is the cytosolic adaptor protein Apaf-1, which when it senses cytochrome c binds to it. This event induces Apaf-1 oligomerization and formation of a wheel-shaped structure, the apoptosome, which will be used as a platform for signaling and apoptosis progression. The apoptosome formation is a complex process, which requires the presence of cytochrome c and ATP. To understand in depth how the apoptosome forms, it is necessary to understand the nature of Apaf-1 [29,30]. Apaf-1 is a multidomain protein, containing three functional regions. It has an N-terminal caspase-recruitment domain (CARD), a nucleotide-binding and oligomerization domain (NB-ARC), and a series of WD40 repeats at its C-terminal. The CARD region of Apaf-1 is able to bind the CARD region of caspase-9 and hence recruit it to the apoptosome. The WD40 repeats are responsible for the cytochrome c binding, and the NB-ARC region, which is found between the CARD and WD40 regions, controls the apoptosome formation. Studies have shown that the NB-ARC region is com-

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posed of an ATPase domain followed by a winged-helix domain and a superhelical domain. In the absence of an apoptotic signal, Apaf-1 is in a monomeric form, where the WD40 repeats confine it in an autoinhibitory state, by folding back on the rest of the protein. It is speculated that the CARD and NB-ARC regions take up a more compact conformation, inhibiting the protein oligomerization, and ATP is bound to the ATPase domain of the NB-ARC region, maintaining this compact conformation. In the presence of an apoptotic signal, cytochrome c is released from the mitochondria and binds to the WD40 repeat region of Apaf-1. This generates a conformational change of Apaf-1 and initiation of the apoptosome formation. There is release of the CARD and NB-ARC regions from the autoinhibitory state, where the CARD region interacts with the ATPase domain and the winged-helix domain of NB-ARC, hindering the recruitment of caspase-9 as well as the blocking the ATPase domain, which is essential for Apaf-1 oligomerization. The structure of the ATPase domain is homologous to the structures of domains found in the AAA+ family of ATPases, characteristic for their property to form oligomers [31]. The principle of this formation is that one ATPase domain settles next to the neighbor molecule, forming a circular structure; in the case of Apaf-1, this is the apoptosome. Although it is not completely clear, ATP appears to be linked to the conformational changes occurring during the apoptosome formation. This is substantiated by the observation that if the ATP binding site is mutated, the apoptosome cannot be formed in the presence of several different apoptotic signals. After the formation of the apoptosome, capsase-9 is recruited and activated. The activation of caspase-9 does not require proteolytic cleavage, but takes place by dimerization, which is a reversible process [32]. Caspase-9 activation has a domino effect, leading to the activation of caspase-3 and caspase-7, and the final phase of apoptosis, the execution phase.

2.2 Extrinsic Apoptotic Pathway The extrinsic pathway involves signal propagation via specific transmembrane receptors, using caspases as the effector molecules. Different death ligands interact with certain receptors on the cell membrane and lead to initiation of an intracellular signaling cascade, involving the activation of caspases [33]. Death receptors are members of the tumor necrosis factor (TNF) receptor superfamily, which all share structural similarities. The different ligands, among which FasL (or CD95L), TNF-a, and TNF-related apoptosis-inducing ligand (Apo2L or TRAIL) by interacting with the receptors Fas (or CD95), TNF receptor 1 (or TNFR1), death receptor 4 (DR4 or TRAILR1), and death receptor 5 (DR5 or

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TRAILR2), respectively, initiate the apoptotic pathway. All the above-mentioned receptors contain the cytoplasmic domain called the death domain, which is essential for the intracellular signal initiation. Furthermore, they all possess a cysteine-rich extracellular domain. TRAILR1 and TRAILR2 are highly homologous with no distinct functions. The ligand–receptor interaction can be regulated by decoy receptors that block the apoptotic signal transmission, due to lack of the death domain or even the whole cytoplasmic domain. For example, the cellular effects of the FasL–Fas interaction are inhibited by the soluble decoy receptor called DcR3. Furthermore, the cell-bound decoy receptors TRAILR3 and TRAILR4 are a drag on the TRAIL–TRAILR1/TRAILR2 signal transduction [34]. The Fas receptor is the most well described receptor from the family of the TNF receptors, and one of its best known functions is the initiation of apoptosis. Binding of FasL to this receptor, which exists in a homotrimeric complex, sparks off the recruitment of adaptor proteins in the intracellular space, such as the Fas associated via death domain (FADD) and the further requisition of inactive procaspases, such as procaspase-8 and procaspase-10, leading to the formation of the death-inducing signaling complex (DISC). First, the death domain of FADD interacts with the death domain of the different cell receptors and, next, the death effector domain (DED) of FADD recruits the initiator procaspase-8 or procaspase-10. The following step in the process of apoptotic signal transmission is the activation of the procaspases to active enzymes. Two different hypotheses have been suggested over the years concerning the mechanism of caspase activation involved in the extrinsic pathway. The first hypothesis suggests that the close proximity of the inactive procaspases within the DISC induces their autoproteolysis and thus activation [35]. According to the second hypothesis, the close proximity of the procaspases in the DISC causes activation, by dimerization. The activated caspase-8 or caspase-10, in turn, will activate the downstream executioner caspases, and finally provoke cell death. Caspase-8 or caspase-10 can be inhibited by the cellular FLICE-like inhibitory protein cFLIP by DED–DED interaction. cFLIPL cFLIPR, and cFLIPS [36] are different cFLIP isoforms. cFLIPR and cFLIPS have been shown to prevent the caspase-8 activation owing to competition with them for FADD binding. The role of the third isoform, cFLIPL, in caspase activation has not yet been clearly established. Some studies have shown that cFLIPL, like cFLIPR and cFLIPS, restrains caspase activation. On the other hand, it has been suggested that it assists procaspase-8 activation and thus is considered a proapoptotic factor. The latter suggestion is supported by the fact that the cFLIP knockout mice have the same phenotype as caspase-8- and FADD-deficient mice, which is death at embryonic day 10.5, due to heart failure [37]. Experiments using different proteasome inhibitors revealed that cFLIP undergoes proteasomal degradation, and Itch is the ubiquitin

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E3 ligase responsible for this [38]. Itch itself is activated by Jun N-terminal kinase (JNK)-dependent phosphorylation, suggesting that the JNK signaling cascade can have a proapoptotic effect [39]. There are also other regulators of DISC formation, for example, protein kinase C, which can block FADD recruitment [40], mitogen-activated protein kinase [41], and protein kinase B [42]. Once caspase-8 or caspase-10 has been activated, there are two paths it can go through. The first one is to activate the executioner caspase-3 and this leads to cell death. The alternative path is the activation of Bid, a Bcl-2 family member, which will induce the release of proapoptotic factors from the intermediate space of mitochondria and thus bring into play the intrinsic apoptotic pathway as well [43]. Thus, cells are classified as type II and type I according to whether the extrinsic pathway requires the activation of the intrinsic pathway to cause cell death or not. In type I cells, possibly owing to excess activation of caspase-8, the cells do not require the intrinsic pathway for apoptosis. In contrast, in type II cells, the DISC formation is weaker, and it requires effector caspase activation via the mitochondria for the execution of cell death. The extrinsic and intrinsic pathways are connected via Bid, which is a caspase-8 substrate.

3 Apoptic Effector Proteins 3.1 Caspases: The Suicide Proteins Regardless of which initiation pathway is activated, apoptosis is a two-step process leading to the activation of highly specialized proteins, the caspases, which are able to amplify the signal and lead to death. Caspases are cysteine proteases that cleave proteins at specific aspartate residues. The fact that caspases themselves are regulated by proteolytic cleavage ensures their rapid activation under different stress conditions [44]. The role of caspases in apoptosis became evident for the first time with the discovery that ced-3, the prototype gene responsible for the induction of programmed cell death in C. elegans, is homologous to the mammalian caspases, and in particular to ICE/caspase-1, which is the protease responsible for the activation of interleukin-1b (IL-1b) and interferon-g [45]. The role of caspases as the main effector enzymes in apoptosis was extensively studied and substantiated by a series of observations. For example, overexpression of caspases in cell lines can induce apoptosis. Experiments using blocking peptides confirmed that caspases are required for most types of apoptosis. Most of the information available today on caspase function comes from studies in caspase-deficient mice [46] (Table  1). Such studies have shown that although caspases are the main regulators of apoptosis, they also have

22  Biochemistry and Cellular Mechanisms of Apoptosis in Vascular Smooth Muscle and Endothelial Cells Table 1  Effects of specific caspase depletion in transgenic mice Caspase knockout

Phenotype

Caspase-1

No IL-1b and IL-18 activationEffects on cytokine activation Oocyte accumulation Defects in brain development. Mice die 3 weeks after birth Defects in development of heart, muscle, and the hematopoietic system Defects in brain development Effects on the immune system homeostasis Effects on cytokine activation and caspase-1 activation Normal development, reduced skin hydration levels

Caspase-2 Caspase-3 Caspase-8 Caspase-9 Caspase-10 Caspase-11 Caspase-14

apoptosis-independent functions. This is why caspases were classified according to their function as proinflammatory and proapoptotic. In humans, for example, caspase-1, caspase-4, and caspase-5, have no role in apoptosis; instead, they control cytokine maturation, such as IL-1b, and therefore are known as proinflammatory. On the other hand, caspase-3, caspase-6, caspase-7, caspase-8, caspase-9, caspase-10, and caspase-12 are known to be a part of the apoptotic machinery and hence are known as proapoptotic [47]. More than 14 family members have been identified up to now. These proteases are initially synthesized as procaspases, in an inactive form, containing a prodomain and a protease domain. The length of the prodomain determines the class of the caspase. Those caspases with long prodomains belong to the class of initiator caspases. For example, caspase-8 and caspase-10 contain two tandem repeats of the DED in their prodomain, which is essential for their function during extrinsic apoptotic cell death. Another group of caspases with long prodomains, such as caspase-1, caspase-2, caspase-4, and caspase-9, contain a CARD, which also greatly influences their function and activity. The caspases with short prodomains, such as caspase-3, caspase-6, and caspase-7 are considered to be downstream effector or executioner caspases that need the initiator caspases to activate them, by proteolytic cleavage. On the other hand, the initiator caspases do not require a proteolytic cleavage to become activated. Their activation depends on the formation and activity of recruitment platforms, which are made of scaffold proteins. Proinflammatory caspases in a way similar to proapoptotic caspases undergo such a kind of activation, in a recruitment platform, called inflammasome, which will eventually lead to an inflammatory response [48]. The role of executioner caspases is to disable processes responsible for cell homeostasis and damage repair and eventually lead to the complete destruction of the dying cell, by caspase-activated DNases that will enter the nucleus and cleave the DNA, leading to the characteristic morphology of a “ladder” upon gel electrophoresis [49].

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Caspases are signaling proteases that are destined for protein cleavage, rather than protein destruction. Usually they do not destroy a protein’s structure, but rather modify it, by creating at maximum a couple of proteolytic cuts at specific aspartate residues. The best example of the proteolytic action of caspases is the caspase signaling itself, where initiator caspases, by irreversible cleavage, activate their downstream effector caspases. Hence, when the executioner caspases are activated, there is no going back; it is an irreversible process leading to the dismantling and final death of the cell [50].

3.2 Inhibitors of Apoptosis Apart from the proteolytic caspase pathway, caspase activity is also regulated by a number of inhibitors of apoptosis (IAP). Most of the IAP have ubiquitin E3 ligase activity, targeting caspases for proteosomal degradation [51]. The best characterized examples are IAP-1, IAP-2, and XIAP. The latter is the stronger inhibitor for activated effector caspase-3 and caspase-7 as well as for the caspase-9, sequestering it in an inactive state. It has been proposed that XIAP might also regulate caspase activity in a rather indirect manner. More specifically, XIAP catalyzes the polyubiquitination of caspase-3 and thus induces its degradation. Similarly, IAP-2 has been shown to monoubiquitinate caspase-3 and caspase-7, which may not lead to their degradation, but affects their stability, in vitro. Last, but not least, survivin is an apoptotis inhibitor, whose role is to interact with XIAP and enhance its antiapoptotic action. De novo survivin synthesis has been linked to cancer progression. The inhibitory action of IAP can be restrained in apoptotic cells by the mitochondrial proteins Smac/Diablo, which when released from the mitochondria accelerate cell death by interrupting the IAP–caspase interaction.

3.3 The Bcl-2 Superfamily Proteins of the Bcl-2 family are the main regulators of the intrinsic apoptotic pathway [52]. The Bcl-2 family is classified into three subfamilies all sharing four domains, the Bcl-2 homology domains (BH1–BH4). The presence of each of these domains is essential for their function. The first Bcl-2 subfamily comprises the antiapoptotic molecules Bcl-2, Bcl-XL, Bcl-w, Mcl-1, A1, and Bcl-B, all of which have the four BH domains. The next subfamily consists of the proapoptotic molecules Bax, Bak, Bok, and Bcl-rambo, which have BH1, BH2, and BH3 domains. The last subfamily is composed of the proapoptotic proteins Bid, Bim, Bad, Bik, Puma, and Noxa; the members of this subfamily share homology only at the BH3 domain, and this is why they are

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also referred to as “BH3-only proteins.” Each subfamily has a different function, which depends on the presence or absence of the different BH domains, although, all Bcl-2 family members control the release of proapoptotic factors from the mitochondria, each in a different way. The antiapoptotic proteins Bcl-2 and Bcl-XL are found on the outer mitochondrial membrane, stabilizing the mitochondrial integrity. Their function is counteracted by the proapoptotic Bcl-2 family members Bax, Bak, and Bok, whose aim is to destabilize the outer mitochondrial membrane and cause the release of cytochrome c and other proapoptotic factors, such as Smac/Diablo, AIF, heat shock protein 60, and endonuclease G, located in the mitochondrial intermembrane space. Cytochrome c and Smac/Diablo are involved in caspase activation, whereas endonuclease G and AIF participate in caspase-independent apoptosis-induced nuclear changes. The function of the third subfamily of proteins, containing the BH3 domain only, target the antiapoptotic molecules Bcl-2 and Bcl-XL, where they neutralize their action, and thus indirectly activate Bax, Bak, and Bok. Examples of such BH3-only proteins include Puma, Noxa, and Bad. On the other hand, Bid and Bim directly activate Bax and Bak. Thus, when caspase-8 activates Bid by proteolysis, during the extrinsic apoptotic pathway, its cleaved form translocates to the mitochondria and leads to the activation of the proapoptotic Bax and Bak and thus initiation of the intrinsic pathway. The remarkable characteristic of the Bcl-2 family members is that they exert effects on each other by forming stable heterocomplexes. Not so long ago it was discovered that caspase-10 can also activate Bid, but via cleavage at another site, resulting in a smaller cleaved form of Bid. It is not clear though whether this differential Bid cleavage happens after the initiation of the extrinsic pathway, and whether it affects Bid’s mode of interaction with the other proapoptotic molecules.

3.4 p53, DNA Damage, and Apoptosis Apart from the Bcl-2 superfamily of proapoptotic factors, several other factors have been shown to regulate the mitochondrial outer membrane permeabilization; such an example is p53. The transcription factor p53 can induce apoptosis by regulating the expression of the proapoptotic BH3-only protein Puma. Further, it has been shown that p53 can provoke apoptosis, via direct interaction with Bax and Bak or even by binding to Bcl-2 and Bcl-XL, and inhibiting their function [53]. Other interesting findings suggest that the nuclear protein Ku70, for example, apart from DNA repair also translocates to the cytosol and inhibits Bax activation. Furthermore, the nuclear protein TR3 also translocates to the cytoplasmic area and interacts with Bcl-2 and thus promotes apoptosis. It has

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also been reported that histone 1.2 is released from the nucleus after DNA damage, interacts with Bcl-2, and causes mitochondrial outer membrane permeabilization.

4 Ion Channels and Apoptosis As already described, one of the morphological signatures of apoptosis is cell shrinkage or in other terms AVD, a process which anticipates caspase activation and cytochrome c release [54]. The process of cell shrinkage depends on ion trafficking through the plasma membrane, and in particular K+ locomotion. Under basal conditions, cytoplasm contains high concentration of K+ ions, which has to be preserved for the maintenance of the cell’s volume. A high concentration of K+ ions is required for the suppression of caspase activity. The intracellular potassium levels, and therefore the cell volume, are regulated by the Na+–K+-ATPase pump, and the K+ channels, leaving the intracellular space with a high concentration of K+ and a low concentration of Na+. On the other hand, in the extracellular space, there are low levels of K+ and high levels of Na+. The opening of K+ channels will cause efflux of K+ ions to the extracellular space, subsequent Cl- release, and finally water leaves the cell via the aquaporins, to maintain the balance between the intracellular and the extracellular space. This causes AVD and imminent apoptosis. There are four different classes of K+ channels, the voltage-gated K+ (Kv) channels, the Ca2+-activated K+ (KCa) channels, the two-pore-domain K+ (K2P) channels, and the inward-rectifier K+ (KIR) channels. Several studies in different cell types have shown that Kv channels are essential for programmed cell death. Kv channels apart from regulating AVD in the early steps of apoptosis, are also able to inhibit caspase activity immediately after the apoptotic stimulus reaches a cell, since loss of intracellular K+ ions leads to activation of caspases [55]. It is now clear that K+ channels are a target of the antiapoptotic factor Bcl-2. Thus, Bcl-2 apart from acting on Bax and Bid also discourages apoptosis progression, by inhibiting Kv channels. Furthermore, the decrease in the level of K+ leads to an overload of cytoplasmic Ca2+, which leads, for example, to increased vasoconstriction of the pulmonary vascular smooth muscle cell and subsequent overproliferation.

5 Endoplasmic Reticulum and Cellular Apoptosis Many studies have indicated that the ER plays a major role in apoptosis induction as well as in other types of cell death, such as autophagy [56]. The ER is the place where proteins

22  Biochemistry and Cellular Mechanisms of Apoptosis in Vascular Smooth Muscle and Endothelial Cells

are folded and secreted proteins undergo posttranslational modifications. In the presence of ER stress leading to alterations in the ER environment, for example, due to oxidative stress, or accumulation of unfolded and misfolded proteins, the unfolded protein response (UPR) pathway is activated, trying to resolve these changes. Three ER kinases participate in this process, protein-kinase-like ER kinase (PERK), inositol-requiring kinase 1 (IRE1), and activating transcription factor 6 (ATF-6). The UPR is mainly a survival pathway; however, if the problem of protein misfolding remains unresolved, it serves as a proapoptotic machinery. The kinases PERK, IRE1, and ATF-6 are involved in both the antiapoptotic and the proapoptotic pathways. In the latter case, it has been shown that PERK and ATF-6 can induce the expression of C/EBI homologous protein (CHOP), which decreases the expression levels of the antiapoptotic factor Bcl-2, and leads to the upregulation of proapoptotic genes [57,58]. Further studies have shown that IRE1, via TRAF2 activation, is able to induce the JNK signaling cascade, and subsequent apoptosis via phosphorylation of key proteins [59]. In most of the cases, the ER-stress-induced apoptosis is regulated by the Bcl-2 family of proteins [59]. It has been also suggested that a pathway similar to the extrinsic and intrinsic pathways takes place at the ER with key initiator molecules, the murine caspase-12 and the human caspase-4. Briefly, activation of caspase-12 or caspase-4 leads to the activation of caspase-9 or caspase-3 in a mitochondria-independent manner [60]. Thus, it has been shown that the murine caspase-12 is indeed involved in the apoptotic cell death. The same is not valid for the human caspase-12. Evidence suggests that human caspase-12 is involved in immune response [61] rather than apoptosis regulation. On the other hand, human caspase-4 is partially located at the ER and is only activated in response to ER stress [60]. Similar pathways have been shown to activate murine caspase-12 and human caspase-4. It is well known that ER stress causes release of the ER-stored Ca2+, leading to an increase in the cytoplasmic Ca2+ concentration and a later increase in the calpain expression levels. Thus, caspase-12 cleavage and subsequent activation have been correlated to calpain activation, induced by the release of ER-stored Ca2+. Further, it has been shown that calpain inhibition or Ca2+ chelation block caspase-12 activation in a series of different experimental settings [62]. Caspase-7 has also been shown to activate caspase-12. ER stress leads to the recruitment of caspase-7 to the ER, where it forms a complex with caspase-12 and the ER chaperone GRP78. Members of the Bcl-2 superfamily also regulate caspase-12 activity. Independent studies have shown that caspase-12 activation occurs in a Bak/Bax-dependent manner. ER-stresstargeted Bak activates caspase-12 in a caspase-7-independent but Ca2+-dependent mode. Less is known about the caspase-4 activation process; however, it is thought that it follows an activation path similar to that of caspase-12.

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Since caspase-12 and caspase-4 are not ubiquitously expressed, in many cases, ER-stress-induced apoptosis happens in a caspase-9-dependent pathway. It also should be stated that many studies have shown that many ER stressors lead to cytochrome c release and thus involve the intrinsic pathway in apoptosis induction. This has been further supported by the fact that Apaf-1 loss inhibits ER-stress-induced apoptosis.

6 Apoptotic Cell Execution and Clearance Finally, during the execution phase of apoptotic cell death, there is proteolysis of specific proteins, such as inhibitors of apoptosis, or other caspases, as well as proteins, which when cleaved lead to the characteristic morphology of apoptotic cells, such as fragmentation of the DNA, membrane blebbing, or formation of apoptotic bodies. Examples of proteins are ICAD/DFF45, the inhibitor of the nuclease responsible for the DNA degradation, nuclear lamina, as well as cytoskeletal regulators [63]. After understanding how important cellular apoptosis is, as well as the diversity of cellular mechanisms controlling this process, it is equally important to get a deeper insight to the process of clearance of the apoptotic bodies, after the cell has committed suicide [64]. The initiating step in this process is the recognition of the apoptotic cell that needs to be removed by either neighboring cells or macrophages and dendritic cells. Thus, the apoptotic cell undergoes some changes that mark it as ready for removal. After the apoptotic cell has been internalized by phagocytes, it goes through a “maturation process” at highly acidified membrane-bound structures called phagosomes, which eventually will fuse with the highly acidic lysosomal structures and lead to complete apoptotic cell destruction. Studies have shown that defects in the removal process of apoptotic bodies can lead to autoimmune disease, for example. The process of apoptotic cell phagocytosis, also known as efferocytosis, is a rather complex and challenging one. In a more detailed view of it, specific plasma membrane receptors on the mammalian phagocyte, such as BAI1, LRP1, MEGF10, TIM4, and MER, are able to recognize the apoptotic cell, in a not that well understood process. Two different pathways have been discovered in both mammals and C. elegans. One pathway suggests that the brain-specific angiogenesis inhibitor 1 (BAI1) on the phagocyte plasma membrane can bind to phosphatidylserine on apoptotic cells and induce their internalization by forming a complex with the guanine nucleotide exchange factors ELMO and Dock180, and further with the small GTPase Rac, regulating the phagocyte cytoskeleton and resulting in better internalization [65]. The second pathway suggests that the protein careticulin on the apoptotic cells induces apoptotic cell engulfment in an low density lipoprotein receptor-related protein (LRP)-dependent manner.

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Upon engulfment and internalization, the apoptotic cell is surrounded by the phagocyte plasma membrane, forming the phagosome. For the degradation process to start, the phagosome needs to reach a sufficiently low pH so that the acidic proteases, belonging to the cathepsin family, can be activated and proceed to the degradation step. The degradation step is very critical because the degraded parts of the cell will be used for presentation on the phagocyte surface via the major histocompatibility complex class I, a process required for the maintenance of self-tolerance. The acidification process of the phagosome is a two-step process. First, there is a small reduction in the pH, which is followed by a translocation of the V-type ATPases into the phagosome, to cause further acidification of its contents. These proteins seem to come with cathepsins from the trans Golgi network. It has been suggested that V-type ATPases might also have a further role in phagosome maturation; however, what this might be is not completely clear and well understood. Most of the knowledge on apoptotic cell clearance and phagosome maturation comes from genetic studies focusing on the receptor-mediated endocytosis in C. elegans, which is responsible for the internalization of particles smaller than 0.5  mm. It has been suggested that the protein dynamin as well as the members of the Rab GTPase family, RAB-5 and RAB-7, whose activity depends on the presence of GTP, are involved in this process. The final step is the acquisition of lysosomal markers such as lysosome-associated membrane proteins 1 and 2 and subsequent fusion to the lysosome, where complete apoptotic cell degradation takes place.

7 Pulmonary Arterial Hypertension and Apoptosis It should be quite clear by now that apoptosis is a very well controlled process. Tissue homeostasis requires a balance between cellular proliferation and apoptosis. Failure to do so inevitably leads to disease. In the context of the pulmonary vasculature, an imbalance between apoptosis and proliferation has been linked to the development of pulmonary arterial hypertension (PAH). PAH is a rare but devastating disease with an annual incidence, one to two per million of population. The hallmark of PAH is excessive vascular remodeling in the small pulmonary arteries, loss of capillaries, and, in severe PAH, plexiform lesions. The process of vascular remodeling is very well orchestrated, with all the cell types of the vessel wall participating. In particular, there is unconfined pulmonary artery smooth muscle cell (PASMC) proliferation resulting from pulmonary artery endothelial cell (PAEC) injury or dysfunction. Extensive research has taken place aiming to elucidate the causes and pathways leading to

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PAH; however, the pathological mechanism still remains unclear. Substantial studies have indicated that endothelial cell dysfunction might be the trigger for the initiation of this disease. Different stimuli have been suggested to cause endothelial cell dysfunction, such as continuous shear stress and strain, an impaired bone morphogenetic protein (BMP)/ transforming growth factor-b (TGF-b) signaling, the proproliferative protein survivin, KV channels, hypoxia, and ROS. In more detail, the discovery that mutations in the bmpr-2 gene [66,67], a member of the TGF-b receptor superfamily, are found in the majority of familiar PAH cases as well as in some sporadic PAH cases turned the attention of researchers toward the BMP/TGF-b signaling cascade. It was thus demonstrated that the BMP signaling, and in particular that of BMP-4, induces proliferation in PAECs and apoptosis in PASMCs. The presence of mutations in the bmpr2 gene, leading to impaired BMP signaling, provoked PAEC apoptosis in a caspase-8- and caspase-9-dependent manner, and PASMC proliferation, a phenotype resembling the PAH development [68]. There is further evidence that the impaired BMP signaling leads to increased TGF-b response, which is responsible for the uncontrolled PASMC proliferation, in the animal model of monocrotaline-induced pulmonary hypertension [69]. Further evidence suggests that shear stress in the pulmonary vasculature induces endothelial cell apoptosis and thus leads to capillary loss and contributes to uncontrolled PASMC proliferation in the larger vessels. On the basis of such studies, apoptotic endothelial cells release cytokines such as TGF-b1, platelet-derived growth factor, and vascular endothelial growth factor, which regulate PASMC proliferation and apoptosis, respectively, in PAH [70]. In addition, there is convincing evidence that the inhibitor of apoptosis, survivin, is de  novo expressed in both the PASMCs and the PAECs in remodeled vessels of PAH patients, indicating that inhibition of apoptosis could be responsible for the uncontrolled proliferation of the vessel wall cells, and thus PAH development [71]. Furthermore, there is a significant decrease in the expression of KV channels in tissue from PAH patients, which induces PASMC proliferation, via the increased intracellular K+ levels, which is known to inhibit caspase activity. As mentioned before, KV channels are important for the volume decrease of apoptotic cells. The decrease of KV channel expression induces efflux of Ca2+ ions, severe vasoconstriction, and increased PASMC proliferation. Hypoxia, which occurs during pulmonary hypertension development, regulates K+ ion concentration by inhibiting several potassium channels [54]. Hence, under chronic hypoxia, reduction of K+ ions and subsequent increase of Ca2+ concentrations leads to inhibition of apoptosis and increased proliferation of PASMCs. Although the role of ROS in these processes is still controversial, a perturbation in ROS signaling also plays a role in disease development owing to increased mitochondrial

22  Biochemistry and Cellular Mechanisms of Apoptosis in Vascular Smooth Muscle and Endothelial Cells

hyperpolarization followed by PASMC depolarization and subsequent increased vasoconstriction and excessive proliferation, a process which is also common in cancer [72].

8 Conclusion Apoptosis is a very well controlled process, essential for cellular and tissue homeostasis. It is clear that disturbances in the apoptotic pathway can lead to disease. In the pulmonary vasculature, for example, an imbalance between cellular proliferation and apoptosis in the vascular cells leads to PAH. As already described, there are several factors that regulate these processes, leading to disease. The next step would be to target such factors and such cellular processes as a therapeutic strategy for pulmonary hypertension.

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Chapter 23

The Coagulation Cascade and Its Regulation James T.B. Crawley, Jose R. Gonzalez-Porras, and David A. Lane

Abstract  Under normal conditions the blood circulates freely within the confines of the vascular system, carrying oxygen, nutrients, and hormonal information around the body and removing metabolic waste. If the blood gains access to extravascular sites or if the vasculature becomes pathologically challenged, the processes associated with hemostasis may be activated. Employing finely regulated positive- and negative-feedback loops, hemostasis represents the process responsible for both generating and regulating the plug that prevents blood loss following vessel injury. The components of hemostasis are both complex and heterogeneous, consisting of cell-associated and plasma-borne proteins, glycosaminoglycans, and platelets. To ensure the maintenance of both blood fluidity and yet also vascular integrity, the hemostatic response must be fast, and yet specifically localized to the site of vessel damage. In this way, the likelihood of further challenges being imposed upon the vascular system may be minimized. In the event of vascular injury, circulating platelets are rapidly recruited to the damaged region. These platelets become activated and aggregate, forming an unstable platelet plug (primary hemostasis) that immediately limits the loss of blood. The coagulation cascade is initiated simultaneously. Blood coagulation ultimately results in the deposition of fibrin, a molecular scaffold that consolidates the platelet plug and allows tissue repair mechanisms to act. This chapter focuses on the coagulation system and its regulation. An important protease generated during coagulation is thrombin, a serine protease with numerous substrates. As the physiological end points of coagulation are the fibrin clot and platelet activation, which are both determined by thrombin, regulation of coagulation can be viewed in its simplest form as the control of the generation and activities of thrombin. Keywords  Haemostasis • coagulant • thrombin • thrombosis • platelet • pulmonary embolism J.T.B. Crawley (*) Department of Haematology, Imperial College London, Hammersmith Hospital Campus, 5th Floor Commonwealth Building, Du Cane Road, London W12 ONN, UK e-mail: [email protected]

1 Introduction Under normal conditions the blood circulates freely within the confines of the vascular system, carrying oxygen, nutrients, and hormonal information around the body and removing metabolic waste. If the blood gains access to extravascular sites or if the vasculature becomes pathologically challenged, the processes associated with hemostasis may be activated. Employing finely regulated positive- and negative-feedback loops, hemostasis represents the process responsible for both generating and regulating the plug that prevents blood loss following vessel injury. The components of hemostasis are both complex and heterogeneous, consisting of cell-associated and plasma-borne proteins, glycosaminoglycans, and platelets. To ensure the maintenance of both blood fluidity and yet also vascular integrity, the hemostatic response must be fast, and yet specifically localized to the site of vessel damage. In this way, the likelihood of further challenges being imposed upon the vascular system may be minimized. In the event of vascular injury, circulating platelets are rapidly recruited to the damaged region. These platelets become activated and aggregate, forming an unstable platelet plug (primary hemostasis) that immediately limits the loss of blood. The coagulation cascade is initiated simultaneously. Blood coagulation ultimately results in the deposition of fibrin, a molecular scaffold that consolidates the platelet plug and allows tissue repair mechanisms to act. This chapter focuses on the coagulation system and its regulation. An important protease generated during coagulation is thrombin, a serine protease with numerous substrates. As the physiological end points of coagulation are the fibrin clot and platelet activation, which are both determined by thrombin, regulation of coagulation can be viewed in its simplest form as the control of the generation and activities of thrombin.

2 Overview of Coagulation The vasculature is lined by a single layer of endothelial cells. These cells do not normally support the initiation and propagation of coagulation owing to the presence of anticoagulant

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Fig. 1  Coagulation cascade. Coagulation is initiated upon vessel damage, which leads to the exposure of tissue factor (TF) to plasma clotting factors (1). TF–activated factor VII (FVIIa) can activate factor X (FX) and factor IX (FIX) (2). Activated FX (FXa) activates prothrombin inefficiently (3) leading to the generation of trace amounts of thrombin. Thrombin can then activate factor VIII (FVIII) and factor V (FV) (4 ), which function as nonenzymatic cofactors for activated FIX (FIXa) and FXa, respectively. FIXa-activated FVIII (FVIIIa) catalyzes the conversion of increased quantities of FXa (5 ), which in conjunction with activated FV (FVa) leads to the enhanced generation of thrombin (6 ). Thrombin at the site of vessel damage converts fibrinogen to fibrin, which is the molecular scaffold of a clot (7 ). Abbreviation: ProT, prothrombin

molecules on their surfaces. The extrinsic or tissue factor (TF)dependent pathway of coagulation represents the major route by which thrombin generation is initiated in response to vessel damage (Fig. 1). Coagulation is activated by the exposure of subendothelial TF to the blood. TF is an approximately 45 kDa integral membrane protein, and is the primary initiator of blood coagulation and, unlike most other procoagulant factors, it does not require proteolytic activation [1]. TF is considered to be normally located at extravascular sites that are not usually exposed to the blood (i.e., on adventitial fibroblasts and variably on vascular smooth muscle cells) [2]. Therefore, only at sites of vascular injury or endothelial disruption may the blood encounter TF-presenting cells, and in turn activate the coagulation cascade. TF is also expressed in a cell-specific manner within certain organs (i.e., lungs, brain, heart, testes, uterus, and placenta). TF in these locations may provide further hemostatic protection against vascular injury in these organs. More recently, circulating microparticle-associated TF has also been identified in blood and implicated in hemostatic plug development and in thrombus formation [3, 4]. Following mechanical injury to the vasculature, blood (containing plasma clotting factors) gains access to cellular and

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matrix components that are normally separated from the blood by the endothelial barrier. The inactive zymogen, factor VII (FVII), circulates in plasma at a concentration of approximately 10 nM. A fraction (about 1%) of total plasma FVII circulates in a potentially active form, FVIIa [5]. TF binds FVIIa with high affinity. The interaction between TF and FVIIa occurs across an extensive interface between the two proteins [6]. This binding reaction occurs in a 1:1 stoichiometry and induces an allosteric change in the FVIIa serine protease domain that modulates its activity (rather than its substrate-binding function) [7]. It is for this reason that although FVIIa can bind its substrate, factor X (FX) (at least in vitro), in the absence of TF, activation of FX does not occur [8]. The trace amount of TF–FVIIa that forms following the exposure of TF-presenting cells to the blood activates the extrinsic pathway (Fig.  1). TF–FVIIa activates the plasma zymogens factor IX (FIX) and FX [9]. Additional TF–FVIIa may then be generated locally from inactive TF–FVII complexes (as TF also binds to FVII with high affinity), either by autoactivation or through positive-feedback loops involving activation by activated FX (FXa) or activated FIX (FIXa). The limited quantities of FXa that are generated via this route facilitate the inefficient conversion of trace quantities of prothrombin to thrombin. The low concentrations of thrombin that arise enable the feedback activation of factor VIII (FVIII) and factor V (FV), nonenzymatic cofactors in the tenase and prothrombinase complexes, respectively [10]. The major site of thrombin generation is on the membrane of activated platelets, which provide a negatively charged surface upon which the components of the coagulation cascade assemble. Here, the prothrombinase complex, consisting of FXa, activated FV (FVa), and prothrombin, accelerates the formation of thrombin. FV, which circulates in plasma but is also released locally by activated platelets, must first be activated by thrombin. Once activated and part of the complex, FVa enhances the ability of FXa to generate thrombin by about 300,000-fold, leading to a rapid burst in thrombin generation at the site of vessel damage. Thrombin has many functions that influence both coagulation and the vascular system (see Sect. 5). Its procoagulant functions include (1) inducing the conversion of fibrinogen to fibrin (thrombin is the only clotting enzyme capable of this function); (2) activating platelets, which provide the procoagulant surface for further thrombin generation; (3) proteolytically activating FVIII, the cofactor for FIXa in the tenase complex; (4) conceivably activating factor XI (FXI), which provides additional FIXa for the tenase complex; (5) activating FV, the cofactor for FXa in the prothrombinase complex; (6) directly activating the TF–FVII complex; and (7) activating factor XIII (FXIII), a transglutaminase, which in its active form cross-links fibrin to form an insoluble clot. In addition to these procoagulant functions, thrombin also activates thrombin-activatable fibrinolysis inhibitor (also termed

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Table 1  Plasma clotting factors and inhibitors. Circulating coagulation proteins are listed with their molecular mass, plasma concentration, domain organization, and role in coagulation Molecular Plasma concentraComponent mass (kDa) tion (mM) Domain structure Major role of active component in hemostasis Factor V 330 0.03 A1, A2, B, A3, C1, C2 Cofactor for activated factor X Factor VII 50 0.01 Gla, 2× EGF, SP Complexes TF to activate factor X Factor VIII 330 0.003 A1, A2, B, A3, C1, C2 Cofactor for activated factor IX Factor IX 56 0.09 Gla, 2× EGF, SP Complexes activated factor VII to activate factor X Factor X 59 0.136 Gla, 2x EGF, SP Complexes activated factor V to activate prothrombin Factor XI 160 0.031 2× (4× Apple, SP) Activates factor IX Factor XIII 320 0.031 2× A, 2× B Cross-links fibrin clot Fibrinogen 340 9.09 A, B, C Clot scaffold Prothrombin 72 1.38 Gla, 2× kringle, SP Deposition of fibrin TFPI 43 0.002 3× Kunitz Inhibitor of TF–activated factor VII Antithrombin 58 2.50 Serpin Inhibits thrombin, activated factor IX, and activated factor X Protein C 62 0.065 Gla, 2× EGF, SP Inhibits activated factor VIII and activated factor V Protein S 69 0.35 Gla, 4× EGF, SHBG Cofactor for protein C TFPI tissue factor pathway inhibitor, EGF epidermal growth factor, SP serine protease domain, SHBG sex-hormone-binding-globulin-like domain TF tissue factor

“thrombin-activatable procarboxypeptidase B”; TAFI), which cleaves C-terminal Lys and Arg residues from fibrin – this inhibits the binding of fibrinolytic enzymes and, in turn, protects the fibrin clot from degradation. Finally, thrombin also confers anticoagulant and cell-signaling actions. Many of the clotting factors and inhibitors of coagulation are listed in Table 1 along with their function and domain organization.

3 Structure of Plasma Clotting Factors Many of the circulating plasma clotting factors are synthesized primarily by the liver. They all have a modular domain organization, and many share several common structural features, which are discussed next. Those of the anticoagulant proteins are discussed in the later sections.

3.1 Gla Domains Several of the clotting factors (prothrombin, FVII, FIX, FX – and also anticoagulant proteins, protein C, protein S, and protein Z) belong to the vitamin K dependent family of proteins. This family of proteins contain a homologous N-terminal domain that generally contains around 45 amino acids, of which between nine and 11 are Glu residues and become g-carboxylated prior to secretion – a process that is dependent upon vitamin K [11]. These g-carboxylated Glu (Gla) residues define the structural and functional properties of this domain, referred to as the Gla domain. This domain is essential for conferring the affinity of this family of proteins for phospholipid membranes. The Gla domains of vitamin K dependent proteins are highly conserved and are all thought to fold in a similar manner. Despite their similarity, the precise affinity for phospholipid

varies quite widely among vitamin K dependent proteins. The additional carboxyl group that is added to Gla residues confers strong Ca2+-binding properties on the domain. The binding of Ca2+ ions induces a dramatic conformational change that reveals the phospholipid-binding site on the Gla domain. This includes the exposure of three hydrophobic residues in what is termed the w-loop [11]. The hydrophobic side chains of these residues insert into phospholipid membranes. The Gla-coordinated Ca2+ ions also interact ionically with the polar head groups of the phospholipid surface to strengthen this interaction. The clinical importance of the Gla domain in the function of clotting factors is demonstrated by the use of vitamin K antagonist drugs, such as warfarin. In the presence of warfarin, vitamin K is inhibited, resulting in inhibition of a specific carboxylase. The Glu residues are then not g-carboxylated, resulting in dysfunctional protein that lacks specific phospholipid binding properties. As membrane binding concentrates/localizes clotting factors to the surfaces upon which coagulation occurs, the lack of Gla domain function essentially ablates assembly of functional complexes on the surface of platelets and reduces thrombin generation.

3.2 Epidermal-Growth-Factor-Like Domains Epidermal growth factor (EGF)-like domains are also common features of coagulation proteins, and are found in FVII, FIX, and FX [as well as in protein C, protein S, thrombomodulin (TM), and protein Z]. These domains are named because of their sequence homology to domains contained in EGF. These domains contain three disulfide bonds that are paired in a generally conserved manner, although occasionally an additional bond (i.e., protein C), or alternative pairing of cysteines (i.e., TM) may occur. EGF-like domains participate in protein–protein interactions and therefore help define

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the functional specificity. These domains can also coordinate Ca2+ ions, most utilizing specific Asp or Asn residues that are posttranslationally b-hydroxylated.

3.3 Serine Protease Domains The serine proteases of the coagulation cascade [thrombin, FVIIa, FIXa, FXa, activated FXI (FXIa), and activated factor XII (FXIIa)] all contain protease domains similar to that of chymotrypsin. All of these proteases are secreted as inactive zymogens and require activating before they can fulfill their enzymatic roles. Activation usually requires a single bond cleavage in the protease domain, inducing a change in its conformation. The cleavage may or may not lead to release of an activation peptide. For all proteases except FVIIa (which requires additional binding to TF), cleavage activation exposes residues that form the active site, Ser193, His57, and Asp102 (numbering based on chymotrypsin). The characteristic serine protease fold also results in a conserved Na+-binding loop, Ca2+-binding sites, and often allows unique charged patches that confer specificity for substrate recognition.

3.4 Structure of Clotting Cofactors (FV and FVIII) FV and FVIII are homologous precursors that become functional cofactors when activated by thrombin [12]. FVa and activated FVIII (FVIIIa) dramatically enhance the enzymatic activities of FXa and FIXa in the tenase and prothrombinase complexes, respectively. Both FV and FVIII contain two types of internal repeats (A and C domains) [13]. The N-terminal region of both procofactors comprises of two A domains (A1 and A2) and the C-terminus consists of a further A domain and two C domains (A3, C1, and C2). In both FV and FVIII the A3, C1, and C2 domains are separated from the A1 and A2 domains by a nonhomologous B domain. High-affinity phospholipid-binding sites (nanomolar range) are present in both FV(a) and FVIII(a) in their C2 domains [14]. Structural data have also implicated the C1 domains in this interaction [15]. Activation of these cofactors occurs by the cleavage of at least three peptide bonds (see later). This converts the proteins into two-chain molecules (heavy and light). The heavy chain of FVa (A1 and A2 domains) remains noncovalently associated with the light chain (A3, C1, and C2 domains) via a highaffinity Ca2+-dependent interaction between the A1 and A3 domains [16]. Although FVIIIa is activated in a similar manner, an additional cleavage between the A1 and A2 domains results in a comparatively weak noncovalent association between the A2 domain and the rest of the molecule.

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4 Anticoagulant Pathways The generation of thrombin and the formation of a fibrin clot occur rapidly at sites of vascular injury through the pathways and feedback mechanisms outlined earlier. However, if these processes were to proceed unhindered as described, systemic blood coagulation might otherwise ensue. For this reason, inhibitory mechanisms [namely, TF pathway inhibitor (TFPI), activated protein C (APC), and antithrombin] exist that serve to limit the extent of coagulation and to localize both the generation and the activity of thrombin to the site of vessel damage. The cumulative actions of these anticoagulant mechanisms contribute to the observed biphasic generation of thrombin. The initiation phase, during which limited quantities of thrombin are formed, arises following TF exposure to blood, and is strongly influenced by the inhibitory actions of TFPI. Subsequently, when the positive-feedback loops are activated, thrombin is generated rapidly. As this propagation phase does not depend upon TF–FVIIa to generate FXa, the inhibitory actions of TFPI become less evident. This provides thrombin with the opportunity to fulfill its myriad functions, including the deposition of fibrin. Regulation is then primarily conferred by the combined actions of the protein C and antithrombin pathways, which localize and subsequently diminish the procoagulant response.

4.1 Tissue Factor Pathway Inhibitor The principal inhibitor of TF-mediated initiation of coagulation is TFPI. TFPI binds and inhibits FXa [17]. The resulting TFPI–FXa complex acts in a negative-feedback loop through the binding and inactivation of TF–FVIIa [18]. The initiating procoagulant stimulus can then be switched off. This ensures that a small procoagulant stimulus does not elicit uncontrolled generation of thrombin. TFPI is a 43-kDa Kunitz-type inhibitor [19] that contains three tandemly arranged Kunitz domains. The major site of TFPI production is in endothelial cells [20], which constitutively express the protein under normal conditions. TFPI is also normally expressed by vascular smooth muscle cells, megakaryocytes/platelets, monocytes, fibroblasts, and cardiomyocytes. TFPI secreted by endothelial cells circulates in plasma at a concentration of approximately 2.5 nM [21]. Most of the plasma pool circulates in truncated forms and/or in association with low-density lipoproteins [22], and as a consequence exhibits markedly reduced inhibitory potential. Only approximately 10% of plasma TFPI circulates in a full-length, free 43-kDa form. TFPI also directly associates with the surfaces of cells that express the protein.

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4.2 TFPI Anticoagulant Function The inhibitory activity of TFPI involves the Kunitz domains mimicking the substrate of the target protease. Once bound, the target enzyme fails to proteolyze the P1–P1¢ peptide bond, which confers a tight-binding, competitive inhibition. The first Kunitz domain of TFPI (K1) interacts directly with TF–FVIIa [23]. K1 binds ionically to the active site of FVIIa in this complex [23]. The second Kunitz domain (K2) mediates the binding and inhibition of FXa [24]. The third Kunitz domain (K3) has no described inhibitory function. The anticoagulant function of TFPI involves the FXa-dependent inhibition of TF–FVIIa (Fig. 2). By targeting FXa and FVIIa, TFPI directly inhibits the initiation phase of coagulation. The first stage in the inhibitory function of TFPI involves the reversible inhibition of FXa (Fig. 2). This occurs via the ionic binding of

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FXa to either full-length plasma or cell-associated TFPI. This interaction involves the P1 residue (Arg107) in TFPI K2 interacting with the active site of FXa [23]. As truncated forms of TFPI or proteolyzed TFPI are less potent inhibitors of FXa, it is likely that other parts of the TFPI molecule are also important for the interaction with this serine protease. Once FXa is assembled in the prothrombinase complex with FVa, it is protected from TFPI inhibition [25], largely owing to competition for the active site of FXa with prothrombin, which is present at much higher concentrations than TFPI. The second stage in the inhibition of TF-dependent coagulation involves the binding of the TFPI–FXa complex to TF–FVIIa (Fig. 2). During this step, the P1 residue in TFPI K1 (Lys36) interacts with the active site of FVIIa. The FXa-dependent inhibition of TF–FVIIa by TFPI results in the formation of an inactive TFPI–FXa–TF–FVIIa quaternary complex on a cell membrane. Although, as presented here, this process is frequently described as a twostage process, first involving the binding of free FXa and thereafter binding to TF–FVIIa, kinetic data favor a model in which TFPI interacts with FXa that has not yet been released from the activating TF–FVIIa complex [26]. Consequently, the amount of FXa that escapes inhibition is directly related to the concentration/availability of TF.

4.3 Overview of the Protein C Pathway

Fig.  2  The inhibition of TF-dependent coagulation by TF pathway inhibitor (TFPI ). Following the formation of the TF–FVIIa complex, the circulating zymogen, FX, binds to TF–FVIIa (1) and thereafter becomes activated (2). Once activated, FXa may dissociate (3a) from the activating complex. FXa may then elicit its procoagulant function or, alternatively, become inactivated by cell-associated or plasma TFPI via its second Kunitz domain. This complex can now reassociate with TF–FVIIa (4), with the first Kunitz domain of TFPI binding to the active site of FVIIa. Although frequently described as a two-stage process, kinetic studies favor a model whereby TFPI binds and inactivates TF–FVIIa–FXa prior to FXa release (3b/4). In each case the resulting inactive quaternary complex is the same

A second coagulation inhibitory pathway is the protein C anticoagulant pathway. Protein C bound to endothelial cell protein C receptor (EPCR) is activated on the surface of endothelial cells by thrombin bound to the integral membrane protein, TM [27]. APC, supported by its cofactor, protein S, proteolytically inactivates the nonenzymatic cofactors FVIIIa and FVa [28], which limits the further generation of thrombin. As the intact endothelium (i.e., adjacent to the site of vascular injury) normally expresses TM, the action of thrombin can be “switched” to impart an anticoagulant function. Conversely, at sites of endothelial disruption/dysfunction, the procoagulant actions of thrombin are favored. The action of thrombin can thereby be modulated depending on its location relative to the site of injury. Once released from EPCR and the thrombin–TM complex, APC can proteolytically inactivate FVIIIa and FVa by cleavage of specific peptide bonds. These APC-mediated reactions are augmented by its cofactor, protein S, which aids in both localizing APC to negatively charged membranes and favourably orienting the active site of APC toward its substrates. The following sections describe the components of this pathway in more detail.

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4.4 Thrombomodulin The primary site of TM expression is on the surface of the vascular endothelium, where it functions as a receptor/cofactor for thrombin in the activation of protein C. TM accelerates activation of protein C by thrombin by approximately 20,000-fold, and protein C almost certainly does not become activated physiologically in its absence. TM has a modular structure that is made up of an N-terminal lectin-like domain, a hydrophobic region, six tandemly arranged EGF domains, a Ser/Thr-rich region, a transmembrane region, and a C-terminal short cytoplasmic tail [29]. EGF domains 5 and 6 contain a charged patch that interacts with anion-binding exosite I on the surface of thrombin [30], whereas EGF domain 4 contains a low-affinity protein C binding site [31]. The Ser/Thr-rich domain, located between TMEGF6 and the plasma membrane, contains an attachment site for chondroitin sulfate, a glycosaminoglycan moiety that is strongly negatively charged. TM may be synthesized with or without this modification and this varies between different vascular beds. The presence of chondroitin sulfate enhances thrombin binding affinity for TM by approximately 13-fold, when compared with unmodified TM [32]. This is manifest by an additional site of interaction between the chondroitin sulfate on TM and exosite II on the surface of thrombin [33]. The lectinlike domain and EGF domains 1, 2, and 3 of TM are not required for protein C activation.

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the serine protease domain following cleavage of the Arg169– Leu170 peptide bond by the thrombin–TM complex, resulting in the activation of the enzyme. An important feature of the serine protease domain is an exosite region of positively charged residues [35], similar to exosite I of thrombin. This exosite is important for recognition of FVa during its inactivation. The importance of protein C in the downregulation of thrombin generation is supported clinically by the strong association between protein C deficiency and thrombosis.

4.6 Endothelial Cell Protein C Receptor EPCR is a type 1 transmembrane glycoprotein, with a molecular size of 46 kDa [36]. EPCR is expressed primarily by vascular endothelial cells, but preferentially by those of large vessels [37, 38]. The primary role of EPCR is as a cellsurface receptor for protein C/APC [39], which it binds with high affinity through its Gla domain [40]. EPCR binds both protein C and APC with similar affinity (Kd 30–60 nM) [39]. EPCR binding of protein C serves to concentrate/localize protein C in the plain of the endothelial cell plasma membrane. In this way, EPCR augments the rate of protein C activation by thrombin–TM on the endothelial surface by approximately 20-fold [41]. Binding of APC to EPCR also facilitates signaling of APC through proteolysis of proteaseactivated receptor (PAR)-1.

4.5 Protein C/APC

4.7 Protein S

Protein C is synthesized primarily in the liver and secreted into the plasma, where it circulates at an approximate concentration of 65 nM. As noted already, protein C shares homology with other vitamin K dependent serine proteases of the coagulation cascade [34]. Protein C zymogen is secreted as a 62-kDa protein, comprising a Gla domain (residues 1–45) at its N-terminal followed by two EGF-like domains linked to a chymotrypsin-like serine protease domain. As mentioned earlier, Gla domains undergo Ca2+dependent conformational changes that present the hydrophobic residues of the w-loop essential for phospholipid binding. In the case of protein C/APC, this region is also critical for binding to its cellular receptor, EPCR. The serine protease domain of protein C shares approximately 70% homology with other chymotrypsin-like serine protease family members. The catalytic triad consists of Ser, His, and Asp residues, which are brought into close proximity by the folding of the serine protease domain. APC contains a Ca2+-binding site and a Na+-binding site within the catalytic domain [35]. A 12-residue activation peptide is released from

Protein S is a 69-kDa vitamin K dependent plasma glycoprotein that functions as a cofactor for APC [42]. It is synthesized in hepatocytes, endothelial cells, and megakaryocytes. The plasma concentration of protein S is approximately 350 nM [43]; however, only about 40% of this is free protein S, with the remaining 60% circulating in a noncovalent, high-affinity complex with C4b-binding protein (C4BP). C4BP is a cofactor of serine protease factor I that ultimately regulates the formation of the C4b2a complex in the classic complement pathway. Only free protein S has full APC cofactor activity. Protein S is a modular protein made up of an N-terminal Gla domain, a thrombin-sensitive region (TSR), four tandem EGF-like domains, and a C-terminal sex-hormone-bindingglobulin-like domain that mediates the C4BP interaction [44]. High-affinity membrane binding is a characteristic of protein S and is mediated through its Gla domain [45]. This is a prerequisite for the recruitment and colocalization of APC on activated surfaces upon which the procoagulant complexes that it inactivates assemble. Protein S has one of the highest affinities for phospholipids of all vitamin K dependent proteins

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(Kd ~10 nM, as opposed to protein C Kd ~1 mM) [46]. The APC interaction site on protein S involves multiple residues in the Gla domain, TSR, and EGF domains [47, 48]. Protein S interacts specifically with residues 36–39 in APC [49].

4.8 Activation of Protein C Protein C activation is initiated when thrombin that escapes the site of injury encounters TM on the endothelial surface (Fig. 3). The site of interaction between thrombin and TM involves exosite I on the surface of thrombin [50]. This is a high-affinity interaction (Kd ~4  nM – without the TM chondroitin sulfate moiety). This basic (positively charged) patch binds to EGF domains 5 and 6 of TM. As exosite I is also involved in thrombin’s interaction with fibrinogen, PAR molecules, FV, and FVIII, the binding of thrombin to TM prevents association with these procoagulant substrates, and switches its role to that of an anticoagulant. The chondroitin sulfate attachment to the Ser/ Thr-rich region of TM increases the affinity of thrombin for TM through interactions with exosite II of thrombin.

Fig. 3  The activation of protein C by thrombin–thrombomodulin (TM). The domain structure of TM is shown, including the chondroitin sulfate attachment. The exosites (I and II) on the surface of thrombin (activated factor II) are labeled, as is the basic exosite (+) on protein C (PC). During the first stage, thrombin generated at the site of vascular injury that escapes the developing thrombus can bind to TM on the surface of the adjacent endothelium (1). This interaction involves exosite I binding to epidermal growth factor domains 5 and 6 on TM. The chondroitin sulfate moiety provides an additional point of interaction with exosite II on thrombin. PC binds via its Gla domain on endothelial cell surfaces to endothelial cell PC receptor, which serves to increase the local concentration of PC. Once bound to TM, thrombin loses its specificity for procoagulant substrates, but can now activate PC. PC interacts ionically with TMEGF4 via its basic exosite (2). This brings the activation peptide into close proximity with the active site of thrombin (3). Once cleaved, the activation peptide is released to yield the active serine protease, activated PC (APC ). This can now dissociate (4 ) and exert its anticoagulant functions

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Just as exosite I on thrombin mediates the binding to TM, a homologous positively charged region on the surface of the protein C zymogen facilitates its interaction with EGF domain 4 (and also 5) of TM [51]. This interaction is relatively weak and on its own represents an inefficient mechanism for bringing protein C into proximity with TM for activation. The binding of protein C by EPCR on the endothelial surface augments the recruitment of protein C and presents it for activation by the thrombin–TM complex. Thrombin bound to TM activates protein C by a single cleavage after Arg169 in the serine protease domain. Once the short activation peptide of protein C has been cleaved, a conformational change takes place, which converts the zymogen is into an active serine protease. The APC molecule can then be displaced by another protein C molecule and the activation can be process repeated.

4.9 APC Anticoagulant Function The anticoagulant function of APC is conferred through proteolytic inactivation of activated cofactors, FVa and FVIIIa. In FVa, APC recognizes two sites, Arg306 and Arg506, which are exposed on the surface of FVa (Fig. 4). Cleavage after Arg306 separates the A1 and A2 domains, whereas Arg506 bisects the A2 domain [52]. APC also proteolyzes FVa after Arg679 [53]. However, the physiological importance of this is unknown as it is neither necessary nor sufficient to inactivate FVa. In the absence of protein S, APC inactivation of FVa is biphasic, which represents the different rates of proteolysis at the two scissile bonds [53]. The first, most rapid cleavage occurs at Arg506 and results in a molecule with intermediate cofactor activity (25–40%). The second, slower cleavage is at Arg306 and is necessary for complete ablation of FVa procoagulant function [53]. This cleavage causes the dissociation of the A2 domain from FVa, which is the cause of its complete inactivation. A naturally occurring mutation, FV Leiden (Arg506Gln) [54], found in up to 5% of certain Caucasian populations, results in a FV molecule that exhibits partial resistance to APC inactivation. In individuals carrying the FV Leiden mutation, the initial cleavage at Arg506 cannot occur [54]. Therefore, as only the slower cleavage at Arg306 is possible, thrombin generation is not regulated as efficiently. Carriers of FV Leiden thus have a phenotype that predisposes them to thrombosis. The inhibition of FVa cofactor function by APC can only proceed prior to its assembly into the prothrombinase complex. Once bound to FXa, FVa is protected against inactivation, primarily by preventing the proteolytic cleavage after Arg506.

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Fig. 4  Anticoagulant function of APC. 1 FV (and FVIII) are proteolytically activated by thrombin, causing the dissociation of the B domain. FVa binds to activated platelet surfaces via its C domains, where the prothrombinase complex assembles. FVa is inactivated by APC via specific proteolysis of the Arg506 and Arg306 peptide bonds. The active site of PC is predicted to be located about 94 Å above the membrane surface to which it binds via its Gla domain, causing preferential cleavage of the Arg506 bond. The cofactor function of proteins serves to increase the affinity of APC for membrane surfaces and to reposition the active site of APC to 84 Å over the membrane surface, enhancing the proteolysis at the Arg306 site. 2 proteolysis of Arg506 causes partial inactivation of FVa, whereas the slower proteolysis of Arg306 (and also the noncritical Arg679) results in complete inactivation and dissociation of the A2 domain. 3 APC inactivates the homologous FVIIIa in similar manner through analogous cleavage of the Arg562 and Arg336 bonds. Inactivation of FVIIIa can be further enhanced by protein S in synergy with FV inactivated by APC in a reaction that is not fully understood

The anticoagulant activities of APC are augmented by the cofactor function of protein S [55] (Fig. 4). The interaction between protein S and APC is phospholipid-dependent, and results in complex formation on the surface of activated endothelial cells and platelets [56]. Protein S enhances

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APC-dependent inactivation of FVa in several ways. Owing to its high affinity for phospholipids, in binding APC, protein S increases its affinity for the phospholipid surfaces. Protein S can also counteract the protective effect that bound FXa has on the Arg506 cleavage [57, 58]. Third, it specifically enhances cleavage at Arg306 approximately 20-fold, so both cleavages occur at comparable rates through relocation of the position of the APC active site relative to FVa. The net effect of protein S cofactor function upon APC is that it increases the kcat of FVa inactivation. Inactivation of FVIIIa by APC occurs through proteolysis after Arg336 and Arg562 [59] (Fig. 4). However, unlike FVa, cleavage occurs preferentially at Arg336, and thereafter at Arg562 (as opposed to Arg506 and then Arg306 in FVa). The dissociation of the A2 domain that is rapidly induced following these proteolytic events results in complete loss of FVIIIa cofactor function [59]. Once assembled as part of the tenase complex, FIXa can protect the Arg562 peptide bond from APC-mediated cleavage [60]. Furthermore, as the substrate for the tenase complex, FX, competes with APC for the same binding site on FVIIIa, in this way it specifically protects the cleavage site at Arg362 [61]. Consequently, once a part of the tenase complex, FVIIIa is considerably more resistant to proteolytic inactivation by APC. The effects of protein S can overcome the protective action of FX. The role that protein S plays in its cofactor-mediated enhancement of FVIIIa cleavage is somewhat more complex that for FVa. For example, unlike FVa, the rate of cleavage at Arg336 by APC is unaltered by protein S. APC alone does not inactivate FVIIIa very efficiently and although protein S increases the efficiency slightly (threefold), intact FV is necessary for maximum augmentation (tenfold) to occur [62]. This interaction is not fully understood, but it would seem that FV must first be cleaved by APC at multiple sites, with the cleavage at Arg506 appearing to be of particular importance for the ability of FV to serve as an APC cofactor with protein S [63].

4.10 Overview of Antithrombin Function Thrombin is rapidly inactivated in plasma. This is largely due to the inhibitory action of antithrombin (the third anticoagulant pathway), but also a2-macroglobulin. Antithrombin is a plasma-borne serine protease inhibitor (serpin) that functions with broad specificity by mimicking target enzyme substrates. As target enzymes that are already assembled in their respective complexes are generally less accessible, antithrombin preferentially inhibits free enzymes. Thrombin, FIXa, FXa, FXIa, and FXIIa may all be inhibited [64], although arguably thrombin and FXa represent its most important physiological targets. It is through the inhibition of these serine proteases that antithrombin exerts its primary anticoagulant functions.

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Antithrombin is the most important direct inhibitor of coagulation proteases. Circulating at a concentration in blood of 2.5 mM, it inhibits the activity of thrombin, FXa, FIXa, FXIa, and FXIIa by forming tight equimolar complexes that block the accessibility of respective substrates to the protease active sites [65]. Despite its wide specificity, the inhibition of thrombin is probably most effective at suppressing coagulation. This is because of the key role of thrombin in accelerating its own generation by participating in FV and FVIII activation. Antithrombin activity is greatly accelerated by heparin, a highly negatively charged linear glycosaminoglycan, structurally related to heparan sulfate, which is found as a proteoglycan on the endothelial cell surface. A subpopulation of vascular heparan sulfate accelerates the inhibitory action of antithrombin against coagulation proteases. It is this mechanism of accelerated protease inhibition that forms part of this natural anticoagulant pathway [66]. Antithrombin is member of the serpin superfamily. Its importance as an inhibitor of coagulation is highlighted by the thrombotic problems of individuals with inherited disorders or with deficiency of this serpin [67].

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be cleaved, causing reactive bond separation to opposite ends of the molecule, with the polypeptide N-terminal to Arg393 inserting into the sheet A structure (Fig.  5 – “cleaved and inactive complex”). A similar complete loop insertion is observed in denatured or “latent” antithrombin, which is not cleaved but is functionally inactive. Alternatively, during normal inhibitory function, the substrate reaction proceeds from the Michaelis complex to an acyl intermediate stage. At this point, a conformational change is induced in antithrombin that stabilizes the complex with thrombin. This stabilizing conformational change involves partial, near complete, insertion of the reactive loop into sheet A (Fig.  5 – “stabilized complex”). This inactive complex circulates until it is cleared from plasma by the liver.

4.11 Antithrombin Structure Antithrombin has a highly ordered tertiary structure that is folded as three b-sheets (A–C) and nine a-helices (A–I). Like all serpins [68], the inhibitory action of antithrombin against proteases involves recognition of the reactive-site bond (Arg393–Ser394, also known as the P1–P1¢ bond) by the active site of the protease. In antithrombin, the reactive site is located in a reactive loop that protrudes above the main body of the inhibitor. Loop mobility appears to be crucial for the inhibitory action of all serpins. The reactive loop can adopt different positions, which have been identified by structural analysis. These are represented in cartoon form in Fig. 5. The reactive loop is shown above the main body of the serpin. One of the sheets (sheet A) is depicted by its several strands (arrowed lines) and one of the helices (helix D) protrudes from its side. In Fig. 5, two natural conformations are depicted, one with a partial insertion into sheet A (termed “inactive”), and one with fully expelled loop (“active”). The active conformation is also that adopted when heparin interacts with antithrombin (see later).

4.12 Inhibition of Thrombin by Antithrombin The first step in the interaction between antithrombin and target protease is the formation of a Michaelis complex between the reactive loop and the protease. A substrate reaction then begins in which the protease attempts to cleave the P1–P1¢ bond. The complex then has one of two fates. The serpin may

Fig. 5  Antithrombin structural changes. Antithrombin has several structural forms. These involve a mobile protruding reactive loop containing the reactive-site bond (P1–P1¢), helix D around which the heparin-binding site is centered, and a sheet A structure with five strands (arrowed). Antithrombin has two normal conformational states, one relatively “inactive,” with its reactive loop partially inserted into sheet A, and the other “active,” with a more accessible P1–P1¢ bond. During the inhibition of serine proteases (e.g., thrombin), the protease attempts to cleave the reactive-site bond, causing the reactive loop to insert strand A (cleaved inactive complex). If proteolysis does not occur, the protease–antithrombin complex is stabilized by incorporation of the reactive loop into sheet A (stabilized complex)

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It appears that for all serpin–protease reactions there is competition between loop insertion and the substrate reaction, the outcome of which determines the ultimate fate of the interaction (cleaved, inactive free serpin and protease on the one hand, or stabilized serpin–protease complex on the other) [69]. Inhibition of proteases by antithrombin is slow in relation to other serpin–protease reactions. It has been suggested that lack of reactivity is due to relative inaccessibility of the Arg393 (P1) residue to the protease.

4.13 Role of Heparin and Heparan Sulfate Heparin has an appreciable accelerating effect upon the formation of antithrombin–protease complexes. The formation of the antithrombin–thrombin complex is accelerated at least 2,000-fold under optimum conditions. Rate enhancements are also observed for the inactivation of FXa and FIXa by antithrombin. Heparin promotes the formation of a ternary complex of antithrombin and protease in which the active site of the protease is brought into contact with the reactive site of antithrombin. Assembly of the ternary complex proceeds preferentially through initial formation of a heparin–antithrombin complex, due to the high affinity of antithrombin for heparin. The binding of heparin to antithrombin is a two-step process with an initial low-affinity step (Kd of the order of micromolar) that, in turn, induces higher-affinity interaction (Kd ~ 20 nM). Thrombin and other proteases that bind with quite high affinity to heparin will bind to the heparin chain adjacent to antithrombin on the relatively nonspecific regions of the polysaccharide chain that adjoin the antithrombin-binding pentasaccharide sequence. In this circumstance, a crucial role of heparin seems to be the bringing together of inhibitor and protease, in a process termed “approximation” [70]. A key property of heparin is its induction of a conformational change in the reactive loop of antithrombin: with respect to accelerated thrombin inhibition, this induced conformational change is less important than the approximation effect. Binding to the heparin chain and approximation are essential for thrombin’s accelerated inhibition: binding is mediated by exosite II on the surface of the protease. In contrast, FXa and other proteases that bind only weakly to heparin are inhibited directly by the heparin–antithrombin complex, and approximation of antithrombin and protease on heparin does not need to take place. Rather, the conformational change induced in the reactive loop of the inhibitor by heparin appears to enable the inhibition reaction to proceed more rapidly. Once a stabilized complex has formed between the reactive site of antithrombin and the active site of the

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protease (FXa or thrombin), a further change in conformation in antithrombin (the loop insertion into sheet A) results in a reduced binding affinity of the complex for heparin. This enables its release and participation in additional antithrombin–protease reactions. Although blood is exposed to heparin only following its administration during clinical use, the vasculature is surrounded by large quantities of heparan sulfate, which is a proteoglycan with core proteins intercalated into cell membranes. Heparan sulfate itself is greatly heterogeneous but is structurally and biosynthetically related to heparin. Endothelial cells synthesize small amounts of anticoagulantly active proteoglycan. However, although the luminal surface of the endothelium contains only a small amount of the active heparan sulfate, the subendothelial space contains appreciably more.

5 Thrombin Thrombin is a central enzyme in coagulation with multiple roles that are critical to the hemostatic response (Fig. 6). The inactive thrombin precursor, prothrombin, circulates in plasma at a concentration of approximately 1.4 mM. Prothrombin contains an N-terminal Gla domain, two kringle domains, and a serine protease domain. Following the initiation of the coagulation cascade, thrombin is generated through FXa activation of prothrombin, albeit inefficiently. Subsequently, feedback activation of the coagulation cascade occurs by thrombindependent activation of FV and FVIII (see below). Cleavage of prothrombin by FXa or the prothrombinase complex (FXa– FVa) first occurs at Arg320 to generate the intermediate, meizothrombin, and then at Arg271 to generate thrombin [71]. This proteolytic activation liberates fragment F1 + 2, containing the prothrombin Gla and kringle domains. In prothrombin, these modules enable efficient localization of plasma prothrombin to activated platelet membrane surfaces at the site of vessel damage. This therefore enables its efficient recruitment to the prothrombinase complex. Once prothrombin has been activated, thrombin can escape the activation complex and is then free to attack its different and varied target substrates. Thrombin is unique among the activated coagulation serine proteases in that once it has been formed, the domains important for its initial recognition and activation are lost. Loss of the Gla and kringle domains allows thrombin to diffuse freely to encounter, recognize, cleave, and dissociate from its many substrates. This mobility is central to its ability to interact with its numerous substrates. Activation of prothrombin induces the exposure of three cryptic functional regions [72], including the active site and two charged binding regions, termed exosite I and exosite II, each of which is a critical determinant of thrombin specificity.

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Fig. 6  Multifunctional roles of thrombin. Once activated by the coagulation cascade, thrombin can function in procoagulant, anticoagulant, antifibrinolytic, and pro/anti-inflammatory pathways

5.1 Hemostatic Substrates of Thrombin A primary function of thrombin is to catalyze the conversion of fibrinogen to fibrin [73]. During this reaction, two short fibrinopeptides (FPA – 16 amino acids, FPB – 14 amino acids) are cleaved from the N-terminal ends of the fibrinogen Aa and Bb chains, respectively. Cleavage of FPA occurs first and exposes a cryptic polymerization domain. The fibrin monomer formed spontaneously polymerizes by interaction of the newly exposed polymerization domain with a preexisting complementary polymerization domain on the C-terminal domains of the molecule, to form protofibrils. Cleavage of FPB exposes additional polymerization sites that facilitate lateral aggregation to form an extensive meshwork that surrounds/encases the aggregated platelets at the site of vessel injury. Thrombin activates FV and FVIII by excision of their central B domains. In FV, Arg709, Arg1018, and Arg1545 are cleaved, leaving an A1–A2 domain fragment, ionically associated with the A3–C1–C2 fragment. In FVIII, the corresponding and analogous cleavages are Arg740, Arg1649, and Arg1689, with an additional cleavage at Arg372. This leaves the ionically associated A1–A2 fragment noncovalently bound to the A3–C1–C2 fragment. Thrombin proteolytically activates FXIII in a procoagulant reaction that is enhanced by fibrin. Once thrombin has cleaved the FXIII A subunit after Arg37, an N-terminal activation peptide is released. This exposes the active-site Cys314 residue to generate the active transglutaminase that stabilizes deposited fibrin fibrils by catalyzing covalent cross-linking [73]. Thrombin specifically activates platelets by binding and cleaving PAR molecules [74]. Cleavage of PAR-1 occurs at Arg41. This proteolytic event untethers an intramolecular

ligand that self-associates with its ligand binding site. This induces receptor-mediated intracellular signaling that leads to platelet activation. Thrombin can also proteolytically activate FXI, albeit under rather defined conditions that require anions such as dextran sulfate. However, in the presence of activated platelets, a more likely physiological activation process occurs that results in accelerated cleavage of factor XI after Arg369 [75]. Distinct to the procoagulant activities described above, thrombin also participates in interactions/reactions that elicit anticoagulant function, as described in earlier sections. For example, thrombin that is generated adjacent to endothelial cells lining the vessel lumen or thrombin that escapes the hemostatic plug can bind to TM on the surface of the endothelium in a high-affinity complex. Once thrombin is bound in the thrombin–TM complex, its substrate specificity is redirected from procoagulant to anticoagulant reactions. Procoagulant reactions are impeded largely by the occupancy of exosite I, whereas activation of protein C is greatly enhanced. APC and its anticoagulant roles were described in earlier sections in more detail. The thrombin–TM complex also activates the carboxypeptidase TAFI by specific proteolysis at Arg92 [76]. The rate of this activation is approximately 1,000 fold greater than by thrombin alone. Activated TAFI (TAFIa) stabilizes fibrin clots through proteolytic removal of terminal Lys residues from fibrin. The clipping of these amino acids disrupts the binding sites for fibrinolytic proteins that destabilize the fibrin meshwork. TAFIa also has an anti-inflammatory function in inactivating C5a in the complement cascade. Antithrombin locks thrombin in an irreversible inactive complex and is then cleared from the circulation. This reaction is accelerated by glycosaminoglycan cofactors (i.e., heparin or heparan sulfate proteolglycans), which approximate thrombin

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and antithrombin [70]. The multiple interactions of thrombin outlined above raise interesting questions. First, how can thrombin be a specific protease, when it has so many substrates? Second, how are its activities specifically and spatially coordinated during hemostasis to produce a hemostatic plug only at the site of injury and not over intact endothelium? [77] The questions may be explained by the specific occupancy of its surface exosites (I and II) by the above-mentioned cofactors for many of its reactions [78], and by competition of these cofactors for their respective exosites [77, 79].

5.2 Thrombin Exosites and Cofactors The major initial procoagulant functions of thrombin (i.e., cleavage of fibrinogen, FV, and FVIII) occur without cofactors. Interestingly, all of these reactions involve extended interactions of both exosite I and exosite II on thrombin with the target substrate [80–85]. In addition, the Na+-binding loop and the active site impart further specificity to these reactions. These multiple sites of interaction ensure efficient proteolysis in the absence of cofactors. The importance of exosites and cofactors can now be appreciated by considering the anticoagulant reactions of thrombin. Protein C activation by thrombin requires thrombin to be bound by its endothelial cell surface cofactor, TM. This is partly because TM localizes thrombin (bound by exosite I) and protein C (bound weakly by TM) together. The association of thrombin with TM is further enhanced if TM has been posttranslationally modified with the chondroitin sulfate moiety [86]. This carbohydrate structure increases the affinity of thrombin for TM through specific interactions with exosite II. Not only does the occupancy of exosite I by TM block its procoagulant functions, but it may also induce a conformational change in the active site that favors its interaction with protein C [87]. A second example of the functional role of exosites and cofactors is provided by the role of heparin (or heparan sulfate) in accelerating the interaction of antithrombin with thrombin. The glycosaminoglycan is able to bind both antithrombin and thrombin, bringing them into proximity (“approximation”). In this case, the functional exosite on thrombin is exosite II. Thrombin uses an adaptive strategy of cofactor utilization to extend its effective range of substrates. The next example of a cofactor is fibrin, which accelerates the activation of FXIII by thrombin. In this instance, fibrin behaves much as TM does as a cofactor, using exosite I of thrombin for binding, although the fibrin polymer must first be generated by thrombin before a functional cofactor emerges [79]. It has been suggested that fibrin polymerization brings FXIII (which also binds fibrin) into proximity with thrombin, enhancing activation of this transglutaminase [77].

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5.3 Cofactor Competition for Exosites: A Mechanism for Restricting Thrombin Action Cofactors extend the range of efficient thrombin interactions, but can only use one of two exosites on its surface. This raises the question of what happens when two cofactors are available for thrombin to interact with? This has been studied in the case of fibrin-enhanced FXIII activation by addition of TM: both fibrin and TM bind to residues within exosite I. However, fewer exosite residues are involved in fibrin-enhanced FXIII activation than in TM-enhanced protein C activation by thrombin [77, 79]. Moreover, the affinity of the thrombin interaction with TM is orders of magnitude higher that that with fibrin. Consequently, when both cofactors are present, TM will successfully compete with fibrin for exosite I. It will therefore terminate fibrin-enhanced FXIII activation by thrombin and remove thrombin from the clot. Competition for exosite II can occur in much the same way. Exosite II is used to increase the efficiency of reactions involving heparan sulfate (on the endothelial cell surface) and GpIba (on the platelet surface). As the affinities of thrombin for these cofactors are similar, the abundance of the cofactor becomes an important determinant in the outcome of any competition. Many proteins are susceptible to proteolytic cleavage by thrombin. However, the ability to cleave does not necessarily translate into a clearly defined physiological role. The specificity of thrombin is further determined by the concentration of each substrate in conjunction with the relative affinity of the exosite for that substrate.

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Chapter 24

Platelets in Pulmonary Vascular Physiology and Pathology Michael H. Kroll

Abstract  Platelets are small, nonnucleated cells that circulate for about 10 days after they are released into the bloodstream by bone marrow megakaryocytes. Platelet production derives from sequential processes termed megakaryopoiesis and thrombopoiesis, the biological basis of which is an intricate nexus of signaling events specified by cytokines and growth factors and organized temporally and spatially via exquisitely fine-tuned nuclear and cytosolic responses. Thrombopoietin (TPO) is the primary regulator of megakaryopoiesis. In conjunction with other cytokines, including stem cell factor, interleukin (IL)-6, IL-11, and erythropoietin, it promotes megakaryocyte lineage commitment from pluripotent hematopoietic stem cells. Counterbalancing these promegakaryopoiesis factors are transforming growth factor b1, platelet factor 4, and IL-4, each of which is considered a negative regulator of platelet production. Megakaryocytes mature into unique platelet-producing cells. Thrombopoiesis begins with a shift in compartmentalization as megakaryocytes migrate from the endosteal stem cell compartment to the vascular zone, where they adhere to the marrow sinusoids. These vascular zone megakaryocytes accumulate genetic material and cytoplasm as they maintain cell cycling that never progresses beyond late anaphase, so they fail to undergo nuclear envelope division and cytokinesis, thus resulting in the typically large irregularly shaped polyploid cells containing up to 64 pairs of chromosomes. As they enlarge, megakaryocytes express a huge surface area derived from an internal reservoir of membrane permeating the cytoplasm – designated the demarcation membrane system – that permits them to remodel their cytoplasm into a series of microtubule-scaffolded extensions termed proplatelets. Finally, via a series of microtubular, cytoskeletal, and contractile responses modulated by hydrodynamic stimuli, the proplatelet elongates and bifurcates, with individual platelets released into the circulation following their being sheared off by the forces of the flowing blood.

M.H. Kroll (*) Department of Benign Hematology, University of Texas, MD Anderson Cancer Center, 1515, Holcombe Blvd, Unit 1464, Houston, TX 77030, USA e-mail: [email protected]

Keywords  Coagulation • Thrombosis • Embolism • Thrombopoiesis • Megakaryocyte • Hemolysis • Thrombo­poietin

1 Platelet Production Platelets are small, nonnucleated cells that circulate for about 10 days after they are released into the bloodstream by bone marrow megakaryocytes. Humans produce about 1 × 1011 platelets daily, and production can be increased at least 20-fold in states such as acute hemorrhage, acute hemolysis, and inflammation. Platelet production derives from sequential processes termed megakaryopoiesis and thrombopoiesis, the biological basis of which is an intricate nexus of signaling events specified by cytokines and growth factors and organized temporally and spatially via exquisitely fine-tuned nuclear and cytosolic responses. Thrombopoietin (TPO) is the primary regulator of megakaryopoiesis. In conjunction with other cytokines, including stem cell factor, interleukin (IL)-6, IL-11, and erythropoietin, it promotes megakaryocyte lineage commitment from pluripotent hematopoietic stem cells [1]. Counterbalancing these promegakaryopoiesis factors are transforming growth factor b1, platelet factor 4, and IL-4, each of which is considered a negative regulator of platelet production. Megakaryocytes mature into unique platelet-producing cells [2]. Thrombopoiesis begins with a shift in compartmentalization as megakaryocytes migrate from the endosteal stem cell compartment to the vascular zone, where they adhere to the marrow sinusoids. These vascular zone megakaryocytes accumulate genetic material and cytoplasm as they maintain cell cycling that never progresses beyond late anaphase, so they fail to undergo nuclear envelope division and cytokinesis, thus resulting in the typically large irregularly shaped polyploid cells containing up to 64 pairs of chromosomes. As they enlarge, megakaryocytes express a huge surface area derived from an internal reservoir of membrane permeating the cytoplasm – designated the demarcation membrane system – that permits them to remodel their cytoplasm into a series of

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_24, © Springer Science+Business Media, LLC 2011

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microtubule-scaffolded extensions termed ­proplatelets. Finally, via a series of microtubular, cytoskeletal, and contractile responses modulated by hydrodynamic stimuli, the proplatelet elongates and bifurcates, with individual platelets released into the circulation following their being sheared off by the forces of the flowing blood [3].

2 Does Thrombopoiesis Occur in the Lungs? Platelets may be released into the circulation from a pulmonary thrombopoietic compartment. Over 40 years ago, data derived from studies of right atrial blood in humans undergoing cardiac catheterization provided the estimate that up to 50% of bone marrow megakaryocytes exit the marrow cavity, enter the blood, and end up in the lung vasculature, where active thrombopoiesis results in the release from the pulmonary capillary bed of nearly one fifth of the circulating platelet mass [4]. These clinical data have been corroborated [5] and contradicted [6], but animal data – principally from the mouse – have fairly consistently shown that megakaryocytes are present and making platelets in the lung [7]. The idea that thrombopoiesis occurs in lungs is particularly interesting when considering that pulmonary fibrosis develops in some patients with a platelet secretory defect in which unpackaged growth factors leak from the megakaryocyte into their surrounding environment (e.g., the gray platelet syndrome) and in some patients with myeloproliferative disorders (see later). One might also speculate that platelet production in the lung is important in maintaining hemostasis within the vast pulmonary microvasculature, estimated to contain over one billion capillaries [8]. It is striking to note that pulmonary hemorrhage is an infrequent manifestation of even extreme disturbances in hemostasis, such as severe underproduction thrombocytopenia and hemophilia.

3 Platelets in Hemostasis and Thrombosis Circulating platelets participate in both physiological hemostasis and pathological thrombosis. Primary hemostasis is defined as the platelet–blood vessel interactions that initiate physiological hemostatic plug formation and prevent superficial microvascular hemorrhage. When the trigger is a pathological event, such as a ruptured atherosclerotic plaque, platelet adhesion to the damaged arterial wall leads to platelet aggregation, resulting in a vasooclusive white thrombus. A platelet-dependent thrombus is the fundamental pathological mediator of arterial ischemia or infarction, such as that causing heart attacks and strokes. Platelets circulate in an inactivated state. They respond to vessel wall injury, alterations in blood flow, or chemical

M.H. Kroll

stimuli with the activation of a functional triad of adhesion, secretion, and aggregation. These linked responses occur via a series of carefully coordinated signals that convert extracellular stimuli into intracellular chemical messengers that direct specific enzymatic reactions leading to changes in cell structure, the expression of a new repertoire of functional adhesion receptors, and the secretion of several proaggregatory and growth-promoting substances. The state of platelet activation is regulated dynamically by the actions of a diverse array of excitatory and inhibitory extracellular stimuli [9]. Platelets are equipped with specific plasma membrane receptors that organize these various stimuli and transform them into biological responses. This transformation occurs via transmembranous signaling that results in the generation of intracellular second messengers. The major activation pathways in platelets are stimulated by collagen, von Willebrand factor (VWF), thrombin, adenosine diphosphate (ADP), and epinephrine. The pathways that are activated downstream of the receptors for these stimuli include phospholipase C, which cleaves membrane phosphatidylinositol 4,5-bisphosphate to from diacylglycerol (which activates protein kinase C) and inositol 1,4,5-trisphosphate (which leads to elevated levels of cytoplasmic ionized calcium); phosphatidylinositol 3-kinase, which phosphorylates phosphatidylinositol 4,5-bisphosphate at the 3-position, leading to phosphatidylinositol 3,4,5-trisphosphate; and phospholipase A2, which hydrolyzes arachidonic acid from the 2-position of membrane phosphatidylcholine. Arachidonic acid is rapidly converted by platelet cyclooxygenase 1 to the proaggregatory and vasoconstricting (and therefore prothrombotic) product thromboxane A2 (TXA2). The major inhibitory signaling pathways are activated by prostacyclin (PGI2) and nitric oxide (NO), two constitutively released endothelial cell (EC) products that serve to maintain blood fluidity principally by maintaining platelets in their basal quiescent state. PGI2 binds to specific cell-surface receptors to activate adenylyl cyclase, whereas NO diffuses through the platelet plasma membrane to activate cytosolic (or soluble) guanylyl cyclase. These convert adenosine triphosphate and guanosine triphosphate into cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP), respectively. cAMP and cGMP cause activation of cAMP- and cGMP-dependent protein kinases, which serve to inhibit platelet activation through pleiotropic downstream effectors.

4 Platelet–Coagulation Factor Interactions Activated platelets are very important in regulating the generation of insoluble fibrin. They release a variety of prothrombotic substances – such as fibrinogen, VWF, and factor

24  Platelets in Pulmonary Vascular Physiology and Pathology

V – and they express receptors for factor XI that enhance its activation by thrombin [10]. Through the “flipping” of phosphatidylserine from the inner to the outer plasma membrane, activated platelets express specific surface binding sites for soluble clotting factors and thereby promote critically important coagulation reactions: the activation of factor X by the activated factor IX–activated factor VIII complex and the activation of prothrombin by the activated factor X–activated factor V complex. In addition to these reactions, activated platelets shed “microparticles” that may promote and disperse procoagulant responses. Activated platelets can also regulate natural anticoagulant mechanisms by promoting the inactivation of activated factor V by activated protein C and by releasing two proteins that inhibit activated factor XI [11].

5 Platelet–Leukocyte Interactions Platelet-dependent hemostasis and thrombosis are directly coupled to leukocyte recruitment and activation [12]. Activated platelets express P-selectin, which engages P-selectin glycoprotein ligand-1 (PSGL-1) on neutrophils and monocytes. This results in leukocyte adherence and activation on and within the developing platelet thrombus. In the leukocyte, PSGL-1-mediated responses are coupled to the upregulation of Mac-1 – which binds to platelet glycoprotein (Gp) Iba – and the activation of CD11b/CD18 – which binds to fibrinogen attached to platelet aIIbb3. This process of “secondary capture” is often followed by leukocyte–EC interactions, the release of inflammatory and procoagulant factors by the platelet–leukocyte aggregates, and amplification of platelet and leukocyte activation, leading to pathological changes in EC permeability and function [13]. Of note, eliminating platelet–neutrophil interactions in a mouse model of acute lung injury (ALI) protects against hypoxemia and enhances survival [14]. Platelet–lymphocyte interactions are directed by chemokines. Megakaryocytes synthesize several chemokines, and these molecules are stored in platelet a-granules and are released upon activation. Platelets contain many chemokines, including the a-chemokines platelet factor 4, growth-regulating oncogene a, platelet basic protein (which is cleaved to b-thromboglobulin), epithelial neutrophil activating protein-78, and IL-8; and the b-chemokines inflammatory peptide-1a, regulated on activation normal T cell expressed and secreted (RANTES), monocyte chemotactic protein-3, and thymus and activation-regulated chemokine. These effect immune responses by binding to specific receptors on lymphocytes or monocytes designated CXCR (for the a-chemokines) and CCR (for the b-chemokines). Platelets themselves express CXCR4, CCR1, CCR3, and CCR4, and these

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r­ eceptors transduce functionally important “priming” signals when they engage their specific chemokine ligand [15].

6 Platelet Secretion Platelets store or synthesize many other bioactive compounds capable of regulating platelet, coagulation protein, leukocyte, and vascular wall responses. The most prominent of these are pharmacological targets: aspirin-inhibitable TXA2, which is proaggregatory and vasoconstricting, and thieneopyridine-inhibitable ADP, which is proaggregatory [11]. TXA2 is synthesized from arachidonic acid by platelet cyclooxygenase 1 and ADP is stored in dense granules and released upon platelet stimulation. In addition, megakaryocytes synthesize and platelets store a variety of growth factors, cytokines, and adhesive proteins, each of which is capable of influencing short- and long-term tissue responses. Platelet a-granules contain platelet-derived growth factor (PDGF), transforming growth factor-b, and basic fibroblast growth factor, and their release into tissue compartments (both vascular and hematopoietic) may cause profound pathological changes, including myelofibrosis and perhaps even pulmonary fibrosis (see later). Platelets also store vascular endothelial growth factor (VEFG) along with a “panoply of endothelial trophogens” [16]. In the steady state they comprise the tools by which platelets accomplish their task as “guardians of the microvasculature.” When they are deficient, such as in thrombocytopenic states, it may not be possible to maintain hemostatic EC junctions, and the break in these junctions results in mucocutaneous bleeding. When they are secreted in excess, they may trigger neovascularization and angiogenesis, and thereby contribute to tissue repair and remodeling, and perhaps tumor cell growth [17].

7 Platelet–Complement Interactions The complement system provides another link between thrombosis and inflammation. Platelets are primed by several complement proteins and are activated to secrete and aggregate by C3a and C5b-9 [18, 19]. Conversely, activated platelets activate the complement system [20]. Complementactivated platelets may be a relatively rich source of procoagulant microparticles [19], which appear to feed-forward activate the complement system [21]. The congenital deficiency of the complement-regulatory protein factor H is associated with a hemolytic uremic syndrome (HUS)-like thrombotic microangiopathy that almost always involves the kidneys and almost never involves the lungs [22]. The role of complement in other thrombotic microangiopathies and the

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basis for sparse pulmonary vasculature involvement in this and other thrombotic microangiopathies [23] are unknown.

8 Pulmonary Vascular Anatomy and Rheology The pulmonary artery is relatively unaffected by atherothrombosis. It is also relatively resistant to bleeding in the face of extreme hemostatic challenge. The basis for these clinical observations is not clear, but it is likely to emanate from the biophysical properties of the pulmonary vasculature. The pulmonary arteries diverge by 15 branch-orders into a vast network of capillaries whose ECs abut alveolar epithelial cells organized into deformable structures that most closely resemble polyhedrons when the alveolar sac is fully inflated [24]. These capillaries, filled with oxygen-rich blood, reconverge about 15 times into increasingly larger pulmonary veins. Vessels of successive orders are connected in series and vessels within each order are connected in parallel. This circuitry design maintains low resistance (high capacitance) and low pressure in the face of high pulsatile blood flow (the entire right ventricular output) moving through a vast circuit estimated to comprise 300 million arteries, 300 million veins, and several billion capillaries [8]. Afferent arterioles are defined as 100 mm descending to less than 10 mm in diameter, with little subendothelium separating the luminal endothelium from the vascular smooth muscle cell layer. The capillary bed comprises an extensive collateral network of tiny (about 6 mm) thin-walled vessels that function in gas and solute exchange. The pulmonary capillaries comprise continuous (without fenestrations) thin (100–200-nm) ECs. There is one capillary per interalveolar septum [24]. Capillaries then converge into widening branches of the efferent venules, which expand in size from tens to hundreds of microns in diameter. Pulmonary venules lack two features found in almost all other venules, and the lack of these features undoubtedly reflects important hemostatic function. The pulmonary venules lack fenestrae – which permit egress of cells and proteins adluminally in other circuits – and valves – which prevent backflux into the capillary network. Rather than valves, pulmonary venules and veins possess a nearly continuous layer of smooth muscle cells surrounding the endothelium. This smooth muscle layer buttresses the barrier function of the return circuit and actively maintains blood flow out of alveolar interstitium all the way back to the left atrium through sequential sphincterlike contractions [25]. It is reasonable to speculate that these features are anatomic elements essential for protecting the lungs against hemostatic challenges that typically lead to hemorrhage from the venular compartment in other tissues and organs.

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9 Virchow’s Triad and the Pulmonary Vasculature Rheological principles govern pulmonary vascular biological processes, physiological hemostasis, and pathological thrombosis, but blood flow is only one component of a complex nexus of variables that modulate prohemostatic and prothrombotic responses. These complexities have been organized for over 150 years according to the model of the eminent German pathologist Rudolph Virchow. Virchow’s triad reminds us that hemostasis and thrombosis are regulated by the simultaneous interactions between blood (cells and soluble constituent), blood vessel (endothelium, subendothelium, and smooth muscle), and blood flow (related to diameter, branching, turbulence, and obstruction). In considering the variables that regulate platelet-dependent hemostasis and thrombosis in the lungs, one can begin by teasing apart Virchow’s triad to see what it looks like within the pulmonary vascular compartment.

9.1 Blood Flow Shear stress is defined as the force per unit area exerted on blood by blood flowing in layers of differential velocity. Blood flow in tubular structures results in a central stream of highest velocity and lowest shear stress and with the lowest velocity and highest shear stress at the vessel wall. Plateletdependent reactions occur in vascular compartments with high wall shear stress, whereas the soluble coagulation protein reactions assemble and generate fibrin under low-shear conditions. The healthy pulmonary circuit maintains relatively low shear stress throughout its expanse because of its ability to accommodate increased flow without changing its intraluminal pressure. This happens because of vasodilation of both arterial and venous elements, and because of the recruitment of capillary reservoirs during episodes of increased alveolar ventilation. It is only in diseased vessels, such as those in primary pulmonary hypertension, that blood flow in the pulmonary artery is accompanied by elevated pressures and generates pathologically elevated wall shear stress. The healthy pulmonary artery and its branches maintain shear stresses below 30 dyn/cm2. Shear stresses may rise to 60 dyn/cm2 in “feed” arterioles, which bifurcate into branch arterioles at about 200-mm intervals, with declining flow velocities and shear stress as branches narrow (for example, a first branch of 20 dyn/cm2 shear stress and a fourth branch shear stress of 9 dyn/cm2). At branch points there is turbulence, increased resistance, and even backflux as cyclical blood flow overcomes afferent arteriolar autoregulatory vasoconstriction triggered with every cardiac diastolic

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r­ elaxation. As blood enters the extensively branched ­capillary bed, shear stress decreases further. There are very few published data that provide one with precise measurements of shear stress – or other rheological parameters – in capillaries, but estimates based on viscosity calculations and flow and pressure measurements at the entrance and exit of capillaries indicate that physiological capillary blood flow has low flow velocity and generates low shear stresses. The branching postcapillary venules then converge into efferent vessels of increasing diameter and capacitance and gradually increasing – but still low – shear stress (less than 5 dyn/cm2). In the pulmonary system, venular backflux is prevented and centripetal flow maintained by smooth muscle “sphincters” in venules 50–100 mm in diameter. Flow turbulence decreases and flow velocity increases as smaller venular branches come together into larger venules and, eventually, form the pulmonary veins.

9.2 Blood 9.2.1 Von Willebrand Factor Pathological shear-stress-dependent platelet adherence in arteries and arterioles is triggered by platelet GpIba binding to plasma or vessel wall VWF. VWF is synthesized by vascular endothelium and by megakaryocytes. It is constitutively released adluminally (into the subendothelium) and abluminally (into the blood) by the endothelium, and it is also stored and secreted following cellular activation (stored in EC Weibel–Palade bodies and in platelets’ a-granules). It is a multimeric protein built up of tens to hundreds of disulfide-bonded multivalent protomeric units. Larger multimers appear to have greater hemostatic and prothrombotic properties. The protomeric subunit comprises two disulfide-linked mature VWF polypeptides, each being divided structurally and functionally into several domains: the A, B, C, and D domains. The A domains are of particular importance because the A1 domain forms the primary GpIba recognition site and binds to type VI collagen, the A2 domain contains the recognition sequence for degradation by the VWF multimer-cleaving protease “a disintegrin and metalloproteinase with thrombospondin type 1 motif ” (ADAMTS)-13, and the A3 domain binds to fibrillar collagens types I and III found in arterial subendothelium, thus allowing soluble VWF to tack down onto the subendothelium of ruptured atherosclerotic plaques. Two other domains of VWF are also important for hemostasis and thrombosis: the C2 domain contains an RGD integrin recognition domain essential for VWF binding to platelet aIIbb3 and an N-terminal D domain binds factor VIII [26].

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9.2.2 Platelet GpIb Complex The GpIb complex is made up four type I proteins: GpIba is disulfide-bound to GpIbb and GpIba/b is noncovalently bound to GpIX. Two GpIba/b–GpIX complexes are noncovalently bound to a single molecule of GpV. The VWF binding site is on a region of the N-terminal extracellular domain of GpIba. The conformation of this region of GpIba is altered by shear stress into a ligand-receptive conformation [27]. Upon ligand binding under high-shear-stress conditions, the cytoplasmic domain of GpIba signals secretion and aIIbb3 activation, possibly through cytoskeletal-based mechanotransduction [28]. VWF binding to GpIba is required for microvascular hemostasis, which appears to be triggered in the injured arteriole – a high-shear-stress microenvironment – by soluble and subendothelial VWF alone or bound to collagen (type VI may be the predominant microvascular collagen) attaching to platelets via GpIba, with the subsequent activation of aIIbb3 to a ligand-receptive conformation. The high shear stress in the arteriole limits fibrin deposition and leukocyte recruitment, and neither soluble coagulation factors nor leukocyte number or function contribute in any clinically important manner to microvascular hemostasis in the epithelium (such as skin, mucous membranes, and the urinary and gastrointestinal tracts). Only the GpIb complex, which forms catch bonds with VWF of very high tensile strength and therefore is capable of withstanding the disrupting effects of elevated shear forces, can capture, adhere to, and accrue platelets in arterioles or stenotic arteries [29]. As shear stress falls in the occlusive hemostatic plug to venular levels, fibrinogen binding to activated aIIbb3 is important for interplatelet cohesion. The importance of fibrinogen is emphasized by clinical observations that fibrinogen or aIIbb3 deficiency causes a severe hemostatic defect. Regional regulation of the VWF-triggered hemostatic plug induced by a bleeding time wound is remarkably fine-tuned, as the platelet-rich thrombi accrue only at the mouths of the transected arterioles, and little or no platelet accumulation occurs within the adjacent arteriole lumen. VWF-dependent arteriolar platelet thrombosis is the hallmark of thrombotic thrombocytopenic purpura (TTP) and HUS. In TTP the primary pathogenetic event may be an acquired deficiency of ADAMTS-13, resulting in persistent “ultralarge” VWF multimers – on EC surfaces and in the blood – effecting GpIb–GpIX–GpV-dependent platelet adhesion, secretion (predominantly dense granules which contain costimulatory ADP), aIIbb3 activation, and aggregation. In diarrhea-associated HUS the primary pathophysiological process may involve an enterotoxin-mediated overstimulation of “ultralarge” VWF multimers from arteriolar endothelial Weibel–Palade bodies. This bolus of large VWF multimers remains attached to the EC surface and thereby

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recruits passing platelets through the shear-dependent ­binding of platelet GpIba to the EC-associated VWF [30]. A similar effect may occur in low-shear-stress postcapillary venules when rapid EC Weibel–Palade body release is stimulated by calcium ionophore or histamine, suggesting that there may be clinical conditions associated with venular inflammation in which part of the pathophysiological process is caused by VWF–GpIb–GpIX–GpV-mediated platelet recruitment within a low-shear-stress microenvironment.

9.2.3 Coreceptors Although the GpIb–IX–V complex is required for the initiation of microvascular hemostasis and is a primary factor contributing to microvascular thrombosis in TTP and HUS, several additional platelet receptors are also involved in both microvascular physiological processes and microvascular pathological changes [11]. In every case, the coreceptor functions to support platelet-mediated microvascular responses after GpIb–IX–V engages ligands (the coreceptor supports postadhesion responses), and in some cases the coreceptor requires activation or expression downstream of VWF–GpIb–IX–V interactions (aIIbb3 and a1b2 are “activated,” whereas P-selectin is expressed following a-granule secretion). Observations of human and/or mouse deficiencies of aIIbb3 (the human defect is called Glanzmann’s thrombasthenia) reveal that it causes a severe bleeding disorder but protects against macrovascular arterial thrombosis. Similar observations have been made with the protease activated receptors that mediate thrombin-induced platelet activation, the TXA2 receptor; and the P2Y12 receptor that binds ADP, is the target of thienopyridine antiplatelet drugs, and appears to be indispensable to platelet aggregation triggered by VWF binding to GpIb–GpIX–GpV. Similar but lesser effects on hemostasis and macrovascular thrombosis are seen in mice lacking the P2Y1 receptor or P-selectin. In contrast, human deficiencies of the collagen receptors – GpVI and integrin a2b1 – result in a mild bleeding diathesis, whereas GpVI- or a2b1-deficient mice have only a small or no hemostatic defect, respectively. Either GpVI or a2b1 deficiency or pharmacological perturbation results in delayed and decreased ex vivo thrombus formation on type 1 collagen under both arteriolar and low-shear-stress conditions, indicating that GpVI and a2b1 (probably to a lesser extent in comparison with GpVI) are important but secondary mediators of microvascular thrombosis. The prototypical example of a platelet coreceptor that appears to effect hemostasis and thrombosis paradoxically is platelet EC adhesion molecule (PECAM)-1, which is expressed by the microvascular endothelium and platelets. Mice deficient in PECAM-1 have a hemostatic defect not because of a loss of platelet expression, but because they lack homotypic interactions between PECAM-1 on

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adjacent ECs needed to regulate the endothelial component of Virchow’s hemostatic response. In fact, PECAM-1deficient platelets are actually hyperresponsive to both VWF and collagen because PECAM-1 is a negative regulator of GpIb–IX–V- and GpVI-dependent signaling [31].

9.2.4 ADAMTS-13 ADAMTS-13 is the VWF multimer-cleaving protease. It is synthesized in the liver, circulates in blood, and may attach to the vascular endothelium. Although the regional distribution of ADAMTS-13 activity is not yet understood, on the basis of the end-organ pathological features of TTP it appears to play an important role in processing “ultralarge” VWF multimers – released constitutively and secreted following EC stimulation – in the cerebral, mesenteric, myocardial, splenic, renal, pancreatic, and adrenal arterioles. It also appears that arteriole-level shear stress is required for ADAMTS-13-mediated cleavage of “ultralarge” VWF multimers: higher levels of shear stress open up or untangle the multimers and thereby expose the ADAMTS-13 cleavage site in the VWF monomer A2 domain. The mechanism by which ADAMTS-13 deficiency leads to TTP is quite well understood [32]. Under physiological conditions, ADAMTS-13 is synthesized, homes to the arteriole EC, and under arteriole levels of shear stress breaks down prothrombotic “ultralarge” VWF multimers. This maintains blood flow within the microvasculature. When ADAMTS-13 is deficient, arteriole-level shear stress triggers platelet GpIba binding to the unprocessed multimers, thus causing the arterioles to become occluded with both EC-attached platelets and platelet clumps that exceed the diameter of the narrowing arteriolar branches. This leads to ischemia and infarction of many organs. Of note, the lungs are usually spared from the thrombotic complications of TTP, HUS, or other thrombotic microangiopathies despite the pulmonary artery and its branches being lined by ECs that synthesize VWF [23]. It may be that ADAMTS-13 is not involved in pulmonary vascular hemostasis and therefore a systemic deficiency of ADAMTS-13 has no impact in the lungs. It is known that ADAMTS-13 is not expressed in pulmonary tissues [33] and it is possible that ADAMTS-13 produced by the liver does not home or bind to the pulmonary endothelium.

9.2.5 Red Blood Cells Platelet GpIb–IX–V-dependent hemostasis is mainly a physiological arteriolar response to injury. In addition to the blood factors that interact directly with platelets, there are blood elements that modify platelet–vascular interactions

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indirectly. Perhaps most important is the red cell. Under ­arteriolar flow conditions the red cells flow centrally and push the platelet stream peripherally toward the arteriolar wall. Because blood flow in a tubular arteriole can be considered to be parabolic and composed of an infinite number of infinitesimal laminae, the centripetal movement of platelets exposes them to flow laminae of lowest velocity and highest shear stress, thus slowing them and making wall collisions more efficient (or sticky). As arterioles narrow and branch into capillaries, their luminal diameter narrows, red cells are excluded from the central stream, flow velocity falls, and platelets become evenly dispersed throughout the bloodstream. If the average pulmonary capillary diameter is calculated to be 5.8 mm, and the size of a platelet is 3 mm and that of a red cell is 7  mm, it becomes intuitively apparent that platelets flow relatively better (e.g., faster) than red cells through these capillaries. This facilitates red-cell-mediated oxygen uptake in the pulmonary alveoli and delivery everywhere else. It also keeps platelets from slowing and sticking because platelet–capillary interactions are minimized simply because fewer platelets are at the wall and more platelets are in the central stream with highest flow velocity. Pulmonary capillary beds may be relatively protected against thrombosis because of rheological factors – which are a direct consequence of capillary diameter and red cell size, shape, and deformability – that keep platelets flowing rapidly through the thin central stream of capillary blood. The calculated velocity of platelet flow through a pulmonary capillary is approximately 500 mm/s. A similar rheological environment is found in venules where the hemostatic response is compartmentalized: until red cells that have squeezed their way through the capillary bed gradually queue back into concentric central stream laminae, platelets remain randomly dispersed throughout the venular lumen even as the flowing blood accelerates up to velocities 3–4 times greater than those found in the capillaries. This means that platelet and venule wall collisions leading to attachment are relatively rare. This is another reason why defects in platelet-dependent hemostasis in most tissues are manifested by postcapillary venular bleeding. Recall that lung venules are relatively protected from red cell leakage by smooth muscle cells that wrap around the endothelium to create a hemostatic barrier that can be enhanced by their constriction. The pulmonary venule barrier may be a double-edged sword, however, as the pulmonary postcapillary venule is the site where nondeformable sickled red cells cause vasoocclusion leading to the acute chest syndrome in sickle cell diseases [34]. In fact, the major defect resulting from red cell sickling that leads to vasoocclusion is a perturbation in the natural anticoagulant properties of the intact vascular endothelium. Sickled cells trapped in the postcapillary venules undergo hemolysis and release free hemoglobin, which

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s­ cavenges NO and stimulates arginase, thereby decreasing the level of arginine, which is the substrate for EC nitric oxide synthase. The sum result is that the pulmonary postcapillary venule suffers a deficiency of vasodilatory and antiplatelet NO, secondarily causing ischemia from imbalanced vasoconstriction and platelet activation. In the long term, these events lead to pathological remodeling of the pulmonary microvasculature associated with pulmonary hypertension in approximately 20% of patients with sickle cell disease [35].

9.3  Blood Vessel The vasculopathy of sickle cell disease is an emphatic example of the importance of an intact vascular endothelium in actively maintaining blood fluidity. A bleeding time wound shows platelet thrombus formation only at the site of arteriolar transection because the adjacent arteriolar, capillary, and venular ECs constitutively secrete or express on their surface molecules that prevent platelet adhesion, secretion, and aggregation. These include PGI2 and NO (both of which inhibit platelet adhesion and activation), the ectoADPase CD39 (which breaks down soluble ADP), thrombomodulin and heparan sulfates (which bind and inactivate thrombin), and urokinase-type plasminogen activator and tissue plasminogen activator (which generate plasmin capable of degrading VWF and fibrinogen). The capillary endothelium is particularly richly endowed with tissue factor pathway inhibitor, which is both secreted into the blood and retained on capillary endothelium, where it binds to and inactivates the tissue factor–activated factor VII–activated factor X complex, thereby eliminating thrombin generation. This suggests that the capillary may be an important gate preventing the initiation of coagulation despite continuous low-level exposure to prothrombotic stimuli. Only urokinase-type plasminogen activator or tissue plasminogen activator deficiency is associated with spontaneous microvascular thromboses, but a deficiency of any one of the vessel-derived natural anticoagulants leads to exaggerated responses to thrombotic stimuli. The exception to this is CD39 deficiency, which causes a severe bleeding diathesis due to compromised hemostasis because elevated blood levels of adenine nucleotides lead to P2Y1-mediated platelet desensitization. This is a useful reminder of the dynamic nature of the complex interactions that occur over time within Virchow’s triad. Subendothelial VWF is most abundant in the macrovasculature large veins and arteries (most large veins > pulmonary artery > cerebral arteries > aorta > coronary arteries > renal arteries > hepatic arteries > pulmonary vein). It is generally less abundant in microvascular subendothelium, and its distribution in the microvasculature is­

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noteworthy – venules > arterioles > capillaries, with very little or no VWF observed in any embryo capillary bed (in mice) and with relatively little VWF observed in adult myocardium (in pigs).

10 Pathological Interplay Between Platelets and the Pulmonary Circulation As the pathogenesis of pulmonary vascular diseases becomes increasingly illuminated, one can find many shadows of platelets, but the links that could direct innovations in diagnosis and treatment are missing. This probably reflects normal physiological function: the lungs are endowed with innate protection against several platelet-dependent disorders, such as atherothrombosis [35], microvascular thrombosis [23], and hemorrhage from severe thrombocytopenia or functional platelet disorders [36]. And it is clear that the pulmonary vascular elements within Virchow’s triad that are most important in effecting this protection are its unique rheological characteristics and vessel wall anatomy (see earlier). To eliminate the umbra, therefore, one must shine a light on platelets to try to see what they are doing within Virchow’s triad in pulmonary vascular diseases.

10.1 Hemorrhagic Interplay 10.1.1 Alveolar Hemorrhage Diffuse alveolar hemorrhage (DAH) develops in stem cell transplant (SCT) recipients. Its overall incidence is estimated at 1–21% and it occurs equally frequently in both auto-SCT and allo-SCT [37]. In one series, DAH was the principal diagnosis in 39% of SCT patients referred for bronchoscopy and nearly half of these patients died from respiratory failure or multiorgan failure [38]. Although DAH is clearly due to severe pulmonary microvascular derangements from radiotherapy, chemotherapy, graft-versus-host disease, and superimposed infection, thrombocytopenia is an important contributing factor and a standard therapeutic target [38]. DAH can develop in association with connective tissue diseases, Goodpasture’s syndrome (which leads to the “pulmonary–renal syndrome”), antiphospholipid antibodies, and many other vasculitides [39–41]. DAH is also part of a rare congenital syndrome of “idiopathic pulmonary hemosiderosis” [42] and a similar acquired disorder sometimes associated with primary pulmonary hypertension designated “pulmonary capillary hemangiomatosis” (PCH) [43]. In most cases of DAH, the primary insult is to the microvasculature and presumably the postcapillary venules. This insult leads to venular leakage of red cells despite normal

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hemostatic function (i.e., normal platelet counts and coagulation activity). Of note, hemoptysis in mitral stenosis is often from DAH, indicating that elevated pulmonary venous pressure is transduced back to the microvasculature and causes EC junction failure despite normal platelet counts and normal peristaltic function of pulmonary venules and veins to move blood centripetally. Although there is little evidence of a role for platelet-dependent hemostasis in most cases of DAH outside the SCT setting, cases of severe and often lethal DAH have been associated with platelet GpIIb/GpIIIa inhibitors given to patients with acute coronary syndromes [44].

10.2 Thrombotic Interplay 10.2.1 Pulmonary Embolism Pulmonary embolism (PE) is not a primary pulmonary vascular disease. Nonetheless, a massive PE can cause right ventricular collapse from acute pulmonary hypertension and recurrent PEs cause chronic pulmonary hypertension. A PE is a fibrin-rich thrombus that almost always develops in the lowflow venous circuit. Platelets become a constituent of the thrombus secondarily, and their role in the pathophysiological process and treatment of PE is considered small [45]. There are older experimental [46] and clinical [47] data demonstrating that platelets are activated in venous thromboembolism (VTE). There are also many clinical data – including data from recent randomized prospective trials –demonstrating a small but significant effect of antiplatelet therapy on preventing VTE in high-risk settings [45]. Aspirin is less effective than other pharmacological interventions; and it also causes less bleeding. Because of its ease of use, inexpensiveness, and safety, aspirin is being examined in two large prospective placebo-controlled randomized trials of secondary VTE prevention. The WARFASA trial (a European study of 1,400 persons examining if low-dose enterically administered coated aspirin given for 2 years after 6 months of warfarin treatment will prevent recurrent VTE and cardiovascular events) and the ASPIRE trial (an Australian trial of 3,000 persons examining if 100 mg aspirin prevents recurrent VTE after standard anticoagulation therapy) are expected to provide conclusive data about aspirin and VTE recurrence. They are not, however, likely to yield anything other than pilot data about the effect of inhibiting cyclooxygenase on pulmonary vascular responses to VTE. 10.2.2 Thrombotic Microangiopathies Pulmonary vascular ECs synthesize, store, and release VWF, perhaps more robustly than EC in other circuits. Yet the regulation of VWF processing in the lungs is largely

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unknown, and VWF-induced platelet-dependent pulmonary vascular thrombosis is considered rare. The paradigm for a VWF- and platelet-dependent thrombotic diathesis is TTP which, as described already, does not typically involve the lungs. There are, however, reported cases of lung involvement in primary or de  novo TTP due to acquired ADAMTS-13 [23]. The pathological investigation of the lungs reveals microvascular hyaline thromboses – containing platelets and fibrin – in the terminal arterioles and capillaries [48]. This is associated in some cases with “noncardiogenic” pulmonary edema from venular capillary leak. It is interesting to note that there are cases of DAH due to microvascular thromboses in transplanted lungs of patients who developed posttransplant TTP due to ADAMTS-13 deficiency, which is an uncommon cause of posttransplant TTP [49]. This is a reminder that the expression of ADAMTS-13 deficiency in any particular organ or tissue depends on other perturbations to Virchow’s triad that develop in that vascular compartment. The physiological processes regulating pulmonary vascular ADAMTS-13 are (to my knowledge) unknown. It is likely that ADAMTS-13 is synthesized and secreted by the liver, enters the circulation though the hepatic vein, and then enters the lungs through the pulmonary artery. Once in the lungs, ADAMTS-13 must home to the vascular endothelial compartment; but the sites of its homing, its recognition and binding motifs, and how its activity is regulated – in short, almost everything about its presence and function in the pulmonary circulation – remain a mystery. It is likely that elucidating the biological processes and pathological changes in pulmonary vascular ADAMTS-13 will teach us a lot about protecting the lung from thromboses and perhaps even provide potential therapeutic insight into common pulmonary inflammatory processes, such as pneumonia and asthma [50, 51].

10.2.3 Sickle Cell Disease There is systemic platelet activation in sickle cell crises. This is a secondary phenomenon of uncertain pathophysiological consequences. It is due in part to shear-induced platelet activation by flow across a regurgitant tricuspid valve (due to pulmonary hypertension), hemolyzed red-cellderived ADP-induced platelet activation, and the decreased endogenous antithrombotic properties of the vascular endothelium due to NO scavenging by free hemoglobin [52]. VWF also participates in the pathogenesis of sickle cell vasoocclusion, but its effect is independent of platelets and ADAMTS-13 deficiency [53]. Freshly secreted large VWF multimers are involved in the adherence of young red cells (“neocytes”), but not sickled red cells, to the microvascular endothelium.

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The two most important pulmonary complications of sickle cell disease are the acute chest syndrome and chronic pulmonary hypertension. Both are common (affect ­approximately 30%), due to many different factors (such as infection and necrotic marrow fat embolism), and are not simply the short- and long-term consequences of vasoocclusion [54]. Within this complex pathophysiological milieu, it is difficult to rank the importance of platelet-dependent thrombosis. One unfavorable prognostic indicator of the acute chest syndrome is a drop in platelet count, but this finding does not appear to represent pulmonary sequestration of platelets or any associated platelet thromboses. There are no data available to assess an effect of antiplatelet therapy in the acute chest syndrome and there appears to be no affect of antiplatelet agents on the natural history of sickle cell disease, including the frequency of the acute chest syndrome and the development of pulmonary hypertension [55–57].

10.2.4 Pulmonary Fibrosis In the USA, idiopathic pulmonary fibrosis affects between three and 29 persons per 100,000 and the median survival for those affected is between 3 and 6 years [58]. Its pathogenesis – including the role of platelets, VWF, and ADAMTS-13 – is not understood. There are, however, two theoretical reasons to examine platelets in idiopathic pulmonary fibrosis. The first is that platelets store and secrete upon activation several cytokines and growth factors allegedly involved in the fibrotic response, such as transforming growth factor-b, fibroblast growth factor, and PDGF [59]. The second is more compelling: “secondary” pulmonary fibrosis is associated with inherited storage pool defects due to abnormal packaging of platelet a-granules and dense granules. Congenital a-granule deficiency is a very rare bleeding disorder designated the “gray platelet syndrome.” The human mutation leading to it is unknown and its inheritance and penetrance are variable, but the platelet phenotype and disease expression are thoroughly cataloged [60]. Circulating platelets do not have a-granules owing to a failure of proteins synthesized by the megakaryocyte (such as growth factors) or endocytosed by megakaryocytes or platelets (such as fibrinogen) to traffic into the secretable pool of a-granule constituents. Most patients with the gray platelet syndrome have myelofibrosis caused by the constitutive release into the bone marrow microenvironment of megakaryocyte-derived growth and fibrosing factors that would normally be packaged into a-granules. There is one case of pulmonary fibrosis reported out of a total of 18 well-characterized gray platelet syndrome families [61]. Pulmonary fibrosis is more common in the inherited platelet storage pool deficiency designated “Hermansky– Pudlak syndrome” (HPS). This is another rare bleeding

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d­ isorder due to homozygous deficiency of one of seven HPS genes involved in the biogenesis of several storage organelles such as platelet dense granules and lysosomes. The clinical expression varies with the specific gene affected, but generally the bleeding disorder is accompanied by oculocutaneous albinism and pulmonary disease. The most common variant is due to a mutation in the HPS1 gene on chromosome 10. A 16-base-pair duplication in exon 15 affects over 400 persons in a small region of northwest Puerto Rico, and other mutations in HPS1 are found in similarly affected people in Europe and Japan. Almost all of these persons develop pulmonary fibrosis by their fourth decade and pulmonary fibrosis is lethal in at least 50% by the fifth decade. None develop myelofibrosis. The mechanisms of pulmonary fibrosis in HPS are not known. One model posits that pulmonary alveolar type II pneumocytes fail to secrete surfactant and that unpackaged lipids accumulate as waxy ceroid deposits in the air spaces. These deposits evoke macrophage-induced lung injury, especially the release of cathepsin L, which stiffens lungs by degrading collagen and elastin [62]. The role of platelets in HPS-associated pulmonary fibrosis has not been investigated and the mechanisms of plateletmediated pulmonary fibrosis in the gray platelet syndrome are uncertain. But the paradigm of myelofibrosis in the gray platelet syndrome should not be overlooked. It is possible that pulmonary fibrosis develops in both HPS and the gray platelet syndrome because the dysfunctional granule biogenesis directly affects megakaryocytes within a pulmonary vascular compartment. In HPS, leakage of unpackaged lysosomal contents may lead to airway and alveolar damage and leakage of dense granule serotonin may lead to smooth muscle cell contraction, proliferation, and neointima formation [63]. In the gray platelet syndrome, leakage of mitogenic, angiogenic, and permeability factors may lead to fibrosing airway and alveolar damage. There is additional evidence in support of a connection between abnormal platelet production and pulmonary pathophysiological processes. For example, biopsy-proven pulmonary interstitial inflammation accompanies immune thrombocytopenia (ITP) and remits along with the ITP [64]. This is germane because megakaryocytopoiesis is almost always abnormal in ITP, with the bone marrow typically showing megakaryocyte hyperplasia, and it is possible that brisker megakaryocytopoiesis in the lungs leads to the inflammatory response. As a second example, there is evidence that patients with idiopathic myelofibrosis develop secondary pulmonary hypertension [65] and that patients with primary pulmonary hypertension develop secondary myelofibrosis [66]. The next section will examine these phenomena and attempt to illuminate where platelets fit into this unexpected bidirectional linkage between the lungs and abnormal hematopoiesis.

M.H. Kroll

10.2.5 Pulmonary Hypertension The example that underlies the basis for considering platelets relevant in the pathogenesis of pulmonary hypertension derives from a rare case of a poorly understood inherited isolated dense granule deficiency (i.e., not associated with lysosomal or melanosomal abnormalities) associated with pulmonary hypertension [67]. The observations that this patient’s platelets leaked serotonin and that there were elevated levels of plasma serotonin are important components of the foundation for the “serotonin hypothesis” of pulmonary hypertension [68]. Although the role of platelets in pulmonary hypertension appears less certain today than when these observations were reported in 1990, data on their potential mechanisms of effect in this disease shed some light on how platelets and pulmonary vascular diseases might be linked [63]. Pulmonary hypertension is associated with thrombocytosis. In many cases it is a reactive thrombocytosis secondary to lung inflammation developing during the natural history of pulmonary hypertension [68, 69]. However, pulmonary hypertension also occurs in patients with postsplenectomy thrombocytosis [70] and myeloproliferative disease (MPD)associated thrombocytosis [71], and it commonly develops in myelofibrosis with myeloid metaplasia [65]. How do these clinical conditions, each of which imposes unique changes on several elements within Virchow’s triad, lead to pulmonary hypertension? Postsplenectomy or MPD-associated thrombocytosis may contribute to pulmonary hypertension through thrombocyte elaboration of growth factors, such as platelet-derived VEGF and PDGF. In fact, platelet-derived VEGF and PDGF are proposed as mediators of vascular changes developing in idiopathic pulmonary hypertension [72]. In postsplenectomy pulmonary hypertension, red cells are also important in the pathogenesis of pulmonary hypertension. When the spleen is absent, there is abnormal red cell processing which causes many weirdly shaped less deformable red cells (poikilocytes) to circulate. The poikilocytes are trapped, ripped up, and hemolyzed within the pulmonary microvasculature. Resulting free hemoglobin shuts down the endogenous NO antithrombotic property of the pulmonary vascular endothelium, whereas free ADP stimulates the platelets, resulting in a prothrombotic imbalance within Virchow’s triad [70]. In the MPDs – including myelofibrosis – extramedullary hematopoiesis may also contribute to pulmonary hypertension. It develops as stem cells leave the dysfunctional marrow compartment and colonize new compartments, including the lungs. This leads to functional changes similar to those in idiopathic pulmonary hypertension, such as arteriolar and capillary obstruction. It also leads to the elaboration of cytokines and growth factors that cause histopathological changes typical of idiopathic pulmonary hypertension, including ­intimal hyperplasia, vascular smooth muscle proliferation,

24  Platelets in Pulmonary Vascular Physiology and Pathology

and neovascularization. In support of the extramedullary hematopoiesis model, there is one series of patients with the MPD chronic myelogenous leukemia in which ten of 21 autopsies showed pulmonary vascular megakaryopoiesis and two out of the ten persons with pulmonary megakaryopoiesis had accompanying pulmonary fibrosis [73]. In support of the humoral model of cytokine and growth factor effects, there is one case of MPD-associated pulmonary hypertension in which blood levels of platelet factor 4 (a bioactive platelet a-granule constituent) were elevated [70] and one fairly large series in which 22 patients with myelofibrosis and pulmonary hypertension were evaluated and determined to have a “distinctive angiogenic phenotype” characterized by increased levels of circulating endothelial progenitor cells, increased levels of serum VEGF, and increased microvascular density in the marrow compartment [65]. How else might platelets contribute to pulmonary hypertension? There are ambiguous data about systemic platelet activation in idiopathic pulmonary hypertension and pulmonary hypertension associated with congenital heart diseases causing right-sided overload [74]. More consistent elevations of platelet activation are observed in anorectic medication (such as fenfluramine)-induced pulmonary hypertension. In these cases the common denominator may be cellular activation – leading to serotonin release by platelets and pulmonary vascular smooth muscle cell contraction – by drug-induced blockade of a voltage-sensitive potassium channel [75, 76]. Consistent with the idea that platelets are involved in anorectic drug-induced pulmonary hypertension, effective pulmonary hypertension treatment with the prostaglandin PGI2 usually leads to decreased platelet activation [77]. In all of these cases it is impossible to determine if platelet activation is a cause or an effect of pulmonary vascular changes in flow and structure, but it is most likely an effect of uncertain consequence. Similarly, elevations of plasma VWF levels in association with both primary and secondary pulmonary hypertension are probably epiphenomena of no pathophysiological significance [78]. Compelling data show that pulmonary hypertension is associated with increased levels of platelet TXA2 and decreased levels of endothelial PGI2, but the pathophysiological importance of these data seems negligible in the face of other data showing that treatment with aspirin or a thienopyridine has no effect on the severity or natural history of pulmonary hypertension, and that there is no evidence that pulmonary hypertension is associated with an EC-specific cyclooxygenase 2 inhibitor, such as rofecoxib or celocoxib, which selectively depletes PGI2 [75]. 10.2.6 Myelofibrosis in Pulmonary Hypertension The connection between bone marrow and pulmonary vascular compartments is as intriguing as it is opaque. This is

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because it is bidirectional: just as primary myelofibrosis leads to pulmonary hypertension, primary pulmonary hypertension leads to myelofibrosis [66, 79, 80]. The association is very strong: when first reported, all 17 primary pulmonary hypertension patients who were evaluated had myelofibrosis, including 11 designated as having severe fibrosis [79]. The myelofibrosis is a reactive process (not due to a clonal proliferation of myeloid elements) and it may be clinically important because it is accompanied by anemia and/or thrombocytopenia in most patients [66]. The cause of myelofibrosis is unknown, and there are no pathophysiological elements that are known to bridge lung and marrow fibrosis. Speculation focuses on pathological angiogenesis, and there is one study showing a paucity of microvascular pericytes in the bone marrow of pulmonary hypertension patients with secondary myelofibrosis [80]. This finding implies that myelofibrosis is related to aberrant angiogenesis and suggests the possibility that this aberrancy also underlies idiopathic pulmonary hypertension. The role of megakaryocytes and platelets has not been explored. One study showed that the TPO level is elevated in the right ventricle, pulmonary artery, and left ventricle of patients with primary pulmonary hypertension [81]. This could reflect decreased pulmonary thrombopoiesis (platelets regulate blood TPO levels by binding TPO; they are a TPO “sink”) as changes in the vascular compartment in pulmonary hypertension drive platelet precursors from the lungs into the bone marrow compartment.

10.2.7 Miscellaneous Lung Diseases Platelets may be significant elements in the pathogenesis of several pulmonary diseases, including some that are not obviously vascular diseases. Pulmonary venoocclusive disease (VOD) is an uncommon complication of SCT and an uncommon toxicity of certain chemotherapies, especially bleomycin, mitomycin, and carmustine [82]. It is triggered by postcapillary venular inflammation causing thrombosis. This leads to pulmonary hypertension and right-sided heart failure. Pulmonary VOD rarely leads to pulmonary capillary hemangiomatosis (PCH), which is an angioproliferative response to postcapillary venular occlusion [83]. For pulmonary VOD and PCH, the pathogenetic factors downstream of venular damage are unknown. The role of platelets in thrombosis and maintaining postcapillary venular integrity [16] and the fact that platelets are rich sources of cytokine and growth factors [17] suggests the possibility of their involvement. There is no evidence, however, that antiplatelet therapy (or any therapy) favorably affects the natural history of pulmonary VOD or PCH. Acute lung injury (ALI) is a vague term for a common condition encompassing pneumonia, aspiration, sepsis, trauma,

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and even massive transfusion; its severest manifestation is known as “acute respiratory distress syndrome” [14]. The heterogeneity of causes defines multiple pathogenetic mechanisms, some of which include platelet-dependent abnormalities. For example, experimental data demonstrate that platelets are essential for establishing leukocyte–capillary endothelial interactions and subsequent alveolar inflammatory responses in animals with acid- or sepsis-induced ALI [14]. These data introduce the hypothesis that platelet P-selectin is a potential therapeutic target in ALI [84]. As another example, there is evidence that platelet lipid debris accumulating during bloodbank storage may contribute to transfusion-associated ALI (TRALI) [85]. Most cases of TRALI are from donors’ antibodies binding to recipient leukocytes resulting in activated host neutrophils being sequestered in the pulmonary microcirculation. Ten percent of TRALI cases, however, are not associated with leukocyte antibodies, and it is believed that most of these cases are due to the release of biologically active lipids from platelets during storage [86]. Platelets also appear to play a role in animal models of asthma in which allergen-induced leukocyte recruitment and airway inflammation are reduced when platelets are selectively depleted [87]. There is evidence of platelet and leukocyte activation and circulating leukocyte– platelet aggregates in humans with asthma [87] and even evidence of hyperreactive platelets, activated platelets, and circulating leukocyte–platelet aggregates in patients with cystic fibrosis [88]. Of note, however, is that there is no evidence for a beneficial effect of antiplatelet therapy with aspirin or a thieneopyridine on ALI, TRALI, asthma, or cystic fibrosis. Chronic high-dose ibuprofen may slow the progression of cystic fibrosis, but this is probably not a platelet-mediated effect [89].

11 Summary There is an impressive breadth and depth of information about platelets and how they function within Virchow’s triad to effect physiological hemostasis and pathological thrombosis. A similarly impressive information base – much of it immediately available within this vast tome – encompasses pulmonary vascular biological and pathophysiological processes. Little is known, however, about how platelets function in the lungs under normal and pathological conditions, or about how the lungs affect platelets in hemostasis and thrombosis. Obviously important and intriguing questions are unanswered. Is platelet production in the lungs important? Why does severe thrombocytopenia not cause hemoptysis? How is the pulmonary circulation protected against atherothrombosis and thrombotic microangiopathies? These questions are unanswered but not unanswerable. And although obtaining their answers is inevitable, they are likely to be most expedient if we continue to illuminate the platelet

M.H. Kroll

within the complexities of Virchow’s triad within the ­pulmonary compartment. By focusing on the platelet, we will elucidate mechanisms of pulmonary vascular hemostasis and thrombosis and develop hypotheses about innovative therapeutics probably relevant to diseases outside the pulmonary compartment. A great adventure awaits the platelet biologist who explores pulmonary vascular biology and pathology.

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384 66. Popat U, Frost A, Liu E et al (2006) High levels of circulating CD43 cells, dacrocytes, clonal hematopoiesis, and JAK2 mutation differentiate myelofibrosis with myeloid metaplasia from secondary myelofibrosis associated with pulmonary hypertension. Blood 107:3486–3488 67. Herve P, Drouet L, Dosquet C et  al (1990) Primary pulmonary hypertension in a patient with a familial platelet storage pool deficiency. Am J Med 89:117–120 68. Farber HW, Loscalzo J (1999) Prothrombotic mechanisms in ­primary pulmonary hypertension. J Lab Clin Med 134:561–566 69. Schafer AI (2004) Thrombocytosis. New Eng J Med 350:1211–1219 70. Hoeper MM, Niedermeyer J, Hoffmeyer F, Flemming P, Fabel H (1999) Pulmonary hypertension after splenectomy? Ann Intern Med 130:506–509 71. Rosenstingl S, Brouland J-P, Zinin J-M, Tobelem G, Dupuy E (2003) Mixed myelodysplastic syndrome and myelproliferative disorder with bone marrow and pulmonary fibrosis: the role of megakaryocytes. Acta Haematol 109:145–149 72. Eddahibi S, Humbert M, Sediame S et al (2000) Imbalance between platelet vascular endothelial growth factor and platelet-derived growth factor in pulmonary hypertension. Am J Respir Crit Care Med 162:1493–1499 73. Yamuchi K, Shimamura K (1994) Pulmonary fibrosis with megakarycytoid cell infiltration and chronic myelogenous leukemia. Leuk Lymphoma 15:253–259 74. Chaouat A, Weitzenblum E, Higgenbottam T (1996) The role of thrombosis in severe pulmonary hypertension. Eur Respir J 9:356–363 75. Lopes AA (2006) Pathophysiological basis for anticoagulant and antithrombotic therapy in pulmonary hypertension. Cardiovasc Hematol Agents Med Chem 4:53–59 76. Weir EK, Reeve HL, Johnson G et al (1998) A role for potassium channels in smooth muscle cells and platelets in the etiology of ­primary pulmonary hypertension. Chest 114:200S–204S 77. Sakamaki F, Kyotani S, Nagaya N et  al (2000) Increased plasma P-selectin and decreased thrombomodulin in pulmonary ­hypertension

M.H. Kroll were improved by continuous prostacyclin infusion. Circulation 102:2720–2725 78. Lopes AA, Maeda NY, Goncalves RC, Bydlowski SP (2000) Endothelial dysfunction correlates differentially with survival in primary and secondary pulmonary hypertension. Am Heart J 139: 618–623 79. Popat U, Frost A, Liu E et  al (2005) New onset myelofibrosis in association with pulmonary arterial hypertension. Ann Intern Med 145:466–467 80. Zetterberg E, Popat U, Hasselbach H et al (2008) Angiogenesis in pulmonary hypertension with myelofibrosis. Haematologica 93:945–946 81. Haznedaroglu IC, Atalar E, Ozturk MA et al (2002) Thrombopoietin inside the pulmonary vessels in patients with and without pulmonary hypertension. Platelets 13:395–399 82. Williams LM, Nelson S, Mason CM et al (1996) Pulmonary venoocclusive disease in an adult following bone marrow transplantation: case report and review of the literature. Chest 109:1388–1391 83. Lantuejoul S, Sheppard MN, Corrin B et al (2006) Pulmonary venoocclusive disease and pulmonary capillary hemangiomatosis. Am J Surg Pathol 30:850–857 84. Kuebler WM (2006) Selectins revisited: the emerging role of platelets in inflammatory lung disease. J Clin Invest 116:3106–3108 85. Silliman CC, Bjornsen AJ, Wyman TH et al (2003) Plasma and lipids from stored platelets cause acute lung injury in an animal model. Transfusion 43:633–640 86. Loonet MR, Gropper MA, Matthay MA (2004) Transfusion-related acute lung injury: a review. Chest 126:249–258 87. Pitchford SC, Yano H, Lever R et al (2003) Platelets are essential for leukocyte recruitment in allergic inflammation. J Allergy Clin Immunol 112:109–118 88. O’Sullivan BP, Linden MD, Frelinger AL III et al (2005) Platelet activation in cystic fibrosis. Blood 105:4635–4641 89. Konstan MW, Byard PJ, Hoppel CL, Davis PB (1995) Effect of high-dose ibuprofen in patients with cystic fibrosis. N Eng J Med 332:848–854

Chapter 25

Lysis and Organization of Pulmonary Thromboemboli Timothy A. Morris and Debby Ngo

Abstract  After an acute pulmonary thromboembolism, thrombotic material in the pulmonary arteries either resolves by fibrinolysis or remodels into scars within the pulmonary arterial lumen. This chapter focuses on the balance between these two processes and how perturbations in this fine balance may contribute to the development of permanent vascular obstruction within the pulmonary circulation. Keywords  Fibrinolysis • Fibrinogen • Fibrin • Pulmonary embolism • Chronic thromboembolic pulmonary hypertension • Thrombosis

1 Introduction After an acute pulmonary thromboembolism, thrombotic material in the pulmonary arteries either resolves by fibrinolysis or remodels into scars within the pulmonary arterial lumen. This chapter focuses on the balance between these two processes and how perturbations in this fine balance may contribute to the development of permanent vascular obstruction within the pulmonary circulation.

reasonable to expect that erythrocytes entrapped within thrombi would disintegrate within this period. The duration for which fibrin persists within thromboemboli may be inferred by plasma levels of fibrin d-dimer, a principal fragment resulting from fibrinolytic digestion. When patients with pulmonary thromboembolism are treated with anticoagulants to prevent the deposition of new fibrin, elevated blood d-dimer levels can be found in about two fifths of patients at the end of 1  month [4]. However, by 3  months only about one tenth of patients remain positive and about half of those are positive due to recurrence. Residual defects after acute pulmonary thromboembolism commonly persist beyond the expected survival of erythrocytes and fibrin [5]. It follows from these observations that long-term residual defects within the pulmonary arteries would be attributed to processes beyond the initial thrombotic episode. Recent experimental data suggest that early after pulmonary thromboembolism, inflammation and associated intimal hyperplasia occur within the pulmonary arteries (Fig. 1b–d) [6], similar to what is observed with chronic thromboembolic pulmonary hypertension [7, 8]. The extent to which this histological process occurs in actual patients must be inferred from clinical outcome studies.

2 Components of Acute Thromboemboli Acute thromboemboli themselves are impermanent structures. Histologically, they are composed of layers of platelets, fibrin, and erythrocytes [1]. Fibrin-entrapped erythrocytes constitute most of the volume in acute thromboemboli (Fig. 1a). The life expectancy of new erythrocytes within the circulation is between 2 and 4  months [2, 3] and it seems

T.A. Morris (*) Division of Pulmonary & Critical Care Medicine, University of California, San Diego, 200 W. Arbor Drive, San Diego, CA 92103, USA e-mail: [email protected]

3 Recovery of Perfusion After Acute Pulmonary Thromboembolism 3.1 Normalization of Perfusion In some patients after an acute thromboembolism, lung perfusion is rapidly restored, suggesting that fibrinolysis had been accelerated [9]. More typically, the resolution in the first week is incomplete and is followed by a slow recovery over the next 1–2  months [10, 11]. After this recovery phase, it is likely that residual perfusion defects reflect persistence of material other than fibrin-enmeshed erythrocytes.

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Fig. 1  Histologic sections from patients with unresolved pulmonary thromboembolism, leading to chronic thromboembolic pulmonary hypertension (CTEPH). Hematoxylin and eosin stained sections of pulmonary thromboemboli at various stages of fibrotic organization. (a) A ×10 view shows an acute thrombus with prominent fibrin, platelets, and

T.A. Morris and D. Ngo

erythrocytes. (b) A ×40 view shows inflammatory cell infiltration into the luminal edge of a thrombus. (c) A ×200 view shows fibrotic organization occurring in a region of the thrombus that had been infiltrated by inflammatory cells. (d) A ×40 view shows thrombus organized into scar tissue with fibroblast infiltration and dense collagen deposition Table 1  Normalization of perfusion after acute pulmonary thromboembolism (PE ) Patients with normalization Time interval after Patients of perfusion lung scan (%) acute PE Study (n)

Fig.  2  Serial lung perfusion scans measure perfusion recovery after 6  months of treatment for acute pulmonary thromboembolism. The patient depicted in the top row resolved 100% of the (34%) perfusion defect he demonstrated upon presentation. In contrast, the patient in the bottom row resolved only 7% of his original (43%) defect

One method of characterizing perfusion recovery after pulmonary thromboembolism (PE) is to examine the proportion of patients who have completely recovered lung perfusion at specific time points (Fig. 2). Several clinical studies disclosed that resolution of perfusion continues throughout the first several months of therapy, but that complete recovery (defined as the development of a scintigraphic perfusion scan with no regional perfusion defects) is far from universal. A retrospective cohort study of 102 patients with first-time submassive PE treated with heparin followed by oral anticoagulation for a minimum of 6 months disclosed complete resolution of defects in 6% of patients (six of 97) at 7–10 days and in only 27% of patients (26 of 96) by 6 months [12]. Another series, in which 74 patients received lung scans at various intervals during the first year after acute PE, reported that complete restoration of perfusion occurred in only two patients [13]. A series reporting serial perfusion scans in 69 patients with confirmed PE demonstrated complete resolution in only 46% of patients (32 of 69) at 4 months [11]. A smaller group

Menendez et al. [12]

96a

Walker et al. [13] Tow et al. [11] Murphy et al. [14] Hvid-Jacobson et al. [15] Palla et al. [16] Winebright et al. [17]

74 69 25 30 69 70

6 27 2 46 60 43

7–10 days 6 months 6 weeks to 1 year 6 months 5 months 6 months

0 3 33 13 34 84 b 75c 27

6 months 5 days 60 days Wartski et al. [5] 157 8 days 3 months UPET [18] 105 1 year Miniati et al. [19] 235 1 year Paraskos et al. [20] 33 1–7 years (mean 29 months) Hall et al. [21] 48 42 1–8 years Sutton et al. [22] 18 56 1–8 years De Soyza et al. [23] 13 69 25–59 months (mean 49 months) a  n = 96 patients evaluated for complete resolution at 7–10 days b  Near-normal perfusion reported and defined as less than 10% perfusion defect c  Near-normal perfusion reported and defined as less than 5% perfusion defect

of patients with angiographically proven PE showed a 60% (15 of 25 patients) incidence of complete resolution at some point during a 5-month follow-up period [14] (see Table 1). There is some discordance among the clinical reports about the prevalence of complete recovery several months after PE. For example, one 6-month follow-up of 30 patients reported complete resolution in 43% of PE patients [15], whereas follow-up for a similar period in another group of 69 PE patients did not identify complete restoration of blood flow in a single patient [16]. However, all studies disclosed a

25  Lysis and Organization of Pulmonary Thromboemboli

significant ­proportion of patients with incomplete perfusion recovery during the first half year after pulmonary thromboembolism. In one long-term follow-up series, perfusion scans were repeated in 70 acute PE patients (without other lung abnormalities) every 5 days for 1 month, then again at 3 months, 6 months, and up to 16 months later [17]. The frequency of perfusion recovery was only 3% (two of 70 patients) at 5 days, but increased to 33% (23 of 70 patients) by 60 days, without further changes after that point. Although perfusion recovery was not precisely defined, which makes the results difficult to compare with those of the other reports, it was clear that some patients recovered relatively rapidly, whereas the rest recovered slowly or incompletely. Several studies have accepted “near-normal” perfusion on follow-up perfusion scans as evidence of complete recovery. Defining a near-normal perfusion scan as showing an overall perfusion defect of 5% or less, the Tinzaparin ou Heparin Standard: Evaluation dans l’Embolie Pulmonaire Study (THESEE) study group reported complete recovery in 13% of patients (21 of 157) at 8 days and in 34% of patients (53 of 157) at 3  months [5]. When a more forgiving cutoff of less than 10% perfusion defect was used to indicate recovery, the recovery rate only increased to 41% (65 of 157 patients) at 3 months. Another series reported only a 3% frequency of completely normal perfusion after 3 months and near-normal recovery (defined as residual perfusion defects of less than 10.5%) in 33% of patients (24 of 74) [13]. The differences in the reported outcomes may reflect different patient populations and different criteria for detecting residual perfusion defects. However, it was typical in these series that more than half of the patients failed to completely resolve their perfusion defects during the first 6 months of follow-up. Some series have demonstrated that many patients fail to recover their perfusion, even years after their acute PEs. Of 105 patients followed out to 1  year in the Urokinase Pulmonary Thromboembolism Trial (UPET), the perfusion scan was near normal (defined as perfusion defect less than 10%) in 84% of patients (88 of 105), with no significant changes in the proportion of near-normal scans between 3  months and 1  year [18]. Another recent study reported near-normal perfusion in 75% of patients 1  year after PE [19]. However, in a different series, repeat lung scans 1–7 years (mean 29 months) after acute PE disclosed complete perfusion recovery in only 27% of patients (nine of 33) [20]. Another study, limited to patients surviving acute massive PE (defined as the involvement of more than half of the major pulmonary arteries), performed repeat lung scans 1–8 years later and reported complete recovery in only 42% of patients (20 of 48) [21]. Somewhat smaller studies have shown similar results. Repeat perfusion scans in a series of

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18 patients 1–8  years after “minor” or “subacute massive” PE demonstrated recovery in 56% of patients (ten of 18) [22], whereas 13 male patients studied 25–59 months (mean 49 months) after PEs associated with pulmonary hypertension showed a perfusion recovery rate of 69% (nine of 13 patients) [23]. Despite the heterogeneity of the clinical data, several generalizations can be made with respect to the time course of perfusion recovery after acute PE. The percentage of patients experiencing complete scintigraphic resolution during the first week is very low. By 1 month, roughly one fifth to one third of patients on average will go on to complete recovery. Between 1 and 3 months, an additional small percentage of patients may recover. After 3 months, additional patients will recover, although the data vary so widely that it is difficult to draw conclusions regarding the proportion who will eventually recover perfusion completely. The likelihood of complete recovery is influenced by the size of the initial defect as well as the existence of other cardiopulmonary diseases. Larger initial perfusion defects lower the incidence of complete recovery at all time points after PE. Short-term recovery of perfusion occurred in one third of patients with small initial perfusion defects (less than 15%), but only in about one fifth of those with larger defects [17]. Likewise, the frequency of complete resolution 4 months after PE varied from 67% in patients with minimal initial perfusion defects (15% or less) to 20% in patients with severe defects (31–50%) [11]. Walker et al. observed complete recovery in 49% of patients whose initial defects were less than 30% compared with only 14% among those with initial defects of 30% or greater, a statistically significant difference [13]. Pre-existing cardiac or pulmonary disease at the time of PE has been associated with a lower likelihood of complete resolution of perfusion defects. One series reported complete recovery by 3 months in 32% of patients (16 of 51) without heart disease compared with only 5% of patients (one of 19) with heart disease [17]. A longer follow-up of 48 patients surviving acute massive PE reported complete scintigraphic normalization in 46% of patients without concomitant cardiopulmonary disease versus 22% of patients with concomitant cardiopulmonary disease [21]. These observations may reflect slower rates of PE resolution in these patients or may be explained by the effect of the concomitant diseases themselves on lung perfusion.

3.2 Partial Recovery of Perfusion Although complete recovery of perfusion is by no means universal after acute PE, relative recovery occurs in most

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patients and continues at least throughout the first 6 months. Perfusion recovery can be characterized by the average amount of perfusion recovered relative to the initial perfusion defect. Relative perfusion recovery = (initial defect-defect on follow-up)/(initial defect), where defects are expressed as proportions of the normal perfusion to both lungs. Most studies disclosed remarkably similar patterns of relative perfusion recoveries after acute PE. In 69 patients with PE followed with serial lung scans, the mean improvement by 6 months was 59% of the initial perfusion impairment [16]. In a similar group of 33 patients with PE, serial lung scans disclosed a mean relative perfusion recovery of about two thirds by 6 months [24]. Serial scans for 1 year in 61 patients with PE showed a mean relative perfusion recovery of 64%, with most of the recovery within the first 6 months [25]. In a series of 96 PE patients, the number of unperfused lung segments improved by 41% at 7–10 days and by 68% at 6  months [12]. Finally, among the 157 patients from the THESEE group, the estimated vascular obstruction decreased by 41% at 8  days and by 61% at 3 months [5]. An earlier and somewhat smaller study [14] reported faster recovery in 25 patients with angiographically proven PE (63% mean relative perfusion recovery by 1  week, 67% by 2  weeks, 80% by 3  weeks, and 80% by 4 weeks), although these results have not been observed in other studies. The rate of recovery is most rapid during the first month [13, 16, 24, 25], which accounts for about three fourths of the entire recovery that will occur [16, 24]. On average, the relative perfusion recovery during this phase falls somewhere between 30 and 50%. During the time period from 1 to 3  months, additional perfusion is recovered, but this improvement is not as substantial or as rapid as that seen during the first month. After 3 months, additional recovery occurs at an even slower rate, if it occurs at all (see Table 2). Table 2  Relative recovery of perfusion after acute PE Mean relative perfusion Time interval Study Patients (n) recovery (%) after acute PE Palla et al. [16] Prediletto et al. [24] Donnamaria et al. [25]

69 33 61

Menendez et al. [12]

96

Wartski et al. [5]

157

Murphy et al. [14]

25

59 60 59 64 41 68 41 61 63 67 80 80

6 months 6 months 6 months 12 months 7–10 days 6 months 8 days 3 months 1 week 2 weeks 3 weeks 4 weeks

The relative perfusion recovery after PE is typically consistent among patients, regardless of the size of the initial perfusion defect. Three separate studies disclosed no significant differences in the relative recovery of perfusion between patients with large initial defects and those with smaller ones when the subjects had repeat perfusion scans at 3 months [5, 17] or 4 months [11]. It follows that the size of the initial PE-related perfusion defect influences the size of the residual defects during follow-up. Among 29 PE patients without other cardiopulmonary disease, those with initial defects of greater than 30% had an average defect of 18.7% after 6  weeks, compared with an average defect of 9.9% for those with smaller initial defects [13]. Two large multivariate analyses of various factors affecting resolution after PE also identified the extent of the defects at 7–10 days as predictive of the extent of residual defects measured at 6 months [12, 26]. Relative perfusion recovery is influenced by the presence of underlying cardiopulmonary disease. One series reported 50% or more relative perfusion recovery in 58% of patients without cardiovascular disease (23 of 40) compared with 44% of patients with cardiovascular disease (12 of 27) [11]. Another series showed the frequency of 50% or greater relative perfusion recovery at 90 days to be 93% in patients without cardiac disease compared with 58% in patients with cardiac disease [17]. Six months after the initial PE, 35 patients without cardiopulmonary disease had a mean relative perfusion recovery of 70% compared with 45% relative perfusion recovery among 34 patients with cardiopulmonary disease [16]. Among 157 patients from the THESEE study scintigraphically followed out to 3 months, those with associated cardiac or pulmonary disease experienced a relative recovery of 57%, with pulmonary vascular obstruction score improving from 53 to 23%, whereas those without associated cardiac or pulmonary disease experienced a relative recovery of 68%, with pulmonary vascular obstruction score improving from 44 to 14% [5]. One may expect that in acute PE patients with pre-existing cardiopulmonary disorders the degree of perfusion recovery will be about one third less than the degree of recovery in other patients with acute PE. It is likely that underlying cardiopulmonary conditions themselves are responsible for some of the perfusion abnormalities observed during PE, but it is also possible that underlying cardiopulmonary disease predisposes to persistent or recurrent thromboembolism. The contribution of each of these factors is difficult to sort through, since the persistence of a perfusion defect in the same position from one scan to another may reflect the failure of an initial thrombus to resolve, the recurrence of thrombus in the same location, or simply decreased perfusion from parenchymal or cardiac disease.

25  Lysis and Organization of Pulmonary Thromboemboli

Fig.  3  Gross pulmonary thromboendarterectomy specimen from a CTEPH patient with bilateral disease, showing segmental and subsegmental extension

3.3 Clinical Importance of Incomplete Recovery of Perfusion In up to 4% of PE patients, pulmonary artery scarring (Fig. 3) develops into symptomatic chronic thromboembolic pulmonary hypertension (CTEPH). CTEPH, however, may be the extreme manifestation of a much more common phenomenon. For example, up to 15% of acute PE patients remain symptomatically compromised 2 years after treatment [27] and may have abnormal pulmonary gas exchange (O2 gradients, dead space, etc.) as well [24]. Some of the hemodynamic manifestations of pulmonary emboli may also persist beyond the acute episode. Serial echocardiography of PE patients suggests a rapid, but often incomplete phase of recovery in the systolic pulmonary artery pressure (as estimated from the tricuspid regurgitation jet velocities) during the first month, followed by a much more gradual rate of recovery in the subsequent year [28, 29]. High pulmonary artery pressures during the initial phase were associated with increased risk of persistent pulmonary hypertension, further supporting the etiological role of incomplete thrombus resolution in the development of chronic disease. These findings suggest that incomplete clot resolution has clinical manifestations in a significant proportion of acute PE patients. Since the most severe manifestation of this phenomenon is CTEPH, it serves as a model to evaluate the mechanisms of unresolved PE.

4 Pharmacologically Enhanced Fibrinolysis The long-term effect of plasminogen-activating drugs on the resolution of acute PE, and the likelihood of CTEPH, is unclear. Management decisions must be made on the basis of

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indirect information rather than by comparative clinical trials in the appropriate patient population. The Urokinase in Pulmonary Thromboembolism Trial (UPET) [18], the largest trial to randomize patients into thrombolytic or standard heparin therapy, was performed on hemodynamically stable patients. There was a very shortterm hemodynamic benefit: cardiac outputs and ­pulmonary pressures were improved on the day after thrombolytics. No positive effects on morbidity or mortality were demonstrated, however, nor was the ultimate degree of embolic resolution any greater. Multiple studies have confirmed that these agents promote rapid embolic resolution [30]. However, there does not appear to be a long-term benefit to more rapid (but not necessarily more complete) clot resolution [31]. Patients with massive PE have much higher mortality rates than the patients evaluated in the UPET. The mortality for patients with high right ventricular pressures and high pulmonary artery pressures is about double the mortality for more stable PE patients. The mortality increases exponentially as patients progress to hypotension, shock, and then the need for cardiopulmonary resuscitation [32]. It is likely that the acute hemodynamic benefits of thrombolytics will have a much higher impact on these patients than on the stable patients in the UPET. The outcome benefit of ­thrombolytics in unstable patients has been studied in uncontrolled, retrospective studies. One study reported a lower mortality in patients treated with thrombolytics [32], whereas another study reported a higher mortality (due to bleeding) [33]. A study of “submassive” PE (i.e., with evidence of right ventricular strain) [34] randomized patients to either thrombolytics plus heparin or heparin plus a placebo. The groups were compared on the basis of a “combined” primary end point including death and escalation of treatment (including subsequent thrombolytic therapy). Although the group receiving thrombolytics had a lower incidence of the combined end point, the only actual “outcome” difference between the groups was that those treated with heparin plus a placebo received more thrombolytic therapy during the follow-up period. No other outcome, such as death and the need for mechanical ventilation or vasopressors, was different between the groups, begging the question of whether “rescue” thrombolytic therapy was necessary. Unfortunately, the results did not answer the question of whether thrombolytic therapy is beneficial initially or even during followup for patients with evidence of right-sided heart strain. Thrombolytics are costly and carry significant risks. A review of clinical trials disclosed a 2.1% intracranial hemorrhage rate and 1.6% fatal intracranial hemorrhage rate when thrombolytics were used for thromboembolic disease [35]. For this reason, thrombolytics should be reserved for management of the patient with massive embolism and

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p­ ersistent hypotension. Even in this situation, patient selection is difficult and only physicians quite familiar with the drugs should use them.

5 CTEPH as a Model of Unresolved Acute PE CTEPH can be induced following an acute PE in an animal model by inhibition of plasmin-mediated fibrinolysis with tranexamic acid. Similar to e-aminocaproic acid, tranexamic acid inhibits fibrinolysis by attaching to lysine-binding sites on plasminogen, preventing its activation [36]. In the absence of pharmacological inhibition, the canine fibrinolytic system is typically so efficient that acute pulmonary emboli would be nearly undetectable after 30 days [37]. However, dogs treated after PE with tranexamic acid and autopsied 30–40 days later had large amounts of chronic, organized material adherent to the pulmonary arterial wall, similar to the material observed in patients with CTEPH. Furthermore, the animals with large amounts of the intra-arterial organized material had persistent perfusion defects on scintigraphic scanning and significantly elevated pulmonary vascular resistances, also similar to patients with CTEPH. Clinically, incomplete recovery of perfusion may predispose patients to developing CTEPH [38]. The reason why a fraction of patients with PE go on to have CTEPH is unclear. Most patients who present with CTEPH who were previously diagnosed with acute pulmonary emboli had initial presentations (clot burden, clinical stability, etc.) that were indistinguishable from those of patients who went on to resolve their acute clots [39]. A prospective study of patients presenting with PE disclosed that, in addition to the size of the initial PE, factors such as recurrent PE, idiopathic PE, and PE at a young age were associated with a higher risk for CTEPH [38]. These findings raise the possibility that an inherited or acquired predisposition to incomplete clot resolution is present in at least some patients who develop CTEPH. Although the specific causes leading to incomplete thrombus resolution after PE are not fully understood, some insights may be gained by laboratory and clinical investigations into the cause of CTEPH. It stands to reason that delayed fibrinolysis could be implicated in the development of CTEPH if the acute pulmonary thromboembolism created a substantial local nidus for remodeling and intravascular scar formation. The resulting persistent large vessel obstruction combined with various degrees of an incompletely understood small vessel reaction lead to progressively worsening pulmonary hypertension [40]. For these reasons, investigation has focused on two areas: (1) the persistence of acute thromboemboli themselves (e.g., inadequacy of or resistance to fibrinolysis) and (2) mechanisms of thrombus replacement by inflammatory cells and connective tissue.

T.A. Morris and D. Ngo

6 Fibrinolytic Enzymes and CTEPH Despite extensive studies, no specific abnormalities of fibrinolytic mediators that could disturb the balance between plasminogen activators and their inhibitors, or regulate the conversion of plasminogen to plasmin have been discovered in CTEPH patients [41]. There have been no abnormalities in the plasma levels of plasminogen or a2-antiplasmin (the primary physiological inhibitor of plasmin) associated with CTEPH. A study of 32 CTEPH patients revealed neither high resting plasma levels of plasminogen activator inhibitor 1 (PAI-1; the primary physiological inhibitor of tissue plasminogen activator, tPA) nor blunted response of tPA to venous occlusion [42]. In a separate study to determine if failure to lyse pulmonary thromboemboli might be caused by an abnormality in the endothelial-cell-associated fibrinolytic system, endothelial cells were scrape-harvested from the pulmonary arteries of 13 CTEPH patients during pulmonary thromboembolism surgery [43]. Conditioned medium from primary cultures of these cells was analyzed for levels of tPA and PAI-1; cells harvested from pulmonary arteries of organ donors were used as controls. No significant differences between patients and controls were noted. Moreover, CTEPH patient cells were observed to increase the secretion of tPA and PAI-1 in response to thrombin in a fashion similar to cells from organ donors.

7 Fibrinogen and Susceptibility to Fibrinolysis There is evidence to suggest the fibrin clots themselves are relatively resistant to lysis in patients with CTEPH [44]. This section will explore the components of thrombus structure and fibrinolysis that have been investigated as possible etiological factors responsible for CTEPH. Fibrinogen is a dimeric glycoprotein, composed of three pairs of nonidentical, disulfide-linked polypeptide chains (Aa2Bb2g2) [45]. The thrombin-catalyzed conversion of fibrinogen to fibrin involves the sequential removal of fibrinopeptides A and B from the N-terminal regions of the Aa and Bb chains, respectively (Fig. 4a, b). The appearance of new N-terminal sequences on the a and b chains creates “A knobs” and “B knobs,” respectively, in the central domain of the fibrin molecule. The A knobs and B knobs fit noncovalently into complementary “holes” in a terminal globular domain of an adjacent molecule (Fig.  4c). Fibrin then spontaneously polymerizes into dimers that form half-staggered protofibrils. The protofibrils are then stabilized by covalent cross-links that occur first between the g chains and then between the a chains [46]. The organization of these protofibrils results in a

25  Lysis and Organization of Pulmonary Thromboemboli

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herin (VE-cadherin) [48]. Fibrin induces expression of interleukin-8 and facilitates the migration of repair cells along endothelial cells by interactions with intracellular adhesion molecule-1 (ICAM-1) [49] and the leukocyte adhesion receptor Mac-1 [50] (see Fig. 5).

7.1 Biological Properties of the Fibrin B Chain N-Terminus (B Knob) That May Affect Thrombus Remodeling

Fig. 4  Fibrin polymerization and fibrinolysis. (a) Fibrinogen contains fibrinopeptide A (FPA) and fibrinopeptide B (FPB) on the N-terminal region of the Aa and the Bb chain, respectively. (b) Thrombin-induced cleavage of fibrinopeptides exposes the fibrin “A knobs” and “B knobs.” The B knobs (arrow) are shown in gray. (c) The exposed A and B knobs fit into complementary holes in a terminal globular domain of an adjacent molecule, allowing fibrin to polymerize into thick network of fibers. (d) Plasmin (arrows) degrades the fibrin polymers during fibrinolysis. Remodeling cells represent endothelial and fibroblast cells interacting within the dense fibrin network and forming intraluminal connective tissue

thick network of fibers that form the structural backbone of the acute thrombus. It is this network that is ultimately degraded by the proteolytic action of plasmin (Fig. 4d) Fibrinogen may also be important to pulmonary artery remodeling. Fibrin activates pulmonary artery endothelial cells [47] via the fibrin receptor vascular endothelial cad-

The fibrin B knob has been implicated in a variety of physiological events such as heparin binding [51], cell signaling [48], and angiogenesis [52] that may be involved with thrombusinduced pulmonary artery scarring. Fibroblastic and endothelial cell growth onto fibrin polymers (the first step in organization of the thrombus into scar tissue) is stimulated by peptides found at the B knob (Fig. 4d) [53]. Endothelial cell proliferation can be stimulated by the E fragment of fibrin, which contains the B knob, as well as peptide analogues of the B knob. Fibrin fragment E stimulates the proliferation, migration, and differentiation of microvascular endothelial cells in vitro, both in the presence and in the absence of additional growth factors, including vascular endothelial growth factor and basic fibroblast growth factor (bFGF) (Fig. 5). The N-terminus of the b chain occurs in a very flexible part of fibrinogen, which cannot be resolved by X-ray crystallography [54]. The only human variant found within this region involves substitution of the N-terminal Gly, possibly owing to the persistence of fibrinopeptide B and consequently unexposed B knob [55]. However, fibrinogen modifications remote from the N-terminus itself may also affect its plasminmediated removal. For example, the prothrombotic fibrinogen Nijmegen [56] involves a b44 Arg → Cys substitution that may result in steric interference with plasmin-mediated release of the B knob through disulfide bonding [55]. Much of the lytic action of plasmin occurs at the coiledcoil regions that connect fibrin’s globular domains. However, some of the fibrin fragments that plasmin releases encompass only residues close to the N-terminus of the b chain, which is not involved in the coiled-coil regions (Fig.  4d). Cleavage of the N-terminus of the b chain would not help separate the globular regions of fibrin from each other, but may influence the balance between fibrinolysis and cellmediated thrombus remodeling. Thrombi would also enhance the stimulation of repair cells if their B knobs were more accessible to binding (even if the number of B knobs themselves were normal). Binding experiments with T2G1 antibodies (specific for the fibrin B knob) disclosed that less opaque “fine” fibrin clots, composed of thinner fibers [57], have greatly increased accessibility of the B knobs, compared with “coarser” fibrin clots [58, 59]. It stands

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Fig. 5  The factors implicated in remodeling of thrombi into intravascular scars and the interactions between fibrin and inflammatory cells and mediators involved in pulmonary vascular remodeling (see the text). MCP1 monocyte chemoattractant protein 1, bFGF basic fibroblast growth factor, EC endothelial cell

to reason that abnormally “fine” clots, with more accessible B knobs, would be more likely to stimulate fibroblastic and endothelial-cell-mediated thrombus organization [53] than would normal, coarser clots, regardless of lysis rates.

7.2 Resistance to Fibrinolysis and CTEPH Fibrinogen variants might be involved with CTEPH development. For example, five heterozygous fibrinogen gene mutations (fibrinogensSan Diego 1-5) with abnormalities in fibrin polymer structure and susceptibility to lysis were discovered in a series of 33 CTEPH patients (Fig. 6) [60]. Another variant, fibrinogenBellingham, includes a g275 Arg → Cys substitution that confers a relative resistance to tPA-mediated fibrinolysis [61]. The mechanism of resistance was presumably related to steric interference with the normal polymerization/cross-linking structure owing to the cysteine residue or a structure bound to it. Interestingly, family studies of fibrinogenBellingham demonstrated that the mutation alone was not sufficient to cause CTEPH. Rather, CTEPH required the combination of acute PE (in the reported case, due to knee replacement surgery as well as an additive thrombogenic genetic risk factor) along with resistance to fibrinolysis. Relatives with the g275 Arg → Cys substitution alone did not develop CTEPH, nor do most other individuals with the g275 Arg → Cys substitution. For this reason, the absence of disease in family members of CTEPH patients does not rule out the presence of fibrinogen abnormalities.

Fig.  6  CTEPH-associated mutations: fibrinogens (SD-1–SD-5). The positions of five CTEPH-associated mutations are illustrated on a ribbon model of fibrinogen. In fibrinogen SD-1 and fibrinogen SD-5, negative charges are imparted to regions within fibrin, promoting the formation of “thin” fibrin fibers. In fibrinogen SD-2 (gY114H) and fibrinogen SD4 (AaL69H), amino acid substitutions result in the insertion of a polar side chains within the “helix-permissive” hydrophobic center of the fibrin coiled coil, which would disrupt the molecular structure. In fibrinogen SD1–SD3, the Bb P235L substitution imparts a structural change to a site that is involved in the activation of tissue plasminogen activator by fibrin

8 Acute Thromboemboli, Inflammation, and Remodeling Incomplete thrombus resolution would result in pulmonary vascular scarring after an acute PE only if there was a persistence of ligands within the residual thrombotic material that would stimulate inflammation and remodeling by connective tissue cells. Interestingly, there is a clinical association between chronic inflammatory conditions and the development of CTEPH after acute PE (Table 3) [62].

25  Lysis and Organization of Pulmonary Thromboemboli Table  3  Conditions that have been clinically associated with the development of chronic thromboembolic pulmonary hypertension (CTEPH) after acute PE Conditions associated with CTEPH Clinical • Idiopathic cause of the PE [38] • Right-sided heart strain during acute PE [28, 38, 81] • Age [38] • Previous splenectomy [82–84] • Ventriculoatrial shunts (treatment of hydrocephalus) [62, 82–84] • Chronic inflammatory disorders (e.g., osteomyelitis, inflammatory bowel disease) [62, 82–84] Laboratory • Lupus anticoagulant [85] • Elevated factor VIII levels [83] • Non-O blood groups [84] • Lipoprotein (a) [86] • Heart-type fatty acid binding protein [87]

Several inflammatory mediators may be stimulated by thrombosis, and may be implicated in the connection between acute pulmonary thromboemboli and the vascular scaring associated with CTEPH [63]. Fibrin(ogen) has been implicated in biological processes such as angiogenesis in wound healing, tissue repair, tumorigenesis, and cardiovascular disease. Fibrin has been linked to mediators that facilitate interaction of endothelial cells with each other and extracellular matrix molecules (Fig. 5).

8.1 VE-Cadherin The interaction between fibrin and VE-cadherin can induce biological changes that lead to remodeling in the vascular wall (Fig. 5) [64]. Endothelial cells cultured on a fibrin-coated surface will form a dense monolayer; and if they are subsequently exposed to a fibrin gel, they will rearrange themselves along the gel’s surface into a network of capillary tubes [48]. This property of endothelial cells appears to require interaction with the fibrin B knob. Fibrinogen treated with thrombin (which removes fibrinopeptides A and B) is capable of inducing the endothelial cell reaction, whereas unaltered fibrinogen, fibrinogen treated with Atroxin (which removes only fibrinopeptide A), or fibrin treated with protease III (which removes the peptide Bb1-42 that encompasses the fibrin B knob) does not. Furthermore, the addition of “soluble B knob” (peptide b15-42) to medium influenced the endothelial cells’ morphological organization. These finding suggests that the fibrin B knob is an important stimulus of endothelial-cellmediated vascular remodeling by endothelial cells. The interaction with fibrin is specific to vascular endothelial cells and stimulation of vascular endothelial cells by the fibrin B knob appears to be mediated by VE-cadherin [48,

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64–66]. Adipose-derived endothelial cells expressing only N-cadherin but no VE-cadherin demonstrated no specific binding of fibrin B knobs [64]. VE-cadherin is a transmembrane cell–cell adhesion receptor with a modular structure that includes cytoplasmic, transmembrane, and five extracellular domains, four of which appear to be necessary for interaction with fibrin [65]. VE-cadherin regulates cell–cell contacts at endothelial intercellular junctions, and therefore plays an important role in maintaining endothelium integrity. The normal, highly avid, binding between endothelial cells and fibrin B knobs is disrupted by antibodies against residues 26 to 156 of VE-cadherin [48]. In addition, monoclonal antibodies against the first extracellular domain of VE-cadherin inhibited capillary tube formation and disrupted preformed capillary tube integrity in human umbilical vein endothelial cell monolayers [66]. Endothelial cells will bind to fragments of fibrin II (e.g., with fibrinopeptides A and B removed) that contain only the “N-terminal disulfide knot” (NDSK II), which contains the fibrin B knob. Endothelial cells do not specifically bind to NDSK fragments that do not contain the b15-42 peptide (Fig. 7) [64]. Furthermore, endothelial cell binding to NDSK II can be inhibited by a b15-42/ovalbumin conjugate as well as monoclonal antibodies to VE-cadherin [64]. Binding of VE-cadherin to fibrin analogues requires the B knob to be present in the configuration in which it typically occurs within the NDSK II. Normally, fibrin B knobs reside as closely apposed pairs (one from each peptide b chain) within NDSK II. Studies with recombinant peptides have demonstrated that VE-cadherin will bind to B knob analogues only if they are present in dimeric form, with the ligands at a distance from each other similar to what is observed in NDSK II (e.g., dimerized b15-66 peptides) [65].

8.2 E-Selectin and P-Selectin The role of selectins in the balance between thrombus resolution and vascular remodeling remains a topic of investigation. Some experimental data appear to implicate selectins in both thrombus formation and the subsequent inflammatory response to thrombosis (Fig. 5). Significant increases in P-selectin messenger RNA (mRNA) expression and blood levels of P-selectin have been observed in C57BL/g mice for 2  days after experimental induction of inferior vena cava (IVC) thrombi [67]. A subsequent rise in E-selectin mRNA and protein levels was observed 6 days after the induced thrombosis. No effect was observed in sham-treated subjects. Experiments with knockout mice for P-selectin and E-selectin also further elucidate the role of selectins after experimental thrombosis [67]. Knockout of E-selectin,

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Fig.  7  Accessibility of the fibrin B knob in various derivatives of fibrin(ogen). The fibrin B knob (shown in gray), which has been implicated in endothelial cell binding, is not exposed in fibrinogen because of the presence of the N-terminal FPB. The B knob is exposed (arrows) after the conversion of fibrinogen to fibrin II, by thrombin-mediated fibrinopeptide cleavage. Proteolysis of fibrinogen with cyanogen bromide generates the “N-terminal disulfide knot” (NDSK), which contains the intact N-termini of the Aa, Bb, and g chains, including FPA and FPB. NDSK treated with Atroxin releases FPA to create NDSK I. Neither product contains exposed B knobs. If NDSK is treated with thrombin to create NDSK II, in which FPA and FPB are cleaved, the B knob is exposed. NDSK treated with protease III to cleave the Bb1-42 peptide produces NDSK 325, which lacks the B knob entirely. Only the molecules that contained exposed B knob (fibrin II and NDSK II) are able to bind to endothelial cells (see the text)

P-selectin, or both resulted in a more rapid and more complete resolution of experimentally induced IVC thrombi. Immunohistochemistry confirmed that the thrombi formed in selectin-deficient mice contained significantly less fibrin content 2 and 6  days after thrombosis than thrombi from wild-type mice. P-selectin may have a specific role in thrombosis-related inflammation (Fig.  5) [68–70]. P-selectin is an adhesion molecule expressed on the surface of activated platelets and

T.A. Morris and D. Ngo

endothelium. It mediates their interaction with neutrophils, monocytes, and other inflammatory cells by binding to its ligands, predominantly the transmembrane sialylated and sulfated glycoprotein PSGL-1. P-selectin expressed on platelet membranes is a mediator of neutrophil and monocyte accumulation onto venous thrombi, which in turn enhances further fibrin deposition on the clot surface [71]. Once expressed on the surface of platelets, some of the P-selectin is released into the circulation. In patients with PE, the levels of both P-selectin on activated platelets and soluble P-selectin in the plasma are elevated [72]. P-selectin activates monocytes to produce tissue factor, further stimulating the clotting cascade [73], and induces neutrophils to activate protein kinases involved in inflammation [74]. P-selectin is also involved in platelet–platelet activation interactions, mediated through the counterreceptor glycoprotein Iba–IX–V [75]. P-selectin antagonism with P-selectin glycoprotein legend (rPSGL-Ig) and low molecular weight heparin significantly decreased the postthrombotic appearance of profibrotic mediators such as interleukin-13, monocyte chemoattractant protein 1, bFGF, and transforming growth factor b in a rat model of thrombosis (Fig.  5) [76]. The decrease in these mediators corresponded to a subsequent decrease in postdeep vein thrombosis vein wall fibrosis, which was independent of the masses of the original thrombi. P-selectin enhances leukocyte–leukocyte, leukocyte–platelet, and leuckocyte– endothelial interactions; it is plausible that these interactions might be implicated in the release of profibrotic mediators. This finding supports the role of P-selectin as a possible mediator of thrombus persistence, through the mechanism of postthrombotic inflammation and thrombosis.

8.3 Heparin, Low Molecular Weight Heparin, and Fondaparinx The role of P-selectin and E-selectin in the persistence of thrombi and the subsequent postthrombotic vascular inflammation may have implications for the pharmacotherapy of thrombosis. Heparin, its derivatives (low molecular weight heparin), and analogues (fondaprinux) are the mainstay of the initial therapy of acute pulmonary thromboembolism, but the mechanism by which these drugs encourage resolution has not been systematically studied. Experimental evidence suggests that, in addition to their anticoagulant properties, heparin-type medications may block selectin and attenuate the inflammation and remodeling that can lead to poor resolution of the thromboemboli within the pulmonary arteries (Fig. 5). However, different formulations of heparin and low molecular weight heparin have markedly different selectinblocking potencies in  vitro, influenced largely by their patterns of saccharide sulfation [77]. Furthermore, the

25  Lysis and Organization of Pulmonary Thromboemboli

s­ electin-blocking potencies of heparin and its derivates appear to correspond with their ability to inhibit selectinmediated cellular growth in vivo [78]. However, the relative effects of these medications on the resolution of pulmonary thromboemboli has not been systematically studied.

395

There is some experimental evidence to suggest that interleukin-10 (IL-10), an anti-inflammatory cytokine, may attenuate perithrombotic inflammation and thrombosis (Fig. 5). A rat model of thrombosis followed by local transfection with viral IL-10 demonstrated that local IL-10 expression can decrease leukocyte extravasation into the adjacent vascular wall caused by thrombus-related inflammation. In addition, immunohistochemistry studies demonstrated a lower concentration of P-selectin and E-selectin at endothelial cell borders. ELISA studies demonstrated a corresponding decrease in the cellular expression of P- selectin, E-selectin, and ICAM-1 [79]. IL-10 expression rises in response to thrombus formation, although it is not clear whether the increase in blood levels causes an attenuation of postthrombotic inflammation and remodeling [67]. An immediate rise after thrombosis, presumably due to the release of preformed IL-10, is followed by elevations in IL-10 mRNA and IL-10 protein expression for several days. However, experiments to date have not shown a consistent effect of IL-10 on thrombus resolution, so the clinical importance of this mechanism remains unclear [80].

exchange and ­elevated pulmonary artery pressures, and, finally, severe pulmonary ­vascular obstruction and hypertension seen in CTEPH. The pathophysiologic mechanisms related to poor thrombus resolution and its eventual organization into scar tissue are incompletely understood. Incomplete thrombus resolution may be due to resistance to fibrinolysis. Organization of the thrombotic material into scar tissue may be due to fibrin-mediated pulmonary vascular remodeling as well as thrombus-mediated inflammation. Mutations of fibrinogen associated with CTEPH may render it more resistant to fibrinolysis. As the initial clot fails to completely lyse, the surrounding pulmonary vascular system is persistently exposed to thrombotic components such as fibrin and platelets. Fibrin, in particular its B knob fragments, has been implicated in several physiologic events such as heparin binding, cell signaling, and angiogenesis that may be involved in thrombus-induced pulmonary artery scarring. Additionally, experimental data suggest involvement of E-selectin and P-selectin in thrombus formation and inflammatory response to thrombosis. Despite these findings, it still remains unclear why some patients with acute PE have poor thrombus resolution and why a few go on to develop CTEPH. There are many factors underlying the development of CTEPH that perturb the complex balance between coagulation and fibrinolysis and the normal processes of pulmonary vascular remodeling and inflammation. Although it is widely believed that there is a thromboembolic basis in the development of CTEPH, the detailed genetic, molecular, and cellular mechanisms underlying its pathogenesis have yet to be fully explained.

9 Conclusion

References

8.4 Interleukin-10

After an acute PE, thromboemboli resolve to varying degrees by fibrinolysis. In rare cases they develop into scars that cause the permanent pulmonary vascular obstructions seen in CTEPH. Although most patients undergo complete perfusion recovery, many patients fail to resolve perfusion defects even months to years after an acute PE. The degree to which resolution occurs is influenced by clinical factors, including the size of initial thrombus as well as coexistence of cardiopulmonary disease. However, the factors predisposing a patient to poor recovery after acute PE and the development of CTEPH have not been completely elucidated. CTEPH can serve as a model to evaluate mechanisms of unresolved pulmonary thromboemboli. It may represent the extreme clinical manifestation of a more common phenomenon of incomplete thrombus resolution. The spectrum of clinical consequences resulting from incomplete thrombus resolution includes no symptoms, persistent symptomatic compromise associated with abnormal pulmonary gas

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Chapter 26

Interactions of Leukocytes and Coagulation Factors with the Vessel Wall Scott Visovatti, Takashi Ohtsuka, and David J. Pinsky

Abstract  Vascular endothelium is a highly specialized, metabolically active organ possessing numerous physiological, immunological, and synthetic functions. As a major capacitance reservoir which must accommodate passage of the entire circulating blood volume every minute, flow regulation and control of blood fluidity in the lung are essential. As a major component of the alveolar–capillary unit, the pulmonary endothelium is vulnerable to injury from multiple sources, including inhaled and circulating noxious agents, reactive oxygen species (ROS), and shear stress. Vascular damage triggers a complex and interconnected series of proinflammatory and procoagulation pathways that reinforce and even amplify one another. For example, in severe sepsis, proin-flammatory cytokines upregulate tissue factor (TF) on endothelium and monocytes, leading to systemic activation of coagulation. Conversely, coagulation proteases such as thrombin can bind to protease-activated receptors (PARs) on endothelial cells, mononuclear cells, platelets, fibroblasts, and smooth muscle cells, resulting in an upregulation of inflammatory mediators such as interleukin (IL)-1. Such interactions between the inflammatory and coagulation systems contribute to the pathogenesis of conditions such as acute lung injury (ALI), ventilator-associated lung injury (VALI), disseminated intravascular coagulation, and pulmonary hypertension. The purpose of this chapter is  to explore intrinsic features and external factors that interact with pulmonary endothelium which cause a shift between a baseline antithrombotic/anti-inflammatory phenotype and an activated state of endothelium, characterized by prothrombotic/proadhesive properties. Given the complexities of these systems, each will be discussed separately in this chapter, with emphasis placed on areas of overlap. Keywords  Coagulation • Blood-endothelium interaction • Thrombosis • Cell-cell adhesion • Barrier function • Lung injury • Reactive oxygen species • Inflammation

S. Visovatti (*) Division of Cardiology, University of Michigan, 1150 W. Medical Center Drive, Rm 7220, MSRB III, Ann Arbor 48109, MI, USA e-mail: [email protected]

1  Introduction Vascular endothelium is a highly specialized, metabolically active organ possessing numerous physiological, immunological, and synthetic functions. From one vascular bed to another, endothelial cells exhibit a phenotypic heterogeneity that allows them to provide tailored functions for specific microenvironments [1]. For example, the spacing between endothelial cells can range from the continuous, nonfenestrated configuration that closely regulates fluid and solute transfer in the vasculature of the lungs and heart, to the discontiguous or sinusoidal endothelium found in the liver [1]. Barrier function varies widely between vascular beds, such as those of the brain and testes, where it is highly restrictive to the passage of macromolecules, and those of the dermis, where there is far less restriction. Other aspects of endothelial heterogeneity include variations in the number of clatherin-coated pits and caveolae [2]; expression of MHC I and II molecules, growth factors, chemokines, and integrins; and release of vasoactive molecules [1]. Such heterogeneity of form and function is especially apparent in pulmonary endothelial cells. As a major capacitance reservoir which must accommodate passage of the entire circulating blood volume every minute, flow regulation and control of blood fluidity are essential. As a major component of the alveolar–capillary unit, the pulmonary endothelium is vulnerable to injury from multiple sources, including inhaled and circulating noxious agents, reactive oxygen species (ROS) [3], and shear stress. Vascular damage triggers a complex and interconnected series of proinflammatory and procoagulation pathways that reinforce and even amplify one another. For example, in severe sepsis, proinflammatory cytokines upregulate tissue factor (TF) on endothelium and monocytes, leading to systemic activation of coagulation [4] (Fig. 1). Conversely, coagulation proteases such as thrombin can bind to protease-activated receptors (PARs) on endothelial cells, mononuclear cells, platelets, fibroblasts, and smooth muscle cells [5], resulting in an upregulation of inflammatory mediators such as interleukin (IL)-1 [6]. Such ­interactions

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_26, © Springer Science+Business Media, LLC 2011

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Fig. 1  Interactions of inflammatory mediators and coagulation factors with vascular endothelium. The complex intersection between intravascular inflammation and coagulation. ADP adenosine diphosphate, AMP adenosine monophosphate, APC activated protein C, ATIII antithrombin III, CD39 ectonucleoside triphosphate diphosphohydrolase-1, eNOS endothelial nitric oxide synthase, EPCR endothelial protein C receptor,

GPIIb/IIIa glycoprotein IIb/IIIa, HSPG heparan sulfate proteoglycan, ICAM-1 intercellular adhesion molecule-1, IL-1 interleukin-1, NO nitric oxide, PAR protease-activated receptor, PGI2 prostacyclin, PSGL-1 P-selectin glycoprotein ligand-1, ROS reactive oxygen species, TF tissue factor, TFPI tissue factor pathway inhibitor, TM thrombomodulin, TNFa tumor necrosis factor-a, VWF von Willebrand factor

between the inflammatory and coagulation systems ­contribute to the pathogenesis of conditions such as acute lung injury (ALI), ventilator-associated lung injury (VALI), disseminated intravascular coagulation, and pulmonary hypertension. The purpose of this chapter is to explore intrinsic features and external factors that interact with pulmonary endothelium which cause a shift between a baseline antithrombotic/anti-inflammatory phenotype and an activated state of endothelium, characterized by prothrombotic/proadhesive properties. Given the complexities of these systems, each will be discussed separately, with emphasis placed on areas of overlap.

2 Interactions of Leukocytes with Pulmonary Endothelium: Proinflammatory Factors It has been recognized that endothelial cells orchestrate the immune response by shifting from their quiescent anti-inflammatory phenotype to an activated state characterized by proadhesive properties [7]. Stimulated neutrophils transmigrate along chemotactic gradients into lung tissue across the endothelium en route to sites of pathogen invasion or tissue damage due to metabolic, toxic, or physical insult. A critical event in this transformation is the ­expression of adhesion

26  Interactions of Leukocytes and Coagulation Factors with the Vessel Wall

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Fig. 2  Effect of ischemia on ICAM-1 expression. Immunohistochemical localization of ICAM-1 antigen expression in the lungs. Sites of binding of a primary mouse monoclonal anti-rat ICAM-1 antibody emit intense green fluorescence when exposed to an excitation wavelength of 475 nm. (a) ICAM-1 immunostaining of fresh (nontransplanted) lung. ICAM-1 appears to be expressed primarily in the structures of the

a­ lveolar wall. (b) ICAM-1 immunostaining of a lung that had been ­preserved for 4 h at 4°C, transplanted orthotopically into an isogeneic recipient, and then reperfused for 6 h. The endothelial lining of small pulmonary vessels stained intensely for ICAM-1, with an increased intensity of alveolar staining as well. Magnification ×3,250. (Reprinted from [15] with permission)

molecules on the endothelium ­surface [8], which serve as cognate ligands for their counterparts on leukocytes.

such as P-selectin glycoprotein ligand-1 (PSGL-1) [14]. E-selectin is rapidly synthesized by endothelium after cell activation by cytokines such as TNF-a and IL-1, or the bacterial wall product, endotoxin [12]. The second phase is firm neutrophil adhesion. This requires the interaction of the b2 (CD18) integrin family (more specifically the CD11/CD18 integrins) expressed on neutrophils, mainly with intercellular adhesion molecule (ICAM)-1, a member of the immunoglobulin superfamily expressed on endothelial cells [12]. ICAM-1 expression on endothelial cells is augmented by inflammatory mediators such as TNF-a, IL-1, g-interferon, and endotoxin. Inhibiting ICAM-1 expression significantly improves lung graft function after lung transplantation [15] (Fig. 2). Oxidant stress promotes neutrophil adhesion [16], and the aerated microenvironment of the lungs, particularly when reperfused, is a highly oxidizing environment. When neutrophils firmly adhere on the pulmonary endothelial layer, they create a microenviroment for injury, mainly via the production of proteases and ROS (i.e., oxidant burst), which induces cell injury and death. Neutrophil adherence to matrix proteins appears to prime the former for a massive burst lasting 1–3  h in response to a stimulus such as TNF-a. Activated endothelium also generates ROS, contributing to maintaining an oxidant-rich environment. In the lungs, therefore, there are multiple cellular sources and a plentiful supply of oxidizing molecules.

2.1 Adhesion Molecules (Intercellular Adhesion Molecule-1, P-Selectin, E-Selectin, Integrin) Pulmonary endothelium–leukocyte interaction is a key step in ALI development since alterations in cell–cell adhesion is an intermediate step in leukocyte migration from the capillaries into the lung parenchyma, and the subsequent inflammatory response. Neutrophils appear to be the key cell type driving pulmonary injury in ALI, although eosinophils and macrophages have also been implicated [9–11]. Neutrophil adhesion to endothelium is a multistage process and is necessary for successful neutrophil migration and extravasation. The initial phase, neutrophils rolling and capture, is mediated by cell adhesion molecules of the selectin family: L-selectin is constitutively expressed on neutrophils, P-selectin is found on platelets and endothelial cells, and E-selectin is expressed solely on endothelial cells [11, 12]. P-selectin is expressed within minutes on the endothelial surface after endothelial activation by a stimulus such as histamine, thrombin, bradykinin, leukotriene C4, or free radicals [13]. P-selectin interacts with neutrophil counterreceptors

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2.2 Cytokines, Soluble Factors (TNF-a , IL-1, IL-8, IL-10) Cytokines are soluble polypeptides serving as chemical messengers between cells. They are involved in processes such as cell growth and differentiation, tissue repair and ­remodeling, and regulation of the immune response [17]. Endothelial cells are both targets and cytokine producers. In the lungs, TNF-a and IL-1 are mainly produced by activated interstitial and alveolar cells (primarily macrophages), as well as endothelial cells, and have a major role in the early stages of ALI [18]. TNF-a and IL-1 share a number of biological properties, and markedly amplify each other’s biological actions [18]. They act on endothelial cells mainly by inducing a functional program that promotes thrombosis and inflammation [19]. Hypoxia, or tissue ischemia, is a stimulus which induces massive endothelial cell synthesis and release of IL-1a, resulting in an autocrine enhancement in the expression of adhesion molecules [20]. Hypoxia of endothelial cells also leads to synthesis of IL-8, which can recruit and activate neutrophils [21]. When proadhesive, proinflammatory paradigms are triggered, countervailing mechanisms swing into action. IL-4 and IL-10 have been described to have suppressive effects on TNF-a, IL-1, and inducible nitric oxide synthase (iNOS) [22, 23]. Recombinant IL-10 given to IL-10-null mice reduced pulmonary vascular fibrin deposition, and rescued mice from ALI [24].

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tone. NO is a potent vasodilator and inhibitor of hypoxic ­pulmonary vasoconstriction as well as platelet aggregation. Animal studies showed that augmenting NO and the cyclic GMP (cGMP) pathway in lung transplantation improves lung graft function and recipient survival [26]. However, other studies seem to support the notion that high-output NO from iNOS, in addition to its bactericidal effects, can worsen lung injury, presumably by direct effects of highly reactive and toxic NO metabolites on various proteins and lipids [27]. Prostacyclin is produced downstream of cyclooxygenase by prostacyclin synthase in endothelial cells and, like NO, is also a potent vasodilator and inhibitor of platelet aggregation, acting primarily through cyclic AMP (cAMP) as opposed to NO, which predominantly activates cGMP production [26]. In a study addressing the mechanism of action of prostacyclin and prostaglandin E2 on endothelial cells, it was shown that these compounds enhanced the barrier properties of cultured endothelial cells at the baseline, attenuated thrombin effects, and conferred lung protection in an experimental model of ventilator injury [28]. In lung ischemia– reperfusion injury, the protection conferred by prostaglandin E1 (PGE1) has been attributed to its vasodilator properties, which leads to a better distribution of the preservation solution throughout the lung graft. However, PGE1 also protects lungs from ischemia–reperfusion injury through its ability to reduce neutrophil and platelet accumulation, and maintain endothelial barrier function [29].

2.4 Activated Protein C 2.3 Pulmonary-Endothelium-Derived Vasoactive Mediators (Nitric Oxide, Prostaglandins) Nitric oxide (NO) modulates pulmonary vascular tone and leukocyte–endothelium interactions. NO released from endothelial cells maintains vascular homeostatic properties by relaxing vascular smooth muscle, inhibiting neutrophil adhesivity and platelet aggregation, and maintaining endothelial barrier properties. Endothelial nitric oxide synthase (eNOS), an enzyme abundantly expressed in the lung, constitutively produces NO from l-arginine and oxygen in an NADPH-dependent fashion [25]. A number of physiologic stimuli can contribute to NO production via eNOS, triggered by calcium flux within endothelial cells: endothelial cell stretch and receptor ligation (bradykinin, histamine, and thrombin are classic examples) are proximate triggers of the calcium pulse leading to NO release. NO released in the lungs takes on special significance in that some believe it to be an essential mediator of oxygen release from hemoglobin in distal tissues, and a key modulator of pulmonary vascular

Protein C, a zymogen made by endothelial cells, when activated by thrombin (a process accelerated by thrombomodulin) has distinct anti-inflammatory and anticoagulant properties. Treatment with activated protein C (APC) had been promising in a number of animal studies and its use relies on solid scientific evidence [30]. APC appears to play a major role in sepsis, being an important regulator of the coagulation system (anticoagulant protein C pathway) and, in addition, numerous investigations have provided evidence that APC exerts direct cytoprotective effects on various cell types, and more specifically on endothelial cells. These cytoprotective effects appear mostly related to modulation of gene expression, antiinflammatory and antiapoptotic activity, and endothelium barrier stabilization [30]. Most cytoprotective effects require the activation of PAR-1, whereas the endothelial protein C receptor (EPCR) serves as a c-receptor. Animal studies using APC inhalation ­documented lung protective effects of the drug [31] despite apparent variations in the mode of APC action probably related to differences in experimental design.

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ALI and the related adult respiratory distress syndrome 3 Interactions of Leukocytes with Pulmonary have an incidence of 200,000 cases per year in the USA [42] Endothelium: Procoagulation Factors Pulmonary artery endothelium is an active participant in regulation of platelet aggregation, coagulation, and fibrinolysis [32]. More than just being barriers that line the interior of vessels which separate circulating blood from prothrombotic subendothelial factors, pulmonary artery endothelial cells are master regulators of hemostasis. Early research advanced the concept of the endothelium existing in either a quiescent, vasodilatory, anticoagulant and antiadhesive phenotype or an activated, vasoconstrictive, procoagulant, proadhesive phenotype [33]. Subsequent work has shown that such an off/on concept of endothelial function is overly simplistic, and that endothelial cells are capable of a spectrum of responses. The role of the endothelium in hemostasis illustrates the ability of endothelial cells to produce a variety of effects and thus tip the balance toward either a procoagulative or an anticoagulative state. The high procoagulant potential of the pulmonary endothelium, observed under conditions of stress, fosters interactions between damaged endothelium and circulating platelets, necessitating the existence of especially robust mechanisms for maintaining blood flow. Quantitative reverse transcription PCR measurements of von Willebrand factor (VWF) messenger RNA in various murine tissues showed a 5–50 times higher concentration of this procoagulant in the lung compared with the kidney and liver [34]. In addition, gene expression of TF, a potent trigger of coagulation, has been shown to be higher in the lung than in the heart and kidney in a murine model of septic shock [35]. Yet at the same time, anticoagulant mechanisms in the lung are highly active – including thrombomodulin expression, NO and prostacyclin generation, and APC synthesis. Seminal work in the areas of ALI and VALI has established a link between these conditions and disruptions of hemostasis [36]. In 1969, Saldeen et al. noted that injection of thrombin produced canine respiratory insufficiency that was linked to emboli in the pulmonary microcirculation [37]. In addition, bronchoalveolar lavage fluid from patients with ALI has shown elevated levels of fibrinopeptide A, factor VII and D-dimer, as well as an increase in the procoagulant plasminogen activator inhibitor (PAI) and a decrease in the thrombolytic urokinase [38]. The importance of active fibrinolytic mechanisms in the lung cannot be overstated, as in the end, the lungs serve to passively capture embolized clot or clot fragments from the peripheral circulation. Under pathologic conditions, native thrombolysis is disrupted. Coagulation also triggers other pathologic processes in ALI, as studies have shown that fibrin contributes to alveolar collapse by inhibiting surfactant production, promotes fibrosis by recruiting fibroblasts, and is a chemoattractant for neutrophils [39, 40]. In addition, coagulation genes have been shown to be among the most highly induced by the mechanical stress of VALI [41].

and a mortality rate of 25–40% [43]. Given that disordered hemostasis contributes to the pathobiological processes of such devastating pulmonary diseases, an understanding of the mechanisms by which coagulation is regulated in the pulmonary vascular bed is vital.

3.1 Platelet Tethering and Activation: The Collagen Pathway The process of coagulation often begins with physical disruption of the endothelium. The resulting exposure of subendothelial collagen and TF leads to an initial tethering of circulating platelets that allows them to attach to the vessel wall despite the shearing force of flowing blood. A new paradigm [44] describes two distinct pathways of platelet tethering and subsequent activation. In the collagen pathway, platelets are first captured through interactions between two sets of ligands: exposed collagen and platelet glycoprotein VI, as well as collagen-bound VWF and the platelet glycoprotein Ib–V–IX complex. Glycoprotein VI subsequently activates platelets and triggers platelet granule release [44]. Collagen types I, II, III, and IV are thought to be the most reactive to platelets, although the shearing forces in the vicinity of tethering may affect the reactivity of each type of collagen [45].

3.2 Platelet Tethering and Activation: The Tissue Factor Pathway The TF pathway of platelet activation is itself dependent upon the extrinsic pathway of coagulation. Exposed TF and activated factor VII (factor VIIa) catalyze the conversion of factor X into its active form (factor Xa), which then cleaves prothrombin to thrombin. In addition to its role in fibrin formation, thrombin activates platelets by cleaving PARs on the platelet surface [44]. Previously hidden epitopes are thereby revealed, and these “tethered ligands” bind to themselves to trigger platelet activation. Although endothelial cells express little or no TF constitutively, cell culture studies have shown that endothelial TF may be induced by stimulation with activated platelets [46], monocytes [47], lipopolysaccharide [48], TNF-a, and IL-1 [49]. The pulmonary vasculature may be especially prone to TF upregulation as evidenced by an in vivo murine model of sickle cell disease which used immunostaining to show that endothelial TF is induced in pulmonary veins and further augmented by hypoxia [50]. This prothrombotic potential is

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Fig. 3  Lung tissue from normoxic mice (a) and mice hypoxic for 6 h (b) was simultaneously immunostained with goat anti-rabbit TF IgG, with TF (pink areas identified by arrows) being more ­prominent in

hypoxic lung. Magnification ×3,600. (Reproduced from [52] with ­permission from the American Society for Clinical Investigation)

supported by studies linking TF to ALI. For example, TF-induced alveolar thrombin generation has been ­documented in a chimpanzee model of endotoxemia [51]. Hypoxia alone is sufficient to trigger TF expression in cells of the lung, as well as microvascular thrombosis [52]. The mechanism by which TF is induced on endothelial cells likely involves activation of at least two promoter regions. The lipopolysaccharide-response element contains binding sites for activator protein-1 and nuclear factor kB, allowing inducibility by inflammatory mediators such as lipopolysaccharide, TNF-a, and IL-1B [53]. The second promoter region, the serum-response region, contains early growth response protein-1 binding sites which mediate TF induction in response to hypoxia, shear stress, low-density lipoprotein, and vascular endothelial growth factor. CD40 ligation on endothelial cells results in a broader activation of the intracellular TF activation pathways, thus providing another example of how the extrinsic milieu can lead to transcriptional regulation of the gene [53]. Recent research has proposed that NO may inhibit TF, although results from various studies are conflicting [54] (Fig. 3).

P-selectin and facilitates adhesion of TF-rich microparticles to activated platelets [63]. The resultant concentration of TF near sites of vascular injury illustrates a putative role for microparticles in the initiation of coagulation [64]. In addition, interactions involving PSGL-1 and CD15 (an adhesion molecule) on microparticles and P-selectin on platelets and endothelial cells may facilitate the transfer of TF from circulating microparticles to platelets [65]. The role of P-selectin in TF-associated thrombus formation is supported by studies showing high levels of circulating of P-selectin associated with increased venous thrombus formation in mice [66], whereas P-selectin-null mice have developed thrombi containing minimal TF and thrombin [63]. It has been proposed that TF on some microparticles exists in an inactive, or cryptic, conformation, which may limit the ability of circulating microparticles to trigger systemic coagulation [44].

3.3 Circulating Tissue Factor In addition to its endothelial-bound form, TF has been found on circulating monocytes, platelets [55], and some microparticles. Microparticles are sub-micron-sized particles composed of cell membrane and associated surface proteins that are shed from activated or injured platelets, leukocytes, erythrocytes, or endothelial cells [56]. Microparticleassociated TF may induce thrombogenesis in diseases such as pulmonary hypertension [57, 58], lupus [59], metabolic syndrome in type 2 diabetes [60], and acute coronary syndrome [61] and in patients with coronary artery disease undergoing bypass surgery [62]. Among other proteins, microparticles express PSGL-1, which binds to platelet

3.4 Platelet Recruitment and Thrombus Growth Following initial platelet adhesion to the endothelium, the platelet aggregation response is amplified and sustained through the effects of mediators including ADP, thrombin, epinephrine, and thromboxane A2 [67]. These agonists activate G-protein-coupled receptors [68] that ultimately facilitate platelet-to-platelet aggregation through VWF and fibrinogen bridges between glycoprotein IIb/IIIa integrins on platelets [69] (Fig. 4) An important example of this type of activation involves the interaction of ADP with its P2Y12-coupled Gi receptor located on the platelet surface. Activation leads to a decrease in the level of intraplatelet cAMP and results in the perpetuation of platelet stimulation. In a model of mesenteric artery injury, P2Y12-null mice were shown to generate only small, unstable thrombi with decreased platelet density and activity [70].

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Fig.  4  (a) Electron micrograph of murine pulmonary vasculature exposed to hypoxia (FiO2 ~ 6%) for 16  h showing endothelium, red blood cells (RBC), platelet clumping (Plt), and platelet-associated fibrin (arrow). (b) Higher magnification demonstrates 22.5-nm periodicity characteristic of fibrin; black bar 112 nm. (Reproduced from [52] with permission from the American Society for Clinical Investigation)

The process of thrombus development requires a ­complex process of platelet coalescence that is dependent upon ­interactions between glycoprotein Iba on free-flowing ­platelets and VWF on the surface of immobilized platelets [69]. Aggregation is a dynamic process during which only a small percentage of platelets that initially tether upon the growing thrombus ultimately adhere [69]. As a thrombus grows, it requires additional stabilization so as to withstand the shearing effect created by blood as it flows with greater velocity at the center of a vessel than near the wall. This difference in velocities creates a shear stress that is greatest near the wall and thus the developing thrombus [71]. Endothelial cells, particularly when activated, secrete from Weibel–Palade bodies a highly thrombogenic form of ultralarge VWF multimers that support stable platelet aggregation [71]. Pulmonary endothelium contributes to global hemostasis in important ways. For example, human adult microvascular endothelial cells have been shown to be an important extrahepatic source of factor VIII, which is vital for factor X activation and the subsequent conversion of prothrombin to thrombin [72]. This may explain why factor VIII levels may increase despite severe liver disease. This could provide a mechanism through which the pulmonary endothelium regulates the common pathway of the clotting cascade.

4 Endothelial Modulation of Coagulation Coagulation occurs after inciting stimuli trigger a cascade resulting in a large fibrin clot. To prevent an explosive spread of clot formation beyond a site of injury, the body relies upon a system of intrinsic anticoagulants to restore the hemostatic balance. Given the central role of the endothelium in the coagulation cascade and the high

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p­ rothrombotic potential of the pulmonary vasculature, it is not surprising that pulmonary endothelial cells maintain an array of potent anticoagulation mechanisms. In keeping with the concept of endothelial heterogeneity, the distribution of these ­mediators of hemostasis varies depending on the vascular bed, thus giving pulmonary endothelial cells a relatively unique anticoagulation phenotype. For example, radioimmunoassay has shown much higher thrombomodulin antigen concentrations in the lungs and placenta compared with the heart, liver, or brain [73]. The endothelium modulates coagulation by producing, sequestering, and modifying various coagulation-inhibitory factors. NO, prostacyclin, tissue plasminogen activator (t-PA), and TF pathway inhibitor (TFPI) are produced and released by endothelial cells. The endothelium supports anticoagulant surface proteins including thrombomodulin, the EPCR, the ectonucleotidase CD39, heparan sulfate proteoglycan, and protein S. Finally, the endothelium contributes to anticoagulation by modifying circulating protein C and antithrombin.

4.1  Anticoagulants Elaborated by Vascular Endothelium 4.1.1 Nitric Oxide NO is a ubiquitous free radical that functions as both an intra- and extracellular signaling molecule. NO is synthesized from precursor l-arginine by three isoforms of nitric oxide synthase, including the endothelial- and plateletbound e-NOS. In addition to its function as a regulator of vascular tone, NO prevents platelet glycoprotein IIb/IIIa binding to fibrinogen by stimulating intraplatelet cGMP production [74]. Inhaled exogenous NO has been shown to modulate platelet aggregation in healthy animals [75] and humans with adult respiratory distress syndrome [76]. The combination of effects driven by NO and its distal effector cGMP, vasodilation and inhibition of platelet aggregation, could serve as a powerful means for disaggregating local thrombus and moving fragments downstream. A potent vasodilatory drug which raises the level of cGMP, sildenafil, is in fact used as treatment for pulmonary arterial hypertension, which may have thrombosis as a contributing mechanism.

4.1.2 Prostacyclin Prostacyclin is a labile prostanoid synthesized by endothelial and smooth muscle cells through the arachidonic acid pathway [77]. Prostacyclin works synergistically with NO

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[78] to inhibit platelet activation, secretion, and aggregation by increasing the level of intracellular cAMP [79]. Thromboxane A2, another cyclooxygenase product, triggers platelet aggregation [80]. Of note, antithrombin III has been shown to stimulate prostacyclin production [81], illustrating a method by which one anticoagulant is capable of triggering or ­amplifying the production of another.

4.1.3 Tissue Plasminogen Activator Thrombin and shear stress induce the production of the serine protease t-PA by the endothelium [82]. In the presence of thrombin, t-PA cleaves plasminogen into plasmin, which subsequently degrades fibrin. Free t-PA can then bind to the endothelium, where it is protected from its inhibitor, PAI-1, itself a product of the endothelium. The net result is lysis of thrombus. This topic is covered in Chap. 24.

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4.2 Membrane-Associated Anticoagulants 4.2.1 Thrombomodulin Thrombomodulin is a transmembrane cofactor on the endothelial surface that forms a reversible, high-affinity complex with thrombin. Estimates of surface expression of thrombomodulin suggest that there are approximately 50,000 active sites per cell [88]. The thrombin–thrombomodulin complex blocks thrombin’s ability to catalyze fibrin formation and activate both factor V and platelets. In addition, thrombomodulin-bound thrombin converts circulating protein C into APC, a potent modulator of coagulation and inflammation. It can therefore be viewed as an important, fixed component of the endothelial anticoagulant machinery. It is interesting to note that upon exposure of endothelial cells to hypoxia or inflammatory mediators expression of thrombomodulin becomes markedly diminished [89].

4.2.2 Protein C Receptor 4.1.4 Tissue Factor Pathway Inhibitor TFPI is an endogenous serine protease inhibitor of coagulation synthesized by the endothelium that forms a quaternary TF–factor VIIa–factor Xa–TFPI complex, thus limiting the amount of factor Xa available to cleave prothrombin into thrombin [83]. Once released, TFPI can bind to either circulating lipoproteins [84] or microvascular glycosaminoglycans capable of later releasing TFPI into the circulation after exposure to heparin [85]. Exposure of blood to TF has been shown to trigger disseminated intravascular coagulation (DIC), the widespread, disseminated occurrence of microvascular thrombosis that compromises blood supply to multiple organs [86]. Again illustrating the overlap that exists between the pathways of coagulation and inflammation, studies have investigated the role of TFPI in regulating the microvascular thrombosis that occurs in the setting of septic shock. Creasey et  al. administered lethal intravenous Escherichia coli infusions to baboons and noted that early postinfusion treatment with purified recombinant TFPI resulted in survival of all animals receiving therapy, whereas all control animals died within 40  h. In addition, there was evidence that TFPI attenuated lung, kidney, and adrenal disease [87]. Such benefits were not found in the OPTIMIST trial, which randomized 1,754 patients with severe sepsis and an elevated international normalized ratio to treatment with or without tifacogin, a recombinant TFPI. Tifacogin therapy was shown to have no effect on all-cause mortality and created an increased risk of bleeding [85]. This area of therapy, albeit promising, remains controversial.

In addition to expressing thrombomodulin, endothelial cells are capable of activating protein C through EPCR. This type 1 transmembrane protein binds to protein C and presents it to the thrombin–thrombomodulin complex for activation [90]. EPCR also binds APC, and although this interaction prevents APC from exerting its anticoagulant activity [91], EPCR-bound APC activates cellular signaling leading to endothelial barrier protection [92]. Conceivably, sustenance of the endothelial barrier limits contact between the circulation and the procoagulant vascular substrata.

4.2.3 CD39 Endothelial ENTPDase1 (CD39) is a Ca2+/Mg2+-dependent ectonucleotidase that hydrolyzes extracellular ATP and ADP to AMP [93]. Through interactions with platelet P2X1, P2Y1, and P2Y12 purinergic receptors [94], ADP released from activated platelets both activates platelets and potentiates the anticoagulation signals of other mediators [95]. In addition, CD39-mediated removal of ADP from the intravascular milieu has been shown to inhibit platelet function [96]. The importance of CD39 in maintaining the anticoagulant endovascular phenotype is illustrated by the finding that it maintains vascular fluidity even in the complete absence of prostacyclin and NO [97]. In a murine model of cerebral infarction, CD39-deficient animals were shown to have increased microvascular thrombosis, reduced postischemic perfusion and increased infarct volumes [98]. The administration of a recombinant soluble form of CD39 following infarction

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was able to rescue the knockout animals, leading to restored cerebral blood flow and markedly decreased infarct volumes [98].

4.2.4 Heparan Sulfate Proteoglycans Heparan sulfate proteoglycan, a linear polysaccharide composed of alternating units of hexuronic acid and d-glucosamine [99], is expressed on the surface of endothelial cells, providing an estimated 50,000 binding sites for antithrombin per cell [100]. This represents an additional anticoagulant defense at the blood–vessel interface.

4.3 Anticoagulation via Endothelial Interaction with Circulating Factors 4.3.1 Protein C and Protein S Protein C is a vitamin K dependent serine protease that is synthesized by the liver as a zymogen. Circulating protein C is activated by the endothelial-bound thrombin–thrombomodulin complex to form APC, which proteolytically inactivates activated factor V (factor Va) and activated factor VIII, thus blocking downstream thrombin generation [32]. Protein S is a vitamin K dependent plasma protein synthesized by the liver and endothelium. Protein S is best known as a cofactor for protein C, and its presence increases the inactivation of factor Va by 20-fold [101], and factor VIIa inactivation by at least threefold [102]. In addition, recent research has identified protein S as a potentially important cofactor of TFPI [103]. APC is an important modulator of both coagulation and inflammation in the lungs. Patients with ALI and decreased plasma levels of APC have higher mortality and more multiorgan system dysfunction [104, 105]. Findings such as these have led to clinical trials involving APC administration in the setting of sepsis. In the landmark PROWESS trial, the administration of recombinant human APC to patients with severe sepsis was associated with a reduction in the relative risk of death of 19.4% and an absolute risk reduction of 6.1% [106]. A trial involving the use of APC in patients with ALI showed no benefit in terms of ventilator-free days, mortality, or lung injury score, although these patients were less critically ill than those in the PROWESS study [43].

4.3.2 Antithrombin Antithrombin III is a plasma serine protease inhibitor that targets activated factor XII, activated factor XI, factor Xa,

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activated factor IX, factor VIIa, and activated factor II (thrombin) [107]. The endothelium plays a role in thrombin inactivation, as it sequesters both thrombin and antithrombin III from the circulation and facilitates formation of an antithrombin–thrombin complex that prevents thrombin activation [32]. Endogenous endothelial-bound or exogenous administered heparin activates antithrombin by facilitating the detachment of a reactive center loop from partial insertion in the A sheet of the molecule [108]. Heparin activation produces a 9,000-fold increase in inhibitory capacity against thrombin and a 17,000-fold increase against factor Xa [109]. A diffuse procoagulant state develops in patients with sepsis [110] as anticoagulant factors are depleted [111] and PAI-1 (which inhibits fibrinolysis) is induced. The KyperSept trial evaluated the use of high-dose antithrombin III administration in severe sepsis. This therapy had no effect on all-cause mortality when administered with heparin, possibly owing to an increased risk of hemorrhage. A subgroup analysis of patients who received antithrombin III without concomitant heparin suggested some benefit [112]. A recent meta-analysis of three randomized controlled trials involving patients with severe sepsis and DIC concluded that the use of antithrombin concentrate for up to 5 days may significantly reduce all-cause short-term mortality [113]. Taken together, these trials suggest a role for novel antithrombotic strategies in treating sepsis or DIC, but much yet remains to be learned.

5 Conclusion The pulmonary endothelium’s unique location at the capillary–alveolar interface makes it vulnerable to injury from a wide variety of sources, including oxygen radicals, sepsis, and barotrauma. Other extracellular signals leading to endothelial cell damage and/or activation include growth factors, cytokines, shear stress, circulating lipoproteins, coagulation factors, and components of the extracellular matrix [107]. The pulmonary endothelium integrates this barrage of external signals into a variety of responses that tip the hemostatic and inflammatory balances in one direction or the other, depending upon the unique and frequently changing requirements of a particular vascular bed. Although the interaction between circulating factors and the endothelium may be the major determinant of pulmonary vascular inflammatory and coagulation phentoypes, it is important to look broadly when considering all the interactive mechanisms involved. For example, environmental factors such as tobacco use, hormone ingestion, air pollution, diet, and medical conditions may modify the mechanisms discussed in this chapter. In addition, intracellular modifiers such as genetic polymorphisms or epigenetic

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changes may alter the interactions between pulmonary endothelium and circulating factors of inflammation and coagulation. For example, inherited deficiencies of protein C [114], protein S [115], and antithrombin, [116] and polymorphisms of ­factor V Leiden [117] and prothrombin 20210A [118] have long been known to increase the risk of venous thrombotic disease. It is reasonable to suspect that alterations in genes encoding for coagulation factors, thrombomodulin, fibrinogen, platelet glycoproteins, and the fibrinolytic system may also contribute to thrombosis, although multiple polymorphisms may be required for a change in phenotype. For example, a TFPI polymorphism has been identified that predisposes carriers to venous thromboembolism, but only when inherited with factor V Leiden or antiphospholipid antibody syndrome [119]. Other challenges to the identification of additional modifier genes include phenomena such as variable expressivity and incomplete penetrance.

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Chapter 27

Interaction of the Plasminogen System with the Vessel Wall Riku Das and Edward F. Plow

Abstract  In this chapter, we use the term ­“plasminogen ­system” to designate the pathway that controls the ­conversion of plasminogen to plasmin and the proximal consequences of plasminogen activation. The “plasminogen system” is synonymous with the “fibrinolytic system,” a nomenclature that emphasizes the primary substrate of plasmin, fibrin, and function, and the outcome of fibrin degradation, fibrinolysis. However, evidence documents vital roles of components of the plasminogen system in physiological and pathological processes which cannot be attributed to and clearly extend beyond its role in clot dissolution. In particular, components of the plasminogen system can influence cell invasion into tissue, which is dependent on the capacity of plasmin to degrade various extracellular matrix (ECM) proteins, including fibronectins, thrombospondins, von Willebrand factor, and laminin, and to activate certain matrix metalloproteases, enzymes that degrade still other ECM constituents. Many components of the plasminogen system also interact with cells to generate intracellular signals that affect cell survival, proliferation, adhesion, and migration. Some of these roles are directly relevant to endothelial cell (EC) and smooth muscle cell (SMC) biology and it is in this context that we consider the interaction of the plasminogen system with these vascular cells. Why should a book that focuses on pulmonary vascular disease have a chapter dedicated to the interaction of the plasminogen system with the vessel wall? The answer is straightforward; there is compelling evidence linking the plasminogen system to pathological processes in the lung. Keywords  Fibrinolysis • Thrombosis • Cell adhesion • Migration • Endothelial cell • Smooth muscle cell • Fibrin • Fibronectin • Plasmin

R. Das (*) Department of Molecular Cardiology, Cleveland Clinic Foundation, 9500 Euclid Avenue NB-50, Cleveland, OH 44195, USA e-mail: [email protected]

1 Introduction In this chapter, we use the term “plasminogen system” to designate the pathway that controls the conversion of plasminogen to plasmin and the proximal consequences of plasminogen activation. The “plasminogen system” is synonymous with the “fibrinolytic system,” a nomenclature that emphasizes the primary substrate of plasmin, fibrin, and function, and the outcome of fibrin degradation, fibrinolysis. However, evidence from enumerable in  vitro studies and numerous studies conducted in mice deficient in various components documents vital roles of components of the plasminogen system in physiological and pathological processes which cannot be attributed to and clearly extend beyond its role in clot dissolution. In particular, components of the plasminogen system can influence cell invasion into tissue, which is dependent on the capacity of plasmin to degrade various extracellular matrix (ECM) proteins, including fibronectins, thrombospondins, von Willebrand factor, and laminin, and to activate certain matrix metalloproteases, enzymes that degrade still other ECM constituents. Many components of the plasminogen system also interact with cells to generate intracellular signals that affect cell survival, proliferation, adhesion, and migration. Some of these roles are directly relevant to endothelial cell (EC) and smooth muscle cell (SMC) biology and it is in this context that we consider the interaction of the plasminogen system with these vascular cells. Why should a book that focuses on pulmonary physiology have a chapter dedicated to the interaction of the plasminogen system with the vessel wall? The answer is straightforward; there is compelling evidence linking the plasminogen system to pathological processes in the lung. In bleomycin-induced pulmonary fibrosis, deficiencies of plasminogen, urokinase plasminogen activator (uPA), uPA receptor (u-PAR), and tissue plasminogen activator (tPA) in mice, all components of the plasminogen system, alter the pathogenic response substantially [1, 2]. Similarly, the response to lung infections and endotoxin-induced lung injury is markedly altered in mice deficient in certain components of the plasminogen system, and expression patterns

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_27, © Springer Science+Business Media, LLC 2011

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Fig. 1  A model depicting various interactions of the plasminogen (Plg) system with the vessel wall. Endothelial cells and smooth muscle cells are the major cellular components of the vascular wall. Plg binding to a Plg receptor (Plg-R) either on the endothelial cell surface or on the fibrin surface enhances its activation to plasmin (Plm). Plm activates growth factors (GFs) and matrix metalloproteases (MMPs) and degrades extracellular matrix (ECM) components, which controls vascular remodeling by enhancing cell migration on the ECM. Plm also degrades fibrin to generate fibrin degradation products and promotes fibrinolysis to maintain vascular patency. Plg activation on cell or fibrin surfaces is mediated by Plg activators, either urokinase Plg activator (uPA) or tissue Plg

activator (tPA). uPA or tPA secreted from endothelial cells or smooth muscle cells binds to uPAR or tPA receptors, respectively, to activate Plg to Plm. Both tPA and uPA, either bound to their receptors or free, are inhibited by Plg activator inhibitor (PAI). uPA binding to uPAR generates intracellular signals in association with integrins. This process can lead to Plm-independent events such as cell adhesion, migration, differentiation, and proliferation. The uPA–PAI-1–uPAR complex formed on the cell surface is internalized and free uPAR is recycled back to the cell surface. Plg binding to Plg-R also generates intracellular signals and contributes cellular functions such as proliferation, migration, and adhesion. Unbound Plm is rapidly inhibited by a2-antiplasmin (A-P) in the blood

of these components change in wild-type mice in such models [3]. In ovalbumin-induced bronchial airway inflammation models of asthma, deficiency of plasminogen or inhibitors of plasminogen functions prevents the recruitment of inflammatory cells into lung tissue and spares tissue damage [4]. In such models, deficiencies of plasminogen activator inhibitor (PAI)-1 also alter the development of lung tissue damage [2, 5]. Such results correlate closely with evidence of activation and changes in the plasminogen system in human pulmonary diseases [6].

Interactions of these components with cells regulate many of their functions, including plasminogen activation and plasmin inhibition [7, 8]. Furthermore, the receptors for these components on ECs and other cell types are subject to modulation, thus controlling the proteolytic potential at the cell surface [14, 18]. EC also have the capacity to synthesize and secrete both plasminogen activators and PAI-1 in response to specific stimuli. These secreted components can interact with the cells in an autocrine and paracrine fashion. Many of the components of the plasminogen system, including plasmin(ogen), a2-antiplasmin, and tPA also bind to the surface of a fibrin clot [19]. Thus, there are many parallels between the regulation of the plasminogen system on the cell surface and that on the fibrin surface. We will briefly describe the molecular properties of the individual proteins of the plasminogen system, consider how these proteins interact with cells via their receptors, and then describe specific functions that relate to their interaction with cells within the vessel wall.

2 Components of the Plasminogen System For the purposes of the chapter, the plasminogen system is confined to the molecules and events depicted in Fig.  1. In vertebrates, there are two plasminogen activators, tPA and uPA, which activate the inactive plasminogen zymogen to the active serine protease plasmin. The activity of plasmin is regulated by PAIs, primarily PAI-1, and by plasmin inhibitors, primarily a2-antiplasmin [7, 8]. All the aforementioned components bind directly to cells or influence the interaction of other members of the system with cell surfaces, including ECs [9–14] and SMCs [15–17].

3 Plasminogen and Plasmin Plasminogen is a 92-kDa single-chain glycoprotein of 791 amino acids. Hepatocytes synthesize plasminogen [20] and

27  Interaction of the Plasminogen System with the Vessel Wall

give rise to the high concentrations of plasminogen ­(approximately 2 mM) in blood, but plasminogen messenger RNA has been detected in other cells as well [21]. The plasminogen molecule can be divided into three regions on the basis of function: (1) N-terminal region: As synthesized, plasminogen has glutamic acid at its N-terminus and is referred to as Glu-plasminogen. The N-terminal region contains several plasmin-sensitive bonds between residues 60 and 80, and their cleavage gives rise to molecules that are referred to collectively as Lys-plasminogen. Proteolytic deletion of the N-terminal region influences the overall conformation of plasminogen, which in turn influences interactions with cells [22]. (2) Kringle region: Plasminogen contains five disulfide-looped kringle domains, each of 80–90 amino acids [23]. Three of the five kringles, KI, KIV, and KV, have lysine-binding sites (LBS). These three kringles display preferences for C-terminal lysine residues, although their specificities are not identical [22]. The LBS mediate plasminogen binding to cell surfaces [24], fibrin [25], and a2-antiplasmin [26]. Therefore, lysine analogues (e.g., e-aminocaproic acid, EACA) [27], tranexamic acid (TXA) [28], and peptides with C-terminal lysines [29] or antibody directed against C-terminal sequences of these binding partners block plasminogen interactions [30]. (3) Protease domain: This domain contains the catalytic triad His603, Asp646, and Ser741A, which is typical of serine proteases. The single-chain plasminogen, which is enzymatically inactive, is converted to two-chain plasmin by cleavage of a single peptide bond (Arg561–Val562) by tPA or uPA. Of the two chains of plasmin, the heavy chain, containing the kringles, and the light chain, containing the protease domain, remain disulfide-linked to each other [31].

3.1 Plasminogen Receptors and Their Modulation Many different cell types have been shown to bind plasminogen in a specific and saturable manner, indicating that they express a discrete numbers of binding sites. These binding sites are heterogeneous in character and are collectively referred to as plasminogen receptors (Plg-Rs). All circulating blood cells, with the exception of erythrocytes [32], express Plg-Rs; the number of Plg-Rs on these cells is great and varies from 104 to more than 106 [7, 33]. Tissue fixed cells also bind plasminogen with high capacity [34]. In general, transformed cells bind plasminogen with higher capacity than their nontransformed counterparts [35–38]. Thus, the almost ubiquitous distribution and the high density of expression are defining characteristics of Plg-Rs. Even though Plg-Rs are heterogeneous, they bind plasminogen with similar affinities; Kd values are generally in the range 0.1–2 mM [33, 35].

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Despite relatively low affinities, the high concentration of plasminogen in local microenvironments ensures that significant occupancy of Plg-Rs occurs on cells. In addition, some much higher affinity Plg-Rs have been identified [9] and some receptors which are selective for plasmin as opposed to plasminogen have been reported [39, 40]. The similarities in the affinities of most Plg-Rs reflect interactions mediated by the LBS of plasminogen. Binding of plasminogen to Plg-Rs via its LBS has several functional consequences which are inherent to LBS engagement [24]: (1) plasminogen activation is enhanced in cell surfaces as compared with solution [32, 41]; (2) plasmin bound to cell surfaces is protected from rapid inactivation by a2-antiplasmin [42, 43]; (3) the transition from Glu-plasminogen to Lys-plasminogen plays an important role in binding and activation at cell surfaces [44, 45]; and (4) plasmin bound to Plg-Rs is catalytically more efficient than free plasmin [46]. Thus, Plg-Rs endow the cell surface with the broad and sustained proteolytic activity of plasmin, which can be utilized for a wide variety of the biological functions of the enzyme, including fibrinolysis, degradation of the ECM, regulation of growth factor activity [47, 48], and generation of intracellular signaling [49, 50]. Many different Plg-Rs have been identified. These can be divided into two groups: those with and those without C-terminal lysines. A BLAST search of the human genome identifies hundreds of proteins with lysine at their C-terminus, and as such they have the potential to interact with the LBS of plasminogen. However, not all these proteins function as Plg-Rs. The residues adjacent to the C-terminal lysine, the availability of the C-terminal lysine at the cell surface, and processing of the C-terminal lysine by proteolytic enzymes, such as the basic carboxypeptidases, which remove C-terminal lysines, can suppress or modulate the ability of proteins with C-terminal lysines to function as Plg-Rs [51]. By the same token, proteolysis to generate a new C-terminal lysine can create a new Plg-R [18, 52]. Cleavage is believed to account for the capacity of p37 (or p36) of the annexin 2 heterotetramer to bind plasminogen [53]. Some of the proteins with C-terminal lysines which have been shown to function as Plg-Rs are listed in Table  1. Of note, many of the known Plg-Rs lack signal sequences and transmembrane domains. Thus, to function as Plg-Rs, they must reach the cell surface by a nonconventional secretory pathway. The Plg-Rs without a C-terminal lysine are also multiple and heterogeneous (Table 1). These include some molecules which bind to the LBS of plasminogen as indicated by the inhibition of binding by EACA or TXA [54–58]. These Plg-Rs contain an internal residue(s) that mimics a C-terminal lysine. Notable in this category are the integrins [56–58]. A few Plg-Rs interact with plasminogen or plasmin and the interactions are not EACAor TXA-sensitive. Heymann nephritis autoantigen, gp330, and a5b1 fall into this category [59, 60]. In the nonprotein

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Table 1  Presence of plasminogen receptors (Plg-Rs) on various cell types and their mechanism of binding Plg-Rs Cell types C-terminal lysine a-Enolase Cytokeratin-8 p11 TATA-binding proteininteracting protein Histone H2B Annexin 2 Actin avb3 aMb2 aIIbb3 Amphoterin

Monocytes, neutrophils, lymphoid cells, myoblasts, neurons, transformed cells Hepatocellular and breast carcinoma Endothelial cells, fibroblasts Monocytoid cells

Present

Neutrophils, monocytoid lineage endothelial cells Endothelial cells, monocyte lineage Endothelial cells, carcinoma, catecholaminergic cells, neuronal cells, fibroblasts Endothelial cells Neutrophils Platelets, rheumatoid arthritis synovial fibroblasts Neuronal cells

Present Absent, but generated by proteolysis Absent, but exposed C-terminal lysine on the cell surface Absent Absent Absent Absent

category are gangliosides [61], which may help to account for the extraordinarily high plasminogen binding capacity of some cells [61]. This interaction is LBS-mediated. Cells may utilize plasminogen in their physiological and pathological responses by modulating their cell-surface expression of Plg-Rs [18]. Processes associated with modulation of Plg-R expression include transformation [24, 36, 37], cell adhesion [62, 63], agonist stimulation [14, 62–65], differentiation [30, 66–68], and apoptosis [69]. Breast tumor with high invasive phenotypes binds more plasminogen than cells lines with lower invasive properties [37]. Thus, the high metastatic potential correlates with high plasminogen binding capacity. Cytokeratin 8 and enolase have been identified as Plg-Rs which are expressed at high levels on tumor cells [38, 70]. With respect to cell adhesion, when cells detach they can display increased capacity to bind plasminogen; when they adhere to substrates, their plasminogen binding capacity is again reduced [62]. These observations indicate that cells are capable of both upregulating and downregulating Plg-Rs; i.e., the modulations can be reversible. Thrombin-stimulated platelets bind fivefold more plasminogen than resting platelets [57], indicating that cells can respond to agonists by enhancing the plasminogen binding capacity. Cells of the monocytoid lineage can also modulate their expression of Plg-Rs in response to maturation and differentiation. When monocytoid cells are differentiated to macrophages, either in  vitro or in  vivo, their plasminogen binding capacity increases three- to four-fold [30, 66]. Cell-surface expression of four most prominent Plg-Rs on these cells, a-enolase, Histone H2B annexin 2 and p11 (the annexin 2 heterotetramer), leads to the increase in plasminogen binding to the differentiating monocytes. Both in vitro and in vivo studies in mice indicate that histone H2B accounts for approximately 50% of the plasminogen binding capacity of macrophages [30]. The higher plasmin activity on the surface of these cells may facilitate migration of these cells across the ECM to sites of inflammation. Plasminogen binding capacity was

Present Present Present

also observed to increase enormously when premature fat cells differentiate into mature adipocytes [68]. Synovial fibroblasts from patients with rheumatoid arthritis have a higher plasminogen binding capacity than normal synovial fibroblasts [71]. The higher plasmin activity may contribute to the destruction of joint cartilage and bone in patients with rheumatoid arthritis [72].

3.2 Interaction of Plasminogen with ECs The endothelium presents a continuous interface to blood, a rich source of plasminogen. In vitro studies indicate that plasminogen influences EC migration and invasion [58, 73, 74], and studies conducted in plasminogen-deficient mice indicate that plasminogen plays an influential role in EC responses, including angiogenesis [75] and wound healing [76]. Plasminogen also influences complex vascular disease responses, such as atherosclerosis [77] and restenosis [78]. Hence, the interaction of plasminogen with ECs has been analyzed in numerous studies. Most studies of plasminogen binding to ECs have been performed with human umbilical vein ECs (HUVEC). Plasminogen binds to the HUVEC surface via its LBS. Dissociation constants have been estimated to range from 0.3 to 2.0 mM [9, 10, 14]. Glu-plasminogen binds to the HUVEC and is rapidly converted to Lys-plasminogen and to plasmin on the cell surface [45, 53]. The number of plasminogen binding sites ranges from 1.4 × 106to 12.4 × 106. This extraordinarily high number makes it likely that binding is mediated by multiple Plg-Rs. Indeed, several different Plg-Rs have been detected on HUVECs (Table 1). The first Plg-R described on ECs was annexin 2 [53]. Annexin 2 was originally reported to express distinct binding sites for tPA and plasminogen/ plasmin. Annexin 2 does not contain a C-terminal lysine, and it was suggested that it had to be cleaved to become a

27  Interaction of the Plasminogen System with the Vessel Wall

p­ lasminogen binding protein [53]. However, other studies have suggested that annexin 2 only binds plasminogen when it is associated with its heterotetrameric partner p11 [79]. p11 is synthesized with a C-terminal lysine. The expression of annexin 2 alone or as a heterotetramer in complex with p11 may be regulated by phosphorylation [80]. A second notable Plg-R on ECs is integrin avb3 [58]. There is a substantial body of data implicating avb3 in angiogenesis [81]. Although neither subunit has a C-terminal lysine, the binding of this integrin to plasminogen is LBS-mediated [58]. Interaction of avb3 with plasminogen promotes plasmin-mediated adhesion and migration of bovine aortic ECs [58]. avb3 also binds the antiangiogenic plasminogen fragment angiostatin via its RGD sequence [58]. Plasminogen binding to the EC surface via actin in a kringle-dependent manner has also been reported [82]. We have also detected histone H2B on the surface of HUVECs (unpublished). Our antibody to the C-terminal lysine region of H2B partially inhibits plasminogen binding to HUVECs [30] (and unpublished observations). Modulation of Plg-Rs and plasmin generation on the EC surface has been demonstrated in various studies. Thrombin or other ligands of the protease-activated receptors enhance expression of both annexin 2 and p11 on the HUVEC surface [65]. The upregulation of these Plg-Rs was associated with increased plasminogen binding and enhanced tPA-mediated plasmin generation. Vascular endothelial growth factor (VEGF) stimulation of HUVECs leads to the enhanced fibrinolytic activity by increasing plasmin generation as a result of increased expression of tPA and some matrix metalloproteases by the cells [83]. Our unpublished results also demonstrated an increase in the levels of Plg-Rs when HUVECs are stimulated with VEGF.

3.3 Role of Plasminogen/Plg-Rs in EC Function Upon blood vessel injury, local coagulation culminates in fibrin deposition to prevent excessive blood loss. Fibrinolysis is initiated to restore blood vessel patency and blood flow. The fibrin surface provides binding sites for plasminogen and tPA and their colocalization enhances tPA-mediated plasminogen activation by approximately 500-fold [84]. The EC surface has the capacity to bring plasminogen close to tPA and uPA and also enhances plasminogen activation. Annexin 2 binding of plasminogen enhances its activation by tPA by approximately tenfold [53, 85]. The biological significance of this effect was demonstrated in  vivo. Treatment of carotid arteries with recombinant annexin 2 reduced vessel thrombosis induced by ferric chloride injury of the vessel wall in rats [86]. Ling et al. [87] further showed that deficiency of annexin 2 in mice reduces both microvascular fibrin homeostasis and

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macrovascular clearance of thrombi. The contribution of other Plg-Rs on the EC to fibrinolysis has yet to be examined. Since the properties of Plg-Rs with regard to plasminogen binding and activation appear to be similar, one could predict that the contributions of individual Plg-Rs to fibrinolysis would correlate with their expression levels within particular vascular beds. Another major role of plasminogen in EC-dependent responses relates to angiogenesis. Three key steps of angiogenesis are proteolytic degradation of basement membrane, EC migration, and EC proliferation. Plasminogen binding to ECs can play a critical role in each of the three steps. Plasmin can directly degrade ECM proteins, can activate various matrix metalloproteases which are required for cell migration across collagenous matrices, and can also activate latent angiogenic growth factors [47, 48, 88, 89]. Basic fibroblast growth factor (bFGF)-induced formation of capillary tubes from ECs was suppressed in a gelatin sponge assay in mice by inhibitors of plasminogen binding to cells (TXA and EACA) [73]. ECs from plasminogen-/- mice have reduced capillary sprouting in culture and from aortic rings on collagen beds compared with their wild-type counterparts [74, 90]. In a widely used corneal implant model to evaluate angiogenesis in vivo, Oh et al. [75] demonstrated that both bFGF- and VEGF-induced corneal blood vessel formation was significantly reduced in plasminogen-/- mice compared with plasminogen+/+ mice. Angiogenesis was also impaired in annexin 2 null mice [87]. Angiostatin (kringles 1–3) binding to integrin aVb3 inhibited bFGF- and plasmin-induced EC migration [58]. Tissue factor not only binds plasminogen but also angiostatin K 1-3, angiostatin K 1-5 and angiostatin K4 in an LBS-dependent manner, and binding of soluble tissue factor to angiostatin abolished the inhibitory effects of angiostatin on the bFGF-mediated EC proliferation [91]. Kringle 5 of plasminogen promotes caspase activity and induces apoptosis on ECs [92] and, therefore, also functions as an inhibitor of angiogenesis. EC-derived microparticles are found at elevated levels in patients with thrombotic disorders [93] and they can serve as a surface for plasmin generation and also can express uPA and uPAR [94]. Functionally, EC-derived microparticles affected EC tube formation from progenitor cells. Thus, the interaction of components of the plasminogen system with EC-derived microparticles has the potential to influence inflammation, angiogenesis, and atherosclerosis.

4 tPA and tPA Receptors tPA is synthesized primarily by ECs, but not all ECs secrete tPA [95, 96]. In the pulmonary circulation, vessels with diameters of 7–30 mm produce tPA, but capillaries or larger vessels

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do not [96]. The release of tPA from ECs is regulated. Rapid release in response to G-protein-coupled-receptor activation and rises in intracellular Ca2+ levels depend upon secretion of a preexisting pool of tPA, which resides in small storage granules [97]. Release over the longer term also can be induced as well and depends upon new protein synthesis [98]. Released tPA is a 70-kDa single-chain, fully active serine protease [99]. The single-chain form can be cleaved to a two-chain form by plasmin without alteration of proteolytic activity [99]. The N-terminal of tPA is composed of a finger domain, an epidermal growth factor (EGF) domain, and two kringle domains. The serine protease domain resides in the C-terminal region of tPA and is composed of a catalytic triad of His322, Asp371, and Ser478. The enzymatic activity of tPA is tightly regulated by inhibitors of the serpin family, particularly PAI-1. Hence, the ratio of tPA to PAI-1 establishes the capacity of tPA to function as a plasminogen activator within local microenvironments [100]. tPA binds preferentially to fibrin rather than fibrinogen, and this interaction, together with the binding of plasminogen itself to fibrin, targets its plasminogen activator activity to fibrin clots [101]. The finger and the second kringle domain of tPA are involved in its binding to fibrin. tPA also binds to the surfaces of cells. Blood cells which bind tPA include platelets [102], monocytes, and neutrophils [103]. These interactions also depend on the finger and kringle domains of tPA. At least some of these sites are the same C-termini of proteins that also bind plasminogen; [104] and, hence, in competitive situations, the higher plasminogen concentration favors its occupancy of these binding sites.

4.1 Interaction of tPA with ECs A portion of the tPA released from the EC is retained on the cell surface, providing evidence for the presence tPA receptors on such cells [105]. Various groups have examined the interaction of tPA with HUVECs, but the results of these studies have not been entirely consistent. Cobbling these results together, there appear to be three classes of tPA binding sites with respect to affinity. The highest-affinity binding sites for tPA are PAI-1-dependent [12, 106]. PAI-1 is synthesized by EC cells and accumulates in the ECM, where it binds to vitronectin [107]. PAI-1 forms a tight complex with secreted tPA on the matrix; but a portion of PAI-1 associates with the EC surface and contributes to tPA binding to the cells [105]. It is this interaction, which is a consequence of the formation of an enzyme–inhibitor complex, that has been interpreted as a high-affinity receptor for tPA. Annexin 2 on the EC surface contribute to tPA binding as an intermediate-affinity interaction. tPA binds to the N-terminal core 1 domain of annexin 2, whereas plasminogen binds to the core

R. Das and E.F. Plow

4 domain [108]. Unlike plasminogen, tPA binding to annexin 2 is insensitive to lysine analogs but is affected by homocysteine [12, 109]. The Kd value of tPA binding to annexin 2 is 18 nM and accounts for up to 800,000 binding sites on HUVECs [110]. The p11 subunit of the annexin 2 tetramer, which is expressed by ECs, binds to tPA with a Kd of 0.45 mM [104] and may contribute to the low-affinity interaction of tPA with ECs. Actin [55] and tubulin [111] also bind to tPA,; these interactions are inhibited by C-terminal lysine analogs. The mechanism(s) for cell-surface expression of these cytoskeletal proteins is unknown. It is also likely that many of the identified Plg-Rs can be expressed by ECs and also bind tPA.

4.2 Roles of tPA/tPA Receptors in EC Function tPA binds both fibrin and cell surfaces. Binding of tPA to fibrin enhances plasmin generation by 500-fold [112], and binding to the EC surface induces a similar enhancement in plasminogen activation by tPA. Binding of plasminogen and tPA to isolated receptors, such as annexin 2, leads to a significant increase in plasminogen activation [113], although not quite as efficient as on cell surfaces. The functions of tPA overlap those of plasminogen (see earlier). Plasminogenindependent functions of tPA have been described in the brain [114] and activation of extracellular-signal-regulated kinase (ERK) has been attributed to the interaction of tPA with brain.

5 Urokinase Plasminogen Activator uPA is a 54-kDa serine protease. The zymogen is synthesized as a single-chain form (sc-uPA). Upon proteolytic cleavage sc-uPA is converted to a two chain form (tc-uPA), which has plasminogen activator activity dependent on a catalytic triad (Asp225, His204, and Ser356) within its C-terminal domain [115]. tc-uPA can be further cleaved by plasmin, which results in the removal of its growth factor domain and kringle domain from the N-terminal region to generate a two-chain low molecular weight form of uPA (33  kDa). This form retains plasminogen activator activity but no longer binds to the urokinase receptor, uPAR, on cell surfaces [42, 116].

5.1 uPA Receptor A specific cellular receptor for uPA was first demonstrated on monocytoid cells by Vassalli et al. in 1985 [116]. Significant contributions to our early understanding of uPAR were made

27  Interaction of the Plasminogen System with the Vessel Wall

by the Blasi [117] and the Dano [118] groups. Initial studies of the uPAR-deficient mouse failed to identify a significant role of the receptor in fibrinolysis as well as a number of other models [119, 120], but subsequent studies have implicated uPAR in inflammatory cell recruitment in certain models, including models of pulmonary infections [3, 121–123]. uPAR is present on many different cell types, including ECs. It is anchored to the plasma membrane by a glycosyl phosphatidylinositol (GPI) linkage, which permits substantial mobility of uPAR within the membrane [124]. Consequently, uPAR localization on the cell surface can be altered as cells respond to stimuli. On migrating cells, uPAR concentrates at the leading edge but is distributed uniformly on the membrane of nonmigrating cells [125]. uPAR is a glycosylated protein of 50–60  kDa composed of three distinct structural domains (D1, D2, and D3) and two short intradomain linker regions [124]. Most studies have implicated the importance of the D1 and D2 domains in uPA binding [126]. uPAR binds to all uPA forms which contain an intact growth factor domain. It has high affinity for uPA, sc-uPA, and the N-terminal fragment (ATF) that contains the growth factor domain [117]. The affinities of the uPA forms for uPAR range from less than 0.1 nM to more than 2 nM [117]. Affinity of uPAR for uPA can be modulated as cells respond to stimulation [127, 128]. The number of uPAR molecules expressed on cells ranges from 50,000 to 200,000 per cell on monocytoid cells, ECs and fibroblasts, and uPAR expression levels and affinity are subject to modulation depending on stimulation of the cells [125, 128]. uPA can cleave uPAR in the linker region between D1 and D2, releasing D2/D3 domain (soluble uPAR), which has chemotactic activity [129]. Besides uPA, uPAR also binds vitronectin, which is found in serum and the ECM and which also complexes with the uPA inhibitor PAI-1 [129]. Moreover, membrane-anchored uPAR can be released from cells by cleavage of its GPI anchor [124]. In paraxomal hemoglobinurea, the enzyme that attaches the GPI anchor to proteins is mutated and uPAR and other GPI-linked proteins are absent from cell surfaces [130]. It has been suggested that the absence of uPAR may contribute to the prothrombotic phenotype in paraxomal hemoglobinurea patients [130]. PAI-1 inactivates uPA whether it is or is not bound to uPAR [131]. Formation of PAI-1 or PAI-2 complexes with uPA bound to uPAR on the cell surfaces leads to internalization of uPAR/uPA/PAI complexes and drives uPAR cycling back to the cell surface. This process is enhanced by several adaptor molecules such as LDL-receptor-related protein, glycoprotein 130, very low density lipoprotein, urokinasereceptor-associated protein, and Endo 180 [132, 133]. The mannose 6-phosphate receptor, another uPAR-binding protein, specifically binds to the D2/D3 fragment of uPAR and targets it to lysosomes for degradation [134]. In the acidic environment of endosomes, sc-uPA and PAI-1 dissociate from the uPAR-containing complex and are degraded,

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whereas uPAR is recycled back to the cell surface in a ­functionally active form. In addition to its function as a uPA receptor, uPAR also affects cell adhesion, migration, differentiation, and proliferation via intracellular signaling. These signaling-dependent functions of uPAR depend on its association with other cellular proteins, including integrins, G-protein-coupled receptors [135], and caveolin [136]. The highest affinity of uPAR binding to integrins is with a5b1 and a3b3 and uPAR can promote adhesion of cells to vitronectin, fibronectin, and collagen by regulating these integrins and can even alter the specificities of these integrins for their ligands [137]. uPARdependent intracellular signaling can be induced by binding of uPA to uPAR or by clustering of uPAR. The signaling pathways/events induced by uPAR include the mitogen-activated protein kinase (MAPK) and focal adhesion kinase pathways and the downstream effectors of these pathways, including cytoskeletal rearrangements. uPAR was also found to coprecipitate with the members of Janus kinase/signal transducer and activator of transcription pathway, phosphatidylinositol 3-kinase/Akt pathway, the Src family kinases, and protein kinase C [129]. In addition to uPAR, some studies have suggested the presence of additional mechanisms for uPA to interact with cell surfaces [138]. The identity of these binding sites has not been fully resolved. It has been shown that gangliosides also provide a relatively low affinity binding site for uPA on cells [61].

5.2 uPAR on ECs uPA interacts with uPAR on ECs with high affinity (Kd < 2 nM). The number of uPA molecules bound per EC varies from 20,000 to 200,000 [139]. This variability may be due to differences in the activation states of the ECs used in the analyses. A number of agonists and cytokines modulate uPAR expression in ECs. For example, thrombin leads to a significant decrease in uPAR expression on HUVECs, whereas phorbol 12-myristate 13-acetate causes a significant increase in receptor number. Of particular relevance to EC biology, stimulation with the angiogenic factors VEGF [140] and bFGF [141] leads to robust induction of uPAR expression on the EC surface. Expression of uPAR on ECs is controlled primarily at the transcriptional level. Transcription factors activating protein-1, activating protein-2, Sp1, hypoxia-inducible factor-1, and nuclear factor kB (NFkB) have all been implicated in regulation of uPAR gene expression [142]. uPAR gene expression is linked to adhesive states of ECs. Among two major integrin subfamilies expressed on ECs, b1 and b3, it is the b3 integrin that has been associated with regulation of

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uPAR expression [143, 144]. Upon b3 integrin ligation with ECM proteins, ­outside-in signaling leads to the binding of b3-endonexin, a b3 cytoplasmic binding partner, to interact with the p50/p65 subunits of NFkB and inhibits NFkB binding to the uPAR promoter. As a consequence, NFkB-mediated uPAR expression is suppressed in ECs [145]. Hypoxia also has been shown to regulate uPAR expression in ECs. In HUVECs, hypoxia-induced uPAR expression was shown to be regulated by an oxygen-binding heme protein [146]. However, in breast cancer and other tumor cells, hypoxiainduced uPAR expression by a transcriptional activation pathway is dependent on ERK [147] and hypoxia inducible factor-1a [148]. VEGF-induced uPAR messenger RNA expression in bovine retinal microvascular ECs is dependent on inactivation of glycogen synthase kinase-2 by phosphatidylinositol 3-kinase/Akt pathways, which leads to the nuclear translocation of b-catenin [149]. In addition to gene expression, redistribution and internalization of uPAR provides another mechanism for control of uPA activity on ECs. uPAR localization at the leading edge assists the directed migration of ECs [150]. Interaction of VEGF165 with VEGF receptor-2 redistributes uPA–uPAR to the leading edge and activates pro-uPA to uPA. This is followed by complex formation with PAI-1 and internalization of the ternary complex uPAR–uPA–PAI-1 in an LDLreceptor-like-molecule-dependent fashion [151]. Although the uPA gene is constitutively expressed by some ECs, a number of cytokines, growth factors, and stress have been shown to enhance uPA expression and uPA-mediated cell functions. VEGF increases cell-bound uPA expression and short double-chain uPA secretion, and this response is diminished by transforming growth factor (TGF)-b [149]. Shear stress represses uPA gene expression in human coronary artery ECs by activating the GATA6 transcription factor [152]. uPA expression by lung ECs induces uPAR gene expression that maintains a feedback loop for degradation of fibrin deposits [153].

R. Das and E.F. Plow

is antiangiogenic but is less potent if linked to the growth factor binding domain [159]. uPA binding to uPAR also induces EC survival by activating NFkB and by inducing the X-linked inhibitor of apoptosis protein [160]. These broad functions of uPA and uPAR on ECs suggest that these molecules or their interaction may be interesting therapeutic targets in a variety of pathogenic settings.

6 Plasminogen Activator Inhibitors The main physiological inhibitor of both tPA and uPA in the vascular system is PAI-1. PAI-1 was first identified in 1984 as a secretory protein that inhibited fibrin clot lysis in ECs [161]. It is a member of highly homologous serine protease inhibitor (SERPIN) superfamily. PAI-1 is synthesized as a 52-kDa single-chain glycoprotein with 379 amino acids. Its reactive site with tPA and uPA is an Arg346–Met peptide bond and the reaction leads to the formation of a very stable biomolecular complex [162]. PAI-1 inhibits single-chain and two-chain tPA and tc-uPA very rapidly with second-order rate constants of the order of 107  M-1 s-1 [131]. It does not react with sc-uPA. PAI-1 can occur in both an active form and an inactive form. The active form, in which it is an effective inhibitor, is extremely labile, with a half-life of 2 h at ambient temperature. The structural basis for the latency of PAI-1 activity has been resolved [163]. The stability of active PAI-1 may be increased by its interaction with its binding partners, vitronectin [107] and heparin [164]. The normal plasma concentration of PAI-1 ranges between 6 and 80 ng/mL. In the vessel wall, PAI-1 is produced by both ECs and SMCs [165]. It is also made in the liver and spleen and by adipose tissue [166, 167]. The elevated levels of PAI-1 that are found in plasma are associated with myocardial infarction and other thrombotic disorders and insulin-resistant type 2 diabetes may be a consequence of its release from activated platelets, which contain substantial amounts of PAI-1 [166, 168, 169], or from ECs.

5.3 Functions of uPA and uPAR on ECs The major function of uPA binding to uPAR is plasminogen activation. uPA can also degrade certain ECM proteins and can activate various growth factors independent of plasmin activity [154–156]. In addition, uPAR mediates cellular adhesion to vitronectin and promotes integrin-dependent migration and initiates intracellular signaling events. Therefore, in addition to fibrinolysis and thrombosis, the uPA–uPAR system has been implicated in various EC functions, including embryogenesis, angiogenesis, tumor metastasis [157], vascular permeability [149], venous thrombosis [158], and atherosclerosis [157]. The kringle domain of uPA

6.1 Functions of PAI-1 PAI-1 inhibits uPA-mediated cell migration by interacting with uPA bound to u-PAR. As noted already, this complex is then internalized via endocytic mechanisms involving the LDR receptor family. The binding of PAI-1 to uPA on the cell surface also leads to deactivation and internalization of integrins that associate with uPAR, inducing cell detachment [170]. The high-affinity interaction of PAI-1 with vitronectin has several functional consequences. PAI-1 in complex with vitronectin acquires the ability to inhibit another serine

27  Interaction of the Plasminogen System with the Vessel Wall

p­ rotease, thrombin [171]. PAI-1 detaches cells from the ECM by disrupting u-PAR–vitronectin and integrin–vitronectin complexes [170]. These mechanisms allow PAI-1 to influence cell adhesion and migration independent of its protease inhibitory action. As previously mentioned, elevated levels of plasma PAI-1 have been linked to an increased risk for thrombosis, myocardial infarction, and obesity. PAI-1 levels are regulated by various stimuli, including angiotensin, TNFa, TGF-b, and hypoxia [172]. In macrophages, PAI-1 expression was induced by complement component C5a [173]. Of these various stimuli, on ECs, TNFa is a particularly powerful inducer of PAI-1 gene expression. At the same time as TNFa upregulates PAI-1 expression, it can downregulate tPA synthesis. TNFa acts via Nur77 in the proximal promoter of the PAI-1 gene [174] and may also involve activation of NFkB via an element located far upstream (approximately 15 kb) from the transcription start site of PAI-1 [175]. TGF-b regulates PAI-1 expression via Smad3/Smad4 and proteins associated with the MAPK/ERK kinase pathways [176]. In response to glucose and angiotensin, an Sp1 element was found to responsible for PAI-1 gene expression [177, 178]. Fibrin deposition within the pulmonary vasculature in hypoxic lungs was found to arise from an increase in PAI-1 expression and a decrease in tPA and uPA expression [179]. Hypoxia and the hypoxia-response element have also been shown to regulate PAI-1 expression [180].

6.2 Functions of PAI-1 in Vascular Wall In homeostasis, PAI-1 in mature blood vessels prevents excessive fibrinolysis, fibrinogenolysis, and other plasminmediated proteolyses. However, increased synthesis of PAI-1 by activated ECs or SMCs or PAI-1 secretion from activated platelets can tip the hemostatic balance and create a prothrombotic state favoring fibrin deposition. A complete deficiency in PAI-1 in humans causes a bleeding diathesis [181]. In an endotoxemia model of thrombosis, PAI-1-deficient mice developed fewer venous thrombi than wild-type mice [182]. PAI-1-deficient mice also showed increased lysis of platelet-rich arterial thrombi [183], and transgenic mice overexpressing PAI-1 showed age-dependent coronary arterial thrombosis with spontaneous thrombi after 6 months of age [184, 185]. However, the neointimal thickening following vascular injury has been variable in various models. PAI-1deficient mice show increased neointimal formation in electrical and mechanical arterial injury models [186], but increased PAI-1 expression promoted neointimal growth in a rat carotid artery injury model [187]. Variable effects of PAI-1 on angiogenesis have also been observed and may reflect the level of PAI-1 expression [90].

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7 The Plasminogen System in Other Cells of the Vascular Wall In the uninjured vessel wall, the EC serves as the major source of plasminogen activator activity by secreting tPA and uPA. In this surveillance mode, the EC controls basal fibrinolysis to sustain vascular patency. However, upon injury, expression of tPA, uPA, and uPAR, not only by ECs but also by SMCs and leukocytes, increases significantly [188, 189]. This results in the robust induction of fibrinolytic activity, even in the face of increased PAI-1 expression. The role of the contribution of other cellular sources of components of the plasminogen system has been deduced from studies of deficient mice. Deficiency of uPA, but not tPA, decreases neointima formation in arterial injury models [190, 191]. This response is consistent with the ability of uPA to induce SMC proliferation and migration and hence the response to arterial wound healing [191–193]. Enhanced expression of uPA in the vessel wall does result in increased SMC proliferation and migration and subsequent neointimal formation [192, 193]. These uPA-mediated SMC responses appear to be independent of uPAR since SMC function and neointima formation were not impaired in uPAR-deficient mice [194]. However, uPA-mediated SMC migration and proliferation were found to be dependent on plasminogen activation [17]. Indeed, the direct effect of plasmin in SMC proliferation has also been demonstrated and has been shown to involve activation of an EGF and ERK1/2 pathway [17]. Nevertheless, a plasmin-independent mechanism for uPAinduced SMC migration has also been described [195]. uPA within injured blood vessels also stimulates production of oxygen free radicals by increasing the activity and expression of both Nox 1 oxidase and Nox 4 oxidase [196]. Since reactive oxygen species regulate vascular tone and SMC proliferation, the production of these species by locally generated uPA may contribute to vascular remodeling. Plasmin-induced activation of growth factors, such as bFGF, platelet-derived growth factor, and TGF-b1, and their release from the degrading ECM, may also be critical in SMC proliferation and migration. Even though intimal thickening was still observed in a vascular injury model in tPA-deficient mice, tPA deficiency was found to influence late development of atherosclerotic lesions [192]. It was suggested that tPA binds to the SMC in a conformation distinct from that in which it binds to fibrin [15], thereby providing a basis for the differences in responses in different vascular injury models. In human atherosclerosis specimens, apoptosis of SMCs was observed in areas of pericellular proteolysis arising from sustained plasmin generation by secreted tPA [197]. Such local proteolysis of the ECM, cell retraction, and finally detachment may ultimately lead to SMC apoptosis. PAI-1 suppresses these events as was seen in PAI-1-deficient mice [198].

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Chapter 28

Endothelial Apoptosis and Repair in Pulmonary Arterial Hypertension Rohit Moudgil, Manoj M. Lalu, and Duncan J. Stewart

Abstract  Apoptosis (or programmed cell death) is a mechanism of cellular destruction that is essential for a variety of physiological events, such as tissue sculpturing during develop­ment and the removal of abnormal or damaged cells. However, untimely activation of this process can contribute to the pathogenesis and progression of a variety of human diseases, and pulmonary arterial hypertension is no exception. Recent evidence indicates that endothelial cell (EC) apoptosis is critically involved in the pathophysiological changes in PAH and that increased or decreased levels of apoptosis may contribute to different stages of the disease. The purpose of this chapter is to provide an overview of this rapidly evolving field and try to reconcile some of the apparent conflicts regarding the role of EC apoptosis in the initiation and progression of pulmonary hypertension. Considerable attention has been paid to the role of reduced apoptosis in other vascular cell types, particularly vascular smooth muscle cells (SMCs), which contributes to the massive medial growth and arterial remodeling characteristic of established disease, and this is reviewed in detail in other chapters. In contrast, this chapter will highlight the current concepts and controversies surrounding the role of abnormal EC growth and survival in the pathogenesis of PAH. In particular, the role of endothelial apoptosis as an initiating event in the development of PAH will be reviewed and current concepts pertaining to the emergence of hyperproliferative and ­apoptosis-resistant ECs in later stages of this disease will be discussed. This chapter also attempts to provide a unifying framework reconciling the apparent discrepancies between early apoptosis and EC cell loss which may represent one of the first events in the initiation of PAH, and the angioproliferative lesions that contribute characteristic pathological features of advanced disease.

D.J. Stewart (*) Department of Medicine, University of Ottawa and Ottawa Health Research Institute, The Ottawa Hospital, 501 Smyth Road, Ottawa, ON K1H 8L6, Canada e-mail: [email protected]

Keywords  Apoptosis • Capillary loss • Regeneration of endothelial cells • Stem cell • Cell therapy for pulmonary hypertension

1  Introduction Apoptosis (or programmed cell death) is a mechanism of cellular destruction that is essential for a variety of physiological events, such as tissue sculpturing during development and the removal of abnormal or damaged cells. However, untimely activation of this process can contribute to the pathogenesis and progression of a variety of human diseases, and pulmonary arterial hypertension (PAH) is no exception. Recent evidence indicates that endothelial cell (EC) apoptosis is critically involved in the pathophysiological changes in PAH and that increased or decreased levels of apoptosis may contribute to different stages of the disease. The purpose of this chapter is to provide an overview of this rapidly evolving field and try to reconcile some of the apparent conflicts regarding the role of EC apoptosis in the initiation and progression of PAH. Considerable attention has been paid to the role of reduced apoptosis in other vascular cell types, particularly vascular smooth muscle cells (SMCs), which contributes to the massive medial growth and arterial remodeling characteristic of established disease, and this is reviewed in detail in other chapters. In contrast, this chapter will highlight the current concepts and controversies surrounding the role of abnormal EC growth and survival in the pathogenesis of PAH. In particular, the role of endothelial apoptosis as an initiating event in the development of PAH will be reviewed and current concepts pertaining to the emergence of hyperproliferative and apoptosis-resistant ECs in later stages of this disease will be discussed. This chapter will also attempt to provide a unifying framework reconciling the apparent discrepancies between early apoptosis and EC cell loss which may represent one of the first events in the initiation of PAH, and the angioproliferative lesions that contribute to characteristic pathological features of advanced disease.

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_28, © Springer Science+Business Media, LLC 2011

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2  Historical Perspective The process now termed “apoptosis” was first identified as “chromatolysis” by Walter Flemming in 1885 from studies of the regression of epithelium in mammalian lymphoid follicles [1]. Half a century later, Glücksmann described “programmed cell death” in a series of articles dating from 1930 to 1947 [2–6] as occurring in three stages: “They may occur in the isolated cell or inside another cell, that is, the degenerating cell may be phagocytosed by a neighbor, and the last stage in particular is often found to proceed within the resorbing cell.” In 1961, Williams likened this process as following a “construction manual” so that “the cell that will die is programmed to do so” [7]. In his Ph.D. thesis, Kerr sought to understand the rapid involution of hepatic parenchyma in response to the acute surgical interruption of the portal venous blood supply of the liver. He found that cells within the ischemic hepatic lobes were transformed into small round vesicles of cytoplasm that often contained specks of condensed nuclear chromatin. These vesicles were then taken up and ingested by surrounding cells as well as by specialized professional (mononuclear) phagocytes [8]. Kerr’s description of this new type of cell death contrasted with the findings of typical necrosis, in which cells lose membrane integrity and become edematous, followed by leakage of their contents into surrounding tissues and the incitement of local inflammation. Since the cells that Kerr observed in the liver actually decreased in size as they died, he referred to this type of cell death as “shrinkage necrosis” [9]. In 1972, Kerr et al. coined the term “apoptosis” [8], derived from Greek roots, meaning “falling off,” like a leaf falling from a tree.

3  Current Concepts Since the initial description of apoptosis, 35 years of intensive research has produced an understanding which, although by no means complete, provides fairly detailed molecular insights into this highly intricate and complex process. A very brief outline of the current concepts of apoptosis will be included below, but for a more in depth treatment of the field the reader is referred to a number of recent and comprehensive reviews [10–12]. Caspases are key effector enzymes in apoptosis and are commonly grouped into either the initiator (i.e., caspases 8 and 10) or execution (i.e., caspases 3, 6, and 7) enzymes [13]. Activation of the execution set of caspases occurs via the extrinsic or intrinsic pathway of apoptosis [13], and is mediated by death receptors on binding of their specific ligands, which include tumor necrosis factor (TNF), TRAIL

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(a TNF-related, apoptosis-inducing ligand that binds to the DR4 and DR5 death receptors), and FasL (a ligand that binds to the Fas receptor) [14]. The association of a ligand and its cognate death receptor leads to the recruitment of adaptor molecules, such as FADD (Fas-associated death domain) and TRADD (tumor-associated death domain), thereby activating the initiator caspases 8 and 10, which in turn cleave and activate the executioner caspases 3, 6, and 7 [14]. On the other hand, the intrinsic or mitochondrial pathway is initiated by the release of cytochrome c from the mitochondria into the cytosol, which then interacts with (apoptosis-activating factor 1, ATP, and procaspase 9) to form a structure known as the apoptosome [15]. The apoptosome cleaves and activates caspase 9, which leads to activation of caspases 3, 6, and 7 and apoptosis [15]. Activation of caspase 9 without involvement of the apoptosome has also been described [10]. Each caspase is associated with a specific inhibitor, allowing the system to be tightly regulated by positive- and negative-feedback mechanisms. Caspase 3 represents the final enzymatic pathway within this cascade, and after activation of caspase 3, the morphologic events of apoptosis quickly follow, resulting in the orderly breakdown of cellular proteins, including the cytoskeleton and nuclear matrix, and the activation of poly-ADP-ribose polymerase, an enzyme that facilitates the degradation of nuclear DNA into 50- to 300-kilobase-sized pieces (DNA ladder formation) [16] (Fig. 1).

4  Endothelial Dysfunction in PAH Numerous studies have shown that the endothelium is central to vascular health. Abnormalities in its biological processes contribute directly to the pathophysiological changes in numerous diseases, notably PAH. The endothelium plays a critical role in vascular homeostasis by determining its permeability, regulating vasomotor tone and growth of the smooth muscle medial layers, and participating in repair and regeneration of blood vessels [17, 18]. Therefore, it is not surprising that early studies identified endothelial dysfunction as an important contributor to PAH, with an imbalance between various endothelialderived vasoconstrictor and vasodilator factors. Specifically, reduced vasodilator substances such as prostacyclin (PGI2) and nitric oxide (NO) and increased vasoconstrictor factors such as endothelin (ET) play a key role in imbalance in vascular homeostasis and tone. In the following paragraph, we briefly explore the role of endothelial dysfunction and alterations in the release of its various mediators in the development of PAH, but again, the reader is referred to several more comprehensive reviews of this topic for further detail [11, 19].

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Fig. 1  Biochemical events in apoptosis. Apoptosis can be activated in two ways. First, at the plasma membrane, a ligand (TNF or FasL) must be bound to a death receptor to activate apoptosis. Binding leads to oligomerization of the receptors, recruitment of an adaptor protein, and activation of TRADD/FADD. Caspase 8 is then bound to this death complex and activated. This enzyme then binds and activates caspase 3, the executioner caspase, which then activates caspase-activated deoxyribonucleases (CAD). These enzymes are responsible for degrading chromatin and generating the DNA fragments that are characteristic of

apoptosis. Conversely, the second pathway requires activation of Bax and Bid, two proapoptotic members of the Bcl-2 family, while inhibiting or inactivating Bcl-2 and Bcl-x, two antiapoptotic proteins. When this occurs, there is release of cytochrome c, apoptosis-activating factor 1 (Apaf-1), and other proteins from the intramembrane space to form an apoptosome that binds procaspase 9. Caspase 9 then activates caspase 3, which also causes the nuclear changes described above. Mitochondria release yet another protein, Smac/Diablo. This protein promotes caspase activation by associating with the apoptosome

4.1  Prostaglandins

could reduce pulmonary arterial and right ventricular enddiastolic pressure. Subsequently, extensive clinical testing established that chronic PGI2 therapy improves pulmonary hemodynamics, clinical status, and survival of patients with severe PAH [25–29]. Thus, studies from cells to animals and humans strongly support a key role for prostaglandins in the biological processes and treatment of PAH.

Prostaglandins were one of the first groups of endothelial factors to be identified. Early studies reported a decrease in prostaglandin H synthase expression in lung tissue from PAH patients [20], and decrease in urinary excretion of (6-ketoprostaglandin F1, a stable metabolite of PGI2, in patients with idiopathic PAH [21]. Moreover, pulmonary ECs isolated from PAH patients exhibited reduced expression of PGI2 synthase [20]. Lung-specific overexpression of PGI2 prevented the development of PAH and pulmonary vascular remodeling induced by chronic hypoxia [22]. Similarly, PGI2 receptor knockout mice exhibited an exaggerated elevation in pulmonary artery pressures and pulmonary arteriolar remodeling in response to chronic hypoxia compared with wild-type mice [23]. Similarly, at the cellular level, ECs cultured from pulmonary arteries of calves with hypoxia-induced PAH demonstrated reduced production of PGI2 [24]. These observations led to the first clinical demonstration in the late 1970s that infusion of PGI2 into patients with PAH

4.2  Nitric Oxide NO is the classic “endothelium-derived relaxing factor,” and was initially identified in the seminal work of Bob Furchgott [30]. Studies examining transgenic mice with targeted disruption of endothelial nitric oxide synthase (eNOS) revealed increased right ventricular mass and a greater percentage of fully remodeled precapillary pulmonary arteries in these animals in response to mild hypoxia compared with wild-type controls [31]. However, at simulated high-altitude conditions (i.e., 17,000  ft), the eNOS knockout and wild-type mice

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developed similar degrees of PAH [32]. Similarly, the first human studies examining eNOS expression in PAH yielded conflicting reports. Whereas, one study reported reduced expression of eNOS in the vascular endothelium of pulmonary arteries [33], this was not reproduced in other reports, which in fact showed increased expression specifically in the plexiform lesions of patients with advanced disease [34]. However, it is well known that reduced NO bioavailability, rather than eNOS messenger RNA or protein expression, is the principal determinant of endothelial dysfunction. Interestingly, patients with PAH exhibited lower levels of exhaled NO [35], and the severity of PAH was inversely correlated with NO levels estimated by measurement of NO reaction products in bronchoalveolar lavage fluid [36]. Furthermore, exogenous administration of NO gas by inhalation was shown to be a highly effective therapy for the short-term treatment of elevated pulmonary vascular resistance, particularly in acute care settings in which it can be easily provided to ventilated patients [37, 38]. These findings suggest that NO bioavailability may be important in the regulation of pulmonary vascular tone and structure in the context of PAH. However, difficulties in achieving convenient and reliable methods for long-term NO administration have limited its general application as a chronic therapy for PAH.

4.3  Endothelin ET-1, a 21-residue peptide that was discovered in 1988 as the first endothelial-derived constricting factor [39], has many important roles in various systemic vascular diseases as is reviewed elsewhere [40–42]. In the lung, increased expression of ET-1 has been demonstrated in animal models of PAH, including chronic hypoxia [43], monocrotaline (MCT) [44], and embolism-induced PAH [45]. As well, a marked elevation in the arteriovenous ratio of plasma ET-1 was demonstrated in patients with PAH, suggesting increased production of this peptide within the abnormal pulmonary vasculature [46]. Pathology studies using immunohistochemistry and in situ hybridization revealed that ET-1 was ­abundantly expressed in the lungs from patients with PAH, highly localized to the endothelium of pulmonary arteries, and correlated with elevations in pulmonary vascular resistance [46, 47]. The clinical relevance of elevated levels of ET-1 in this disease was confirmed by the first clinical trials, showing the benefit of the dual receptor antagonist bosentan in the treatment of iodiopathic PAH [48], as well as PAH associated with scleroderma [49] and congenital heart disease [50]. Similarly, ETA-selective antagonists, such as sitaxsentan [51] and ambrisentan [52], have been shown to provide similar benefits in clinical trials. In a 12-week, open-label

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study of sitaxsentan in patients with PAH, congenital heart disease, or connective-tissue-disease-associated PAH, sitaxsentan improved pulmonary hemodynamics and slightly improved a 6-min walk distance [51].

5 Genetics of PAH In 1984 a familial pattern of idiopathic PAH was noted, with approximately 10% of patients demonstrating a family history for the disease [53], characterized by an autosomal dominant pattern of inheritance with reduced penetrance and some degree of genetic anticipation. In 1997, the “PPH gene” was mapped to a single locus on chromosome 2q31-32 [54] and in 2000, two independent groups identified mutations of the bone morphogenetic protein receptor-2 (BMPR2) gene located within this region [55, 56]. In total, over 145 distinct mutations have been identified in 210 independent patients with heritable (H)PAH [57]. Approximately 80% of cases of familial PAH have now been associated with a mutation within one of the 13 exons of BMPR2 [58]; 70% of these mutations are nonsense or frameshift mutations that likely result in a nonfunctional product or transcript degradation [56, 59]. The remaining 30% of mutations lead to missense mutations in the functional domains of the receptor that interrupt trafficking of the receptor to the cell surface [60]. Thus, haploinsufficiency for BMPR2 appears to represent the principal molecular mechanism that underlies most patients with HPAH. Despite this major advance in our understanding the genetic basis of PAH, it is still very unclear how mutations of BMPR2 are in fact mechanistically related to the develop­ ment of PAH. BMPR2 is a ubiquitously expressed gene that is necessary for the activities of the various bone morphogenetic proteins (BMPs), involving a plethora of critical processes throughout the body, including heart development [61], bone remodeling, and tissue healing [62, 63], among many others (for reviews see [57, 64]). Thus, it remains a mystery why mutations in this ubiquitously expressed gene should result in such a specific phenotype, one that is limited to vascular disease localized in only a single organ of the body – the lung. Studies to date have suggested that, regardless of the specific BMPR2 mutation, the end result is a reduced capacity of the receptor to signal normally. The essential role of BMPR2 in PAH has been further highlighted by a study showing reduced expression of BMPR2 even in lungs of pulmonary hypertension patients without BMPR2 mutation [65]. Thus, altered BMPR2 signaling may underlie many forms of PAH, even those that are not the direct result of a known BMPR2 mutation.

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6 BMPR2 Signaling Within the lung vasculature, BMPR2 receptor messenger RNA and protein are expressed in the endothelium, macrophages, and medial SMCs [65]. Signaling of BMPR2 is reviewed in detail elsewhere [66]. However, in brief, this receptor is a constitutively active serine-threonine kinase that binds a series of ligands (BMP-2, BMP-3, BMP-4, BMP-6, BMP-7, BMP-9, and growth differentiation factors 5 and 6) and subsequently heterodimerizes with a type I receptor (BMPR1A or BMPR1B, also known as activin-receptor-like kinase-3 or activin-receptor-like kinase-6). The exact composition of the heterodimer formed likely determines the exact nature of the downstream signaling process. However, it has been established that the activated type I receptor can phosphorylate downstream signaling molecules such as receptoractivated Smads 1, 5, and 8 (R-Smads; small mothers against decapentaplegic). R-Smads translocate to the nucleus, where they complex with Smad-4 (co-Smad) and regulate positively and negatively the transcription of several genes. Smadregulated genes control differentiation, apoptosis, and repair in a wide variety of tissues. Interestingly, by themselves Smads bind weakly to DNA, which can be modulated by a number of corepressors and coregulators [67]. Aside from Smad signaling, BMPR2 is also recognized to activate a number of mitogen-activated protein kinases (MAPKs), including extracellular-signal-regulated protein kinase (ERK1/2) c-Jun NH2-terminal protein kinase (JNK), and p38 MAPK. The complicated signaling of BMPR2 ultimately allows many potential points of regulation along this pathway. The genes regulated downstream of BMPR2 play an important role in normal vasculogenesis and soft tissue development. The importance of these pathways is underscored by the embryonic lethal phenotype of the BMPR2-null homozygote mouse at the earliest stages of development [68]. Moreover, BMPR2 signaling is highly context- and cell-typedependent and, as discussed later, produces divergent consequences in the two principal cell types implicated in PAH.

6.1 BMPR2 and Pulmonary SMCs The first studies characterized a role of BMPR2 in regulating pulmonary artery SMC growth. BMPs were shown to induce apoptosis [69] and inhibit growth [70] in pulmonary artery SMCs isolated from healthy patients, but not in cells from patients with primary (idiopathic) PAH [70]. In further studies, the downstream mediators of this growth regulatory effect were explored. Pulmonary SMCs isolated from three patients with different BMPR2 mutations exhibited reduced Smad-1 phosphorylation and decreased activation of a Smadresponsive luciferase reporter transcription [71]. A decreased

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level of phospho-Smad-1 was also noted in medial and intimal cells of vessels in lung tissue from PAH patients when compared with vessels from healthy patients. Further downstream targets of BMPR2 include the inhibitor of the DNA-binding (Id) family of proteins [72]. Id proteins interact with basic helix–loop–helix transcription factors and reduce their ability to bind DNA and thereby regulate transcription. In pulmonary artery SMCs, BMP-4 increased Id gene expression in cells from healthy patients, resulting in reduced cellular proliferation [72]. This response was impaired either in cells from PAH patients or in cells from healthy patients subjected to small interfering RNA silencing of BMPR2. Taken together, these studies suggest that reduced BMPR2 loss-of-function mutations alter the balance of growth-stimulatory and growth-inhibitory signaling leading to unchecked pulmonary SMC growth.

6.2 BMPR2 and the Pulmonary Vascular ECs The potential importance of BMPR2 in the endothelium is underscored by its preferential expression in the vascular endothelium layer of small pulmonary arteries [65]. However, in contrast to their effect on SMCs, BMPs appear to have the opposite effect on ECs. The first evidence for a role of BMPR2 signaling in promoting EC survival came from the demonstration that BMP-2 protected against apoptosis [measured by ­terminal deoxynucleotide transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) and caspase-3 activation] induced by either serum-free conditions or the proinflammatory cytokine TNF-a [73]. Furthermore, BMPR2 gene silencing increased EC apoptosis even in basal culture conditions in the presence of serum and growth, factors, consistent with a survival effect mediated through BMPR2. Moreover, “early growth” endothelial progenitor cells (EPCs) derived from patients with idiopathic PAH displayed a heterogeneous response to BMP-2, with some cells exhibiting substantial increases in apoptosis, whereas BMP-2 consistently reduced apoptosis in EPCs from healthy subjects. There was also a direct correlation between the hemodynamic severity of PAH and the increase in apoptosis, such that cells from patients with the highest pulmonary artery pressure demonstrated the greatest increases in apoptosis resulting from BMP-2 stimulation. The role of the BMP pathway in promoting EC survival and growth has now been confirmed by other groups [74, 75], and the opposing effects of BMPR2 signaling on the two major cell types of the pulmonary vascular wall may provide insight as to why mutations of this gene are so integrally linked to PAH (Fig. 2). Together, these divergent consequences of BMPR2 haploinsufficiency may synergize to produce the characteristic structural and functional abnormalities of this disease. Thus, reduced BMPR2 activity predisposes to EC damage and apoptosis, and may be a central mechanism in

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Fig. 2  Role of bone morphogenetic protein receptor-2 (BMPR2) mutation in the pathogenesis of pulmonary arterial hypertension (PAH). The “double jeopardy” hypothesis. Loss-of-function mutation in BMPR2 results in disruption of the signal transduction mechanism that culminates in endothelial apoptosis and smooth muscle cell (SMC) proliferation. The early event precipitates the drop-out at the level of the

precapillary arterioles and reduced perfusion of arteriolar–capillary units, contributing to a progressive rise in pulmonary vascular resistance. Conversely, proliferation of smooth muscle cells results in medial hypertrophy and mechanistic increase in pulmonary vascular resistance. Cumulatively, both processes lead to pulmonary arterial hypertension (Reproduced with permission from Southwood et al. [75])

the initiation of this disease, since EC loss at the level of the fragile precapillary arterioles may reduce perfusion of arteriolar–capillary units, and contribute to a progressive rise in pulmonary vascular resistance as more and more and more units are affected. At the same time, the loss of regulatory influences on SMC growth may contribute to an exaggerated proliferative response to growth factors (e.g. platelet-derived growth factor) or elevated wall tension, leading to the massive medial hypertrophy and distal muscularization (Fig. 2). Although this may explain in part why BMPR2 is so central to the pathogenesis of PAH, it does not explain why this is a phenomenon unique to lung, and why these mutations are not associated with a general abnormality in vascular remodeling and function that might be expected to lead to diseases of the systemic vasculature including hypertension,

atherosclerosis, and restenosis. The answer to this paradox may lie in the unique structure of the pulmonary microvasculature. The lung represents the largest vascular bed of the body, and must accommodate the entire cardiac output, even at peak exercise, offering minimal resistance to blood flow. As well, unlike most systemic vascular beds, the distal arteriolar beds are poorly muscularized and, at the level of smallest intra-acinar arterioles, there are few supporting mural cells (i.e., no SMCs and only rare pericytes). Thus, the endothelium of the precapillary pulmonary arteriole may be particularly dependent on survival signaling, such as that provided by BMP/BMPR2, for the maintenance of vascular homeostasis. In support of this concept, inhibition of other endothelial survival pathways such as vascular endothelial growth factor (VEGF) [76] and Tie2 [77] have been reported to induce or

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aggravate experimental pulmonary hypertension by a mechanism dependent on increased EC apoptosis (see section 7).

6.3 BMPR2 Haploinsufficiency – Need for a “Second Hit”? Despite the evidence supporting a central role for BMPR2 mutations in this disease, only 20% of individuals who carry the BMPR2 mutation will actually develop clinical ­pulmonary hypertension [78]. The lack of direct correlation between genotype and phenotype is supported by animal studies demonstrating that the heterozygote BMPR2+/- mouse models show only a mild phenotype with only modest [79] or no pulmonary hypertension under basal conditions [80, 81]. Such a low penetrance suggests that other factors (i.e., genetic or environmental) are necessary for the full phenotype to be manifested. Indeed, in BMPR2 heterozygous knockout mice, the addition of a well-characterized pulmonary hypertensive stimulus, such as serotonin or inflammatory mediators [72, 80, 81], results in the unmasking of a severe PAH phenotype.

7  Endothelial Growth and Survival in Experimental Models of PAH The advanced stages of PAH are characterized by proliferative intimal and plexiform lesions [82, 83], which are suggestive of dysregulated EC growth. These so-called angioproliferative lesions may contribute to elevation in pulmonary vascular resistance by occluding the lumen of intra-acinar pulmonary arterioles [84]. Direct evidence in support of abnormal EC growth has been obtained from the analysis of cells isolated from lungs of patients with idiopathic PAH, which showed a greater response to growth factors in culture and resistance to apoptosis [85]. The pulmonary expression of VEGF and its receptor, Flk-1, is increased in response to hypoxia in vitro [86] and in vivo [87], and an increase in VEGF expression was found associated with intimal and plexiform lesions in lungs of patients with severe PAH [82], suggesting a possible causal relationship between VEGF expression and the development of these angioproliferative lesions [88]. Further evidence of VEGF involvement in development of plexiform lesions came from studies of lungs from patients with PAH associated with congenital heart disease [89]. VEGF was detected in pulmonary artery SMCs and ECs more frequently in patients with the histological lesions of advanced pulmonary plexogenic arteriopathy [90]. These observations suggest that increased VEGF expression and release may contribute to the development of PAH, possibly by promoting abnormal vascular EC growth.

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However, these largely observational studies do not establish causality of VEGF in promoting vascular disease and progression of PAH, and indeed experimental studies manipulating VEGF expression and activity have suggested a more complex mechanism of PAH. Thus, rather than preventing pulmonary hypertension, VEGF receptor blockade with SU5416, a semiselective VEGF receptor-2 (VEGFR2) antagonist, resulted in a marked potentiation of pulmonary artery pressures in the chronic hypobaric hypoxia rat model [76]. Of note, this was associated initially with a significant increase in pulmonary EC death, as demonstrated by activated caspase 3 immunostaining and TUNEL assay [76]. Also, VEGF blockade in this model increased vascular remodeling, with evidence of precapillary arterial occlusion by proliferating ECs (or endothelial-like cells) [76], reminiscent of the plexiform lesions seen in human disease. Of interest, these hemodynamic and vascular abnormalities could be abrogated by treatment with a pancaspase inhibitor, Z-AspCH2-DCB, confirming that EC apoptosis induced by loss of VEGF survival signaling was the initiating mechanism in this interesting experimental model of severe PAH [76]. Similarly, the overexpression of VEGF-A using a cellbased gene transfer approach did not worsen, but rather prevented, the development of pulmonary hypertension in inbred Fisher 344 rats induced by the endothelial toxin MCT [91], an effect which was associated with reduction in arteriolar EC apoptosis. In addition, delay of cell-based gene transfer until after the development of established pulmonary hypertension significantly reduced the progression of right ventricular hypertension and hypertrophy [91]. Other groups have reported that adenoviral VEGF gene transfer also prevented pulmonary hypertension in both the MCT and the chronic hypoxia rat models [86, 92]. In addition, pretreatment with the adenovirus VEGF vector 2 days prior to an hypoxic insult increased eNOS activity in lung tissue [92], and resulted in partial restoration of endothelium-dependent vasodilation in isolated lungs from chronically hypoxic rats, as assessed by improvement of ionophore A23187-induced vasodilation and attenuation of ET-1-induced vasoconstriction [92]. These studies suggest that rather than being part of its causation, VEGF overexpression in the lungs of patients with severe PAH may represent a compensatory mechanism that attenuates development of PAH, in part by protecting endothelial function and preventing EC apoptosis. As well, overexpression of another important endothelial survival factor, angiopoietin-1 (Ang-1), was shown to prevent pulmonary hypertension in both the MCT [93] and the chronic hypoxia rat [94] models. Survival studies revealed that Ang-1 also reduced mortality in animals receiving MCT from 77% mortality at 28 days to only 14%. We have recently confirmed a key protective role for the Ang-1/Tie pathway in PAH using loss and gain of function transgenic mouse models [95], and we believe these new results, together with those of

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the previous gene transfer studies, strongly refute the suggestion by another group that Ang-1 may play a causal role in this disease [77, 96–98].

8 Relationship Between EC Apoptosis and Proliferation in PAH Thus, the evidence so far suggests a complex interaction between pulmonary EC damage and loss, on the one hand, and increased proliferation, on the other, possibly induced initially as part of a mechanism of endothelial repair. However, the mechanistic link between damage (apoptosis) and repair is not clear. Of interest, the engulfment of apoptotic bodies by phagocytes has been reported to stimulate the secretion of bioactive substances, including VEGF, that lead to EC proliferation [99]. The role of VEGF in this process was further confirmed by VEGF-blocking antibodies, which inhibited the proliferation of ECs elicited by the conditioned medium [99]. But, as already discussed, VEGF blockade (SU5416) potentiated the functional and pathological abnormalities in the chronic hypoxic rat PAH model [100]. The Voelkel group developed an in vitro model to better examine the underlying mechanisms by which apoptosis can lead to the emergence of hyperproliferative and apoptosis-resistant ECs [76, 101]. Using a microcapillary perfusion culture system, they showed that hyperproliferative, phenotypically altered ECs were generated 7 days following induction of apoptosis with VEGF receptor blockade coupled with high fluid shear stress [101]. These altered ECs expressed the antiapoptotic proteins survivin and Bcl-XL, typically found in neoplastic tumors, and were resistant to induction of apoptosis after challenge with TNF-a plus cycloheximide or hydrogen peroxide [100]. In addition, the cells demonstrated survival in a serum-free culture medium [100]. On the basis of these data, they suggested that sustained apoptotic EC stress creates conditions which allow for the emergence of EC populations that are resistant to apoptosis and have a greater growth potential.

9 Role of Resident or Circulating EPCs in Lung Vascular Repair The integrity of the endothelial lining is critical to normal vascular function, and therefore mechanisms for rapid and efficient repair are required to minimize the structural and functional consequences of EC damage. Two possible ­mechanisms may be involved in endothelial repair; the first is local and the other involves the “drop in” repair by circulating bone-marrow-derived endothelial progenitor cells (EPCs).

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In addition to the contribution of neighboring cells that can divide and migrate to restore the continuity of the endothelial lining, it has recently been suggested that resident endothelial stem cells, or endothelial colony-forming cells (ECFCs), may also contribute to vascular repair [102, 103]. ECFCs are rare among mature ECs and may actually originate from the vascular endothelium itself [104]. Ingram et al. reported that a single ECFC has significant potential for clonogenic expansion [105], and colonies can be serially replated to generate a monoclonal population of ECs. This was further confirmed in cells freshly isolated from human umbilical veins [103]. Furthermore, the ability of these cells to form viable vessels was tested by their incorporation within collagen gels and implantation into immunodeficient mice [106, 107]. Upon excision, 14 days later, ECFCs could be identified in de novo vessels which were perfused by the host murine circulation [106]. Recently, Stevens et al. [108]. reported that the lung microvasculature may be particularly rich in endothelial progenitors that exhibit very high growth and regenerative potential, consistent with the concept that the lung is endowed with a mechanism for rapid endothelial repair. The presence of circulating EC precursors was first suggested in the 1960s when Stump et al. [109] showed that a piece of Dacron material suspended intravascularly within a pig aortic graft could be endothelialized despite the lack any continuity with the vessel wall. The authors concluded that circulating endothelial precursors attached and proliferated to cover the suspended material [109]. More recently, the work of Asahara et al. [110] popularized the concept of bonemarrow-derived EPCs that home to regions of injury and function to repair and regenerate the systemic vasculature. However, the role of these cells in the pulmonary circulation has been less well studied. To the extent that these cells contribute to lung endothelial repair, it may be possible to enhance this process by the therapeutic administration of EPCs. One of the difficulties in this field is the confusion that surrounds what constitutes an “EPC” and how best to identify and select truly active cell populations. Again the reader is referred to more comprehensive reviews of this topic [111, 112]. Briefly, there are two main methods for isolating EPCs from the general mononuclear cell (MNC) population. The first relies on the presence of surface markers, such as CD34 (hematopoietic stem cell marker), which was originally used in the pioneering work of Asahara [110], with or without CD133 [113] and often coupled with an endothelial determinant such as VEGFR2 [114]. However, there is no general agreement as to what are the most appropriate markers to use and therefore other groups have relied on a selection strategy based on the functional characteristics of these cells under specific conditions of culture. In this method, circulating or bone-marrowderived MNCs are plated on an appropriate matrix (often

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fibronectin) in the presence of a cocktail of endothelial growth factors (VEGF-A, insulin-like growth factor-1, epidermal growth factor, etc.) [115]. This results in the appearance of rod-like cells of low growth potential after about 3–5 days of culture, which are termed “early” EPCs. Although these cells express a variety of endothelial genes (VEGFR2, CD31, Tie2, eNOS), they retain the panleukocyte (CD45) and MNC (CD14) markers, and are probably better considered to be “culture-modified” or “angiogenic” MNCs [116]. Nonetheless, these cells are highly active in inducing neovascularization in a number of different models in vitro and in  vivo, likely by paracrine as well as direct vasculogenic mechanisms [110]. If these cultures are maintained beyond 1–2 weeks, cell aggregates appear that resemble colonies and give rise to rapidly proliferative, spindle-shaped cells that quickly over grow the dish, resulting in a cobblestone lawn of cells that have a very strong endothelial phenotype [117]. These “late-growth” EPCs have lost all leukocyte markers, and, apart from their high proliferative potential, are nearly indistinguishable from mature ECs [104]. However, the regenerative potential of late-outgrowth cells has been less well studied, although it has been suggested that they may act primarily as building blocks in the production of new vessels [104, 117]. Our laboratory has shown that fluorescently labeled endothelial-like progenitor cells (ELPCs) resembling early EPCs engrafted at the level of the distal pulmonary arterioles and incorporated into the endothelial lining in the MCTinjured lung prevented the increase in right ventricular systolic pressure seen at 3 weeks with MCT alone [118]. Delayed administration of progenitor cells 3 weeks after MCT injury prevented the further progression of PAH 2 weeks later (i.e., 5 weeks after MCT injury), whereas only rats receiving progenitor cells transduced with human eNOS exhibited significant reversal of established disease at day 35 [118]. Fluorescent microangiography revealed widespread occlusion of pulmonary precapillary arterioles 3 weeks after MCT injury, whereas arteriolar–capillary continuity and microvascular architecture were preserved with the administration of syngenic ELPCs. This suggests that the engrafted progenitor cells can restore microvasculature structure and function in MCT-induced PAH [118]. Thus, circulating ECs have the ability to participate in vasculogenesis and angiogenesis.

9.1 A “Unifying” Hypothesis Although some of the concepts described herein about role of EC apoptosis and proliferation in PAH may seem contradictory, the current evidence suggests a logical sequence of events that could provide insight into mechanisms underlying the initiation and progression of this

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disease (Fig. 3). Under normal conditions the lung vasculature is constantly exposed to the environment, particularly at the level of the fragile precapillary arterioles that are in direct contact with the inspired air. As a result, it is likely that lung microvascular EC damage is part of a normal cycle of loss and repair that occurs periodically throughout life, involving EC apoptosis and proliferation, possibly enhanced by resident or circulating endothelial progenitors. However, in the face of overwhelming environmental stress (i.e., toxic oil syndrome, severe left–right shunt), or a genetic predisposition to EC loss (e.g., BMPR2 mutation), the reparative capacity of the lung may be overwhelmed, and this may have two important consequences (Fig. 3). First is the “drop out” of ECs, especially at the level of the precapillary arterioles (which are essentially endothelial tubes with scant matrix and few or no mural cells). This may lead to a loss of ­functional continuity between the arterial and distal alveolar capillary beds. A progressive loss of efficient (i.e., low-pressure) perfusion of these alveolar–capillary units could contribute directly to an increase in the overall pulmonary vascular resistance, and hence PAH. Second, widespread and persistent (or recurrent) pulmonary vascular EC apoptosis creates conditions that favor the selection of apoptosis-resistant and hyperproliferative ECs that have been demonstrated in the in vitro and in vivo models discussed earlier. These abnormal ECs may ultimately contribute to formation of plexiform lesions and occlusive intimal lesions that characterize the clinical pathological manifestations of advanced PAH. These cells may thus may be involved in the progression of PAH, particularly in the late stages of the disease, but the extent to which this process is central to its pathogenesis rather than being a manifestation of the underlying abnormalities in survival of normal lung ECs is not clear.

10  Therapeutic Implications This paradigm for the pathogenesis of PAH has several important implications for the prevention and treatment of this disease. The first is that novel therapies aimed at reducing EC damage and loss or enhancing reparative mechanisms may be effective in preventing the initiation and progression of PAH. These may include the use of selective EC survival factors (i.e., VEGF and Ang-1) and the administration of cells such as EPCs that may enhance endothelial repair. Indeed, the preclinical data reviewed herein support the potential efficacy of these strategies, some of which are now being tested in early-phase clinical studies such as the Pulmonary Hypertension and Cell Therapy (PHACeT) trial. However, there is a potential concern that a strategy aimed

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Fig.  3  Endothelial injury in health and disease. During the time of minor injuries such as environmental stresses, endothelial apoptosis triggers a cascade of mechanisms resulting in endothelial repair by endothelial progenitor cells, thus maintaining vascular homeostasis.

However, in events of overwhelming stress reparative mechanisms become overburdened thus precipitating an exaggerated response possibly contributing to the pathogenesis of pulmonary arterial hypertension (PAH)

at inhibiting EC apoptosis would need to be highly selective, since there is considerable evidence that reduced SMC apoptosis contributes to arterial remodeling and abnormal muscularization in PAH. Indeed, strategies to enhance SMC apoptosis are being explored as another novel therapeutic strategy for this disease [119, 120], although again, such approaches would need to be selective, since induction of EC apoptosis would likely be counterproductive. As illustrated in Fig. 4, nonselective induction of cellular apoptosis would only be safe if there was a clear temporal separation between increased EC apoptosis as an early event and reduced apoptosis of both ECs and SMCs as a later and dominant mechanism of arteriolar remodeling and occlusion (model A). However, if EC loss continues in an episodic fashion, possibly as a result of repeated injury, throughout the course of this disease (model B), then a treatment that increases EC apoptosis may actually worsen the progression of disease. In this case, selective strategies will be needed to target particular cell populations, such as the

use of endothelial-specific growth and survival factors, or possibly the modulation of SMC metabolic state [119, 120]. Imatinib, a platelet-derived growth factor receptor antagonist approved for the treatment of chronic myeloid leukemia, may represent a promising strategy to target SMC proliferation, and an initial case report showed dramatic improvement 3 months after initiation of treatment [121]. A randomized trial is now under way; however, toxic effects on the heart could limit its long-term use [122]. Similarly, strategies to reduce the growth of hyperproliferative ECs in the later stages of PAH by inhibiting angiogenesis are being explored. For example, a multikinase inhibitor, sorafenib, which targets VEGFR2 among other receptor tyrosine kinases, is being studied in early clinical trials and may have promise for the treatment of PAH [123, 124]. However, although targeting the “angioproliferative” lesions, this may be at the cost of accelerating the fundamental loss of the normal microvascular ECs, depending on which of the models illustrated in Fig. 4 proves to be more accurate.

28  Endothelial Apoptosis and Repair in Pulmonary Arterial Hypertension

Fig. 4  Endothelial cell apoptosis: a tale of two models. The current concepts dictate that there are two possibilities of endothelial apoptosis. Model A describes a situation when a single catastrophic endothelial cell (EC) apoptotic event at early stages triggers an increase in pulmonary artery pressure (PAP) and eventual emergence of hyperproliferative endothelial cells in the late stage. Model B presents a situation where multiple endothelial apoptotic events underscore the elevation in pulmonary artery pressure and the eventual emergence of hyperproliferative endothelial cells in late stages

11  Conclusion Although PAH is a disease of diverse origins, it shares common pathological features, which may reflect common underlying pathogenic mechanisms regardless of the underlying cause (i.e., genetic mutation vs. cardiac shunt). Therefore, understanding the key elements in its pathogenesis will likely yield valuable therapeutic insights and novel strategies to prevent the progression or even regression of this cancer of pulmonary vasculature, some of which were discussed herein. In this review we presented a paradigm for the pathogenesis of PAH that reconciles many of the seemingly divergent concepts that are being explored in the literature, and hopefully such a synthesis can inform a rational search for truly effective novel therapies for this lethal condition.

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Chapter 29

Bronchial Arterial Circulation in the Human Gabor Horvath and Adam Wanner

Abstract  The bronchial vasculature derives oxygenated blood from the systemic circulatory system and serves as the principal vascular supply of the conducting (nonrespiratory) portions of the airways. Its predominant function is to supply nutrition to the tracheal and bronchial wall, large pulmonary blood vessels, some hilar structures, including lymph nodes, lung parenchyma, and visceral pleura. In addition, the bronchial circulation has important roles in airway heat and water exchange with the environment, the clearance of locally released biologically active substances in the airway wall, and possibly the regulation of airway caliber in the lung periphery. Furthermore, the bronchial circulation functions as the conduit for inflammatory cell recruitment in inflammatory airway diseases such as bronchial asthma and chronic bronchitis, and participates in the clearance of inhaled drugs from and the distribution of systemic drugs to the airway. For decades, the technical challenges of assessing the bronchial circulation in vivo have limited the amount of information on the bronchovascular components of airway diseases. Thus, bronchial blood flow measurements have been the focus of researchers only, and several techniques have been used. Because of the availability of recent noninvasive methods to measure blood flow in the airway mucosa, a better understanding of the bronchial circulatory system’s functional and pathophysiological importance is slowly emerging. Keywords  Bronchial circulation • Systemic vasculature • Nutrient and oxygen delivery • Inflammatory airway disease

1 Introduction The bronchial vasculature derives oxygenated blood from the systemic circulatory system and serves as the principal vascular supply of the conducting (nonrespiratory) portions G. Horvath (*) Department of Pulmonology, Semmelweis University School of Medicine, Diosarok 1/C, Budapest 1125, Hungary e-mail: [email protected]

of the airways. Its predominant function is to supply nutrition to the tracheal and bronchial wall, large pulmonary blood vessels, some hilar structures, including lymph nodes, lung parenchyma, and visceral pleura [1, 2]. In addition, the bronchial circulation has important roles in airway heat and water exchange with the environment [3, 4], the clearance of locally released biologically active substances in the airway wall [5, 6], and possibly the regulation of airway caliber in the lung periphery [7–9]. Furthermore, the bronchial circulation functions as the conduit for inflammatory cell recruitment in inflammatory airway diseases such as bronchial asthma and chronic bronchitis, and participates in the clearance of inhaled drugs from and the distribution of systemic drugs to the airway [10]. For decades, the technical challenges of assessing the bronchial circulation in  vivo have limited the amount of information on the bronchovascular components of airway diseases. Thus, bronchial blood flow measurements have been the focus of researchers only, and several techniques have been used. Because of the availability of recent noninvasive methods to measure blood flow in the airway mucosa [11–13], a better understanding of the bronchial circulatory system’s functional and pathophysiological importance is slowly emerging.

2 Anatomy of the Bronchial Circulatory System The two distinct circulatory systems of the lung have been known for centuries. However, in contrast to the extensively investigated pulmonary circulation, anatomical details and the functional importance of the tracheobronchial circulatory system have received attention only recently. The anatomy of the bronchial vasculature has been studied in humans and other mammals, with somewhat different findings among investigators. Moreover, the studies revealed that the bronchial vascular anatomy, including vessel origin, distribution, and density, vary significantly from species to species [2].

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_29, © Springer Science+Business Media, LLC 2011

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2.1 Arterial System The bronchial circulation is a high-pressure, small-volume system that derives arterial blood from the systemic circulation (Fig. 1). The origin of the bronchial arteries is quite variable. The major bronchial arteries usually arise either directly from the descending aorta at the level of the tracheal bifurcation or indirectly via an intercostal artery. Infrequently, accessory bronchial arteries may arise from the innominate artery, the subclavian artery, and the internal mammary, pericardiophrenic, and esophageal arteries [14, 15]. Before entering the lung through the hilum, bronchial arteries form the bronchomediastinal plexus that supplies most of the mediastinal structures, such as the esophagus, hilar lymph nodes, vasa vasorum of the thoracic aorta, mediastinal pleura, and pericardium. Upon reaching the main bronchus on each side, bronchial arteries divide and follow the branches of the bronchial tree. The bronchial artery diameter ranges from approximately 1.5  mm at the hilum to approximately 0.5– 0.75 mm at the point of entry into a bronchopulmonary segment. Bronchial arteries surround the airway in the form of an annulus (i.e., the peribronchial plexus), which supplies branches to the bronchi and vasa vasorum. Smaller branches penetrate the muscular layer of the bronchial wall and form an extensive vascular network underneath the airway epithelium (i.e., the mucosal plexus). The interconnected peribronchial and mucosal plexuses follow airways as far as the terminal bronchiole. Some species have sinuses under the subepithelial plexus; however, the significance of this capacitance vessel system is not known.

Fig. 1  The bronchial circulation as the systemic circulatory supply to the intrathoracic airways and lung. The blood flow from the lower trachea and airways outside the lung returns to the right atrium, whereas blood from the intrapulmonary airways and the rest of the lung drains into the left atrium. (Reproduced from Deffebach et al. [1] with permission. Copyright American Thoracic Society)

G. Horvath and A. Wanner

2.2 Capillary Beds and Bronchopulmonary Connections Within the airway wall, the majority of blood flow is distributed to the subepithelial tissue, where the microvasculature comprises a significant percentage of tissue volume [16] (Fig. 2). The extensive arteriolar network around the bronchi terminates in a dense system of subepithelial capillaries and bronchial venous plexuses in the mucosa close to the airway lumen. This subepithelial network of microvessels lying between the smallest arteries and veins is formed by ramifications of the arterioles, capillaries, and postcapillary venules. The terminal arterioles give rise to capillaries located just beneath the epithelial basement membrane and tend to run parallel to the long axis of the airway. In the human airway, the total number of vessels residing in the submucosa within 200 mm of the bronchial epithelial basement membrane is about 120 per millimeter [2, 17]. The bronchial microvascular network is obvious even in small bronchi (0.5-mm diameter) and represents the terminal portion of the bronchovascular system. This network plays an important role in the transfer of nutrients, gases, and metabolic waste products in the airway. The bronchial microvascular network anastomoses with the pulmonary microvasculature along the airways in the lung parenchyma, and is also known as the “pulmonary collateral circulation” or “systemic blood supply to the lungs” [18]. Anastomoses have been demonstrated at precapillary, capillary, and postcapillary levels of the bronchial vasculature [2]. Thus, in the distal airways, oxygenated blood can flow from the bronchial circulation via bronchial capillaries to pulmonary vessels [1]. Despite the extensive anastomoses

Fig.  2  Cross section of bronchial wall showing the subepithelial microvasculature and its proximity to the airway lumen in sheep. Tissue fixation with glutaraldehyde was completed at 100-mmHg and 20-mmHg pulmonary arterial pressure aortic pressure. A bronchial artery, C capillaries, V venule, SG submucosal gland, N autonomic nerve, E epithelium. (Courtesy of Andrew Mariassy)

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Fig. 3  The bronchial circulation including its venous drainage. BA bronchial artery, BV bronchial vein, LA left atrium, PA pulmonary artery, PV pulmonary vein, SVC superior vena cava. (Reproduced from Chediak et al. [20] with permission from Elsevier)

between the bronchial and pulmonary vessels, most of the blood supply to the intraparenchymal airways down to 1 mm in diameter is from the bronchial circulation [19].

2.3 Venous System Venous blood from the extrapulmonary airways drains successively through the bronchial veins, the vena azygos, and superior vena cava to the right side of the heart [20, 21] (Fig. 3). In contrast, venous blood from the intraparenchymal airways mainly drains into the pulmonary circulation and subsequently into the left side of the heart [22, 23]. Normally, approximately 66–80% of the bronchial venous flow empties into the left side of the heart. Part of the bronchial circulation drains into the systemic venous return, and another part connects with the pulmonary venous drainage, thereby constituting a physiological right-to-left shunt.

3 Physiological Functions Since the two circulatory systems of the lung have different anatomic structures, their functions are also different. The bronchial circulatory system is ideally located to perform a number of functions, including nourishing the bronchi, heating and humidifying the inspired air, and distributing and clearing mediators or drugs to and from the airway [1, 24]. Airway blood flow seems to support the mucociliary apparatus through as yet unknown mechanisms [25]. However, the physiological processes and clinical relevance of the bronchial circulation continue to be under active investigation.

3.1 Nourishment of the Bronchial Mucosa Under physiological conditions, total blood flow through the tracheobronchial vasculature comprises 0.5–1% of cardiac

output in mammals, including humans [1]. Although this is a trivial flow in absolute terms, it assumes a significant value when normalized for the relatively small tissue volume it perfuses. The majority of bronchial blood flow is distributed to the inner airway wall in vessels that occupy a significant volume of the subepithelial tissue. For example, the microvasculature has been found to accommodate approximately 85% of total tracheobronchial blood flow and to occupy 10–15% of the subepithelial tissue in sheep trachea and bronchi [16]. Subepithelial (mucosal) blood flow has been reported to range between 30 and 95 mL/min/100 g wet tissue in different species, including humans [26]. The primary function of the bronchial circulation presumably is to supply oxygenated blood and to nourish the epithelium and submucosal glands. The airway epithelium is active both in ciliary beating and active transport of ions and macromolecules. The metabolic rate of cultured sheets of tracheal epithelia is high, corresponding to an oxygen consumption of 5.13 mmol/min/g. This value may be compared with 1.0 mmol/ min/g for liver and 4.5 mmol/min/g for the beating heart [27]. Therefore, the main function of this network presumably is to provide adequate nourishment to this tissue, which has one of the highest metabolic rates in the body.

3.2 Air Conditioning During quiet nasal breathing, the inspired air is fully water saturated at body temperature by the time gas reaches the alveoli. One of the major physiological duties of the airway mucosa is to warm and humidify inspired air [1, 28]. As the air is warmed, its capacity to hold water increases, and it is humidified by evaporation from the airway lining fluid. Thus, heat and water exchange within the respiratory tract determines the temperature of the bronchial mucosa and the osmolarity and viscosity of the airway lining fluid [29]. The heat and water that are transferred to condition the ambient air come from the walls of the bronchial tree, and they need to be replenished by bronchial blood flow. This requires an increase in airway blood flow, i.e., vasodilatation, in response to thermal challenge, especially during

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hyperventilation. Thus, heat exchange and water control inside the tracheobronchial airways is modulated by changes in airway mucosal blood flow. Complete air conditioning inside the upper airway might not be achieved during certain conditions such as mouth breathing and hyperventilation or during cold air breathing [28, 29]. Moreover, the airway mucosal blood flow represents such a small fraction of the total (pulmonary and bronchial) circulation to the lung that it can only be effective in heating the airway during a low thermal burden [29]. Indeed, it has been shown that the response exhibited by the bronchial circulation depends on the magnitude of the thermal stress to which it is exposed [4, 30]. Dry air hyperpnea leads to significant heat and water losses from the conducting airway. Since the bronchial circulation replaces relatively little of the heat lost to inspired air, the pulmonary circulation plays the primary role in opposing bronchial cooling, transferring heat to the airway by lateral conduction or by direct perfusion. Nonetheless, the bronchial circulation may be important in minimizing airway dehydration that would otherwise accompany respiratory water loss.

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soluble gases [33]. If any soluble gas is present in the inspired air, the distance of penetration into the airway tree before its complete loss (i.e., mucosal absorption) largely depends on gas solubility (blood–air partition coefficient) and bronchial blood flow. The diffusion gradient for soluble gas exchange with the airways reverses from inspiration to expiration. If the soluble gas is absent from the inspired air but present in the airway lining fluid, the gas desorbs and is added to the inspired air. As a result, the partial pressure of these soluble gases in the breath is always less than that in the blood. Alcohol is also exchanged entirely within the conducting airways via diffusion from the bronchial circulation, not within the alveoli [34]. During expiration, about 20–50% of the alcohol released from the airways on inspiration is redeposited to the airways and reabsorbed by the airway circulation. The airway alcohol exchange process is largely limited by diffusion (airway tissue) and perfusion (bronchial circulation) [35]. In addition, breathing conditions, exhalation flow rate, and exhaled volume can all alter breath alcohol concentration by as much as 20% [34].

3.3 Gas Exchange in the Airway The theory of gas exchange from the bronchial circulation is supported by various animal models and human studies in congenital absence of a pulmonary artery. In physiological conditions, the bronchial circulation contributes little to oxygen and carbon dioxide exchange between the atmosphere and the blood. Since the bronchial blood flow is only a small fraction of total lung perfusion, its role in quantitative gas transfer is very small. Moreover, the partial pressures of oxygen (PaO2) and carbon dioxide (PaCO2) in arterial blood are equilibrated with alveolar gas pressures. With hypertrophy of the bronchial circulation in lung diseases, such as chronic pulmonary embolism and cystic fibrosis [31], the proportion of cardiac output delivered to the lung via the bronchial circulation can be increased manyfold. As a consequence, increased bronchial blood flow delivers arterial blood with high PaO2 and low PaCO2, providing a second opportunity to exchange gas in the affected portion of the lung. Although little or no oxygen uptake occurs except under conditions of systemic hypoxia, the recirculation of blood with increased PaCO2 augments the elimination of carbon dioxide. This notion is supported by studies on animal models in which 30% of carbon dioxide production can be eliminated through one lung via a hypertrophied bronchial circulation in sheep [32]. The lung exchanges, however, many more gases than simply the respiratory gases. Many nonrespiratory gases that are exchanged in the lung are soluble inert gases such as ethane, ether, toluene, acetone, and ethanol; conducting airways play an important role in the exchange of these highly

3.4 Autonomic and Nonautonomic Regulation of Bronchial Vasculature In animal studies, electrical nerve stimulation [36], close-arterial injection of adrenergic agonists [37], systemic adrenergic and cholinergic intravenous administration [38, 39], and inhalation of selective adrenergic and cholinergic agonists [40] have been used to assess autonomic control [41]. Although adrenergic and cholinergic nerves have been demonstrated in the airway wall [42, 43], pharmacology studies have demonstrated that the main nervous control of the bronchial circulation is provided by the sympathetic nervous system. Sympathetic (adrenergic) nerve fibers have been identified in close association with bronchial arteries [42, 43], whereas a-adrenergic receptors (predominantly a1 subtype) have been localized to the vascular smooth muscle. Sympathetic nerves release norepinephrine to cause vasoconstriction through the activation of a1-adrenoreceptors on airway vascular smooth muscle [36, 37, 40]. In contrast to the vasoconstriction induced by a-adrenergic receptor activation, b2-adrenergic agonists cause vasodilation and increase bronchial blood flow in sheep [44], a finding subsequently confirmed in humans [12, 45] (Fig. 4). In comparison with a-adrenergic receptors, b-receptors are less numerous and predominantly of the b2 subtype [46]. Although vessels of the airway mucosa appear to lack local cholinergic control, total bronchial blood flow is increased by vagal nerve (parasympathetic) stimulation [47] and by intra-arterial infusion of exogenous cholinergic agonists

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Fig. 4  The effects of the inhaled adrenergic agonists methoxamine (selective a1-adrenoceptor agonist) and albuterol (selective b2-adrenoceptor agonist) on airway mucosal blood flow (Qaw) in healthy (a) and asthmatic (b) subjects. Responses were measured with a soluble inert gas (dimethyl ether, DME) uptake method. Values are means ± the standard error. *p < 0.05 versus the baseline. (From Brieva et al. [45] with permission)

[39], whereas aerosolized acetylcholine has only minimal effects [48]. This suggests that airway mucosal blood flow is regulated independently of total bronchial blood flow as previously evidenced by demonstrating a different response of airway blood flow (decrease) and total bronchial blood flow (increase) to hypoxemia [49]. Autonomic nonadrenergic, noncholinergic mechanisms have also been described. Vasoactive intestinal polypeptide, polypeptide histidine isoleucine, and polypeptide histidine methionine released by parasympathetic nerves are vasodilators, whereas neuropeptide Y released by sympathetic nerves is a vasoconstrictor in the tracheobronchial circulation [36, 37]. Sensory neuropeptides, as substance P, neurokinin A, and calcitonin gene related peptide released from by unmyelinated sensory afferents induce vasodilation [41]. Nitrergic mechanisms have been reported to regulate bronchovascular tone and partially mediate b-adrenergic vasodilator responses [40, 50, 51]. Physicochemical factors including the mechanical effects of lung inflation, systemic blood pressure, pulmonary arterial and venous pressures, temperature, humidity, hypoxia, hypercarbia, alterations of acid–base balance, and changes of the airway surface liquid osmolarity have been reported to influence airway blood flow in different ways; however, controversies still exist, reflecting the need for further investigation in the field.

4  M  ethods of Measuring Bronchial Blood Flow Functional assessment of the bronchial circulation is technically difficult, as indicated by the large number of different techniques that have been developed to evaluate bronchial hemodynamics [52]. Bronchial blood flow is only a small

fraction of cardiac output, and vessels are located deep in the thorax adjacent to vital structures. As many reliable techniques are too invasive for human applications, most of the physiological data have been obtained in animal models, such as sheep and dogs [1], and isolated vessel preparations, whose relevance to humans may be uncertain [53]. In addition, most investigations have focused on the assessment of total bronchial blood flow; less is known about the circulation of the mucosal (subepithelial) tissue that plays major physiological and pathological roles in the airway. In summary, measurements of the bronchial hemodynamics remain a continuous challenge to the investigator. Berry and Daly [54] made the first measurements of bronchial blood flow in 1931 using isolated perfused dog lungs. Since that time, techniques have been used to measure the amount of blood that originates from the systemic circulation and returns through the pulmonary veins to the left atrium (referred to as bronchial systemic-to-pulmonary blood flow), total bronchial blood flow (i.e., blood returning to the right and the left atrium), and blood flow to the airway mucosa. The bronchial systemic-to-pulmonary blood flow can be estimated indirectly by subtracting right from left ventricular outputs measured simultaneously using oxygen uptake and Fick’s principle. The difference represents systemic-to-pulmonary shunting and was assumed to be bronchopulmonary anastomotic blood flow [55, 56]. In a different approach, in patients undergoing cardiopulmonary bypass surgery, when there is no pulmonary blood flow, the only blood flowing to the pulmonary circulation is the systemic-to-pulmonary bronchial blood flow [57]. The volume of blood returning to the left side of the heart was measured in the absence of pulmonary and coronary flows as an estimate of bronchial blood flow by placing a cannula in the left ventricle during bypass procedures. Measurements of the total bronchial arterial blood flow include the application of some form of flow probes to a vessel, or measurements involving the injections of markers or

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indicators of flow. These techniques involve the surgical isolation (through thoracotomy) of a small segment of the descending aorta to form an aortic pouch from which bronchial arteries arise or direct cannulation of the common bronchial artery [58], placement of electromagnetic [59] or ultrasonic [60] flow probes around the bronchial artery, combination of arteriography with the insertion of intravascular Doppler guide wires [61], video densitometry using contrast medium as the flow indicator [62], and the intravascular injection of radioactive or fluorescent microspheres [19]. These techniques require extensive surgical preparation, which can affect the measurements by changing the physiological conditions. Moreover, some of these require postmortem examination of the airways. More recently, less invasive techniques have become available that allow measurements of blood flow to the airway mucosa. With use of laser Doppler velocimetry, mucosal blood flow can be measured using a probe inserted through a fiberoptic bronchoscope and applying it to the membranous portion of the airway. The method measures microvascular perfusion; however, since it provides relative values of mucosal airway blood flow, it requires calibration by an independent method [63]. One of the non-invasive techniques developed is the measurement of airway blood flow with dimethyl ether (DME), a highly soluble inert gas. The technique allows calculation of airway blood flow in the mucosa (to a depth of 200 mm) from the steady-state uptake of DME from a defined airway segment [64]. Validation of the technique was accomplished by correlating the results to those obtained with microsphere injection [64]. Several studies in sheep [11, 44] and subsequently in humans [12, 45] have employed this technique to determine airway blood flow, and showed that mucosal blood flow can undergo large changes under certain conditions (e.g., thermal stress) [4, 30]. This noninvasive technique has also provided information on bronchovascular responses to inhaled drugs, such a-adrenergic and b-adrenergic agonists [45], and corticosteroids [65–67].

5 Clinical Aspects 5.1 The Bronchial Circulation in Lung Diseases The bronchial circulation plays an important role in lung defense and may contribute to the pathogenesis of several forms of lung disease [68]. Inflammation of the bronchial wall has been identified as a critical factor in the response to airway injury and infection. The microvasculature typically participates in tissue inflammation; hyperemia, endothelial hyperpermeability, and tissue edema, the three major

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manifestations of inflammation, have been demonstrated in the airway mucosal circulation [10, 53]. The increase in airway mucosal blood flow in the inflamed airway is generally believed to result from arterial vasodilatation and new vessel formation (i.e., angiogenesis). However, changes in the venomotor tone can also influence the local vascular response to inflammation. In addition to responses to acute inflammation, the airway circulation is known to proliferate in a variety of chronic pathological conditions, such as chronic inflammatory airway diseases [69], pulmonary artery occlusion [70], lung abscess [71], pulmonary tuberculosis [72], and lung tumors [73]. Chronic bronchovascular responses include growth of new blood vessels from existing ones (angiogenesis) and alterations of the existing blood vessels (microvascular remodeling).

5.1.1 Asthma As an airway disease characterized with continuous inflammation and repair processes simultaneously, asthma is known to lead to both functional (i.e., vasodilation, hyperperfusion, increased microvascular permeability, and edema formation) and structural (i.e., angiogenesis) changes in the airway vasculature [10, 53]. Among other functional consequences, these factors may contribute to a widespread narrowing of the air passages, and hence to an increased airflow resistance. Airway mucosal blood flow has been shown to be increased in patients with stable bronchial asthma [45, 65, 66, 74]. Measurements of airway blood flow with the uptake of DME showed that mean blood flow values were 24–77% higher in asthmatics than in healthy controls [45, 74]. Inhalation of allergen produces both immediate and latephase inflammatory responses with plasma extravasation from the airway microcirculation in patients with allergic asthma [75–77]. Plasma leakage is believed to occur through the formation of intercellular gaps between otherwise tightly associated endothelial cells in postcapillary venules [78, 79]. Extravasated plasma can collect in the airways, compromise epithelial integrity and ciliary function, and thereby contribute to excessive airway secretions to form luminal mucus plugs [80]. Plasma leakage can also lead to mucosal edema and bronchial wall swelling, which could reduce airway caliber and cause airflow limitation. As evidence of active angiogenesis, a number of studies have demonstrated increased vascularity (i.e., number and size of vessels, cross-sectional vascular area) of the bronchial mucosa in patients with asthma [81–85]. The cellular mechanisms leading to airway angiogenesis and its relative contribution to airway hyperperfusion still need to be clarified. A correlation between airway vascularity and airway hyperresponsiveness has also been found in asthmatic subjects [84, 86, 87]; whether a cause–effect relationship exists remains to be demonstrated.

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5.1.2  Chronic Bronchitis and Emphysema Early reports on the structure of the airway circulation in chronic obstructive pulmonary disease (COPD) suggested that intrapulmonary bronchial artery branches are obliterated or narrowed in emphysematous regions of the lungs and that the bronchial venous system is greatly expanded [88]. These findings have not been supported by recent studies that showed increased airway wall vascularity in stable COPD with a slight increase in the number of vessels in small airways, but to a much lesser degree than in asthma, where there is an increased vascularity throughout the bronchial tree [89, 90]. Airway mucosal blood flow rates have been shown to be not significantly higher [91] in patients with COPD. These findings are consistent with the morphological observations that, in contrast to asthma, angiogenesis in the airway wall does not appear to be a typical feature of stable COPD.

5.1.3 Bronchiectasis and Tuberculosis A marked proliferation and enlargement of the bronchial arteries accompanied by numerous precapillary anastomoses between the pulmonary and bronchial circulation has been described in patients with bronchiectasis and tuberculosis [68]. This vascular remodeling is a likely cause of hemoptysis encountered by patients with bronchiectasis. The primary stimuli for the development of bronchiectasis and airway vascular hypertrophy presumably are suppurative airway infections that typically are seen in these patients. In support of this assumption, chronic lung abscesses also have been shown to lead to a localized hypertrophy and hyperplasia of the airway circulation [71]. In tuberculosis, thrombosis of the pulmonary arteries supplying the infected area has been described [92]. The bronchial arteries surrounding the walls of the cavitary lesions are tortuous, distorted, and extensively ramified. Bronchial blood flow in patients with unilobar pulmonary tuberculosis was found to have increased to about 4% of the cardiac output.

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context of airway inflammation. This causes the formation of endothelial pores that are responsible for the leakage and extravasation of plasma proteins. A passive water movement due to an osmotic effect follows the release of these plasma proteins into the nearby connective tissue, contributing to edema formation in the airway mucosa and submucosa [94].

5.1.5 Tumors Primary bronchogenic carcinomas and bronchial adenomas derive a significant portion of their blood supply from the bronchial circulation. In contrast, metastatic carcinomas derive their blood supply principally from the pulmonary circulation. Thus, by postmortem bronchial arteriography, it has been demonstrated that “primary malignancies of the lung are uniformly accompanied by the development of a rich bronchial artery supply” and that “any bleeding which occurs is probably from bronchial arterial origin” [1, 68]. In contrast to the relatively high incidence of hemoptysis in bronchogenic carcinoma, the incidence is low in metastatic carcinoma, which lacks an expanded bronchial circulation [95]. In view of the finding of a bronchial blood supply to lung tumors, attempts have been made to infuse chemotherapeutic agents directly into the bronchial arteries supplying the tumors.

5.1.6  Tissue Repair After Lung Transplantation Bronchial anastomotic complications remain a significant cause of morbidity after lung transplantation. The lack of an adequate blood supply to the bronchus during the immediate postoperative period impairs bronchial anastomotic healing [96]. Postoperatively, the transplanted bronchus is deprived of its systemic blood supply, and depends on retrograde blood flow from bronchopulmonary collaterals [97]. Bronchial artery regeneration requires 2–4 weeks after lung transplantation. In animal models, bronchial mucosal blood flow has been shown to be significantly reduced during acute rejection after lung transplantation [98]. Thus, evaluation of bronchial blood flow as a surveillance tool in patients who have undergone lung transplantation has been suggested [61, 98].

5.1.4 Pulmonary Edema Pulmonary edema can result from fluid leakage from the pulmonary circulation due to hydrostatic forces, pulmonary vascular hyperpermeability, or both. The bronchial circulation plays a significant role in the formation and reabsorption of both hydrostatic and permeability edema [93]. Owing to the dual venous drainage of the bronchial circulation, an elevation of either the right or the left atrial pressure would be expected to promote extravasation of fluid from the bronchial circulation. Moreover, microvascular endothelial cells have the ability to contract when stimulated, especially in the

5.2 The Impact of Bronchial Vascular Changes 5.2.1 The Influence of Mucosal Thickness on Airway Caliber Since vessels occupy a significant portion of the inner airway wall, it is possible that the vascular component of inflammation increases mucosal thickness enough to increase airflow resistance. Airway vascular engorgement, submucosal

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edema, and luminal fluid accumulation have all been proposed to contribute to airway narrowing and enhanced airway responsiveness in asthma [99]. From the use of highresolution computed tomography (resolution of small morphologic structures down to 200 mm) to assess the short-term effects of intravascular volume expansion on airway caliber, a rapid, reversible bronchial mucosal vascular engorgement has been suggested to increase airway resistance evidenced in patients presenting with heart failure [100]. Supporting this theory, rapid infusion of intravenous fluids has been shown to cause airway obstruction [9]; however, others have not confirmed this finding [99]. Collectively, despite the numerous animal and human studies, the results are conflicting with respect to the effects of vascular congestion on airway caliber. There is less controversy on the effect of mucosal edema on airway caliber. Edema of the inner wall internal to the smooth muscle does at least contribute to airway hyperresponsiveness as the same degree of muscle shortening, causes greater luminal narrowing than in the normal airway.

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inflammatory substances (e.g., spasmogens) and decreasing the magnitude and duration of the effect of inhaled bronchoactive drugs [10, 53].

5.2.3  Airway Heat and Water Exchange Exercise-induced bronchoconstriction is a common condition in asthmatics. If the bronchovascular responsiveness to cooling and drying of air were of clinical importance, it would be expected to be most relevant in exercise-induced asthma [101]. Indeed, cold air, exercise, and hyperventilation have been shown to induce bronchoconstriction, and a role for the lowerairway vasculature in this response has been postulated [102]. Reactive hyperemia in response to airway cooling and increased airway liquid and interstitial osmolarity caused by water loss could have a role [4]; the former may narrow the airway, whereas the latter may promote inflammatory mediator release. The magnitude of the exercise-induced bronchoconstriction in asthmatics is largely determined by the level of ventilation during exercise and the temperature and humidity of the air being inspired. Hyperventilation, and low temperature and water content exacerbate the severity of the obstruction [28].

5.2.2 Drug Absorption and Metabolism (Drug Distribution Within the Lung) 5.2.4 Mucociliary Clearance As the principal source of blood to the conducting airways, the airway circulation can influence the metabolism and clearance of biologically active, locally released molecules, the distribution of systemically administered drugs to the airways, and the absorption of inhaled pharmacological agents from the airway. Therefore, alteration of airway perfusion may have important consequences in terms of the therapeutic effect of aerosol medications in the airway [24]. Pharmacological agents that enhance airway blood flow (i.e., vasodilators) may be rapidly cleared from the airway lumen, thereby producing local effects of short duration compared with similar agents that lack a vascular effect. Conversely, augmenting airway blood flow may improve the distribution of systemically administrated pharmacological agents to the airway. Theoretically, an inhaled vasodilator may enhance the effect of a systemically administered drug by increasing its effective delivery to its final target, i.e., the airway [20]. Furthermore, combining an inhaled airway drug with a vasoconstrictor could enhance and prolong the local action of the airway drug. In support of this theory, the magnitude and duration of the airway smooth muscle response to an aerosol antigen challenge in sheep could be increased or decreased with pharmacological agents that decrease or increase the airway circulation, respectively [6, 8]. In relation to airway inflammation, it has been suggested that an inflammatory increase in airway blood flow could favor the recruitment of inflammatory cells to the airway wall while increasing the clearance of locally released

The role of tracheobronchial circulation in supporting the mucociliary apparatus has been suggested by animal models and studies on lung transplantation patients; however, is still incompletely understood. For example, mucociliary clearance impairment has been confirmed in the immediate postoperative period after lung transplantation [103]. Furthermore, the influence of the bronchial circulation on mucociliary transport of insoluble particles has been confirmed in sheep, as the clearance of small insoluble radiolabeled aerosol particles from the airway was impaired when bronchial blood flow was stopped [104].

5.2.5 Airway Blood Flow as a Noninvasive Marker of Inflammation There is an ongoing search for markers of airway inflammation, which can be assessed independent of lung function as an index of disease severity and to quantitate the response to anti-inflammatory therapy in patients with asthma [105]. Several invasive and noninvasive methods have been used, including the analysis of cellular and acellular components of induced sputum and bronchoalveolar lavage fluid, histological examination of bronchial biopsy specimens, assays of inflammatory serum markers, measurements of the concentrations of exhaled gases (e.g., NO), and inflammatory mediators in exhaled breath condensate. With the advent of a noninvasive method to measure airway blood flow [11, 74],

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airway hyperperfusion could be an important biomarker of asthma-associated inflammation. Reported reduction of airway blood flow by anti-inflammatory therapy in asthmatics lends support to this concept [65, 106].

5.3 The Effects of Inhaled Drugs on the Bronchial Vasculature 5.3.1 Inhaled Bronchodilators Adrenergic agonists have a profound effect on the airway mucosal vasculature. Inhaled b2-adrenergic agonists, the most widely used bronchodilators in obstructive airway disease, cause vasodilation and increase airway mucosal blood flow [45, 91, 107]. However, although the blood flow response is dose-dependent in healthy subjects, the vasodilator effect of inhaled albuterol, a b2-adrenergic agonist, is blunted in asthmatics (Fig. 4) [45]. Similar observations have been made in smokers and patients with COPD [91, 108]. The mechanisms for the blunted airway vascular response in patients with airway disease compared with normal subjects remain unknown. In contrast to b2-adrenergic agonists, inhaled a-adrenergic agonists cause vasoconstriction and decrease bronchial blood flow [45], interestingly with a greater response in asthmatics than in healthy subjects (Fig. 4). However, these latter drugs have no indication in the inhalation therapy of airway disease. Inhaled cholinergic antagonists have been shown to lack a noticeable effect on airway blood flow in various animal models, indicating an insignificant cholinergic (vagal) tone in the airway vasculature [39, 109, 110].

5.3.2  Corticosteroids Inhaled corticosteroids, the most effective drugs to suppress airway inflammation in asthma, have complex rapid (nongenomic) and delayed (genomic) actions on the airway vasculature [10]. They have been shown to counteract all the vascular manifestations of airway inflammation (i.e., increased blood flow and permeability, edema formation, angiogenesis) by inhibiting the release of vasoactive inflammatory mediators or acting directly on vascular smooth muscle or the endothelium. Among these effects, inhaled corticosteroids acutely (within 30  min) reduce airway mucosal blood flow both in healthy and in asthmatic subjects [66, 111, 112]. Although the mechanism has still not been fully explored, recent studies suggest that corticosteroids are likely to facilitate sympathetic (noradrenergic) signal transmission by inhibiting extraneuronal catecholamine uptake to induce neurogenic vasoconstriction [113, 114].

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In addition to vasoconstriction, a number of studies have demonstrated that inhaled corticosteroid therapy cause a long-lasting decrease in airway mucosal blood flow in patients with asthma. In a cross-sectional study, airway mucosal blood flow was higher by about 23% in corticosteroid-naive than in corticosteroid-treated asthmatics [74]. In interventional studies, inhaled fluticasone propionate (440  mg/day) therapy for 2  weeks reduced blood flow by about 12 and 21% in two groups of asthma patients [65, 106], and additionally showed that airway blood returned to the pretreatment level 2 weeks after cessation of therapy. Inhaled corticosteroids also have a potential therapeutic role of reversing airway angiogenesis in asthma. In a crosssectional study, therapy with high doses (800  mg or more per day) of beclomethasone was associated with a reduced vascular area of the lamina propria [84]. In interventional studies, beclomethasone (800 mg/day) therapy for 6 months reduced the number of blood vessels and the vascular area [115], whereas a 6-week treatment with fluticasone was effective at high inhaled dose (1,000 mg/day) in reducing the number of blood vessels and the vascular area [116]. Collectively, inhaled corticosteroids seem to reverse both functional and structural abnormalities of the airway vasculature in asthma. Although the respective roles of these actions are still incompletely understood, they are expected to have significant diagnostic and therapeutic implications.

5.3.3  Drug Interactions The airway mucosal circulation plays a major role in the clearance of inhaled drugs from the respiratory tract. The corticosteroid-induced decrease in airway blood flow is likely to enhance the action of the simultaneously inhaled bronchodilators by diminishing their clearance from the airway. Although the underlying mechanism has not yet been verified directly, recent studies support the rapid interaction of inhaled bronchodilators and corticosteroids in patients with COPD [117] and bronchial asthma [118]. Since the corticosteroidinduced vasoconstriction peaks rapidly (about 30  min after drug inhalation), simultaneous administration of inhaled corticosteroids and bronchodilators is likely to be of clinical significance.

6  Summary The mucosal vessels in the airways are ideally located to perform a number of functions, including nourishing the bronchi, heating and humidifying the inspired air, and distributing and clearing mediators or drugs to and from the airway, and are likely to play an important role in lung defense and in the

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pathogenesis of a number of airway diseases. The airway circulation may act as a capacitance system that could alter airway wall thickness, and therefore airway caliber over short periods of time under the influence of a variety of factors. Bronchial capillaries can hypertrophy, regenerate, and form new vessels very rapidly, by a process called angiogenesis. However, the anatomy, physiological function, and biological relevance of the bronchial circulation are still under active investigation. Although the role of the pulmonary circulation in lung disease has been extensively studied and well characterized, the same cannot be said for the bronchial circulation. It is technically difficult to assess the function of the airway circulation in intact humans, and this has limited the amount of information on the airway vasculature in airway disease. The anatomical development of a collateral circulation between the bronchial and the pulmonary circulation after chronic obstruction of the pulmonary arterial system is well recognized. Soon after pulmonary arterial occlusion, bronchial arteries have been shown first to dilate and subsequently to proliferate, vicariously providing nutrients to the lung and contributing to gas exchange. Despite the growing appreciation of the changes of the bronchial vasculature in inflammatory airway diseases, there remain significant gaps in our understanding of their pathophysiological roles in asthma and COPD, including disease severity and persistence. In particular, specific data on transudative and exudative processes and inflammatory cell diapedesis in the airway microvasculature are scarce. Until recently, it has not been possible to quantitate tracheobronchial blood flow noninvasively in humans, precluding the obtaining of information on the hemodynamic effects of many pharmacological agents and their clinical consequences. This is likely to change with the advent of new noninvasive methods, and new drugs with therapeutically beneficial vascular effects may emerge in the future.

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450 76. Svensson C, Grönneberg R, Andersson M et  al (1995) Allergen challenge-induced entry of a2-macroglobulin and tryptase into human nasal and bronchial airways. J Allergy Clin Immunol 96:239–246 77. Greiff L, Andersson M, Erjefält JS, Persson CG, Wollmer P (2003) Airway microvascular extravasation and luminal entry of plasma. Clin Physiol Funct Imaging 23:301–306 78. McDonald DM (1994) Endothelial gaps and permeability of venules in rat tracheas exposed to inflammatory stimuli. Am J Physiol 266:L61–L83 79. Baluk P, Bolton P, Hirata A, Thurston G, McDonald DM (1998) Endothelial gaps and adherent leukocytes in allergen-induced early- and late-phase plasma leakage in rat airways. Am J Pathol 152:1463–1476 80. Goldie RG, Pedersen KE (1995) Mechanisms of increased airway microvascular permeability: role in airway inflammation and obstruction. Clin Exp Pharmacol Physiol 22:387–396 81. Vrugt B, Wilson S, Bron A, Holgate ST, Djukanovic R, Aalbers R (2000) Bronchial angiogenesis in severe glucocorticoid-dependent asthma. Eur Respir J 15:1014–1021 82. Carroll NG, Cooke C, James AL (1997) Bronchial blood vessel dimensions in asthma. Am J Respir Crit Care Med 155:689–695 83. Li X, Wilson JW (1997) Increased vascularity of the bronchial mucosa in mild asthma. Am J Respir Crit Care Med 156:229–233 84. Orsida BE, Li X, Hickey B, Thien F, Wilson JW, Walters EH (1999) Vascularity in asthmatic airways: relation to inhaled steroid dose. Thorax 54:289–295 85. Tanaka H, Yamada G, Saikai T et  al (2003) Increased airway vascularity in newly diagnosed asthma using a high-magnification bronchovideoscope. Am J Respir Crit Care Med 168: 1495–1499 86. Hoshino M, Nakamura Y, Hamid QA (2001) Gene expression of vascular endothelial growth factor and its receptors and angiogenesis in bronchial asthma. J Allergy Clin Immunol 107:1034–1038 87. Hoshino M, Takahashi M, Aoike N (2001) Expression of vascular endothelial growth factor, basic fibroblast growth factor, and angiogenin immunoreactivity in asthmatic airways and its relationship to angiogenesis. J Allergy Clin Immunol 107:295–301 88. Cudkowicz L, Armstrong JB (1953) The bronchial arteries in pulmonary emphysema. Thorax 8:46–58 89. Hashimoto M, Tanaka H, Abe S (2005) Quantitative analysis of bronchial wall vascularity in the medium and small airways of patients with asthma and COPD. Chest 127:965–972 90. Jeffery PK (2004) Remodeling and inflammation of bronchi in asthma and chronic obstructive pulmonary disease. Proc Am Thorac Soc 1:176–183 91. Mendes ES, Campos MA, Wanner A (2006) Airway blood flow reactivity in healthy smokers and in ex-smokers with or without COPD. Chest 129:893–898 92. Cudkowicz L (1952) The blood supply of the lung in pulmonary tuberculosis. Thorax 7:270–276 93. Baier H (1986) Functional adaptations of the bronchial circulation. Lung 164:247–257 94. Dinh Xuan AT (1990) Bronchial blood flow and microvascular permeability in the pathophysiology of asthma. Med Hypotheses 32:207–209 95. Fishman AP (1961) The clinical significance of the pulmonary collateral circulation. Circulation 24:677–690 96. Mills NL, Boyd AD, Gheranpong C (1970) The significance of bronchial circulation in lung transplantation. J Thorac Cardiovasc Surg 60:866–878 97. Zheng L, Orsida BE, Ward C et al (1999) Airway vascular changes in lung allograft recipients. J Heart Lung Transplant 18:231–238

G. Horvath and A. Wanner 98. Takao M, Katayama Y, Onoda K et  al (1991) Significance of bronchial mucosal blood flow for the monitoring of acute rejection in lung transplantation. J Heart Lung Transplant 10:956–966 99. Blosser S, Mitzner W, Wagner EM (1994) Effects of increased bronchial blood flow on airway morphometry, resistance, and reactivity. J Appl Physiol 76:1624–1629 100. Wetzel RC, Herold CJ, Zerhouni EA, Robotham JL (1993) Intravascular volume loading reversibly decreases airway crosssectional area. Chest 103:865–870 101. Solway J (1992) Respiratory air conditioning and the bronchial circulation. In: Butler J (ed) The bronchial circulation. Dekker, New York, pp 291–336 102. McFadden ER Jr (1990) Hypothesis: exercise-induced asthma as a vascular phenomenon. Lancet 335:880–883 103. Paul A, Marelli D, Shennib H et al (1989) Mucociliary function in autotransplanted, allotransplanted, and sleeve resected lungs. J Thorac Cardiovasc Surg 98:523–528 104. Wagner EM, Foster WM (1996) Importance of airway blood flow on particle clearance from the lung. J Appl Physiol 81:1878–1883 105. Kharitonov SA, Barnes PJ (2004) Effects of corticosteroids on noninvasive biomarkers of inflammation in asthma and chronic obstructive pulmonary disease. Proc Am Thorac Soc 1:191–199 106. Mendes ES, Campos MA, Hurtado A, Wanner A (2004) Effect of montelukast and fluticasone propionate on airway mucosal blood flow in asthma. Am J Respir Crit Care Med 169:1131–1134 107. Pereira A, Mendes E, Ferreira T, Wanner A (2003) Effect of inhaled racemic and (R)-albuterol on airway vascular smooth muscle tone in healthy and asthmatic subjects. Lung 181:201–211 108. Mendes ES, Horvath G, Rebolledo P, Monzon ME, CasalinoMatsuda SM, Wanner A (2008) Effect of an inhaled glucocorticoid on endothelial function in healthy smokers. J Appl Physiol 105:54–57 109. McIlveen S, White S, Parsons G (1997) Autonomic control of bronchial circulation in awake sheep during rest and behaviour. Clin Exp Pharmacol Physiol 24:940–947 110. Matran R, Alving K, Lundberg JM (1991) Differential bronchial and pulmonary vascular responses to vagal stimulation in the pig. Acta Physiol Scand 143:387–393 111. Clarke GW, Rafferty GF, Ledbetter CL et al (2004) The effects of inhaled spasmogens on airway mucosal blood flow in asthmatic subjects. Am J Respir Crit Care Med 169:A185 112. Paredi P, Kharitonov SA, Barnes PJ (2005) Correlation of exhaled breath temperature with bronchial blood flow in asthma. Respir Res 6:15 113. Horvath G, Lieb T, Conner GE, Salathe M, Wanner A (2001) Steroid sensitivity of norepinephrine uptake by human bronchial arterial and rabbit aortic smooth muscle cells. Am J Respir Cell Mol Biol 25:500–506 114. Horvath G, Sutto Z, Torbati A, Conner GE, Salathe M, Wanner A (2003) Norepinephrine transport by the extraneuronal monoamine transporter in human bronchial arterial smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 285:L829–L837 115. Hoshino M, Takahashi M, Takai Y, Sim J, Aoike N (2001) Inhaled corticosteroids decrease vascularity of the bronchial mucosa in patients with asthma. Clin Exper Allergy 31:722–730 116. Chetta A, Zanini A, Foresi A et al (2003) Vascular component of airway remodeling in asthma is reduced by high dose of fluticasone. Am J Respir Crit Care Med 167:751–757 117. Cazzola M, Santus P, Di Marco F et al (2004) Onset of action of formoterol/budesonide in single inhaler vs. formoterol in patients with COPD. Pulm Pharmacol Ther 17:121–125 118. Mendes ES, Horvath G, Campos M, Wanner A (2008) Rapid corticosteroid effect on b2-adrenergic airway and airway vascular reactivity in patients with mild asthma. J Allergy Clin Immunol 121:700–704

Part II

Methodological Approaches for Research

Chapter 30

Animal Models of Pulmonary Hypertension Jocelyn Dupuis and Norbert Weissmann

Abstract  A variety of animal models have been used to study the pathobiological mechanisms of or to test new treatment strategies for pulmonary hypertension (PH). The most commonly utilized animal models of PH are the monocrotaline injury model in rats and the chronic hypoxia model in different species. Besides their wide availability and welldescribed histopathological characteristics, these models have been popular because of their good reproducibility, allowing for the evaluation of various therapeutic strategies or the detection of pathological principles underlying PH. With the constantly progressing knowledge of the pathophysiological mechanisms of PH, newer models are being developed in an attempt to more accurately reproduce the human pathobiological processes of the different forms of the disease. Animal models can provide information on the pathophysiological mechanisms of PH and on the potential of new therapeutic approaches. In general, it is recognized that these models suffer from various limitations, do not always reproduce human PH, and, as for all animal models, the question arises whether findings made in the animal model can be transferred to the human disease. However, new therapeutic strategies which have successfully been used as a curative approach in different animal models of PH have subsequently proven to be useful in human PH. With these limitations in mind, this chapter gives an overview of the various animal models of PH according to the pathophysiology-based human classification of PH and delineate some salient characteristics of these models. Keywords  Monocrotaline • Endothelium injury • Chronic hypoxia • Animal models • Histology • Vascular remodeling

1  Introduction A variety of animal models have been used to study the pathobiological mechanisms of or to test new treatment strategies for pulmonary hypertension (PH). The most ­commonly J. Dupuis (*) Department of Medicine, Montreal Heart Insitute, 5000 Belanger est., Montreal, QC H1T 1C8, Canada e-mail: [email protected]; [email protected]

utilized animal models of PH are the ­monocrotaline injury model in rats and the chronic hypoxia model in different ­species. Besides their wide availability and well-described ­histopathological characteristics, these models have been popular because of their good reproducibility, allowing for the evaluation of various therapeutic strategies or the detection of pathological principles underlying PH. With the constantly progressing knowledge of the pathophysiological mechanisms of PH, newer models are being developed in an attempt to more accurately reproduce the human pathobiological processes of the different forms of the disease. Some of these incorporate a “multiple hit” approach by combining more than one intervention, be it a toxic agent, selective over expression, or selective underexpression of biologic pathways or modifications of normal hemodynamics. With the recent sequencing of human and animal genomes and the availability of microarray technology, newer models developed form potential candidate genes are to be expected. Animal models can provide information on the pathophysiological mechanisms of PH and on the potential of new therapeutic approaches. In general, it is recognized that these models suffer from various limitations, do not always reproduce human PH, and, as for all animal models, the question arises whether findings made in the animal model can be transferred to the human disease. However, new therapeutic strategies which have successfully been used as a curative approach in different animal models of PH have subsequently proven to be useful in human PH. With these limitations in mind, we will attempt to give an overview of the various animal models of PH according to the pathophysiology-based human classification of PH and delineate some salient ­characteristics of these models.

2 The Animal Model of MonocrotalineInduced PH Monocrotaline is a toxic pyrrolizidine alkaloid agent ­present in the plant Crotalaria spectabilis. Ingestion of

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­ onocrotaline results in the progressive development of m PH in some animal species and was described after repeated oral ­ingestion in laboratory rats 40 year ago [1]. The injury is, however, attainable with only one single subcutaneous or intraperitoneal injection of the drug [2], making this a technically appealing and readily accessible model. However, the damage is variable between species; not all species develop PH after monocrotaline application. This is suggested to be in great part because the toxin requires hepatic metabolism of monocrotaline by cytochrome P450 to pyrrolic metabolites [3, 4]. Although the model has been used in different species, the preferred species is the rat. The model has also been used in larger mammals such as dogs by directly injecting­ the toxic metabolite dehydromonocrotaline [5]. Although the exact mechanism of how PH is triggered in the monocrotaline model is not known, initial endothelial injury is thought to be a key step in this process. Monocrotaline pyrrole can cause direct endothelial damage [6]. There is ­evidence of dysregulation of the bone morphogenetic protein signaling pathway in monocrotaline-induced PH with impairment of the transforming growth factor-b/ Smad2,3 signaling axis, lending support to their contribution to the development of pulmonary arterial hypertension (PAH) [7, 8]. Although lung damage is predominant presumably because the lung is the first downstream target organ of the hepatic metabolite, the toxin is also responsible for some liver and kidney ­damage [9]. The temporal evolution of hemodynamic and histopathological modifications following monocrotaline pyrrole injury is well characterized [2, 4, 10–13]. In the hours following administration, there is inhibition of endothelial cell proliferation followed by vacuolization, vascular leak, and pulmonary edema. Adventitial inflammation is also present. Extension of smooth muscle cells into normally nonmuscularized pulmonary arterioles occurs within approximately 1 week, followed by medial hypertrophy of muscular arteries. In rats, PH is present after approximately 14 days and its severity continually progresses in parallel with progression of medial hypertrophy with eventual loss of pulmonary perfusion (Fig. 1). If left untreated, rats with monocrotaline-induced PH develop severe PAH with right ventricular systolic pressures reaching 80 mmHg after 5 weeks, and a low survival rate of 35% [6]. The severity of this model and the rapid progression of the disease process are comparable to those of human PAH (equivalent to New York Heart Association classes III–IV) and makes it an attractive model to test therapeutic agents or strategies. By itself, however, the monocrotaline injury model of PAH lacks the intimal proliferation found in human idiopathic PAH. Interestingly, however, combination of monocrotaline injection with another trigger of PH can ­generate the intimal proliferation lesions, suggesting that

J. Dupuis and N. Weissmann

Fig.  1  Comparison of histopathological changes in different animal models of pulmonary hypertension compared with human pulmonary hypertension. (a) Human idiopathic pulmonary arterial hypertension. (b) Hypoxia-induced pulmonary hypertension in mice. Mice were exposed to normobaric chronic hypoxia (10% O2). (c) Monocrotalineinduced pulmonary hypertension in rats 5 weeks after a single monocrotaline injection. (d) Pulmonary hypertension in rats 5 weeks after combined application of hypoxia (normobaric, 10% O2) and the ­vascular endothelial growth factor receptor antagonist SU5416. The images were taken after a double staining against von Willebrand factor (brown) and a-smooth muscle actin (violet)

alterations of other pathways are needed. This has been achieved by concomitantly increasing pulmonary flow either by contralateral pneumectomy or by subclavian artery to pulmonary artery shunting [14, 15]. Intimal proliferation has also been demonstrated in endothelin B receptor deficient rats after monocrotaline injury [16].

30  Animal Models of Pulmonary Hypertension

3 The Animal Model of Chronic HypoxiaInduced Pulmonary Hypertension Normobaric and hypobaric hypoxia are also frequently used to induce PAH. This model is very predictable and reproducible, with some variability between species, and is also affected by age, as younger individuals with maturing lungs are more susceptible to this trigger [17–20]. However, in contrast to the monocrotaline model, hypoxiainduced PH in almost all mammals investigated so far showed quite identical structural changes (Fig. 1). The model is generally used in rodents with hypoxic exposure for 2 weeks or more. Following the initial hypoxic vasoconstriction with reactive PH, pulmonary structural changes are rapidly evident and progressive [21]. Muscularization of small normally nonmuscular arterioles and alveolar wall starts after only 3 days of exposure, with subsequent increased medial hypertrophy of the muscular vessels. Whether isolated chronic hypoxia effectively results in the loss of pulmonary circulation is currently a subject of debate and the interested reader is referred to a recent article exposing the pros and cons on this topic [22]. After 2 weeks of hypoxia, rats develop moderate PH [21] that ­correlates with the progression of structural changes. As is the case for the monocrotaline model, there is no neointimal­ proliferation in the hypoxia model of PH. The severity of the structural modifications in hypoxic lungs is influenced by hemodynamics as variations of blood flow by pulmonary artery banding can modulate the changes [23]. Importantly, pharmacologic inhibition of the VEGF receptor 2 with combined chronic hypoxia has resulted in severe PAH with the added histological finding of intimal ­proliferation [24, 25]. This again exemplifies in animal models the importance of multiple triggers in the generation of PAH. The chronic hypoxic model of PH can be regarded as a model for less severe PH (equivalent to New York Heart Association class II) and has relevance to human PH associated with hypoxia, as it occurs in pulmonary parenchymal disease, sleep-disordered breathing, severe COPD, and residence at high altitude. Besides the monocrotaline and the hypoxia model, a variety of different but less frequently used models exist.

4 Pulmonary Hypertension Induced by Left-to-Right Shunt Surgical creation of a left-to-right shunt to mimic some congenital heart diseases causes an increase in pulmonary blood flow, leading to PAH. The preferred approach is to create an

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aortopulmonary shunt in piglets, although the procedure can also be performed in lambs in utero or in rodents with an abdominal aortocaval fistula [26–28]. Three months after the procedure, piglets develop moderate PAH with medial hypertrophy. There is no intimal proliferation at this early stage, although it has been described in animals kept for a longer period of up to 4 years. This model has the advantage of reproducing the exact pathophysiological process of left-toright shunting, but has the limitations inherent in a more sophisticated surgical model necessitating many months to produce pathological changes.

5 Pulmonary Hypertension of the Newborn Persistent PAH of the newborn can be created by surgical animal models by in utero ligation of the ligamentus arteriosus or by creation of a diaphragmatic hernia [29, 30].

6 Pulmonary Embolism To Induce Pulmonary Hypertension Multiple pulmonary emboli with subsequent development of PAH can be accomplished by the intravenous injection of microspheres, of blood clots, or by air or fat embolism. These models have generally been used for the study of acute ­pulmonary embolism but have also been utilized for the study of chronic PH. Surgical ligation of lung lobes can also be used to study postobstructive pulmonary vasculopathy changes.

7 Genetic Models of Pulmonary Hypertension There are now numerous animal models that confer a genetic predisposition to the development of PH. These models can be used alone or in combination with other triggers of PH. Fawnhooded rats have a genetically determined deficient platelet uptake of serotonin, leading to PH after 4 weeks of life. The defect, which resembles that induced by ­dex­fenfluramine, causes lung vascular remodeling without intimal proliferation and is accentuated by exposure to hypoxia [31–33]. Recently a contribution of mitochondrial dysfunction has been suggested in this model. In the past few years, numerous transgenic animal models have been evaluated. These models exemplify the apparent complexity and the probable importance of numerous ­pathways in the development of PH, including genetic ­predispositions.

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They are helpful in our understanding of the interactions between these pathways. For example bone morphogenetic protein receptor 2 (BMPR2) heterozygous mice do not develop PH in room air or in hypoxic conditions, but have increased susceptibility to hypoxic PH when additionally infused with serotonin [34]. A dominant-negative transgenic model of BMPR2 mutation with specific smooth muscle repression of the gene after birth however developed PH in room air, thus linking for the first time a single human genetic defect to an animal model of PH [35]. Transgenic animals with dysregulation of serotonin synthesis and of serotonin receptors, adrenomedullin, prostacyclin, and nitric oxide synthase have also demonstrated their predisposition to or relative protection against the development of PH as do those with genetic deletion of the hypoxia-inducible transcription factors [36–41].

8 Models of Pulmonary Hypertension with Other Conditions Although they have not classically been used primarily to study the pulmonary circulation, there are numerous animal models that are associated with the development of PH. With the recent introduction of oral therapies for the treatment of PH and preliminary data demonstrating their effectiveness in PH associated with other conditions, we can expect that investigators will increasingly use these models and primarily focus on the pulmonary circulation and PH. There are many models of PH associated with left-sided heart disease. The most frequently used model is the myocardial infarction model in rats by left anterior descending coronary artery ligation. Although left ventricular function has primarily been the focus of this model, animals with moderate to large infarcts develop lung structural remodeling with PH and right ventricular hypertrophy [42–44]. Other heart-failure models include the overdrive pacing model, usually performed in dogs, as well as models of inherited cardiomyopathy in rodents. There are also numerous models of lung parenchymal disease that are associated with the development of PH. These are usually done in rats or mice. They include models of lung fibrosis such as the bleomycin injury model [45] and models of chronic airway disease such as the elastase emphysema model [46] or models of smoke-induced emphysema [47]. These models are generally used to evaluate lung function and airway disease. They, however, will probably be more frequently used in the future to more specifically focus on the pulmonary circulation. Moreover, it has been shown that animals exposed to tobacco smoke inhalation for several months to mimic COPD can develop PH.

J. Dupuis and N. Weissmann

9 The Value of Animal Models for Investigation of Pulmonary Hypertension Animal models can provide invaluable insight into the numerous possible pathways that contribute to the ­develop­ment and maintenance of PH, but especially into the important ­interactions between these various triggers and their temporal­ evolution in the disease process. Researchers must not be disenchanted by occasional conflicting results that could suggest a lack of implication of a specific pathway in the pathogenesis of PH. For example, on the basis of transgenic mice overexpressing endothelin-1 (ET-1) in their lungs but that do not develop PH but develop perivascular inflammation and fibrosis [48], one would unfortunately be tempted to conclude that ET-1 does not contribute to PH. The monocrotaline model of PH would further support this assumption since these animals have reduced ET-1 expression in their lungs [49]. Nevertheless, administration of endothelin receptor antagonist has been demonstrated to be highly effective in this model as well as in many other models of PH, and these drugs have proven their effectiveness in randomized clinical studies, leading to the approval of this new class of agents for the therapy of PH. Conversely, some therapies were demonstrated to be effective in animal models of PH with no evidence of efficacy in human. On the other hand (1) identification of pathophysiological processes that lead to the development of PH in ­animal models and (2) successful treatment of PH in animal models have recently led to new therapeutic concepts to treat PH in humans, demonstrating the value of animal models. We, however, have to be aware that there is no single ­perfect animal model covering all forms of PH in humans. For example, chronic hypoxia results in similar pathological findings in most mammals, including humans, with only a difference in the severity of the disease in different species. Thus, such models perfectly mimic hypoxia-induced PH in humans, whereas there is no ideal model mimicking other forms of PH (e.g., idiopathic PAH). In this regard, however, one has to bear in mind that PH in humans is also very diverse and can be caused by different triggers even within one ­category, like idiopathic PAH (Fig. 2). In addition to the lack of a perfect animal model reproducing each subtype of human PH, there are inherent differences between species that are still incompletely understood. For a given biologic pathway, the relative importance in a specific animal strain could be influenced, for example, not only by the disease state but also by age, gender, environment, and counterregulatory modifications that may differ from those in humans. With regard to proof-of-principle studies for evaluation of new therapeutic approaches to treat PH, one should consider starting treatment only after PH has been established in an

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Fig. 2  Assignment of different animal models of pulmonary hypertension to the classification of human pulmonary hypertension

animal model as interference with the induction of the ­disease may lead to false-positive results. It is the authors’ opinion that if a therapeutic approach performed in a curative fashion is successful in different models of PH, such as monocrotaline and chronic hypoxia, there is likelihood that this can be translated to human PH. Nevertheless we believe that there should be continued efforts aiming at the development of animal models that specifically mimic the respective forms of human PH.

References 1. Kay JM, Harris P, Heath D (1967) Pulmonary hypertension produced in rats by ingestion of Crotalaria spectabilis seeds. Thorax 22:176–179 2. Hilliker KS, Bell TG, Roth RA (1982) Pneumotoxicity and ­thrombocytopenia after single injection of monocrotaline. Am J Physiol 242:H573–H579 3. Reindel JF, Ganey PE, Wagner JG, Slocombe RF, Roth RA (1990) Development of morphologic, hemodynamic, and biochemical changes in lungs of rats given monocrotaline pyrrole. Toxicol Appl Pharmacol 106:179–200 4. Roth RA, Reindel JF (1991) Lung vascular injury from monocrotaline pyrrole, a putative hepatic metabolite. Adv Exp Med Biol 283:477–487 5. Okada M, Yamashita C, Okada K (1995) Establishment of canine pulmonary hypertension with dehydromonocrotaline. Importance of larger animal model for lung transplantation. Transplantation 60:9–13

6. Jasmin JF, Lucas M, Cernacek P, Dupuis J (2001) Effectiveness of a nonselective ETA/B and a selective ETA antagonist in rats with monocrotaline-induced pulmonary hypertension. Circulation 103:314–318 7. Zakrzewicz A, Kouri FM, Nejman B et al (2007) The transforming growth factor-beta/Smad2,3 signalling axis is impaired in experimental pulmonary hypertension. Eur Respir J 29:1094–1104 8. Morty RE, Nejman B, Kwapiszewska G et al (2007) Dysregulated bone morphogenetic protein signaling in monocrotaline-induced pulmonary arterial hypertension. Arterioscler Thromb Vasc Biol 27:1072–1078 9. Roth RA, Dotzlaf LA, Baranyi B, Kuo CH, Hook JB (1981) Effect of monocrotaline ingestion on liver, kidney, and lung of rats. Toxicol Appl Pharmacol 60:193–203 10. Meyrick B, Gamble W, Reid L (1980) Development of Crotalaria pulmonary hypertension: hemodynamic and structural study. Am J Physiol 239:H692–H702 11. Ghodsi F, Will JA (1981) Changes in pulmonary structure and function induced by monocrotaline intoxication. Am J Physiol 240:H149–H155 12. Lappin PB, Ross KL, King LE, Fraker PJ, Roth RA (1998) The response of pulmonary vascular endothelial cells to monocrotaline pyrrole: cell proliferation and DNA synthesis in vitro and in vivo. Toxicol Appl Pharmacol 150:37–48 13. Lappin PB, Roth RA (1997) Hypertrophy and prolonged DNA ­synthesis in smooth muscle cells characterize pulmonary arterial wall thickening after monocrotaline pyrrole administration to rats. Toxicol Pathol 25:372–380 14. Tanaka Y, Schuster DP, Davis EC, Patterson GA, Botney MD (1996) The role of vascular injury and hemodynamics in rat pulmonary artery remodeling. J Clin Invest 98:434–442 15. Okada K, Tanaka Y, Bernstein M, Zhang W, Patterson GA, Botney MD (1997) Pulmonary hemodynamics modify the rat pulmonary artery response to injury. A neointimal model of pulmonary hypertension. Am J Pathol 151:1019–1025 16. Ivy DD, McMurtry IF, Colvin K et al (2005) Development of occlusive neointimal lesions in distal pulmonary arteries of endothelin B

458 receptor-deficient rats: a new model of severe pulmonary arterial hypertension. Circulation 111:2988–2996 17. Meyrick B, Reid L (1982) Normal postnatal development of the media of the rat hilar pulmonary artery and its remodeling by chronic hypoxia. Lab Invest 46:505–514 18. Meyrick B, Reid L (1981) The effect of chronic hypoxia on pulmonary arteries in young rats. Exp Lung Res 2:257–271 19. Rabinovitch M, Gamble WJ, Miettinen OS, Reid L (1981) Age and sex influence on pulmonary hypertension of chronic hypoxia and on recovery. Am J Physiol 240:H62–H72 20. Stenmark KR, Fagan KA, Frid MG (2006) Hypoxia-induced pulmonary vascular remodeling: cellular and molecular mechanisms. Circ Res 99:675–691 21. Rabinovitch M, Gamble W, Nadas AS, Miettinen OS, Reid L (1979) Rat pulmonary circulation after chronic hypoxia: hemodynamic and structural features. Am J Physiol 236:H818–H827 22. Rabinovitch M, Chesler NC, Molthen R (2007) Chronic hypoxiainduced pulmonary hypertension does/does not lead to loss of pulmonary vasculature. J Appl Physiol 103:1449–1451 23. Rabinovitch M, Konstam MA, Gamble WJ et al (1983) Changes in pulmonary blood flow affect vascular response to chronic hypoxia in rats. Circ Res 52:432–441 24. Taraseviciene-Stewart L, Kasahara Y, Alger L et al (2001) Inhibition of the VEGF receptor 2 combined with chronic hypoxia causes cell death-dependent pulmonary endothelial cell proliferation and severe pulmonary hypertension. FASEB J 15:427–438 25. Kwapiszewska G, Wygrecka M, Marsh LM et al (2008) Fhl-1, a new key protein in pulmonary hypertension. Circulation 118:1183–1194 26. Rondelet B, Kerbaul F, Motte S et al (2003) Bosentan for the prevention of overcirculation-induced experimental pulmonary arterial hypertension. Circulation 107:1329–1335 27. De Canniere D, Stefanidis C, Brimioulle S, Naeije R (1994) Effects of a chronic aortopulmonary shunt on pulmonary hemodynamics in piglets. J Appl Physiol 77:1591–1596 28. Reddy VM, Meyrick B, Wong J et al (1995) In utero placement of aortopulmonary shunts. A model of postnatal pulmonary hypertension with increased pulmonary blood flow in lambs. Circulation 92:606–613 29. Black SM, Johengen MJ, Soifer SJ (1998) Coordinated regulation of genes of the nitric oxide and endothelin pathways during the development of pulmonary hypertension in fetal lambs. Pediatr Res 44:821–830 30. Thébaud B, de Lagausie P, Forgues D, Aigrain Y, Mercier JC, ­Dinh-Xuan AT (2000) ETA-receptor blockade and ETB-receptor stimulation in experimental congenital diaphragmatic hernia. Am J Physiol Lung Cell Mol Physiol 278:L923–L932 31. Kentera D, Susic D, Veljkovic V, Tucakovic G, Koko V (1988) Pulmonary artery pressure in rats with hereditary platelet function defect. Respiration 54:110–114 32. Sato K, Webb S, Tucker A et al (1992) Factors influencing the idiopathic development of pulmonary hypertension in the fawn hooded rat. Am Rev Respir Dis 145:793–797 33. Le Cras TD, Kim DH, Gebb S et al (1999) Abnormal lung growth and the development of pulmonary hypertension in the fawn-hooded rat. Am J Physiol 277:L709–L718

J. Dupuis and N. Weissmann 34. Long L, MacLean MR, Jeffery TK et al (2006) Serotonin increases susceptibility to pulmonary hypertension in BMPR2-deficient mice. Circ Res 98:818–827 35. West J, Fagan K, Steudel W et al (2004) Pulmonary hypertension in transgenic mice expressing a dominant-negative BMPRII gene in smooth muscle. Circ Res 94:1109–1114 36. Izikki M, Hanoun N, Marcos E et al (2007) Tryptophan hydroxylase 1 knock-out and tryptophan hydroxylase 2 polymorphism: effects on hypoxic pulmonary hypertension in mice. Am J Physiol Lung Cell Mol Physiol 2293:L1045–L1052 37. Keegan A, Morecroft I, Smillie D, Hicks MN, MacLean MR (2001) Contribution of the 5-HT1B receptor to hypoxia-induced pulmonary hypertension: converging evidence using 5-HT1B-receptor knockout mice and the 5-HT1B/1D-receptor antagonist GR127935. Circ Res 89:1231–1239 38. Fagan KA, Fouty BW, Tyler RC et al (1999) The pulmonary circulation of homozygous or heterozygous eNOS-null mice is hyperresponsive to mild hypoxia. J Clin Invest 103:291–299 39. Matsui H, Shimosawa T, Itakura K, Guanqun X, Ando K, Fujita T (2004) Adrenomedullin can protect against pulmonary vascular remodeling induced by hypoxia. Circulation 109:2246–2251 40. Hoshikawa Y, Voelkel NF, Gesell TL et  al (2001) Prostacyclin receptor-dependent modulation of pulmonary vascular remodeling. Am J Respir Crit Care Med 164:314–318 41. Wanstall JC, Gambino A, Jeffery TK et al (2002) Vascular endothelial­ growth factor-B-deficient mice show impaired development of hypoxic pulmonary hypertension. Cardiovasc Res 55:361–368 42. Jasmin JF, Calderone A, Leung TK, Villeneuve L, Dupuis J (2003) Lung structural remodeling and pulmonary hypertension after myocardial infarction: complete reversal with irbesartan. Cardiovasc Res 58:621–631 43. Nguyen QT, Cernacek P, Calderoni A et  al (1998) Endothelin A receptor blockade causes adverse left ventricular remodeling but improves pulmonary artery pressure after infarction in the rat. Circulation 98:2323–2330 44. Nguyen QT, Colombo F, Rouleau JL, Dupuis J, Calderone A (2000) LU135252, an endothelinA receptor antagonist did not prevent pulmonary vascular remodelling or lung fibrosis in a rat model of myocardial infarction. Br J Pharmacol 130:1525–1530 45. Williams JH Jr, Bodell P, Hosseini S, Tran H, Baldwin KM (1992) Haemodynamic sequelae of pulmonary fibrosis following intratracheal bleomycin in rats. Cardiovasc Res 26:401–408 46. Shigemura N, Sawa Y, Mizuno S et al (2005) Amelioration of pulmonary emphysema by in vivo gene transfection with hepatocyte growth factor in rats. Circulation 111:1407–1414 47. Lee JH, Lee DS, Kim EK et al (2005) Simvastatin inhibits cigarette smoking-induced emphysema and pulmonary hypertension in rat lungs. Am J Respir Crit Care Med 172:987–993 48. Hocher B, Schwarz A, Fagan KA et al (2000) Pulmonary fibrosis and chronic lung inflammation in ET-1 transgenic mice. Am J Respir Cell Mol Biol 23:19–26 49. Miyauchi T, Yorikane R, Sakai S et al (1993) Contribution of endogenous endothelin-1 to the progression of cardiopulmonary alterations in rats with monocrotaline-induced pulmonary hypertension. Circ Res 73:887–897

Chapter 31

Transgenic and Gene-Targeted Mouse Models for Pulmonary Hypertension James D. West

Abstract  Transgenic and gene-targeted models are ­capable of providing a much more specific molecular stimulus than models based on hypoxia, toxins, or surgery. In a reductionist approach to understanding the roles of specific pathways in disease development, this ability to isolate the consequences of deletion, haploinsufficiency, or mutation in particular genes provides extraordinary power. This chapter is intended to provide an introduction to the use of gene-targeted and transgenic mice in the study of pulmonary arterial hypertension (PAH), in sufficient detail to allow proper understanding and use of existing models, and as an overview of methods used to create new models. In PAH, there is no “mouse model of disease” – the human disease is multifactorial, almost certainly combining major and minor genetic defects with environmental stimuli and  a  long time course. The important factors are poorly ­understood, and are certainly not reproduced in any mouse. One should thus approach these models as models of gene function, rather than disease. These models have been grouped into those primarily impacting vasoreactivity, those primarily involving dysregulation of the familial PAH  gene bone morphogenetic protein (BMP) type 2 ­receptor (BMPR2), and those primarily impacting plexogenesis. However, these divisions are crude; as discussed herein, there is considerable interaction between pathways, and many of these genes impact proliferation, inflammation, and vasoreactivity simultaneously. Keywords  Animal models • Pulmonary hypertension • Bone morphogenetic protein receptor type II • Genetic modification

1 Introduction Transgenic and gene-targeted models are capable of providing a much more specific molecular stimulus than models based on hypoxia, toxins, or surgery. In a reductionist approach to understanding the roles of specific pathways in disease development, this ability to isolate the consequences of deletion, haploinsufficiency, or mutation in particular genes provides extraordinary power. This chapter is intended to provide an introduction to the use of gene-targeted and transgenic mice in the study of pulmonary arterial hypertension (PAH), in sufficient detail to allow proper understanding (Sect. 2) and use (Sect. 3) of existing models, and as an overview of methods used to create new models. In PAH, there is no “mouse model of disease” – the human disease is multifactorial, almost certainly combining major and minor genetic defects with environmental stimuli and a long time course. The important factors are poorly understood, and are certainly not reproduced in any mouse. One should thus approach these models as models of gene function, rather than disease. These models have been grouped into those primarily impacting vasoreactivity (Sect. 4), those primarily involving dysregulation of the familial PAH gene bone morphogenetic protein (BMP) type 2 receptor (BMPR2) (Sect.  5), and those primarily impacting plexogenesis (Sect.  4). However, these divisions are crude; as discussed herein, there is considerable interaction between pathways, and many of these genes impact proliferation, inflammation, and vasoreactivity simultaneously.

2 Methods of Generating Models J.D. West (*) Division of Allergy, Pulmonary and Critical Care Medicine, Vanderbilt University School of Medicine, 1161 21st Ave. S., Suite T-1218 MCN, Nashville, TN 37232-2650, USA e-mail: [email protected]

There are two distinct methods of creating genetically modified mice: site-specific recombination, also known as knockout or knock-in, and transgenics. Each of these has a host of potential refinements, including mechanisms for spatial and temporal control. The essential difference is that site-specific

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recombination is a bit more complex, but allows modification of an existing gene within the murine genome, whereas creation of transgenic constructs is simpler, and to some extent allows greater versatility, but has problems associated with random insertion sites. The goal of this section is to provide an overview of these methods, as well as some practical details not found in more technical sources.

2.1 Knockouts and Knock-ins The primary advantage of knockout technology is consistency of effect: there are no secondary effects from the insertion site or expression level. The primary disadvantage is the potential for uninteresting developmental defects, such as fetal lethality, which prevent study of more interesting later stages. The central technologies which allow the creation of knockout mice are the creation of embryonic stem (ES) cell lines, homologous recombination, and blastocyst microinjection [1]. Creation of the construct is the central concern of the researcher. This must contain 5–10 kb of flanking sequence homologous to the site to be modified, surrounding two elements: (1) modification to the gene of interest, such as introduction of a key mutation or deletion of an exon, and (2) a cassette allowing selection of positively transfected ES cells, such as neomycin resistance (Fig. 1). Note that just about anything can be placed between the homologous flanking sequences, allowing for some very clever knock-in strategies. Aside from the Cre–loxP system discussed in the next section, a standard modification is to knock in a marker, such as b-galactosidase or green fluorescent protein, to mark cells which would normally

Fig. 1  Homologous recombination is the mechanism by which knockout and knock-in genetically modified mice are created. A sample gene is depicted (original sequence), consisting of a promoter (arrow), noncoding exons (open boxes) and coding exons (filled boxes). The researcher selects an exon to delete or modify and creates a plasmid construct containing a minimum of 5-kb flanking sequence surrounding the genetic modification, in this case deletion of a critical exon with replacement with a selection cassette. After insertion into embryonic stem cells, the construct undergoes homologous recombination with the native DNA sequence, leaving the recombined sequence, missing the critical exon but with addition of a selection cassette to allow selection of recombined embryonic stem cells

J.D. West

express the gene knocked out. Alternatively, any other gene could be added, potentially driven by the promoter native to the gene knocked out. Aside from the creation of the initial construct, steps in the creation of the knockout mouse are usually entirely handled by a core laboratory. The construct is transfected into the ES cells using any standard technology, and cells in which homologous recombination has occurred are isolated using the selection cassette. The genetically modified ES cells are microinjected into a blastocyst, which is transferred into a pseudopregnant mother. The first mice created in this way will be chimeras, consisting of a mixture of genetically modified ES cells and the wild-type cells which made up the blastocyst. However, some germ cells will be ES cells; crosses between this first-generation mouse and a normal mouse will result in some offspring in which 100% of cells are heterozygous for the genetic modification. Often, it is the researcher’s responsibility to screen these offspring for the modification.

2.2 Cre–loxP To allow spatial or temporal control of the knockout, an increasingly popular modification of the knockout strategy is use of the Cre–loxP system [2]. This system is similar to the knockout strategy described already, except that instead of deletion of the problem exon, it is flanked by 34-bp loxP sites. In the presence of Cre recombinase, these will recombine and delete the section in between (Fig. 2). This allows control of when or where the gene will be knocked out. Cre is a bacterial gene, with no native expression in mammals, and so it can be driven by any promoter, commonly tissue specific, developmental-stage-specific, or drug-inducible. This versatility is the central advantage to the Cre–loxP system, as it allows the lethality issue associated with traditional knockouts to be overcome. In general, a mouse line with tailored expression of Cre is bred to a line with loxP sites designed to knock out a gene of interest. Offspring which have at least one copy of the Cre transgene and are homozygous for the loxP modification will result in tissue- or time-specific knockout of the gene upon Cre expression. Mice with Cre expression are usually produced by transgenic methods, although there are exceptions with knock-ins of Cre behind native promoters. A wide library of tissuespecific or drug-inducible Cre expression mice are available from the research community or mouse repositories. There are several important issues to be considered in the use of this system. First, loxP sites are not 100% efficient; the farther apart they are, the less efficient they become. In many systems, only about 50% of cells have homozygous excision of the region flanked by loxP. In some systems this is sufficient to see phenotype; in others it is not. Further, presence of

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Fig. 3  A transgenic construct consists of a promoter, complementary DNA with a Kozak sequence and sometimes a tag, and a 3¢ untranslated region which requires a polyadenylation sequence and may include a noncoding intron–exon pair to improve translation. This is inserted into the genome in a random location

Fig. 2  Use of loxP sites to create a tissue-specific knockout requires two copies of loxP and Cre expression. In this example, a mouse homozygous for brain-specific expression of Cre is crossed to a mouse homozygous for a knock-in with loxP sites around a gene of interest (generation 1). The level of gene-of-interest expression is shown by shading in the mouse. In generation 2, all mice are heterozygous for each transgene, resulting in loss of one allele of the gene of interest. In generation 3, with this breeding strategy, half of mice lack Cre, and so have normal expression of the gene of interest regardless of how many loxP alleles they have. This is true even though their parent had loss of one allele in the brain, since the loss was not in the germ line. Only one quarter of mice have two copies of the loxP allele and one copy of the Cre allele, resulting in complete knockout of the gene of interest in brain. If this is not lethal, these proportions can be increased in future generations by crossing these mice to each other

the loxP sites themselves or the selection cassette may impact expression or translation of the gene, even before Cre activation. To reduce this problem, in some systems the selection cassette is itself flanked by loxP sites and excised in the ES cells through transient induction of Cre; however, the effect of loxP sites must be experimentally determined. Next, specificity of Cre expression is usually not quite so precise as one might hope; often, nominally tissue-specific promoters are not expressed in all of the tissues one might think they would be, and they are expressed in tissues one would hope they would not be. If Cre is expressed in germ cells, then future generations will just be knockouts. In inducible systems, there is often a problem with leak, resulting in some premature activation of the loxP sites, and the drug used to activate the loxP sites sometimes has effects independent of target gene deletion. Both tamoxifen and doxycycline can have substantial independent effects on estrogen-responsive organs and on matrix structure, respectively.

2.3 Transgenics Transgenic mice are substantially easier to produce than knockouts, both for the scientist creating the construct, as

well as for the core laboratory which produces them. The construct for the transgenic is substantially smaller, as it does not require the flanking sequence. Key elements include a promoter, complementary DNA led by a Kozak sequence to improve translation, and a 3¢ untranslated region and polyadenylation sequence (Fig.  3). This 3¢ untranslated region and polyadenylation sequence can usually be taken from any eukaryotic expression vector, and they have already been optimized to some extent. The construct is cut from the backbone to include only the elements listed, and a core facility microinjects it into a fertilized oocyte, which is then transferred into a pseudopregnant female. Offspring are tested for the presence of the transgene: in our experience, between 5 and 20% of offspring will carry the transgene, providing multiple potential founder lines. Unfortunately, the presence of multiple founder lines is necessary because of the central drawback to transgenics: a random insertion site. The transgenic construct is integrated into the host genome in a random location, usually with multiple copies at the same site. Depending upon the integration site, this may interrupt host genes, it may result in expression in undesired tissues, or it may result in silencing of the promoter. It is critical, then, to test all of the founder lines for specificity and level of expression, and for ability to be bred to homozygosity. Further, if unexpected effects arise, it is important to demonstrate that they can be produced by more than one founder line, since otherwise they could be the result of integration effects. Because of the need to ensure correct localization of expression, it is useful to tag the transgene (although one needs to verify in cell culture that the tag does not impact gene function). Note, however, that the Myc tag can have cross-reactivity to native Myc and that mice appear to have a cell-surface protein that cross-reacts to FLAG. An interesting variant on transgenic mice which has traditionally been used for overexpression of genes or expression of dominant negatives is expression of short hairpin loops which in  vivo act as small interfering (siRNA), thus knocking down expression of the target gene [3].

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2.4 Doxycycline-Inducible Systems Doxycycline (or tetracycline)-inducible systems are a key refinement of transgenic technology, which allows control of transgene expression based on addition of the antibiotic doxycycline to food or water. The tetracycline transactivator (tTA) is a bacterial gene which drives transcription from the Tet operon only in the absence of doxycycline. An artificial modification of this, the reverse tTA (rtTA) drives transcription from the Tet operon only in the presence of doxycycline. In practice, this system requires two transgenes: a tissue-specific promoter driving rtTA (or tTA), and the promoter to which it binds in the presence of doxycycline (TetO)7–CMV, driving the transgene of interest (Fig.  4). (TetO)7–CMV is a promoter consisting of seven copies of the Tet operon, followed by a minimal CMV promoter. Transcription from this promoter is driven only by either rtTA in the presence of doxycycline, or tTA in the absence of doxycycline. Overall, then, this system allows simultaneous tissue- and time- specific expression of transgenes; tissue specificity is gained by the promoter driving rtTA, but only when mice are fed doxycycline. Doxycycline can be administered to mice in either food or water; after extensive testing (unpublished), we have found that it makes little difference to the speed or level of transgene activation. The form of doxycycline also makes no difference; doxycycline hyclate is as effective as doxycycline hydrochloride. Food can be purchased with doxycycline premixed with mouse chow; we use 1  g/kg, but as little as 250 mg/kg is probably sufficient in some systems. Doxycycline can also be mixed with water; we use 1 mg/mL, but fourfold less will work in some systems. Doxycycline is light-sensitive,

J.D. West

and has moderate stability in water, so bottles must be amber- or foil-wrapped, and must be changed at least twice per week. Doxycycline in water has a bitter taste, and so it is sometimes sugared, but we have tested and found that sugar makes no difference to the amount consumed (an adult mouse drinks 4–5 mL/day), and can cause mold growth in bottles. Generally, using premixed food is far more convenient. In addition to the drawbacks associated with any transgenic, there are two primary disadvantages to this system; leak and breeding. All doxycycline-inducible genes will have some level of expression even in the absence of doxycycline. In our experience, the level of leak is different in different tissues, probably reflecting an integration-site effect. We have found this to be true even when using next-generation transactivators such as rtTA2-M2 in vivo [4]. Whether or not the leak is a problem depends upon the precise system used. Inducibility in vivo, in our experience, ranges from 7 times to more than 100 times, depending on the transgenes and tissues used, averaging about 15–20 times. When founders are examined, it is essential in addition to the factors discussed previously to look for low leak and high inducibility. It is possible to reduce leak using the transcriptional silencer tTS [5], but this also somewhat reduces maximum induced expression, and adds yet a third transgene. This is the second major problem with inducible systems; the need for two transgenes. If these are kept in the same mouse for breeding, leak will eventually select against expression. This can be overcome by keeping them in separate homozygous lines and only crossing for experiments, but heterozygotes may have lower expression, and crossing in a third transgene is very time consuming. It may be possible to overcome these problems by coinjecting multiple constructs when first generating the transgenic animal. Constructs coinjected into the oocyte tend to integrate into the same site, and segregate together during subsequent generations. Thus, triple transgenics with an rtTA construct, a (TetO)7–transgene construct, and a tTS construct can be made, although this reduces the versatility of the system.

3 Husbandry

Fig. 4  Doxycycline-inducible transgenics require two transgenes, usually created separately; a tissue-specific promoter driving the reverse tetracycline transactivator (rtTA; shaded mouse), and the (TetO)7–CMV promoter driving the gene of interest (cross-hatched mouse). When these two mice are bred, offspring positive for both transgenes will express the gene of interest, but only when fed doxycycline, and only in the tissues in which rtTA is expressed

In most universities within the USA, mouse research is largely governed by standards set by the Institute for Laboratory Animal Resource’s (ILAR) Guide for the Care and Use of Laboratory Animals [6] and by the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC). However, the guidelines and training provided by these groups tends to be of a general nature. The purpose of this section is to provide some more practical details associated with mouse husbandry. Note that there is substantial strain-to-strain variability in behavior and development.

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3.1 Caging Cages used in most animal care facilities allow five mice to be housed together. Same-sex mice from different litters should not be housed together; they will likely fight. Mice from the same litter that have previously been housed separately should not be housed together; they will likely fight. Males will often fight, and cause each other injury, requiring euthanasia, even if from the same litter; if so, the aggressor needs to be isolated. Mice housed individually sometimes will injure themselves, whether male or female, so solitary housing is neither costeffective nor particularly humane, if sometimes unavoidable. We have found that these restrictions mean that our average number of mice per cage is only 2–3. Use of enrichment devices, particularly nesting biscuits or cardboard tubes, and possibly wheels, seems to reduce fighting and self-inflicted injury. Horizontal exercise wheels that will fit within institutional cages are commercially available, although if these are not washed by the facility, they become impractical.

3.2 Breeding When transgenic strains are used, it is usually necessary to maintain a colony. The optimal age for first breeding of mice starts at about 8 weeks of age and lasts to about 16 weeks, although mice can become fertile as early as 5  weeks, and mice that are continuously bred can remain fertile until over 12 months. However, in our experience, fewer than half of new breeder pairs over the age of 6 months will produce litters. When establishing a breeder pair, always use a new cage, to avoid territoriality issues, and provide nesting biscuits or cardboard tubes, so that the mice can tear these up instead of each other. With some strains, it is necessary to remove the male after a few days, to prevent fighting with the female or injury to the pups. Some pulmonary-hypertension-related strains, including neuronal nitric oxide synthase (nNOS) knockouts [7] and our BMPR2delx4+ lines [8], are particularly aggressive. The gestational period for a mouse is 3 weeks. This is conveniently also the weaning age, which is particularly important since mice are fertile for the first 3  days after producing a litter. Some breeder pairs will thus produce a new litter continuously every 3 weeks for many months. A new litter in a cage with an old litter will usually die of neglect, so it is important to be vigilant and wean old litters immediately. Different strains grow at different rates; BL6 mice are quite large by 3 weeks and rarely need support in weaning, whereas FVB/N mice are still small, and may need gel packs or wetted food placed in the bottom of the cage.

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First-time mothers sometimes, and mothers of some strains often, are bad mothers, and will not properly feed or care for their pups. For instance, serotonin transporter (SERT) knockout mice are usually bad mothers. Pups should be examined about 1 day after they are born, without opening the cage. They are translucent, and so it should be possible to see a horizontal white stripe indicating milk in their stomachs. If there is no milk, or if they are scattered around the cage, they will probably die if not fostered. Remove the foster mother temporarily from her pups, mix in the new pups with the original pups, and replace the foster mother. In our experience, foster mothers will accept these pups even if they are from a different strain, and are capable of caring for almost twice as many pups as their standard litter size (up to 14 pups for FVB/N mice).

3.3 Genotyping Another necessity for maintaining a transgenic colony is identifying and genotyping animals. The three standard methods of identifying animals are use of metal ear tags, use of distinctive patterns of ear punches, and use of tracking chips. The advantage of ear tags is their unambiguity; the disadvantage is that they can be torn out. The advantage of ear punches is simplicity; the disadvantage is that they can be ambiguous, or they can heal until they are invisible (we have found that a minimum 1.5-mm-diameter punch is required to avoid this). Tracking chips’ main drawback is expense, and inability to distinguish mice at a glance. Amputation of toes has occasionally been used as an identifier, but this is currently discouraged. DNA for genotyping can be extracted from blood, or more commonly from the tip of the tail, collected when weaning and marking pups. Only approximately 4 mm from the tip of the tail is required, and can be collected without anesthesia at weaning. Genotyping was historically done by Southern blot, but PCR, or quantitative PCR, with allele-specific primers, is now more common and simpler.

4 Vasoreactivity-Related Models By 1960, Wagenvoort had proposed aberrant vasoconstriction as causative in PAH [9]. Although it had become apparent by the 1980s that treating vasoconstriction alone was unable to resolve idiopathic PAH [10], vasodilators are still our most effective treatment of idiopathic and familial PAH, and recent studies have confirmed fundamental defects in vasoreactivity in both mouse models carrying the familial PAH gene [11] and in asymptomatic carriers of the PAH gene [12]. Moreover, defects in expression or function of vasoreactivity- and reactive oxygen species (ROS)-related genes seem likely to be

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central to persistent pulmonary hypertension of the neonate and PAH related to COPD. Although there are an increasing number of factors related to vasoreactivity, in this section knockout and transgenic mice related to several central pathways are discussed, including nitric oxide synthases (NOSs), caveolins, endothelin, superoxide dismutases (SODs), and serotonin. One of the most interesting findings from these animal models is the role that these pathways play not just in vasoreactivity, but also in development, angiogenesis, and inflammation.

4.1 Nitric Oxide Synthases Nitric oxide has a variety of activities in the lung in addition to its well-known role in endothelium-mediated vasodilation, including bronchodilation, antiadherence, antimitogenic, and anti-inflammatory properties [13], as well as playing an important part in angiogenesis [14]. It is generated from l-arginine by three NOS enzymes, nNOS, endothelial NOS (eNOS), and inducible NOS (iNOS). Both knockout and transgenic overexpression mice exist for all three isoforms, all of which are present in the lung. In vivo and isolatedperfused lung studies have shown that in the pulmonary circulation, nNOS does not modulate tone, eNOS is most responsible for endothelium-dependent vasodilation, and both iNOS and eNOS set basal tone [15]. The NOS knockout and transgenic mice are mature models; the primary phenotype in each was described more than 10 years ago, and contained few surprises. eNOS knockout mice have systemic hypertension [16], whereas transgenic mice overexpressing eNOS in the vascular wall have systemic hypotension [17]. However, more recent studies have found that NOS genes do much more than regulate tone. eNOS is essential for neovascularization [18] and for fetal lung development [19]. iNOS knockout mice had enhanced inflammatory responses to lipopolysaccharide [20] and in allergen models; its role in the allergen models but not in lipopolysaccharide models requires intact nNOS [21]. Because nitric oxide seems to interact with many functions involved in lung growth, function, and repair, overexpression or underexpression of nitric oxide in a mouse is a useful model for many areas of study.

4.2 Caveolin Caveolae are abundant vesicular organelles present in most cell types, with a primary role in trafficking; they are made up of proteins called caveolins, of which there are three. Caveolin-1 and caveolin-2 predominate in nonmuscle cells, whereas caveolin-3 is restricted to muscle cells [22].

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Caveolin-1 knockouts result in complete loss of caveolae in endothelium and epithelium and vascular smooth muscle, in lungs as well as other organs, but this results in a relatively subtle phenotype, consisting of malformations of the lungs, abnormalities in vasoreactivity, and cardiomyopathy [23]. NOS inhibition reversed many of the abnormalities found in Cav1-/- mice [24], suggesting that NOS inhibition is one of the primary functions of caveolae. Study of the role of caveolae and caveolin-1 has progressed rapidly, but questions remain about the mechanism by which caveolin-1 regulates NOS activity, and the presence of other proposed caveolin functions such as intracellular trafficking and calcium regulation.

4.3 Endothelin Endothelin-1 (ET-1) is a 21 amino acid peptide, expressed primarily in endothelial cells, made from a 213 amino acid propeptide by endothelin-converting enzyme (ECE). ET-1 is principally known as an extremely potent vasoconstrictor, acting through two G-protein-coupled receptors, endothelin receptor A and endothelin receptor B. Bosentan, a current treatment for PAH, is an endothelin receptor blocker. Knockout mice for ET-1, ECE, and both receptors have been made. ET-1-/- mice die of respiratory failure at birth, with additional craniofacial developmental abnormalities [25]. ET-1+/- mice have slightly elevated systemic pressures [25] and are hyperresponsive to methacholine in asthma models [26]. Conversely, overexpression of ET-1 in transgenic mice does not cause elevated pressure, but does lead to hypertrophy, increased oxidant stress [27], and vascular inflammation [28]. It appears, then, that with either constitutive overexpression or deletion of ET-1, a new set point is reached which prevents a role in vascular tone. However, both overexpression and deletion still have important roles in regulating development, angiogenesis, and inflammation. Endothelin receptor A knockouts have defects in craniofacial development that mimics ET-1 knockouts, in addition to defects in the cardiovascular outflow tract [29]. By contrast, endothelin receptor B knockouts have a phenotype dominated by colon and coat developmental abnormalities [30]. Knockout of ECE-1 produces a phenotype indistinguishable from either ET-1 or endothelin receptor A knockout [31]. Although both endothelin receptors apparently have important roles in regulating different populations of neural crest cells, the phenotype in knockouts is not particularly relevant to PAH. It seems likely that conditional and tissue-specific abrogation, possibly with siRNA constructs, would be needed to produce a mouse with a phenotype more relevant to PAH, by avoiding developmental defects. ET-1 has important roles in

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regulating many PAH-related activities, including inflammation and angiogenesis, and so conditional expression or knockout of ET-1 and its receptors would likely add significantly to our understanding of details of its function.

4.4 Superoxide Dismutases Superoxide (O2−) is produced in the lung through a myriad of processes, including hyperoxia, a variety of environmental toxins, normal function of inflammatory cells, and the normal action of NOS proteins and mitochondria [32]. Superoxides, and ROS in general, are highly damaging and play an important role in several lung diseases, possibly including PAH. In particular, ROS stimulates vascular smooth muscle cell proliferation, increases vascular tone, and may directly inhibit eNOS and NO [33]. SODs are one of the primary mechanisms for converting superoxide into peroxide, which is later converted to water. There are three SODs: intracellular copper–zinc SOD (SOD1), mitochondrial manganese SOD (SOD2), and extracellular SOD (SOD3). There are both knockout and transgenic overexpression models for all of these. The phenotypes of knockout mice are relatively mild: knocking out SOD1 and SOD3 either individually or together has little obvious phenotypic effect aside from increasing the levels of ROS [34]. Several types of SOD2 knockout or knockdown mouse have been produced, all of which die within days or weeks of birth, owing to a variety of symptoms all associated with mitochondrial damage [35]. SOD2 heterozygote mice, however, have a subtle phenotype associated with increased susceptibility to mitochondrial damage. The mild phenotype in the SOD1 and SOD3 knockout and the SOD2 heterozygote can actually be considered an advantage; they are suitable templates on which the effects of hypoxia or inflammatory stress in the lung can be tested. Transgenic overexpression of SOD2 is, as expected, protective against mitochondrial injury caused by drugs [36], diabetic stress [37], and ischemia/reperfusion [38]; however, regulation of the cellular antioxidant/prooxidant balance was sometimes regulated not quite as would be expected from a simple model. Transgenic overexpression of SOD3 is protective against remodeling caused by chronic hypoxia in mice [39], as well as protecting against hyperoxia-induced damage [40]. Transgenic mice with threefold overexpression of SOD1 have a similarly subtle phenotype [41], although mice transgenic for mutant SOD1 develop amyotrophic lateral sclerosis [42]. Broadly, both knockout and overexpression of SOD genes have a sufficiently subtle phenotype that they are suitable substrates for other experiments.

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4.5 Serotonin Serotonin has been linked to PAH since the epidemic associated with Aminorex diet pills in the 1960s [43]. There are multiple mechanisms by which serotonin could predispose to PAH; it can drive both proliferation and constriction [43], and is intimately linked to cytokine signaling [44]. SERT polymorphisms are associated with increased risk of disease or earlier onset in some families [45, 46]. There is no gene for serotonin; rather, it is synthesized from the amino acid tryptophan by one of two tryptophan hydroxylases (TPH1, TPH2). It is cleared by SERT and signals through both SERT and 14 different receptors grouped into seven families [43]. SERT-/- mice are viable and healthy, if a bit difficult to breed, and are slightly protected against hypoxic PAH [47]. Transgenic mice overexpressing SERT in smooth muscle [48] or under its own promoter [49] spontaneously develop PAH, although the mechanism is still unclear. Many serotonin receptor models have been created by psychology researchers, but to date only one appears to have been studied in the context of pulmonary hypertension. HTR1B knockouts are protected against remodeling caused by chronic hypoxia, probably because of effects on constriction [50]. TPH1 knockout mice have dramatically lower serotonin production, and are protected against hypoxic [51] and dexfenfluramine-induced [52] PAH. TPH1 is a particularly interesting target, because it is a potential small molecular inhibitor target, and so could be effectively attacked therapeutically. Overall, these studies suggest that elevated serotonin levels can contribute to human PAH, as evidenced both by epidemiology, studies of human tissues, and multiple animal models, through an as yet unspecified mechanism possibly combining immune, proliferative, and vasoconstrictive effects, and that this might be attacked through inhibition of TPH1.

5 BMP Pathway Models At least 80% of familial PAH patients and about 25% of sporadic PAH patients have mutations or rearrangements in the only type 2 receptor for the BMP pathway, BMPR2 [53–57]. This is the strongest evidence yet discovered for the early molecular cause of disease. Unfortunately, as of its discovery as the PAH gene, BMPR2 had primarily been studied as a developmental gene [58]. The bulk of our knowledge about its role in adult lung has been developed through mouse models in the last few years. The BMP pathway is part of the transforming growth factor b superfamily. It consists of more than a dozen ligands, perhaps

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a half-dozen secreted inhibitors, several intracellular inhibitors and modifiers, multiple downstream signaling pathways, including Smad, X chromosome-linked inhibitor-of-apoptosis protein [59], LIM kinase [60], Src [61], and Tctex [62], two or three type 1 receptors depending upon cell type, but only one type 2 receptor, BMPR2. That the mutations have to date been found in BMPR2, rather than other pathway genes, implies that it is the integration of these functions that is important, rather than any specific downstream pathway. BMPR2 is a single-pass transmembrane protein with four broad domains: an extracellular ligand binding domain, a short transmembrane domain, a kinase domain, and a long cytoplasmic tail. Mutations in PAH patients are found throughout the gene, many of which clearly lead to haploinsufficiency, but many of which lead to proteins predicted to act as dominant negatives.

5.1 BMPR2 Knockout and Hypomorph The first BMPR2 mouse model published was a constitutive knockout of exons 4 and 5, resulting in truncation late in the ligand binding domain and effectively a null allele. Homozygote BMPR2-/- mice die during early development owing to failure to form mesoderm [63], but heterozygote BMPR2+/- mice are relatively phenotypically normal. They show a mild increase in normoxic pulmonary vascular resistance through an as yet unspecified mechanism [64], but no change in right ventricular systolic pressure (RVSP) compared with wild-type mice at the baseline [65] (note that because of excess capacity, pulmonary vascular resistance can increase substantially before RVSP increases). BMPR2+/mice had increased susceptibility to inflammatory challenge by adenovirus expressing 5-lipoxygenase, with more elevation in RVSP and a longer recovery time [65]. However, whereas in one study they had slightly lower susceptibility to chronic hypoxia [64], in another they did not [66], although slightly different metrics were used. BMPR2+/- mice did show increased response to serotonin, suggesting interesting interactions between the two pathways [66]. An alternative BMPR2 knock-in mouse, the hypomorph, has a deletion of exon 2, causing loss of half of the extracellular binding domain but an otherwise normal protein. When homozygous, these mice survive gastrulation, but die later in development with cardiovascular and skeletal defects [67]. Heterozygote mice have mild endothelial dysfunction, which can be brought out in the phenotype by extended chronic hypoxia; 5  weeks, but not 3  weeks, of hypoxia results in increased RVSP in these mice compared with controls [68]. Broadly, the BMPR2+/- and BMPR2Dx2/+ mice are probably the best available model of the effect of human mutation, since many human mutations produce simple haploinsufficiency. However, BMPR2+/- and BMPR2Dx2/+ mice do not have a phenotype at the baseline, and their phenotype even

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with hypoxia is very subtle. The primary problem with these models is that they lack the currently unknown cofactors, either environmental or genetic, which lead to PAH in human BMPR2 mutation carriers. Since these are largely unknown in humans, adding back a good guess to bring out the phenotype risks producing a largely irrelevant model. A stronger understanding of the cofactors in humans would make these models more useful.

5.2 BMPR2 siRNA and Cre–loxP The knockout and hypomorph mice already discussed are fetal lethal when homozygous, and largely asymptomatic when heterozygous. Attempts to circumvent these problems produced the siRNA and Cre–loxP models. Novartis’s mouse knocked a short hairpin RNA (shRNA) BMPR2 construct into the Rosa26 locus using a Cre–lox system in development of the model but not in its final form [3]. This shRNA, when cleaved, produces a siRNA for BMPR2, resulting in universal knockdown of BMPR2 function to about 20% of that in wild-type mice. Unfortunately, although the design of these mice was quite clever, they were also dominated by nonpulmonary and likely developmental abnormalities, including severe mucosal hemorrhage, incomplete mural cell coverage on vessel walls, and gastrointestinal hyperplasia. These mice also showed a defect in angiogenesis, assayed by Matrigel plugs [3]. A more successful attempt to model PAH resulted from use of an endothelial-specific promoter driving Cre crossed to native BMPR2 gene with loxP sites knocked in, resulting in an endothelial-specific BMPR2 knockout. A subset of these mice developed elevated RVSP, combined with perivascular inflammation and thrombosis, with penetrance increasing with age [69]. Although the mechanism for this phenotype has not yet been explored, it is a clear indication that BMPR2 mutation in endothelium does not have as its primary consequence loss of control of proliferation. Although it suffers mildly from a low-penetrance problem, this can be resolved by using large animal numbers: this model appears to be an excellent model with which to study BMPR2 function in pulmonary endothelium. Moreover, combined with other tissuespecific Cre expression, it could be used for tissue-specific knockouts in other pulmonary tissues.

5.3 Inducible BMPR2 Mutants Our group has a system of models allowing BMPR2 knockdown by doxycycline-inducible tissue-specific overexpression of dominantly acting BMPR2 mutations. We have published extensively on smooth-muscle-specific expression of two

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mutations: a truncation mutation just after the transmembrane domain, which is a dominant negative for all BMPR2 functions (BMPR2Dx4+), and a truncation mutation at amino acid 899 (BMPR2R899X) which leaves Smad functions intact. In addition, we have discussed at conferences two new models: a universal expression of BMPR2R899X, with a phenotype similar to that of the smooth-muscle-specific version discussed below, although more penetrant, and endothelial-specific expression of BMPR2Dx4+, with a phenotype remarkably similar to that of the endothelial Cre–loxP model described already. Smooth-muscle-specific expression of BMPR2Dx4+ produces elevated RVSP with as little as 1 week of induction in adult animals likely as the result of defects in vasoreactivity [11], although the model never develops substantial pulmonary vascular remodeling [8]. The dominant phenotype appears to be a switch from a contractile to a synthetic phenotype in smooth muscle [70], with attendant defects in reactivity, combined with an increase in the expression of specific cytokines [71]. Unfortunately, the loss of full differentiation of smooth muscle results in downregulation of the transgene, limiting the phenotype. Smooth-muscle-specific expression of BMPR2R899X produced a rather different phenotype, dominated by vascular pruning, rather than defects in vasoreactivity: even those mice with normal RVSP still showed extensive loss of vessels by fluorescent microangiography [72]. These mice showed substantial perivascular inflammation, obliteration of small vessels by cells of still uncertain origin, and in some animals with elevated RVSP, complex vascular lesions consisting of concentric rings of smooth muscle, disorganized endothelium, or both. By gene array, most of the increased cytokine expression seen in the earlier BMPR2Dx4+ model was missing in the BMPR2R899X model; this seemed odd, because histologic indications of inflammation, such as the presence of inflammatory cells, were far more extensive. These mice also had extensive changes in collagen, matrix, actin-organization, mitogen-activated protein kinase, and adhesion-related genes, even in mice with normal RVSP. Comparison of these two models suggests that Smad signaling downstream of BMPR2 is responsible for vasoreactivity defects and some cytokines, whereas cytoplasmic tail domain signaling causes defects in inflammatory cell recruitment, adhesion, migration, and likely proliferation. Oddly, both BMPR2Dx4+ and BMPR2R899X models seem to have strong interaction with even mild hypoxia; a chronic drop in ambient pressure of even 15% caused much higher RVSP in the former model and much greater penetrance in the latter model. These models have several mild drawbacks. First, they suppress BMPR2 to a greater extent than any human mutation. Second, they probably have a stoichiometry problem; there is far more mutant BMPR2 than native BMPR2 than is present in the human disease. Third, they do not require cofactors to achieve disease; this is an advantage in the sense that it allows examination of the effect of strong downregulation of

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the BMP pathway, but a disadvantage in that it probably does not reflect the human disease. However, these models do show that suppression of BMPR2 alone is sufficient to cause a close approximation of end-stage human disease, which strongly implies that these cofactors may function to suppress the BMP pathway.

5.4 Conclusions About BMPR2 Function A common theme in the majority of these models is either direct inflammation or increased expression of cytokines, as in the endothelial Cre–loxP BMPR2, BMPR2delx4+, and BMPR2R899X models, or increased susceptibility to inflammation, as in the BMPR2+/- mouse. It is thus clear that the BMP pathway plays a role in regulating inflammation, with loss of BMPR2 leading to increased inflammation. Second, it is clear that the BMP pathway is required for proper angiogenesis (as in the shRNA BMPR2 model), endothelial function (as in the Cre–loxP model), and smooth muscle function (as in the BMPR2delx4+ model). BMP signaling drives terminal differentiation of numerous cell types, and loss of it leads to defects in function. Finally, and equally important, are defects that were not seen. In no model did loss of BMPR2 lead directly to uncontrolled proliferation, regardless of how thoroughly it was suppressed. Further, neither the Cre–loxP model nor the transgenic models had any obvious defects in angiogenesis while at normal RVSP, but still developed elevated RVSP. Next, even though the Cre–loxP model had the BMPR2 defects directly in endothelium, it did not have a baseline increase in pressure; the same is true of the BMPR2R899X models, which develop elevated RVSP even without apparent defects in vasoreactivity. The lessons learned from the models so far seem to point to the primary role for BMPR2 in adults as suppressing inflammation and driving differentiation, and as having a role in proliferation, angiogenesis, or vasoreactivity only as a secondary effect.

6 Models of Plexogenesis One of the characteristic markers of PAH is the generation of plexiform lesions, complex vascular lesions that appear to be defective attempts at angiogenesis. These lesions occur even in secondary forms of PAH, and animal models suggest that angiogenesis and defective angiogenesis are not part of the initial cause of disease, as discussed earlier, but resolution of the consequences of abnormal angiogenesis, present in all adult PAH patients but not children [73], is likely to be important in treating most human PAH.

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6.1 Vascular endothelial growth factor

7 Conclusions

Vascular endothelial growth factor A (VEGFA) and its receptors are essential for angiogenesis, both in development and in adults. There are two reasons to suspect angiogenesis is important in PAH; plexiform lesions found in human PAH patients are almost certainly the result of a defective attempt at angiogenesis, and hypoxic rodents with inhibition of angiogenesis develop more extensive disease [74]. Knockout mice have been made for VEGFA and its three receptors, VEGFR-1 (Flt-1), VEGFR-2(Flk-1), and VEGFR3. All are embryonic lethal at between 9.5 and 12 days into development [75–78] owing to defects in angiogenesis; even heterozygous VEGFA knockouts are embryonic lethal. However, receptor heterozygotes and mice with knock-ins slightly altering VEGFA expression are available, and have been used successfully in systemic organs to study angiogenesis [79]. These had roughly the anticipated results, with slight inhibition of angiogenic response when receptors were heterozygous, and slightly increased angiogenic response with a slight increase in VEGFA expression. In general, the vascular endothelial growth factor system is so critical to survival and development, and so tightly regulated, that it seems likely that proper study of these on a genetic basis will require creation of an inducible and tissue-specific system. To date no such system exists for pulmonary research, which has been restricted to either nonspecific pharmaceutical inhibition or largely descriptive studies in vivo.

Decades ago, before any of the molecular causes of the disease were known, and before there were any genetically modified animal models, the central hypothesis for the cause of PAH focused on three factors: vasoconstriction, proliferation, and thrombosis. It was unclear which of these were causative, and which were side effects. The purpose of genetic animal models was to approach this issue in the most reductionist approach possible. The most interesting finding from the animal models is that, to a large extent, the problem cannot be reduced: even genes which are thought to primarily affect vasoreactivity, or angiogenesis, or inflammation, or proliferation, impact all of the other elements as well. It is this deep interconnectivity between pathways in the lung that is one of the most interesting findings to date from animal models. We understand a large amount now about the pathways relevant to PAH, their broad functionality, and their interconnectedness. The important challenge remaining is to determine not just which genes can contribute to PAH, because just about all of those studied contribute with the correct model under the correct circumstances, but the controlling nodes at which intervention is both possible and meaningful in human PAH patients. This will require not just a stronger interface with human data, but much more detailed models – with tissue and temporal control and readouts. We understand broadly how these pathways work, but we do not understand the details well enough yet to produce the sorts of data that will actually help us cure human disease. There is thus broad scope remaining for more comprehensive examination of existing models as well as for the creation of more tightly controlled models. Genetically modified mouse models are an excellent method of generating relevant preclinical data, and a resource that has only begun to be tapped.

6.2 S100A4 S100A4 is a calcium-binding gene with many functions, originally isolated as overexpressed in highly metastatic mouse mammary adenocarcinoma cells [80]. The S100 family members have diverse functions, including cell proliferation, differentiation, signal transduction, adhesion, and cell motility [81]. S100A4 specifically appears to be involved primarily in motility, with direct interactions with stress fibers, F-actin, and tropomyosin [82]. Transgenic overexpression of S100A4 under the 3-hydroxy3-methylglutaryl coenzyme A reductase promoter results in plexiform-like lesions in about 5% of mice with advanced age [83]. Infection with a vasculotropic virus increased this percentage substantially, possibly by increasing elastase activity [84]. This arteriopathy occurred without the presence of any earlier stages of vascular remodeling, suggesting that S100A4 is directly involved in the creation of complex vascular lesions, and that this phenomenon is entirely mechanistically separable from pulmonary hypertension itself.

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Chapter 32

Animal Models of Increased Lung Vascular Permeability Sara Hanif Mirza, M. Kamran Mirza, and Asrar B. Malik

Abstract  The pulmonary microvascular endothelium regulates vascular water and solute flux into tissues. Vascular barrier function is physically established by tight junctions and adherens junctions between neighboring endothelial cells. Endothelial permeability together with vascular oncotic and hydrostatic pressures control fluid and solute movement into the pulmonary interstitium via paracellular and transcellular pathways. Transvascular fluid flux into the interstitium is constantly counteracted by a slow lymphatic reabsorption from the extravascular space. Failure of pulmonary fluid homeostasis results in the accumulation of interstitial fluid which then invades the alveolar air spaces. Disruption of the endothelium leading to pulmonary edema is the cardinal feature of acute lung injury (ALI) and its severest counterpart acute respiratory distress syndrome (ARDS) manifesting clinically as alveolar edema, impaired gas exchange, and hypoxemia. Despite extensive research, the fundamental mechanisms that initiate and propagate the increase in lung vascular permeability have not been esta­ blished. Modeling the complex regulation of fluid flux in the lung demands the use of ex vivo and in vivo experimentation and animal models, which are useful tools in studying the permeability responses. In this chapter, we provide a perspective on the field by focusing on animal models of lung vascular permeability. We will in particular address the “known unknowns” and identify wherever possible the clini­cal import of the cited studies. Keywords  Animal models • Acute lung injury • Respiratory distress syndrome • Lung vascular permeability • Endothelial function • Inflammation

M.K. Mirza (*) The University of Chicago Medical Center, 5841 S. Maryland Avenue, MC 6101, Chicago, IL 60637, USA e-mail: [email protected]

1 Introduction The pulmonary microvascular endothelium regulates ­vascular water and solute flux into tissues. Vascular barrier function is physically established by tight junctions and adherens junctions between neighboring endothelial cells. Endothelial permeabi­ lity together with vascular oncotic and hydrostatic pressures control fluid and solute movement into the pulmonary interstitium via paracellular and transcellular pathways. Transvascular fluid flux into the interstitium is constantly counteracted by a slow lymphatic reabsorption from the extravascular space. Failure of pulmonary fluid homeostasis results in the accumulation of interstitial fluid which then invades the alveolar air spaces. Disruption of the endothelium leading to pulmonary edema is the cardinal feature of acute lung injury (ALI) and its severest counterpart acute respiratory distress syndrome (ARDS) manifesting clinically as alveolar edema, impaired gas exchange, and hypoxemia. Despite extensive research, the fundamental mechanisms that initiate and propagate the increase in lung vascular permeability have not been established. Modeling the complex regulation of fluid flux in the lung demands the use of ex vivo and in  vivo experimentation and animal models, which are useful tools in studying the permeability responses. In this chapter, we provide a perspective on the field by focusing on animal models of lung vascular permeability. We will in particular address the “known unknowns” and identify wherever ­possible the clinical import of the cited studies.

2 What Is Being Modeled? The purpose of developing animal models is to mimic the in vivo milieu featured in the setting of abnormal pulmonary vascular permeability. Under resting conditions, equilibrium exists between filtration and reabsorption at proximal and distal segments of capillaries such that the net filtration into the interstitium is negligible. Many factors may mediate loss of this homeostasis and before defining the models of increased lung vascular permeability, we review the main features of edema fluid formation.

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_32, © Springer Science+Business Media, LLC 2011

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2.1 Formation of Edema Fluid Starling forces across the microvessel wall govern the process of edema formation. The fluid filtration rate JV is written as

J V = LP S ( PC − PI ) − s (p C − p I )1

where LP is the hydraulic conductivity, S is the vessel surface area, P are the hydrostatic pressures, p are the oncotic pressures, with the subscripts C and I signifying the capillary and interstitial compartments, respectively, and s is the osmotic reflection coefficient for albumin. PC – PI gives the transmural hydrostatic pressure difference and the product s (pC – pI) gives the effective transmural oncotic pressure difference. A higher PC – PI favors fluid filtration, whereas higher s (pC – pI) favors reabsorption. A large increase in fluid filtration over reabsorption overrides the capacity of the lymphatic system to remove excess fluid from the pulmonary interstitium, leading to edema. “Hydrostatic pressure edema” results from increase in the pulmonary capillary pressure, whereas “permeability edema” is a result of barrier rupture or breakdown creating a protein-rich transexudate. In the latter case, fluid flux can increase secondary to increased Jv in the absence of any change in pulmonary vascular hydrostatic pressure. This condition resulting in pulmonary edema is a characteristic feature of ALI [2]. These two mechanisms can be separated rigorously in animal models by measuring the constants s (which measures transendothelial albumin permeability) and LP (which measures transendothelial fluid permeability [3]). Many forms of ALI have a key confounding factor; ­simultaneous pulmonary capillary hypertension coupled to the increased lung vascular permeability leads to a greater increase in the net filtration across the pulmonary exchange barrier and amplification of edema formation than occurs in the absence of the hypertension.

increase vascular permeability in a segment-specific fashion. This results in a nonuniform distribution of fluid accumulation in lungs.

2.3 The Ideal Model of Increased Lung Vascular Permeability Modeling lung vascular permeability is a challenge owing to (1) a variety of inciting mechanisms that increase vascular permeability, (2) the broad range of endothelial dysfunction achieved by different permeability-increasing agents, and (3) the wide variety of segment-specific and species-specific responses. What constitutes an ideal model of increased lung vascular permeability? Ideally, animal models should reproduce an increase in lung vascular permeability, increase in EVLW, and mirror impairment of gas exchange and hypoxemia accompanying alveolar flooding. Loss of this fluid balance can be initiated by multiple factors: direct injury to the endothelium, activation of coagulation, sepsis, changes in vascular oncotic and hydrostatic pressures that increase transcapillary fluid filtration, agents that increase actin–myosin contraction coupling in endothelial cells, oxidant production, release of proinflammatory cytokines, and direct alveolar epithelial injury leading to epithelial barrier breakdown. Thus, in modeling lung vascular permeability, one can adopt many approaches. Although lung epithelial permeability is an important entity unto itself, our focus will be agonists that have been used in animal models to induce an increase in lung vascular permeability.

3 Models of Increased Lung Vascular Permeability

2.2 Fluid Accumulation in Lungs

3.1 Thrombin

Accumulation of fluid begins in perivascular and peribronchiolar regions of the lung, followed by buildup in the septal interstitium and eventually alveolar flooding [4]. It has been shown that alveolar flooding occurs after extravascular lung water (EVLW) content increases by more than 35%, ­corresponding to an interstitial pressure of 2  mmHg [1]. Alveolar edema is not homogeneously distributed throughout the lung [5]. Permeability-increasing agonists introduced in  vivo may not increase vascular permeability in all ­segments of the pulmonary vasculature yet still show an overall increase in net fluid filtration [2]. There are also agents that show no difference in net fluid filtration, but may

The role of thrombin is underscored by its multifaceted effects in promoting edema formation, platelet aggregation [6–8], and endothelial cell activation that is defined as loss of ­vascular integrity, expression of leukocyte adhesion ­molecules, change in phenotype from antithrombotic to prothrombotic, release of cytokines, and upregulation of HLA molecules [9–11]. In endothelial cells, thrombin plays an important role in cell contraction, paracellular gap formation, and vascular barrier disruption through disassembly of adherens junctions [12, 13]. Thrombin is generated as the effector protease after a series of zymogen conversions initiated by exposure to ­tissue factor [14, 15]. Thrombin activity is short-lived, with a

32  Animal Models of Increased Lung Vascular Permeability

half-life of 5 min [15], and thrombin is rapidly removed by activated protein C [15]. Thrombin increases endothelial permeability by binding to and cleaving protease-associated receptor-1 (PAR-1) on endothelial cells [7]. PAR-1 cleavage creates a tethered ligand that binds the body of the receptor to initiate downstream effects via phospholipase C-b, increasing intracellular Ca2+ levels and actin–myosin cross-bridging, leading to adherens junction disruption [16]. The synthetic 14 amino acid peptide SFLLRNPNDKYEPF (TRP-14) substitutes for the tethered ligand created by thrombin on PAR-1 [17] and has been shown to mimic the effects of thrombin in in vivo studies [18] wherein lung vascular permeability was measured. This mimicry lacks the prothrombotic response of thrombin, which would have complicated the use of this agent to increase vascular permeability. Studies using transendothelial resistance measurements to assess changes in the dimensions of junctions have shown that thrombin increases junctional opening within minutes and the response is restored in 2 h [19, 20]. Thrombin application also induces protein kinase C (PKC)-z dependent Na+,K+-ATPase endocytosis in epithelial cells of intact rabbit lungs [21], suggesting the possibility of impaired alveolar fluid clearance. Thrombin likely mediates a twofold increase in lung vascular permeability by inducing endothelial ­leakage and impeding fluid clearance. Thrombin causes RhoA- and Rac1-dependent actin stress fiber formation [22] and increases endothelial expression of P-selectin, which ­promotes the migration of neutrophils [23]. Neutrophil sequestration and injury may therefore represent another route of increased endothelial permeability. Thrombin levels are increased in plasma and lavage fluids of patients with pulmonary edema [24], suggesting an important role of the coagulation cascade in ALI [24]. Thrombin and the PAR-1 cognate effector peptide have also been shown to increase pulmonary vascular resistance in conjunction with a rise in the coefficient of capillary filtration (Kf,c) [9, 18] and lung water content [18]. Intravenous administration of 100  nM a-thrombin or 5  mM PAR-1 peptide (TFLLRNPNDK-NH2) for 20  min increases the pulmonary vascular 125I-albumin permeability–surface area product (PS), a measure of endothelial albumin permeability, by sevenfold, and a 1.5-fold increase in lung microvascular Kf,c [18] is seen, indicating that thrombin induces the opening of junctional gaps that permit a large flux of albumin (Fig. 1). Using PAR-1 knockout mice (PAR-1−/−), Vogel et al. [18] reported the critical role of PAR-1 signaling in thrombin-mediated increase in endothelial permeability as well as pulmonary vasoconstriction, suggesting that the rise in capillary hydrostatic pressure also contributes to the formation of pulmonary edema. Responses to thrombin may vary depending on the level of PAR-1 expression and the preactivation state of endothelial cells (i.e., whether the cells or animals have been challenged with endotoxin or other agents that induce PAR-1 expression)

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Fig. 1  Thrombin as a model of increased lung vascular permeability. (a) Effect of a-thrombin (0.1 mM) and protease-associated receptor-1 (PAR-1) agonist peptide (5 mM) or control peptide (5 mM) on 125I-bovine serum albumin permeability–surface area product (PS) in isolated ­perfused mouse lung preparations. PAR+/+ or PAR-1−/− preparations were exposed to the specified additive for a 20-min period; then, the 3-min 125I-labeled albumin PS was plotted. Effects induced by thrombin or PAR-1 agonist peptide were ablated in PAR-1−/− lung prepara­ tions. (b) Reversible action of selective PAR-1 agonist peptide (TFLLRNPNDK-NH2; 5 or 10  mM) on the capillary filtration coefficient (Kf,c) in PAR+/+ lung preparations. Note complete absence of an effect on Kf,c with scrambled (control) peptide (FTLLRNPNDK-NH2; 10 mM). (Reproduced from [18] with permission)

[25]. In addition, there may be variations in the expression of PAR-1 in microvessel compared with large vessel endothelial cells, leading to a variable thrombin-mediated increase in vascular permeability in different lung vascular segments. Receptor expression profiling in endothelial cells from different segments of the pulmonary vascular bed may be useful in identifying the most susceptible segments for a robust thrombin response. It is also important to distinguish whether increase in endothelial permeability alone is sufficient or whether a concomitant rise in pulmonary capillary hydrostatic pressure is required to induce pulmonary edema. Using different concentrations of thrombin or PAR-1 peptide, Vogel et al. [18] differentiated between thrombin’s direct permeability-increasing

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effect and the edemagenic response secondary to the rise in capillary hydrostatic pressure induced by pulmonary vasoconstriction. The results indicated that the rise in hydrostatic pressure was required for accumulation of lung water content. Troyanovsky et  al. [26] reported an unexpected role for thrombin in actually enhancing barrier function in the rat endothelium. Their results showed that PAR-1 activating peptide (100 mg/mL) induced an increase of Kf,c (a function of hydraulic conductivity and vascular surface area) in the isolated perfused rat lung, pointing to a barrier enhancement role of thrombin in lung vessels. Thus, the in vivo effects of thrombin on endothelial cells may differ in both a concentration- and a lung-vascular-segmentspecific fashion. The signaling regulation of thrombin-mediated increase in endothelial permeability is understood to some extent, justifying its use as a model of lung vascular permeability. Further studies are needed to determine the segmental variability of lung vascular permeability to thrombin.

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infusion, however, fails to increase vascular permeability. This injection-route-dependent difference in edemagenic response is not due to degradation of histamine or differences in blood flow [39], but is the result of antagonistic activity of endogenously released catecholamines by direct b-adrenergic stimulation [39]. This finding suggests that the direct actions of edema-producing agents may differ as a function of the route of administration owing to a possible interaction with other regulatory systems. It is unclear whether infusion of histamine alone leads to a full-blown increase in pulmonary vascular permeability since the increase in vascular permeability is small and shortlived. Since the main receptor for histamine in endothelial cells, the H1 receptor, is involved in the pathogenesis of allergy [30], H1 receptor antagonists may be of value in modeling and understanding allergy-induced increase of pulmonary vascular permeability.

3.2 Histamine

3.3 Oxidant Generation and Ischemia/ Reperfusion Injury

Histamine, an amine stored in mast cells is a well-known activator of vascular permeability [27]. Histamine has been shown to mediate both dilation and constriction of the vasculature [28] by binding to one of three receptor ­subtypes; H1, H2, and H3 [29]. Both H1 and H2 receptors are present in large-vessel and microvessel endothelial cells [28]; however, the effect of histamine on vascular ­permeability occurs through the activation of H1 receptors [30]. Sepsis-induced increase in lung vascular permeability in animals is associated with elevated histamine concentrations in plasma [31], justifying its use as a model for pulmonary fluid gain. Administration of histamine induces the formation of ­interendothelial junction gaps [27] secondary to a rise in intracellular Ca2+ concentration. This results in changes to the phosphorylation state and destabilization of adherens junctions and the tight junctional proteins vascular endothelial cadherin [32], p120 catenin [33], and zona occludins 1 [34]. This finding has been reproduced in both in vivo [35] and in vitro [22, 32–34] models. Like thrombin (discussed already), histamine causes RhoA- and Rac1dependent actin stress fiber formation and increases endothelial expression of P-selectin, promoting the migration of neutrophils [22, 23]. This increase represents another mechanism for increasing lung transvascular fluid flux. Histamine infusion increases Kf,c in a rat model of pulmonary microvessels [35] and porcine coronary vasculature [36]. Histamine has also been shown to induce interstitial edema secondary to an increased permeability of venules in the canine bronchial circulation [37]. Intra-arterial infusion of histamine increases protein and fluid leak owing to ­microvascular permeability to plasma proteins, reflected in a higher lymph total protein concentration [38]. Intravenous

The pulmonary vascular response to oxidants such as H2O2 is characterized by an increased pulmonary artery pressure (Ppa), pulmonary capillary pressure (Ppc), and pulmonary edema [40, 41]. Oxidants affect lung fluid balance by (1) the vasoactive reaction resulting in an increase in capillary pressure and (2) permeability increase which alters fluid exchange [42]. Oxidants have been shown to directly increase vascular permeability as shown with H2O2- and O2.--dependent albumin flux across endothelial monolayers [43]. The mechanism of increased endothelial permeability involves oxidant-mediated endothelial contraction via RhoA-mediated myosin light chain kinase activation [8, 44] and increase in intracellular Ca2+ concentration [45]. Johnson et al. [41] studied the role of PKC in H2O2-mediated increased Ppa, Ppc, and Kf,c using inhibitors and implicated PKC in signaling the oxidantinduced increase in vascular permeability and pulmonary edema. At higher concentrations, oxidants directly interact with endothelial cell membranes, causing lipid peroxidation and resultant endothelial injury [46]. The lung is susceptible to high concentrations of oxygen. Increasing inhaled oxygen concentration has the potential of increasing the rate of radical production [47]. Modeling increased lung vascular permeability induced with oxidants is difficult owing to the immediate breakdown of free radicals and the inability to accurately control a given dose. Oxidant injury can, however, be modeled using hyperoxia and indirect means such as administering activated neutrophils, chemical generation of oxidants such as xanthine–xanthine oxidase, injection of reducing agents, and ischemia/reperfusion injury. Perfusion of guinea pig lungs with H2O2 [41], rabbit lungs with xanthine–xanthine oxidase [48], or activated

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­neutrophils [49] all resulted in increases of Kf,c and edema formation. Ischemia followed by reperfusion (I/R) leads to increased lung vascular permeability as illustrated by the reimplantation response following lung transplantation [50]. The edema is of the noncardiogenic type involving increased vascular permeability, inflammatory cell infiltrates, and hypoxia and is unrelated to rejection of the transplanted organ [51, 52]. In the lung I/R injury model, ischemia is produced by either clamping the pulmonary artery (while preserving the ­bronchial circulation) or impeding both pulmonary and bronchial circulations by clamping the hilum [53]. Thus, the bronchial circulation does not appear to be important for the response. I/R induces a twofold lung weight gain after 120 min of ischemia in the rat lung model [54]. I/R in nonpulmonary circulations such as gastrointestinal and hepatic circulations also elevates pulmonary vascular permeability in rats [54], rabbits [55], and sheep [56]. This is perhaps secondary to the release of mediators that affect the lung vascular endothelium. The pathogenesis of vascular leak in I/R is still unclear but a key role of oxidants has been invoked. Hypoxia and lack of blood flow induces the generation of reactive oxygen species, activates the NADPH oxidase [57] (Nox-2) family of enzymes, which in turn results in the activation of nuclear factor kB (NF-kB), and promotes production of proinflammatory cytokines [58]. Srinavasan et al. [54] showed that alveolar collapse ­during lung ischemia is a key determinant of reperfusion-induced lung vascular injury because ventilation of the lungs during ischemia or instillation of surfactant prevented this response. The rise in pulmonary capillary hydrostatic pressure and increased surface area cannot account for the increased albumin PS observed during reperfusion. Mechanical factors resulting from the loss of alveolar tethering forces during alveolar collapse may injure the pulmonary capillaries on reperfusion.

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VEGF induction of Flt-1 was demonstrated to be sufficient for hyperpermeability in porcine microvascular endothelial cells [63]. Upon binding to its receptor, VEGF signals via phospholipase g, thereby activating diacylglycerol and inositol triphosphate production [60]. Intracellular Ca2+ concentration is increased owing to Ca2+ release from both intracellular stores as well as store-dependent and store-independent channels [60], resulting in increased paracellular and transcellular permeability [60]. VEGF has been shown to increase permeability in both intact vessels [64] and endothelial cells in culture [63]. Lee et al. [64] found that inhibition of p110d, the catalytic subunit of phosphatidylinositol 3-kinase, reduced ovalbumin-induced upregulation of VEGF concentrations, attenuating antigen-induced vascular leakage. VEGF expression increases in rat lung pericytes upon lipopolysaccharide (LPS) stimulation [65] and phosgene exposure [66]. Kaner et al. [67] showed that overexpression of human VEGF165 complementary DNA in the lung (­adenovirus gene transfer vector 10 [9] pfu) increases transendothelial permeability to albumin as measured by the Evans blue dye assay (Fig. 2). Despite the original description of VEGF as vascular permeability factor [68], the

3.4 Vascular Endothelial Growth Factor Vascular endothelial growth factor (VEGF), a well-known endothelial mitogen and initiator of angiogenesis [59], mediates its effects by the specific kinase domain receptors fmslike tyrosine kinase (Flt-1) and fetal liver kinase (Flk-1) [the mouse homologue to kinase-insert-domain-containing receptor (KDR)] [59]. Activation of KDR/Flk-1 has been shown to be the predominant signaling pathway implicated in VEGFinduced phosphorylation of junctional targets [60]. The adherens junction components vascular endothelial cadherin, b-catenin, plakoglobin, and p120-catenin [61, 62] are tyrosine-phosphorylated 15 min to 1 h after VEGF stimulation in human umbilical vein endothelial cells [61]; a-catenin is not modified [61]. Flt-1 activation did not affect tyrosine phosphorylation of cadherins and catenins [61]. However,

Fig.  2  Vascular endothelial growth factor (VEGF) overexpression increases lung vascular permeability. Effect of AdVEGF165 on extravascular lung water and albumin permeability. (a) Lung wet to dry weight ratios corrected for intravascular volume 5 days after intratracheal administration of 10 [9] pfu AdVEGF165 or Adbgal. (b) Lung vascular permeability expressed as plasma equivalents (mL) in the extravascular space of the lung. (Reproduced from [67] with permission. The official journal of the American Thoracic Society copyright American Thoracic Society)

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p­ roangiogenic properties of VEGF have been heavily researched, whereas its role in modeling increased lung ­vascular permeability in sepsis remains less understood. There are conflicting­ results concerning the lung vascular response to overexpression and inhibition of VEGF in ­animal models. Koh et  al. [69] demonstrated a protective role for exogenous VEGF treatment in sepsis-induced albumin leakage­and edema formation in lungs, whereas other studies have shown that VEGF ­mediates an increased endothelial permeability response [67]. The basis of this discrepancy is not clear, but it may point to a dual role for VEGF in animal models of lung vascular permeability, perhaps resulting from concentration-dependent differences.

3.5 Endotoxin and Polymicrobial Sepsis LPS or endotoxin is a structural glycolipid present in the outer membrane of Gram-negative bacteria composed of a polar lipid head group (lipid A) and a chain of repeating disaccharides [70]. LPS mediates many of the important host responses to Gram-negative bacteria. Systemic administration of LPS was one of the earliest approaches used to model the consequences of bacterial sepsis and shock. In serum, LPS binds to a specific LPS binding protein (LBP) [71], forming an LPS– LBP complex that activates CD14 antigen. This complex interacts with toll-like receptor-4 (TLR4) in an unknown manner triggering downstream signaling events [72]. In lungs, TLR4 is expressed by several different cell types, including professional host defense (dendritic cells, macrophages) as well as airway epithelial and endothelial cells [73]. When administered in  vivo, LPS induces profound lung vascular leakage [74] (Fig. 3) and releases proinflammatory mediators such as TNFa, IL-1, and IL-8 [72] by a transcriptional mechanism involving the activation of NF-kB [72] via MyD88dependent and MyD88-independent signaling pathways. LPS induction of increased endothelial cell permeability is RhoAdependent and induces myosin light chain phosphorylation and actin reorganization [13], suggesting that it is in part the result of an endothelial contraction mechanism. The LPS permeability response has been measured by increases in lung wet weight, bronchoalveolar lavage (BAL) fluid polymorphonuclear neutrophils, albumin extravasation, Kf,c, and uptake of Evans blue dye. Transcriptional activation of NF-kB-dependent proinflammatory mediators complicates the evaluation of direct LPS-mediated increases in vascular permeability, since a complex inflammatory response is triggered involving multiple signaling pathways, many of which lead to increased endothelial permeability. An important caveat of modeling LPS-induced increase in lung vascular permeability is the considerable variability of the response in species. TLR4 profiles and vascular reactivity

Fig. 3  Lipopolysaccharide (LPS) increases lung vascular permeability as determined by Kf,c measurement. Typical lung weight and ­pulmonary artery pressure changes with LPS treatment versus the control. (a) Weight change recordings for wild-type mice treated with 2  mg/kg body weight LPS (2  h pretreatment). (b) Wild-type control trace. (c) Pressure trace for the experiment. A 10-cmH2O pulse was applied for 20  min. Kf,c values estimated as given in the text. (Courtesy of Stephen Vogel, University of Illinois at Chicago)

to endotoxin differ in different species [75]. Similarly, it is also necessary to consider background differences within strains; for example, mice show widely varying tolerance to LPS concentrations (e.g., for C57/B6 mice the lethal dose is between 25 and 30 mg/kg body weight, whereas for CD-1 mice it is less than 10  mg/kg body weight). LPS-induced increase in lung vascular permeability has also been studied in ovine, porcine, feline, canine, and murine models, with all studies showing essentially similar responses but requiring different doses of LPS. An important consideration in animal modeling of ­LPS-mediated permeability is how the LPS is generated. Pyrogenicity of bacterial colonies depends on the presence or absence of the O-chain [76]. LPS stocks from different sources may have different contaminants, which may alter the biological effects in  vivo by interacting with signaling pathways other than TLR4 [73]. In studies endotoxin has been administered by intratracheal, intravenous and intraperitoneal routes. In each case, the dose and timing/severity of the response varies and pilot studies are needed to determine LD50/LD80 and evaluate the time course of pulmonary transvascular fluid flux. In sublethal doses, intraperitoneal LPS causes increased vascular permeability within 6 h (as measured

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by Kf,c) that reverses back to normal within 48  h [77]. However, intratracheally administered LPS is also a useful model of pneumonia wherein the infection is mostly restricted to the lungs Sepsis-related inflammation is the primary cause of increased lung vascular permeability seen in ARDS patients [78]. LPS mimics the molecular exterior of Gram-negative bacteria; however, it does not produce the whole array of effects generated by bacterial invasion. Bacterial induction of increased lung vascular permeability has been studied by the model of direct intravascular inoculation and cecal ­ligation and puncture (CLP). Intravascular bacterial ­administration even at relatively low doses leads to an immediate hypotension and mortality [53]. Challenge with Pseudomonas aeruginosa in sheep [79] and Mannheimia haemolytica in cows has been shown to increase lung vascular permeability and polymorphonuclear neutrophil recruitment [80]. CLP is a widely used model that creates a polymicrobial sepsis secondary to a peritonitis caused by surgical ligation and puncture of the cecum in mice. Lung vascular permeability typically increases within 18–72  h after surgery [81]. Blood cultures are positive for multiple enteric organisms [82]. CLP results in increased pulmonary transvascular fluid flux and EVLW [83]. Because of its clinical relevance, CLP is considered an excellent mouse model of sepsis-induced lung injury [53]. However, the need for surgery and its consequences may confound the extrapolation of data in this model to human ALI.

3.6 Oleic Acid Oleic acid (OA) is an endogenous unsaturated fatty acid that represents around 60% of the free fatty acid pool in mammals [84]. An intravenous injection of OA produces lung injury associated with pulmonary vascular hyperpermeability and hypoxemia leading to ARDS [53, 85]. The basis of increased permeability is not clear, although it has been reported that OA increases endothelial permeability secondary to superoxide production [85]. Studies showed that OA-induced increase in pulmonary vascular permeability is attenuated with elastase inhibition as well as the antioxidant N-acetyl cysteine [85], suggesting a role for oxidant generation and perhaps oxidant activation of elastase [85] in the mechanism of the permeability response. OA has a profound toxic effect on endothelial cells at concentrations of 5 × 10–4 M in  vitro, producing vascular leakage [86], and has been used as a model of fatty acid embolism in animals [87]. OA (15 mL/kg) also causes a significant decrease in PaO2 within 10 min after injection [88]. Pulmonary microvascular permeability increases almost immediately after administration, with a dose-dependent elevation of EVLW and leakage of

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protein-rich fluid into the interstitium and air space [85, 89]. OA (25 mM) also blocks active transepithelial [22] Na+ transport [90], suggesting it may have an additional effect in clearance of alveolar fluid. OA does not alter sodium channel or Na+,K+-ATPase surface expression; however, it covalently associates with the pump directly, inhibiting the catalytic activity of purified Na+,K+-ATPase [90]. Hence, OA impairs the essential active Na+ transport mechanisms of the lung and thus promotes alveolar edema formation and prevents edema resolution.

3.7 Mechanical Ventilation Mechanical-ventilation-induced lung injury is termed ­ventilator-induced lung injury (VILI) [91]. In contrast to other models of increased lung vascular permeability, VILIinduced increase in lung water is iatrogenic [53]. The mechanical ventilation model is the model of lung injury most relevant to clinical practice and its understanding may lead to improved human survival in ALI [92]. VILI is caused by mechanical stretch of alveolar epithelial cells and endothelial cells in capillaries and direct tissue damage, with subsequent endothelial breaks resulting in interstitial and alveolar edema [93]. However, the precise mechanisms remain enigmatic since it is possible that humoral and cellular factors may also be involved [94]. Endothelial cell detachment from the basement membrane is seen within 20 min of 45-cmH2O pressure ventilation in rats [95], consistent with the role of mechanically induced injury to the epithelial and endothelial barriers. Measurements of Kf,c values in 4–6-week-old rabbits subjected to ventilator barotrauma showed that high peak inspiratory pressure (50–53  cmH2O) in combination with a high gas flow rate (more than 8 L/min) induces microvascular injury [96]. Other studies in isolated perfused rat lungs showed that Kf,c increases up to 4 times over the baseline after 35-cmH2O peak inspiratory pressure ventilation for 30 min [97], further supporting the barotrauma hypothesis. Studies have also suggested a role of mechanically induced activation of intracellular pathways that may be involved in the mechanism of cell injury [98]. Mechanical stress results in  increased intracellular Ca2+ concentration which may ­mediate increased lung vascular permeability [99]. In addition, VILI-induced vascular permeability may require lung neutrophil sequestration [100], pointing to a concomitant role of proinflammatory mediators in this response. Mechanical ventilation and bacterial products may act synergistically in causing lung injury [101]. A combination of mechanical ventilation, high lung volume, and low-dose endotoxin or bacteria is likely the best model of VILI since it takes into account two important pathogenic mechanisms.

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4 Cross-Talk Mechanisms Between Endothelial Barrier Enhancement and Disruption In contrast to the lung endothelial barrier disruption pathways discussed already, it is also important to consider the intrinsic barrier-enhancing mechanisms which are involved in the control of lung fluid homeostasis. Activation of these mechanisms is important in modulating the severity of the permeability increase described already and thus it is important to consider these pathways in designing models of increased lung vascular permeability. Sphingosine 1-phosphate (S1P) is the product of sphingosine kinase (SPHK)-induced phosphorylation of sphingosine [102]. S1P is a normal component of plasma, present at concen­trations between 0.2 and 0.9 mM [103]. The serum concentration of S1P is higher because of release from platelets. S1P stimulation of S1P receptors triggers an endothelial barrier-protective response that attenuates thrombin- and LPS-mediated increases in lung vascular permeability [104]. A recent review described the dual regulation of endo­thelial permeability in response to PAR-1 stimulation; that is, endothelial barrier-disruptive and barrier-protective signaling activated by PAR-1 signaling [12]. With respect to barrier protection, PAR-1 signaling activates the isoform SPHK1, inducing the production of S1P, which counteracts the increase in endothelial permeability [12]. Thus, S1P generation is an important negative-feedback mechanism for maintaining barrier homeostasis and restoration of the permeability response. SPHK1 depletion in a murine model increases basal endothelial permeability by lowering S1P production [105], consistent with this concept. S1P treatment of SPHK1 knockout lungs or SPHK1-deficient endothelial cells rescues the defective endothelial barrier function [105] (Fig. 4d, e). A similar cross talk has been seen with angiopoetin-1, which blocks the VEGF-mediated increase in lung vascular permeability [106]. Angiopoetin-1 functions by preventing transient receptor potential channel-1 dependent Ca2+ influx induced by VEGF [106]. This cross-talk mechanism requires further analysis of the cellular signaling pathways responsible for overall lung fluid homeostasis. A deeper understanding of these opposing mechanisms regulating lung endothelial permeability is likely to advance our understanding of endothelial barrier regulation in disease states and perhaps identify new therapeutic targets.

5 Evaluation of Increased Lung Vascular Permeability Modeling changes in lung vascular permeability requires the use of scientific techniques that effectively quantify fluid gain in the lung. Evaluation may be invasive or noninvasive.

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Noninvasive estimation of pulmonary edema formation from either increased hydrostatic pressure or vascular permeability­ has been an important goal owing to its obvious benefits over invasive techniques.

5.1 Gravimetric Quantification Gravimetric quantification of pulmonary extravascular fluid accumulation is the most direct way of evaluating lung fluid gain. Assessment requires excision of lungs followed by the determination of the EVLW content, the true measure of pulmonary edema [107]. Lung tissue is homogenized with a known amount of water and aliquots of blood, homogenate, and homogenate supernatant are weighed wet and dry. By comparison with hemoglobin values in the blood, lung wet weight is corrected for blood weight. This method is based on correcting for lung water due to residual blood volume in lungs; thus, EVLW is determined by subtracting the blood water volume and is expressed as the ratio of EVLW to bloodfree dry lung weight. EVLW is the most sensitive weightbased method of assessing lung fluid gain, with normal values between 3 and 7 mL/kg (water content of the pulmonary interstitium) and usually more than 10 mL/kg in the case of cardiogenic or permeability pulmonary edema. Kirov et  al. [108] compared the quantification of EVLW by the transpulmonary single thermodilution (EVLWST) technique (a noninvasive technique, discussed later) and the invasive EVLW calculation by the postmortem gravimetric (EVLWG) technique in sheep and found that although EVLWST overestimated EVLW as compared with EVLWG, it closely correlated with the gravimetric method. Other considerations to be kept in mind regarding gravimetric measurements include the minimization of evaporative losses from fresh tissue and effects of perfusion and or ventilation as part of the experimental protocol. Gravimetric assessment does not distinguish between hydrostatic edema and vascular permeability edema and yields a quantification that pools lung water content into one measurement irrespective of segmental differences in fluid flux.

5.2 Noninvasive Quantification of Pulmonary Fluid Accumulation 5.2.1 Thermo-Dye Dilution Method The thermo-dye dilution method allows calculation of the volume of distribution of an indicator injected into the circulation. On the basis of the exponential nature of blood heat loss following intravenous injection of a thermal indicator, the total volume of distribution is determined. This volume includes both the intravascular and the EVLW space (without distinguishing between interstitial and alveolar water). EVLW

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Fig. 4  Cross talk between endothelial barrier enhancement and disruption mechanisms. Role of the G-protein-coupled receptors PAR-1 and shingosine 1 phosphate receptor 1 (S1P1) in regulation of endothelial barrier function. (a) Signaling mechanism of endothelial barrier disruption. Thrombin induces an increase of intracellular Ca2+ concentration followed by activation of kinases and phosphatases, which in turn modify constituents of adherens junctinss, resulting in junction disassembly. RhoA activation is mediated by phosphorylation of GDP dissociation inhibitor 1 and p115RhoGEF and by the interaction of G 12/13 with p115RhoGEF. (b) Signaling mechanisms of endothelial barrier protection. Activated platelets secrete S1P. Binding of S1P to the receptor S1P1 stimulates Gi-mediated increases in intracellular Ca2+ concentration and activation of Rac1 and Cdc42. (c) Signaling cross talk between PAR-1 and S1P1 as a mechanism of endothelial barrier repair. Activated protein C decreases generation of thrombin and stimulates PAR1-mediated

intracellular synthesis of S1P from sphingosine (Sph) through the activation sphingosine kinase 1 (SphK-1). Additionally, activation of endothelial protein C receptor (EPCR) by activated protein C mediates activation of S1P1 through Akt-dependent and phosphatidylinositol 3-kinase dependent phosphorylation and thereby barrier stabilization. [12] (d, e) Sphingosine 1-phosphate (S1P) prevents PAR-1-induced lung microvascular permeability increase in SphK1−/− mice. Mice were challenged with either phosphate-buffered saline (PBS) or PAR-1 peptide for 15 min, followed by administration of 1 mM S1P. Lung vascular permeability was determined by quantifying EBAE (d) or the wet weight to dry weight ratio (e). The data are given as the mean ± the standard deviation of four experiments. Asterisk different from PBS control group. Pound sign different from the wild-type group after PAR-1 challenge (P < 0.05). (a–c Reproduced from [12] with permission. (d, e) Reproduced from [105] with permission)

content can be estimated by injection of a thermal indicator and a strictly intravascular indicator (e.g., indocyanine) simultaneously followed by detection and comparison of their respective dilution curves in the femoral artery. This method has been validated against the reference gravimetric methods in humans [109] and yields EVLW measurements with good reproducibility [110]. Limitations of this method include situations where there is vascular obstruction in pulmonary vessels. Anatomical differences and vascular malformations in genetically altered animals also need to be taken into account.

5.2.2 Whole-Body Scintigraphy Bouvet et al. [111] have used [111]111In-transferrin accumulation to detect leakage of pulmonary vessels. Such measurements need to be corrected for local blood flow and have good correlation with increased lung vascular permeability. However, the main concern is that they are unable to distinguish whether this increase in tracer uptake is the result of increased edema due to permeability or vascular surface area, or both.

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Gaar et al. [117] first introduced the lung microvascular filtration coefficient Kf,c measurement, which has become a useful tool for rigorously quantifying permeability of the

lung vascular barrier. Isolated perfused lungs allow the ­measurement of small changes in transvascular filtration in which increased vascular filtration pressures can be separated from increased vascular permeability. In this method, the lungs are typically perfused in situ using a peristaltic pump (Fig.  5). The heart and exsanguinated lungs are excised and attached to the perfusion apparatus and the lung preparation is suspended from a sensitive force–displacement transducer. Pulmonary artery pressure is monitored throughout the experiment. Changes in lung wet weight due to gain or loss of fluid from the lung are recorded. Isogravimetric Kf,c is determined by comparing the rate of lung weight gain during the baseline period with that after rapidly elevating the perfusion pressure by 5–10 cmH2O for 5–10 min (DP). Alternatively, a sustained step increase in left atrial pressure may be applied. The lung wet weight increases in a ramplike fashion, reflecting net fluid extravasation. Lung dry weight is determined and the Kf,c (mL·min–1·cmH2O·g·dry wt–1) is calculated from the slope of the recorded weight change normalized to the capillary pressure change and to lung dry weight. The Kf,c calculation is based on certain assumptions, outstandingly reviewed by Bhattacharya [118] and Parker and Townsley [2]. Some important assumptions are: (1) DP results in vascular engorgement that may independently change surface area and lung weight and thus confound Kf,c evaluations; (2) collapsed versus patent vasculature (“recruitment status”) at the beginning of the experiment needs to be controlled since this can interfere with the vascular surface area. DP will also recruit previously collapsed vessels and therefore for baseline readings sufficient pressure should be

Fig.  5  The isolated-perfused lung model for the assessment of ­vascular permeability. (a) Isolated-perfused lung apparatus used for Kf,c measurements. (b) Intraoperative picture of exposed thoracoabdominal cavity. Heart and lung are visible together with a tracheal cannula in place for ventilation (white arrow) and a cannula for ­perfusion of the pulmonary artery via the right ventricle (black arrow).

(c) Photograph of perfused lung preparation during the experiment. Perfusate is pumped into the pulmonary artery and exits via the left atrial cannula. A highly sensitive weight transducer (red arrow) is used to measure changes in the weight of the ex vivo lung. See the text for the experimental protocol. (Courtesy of Stephen Vogel, University of Illinois at Chicago)

5.2.3 Other Imaging Techniques Imaging methods are also available for the assessment of vascular leakage and lung edema. Flat chest X-ray films in larger subjects allow assessment of the severity and distribution of edema, based on defined scoring [112]. Methods such as micro-CT and micro-PET scanning improve the resolution range. Thin-section CT scanning showed sensitivity in measuring lung fluid in a canine model of OA injury [113] and dynamic contrast material enhanced the usefulness of CT in being able to differentiate between permeability and hydrostatic edema in piglets [114]. PET analysis of the pulmonary transcapillary escape rate for [68]68Ga-transferrin also shows good correlation to lung injury in dogs [115]. MRI, traditionally regarded as a suboptimal modality to visualize air (hence, the lung), provided an assessment of edema in 3-D images of lungs. MRI of animal lungs is sensitive enough to measure relatively small changes in lung water with adequate temporal resolution to resolve the time course of the edema formation [116]. However, measurements are inaccurate in conditions such as atelectasis that often accompany pulmonary edema [116].

5.3 Vascular Permeability Measurements in Lungs

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maintained to control for potential differences in vascular surface area; (3) DP increases both vascular and interstitial pressures, potentially affecting the vessel–interstitium pressure gradient. Changes in this gradient would affect Starling’s equation governing fluid flux and confound Kf,c evaluations.; (4) It is possible that DP causes endothelial cell activation due to shear and mechanical forces. For example, changes in endothelial Ca2+ concentration and vascular barrier disruption may lead to a proinflammatory state and thus affect Kf,c readouts.

5.4 Vascular Permeability to Protein

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Lung albumin extravasation has also been measured with a dual-isotope technique using both 125I-albumin and 131 I-albumin. 131I-albumin is used as a plasma volume marker injected immediately before death. This tracer is used to measure blood volume and hence it enables the determination of the tracer that has leaked out of the circulation. Kern and Malik [3] utilized this technique to evaluate the consequences of EDTA administration in rabbit lungs. 125I-albumin was used to measure the diffusive flux during infusion and 131 I-albumin measured total protein flux (diffusive and convective). Samples of perfusate and lung homogenate were counted for tracer activity. In this experiment, PS was calculated as before and the solute flux Js is calculated as

The intact endothelial junctional barrier is size-selective and impermeable to albumin; therefore, transvascular transport of labeled proteins such as albumin is used to determine vascular junctional permeability increase in lungs. The typical tracers used are 125I-bovine serum albumin, radiolabeled albumin, and Evans blue dye with conjugates that have high affinity for albumin. Experimentally, perfusate containing 125 I-albumin is infused at 200  mL/min into the pulmonary artery for 3–5  min and a sample of the venous effluent is saved for determination of perfusate tracer concentration. Unlabeled perfusate is used to flush the lung completely. Lung tissue and venous effluent samples are counted for 125 I  radioactivity and 125I-albumin activity and PS is calculated with the following equation:

PS = A(Ct )−1 ,

where A represents measured tissue counts per gram of dry lung tissue, t is duration of exposure to tracer albumin (in minutes), and C is concentration of 125I-albumin in the perfusate [3]. To assess microvascular permeability to albumin using the Evans blue dye assay in mice, 20 mg/kg Evans blue dye– albumin is intravenously injected and allowed to circulate for approximately 30 min. The lungs are then perfused free of blood and excised. Spectrophotometric differences in lung supernatant read at 620 nm after formamide incubation form the basis of this assay. The PS index has been a useful tool for detection of lung vascular injury in animal models such as tracking permeability response to LPS, pancreatitis-induced lung injury, reperfusion-induced injury, murine sepsis, and after acid aspiration injury. Vascular permeability to albumin may be underestimated in the presence of vascular occlusion with derecruitment or vascular collapse. Pulmonary lymphatic reabsorption of the tracer may also underestimate albumin permeability.

J s = ( PS δ C ) + (1 − s ) J vC ,

where Jv is the water flux across the endothelium, s is the ­albumin reflection coefficient, and C is an average concentration of the solute across the endothelium. When rewritten together, both (PS and Js) equations can be combined to solve for s.

5.5 Bronchoalveolar Lavage Lavage of the bronchial airways has been used extensively to detect lung injury. Plasma proteins and inflammatory cells are normally present in undetectable amounts in the alveolar space owing to highly selective permeability across the endothelial and epithelial barriers. Significant increases in BAL fluid protein concentration approaching that of the plasma protein concentration indicates the loss of endothelial barrier and epithelial barrier functions and hence may be used as a marker of increased permeability of both barriers. BAL in animal models is performed by cannulating the trachea and instilling a known volume of sterile solution followed by ­collection of the fluid by gentle aspiration. BAL analysis is a simple and reproducible method of quantifying lung vascular leak. Recently, the application of proteomic analysis of BAL fluid [119] was used to provide a more comprehensive report of changes in protein profile after lung injury; hence, it may be useful as a marker for analysis of injury of specific lung cell types, but its utility has not been fully exploited.

6 Summary We have reviewed several animal models of lung vascular permeability and discussed advantages and disadvantages of each. Although each model contributes important facts, none of these models adequately mimic the full spectrum of human ALI and ARDS. Although the “perfect” model of studying

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increased lung vascular permeability remains out of reach, the different models described have their strengths. Thus, in choosing an experimental animal model, the first question to ask is what is being modeled? For example, if the answer is increased endothelial permeability as the result of alterations at the level of adherens junctions, then the model of choice should be a mediator that results in disassembly of adherens junctions. As we have highlighted, the regulation of endothelial permeability is still incompletely understood; however, certain models of lung vascular permeability have brought us closer to unlocking the in vivo mechanisms involved in pathological lung fluid gain. One theme which has emerged is that homeostatic regulation of lung fluid permeability is governed by a complex interplay of mechanical, chemical, humoral, neural, and intracellular signaling mechanisms. Recently, a great deal of emphasis has shifted to genetic knockout models of disease. With the use of these mouse models, it is hoped that immense progress will be made in clarifying the mechanisms of increased lung endothelial permeability as mediated by cytoskeletal proteins and enzymes, receptors and channels, transcription factors, and coagulation cascade molecules. Acknowledgements  The authors would like to thank Stephen M. Vogel for invaluable comments and providing Figs. 3 and 5. We are also grateful to Cynthia Sanders for manuscript preparation.

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S.H. Mirza et al. 99. Wirtz HR, Dobbs LG (1990) Calcium mobilization and exocytosis after one mechanical stretch of lung epithelial cells. Science 250:1266–1269 100. Kawano T, Mori S, Cybulsky M et al (1987) Effect of granulocyte depletion in a ventilated surfactant-depleted lung. J Appl Physiol 62:27–33 101. O'Mahony DS, Liles WC, Altemeier WA et al (2006) Mechanical ventilation interacts with endotoxemia to induce extrapulmonary organ dysfunction. Crit Care 10:R136 102. Hla T (2003) Signaling and biological actions of sphingosine 1-phosphate. Pharmacol Res 47:401–407 103. Okajima F (2002) Plasma lipoproteins behave as carriers of extracellular sphingosine 1-phosphate: is this an atherogenic mediator or an anti-atherogenic mediator? Biochim Biophys Acta 1582:132–137 104. Peng X, Hassoun PM, Sammani S et al (2004) Protective effects of sphingosine 1-phosphate in murine endotoxin-induced inflammatory lung injury. Am J Respir Crit Care Med 169:1245–1251 105. Tauseef M, Kini V, Knezevic N et al (2008) Activation of sphingosine kinase-1 reverses the increase in lung vascular permeability through sphingosine-1-phosphate receptor signaling in endothelial cells. Circ Res 103:1164–1172 106. Jho D, Mehta D, Ahmmed G et al (2005) Angiopoietin-1 opposes VEGF-induced increase in endothelial permeability by inhibiting TRPC1-dependent Ca2+ influx. Circ Res 96:1282–1290 107. Pearce ML, Yamashita J, Beazell J (1965) Measurement of pulmonary edema. Circ Res 16:482–488 108. Kirov MY, Kuzkov VV, Kuklin VN, Waerhaug K, Bjertnaes LJ (2004) Extravascular lung water assessed by transpulmonary single thermodilution and postmortem gravimetry in sheep. Crit Care 8:R451–R458 109. Bock JC, Lewis FR (1990) Clinical relevance of lung water measurement with the thermal-dye dilution technique. J Surg Res 48:254–265 110. Godje O, Peyerl M, Seebauer T, Dewald O, Reichart B (1998) Reproducibility of double indicator dilution measurements of intrathoracic blood volume compartments, extravascular lung water and liver function. Chest 113:1070–1077 111. Bouvet F, Dreyfuss D, Lebtahi R, Martet G, Le Guludec D, Saumon G (2005) Noninvasive evaluation of acute capillary permeability changes during high-volume ventilation in rats with and without hypercapnic acidosis. Crit Care Med 33:155–160 112. Gluecker T, Capasso P, Schnyder P et  al (1999) Clinical and radiologic features of pulmonary edema. Radiographics 19: 1507–1531 113. Scillia P, Kafi SA, Melot C, Keyzer C, Naeije R, Gevenois PA (2001) Oleic acid-induced lung injury: thin-section CT evaluation in dogs. Radiology 219:724–731 114. Schueller-Weidekamm C, Wassermann E, Redl H et  al (2006) Dynamic CT measurement of pulmonary enhancement in piglets with experimental acute respiratory distress syndrome. Radiology 239:398–405 115. Velazquez M, Weibel ER, Kuhn C 3rd, Schuster DP (1991) PET evaluation of pulmonary vascular permeability: a structure-function correlation. J Appl Physiol 70:2206–2216 116. Caruthers SD, Paschal CB, Pou NA, Roselli RJ, Harris TR (1998) Regional measurements of pulmonary edema by using magnetic resonance imaging. J Appl Physiol 84:2143–2153 117. Gaar KA Jr, Taylor AE, Owens LJ, Guyton AC (1967) Pulmonary capillary pressure and filtration coefficient in the isolated perfused lung. Am J Physiol 213:910–914 118. Bhattacharya J (2007) Interpreting the lung microvascular filtration coefficient. Am J Physiol Lung Cell Mol Physiol 293:L9–L10 119. Noel-Georis I, Bernard A, Falmagne P, Wattiez R (2001) Proteomics as the tool to search for lung disease markers in bronchoalveolar lavage. Dis Markers 17:271–284

Chapter 33

Isolation and Culture of Pulmonary Vascular Smooth Muscle and Endothelial Cells Carmelle V. Remillard, Ayako Makino, and Jason X.-J. Yuan

Abstract  Experimental quality is directly proportional to cell quality. This might not be a valid mathematical equation, but it very succinctly and elegantly describes the work of biologists from all fields of research: molecular biology or cell biology, proliferation or apoptosis assays, patch clamp electrophysiology or flow cytometry. One element lies at the heart of the success of each of these procedures: viable and healthy cells. Many days and weeks of valuable experimentation time have been spent perfecting cell isolation techniques, simply to guarantee that we can gather reliable data. Although it is possible to purchase cells from various sources, most research groups have developed their own techniques for isolating cells from tissues, techniques which can be adapted relatively easily from one tissue type to another. In addition, although it is always desirable to use freshly isolated cells (less than 8 h after isolation), many investigators have turned to cell culture as a viable ­alternative to freshly dissociated cells, although this may present some scientific challenges and dilemmas. The current ­chapter addresses the isolation of pulmonary artery smooth ­muscle cells (PASMCs) and pulmonary artery endothelial (PAECs), particularly from humans, rats, and mice. Generally speaking, the methods we outline can also be applied to PASMCs and PAECs from other species, although some modifications may be required. Readers are advised to consult the extensive literature to identify the technique most applicable to their needs. Keywords  Cell preparation • Tissue dissection • Pulmonary artery • Freshly dissociated cells • Primary culture • Cell physiology

1 Introduction Experimental quality is directly proportional to cell quality. This might not be a valid mathematical equation, but it very succinctly and elegantly describes the work of biologists from all fields of research: molecular biology or cell biology, proliferation or apoptosis assays, patch clamp electrophysiology or flow cytometry. One element lies at the heart of the success (and reproducibility!) of each of these procedures: viable and healthy cells. Many days and weeks of valuable experimentation time have been spent perfecting cell isolation techniques, simply to guarantee that we can gather reliable data. Although it is possible to purchase cells from various sources (e.g., Lonza, Invitrogen, and ATCC in North America), most research groups have developed their own techniques for isolating cells from tissues, techniques which can be adapted relatively easily from one tissue type to another. In addition, although it is always desirable to use freshly isolated cells (less than 8 h after isolation), many investigators have turned to cell culture as a viable alternative to freshly dissociated cells, although this may present some scientific challenges and dilemmas. The current chapter addresses the isolation of pulmonary artery smooth muscle cells (PASMCs) and pulmonary artery endothelial (PAECs), particularly from humans, rats, and mice. Generally speaking, the methods we outline can also be applied to PASMCs and PAECs from other species, although some modifications may be required. Readers are advised to consult the extensive literature to identify the technique most applicable to their needs.

2 Cell Isolation Theory and Use of Enzymes for Tissue Dissociation and Cell Harvesting C.V. Remillard (*) Division of Pulmonary Critical Care Medicine, University of California, San Diego, 9500 Gilman Drive, MC 0725, San Diego, CA 92093, USA e-mail: [email protected]

Enzymes are the primary tools in tissue culture research insofar as it concerns the isolation of cells and dissociation of intact tissue. Despite the widespread use of enzymes such as collagenase, elastase, papain, etc., their mechanisms of action

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_33, © Springer Science+Business Media, LLC 2011

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C.V. Remillard et al. Table 1  Parameters affecting cell isolation outcome 1. Type of tissue 2. Species of origin 3. Age of the animal 4. Dissociation medium used 5. Enzyme(s) used 6. Impurities in crude enzyme preparation used 7. Concentrations(s) of enzymes used 8. Temperature 9. Incubation time

in various preparations are poorly understood. As a result, the choice of one technique or enzyme over another is arbitrary and based on experience rather than scientific theory. As mentioned already, the ultimate goal of cell isolation is to yield viable, functional dissociated cells. A number of factors which can determine the outcome of the cell isolation procedure. These are outlined in Table 1. Of these, only the type of tissue and species and the age of the animal are not a matter of choice. The other six parameters are highly variable and can greatly influence cell quality; as such, these are often manipulated to optimize individual cell isolation protocols. If one examines the literature for isolation techniques for a particular cell type (e.g., PASMCs), the results are varied because of individual experience with different enzyme combinations. Even manuals offer conflicting information, mostly due to a combination of (1) lack of information about the structure of the extracellular matrix and (2) the presence of unknown impurities in the crude enzyme preparations. Although we, as investigators, cannot control the ultimate purity of individual enzyme preparations (we rely on reputable companies to oversee that task), we can inform ourselves about the extracellular matrix components for our tissues of interest.

3 Tissue Collection and Isolation of Vascular Myocytes 3.1 Collection of Lung Tissue Samples Human lung tissue samples can be obtained from patients undergoing lung transplantation, lobectomy, and biopsy. Typically, a small (approximately 1 cm3) piece of the peripheral lung tissues is removed from the lung and placed in cold (4°C) saline until dissection can be performed (within 3 h of transplantation). For mice and rats, lungs and heart are removed en bloc following euthanasia (decapitation or cervical dislocation are ideal to reduce trauma to the lungs), and placed in warm N-(2-hydroxyethyl)piperazine-N¢-ethanesulfonic acid buffered saline solution (HBSS). Once the tissues have been obtained, fat, blood, and connective tissue are removed using aseptic techniques so that vascular tissues can be isolated.

Fig. 1  Structure of the pulmonary arteries. Cartoon description of the pulmonary vasculature starting from the whole lung and at successive magnifications. Close-up structure identifies the three main layers of the artery: the intima, containing pulmonary artery endothelial cells (PAEC), the media, containing pulmonary artery smooth muscle cells (PASMC), and the adventitia, composed primarily of fibroblasts (PAFB) and fibers such as collagen and elastin

3.2 Preparation of Primary Culture PASMCs There are two general methods for the preparation of primary cultures of vascular smooth muscle cells. One involves the direct isolation of cells from enzymatically digested vessels and the other involves isolating cells that migrate out from small tissue samples. Since enzyme dissociation is the most commonly used approach, we will describe procedures that have successfully yielded viable healthy human, mouse, and rat PASMCs. The vascular wall is organized into three layers: an inner layer composed of endothelial cells, a middle layer composed of smooth muscle cells, and an outer adventitial layer composed of fibroblasts, connective tissues, and nerve endings. (Fig.  1) The smooth muscle cells that make up the medial layer are involved in processes such as vascular growth and repair, response to injury, and vascular contraction. For the enzymes to reach the medial PASMCs and to isolate pure smooth muscle cells, the first steps of PASMC isolation involve removing the adventitia and intima (Fig. 2a, b). The vessel is placed in fresh HBSS, carefully stretched and pinned onto a dissecting dish. The adventitia (which mainly contains fibroblasts) can be dissected away from the media with forceps. We have also found that a brief treatment (approximately 10  min) with collagenase can significantly “loosen” the adventitia so that it can be “peeled” off the vessel. After removal of the adventitia, the vessel is cut open longitudinally so the endothelial layer can be gently scraped away. The pulmonary artery, now mostly vascular media, is then cut into pieces and placed in collagenase–HBSS (Fig. 2c) at 37°C for 20 min, after which the tissue is allowed to recover overnight for 10–12 h at 37°C in Dulbecco’s modification of Eagle’s medium (DMEM) supplemented with fetal bovine serum (FBS), penicillin/streptomycin, and l-glutamine. The vessel is then transferred to a fresh enzyme solution containing collagenase, elastase, and albumin, and is incubated at

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10% FBS, insulin, human epidermal growth factor , human fibroblast growth factor B, gentamicin, and amphotericin–B. Cells are incubated at 37°C for further growth. The cell medium should be changed initially after 24 h and every 48 h subsequently. Although we have placed emphasis on DMEM and SMGM, several media are available to support SMC growth, including Eagle’s minimal essential medium), medium 199 with Earle’s or Hank’s salt (M199), and Ham’s F12. Many researchers now use plastic, rather than glass, dishes to plate smooth muscle cells. Cell attachment to plastic dishes or coverslips may be enhanced by coating the surface with fibronectin, vitronectin, laminin, collagen, or lysine [1–3].

3.3 Preparation of Primary Culture PAECs

Fig. 2  Overview of enzymatic isolation of pulmonary artery smooth muscle cells (PASMC). (a) Pulmonary arteries are removed from the lung and the adventitia is stripped using a combination of enzymatic digestion and forceps. (b) The arterial ring is cut open and the endothelium is removed with gentle rubbing. (c) The tissue is cut into small pieces for initial digestion by collagenase. (d) Following an overnight rest period in enzyme-free solution, new enzymes are added, the tissue is digested, and trituration releases isolated cells

37°C for approximately 50 min. Finally, the tissue is gently triturated to release cells (Fig. 2d) and DMEM (containing 20% FBS) is added to halt digestion. Cells are separated from tissue debris by filtration through a nylon mesh. The enzyme-containing cell suspension is then centrifuged, and the pellet containing the PASMCs is resuspended in fresh 10% FBS–DMEM. Dissociated cells can now be refrigerated for immediate use, or plated in tissue culture dishes containing prewarmed growth medium. For rat PASMCs, we use 10% FBS–DMEM. For human PASMCs, we use a smooth muscle growth medium (SMGM; Lonza) which contains

As with PASMCs, there are a number of isolation techniques which can yield viable endothelial cells. Probably the simplest technique is to recover endothelial cells by rubbing them off the intimal surface of the pulmonary arteries. In this technique (Fig.  3a), the isolated and cleaned pulmonary artery is excised, then inverted to expose the intimal layer. Endothelial cells are obtained by gently scraping the intimal layer with a sterile scalpel or a plastic cell lifter or scraper. Harvested cells are then plated with culture medium (e.g., F12 nutrient mixture, DMEM) supplemented with 10–20% FBS and antibiotics [4]. The latter technique can be used only with main pulmonary artery segments; however, King et  al. [5] have since described a modified method to isolate rat microvascular PAECs (PMVECs). In this technique (Fig. 3b), thin strips are removed from the lung periphery adjacent to the pleural surface, finely minced, and treated with a collagenase (type 2, Worthington) and bovine serum albumin–DMEM at 37°C. After approximately 15 min, the mixture is filtered through a sterile 80-mesh sieve and centrifuged to pellet PMVECs. PMVECs are resuspended with complete medium composed of one part microvascular conditioned medium and three parts incomplete medium [80% RPMI, 20% FBS, heparin, Endogro (Vec Technologies), and penicillin–streptomycin]. The centrifugation–resuspension process is repeated once and the final cell pellet is resuspended in complete medium and allowed to rest at 37°C prior to plating. Finally, PMVEC suspension drops are placed on coverslips, and the cells are allowed to settle and attach for 1 h at 37°C (5% CO2, 95% O2) before complete medium is added. As shown above, enzymatic dissociation is effective for PMVEC isolation. However, endothelial cells from the main pulmonary artery can also be isolated using enzymes, although the process itself is somewhat different from that for PASMCs or PMVECs. Stevens et al. [6] isolated bovine PAECs using

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Fig.  3  Different techniques used for isolation of pulmonary artery endothelial cells. Each procedure ultimately begins with isolated pulmonary arteries which are excised from the lung parenchyma. (a) Cell scraping: arteries are inverted and exposed endothelial cells are scraped off and plated. (b) Enzymatic dissociation: arteries are minced, digested, and filtered to remove pulmonary artery smooth muscle cells and other cellular contaminants. Retrieved pulmonary artery endothelial cells are then purified for culture and/or experimentation. (c) Isolated cells are filled with enzyme solution and ligated proximally and distally. Perfusate containing cells [including pulmonary artery smooth muscle cells, pulmonary artery endothelial cells, and fibroblasts] is filtered

to isolate pulmonary artery endothelial cells. (d) Magnetic beads: minced arteries are digested, then exposed to magnetic beads coated with anti-IgG antibodies specific for pulmonary artery endothelial cells. Pulmonary artery endothelial cells with bound beads are isolated using a magnet, then resuspended and plated. (e) Endothelial adhesion: arteries are cleaned and cut open, then laid in culture dishes with pulmonary artery endothelial cells facing down. Pulmonary artery endothelial cells gradually adhere to the culture surface, and attached pulmonary artery smooth muscle cells and adventitia are “peeled” off to leave adhered pulmonary artery endothelial cells, which are then plated and cultured

an enzymatic dispersion technique first described for aortic endothelial cells by Coughlin et al. [7]. A similar approach has also been used to isolate PAECs from lambs [8] (Fig. 3c). Briefly, pulmonary arteries are dissected into the lung parenchyma, adventitia is removed as shown in Fig.  2a, and the lumen is washed with phosphate-buffered saline (PBS). The intercostal and distal branch ends are ligated or clamped, cut

distally, and the lumen is filled with 0.1–0.25% collagenase (type A from Roche or type 2 from Worthington) in Hank’s balanced salt solution. The digesting vessel (with ends occluded) is then immersed at 37°C. After approximately 20  min, the collagenase solution is collected, the lumen is washed gently with culture medium, and the wash and collagenase solutions are pooled and centrifuged to pellet PAECs.

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The pellet containing endothelial cells is then resuspended in culture medium and plated. For this technique, the culture medium consists of a minimal essential medium (d-valine) supplemented with 20% calf serum, l-glutamine, penicillin, and streptomycin. We have isolated human PAECs via a modified enzymatic technique first developed for mouse lung PAEC isolation [9] (Fig. 3d). In our procedure, tissues are minced and incubated with M199 containing type 2 collagenase and type 2 dispase for approximately 1 h at 37°C. The digested material is then filtered through a sterile 40-mm mesh and washed in 2% fetal calf serum–M199. The cells are then incubated with Dynabeads (Invitrogen), which are beads coated with sheep anti-rat IgG and incubated with purified anti-platelet/endothelial cell adhesion molecule (PECAM) or anti-CD31 monoclonal antibody. Cells are exposed to the antibody-coated beads for 1 h at 4°C and bead-covered cells are captured by a Dynal magnet. Once the supernatant containing unbound PAECs and contaminating PASMCs or fibroblasts has been aspirated, bound PAECs are resuspended in M199 and plated on glass coverslips. As with human PASMCs, medium is changed every other day until cells are confluent and ready for splitting or experimentation. In our experience, the yield of purified human PAECs via this technique exceeds 90%. A similar procedure can also be used to isolate PASMCs or fibroblasts, using the appropriate cell-specific antibodies. One final PAEC isolation technique involves neither enzymatic dispersion nor scraping PAECs off the arterial lumen, and has been described for rat and bovine PAECs [10] (Fig.  3e). The cleaned arteries are cut lengthwise and the lumen is rinsed clean with PBS. The vessels are cut into 3–5mm2 pieces and placed lumen side down into a culture dish for a few minutes until the tissues adhere to the dish surface. Tissues are then covered with 20% FBS–RPMI medium and allowed to grow for 3 days in a normoxic (5% CO2, 37°C) incubator. Tissue pieces are then lifted carefully out of the medium and adherent PAECs are allowed to grow. After several days, PAECs are trypsinized, diluted, and sparsely plated (one or two cells per well in a 96-well dish) to select for pure cobblestone-shaped PAECs. Selected cell populations are then expanded, and cultured for experimentation.

4 Cell Subculture and Storage 4.1 Subculturing Cells Because large quantities of primary tissues can be difficult to obtain, subculturing of cells can be used to amplify the sample size for future experimental use. Cultured cells should be passaged when they are subconfluent (approximately

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70–80% confluent). The process itself is quite straightforward, and the same procedure can be applied for many different cell types, including PASMCs and PAECs. Cultured cells are first washed with sterile HBSS or Dulbecco’s PBS to remove any dead cells or other cellular detritus. Washed cells are then treated with a trypsin–EDTA solution, which causes the cells to “ball up” and detach from the culture dish; full detachment typically takes less than 5 min, although this will vary according to temperature, cell confluence, cell type, and trypsin concentration. The trypsin exposure time should be kept as short as possibly as prolonged trypsin exposure may damage cell membranes and compromise cell viability. When most of the cells are floating freely, trypsin inhibitor or a trypsin neutralization solution is added to stop trypsinization. The cell suspension is then centrifuged and the pellet containing cells is resuspended in warm medium for renewed plating. As with freshly isolated and plated cells, medium should be changed initially after 24 h and every 48 h thereafter following each cell passaging. The optimal cell density for passaging and plating varies with the size (i.e., surface area) of the plating dish. With commercially available cells, the supplier normally provides an estimate of the basal cell density. For those using freshly dissociated cells, the cells must be counted to obtain an approximation of the cell number. The optimal seeding density is also dependent on the cell type. For PASMCs, we have determined cell proliferation is optimal at an initial seeding density of approximately 3,500 cells/cm2. Cells seeded at a higher initial density begin to proliferate earlier than those seeded at low density. Proliferation, if plotted as a function of time, is sigmoidal. In the initial lag phase, cell growth is minimal. However, within 2–3  days, cell growth increases exponentially during the logarithmic growth phase until confluence is attained, at which point growth is maximal and cells may begin to differentiate. In the presence of the required growth factors to stimulate cell proliferation, initial seeding density and seeding efficiency regulate the rate of cell proliferation.

4.2 Cell Storage Cell storage in liquid nitrogen is the most efficient way to keep cells for long-term storage or for later use, especially those cells originating from a rare tissue source. When the cultured cells are approximately 70–80% confluent, they are passaged as described earlier. However, instead of using medium, one resuspends the final cell pellet in a freezing solution consisting of dimethyl sulfoxide (5–10%) and medium (FBS–DMEM for rat PASMCs, FBS–SMGM for human PASMCs, Endothelial Growth Medium (EGM) for PAECs). Cells in the freezing solution should be aliquoted in cryogenic tubes, and gradually frozen until they can be placed in liquid nitrogen. Many core cell

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facilities have equipment that can gradually freeze samples by decreasing the freezer temperature by 1°C every minute down to −196°C. A simple, and less expensive, alternative also exists for individuals who prefer to handle cell freezing personally. A number of suppliers offer low-technology (but efficient) −80°C freezer-safe containers equipped with plastic inserts designed to hold cryovials. The technology is simple. The lower chamber of the container is filled with 2-propanol and the plastic insert containing cryovials rests upon the alcohol. The container is then placed in a −80°C freezer. The freezing point of 2-propanol is −89°C. Therefore, when placed in a −80°C freezer, cell suspensions are gradually frozen in just 3–4 h and can then be transferred to liquid nitrogen for prolonged storage.

4.3 Cell Thawing and Reseeding After cells have been frozen, they can be thawed and plated for use later. Cells should be thawed quickly (in under 3 min) in a 37°C water bath. The vial should be sterilized with ethanol before it is opened to avoid contamination of this cell stock. The cell solution should be gently aspirated to evenly distribute the cells in their medium. Culture dishes containing prewarmed medium are then seeded, gently rocked to disperse the cells, and incubated at 37°C. As always, after the initial seeding, cell medium should be changed initially after 24 h and subsequently every 48 h.

5 Morphological Characteristics and Identification of PASMCs Epithelioid and the spindle-shaped morphological phenotypes of vascular smooth muscle cells have been defined [11], corresponding to the synthetic and contractile smooth muscle cell types, respectively. Contractile (i.e., quiescent) vascular smooth muscle cells express high levels of contractile proteins, including myosin, and low levels of a-actin. In contrast, synthetic (i.e., proliferating) vascular smooth muscle cells express high levels of a-actin, extracellular matrix proteins, and low levels of myosin. Typically, spindle-shaped contractile vascular smooth muscle cells are nonproliferating or nonmigratory, whereas the epithelioid synthetic vascular smooth muscle cells have entered the cell cycle and are actively proliferating [12]. With phase-contrast microscopy, smooth muscle cellss appear flattened and spindle-shaped with central oval nuclei and long cytoplasmic extensions. Freshly dissociated cells, although smaller and thicker, have a similar appearance.

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Confluent cells appear aligned in parallel so that the broad nuclear region of a cell lies adjacent to the thin cytoplasmic area of another, forming a “hill-and-valley” appearance. Unlike endothelial cells, smooth muscle cells do not show contact inhibition and will therefore grow as multilayers [1]. The time of phenotypic change (i.e., from synthetic to contractile phenotypes) is dependent on the tissue type, donor age, and species. It is not dependent on the presence or absence of serum or serum components in the culture medium [13]. Figure  4a shows a photomicrograph of subcultured human PASMCs and primary cultured rat PASMCs. a-Actin is produced from a smooth-muscle-specific gene. During development, when vascular smooth muscle cells exhibit a “synthetic” phenotype, the main actin isoform expressed is b-actin [14, 15]. As the vessel matures, there is a decrease in expression of b-actin and an increase in smoothmuscle-specific a-actin expression [15]. Smooth muscle a-actin expression persists in smooth muscle cells even after extensive passaging in culture [16]. Figure 4a shows fluorescence images of cultured human and rat PASMCs stained with the membrane-permeable nucleic acid stain 4¢,6¢-diamidino-2-phenylinodole (DAPI) and smooth muscle a-actin. The blue fluorescence emitted at 461 nm by DAPI is used to estimate the total cell number in the cultures as it stains each nucleated smooth muscle cell. The specific monoclonal antibody raised against smooth muscle a-actin is used to evaluate the cellular purity of the cultures, and the secondary antibody fluorescein isothiocyanate (FITC)-conjugated antimouse IgG displays the fluorescence image based on emissions at 529 nm, as depicted in the overlay images of DAPI and smooth muscle a-actin staining (Fig. 4a, right panels). The specificity of smooth muscle a-actin for smooth muscle cells and not for fibroblasts is clearly shown in the rat PASMC micrograph (lower-right panel); PASMCs show clear a-actin filament staining (nucleus and filaments appear), whereas the pulmonary fibroblasts do not (nucleus only).

6 Morphological Characteristics and Identification of PAECs PAECs have a very characteristic cobblestone appearance, with the cells significantly smaller and more block-like than the neighboring PASMCs (Fig. 4b); this appearance persists with prolonged cell culture. When organized as a syncitium, PAEC junctures are relatively impermeable and provide what is termed an “endothelial barrier” that protects the vascular wall from circulating factors. Endothelial barrier dysfunction underlies pulmonary vascular edema and, potentially, pulmonary vascular remodeling.

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One of the main problems associated with PAEC isolation, regardless of the technique used, is contamination of the cell preparation with PASMCs and fibroblasts. Therefore, it is important to be vigilant and check PAEC cultures regularly and remove contaminating cells by scraping and aspiration. Once “clean” primary cultures have been established, PAEC purity is verified by uptake of 1,1¢-dioctadecyl-3,3,3¢,3¢tetramethylindocarbocyanine perchlorate labeled acetylated low-density lipoprotein (acLDL) [4], by Bandeiraea simplicifolia (BS-1) lectin–FITC staining or binding panel [5], or by positive factor VIII or PECAM-1 immunocytochemical staining and negative immunostaining with smooth muscle a-actin antibodies. Alternatively, PAECs can be identified by positive staining and agglutination with Helix pomatia and PMVEC can be identified by positive staining and agglutination with the lectins Glycine max and Griffonia simplicifolia [17]. Both PMVECs and PAECs stain positive for endothelial nitric oxide synthase, PECAM-1, and vascular endothelial cadherin. Figure 4b depicts typical results for factor VII and PECAM-1 immunocytochemistry in endothelial cells from the main pulmonary artery from different species. An example of Griffonia simplicifolia agglutination of PMVECs is also shown. Figure 5a shows typical PAECs prepared from mouse lung tissues using magnetic beads (Dynabeads) coated with the specific antibody against the endothelial cell marker, CD31/PECAM-1.

7 Functional Characteristics of Mouse PAECs

Fig. 4  Appearance and identification of pulmonary artery smooth muscle cells (PASMC) and pulmonary artery endothelial cells (PAEC). (a) Micrographs of subcultured human (top) and primary cultured rat PASMC (bottom). Phase-contrast images and 4¢,6¢-diamidino-2-phenylinodole (DAPI)/smooth muscle a-actin (SMA) overlays are shown for both cell types. (b) The rat pulmonary artery endothelial cell phase-contrast image clearly shows the less elongated block-like morphology of pulmonary artery endothelial cells. Other panels show examples of pulmonary artery endothelial cell identification techniques. Factor VIII appears as brown staining in the cytoplasm (shown by an arrow). Platelet/endothelial cell adhesion molecule 1 stains primarily at the intercellular junctions (shown in green, DAPI-marked nucleus is in blue). Griffonia simplicifolia staining and agglutination is typical for pulmonary microvascular endothelial cells. (Images reproduced from [5, 20–23] with permission)

PAECs isolated from mouse lung tissues using magnetic beads (Dynabeads) coated with anti-CD31 antibody shows a high purity of the endothelial cell population determined by acLDL uptake and BS-1 staining (Fig. 5a). Passive depletion of intracellular Ca2+ stores (e.g., endoplasmic reticulum/­ sarcoplasmic reticulum) by cyclopiazonic acid (10  mM; a reversible sarcoplasmic/endoplasmic reticulum Ca2+-ATPase inhibitor) in the absence of extracellular Ca2+ (for 10–15 min) causes a rapid increase in cytosolic Ca2+ concentration (owing to store-operated Ca2+ entry) when extracellular Ca2+ is reintroduced (Fig. 5b). These data indicate that using magnetic beads coated with specific antibodies against the endothelial cell surface markers is an efficient method to sufficiently isolate pure PAECs from mouse lung tissues. The cells isolated using the Dynabeads technique not only exhibit typical morphology of endothelial cells similar to that of cells isolated from other methods, but also maintain normal cell functions, such as store-operated Ca2+ entry and changes in intracellular Ca2+ concentration in response to agonists. Therefore, mouse

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Fig.  5  Mouse pulmonary artery endothelial cells (PAEC). (a) Fluorescence images (upper panels) show that pulmonary artery endothelial cells isolated from mouse lungs exhibit high purity of the endothelial cell population. The percentage of cells positively stained with acetylated low-density lipoprotei (red) and BS-1–lectin (green) in the total cells stained with Hoechst 33342 (blue) was over 90%. Human

smooth muscle cells were used as a control. (b) Cyclopiazonic acid (CPA)-induced store depletion increases cytosolic Ca2+ concentration by inducing store-operated Ca2+ entry in pulmonary artery endothelial cells; restoration of extracellular Ca2+ in the presence of 10 mM cyclopiazonic acid induced a rapid rise in cytosolic Ca2+ concentration due to store-operated Ca2+ entry

PAECs isolated with the magnetic beads can be used in ­various experiments (e.g., electrophysiology, fluorescence microscopy, cell and molecular biology experiments) to study endothelial cell function and its contribution to the regulation of pulmonary vascular remodeling as well as cell proliferation, migration, and apoptosis.

expression and function is ­rapidly ­becoming an important concept in explaining the underlying mechanisms for a number of forms of pulmonary vascular disease, further emphasis may be placed on the role of autocrine (a cell synthesizes and/or secretes a substance that stimulates that same cell type to undergo a growth response) [19] and paracrine (cells responding to the growth factor synthesize and/or secrete a substance that stimulates neighboring cells of another cell type) growth factors in vascular smooth muscle cell growth [12]. Therefore, further modifications in current cell isolation and growth techniques may hold the key to elucidating the physiological basis of pulmonary vascular disease.

8 Summary Smooth muscle cell culture has been in use since 1913 [18], but it has undergone some significant changes over the decades. As can be attested by the scope of this monograph, the use of ­cultured and freshly dissociated PASMCs and PAECs has allowed for a thorough examination of the physiological processes and pathological changes in pulmonary hypertension. Mechanisms ranging from determination of cell phenotype and function, regulation of ion channels and transporter activity and gene expression have already been elucidated which may explain, at least, in part the physiological regulation of the pulmonary ­circulation under “normal” and pathogenic conditions. Because  heterogeneity of cell

References 1. Gallichio MA (2001) Culture of human smooth muscle cells. In: Drew AF (ed) Atherosclerosis: experimental models and protocols. Humana, Totowa, pp 137–146 2. Wagner RC, Matthews MA (1975) The isolation and culture of capillary endothelium from epididymal fat. Microvasc Res 10:286–297 3. MacPhee MJ, Wiltrout R, McCormick KL, Sayers TJ, Pilaro AM (1994) A method for obtaining and culturing large numbers of purified organ-derived murine endothelial cells. J Leukoc Biol 55:467–475

33  Isolation and Culture of Pulmonary Vascular Smooth Muscle and Endothelial Cells 4. Moore TM, Brough GH, Babal P, Kelly JJ, Li M, Stevens T (1998) Store-operated calcium entry promotes shape change in pulmonary endothelial cells expressing Trp1. Am J Physiol 275:L574–L582 5. King J, Hamil T, Creighton J et al (2004) Structural and functional characteristics of lung macro- and microvascular endothelial cell phenotypes. Microvasc Res 67:139–151 6. Stevens T, Cornfield DN, McMurtry IF, Rodman RM (1994) Acute reductions in PO2 depolarize pulmonary artery endothelial cells and decrease [Ca2+]i. Am J Physiol 266:H1416–H1421 7. Coughlin SR, Moskowitz MA, Zetter BR, Antoniades HN, Levine L (1980) Platelet-dependent stimulation of prostacyclin synthesis by platelet-derived growth factor. Nature 288:600–602 8. Konduri GG, Ou J, Shi Y, Pritchard KA Jr (2003) Decreased association of HSP90 impairs endothelial nitric oxide synthase in fetal lambs with persistent pulmonary hypertension. Am J Physiol Heart Circ Physiol 285:H204–H211 9. Hartwell DW, Mayadas TN, Berger G et al (1998) Role of P-selectin cytoplasmic domain in granular targeting in  vivo and in early inflammatory responses. J Cell Biol 143:1129–1141 10. Zhu D, Zhang C, Medhora M, Jacobs ER (2002) CYP4A mRNA, protein, and product in rat lungs: novel localization in vascular endothelium. J Appl Physiol 93:330–337 11. Bochaton-Piallat M-L, Ropraz P, Gabbiani F, Gabbiani G (1996) Phenotypic heterogeneity of rat arterial smooth muscle cell clones: implications for the development of experimental intimal thickening. Arterioscler Thromb Vasc Biol 16:815–820 12. Berk B (2001) Vascular smooth muscle growth: autocrine growth mechanisms. Physiol Rev 81:999–1030 13. Chamley-Campbell JH, Campbell GR (1981) What controls smooth muscle phenotype? Atherosclerosis 40:347–357 14. Gabbiani G, Kocher O, Bloom WS, Vandekerckhove J, Weber K (1984) Actin expression in smooth muscle cells of rat aortic intimal

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thickening, human atheromatous plaque, and cultured rat aortic media. J Clin Invest 73:148–152 15. Kocher O, Gabbiani G (1987) Analysis of alpha-smooth-muscle actin mRNA expression in rat aortic smooth-muscle cells using a specific cDNA probe. Differentiation 34:201–209 16. Pauly RR, Bilato C, Cheng L, Monticone R, Crow MT (1997) Vascular smooth muscle cell cultures. Methods Cell Biol 52:133–154 17. Solodushko V, Fouty B (2007) Proproliferative phenotype of pulmonary microvascular endothelial cells. Am J Physiol Lung Cell Mol Physiol 292:L671–L677 18. Champey C (1913–1914) Quelques résultats de la méthode de culture des tissus. Arch Zool Exp Gen 53:42–51 19. Frid MG, Aldashev AA, Nemenoff RA, Higashito R, Westcott JY, Stenmark KR (1999) Subendothelial cells from normal bovine arteries exhibit autonomous growth and constitutively activated intracellular signaling. Arterioscler Thromb Vasc Biol 19: 2884–2893 20. Platoshyn O, Golovina VA, Bailey CL et  al (2000) Sustained membrane depolarization and pulmonary artery smooth muscle cell proliferation. Am J Physiol Cell Physiol 279: C1540–C1549 21. Sehgal PB, Mukhopadhyay S, Xu F, Patel K, Shah M (2007) Dysfunction of Golgi tethers, SNAREs, and SNAPs in monocrotaline-induced pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 292:L1526–L1542 22. Pan J-Q, Li J-C, Tan X, Sun W-D, Wang J-Y, Wang X-L (2007) The injury effect of oxygen free radicals in  vitro on cultured pulmonary artery endothelial cells from broilers. Res Vet Sci 82:382–387 23. Moldobaeva A, Wagner EM (2002) Heterogeneity of bronchial endothelial cell permeability. Am J Physiol Lung Cell Mol Physiol 283:L520–L527

Chapter 34

Conventional Patch Clamp Techniques and High-Throughput Patch Clamp Recordings on a Chip for Measuring Ion Channel Activity Carmelle V. Remillard and Jason X.-J. Yuan

Abstract  The term “channelopathy” refers to a disease whose primary underlying mechanism(s) involve dysfunctional ion channel activity. Long QT syndrome and cystic fibrosis are two prime examples of channelopathies, where altered transport of K+, Na+, and/or Cl− ions, respectively, leads to pathogenesis. Such findings have aimed a new spotlight at the overall physiological relevance of transmembrane ion flux in disease development and became of critical importance to industry with discoveries that many drugs can unintentionally induce various channelopathies such as epileptic seizures (often from inhibition of neuronal human NaV1.1 Na+ channels), or torsade de pointes (a lethal long QT syndrome produced most often by inhibition of cardiac human ERG K+ channels). As discussed extensively elsewhere in this monograph, ion channels with selective permeability to Na+, K+, and Ca2+ ions modulate pulmonary vasoreactivity, remodeling, and vascular permeability. Although biochemical and molecular biology techniques have contributed, a significant proportion of our knowledge of the role of ion channels in pulmonary physiology has been garnered by measuring transmembrane ionic currents in intact cells using patch clamp electrophysiology. The purpose of this chapter is to introduce the basics of conventional and high-throughput patch clamp technology and how this can be used to advance the research into the cause of pulmonary vascular diseases. Keywords  Methodology • Electrophysiology • Whole-cell recording • Single channel current • Ion channels • Membrane potential

1  Introduction The term “channelopathy” refers to a disease for which primary underlying mechanism(s) involve dysfunctional ion channel activity. Long QT syndrome and cystic fibrosis are two prime examples of channelopathies, where altered transport of K+, Na+, and/or Cl− ions, respectively, leads to pathogenesis (Fig.  1). Such findings have aimed a new spotlight at the overall physiological relevance of transmembrane ion flux in disease development and have gained critical importance in industry with discoveries that many drugs can unintentionally induce various channelopathies such as epileptic seizures (often from inhibition of neuronal human NaV1.1 Na+ channels), or torsade de pointes (a lethal long QT syndrome produced most often by inhibition of cardiac human ERG K+ channels). As discussed extensively elsewhere in this textbook, ion channels with selective permeability to Na+, K+, and Ca2+ ions (to name just a few) modulate pulmonary vasoreactivity, pulmonary vascular remodeling, and pulmonary vascular permeability. Although biochemical and molecular biological techniques have contributed, a significant proportion of our knowledge of the role of ion channels in pulmonary physiological and pathophysiological functions has been garnered by measuring transmembrane ionic currents in intact cells using patch clamp techniques. The purpose of this chapter is to introduce the basics of conventional and high-throughput patch clamp technology and to describe how this can be used to study pathogenic mechanisms of pulmonary hypertension and advance the research into the cause of pulmonary vascular diseases.

2  P  atch Clamp Electrophysiology: A Study in Time and Technology Development C.V. Remillard (*) Division of Pulmonary Critical Care Medicine, University of California, San Diego, 9500 Gilman Drive, MC 0725, San Diego, CA 92093, USA e-mail: [email protected]

2.1  The First Century: 1900s–2000 The study of electrical signaling and excitability of tissues can be traced back to the nineteenth century (Höber [1],

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and native membrane action potentials under unclamped conditions. The development of different membrane patch configurations (described in Sect.  3.2) has also enabled experimenters to fully investigate the cellular environment and, therefore, channel modulation.

2.2  N  ew Millennium Improvements and High-Throughput Electrophysiology

Fig. 1  Examples of known channelopathies in the human. The underlying channel gene (NCBI appellation) is indicated for each condition

as quoted by Hille [2]). As early as the 1950s, it was believed that positive and negative ions flowed along their electrochemical gradients across the biological membrane via pores. In the early part of the decade, Hodgkin and Huxley were the first to develop a comprehensive model to describe membrane electrical circuits [2–5]. Almost 20 years later, Noble and Tsien [6] described a slow outward current carried by K+ in loose membrane patches from cardiac Purkinje fibers. A year later, Bean et al. [7] recorded discrete electrical events from bacterial membrane proteins embedded in artificial lipid bilayers. In 1976, Neher and Sakmann published the first report of unitary cationic currents recorded from a denervated frog muscle fiber membrane patch using glass micropipettes [8]. The next 5 years saw the optimization of cell isolation techniques, pipette fabrication techniques (to control pipette geometry, size, and composition) (reviewed in [9]), and electronic component designs which improved recording capability and quality. In 1981, Hamill et al. [10] published the breakthrough discovery which revolutionized patch clamping and led to present-day applications; the application of suction to the membrane patch via the glass pipette increased seal resistance into the gigaohm range. Combined with the improved electronic detection and acquisition components, the gigaohm seal allowed for (1) improved signal-to-noise ratio to enable resolution of individual components of currents, (2) clamping the membrane voltage or applying controlled current pulses directly to membrane proteins within the patched membrane area, and (3) improved mechanical stability of the patch during excision. More improvements, albeit minor in terms of technique, but major in terms of the accessibility and precision of the technology, have led to the conventional patch clamp techniques which allow for the measurement of subpicoampere unitary ­(single-channel) currents, macroscopic (whole-cell) currents,

As with any technique that is laborious, tedious, and technically demanding, there have been a great number of attempts to automate voltage-clamp electrophysiology techniques. However two simultaneous events finally precipitated successful automation methods: (1) the evolution of microelectrode manufacturing system (MEMS) techniques to enable nanometer-sized technologies, specifically the creation of micron-level holes in various substrates (i.e., a manufacturing-friendly method became available), and (2) the demonstration of channelopathies and particularly of ionchannel-related pharmaceutical liabilities (i.e., the market revenue potential increased). Ion channels are considered to be relatively accessible drug targets by virtue of their being located on the plasma membrane. From genomic data, it is estimated that ion channels represent up to 25% of the available drug targets, yet, despite this, presently only 5–7% of compounds on the market are targeted against ion channels [11, 12]. A 2006 report suggested that ion channels and transporters represented 6% (76 targets out of a total of 1,267) of all research targets; they represented 12% (32 targets out of a total of 268) of the successful targets, meaning that they were actively being pursued for clinical ­applications [13] (Fig. 2). The overall distribution patterns of successful and research targets were similar to those reported in independent studies in 2000 and 2002 [11, 14], and the numbers are comparable to the distribution of the drugable human genome published by Venter et al. in 2001 [15]. The pharmaceutical industry needs to screen hundreds to thousands of compounds per day, but an electrophysiologist can only perform ten to 15 experiments per day. In response to the demand, other techniques have been developed that estimate ion channel activity without having to voltageclamp the cells, such as fluorescence techniques, radioligand displacement, and radioisotope-uptake assays. These secondary techniques are quite limited in their ability to resolve active from inactive compounds because they are unable to clamp the membrane voltage, are less accurate, and hence have high risk of false positives or false negatives. False ­positives can cost a company tens to hundreds of thousands of dollars annually, but false negatives can cost billions in  lost revenue. The evolution of voltage-clamp to the ­high-throughput forum has been slow, and impeded mainly by the slow development of the necessary technological

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will find a description of the basic techniques involved in ­forming the gigaohm seal. For discussions of other factors, including pipette fabrication, microcircuitry, electronics, and voltage-clamp protocols, of patch clamping readers should consult other textbooks and monographs on these subjects [17, 18].

3.1  Forming the Gigaohm Seal A pipette with a tip resistance ranging from 3 to 6 MW is maneuvered toward the cells, typically using a motorized micromanipulator. When the pipette filled with electrolyte solution is immersed in the bathing medium, a junction potential is created which should be nulled before touching the cell. As the pipette approaches the cell, the tip resistance increases. With the tip of the pipette firmly pressed down on the cell (near the center of the cell to satisfy the “space clamp” criterion, which requires that all regions of the membrane be polarized simultaneously to avoid voltage escape, however travel of the electrons may be physically hindered by thin spaces or dense cytosol), negative pressure (suction) is applied via a port in the pipette holder, and the tip resistance increases gradually into the gigaohm range (Fig. 3a). Fig. 2  Biochemical classes of research (a) and successful (b) drug targets. (a) A sampling of 1,267 research target drugs were screened for classification. (b) A sampling of 268 clinically successful drugs sorted by biochemical class. For (a) and (b), the percentage given represents the fraction of the total number of compounds screened. Numbers in parentheses represent the actual number of compounds for each classification. (From [13]. Adapted with permission from the RHW American Society for Pharmacology and Experimental Therapeutics)

capabilities. A case in point is the introduction of planar voltage-clamp electrodes. Development of the latter was attempted in 1975 [16] but, at the time, could not be pursued  further owing to the unavailability of the necessary technologies or of driving forces. As of July 2009, we counted at least five different automated conventional electrophysiology platforms that use glass pipettes (Table 1) and eight automated planar-array electrophysiology platforms (Table 2). These systems will be discussed in Sect. 4.

3  B  asic Premise of the Patch Clamp Technique Successful patch clamping depends on many factors, including good hand–eye coordination, cell quality, pipette geometry, and solution composition. Herein, the reader

3.2  Patch Clamp Configurations The flexibility of the patch clamp configurations provides the researcher with a selection of tools available to study current activity. The patch is said to be in the cell-attached configuration once the gigaohm seal has been formed on the intact cell membrane. Voltage pulses applied via the pipette can modulate the activity of channels located within the patched membrane area. The output measurement will reflect the steplike unitary activity of individual channels, generally referred to as “single-channel openings.” The current amplitude of individual channel openings varies greatly (picoamperes to milliamperes). However, different types of channels can be identified by their unique channel conductance, which is a reflection of the ease of flow of current across the membrane via the ­channel. With the advent of the molecular era, we now also know the conductance of a number of different channel subunits, enabling us to putatively identify the molecular components of multi-subunit channels. A prime example of this is the large family of K+ channels, whose individual subunit properties have been elegantly collected and described [19]. From the cell-attached configuration, a number of different “holding” modes can be achieved to measure and modulate  ion channel activity. These are illustrated in Fig.  3b.

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Table 1  Automated conventional electrophysiology platforms. (Adapted from [22] with permission from Macmillan Publishers Ltd, copyright 2008) Method Configuration Format Comments* Autopatch (Xention) Glass pipette

Interface, WC

1 or 48 channels

• Not commercially available • Overall daily throughput of 12–17 six-point dose–response curves

Flyscreen (Flyion) Glass pipette

Cells in pipette, WC

2 or 4 channels

• Overall daily throughput of 12–17 6-point dose–response curves • Preliminary validation for different channels/cells, but lacking extensive pharmacological characterizations

RoboPatch (Wyeth) Glass pipette

Cells in pipette, WC

1 channel

Robocyte (ALA Scientific) Two glass pipettes

• Not commercially available • Overall daily throughput of 12–17 6-point dose–response curves • Validated for ligand-gated and voltage-gated channels (human ERG, a 7 nicotinic receptor channel)

TEVC, Xenopus

96 wells, sequential

• Complementary DNA injection possible • Validated for higher-throughput electrophysiological screening

OpusXpress (Molecular Devices) Two glass pipettes

TEVC, Xenopus

8 chambers, parallel

• Closely emulates manual oocyte recordings • Successfully used to rank-order a7 nicotinic receptor channel agonists *All systems are equivalent to manual patch clamp recordings in terms of data quality: true gigaohm seal formation, small access resistance, recording stability. TEVC two-electrode voltage clamp; WC whole-cell patch clamp

Table 2  Automated planar-array electrophysiological platforms. (Adapted from [22] with permission from Macmillan Publishers Ltd, copyright 2008) Method Configuration Format Advantages Shortcomings QPatch (Sophion Bioscience) • Drug EC50 influenced by • Laminar solution flow WC; gigaohm seals 16 or 48 wells in Planar QPlate with parallel; 1 cell/ adherence to plate surfaces embedded • Ligand addition during recordings hole/well recording • No intracellular perfusion • Cumulative/multiple compound and ground additions • No simultaneous ­visualization contacts of channels • Continuous voltage clamp and current vs. time experiment flow • Validated for human ERG • Electrophysiological responses similar to those of conventional techniques PatchXpress (Molecular Devices) • Continuous voltage clamp vs. time • Drug EC50 influenced by WC; gigaohm seals 16 wells in Planar SealChip plots parallel; with adherence to plate surfaces 1 cell/hole/well • Cumulative/multiple compound electrodes • No intracellular perfusion separate additions from chip • Ligand addition during recording • Simultaneous visualization of all 16 channels • Validated for human ERG • Electrophysiological responses similar to those of conventional techniques (continued)

34  Conventional Patch Clamp Techniques and High-Throughput Patch Clamp Recordings on a Chip Table 2  (continued) Method Configuration

Format

Advantages

Shortcomings

• Highest-throughput unit • Allows for collection of 384 data points: ~60% success rate for IonWorks HT and >95% for IonWorks Quattro

• Drug EC50 influenced by adherence to plate surfaces • No high-resistance membrane seal • No simultaneous ligand addition and recording • No intracellular perfusion • No voltage clamp between reads

2, 4, or 8 wells in parallel; 1 cell/ hole/well (Port-a-Patch)

• Borosilicate chip surface • Primary cell use possible (single-well version) • Internal and external perfusion • Continuous voltage vs. time plots • Ligand addition during recordings • Cumulative/multiple compound additions

• System performance information limited

96-well plates; 16 channels sequentially; 1 cell/hole/well

• • • • •

• Requires a proprietary add-in to solutions to achieve higher seal rate • Though meant for 96x, does not contain 96 amplifiers

1 cell/hole/well

• Constant laminar flow • Positive pressure on electrode is independent of suction in surrounding aperture (mimics manual patch clamp)

IonWorks HT and Quattro (Molecular Devices) 384 wells ; 48 Planar 384-well Perforated WC; channels PatchPlate ~100 MW (HT) sequentially; 1 or 30–50 MW cell/hole/well (Quattro) (HT) or 64 seals cells/holes/well (Quattro)

Patchliner (Nanion Technologies) Planar WC; gigaohm seals NPC-16 chip

SyncroPatch96 (Nanion Technologies) Planar chip WC; gigaohm seals

CytoPatch (Cytocentrics) WC Planar, electrode tip shape surrounded by aperture in borosilicate glass surface IonFlux HT (Fluxion Biosciences) Microfluidic WC; megaohm well plate seals

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Borosilicate chip surface Ligand addition during recordings Internal and external perfusion Continuous voltage vs. time plots Cumulative/multiple compound additions

• Laminar solution flow • Ligand addition during recordings • Cumulative/multiple compound additions • Continuous voltage vs. time plots • Tested with voltage- and ligandgated channels • Separate cell and ligand wells Dynaflow HT (Cellectricon / The Automation Partnership / Astra Zeneca) Microfluidic WC; megaohm 96 channels/plate • Ligand addition during recordings well plate seals • Cumulative/multiple compound additions • Silica microfluidic chip minimizes compound adherence and consequent EC50 shifting • Validated for human ERG and GABAA 96- or 384-well; 16 or 64 (HT) amplifier arrays; 20 cell ensemble recordings

For each, one can control channel activity by altering voltage pulses or the solutions’ (intracellular and extracellular) ionic composition. In the inside-out configuration, the outer face of the membrane patch is sealed within the pipette while the previously inner membrane is exposed to the perfusing solution. This configuration is achieved by rapidly pulling up the

• Not commercially available; used only for in-house screening

• Drug EC50 influenced by adherence to polymer plate surfaces? • No high-resistance membrane seal

• No high-resistance membrane seal

pipette from the attached cell, without breaking the gigaohm seal. The perforated patch configuration allows for the diffusion of small molecules, but not of cytoplasmic proteins and larger molecules. Partial permeability of the membrane (without total rupture) is achieved by the addition of an ­antibiotic ionophore (e.g., nystatin, amphotericin B) to the

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Fig.  3  Basics of patch clamp electrophysiology. (a) A cell in a gigaohm cell-attached configuration. Basic electronics are depicted: ground (reference) electrode, borosilicate glass microelectrode, AgCl electrode connected to a downstream amplifier which controls voltage output (voltage-clamp mode) or current output (current-clamp mode).

(b) Different patch clamp configurations achievable using conventional, automated, or planar technology; outside-out and inside-out configurations can only be achieved with conventional patch clamp technology. Steps involved in achieving each configuration are outlined in the text

pipette solution and the latter’s subsequent diffusion toward and embedding in the membrane, where it creates small transmembrane openings. Perhaps one of the most widely used configurations involves accessing the entire cytosolic space: the whole-cell configuration. Following stabilization of the gigaohm seal, additional suction or a strong and fast voltage surge (“zap”) applied via the pipette ruptures the membrane patch, giving the user access to the intracellular medium. In this mode, the user can control ion flow across the plasma membrane by modifying the ionic composition in both the pipette and the bath solutions. Macroscopic currents recorded from cells represent the cumulative activity of all the ion channels contained within the cell membrane. The outside-out configuration is generated from the whole-cell configuration by gently pulling up the pipette, thereby stretching the membrane outside the pipette until it reseals itself, forming a miniature cell. It is important to note at this point that only the cell-attached and whole-cell configurations (ruptured and perforated patch) can be achieved using either planar chips or automated patch clamp systems. Each patch clamp configuration has its experimental applications, as evidenced by the sheer volume of articles published in which the patch clamp technique was the tool of choice. Singlechannel current recordings from cell-attached patches provide vital information about the gating and kinetics of individual channel proteins, and allow scientists to distinguish between currents flowing through related channels (e.g. K+ ions flowing through maxi KCa vs. KV channel proteins) on the basis of their  unitary conductance. Unfortunately, successful singlechannel recordings require that at least (and preferably only)

one channel protein be present within the membrane patch, which is not always possible depending on the overall protein density of certain channels. The ability to simultaneously record the activity of a whole population of the same channel on the overall plasma membrane is the greatest advantage provided by the whole-cell configuration. Most particularly, the whole-cell mode allows one to record currents generated by ion channels with either small individual conductances or low densities and currents which may be difficult to resolve in cellattached patches. As a disadvantage, the large pipette volume relative to the small cell acts as a “sink” into which normally intracellular metabolic factors can dissolve, potentially leading to channel rundown during the course of an experiment. The prime advantage of the inside-out and outside-out excised-patch configurations is the ability to precisely control the cellular environment. In both scenarios, the operator controls the ionic compositions of solutions on either side of the membrane; this total control of the membrane environment allows for a better understanding of the channels’ permeation processes (pore selectivity and conductance), gating properties (channel opening and closure), and regulation (e.g., by intracellular signaling systems).

3.3  Troubleshooting and Limitations The relative inability to control the cellular environment without modifying the cell’s physiological properties is perhaps the most serious limitation of the patch clamp technique. Because we can only speculate as to the exact composition of the

34  Conventional Patch Clamp Techniques and High-Throughput Patch Clamp Recordings on a Chip

i­ntracellular and extracellular media, the ionic composition of the solutions used in patch clamp experiments becomes a determining factor in isolating and identifying currents. Currents can be identified using ion-selective solutions. For example, in studying voltage-dependent Ca+ currents, K+ may be replaced by Cs+ (or tetraethylammonium) in both the pipette and the bath solution to minimize K+ channel activity. Alternatively, Ba2+ can be used as a replacement for Ca2+ since these channels are more highly permeable to Ba2+. Pharmacological tools are also indispensable for isolating specific currents. For example, tetrodotoxin can be used to selectively block Na+ channels or 4-aminopyridine can be used to block KV channels. The ability to clamp Em also allows for the selective regulation of channel activation. For example, rapidly inactivating Na+ currents are elicited by membrane depolarizations from very negative potentials (–80 to –60 mV), whereas their activation is minimized by using a holding potential of –40  mV since many of the channels are inactivated at this relatively negative Em. Similarly, L-type and T-type Ca2+ channels can be differentiated from each other by using different holding potentials (–70 and -100  mV, respectively). However, the ability to regulate channel activity by modifying the holding potential does not alleviate the need to minimize the activity of other “contaminating” currents. Therefore, ion replacement and pharmacological approaches are still essential in most experiments. Electrophysiologists agree that optimal cellular environment and cell quality, although critical, are not the only limiting factors in completing an experiment. Successful patch clamping is highly dependent on the technical ability of the user, effective cancellation of external electrical noise, attenuation of vibration within the system, and pipette reliability; these factors apply to both manual and automated systems unfortunately. Conventional patch clamp electrophysiology relies on the use of glass microelectrodes maneuvered onto a cell to form a highresistance seal on its membrane. More concisely, a cell is sandwiched between a 150-mm-thick cover glass and a pipette attached to a mechanical micromanipulator positioned inches away (a light year away as far as a cell is concerned!) to achieve the gigaohm seal. The number of things that can go wrong is amazingly high. (1) Moving the pipette toward the cell membrane and forming a seal requires an operator with a high level of training and a steady hand. Because the cell is relatively flat on the cover glass (especially plated and cultured cells), a few extra micrometers of vertical displacement toward the cell may transform a potential gigaohm seal into cell impalement or shattering of the pipette tip. (2) The fabrication of glass microelectrodes is not entirely reliable despite the vast improvements in pipette puller technology. Consequently, the size and geometry of the pipettes can vary from one glass capillary to the next and between pulls on the same day. Correcting for this variation is a time-consuming and tedious process that must be constantly addressed. (3) Most conventional patch rigs are housed within a Faraday cage to insulate them against random external

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electrical noise. The electrode being held high above (and ­connected to) the cell acts as an effective antenna for electrical noise. Therefore, all components connecting to electrical ­supply systems must be effectively grounded, especially to allow for the resolution of small currents. (4) Vibration in the system can reduce the success and longevity of an experiment; conventional patch clamp setups are mounted on air tables that absorb floor vibrations and allow maintenance of the seal.

4  T  he Advent of Automated Technologies: From Pipettes to Planar Chips 4.1  S  tepping Stones to the Development of Planar Electrodes Four significant advances in technology have provided the stepping stones leading to the development of planar ­electrodes: computation, MEMS, cell lines, and synergy with other high-throughput screening technologies [20]. High-speed data acquisition in parallel across 16–48 channels requires a data throughput of 800  kB/s, with a storage capacity of 1–5 GB each day. Also, analog-to-digital signal conversion with the small cells that are typically used for ion channel screening requires a 500 kHz sampling rate to capture the short capacitative transient during a voltage step in 16 data channels (approximately 30  kHz per channel). This is required to successfully detect the transition from the gigaohm seal to the whole-cell access configuration. Today’s computers can handle the level of multitasking, speed, and storage space required to automate high-throughput data handling and analysis. The development of new micromanufacturing technologies with submicron resolution represents the second significant advance. Advances in microfabrication techniques (reactive ion beam milling, etching, laser ablation) and substrate technologies (silicon, fused silica or quartz, SiO2, glass, plastics, doping and deposition methods) have enabled the production of a 1-mm hole on a planar substrate. Chemical modifications have also improved the “odds” of successful gigaohm seal formation; one example is the high-temperature oxidation of silicon (a semiconductor) to SiO2 (high-resistance dielectric) which is suitable for performing high-quality electrophysiological measurements across the aperture. The use of planar electrodes enables “miniaturizing” and parallelizing the patch clamp technique in an automationready manner. The increased availability of engineered transgenic cell lines has propelled the development of highthroughput devices. Otherwise electrically quiescent cells have been immortalized as perpetual cell lines which may then be transgenically modified to stably express target ion

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channel genes, providing a continuous supply of isolated and uniform cells for automated processing. The emergence of prepackaged automation component technologies (robotic fluidic handling systems, high-precision robots, standardized multiwell plate formats, automation software) under a high-throughput-screening paradigm has facilitated the growth of planar electrode voltage-clamp technology. Their commercial availability has significantly reduced the development time for instrumentation, and made available interfacing technologies and commercial products, as well as the financial burden of developing the instrumentation necessary to interface with the newly developed biochips.

4.2  “ Automatic” Cell Positioning: The First Step to Automation Automated electrophysiology technologies rely on the quality of cells that are isolated and delivered to the electrode in suspension. Cells must be relatively uniform in size and the suspension must be free of debris that could occlude the aperture during cell positioning, a step that is most easily accomplished by negative pressure. However, two exceptions are worth mentioning. Apatchi-1™ moved the pipette to a cell that is already settled onto a surface; therefore, negative pressure is not required to locate the cell onto the electrode. This device is no longer being manufactured. Cytocentrics’s CytoPatch™ places the recording aperture within a larger hole that draws the membrane down onto the recording aperture, thereby mimicking the effect of applying positive pressure with the recording aperture.

4.3  A  utomating the System: Glass Pipette Interfaces Throughout the last 25 years, there have been many efforts to provide automation to voltage-clamp experiments. One direction of automation started with fast solution exchangers and picospritzers, and culminated in a high-throughput device from Cellectricon (Dynaflow) which is able to expose a manually voltage clamped cell to very high speed solution changes by moving the cell (clamped on a standard glass pipette) near and across an array of up to 48 microchannels each outflowing solution from a different compound well. Another direction started with specialized chambers to always bring a cell of interest into a predetermined position to simplify the hunt, and had its pinnacle with the development of the Apatchi-1™ system from Sophion [21], a system using computer vision to completely automate the hunting

C.V. Remillard and J.X.-J. Yuan

for an appropriate cell, patch pipette handling, gigaohm seal formation, and rupture of the membrane into the whole-cell configuration. In one particular embodiment of this approach, a group in Japan automated the hunt by placing the cells into the glass pipette instead of painstakingly chasing one down in the solution outside the pipette, a technique later repeated by Flyion for its Flyscreen product. The early field innovators include the Autopatch (Xention), Flyscreen (Flyion), RoboPatch (Wyeth), Robocyte (ALA Scientific), and OpusXpress (Molecular Devices, formerly Axon Instruments); the last three are for Xenopus laevis oocytes or similar cells, whereas the others were specialized for mammalian cells but were either poorly adopted or never achieved the necessary success rates for drug screening. Table  1 provides a brief description of the individual platforms. Other publications and reviews discuss the operation of these systems and should be referred to for more information [22–26]. Each of these apparatus (as well as the high-throughput devices discussed in Sect. 4.4) contain the six essential elements for any electrophysiology apparatus: an intracellular chamber, an intracellular electrode, an extracellular chamber, an extracellular electrode, a hole between the two chambers that can form a high-resistance seal with a cell, and a conductive medium in each chamber and within the hole. The most important finding with all of these systems is the following: all are equivalent to manual patch clamp recordings in terms of data quality, including true gigaohm seal formation, small access resistance, and stability of recordings. Yet another direction in the automation of electrophysiology started among academia with the making a micron-sized hole in a relatively flat section of a thin plastic tube [16], and culminated with a micromanufactured hole in a planar substrate. It was the planar substrate that finally removed the barrier to the modern higher-density and higher-throughput automation devices that are presently the new standard in electrophysiology.

4.4  Planar Array-Based Approaches Early attempts to produce planar electrodes for voltage clamping failed because of crude and unreliable manufacturing techniques (e.g., poke a fragile glass electrode into plastic, molding around delicate micron-sized features); however, the evolution of modern MEMS produced new methods, such as reactive ion drilling and laser drilling facilitated etching, and finally led to the production of a micronsized hole. “Planar array” refers to the use of multiwell configurations in either a plate-based or a chip-based format to record multiple channels in parallel. For the most part, the systems have integrated robotics which handle cells, solutions, and compounds. Table  2 outlines the major systems

34  Conventional Patch Clamp Techniques and High-Throughput Patch Clamp Recordings on a Chip

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Fig.  4  Chamber configurations for automated and planar patch clamp technologies. (a) PatchPlate used in IonWorks HT. (b) PatchPlate population chip used in IonWorks Quattro. (c) QPlate used in QPatch. (d) SealChip used in PatchXpress. (e) NPC-16 chip used in Patchliner

NPC-16. (f) CytoPatch chip used in CytoPatch. (g) Microfluidic bath used in the IonFlux HT system. (h) Microfluidic bath used in the Dynaflow HT system

which are currently available (as of July 2009) and/or in development. Schematic representations of system assemblies (including bath, perfusion, cell trapping, and electrodes) are provided for each in Fig. 4. The IonWorks HT system from Molecular Devices was the first screening system to come into use [27]. It uses a disposable PatchPlate™ electrode (Fig. 4a) which receives the cells and then experimental compounds. Although the consumable contains 384 wells (recording locations), the recording head has only 48 amplifiers and hence the instrument was designed to sample the clamped cells sequentially rather than simultaneously, imposing discontinuity in the voltage clamp of the cells. Forty-eight currents (perforated patch only) can be measured simultaneously. The technology for the IonWorks Quattro system is identical, except that the amplifier is able to drive a larger current to each chamber. This is because whereas the IonWorks HT consumable has one cell in one hole in each well, the Quattro system has 64 cells in 64 holes (in an 8 × 8 array) for each well (Fig. 4b). Each 384-well plate is capable of patching 150–200 cells for up to 6 min. Neither the HT version nor the Quattro version is able to achieve gigaohm seals;

­however the 50–200-MW seals (quasi-voltage-clamped) that are achieved appear to be sufficient to study the timeindependent, or steady-state, currents and the throughput has been unmatched by other devices on the market at the time of this writing. Because of the slow compound delivery and interrupted voltage-clamp methods implemented, this system is not ideally suited to recording fast transient currents or ligand-gated currents. The second screening system released was the Patch­ Xpress (Molecular Devices), using AVIVA Bioscience’s SealChip16, a 16-well disposable that is capable of patching more than 12 cells (one cell per well) simultaneously for more than 15 min (Fig. 4d). Unlike the IonWorks systems, true gigaohm seals can be achieved (the first to be able to do so) [28, 29]. The PatchXpress became the golden standard in the field for automated electrophysiology because of its success rates with gigaohm seals, the longevity of seals matching that of glass pipettes, and its versatility, allowing the user to program virtually any experimental protocol. At the time of this writing, it is still unmatched in its versatility. The PatchXpress is ideal for all kinds of ion channels, and has very efficient data analysis methods;

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however, its throughput, compared with the throughputs of upcoming systems, is too slow for true high-throughput screening. The third system on the market to automate voltage clamping was a single-cell, low-throughput device from Nanion, Port-a-Patch, engineered for academic users, but lacking any fluidics automation. Nanion later attempted a higher-throughput device, the NPC-16 Patchliner, which was capable of microfluidic exchange of both the intracellular and the extracellular solutions [30] (Fig. 4e). Although the consumable had 16 recording chambers, the instrument only had two to eight parallel recording heads designed to perform an entire uninterrupted experiment on a well before moving to the next well on the consumable. The throughput for this device was insufficient to compete successfully on the market and has remained unpopular. Nanion recently developed the SyncroPatch 96, a large-scale version of the Patchliner which uses a 96-chamber consumable and has 96 simultaneous recording heads. Its fluidics system is designed to handle 384-well plates. This system is designed with one hole per and can achieve gigaohm seals with longevity comparable to that of the gigaohm seals of the PatchXpress, and throughput comparable to that of the IonWorks Quattro. Nanion has validated its application for measuring both ligand-gated and voltage-gated channels. The CytoPatch (Cytocentrics) was the fourth system to be released on the market, with a slightly different design from previous systems. Although it is a planar-based array, an electrode tip shape is micromachinned inside a larger aperture (Fig. 4f). The latter enables easier “capture” and ­positioning of cells via applying negative pressure to the larger aperture to position the cells while simultaneously applying positive pressure to the “electrode tip” to keep it clean and also to clean the cell surface prior to contact with the tip for the formation of the gigaohm seal, similar to what is done in conventional patch clamping [31]. Although this device has been launched, it is unclear how many instruments have been sold. No validation data are available at this time. The QPatch (Sophion) was the fifth system released (Fig. 4c). The first release had a 16-chamber consumable that could accommodate up to 48 chambers on the same platform. This was provided with 16 simultaneous headstages for a continuous clamp. All the fluidics are retained on the consumable and hence it is limited to using only small volumes, with all compound additions and washouts being left on the consumable [21]. This device is similar to the PatchXpress in many functions; however, it lacks the versatility and the fast washout method, requiring the use of the compound-delivery robotics to provide not only the drug additions but also the washouts. In spite of this, the availability of multiple-compound delivery nozzles allows the QPatch to be used with most ligandgated channels (which are usually more challenging because of the need for a fast washout). The initial release of QPatch

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has now undergone two upgrades, each costing almost as much as the original instrument. The first upgrade increased the number of consumable chambers and the number of headstages to 48, which made its throughput higher than that of the PatchXpress, but still lower than that of the IonWorks systems. The second upgrade increased the current driving capacity of the amplifiers, and there are multiple holes in each well, similar to the IonWorks Quattro. Although the presence of multiple holes prevents the formation of gigaohm seals, it increases the number of useable chambers to nearly 100%. Between 2007 and 2009, two new systems have been announced for primary and secondary drug screening [32]. Fluxion Biosciences introduced the IonFlux, a 16× precursor to the upcoming IonFlux HT which looks like a traditional plate reader, but can, according to the company, deliver between 8,000 and 10,000 assays per day with a 96× consumable. The consumable has a standard 384-well microplate footprint into which cells, compounds, and reagents are loaded. (Fig. 4g). A microfluidic channel network is built into the well plate bottom, allowing for real-time compound addition and cumulative dose–response curves. Electrodes are integrated into the bath as well, and the whole assembly is linked up with 64 amplifiers. Although the system appears to be capable of only loose megaohm seals (20-cell ensemble recordings), the full-time voltage clamp and ability to wash out in the same fluidics channels makes it effective for both voltage- and ligand-gated channels. In partnership with The Automation Partnership (TAP) and AstraZeneca, Cellectricon recently announced the Dynaflow®HT. The system contains cell and compound storage, as well as robotic liquid and plate handling, all of which allow the cells to be prepared in one place (Fig. 4h). The cells are then sucked from 96-well plates into a microfluidic chip. Here the cell membrane forms a seal with the chip where the current changes are monitored. Originally, the system achieved over 7,500 data points per day. With the introduction of a new chip late in 2009, the Dynaflow HT system generates 18,000 data points per day. As with the IonFlux HT system and many other high-throughput planar devices, the system has been developed with multiple cell lines with both voltage- and ligand-gated ion channels.

4.5  W  hat About Channels Not on the Plasma Membrane? A number of ion channels are located on the membranes of intracellular organelles such as the sarcoplasmic/endoplasmic reticulum, the mitochondrion, synaptic vessels, and lysosomes. None of the patch clamping technologies described up to this point can possibly measure currents from such channels because the membranes are inaccessible. Therefore, these

34  Conventional Patch Clamp Techniques and High-Throughput Patch Clamp Recordings on a Chip

intracellular channels remain a drug target inaccessible to the automation techniques described so far owing to technological limitations. Furthermore, an important subset of ion channels are electroneutral exchangers, such as Na+–H+ exchanger, or generate a very small current that is difficult to measure under standard voltage-clamp conditions, such as the Na+–Ca2+ exchanger or Na+–K+-ATPase. These also remain inaccessible to planar electrodes because of small currents that may be hidden in the electrical noise from vibratory sources. The SURFE2R (surface electrogenic event reader) developed by IonGate has overcome these limitations (http:// www.iongate.de/index.php?lang=en&cont=4_0). Its technology does not use whole cells, but instead reads the charge displacement from membrane preparations isolated from cell culture. The membranes are coupled via an alkaline thiol and lipid bilayer to gold-surface electrodes, and the latter detect changes in the electrical signal that are produced by a halfreaction step of an exchanger without the need to use reporter molecules. The SURFE2R system is as efficient at measuring the electrical activity of electrogenic pumps such as the Na+–Ca2+ exchanger and the Na+–K+-ATPase pump as it is at measuring the electrical activity of electroneutral channels such as the Na+–H+ exchanger. Its application has been expanded through the use of specialized techniques to include the measurement of ion channels.

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However, this is difficult to do using macroscopic currents (I ) recorded in the whole-cell configuration because I is a function of the single-channel current (i), the number of channels (N ), and the probability of the channel being in the open state (Po) (Eq. 1), none of which can be determined for individual channels in whole-cell patches.

I = iNPo

(1)

Single-channel recordings are typically obtained from cellattached patches, although they can also be obtained from inside-out or outside-out membrane patches. In the simplest scenario, the small membrane patch surrounded by the walls of the glass pipette contains a single channel protein. Nature rarely cooperates this readily, so a more common occurrence is the measurement of multiple step-like channel openings within a membrane patch (Fig. 5a). From these openings, we can measure or calculate the following channel parameters: conductance/amplitude, open probability, and gating.

5  Characterizing Ion Channel Currents Permeation and gating mechanisms control the flux of ions through channel pores. The channel’s pore region contains amino acid sequences which determine the size, geometry, and charge within the channel pore, thereby acting as a filter to select which ions can be conducted and controlling the net rate of ion flux through the channel pore. Closure and opening of the channel (i.e., gating) occurs randomly between the open and closed states. The section that follows provides a brief description of how patch clamping applies to the study of the permeability and gating characteristics of ion channels.

5.1  Unitary Currents Ion channels open and close in a stochastic fashion. However, the probability of finding the channel closed or open is not a fixed number but can be modified/modulated by an external stimulus, such as voltage (i.e., voltage-gated channels), stretch (i.e., mechanosensitive channels), or an agonist (i.e., ligand-gated channels). Patch clamping can provide information on a channel’s open probability and conductance.

Fig.  5  Single-channel analysis. (a) The cell-attached configuration. Although many channel proteins are likely present within the patched area, a single channel has been represented here. The right panel depicts a sample single-channel current recording from a human pulmonary artery smooth muscle cell recorded at +50 mV. The three open channel levels (Ox – representing at least three different channels within the patch) and the closed level (C) are indicated. (b) Amplitude histogram with peaks for open levels 1–3 indicated. An I–V curve from which conductance is derived is shown on the right. (c) Open probability (Popen) is shown as a box plot of Popen as a function of time. Popen can be plotted as a function of patch potential (Epatch) to illustrate the voltage dependence of single-channel gating (right panel)

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5.1.1  C  hannel Conductance, Selectivity, and Current Amplitude Each channel opening enables the passive flow of ions through the protein along their electrochemical gradient. The ease of flow of current across the membrane, or ­conductance (measured in siemens), cannot be directly measured, but it can be approximated from the amplitudes of the channel openings at different voltages. The amplitude, the differential amplitude between the maximum open level of one channel (O1) and the closed level (C), is generally determined from a raw-data-based histogram which reports the number of openings as a function of current amplitude (Fig.  5b, left). Fitting the histogram peaks with Gaussian distributions provides the mean amplitude of each open level (O1 – O3). In the example shown, the amplitudes of the openings were multiples of each other, suggesting that the channel proteins responsible were of the same type. When amplitude values are then plotted as a function of the applied patch potential [Epatch, = –(Eapplied)-|Em|, where Em is –40  mV], a linear regression fit of the data provides the calculated slope (g ), which approximates the conductance of the channel (Fig. 5b, right). The current–voltage relationship can also provide some indication as to the selectivity of the channel. For each ­concentration gradient, the reversal potential EX ss defined by the Nernst equation (Eq. 2):

EX =

[X ]o RT log , zF [X ]i

(2)

where R is the gas constant, T is the absolute temperature (Kelvins), z is the valence of the ion, F is the Faraday constant, and [X]o/[X]i is the ratio of extracellular to intracellular concentrations of the ion. EX can be determined by measuring the voltage at which the unitary current changes direction (i.e., amplitude goes from positive or negative,or vice versa, on the current–voltage curve).

5.1.2  Channel Open Probability Assuming that single-channel conductance is independent of Em, the measured current can also be described by Eq. 3:

i(t ) = NγPo (E, t )(E − EX ),

(3)

where i(t) is the time-dependent ionic current measured, N is the number of channels in the patch, g is the single-channel conductance, Po(E,t) is the probability that a channel is in the open state, E is the patch potential, and EX is the reversal potential of the current, determined using a double-pulse

protocol or the Nernst equation. As suggested by the above equation, the open probability of a channel protein varies with time and membrane potential. For each patch, the total single channel open probability (NPo) is calculated as follows (Eq. 4):

NPo =

∑ (O n), n

T



(4)

where n is the number of channels in the patch (1, 2, 3…), On is the time spent at the open level for each channel (i.e., 1–n), and T is the total recording time. The fraction of channels in the open state varies between 0 (closed) and 1 (open) depending on the nature of the channel and the stimulus. Figure 5c shows a representative NPo histogram. 5.1.3  Channel Gating Unitary recordings provide information regarding the behavior of the channel protein(s). A dwell level refers to the time a channel spends in the open or closed states. Like amplitude measurements, the durations of the channel transitions from the closed to open states can be measured and sorted into histogram bins from which we can estimate the number of closed and open states and their respective time constants. Typically, dwell times are plotted as a function of the number of observations. Alternatively, dwell times can be plotted as a function of time, as for a time course, to monitor the behavior of a channel over time (e.g., during drug application or in response to an external stimulus). Dwell-time analysis should only be performed in patches with a single open level to better approximate channel behavior, simply because channel closure may be masked by opening of another channel simultaneously. In some cases, the openings are “flickery,” as shown by the left-side openings in Fig. 5d. Dwell-time analysis of these recordings should be done using burst analysis. The latter applies to recordings where consecutive channel openings are separated by a dwell time less than the interburst (definite channel closure) interval. Only the duration of events (openings or closings), not their amplitude, is measured in this mode; therefore, burst analysis cannot be performed on data with more than amplitude level.

5.2  Macroscopic Currents As mentioned earlier, the whole-cell configuration provides a certain degree of experimental flexibility since the user can simultaneously control both intracellular and extracellular ion content and voltage. It allows for the determination of the

34  Conventional Patch Clamp Techniques and High-Throughput Patch Clamp Recordings on a Chip

function of a channel type as a whole family, Therefore, it can be used to successfully characterize a channel protein type in terms of both its permeability and its gating characteristics. 5.2.1  Passive Cell Membrane Properties



I x = g ∆E ,

Since many membrane channels are voltage-dependent, the membrane resistance likewise varies with changes in Em. Experimentally, cell membrane capacitance (Cm) is determined by software based on Eq. 6:



Likening the whole-cell configuration to an electrical circuit allows electrophysiologists to determine passive cell membrane properties, i.e., membrane capacitance, membrane resistance, and cell surface area. The pipette, with its two charged surfaces, is a dielectric substance and therefore acts as a capacitor in the circuit. Pipette capacitance (Cp, measured in Farads) is complicated in character, but its contribution to the overall circuit is usually minimized electronically by injecting a current transient designed to precharge the glass surface to the desired potential. The pipette pore presents a resistance to current flow that may be easily measured before seal formation (Rp, measured in ohms). During wholecell access, however, this resistance is increased by further resistance to current flow due to the contents or geometry of the cell itself (“series resistance,” or “access resistance”), i.e., resistance to filling the entire cytosolic space with the desired amount of charge, or potential due to interaction of charges with proteins or due to limited flux through long cell processes or narrow cell geometry. Once the cytosolic space of the cell has been voltageclamped, the cell membrane also presents its own capacitance. Unlike the pipette, the cell membrane is of relatively uniform thickness and uniform dielectric content. Therefore, in most cells the specific membrane capacitance (Cm), which is normalized by the area of the plasma membrane, is approximately 1 mF/cm2 [2]; cell capacitance is a good indicator of cell size (i.e., the surface area of a cell). The cell membrane itself is a very good dielectric, presenting a resistance of several gigaohms, in effect stopping the flow of charge across the membrane. However, the membrane resistance (Rm) is strongly influenced by the presence of ion conductances through membrane ion channels. Ion channels are selectively permeable to specific cations and have a gating mechanism that may be controlled by voltage or other methods. Ion channels produce a conductance (g, inverse of R) that is dependent on the transmembrane electrical potential energy (DE, measured in volts), and defined by Ohm’s law, which can be alternatively expressed as Eq. 5: (5)

where Ix is the conductance through a channel. The overall membrane resistance is the inverse of the sum of all the conductances present on the membrane. Overall membrane resistance is therefore a good indicator of the amount of current carried through all the open channels on the membrane.

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Cm =

∫I

tr

∆Vcomm

,

(6)

where DVcomm is the amplitude of a small hyperpolarization applied to induce a current transient (Itr). Similarly, membrane input resistance (Rm) can be calculated from Eq. 7:

Rm =

Rtotal × Rseal , Rseal − Rtotal

(7)

where Rseal and Rtotal are the resistances determined, respectively, from the steady-state currents of Itr in response to DVcomm (-5 mV) before and after break-in. The resting membrane potential (RMP) is also a very important property of quiescent cells. Although other techniques may also be used, RMP can be measured in the current-clamp mode once whole-cell access has been established. In many excitable cells, RMP ranges between –85 and –65 mV, which is close to the reversal potential for ­potassium ions (EK). Coincidentally, as discussed in other chapters, the activity of K+-permeable channels is the main determinant of RMP in many excitable cells, including pulmonary artery smooth muscle cells.

5.2.2  Channel Permeation and Ion Selectivity Channel selectivity can be studied by using the relative permeability (Px/Py) of a channel to different permeant ions. Because the driving force for ions in a cell membrane is dependent on voltage, a simplified voltage form of the Goldman–Hodgkin–Katz equation (Eq.  8) can be used to approximate Px/Py:

Erev =

Px [X ]o RT log . zF Py [Y ]i

(8)

With use of either the outside-out or the whole-cell patch clamp configurations to fix the ionic gradients, this ratio can be easily determined and the reversal potential of a current (Erev) can be measured, providing that ions X and Y are the only permeating ions on the external and internal sides of the membrane. Under whole-cell conditions, the reversal potential (therefore ion selectivity) can be established in two ­different ways. First, rapid current–voltage (I–V) relationships can be established by using ramp protocols under whole-cell conditions. In this scenario, the reversal potential

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is determined in a fashion similar to that using single-­channel analysis. Second, a double-pulse protocol can be utilized whereby cells are depolarized to a single potential, then hyperpolarized to a range of potentials in which the voltage dependence and Erev of the current overlap. A current–voltage relationship generated by plotting the amplitude of the tail current (deactivating current induced by the hyperpolarizing step) as a function of its voltage can clearly reveal Erev and the selectivity of a particular channel.

5.2.3  Channel Gating Properties The stochastic behavior of a channel dictates that it oscillate between its open and closed states, with a jump of an energy barrier required for each state transition (Fig. 6). In some cases, the channels may also be found in intermediate inactivated or subconductance states. Transitions between these conformational states can be dependent on time, voltage, or ligand binding; the speed of these transitions is given by rate constants (v, v¢, w, w¢, x, and z). Establishing a channel’s voltage dependence is typically achieved by a simple voltage step protocol by which the cell membrane is depolarized to a range of potentials before being repolarized to a common negative potential near the cell’s resting Em. Plotting current amplitude as a function of the test potential establishes the current–voltage (I–V) relationship. Analysis of the shape of the currents also provides some information as to the activation (time-to-peak, tact) and inactivation (peak-tosteady state, tinact) kinetics, at least for time-dependent currents, which can be used to derive the rate constants for the channel state transitions. One problem associated with using simple I–V protocols to study the gating properties of a channel is that one cannot discriminate between the combined effect of the gating mechanisms and electrochemical driving force for the ion on channel gating. The simplest way to bypass this issue is to use a double-pulse protocol. An initial voltage step is applied to activate the channels, followed by a step down to a negative potential to allow time-dependent channel closure. Plotting the relative amplitude of the tail current as a function of the step voltage provides an indication of the voltage sensitivity and activation thresholds of the current in question. Relative availability of the channels following current inactivation can also be determined using a similar doublepulse protocol. The slope of the mathematical fit (Boltzmann function) to the activation (as given in Eq. 9) and ­inactivation C

C

v w

O

z

v w x

I

O y

C

v w

O

v w

O

Fig. 6  Putative state transitions of channels between open and closed states

(use V–V½ in lieu) curves provides an indication of how voltage controls the gating mechanisms, information that is particularly useful when one is studying the functional effect of a drug on channel activity. In this equation, V½ represents the half-maximal activation or inactivation potential and k is the slope factor, which is a good indicator of the steepness of the voltage dependence of channel opening and closing. Many channel-modulating compounds shift or alter V½ and/ or k values to produce their physiological effects. One example is 4-aminopyridine, which shifts the steady-state activation curve of KV currents to more positive potentials, steepens the voltage dependence (k) of activation, and shifts channel availability to more positive potentials in isolated rabbit coronary smooth muscle cells [33]. −1



 V −V  Y = 1 + exp  ½  k   

(9)

Finally, one property of inactivating currents remains to be investigated: the recovery from inactivation (I → C transition). The protocol to study recovery also employs a doublepulse strategy, with the determining factor being the delay between two pulses (the latter two being of similar amplitude and duration). A sample protocol is as follows: the membrane is depolarized from the holding potential to a positive membrane potential for 20 s (P1, to allow for full channel inactivation), repolarized to the holding level (approximately RMP), and depolarized again (P2) to the same potential as P1 for a shorter time. The pulses are repeated with the time delay between P1 and P2 varying for each episode of the stimulus train. Percentage recovery (P2/P1 × 100) is then plotted as a function of the time delay between P1 and P2. A recovery time constant (treact) derived from a mathematical fit of the resulting data points describes the recovery kinetics of the current. Repeating this protocol using different holding potentials can be useful in interpreting the voltage dependence of the transition between the inactivated and closed states, i.e., the change in treact may be dependent on voltage.

6  Summary and Future Directions The discovery of channelopathies and the increasing knowledge of the molecular composition of ion channels has placed a heavy burden on research scientists to understand the basic principles regulating ion channel function. As basic scientists learn more, the onus is being put on the pharmaceutical industry to develop compounds which target ion channels and transporters. Ion channels and transporters represent 6% of the overall research drug targets today, a number which has not changed dramatically since the beginning of this millennium. Currently, compound development for ion channel research is at a

34  Conventional Patch Clamp Techniques and High-Throughput Patch Clamp Recordings on a Chip

s­ tandstill, in part because of the relatively slow ­development of reliable, efficient, and cost-effective high-throughput devices to replace conventional patch clamping systems. The main goal of this chapter was to provide a simple, yet comprehensive description of the patch clamp technique and its evolution from the motor-driven pipette of conventional patch clamping rigs to the planar chip technology which has rendered manual patching somewhat obsolete. A secondary goal was to elaborate upon the information provided from current recordings and how it can be interpreted on a physiological basis. Advanced molecular biology techniques, as described in other chapters, have provided much information about the molecular composition of ion channels. However, patch clamp electrophysiology remains the tool par excellence for characterizing ion channels and for ascribing biological properties to channel subunits and differentiating between their contributions to the overall physiological state. Ongoing improvements in the high-throughput screening process and technology will no doubt bring to light much more clearly the relative importance of ion channel dysfunction in disease development and treatment. Acknowledgement  The authors wish to thank António Guia at Aviva Biosciences for his critique and thoughtful insights regarding automated and high-throughput voltage-clamp devices.

References 1. Höber R (1905) Über den Einfluss der Salze auf den Ruhestrom des Froschmuskels. Pflugers Arch Eur J Physiol 106:599–635 2. Hille B (1992) Ionic channels of excitable membranes, 2nd edn. Sinauer Associates, Sunderland 3. Hodgkin AL, Huxley AF (1952) Propagation of electrical signals along giant nerve fibers. Proc R Soc Lond B Biol Sci 140:177–183 4. Hodgkin AL, Huxley AF (1952) A quantitative description of membrane current and its application to conduction and excitation in nerve. J Physiol 117:500–544 5. Hodgkin AL, Huxley AF (1952) Movement of sodium and potassium ions during nervous activity. Cold Spring Harb Symp Quant Biol 17:43–52 6. Noble D, Tsien RW (1968) The kinetics and rectifier properties of the slow potassium current in cardiac Purkinje fibres. J Physiol 195:185–214 7. Bean RC, Shepherd WC, Chan H, Eichner J (1969) Discrete conductance fluctuations in lipid bilayer protein membranes. J Gen Physiol 53:741–757 8. Neher E, Sakmann B (1976) Single-channel currents recorded from membrane of denervated frog muscle fibers. Nature 260:799–802 9. Rae JL, Levis RA (2003) Fabrication of patch pipets. Curr Protoc Neurosci Unit 6.3 10. Hamill OP, Marty A, Neher E, Sakmann B, Sigworth FJ (1981) Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch 391:85–100 11. Drews J (2000) Drug discovery: a historical perspective. Science 287:1960–1964

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12. Overington JP, Al-Lazikani B, Hopkins AL (2006) How many drug targets are there? Nat Rev Drug Discov 5:993–996 13. Zheng CJ, Han LY, Yap CW, Ji ZL, Cao ZW, Chen YZ (2006) Therapeutic targets: progress of their exploration and investigation of their characteristics. Pharmacol Rev 58:259–279 14. Hopkins AL, Groom CR (2002) The druggable genome. Nat Rev Drug Discov 1:727–730 15. Venter JC, Adams MD, Myers EW et al (2001) The sequence of the human genome. Science 291:1304–1351 16. Kostyuk PG, Krishtal OA, Pidoplichko VI (1975) Effect of internal fluoride and phosphate on membrane currents during intracellular dialysis of nerve cells. Nature 257:691–693 17. Purves RD (1981) Microelectrode methods for intracellular recording and ionophoresis. Academic, London 18. Sakmann B, Neher E (1995) Single-channel recording, 2nd edn. Plenum, New York 19. Coetzee WA, Amarillo Y, Chiu J et al (1999) Molecular diversity of K+ channels. Ann N Y Acad Sci 868:233–285 20. Guia A, Xu J (2005) Planar electrodes: the future of voltage clamp. In: Yuan JX-J (ed) Ion channels in the pulmonary vasculature. Taylor & Francis, Boca Raton, pp 635–651 21. Asmild M, Oswald N, Krzywkowski KM et  al (2003) Upscaling and automation of electrophysiology: toward high throughput screening in ion channel drug discovery. Receptors Channels 9:49–58 22. Dunlop J, Bowlby M, Peri R, Vasilyev D, Arias R (2008) ­High-throughput electrophysiology: an emerging paradigm for ion-channel screening and physiology. Nat Rev Drug Discov 7:358–368 23. Lepple-Wienhues A, Ferlinz K, Seeger A, Schafer A (2003) Flip the tip: an automated, high quality, cost-effective patch clamp screen. Receptors Channels 9:13–17 24. Schnizler K, Kuster M, Methfessel C, Fejtl M (2003) The roboocyte: automated cDNA/mRNA injection and subsequent TEVC recording on Xenopus oocytes in 96-well microtiter plates. Receptors Channels 9:41–48 25. Vasilyev D, Merrill T, Iwanow A, Dunlop J, Bowlby M (2006) A novel method for patch-clamp automation. Pflugers Arch 452:240–247 26. Vasilyev DV, Merrill TL, Bowlby MR (2005) Development of a novel automated ion channel recording method using "inside-out" whole-cell membranes. J Biomol Screen 10:806–813 27. Schroeder K, Neagle B, Trezise DJ, Worley J (2003) Ionworks HT: a new high-throughput electrophysiology measurement platform. J Biomol Screen 8:50–64 28. Tao H, Santa Ana D, Guia A et al (2004) Automated tight seal electrophysiology for assessing the potential hERG liability of pharmaceutical compounds. Assay Drug Dev Technol 2:497–506 29. Xu J, Guia A, Rothwarf D et  al (2003) A benchmark study with SealChip™ planar patch-clamp technology. Assay Drug Dev Technol 1:675–684 30. Farre C, Stoelzle S, Haarmann C, George M, Brüggemann A, Fertig N (2007) Automated ion channel screening: patch clamping made easy. Expert Opin Ther Targets 11:557–565 31. Stett A, Burkhardt C, Weber U, van Stiphout P, Knott T (2003) CYTOCENTERING: a novel technique enabling automated cellby-cell patch clamping with the CYTOPATCH chip. Receptors Channels 9:59–66 32. Pearson S (2009) Investigating and focusing on ion channels as drug targets. Genetic Eng Biotechnol News 1–3 33. Remillard CV, Leblanc N (1996) Mechanism of inhibition of delayed rectifier K+ current by 4- aminopyridine in rabbit coronary myocytes. J Physiol 491:383–400

Chapter 35

Measurement of Pulmonary Vascular Structure and Pulmonary Blood Distribution by Multidetector-Row Computed Tomography and Magnetic Resonance Imaging Techniques Sara K. Alford and Eric A. Hoffman

Abstract  Pulmonary-based X-ray computed tomography (CT) and magnetic resonance (MR) imaging techniques have been developed for structural and functional assessment of the lungs. CT methods, taking advantage of newer multidetectorrow CT (MDCT) technology, provide a means of quantitatively studying regional pulmonary perfusion (via iodine-based contrast agents) in conjunction with the acquisition of highly detailed information regarding the lung parenchyma and vasculature structure. Additional scanning protocols also allow for regional assessment of ventilation via the use of xenon gas, which is radiodense. Simultaneous to advances in imaging and imaging protocols, software has emerged which provides reliable quantitative tools for automated assessment of structure and function. Texture measures provide information at levels beyond the ability to resolve anatomic structures. CT-based pulmonary vascular structure and assessment for acute and chronic pulmonary embolism (PE) is now common clinical practice, and new functional perfusion techniques are being applied in research studies focusing on pathological changes and the causes of COPD and pulmonary hypertension (PH). Advances in technology have also dramatically improved the image quality and diversity of pulse sequences used for the lung in MR imaging, providing for another powerful imaging technique for the assessment of lung function. This diverse array of MR pulse sequences can be performed back to back to obtain a comprehensive assessment of the lung vasculature with MR angiography (MRA), perfusion imaging, and assessment of the right side of the heart. New functional measures including hyperpolarized noble gas imaging provide new measures of lung function, including ventilation and regional partial oxygen measurements.

S.K. Alford (*) Department of Radiology, University of Iowa, 200 Hawkins Dr., Iowa City, IA 52242, USA e-mail: [email protected]

Keywords  X-ray computed tomography • Magnetic resonance imaging • Lung structure • Pulmonary embolism • Angiography

1 Introduction Pulmonary-based X-ray computed tomography (CT) and magnetic resonance (MR) imaging techniques have been developed for structural and functional assessment of the lungs. CT methods, taking advantage of newer multidetector-row CT (MDCT) technology, provide a means of quantitatively studying regional pulmonary perfusion (via iodine-based contrast agents) in conjunction with the acquisition of highly detailed information regarding the lung parenchyma and vasculature structure. Additional scanning protocols also allow for regional assessment of ventilation via the use of xenon gas, which is radiodense. Simultaneous to advances in imaging and imaging protocols, software has emerged which provides reliable quantitative tools for automated assessment of structure and function. Texture measures provide information at levels beyond the ability to resolve anatomic structures. CT-based pulmonary vascular structure and assessment for acute and chronic pulmonary embolism (PE) is now common clinical practice, and new functional perfusion techniques are being applied in research studies focusing on pathological changes and the causes of COPD and pulmonary hypertension (PH). Advances in technology have also dramatically improved the image quality and diversity of pulse sequences used for the lung in MR imaging, providing for another powerful imaging technique for the assessment of lung function. This diverse array of MR pulse sequences can be performed back to back to obtain a comprehensive assessment of the lung vasculature with MR angiography (MRA), perfusion imaging, and assessment of the right side of the heart. New functional measures including hyperpolarized noble gas imaging provide new measures of lung function, including ventilation and regional partial oxygen measurements.

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_35, © Springer Science+Business Media, LLC 2011

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2 CT Imaging Techniques 2.1 Development of CT Scanners CT serves as the modality of choice for lung imaging owing to its widespread availability, resolution, high signal-to-noise ratio (SNR) for lung tissue, and fast image-acquisition speed. CT technology has rapidly advanced since its introduction into the medical field in the early 1970s. An early, experimental scanner, known as the Dynamic Spatial Reconstructor, built at the Mayo Clinic housed 14 X-ray sources aimed at a hemicylindrical fluorescent screen, in turn monitored by 14 television cameras. The 20 t gantry rotated at 15 rpm and provided enough projection data to reconstruct up to 240 slices of the body every 1/60 s [1–3]. Many of the early quantitative imaging analysis tools evolved from the efforts to evaluate data obtained with this CT scanner. Data demonstrated the utility of CT imaging in providing regional lung density measures [4–6], lung volumes [7], as well as anatomic detail of the airway and vascular trees [8–11]. Progress regarding volumetric imaging of the lung was delayed largely owing to the clinical availability of volumetric scanning modalities as well as computational resources to evaluate the large data sets derived from volumetric imaging. Spiral CT was introduced by Kalender et al. [12], allowing for volumetric imaging of the lung in a breath-hold. Although single-slice spiral CT scanners with one X-ray source/detector pair were rapidly adopted to acquire volumetric images of the lung, breathhold times were often too long for patients to sustain a breath-hold and slice thicknesses were large relative to the anatomic structure of interest. Thin-slice, high-resolution, step-and-shoot modes, gathering slices widely spaced from the lung apex to the base, were introduced and remained the method of choice for assessing peripheral lung anatomy [13, 14] and functional data were not available clinically. Fourslice MDCT systems became available in 1998, followed by 16-slice and 64-slice systems in 2001 and 2004, respectively. At the time of this writing, scanners are available with 320 slices covering approximately 12 cm of the apical–basal lung axis. MDCT scanners use cone-beam X-ray geometry and multiple detector rows to collect more slices per gantry rotation, thereby allowing for increased z coverage. Their increased speed and ability to image thin slices of isotropic (voxels with the same x, y, and z dimensions) resolution provide significant benefits compared with the earlier generation of single-detector or limited multidetector-row scanners. With isotropic voxels and a voxel dimension on the order of 1 mm or less, one is able to reslice the volumetric image according to anatomic structure of interest. Once 16-slice scanners had been developed, full lung volumes in a breath-hold (10–20 s compared with 40  s for the four-slice scanner) and contrast-enhanced images during multiple phases of the cardiac cycle were now

S.K. Alford and E.A. Hoffman

feasible. As technology improved, the gantry rotation speed also increased, leading to improvements in technical scanner performance. As detector arrays advanced, 64 slices could be acquired in one gantry rotation in as little as 0.33  s. With MDCT systems, the scanner can also be used in an axial mode where the detector and X-ray source do not spiral through the patient, but rather remain in the same axial position and provide data for lung in the detector plane. This allows for imaging of a slab of lung slices over time to provide functional imaging such as of perfusion. As detector arrays widen, more lung can be covered by the slab using the axial mode. One limitation may be the use of a very broad X-ray beam needed to cover the lung volume without spiraling. The effect of a cone-beam X-ray source on the image reconstruction is currently being explored and new reconstruction algorithms are being developed [15–19].

2.2 Dual-Energy CT A new generation of CT scanner has been introduced by Siemens Medical Systems (Somatom Definition and Somatom Definition Flash) that includes two X-ray sources and two detectors mounted on a rotating gantry and angularly offset approximately 90° from one another [20]. The first dual-source CT (DSCT) scanner system specifications were similar to those of the 64-slice scanner, but could either be used as a dual source, providing twice the coverage for the time, or for dual-energy CT (DECT) imaging, where X-ray sources are set to different energies (kilovolts). DSCT utilizes both sources, thereby increasing temporal resolution, which allows for less motion artifact, which is important for cardiac and lung imaging applications. DECT imaging acquires data from two X-ray guns set to a different energies such as 80 and 140 kV. At the two energy levels, there is a density shift in regions with iodinated or xenon gas contrast agents, but little to no concomitant shift is observed in normal body tissues, allowing for sensitive discrimination between tissue types and contrast agent [21, 22]. The iodine contrast component can be separated out by subtracting lung tissue from the two images and the automatic removal of bone. This iodine map provides a perfused blood volume or microcirculation map. Although the enhancement is also dependent on external factors such as contrast volume, flow rate, and administration site, these maps can serve as an index of regional lung perfusion. In addition, a “diagnostic scan” can be determined from an averaging of the data collected by the two X-ray sources. Alternatively, the iodine signal can be subtracted from the reconstructed image to provide for the ability to assess a virtual unenhanced image for regional lung density analysis. The second-generation DSCT scanner (Siemens Definition Flash) rotates at 0.28 s, provides 128 slices

35  Measurement of Pulmonary Vascular Structure and Pulmonary Blood Distribution by Multidetector-Row

per rotation, and spirals through the entire thorax in as little as 0.6 s with a table feed of up to 43 cm/s. Added filters placed in front of each X-ray source provide improved separation of the delivered photon energy spectra from the X-ray tubes set at 80 and 140 kV, providing increased dual-energy-based characterizations.

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voxel value of the 2D maximum intensity projection image. Misrepresentation (an over- or underrepresentation) occurs more frequently with this technique, especially if there are overlapping structures or high-intensity regions in the vasculature such as calcification.

2.4 CT Angiography 2.3 Quantitative Assessment of Volumetric Chest MDCT Images The structure and integrity of the pulmonary vasculature can be assessed with a noncontrast spiral lung volume scan obtained typically at full inspiration. This lung volume provides the best contrast between the air-filled spaces and the parenchyma, airways, and most pathological lung processes. CT contrast is based on the attenuation of different tissues, approximately related to tissue densities. Each voxel reflects the tissue density or X-ray attenuation coefficient by its Hounsfield unit (HU) value. The HU scale is defined as -1,000 HU for air, 0 HU for water, and 1,000 HU for bone. For a noncontrast volumetric lung scan, if one considers the lung as composed of two materials, air at -1,000 HU and “tissue” (including blood volume) at approximately 55 HU, then the density in HU can be converted to the amount of air and “tissue” content per voxel [23]. Volumes are determined on the basis of the product of the segmented in-plane cross-sectional area by the slice thickness, summed over all the slices spanning the segmentation. By combining these two measures, one can determine the absolute volumes of air or tissue in the lung from the region volume multiplied by the fraction of air or tissue. With the recognition that many peripheral pulmonary processes either increase or decrease regional lung density, there is a growing focus on quantifying the distribution of lung attenuation values within CT images. Lung parenchyma voxels can be categorized on the basis of their attenuation or HU values and according to their place in the lung attenuation histogram. This has been applied to emphysema, with density masks of −950 and −910 HU used to determined the percentage of severe and moderate emphysema present in the lung parenchyma [24]. Postprocessing techniques have been developed to aid with management and assessment of large volumetric CT data sets. The near isotropic resolution of the MDCT data allows for 2D and 3D visualization. Three-dimensional volume rendering images of the lung, lobes, airway, or vasculature tree segmentations can be used to visualize the anatomy in three dimensions. Multiplanar reconstructions in the coronal, sagittal, or oblique views are used to evaluate the pulmonary vasculature, specifically for defining the extent of embolization or stenosis. In maximum intensity projection images, the highest HU value attenuation along a user-selected ray becomes the

As a result of the increased image-acquisition speed, improved spatial and temporal resolution, and greater scan volume with newer MDCT scanners, CT angiography (CTA) has become an excellent clinical assessment technique for the pulmonary vasculature and has become the first-line imaging test for the clinical assessment of a suspected acute PE. CTA protocols obtain high-resolution images of the entire chest by acquiring thin high-resolution slices during enhancement from an iodinated contrast medium delivered intravenously with a contrast injector. The timing of the peak contrast enhancement with imaging is crucial and therefore a bolus-triggering software program or a test bolus is used to determine the scan delay time. If images are acquired too early, proper enhancement may not have occurred, resulting in a filling defect. This lessens the contrast between the blood and clot, leading to more difficulty in distinguishing the two. When abnormal hemodynamics are present, such as in the case of low cardiac output or right-sided heart dysfunction, the bolus time delay can vary significantly. Short scanning times help minimize the respiratory motion artifact and ensure high image quality. With scanners acquiring images in as short a time as 0.6 s, dyspneic patients will no longer have trouble with breathholding. In fact, it is possible to acquire full chest scans with no breath-hold at all. For quantitative studies, it will remain a requirement that images be acquired at standardized lung volumes. With the use of slower scanners, respiratory motion results in global parenchymal blurring and can be detected by its presence in all vessels in a region of lung.

2.5 Four-Dimensional X-Ray CT Perfusion Measurements Dynamic perfusion imaging with 4D MDCT scanners allows for quantitative in vivo measurements of regional pulmonary blood flow (PBF) and mean transit time (MTT) [25, 26]. A series of temporal, ECG-gated, breath-hold images are acquired in axial scanning mode (nonspiral scanning mode with fixed z coverage) during the delivery of a central bolus of iodinated contrast, thereby tracking the first pass of iodinated contrast through the pulmonary circulation. A catheter is placed in the superior vena cava and a power injector is used to deliver a fast, sharp bolus. Images are acquired during

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a single breath-hold, allowing for the acquisition of data at a constant lung volume, typically functional residual capacity. ECG-gating minimizes cardiogenic motion by scanning at the same point in each cardiac cycle. As the bolus passes through the pulmonary vasculature and lung parenchyma, a change in tissue radiodensity is reflected by a change in the measured CT voxel HU value. HU values directly increase with increasing concentration of iodinated contrast agent, thereby reflecting the concentration of iodinated contrast in the region at that time point. Lung parenchyma voxels are segmented and filtered to remove airways and vessels. Time– attenuation curves for the pulmonary artery and regions of lung parenchyma are used to calculate regional MTT and PBF using indicator-dilution theory. Wolfkiel et al. measured myocardial blood flow by electron-beam CT, using a singlecompartment model of indicator transport [27]. The basic concept of their model is that regional flow is proportional to maximum enhancement or the total accumulated contrast in the lung region of interest. Therefore, the peak height of the time–attenuation curve is a measure of the flow within that tissue region of interest. The indicator must satisfy several criteria to allow the application of first-pass indicator-dilution theory: (1) contrast indicator should not affect flow by physiologic or volume effects, (2) the indicator must mix with the blood uniformly, and (3) the indicator must remain in the intravascular space. Nonionic iodinated contrast agents used in CT are a suitable indicator. Wu et al., using the DSR scanner, applied this model in pigs to measure PBF and showed a good correlation with microsphere-based flow measurements [28]. The model assumes that tissue accumulation of indicator must be nearly complete before the onset of indicator washout. Wolfkiel and Rich tested this assumption by comparing time–density curves of the pulmonary outflow tract, lung parenchyma, and aortic outflow tract, and determined with a central intravenous, sharp bolus injection the washout was minimal and the assumption was valid [29]. Time–attenuation curves are obtained for regional lung parenchyma and pulmonary artery by plotting the mean HU value for a region of interest with respect to time. Flow per volume is obtained from the ratio of the peak HU value in the parenchymal region of interest to the area under the arterial input curve. In CT imaging, this equation can be rewritten in terms of HU value changes in the lung parenchyma and pulmonary artery. MTT measurements can be obtained by applying the residue-detection model for a bolus injection of contrast. Deconvolution techniques can recover the regional residue function. MTT can be calculated as the ratio of the area under the residue curve to the maximum height of the residue curve. Figure 1 demonstrates a typical time–intensity curve obtained from the region of the pulmonary artery as well as a dependent and nondependent parenchymal lung region. Quantitative measures of MTT and PBF are acquired for each predefined sized region of interest throughout the lung. Also

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shown in Fig. 1 is the ability to detect, without use of contrast agent, detailed anatomic structure of the pulmonary arterial tree. Recently, image processing software has been shown to have the ability to label the arteries and veins separately from the detected pulmonary vascular tree structure [30]. Partial volume effects observed in perfusion images suggest that the time–attenuation curves obtained from voxels of lung parenchyma do not represent flow solely at the capillary level, but may also represent flow from the small arteries. Won et al. demonstrated the ability to use dynamic CT imaging to extract the signal within a single voxel related to microvascular blood flow, thus demonstrating the ability to estimate perfusion parameters at or near the level of gas exchange [26]. This was done by assessing the time–attenuation curve in the feeding pulmonary artery and in the peripheral parenchymal regions to then extract a transfer function, which expresses the mathematical function serving to convert the arterial curve into the parenchymal curve. Deconvolution of the pulmonary artery and regional parenchymal time–attenuation curves consistently yields a bimodal transfer function consisting of a sharp, narrow peak and a second, overlapping more dispersed curve component. It was concluded that the first mode reflected the partialvolume small arteries in the voxel, and the second mode reflected the partial-volume microvascular bed. By separating out the first sharp, narrow curve component from the second, more dispersed component, and reconvolving the arterial input curve with only the second component of the transfer function, they obtained a parenchymal curve reflecting the microvascular perfusion. Therefore, the CT-based approach has the ability to determine the microvascular and arterial curves within peripheral regions of interest and correct for the partial-volume effect of the small arteries within the voxel.

3  MR Imaging Techniques MR imaging relies on the manipulation of gradient fields to create contrast based on the precession of hydrogen atoms when placed in an external magnetic field. These techniques do not use ionizing radiation; therefore, unlike CT, longitudinal studies to follow disease and studies in children are feasible without dose concerns. Traditionally, the lung has been a difficult organ to image with MR imaging. The lung is compose mainly of air, meaning it has a low proton density, resulting in low signal. In addition, the lung has an extremely heterogeneous magnetic susceptibility owing to multiple air–soft tissue interfaces which produce large local gradients that dephase the signal, resulting in very short T2* values [31, 32]. The signal is further reduced owing to respiratory motion, cardiac motion, pulsatility of PBF, and molecular diffusion. Fast imaging sequences are necessary to capture the signal before it decays by using ultrashort echo times (TE) [33]. A fast gradient echo

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Fig. 1  Upper left: Computed tomography (CT)-based regional pulmonary blood flow (PBF, mean normalized) measurements for a 46-year-old male smoker with pack-year history of 31  years. His spirometry test results were normal but CT imaging studies demonstrated early signs of emphysema. CT perfusion imaging studies demonstrated increased heterogeneity in regional PBF and mean transit time (MTT) measurements in subjects with early signs of emphysema compared with never smokers or smokers with normal spirometry and

imaging studies. Upper right: An example of time–intensity curves for the pulmonary artery (top graph) and a dependent (purple) and nondependent (orange) parenchymal region used to calculate PBF and MTT. Lower left: Detailed vascular anatomy is detectable in a nonenhanced CT scan of the lung obtained at a total lung capacity breath-hold. Lower right: New algorithms are being developed with the ability to separate arteries from veins. (Reprinted from [30] with permission)

sequence with ultrashort TE utilizing high-speed, high-strength gradient systems can be used to acquire structural and functional information about the lung. High-performance gradient systems with amplitudes in excess of 20 mT/m and slew rates over 120 mT/m/ms have allowed for ultrafast MR sequences, including ultrafast MRA and significant reduction in the imaging time. Fast imaging sequences typically utilize a fractional echo sampling scheme, high-bandwidth data acquisition, and a truncated RF pulse to minimize the TE.

allowing for the reduction of minimum TE and repetition time (TR). A short TR allows for shorter breath-holds, whereas a short TE minimizes the background signal and susceptibility artifacts. Gadolinium contrast is injected intravenously with a power injector at rates between 2 and 5 mL/s, followed by injection of a saline chaser, causing the blood to appear bright in images. The development of parallel imaging has further improved the ability of 3D MRA to capture the pulmonary vasculature, especially in dyspneic patients who breath-hold poorly. Parallel imaging uses the geometry of numerous surface array coils and sensitivity maps to create k-space information from undersampled regions. The trade-off for faster data-acquisition speeds (temporal resolution) or higher spatial resolution is a reduction of the SNR. By combining parallel imaging with nonCartesian k-space filling pulse sequences such as simultaneous acquisition of spatial harmonics (SMASH) and sensitivity encoding (SENSE), one can further improve image quality. Depending on the clinical indication or research question, parameters can be adjusted to obtain either a set of highspatial-resolution, monophasic images or a multiphasic image set with high temporal resolution, referred to as dynamic time-resolved contrast-enhanced 3D MRA. To obtain higher

3.1 MR Angiography Contrast-enhanced 3D MRA was introduced by Prince et al. [34] and is now the most commonly used MR technique to image the pulmonary vasculature for the assessment of congenital anomalies, PH, or acquired conditions such as PE, specifically in the subset of patients unable to undergo CT (pregnant women or patients who would need following up). MRA images are obtained during a breath-hold using T1-weighted gradient echo MR imaging utilizing a high-performance gradient system,

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temporal resolution with the same image time (ensuring breath-hold), spatial resolution is sacrificed. A high-spatial resolution enables the evaluation of the peripheral pulmonary vasculature including the subsegmental branches, whereas high temporal resolution allows for discrimination of arterial and venous vessels through separation into various phases (arterial phase, parenchymal phase, venous phase). Regional perfusion can be assessed and quantified with contrast-enhanced images obtained from time-resolved angiographic imaging. Dynamic contrast-enhanced MR imaging of pulmonary perfusion acquires a series of images over time, tracking the first pass of a bolus of contrast through the pulmonary circulation. Images are acquired for a single plane (2D) or of the entire lung (3D) using T1-weighted ultrafast imaging sequences with short TR and TE during a low-dose bolus of gadopentetate dimeglumine (Gd-DTPA) contrast agent injected via an intravenous line in the antecubital vein in the arm. This technique uses ultrashort TE that then allow for higher temporal resolution, thereby allowing collection of dynamic information for the direct visualization of regional perfusion. Images are obtained approximately every second over 20–25 s at a slice thickness of 12–20 mm. Quantitative perfusion measurements are obtained on the basis of indicatordilution principles for first-pass bolus studies [35–37]. From signal intensity–time curves, PBF, MTT, and pulmonary blood volume (PBV) measurements are obtained using indicatordilution theory, deconvolution analysis, and the centralvolume principle on a voxel-by-voxel basis. For measurements to be accurate, the measured signal intensity change must be linearly correlated with the local concentration of Gd-DTPA [38]. It is therefore important to use a lower dose of contrast to not cause saturation. The regional PBV is calculated directly from the area of the MR signal intensity–time curve for a region of interest, normalized to the integrated arterial input function from the pulmonary artery. Regionally, PBF is calculated using a relationship between the concentration–time curve for the region of interest and the pulmonary artery. The residue function is determined by deconvolution. The initial height of the deconvolved time–concentration curves, or the residue function evaluated at time zero, equals the PBF. Using the central-volume principle, one determines MTT by PBV divided by PBF. Perfusion maps of the lung can be displayed to demonstrate perfusion deficits.

3.2 Cardiac Imaging for Right Ventricular Assessment Cardiovascular MR imaging techniques have been developed for quantitative measurements of right ventricular anatomy, shape and function, velocity profiles, stress and strain measurements, coronary artery anatomy, and myocardium viability. The

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assessment of the right ventricular anatomy and function is necessary along with pulmonary vasculature measurements to fully understand pulmonary vasculature diseases, specifically for pulmonary arterial hypertension. Protocols combining MRA, lung perfusion imaging, and the assessment of rightsided heart function with cardiovascular MR imaging allow MR protocols to provide a comprehensive evaluation of the lung and heart. Delayed contrast enhancement imaging is widely used to assess myocardium viability following myocardial infarctions, but different delayed enhancement patterns have also been documented in subjects with PH. In addition to the first-pass kinetics and demonstration of pulmonary vasculature anatomy from MRA, images taken 10–15 min following contrast administration demonstrate a pattern of delayed enhancement which allows for the assessment of myocardial viability. In delayed-enhancement imaging, there is hyperenhancement of nonviable tissue, reflecting irreversibly damaged regions of myocardium necrosis and fibrosis [39, 40]. MR quantitative volumetric assessment of the ventricles has become the gold standard and work has been done to determine normative values for cardiac parameters based on age and sex [41]. Many pulse sequences, including spin echo (“black blood”) and steady-state free precession (“bright blood”), have been developed for the visualization of the heart chambers and anatomy. Static image sets are acquired at enddiastole and end-systole while the subject is holding his/her breath at end expiration. A dynamic cine image set can capture multiple phases of the cardiac cycle. Although 2D static images can provide data on the anatomy, a 3D contiguous stack of short-axis or transverse images with at least two phases of the cardiac cycle is necessary for volumetric measurements. Manual or semiautomatic segmentation of the endocardial and epicardial contours is performed and cardiac parameters including ventricular volumes, stroke volume, ejection fraction and myocardial mass can be calculated. Right ventricular volume for end systole (ESV) and right ventricular volume for end diastole (EDV) are determined on the basis of Simpson’s rule. Stroke volumes can be determined by subtracting ESV from EDV. Ejection fraction is the stroke volume divided by the EDV. Ventricular mass is the myocardial volume multiplied by the specific weight of muscle (1.05 g/cm3). Phase-contrast MR imaging is capable of determining velocity and blood flow in the pulmonary vasculature and has been used to differentiate pulmonary disease such as pulmonary arterial hypertension [42]. Images are obtained in a plane perpendicular to the dominant flow and voxels represent the signal phase, which is velocity-encoded. Volumetric flow measurements are obtained by multiplying the spatial mean velocity (cm/s) of blood flow with the cross-sectional area of the vessel (cm2). Measurements of the velocity and flow of the pulmonary artery can be used to determine stroke volume, cardiac output, ejection fraction, and regurgitation fraction. Many new techniques and applications of cardiovascular MR

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imaging are currently in development. Myocardial tagging sequences track the motion of the heart by using RF pulses to form grid lines across images which deform as the heart contracts. Three-dimensional global and local deformation such as strain patterns can be determined for the right ventricle [43]. Real-time MR imaging is being used to perform MR-guided endovascular catheterization procedures. Catheter placement is visualized by susceptibility artifacts and right ventricle and pulmonary artery pressures can be determined and correlated with ventricular volume measurements to construct right ventricular pressure–volume curves [44, 45].

3.3 Arterial Spin Labeling Arterial spin labeling (ASL) is an MR perfusion method for quantitatively measuring regional PBF by using arterial blood as an endogenous tracer. It is completely noninvasive, does not require any contrast injection, and is repeatable. The protons in the water of pulmonary arterial blood are magnetically tagged, and the quantity of tracer in the lung parenchyma can be determined on the basis of measurable magnetization effects of the water present in the lung. ASL magnetizes the blood by applying an inversion RF pulse proximal to the imaging slice, thereby tagging the blood. MR imaging for ASL is typically performed using a 1.5-T scanner with a phased-array coil and ECG gating to minimize cardiac motion. Breath-holding at functional residual capacity ensures consistent lung volume, minimizes respiratory motion, and optimizes the volume of blood relative to the air in the lung and thus improves the SNR. ASL techniques can be divided into two categories: steady-state and pulsed sequences. Steady-state ASL was introduced by Detre et al. [46, 47] and applied to the lungs of human subjects by Roberts et al. [41, 48, 49]. The steady-state ASL technique measures perfusion on the basis of a detected change of steady-state magnetization due to the labeling of arterial water supplying an organ. Pulsed ASL sequences were introduced by Edelman et  al. [50] and now several specialized pulsed ASL sequences have been developed, including signal targeting alternating RF (STAR) [51–53], flow-sensitive alternating inversion recovery (FAIR), and more recently, FAIR with an extra RF pulse (FAIRER) [54]. These techniques provide a perfusionweighted image generated by the subtraction of a tagged and nontagged image. The pulsed ASL technique labels the arterial blood by inverting the protons proximal to the imaging slab by applying a selective inversion recovery RF pulse. For the lung, spins are typically magnetized in the right ventricle or main pulmonary artery. After a transit time delay, a tagged image is acquired. This delay allows for spins to distribute throughout the pulmonary circulation. A control image (no tagging) is also obtained. These two images are then pair-

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wise subtracted, yielding a perfusion-weighted image. The signal intensity of each voxel in the perfusion-weighted image is proportional to the volume of arterial blood delivered to the voxel during the time delay, which in turn is proportional to the local perfusion. Quantification of regional blood flow can be determined from ASL images by the application of mathematical models that take into account other physiological and MR factors. The Bloch equation has been modified to include flow effects and kinetic models have been developed for the various pulse sequences [55]. Perfusion-weighted images have near-complete background suppression since the signal from stationary protons present in both images will cancel out during the subtraction. This subtraction is extremely sensitive to motion. Another consideration is that although ASL provides perfusion-weighted images, it provides a snapshot of blood flow located in many vessels, not just the capillary bed. All flowing blood entering the image slice from outside the tagged region will contribute to the signal intensity of the perfusion-weighted images; therefore, changes in signal intensity could reflect alterations in local arterial blood flow, venous blood flow, or both.

3.4 Hyperpolarized 3He MR Imaging The use of a hyperpolarized gas such as 3He with MR imaging was introduced in the mid 1990s, and hyperpolarized 3He MR imaging provides a variety of techniques capable of providing functional and morphological information on the lung. 3He is an inert, nontoxic noble gas, not absorbed in the body after inhalation. To enhance the signal by 4–5 orders of magnitude, the gas can be hyperpolarized by optical pumping techniques (spin-exchange method or metastability-exchange method). Hyperpolarization is a process in which atoms are brought to a higher energy level by the introduction of laser light at appropriate wavelengths. Depending on method, 30–60% polarization can be achieved. The hyperpolarized gas is inhaled and diffuses rapidly into the air spaces of the lung, acting as a contrast agent for the ventilated airways and alveolar spaces. To image with hyperpolarized 3He, the MR scanner must have broadband RF transmit/receive hardware and have a coil tuned to the Larmor frequency of 3He, not 1H. 3He is currently an investigational new drug and has not been approved by the Food and Drug Administration for general clinical use. No adverse effects have been reported to date, but continuous pulse oximetry is performed to monitor oxygen saturation levels to ensure oxygenation does not fall with the breathing of anoxic or hypoxic gas mixtures during scanning. Four techniques for hyperpolarized 3He MR imaging have been developed: static-ventilation, dynamic-ventilation, diffusion-weighting, and oxygen-sensitive sequences. For a static representation of lung ventilation, the lung is imaged

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during a 10–12-s breath-hold following a single inhalation of 3 He gas. A normal lung will have a homogenous gas distribution, demonstrating ventilation (or delivery of the gas) throughout the lung, whereas a diseased lung will have multiple signal voids or ventilation defects. Dynamic imaging sequences with high temporal resolution can be performed while a subject continuously breathes following a single breath of gas. A series of images are obtained, demonstrating the dynamic distribution of ventilation, allowing for the calculation of transit times of the gas to the trachea, bronchi, airways, and alveolar spaces. On the basis of normal transit times and prolonged washout times, impaired ventilation patterns and air trapping can be detected. Diffusion-weighting imaging provides information about the pulmonary microstructure by measuring the Brownian motion or the random microscopic molecular movement of the gas. To perform this, bipolar gradients are applied to dephase and rephrase spins prior to signal acquisition, and therefore only diffusing spins will contribute to the signal. From a diffusion-weighted image set, the apparent diffusion coefficient (ADC) is calculated regionally, providing ADC maps. Diffusion, assessed by the ADC, is related to the dimensions of the air spaces. Typically, the gas is highly restricted by the walls and alveoli in the lung. But, if the gas is less restricted, it can diffuse over greater lengths. In emphysema, there is destruction of lung parenchyma, leading to an increase in air spaces. The gas is now less restricted, which is reflected by higher ADC values. Oxygen-sensitive sequences can determine the regional alveolar oxygen pressures (pAO2), which reflect the regional pulmonary perfusion, V/Q ratio, and oxygen uptake in the lung. This technique is based on the linear correlation between the partial pressure of oxygen and the loss of hyperpolarization of 3 He gas. Depolarization rates of the hyperpolarized 3He gas are significantly affected by the RF pulses and the paramagnetic properties of oxygen [56]. The dipolar interaction of oxygen will lead to a gradual decrease in the magnetization of polarized 3He. This effect, once separated from the affect of RF pulses, can be used to determine regional measurements of partial pressure of oxygen (pAO2) and depletion rates. Different pulse-sequence techniques have been developed, including a double-acquisition method [57, 58], a single-breath-hold acquisition [59, 60], and a 3D acquisition scheme [61]. The doubleacquisition technique allowed for determination of pAO2 by distinguishing RF from O2 effects with two identical sequences performed with different timing delays. A limitation of this technique was the necessity for identical breath-holds (i.e., equal lung volumes) during the two inhalations of hyperpolarized 3He gas for reliable analysis. The single-breath-hold acquisition scheme uses a pulse sequence with a changing interscan time to acquire information on the flip angle and oxygen effect during one breath-hold. A very short interscan time is used for the acquisition of the first two images, which are used to

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Fig. 2  (a) A series of hyperpolarized 3He images over time from one slice of a 3D single-scan, pO 2 imaging sequence demonstrate loss of signal which is both time- and oxygen-concentration-dependent. (b) From this longitudinal series, a color map with partial pressures of oxygen (pAO2) can be determined. (Adapted and reprinted with permission from [55])

d­ etermine the effect from the flip angle. The remaining images of the series are acquired with a longer interscan time and are used to calculate the molecular oxygen relaxation rate. In healthy subjects, the pAO2 distributions are very homogeneous. An example of this is shown in Fig. 2. The relaxation time, T1, is a function of time because pulmonary gas exchange leads to a fall in alveolar pO2 during a breath-hold. For a breath-hold of up to 35 s, the oxygen uptake is perfusion-limited, not diffusion-limited, and therefore the drop in pO2 is assumed to be a linear process; therefore, the loss of hyperpolarized gas polarization over time is proportional to the concentration of alveolar pO2. A method to derive regional V/Q ratios from pAO2 measurements has been developed from mass-balance relationships for alveolar CO2 and O2 gas exchange [62]. In addition to pAO2 MR measurements, this method requires venous blood obtained by a pulmonary artery catheter for calculations.

4 CT- and MR-Based Blood Pool Agents Blood pool agents provide contrast in the vasculature over a longer time so visualization of structure can occur without reinjection of contrast. These agents could provide the ability to use imaging for the assessment of treatment, such as tissue plasminogen activator therapy in acute PE, without the nephrotoxic effect of contrast agents. The ideal blood pool agent should have the following properties: a long half-life, remain exclusively in the intravascular space, low immuno-

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Fig. 3  Three-dimensional (3D) cardiac reconstructions extracted from an multidetector-row CT (MDCT) (Siemens Sensation 64) image data set acquired from an anesthetized supine sheep 3  h after intravenous infusion of liposomal contrast agent [63–66]. (Image data from an unpublished study by the authors in collaboration with Ananth Annapragada, Ketan Ghaghada, and Edwin van Beek)

genicity, low toxicity, be excretable, and have a high number of reporter groups. Two methods have been developed for blood pool agents. The first group consists of in vivo or ex vivo labeling of blood cells or plasma components. The second category includes synthetic macromolecules, colloids, or liposomes carrying a diagnostic label. Liposome-based blood pool agents are currently being developed for CT and MR imaging [63–66]. The liposomes contain contrast agent, iodinated contrast for CT and Gd-DTPA for MR imaging, in the central core of the liposome. This way, the contrast is protected from breakdown and the stealth coating of the liposome, coupled with its 100-nm size, prolongs its residence time and keeps it from being cleared by the kidney. Instead of renal clearance of the contrast agent, the blood pool agent is taken up and cleared by the reticuloendothelial system. A first application for these agents is envisioned to be in a so-called triple rule out whereby a patient with chest pain can be given a single dose of the liposomal contrast agent and evaluated for pulmonary emboli, peripheral sources of clot, aortic aneurysm, and coronary artery disease coupled with a dynamic study of myocardial function. An example image from a sheep study is provided in Fig. 3, which shows a cardiac image acquired from a sheep into which the liposomal agent was injected and which was imaged 3 h after injection via a Siemens Sensation 64-slice MDCT scanner.

5 Diagnosing Pulmonary Vascular Disease with CT or MR Imaging CT and MR imaging are emerging as complementary imaging modalities for the assessment of the lung. They both have strength in the assessment of the pulmonary anatomy, including

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the structure of the pulmonary vasculature and assessment of abnormalities suggestive of pulmonary vasculature disease (pulmonary emboli, PH, vascultitis, and other anomalies). In addition, imaging studies have proven helpful for a variety of less common conditions, congenital and acquired, such as pulmonary sequestration, pulmonary arteriovenous malformations, and complex congenital heart defects such as partial anomalous pulmonary venous return. Although qualitative methods may provide some information such as in the case of acute PE, quantitative methods to evaluate regional pulmonary perfusion can be used to more precisely assess the pulmonary function and may help with the diagnosis of certain disease states, such as pulmonary arterial hypertension.

5.1 Pulmonary Embolism CTA has been the first line for assessment of subjects suspected of having acute PE, and MRA has provided another excellent imaging modality in the case when CTA is not possible (pregnant woman, contrast allergy, etc.). In 1992, Remy et al. introduced pulmonary CTA with the assessment of 42 patients with central artery emboli [67]. They demonstrated 100% sensitivity and 96% specificity with 5-mm collimation. With the improved speed and thinner submillimeter collimation of newer scanners, CTA of the pulmonary arteries is capable of imaging the pulmonary arteries at the segmental and subsegmental levels, thereby detecting peripheral, small pulmonary emboli. Studies have demonstrated that the clinical validity of using CTA to rule out PE is similar to that of using conventional pulmonary angiography [67–70]. The high negative predictive value of a normal CTA study and its association with beneficial patient outcome has been demonstrated [71]. The value of CTA for acute PE assessment is not solely due to its ability to visualize the pulmonary arteries down to the subsegmental and fifth generation, but also in its ability to provide clinicians with alternative diagnostic information. CT offers a complete assessment of the lung parenchyma and mediastinal structures, which has led to alternative diagnoses in a large subset of patients initially studied for suspected PE. Utilizing mediastinal (soft tissue) window leveling, one can assess acute PE by the presence of a pulmonary arterial filling defect. Regions of hypoenhancement may be central, marginal, or complete. To confirm the defect is arterial, one can switch to lung windows to demonstrate an accompanying bronchus or follow the course of the vessel back centrally. Lung parenchyma findings include wedgeshaped, peripheral or pleural-based consolidation, representing an area of hemorrhage or possible infarction. Dual-source MDCT imaging has been applied to the assessment of pulmonary emboli [71]. Endoluminal clots can be detected with an average of images from the two-tube X-ray sources as

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Fig. 4  A 60-year-old male with a smoking history of 30 pack-years with small-sized peripheral clots present in the right upper lobes with a corresponding perfusion defect. On the contralateral side, there is a small perfusion defect without clot present on transverse images. Image parameters: tube A, 140  kV; tube B, 80  kV;

effective milliampere seconds, 265/46; 120  mL of 35% contrast agent at an injection rate of 4  mL/s. Dose–length product, 281 mGy cm. (Courtesy of Martine Remy-Jardin and Jacques Remy, Department of Thoracic Imaging, University Center of Lille, Lille, France)

efficiently as with single-source CTA, with the perfusion map providing additional information regarding the lung parenchyma. Typical triangular-shaped wedge defects, as seen in Fig. 4, can be demonstrated and are helpful in distinguishing acute PE from a nonspecific lung consolidation.

right side of the heart. Although both MDCT and MR imaging are used to assess the heart, cardiovascular MR imaging is the preferred method for assessment of the right side of the heart. Follow-up of subjects with PH provides information on quantitative ventricular mass, ventricular volume, and ejection fraction, which can be tracked over time to determine disease progression. Evaluation of septal bowing and the curvature from short-axis cine images can be determined. Short-axis curvature measures have been found to have a clear linear relationship to invasive systolic pulmonary artery pressure [73]. Delayed-enhancement imaging provides information on the functionality of the myocardium, with infarcted tissue demonstrated as bright hyperenhanced regions of myocardium.

5.2 Pulmonary Hypertension In the case of PH, CTA or MR imaging studies are required to make a diagnosis, to identify the type of PH, to evaluate the functional and hemodynamic impairment, and to monitor changes in disease in response to therapy. MR imaging is increasingly used for the evaluation of PH owing to its ability to provide a good assessment of the pathological and functional changes in both the heart and pulmonary circulation. Imaging can discriminate between the two main contributing factors: a primary lung parenchymal disease and a primary cardiac process. Imaging signs indicative of PH include central pulmonary artery dilatation, tapering of the peripheral pulmonary arteries, and enlargement of the right side of the heart. Measurement of the diameter of the main pulmonary artery to assess for dilatation is done with either CT or MR imaging. The enlargement of the lobar arteries to a diameter greater than that of the accompanying bronchus is another useful sign of pulmonary arterial hypertension. To help discriminate the type of PH, the assessment of the diameter and distribution of the pulmonary veins and venous flow can aid in the diagnosis of postcapillary PH. Enlarged pulmonary veins indicate chronic pulmonary venous hypertension secondary to left-sided heart disease. Perfusion imaging has been applied to PH in a few small studies. Contrast-enhanced MR perfusion imaging demonstrated long time to peak enhancement times in a group of PH subjects compared with normal subjects [72]. Right-sided heart disease, including ventricular hypertrophy and enlargement, is a common and expected secondary finding of PH due to the increased workload placed on the

5.3 Hypoxic Pulmonary Vasoconstriction The normal response of the lung to hypoxia is hypoxic pulmonary vasoconstriction (HPV) resulting in the shunting of blood toward a better-ventilated region of lung for improved oxygenation. CT-based perfusion methods have been used for the characterization of acute lung injury and HPV. There are suggestions in the literature that, in response to inflammatory processes, HPV is blocked [74, 75]. Easley et al. [76] used MDCT to demonstrate in a sheep model the regional failure of HPV with an endotoxin injury (known to inhibit HPV) compared with lung lavage injury where PBF redistributed consistent with a preserved HPV. Hoffman et al. showed the coexistence of HPV failure and normal HPV response in a sheep with both pneumonia and an endobronchial valve that blocked ventilation to an unaffected lung region (Fig. 5) [61]. MR perfusion imaging techniques have been applied to highaltitude pulmonary edema (HAPE) and provided insights into its pathogenesis. Dehnert et  al. demonstrated with dynamic contrast-enhanced MR imaging that the inhomogeneity of HPV increases with hypoxia in all subjects, but more so in HAPE-susceptible individuals [77]. Hopkins et al. [78] used ASL MR imaging to spatially quantify PBF

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Fig. 5  Before (top row) and after (bottom row) endobronchial valve placement. The left column provides the view of one CT section at the level of the diaphragm dome. Note the significant dependent pneumonia (a). The center column demonstrates ventilation overlaid in color as assessed by xenon-CT. Note that there is little ventilation in either the baseline or the post valve in the region of the dependent pneumonia. By design, there is a large region where ventilation was eliminated by the valve placement (b). The right column shows a color overlay of the perfusion measurements. Note that in region (c) coinciding with region (b) from the central column (no ventilation due to valve placement) there is a regional loss of perfusion, indicating an intact hypoxic pulmonary vasoconstrictor response. At the

same time, in the region of pneumonia (d), where there is little or no ventilation, blood flow is enhanced after valve placement. Presumably, blood flow shunted away from the valve-based hypoxic pulmonary vasoconstriction is shunted straight toward the region of inflammation, presumably because the inflammation has served to blunt hypoxic pulmonary vasoconstriction in this region, leaving this region as the path of least resistance for the blood flow diverted from the region affected by hypoxic pulmonary vasoconstriction (HPV). Thus, this image demonstrates that lung has the ability to locally modulate HPV on the basis of local inflammation. (Adapted from [61] with permission. Official journal of the American Thoracic Society copyright American Thoracic Society)

heterogeneity in HAPE-susceptible individuals, HAPEresistant individuals, and individuals without altitude exposure. Hopkins et al. found that HAPE-susceptible individuals have increased PBF heterogeneity in acute hypoxia, consistent with uneven HPV. It has been hypothesized that uneven HPV in HAPE-susceptible individuals may lead to overperfused regions of lung with increased capillary pressures, resulting in hydrostatic edema to those regions.

the vascular bed related to the severity of the parenchymal destruction has been demonstrated in COPD subjects [79], changes in the early COPD have just recently been explored. Increased heterogeneity of perfusion measures such as the PBF and MTT has recently been demonstrated in smokers with normal PFTs but with very early signs of emphysema, possibly indicating the predilection of these subjects to the disease [80, 81]. Data in animals demonstrate that, in the presence of inflammation, HPV is blocked [61], thus preserving the normal cascade of events involved in the inflammatory process, including the delivery of bone-marrow-derived progenitor cells to repair collateral damage. Hoffman et al. have hypothesized that the failure of inherent mechanisms to block the normal HPV response in the presence of inflammation may lead to a failure of the normal mechanisms serving to limit the inflammatory response and reparative process, thus leading to the emphysema [61]. We are taught that the normal response to regional hypoxia in the lungs is to shunt blood toward better-ventilated regions. However, when local hypoxia

5.4 Emphysema Recent evidence suggests that vascular changes may be important to the pathogenesis and progression of emphysema and may relate to an abnormal HPV response in the presence of inflammation. Dynamic perfusion measurements with CT and MR imaging can provide valuable regional information about vasculature changes related to COPD. Although a reduction of

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is induced by smoking-related inflammatory processes which flood alveoli such as in peripheral bronchiolitis (micro nodules) and/or local interstitial edema (ground glass), it becomes counterproductive to shut down blood flow to that region needing flow to maintain the cascade of defense and reparative mechanisms such as the enhanced delivery of bone-marrowderived mesenchymal stem cells. In support of the above hypothesis is the observation by Remy-Jardin et al. [62] that there is an increased propensity for the lung to develop emphysema in regions where prior inflammatory processes (illdefined ground-glass opacities/micronodules) have been observed. In an individual unable to block HPV in the presence of inflammation, increased vascular resistance to inflamed lung regions is compounded by gravitational effects which further limit flow to the lung apices, thus providing an explanation as to why smoking-related emphysema tends to preferentially occur at the apices.

6 Conclusion CT and MR imaging techniques for the lung have seen great advancements over a short time and can now be used to provide structural and functional information regarding the pulmonary vasculature. Imaging techniques for the assessment of PE have become first-line choices for physicians. New imaging techniques including perfusion imaging capable of regional blood flow assessment are being developed along with new scanner technologies, including DECT, which holds promise for new methods to better explore pulmonary vascular diseases. With the advancements in imaging to assess both structure and function, and to correlate regional structural changes in the lungs to regional functional alterations, and to quite possibly image functional changes as a precursor to structural alterations, there is a growing effort to utilize imaging-based phenotypes to understand the pathological processes leading to disease, to identify underlying genotypes associated with disease susceptibility, and to utilize quantitative imaging tools for drug and interventional device discovery, safety testing, and outcome assessment.

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524 69. Teigen CL, Maus TP, PFd S, Johnson CM, Stanson AW, Welch TJ (1993) Pulmonary embolism: diagnosis with electron-beam CT. Radiology 188:839–845 70. van Rossum AB, Pattynama PM, Ton ER et al (1996) Pulmonary embolism: validation of spiral CT angiography in 149 patients. Radiology 201:467–470 71. Thieme SF, Becker CR, Hacker M, Nikolaou K, Reiser MF, Johnson TRC (2008) Dual energy CT for the assessment of lung perfusion - correlation to scintigraphy. Eur J Radiol 68:369–374 72. Ohno Y, Hatabu H, Murase K et  al (2007) Primary pulmonary hypertension: 3D dynamic perfusion MRI for quantitative analysis of regional pulmonary perfusion. Am J Roentgenol 188:48–56 73. Roeleveld RJ, Marcus JT, Faes TJC et  al (2005) Interventricular septal configuration at MR imaging and pulmonary arterial pressure in pulmonary hypertension. Radiology 234:710–717 74. Hlastala MP, Lamm WJ, Karp A, Polissar NL, Starr IR, Glenny RW (2004) Spatial distribution of hypoxic pulmonary vasoconstriction in the supine pig. J Appl Physiol 96:1589–1599 75. Lamm WJ, Starr IR, Neradilek B, Polissar NL, Glenny RW, Hlastala MP (2004) Hypoxic pulmonary vasoconstriction is heterogeneously distributed in the prone dog. Respir Physiol Neurobiol 144: 281–294

S.K. Alford and E.A. Hoffman 76. Easley RB, Fuld MK, Fernandez-Bustamante A, Hoffman EA, Simon BA (2006) Mechanism of hypoxemia in acute lung injury evaluated by multidetector-row CT. Acad Radiol 13:916–921 77. Dehnert C, Risse F, Ley S et al (2006) Magnetic resonance imaging of uneven pulmonary perfusion in hypoxia in humans. Am J Respir Crit Care Med 174:1132–1138 78. Hopkins SR, Levin DL (2006) Heterogeneous pulmonary blood flow in response to hypoxia: a risk factor for high altitude pulmonary edema? Respir Physiol Neurobiol 151:217–228 79. Ley-Zaporozhan J, Ley S, Eberhardt R et al (2007) Assessment of the relationship between lung parenchymal destruction and impaired pulmonary perfusion on a lobar level in patients with emphysema. Eur J Radiol 63:76–83 80. Alford S, van Beek E, Hudson M, Baumhauer H, McLennan G, Hoffman E (2008) Characterization of regional alterations in pulmonary perfusion via MDCT in nonsmokers and smokers. Eur Radiol 18:330–331 81. Alford S, Van Beek E, McLennan G, Hoffman E (2006) CT-based blood flow measurements in lung parenchyma of normal controls, normal smokers and smokers with emphysema [abstr]. Paper presented at: Radiological Society of North America 92nd Annual Meeting, Chicago, IL

Chapter 36

Quantification of DNA, RNA, and Protein Expression Fiona Murray, Jason X.-J. Yuan, and Paul A. Insel

Abstract  Advances in molecular biology and biochemistry have provided tools with which to explore and increase our understanding of mediators that regulate development and pathological changes in the pulmonary circulation. Combinations of techniques have allowed researchers to start to unravel complex biological pathways that have direct relevance to pulmonary research, such as those that contribute to cellular proliferation and vascular permeability. The aim of this chapter is to describe some of the established protocols to quantify DNA, RNA, and protein expression, discuss advances in these techniques, and illustrate how they can be applied. Expression levels of cell signaling components can be detected at the level of DNA, RNA, and protein. Southern blotting (also termed “Southern hybridization” and named for Edwin Southern, who developed the technique) allows for the detection of a specific DNA sequence in a preparation of DNA, typically from cellular nuclei. This method was modified and extended later for the detection of RNA and protein, named Northern and Western blots, respectively. All three of these protocols are based on similar principles and utilize similar methods. Alternatively, measurement of gene expression [using messenger RNA (mRNA) as a starting material for the generation of complementary DNA (cDNA)] can also be performed via reverse-transcription (RT) polymerase chain reaction (PCR), followed by agarose gel electrophoresis. A modification of this technique, real-time PCR (also termed “quantitative PCR”), allows for quantitative analysis of gene copy number. A number of other high-throughput techniques exist for the profiling of gene and protein expression, such as by use of DNA and protein microarrays; however, these will be discussed in more detail in subsequent sections. Many variations exist for the protocols outlined below and most buffers can be made using a variety of recipes or can be purchased from a number of suppliers. However, the principles remain the same; F. Murray (*) Departments of Medicine and Pharmacology, University of California, San Diego, BSB 3073, 9500 Gilman Drive, La Jolla, San Diego, CA 92093-0636, USA e-mail: [email protected]

understanding the stages in each process and their purpose is key. This chapter outlines the methods that have proven to be versatile, reliable, and sensitive. Keywords  Molecular biology • Methodology • Western blot • Northern blot • Southern blot • RT-PCR • mRNA expression

1  Introduction Advances in molecular biology and biochemistry have provided tools with which to explore and increase our understanding of mediators that regulate development and pathological changes in the pulmonary circulation. Combinations of techniques have allowed researchers to start to unravel complex biological pathways that have direct relevance to pulmonary research, such as those that contribute to cellular proliferation and vascular permeability. The aim of this chapter is to describe some of the established protocols to quantify DNA, RNA, and protein expression, discuss advances in these techniques, and illustrate how they can be applied. Expression levels of cell signaling components can be detected at the level of DNA, RNA, and protein. Southern blotting (also termed “Southern hybridization” and named after Edwin Southern, who developed the technique) allows for the detection of a specific DNA sequence in a preparation of DNA, typically from cellular nuclei [1]. This method was modified and extended by Stark and Towbin for the detection of RNA and protein, named northern and western blots, respectively [2–4]. All three of these protocols are based on similar principles and utilize similar methods, composed of five main steps: (1) sample preparation, (2) size-based separation by gel electrophoresis, (3) transfer to a solid support, (4) hybridization of a probe, and (5) detection (Fig.  1). Alternatively, measurement of gene expression [using messenger RNA (mRNA) as a starting material for the generation of complementary DNA (cDNA)] can also be performed via reverse-transcription (RT) polymerase chain reaction

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_36, © Springer Science+Business Media, LLC 2011

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sequence variation. Such polymorphisms are sometimes used to track the association of a DNA sequence variation with a genetic disease and to measure recombination rates that allow the generation of maps that measure the distance between RFLPs. Examples of the use of Southern blots include (1) the assessment of patients for mutations in bone morphogenetic protein receptor type II, which can contribute to the development of primary pulmonary hypertension, (2) the identification of hypoxia-induced oxidative base modifications in the vascular endothelial growth factor hypoxia-response element, and (3) characterization of the decreased ratio of mitochondrial DNA to nuclear DNA in the left and the right ventricle after 21 days of chronic hypoxia [14–16].

Fig. 1  Flow diagram depicting the five main steps common to Southern, northern, and western blotting, sample preparation, size-based separation by gel electrophoresis, transfer to a solid support, hybridization of the probe, and its detection

(PCR), followed by agarose gel electrophoresis [5, 6]. A modification of this technique, real-time PCR (also termed “quantitative PCR”), allows for quantitative analysis of gene copy number [7, 8]. A number of other high-throughput techniques exist for the profiling of gene and protein expression, such as by use of DNA and protein microarrays; however, these will be discussed in more detail in subsequent sections. Many variations exist for the protocols outlined below and most buffers can be made using a variety of recipes or can be purchased from a number of suppliers. However, the principles remain the same; understanding the stages in each process and their purpose is key. Below we outline the methods that have been or are used in our laboratory and have proven to be versatile, reliable, and sensitive. More detailed or alternative methods have been published [8–13].

2  DNA Quantification 2.1  Southern Blotting Southern blotting is most commonly used to detect specific DNA sequences in the genome, typically in DNA prepared from tissue or cells. Southern blotting can identify if a specific gene is present, the number of copies, the degree of similarity between the chromosomal gene and the probe, and if abnormalities exist in the structure of the gene. Mutations that result in DNA deletion, insertion, or rearrangement are readily detectable by Southern blotting and hence it is routinely used in the initially genotyping of transgenic animals. The procedure for Southern blotting can be used to detect restriction fragment length polymorphisms (RFLPs), which are indicative of DNA

2.1.1 DNA Isolation Approximately 200–250 mg of DNA can be purified from blood (10  mL), cultured pulmonary artery cells (approximately 5 × 107), and lung tissue biopsies (up to 1 cm3) using a variety of buffers to extract the DNA. Many such buffers are available from commercial suppliers and avoid the use of phenol–chloroform extraction, which was a classically used approach to prepare DNA. Extraction works best if tissues are homogenized or ground to a fine powder under liquid nitrogen before the extraction process. Tissue or cells are suspended in TE buffer [10  mM tris(hydroxymethyl) aminomethane–hydrogen chloride (Tris–HCl) pH 8.0, 1 mM ethylenediaminetetraacetic acid (EDTA)] then mixed with 10 mL/mL DNA extraction buffer [10 mM Tris–HCl (pH 8.0), 1  mM EDTA, 20  mg/mL RNAse, 0.5% sodium dodecyl sulfate (SDS)] and incubated for 1  h at 37°C. Proteinase K is added to give a final concentration of 100 mg/mL and the samples are incubated for a further 3 h at 50°C. An equal volume of phenol equilibrated with 0.5 M Tris–HCl (pH 8.0) is then added and the two phases are mixed for 10  min. The viscous phase is removed using a wide-pore pipette and the phenol extraction is repeated. Two preferred approaches can then be used to recover DNA: either dialyze the aqueous phase four times at 4°C against 4 L of 50 mM Tris–HCl (pH 8.0), 10 mM EDTA (pH 8.0) or add 0.2 vol of 10 mM ammonium acetate followed by 2 vol of ethanol, after which the DNA is removed using a Pasteur pipette, washed with 70% ethanol, and then resuspended in TE buffer (pH 8.0) at approximately 5 × 106 cells/ml. The DNA is left to dissolve overnight at 4°C; the concentration and purity are then measured using a spectrophotometer (to quantify A260/A280). Ideally this ratio should be 1.75–2.0; if A260/A280 is less than 1.75, protein contamination is present and the sample should be repurified. Commercial kits such as the QIAamp DNA mini kit (Qiagen) are available for rapid purification of DNA, which is increasingly the preferred choice of many researchers.

36  Quantification of DNA, RNA, and Protein Expression

2.1.2 Gel Electrophoresis and Transfer Once purified, the DNA (approximately 10  mg) is digested with restriction endonuclease enzymes [e.g., HindIII, EcoRI together with its appropriate 1× enzyme buffer and 1× acetylated bovine serum albumin (BSA) (New England Biolabs)]. At the end of digestion, gel-loading buffer [10× sample buffer: (65% (w/v) sucrose, 10 mM Tris–HCl (pH 7.5), 10 mM EDTA, and 0.3% (w/v) bromophenol blue] is added and the DNA is separated by electrophoresis alongside an appropriate DNA marker on a 0.7% agarose gel cast in 0.5× stock TBE buffer containing 0.5  mg/mL ethidium bromide [10× stock TBE buffer: 0.9 M Tris base, 0.9 M boric acid, 0.25 M EDTA, (pH 8.3)]. After adequate separation has been achieved, the DNA is visualized using UV light, and then the gel is directly soaked in an alkaline buffer (1.5 M NaCl, 0.5 M NaOH) to denature the double-stranded fragments for 45 min. This is followed by incubation with neutralization buffer (1.5 M NaCl, 1 M Tris–HCl, pH 7.4) for 30 min before transfer onto a membrane of choice. Nylon-based (e.g., Amersham Hybond N, N+, GE Healthcare), nitrocellulose, or poly(vinylidene fluoride) (PVDF) membranes can be employed. Nylon membranes are stronger, easier to recycle, and bind small DNA fragments more efficiently; however, they tend to give higher background values. Capillary transfer, which runs overnight, was used in Southern’s original method and still remains popular, at least in part owing to its low cost. For capillary transfer, the gel is laid on Whatman 3MM paper, placed in a reservoir of transfer buffer (10× stock standard saline citrate (SSC): 1.5 M NaCl, 0.15 M sodium citrate, pH 7.0) and covered with the membrane. To aid in removing air bubbles and ensuring complete contact with the Whatman 3MM paper, a stack of paper towels and a weight are used to hold the stack down. Transfer buffer moves from a reservoir below the gel into the dry stack of paper towels above the membrane, thereby carrying the nucleic acid with it until it is trapped on the membrane. The ­transfer process can be ­shortened to 30–60  min by using vacuum blotting or ­electrophoresis (according to the manufacturer’s instructions). The transferred DNA is fixed to the membrane by baking at high ­temperature (approximately 80°C for 0.5–2  h for both nitrocellulose and nylon membranes) or through cross-linking by exposure to UV radiation (nylon membranes).

2.1.3 Hybridization of Probe and Detection For prehybridization the membrane is incubated for 1–2 h at 68°C in hybridization buffer (6× stock SSC, 5× stock Denhardt’s reagent, 0.5% SDS, 100 mg/mL denatured, fragmented salmon sperm DNA) to block nonspecific attachment of the probe to the membrane. One then adds a radiolabeled probe specific for the gene of interest, either a DNA probe (cDNA clone, genomic

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fragment, oligonucleotide) or an RNA probe for the required period. Preparation of radiolabeled DNA and RNA probes is not within the scope of this chapter; however, other publications provide detailed methods [9]. Radioactive probes, most commonly with 32P, 33P, and 35S, which all produce b particles, can readily be detected on X-ray film. Washing the membrane to remove unbound probe is performed in several steps of increasing degrees of stringency depending on the application and degree of sensitivity required: typically, several washing steps are carried out in buffers starting with high salt to low salt concentrations (2× stock SSC to 0.1× stock SSC, 0.5% SDS at 65°C) and stringent conditions. Hybridization is detected by visualization on X-ray film by autoradiography, which allows for the identification of the sizes and the number of fragments of genes that bind to the probe. A major advance in Southern blotting is that nonradioactive probes, such as fluorescent or chromogenic dyes, are now available that allow detection of hybridization by the development of color on the membrane. These nonradioactive probes, such as digoxogenin, biotin, and fluorescein-11dUTP, are sensitive yet much safer, easier to dispose of, and less expensive than radioactive probes [13].

3 RNA Quantification The variety of methods that exist for the quantification of the transcript are northern blotting, in situ hybridization, RNase protection assays, RT-PCR, and cDNA arrays, all of which require the isolation of high-quality RNA.

3.1 RNA Isolation RNA can be readily purified from lungs and isolated pulmonary cells; however, since RNA is unstable compared with DNA, a number of steps need to be employed to ensure that its integrity is maintained throughout the procedure. It is important to inhibit the activity of RNases, avoid introduction of RNases from other sources, and work quickly: RNase-free glassware and plastics should be used or alternatively autoclaved, rinsed with chloroform, or treated with diethyl pyocarbonate (DEPC) before use, and addition of RNase inhibitors is recommended during cell/tissue lysis procedures. Furthermore, when tissue or cells are harvested for extraction of RNA at a later date, samples must be snap-frozen in liquid nitrogen or treated in a way to stabilize the RNA, e.g., with the addition of RNAlater (Qiagen). A number of methods exist for isolating RNA from cells and tissues, such as acid–phenol extraction coupled with alcohol precipitation, and guanidine isothiocyanate extraction

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coupled with cesium chloride ultracentifugation [17–19]. Commercially available RNA extraction kits and reagents use a combination of the above-mentioned extraction techniques or patented technology and are generally thought to be safer, yet effective, for extracting high-quality RNA from both tissue and cells; TRIzol® (a monophasic solution of phenol and guanidine isothiocyanate, Invitrogen) and Qiagen RNeasy kits are both routinely used in our laboratory. The choice of kit depends on downstream application and price; it is recommended to incorporate a DNase treatment step (available from each supplier) to remove genomic contamination. For isolation of RNA from pulmonary artery branches and lungs it is recommended to grind them to a fine powder in liquid nitrogen with a mortar and pestle and place them in lysis buffer, followed by further homogenization and then either to pass the lysate through a 25-gauge needle five times or to pipet samples onto a QIAshredder (Qiagen, Valencia, CA, USA) column (to disrupt the RNA. The expected yield of RNA using these kits is roughly 2 mg/mg of lung tissue and 5 mg per 1 × 106 cultured pulmonary artery smooth muscle cells (PASMCs). Purified RNA is dissolved in RNase free water and its concentration and purity are determined using a spectrophotometer (quantified A260/A280). RNA absorbs UV radiation at 260  nm with an extinction coefficient of 40  mg/mL when using a cuvette with a path length of 1 cm. The ratio A260/A280 for pure RNA is roughly 2 (range 1.75–2.0 is usually deemed an appropriate value to use the RNA for subsequent studies) but this ratio decreases in the presence of protein (which is preferentially detected at 280  nm). RNA integrity should also be confirmed by assaying 2–4 mg of the RNA sample by gel electrophoresis on a denaturing 0.7% agarose gel cast in 0.5× TBE containing 0.5  mg/mL ethidium bromide (see above), especially when the RNA is to be used for applications such as microarrays. Good-quality RNA should reveal (as identified with ethidium bromide) discrete 18S and 28S ribosomal RNA (rRNA) bands with the 28S rRNA band approximately twice as intense as the 18S rRNA band. If desired, poly(A) + RNA (mRNA, 1–2% of total RNA) can be purified from total cellular RNA by chromatography on oligo(dT)-cellulose, since for gene expression this is the RNA required [20, 21]. RNA selection is not always necessary, although for some uses, e.g., as a template to construct cDNA libraries, mRNA is required. Next we discuss the downstream applications of northern blotting and RT-PCR.

3.2 Northern Blotting Northern blotting employs a similar approach to that of Southern blotting but is designed to detect RNA rather than DNA. Northern blotting relies on the hybridization of a

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labeled probe to a target mRNA and is valuable for examining expression of specific genes in development, disease in response to various stimuli, and for detecting alternatively spliced gene variants. Northern blotting is still commonly performed and, for example, has been used to demonstrate the upregulation of genes such as angiopoietin-1 and vasoactive intestinal peptide receptor in patients with pulmonary hypertension [22, 23].

3.2.1 Gel Electrophoresis and Transfer Denaturation of RNA (by glyoxal and dimethyl sulfoxide, methylmercuric hydroxide, or formaldehyde) allows for its tight binding to nitrocellulose membranes and the hybridization of radioactive probes [24–26]: below we describe a method for the electrophoresis of RNA through gels containing formaldehyde. Samples are prepared by mixing 20  mg of purified RNA (4.5  mL), formaldehyde gel-running buffer [2.0  mL, 5× stick formaldehyde gel-running buffer: 0.1 M 3-morpholinopropanesulfonic acid sodium salt (pH 7.0), 40 mM sodium acetate, 5 mM EDTA (pH 8.0)], formaldehyde (3.5  mL), and formamide (10.0  mL), followed by incubation at 65°C for 15 min. The samples are mixed with loading buffer [10× stock sample buffer: 65% (w/v) sucrose, 10 mM Tris–HCl (pH 7.5), 10 mM EDTA, and 0.3% (w/v) bromophenol blue] and together with a molecular weight marker are run on a denaturing 1% agarose gel (the percentage of agarose depends on the degree of separation required) cast in 1× stock formaldehyde gel-running buffer. After adequate separation, the gel is removed, cut, and stained (in the marker lanes that contain size standards) with ethidium bromide (0.5 mg/mL) and photographed so that the size of the bands on the membrane after hybridization can be estimated. Before transfer the formaldehyde must be removed from the gel by washing with DEPC-treated water, after which the RNA is transferred (by capillary elution, vacuum transfer or electroblotting) in 20× stock SSC (3 M NaCl, 0.3 M sodium citrate, pH 7.0) to a solid support (either nitrocellulose or charged nylon membrane) similar to that outlined earlier for Southern blotting, and then the membrane is dried and baked at 80°C (or by UV cross-linking).

3.2.2 Hybridization of Probe and Detection Membranes are incubated for 1–2 h at 68°C in hybridization buffer (6× stock SSC, 2× Denhardt’s reagent, 0.1% SDS) and then with the radiolabeled probe for 16–24 h. After the membrane has been washed (0.2× SSC: 30  mM NaCl, 3  mM sodium citrate, pH 7.0 containing 0.1% w/v SDS, twice at 50–60°C for 10 min, depending on the probe), the bands on the gel are visualized by autoradiography (exposed for

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24–48  h at –70°C). The membrane can be incubated for 10 min at 96°C in “stripping buffer” (10 mM Tris–HCl, pH 7.5 containing 1 mM EDTA, and 1% SDS) and then washed three times for 10 min to allow for hybridization of a new probe. Stripping and reprobing is, however, only recommended on the stronger Nylon+ membranes and although this can be done repeatedly, we recommend no more than five cycles. Quantification of mRNA expression is measured by band intensity via densitometery. The gel is either scanned or photographed and the pixels in each band are measured using image quantification. We use ImageJ, free software available from NIH (http://rsb.info.nih.gov/ij/). A shape is drawn round the largest band and then copied to each band that requires quantification. It is important to determine the background around each band so as to normalize the intensity and always to run samples on the same gel and use different exposure times to confirm that the signal is not saturated. As controls for equal RNA loading in each lane on the gels, one uses a “housekeeping gene” such as glycerol 3-phosphate dehydrogenase (GAPDH) or b-actin and corrects results for differences in loading. As will be discussed below, no single housekeeping gene is ideal for such studies. Advances in northern blotting methods have included the introduction of nonradioactive probes such as those labeled with digoxogenin and biotin [13]. However, the greatest advance in detecting changes in gene expression has been the introduction of RT-PCR and microarray approaches, which have advantages compared with northern blots for studies of RNA.

3.3 Reverse-Transcription PCR PCR allows the amplification of specific DNA sequences in  vitro by primer extension of complementary strands of DNA [5, 6]. PCR is generally less expensive, easier, quicker, and more sensitive than other methods currently used to assess gene expression. The general method of PCR involves ­thermostable DNA (taq) polymerase-catalyzed amplification by multiple rounds of melting, annealing primers, and extension. Template cDNA mixed with a buffer that allows for maximal polymerase activity, primers specific for the gene of interest, and all four deoxynucleotides (dNTPs) are necessary for the reaction: it is recommended that a “master mix” be used to increase the accuracy of the procedure. Specific PCR conditions should be optimized for each application. Therefore, as the general method is outlined below, each parameter that influences the specificity of the PCR will be discussed. We have used PCR to show the expression pattern of ion channels and phosphodiesterases (PDEs) in PASMCs and changes with hypoxia or the development of pulmonary hypertension [27–29]. To use PCR to measure gene expression, isolated RNA must be converted into cDNA. RT converts almost exactly

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the same proportion of RNA into cDNA (e.g., addition of 1 mg RNA in a 20-mL RT reaction gives cDNA with a final concentration of 50  ng/mL), using dNTP (10 mM each) building blocks, enzymes such as Moloney murine leukemia virus or avian myeloblastosis virus, primers for the reaction, and RNase H to eliminate residual RNA [30]. An identical reaction without addition of reverse transcriptase should always be included to verify the absence of genomic DNA (“no-RT control”). Primers routinely used to aid in the synthesis cDNA are generic sequences (2 pmol), random hexamers (50 ng) or oligo(dT) (200 ng). Oligo(dT) binds to the poly(A) tail of mRNA and thus only synthesizes cDNA from mRNA, whereas random hexamers nonspecifically prime cDNA synthesis from nonpolyadenylated transfer RNA and rRNA. We use random hexamers since we generally normalize our data to 28S RNA (see later for the choice of housekeeping gene). Commercial kits are available for RT, and provide all the reagents required for the reaction: we routinely use both Iscript (Bio-Rad Laboratories) and Superscript III (Invitrogen).

3.3.1 Primer Design PCR amplifications are performed using gene-specific forward and reverse primers. Primer length, percent G–C content, melting temperature, and 3¢ end sequence needs are optimized for successful PCR [31, 32]. We generally design primers on the basis of the product of interest’s mRNA sequence (http:// www.ncbi.nlm.nih.gov/sites/entrez?db = nuccore&itool = toolbar). Primer specificity can be confirmed via NCBI BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Many Web sites exist to aid in the design of primers (e.g., Primer3, http://frodo.wi. mit.edu). A number of general rules should be followed to prevent mispriming or the generation of primer dimers: • Primers should be 18–25 bases in length. • Base composition should be 50–60% (G + C). • Melting temperature (Tm) is typically 50–70°C (same Tm for both primers). • Primers should not be self-complementary (form hairpins). • The 3¢ end should end in a G or a C, or CG or GC (as this increases the efficiency of priming); however, the 3¢ end of each primer should not be complementary (leading to the formation of primer dimers) or contain runs of three or more Cs or Gs (which promote mispriming at G- or C-rich sequences). • Primers should span exons so genomic contamination can be easily detected (since the product size will be much larger). • The optimal primer concentration for PCR is approximately 0.2 mM. As an important positive control we validate all our primers using species-specific reference RNA (Stratagene). If no

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product appears in PCRs that contain cDNA from our cells of interest but the positive control yields a PCR product, the lack of product from the cells of interest is thus not the result of poor reaction conditions.

3.3.2 PCR Reagents and Buffers All reagents should be in excess in the PCR, thereby allowing the cDNA to be limiting. PCR is performed in a standard buffer (10 mM Tris–HCl, pH 8.0, 50 mM KCl, 1.5 mM MgCl2, 0.01% Tween 20): increasing the MgCl2 concentration can increase the efficiency of the reaction, and addition of dimethyl sulfoxide (2%v/v) helps prevent the formation of secondary structures. A dNTP mixture (final concentration, 0.2 mM) is essential to provide the nucleotides for efficient extension from the primers. The main component of the PCR is the enzyme Taq DNA polymerase, which can be obtained from numerous commercial sources (1 unit of Taq per reaction is usually sufficient). Modifications to Taq have allowed it to be kept inactive at room temperature, such as Platinum® Taq (Invitrogen), which is only active after it has been denatured at 94°C, thus preventing nonspecific primer annealing at room temperature.

3.3.3 PCR Cycles PCRs are performed in programmable thermal cyclers that have the ability to quickly ramp up and down in temperature. Typical PCR protocols begin with a 95°C denaturation for 10 min to fully melt the template, exposing single-stranded template DNA for primer annealing, followed by 35–40 cycles of the following three steps (example given for primers with a Tm of 60°C for a 250-bp product): • Denaturation: 95°C for 15 s (denatures the newly synthesized products) • Annealing: 55°C for 30 s (binding of the primers to their target site) • Extension: 72°C for 1 min (approximately 1 min per kilobase DNA synthesis of primer) A final extension of 72°C for 5  min is not essential but is usually included to allow complete synthesis of any partial products that may have formed in the later stages of PCR. Since temperatures of 95°C can lead to evaporation, this should be prevented by overlaying the reaction with oil or performing the PCR in a thermal cycler with a heated lid. The annealing temperature is dependent on the Tm of the primers and is the most variable parameter that requires adjustment: the annealing temperature can be easily optimized using a gradient thermal cycler. In general, the annealing temperature is about 3–5°C below the Tm.

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3.3.4 Quantification of PCR Products To analyze the PCR products 10× stock sample buffer [65% (w/v) sucrose, 10 mM Tris–HCl (pH 7.5), 10 mM EDTA, and 0.3% (w/v) bromophenol blue] is added to give a final concentration of 1× and then this mix is electrophoresed together with appropriate molecular weight standards on a nondenaturing gel (1% agarose gel cast in 0.5× TBE containing 0.5 mg/mL ethidium bromide) and visualized using UV light. The first time primers are used, it is important to excise and sequence each product and verify its identity; we use the QIAquick gel extraction kit (Qiagen) for this procedure. Gene expression is quantified by measuring the band intensity as described earlier for northern blot. For semiquantification of the PCR products, GAPDH, b-actin, rRNAs, or other housekeeping genes are used as an internal control. The inclusion of a loading or internal control (i.e., an RNA that does not change with treatment of cells) is required for all techniques that measure expression. The choice of a housekeeping gene is somewhat controversial [33, 34]. For example, expression of GAPDH mRNA can be upregulated in proliferating cells, including aggressive cancers [35]. Furthermore, the use of 18S or 28S to normalize the results (which can only be used if cDNA synthesis was primed using random hexamers) may not always represent the overall cellular mRNA population since it is a rRNA [36]. We believe that it is best to assess several different loading controls until one has been validated for use with a particular target cell or tissue. In our experience, 28S rRNA is optimal for normalizing RNA expression in human PASMCs and lung tissue [36]. It is important to note that PCR is at best semiquantitative since one measures generated product at an end point (e.g., after 40 cycles) when the product formation will be at a plateau. Quantification of the PCR product at the end of each cycle would be required to enhance sensitivity, a process that would be extremely time consuming. A key advance in PCR has been the development of real-time PCR, which is more sensitive and accurate for quantification of mRNA and is discussed in more detail next [7, 8].

3.4 Real-Time PCR The general principles of real-time PCR are similar to those of traditional PCR but fluorophore probes that bind to double-stranded DNA are used for the detection and quantification of the PCR product. These fluorescent probes include nonspecific DNA intercalating dyes (SYBR Green I) or sequence-specific fluorescent oligonucleotide probes

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(TaqMan, molecular beacons, and Scorpion probes that use Förster resonance energy transfer [8, 37–42]). Fluorescence intensity is measured after each cycle; the intensity increases proportional to the increase in the amount of PCR product. Fluorescence versus cycle number is then plotted for each sample and a threshold fluorescence is set within the linear amplification range (Fig. 2). The cycle number at which the threshold fluorescence crosses the amplification plot is known as the Ct or threshold cycle. The greater the amount of template, the lower the number of cycles required to reach the Ct value. Because real-time PCR offers a 107-fold dynamic range, a variety of ratios of target and normalizer can be assayed with equal sensitivity and that eliminates post-PCR processing. Real-time PCR is the preferred technique in our laboratory to measure gene expression. We have used it to demonstrate the relative expression of PDEs and caveolins in PASMCs and how the expression of PDE isoforms is altered in PASMCs isolated from patients with pulmonary hypertension [43, 44].

3.4.1 Choice of Probe The first step in real-time PCR is to decide which dye/probe is best for a particular experiment. The four different probes that are currently available for real-time PCR are TaqMan, molecular beacons, Scorpion probes, and SYBR Green I, each of which allows the detection of PCR products via the generation of a fluorescent signal. SYBR Green I (an intercalating dye that fluoresces upon binding to double-stranded DNA) is the cheapest, most widely used, and most flexible

Fig. 2  Example of amplification plots obtained from a real-time PCR experiment showing 28S and Epac-1 in human pulmonary artery smooth muscle cells (PASMCs). The threshold cycle (Ct) is inversely proportional to the initial copy number. DCt is the difference between the Ct of 28S and the Ct of Epac-1 for both samples

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probe. SYBR Green fluoresces when it binds to doublestranded DNA, which ­facilitates its use with any gene-specific primer. However, it also can bind to nonspecific products and primer dimers, although melt curves allow the detection of the latter [45]. TaqMan, molecular beacons, or Scorpion gene-specific probes can be designed, and are oligonucleotides (20–30 bases long with a Tm 10°C higher than for the primers) with a reporter dye such as 5¢ FAM (5-carboxyfluorescein) attached to the 5¢ end and a quencher dye such as 3¢ MGB (minor groove binder) attached to the 3¢ end. TaqMan probes specifically anneal between the forward and reverse primers and prior to replication of the template. The 5' exonuclease activity of the polymerase cleaves the probe, releasing the reporter molecule from its close proximity to the quencher, thereby increasing fluorescence intensity [46]. Use of any these probes can allow for multiplexing, for example, so the housekeeping gene is amplified in the same tube as the gene of interest, as long as each probe is designed with a spectrally unique fluorophore [39, 47, 48]. TaqMan probes require very little optimization and have recently been used to define the anatomic distribution of G-protein-coupled receptors (GPCRs) [49].

3.4.2 General Protocol The general protocol for real-time PCR is similar to that for PCR, although a few modifications are required to optimize the efficiency of the reaction. It is important to keep the PCR products optimally sized between 100 and 150 bp, since it is believed longer products create a stronger signal. It has been suggested that products can be as small as 50 bp; however, this can make sequencing of products more difficult. Owing to the increased sensitivity of the real-time reaction, it is usually best to use lower concentrations of both cDNA and primers: a general guide is 8 ng and 100 nM per reaction, respectively; however optimization is usually required. One needs to determine the efficiency of each reaction so as to allow comparison of gene expression within a tissue or cell (see below). We currently use the Bio-Rad DNA engine Opticon 2 thermocycler that allows for reactions to be performed in both 96-well and tube format, and qPCR MasterMix for SYBR Green I [dNTPs, HotGoldStar DNA polymerase, MgCl2 (5 mM final concentration), uracil-N-glycosylase, SYBR Green I] from Eurogentec: many real-time thermocylers and SYBR Green master mixes are currently commercially available. As with PCR, controls that lack the template and RT are required for each experiment and multiple replicates should always be run. If the Ct between replicates differs by more than 0.5, the values should be excluded. Below we outline an example of the real-time PCR protocol using qPCR Mastermix for SYBR

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Green I for primers with a Tm of 60°C that detect a product of 120 bp: 1. Uracil-N-glycosylase step: 50°C for 2  min (cleaves contaminating template) 2. Initial denaturation: 95°C for 10 min (activates Taq and denatures uracil-N-glycosylase) 3. Denaturation: 95°C for 15 s (denatures newly synthesized products) 4. Annealing: 55°C for 30 s (binding of primers to their target site) 5. Extension: 72°C for 1 min (approximately 1 min per kilobase, DNA synthesis off the primer) 6. Plate-read, and repeat steps 3–5, 39 times 7. Melt curve 60–95°C, reading every 0.2°C, hold 0.1 s The last step in any real-time PCR that uses SYBR Green should be the inclusion of a melt curve, which provides information on specificity. The temperature is increased from 60 to 95°C and the fluorescence is continuously measured. At a specific temperature the product breaks, thereby resulting in a drop of fluorescence as the SYBR Green dissociates from the double-stranded DNA: the dissociation temperature depends on the length and composition of the product. A good melt curve should yield a single peak, as shown in Fig.  3. If primer dimers are formed, a small peak around a lower temperature (e.g., 70°C) is observed.

3.4.3 Reaction Efficiency The efficiency of the reaction needs to be calculated for each target [50]. To calculate the efficiency, we use reference

Fig. 3  The first derivative of raw fluorescence plotted against increasing temperature in the melt curve. The single melt peaks at 78°C (28S) and 81.5°C (Epac-1) indicate that a single PCR product from PASMCs has been amplified in each well

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cDNA (Stratagene) as the template, since some primers may show poor efficiency in a tissue of interest if the gene is poorly expressed. The easiest way to determine efficiency is to construct a standard curve by diluting the template (four or five points on the standard curve can accurately determine the efficiency) [51]. We use the dilutions 1:8, 1:64, and 1:512 since each should be exactly three Ct different if the reaction is 100% efficient (23, 26, and 29 respectively). Plotting the logarithm of the cDNA concentration against the Ct number will determine the linearity and sensitivity of the reaction. The efficiency, which should be 90–100% (slope between −3.1 and −3.6, where −3.32 represents most efficient), can be calculated by the following equation:

efficiency = 10 (−1 / slope ) − 1.

(1)

The linearity of the PCR assay is determined by R2 (the ­correlation coefficient, where 1 represents a perfect fit). If R2 is less than 0.98, this indicates that no linear relation exists between Ct and the concentration of the DNA and suggests the reaction is not efficient.

3.4.4 Normalization and Quantification Methods The main problems associated with real-time PCR are the uncontrolled variables and the interpretation of the results: normalization can correct for some of these variables. As outlined already, data should be normalized to a housekeeping gene whose expression is abundant and remains constant between samples. The same problems exist for real-time PCR as with PCR for determining the best housekeeping gene; however, we believe that the best option is to use multiple housekeeping genes for normalization. Absolute quantification measures the exact number of copies of the gene, whereas relative quantification defines the level of expression of the gene of interest relative to that of another gene, typically that of a housekeeping gene [52, 53]. Absolute quantification requires a standard curve constructed with known concentrations of the target (using a standard such as cDNA plasmids). Unknown concentrations are determined by interpolating values from the standard curves. In our laboratory, for example, we have used this type of quantification to investigate the stoichiometry of GPCR signaling pathways [54]. The most widely used quantification technique is relative or comparative quantification, in which one quantitates target genes without the need for a standard curve. Gene expression is calculated relative to a calibrator, which can be, for example, control patient samples, untreated cells, or the lowest expressed gene in the sample studied. Usually the DDCt method is used, which compares the normalized Ct level between the gene in the sample versus the calibrator. Initially,

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the DCt for the target gene is calculated for both the sample and the calibrator by

∆Ct = Ct target gene − Ct housekeeping gene .

profile gene expression and microRNA expression in single cells [55, 56].

(2)

The difference between the DCt of the sample and that of the calibrator is then calculated, giving the DDCt value: Relative quantity to the calibrator = 2-DDCt, where

4 Protein Quantification

Tools for analyzing mRNA expression are now widely available. However, the quantification of protein levels is ∆∆Ct = (Ct target gene − Ct housekeeping gene )calibrator necessary for optimal understanding of physiological and − (Ct target gene − Ct housekeeping gene )sample . (3) pathological states, including in the pulmonary circulation: mRNA expression does not always correlate with protein As long as the target and normalizer have similar dynamic expression and therefore one can draw erroneous concluranges, the comparative Ct method (DDCt method) is the most sions if only RNA expression is assayed [57]. practical method. Figure  4 shows the expression (whereby One of the most widely used techniques to identify prowhen one uses DCt, a lower Ct yields higher expression) of a tein expression is immunoblotting, commonly termed “westnumber of PDEs in PASMCs isolated from control subjects ern blotting.” Western blotting not only detects an individual and patients with pulmonary hypertension (n = 3). These data protein in a complex mixture, but it also provides informashow the relative expression of these PDEs, since primer effi- tion on its weight, abundance and can provide accurate ciency is the same for each product, in control PASMCs to be quantification [4, 9, 58]. Western blotting involves the sepaPDE3A > PDE5A > PDE4B > PDE4A > PDE4C > PDE2A > P ration of proteins (on the basis of molecular mass) on SDS– DE1C > PDE1A > PDE4D > PDE3B. However, this pattern polyacrylamide gels, then transfer to a membrane on which changes in PASMC samples from patients with pulmonary the protein can be detected using specific primary antibodies hypertension: PDE5A > PDE1C > PDE1A > PDE3A > PDE4B  followed by labeled secondary antibodies. Western blotting > PDE4A > PDE4C > PDE3B > PDE2A > PDE4D. Thus mRNA is highly dependent on the quality of primary antibody that is expression of PDE1A, PDE1C, PDE3B, and PDE5A signifi- used and its specificity for the protein of interest. Although cantly increases with pulmonary hypertension. many antibodies are obtainable from commercial sources, if Advances in real-time PCR have allowed this technology a particular protein is novel, one will need to generate an to be assessable to most laboratories. More recently, improved antibody, which can sometimes be difficult and costly in performance of real-time PCR has allowed it to be adapted to terms of both time and money. Western blotting has been

Fig.  4  Messenger RNA expression of multiple phosphodiesterase (PDE) isoforms in control and pulmonary arterial hypertension PASMCs expressed as Ct normalized to 28S rRNA (mean ± standard error of the mean)

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used in many published works to identify proteins that are expressed in the pulmonary circulation and/or whose expression changes with disease or stimuli [29, 43, 44, 59].

4.1 Protein Isolation Before solutions of proteins can be separated by gel electrophoresis, tissues and cells need to be lysed. A number of lysis buffers and conditions can be used; these differ in ionic strength, pH, and nature/concentration of the detergent depending on the protein of interest. Use of detergents is essential for studies of membrane-bound proteins, but different types of detergents (e.g., ionic, nonionic, zwitterionic) can have widely differing effects in terms of yield, denaturation, and impact on charge of solubilized proteins and thus on migration patterns when the protein solutions are subsequently electrophoresed [9]. It is important to keep the sample on ice at all times and add protease and phosphatase inhibitors (if one is studying proteins that are phosphorylated) to the lysis buffer to minimize protein degradation. Unless specialized methods of extraction are required, owing to its broad utility we typically use the detergent NP40 in our lysis buffer [50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 100 mg/mL phenylmethylsulfonylfluoride (a protease inhibitor), 1  mg/mL aprotinin (a protease inhibitor), and 1% NP-40]. For PASMCs, we typically add 100 mL lysis buffer per well of a six-well plate and incubate on ice for 10 min, after which the cells are scraped and sonicated three times for 15 s; it is advisable to wash the cells with ice-cold phosphate-buffered saline to remove residual serum. Lung tissue should be ground under liquid nitrogen before the addition of 200 mL lysis buffer per 100 mg tissue (in general 2 vol of buffer for each volume of tissue), after which the sample should be homogenized using a handheld rotor homogenizer. Lysates are centrifuged at 12,000 rpm for 2 min at 4°C to remove debris and the supernatant is assayed for protein content so as to ensure equal loading of samples onto gels. One of the most commonly used methods for protein quantification is the Bradford assay (available commercially from Bio-Rad) [60]. The protein content of the sample is estimated using a standard curve constructed with known concentrations of BSA. Samples are prepared by heating them at 70°C for 10 min in sample buffer (50 mM Tris–HCl (pH 6.8), 2% SDS, 0.1% bromophenol blue, 10% glycerol, 2.5% b-mercaptoethanol): in general we recommend loading 10–15 mg protein per well. Both the high temperature and the reducing agent (b-mercaptoethanol) destroy tertiary protein folding and quaternary protein structures by breaking disulfide bonds and preventing their reformation. SDS denatures secondary structures and non-disulfide-linked tertiary structures, thus unfolding and linearizing the proteins.

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4.2 Gel Electrophoresis and Transfer Proteins are typically separated by SDS–polyacrylamide gel electrophoresis using a discontinuous buffer system in a vertical gel system (e.g., Invitrogen). Precast gels are commercially available (NuPage® Novex® Bis-Tris mini gels, Invitrogen) or gels can be poured individually. The concentration of acrylamide can vary depending on the size of the target protein; typically separation gels are 8–15%: a higher percentage of acrylamide is used to separate proteins with lower molecular weight. We prepare separating gels with a final concentration of 10 or 12% acrylamide [components of separating gel: 10/12% acrylamide, 0.375 M Tris-Base (pH 8.8), 0.1% (w/v) SDS, 0.05% (w/v) ammonium persulfate, 0.05% (v/v) N,N,N¢,N¢,-tetramethylethylenediamine (TEMED), and distilled H2O]. After setting of the separation gel, a 6% acrylamide stacking gel [6% acrylamide, 0.125 M Tris base (pH 6.7), 0.1% (w/v) SDS, 0.05% (w/v) ammonium persulfate, 0.1% (v/v) TEMED, and distilled H2O] is poured on top and a tenwell comb or a 12-well comb is inserted. After complete polymerization has occurred, the combs are removed, the gel is mounted in electrophoresis running buffer [25 mM Tris, 250 mM glycine (pH 8.3), 0.1% SDS], and the samples are loaded together with molecular weight standards (colored Novex sharp prestain protein standards are useful as they also determine transfer efficiency; Invitrogen). Gels are then run at a constant voltage (200 V) until the dye front is at the bottom. Transfer from the gel to a solid support (typically either nitrocellulose or PVDF) takes advantage of the negative charge of the proteins (which has been imparted by the SDS). For the conventional apparatus, the gel and membrane are sandwiched between pieces of Whatmann 3MM paper and sponges that had been presoaked in transfer buffer (39 mM glycine, 48 mM Tris-base, 0.037% SDS, 20% methanol, pH 8.3) with the membrane on the anodic side. The chamber is filled with transfer buffer and the gel is run at 100 V for 60  min. We currently use the Iblot dry blotting system (Invitrogen), which allows transfer in as little as 8 min without the need to prepare buffers. After transfer, the membrane can be stained (for protein) with Ponceau S to ensure good transfer, although we use the colored marker as a guide.

4.3 Hybridization of Probe and Detection To optimize the sensitivity of western blotting, it is important to block nonspecific binding. Typically, 5% nonfat dry milk or BSA in TBST [0.05% Tween 20 (a nonionic detergent) in phosphate-buffered saline or TBS (Tris-buffered saline)] is used depending on the compatibility of the detection system. The membrane is typically blocked for 2–3 h at room temperature, after which the primary antibody [at the optimal

36  Quantification of DNA, RNA, and Protein Expression

concentration (generally the one recommended by the manufacturer) in 2% milk or BSA in TBST) is incubated with the membrane overnight at 4°C. The membrane is then washed three times for 10 min with TBST, incubated with the secondary antibody (according to the manufacturer’s instructions) for 1 h at room temperature, and then the washes are repeated. All stages of the protocol are carried out on an orbital shaker to ensure equal coverage and washing of the membrane. Most secondary antibodies are conjugated with horseradish peroxidase, which, if exposed to hydrogen peroxide and an appropriate chemiluminescent agent (luminol), produces fluorescence in proportion to the amount of bound protein. Enhanced chemiluminescence (ECL) detection is a sensitive method that avoids use of radioactivity. Equal volumes of ECL detection reagents (2.5 mM luminol, 1.1 mM p-coumaric acid, 0.1 M Tris base, pH 8.5, and 0.02% hydrogen peroxide, 0.1M Tris base, pH 8.5) are mixed to give a final volume of 125 mL/cm2 on the membrane and are applied to the membrane for 1  min: we also use ECL Advance (Amersham), which provides increased sensitivity. To visualize immunoreactive bands, the membrane is then exposed to X-ray film for 10 s to 10 min depending on the intensity of the signal. Furthermore, we and many other laboratories use the UVP Bioimaging system (CCD camera), which captures images of the developed membrane without the need for film, developer, or fixer and which allows one to document the migration pattern of proteins on the membrane and save this information on a computer for subsequent analysis and presentation of the results. Membranes can be stripped to remove the initially used primary antibodies and then reprobed with other antibodies by incubating the blot with gentle agitation in a buffer [100 mM b-mercaptoethanol, 2% SDS, 62.5 mM Tris–HCl, pH 6.7, or RestoreTM stripping buffer (Thermo Scientific)] at 37°C for 1 h. After removal of the stripping buffer and washing of the membrane (three times for 10 min in the wash buffer), the membrane is reblocked and probed with a new antibody as outlined above. Protein quantification is measured by band intensity via densitometry, using ImageJ. Samples to be compared should always be run on the same gel and several different exposure times in rapid succession should be used to ensure the signal is not saturated so as to prevent misinterpretation of results. Protein-loading controls such as GAPDH and b-actin should be used to normalize the data to ensure equal loading of samples. If the target protein and the loading control have different molecular weights, the membrane can be cut and probed for both antibodies simultaneously; otherwise the membrane should be stripped and reprobed. Figure 5 shows the protein expression by immunoblotting of PDE5A, GAPDH, and caveolin-1 in whole cell lysates from lungs isolated from control and monocrotaline-treated rats. These data show that protein expression of PDE5A increases, whereas that of caveolin-1

535 PDE5A 100kDa

α β

Cav-1 21-24 kDa

GAPDH 36 kDa Control 2

MCT-treated

*

1 0

PDE5A

CAV-1 α

CAV-1 β

−1 −2 *

−3

n=5 −4

*

Fig. 5  Example of an image obtained by western blotting that depicts increased expression of PDE5A protein and decreased expression of caveolin-1 protein in whole lung from monocrotaline-treated rats compared with the control. Glycerol-3-phosphate dehydrogenase was used to normalize the data for equal protein loading and ImageJ was used to quantify the changes. Cav-1a and cav-1b refer to the two bands of caveolin 1 shown on the western blot

decreases, with monocrotaline treatment. These changes ­associated with monocrotaline are verified by showing equal ­protein loading via GAPDH and by measuring the band intensity using ImageJ. The technique described above provides a semiquantitative analysis of protein expression; however, for more accurate quantification, one needs to load a series of diluted protein standards (i.e., purified protein that is commercially available or obtained by cloning, expression, and purification). A dilution range of up to 50-fold is recommended; however, this depends on the dynamic range, and gels should be run in duplicate to provide greater accuracy. To calculate the concentration of the unknown, the band intensity (using the image analysis program) of each diluted purified protein is measured to generate a standard curve to define the protein content of unknowns. Our laboratory has used this method along with radioligand binding to determine the stoichiometry of GPCR signaling in rat myocytes [61]. Advances in the detection and quantification of proteins with disease or stimuli have included the development of many commercially available products that reduce nonspecific binding, increase the sensitivity, and reduce the time of the procedure. One recent approach is the use of secondary antibodies that are directly conjugated with a nearinfrared (NIR) fluorophore such as IRDyeTM800 or Alexa

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Fluor 680, which can even be used simultaneously for ­multiplexed analysis of two proteins (e.g., to detect total and ­phosphorylated protein at the same time). This technique has been shown to give a wider range of linear detection and unlike chemiluminescence the signal is measured in a static state [62]. Furthermore, since the NIR fluorescent blots are stable, they can be rescanned months later without any loss of the signal. One disadvantage is the need to buy a new detection system, such as the Odyssey infrared imaging system from LI-COR Biosciences, which is currently quite expensive.

5 Summary We have described techniques, experimental procedures, and some of the applications for the study of DNA, RNA, and protein, in particular as relevant to pulmonary research and medicine. The availability of new types of instrumentation as well as kits and standard reagents from commercial suppliers has helped simplify a number of the methods. In effect, the availability of these instruments and reagents helps create “best practices” that will help minimize interlaboratory differences in results. The application of these techniques should not only allow researchers to gain more insight into the correlation of gene expression with physiological function, but will hopefully also aid in the generation of new therapeutic targets as well as improvements in diagnostic techniques. Ultimately, their application and use may help guide the management of patients who are at high risk of developing or who present to physicians with diseases such as pulmonary hypertension. Acknowledgements  This work was supported by grants from the National Heart, Lung and Blood Institute of the National Institutes of Health, the Lymphoma and Leukemia Society, and the Ellison Medical Foundation.

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36  Quantification of DNA, RNA, and Protein Expression 27. Murray F, MacLean MR, Pyne NJ (2002) Increased expression of the cGMP-inhibited cAMP-specific (PDE3) and cGMP binding cGMP-specific (PDE5) phosphodiesterases in models of pulmonary hypertension. Br J Pharmacol 137:1187–1194 28. Platoshyn O, Yu Y, Ko EA, Remillard CV, Yuan JX-J (2007) Heterogeneity of hypoxia-mediated decrease in IK(V) and increase in [Ca2+]cyt in pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 293:L402–L416 29. Yu Y, Fantozzi I, Remillard CV et al (2004) Enhanced expression of transient receptor potential channels in idiopathic pulmonary arterial hypertension. Proc Natl Acad Sci U S A 101:13861–13866 30. Gerard GF, Fox DK, Nathan M, D'Alessio JM (1997) Reverse transcriptase. The use of cloned Moloney murine leukemia virus reverse transcriptase to synthesize DNA from RNA. Mol Biotechnol 8:61–77 31. Dieffenbach CW, Dveksler GS (1993) Setting up a PCR laboratory. PCR Methods Appl 3:S2–S7 32. Mullis KB (1990) Target amplification for DNA analysis by the polymerase chain reaction. Ann Biol Clin (Paris) 48:579–582 33. Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25:169–193 34. Huggett J, Dheda K, Bustin S, Zumla A (2005) Real-time RT-PCR normalisation; strategies and considerations. Genes Immun 6:279–284 35. Goidin D, Mamessier A, Staquet MJ, Schmitt D, Berthier-Vergnes O (2001) Ribosomal 18S RNA prevails over glyceraldehyde-3-phosphate dehydrogenase and beta-actin genes as internal standard for quantitative comparison of mRNA levels in invasive and noninvasive human melanoma cell subpopulations. Anal Biochem 295:17–21 36. Barbu V, Dautry F (1989) Northern blot normalization with a 28S rRNA oligonucleotide probe. Nucleic Acids Res 17:7115 37. Heid CA, Stevens J, Livak KJ, Williams PM (1996) Real time quantitative PCR. Genome Res 6:986–994 38. Solinas A, Brown LJ, McKeen C et  al (2001) Duplex Scorpion primers in SNP analysis and FRET applications. Nucleic Acids Res 29:E96 39. Vet JA, Marras SA (2005) Design and optimization of molecular beacon real-time polymerase chain reaction assays. Methods Mol Biol 288:273–290 40. Wittwer CT, Herrmann MG, Moss AA, Rasmussen RP (1997) Continuous fluorescence monitoring of rapid cycle DNA amplification. Biotechniques 22(130–131):4–8 41. Lee LG, Connell CR, Bloch W (1993) Allelic discrimination by nick-translation PCR with fluorogenic probes. Nucleic Acids Res 21:3761–3766 42. Livak KJ, Flood SJ, Marmaro J, Giusti W, Deetz K (1995) Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl 4:357–362 43. Murray F, Patel HH, Suda RY et al (2007) Expression and activity of cAMP phosphodiesterase isoforms in pulmonary artery smooth muscle cells from patients with pulmonary hypertension: role for PDE1. Am J Physiol Lung Cell Mol Physiol 292:L294–L303 44. Patel HH, Zhang S, Murray F et al (2007) Increased smooth muscle cell expression of caveolin-1 and caveolae contribute to the

537 pathophysiology of idiopathic pulmonary arterial hypertension. FASEB J 21:2970–2979 45. Morrison TB, Weis JJ, Wittwer CT (1998) Quantification of lowcopy transcripts by continuous SYBR Green I monitoring during amplification. Biotechniques 24:954–958, 960, 962 46. Hiyoshi M, Hosoi S (1994) Assay of DNA denaturation by polymerase chain reaction-driven fluorescent label incorporation and fluorescence resonance energy transfer. Anal Biochem 221:306–311 47. Rickert AM, Lehrach H, Sperling S (2004) Multiplexed real-time PCR using universal reporters. Clin Chem 50:1680–1683 48. Vrettou C, Traeger-Synodinos J, Tzetis M, Palmer G, Sofocleous C, Kanavakis E (2004) Real-time PCR for single-cell genotyping in sickle cell and thalassemia syndromes as a rapid, accurate, reliable, and widely applicable protocol for preimplantation genetic diagnosis. Hum Mutat 23:513–521 49. Regard JB, Sato IT, Coughlin SR (2008) Anatomical profiling of G protein-coupled receptor expression. Cell 135:561–571 50. Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3:1101–1108 51. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45 52. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-DDCT) method. Methods 25:402–408 53. Sellars MJ, Vuocolo T, Leeton LA, Coman GJ, Degnan BM, Preston NP (2007) Real-time RT-PCR quantification of Kuruma shrimp transcripts: a comparison of relative and absolute quantification procedures. J Biotechnol 129:391–399 54. Fuhs SR, Bundey RA, Ling Z, Insel PA (2007) Stoichiometry of GPCR-Gq/11 signaling components in MDCK cells: Are there “spare receptors”? FASEB J 21:568.17 55. Edvardsson L, Olofsson T (2009) Real-time PCR analysis for blood cell lineage specific markers. Methods Mol Biol 496:313–322 56. Schmittgen TD, Lee EJ, Jiang J et  al (2008) Real-time PCR quantification of precursor and mature microRNA. Methods 44:31–38 57. Richter W, Jin SL, Conti M (2005) Splice variants of the cyclic nucleotide phosphodiesterase PDE4D are differentially expressed and regulated in rat tissue. Biochem J 388:803–811 58. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685 59. Rabinovitch M (2008) Molecular pathogenesis of pulmonary arterial hypertension. J Clin Invest 118:2372–2379 60. Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254 61. Post SR, Hilal-Dandan R, Urasawa K, Brunton LL, Insel PA (1995) Quantification of signalling components and amplification in the beta-adrenergic-receptor-adenylate cyclase pathway in isolated adult rat ventricular myocytes. Biochem J 311:75–80 62. Picariello L, Carbonell Sala S, Martineti V et al (2006) A comparison of methods for the analysis of low abundance proteins in ­desmoid tumor cells. Anal Biochem 354:205–212

Chapter 37

Gene Cloning, Transfection, and Mutagenesis Ellen C. Breen and Jason X.-J. Yuan

Abstract  Mutagenesis is a change or alteration in the DNA sequences of a gene. Mutagenic events may occur spontaneously within the genome of an organism and many times do not lead to functional consequences or an altered phenotype. However, at times a change in the coding sequence of a gene manifests itself as a dysfunctional phenotypic trait or predisposes an individual to a particular disease. The human genome may contain variations or mutations in an individual or in a related group of individuals that predispose them to a particular disease. Identifying and understanding these mutations (or polymorphisms) in gene structure can aid in the understanding, diagnosis, and treatment of patients. Quite the opposite to the mutations that spontaneously occur and may be difficult to identify, purposefully introducing mutations into a candidate gene that is then expressed in a cell or mouse model systems can readily reveal important information. This chapter provides an overview of the strategy to clone a gene, express it in a cultured cell system, and elucidate the function of the expressed gene product using mutagenesis methods. The value of this type of experimental approach will be highlighted by reviewing two pulmonary genes that have been analyzed using in vitro mutagenesis methods, the potassium channel Kv1.5 and the bone morphogenetic protein receptor II. Keywords  Gene mutation • Polymorphism • Heritable pulmonary arterial hypertension • KCNA5 • BMPR2 • Genotype and phenotype

1  Introduction Mutagenesis is a change or alteration in the DNA sequences of a gene. Mutagenic events may occur spontaneously within the genome of an organism and many times do not lead to E.C. Breen (*) Division of Physiology, Department of Medicine, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0623, USA e-mail: [email protected]

functional consequences or an altered phenotype. However, at times a change in the coding sequence of a gene manifests itself as a dysfunctional phenotypic trait or predisposes an individual to a particular disease. A classic example is the gene mutation first described in 1917 by Emmel found in those with sickle cell anemia [1]. In this rare genetic disease, the recessive gene for hemoglobin S is inherited. Expression of hemoglobin S leads to fragile “sickle-cell”-shaped red blood cells that are unable to adequately transport oxygen to organs throughout the body. Although autosomal recessive diseases in general are rare in the overall population, the human genome may contain variations or mutations in an individual or in a related group of individuals that predispose them to a particular disease. Identifying and understanding these mutations (or polymorphisms) in gene structure can aid in the understanding, diagnosis, and treatment of patients. Quite the opposite to the mutations that spontaneously occur and may be difficult to identify, purposefully introducing mutations into a candidate gene that is then expressed in a cell or mouse model systems can readily reveal important information. For instance, specifically designed mutations within the coding region of a gene can lead to the elucidation of how that particular expressed gene product functions within a cell to produce a biological effect. Systematic mutational analysis of untranslated gene regulatory regions (cis-acting DNA elements located in promoters and introns) is often used as a tool to identify the interaction between DNA binding sites and the transcription factors that recognize these specific DNA elements and determine when and how a gene is transcribed. Finally, the re-creation of gene mutations (polymorphisms) that appears at a higher frequency in human populations with lung diseases, such as primary pulmonary hypertension, chronic obstructive pulmonary disease, and asthma, can allow investigators to determine if these mutations may contribute to the early onset or progression of disease symptoms. This chapter will provide an overview of the strategy to clone a gene, express it in a cultured cell system, and elucidate the function of the expressed gene product using mutagenesis methods. The value of this type of experimental approach will

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_37, © Springer Science+Business Media, LLC 2011

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be highlighted by reviewing two pulmonary genes that have been analyzed using in vitro mutagenesis methods, the potassium channel Kv1.5 and the bone morphogenetic protein (BMP) receptor II (BMPRII) [2–24]. The first gene to be discussed is one of the oxygen-sensitive potassium channels, Kv1.5, expressed in a subset of pulmonary artery smooth muscle cells (PASMCs) located in the precapillary resistance vessels [25, 26]. The importance of the second gene product, BMPRII, in pulmonary biology was brought under scrutiny owing to the identification of a high percentage of polymorphisms or mutations in patients who exhibit pulmonary arterial hypertension (PAH) [10, 11, 27–33]. Review of the analysis of these two genes using mutagenesis approaches will provide examples of how these methods can be applied to further our understanding of pathogenic mechanisms of lung vascular diseases and lay a foundation for rationale-based drug design to treat pulmonary hypertension.

2  Gene Cloning To study the function of a gene, the first requirement is to know the composition and order of the base pairs that make up the DNA gene sequence. A sensible strategy to clone and sequence a gene of interest will depend on how much information is already known about the particular gene, genes within the same family, or the same gene in related species.

E.C. Breen and J.X.-J. Yuan

2.2  PCR Cloning With the advent of recombinant DNA technology and the thermostable DNA polymerase (Taq enzyme), PCR cloning is probably the easiest and fastest approach for cloning a gene sequence today [35]. The PCR is a reaction in which a region of DNA can be amplified (or exponentially synthesized) between two known primer regions (approximately 20–30  bp in length) of DNA [38]. If regions of DNA are highly conserved among species or within similar gene products in the same species, these conserved regions may be used as the initial primers to amplify or generate the intervening unknown DNA sequence from the species of interest. Usually the amplified DNA fragment produced is not long enough to contain all the sequence information to encode the gene product. This partial DNA fragment can then be used to screen a DNA library [36, 37] or the complete gene can be synthesized using one-sided anchored PCR methods or rapid amplification of cDNA ends [39]. The later method relies on knowing enough of the DNA sequence information to design one sequence-specific primer and utilizes a generic primer (such as the poly A sequence) to accomplish the exponential amplification of the unknown intervening sequence through cycles of DNA synthesis with the thermostable DNA polymerase (Taq).

2.3  Library Screening 2.1  Computer Resources The NIH GenBank provides a tremendous resource to start this process. Many genes have already been cloned or partially cloned and are available from several commercial vendors (http://www.ncbi.nlm.nih.gov/Genbank34). In addition BLAST (which stands for “basic local alignment search tool”) is a computer algorithm that allows comparison of gene sequences across species or with an unknown DNA sequence and identifies regions that are similar (http://www. ncbi.nlm.nih.gov/blast). The main advantage of having some or all of the base pair information about a gene of interest is that this can be used to “read” the DNA sequence, the coding region, which will be used to design specific mutations for functional analysis. If only part of the sequence information is known or similar sequences have been cloned in other species or genes within the same family of proteins, conserved and/or known regions may be used to design primers to screen for the gene of interest using PCR cloning [35]. Alternatively, partial DNA fragments may be used as hybridization probes to screen a genomic DNA or complementary DNA (cDNA) library and identify, isolate, and amplify the full-length gene sequence [36, 37].

A second method to isolate the full-length gene sequence, if a partial region of the gene is known, is to screen a DNA library. DNA libraries are collections of DNA fragments from a particular tissue or species that in total contain all the sequence information from the organs or species from which they were derived. The DNA may be genomic, containing all the intron and exon information, or a cDNA library can be assembled from cDNA that is reverse-transcribed from isolated messenger RNA (mRNA). Many types of genomic and cDNA libraries from a variety of organs and species are commercially available for screening a given gene within a particular species. The collection of DNA fragments that make up a library are “housed” in vectors (plasmids, cosmids, and phage) which are vehicles for amplifying the collection of DNA sequences in a bacterial strain that is then screened for the correct DNA containing clone. Once identified, this gene-containing bacterial colony can be grown in larger quantities to further amplify, isolate, and purify the DNA clone of interest. If a partial DNA fragment or short oligonucleotide is available, this DNA sequence information can be used to screen for full-length sequences by filter hybridization [36, 40]. If no DNA sequence information is known, then an alternative screening method must be designed, and

37  Gene Cloning, Transfection, and Mutagenesis

this experimental strategy is referred to as “expression cloning.” This could be the use of an antibody to screen for the expressed protein or possibly a functional assay, for instance, an enzyme activity or unique biological response [41]. By whichever method is chosen to identify, isolate, and purify the gene of interest, the final cloned DNA is sequenced to determine the precise composition and order of base pairs that will be transcribed and translated into the final gene product.

3 Gene Expression in Cell Systems 3.1 Expression Vectors and Cell Systems Once the coding region of the gene is known, it is subcloned into an expression vector, which will allow the gene product to be produced within a cell. The key features of expression plasmids are (1) a strong (or efficient) promoter that will allow expression of the gene using the endogenous factors present in the cell system chosen, (2) a translational start site to allow expression of both the cloned gene and a bacterial selectable marker gene (i.e., antibiotic resistance gene), and (3) a poly A sequence at the 3¢ end to allow for efficient transcription and stabilization of the transcribed mRNAs. In addition, often an intervening sequence (intron) that is spliced out in the final mRNA is included for the purpose of enhancing expression efficiency. These are the basic elements that will allow the gene to be expressed within a cell. In addition, a mammalian selectable marker gene is very often included. This allows the separation of cultured cells, which express the cloned cells from a mixed population of cells that are both positive (expressing) and negative (nonexpressing) for the introduced gene. Included in the design of the vector may be the further option to remove this selectable gene sequence at a later stage. Thus, the next consideration is what type of cell system will work best for studying the gene of interest. Several heterologous cell systems have been routinely used for gene expression, including HEK 293, COS-7 cells, CHO cells, and Xenopus oocytes. In these heterologous cell systems, the gene of interest is often not endogenously expressed or is expressed at low levels, and this feature allows the newly incorporated gene to be more easily studied. However, often these more generic cell systems do not contain all the accompanying cellular factors to allow true gene expression under basal or experimental conditions. For example, heterologous expression of the Kv1.5a subunit in HEK 293 cells and mouse L cells differs in the voltage-dependent activation and slow inactivation kinetic profile monitored by in vivo patch clamp experiments. In this case an associated b subunit, endogenous Kvb2.1, is expressed in HEK 293 cells

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but is not expressed in mouse L cells [42]. Thus, in the interpretation of data from these types of heterologous expression experiments, one must consider the type of cell system. Cell lines derived from more differentiated organs or even primary cells from patients carrying a disease-related polymorphism may provide an environment that much more closely represents the conditions that occur in vivo in an intact organism. In the case of primary pulmonary hypertension, PASMCs and endothelial cells are often studied. The disadvantage of using a primary cell culture is that these cells may be limited in the number of times they can be passaged before they start to lose their unique phenotype or proliferative capacity. Thus, both cell lines and primary cell cultures are valuable systems and deciding which one is most appropriate will depend on the type of mutagenesis method utilized and the experimental question being addressed.

3.2 Methods of Gene Delivery 3.2.1 Transient Transfection Methods Once the gene or interest has been cloned into an expression vector, there are several methods for expressing that gene within a cultured cell [43]. The choice of gene delivery will depend on the cell type, the proliferative or quiescent state of the cell culture, the length of the gene, the required efficiency (number of cells that take up the gene), and the expression levels required for end analysis. The process of introducing an expression plasmid into cells in culture is referred to as “transfection.” In cell culture studies several basic methods that employ agents or chemicals that encourage plasmid DNA to be taken up by the cell are routinely used, including diethylaminoethyl (DEAE)-dextran, calcium phosphate, and lipofectin. In the DEAE-dextran and calcium phosphate methods, the DNA forms a precipitate on the surface of the cell that is engulfed by a form of “endocytosis” [44, 45]. A requirement for these chemical-based transfection methods is that the cells in culture must be actively dividing or proliferating to incorporate the plasmid DNA. Calcium phosphate and DEAE-dextran routinely provide a transfection efficiency of 50% or greater. For liposomes to mediate transfection of foreign DNA into the cell, plasmid DNA is mixed with cationic and neutral lipids,which forms a compact structure through electrostatic interaction with the nucleic acid backbone. The cationic “charged” DNA–lipid complexes are able to cross the negatively charged, hydrophobic cell membrane and enter the cells [46]. For cell types that divide more slowly (i.e., fully differentiated or primary culture cells) or for transfection into quiescent cells, alternative methods must be considered. One of these alternative transfection methods is electroporation. In this method a current of

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specific amplitude and duration, depending on the cell type, is applied to a cell in contact with the plasmid DNA and temporarily creates pores or openings in the cell membrane that allow DNA uptake. Once the plasmid DNA has been incorporated into the cell, by any of these described methods, it is generally only transiently expressed over the next several days (72 h), and only a small percentage cells (on the order of 1 in 104 depending on the cell type) actually incorporate the foreign DNA into the cell chromosomes. For many experimental designs, this type of “transient” expression pattern is perfectly adequate. However, if long-term consistent expression is desired, cells that integrate the transgene and maintain expression may be selected in a process that establishes a “stably” expressing cell line.

3.2.2 Selection of Stable Transfected Cells There are two basic ways to create populations of cells that stably (or continuously) express a transfected gene through incorporation into the cell’s genome. The first is to use a dominant selectable marker. This is a gene that is usually expressed along with the subcloned gene in the expression vector. After transfection, the cells that expresses both the gene-coding region and the selectable marker are cultured in a medium that contains a gene-specific agent that only allows transfected cells to survive. There are several types of selectable marker genes, such as the commonly used neomycin resistance (NeoR) or hygromycin resistance (Hyg or hph) genes. Cells then cultured with aminoglycoside antibiotics, gentamicin (G418), or hygromycin, which interferes with eukaryotic cell protein translation, do not survive unless they also express the corresponding resistance gene, NeoR or Hyg. After several rounds of selection and propagation, a cell line which expresses high levels of the transfected gene is obtained. One potential drawback of this type of approach is that if the expressed gene has a negative effect on cell growth or proliferation, it will be impossible to select a stable population. Furthermore, this process usually takes several weeks, during which time primary cell types may change or lose the expression of some of their unique differentiated genes before a stable, high-expressing population of cells is selected. The other possibility is to select the genes shortly after the initial transfection procedure. This can be done by expressing a selectable “tag.” For instance, fluorescent molecules may be expressed as part of a fusion product included in the cloned gene product or cells may be cotransfected with a second plasmid encoding a fluorescent reporter molecule. Another option is to coexpress a marker gene or a gene “label” in tandem on the same plasmid using an internal ribosomal entry site element. This allows two genes to be translated from the same vector. Thus, if the second gene is a fluorescent protein, cells can be readily identified and isola-

E.C. Breen and J.X.-J. Yuan

tion of the cells expressing the newly incorporated DNA is possible by fluorescence-activated cell sorting. This also eliminates the possibility that the introduced gene will be silenced by methylation modification over the extended time it would take to purify the transfected population with a dominant selectable marker gene.

3.2.3 Viral Vectors In addition to transfection of expression plasmids, viral vectors provide a highly efficient way to express foreign DNA in cultured cells. Viral vectors are particularly useful for transfecting primary cultures, which are quiescent or slowly proliferating, and are therefore not easily transfected (i.e., with calcium phosphate, DEAE-dextran, or cationic lipid reagents). The production of virus vectors is a bit more involved as it requires viral proteins and target genes to be expressed and assembled in specialized packaging cell lines from which the recombinant virus is subsequently isolated and purified. Once again, the choice of viral vector will depend on whether the cells are actively dividing, whether “transient” or “stable” transfection is required, and the length of the coding sequence expressing the gene of interest. Three viral vectors, among those commonly used in cell culture systems, are retroviruses, adenoviruses, and adeno-associated viruses (AAVs). As the name implies, retroviruses are RNA viruses [47]. Retroviral particles require actively dividing cells for the viral complex to enter into the host nucleus and stable incorporation into the host cell genome. Once infected with a retrovirus expressing recombinant DNA, the gene product may then be studied in transiently expressing cells or selected for stable expression. A limitation to consider when working with a recombinant retrovirus is that very large coding regions (more than 8–11  kb) are too large to be packaged into the recombinant retroviral particle [48]. An adenovirus is a DNA virus that also provides a high level of expression in mammalian cells. Recombinant adenoviruses have been engineered to express genes of interest along with fluorescent or selective genes and readily infect both dividing and nondiving cells. However, this expression is transient and the introduced DNA does not stably integrate into the cells genome. Commercially, adenovirus vectors can incorporate coding sequences up to about 8 kb. The last virus discussed here is the AAV, which contains viral elements similar to those of the recombinant adenovirus except it uses an additional helper virus sequence to provide the viral genes used in the production and assembly of the recombinant AAV. In addition, recombinant AAVs will infect nondividing cells, a feature that may be useful for expression in primary or fully differentiated cells. Recombinant AAVs provide high-level long-term expression and roughly 5–10% of the transduced cells will stably integrate the foreign gene into the cell’s

37  Gene Cloning, Transfection, and Mutagenesis

chromosomes. A limitation of the AAV system is it will not accommodate large coding regions (more than 3  kb) [49]. All these recombinant-type viral tools have been designed to be replication-incompetent by removing several of the viral encoded proteins (i.e., E1 and E3). This also provides an increased capacity for including the nonviral coding sequence (the target gene). Since these recombinant viruses can infect a cell once and produce the gene product without repeatedly infecting many cells, they are safer to work with than wildtype viruses. Although this design provides a degree of biosafety when working with these viral vectors, it is necessary to implement proper safety procedures when working in the laboratory.

3.2.4 Heterologous Overexpression Overexpression of the introduced DNA is then measured using standard methods to quantify gene expression (Fig. 1). These methods include reverse-transcription PCR to measure mRNA levels and western blot analysis and enzymelinked immunosorbent assays for assessment of the final protein level. Fluorescence microscopy or standard immunohistochemistry may also be used to detect the subcellular location, particularly if the expressed gene is a fluorescent fusion product or contains a generic tag (i.e., a FLAG), given that the resulting chimeric protein does not interfere with the protein or lipid interactions of the native protein. Finally, the cellular function is ready to be tested.

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Fig. 1  Characterization of expression and function of the human Kv1.5 potassium channel heterologously expressed in HEK293 cells. The construct [85] includes a human Kv1.5 encoding sequence (nucleotides –22 to +1,895) subcloned into XbaI and KpnI sites of the pBK-CMV expression vector. (a) DNA products of the regular (upper) and singlecell (lower) reverse-transcription PCR performed with messenger RNA isolated from native HEK293 cells (WT) and from HEK293 cells transfected with the human Kv1.5 expression construct (Kv1.5). The Kv1.5 transcription level in Kv1.5-transfected cells is much higher than in wild-type cells. (b) Western blots of total cell protein samples prepared from native HEK293 cells (WT) and from HEK293 cells transfected with human Kv1.5 expression construct (Kv). The amount of Kv1.5 protein product heterologously expressed in Kv1.5 transfected cells is very high compared with nontransfected cells. (c) Whole-cell currents from HEK293 cells transfected with empty pBK-CMV vector (-hKv1.5) and from HEK293 cells transfected with humans Kv1.5 expression construct (+hKv1.5). The current amplitude from Kv1.5-transfected cells is almost 25 times larher than in nontransfected cells. (d) Reversible inhibition by 4-aminopyridine (4-AP) of Kv1.5 channels heterologously expressed in HEK293 cells. The significant and reversible current decrease in Kv1.5-transfected cells treated with 4-AP reflects reversible inhibition of expressed ion channels by the voltage-dependent potassium channel blocker and shows the specificity of this channel. (Reproduced from [102] with permission)

4 Design of Recombinant Mutant Clones Over the last 10–20 years, several very clever and creative methods have been invented, and modified as molecular biology methods have evolved, to incorporate a mutation into cloned DNA and then select and amplify the mutated DNA clones for further analysis. Mutations may be randomly incorporated using chemicals or nucleases and then screened for changes in phenotype or function. However, there are several advantages of being able to precisely incorporate specific mutations in precise locations within a DNA sequence. Large changes in gene structure may be accomplished using restriction enzymes to remove or insert regions from a DNA clone that is then religated and transformed into bacteria. Using a variety of techniques, one can also incorporate multiple mutations over much more defined regions using a method such as linker scanner mutagenesis or scanner saturation mutations [50, 51]. Such methods are often used to elucidate the functional regions within 5¢ promoter regions or scan for peptide binding sites in expressed proteins. PCR either with incorporated restriction enzyme sites

or with gene splicing using overlap extensions can be used to introduce or exchange regions of DNA from another gene or to reintroduce DNA regions which contain specific deletions or insertions, or clusters of mutations. In addition to gross changes in gene structure (deletions, duplication, rearrangement, and multiple mutations over a region), precise mutations directed to a single or a few base pairs are also possible through site-directed mutagenesis methods [52, 53]. Practically, any type of change can be made with precision within the DNA sequences. A directed mutagenesis approach is very useful for studying proteins localized in the membrane (i.e., receptors and channels) since these proteins are not easily secreted and isolated for analysis. In addition, more information may be gained from studying their function in the context of native membranes (plasma membranes or endoplasmic reticulum) in which channel proteins or receptors interact with associated proteins and/or lipids. The next section will describe the design of methods to incorporate gross and site-directed single mutations (Fig. 2).

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Fig.  2  Designs for mutagenic clones: large deletion, chimera, and site-directed mutant. (a) Gene deletions may be created by restriction digestion of the cloned gene using sites that flank the region or subunit to be removed. The clone may be digested with one enzyme that cuts twice, two enzymes with compatible ends, or unique enzymes that are then filled in to form blunt ends. The resulting DNA fragments are separated by electrophoresis. The remaining cloned DNA is religated with T4 ligase and used to transform competent bacteria. (b) Overlapping PCR chimeric proteins may be created by interchanging subunits from similar or related genes (e.g., Kv1.2. Kv1.5, and Kv1.2). The two target genes (gene 1 and gene 2) are first amplified separately by PCR using four different primers (P1, P2, P3, and P4). P2 is designed to be homologous to gene 2 and P3 is homologous to gene 1. The outside primers P1 and P4 include restriction enzyme sites to allow the joined fragments to be reintroduced into the full-length gene clone. The two newly synthesized double-stranded DNA PCR

products are denatured to separate the DNA stands and allowed to anneal. This allows the homologous end regions to bind. Then Taq polymerase is used to extend the attached linear DNA. The resulting chimeric DNA is amplified using end primers (P1 and P4) and subcloned back into the original gene clone. (c) Site-directed mutants can be created using primers that contain the precise change in one or two base pairs. The target gene clone is denatured and the primers are allowed to anneal to the target sequence. The new strand is synthesized using a high-fidelity DNA polymerase (i.e., Pfu) in a linear reaction. The original DNA template contains methylated and hemimethylated sites, whereas the new DNA synthesized off the parent template is unmethylated. Incubation with the restriction enzyme DpnI selectively digests the parental methylated DNA. The mutated gene remains but this DNA also contains several “nicks” or gaps in the sequence. This new mutated DNA is transformed into a bacterial strain that allows the “nicked” DNA to be repaired

4.1 Large Deletions

that recognizes and cleaves sites near the ends of the DNA fragment, which will be removed. The resulting restriction digest fragment is separated by agarose electrophoresis from the remaining plasmid DNA (the plasmid backbone and remainder of the cloned gene), which is isolated from the agarose gel, religated with T4 DNA ligase, and transfected into the appropriate bacterial strain for amplification, isolation,

If the gene has already been cloned into a plasmid, a straightforward approach to creating a large deletion is to use the endogenous restriction enzyme sites that are encoded in the cloned DNA sequence. The plasmid, containing the gene of interest, is isolated and digested with a restriction enzyme

37  Gene Cloning, Transfection, and Mutagenesis

and eventual delivery into a mammalian cell system. If the identical restriction sites are not conveniently located at the ends of the region to be deleted, then two unique restriction enzymes with compatible ends may be used or the “sticky” ends can be filled in with a DNA polymerase (i.e., Taq or Klenow) to create blunt ends that can be ligated. Alternatively, directed mutagenesis using the PCR may be utilized to incorporate a particular restriction enzyme within the gene sequence.

4.2  C  onstruction of Chimeras with Overlapping PCR Another useful mutation to define the subunit specificity of a protein is to replace one functional region with another and create a hybrid protein [54]. For instance, in Kv1.5 it has been shown that the T1 domain in the N-terminal region dictates the kinetics of channel inactivation. Furthermore, this property is conserved between channels as a chimeric protein containing the N-terminal of Kv1.1 or Kv1.3 and a shortened form of Kv1.5 were able to reproduce the inactivation kinetics found in full-length Kv1.5 [21]. One way to create a chimera is to employ a method based on sequential PCRs using overlapping PCR. In this scheme two genes or regions of DNA to be joined are first amplified in separate reactions that incorporate ends suitable for joining and reinserting the newly created DNA into the appropriate expression vector. One primer for each of the two reactions is designed to include end-restriction sites used for reinsertion of the joined DNA fragments into the expression vector. The “overlapping” component of this technique is to design internal primers that are homologous to the other target gene. In other words, primer 2 used to synthesize target gene 1 would be homologous to the end of target gene 2 and primer 3 would be homologous to the end of target gene 1. The resulting PCR-amplified target genes are mixed, denatured, and reannealed so the homologous primer can bind. Single-stranded connected DNA fragments are then extended with Taq polymerase to form double-stranded products that are subsequently amplified by PCR using the external primer (primers 1 and 4). This new chimeric region is now ready to be recloned into an expression vector for functional analysis.

4.3  Site-Directed Mutagenesis Kunkel et al. were the first to describe an elegant method to create single base pair changes in DNA [53, 55]. In this design a new mutated strand is synthesized using a mis-

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matched “mutated” primer and a single-stranded M13 DNA template containing several dUTP residues in place of TTP. This special uracil-containing template is isolated from M13 phages that have been amplified in Escherichia coli deficient in the enzyme dUTPase. This –dut –ung E. coli strain (E. coli CJ236 or equivalent dut − ung − F¢ strain) is also deficient in the glycolase that would normally catalyze the removal of uracil incorporated into DNA, uracil N-glyosylase (ung). The new mutated strand that complements this UTP-containing template is synthesized in a reaction containing the normal proportion of each dNTP and results in a new strand without uracil resides. The resulting double-stranded DNA product is then reintroduced into a wild-type bacterial strain (E. coli +dut, +ung) which will selectively amplify the mutated DNA [53, 55]. An obvious improvement on this method is to synthesize the new mutated DNA again using a mismatched primer but in this case the PCR is used to amplify the mutated DNA. This design results in incorporation of the precise mutation with a very high efficiency [52]. The resulting DNA fragment is then recloned into the original expression vector using either restriction enzyme sites (endogenous or incorporated into the PCR primer design) or by overlapping PCR (as described earlier). One caution with PCR-based methods is that there is an inherent mutation rate in Taq polymerase. Several high-fidelity thermostable polymerases are currently commercially available that synthesize DNA with a lower mutation rate and thus reduce the probability of creating unwanted mutations in the cloned gene. Yet another variation of using mismatched primers, but eliminating the PCR amplification step, has been developed to introduce site-directed changes in a gene sequence and to further reduce the risk of incorporating random spurious mutation (developed by Stratagene, “Quick Exchange” [56]). In this design, primers which incorporate the precise change in DNA sequence are annealed to a plasmid template. New double-stranded products are produced by extending the primers with the high-fidelity DNA polymerase (PfuTurbo) in a linear fashion from each primed end. This step differs from the repeated cycles of DNA polymerase synthesis used to exponentially amplify the DNA in a standard PCR. This DNA synthesis reaction results in a new mutated plasmid containing the precise mutation. However, staggered DNA nicks are also present throughout this newly synthesized strand. The reaction product containing the new “nicked” DNA and original template are then incubated with DpnI endoculease, which selectively recognizes methylated and hemimethylated sites and digests the parental DNA. The nicked plasmid containing the desired mutation is then transformed into a bacterial strain able to repair the “nicked” DNA strand and amplify the new construct.

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5  The Kv1.5 Potassium Channel and BMPRII: Key Genes Implicated in Pulmonary Hypertension This section will describe examples of two genes that are important for pulmonary vascular function. Analysis of these two pulmonary genes, using experimental mutagenesis approaches, has been valuable in both revealing their biological function in the lung as well as understanding how dysfunction of these expressed pulmonary proteins may contribute to pulmonary hypertension. The first gene to be discussed is an oxygen-sensitive K+ channel expressed by PASMCs located in the resistance (or precapillary) vessels. In this situation mutagenesis approaches have provided a wealth of knowledge on how this related Kv1.5 channel functions to maintain vasomotor tone and contributes to the hypoxic pulmonary vasoconstriction (HPV) response to acute hypoxia. In addition to understanding the conformation and function of Kv1.5 and related Kv channel a subunits, this same mutagenesis approach has allowed a rational approach to the design of drugs for the treatment of pulmonary hypertension [57–59]. The second pulmonary gene discussed in the following sections is BMPRII. This tyrosine kinase receptor is also implicated in regulating PASMC proliferation, and heterozygous germ line mutations have been identified and implicated in predisposing patients to familial and idiopathic pulmonary hypertension [10, 11, 27–30, 33, 60–65]. In this case, mutagenesis has been used as a very important tool for analyzing the mutation already found in patients that are predisposed to or exhibiting familial or idiopathic pulmonary hypertension.

5.1 The Oxygen-Sensitive Potassium Channel, Kv1.5 Kv1.5 belongs to a family of related potassium channels (for reviews, see [25, 26, 66]). Voltage-sensitive Kv channels conduct potassium currents at or near the diffusion-limited rates and this property contributes to the maintenance of membrane potential (Em) in PASMCs and consequently resting vasomotor tone. In response to a decrease in the level of environmental O2, hypoxia, the pulmonary resistance vessels very rapidly constrict in a response that is thought to divert blood flow away from poorly ventilated alveoli. The benefit of this adaptive response may be to limit ventilation perfusion inequalities and maintain systemic oxygen levels [67]. PASMCs express a variety potassium channels, including voltage-gated K+ channels (Kv), inward rectifier K+ channels (KIR), calciumactivated K+ channels (KCa), and tandem-pore two-domain K+ channels (K2P) [68]. However, certain members of the Kv family of delayed rectifier (IK,DR) and noninactivating (IK,N) channels (Kv1.2, Kv1.5, Kv2.1, Kv3.1b, and Kv9.3) are selectively expressed in resistance vessel PASMCs [69, 70],

E.C. Breen and J.X.-J. Yuan

and in particular Kv2.1 and Kv1.5 are thought to be major contributors to the oxygen-sensitive change in the voltage sensitivity and activation kinetics that regulate K+ current across the PASMC membrane [26, 71, 72]. In response to an acute hypoxic exposure, the resistance vessel PASMCs are thought to generate less reactive oxygen species (ROS) [25]. This decrease in the amount of ROS leads to an inhibition of the Kv1.5-gated K+ current. Kv channels close, preventing the tonic efflux of K+ ions and the cell depolarizes as positive charge accumulates within the cell. This new membrane potential favors Ca2+ channel opening and Ca2+ ions move into the PASMC down a steep concentration gradient. This rise in the level of intracellular calcium signals myocytes to contract around the vessel, or vasoconstriction. The entire process is referred to as “hypoxic pulmonary vasoconstriction” (HPV) [67, 69, 73, 74]. The inhibition of ROS in response to hypoxia is unique to pulmonary arteries and although the oxygen sensor in this system is not yet definitively known, the release of ROS (from mitochondrial or NADPH oxidoreductase) is thought to signal channel activity inhibition and downregulate Kv1.5 expression through a hypoxia-inducible factor 1a dependent pathway [25]. Interestingly, upon exposure to chronic hypoxia (greater than 3  weeks), a blunted HPV response is observed upon return to hypoxia that is accompanied by decreased expression of Kv 2.1, Kv 1.5, and Kv 9.3 [75, 76]. However, the HPV can be rescued in chronically exposed mice by overexpression of adenovirally delivered Kv1.5 [77]. However, unlike exposure to chronic hypoxia, in which pulmonary artery remodeling and HPV are eventually restored to normal upon return to a normoxic environment, pulmonary hypertension is associated with a persistent decrease in Kv1.5 expression and K+ current that lead to sustained vessel tension [78, 79]. In addition, the role of Kv1.5 channels in regulating PASMC proliferation and apoptosis could contribute to the aberrant vascular remodeling and the formation of plexiform lesions in the precapillary arteries, two hallmarks of idiopathic PAH (IPAH) and familial PAH (FPAH) [66, 80, 81]. Recently, a potential dysfunction role of Kv1.5 in PAH has been supported by the finding of several single nucleotide polymorphisms in the Kv1.5 gene in PASMCs from patients with IPAH [82]. A great amount of information has been gained through the functional analysis of cloned Kv1.5 channels designed with a strategically positioned mutation. Most of the studies used the patch clamp technique first described by Neher and Sakmann [83] to record the current through single K+ channels and plot the kinetics of channel activation and inactivation in response to a voltage change. The Kv1.5 channel was first identified and cloned from rabbit vascular smooth muscle cells [84] and the human gene was isolated and cloned from the heart in 1993 [85]. As illustrated in Fig. 3, Kv1.5 is composed of six subunits or domains the span the plasma member (S1–S6) along with both C-terminal and N-terminal tail

37  Gene Cloning, Transfection, and Mutagenesis

Fig.  3  Structure of KCNA5 and characteristics of Kv1.5 channel currents. (a) KCNA5 gene organization showing the start codon (dotted line with arrow), the heteromerization domain (Het dom), and six transmembrane (TM) segments (TM1–TM6). Noncoding flanking 5¢- and 3¢-untranslated regions (UTR) are also indicated by hatched boxes. Right, pictogram of human chromosome 12 highlighting the p13 region containing KCNA5 (see the arrow at top). (b) Kv channel subunit protein folding motif and regulatory subunit binding; the pore region (P) is shown between TM5 and TM6. (c) Cross-sectional view of the arrangement of a and bsubunits into a tetrameric Kv channel with an ion-selective pore. (d) Representative single-channel Kv1.5 currents (left) at different test potentials (TP) and the current–voltage (I–V) relationship curve (middle) recorded

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from a cell-attached membrane patch of a human pulmonary artery smooth muscle cell (PASMC) transiently transfected with KCNA5. The fluorescence microscopy image (right) depicts human a PASMC transfected with KCNA5, identifiable by the green fluorescence. e Representative single-channel Kv1.5 currents at +100  mV from a COS-7 cell, a rat PASMC, a HEK293 cell, and a human coronary artery smooth muscle cell (CASMC) transfected with KCNA5 (left). The summarized I–V curve is constructed from single-channel Kv1.5 currents recorded from multiple cell types. The images identify KCNA5-transfected COS-7 cells, rat PASMCs, HEK-293 cells, and rat mesenteric artery smooth muscle cells (rMASMC) by their green fluorescence (right and bottom). (Reproduced from [82] with permission)

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regions that confer distinct biological properties. The last two helices (S5 and S6) are connected by a linker region. Four Kv a subunits assemble to form homomeric or heteromeric channels and are associated with b subunits, which bind to the N-terminal region of the channel and modulate channel function. Much of the early work on the organization of Kv channels has come from study the drosophila prototype, Shaker, and the Kv 1.2 channel crystallographic structure was solved in 2005 [86]. However, in the small resistance pulmonary

arteries, Kv1.5 has a major role in the rapid HPV response to acute hypoxia and altered Kv1.5 function and expression are implicated in the pathogenesis of PAH [26, 70, 87]. Mutagenesis studies using cloned Kv1.5 have been utilized to elucidate the subunit domains that sense a change in voltage to activate the channel as well as the functional domains responsible for “gating” the selective flux of K+ ions across the PASMC membrane (Table 1). For instance, creation of a chimeric construct in which Kv1.5 and Kv2.1 are

Table 1  Characterization of Kv1.5 functional domains through expression of cloned recombinant mutants Cell system and Mutations Method of mutagenesis functional assay Finding

References

N-terminal mutations Kv1.5/Kv1.2

[71]

Overlapping PCR to express both a subunits in tandem

Mouse L cells Patch clamp

Kv1.5DN209

N-terminal deletion using restriction enzyme sites

HEK 293 Patch clamp

Kv1.5DN19 Kv1.5DN91 Kv1.5DN119 Kv1.1 N/Kv1.5 Kv1.3 N/Kv1.5 KV1.5AAQL Kv1.5 N/Kv1.2 Kv1.2 N/Kv1.5

Bal31 5¢ nuclease digestion and PCR subcloning; subcloning using restriction enzymes and PCR; and primer exchange

HEK293 Xenopus laevis oocytes Patch clamp

Site-directed primer exchange

Mouse Ltk– cells Patch clamp

Established the importance of the first proline in the Pro-X-Pro “hinge” motif for channel gating

[13]

PCR site-directed mutagenesis

Mouse Ltk– cells Patch clamp

Residues near the Pro-X-Pro “hinge” sequence affect the stability of the activation pore

[16]

Site-directed primer exchange

HEK293 Patch clamp

[17]

Site-directed primer exchange

HEK293 Ltk– cells Patch clamp

Excess H+ or Zn2+ can bind to H463 in the pore turret and stabilizes a channel conformation that is in an inactivated state Identified a group of amino acid residues (including but not solely H463) in the pore turret that modulate slow inactivation

S6 mutations PVPV (WT) AVAV AVPV AVPP PVAV PVPP T505I T505V V512A V512M S515E Y519F Y519N H463Q H463G (H463Q, R487V) Small, neutral H463G H463A Large, charged H463R H463K H463E Cysteine substitutions T462C⇒P468C

Kv1.2 and Kv1.5 associated to form functional O2-sensitive heteromeric K+ channel The truncated from of Kv1.5 displays accelerated inactivation from closed states relative to full-length Kv1.5 Established interactions with the Kv1.5T1 subunit imparts U-type slow-inactivation channel kinetics

[20]

[21]

[18]

(continued)

37  Gene Cloning, Transfection, and Mutagenesis

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Table 1  (continued) Mutations S4 and S6 mutations S4 domain R410N S6 domain P511G

Method of mutagenesis Coexpressed as monomers or in tandem within a subunit

(V407I, I410L, S414T) Site-directed primer exchange W472F R487V H463G C-terminal membrane localization Kv1.5∆C10 Kv1.5∆C13 Kv1.5∆C18 Kv1.5∆C28 Kv1.4/Kv1.5 Kv1.4∆C37 +1.5 Kv1.5∆C28 + 1.4 Interaction with PDZ domain FL-Kv1.5-A

FL-Kv1.5-A FL-Kv1.5Gly- ∆SAP97

Src adaptor Kv1.5-DPro Kv1.4-Kv1.5 Kv1.4-Pro

Cell system and functional assay Ltk– cells

Xenopus oocytes Voltage clamp fluorimetry

Finding

References

Coupling between voltage sensing (S4 domain) and gating (S6 domain) occurs by intrasubunit interactions Dynamic monitoring of voltage sensing by S4 coupled to channel opening and closing of the K+-selective filter

[14]

[12, 90, 91, 93, 94]

Mutations using endogenous restriction enzymes and PCR site-directed mutagenesis

GFP-tagged constructs monitored by confocal microscopy and patch clamp recording

Surface expression governed by C-terminal motif VXXSL

[23]

Mutations generated using PCR to replace the terminal TDL residues with AAA and tag the construct with the FLAG epitope Kv1.5 N-terminal deletion and glycine. Series of SAP97 deletions

Cardiac myocytes COS-7 Xenopus oocytes

SAP97 and Kv1.5 C-terminal functional interactions

[101]

Cos-7 CHO

Caveolin 3 and SAP97 in PDZ2 and PDZ3 recruit Kv1.5 and regulate K currents.

[15]

Deletions and insertions of proline-rich motif RPLPPLP

HEK293 Patch clamp

Kv1.5 subunits function as an Src homology domain (SH3)dependent adaptor allowing phosphorylation of associated a subunits that lack an SH3 domain

[95]

expressed in tandem in heterologous cells has determined that both of these Kv channels confer oxygen sensitivity, although the voltage-gating properties of these Kv proteins expressed separately differ from those of heteromeric channels. In L cells expressing Kv1.2/Kv1.5 the whole-cell current is reversibly inhibited by hypoxia in the voltage range of the PASMC resting Em [71]. As stated previously, Kv1.5 is a rapidly activating delayed rectifier channel which is inactivated by a slower pore-dependent mechanism termed “C-type inactivation” that involves the constriction of the outer mouth of the channel pore [6]. Realization that the N-terminal tail region of the channels plays a role in this slow inactivation process came from the comparison of a truncated N-terminal Kv1.5 isoform, found to occur naturally in cardiac myocytes, with full-length Kv1.5. The truncated Kv1.5 isoform (Kv1.5D209) displays an accelerated inactivation from

closed states in patch clamp experiments. The voltage–inactivation relationship for Kv1.5D209 is U shaped, in which more complete inactivation occurs at intermediate changes in membrane potential and relief of inactivation takes place at very positive potentials. Systematic deletion and sitedirected mutagenesis of the N-terminal region narrowed down a specific T1 domain within the N terminus that imparts U-type inactivation. This region is conserved among a subset of Kv channels, including Kv2.1, Shaker, and Kv3.1 [21]. Thus, creation of chimeras that include the N-terminal domain of Kv2.1 plus transmembrane segments and the C-terminal region of Kv1.5 result in a U-shaped inactivation–voltage relationship. The opposite effect was found when the Kv1.5 N-terminal tail was combined with Kv2.1 transmembrane domains, and the U-type inactivation profile was attenuated compared with a native Kv2.1 channel.

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However, for voltage-dependent activation of the channel to occur there must be a mechanism to sense a change in membrane potential. In Kv channels this is thought to occur predominantly through the S4 domain, which contains several charged residues (arginines) located in the membrane electric field which could potentially change their position in response to a field change [88]. In Kv1.5, precise mutations support S4 as the voltage-sensing domain, whereas S6 controls activation–inactivation kinetics by controlling the equilibrium between an open and a closed pore region [16]. Furthermore, these voltage-sensing and gating functions are coupled through intrasubunit interactions (not intersubunit interaction within individual Kv channels) [14]. A conserved Pro-X-Pro sequence is located in the region between helices S5 and S6 that forms a “hinge” allowing the gate to close and open. To understand how the S6 transmembrane region modulates channel activation and deactivation, site-directed mutagenesis was used to change residues in and around this Pro-X-Pro “hinge” motif. The first proline residue in the ProX-Pro “hinge” motif was found to be vital for channel gating and replacing a nearby proline partially restores open and closing of the channel although it does so with altered activation kinetics [13]. Additional studies have systematically substituted cysteine residues in the “turret” of the pore with neutral or charged amino acids to identify residues that modulate the conductance of K+ through the permeation pore. Specific functional domains or a group of particular amino acid residues (including but not exclusively H463) in the pore turret regulate the slow inactivation properties of the Kv1.5 channels [17, 18]. A very interesting study was recently reported by Vaid et al. in which a fluorophore, attached to particular amino acids in the S3–S4 linker, was used to dynamically monitor the macroscopic voltage-sensitive movements of the channel during the time course of activation and inactivation [12]. By recording both the voltage-dependent K+ current and the change in fluorescence, they could decipher macroscopic movement of the S4 voltage-sensing domain and conformational rearrangements that couple opening of the intracellular pore gate (Fig.  4). In this study, S4 movement was reflected as rapid fluorescence quenching associated with activation of the voltage sensors upon depolarization. In the case of Kv1.5, this is followed by a later dequenching of the fluorescent signal. This later dequenching phase is not present when the same technique is used to monitor Shaker channels [89]. To further understand this late dequenching phase of the voltage-dependent fluorescent signal, previously well characterized site-directed mutations were again studied. The triple ILT mutation (V407I,/I410L/S414T [90]) was used as a way to dissociate channel opening from voltage sensing and led to the suggestion that Kv1.5 fluorescence dequenching reflects the channel opening late in the activation pathway. Immobilization of the outer pore by sitedirected mutation (W472F, H463G or R487V [91–94]) or

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raising the extracellular K+ concentration revealed that this dequenching is likely associated with a K+ selectivity filter in the outer pore as opposed to the intracellular pore gate [12]. For the Kv channels to sense and gate K+ currents, they must be translocated (or be expressed) on the cell membrane. This cellular trafficking was first attributed to a motif (VXXSL) in the C-terminal domain [23]. Within the membrane, Kv1.5 a subunits interact with additional proteins and lipids that modulate the amplitude and duration of the K+ current. Specifically the last three amino acid residues (TDL) of the N-terminal tail have been shown to interact with SAP97 located in the PDZ membrane complex [5]. More recently it has been shown that both N-terminal and C-terminal regions interact with components of PDZ domains and influence the magnitude of the K+ current across the channel [19]. In particular, caveolin 3 and SAP97 are thought to play a role in recruiting Kv1.5 to the membrane and stabilize its location in a tripartite structure [15]. Kv1.5 has also been shown to interact with Src tyrosine protein kinase and serve as an adaptor to initiate the phosphorylation of heteromeric Kv channels that do not contain a similar SH3 domain [95]. Thus, mutagenesis approaches have been extensively used as a tool to elucidate the complex structure–function relationships within the a subunit of Kv1.5 and also its interactions with other Kv a subunits, associated proteins, and lipids that control the flux of K+ through the membrane.

5.2 BMP Receptor Polymorphisms Associated with Pulmonary Hypertension In 2000, two separate laboratories reported results suggesting that familial primary pulmonary hypertension was caused by mutations in BMPRII [27, 28]. The study by Deng et al. [28] used positional cloning to identify five mutations that predicted premature termination and two missense mutations in the BMPRII gene from a screen of individuals from 19 families [28]. In a separate publication form the International PPH Consortium, two frame-shift, two nonsense, and three missense mutations were found in a panel of eight kindreds studied [27]. Over the years, several studies of large cohorts of patients around the world have uncovered a strong link between BMPRII mutations and a predisposition to FPAH or IPAH. It is estimated that heterozygous germline mutations are present in more than 50% of patients with FPAH and in approximately 20% of those with sporadic IPAH [29, 60, 61]. In addition, these patients often have disease symptoms an average of 10 years earlier than noncarriers and exhibit more severely compromised hemodynamic dysfunction [64]. BMPRII is member of the transforming growth factor b (TGF-b) receptor superfamily that includes several type II

37  Gene Cloning, Transfection, and Mutagenesis

551

Fig. 4  Characteristics of the tetramethylrhodamine maleimide (TMRM) fluorescence report from Kv1.A397C channels. Representative ionic current (a, d) and fluorescence (b, e) traces recorded from oocytes expressing Shaker A359C (a, b) or Kv1.5 A397C (d, e) channels labeled with TMRM. Voltage-clamp pulses were applied from −80 to +60 mV from a holding potential of −80 mV (only highlighted traces are shown for clarity). (c, f) Mean G–V and F–V relations for Shaker A359C (c) and Kv1.5 A397C (f). V1/2 and k values for the G–V relation of Shaker A359C channels were −16.2 ± 1.3 and 16.8 ± 1.1 mV, respectively, and the corresponding values for the F–V relation were −40.9 ± 1.9

and 17.0 ± 1.7 mV, respectively. V1/2 and k values for the G–V relation of Kv1.5 A397C were 7.3 ± 1.6 and 16.4 ± 1.2  mV, respectively, and the corresponding values for the F–Vpeak relation were 1.9 ± 1.9 and 19.0 ± 1.3 mV, respectively. The F–Vpeak relation was not significantly shifted from the G–V relation. The voltage dependence of the dequenching component of fluorescence from Kv1.5 A397C (calculated as the peak minus the end fluorescence amplitude, F–Vdecay) is also shown in (f); V1/2 and k values were 31.0 ± 2.4 and 15.5 ± 1.9  mV, respectively (Copyright Vaid et  al. [103]. Originally published in the Journal of General Physiology. doi:10.1085/jpg.200809978)

receptors [BMPRII, and activin receptors (ActR-IIA and ActR-IIB)] and type I receptors [ActR-I (ALK2), BMPR-IA (ALK3) and BMPR-IB (ALK6)] that function as heterodimers in both vascular endothelial cells and PASMCs. In the small pulmonary arteries, selective binding of BMP ligands is thought to maintain the balance of PASMC and endothelial cell turnover. In contrast, in human PASMCs from patients with PAH carrying a BMPRII mutation, uncontrolled PASMC proliferation contributes to the aberrant pulmonary artery remodeling that occludes vessels and leads to persistent elevated pulmonary artery pressures. Thus, several in vitro experiment have been designed to recreate the missense and frame-shift mutations that have been identified in FPAH and IPAH to better understand how these polymor-

phisms could predispose an individual to a disease phenotype (Table 2). The first BMPRII mutants to be analyzed in vitro recreated some of the common polymorphisms found in patients with FPAH [27, 60]. In vitro expression of a kinase domain missense mutation (D485G) and the frame-shift mutation [1,860  fs(+10)] in HeLa cells and the mammary epithelial cell line NMuMG (a cell type known to be competent for TGF-b signaling) showed decreased basal and BMP4induced transcriptional activation of Smad binding element reporter constructs [60]. This early study was followed the analysis of a more extensive number of mutant BMPRII by Rudarakanchana et al. [1]. A series of site-directed mutations were created in the ligand binding, kinase domain and

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Table 2  Functional analysis of bone morphogenetic protein (BMP) receptor II (BMPRII) mutants Mutation Method of mutagenesis Cell system Functional consequence

References

Mammary-gland epithelium cell (NMuMG) line that is constitutively competent for TGF-b signaling HeLa cells, primary PASMCs and NMuMG cells

Mutants revealed decreased transcriptional activation of a Smad binding element transcriptional reporter

[60]

Revealed different effects of mutants expressed in heterologous cells and NMuMG on Smad-dependent transcription, MAPK activity, and cell proliferation

[1]

Recombinant adenovirus

Human PASMCs

Suggest that the kinase domain is essential for Smad signaling and that the C-terminal tail plays a role in BMP7-induced apoptosis

[8]

Recombinant adenovirus

PASMCs from BMPRIILox/Lox mice and delivered AdvCre to delete the BMPRII gene

Short and long isoforms of BMRPII were both found to restore BMP-mediated growth inhibition and osteogenic differentiation of PASMCs in response to BMP4 and BMP7

[7]

D485G1,860 fs(+10)

Restriction digest Site-directed primer exchange

Ligand binding C(60)Y C(117)Y C(118)W C(123)R/S Kinase domain C(347)Y C(420)R C(483)R D(485)G R(491)W/Q Cytoplasmic tail K(512)T N(519)K S(532)X R(899)X Kinase domain C347Y Truncated C-terminus I860X Deleted C-terminus ∆T AdvLF – long AdvSF – ∆T

Site-directed primer exchange

Fig. 5  Functional domain structure of the human bone morphogenetic receptor II (BMPRII). BMPRII consists of a signal peptide (SP) followed by the ligand binding domain, transmembrane domain (TM), kinase domain, and a unique C-terminal cytoplasmic tail

C-terminal cytoplasm tail of BMPRII that were expressed in HeLa cells, PASMCs, and NMuMG cells (Fig. 5). Cysteine substitutions in the ligand binding and kinase domain were found to prevent trafficking of the BMPRII receptor to the cell surface and consequently basal and BMP4-induced transcription activation of a BMP/Smad reporter construct. Interestingly non-cysteine substitutions in the kinase domain localized to the cell membrane but transcriptional activation of the BMP/Smad reporter construct was suppressed. Finally, cytoplasmic terminal mutants translocated to the cell surface

but in this case retained BMP signaling. When any of the above-mentioned mutants were transfected into NMuMG epithelial cells, activation of p38MAPK signaling and increased proliferation occurred even in the absence of a BMP4 ligand. This study was the first to indicate that BMPRII can signal through both the Smad and the mitogen-activated protein kinase pathways, but even more intriguing was that in the absence of a BMP ligand, the cues to prevent proliferation were lost. Subsequent in vitro mutagenesis studies focused on the cytoplasmic C-terminal domain, which is unique to BMPRII. Using a PASMC cell system in which the BMPRII gene was conditionally deleted in vitro, Yu et al. demonstrated that the ability of BMP2 and BMP4 to inhibit PASMC growth was lost. However, in these same BMPRII-deficient cells BMP6 and BMP7 signaling was augmented. The BMPRIIdeficient PASMCs could transduce BMP6 and BMP7 signals through an altered set of receptors, in this case the activinIIA receptor and a subset of type I receptors that do not normally interact with BMPRII [3]. In a subsequent study, reintroduction of the BMPRII gene as either a complete long

37  Gene Cloning, Transfection, and Mutagenesis

form or short form (not including the C-terminal cytoplasmic tail) was able to restore BMP4- and BMP7-mediated growth inhibition of platelet-derived-growth-factor-activated SMCs and restore osteogenic differentiation [7]. In a study by Lagna et  al. [8], mutations in the serine/threonine kinase mutation (C347Y), a truncated COOH tail domain (I860X), or complete deletion of the C-terminal domain revealed a PASMC population that was resistant to apoptosis [8]. So although it was first thought that BMPRII mutations, which in most cases abrogate BMPRII expression, were due to haplotype insufficiency [60], it appears that some mutations actually lead to a gain of function [3, 8]. However, both PASMCs and vascular endothelial cells express BMPRII receptors as well as additional type II and type I receptors that compete for BMP ligand binding and these precise cellspecifc ligand binding mechanisms are just beginning to be elucidated [4, 96]. However, irrespective of whether mutations in human PASMCs result in altered signaling owing to haplotype insufficiency or to a new dominant phenotype that works through an alternative set of BMP receptors and ligands, it is still unknown why these mutations are silent for many years until the symptoms associated with PAH begin to surface in young adults. A “second hit” hypothesis suggests that additional genetic or environmental factors are necessary to provide the conditions in which BMPRII mutated cells begin to express an abnormal phenotype. These secondary cues may be inflammatory stimuli and/or possibly altered interactions with a modifying factor, such as RACK1 or Src tyrosine kinase [9, 97, 98]. In vivo, mice with a heterozygous deletion of BMPRII require an additional inflammatory stress to develop PAH [99]. However, heterozygous or homozygous BMPRII gene deletion targeted to endothelial cells is sufficient to predispose a subset of mice to PAH [100]. Thus, current research is focused on understanding the cell-specific additional genetic and environmental factors that trigger the onset of PAH. Further understanding these pathways through mutant phenotypes that disrupt the balance of PASMCs and endothelial cells will hopefully allow for better design of treatment strategies that target the abnormal cellular processes unique to each susceptible individual.

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Chapter 38

Approaches for Manipulation of Gene Expression Ying Yu and Jason X.-J. Yuan

Abstract  Disease phenotypes are typically a result of interactions between both genetic variation and environmental conditions. More than 25,000 genes that make up the human genetic blueprint have been decoded. This extensive gene database along with the advent of DNA microarray technology and bioinformatics enable researchers to generate gene expression profiles in any given cell or tissue of interest. It is therefore possible to identify altered gene expression that occurs in a particular disease condition. These molecular breakthroughs open up new avenues for accurate diagnoses and genetic counseling, as well as opportunities for gene or protein therapy. Manipulating the expression of specific genes into their respective final ­protein products is an important tool for understanding the function of a specific gene that may have potential as a gene therapy. Traditional gene manipulation has focused on introducing a target gene into the cells and tissues through recombinant DNA and gene transfer techniques to express the gene of interest. To downregulate or inhibit the expression of a gene, antisense DNA and RNA interference (RNAi) fragments, which block RNA processing or translation of specific messenger RNAs (mRNAs), are methods that provide powerful and specific tools for studying gene regulation. This chapter discusses some of the most widely used molecular biology tools for downregulating gene expression: antisense-oligonucleotidemediated gene silencing and RNAi-mediated gene silencing. Keywords  Gene expression profile • Gene manipulation • Gene silencing • Small interference RNA • Molecular biology • Methodology

1 Introduction Disease phenotypes are typically a result of interactions between both genetic variation and environmental conditions [1]. The completion of the Human Genome Project in 2003 is a historic achievement and a fundamental milestone in life science research. More than 25,000 genes that make up the human genetic blueprint have been decoded. This extensive gene database along with the advent of DNA microarray technology and bioinformatics enable researchers to generate gene expression profiles in any given cell or tissue of interest. It is therefore possible to identify altered gene expression that occurs in a particular disease condition. These molecular breakthroughs open up new avenues for accurate diagnoses and genetic counseling, as well as opportunities for gene or protein therapy [2]. Manipulating the expression of specific genes into their respective final protein products is an important tool for understanding the function of a specific gene that may have potential as a gene therapy. Traditional gene manipulation has focused on introducing a target gene into the cells and tissues through recombinant DNA and gene transfer techniques to express the gene of interest. To downregulate or inhibit the expression of a gene, antisense DNA and RNA interference (RNAi) fragments, which block RNA processing or translation of specific messenger RNAs (mRNAs), are methods that provide powerful and specific tools for studying gene regulation. In this chapter, we will discuss some of the most widely used molecular biology tools for downregulating gene expression: (1) antisense-oligonucleotide-mediated gene silencing; and (2) RNAi-mediated gene silencing.

2 Antisense-Olignucleotide-Mediated Gene Silencing 2.1  Concept and Mechanism Y. Yu (*) Department of Medicine, University of California, San Diego, 9500 Gilman Drive, MC 0725, La Jolla, CA 92093, USA email: [email protected]

The concept underlying antisense-oligonucleotide-mediated gene silencing is relatively straightforward. Based on

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_38, © Springer Science+Business Media, LLC 2011

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Watson–Crick base-pairing interactions, an exogenous DNA sequence is employed which specifically hybridizes to the complementary RNA of the target gene and sterically inhibits the mRNA translation. As the transcribed RNA molecule is the sense strand of a given gene, any DNA sequence that hybridizes to that RNA molecule must be on the antisense strand. Therefore, the antisense oligonucleotide would, via the formation of an mRNA–DNA duplex, specifically prevent the translation of that mRNA into protein [3]. There are two major mechanisms by which antisense oligonucleotides may inhibit gene translation: (1) Direct antisense oligonucleotide DNA–RNA hybridization. In this case, the DNA–RNA duplex exerts a direct steric effect and physically blocks the RNA splicing and translational machinery. These types of antisense oligonucleotides are efficient only when targeted to the 5¢ or AUG initiation codon region. (2) Antisense targeted RNase H cleavage. RNase H is a ubiquitous enzyme required for DNA replication [4]. The RNase H enzyme recognizes the DNA–RNA hybrid and cleaves the RNA strand, resulting in fragmentation of the original full-length mRNA into two fragments, one lacking a 5¢-cap structure and one lacking a 3¢-poly(A) tail. Such fragments are usually rapidly degraded by exonuclease activity, and are thus no longer available for subsequent translation and protein synthesis [5]. Compared with antisense oligonucleotides designed as a steric blocker, RNase H-dependent oligonucleotides can inhibit protein expression when targeted to virtually any coding region of the mRNA [6].

2.2  Design and Synthesis Antisense oligonucleotides are usually designed to contain ten to 30 nucleotides complementary to a target mRNA by standard Watson–Crick base-pairing rules. However, if the target site in the mRNA is picked randomly, only a few complementary oligonucleotides can effectively inhibit targeted mRNA expression. The criteria for selection of antisense oligonucleotides are as followed: (1) specific target recognition by Watson–Crick base pairing; (2) good structural mimicry to the natural DNA–RNA; and (3) activation of RNase H to promote the target mRNA cleavage. To date, computational approaches have been developed to predict biological active antisense oligonucleotides for a precise target sequence. A computational approach significantly increases the probability for selecting an efficacious antisense oligonucleotide.

2.3 Limitations and Prospects in Therapeutic Application The study of the use of short antisense oligonucleotides to block translation of specific mRNAs was first reported by

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Zamecnik and Stephenson in 1978. It was demonstrated that antisense oligonucleotides complementary to the 3¢- and 5¢-terminal repeat sequences of Rous sarcoma virus inhibit viral replication and the expression of viral proteins in chicken embryo fibroblasts [7, 8]. Since then, antisense oligonucleotides have continued to be developed to selectively inhibit expression of a wide variety of genes at the translational level, and potentially treat a broad range of diseases, including human types of cancer, diabetes, asthma, and pulmonary vascular diseases. In 1998, the first antisense drug, Vitravene, an inhibitor of cytomegalovirus replication, was approved by the Food and Drug Administration for the treatment of retinitis in AIDS patients [9]. However, to date, most antisense therapies have not produced significant clinical results [10, 11]. A distinct disadvantage of the antisense approach is the instability of the single-stranded antisense oligonucleotides. Several modifications are being used to increase nuclease resistance (or antisense stability) as well as improving membrane permeability and binding affinity. One of the most frequently used modifications is the incorporation of a phosphorothioate group [12]. A phosphorothioate modification of antisense oligonucleotides is accomplished by replacing the nonbridging oxygen on the phosphate linkage with a sulfur atom. This type of modification significantly increases the stability of antisense oligonucleotides as a result of enhanced resistance to enzymatic hydrolysis. However, some phosphorothioate-modified antisense oligonucleotides may also fail to activate RNase H, which is considered to be an important step in the antisense mechanism of action [13]. Overall, the optimal use of antisense oligonucleotides for inhibiting target gene expression requires the resolution of problems relating to effective design, enhanced biological activity, and efficient target delivery. These issues are currently being actively addressed and will hopefully continue to shed light on ways to increase therapeutic efficacy and specificity [3].

3 RNAi-Mediated Gene Silencing 3.1 Concept and Mechanism RNAi represents an innovative new strategy for using small RNA molecules to manipulate target gene expression [14]. The concept for the RNAi-dependent gene regulation process was originally derived from the discovery of doublestranded RNA (dsRNA)-induced gene silencing in Caenorhabditis elegans [15]. Studies of the dsRNA-mediated gene silencing mechanism demonstrated it was initiated by an ATP-dependent, progressive cleavage of the long dsRNA template into short double-stranded fragments of 21–23 base pairs by the RNase Dicer [16–18]. These short

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double-stranded fragments are called small interfering RNA (siRNA) and are incorporated into a protein complex called RNA-induced silencing complex (RISC). ATP-dependent unwinding of the siRNA duplex generates an active RISC with single antisense strands. Once unwound, the singlestranded antisense guides RISC to the target mRNA that has a complementary sequence. Argonaute, the catalytic component of the RISC, endonucleolytically cleaves the target mRNA in the cytoplasm of cells and, thereby, prevents it from being used as a translation template [19–21] (Fig.1). Further studies have shown that RNAi is not only an important regulator of gene expression in many eukaryotic cell types, but also plays important roles in defending cells against parasitic genes, viruses, and transposons [22].

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RNAi was not initially considered a useful experimental or therapeutic tool in mammalian cells owing to highly toxic effects of long dsRNA and dsRNA-triggered interferon responses [23]. To eliminate nonspecific side effects of long dsRNA and exclude long dsRNA from the RNAi process, Elbashir et al. initiated the use of siRNAs that were 21-mer duplexes with 5¢ phosphates and two-base 3¢ overhangs on each strand. These shorter siRNAs were demonstrated to selectively and efficiently degrade targeted mRNAs upon introduction into several mammalian cell lines [24]. Recently, siRNA has been shown not only to be a valuable experimental tool in a variety of biological systems, but is also a potential therapeutic method for several diseases [25].

3.2 siRNA Design and Synthesis 3.2.1 siRNA Design

Fig. 1  RNA interference (RNAi) pathway. Long double-stranded RNA (dsRNA) molecules, which share sequence-specific homology with a target messenger RNA (mRNA), are cleaved as 21–23-nucleotide small interfering RNA duplexes (siRNAs) by an RNase III like enzyme known as Dicer. The siRNAs are recruited into a multiprotein siRNA–protein complex called RNA-induced silencing complex (RISC). Once activated by ATP, an activated RISC is generated with a single antisense strand of siRNA, which serves as a probe to target mRNA that has a complementary sequence. When a complementary sequence is found, silencing is mediated via endonucleolytic cleavage

Exogenous siRNA typically consists of a 19–23 base-paired duplex with two-nucleotide 3¢ overhangs. In principle, any siRNA containing the appropriate antisense sequence can be directed against the target mRNA that has a complementary sequence. However, only a few randomly selected siRNAs show effective silencing activity and lead to detectable altered phenotypes in functional studies [26, 27]. Specific criteria for designing the most efficient siRNA have been established [24, 28–31]. Ideally, the target mRNA sequence should begin with an AA dinucleotide as AA(N19)TT. N19 represent any 19 nucleotides of the target mRNA. The 3¢-UU overhangs, or their DNA counterparts, 3¢-TT, are often included to stabilize the siRNAs against RNases and are generally preferred over other sequences [24]. Additional important criteria for selecting the target gene sequence are a low G/C content, lack of inverted repeats, low internal stability at the sense strand 3¢ terminus, and sense strand base preferences (positions 3, 10, 13, and 19) [31]. Ui-Tei et al. further developed the siRNA selection rules to include (1) A/U at the 5¢ end of the antisense strand; (2) G/C at the 5¢ end of the sense strand; (3) at least five A/U residues in the 5¢ terminal one third of the antisense strand; and (4) the absence of any GC stretch of more than nine nucleotides in length [32]. Current criteria for siRNA design have also been embedded in the development of computer algorithms to predict effective siRNA sequences. Several siRNA design tools and search engines are available on the Internet to help select the highly efficient siRNAs for silencing experiments [27]. An investigator can simply input the target mRNA sequence in any of the siRNA design Web sites and generate lists of candidate siRNAs. As a practical strategy for obtaining the most effective siRNA, several siRNAs need to be designed according to the empirical rules and tested in cellular systems.

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3.2.2 siRNA Synthesis Several methods are used for preparing or synthesizing siRNAs. These methods include chemical synthesis, in vitro transcription, generation of a mixture of siRNAs by using long dsRNA and recombinant RNase III or Dicer [33], endogenous expression as short hairpin RNAs (shRNAs) from expression plasmids or viral vectors, and endogenous expression from PCR-derived siRNA expression cassettes. Moreover, functional validated siRNA or shRNA libraries are also commercially available. Although chemically synthesized siRNAs provide a promising tool for studying siRNA functions, this approach is limited by its high cost. To reduce the cost of siRNA selection, many investigators generate siRNAs using a less expensive preparation method such as in vitro transcription [34, 35]. siRNA Synthesis via In Vitro Transcription. The key to this method is to use RNA polymerase (e.g. T7) to generate individual strands of the siRNA. Templates for the transcription reactions are the sense and antisense strands of DNA oligonucleotides encoding the desired siRNA strands followed by a T7 promoter region, respectively. The RNA polymerase binds the T7 promoter and initiates RNA synthesis. After transcription, the reactions are combined to anneal the two siRNA strands. Then, the siRNA preparation is then treated with DNase (to remove the DNA template), RNase (to polish the ends of the dsRNA), and purified. In vitro transcription methods provide a rapid, convenient, and highly effective way to produce sequence-specific siRNAs. Furthermore, several commercially available kits, such as the silencer® siRNA construct kit (Ambion) and the BLOCKITTM RNAi TOPO transcription kit (Invitrogen), are available to aid investigators in the production of sequence-specific siRNAs. DNA-Based Vectors for Expression of shRNAs. Although the in vitro transcribed siRNA method provides investigators with a simple method to screen and identify effective siRNAs, two concerns have been raised regarding its further application: (1) a single transient transfection of siRNA into cells may not provide a sufficient window for functional gene inhibition to study proteins with a long half-life; (2) the transcribed siRNAs in functional genomics studies are easily affected by the variable transfection efficiencies in difficult-to-transfect cell lines [36, 37]. These issues have been addressed by the development of DNA-based plasmid vectors or viral vectors that express siRNA oligonucleotides [38–40]. This method is based on siRNAs that can be transcribed under the control of RNA polymerase III (pol III) promoters. Presently, U6 and H1 pol III promoters have been preferentially exploited for producing siRNAs [41]. Two types of pol III promoter based siRNA expression system are widely used. In the shRNA expression system, the vector contains a single pol III promoter followed by a segment of DNA

Y. Yu and J.X.-J. Yuan

that encodes a short hairpin structure with a stem of 19–29 base pairs and a short loop of four to ten nucleotides. The transcribed shRNA is then processed into siRNAs by Dicer within the transfected cell. In the tandem siRNA expression system, the vector contains dual pol III promoters arranged in a convergent manner that drive the expression of two short complementary RNA strands from a single 19-base-pair target region. Then two RNA strands can anneal and form siRNA duplexes with overhangs of four U residues at each 3¢ end. Since the target sequences that work well as synthesized siRNAs also work when incorporated as stem–loop hairpins into vectors [42], investigators usually construct the siRNA expression vectors on the basis of their target sequence selection from the inhibitory effects of the in  vitro transcribed siRNA candidates. Vector-based siRNA expression systems not only allow for the creation of stable cell lines depleted in a specific target gene [43], but also permit coexpression of reporter genes such as green fluorescence protein and luciferase, which facilitates tracking and/or selection/enrichment of transfected/transduced cells. Viral vectors have been used extensively in gene therapy to deliver DNA to target cells. Viruses evolved to specialize in gene transduction can also be used to ferry siRNA into cells. The use of viral vectors, such as lentiviruses [44, 45] and adenovirus [46, 47], allows the expression of siRNAs in difficultto-transfect cell lines and primary cell cultures [48].

3.3  siRNA Delivery The major bottleneck of siRNA application, as for most antisense or nucleic acid based strategies, is their poor cellular uptake associated with negatively charged siRNA and the hydrophobic nature of the cellular membrane [49, 50]. How to realize sufficient siRNA delivery is a major challenge for its application [51]. siRNA delivery systems are required to facilitate siRNA diffusion across the cell membrane, escape from endosome and/or lysosome degradation or errant siRNA compartmentalization, and target siRNA bioactivity to the appropriate intracellular site of action [51]. Several delivery systems have been developed, including: (1) Cationic liposomal delivery system: Commercially available cationic liposomes such as Lipofectamine (Invitrogen), RNAifect (Qiagen), and DharmaFECT (Dharmacon) are more commonly used for delivery of synthesized siRNAs and siRNA expression plasmid vectors into cultured cells. However, the large size and the toxicity of cationic lipid particles significantly affect their utilization in vivo [52]. (2) Viral delivery systems: There is no doubt that viral vector-based shRNAs are highly efficient in both in  vitro and in  vivo studies. However, the use of viruses in vivo has significant safety

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concerns and limitations, including toxic immune responses and inadvertent gene expression changes following random integration into the host genome [53, 54]. (3) Cationic cell-penetrating peptides (CPPs) delivery system: CPPs are a class of small cationic peptides derived from protein transduction domains and membrane transduction sequences. These molecules are composed of about ten to 30 amino acids and have the ability to engage the anionic cell surface through electrostatic interactions and rapidly induce their own cellular internalization through various forms of endocytosis [55–57]. The most well characterized CPPs include TAT peptide (the HIV-1 TAT protein), penetratin, transportan, polyarginine, and MPG (a short amphi­ pathic peptide) [58–62]. These cationic peptides are powerful carriers for cellular uptake for a variety of macromolecules, including proteins, peptides, and oligonucleotides, making them attractive candidates for siRNA delivery in vitro and in vivo [57]. For the CPP-based siRNA delivery, however, most of the siRNA internalized to the cell is entrapped in endosomes [56]. Strategies for endosomal escape of siRNAs must therefore be included when designing and synthesizing CPPs. It has been reported that histidine polymers are able to absorb protons in the acidic environment of the endosome, leading to osmotic swelling and membrane disruption, which promote the escape of plasmid DNA from the endosomes [63]. We propose that the CPP fused to five histidine residues and one cysteine residue at both the C-terminus and the N-terminus (C-5H-CPP-5H-C) is an effective peptide model for siRNA delivery. Two cysteine residues at the C- and N-terminal ends of the ­delivery peptide are used to enhance stability of peptide–siRNA complexes through the formation of interpeptide disulfide bonds. Selection of the most effective CPP is another challenge for CPP-based siRNA delivery. High transduction efficacy and low cellular cytotoxicity are major characters for effective CPPs. Some novel CPPs have been generated as chimeras of natural CPPs, such as YM2 peptide “PVRRPRRRRRR” and YM3 peptide “THRLPRRRRRR” with improved cell penetrating activity over wild-type TAT peptide “YGRKKRRQRRRR” [64]. In addition to further improvement in the penetration ability of CPPs, the most challenging task will be searching for effective CPPs for tissue- or cell-specific delivery. (4) Nanoparticle delivery systems: The cationic polymer such as polyethyleneimine have been used to successfully deliver siRNA to target cells. Safety evaluation of this potential approach is essential for its in vivo application [65]. (5) Cholesterol-conjugated siRNA delivery systems: Cholesterol-conjugated siRNA was successfully applied for tissue-specific siRNA delivery based on conjugation of lipophilic groups that can enhance cell uptake and enhance the pharmacokinetics and tissue biodistribution of oligonucleotides [66]. This approach may have high clinical significance, particularly as it relates to the development of new siRNA therapeutic

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strategies. (6) Specific siRNA delivery systems: Antibodymediated siRNA delivery system [67] and cell-type-specific delivery system [68, 69]. It is believed that siRNA delivery strategies will soon be further improved and developed for any given clinical condition [51].

3.4 Limitations and Prospects in Therapeutic Application siRNA has been widely adopted by investigators as a standard tool to investigate gene function in molecular and cell biology studies. siRNA approaches also have many very promising potential therapeutic applications to combat disorders with identified molecular targets [70]. However, a number of problems and hurdles remain for siRNA-based therapeutics, such as the instability of siRNA, efficient siRNA delivery, and siRNA-mediated unspecific immune response. As naked siRNAs are easily degraded in human plasma with a half-life of minutes [71], the instability of siRNA was an important obstacle for its application in humans. Fortunately, recent chemical modifications of siRNA have made significant progress to overcome this issue of instability [72, 73]. Currently, the biggest hurdle in developing RNAibased therapies is the in vivo barriers issue. This includes (1) rapid clearance by the kidneys, (2) degradation by serum and tissue nuclease, (3) uptake by phagocytes of the reticuloendothelial system leading to sequestration in liver and spleen, (4) failure to cross the capillary endothelium, (5) slow diffusion/ binding in extracellular matrix, (6) inefficient endocytosis by tissue cells, and (7) inefficient release from endosomes [50]. As we described above, following rapid advancement in understanding the details of the RNAi biological pathway, novel siRNA delivery systems are being developed and traditional delivery systems will continue to improve. It is expected that more exciting discoveries and applications of siRNAmediated therapy will emerge soon. Human clinical trials of RNAi-based drugs by several pharmaceutical companies are currently under way. Intravitreal administration of siRNA targeting vascular endothelial growth factor (VEGF) for treating age-related macular degeneration (Sirna Therapeutics) and intranasal administration of siRNA for treating respiratory syncytial virus infection (Alnylam Pharmaceutics) are just two examples [74]. As siRNA biology is complex, it is essential to validate the mechanisms underlying the observed biological effects before attributing them to RNAi-mediated therapeutic effects. siRNAs have been shown to activate immune response in a sequence- and concentration-dependent manner, leading to nonspecific gene silencing [75, 76]. An example of this type of concern comes from a recent study of siRNAs targeting VEGF or its receptor to inhibit retinal angiogenesis in an animal model of macular degeneration [77].

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A comprehensive analysis revealed that the observed reduction of angiogenesis by “naked” VEGF siRNA was not due to RNAi-mediated inhibition of VEGF expression, but rather was caused by sequence-independent stimulation of Toll-like receptor 3 and downstream interferon-g and IL-12 mediated suppression of neovascularization [77]. Thus, the possible interplay of siRNA with various members of the Toll-like receptor family of cell-surface proteins must be considered in the design of experiments [50]. Although it remains an open question concerning the safety, tolerability, tissue biodistribution, and biological effectiveness of RNAi therapy, it is almost certain that ­RNAi-based therapy will have a profound effect in the coming decades.

4 The Application of siRNA to Elucidate the Role of the Canonical Transient Receptor Potential 6 Channel in Idiopathic Pulmonary Arterial Hypertension 4.1 Background Idiopathic pulmonary arterial hypertension (IPAH) is a progressive and ultimately fatal lung disorder characterized by increased resting pulmonary arterial pressure [78–80]. Sustained contraction and excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs), mediated by persistent Ca2+ entry through Ca2+ channels, may play critical roles in the development of IPAH [81]. Recently, we demonstrated that canonical transient receptor potential 6 (TRPC6), an essential component of the receptor-operated Ca2+ channels, is significantly upregulated in PASMCs isolated from IPAH patients (IPAH-PASMCs) [82]. As upregulation of TRPC6 channel expression is an important initial step in the elevation of cytosolic free Ca2+ concentration required for mitogen-mediated PASMC proliferation [83], we hypothesized that upregulation of canonical TRPC6 channels in IPAH-PASMCs may serve as a critical Ca2+ entry pathway, raise intracellular Ca2+ concentration, and stimulate PASMC proliferation. To evaluate the potential therapeutic effects of TRPC6 inhibition on the abnormal PASMC proliferation in IPAH patients, we generated siRNA to specifically target TRPC6.

4.2 Synthesis and Selection of Efficient TRPC6 siRNA Using In Vitro Transcription Four sets of sense and antisense oligonucleotide primers were designed using Ambion’s siRNA Target Finder

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(http://www.ambion.com/techlib/misc/siRNA_finder.html). By using the silencer siRNA construction kit (Ambion) with these four sets of primers, we synthesized four siRNAs which targeted the following 21 nucleotides of the human TRPC6 mRNA sequence (GenBank accession no. NM_004621). The target sequences corresponding to siRNAs were as ­follows: siRNA1 (AAGTGCAATGACT-GCAACCAG); siRNA2 (AAGGTCTTTATGCAATTGCTG); siRNA3 (AAGTTCCTTGTGGTCCTTGCT) and siRNA4 (AAGCC­ TTCACAACAGTTGAAG). A scrambled 21-nucleotide control oligonucleotide (AAGCGCGCTTTG-TAGGATTCG) was also generated which bore no significant homology to any mammalian gene sequence. To identify the most effective siRNA for the inhibition of TRPC6 gene expression, COS-7 (African green monkey SV40-transfected kidney fibroblast cell line) cells were cotransfected with 2 mg of TRPC6-expressing plasmid and 100  nM siRNA or 100  nM scrambled control using Lipofectamine (Invitrogen). Protein levels were evaluated by western blot analysis after 72 h. As shown in Fig. 2a and b, two siRNA candidates (siRNA1 and siRNA2) suppressed TRPC6 protein expression levels, with siRNA2 being more effective [82].

4.3 Generation of Recombinant Adenovirus Expressing TRPC6 siRNA On the basis of the previously presented selection data, we firstly constructed a DNA-based vector (pSilencer 2.0-U6-TRPC6) that expressed TRPC6 siRNA targeting AAGGTCTTT ATGCAATTGCTG (nucleotides 2212–2232 of TRPC6 mRNA, NM_004621). Efficient delivery of siRNA or siRNA expression vector into cells is a critical step for RNAi-based gene silencing experiments. As IPAH-PASMCs are primary cultured cells, traditional transfection reagents may be inefficient for siRNA delivery. Recombinant adenovirus carrying siRNA targeting human TRPC6 was generated using an Invitrogen ViraPower adenoviral expression kit. The siRNA expression DNA cassettes, including RNA pol III promoter U6, a hairpin siRNA target sequence and terminator, were subcloned into pENTR1A entry vector. Scrambled control vector was also constructed as a negative control after efficient recombination of the entry vector into the promoterless pAd/PL-DEST Gateway® vectors, followed by viral production and transduction. The adenoviral particles for TRPC6 siRNA (Ad-siRNA-TRPC6) and the scramble control siRNA (Ad-siRNA-scramble) were plaque-purified and amplified in 293A cells. The titer of the adenoviral stock was measured by a spectrophotometer. Reverse transcription PCR and western blot data showed that adenoviral TRPC6 siRNA effectively knocked down TRPC6 expression in IPAHPASMCs [82] (Fig. 2c, d).

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Fig.  2  Downregulation of canonical transient receptor potential 6 (TRPC6) with siRNA inhibits 1-oleoyl-2-acetyl-sn-glycerol (OAG)induced Ca2+ influx and cell proliferation in pulmonary arterial smooth muscle cells isolated from idiopathic pulmonary arterial hypertension patients (IPAH-PASMCs). (a) COS-7 cells were cotransfected with pCMS-EGFP-TRPC6 (human TRPC6 expression vector) and one of four TRPC6 siRNA candidates (each siRNA at 100 nM) or scrambled siRNA. Western blot analysis demonstrates the effect of siRNA candidates and scrambled siRNA on TRPC6 protein level in COS-7 cells. The control lane indicates cells transfected with an empty vector. b-Actin controls are shown below. (b) A bar graph showing the normalized (to b-actin) TRPC6 protein expression in the presence of the different siRNA candidates. Among them, siRNA2 significantly decreased TRPC6 protein expression. (c, d) On the basis of the selection data, recombinant adenovirus expressing siRNA2 targeting human TRPC6 (TRPC6 siRNA) was generated. As a control, recombinant adenovirus expressing siRNA targeting human glyceraldehyde

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3-phosphate (GAPDH; GAPDH siRNA) or without targeting (scrambled siRNA) were used. mRNA (c) and protein (d) expression levels of TRPC6, canonical transient receptor potential 4, and GADPH are shown in IPAH-PASMCs treated with scrambled siRNA, GADPH siRNA, or TRPC6 siRNA. (e) Representative records of cytosolic free Ca2+ concentration changes in response to OAG in IPAH-PASMCs treated with scrambled siRNA or TRPC6 siRNA (upper panel). Summarized data of the resting cytosolic free Ca2+ concentration (left) and OAG-induced cytosolic free Ca2+ concentration increases (right) in IPAH-PASMCs treated with scrambled siRNA or TRPC6 siRNA. **P < 0.01, ***P < 0.001 vs. scrambled siRNA treated control cells (lower panel). (f) Cell proliferation was measured by 5-bromodeoxyuridine (BrdU) incorporation. Compared with the untreated or scram­ bled siRNA treated IPAH-PASMCs, TRPC6 siRNA significantly inhibits BrdU incorporation. *P < 0.05 and **P < 0.01 vs. scrambled siRNA treated IPAH-PASMCs. (Reprinted from Yu et  al. [82] with permission)

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4.4 Downregulation of TRPC6 via siRNA Attenuates the Enhanced Ca2+ Influx and Abnormal Cell Proliferation in IPAH-PASMCs Since the resting cytosolic free Ca2+ concentration and the increase in cytosolic free Ca2+ concentration induced by 1-oleoyl2-acetyl-sn-glycerol (OAG; 100 mM), a membrane-permeable diacylglycerol analog that is known to activate TRPC6 channels, were both significantly enhanced in IPAH-PASMCs, we employed adenovirus expressing TRPC6 siRNA to evaluate whether downregulation of TRPC6 inhibits Ca2+ entry activity, reduces the elevated resting cytosolic free Ca2+ concentration, and attenuates cell proliferation in IPAH-PASMCs. As shown in Fig. 2e and f, inhibition of TRPC6 expression in IPAH-PASMCs using siRNA markedly attenuated the OAGmediated increase in cytosolic free Ca2+ concentration (p < 0.001) and reduced the resting cytosolic free Ca2+ concentration (p = 0.003). Furthermore, inhibition of TRPC6 expression in IPAH-PASMCs also significantly inhibited DNA synthesis in proliferating IPAH-PASMCs [82, 84] (Fig. 2g).

5 Summary Manipulation of gene expression constitutes an essential strategy in both basic and applied pharmaceutical research. Several nucleic acid based methods for sequence-specific manipulation of gene expression, including antisense oligonucleotides [85, 86], aptamers [87], DNAzymes [88, 89], and ribozymes [90], have been developed over the past couple of decades. Arguably, none of these have been more promising or generated more interest than RNAi [48]. A major advantage of RNAi relative to other antisense-based approaches for therapeutic applications is that it exploits the cellular machinery that efficiently allows targeting of complementary transcripts, resulting in highly potent downregulation of gene expression [91]. Another distinguishing factor and advantage of RNAi compared with antisense technology is that silencing is dependent on antisense target site ­accessibility and is determined by the local mRNA ­conformation [92] (Table 1). Because of the ability of RNAi to silence any gene once the sequence is known, the application of RNAi has already contributed to an expanded knowledge of gene function [93]. With the arrival of the postgenomic era, RNAi offers a therapeutic platform on which to develop potential picomolar active drug candidates against any human disease, including those respiratory diseases that are poorly responsive to pharmaceutical interventions [70, 94, 95]. Technical developments in science occur that enable leaps forward and enable acceleration of innovation [96]. In addition to siRNAs, there is another abundant and important class of

Y. Yu and J.X.-J. Yuan Table 1  Comparison of antisense oligonucleotides and small interfering RNAs (siRNAs) for inhibition of gene expression Antisense oligonucleotides siRNA Molecular structure Sequence length Selectivity Stability Inhibition of target protein synthesis RNA-structure- dependent activity Catalytic mechanism of action Specificity at therapeutic doses Potency Delivery options

Single-stranded DNA ~ 20 bases High Low–medium Yes

Double-stranded RNA 19–23 bases High High Yes

Yes

No

No

Yes

Low–medium Low–medium Limited

High High Multiple

small approximately 22 nucleotide noncoding endogenous RNA species in cells, the microRNAs (miRNAs) [97]. miRNAs target both mRNA degradation and suppression of protein translation on the basis of sequence complementarity between miRNA and its target [98]. Since miRNAs naturally regulate gene expression posttranscriptionally by cells, this group of silencer RNAs is particularly effective [99]. Currently, more and more biological roles for miRNA in regulation of cancer, metabolic disease, and viral infection are being identified. This accumulating evidence further suggests that miRNA may a represent potential molecular target in these therapeutic areas. Antisense oligonucleotide approaches for inhibiting miRNA function and siRNA technologies for replacement of miRNAs are currently being explored as tools for uncovering miRNA biological functions and developing potential therapeutic agents [100]. Further research into the fundamental mechanisms of miRNAs may unveil new approaches for gene silencing [101]. Acknowledgements  The authors sincerely thank Ellen Breen (Division of Physiology, University of California, San Diego) for her critical revision of the manuscript. We also thank Carmelle V. Remillard and Steve Keller (Department of Medicine, University of California, San Diego) for their helpful comments. This work was partially supported by an American Heart Association Scientist Development Grant (0630117N to Y.Y.). Ying Yu is currently working at Alcon Laboratories, Inc. to develop efficient and nontoxic in  vivo siRNA delivery agents for the treatment of eye diseases.

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Chapter 39

Bioinformatics, Genomics, and Functional Genomics: Overview Ali Torkamani, Eric J. Topol, and Nicholas J. Schork

Abstract  Recent advances in high-throughput genetic and genomic technologies, such as efficient DNA sequencers, multiplex genotyping platforms, microarrays, and proteomics and metabolomics assays, have provided researchers with an unprecedented ability to seek out and characterize the genetic determinants of diseases and clinical outcomes of all sorts. In fact, the application of these technologies has led to the identification of hundreds, if not thousands, of genomic loci that are associated with or even responsible for many different disease conditions, clinical outcomes, and responses to medications. As useful as these technologies are, however, their ability to generate data easily outpaces an ability to draw compelling inferences from those data. The field of bioinformatics evolved out of a need to manage, analyze, and interpret high-dimensional data of the type generated from the application of high-throughput genetic and genomic technologies. Necessary bioinformatics tools include those that enable one to test statistical associations between DNA sequence variations and phenotypes, find patterns in gene or protein expression data, understand how specific perturbations in a protein may affect the functioning of that protein, and determine which fundamental processes, pathways, or genetic networks may harbor the molecular “lesions” causing disease. In this chapter we provide an overview of available bioinformatics strategies and tools that have either been applied or are simply applicable to the genetic and genomic dissection of complex pulmonary vascular diseases (PVD) and other complex diseases. We start with a brief summary of the motivation for examining the genetic basis of PVD, consider various strategies for identifying genetic factors contributing to PVD, and then describe the bioinformatics tools and resources available to facilitate these analyses. We provide relevant Web resources in addition to references, and also provide example analyses and results.

A. Torkamani (*) Scripps Genomic Medicine, Scripps Translational Sciences Institute, 3344 North Torrey Pines Court, La Jolla, CA, 92037, USA e-mail: [email protected]

Keywords  High-throughput • Gene expression profile • DNA sequencing • Genomics • Proteomics • Bioinformatics • Genetic network

1 Introduction 1.1 Bioinformatics and the Contemporary Genetic Research Landscape Recent advances in high-throughput genetic and genomic technologies, such as efficient DNA sequencers, multiplex genotyping platforms, microarrays, and proteomics and metabolomics assays, have provided researchers with an unprecedented ability to seek out and characterize the genetic determinants of diseases and clinical outcomes of all sorts. In fact, the application of these technologies has led to the identification of hundreds, if not thousands, of genomic loci that are associated with or even responsible for many different disease conditions, clinical outcomes, and responses to medications [1]. As useful as these technologies are, however, their ability to generate data easily outpaces an ability to draw compelling inferences from those data. The field of bioinformatics evolved out of a need to manage, analyze, and interpret high-dimensional data of the type generated from the application of highthroughput genetic and genomic technologies. Necessary bioinformatics tools include those that enable one to test statistical associations between DNA sequence variations and phenotypes, find patterns in gene or protein expression data, understand how specific perturbations in a protein may affect the functioning of that protein, and determine which fundamental processes, pathways, or genetic networks may harbor the molecular “lesions” causing disease. In this chapter we provide an overview of available bioinformatics strategies and tools that have either been applied or are simply applicable to the genetic and genomic ­dissection of complex pulmonary vascular diseases

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_39, © Springer Science+Business Media, LLC 2011

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(PVD)  and other complex diseases. We start with a brief summary of the motivation for ­examining the genetic basis of PVD, consider various strategies for identifying genetic factors contributing to PVD, and then describe the bioinformatics tools and resources ­available to facilitate these analyses. We provide relevant Web resources in addition to references, and also provide example analyses and results.

1.2 Heritable Factors in Pulmonary Vascular Disease Although the genetic basis of some forms of PVD have been worked out, such as certain subtypes of familial pulmonary arterial hypertension (FPAH), these forms of PVD tend to be rare and follow overtly Mendelian, monogenic inheritance patterns, making the identification of the genes ­responsible somewhat straightforward with contemporary family-based linkage analysis and positional cloning strategies [2–5]. For example, it is known that mutations in bone morphogenetic protein receptor II (BMPR2) gene are found in more than 70% of all FPAH cases and provide an explanation for the autosomal dominant pattern of inheritance exhibited in families with members with FPAH [5]. However, the vast majority of the different types of PVD are more complex, with multiple genetic and nongenetic determinants contributing to them, making the identification of each and every contributing factor difficult. This has led researchers to adopt more sophisticated, biologically integrated, wholegenome approaches to the identification of relevant genetic factors. To motivate genetic studies of more complex forms of PVD, it has been noted, for example, that a family history of PVD has consistently proven to be a strong predictor for future cardiovascular events [6]. This suggests that a strong genetic, or inborn, biological mechanism contributes to the development of PVD. The desire to identify and characterize these mechanisms has led to the development of clever strategies that are being applied to the study of pulmonary and vascular diseases to an increasingly large extent. These methods interrogate the disease state at a variety of biochemical levels, including the genomic level (i.e., DNA sequence variations), whole-genome RNA expression levels, and wholegenome protein levels. In addition, bioinformatic methods for integrating the data and results of these, and related, approaches have been developed, which essentially allow researchers to link genes and proteins at the DNA sequence, expression, and biochemical network and/or pathway level. We briefly describe each of these approaches and their application to PVD, with an emphasis on available bioinformatics tools for this purpose.

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1.3 Genetic or Meiotic Mapping A traditional approach to the identification of genetic factors that influence a particular phenotype is, as noted in the previous section, genetic or meiotic mapping [3, 7]. Genetic mapping involves tracing genetic variations that appear to “cosegregate,” or are inherited together, with a particular phenotype. Once a genomic region that appears to cosegregate with a phenotype has been identified – either within families as in traditional “linkage” analysis or simply among individuals with and without the phenotype, as in association or linkage disequilibrium (LD) mapping (as described below) – then that genomic region can be further scrutinized to identify the offending causal variations [3, 7, 8]. In this way, genetic mapping exploits the fact that genetic variations near each other are typically inherited together because during meiosis recombination events between homologous chromosomes tend to occur only a few times and hence make it unlikely that unique parentally derived variations or “alleles” at loci near each other on the genome will be shuffled across homologous chromosomes over one or a few generations (Fig. 1). Before considering some of the details behind genetic mapping and its relevance to PVD, we should make clear that there are currently two major designs or strategies for considering and identifying genetic variations that influence a particular phenotype, with each rooted in a slightly different orientation for characterizing the association of those variations with a disease or phenotype of interest: one strategy assumes that the variations influencing a particular trait are fairly common (but may have low “penetrance,” or a low probability of actually inducing the phenotype in carriers of the variation) and the other assumes that the variations may be numerous but rare (but may have high penetrance) within genomic regions of relevance. The first strategy has been used to search for variations throughout the entire genome in what have been termed “genome-wide association (GWA) studies” [1]. These studies essentially test as many genetic variations for association with a phenotype as possible using sophisticated statistical analysis strategies and clever choices of which variations to test. The second strategy exploits DNA sequencing of individual genomes to uncover rare variations that collectively are more pronounced in individuals with or without the disease or phenotype of interest [7]. This second strategy has largely been confined to searches within predefined candidate genomic regions owing to technological limitations in available sequencing technologies [9]. GWA studies build on the fact that the human genome is populated with numerous common single-nucleotide polymorphisms (SNPs). SNPs are single base pair changes in the DNA sequence and many have appreciable allele frequencies across individuals. Many are common to individuals within different ethnic or racial groups, whereas others occur more

39  Bioinformatics, Genomics, and Functional Genomics: Overview

Fig. 1  The inheritance of chromosomal segments harboring a unique set of variations that forming haplotypes that may surround a disease influencing variation. Individual labeled 1-2 possesses a chromosome (shaded black) that harbors a mutation (the “A” nucleotide) that causes a certain disease phenotype. At loci neighboring the locus harboring the mutation, individual 1-2 has a particular set of nucleotides: A-T-A-G-C. The loci are polymorphic such that on individual 1-2’s homologous chromosome, which does not harbor the mutation (the light shaded chromosome), there are a different set of alleles: T-C-T-A-G. Mate pair 1-1 and 1-2 have four offspring: 2-1, 2-2, 2-3, and 2-4. Owing to recombination, the offspring receive differently sized chunks of the black shaded chromosome from parent 1-2. However, the individuals that received the disease-causing mutation (individuals 2-1 and 2-2), also received the neighboring alleles. The individuals that did not receive the disease-causing mutation (individuals 2-3 and 2-4) received the alleles at the site of the mutation and the neighboring loci on parent 1-2’s homologous chromosome (i.e., T-C-T-A-G). Individuals 2-1, 2-2, 2-3, and 2-4 go on and start lines of descent with some of their descendants being transmitted the mutation G generations later. Owing to further recombination events occurring during this line of descent, the size of the original chromosome from individual 1-2 harboring the mutation will be diminished such that only alleles at loci very close to the site of the mutation will be preserved (e.g., haplotype T-A-G) in individuals G-1, G-2, G-3, and G-4. Individuals descending from individuals not possessing the disease-causing mutation (i.e., G-5, G-6, G-7, and G-8) will receive a haplotype different from that received by those inheriting the disease-causing mutation. Thus, the offspring inheriting the diseasecausing mutation will share a large haplotype surrounding the mutation that might be identified by genotyping them on a specific set of markers. Individuals in later generations that harbor the disease and hence disease-causing mutation would have to be genotyped at a greater number (i.e., density) of markers to identify the shorter haplotype they carry that contains the disease-causing mutation

widely across all human populations, albeit at different ­frequencies. “Common” SNPs are defined as those SNPs having a major allele frequency of 5% or more in the population as a whole. The identification of common SNPs that can be used with great efficiency in GWA studies was the motivation for the International HapMap Project (http://www. hapmap.org) – an initiative that resulted in detailed knowledge of the genomic location of SNPs as well as the LD relationships between their alleles (i.e., correlations) [10, 11].

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Given a panel or map of genetic markers (such as ­ apMap-derived SNPs), for GWA studies, individuals with H (cases) and without (controls) a phenotype are genotyped on this panel and the frequencies of the alleles at each SNP locus are compared across these individuals. The ultimate number of markers used in such studies varies, but is often on the order of 500,000 or more, since this number has been shown by the HapMap researchers and others to adequately cover most of the common variations in the genome on the basis of LD relationships. About 20% of the genome is not “SNP-able.” Genotyping is usually pursued with what have been referred to as “SNP chips.” A SNP chip is typically a high-density oligonucleotide array, which contains short sequences of DNA matching genomic sites which are known to contain common SNP variants. DNA from individuals in the study can be isolated, applied to the chip, visualized by specialized chip readers, and ultimately a readout of the DNA variations possessed by each of those individuals at the SNP loci can be provided [12]. The resulting patterns of genetic variation can be analyzed in families by tracing overt evidence for cosegregation of a particular set of variations with a phenotype from generation to generation if a family is studied, or by contrasting the frequency of a single variation, or set of variations, between individuals with and without a particular phenotype (Fig. 2). The design of genotyping chips as well as GWA studies in general is extremely important, as is careful analysis of the genotype data [13] (Fig. 3). Data analysis strategies must not only be able to appropriately model gene–phenotype associations, but must also be able to accommodate the huge multiple-comparison problems arising from testing hundreds of thousands of SNPs for association [7]. In addition, one of the primary reasons why the design of the genotyping chips is so important is that other forms of variation, such as insertion/ deletion polymorphisms and other forms of copy-number variations, may actually influence the phenotype of interest and, unless these forms of variation are in LD with (i.e., are correlated with) the SNPs used in a study, their effects would not be discovered [14]. It is quite possible, if not very probable, that instead of, or in addition to, common variations influencing a particular disease phenotype, multiple, possibly independent, rare variations contribute to the expression of that phenotype as noted above [15]. In fact, it is well known that individual human genomes contain a large number of rare variants whose functional or phenotypic significance are unknown. These rare variations may have arisen de novo in specific individuals, or may have been inherited but simply are not shared by a large portion of the population. The identification of rare variations that might influence a disease phenotype requires the application of sequencing technologies to essentially discover the variations, since they are not likely to have been observed and catalogued before. For example, rare alleles

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Fig. 2  The linkage disequilibrium (LD) relationships between singlenucleotide polymorphisms (SNPs) within the bone morphogenetic protein receptor II (BMPR2) gene region. The plot was generated from genotype data on the northern European individuals in the International HapMap database (i.e., the “CEPH” panel of individuals; http://www. hapmap.org). The plot was made with the much-used program Haploview [76]. The diamond-shaped figures at the top denote positions of SNPs within the chosen BMPR2 gene region. The horizontal line highlighted with yellow depicts elements with the BMPR2 gene

Fig. 3  The results of a mock genome-wide association (GWA) study. The x-axis denotes the genomic positions of SNPs tested for association. The y-axis denotes the strength of the association based on –log p of a c2 test of the association of the SNP alleles and disease status. Each circle represents a different SNP location. The horizontal lines denote significance levels that may be meaningful given the large number of tests pursued (e.g., that can be used to control for multiple comparisons – a –log p of 10−8 is often seen as significant in GWA studies). This kind of plot is often called a “Manhattan” plot since it resembles the Manhattan skyline

have been associated with HDL cholesterol levels [16]. Currently, sequencing technologies do not permit the sequencing of the entire human genome in a large enough

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(e.g., exons as small vertical lines) that can be related to SNP positions. The inverted triangle depicts the strength of the LD relationships between SNP alleles by position. Red indicates stronger LD and gray indicates weaker LD. White indicates no information. Note that in this region some SNPs that are far apart exhibit strong LD and some SNPs in close proximity exhibit weak LD. This is consistent with the existence of recombination hot spots, mutation and gene conversion events, and a whole host of other factors that may interrupt the association between strong LD and the proximity of loci [77]

collection of individuals to statistically identify the small subset of rare variations that might be associated with a particular disease, although success with sequencing the entire genomes of specific individuals has been achieved, suggesting that efficient whole-genome human sequencing is on the horizon [17, 18]. Therefore, contemporary studies seeking to identify collections of rare variations that might contribute to a particular phenotype have been confined to specific genomic regions [19, 20]. Importantly, DNA sequencing studies motivated by the desire to identify rare variations that contribute to phenotypic expression are often coupled with bioinformatics approaches to characterizing the likely functional significance of identified variations, since many variations will be identified and only a small subset are likely to be meaningful. Differentiating the likely functional or causal variations from others that simply arose over time is often nontrivial. When faced with a series of potential nonsynonymous polymorphisms that may underlie the phenotype of interest, it can pay to prioritize mutations before testing them further. A number of computational techniques aimed at differentiating neutral from functional nonsynonymous polymorphisms have been developed [21]. These methods could be applied to narrow down a series of common polymorphisms in candidate genes before an association study, to increase the power of the study, or these methods may be helpful in narrowing down the number of mutations to be tested for

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functional significance in in vitro or in vivo settings, sparing the investigator a lot of time, energy, and money. Most computational methods will take advantage of sequence conservation in some form. SIFT, arguably the most popular of these methods, takes as its input the protein of interest, searches for similar proteins, generates multiple alignments with these proteins, and uses these alignments to determine the degree of conservation at any residue, which in turn is used to output a probability that an amino acid substitution is deleterious [22]. SIFT is available at http://blocks. fhcrc.org/sift/SIFT.html. Other conservation-based methods, such as subPSEC, are also available online (http://www.pantherdb.org/tools/csnpScoreForm.jsp) [23]. Other methods, such as Polyphen (http://genetics.bwh.harvard.edu/pph/) [24] and PMut (http://mmb2.pcb.ub.es:8080/PMut/) [25], take advantage of other sequence-based or physiochemical properties in addition to conservation. Other methods, such as SNPs3D, (http://www.snps3d.org/), take full advantage of available crystallographic data when performing predictions [26]. It should be noted that PMut and Polyphen also use crystallographic data in some instances. In some cases, predictors specific to a protein family, or even a specific protein, have been generated. For example, predictors have been developed for DNA repair genes [27], protein kinases [28], and G-protein-coupled receptors [29].

lengths of DNA corresponding to specific gene transcripts, and the number of complementary strands of RNA – preprocessed for application to the chip – is determined by the intensity of the signal on the chip. The intensity read off the chip gives a quantitative measure of the number of strands of RNA belonging to a specific gene transcript present in a tissue or cell sample. Proteomics approaches investigate the same quantitative information as gene expression experiments, but interrogate the levels of a gene at a more terminal level of gene activity. In this case, the levels of protein, which are encoded in the genome, transcribed to RNA, and translated to protein, are determined by a variety of means. Generally, proteins are collected in bulk from a sample, and then separated into individual proteins by physiochemical properties. For example, in two-dimensional electrophoresis, proteins are separated first by their isoelectric point (the pH at which their net charge is zero), then by size. Proteins are then visualized, and their levels determined by a number of means, including staining, interrogating them with specific antibodies, determining the density of a “spot” (or imprint based on imaging) corresponding to a specific known protein, or identification by mass-spectroscopic techniques [31]. The development of gene and protein expression technologies for discovering genes that influence diseases and phenotypes has led to a series of important discoveries in the mechanisms underlying, e.g., cardiovascular pulmonary diseases in ways have that have complemented studies involving DNA sequence variations. For example, recent GWA studies have identified genetic variations that confer risk of developing a range of vascular diseases [32, 33]. At times, as in the case of sequence variants in the chromosome 9p21 region and their association with numerous cardiovascular diseases, the associations literally raise more questions than they answer, since there is no known gene or regulatory element in the genomic vicinity (e.g., less than 100 kb) of the associated variations whose expression levels can be readily interrogated. In other cases, clear associations with specific genes are found to underlie genetic predisposition to disease, and provide interesting targets for therapeutic intervention. In this context, the expression levels of the relevant genes and proteins harboring the variations can be interrogated to see if they are also associated with the disease. Gene expression or protein levels may also act as predictors of an outcome after intervention to ameliorate the disease (e.g., as a “biomarker” of that outcome), given the association between abnormal expression levels of the gene or proteins in question and the disease. For example, there is evidence to suggest that elevated C-reactive protein levels after a stroke predict unfavorable outcomes [34]. It should be noted that such prognostic indicators are almost always suggestive, and, as in the case for C-reactive protein, the clinical value of such predictive biomarkers is continuously under scrutiny [35].

1.4 Gene and Protein Expression Analysis In contrast to association studies linking DNA sequence variations with a trait or disease, researchers can also contrast expression levels of genes or proteins between individuals with and without a particular disease and potentially implicate a differentially expressed gene in disease pathogenesis. Thus, when considering the use and study of the expression levels of genes as a means to identifying genes that might be of relevance to a disease, researchers are essentially interested in the differences in the abundance of the RNA between individuals with and without a particular disease or phenotype. RNA abundance gives some indication of the response of, or perturbations in, a particular gene in, or to, specific conditions. For example, the expression of genes involved in the response to wound healing would be to increase shortly after a wound is inflicted. If someone has a deficiency in wound healing, that deficiency might be attributed to a lack of normal expression of wound-healing genes. Gene expression levels can be determined on an individual gene-by-gene basis, by quantitative polymerase chain reaction strategies, or they can be determined in a much broader scale through high-throughput “microarray” approaches [30]. High-throughput approaches leverage technology similar to that of the SNP chip. However, in the case of expression levels, the chip is designed with numerous

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As the amount of information relating to genetic susceptibility to cardiovascular diseases explodes, the ability to access this information in a comprehensive and easily digestible manner becomes increasingly important. There are numerous resources available to physicians and basic researchers interested in exploring the genetic underpinnings of PVD and related disease. In the sections that follow, we will review some repositories of information, and discuss some bioinformatics techniques and tools that can be applied to develop and to test new hypotheses utilizing relevant study designs and/or publicly available data.

2 Bioinformatic Resources in the Public Domain 2.1 DNA Sequence Variation A number of databases are dedicated to the association of genetic variations with disease phenotypes. A major repository of this information is the Online Mendelian Inheritance in Man (OMIM) (http://www.ncbi.nlm.nih.gov/sites/entrez? db=omim) database [36]. The OMIM database, originally a physical catalog of Mendelian disease mutations, has been maintained electronically for over 40  years by Victor McKusick, until his recent passing, and colleagues. The database has two basic types of entries, gene-centric entries and disease-centric entries. Disease-centric entries generally begin with a basic phenotypic description of the disease, then describe clinical features, inheritance patterns, biochemical features, initial mapping of risk loci, molecular genetics, and animal model information. In some cases, information about clinical management or other clinical or molecular features is also available. Gene-centric entries also begin with a general description, describe initial cloning, gene structure, mapping, biological functions, early studies and history, population genetics, and eventually end in specific mutational information regarding the gene or disease. Note that each entry is not limited to the above-mentioned information, nor is the entirety of this information available for all diseases/genes. The database is easily searchable by keywords and is replete with cross-links to other related disease or gene entries as well as references to primary literature. Additionally, OMIM entries are not restricted to genes with variation information. Suggestive disease links without mutational evidence are also included. The OMIM database is an excellent resource for a disease overview and history; however, the mutational information is not comprehensive. Although one will find a good deal of mutational information, many of the

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entries contain a select set of representative or classical variations. Another, more comprehensive, mutational resource is the Human Gene Mutation Database (HGMD) (http://www. hgmd.cf.ac.uk/ac/index.php) [37]. Although this resource has less descriptive information, it represents a comprehensive, or nearly comprehensive, catalog of disease-causing mutations. Mutations are broken down into categories, such as missense, splicing, or regulatory variations, and are searchable by a number of specific identifiers such as gene symbol, OMIM number, and disease name. Importantly, unlike OMIM, the mutation annotation is accompanied by flanking sequence, significantly increasing the ease and accuracy of mapping variations to any other reference sequence. The database contains both publicly available data and proprietary information, which can be accessed by paying a subscription fee. Note that both HGMD and OMIM are hand-curated, using a different set of evidence requirements for entry into the database. Some mutations reported in the literature may not pass these quality requirements and thus may not be included in one, or both, of the databases. Other mutation databases exists, such as Mutation Discovery (http://www.mutationdiscovery.com/ md/MD.com/home_page.jsp) and HGVBase (http://www. hgvbaseg2p.org/index); however, they do not appear to be nearly as comprehensive as OMIM or HGMD. General mutation databases containing commonly occurring, and not necessarily disease associated, mutations also exist. The most useful and comprehensive of these is the single-nucleotide polymorphism database (dbSNP), maintained at the NCBI Web site (http://www.ncbi.nlm.nih.gov/ projects/SNP/) [38]. Investigators may submit any sequence variations they discover to this database, which aims to catalogue all known DNA variations, including those falling into regions of the genome not mapping to any particular gene. The interface of this database is similar to that of OMIM, and is searchable by simple keyword entries. Additionally, each SNP is designated an identifier, which can be used to query a particular SNP in other databases or search for phenotypic information in primary literature. SNPs in dbSNP are also accompanied by flanking sequence information, gene information, population frequencies if they are known, and validation status, among other pieces of useful information. dbSNP is particularly useful for identifying the known genetic variations in a gene of interest. Another general SNP database is, as noted earlier, the HapMap database (http://www.hapmap.org/) [39]. All HapMap SNPs are contained within dbSNP. However, HapMap contains in-depth information regarding the population distribution of SNPs, as well as the linkage structure around each SNP. Linkage structure reflects the co-occurrence of SNPs with one another, that is, how well possession of one SNP predicts the possession of another neighboring SNP. Many SNPs are

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tightly linked with one another because DNA is inherited in large blocks, called “haplotype blocks,” and thus the genetic variations within a block travel together from parent to child. The HapMap project essentially provides a map of genetic information so that investigators may pursue studies linking particular diseases or other phenotypes to inherited genetic information. This haplotype block information is leveraged to design association studies. In an association study, SNPs which can be accurately used as surrogates for the majority of SNPs within a single haplotype block are used to represent, or tag, a particular haplotype block, and are thus named “tag SNPs.” In carrying out an association study, investigators genotype large groups of individuals with or without a phenotype of interest, and analyze the resultant genotype information for frequency differences among the disease and control groups. Tag SNPs with a statistically elevated frequency within the disease group are likely to be in close proximity to a SNP which contributes to disease risk. The data collected from such large studies are available in the primary literature, or may be deposited in the dbGAP database (http://www.ncbi.nlm.nih.gov/sites/entrez?db=gap) [40]. Some information within the database is available publicly; however, in general, prior permission or authorization is required to gain access to the bulk of the information contained within the database. Access can be requested by applying to the NIH Data Access Committee. Data from the Framingham Heart Study, a study that established many of the common risk factors for cardiovascular disease, is available from dbGAP [41]. A number of tools exist for aiding in the design, analysis, and interpretation of GWA studies. Not only are there repositories for GWA study data and results, such as dbGAP (http:// www.ncbi.nlm.nih.gov/gap) and the NHGRI-run databases for GWA studies (http://www.genome.gov/gwastudies/) and related databases [42], but there are tools to ease the implementation of such studies. For example, a number of resources exist for calculating appropriate sample sizes for GWA studies [43, 44]. In addition, software suites that implement many of the state-of-the-field data analysis tools for GWA studies have been devised, such as the PLINK genetic association analysis software [45].

arrayexpress) [47], and the Center for Information Biology Gene Expression Database (CIBEX) (http://cibex.nig.ac.jp/ index.jsp) [48]. Data adhere to the “minimum information about a microarray experiment” (MIAME) guidelines, which should guarantee access to adequate information to conduct additional analyses using third-party-generated data. The information generally presented is the experimental design, array design, samples, hybridization protocol, measurements, and normalization controls [49]. For the purposes of this chapter, we will focus on navigating through the GEO database. The GEO database allows you either to browse through the available information or to search it using keywords. Each entry within the database has a unique identifier, known as the GEO accession. This identifier is generally published within the original manuscript describing the experiments and analyses carried out to generate the expression data, and can be used within GEO to gain access to primary data. The data within GEO are organized, and can be investigated, in multiple ways. Upon submission, some datasets are assembled within GEO as a GEO DataSet, or GDS. A GDS is precompiled for easy access to simple statistics within a Web interface. Similar individual experiments, such as duplicates of the control experiment, are combined together and can be compared using simple statistics to identify gene expression changes between the different types of samples contained within the dataset. Not all datasets are compiled as a GDS. Upon initial submission, each dataset is entered as a GEO series, or GSE. The GSE contains all MIAME information, citation of the primary literature, and provides raw experimental data for download. Each sample within the series is assigned a GEO sample identifier, or GSM. In most cases, normalized data for each GSM are available for download from the GSE view. This information is generally easier to work with than the raw data and is recommended for exploratory work. In most cases the GSM data are presented as a table with the probe identifier, and signal intensity is presented at a minimum. To be informative, probe identifiers should be converted to gene names. This can be done by referring to the GEO platform. Each platform, i.e., each type of chip (Affymetrix U133A, Illumina Sentrix Human-6, etc.), is assigned a GEO platform identifier, or GPL. Within the platform view, a cross-reference table with probe identifiers and with gene identifiers of various types is presented to enable conversion between probe identifiers and many gene identifiers. Therefore, inhouse analyses can be carried out using this publicly available data, probes of interest can be mapped to their corresponding genes, and hypotheses regarding the function of genes of interest in contributing to a disease phenotype can be developed and tested further.

2.2 Expression Analysis Gene expression datasets are scattered more widely throughout a large number of gene expression data repositories. The major gene expression repositories are the Gene Expression Omnibus (GEO) (http://www.ncbi.nlm.nih.gov/geo/) [46] maintained by NCBI, ArrayExpress (http://www.ebi.ac.uk/

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2.3 Protein Structure and Proteomics Resources Unfortunately, proteomics has lagged behind the gene expression field in establishing standards and requiring data deposition into public repositories upon publication. Only in March 2008 did proteomics data deposition become “strongly recommended” for publication in leading scientific journals [50]. As a result, no clear gold standard database or data format has emerged as of yet. A standard output format for proteomics data, mzML, developed by the Human Proteome Organization Proteomics Standards Initiative and the Institute for Systems Biology, has been set, but its implementation is not yet complete. A number of public data repositories are available, including, PRIDE (http://www.ebi.ac.uk/pride), PeptideAtlas (http://www.peptideatlas.org), Global Proteome Machine Database (http://www.thegpm.org/GPMDB/index. html), and the file distribution system Tranche (http://www. proteomecommons.org/dev/dfs/users/index.html). Proteomics data are incredibly large and complex, and are only likely to be useful to nonproteomics investigators in ­preprocessed form. A few of the above-mentioned resources provide simple Web interfaces for performing queries. For example, the PRIDE database can either be searched by ­keywords or protein names, or browsed to identify datasets of interest. Once a dataset has been identified, proteins across samples can be easily compared using built-in comparison features in the PRIDE Web page. Comparisons allow the user to identify proteins unique to, or common across, multiple tissue samples, and a simple Venn diagram representation of the data structure is provided. Other databases allow for in-depth exploration of protein mass spectra, posttranslational modifications, and other exploration of other complex biochemical features well beyond the scope of the discussion in this chapter. Human Proteinpedia (http://www.humanproteinpedia. org) represents a more user-friendly interface for exploring proteomics data, and provides a highly integrated view of proteomics data, including posttranslational modifications, tissue expression, cell line expression, subcellular localization, enzyme substrates, and protein–protein interactions [51]. Interrogating “omics” data at the protein level is still in its infancy, but should provide an incredibly in-depth and complex view of protein localization and function for investigators interested in understanding the contribution of a small handful of proteins to disease pathogenesis. Protein structures themselves are organized by the WorldWide Protein Data Bank (http://www.wwpdb.org/), a federation of organizations which act as deposition, data processing, and distribution centers for protein structural information. The RCSB Protein Data Bank (http://www.rcsb.org/ pdb/home/home.do) contains a simple Web interface for performing searches for investigation of a protein of interest.

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More advanced searches by sequence, motifs, structural domains, and a whole host of other filtering parameters are also possible. PDB information can be accessed by numerous Web portals. Another example is the Macromolecular Structure Database (E-MSD) from EBI (http://www.ebi.ac. uk/msd/) [52]. A tutorial for E-MSD is available on the Web (http://www.ebi.ac.uk/msd-srv/docs/roadshow_tutorial/).

3 Example of an Analysis To demonstrate the utility of the aforementioned data resources to physicians and investigators, we will describe, step by step, analyses of data derived from hypertensive patients. First, we need to identify a dataset of interest. Bull et al. carried out microarray expression experiments, comparing expression levels in the peripheral blood cells of pulmonary arterial hypertensive patients and those in normal control subjects [53]. Within the methods section of their manuscript they provided the GSE identifier for public access to their data – GSE703. Armed with this information, we may navigate to the GEO Web site (http://www.ncbi.nlm.nih.gov/geo/) and search for GSE703 under “GEO accession.” Executing this search will take us directly to the GSE accession display, where we are provided with background information, contact information for the primary investigators, citations of the primary literature, the platform used, a list of samples, their GSM identifiers, links to the processed data, and a variety of other data formats. However, assuming we did not know about the existence of this dataset, we could easily find it through another search query. Starting again from the GEO homepage, we could enter the keyword “hypertension,” under the “DataSets” search box. This search, at the time of this writing, provides 26 datasets, performed on 23 different ­platforms, representing 56 different experiment series. If we select the “DataSets” tab, we are provided with a rundown of  datasets matching our search criteria. The 23rd entry, “GDS504,” corresponds to our dataset of interest. Clicking on the GDS504 record link, we are taken to the GDS view, where we can perform some simple analyses. We are first presented, again, with background information, including accession numbers, sample types, platform type, primary authors, etc. Under “Subset” and “Sample Info,” we can compare expression levels of hypertensive versus normal peripheral blood mononuclear cells (PBMCs). With the normal and the hypertensive samples presorted and selected for us, clicking “subset effects” takes us directly to genes with differential expression between the two sample types. Note that before performing the analysis, we could uncheck individual samples in the bottom panel if we had reason to believe some sample was invalid for our analyses. For our purposes,

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Fig. 4  A “heatmap” of the similarities in gene expression profiles of 20 individuals participating in a study of pulmonary arterial hypertension. The columns represent gene expression profiles for each individual across 7,129 gene probes and the rows represent gene expression levels for each of the 7,129 probes across the 20 individuals. Red indicates a high (normalized) gene expression and green indicates low gene expression

levels. Black indicates no information. The dendrograms on the upper x-axis and left of the figure indicate clustering of the individuals and genes, respectively, with shorter branch (i.e., connecting) lengths indicating greater similarity in profile. Thus, for example, the six leftmost columns (individuals) have more similar profiles (clustering of gene expression levels based on the color coding) among themselves than with others

we will keep all samples selected and proceed. Often an overall picture of the expression dataset is represented as a heatmap (Fig. 4). By visualizing expression data as a heatmap, we can explore gene sets underexpressed or overexpressed in individuals displaying the phenotype of interest. Individual genes can be explored by looking at subset effects. Subset effects provide us with a long list of genes differentially expressed between PBMCs of hypertensive versus normal individuals. For example, the interleukin 7 receptor (IL7R) is expressed at higher levels in normal PBMCs, whereas human hemopoietic cell protein-tyrosine kinase (HCK) is more highly expressed in PBMCs from hypertensive patients. Information regarding these two genes can be queried in OMIM, where we learn IL7R is an important receptor for regulating lymphopoiesis and HCK is likely to be an important factor in hemopoietic cells, especially those of the myeloid lineage. On the basis of simple observations such as these, we may hypothesize that proliferative

differences in PBMCs from hypertensive versus normal patients may by an indication of pulmonary arterial hypertension. This may be a response to known proliferative signals involved in the pathogenesis of pulmonary arterial hypertension [54]. Of course, such judgments should not be made on the basis of expression changes in a small handful of genes, and more sophisticated bioinformatics approaches may be used to tease out system-level effects. Therefore, we need to download the primary expression data to perform our own analyses. We navigate to the series view, either by clicking the series accession (GSE703) from the GDS504 record or by performing the initial GSE703 search described previously. The portions we are interested in here are the platform, GPL80 (Affymetrix GeneChip HuGeneFL human full-length array), and the sample data. Sample data can be downloaded as individual samples identified by the GSM prefix, or the whole dataset can be downloaded as a Series Matrix File at the bottom of the page.

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Determining which samples ­correspond to what phenotypes can be tricky and or difficult at times; however, in this case the sample names are clearly labeled “PAH” for pulmonary arterial hypertension, or “NOR” for normal. Clicking on the GPL80 link will take us to an informational page describing the platform used for our experiments. Scrolling down takes us to the piece of interest, a cross-reference table which links probe identifiers to genes. Clicking the “View full table…” button allows us to download the cross-reference table. It will require some simple scripting, or knowledge of Excel functions, to convert probe identifiers to gene names. Additionally, raw data from gene expression analyses must be normalized for comparisons. There are a large number of ways to do this; some methods are specific to the platform used. For example, a number of chips have internal standards for normalization; thus, the proper normalization technique will differ from chip to chip. Many of these proprietary normalization techniques are part of a software package provided by the platform manufacturer. A simple technique is to standardize the average, or median, signal across all experiments. Normalization is required to deal with various sources of noise, for example, signal intensities may be higher in one experiment than in the next simply because a larger amount of DNA was hybridized to the chip. In our example, we will simply normalize the average signal across all experiments. Once a normalization technique has been decided on and executed, again we are faced with the choice of how to analyze the given data. There are a large number of ­pathway, or gene ontology, based analyses packages available, and numerous methods for transforming the data for analysis. A few popular pathway analysis suites include Metacore (http://www.genego. com/metacore.php) [55]. Ingenuity (http://www.ingenuity. com/index.html) [56], and Gene Set Enrichment Analysis (http://www.broad.mit.edu/gsea/) [57]. Note that the Metacore and Ingenuity software packages are propriety and require a subscription (there is a free trial), whereas Gene Set Enrichment Analysis is publicly available. In our example, we will consider the pathways and molecular functions enriched in hypertensive PBMCs and in normal PBMCs. To keep our analyses simple, we average the signal across all pulmonary arterial hypertensive samples, average the signal across all normal samples, determine the fold change for pulmonary arterial hypertensive samples over normal samples, and filter for genes with at least a twofold increase in gene expression in the hypertensive samples. These genes will act as the input set for analyses in Metacore. The first view, after signing in at http://portal.genego. com, is the data manager view. Here we can upload our gene list by choosing “file > upload data.” We browse to the data file, and upload it. Upon completion, we need to refresh the data manager view. At this point we need to filter the data list for genes with a twofold or greater gene expression change. To do this, we choose, “Tools > Filter experiment.” We check the thresholds box, enter a threshold value greater than or

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equal to 2, and choose “calculate.” Back at the data manager view, refreshing the page again will show the filtered list as the active gene list. Now we are ready to investigate what pathways and functions are upregulated in hypertensive PBMCs versus normal PBMCs. There are a number of ways to get to this view, but if we select “View > Experiment Statistics,” we get an overview of the maps (pathways), disease pathways, and GeneGO processes. Following any one of these links will give details about the enriched processes. For example, if we are interested in maps (pathways), selecting this link gives a rundown of the significantly ­overexpressed pathways in hypertensive PBMCs. The major pathways upregulated in this gene list are immune response pathways, particularly inflammatory pathways. An example pathway is presented in Fig. 5. This observation is in good agreement with the view that persistent inflammation may be a cause of pulmonary arterial hypertension, at least in some cases, and that the systemic inflammation may be a marker of bad outcomes in pulmonary arterial hypertension cases [58, 59]. If, instead, we choose to look at disease pathways unregulated in hypertensive PBMCs, we observe many disease pathways we would expect. There are a number of heart disease conditions, including heart disease, cardiovascular disease, vascular disease, and myocardial disease. Inflammation is among the top disease pathways activated, as is ischemia, which is also associated with pulmonary arterial hypertension [60]. GeneGO processes are also overwhelmingly indicative of an inflammatory response. Additionally, cell adhesion, cell proliferation, and chemotaxis processes are upregulated. Again, upregulation of these processes in PBMCs is likely to be a biomarker of endothelial and smooth muscle cell dysfunction in pulmonary arterial hypertension, which includes proliferation, angiogenesis, and structural remodeling of vasculature [25]. There have been attempts to integrate data collected in various ways to make sense of human disease pathogenesis, but these have largely been confined to either assay results obtained on easily accessible tissues, such as blood, or very small sample sizes [61, 62]. In addition, some attempts have been made to comprehensively reconstruct important processes in humans, such as metabolic processes, using computational methods, so that in silico or bioinformatics predictions about perturbations in these system can be made [63, 64].

4 The Future We have surveyed the most widely used approaches to identifying genetic factors that contribute to disease ­susceptibility and the bioinformatics tools that facilitate these approaches. It is quite likely that very soon more sophisticated approaches will be necessary. On the basis of recent technological developments, many of these approaches can be anticipated. We describe some of the most salient next.

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Fig. 5  A “pathway” diagram depicting the relationships between genes involved in pulmonary arterial hypertension. This diagram was produced using the software package MetaCore (http://www.genego.com).

Red thermometer heights indicate the level of expression, arrows indicate different gene–gene interactions, the large oval represents the cell nucleus, and the uppermost area represents the extracellular space

4.1 Complete Genome Sequencing As noted previously, the entire genomes for a few individuals have been sequenced [17, 18]. Although the costs associated with these efforts would prohibit large-scale use of wholegenome sequencing in the identification of, e.g., rare sequence variations that mediate disease susceptibility, these studies

do indicate the near-term availability of whole-genome sequencing technologies. In fact, a recent NIH initiative termed the “1000 Genomes Project” will focus on the sequencing of 1,000 individual genomes in an effort to characterize the complete allelic spectrum of variations that populate human genomes and anticipate a time when whole-genome sequencing is no longer cost-prohibitive in

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gene mapping studies (http://www.1000genomes.org). The manipulation, analysis, integration, and ultimate interpretation of massive amounts of DNA sequence information collected on thousands of individuals in an effort to identify genetic variations associated with disease will require very sophisticated database, bioinformatics, and data analysis tools [65].

4.2 Epigenetics The discovery of non-DNA-sequence-mediated, yet heritable, phenotypes has led to an explosion of interest in “epigenetic” factors in health and disease [66–68]. Many of the technologies designed to query epigenetic factors (e.g., methylation and histone modification), such as chromatin immunoprecipitation sequencing [69] are very much in their infancy, making their immediate application to human disease studies impractical on a large scale. However, as these technologies are refined, the results produced from their application in relevant studies will require integration with the results produced by other, more traditional assays if a complete picture of the genomic landscape with respect to disease is to be found.

4.3 Integration of Genome Data via Systems Biology Approaches The use of any single modern high-throughput genomic technology alone to identify the genetic determinants of a particular disease is not likely to yield insights compelling enough explain the complexities of disease phenotype expression. Therefore, as made clear in this chapter, moreintegrated approaches to the identification and characterization of the genetic basis of complex diseases are in order. One set of combined experimental and computational approaches to understanding how gene networks, when perturbed, give rise to disease phenotypes are “systems biology” approaches [70, 71]. Systems biology approaches are rooted in a strategy whereby every element in a particular system (i.e., gene network, pathway, process, etc.) is systematically perturbed, the results of the perturbation recorded, and then sense is made of the various interactions and mechanisms within the system that lead to its function or dysfunction. Although a gross oversimplification, this strategy has been used with great success to understand biological processes within genetically simple lower species, but is motivating studies in humans in an effort to disentangle complex gene– phenotype relationships that work with natural, as opposed to artificially induced, perturbations [61, 72], as well as the

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need to further integrate biological data of all sorts that might avail themselves to understanding complex human systems of the type at play in mediating disease [73].

4.4 Translational Genomic Studies Obviously, the “end game” of all biomedical research is ­clinically useful insights. The “translation” of basic findings – such as the discovery of a gene associated with a disease – into something clinically useful is far from trivial [74], although a number of very thoughtful reviews of the types of study that need to be pursued, as well as impediments to implementing appropriate studies, have been published [75]. However, as more and more genes and genetic factors are identified that influence a particular disease, a greater need for bioinformatics tools that can facilitate the translation of these discoveries into, e.g., drug targets, biomarkers, and diagnostics, will arise.

5 Clinical Conclusions Like virtually all complex traits, PVD will ultimately be dissected into multiple gene pathways, representing different molecular subphenotypes that can lead to the clinical PVD henotype. For this dissection to be accomplished, bioinformatics applied to DNA sequencing, transcriptome, proteome, and epigenome information will be required. Via enhanced understanding, it is hopeful that new and effective approaches to prevent PVD will be possible in the future.

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33. Wellcome Trust Case Control Consortium (2007) Genome-wide association study of 14,000 cases of seven common diseases and 3,000 shared controls. Nature 447:661–678 34. Montaner J, Fernandez-Cadenas I, Molina CA et  al (2006) Poststroke C-reactive protein is a powerful prognostic tool among candidates for thrombolysis. Stroke 37:1205–1210 35. Topakian R, Strasak AM, Nussbaumer K et  al (2008) Prognostic value of admission C-reactive protein in stroke patients undergoing IV thrombolysis. J Neurol 255:1190–1196 36. McKusick VA (2007) Mendelian inheritance in man and its online version, OMIM. Am J Hum Genet 80:588–604 37. Stenson PD, Ball EV, Mort M et al (2003) Human gene mutation database (HGMD): 2003 update. Hum Mutat 21:577–581 38. Sherry ST, Ward MH, Kholodov M et al (2001) dbSNP: the NCBI database of genetic variation. Nucleic Acids Res 29:308–311 39. The International HapMap Consortium (2003) The International HapMap Project. Nature 426:789–796 40. Mailman MD, Feolo M, Jin Y et al (2007) The NCBI dbGaP database of genotypes and phenotypes. Nat Genet 39:1181–1186 41. Dawber TR, Meadors GF, Moore FEJ (1951) Epidemiological approaches to heart disease: the Framingham Study. Am J Public Health 41:279–286 42. Johnson AD, O’Donnell CJ (2009) An open access database of genome-wide association results. BMC Med Genet 10:6 43. Zondervan KT, Cardon LR (2007) Designing candidate gene and genome-wide case-control association studies. Nat Protoc 2: 2492–2501 44. Skol AD, Scott LJ, Abecasis GR, Boehnke M (2007) Optimal designs for two-stage genome-wide association studies. Genet Epidemiol 31:776–788 45. Purcell S, Neale B, Todd-Brown K et al (2007) PLINK: a tool set for whole-genome association and population-based linkage ­analyses. Am J Hum Genet 81:559–575 46. Edgar R, Domrachev M, Lash AE (2002) Gene expression omnibus: NCBI gene expression and hybridization array data repository. Nucleic Acids Res 30:207–210 47. Parkinson H, Kapushesky M, Shojatalab M et  al (2007) ArrayExpress – a public database of microarray experiments and gene expression profiles. Nucleic Acids Res 35:D747–D750 48. Ikeo K, Ishi-i J, Tamura T et al (2003) CIBEX: center for information biology gene expression database. C R Biol 326:1079–1082 49. Brazma A, Hingamp P, Quackenbush J et  al (2001) Minimum ­information about a microarray experiment (MIAME)-toward ­standards for microarray data. Nat Genet 29:365–371 50. Thou shalt share your data (2008) Nat Methods 5:209 51. Mathivanan S, Ahmed M, Ahn NG et al (2008) Human Proteinpedia enables sharing of human protein data. Nat Biotechnol 26: 164–167 52. Golovin A, Oldfield TJ, Tate JG et al (2004) E-MSD: an integrated data resource for bioinformatics. Nucleic Acid Res 32: D211–D216 53. Bull TM, Coldren CD, Moore M et  al (2004) Gene microarray ­analysis of peripheral blood cells in pulmonary arterial hypertension. Am J Respir Crit Care Med 170:911–919 54. Runo JR, Loyd JE (2003) Primary pulmonary hypertension. Lancet 361:1533–1544 55. Ekins S, Nikolsky Y, Bugrim A et  al (2007) Pathway mapping tools for analysis of high content data. Methods Mol Biol 356: 319–350 56. Ganter B, Zidek N, Hewitt PR et al (2008) Pathway analysis tools and toxicogenomics reference databases for risk assessment. Pharmacogenomics 9:35–54 57. Mootha VK, Lindgren CM, Eriksson KF et  al (2003) PGC-1aresponsive genes involved in oxidative phosphorylation are ­coordinately downregulated in human diabetes. Nat Genet 34: 267–273

580 58. Dorfmüller P, Perros F, Balabanian K et al (2003) Inflammation in pulmonary arterial hypertension. Eur Respir J 22:358–363 59. Humbert M, Monti G, Brenot F et al (1995) Increased interleukin-1 and interleukin-6 serum concentrations in severe primary pulmonary hypertension. Am J Respir Crit Care Med 151:1628–1631 60. Gómez A, Bialostozky D, Zajarias A et al (2001) Right ventricular ischemia in patients with primary pulmonary hypertension. J Am Coll Cardiol 38:1137–1142 61. Chen Y, Zhu J, Lum PY et al (2008) Variations in DNA elucidate molecular networks that cause disease. Nature 452:429–435 62. Emilsson V, Thorleifsson G, Zhang B et al (2008) Genetics of gene expression and its effect on disease. Nature 452:423–428 63. Shlomi T, Cabili MN, Herrgård MJ, Palsson BØ, Ruppin E (2008) Network-based prediction of human tissue-specific metabolism. Nat Biotechnol 26:1003–1010 64. Duarte NC, Becker SA, Jamshidi N et al (2007) Global reconstruction of the human metabolic network based on genomic and bibliomic data. Proc Natl Acad Sci U S A 104:1777–1782 65. Cochrane G, Akhtar R, Bonfield J et al (2009) Petabyte-scale innovations at the European Nucleotide Archive. Nucleic Acids Res 37:D19–D25 66. Feinberg AP (2007) Phenotypic plasticity and the epigenetics of human disease. Nature 447:433–440 67. Jirtle RL, Skinner MK (2007) Environmental epigenomics and disease susceptibility. Nat Rev Genet 8:253–262 68. Hatchwell E, Greally JM (2007) The potential role of epigenomic dysregulation in complex human disease. Trends Genet 23: 588–595 69. DeAngelis JT, Farrington WJ, Tollefsbol TO (2008) An overview of epigenetic assays. Mol Biotechnol 38:179–183 70. Ideker T, Galitski T, Hood L (2001) A new approach to decoding life: systems biology. Annu Rev Genom Hum Genet 2:343–372 71. Mo ML, Palsson BØ (2009) Understanding human metabolic physiology: a genome-to-systems approach. Trends Biotechnol 27:37–44

A. Torkamani et al. 72. Rockman MV (2008) Reverse engineering the genotype-phenotype map with natural genetic variation. Nature 456:738–744 73. Jenkinson AM, Albrecht M, Birney E et al (2008) Integrating biological data – the Distributed Annotation System. BMC Bioinformatics 9:S3 74. Butler D (2008) Translational research: crossing the valley of death. Nature 453:840–842 75. Ginsburg GS (2008) Genomic medicine: “grand challenges” in the translation of genomics to human health. Eur J Hum Genet 16:873–874 76. Barrett JC, Fry B, Maller J, Daly MJ (2005) Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 21:263–265 77. Weir BS (2008) Linkage disequilibrium and association mapping. Annu Rev Genom Hum Genet 9:129–142

Additional Reading 78. Bull TM, Coldren CD, Geraci MW, Voelkel NF (2007) Gene expression profiling in pulmonary hypertension. Proc Am Thorac Soc 4:117–120 79. Sampsonas F, Karkoulias K, Kaparianos A, Spiropoulos K (2006) Genetics of chronic obstructive pulmonary disease, beyond a1-antitrypsin deficiency. Curr Med Chem 13:2857–2873 80. Pettersson F, Morris AP, Barnes MR, Cardon LR (2008) Goldsurfer2 (Gs2): a comprehensive tool for the analysis and visualization of genome wide association studies. BMC Bioinform 4(9):138 81. Aulchenko YS, Ripke S, Isaacs A, van Duijn CM (2007) GenABEL: an R library for genome-wide association analysis. Bioinformatics 23:1294–1296 82. Melzer D, Perry JR, Hernandez D et al (2008) A genome-wide association study identifies protein quantitative trait loci (pQTLs). PLoS Genet 4:e1000072

Chapter 40

Genomic Applications to Study Pulmonary Hypertension Todd M. Bull and Mark W. Geraci

Abstract  As part of the World Health Organization (WHO) symposia on pulmonary hypertension, experts from both clinical and basic science arenas recommended revision of the classification scheme for pulmonary hypertension. This scheme was designed to “individualize different categories sharing similarities in pathological mechanisms, clinical presentation and therapeutic options.” The diseases that comprise pulmonary arterial hypertension (PAH) were stratified in a manner consistent with current understanding of the pathobiological processes, facilitating discussion and the enrollment in clinical trials. However, these changes simultaneously highlight our lack of understanding regarding the basic pathological mechanisms underlying the development of PAH. For example, why do radically diverse conditions such as systemic lupus erythematosus, anorexigen exposure, and HIV-1 infection result in similar pathologic changes in the lung vasculature? Why does one individual with a connective tissue disease such as scleroderma develop pulmonary vascular disease whereas another develops pulmonary fibrosis? Why do patients with PAH associated with congenital cardiac shunts have better survival than patients with idiopathic PAH (IPAH)? Clearly, the pathogenesis of PAH is complex, involving multiple modulating genes and environmental factors. Such complexity lends itself to the use of microarray technology, allowing the efficient and accurate simultaneous expression measurement of thousands of genes. Gene microarray technology has most successfully been employed in the investigation of cancer, including hematologic malignancies, and in the classification of histologically indistinct tumor types with divergent natural histories. The power of this technology has recently been directed toward the study of PAH.

T.M. Bull (*) Department of Medicine, Division of Pulmonary Sciences & Critical Care Medicine, University of Colorado, 12700 East 19th Ave, Research 2, 9th Flr., Aurora, CO 80045, USA e-mail: [email protected]

Keywords  Microarray • Gene expression profile • Method­ ology • Molecular biology • Phenotype

1 Introduction As part of the second (Evian), third (Venice), and fourth (Dana Point) World Health Organization (WHO) symposia on pulmonary arterial hypertension (PAH), experts from both clinical and basic science arenas recommended revision of the classification scheme for pulmonary hypertension [1–4]. This scheme was designed to “individualize different categories sharing similarities in pathological mechanisms, clinical presentation and therapeutic options.” The diseases that comprise PAH were stratified in a manner consistent with current understanding of the pathobiological processes, facilitating discussion and the enrollment in clinical trials. However, these changes simultaneously highlight our lack of understanding regarding the basic pathological mechanisms underlying the development of PAH. For example, why do radically diverse conditions such as systemic lupus erythematosus, anorexigen exposure, and HIV-1 infection result in similar pathologic changes in the lung vasculature? [5–9] Why does one individual with a connective tissue disease such as scleroderma develop pulmonary vascular disease whereas another develops pulmonary fibrosis? [10–13] Why do patients with PAH associated with congenital cardiac shunts have better survival than patients with idiopathic PAH (IPAH)? [14] Clearly, the pathogenesis of PAH is complex, involving multiple modulating genes and environmental factors. Such complexity lends itself to the use of microarray technology, allowing the efficient and accurate simultaneous expression measurement of thousands of genes. Gene microarray technology has most successfully been employed in the investigation of cancer, including hematologic malignancies, and in the classification of histologically indistinct tumor types with divergent natural histories [15–19]. The power of this technology has recently been directed toward the study of PAH.

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2 Gene Expression Profiling in Pulmonary Hypertension In the investigation of pulmonary hypertension, gene microarrays have been employed in a variety of study designs performed on a diverse array of cell types and animal species. Human studies have examined the gene expression profile of whole lung homogenates as well as individual cell types, such as smooth muscle cells isolated from the pulmonary arteries of patients and mononuclear cells isolated from peripheral blood [20, 21]. Animal microarray studies have been performed on both whole lung tissue and microdissected pulmonary vasculature of hypoxic and monocrotaline (MCT)-induced PAH [22, 23]. In aggregate, these studies have employed “hypothesis-building” strategies, helping to focus attention on potentially novel pathologic pathways. However, gene expression has also been utilized as a biomarker, potentially useful in classification or identification of an individual’s risk of disease [24]. In the following, we will examine traditional gene expression profiling (3¢-biased amplification strategies) that have been utilized as hypothesis-building efforts to understand pulmonary hypertension. The first set of examples involves exposure of distinct animal species to chronic hypoxia with the subsequent development of pulmonary hypertension. Gene expression patterns in the lungs of patients with pulmonary hypertension have only been obtained in one study and this study is examined. The analysis of peripheral blood mononuclear cells (PBMCs) as a surrogate to tissue will be detailed. Distinct cellular analysis, including pulmonary artery smooth muscle (PASMCs) and endothelial (PAECs) cells, will be ­considered. Analysis of the alterations in the right ventricle has been performed. We will conclude with two distinct genetically engineered murine models, each possessing distinct potential to define pathogenic mechanisms in pulmonary hypertension. Most recently, the analysis of progenitor cells in a rat model of pulmonary hypertension has been performed.

3 Chronic Hypoxic Pulmonary Hypertension Both species and strain differences exist in the response of the lung circulation to chronic hypoxia. Mice, for example, have significantly less pulmonary vascular remodeling in response to hypoxia than do rats. In support of this observation, Hoshikawa et al. demonstrated that whole lung gene expression patterns of these two species differ significantly when exposed to hypoxia [22]. In this series of experiments SpragueDawley rats and C57BL/6 mice were exposed to chronic hypobaric hypoxia for 1 and 3 weeks. Although both species developed pulmonary hypertension, microscopically the mice

T.M. Bull and M.W. Geraci

had significantly less pulmonary vascular remodeling than did the rats. Microarray analysis of the lung tissue demonstrated distinct differences in gene expression induced by hypoxia between the species. Hypoxic conditions in the rat increased expression of genes involved in endothelial cell proliferation and decreased expression of genes associated with apoptosis. The lung tissue from the rats showed a greater than sevenfold upregulated gene expression in both major histocompatibility antigen class 2 antigens and osteopontin, whereas tissue expression of these genes in mice did not change. The phosphoinositide 3-kinase gene was greatly upregulated (sixfold) in its expression in rat lungs but not in mouse lungs. This kinase regulates the expression of p21, enhancing G1/S transition in vascular smooth muscle cells (VSMCs) and insulinlike growth factor (IGF)-1-stimulated VSMC migration and proliferation. The gene expression patterns of the mouse lungs were notable for downregulation of genes responsible for VSMC proliferation such as IGF binding protein [25]. More recently, Kwapiszewska et al. performed microarray experiments using hypoxic mouse models, furthering our understanding of hypoxia-induced pulmonary hypertension. Several differences exist between the rodent models employed by Kwapiszewska et  al. and Hoshikawa et  al., including mouse strain differences, degree of hypoxia, and hypobaric versus normobaric hypoxia [22, 23]. Although pulmonary artery pressures were not presented in this study, this model should induce increases in pulmonary artery pressures with a modest extent of vascular remodeling. Gene expression changes within the pulmonary arteries were measured by first employing laser microdissection to isolate intrapulmonary arteries, thereby avoiding the dilution of the expected changes by the bulk lung [26]. By sharpening their focus in this manner, they were able to detect increases in 20 genes following 1 day of hypoxia, 75% of which were subsequently found to bear putative hypoxia-inducible-factor-responsive elements within upstream and downstream regulatory regions. These immediate changes in gene expression stand in contrast to those observed following 7 and 21 days of hypoxia, which include matrix-associated genes such as procollagens and matrix g-carboxyglutamate. This time-dependent change was further characterized through immunohistochemical staining of S100A4, and CD36, which were localized in VSMCs, and FKBP1a, which was localized in fibroblasts. In a recent study utilizing hypoxia exposure (10% O2) for differing periods of time (3 h to 2 weeks), adult male mice were examined using microarray analysis on whole lung samples. This work by Leonard et al. represents a unique bioinformatic analysis [27]. In effect, genes differentially expressed over the course of 3 h, 6 h, 1 day, 1 week, and 2 weeks, were evaluated by k-means clustering to define a group of hypoxiaregulated genes within the lung. By utilizing a bioinformatic approach for transcription factor binding to regions of DNA common among each of these clusters, the group was able to

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identify cyclic AMP responsive element binding (CREB) family members as candidates for lung selective hypoxiaresponsive transcription factors. Furthermore, the performance of in  vivo experiments demonstrated activation of CREB and activating transcription factor 1 (ATF1). The work demonstrates that upregulation of CREB family genes in the hypoxic lung was independent of other organs examined in this extensive organ-specific analysis. CREB and ATF1 represent transcription factors that are immediately affected by the exposure to hypoxia and are selective for lung-specific responses to hypoxia. Lung microvascular endothelial cells are important in this response, and the proximal effector cells are important in the specific responses of the pulmonary circulation to hypoxia. The findings of functional assays in vitro support these claims in this elegant work [27].

4 Gene Expression Patterns in the Lungs from Patients with Severe Pulmonary Hypertension Geraci et al. analyzed the gene expression profile of lung tissue obtained from six patients with IPAH (two with familial PAH; FPAH) versus six “normal” controls [28]. The normal lung tissue was obtained during surgery and was free from disease on histologic review. All the patients included in the IPAH cohort had severe PAH (mean pulmonary artery pressure greater than 50 mmHg). Two of the IPAH patients had been diagnosed with FPAH. Unsupervised cluster analysis demonstrated significant and reproducible differences in gene expression between patients with IPAH and normal controls. From a total of approximately 6,800 genes analyzed, 307 were differently expressed between the cohorts. There was significant downregulation of genes encoding for a variety of kinases and phosphatases, whereas several oncogenes and genes encoding ion channel proteins were upregulated. A highly intriguing finding was that the gene expression pattern of the lung tissue from patients with FPAH was distinct and distinguishable from that of the samples of nonfamilial IPAH patients. One of the IPAH samples clustered with the FPAH samples. The patient from whom this sample had been obtained had been adopted and her family history was unknown. It was hypothesized that she may have an unrecognized familial form of the disease. The bone morphogenic protein (BMP) receptor-2 (BMPR2) status of these lung samples was unknown. The ability of the gene expression pattern to discriminate between IPAH lung tissue and lung tissue from patients without PAH is remarkable. Furthermore, the ability to distinguish between FPAH and IPAH by microarray analysis demonstrates the potential power of this approach. One of the concordantly differentially expressed genes in the study by Geraci et al. was caveolin-1. The expression of

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gene was decreased in IPAH when compared with normal lung tissue. Calveolin-1 is downregulated in transformed cells and may be a tumor-suppressor protein [29, 30]. On the basis of the microarray experiment results, immunohistochemical analysis of lungs from patients with severe PAH was undertaken and a decrease in caveolin-1 expression in the plexiform lesions was noted [31]. There was no significant difference in calveolin-1 detected by western blot analysis of PAH lung tissue compared with normal lung tissue. The authors hypothesized that the loss of protein detected by immunohistochemical analysis was predominately in the plexiform lesions, with undiminished expression in the surrounding tissue; therefore, decreased total protein levels for calvolin-1 could not be detected by immunoblotting. The study of Geraci et al. provided a wealth of data that require further investigation. For example, this study indicated increased expression of the gene encoding the antiapoptotic protein BCL-2. Indeed, immunohistochemical analysis again confirmed overexpression of BCL-2 protein in the plexiform lesions from patients with IPAH (unpublished data, personal communication with Norbert Voelkel). Zhang et  al. have demonstrated that BMP-2 and BMP-7 induce apoptosis in cultured PASMCs associated with a marked downregulation of BCL-2 [32]. When exposed to BMP-2 and BMP-7, PASMCs isolated from patients with IPAH had decreased apoptosis as compared with PASMCs from patients with other secondary causes of PAH.

5 Microarray Gene Expression of Peripheral Blood Monocytes Inflammation and autoimmunity are possible contributing factors in the development of PAH [33–36]. Bull et  al. hypothesized that the gene expression of PBMCs would be altered in patients with PAH as compared with normal controls. Furthermore, we hypothesized that PBMCs could serve as a readily available surrogate tissue in patients with PAH, and that the gene expression profile of these cells could act as a biomarker of disease. The gene expression of PBMCs from 15 patients with PAH (including IPAH and PAH associated with a variety of other conditions such as CREST, portal hypertension, and thromboembolic disease) was compared with the PBMC gene expression of six normal controls. We identified a signature set of 106 genes which discriminated with high certainty (p £ 0.002) between patients with PAH and normal controls. A subset of these genes was then validated by quantitative PCR both retrospectively on the initial group and prospectively on a novel cohort of patients. The 106 gene signature identified genes previously recognized to be associated with PAH (e.g., adrenomedullin) as well as genes, such as endothelial cell growth factor-1, which may

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play a currently unrecognized role in the disease [37]. Notably, we were unable to identify a gene expression signature which discriminated between IPAH and PAH associated with other conditions such as scleroderma and thromboembolic disease by unsupervised analysis in this study. This inability may have been due to inadequate sample size the diverse nature of diseases included in the non-IPAH cohort [38]. A third possibility is that a unique expression profile for IPAH does not exist in peripheral blood cells; however, a supervised cluster analysis performed on these samples identified genes that may be differentially expressed between the groups. This strategy identified a list of 28 genes differentially expressed between IPAH and PAH associated with secondary conditions such as CREST syndrome, thromboembolic disease, and portal hypertension [21]. One of these genes, herpesvirus entry mediator, was retrospectively and prospectively confirmed by quantitative PCR. This study again represents two potential uses of microarray gene expression in PAH. These include (1) a hypothesis-generating tool to identify previously unsuspected genes or pathways that contribute to disease and (2) gene expression as a biomarker of disease. If a gene expression pattern predictive of PAH could be identified, then “at risk” populations could be screened. Patients who are identified as being at high risk for disease could initiate therapy early in the hope of impacting the natural history of PAH. The strategy of gene expression as a biomarker has been successfully demonstrated in acute myeloid leukemia [18, 19]. A study by Grigoryev et al. confirmed the previous findings of Bull et al. but also extended the study to unique facets of scleroderma-associated PAH [39]. In this study, PBMC samples from nine patients with IPAH, ten patients with scleroderma-associated PAH, and five healthy controls were analyzed using gene expression analysis on the Affymetrix U133A platform. In addition to differentially expressed genes between IPAH and scleroderma-associated PAH, the investigators were able to classify further the severity of PAH as either mild or severe. By analyzing different degrees (mild vs. severe) of scleroderma-associated PAH, the investigators were able to examine the potential for discovering novel candidate genes that are specifically associated with the severity of PAH. These genes could be further investigated as potential biomarkers or therapeutic targets. Differentially expressed candidate genes were analyzed by Gene Ontology terms using both GenMAPP and MAPPFinder tools. This type of analysis defines prominent pathways that are differentially regulated in patient versus control, contributing to angiogenesis, chemotaxis and inflammatory response. To define the severity of PAH genes, the scleroderma group was subcategorized into mild and severe. Genes that appeared to correlate with the severity of disease included ILA, vascular endothelial growth factor (VEGF), ILb, and matrix metalloproteinase (MMP)-9. Moreover, confirmation of previously differen-

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tially expressed genes by the study of Bull et  al. included ADM, IL7R, ZFP36, GLUL, JUND, and BCL-6. Importantly, this study took into account stratification by drug treatment and found, in a global sense, no overall effects in gene expression profiling from treatment with calcium channel blockers, and minimal effects from treatment with both intravenously administered epoprostenol and orally administed bosentan. The work by Grigoryev et  al. represents an important step forward in the development of biomarkers and indicators of disease severity from peripheral blood profiling [39].

6 Gene Expression of Pulmonary Artery Smooth Muscle Cells BMPs regulate cell proliferation and apoptosis. Mutations of BMPR2 have been associated with the development of severe PAH. In the work of Zhang et al. and Morrell et al., smooth muscle cells isolated from patients with BMPR2 mutations and cultured in vitro behaved very differently in response to exogenous BMP-2 than did PASMCs from normal individuals [32, 40]. BMP-2 inhibits apoptosis and reduces proliferation in normal PASMCs while inducing proliferation in smooth muscle cells from patients with IPAH. Fantozzi et al. examined differences in gene expression of cultured human PASMCs from normal individuals and from patients with IPAH [20]. In this interesting study, PASMCs were isolated from two normal lungs and from two patients diagnosed with severe IPAH (mean pulmonary artery pressure greater than 50 mmHg, BMPR2 status unknown). The cultured cells were incubated with 200 nM BMP-2 in vitro for 24 h and changes in gene expression were assessed by microarray analysis. BMP-2 treatment altered the expression of 6,206 genes. Of these, 1,063 genes were divergent in their expression in normal PASMCs compared with the PASMCs from patients with IPAH, (i.e., increased expression in cells from normal individuals and decreased expression in IPAH, or decreased expression in cells from normal pulmonary arteries and increased expression in IPAH). These divergent genes were hypothesized to be relevant to the previously identified divergent phenotypic response of the cultured smooth muscle cells to BMP-2. Fantozzi et al. also attempted to correlate gene expression of the genes altered by BMP-2 exposure either negatively or positively with the pulmonary artery pressure of the patients. Interestingly, several of these genes, such as caveolin-1 and voltage-gated Na+ and K+ channels, are consistent with those reported by Geraci et al. in the whole lung studies of patients with IPAH compared with normal controls [28]. Although the authors admitted there are significant potential problems of interpreting data with such a very small sample size (two patients in each group), this study again demonstrates the power of this approach.

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7 Gene Expression Analysis of the Right Ventricle Henk et al. utilized spotted oligonucleotide arrays to examine the difference in gene expression between right ventricular (RV) failure and RV compensation in response to pulmonary hypertension [24]. Compensated RV hypertrophy or uncompensated RV failure was induced in Wistar rats by varying the amount of MCT administered. MCT is an established means of inducing pulmonary vascular disease, PAH, and RV failure [41]. The gene expression of the hypertrophied right ventricle was compared between these two groups (compensated vs. uncompensated) and with the RV gene expression of control animals. The ventricles “destined” to progress to failure showed activation of proapoptotic pathways, whereas the rats which developed compensated RV hypertrophy had significant upregulation of mitogen-activated protein kinase (MAPK) phosphatase-1, presumably blocking the apoptotic pathway via p38 MAPK. Western blot analysis of phosphorylated p38 MAPK demonstrated marked activation in the compensated RV hypertrophied group only. The microarray expression profiles showed both quantitative and qualitative differences in gene expression, implying a fundamental shift in the balance of proapoptotic versus antiapoptotic signaling in the hypertrophic phenotypes studied. These observations serve to improve our understanding of the basic mechanisms underlying RV failure as well as potentially providing new therapeutic targets for this clinical problem.

8 Gene Expression Analysis of the Progenitor Cells Using a unique animal model examining bone marrow progenitor cells, Spees et al. were able to use sex-mismatched donors, as well as green fluorescent protein (GFP) transgenic animals in a model of progressive pulmonary hypertension utilizing a MCT model of pulmonary hypertension [42]. This model is significant in several respects. An extensive analysis of the cell fate of the bone-marrow-derived transplants was performed. In effect, bone-marrow-derived cells in the lung emerged as (1) hematopoietic cells, (2) VSMCs, (3) endothelial cells, or (4) bronchial epithelial cells, including type 1 pneumocytes. Microarray analysis was performed from flow-cytometry-sorted GFP-positive cells from the blood, bone marrow, or lung digest of the injured animal. Interestingly, the bone-marrow-derived and blood-derived GFP-positive cells were most alike, as determined by global gene expression profiling. GFP-positive cells from the lung were quite distinct and had differential expression of genes reflecting specific cell types. During the development of pul-

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monary hypertension from the MCT model, it is apparent that bone-marrow-derived cells are important in the reparative and disease progression paradigms. Prior to exposure to MCT, bone-marrow-transplanted animals demonstrated that approximately 15% of the cells in the lungs are GFP-positive, whereas the MCT-treated chimeric rats displayed, on average, between 35 and 45% of the cells resident in the lungs being GFP-positive. Extensive control analysis for cell fusion, nuclear fusion, and independent cell analysis is contained throughout this work. This work represents supporting evidence that bone-marrow-derived cells are significant in the pathogenesis and development of pulmonary hypertension [42].

9 Endothelial Cell Analysis and Infection Human herpesvirus 8 (HHV8) is a gammaherpesvirus, also known as Kaposi’s sarcoma (KS)-associated herpes virus [43, 44]. Evidence of HHV8 was found in a large percentage of plexiform lesions of pulmonary hypertension patients examined in Denver, suggesting for the first time that this virus was a contributing factor [45]. There are several proangiogenic or oncogenic genes in its genome, including a viral IL-8 and a viral IL-6 which may play a role in IPAH. In addition, the genome encodes a seven-transmembrane-spanning G-protein-coupled receptor (GPCR) with extensive sequence similarity to cellular chemokine receptors [46]. When expressed in NIH 3T3 fibroblasts, this gene increases their ability to grow in soft agar and to induce tumor formation in nude mice [47]. GPCR increases secretion of VEGF and activation of the ERK1/2 (p44/42) mitogen-activated protein kinase signaling pathway [48]. Endothelial cells that express this gene become immortalized with constitutive activation of VEGF receptor 2 (KDR) [49]. In addition, this gene can cause KS-like lesions in nude mice [47], and overexpression within hematopoietic cells results in angioproliferative lesions, resembling those found in KS [50]. These are yet more examples of viral factors with the potential of altering cellular phenotype in the absence of viral replication. Nevertheless, in spite of their recognized angioproliferative potential and the initial association of HHV8 with plexiform lesions, several groups did not reproduce these results with HHV8 [51]. Studies of patients from a San Francisco clinic along with Japanese and German cohorts found no evidence of latent virus in the lesions or serum antibodies against viral antigens [52–55]. The discrepancy between groups may be a reflection of the methods used to detect the virus or of regional (genetic/environmental) differences in the study population. Of note, latency-associated transcripts may be undetectable if the virus is going through a lytic replication cycle. In addition, serological tests for viral

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antibodies are notoriously difficult and, in many cases, hard to interpret. Thus, further studies are necessary to address these questions. Recent sentinel studies suggest that, indeed, HHV8 can infect pulmonary vascular endothelial cells in vitro and that cells infected with HHV8 downregulate the BMPR-2 pathway and acquire an apoptosis-resistant phenotype upon infection, lending credence to the possible causative role for this infection [56]. HHV8 infection of human pulmonary microvascular endothelial cells altered gene expression as measured by microarray analysis. Of the 54,675 genes represented on the microarray, 25,899 passed filtering criteria. A 1,128-gene expression signature distinguished HHV8infected cells from mock infected cells. A number of genes of interest in relation to the development of PAH were identified by this microarray analysis. These included a decrease in the expression of BMP-4 and BMP receptor-1A in infected cells, an increased expression of noggin (an endogenous inhibitor of BMPs), increased expression of IL-6, and increased expression of MMP-1, MMP-2, and MMP-10. A number of genes involved in apoptosis were also altered in their expression in HMVEC-L cells infected with HHV8 as compared with control cells.

10 Gene Expression Analysis Utilizing Genetically Engineered Animal Models of Pulmonary Hypertension The utilization of sophisticated, genetically engineered murine models of pulmonary hypertension may prove useful in examination of disease pathogenesis. Examination of gene expression profiling from the lungs of two genetically engineered mouse models provided important clues regarding the pathogenesis of transforming growth factor a (TGF-a)induced pulmonary fibrosis and disruption of the BMPR2 pathway. The work by Hardie et  al. utilized an advanced tetracycline-inducible transgenic induction of TGF-a with lung specificity [57]. The transgenic animal was constructed with CCSP-rtTA mice in which treatment with doxycycline induces transgene overexpression. Removal of doxycycline extinguishes transgene expression. The investigators were able to induce pulmonary fibrosis and pulmonary hypertension with activation of the TGF-a transgene. Fibrosis and vascular changes resolved upon cessation of doxycycline treatment. Importantly, gene expression analysis was performed in a time course of both induction and removal of the TGF-a transgene at days 1, 4, 21, and 42 of induction and days 1, 4, 12, 21, and 42 of removal of the transgene overexpression. Six major clusters of genes resulted, with differentially expressed genes numbering between 21 and 159 in each of the clusters. Cluster one, induction of TGF-a, included both TGF-a and extracellular matrix structural

T.M. Bull and M.W. Geraci

constituents as well as cell adhesion molecules. Through the early termination effects, genes upregulated included cell adhesion molecules in response to stress, as well as defense and immunity classifications. During the resolution phase, 50 genes were differentially regulated, including genes involved in development and cell motility. Finally, in the resolution phase after extinction of a transgene for 42 days, blood vessel development genes, signal transducer activities, IGF binding, and cell adhesion classes were the most overrepresented classifications of differentially expressed genes. Comparisons between the genetically altered mice and bleomycin-instillation models were performed, and demonstrated, in hierarchical clustering, that the TGF-a model more closely mimicked human idiopathic pulmonary fibrosis. This orthologous analysis lends credence to this timedependent transgene activation model as a more accurate model for pulmonary fibrosis than the commonly used bleomycin-instillation model [57]. Vascular changes were noted by measurement of RV systolic pressure analysis as well as RV over left ventriclar plus septum hypertrophy measurements, demonstrating that all animals eventually developed pulmonary hypertension. The group of West et al. generated an important transgenic animal in which disruption of the BMPR2 pathway can be induced by the dominant negative expression of mutant forms of BMPR2 in a transgenic animal with SM22rtTA-selective inducible transgenesis [58]. Linking the loss of BMPR2 to downstream molecular and hemodynamic effects can be performed in this manner. In work by West et  al., expression analysis was performed over a time course and distinctions between male and female mice (in terms of gene expression and severity of disease) were also established. To validate expression changes, BMPR2 was knocked-down with small interfering RNA in cell cultures of PASMCs, and the results established that changes in the whole-lung homogenous expression analysis experiments mirrored the changes in cell culture experiments in terms of pathways affected by BMPR2 loss. Differential effects between male and female mice were studied. The time points included an analysis at both 1 week and 8 weeks. Both time points had significant numbers of immune-regulated, developmental, matrix-related, cell cycle, and signal transduction as well as muscle-development-contraction-related gene changes. At the later time point of 8 weeks, cell motility, angiogenesis, and endocytosis-related groups predominated. Changes in the immune-related genes were more pronounced in female mice than in male mice. Importantly, the levels of smooth muscle markers decreased by the 8-week time point for all smooth muscle cell markers examined on the microarray [58]. This work is of great consequence in defining several important findings. These notable findings include the reduction in smooth-muscle-associated genes as the disease process progresses, and the finding that female mice, in particular, have higher levels of a variety of immune-regulated genes than male mice.

40  Genomic Applications to Study Pulmonary Hypertension Table 1  Major components of genomic analysis Gene expression Traditional(3¢ bias amplification) Exon-level(whole transcriptome amplification) Tiling arrays(the true transcriptome)

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Regulation of transcription

Comparing genomes (DNA variation)

Epigenomics and the “methylome” MicroRNA Nucleosome positioning (ChIP on chip, ChIP-seq)

Single-nucleotide polymorphisms Copy-number variation Nex Gen sequencing

11 The Future of Genomics Studies in Pulmonary Hypertension All of the work previously reported utilized traditional (3¢-biased amplification) microarray strategies for the analysis for gene expression. Over the past 5 years, there has been a veritable explosion of the technology for genomics. Three major areas have been accelerated: (1) gene expression analysis using microarray platforms; (2) regulation of gene transcription using arrays and sequencing; and (3) comparing genomes for DNA variation using arrays and sequencing. The description of the comprehensive analysis possible using currently available technologies is detailed in Table 1. Each of these technologies has yet to be applied aggressively and comprehensively to the disease pulmonary hypertension. However, the evolution of these technologies holds great promise for future insight into the pathogenesis of disease, including therapeutic target identification, biomarker development, and modifier gene identification. In terms of gene expression, new elements of the microarray permit the analysis of even greater detail than previously available. Exon-level analysis, utilizing whole-transcriptome amplification, is available through interrogation of selected Affymetrix arrays. Tiling arrays represent an unbiased analysis of the entire true transcriptome, but are mainly utilized in model systems such as yeast or cell lines, and have not yet been applied to disease-based interrogation. In terms of the regulation of transcription, significant strides have been made. The field of epigenomics focuses on modifications of the genome which either augment or silence adjacent genes. Several prominent mechanisms are involved in this intricate process, including methylation of CpG motifs and histone modifications, such as acetylation. Examining the methylation patterns within DNA yields significant insight into the transcriptional activity of adjacent genes. By utilizing high-throughput microarray technologies, Irizarry et al. discovered that the traditional concepts of CpG islands regulating nearby gene transcription may significantly underestimate the importance of methylation in the process of gene transcription [59]. Methylation of regions, much less GC-rich than CpG islands, can occur in regions up to 2,000 base pairs away from the transcriptional start site. These regions, termed “CpG shores,” may represent the most proximal and strongest modulators of gene transcriptions [60].

The global assessment of methylation changes throughout the genome is now possible by the specific interrogation of DNA arrays. Moreover, microRNA species, which regulate RNA stability, RNA transcription, and RNA degradation, can now be assessed by the utilization of microRNA microarray technologies, or multiplexed PCR. These technologies have yet to be applied to the field of pulmonary hypertension. For a review of microRNA analysis, refer to the work of Li and Ruan [61]. Nucleosome positioning can be assessed by chromatin immunoprecipitation (ChIP). By utilizing promoter tiling arrays, one can perform ChIP across the genome for selected transcription factors (so-called ChIP on chip). More recently, high-throughput sequencing applications have enabled the utilization of unbiased ChIP techniques to be utilized in a complex ChIP sequencing paradigm. The variation of several mammalian genomes, including the human genome, has led to the interrogation of the human genome for DNA variation. One of the most common variations is the single-nucleotide polymorphisms (SNPs). Several commercial entities now have available very high density oligonucleotide arrays of the genomes of many animals, including significantly dense human SNP arrays. By analysis of over one million markers per array, genomewide association studies have moved to the forefront of genomic analyses. In addition to the SNPs, copy-number variations of repeat elements within the genome have now been proven to be as significant genetic markers as SNPs. Neurologic diseases such as autism and schizophrenia have now been identified to be heavily associated with selected copy-number variations in certain regions of the genome [62]. Copy-number variations can also be assessed by utilizing this same SNP oligonucleotide arrays meant for SNP interrogation [63]. Three companies have moved to the forefront with highthroughput DNA sequencing modalities. These include the Roche 454, the ABI Solid, and the Illumina Genome Analyzer 2. The Illumina Genome Analyzer 2 and the ABI Solid instruments typically produce reads of between 20 and 40 base pairs in length and the Roche 454 instrument typically can give sequence reads of up to 300 base pairs [64]. These highthroughput sequencers now have the capacity to perform not only DNA sequence analysis but also quantitative RNA transcriptional analysis. The output of information which will come from these qualitative and quantitative sequencing

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technologies in the future looks almost overwhelming. By the utilization of quantitative RNA sequence analysis, quantitation is both accurate and highly reproducible. In addition, genetic variation is assessed directly from the information resulting from sequence reads. Moreover, altered alternative splice variants of transcripts, as well as noncoding and small RNA species, are captured within this technology.

12 The Pulmonary Hypertension Breakthrough Initiative An exciting new initiative is under way that should augment our ability to study tissues and cells from patients with PAH. The Pulmonary Hypertension Breakthrough Initiative results from funding obtained through the Cardiovascular Medical Research Education Fund. The consortium members are detailed in Table 2. The overall goal of the consortium is to collect explanted lungs at the time of transplant from patients with PAH, including control lungs with disease-specific controls and failed normal donor lungs, as well. Each of the cases will be extensively phenotyped in terms of disease severity, physiologic parameters, drug history, quantitative histology, and demographic information. Cells, including pulmonary artery endothelial cells, smooth muscle cells, adventitial fibroblasts, and epithelial cells, are isolated from each case. Extensive genomic, proteomic, and hypothesis-driven research is anticipated within the consortium and beyond. With the wealth of sophisticated technological advances, it is believed that highly phenotyped tissues, collected under standardized protocols, will yield a tremendous resource for the broader community of investigators interested in pulmonary hypertension.

13 Summary Microarray technology has successfully been employed in the investigation of a diverse array of human diseases. The potential of high-throughput expression analysis to improve our understanding of the pathogenesis of PAH is clear from the studies presented in this review. However, it is equally clear that we are at an early stage in the employment of microarray analysis for the study this disease. Although many studies to date have used microarray analysis as a “hypothesis-generating” tool, this technology also has vast potential as a means of identifying and quantifying biomarkers of disease. The oncology literature has demonstrated the utility of gene microarray analysis to predict important outcomes such as response to therapy and survival [65, 66].

T.M. Bull and M.W. Geraci Table 2  Pulmonary Hypertension Breakthrough Initiative research network Center/Institute

Location

Contact

Data Bank and Preparation Center Transplant and Preparation Center Transplant and Preparation Center Transplant and Preparation Center Transplant and Preparation Center Transplant and Preparation Center Transplant and Preparation Center Transplant and Preparation Center Transplant and Preparation Center Transplant and Preparation Center Transplant and Preparation Center Center for Cell Studies

University of Michigan, Ann Arbor

Vallerie V. McLaughlin

Duke University, Durham

Jerry Pang-Chieh Eu

University of Colorado, Denver

Martin R. Zamora

University of California, San Diego

Patricia A. Thistlthwaite

Center for Genomics Center for Proteomics Center for Tissue Processing Center for Tissue Processing ResearchGenomics ResearchProteomics Research-Cell Studies Research-Cell Studies

Cleveland Clinic Serpil C. Erzurum Foundation, Cleveland Stanford University School of Medicine, Stanford Baylor College of Medicine, Houston

Marlene Rabinovitch

University of Michigan, Ann Arbor

Douglas A. Arenberg

Vanderbilt University, Nashville

Barbara Meyrick

University of Alabama, Birmingham

Keith Wille

West Penn Alleghany Health System, Pittsburgh University of Pennsylvania, Philadelphia University of Colorado, Denver Johns Hopkins University, Baltimore University of Alabama, Birmingham University of Colorado, Denver University of Southern California, Los Angeles Vanderbilt University, Nashville University of South Alabama, Mobile University of Michigan, Ann Arbor

Raymond L. Benza

George P. Noon

Peter L. Jones

Mark W. Geraci Allen D. Everett William E Grizzle Rubin M. Tuder James A. Knowles

Barbara Meyrick Jack W. Olson David J Pinsky

Although many of these studies have examined the gene expression of tissue biopsies, others have examined less invasive options such as expression analysis of cells obtained from peripheral blood [67, 68]. It is likely that soon gene

40  Genomic Applications to Study Pulmonary Hypertension

microarrays will also be employed in a pharmacogenomics approach in PAH, helping to identify the most appropriate therapies for individual patients [69, 70]. These goals are ambitious, but certainly accomplishable. Such approaches will significantly increase our understanding of the pathobiological processes of PAH and aid in our struggle against this disabling and deadly disease.

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Chapter 41

Proteomics and Functional Proteomics Dayue Darrel Duan

Abstract  The study of the protein complement of the genome (or proteome), including the characterization of proteins derived from the genetic code to the comparison of variations in their expression patterns and levels under different conditions, study of their interactions, and identification of their functional role in the context of health and disease, is coined “proteomics”. Proteomics differs from genomics owing to the complexity and dynamic variability of the proteome. The human proteome may be formed by a much larger number of proteins (as many as one million proteins) than the human genome (approximately 30,000 genes). Owing to the complexity of gene expression in a living cell, a given gene can make many different proteins and proteins can also affect the relation between transcript and protein levels at the steady state. Therefore, a large-scale analysis of proteins is obviously necessary for our understanding of gene function. This chapter focuses on describing the application of proteomic methods in functional proteomics. Keywords  Proteome • Protein expression • Methodology • Posttranslational modification • Expression profile • Highresolution 2D electrophoresis

1  Introduction The development of high-resolution 2D electrophoresis (2DE) in 1975 by at least three independent laboratories [1–3] not only provided a powerful tool for the large-scale analysis and detection of proteins from complex biological sources but also stimulated the attempt to map the human complement of protein expression. Twenty years later, the “protein complement of the genome” was termed “proteome” [4].

D.D. Duan (*) Department of Pharmacology, University of Nevada School of Medicine, Manville Medical Building Room #9, 1664 N. Virginia Street, MS 318, Reno, NV 89557-0270, USA e-mail: [email protected]

Soon the study of the proteome, including the characterization of proteins derived from the genetic code to the comparison of variations in their expression patterns and levels under different conditions, study of their interactions, and identification of their functional role in the context of health and disease, was coined “proteomics” [5]. Proteomics differs from genomics owing to the complexity and dynamic variability of the proteome [6]. The human proteome may be formed by a much larger number of proteins (as many as one million proteins) than the human genome (approximately 30,000 genes) [6–8]. As a result of the complexity of gene expression in a living cell, a given gene can make many different proteins and proteins can also affect the relation between transcript and protein levels at the steady state. In addition, the abundance of alternative splicing variants, differential turnover of messenger RNA, and posttranslational modifications (PTMs), such as proteolysis, phosphorylation, and glycosylation, all contribute to the diversity of the proteome. Therefore, a large-scale analysis of proteins is obviously necessary for our understanding of gene function. Whereas the genome is relatively static, the proteome is not a stable mixture of proteins. The proteome may constantly change according to the moment-to-moment interactions between the genome and the environment. It is estimated that 1–2% of these polypeptides can be expressed at a given time in a cell, some of them at a very low copy number, whereas others occur at high abundance. In addition, proteomics also differs from traditional protein chemistry in that the latter studies mainly the chemical properties of specific families of proteins, whereas proteomics studies the largescale interaction of the protein repertoire within an organism. It is now known that proteins do not act as single players but act as part of functional complexes whose composition, subcellular localization, and interaction orchestrate their biological role under different conditions. Therefore, it is an enormous challenge to obtain comprehensive expression profile and interaction maps of proteins in various cellular systems. Proteomics can be divided into four main areas: (1) structural proteomics for mapping out the 3D structure of proteins and protein complexes, and large-scale identification of proteins and their PTMs; (2) expression proteomics for quantitative

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study and comparison of protein expression levels between samples that differ by some variable conditions with potential application in a wide range of diseases; (3) functional proteomics for profiling of protein functional state or enzymatic activity and studies of protein–protein interactions using techniques such as mass spectrometry (MS) or the yeast twohybrid (Y2H) system and manipulations of proteins to evaluate their function; and (4) protein informatics for the collection, organization, and analysis of biological data and molecular modeling of manipulation of the structure of proteins. Current proteomic studies are essentially focused on expression proteomics and functional proteomics. Expression proteomics investigates the protein expression patterns in cells under pathological conditions, in comparison with those in cells under physiological conditions. This comparative approach is usually employed to identify proteins that are upregulated or downregulated in a disease-specific manner for use as diagnostic markers or therapeutic targets. In these studies, a reliable analysis of quantitative changes in protein expression is crucial. Functional proteomics focuses on proteins involved in a particular biological function of interest. The functional proteomics approach is used to elucidate biological functions of unknown proteins, to define the cellular mechanisms underlying the specific biological function at the molecular level, and to monitor and analyze the spatial and temporal properties of the molecular networks and fluxes involved in living cells. This chapter will focus on the ­application of proteomic methods in functional proteomics.

2 Proteomic Methods Sophisticated protein isolation, cellular subfractionation, and multidimensional separation methods are required to carry out large-scale studies of gene expression at the protein level and characterize PTMs. There is, however, an inverse correlation between the complexity of the proteome and the maturity of the available technology. Currently, multidimensional gelbased electrophoretic and liquid-based chromatographic techniques are used for protein separation. These two analytical approaches are complementary owing to the unique capacities of each method. Quantitative protein expression analysis is one of the major challenges in proteomics. To quantify the abundance of a protein, separated proteins need to be visualized by means of ultraviolet (UV) detection, which are able to excite the backbone of proteins in chromatographic techniques or protein dyes in the electrophoretic technique. Finally, MS is used to identify the purified protein and the MS ion signal intensity can be used for either absolute or relative quantitation of the protein. Figure 1 presents an example of the possible strategies of a typical proteomics experiment.

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2.1 Multidimensional Separation of Protein Mixtures 2.1.1 Extraction and Solubilization of Proteins for Proteomic Studies Prior to analysis of proteins of interest by using either gelbased or gel-free chromatographic methods and by MS, all proteins must be extracted and completely soluble. In the case of 2DE, proteins must remain soluble as they approach their isoelectric points. The solubilization process should extract all proteins reproducibly. To accomplish this goal, various solubilization cocktails have been used for different purposes. The improved solubilization has increased the total number of proteins able to be visualized on 2DE gels and also allowed the separation of hydrophobic proteins, such as integral membrane proteins [9]. However, it should be noted that there is still no single solubilization cocktail that works perfectly for all conditions and samples owing to sample-source-related interfering compounds and a high degree of heterogeneity among samples. Also, the extraction, solubilization, and analysis of low-abundance proteins can often be complicated by the presence of highly abundant protein species. In addition, the solubilization agents used must be compatible with the subsequent fractionation and analytical methods. To date, the most efficient solubilization cocktails usually contain a mixture of detergents with 13~15-carbon-long hydrophobic chains, chaotropic agents, and a reductant [10, 11]. To extract and solubilize proteins, the following factors must be addressed: protein abundance and total protein concentrations, membrane proteins, detergents, temperature, pH, time, salts, metal ions, and cofactors. Each of these factors may specifically contribute to the outcomes of protein extraction and solubilization for a given application. Therefore, it is important to optimize the conditions with these factors as considerations. A major problem for whole proteome analysis is the largescale and dynamic range of proteins in the proteome, which may be formed by as many as one million proteins [6–8]. Because of total protein load limitations by proteomic methods, the most abundant proteins overwhelm the assay and limit or prevent the detection of low-abundance proteins. To address this issue, depletion strategies [12] and fractionation procedures [13–16] have been used. However, removing and discarding abundant proteins in proteomic studies may lead to the loss of important biomarkers in clinical proteomic studies [17, 18]. Compared with soluble proteins, hydrophobic proteins, in particular membrane proteins, are an underrepresented protein species on 2DE gels. One possibility is that many hydrophobic proteins are simply not extracted from the sample prior to 2DE separation. Therefore, it is very important to choose the right detergents and extraction

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Fig.  1  Proteomics strategy and workflow. Protein samples are prepared through isolation, subcellular fractionation, extraction, and enrichment if necessary. The protein extracts can be submitted to gelbased 1D electrophoresis (1DE) or multidimensional electrophoresis (such as 2D electrophoresis), or gel-free separation (liquid chromatography, liquid isoelectric focusing, capillary electrophoresis, free-flow electrophoresis) or a combination of both separation techniques. Protein spots on the gels can be detected by nonfluorescent (Coomassie, silver, etc.) or fluorescent (CyDye dyes) staining. Relative quantitation

(comparison) can be achieved by using fluorescent dyes (differential in-gel electrophoresis) in gel-based techniques. Peptides of enzymatic (trypsin, etc.) digestion of the purified protein are used for identification through mass spectrometry (MS) or tandem mass spectrometry (MS/MS). Stable isotopes are used for gel-free separation and protein identification and abundance comparison through MS. Higher confidence in protein identification as well as posttranslational modification location and characterization can be obtained by MS and database analysis

methods for membrane proteins. Although detergents such as sodium dodecyl sulfate (SDS) are extremely efficient at solubilizing hydrophobic proteins, their anionic nature greatly limits their effectiveness for conventional proteomic analysis. Therefore, zwitterionic and nonionic detergents have been synthesized and are widely used to solubilize membrane proteins for proteomic studies [19, 20]. Extraction with a mixture of chloroform and methanol, followed by solubilization using a combination of urea, thiourea, sulfobetaine detergents, and tributyl phosphine resulted in the identification of proteins that had not previously been

i­dentified on 2DE gels [14]. It has been recently reported that solubilization and tryptic digestion of membrane proteins in a buffer containing 60% (vol/vol) methanol followed by strong-cation-exchange chromatography and reversed-phase chromatography coupled online with MS/MS for protein identification is compatible with stable isotope labeling at the protein and peptide level and can be used to identify and quantitatively study integral membrane proteins [21]. However, no single perfect method or solution has been identified that solubilizes all proteins from different sample sources completely and reproducibly.

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2.1.2 Gel-Based Methods The classical method for the separation of a large amount of proteins is 2DE, which begins with 1D electrophoresis (1DE) but then separates the proteins by a second property in a direction 90° from the first field. In the first dimension, isoelectric focusing (IEF) is used to separate proteins in 1DE gel, so that all the proteins will lie along a lane but be separated from each other according to their isoelectric point (pI). Originally, carrier ampholytes were used to create the pH gradient within the gel. Recently, the reproducibility of this technique was greatly improved by the introduction of an immobilized pH gradient (IPG) which stabilizes the gradient, preventing cathodic drift during the separation [22]. In the second dimension, proteins are separated according to their molecular weight, or relative molecular mass (Mr). The proteins are treated with SDS along with other reagents. This denatures the proteins and unfolds them into long, straight molecules and binds a number of SDS molecules roughly proportional to the protein’s length. Because an unfolded protein’s length is roughly proportional to its mass, it attaches a number of SDS molecules roughly proportional to the protein’s mass. Since the SDS molecules are negatively charged, the result of this is that all of the proteins will have approximately the same mass-to-charge ratio. In addition, proteins will not migrate when they have no charge (a result of the IEF step); therefore, the coating of the protein in SDS (negatively charged) allows migration of the proteins in the second dimension. In the second dimension, an electric potential is again applied, but at a 90° angle from the first field. The proteins will be attracted to the more positive side of the gel proportionally to their mass-to-charge ratio. This ratio will be nearly the same for all proteins. The proteins’ progress will be slowed by frictional forces. The gel therefore acts like a molecular sieve when the current is applied, separating the proteins on the basis of their molecular weight, with larger proteins being retained higher in the gel and smaller proteins being able to pass through the sieve and reach lower regions of the gel. The proteins are separated and spread out across a 2DE gel according to their pI and their Mr (Fig. 2). Therefore, proteins are more effectively separated in 2DE than in 1DE. Depending on the sample complexity, a 2DE gel can resolve as many as tenfold more polypeptides than an equivalent SDS polyacrylamide gel electrophoresis (PAGE) gel. Thousands of protein spots can be routinely separated in a 20 × 20 cm2 area of highresolution 2DE gel [1–3]. Therefore, 2DE analysis provides a high-resolution map of protein complexity (e.g. isoforms, splice variants), PTMs and relative abundance. Although 2DE remains the highest-resolution technique for protein separation and is the method of choice when complex samples need to be arrayed for characterization in proteomics, the total number of proteins identified from 2DE gels is often only a small percentage of the predicted number in the proteome. Whereas mainly high-abundance

Fig. 2  Comparative 2D electrophoresis analysis of protein expression patterns in membranes of cardiac cells from Clcn3+/+ and Clcn3−/− mice. (a) Representative 2D gel depicting Coomassie-stained proteins from wild-type (Clcn3+/+) mouse heart. (b) Representative 2D gel depicting Coomassie-stained proteins from Clcn3−/− mouse heart. (c) Spot sets created from images of 2D gels of both wild-type and Clcn3−/− mouse heart run under the same conditions as the gels in (a) and (b) and compared using Bio-Rad PDQuest version 7.1.1. Three gels were run for each mouse heart type; two hearts were pooled to provide proteins for each gel. The filled symbols indicate changes in protein patterns in Clcn3−/− mouse heart compared with wild-type mouse heart. A total of 35 proteins consistently changed (minimum criterion, more then twofold change) in membranes from Clcn3−/− mouse heart in all three experiments (six missing proteins, two new proteins, nine upregulated proteins, 15 downregulated proteins, and two translocated proteins). The open squares indicate the location (molecular mass 85 kDa and pI 6.9) of the ClC-3 protein spot (no. 3812) in the 2D gels, which was independently confirmed by western blotting using a specific antiClC-3 C670−687 antibody. (Reproduced from [53] with permission)

proteins are detected on the gel, the more physiologically significant low-abundance proteins are usually excluded. In addition, there is an almost complete lack of hydrophobic proteins on 2DE gels, especially those using IPGs. Obviously, severe solubility problems hamper the application of 2DE

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analysis to many classes of proteins, especially membrane proteins [23]. A strong limitation of IEF, for example, is its incompatibility with charged detergents, which may add charges and thus modify the electrophoretic mobility of proteins. A nonionic or zwitterionic detergent needs to be used in the first dimension. The poor solubility of basic proteins causes instability of basic ampholytes and reductant migration and reduces the resolution of these proteins on the basic side of the proteome [23]. A common approach is to first use a wide pH gradient, typically 3–10, for an overview of the proteome of interest and then employ overlapping zoom pH gels (with narrower pH gradients, for example, the acidic pH 4–7 or the basic pH 6–11 ranges) to zoom at one protein level for better resolution and sensitivity of detection of proteins of interest [24]. Precast IPG gels, called strips, in a number of pH gradients with different lengths (7, 11, 13, 18, and 23  cm) are now commercially available. Recently, a paper-bridge loading method was applied on the basic (pH 6–11) range to optimize resolution of protein spots in the basic pH range up to 11 and consistently produced improved the resolution of both analytical and preparative protein loads [25]. This method may be particularly valuable for mitochondrial proteomics since more than 60% of known mitochondrial proteins have pI > 7.0 [26]. For the second dimension, SDS-PAGE is a more robust technique with fewer limitations in terms of protein solubility and resolution. To enhance the resolving power within particular Mr intervals in the second dimension, different acrylamide gradients and running buffers can be used. For the analysis of the functioning of proteins in a cell, knowledge of their cooperation is essential. Most often proteins act together in complexes to be fully functional. The analysis of this suborganelle organization of the cell requires techniques conserving the native state of the protein complexes. In native PAGE, proteins remain in their native state and are separated in the electric field following their mass and the mass of their complexes, respectively. The result of 2DE is a gel with multiple separated proteins spread out on its surface. These proteins can be detected by a variety of means, but the most commonly used stains are Coomassie and silver because of their low cost and ease of use. Figure  2 shows representative Coomassie-stained proteins on 2DE gels. The advantage of Coomassie staining versus silver staining is its compatibility with MS owing both to the higher amount of protein present within a Coomassiestained spot and to the modifications of proteins introduced during the silver staining procedure. Nonetheless, colloidal Coomassie staining suffers from poor sensitivity, with a detection limit around 8–10 ng of protein, which is a major issue in the application of the staining in proteomic studies. Recently Candiano et al. developed a new enhanced version of colloidal Coomassie staining (blue silver staining) with a detection limit of 5 ng [27].

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In silver staining, a silver colloid is applied to the gel. The silver binds to cysteine groups within the protein. The silver is darkened by exposure to UV light. The darkness of the silver can be related to the amount of silver and therefore the amount of protein at a given location on the gel. This measurement can only give approximate amounts, but is adequate for most purposes. Silver staining has a much higher sensitivity (0.1  ng) than Coomassie staining but it is not compatible with MS. Recently, an ammoniacal silver staining procedure was reported to be MS friendly [28]. Fluorescent stains with a much wider dynamic range and higher sensitivity are becoming more and more popular. However, their use is limited by the cost of the dye and of the laser scanner needed to acquire fluorescent gel images. Besides general dyes that stain all proteins, new dyes able to stain a class of protein such as a unique PTM (specifically glycosylation or phosphorylation) are available. These can be conveniently used in combination with a nonspecific dye enabling superimposition and recognition of PTM forms within the same gel. Differential in-gel electrophoresis (DIGE), another fluorescent approach, is a method that labels protein samples with fluorescent dyes before 2DE. Two or three separate protein samples are labeled with different fluorescent dyes prior to separation, enabling accurate analysis of differences in protein abundance between samples (Fig. 3). This technology allows for simultaneous separation and comparison of up to three samples within the same 2DE gel. This permits the inclusion of up to two samples and an internal standard in every gel. The 2D DIGE technology is based on the specific properties of spectrally resolvable dyes, the CyDye™ DIGE Fluor dyes. Two sets of dyes are available Cy™2, Cy3, and Cy5 minimal dyes (three dyes), and Cy3 and Cy5 saturation dyes (two dyes that have been designed to be both mass-matched and chargematched). Therefore, identical proteins labeled with each of the CyDye DIGE Fluor dyes will migrate to the same position on a 2DE gel. CyDye dyes offer great sensitivity, detecting as little as 125  pg of protein and giving a linear response to a protein concentration of up to 4 orders of magnitude. In comparison, silver staining detects 1–60  ng of protein with less than 100-fold dynamic range. Labeling does not interfere with subsequent identification by MS of proteins excised from 2D DIGE gels, because most peptides will not contain a label. However, both the mass and the hydrophobicity of the CyDye dyes influence protein migration during the second dimension of electrophoresis, SDS-PAGE. As a result, minimally labeled 2D DIGE gels are often poststained, for example, with Deep Purple™ total protein stain, to ensure that the maximum amount of protein is excised for subsequent in-gel digestion and MS. The most established 2D DIGE chemistry uses N-hydroxysuccinimide ester reagents for low-stoichiometry labeling of amino groups of lysine side chains. Labeling reactions are optimized so that only 2–5% of the lysine residues are labeled. This ensures that quantitation is performed using

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carried out for each new sample set to ensure complete reduction and stoichiometric labeling of cysteine residues. In addition, proteins that do not contain cysteine will not be labeled and will therefore not be imaged. Running repetitive gels that swap the dyes used to label samples will control for any dyespecific effects that might result from preferential labeling or different fluorescence characteristics of the gel or glass plates at the different excitation wavelengths of Cy2, Cy3, and Cy5. For example, one set of gels might use Cy3 to label controls and Cy5 to label drug-treated samples. A duplicate set of gels should be run that uses Cy5 to label controls and Cy3 to label drug-treated samples. Protein separation in 2D DIGE is performed similarly to that in conventional 2DE. Images are acquired on a multiwavelength scanner capable of resolving the three CyDye dyes. Besides the typical sensitivity and dynamic range of other fluorescent stains, DIGE introduced the unique possibility to treat different samples with dyes with different emission wavelengths and separate different-colored proteomes within the same gel [29]. Currently, 2D DIGE is rapidly increasing in popularity to monitor the differences in proteomic profile between cells in different functional states.

2.1.3 Gel-Free Methods

Fig. 3  Workflow of 2D differential in-gel electrophoresis (2D DIGE) with three dyes. The protein extracts are labeled with fluorescent dyes, e.g. Cy3 (green) and Cy5 (red), whereas the internal standard is typically labeled with Cy2 (yellow). The mixed samples are then resolved with the use of triphenyltetrazolium chloride (TTC) staining on a 2D electrophoresis gel. Protein spots of interest are excised from the gel, and the ions are analyzed by liquid chromatography–mass spectrometry (LC/MS). Mass spectrometers set for collision-induced dissociation are used for protein identification, whereas those set for electron-transfer dissociation (ETD) are used for the identification of phosphorylation sites. Target candidate proteins are further validated and screened through database analysis

protein molecules that have been labeled only once. In minimal labeling, the internal standard is typically labeled with Cy2, whereas samples are labeled with Cy3 and Cy5. Saturation labeling uses maleimide reagents for labeling all cysteine sulfhydryls in a sample. It is designed for use in situations where sample abundance is limited. Saturation labeling is much more sensitive than minimal labeling, as more fluorophore is incorporated into each protein species. In saturation labeling, where a Cy2 fluor is not available, the internal standard is labeled with one of the CyDye dyes and samples are labeled with the other CyDye dye. For similar experiments, saturation labeling will therefore require twice as many gels as minimal labeling. Careful optimization of saturation labeling conditions must be

Multidimensional separation of proteins or peptides obtained after a global protein digestion with proteases can be achieved in the liquid phase through chromatographic techniques [30] (Fig. 1). Proteomic approaches based on protein digestion prior to separation are also referred to as “shotgun proteomics.” As mentioned already, the traditional approach using gel-based methods followed by MS identification has a number of disadvantages, including its labor-intensive protocol, difficulty in automation, and the preference of the method for certain proteins over others on the basis of charge and size. To overcome these problems, numerous gel-free proteomics techniques have been developed and become more widely used for quantitative proteomics over the past few years. A number of liquid chromatography (LC) techniques allow multidimensional protein separation on the basis of pI (chromatofocusing), size (size-exclusion chromatography), or hydrophobicity (reversed-phase high-performance LC, RP-HPLC) [31]. The use of gel-free systems, which couple high-efficiency LC separation procedures with automated tandem MS (MS/MS or MSn) allows for large-scale sequencing of complex peptide mixtures. Usually an RP-HPLC system is used downstream (second dimension), interfaced with a tandem mass spectrometer. Among the major advantages over gel-based techniques, gel-free methods offer a higher degree of automation through direct online injection into a mass spectrometer without the attendant sample loss and contamination and the unique possibility to detect

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low-abundance proteins as well as very acidic or basic and hydrophobic membrane proteins. Online coupling of LC with MS [e.g. electrospray ionization (ESI)–time-of-flight (TOF) MS, matrix-assisted laser desorption/ionization (MALDI)–TOF MS] is able to separate and analyze minute samples (low femtomole range) without sample loss. The systems consist of a micropump for accurate and reproducible microflow and nanoflow delivery with an autosampler for automated injections and the short connecting tubing provides the smallest possible dead volume and high reproducibility with microliter and nanoliter gradients. High-precision reciprocating pumps with the microflow process and stream splitting generate highly reproducible flow rates ranging from 50 nL/min to 200 mL/min. Automated sample preparation and multidimensional (1D, 2D, 3D) LC can be carried out with column-switching modules. Both 1D LC and 2D LC have been used to separate large sets of protein or peptides. In combination with gel separation, 1D LC is commonly used as a convenient interface to inject liquid samples into a mass spectrometer equipped with an ESI source. As a separation technique complementary to 2DE, 2D LC has been developed to separate and identify membrane proteins and very acidic or basic proteins. The 2D LC/ MS method has a much greater sample dynamic range than 2DE. Multidimensional chromatographic approaches can be used for the separation of more complex peptide mixtures. The archetypal peptide-based approach, termed “multidimensional protein identification technology” (MudPIT) [32], combines a strong-cation-exchange separation in the first dimension and reversed-phase chromatography as the second dimension [21]. The total protein extract is initially digested and the peptides obtained can be separated and injected directly in a tandem mass spectrometer for peptide sequencing. The main advantage is that the initial digestion increases the overall solubility of the sample, thus limiting losses and poor recovery of sample. MudPIT has proven to be effective for investigating global changes in protein expression as a function of development and disease. An analogous approach for wide proteome detection on digested samples and named “combined fractional diagonal chromatography” (COFRADICTM) has recently been developed on the basis of the core technology of diagonal chromatography [33]. Diagonal electrophoresis or chromatography was described 40 years ago and was used to isolate specific sets of peptides from simple peptide mixtures such as protease digests of purified proteins. Gevaert et al. have adapted the core technology of diagonal chromatography so that the technique can be used in so-called gel-free, peptide-centric proteome studies. Subsets of highly representative peptides obtained from the whole protein extract can be selected and separated after digestion, giving a reliable representation of the parent proteins. Initially, methioninecontaining peptides or N-terminal ones were alternatively

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selected in COFRADIC [34, 35]. Theoretically, however, peptide-based proteomic approaches can be applied to profile any PTM that can be specifically and quantitatively modified, including, but not limited to, phosphorylation, glycosylation, and ubiquitination. Over the past few years different procedures have been developed: sorting of methionyl, cysteinyl, N-terminal, and phosphorylated peptides. COFRADIC was used in a peptide-centric analysis of a sputum sol-phase sample of a patient suffering from chronic obstructive pulmonary disease (COPD) and an unexpectedly high number of intracellular proteins next to known biomarkers were identified [33]. Diagonal reversed-phase chromatography was applied to study two different types of protein modification: protein processing and protein N-glycosylation [36]. Another convenient approach to the election of peptide subpopulations prior to RP-HPLC is affinity chromatography. For example, phosphopeptides can be selected by derivatizing these residues in the entire proteins with biotinylated tags for phosphothreonines or phosphoserines. After digestion, the biotinylated phosphopeptides can be purified on a streptavidin column and separated by RP-HPLC prior to MS/MS identification [37]. Protein/peptide visualization in gel-free techniques is usually achieved by UV detection at 214 nm. Since peptide bonds absorb light at this wavelength, the signal produced is dependent on the mass of the protein (the number of peptide bonds) and the protein concentration. Peak display may represent an advantage, but differential display analysis is not yet available for gel-free techniques owing to run shifts and the lack of appropriate software for chromatogram alignment. Many shotgun proteomics techniques randomly sample thousands of peptides in a qualitative and quantitative manner but overlook the vast majority of protein modifications that are often crucial for proper protein structure and function. Peptide-based proteomic approaches have been developed to profile a diverse set of modifications, including phosphorylation, glycosylation, and ubiquitination. Various liquid-phase approaches have been applied also to the separation of intact proteins.

2.1.4 Hybrid Approaches Both gel-based and gel-free approaches are complementary and both types of analysis are required for an extensive representation of the proteome. A number of hybrid approaches utilizing both gel-based and liquid-phase techniques are routinely used. One of the most common examples is 1D SDSPAGE separation followed by RP-HPLC prior to MS. Other liquid-phase nonchromatographic techniques such as liquidphase IEF, capillary electrophoresis, and free-flow electrophoresis have been used alone or in combination with

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chromatographic or gel-based separation methods. In contrast to 2DE techniques, in which high-abundance protein species predominate, the gel-free shotgun profiling procedures provide much more extensive coverage with orders of magnitude increase in resolution. The tandem approach permits peptide species identification and also semiquantitative estimation of the relative abundance. Current efforts are focused on adapting data mining algorithms, which can be trained to find specific features to distinguish between or classify new samples. Shotgun techniques require typically rigorous statistical approaches to evaluate the significance of any predicted patterns, owing to the volume of data analyzed and the hazards of multiple comparisons. The shotgun tandem MS technique currently provides the greatest depth of protein coverage and the widest dynamic range of size of proteins identified. Once the candidates have been identified, validation can be done with gel-based approaches or dedicated protein arrays.

2.2 Identification and Characterization of Proteins The next step following separation of proteins in the proteomic workflow is to identify the proteins and to characterize any modified amino acid residues (Fig.  1). The technologies described so far may provide information on protein properties such as Mr, pI, and hydrophobicity but unambiguous protein identification requires other analytical tools. MS is one of the oldest analytical techniques for the determination of the elemental composition of a molecule. It is also used for elucidating the chemical structures of molecules, such as peptides and other chemical compounds. The principle of MS is to ionize chemical compounds and generate charged molecules or molecule fragments and then measure their mass-to-charge ratios (m/z). MS devices consist of three main modules (Fig. 1): an ion source, which transforms the analytes into charged ions; a mass analyzer, which sorts the ions by their masses by applying electromagnetic fields and orders them according to their m/z; and a detector, which measures the value of an indicator quantity and thus provides data for calculating the abundances of each ion present with a specific m/z. In a typical MS procedure, a sample is loaded into the MS instrument, and its compounds are ionized by different types of sources (e.g. by impacting them with an electron beam), resulting in the formation of charged particles (ions). The m/z of the particles is then calculated from the motion of the ions as they transit through electromagnetic fields. Counts can be plotted as a function of the m/z value, thus generating peaks. Depending on the charge state of the generated ions, the mass of each analyte can be deducted. The technique has both qualitative and quantitative uses. These include identifying unknown compounds, ­determining

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the isotopic composition of elements in a molecule, and determining the structure of a compound by observing its fragmentation. Other uses include quantifying the amount of a compound in a sample or studying the fundamentals of gas-phase ion chemistry (the chemistry of ions and neutrals in a vacuum). MS is an important emerging method for the characterization of proteins. Techniques for ionization have been key to determining what types of samples can be analyzed by MS. The development of soft ionization methods (such as ESI and MALDI) to ionize intact, large, organic compounds and biomolecules greatly extended the biological application of MS. ESI (working in the liquid phase) and MALDI (starting from crystallized material) can be coupled with one or more analyzers and are the two primary methods for ionization of whole proteins. There are many types of mass analyzers, using either static or dynamic fields, and electric or magnetic fields, but all operate according to the following two laws that govern the dynamics of charged particles in electric and magnetic fields in a vacuum:

Lorentz force law : F = Q( E + v × B), Newton ′s second law of motion : F = ma,



where F is the force applied to the ion, Q is the ion charge, E is the electric field, v × B is the vector cross product of thevion velocity and the magnetic field, m is the mass of the ion, and a is the acceleration. Equating the above expressions for the force applied to the ion yields

(m / Q)a = E + v × B. This differential equation is the classic equation of motion for charged particles. Together with the particle’s initial conditions, it completely determines the particle’s motion in space and time in terms of m/Q. Thus, mass spectrometers can be thought of as “mass-to-charge spectrometers.” When data are presented, it is common to use the (officially) dimensionless m/z, where z is the number of elementary charges (e) on the ion (z = Q/e). This quantity, although it is informally called the mass-to-charge ratio, more accurately speaking represents the ratio of the mass number and the charge number, z. The mass analyzers can be generally classified as TOF, quadrupoles, ion traps, or Fourier transform ion cyclotron analyzers. Each analyzer type has its strengths and weaknesses. More than one analyzer can be combined downstream of a source in tandem MS (MS/MS or MSn). Different levels of information can be alternatively obtained by MS or MS/MS. In recent years, MS has undergone exponential advances with increased emphasis and application to proteomic studies. The TOF analyzer uses an electric field to accelerate the ions through the same potential, and then measures the time they

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take to reach the detector. If the particles all have the same charge, the kinetic energies will be identical, and their velocities will depend only on their masses. Lighter ions will reach the detector first. When paired with so-called hard ionization sources that break molecules into fragments during ionization, TOF analyzers have limited use in protein, peptide, and nucleic acid analysis. When coupled with two “soft” ionization methods that generate few fragment ions (MALDI and ESI), TOF systems can be used to analyze large biomolecules. The quadrupole mass analyzers use oscillating electric fields to selectively stabilize or destabilize ions passing through a radio-frequency (RF) quadrupole field. A quadrupole mass analyzer acts as a mass-selective filter and is closely related to the quadrupole ion trap, particularly the linear quadrupole ion trap, except that it operates without trapping the ions and is for that reason referred to as a transmission quadrupole. A common variation of the quadrupole is the triple quadrupole. The quadrupole ion trap [38] works on the same physical principles as the quadrupole mass analyzer, but the ions are trapped and sequentially ejected. Ions are created and trapped in a mainly quadrupole RF potential and separated by m/Q, nondestructively or destructively. There are many mass-tocharge separation and isolation methods but the most commonly used method is the mass instability mode in which the RF potential is ramped so that the orbit of ions with mass a > b are stable while ions with mass b become unstable and are ejected on the z-axis onto a detector. Ions may also be ejected by the resonance excitation method, whereby a supplemental oscillatory excitation voltage is applied to the end-cap electrodes, and the trapping voltage amplitude or excitation voltagevfrequency is varied to bring ions into a resonance condition in the order of their mass-to-charge ratio. A linear quadrupole ion trap [39] is similar to a quadrupole ion trap, but it traps ions in a 2D quadrupole field, instead of a 3D quadrupole field as in a quadrupole ion trap. Thermo Fisher’s LTQ (“linear trap quadrupole”) is an example of the linear ion trap. A Fourier transform ion cyclotron resonance (FT-ICR) [40] mass spectrometer measures mass by detecting the image current produced by ions cyclotroning in the presence of a magnetic field. Instead of measurement of the deflection of ions with a detector such as an electron multiplier, the ions are injected into a static electric/magnetic ion trap (Penning trap), where they effectively form part of a circuit. Detectors at fixed positions in space measure the electrical signal of ions which pass near them over time, producing a periodic signal. Since the frequency of an ion’s cycling is determined by its mass-to-charge ratio, this can be deconvoluted by performing a Fourier transform on the signal. Since each ion is “counted” more than once, FT-ICR MS has the advantage of high sensitivity and much higher resolution and thus precision than TOF or quadrupole ion trap mass analyzers.

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In keeping with the performance and mass range of available mass spectrometers, two approaches are used for characterizing proteins. In the first, intact proteins are ionized by the techniques described already, and are then introduced into a mass analyzer. This approach is referred to as the “topdown” strategy of protein analysis. In the second, proteins are enzymatically digested into smaller peptides using a protease such as trypsin. Subsequently these peptides are introduced into the mass spectrometer and are identified by peptide mass fingerprinting (PMF) or tandem MS. Hence, this latter approach (also called “bottom-up” proteomics) uses identification at the peptide level to infer the existence of proteins.

2.2.1  MALDI-TOF Mass Spectrometry MALDI is a soft ionization technique used in MS, allowing the analysis of intact and large biomolecules (biopolymers such as proteins and peptides), which tend to be fragile and fragment when ionized by more conventional ionization methods. The ionization is triggered by a laser beam (normally a nitrogen laser). A matrix consisting of crystallized molecules is used to protect the biomolecule from being destroyed by the direct laser beam and to facilitate vaporization and ionization. MALDI is most widely used with the TOF analyzer mainly owing to its large mass range. MALDI-TOF instruments are typically equipped with an “ion mirror,” deflecting ions with an electric field, thereby doubling the ion flight path and increasing the resolution. Today, commercial reflectron TOF instruments reach a resolving power m/Dm of well above 20,000 full width at half maximum (Dm defined as the peak width at 50% of the peak height). In proteomics, MALDI-TOF is used for the identification of proteins isolated through gel electrophoresis such as SDSPAGE, size-exclusion chromatography, and 2DE.

2.2.2  ESI-TOF Mass Spectrometry Although MALDI-TOF mass spectrometers are commonly used in most proteomics facilities, MALDI has its limitations. Most notably, MALDI is a solid-state, pulsed technique that cannot easily be coupled online with liquid-based, continuous purification methods such as HPLC. For HPLCbased applications, ESI mass spectrometers are generally the tools of choice. ESI was first described by Malcom Dole of Northwestern University in the 1960s and gained its prominence as a method for biomolecular analysis in the late 1980s through the efforts of John Fenn of Yale University, who was awarded a shared Nobel Prize in chemistry in 2002 for his work in this field. In ESI, a liquid sample is forced through a capillary tip

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in the presence of an electric field. As the liquid becomes charged, its molecules begin to repel each other, forming a fine mist of charged droplets. Solvent is evaporated using a neutral carrier gas, concentrating the charged analyte molecules into smaller droplets that then explode owing to repulsive forces between like charges. The process continues until analyte ions are completely stripped of solvent and only multiply charged ions remain. The resulting spectrum comprises peaks representing different charge states of the analyte. Scientists analyzing very large molecules, such as proteins and nucleic acids, cite ESI’s ability to generate these multiply charged ions as a major advantage, as it reduces the m/z ratio of large ions, effectively extending the mass range of the spectrometer. ESI mass spectrometers can be coupled to an HPLC system or interfaced with a nanoelectrospray apparatus. The latter alternative reduces the high flow and sample consumption rates typical of conventional HPLC but can be labor-intensive; nano-LC systems, although they employ lower flow rates, can be complicated to use even for the most experienced technician. To address these problems, several vendors have developed novel workarounds. Agilent Technologies, for example, developed a microfluidic chip that integrates sample preparation columns with a nanospray tip that can be readily interfaced with a mass spectrometer. Additionally, Advion BioSciences introduced an automated nanoelectrospray system comprising an array of nozzles that ionize individual samples from a 96-well plate and infuse them directly into an ESI mass spectrometer. Recently, Waters introduced a new nanoflow LC technology featuring higher speed, throughput, and sensitivity than conventional HPLC. A range of ESI-TOF mass spectrometers – from benchtop and LC-integrated models to complex MS “hybrids” – are available from most of the major mass spectrometer vendors. ESI is the standard ionization source for quadrupole TOF (Q-TOF) mass spectrometers; to increase resolution, many ESI-Q-TOF instruments feature orthogonal geometry, in which the source is perpendicular to the axis of the TOF analyzer. This configuration decouples the ionization source from the analyzer, making it possible to efficiently pair the continuous electrospray source to the TOF analyzer, which operates in a pulsed mode.

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mass spectrometer such as a MALDI-TOF or an ESI-TOF mass spectrometer. These masses are then in silico compared with either peptide masses in a database containing known protein sequences or peptide masses of each protein encoded the genome. This is achieved by using computer programs that translate the known genome of the organism into proteins, then theoretically cut the proteins into peptides, and calculate the absolute masses of the peptides from each protein. Then the masses of the peptides of the unknown protein are compared with the theoretical peptide masses of each protein encoded in the genome. The results are statistically analyzed to find the best match by matching their constituent fragment masses (peptide masses) to the theoretical peptide masses generated from a protein or DNA database. PMF identification relies on observing a large number of peptides (more than five) from the same protein at high mass accuracy. This technique does well with 2D gel spots where the protein purity is high. PMF protein identification can run into difficulties with complex mixtures of proteins. Low-level identification also becomes difficult owing to commonplace contamination by keratin. The premise of PMF is that every unique protein will have a unique set of peptides and hence unique peptide masses. If a protein sequence in the reference list gives rise to a significant number of predicted masses that match the experimental values, there is some evidence that this protein was present in the original sample. The advantage of PMF is that only the masses of the peptides have to be known, and therefore de  novo peptide sequencing (which can be time-consuming) is not necessary. A disadvantage is that the protein sequence has to be present in the database of interest. Additionally, most PMF algorithms assume that the peptides come from a single protein. The presence of a mixture can significantly complicate the analysis and potentially compromise the results. Typical for the PMF-based protein identification is the requirement for an isolated protein. Mixtures exceeding a number of two to three proteins typically require the additional use of MS/MS-based protein identification to achieve sufficient specificity of identification. Therefore, the typical PMF samples are isolated proteins from 2DE or isolated SDS-PAGE bands. Additional analyses by MS/MS can be either direct, e.g. MALDI-TOF/ TOF analysis, or downstream nano-LC-ESI-MS/MS analysis of peptides eluted from gel spots.

2.2.3  Peptide Mass Fingerprinting 2.2.4  Tandem MS Peptide mass fingerprinting (PMF) (also known as protein fingerprinting) is an analytical technique for protein identification that was developed in 1993 independently by several groups, including Henzel et al. [41–43]. The first step in this method is that an intact, unknown protein is cleaved with a proteolytic enzyme (such as trypsin) into smaller peptides, whose absolute masses can be accurately measured with a

Tandem MS involves multiple rounds of MS, usually separated by some form of molecule fragmentation. There are various methods for fragmenting molecules for tandem MS, including collision-induced dissociation, electron-capture dissociation, electron-transfer dissociation, infrared multiphoton dissociation, and blackbody infrared radiative

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d­ issociation. Tandem MS enables a variety of experimental sequences. Many commercial mass spectrometers are designed to expedite the execution of such routine sequences as single reaction monitoring (SRM), multiple reaction monitoring (MRM), and precursor ion scan. In SRM, the first analyzer allows only a single mass through and the second analyzer monitors the ions for a single user-defined fragment ion. MRM allows for multiple user-defined fragment ions. SRM and MRM are most often used with scanning instruments where the second mass analysis event is duty-cyclelimited. These experiments are used to increase the specificity of detection of known molecules, notably in pharmacokinetic studies. Precursor ion scan refers to monitoring for a specific loss from the precursor ion. The first and second mass analyzers scan across the spectrum as partitioned by a user-defined m/z value. This experiment is used to detect specific motifs within unknown molecules. As shown in Fig. 4, one mass analyzer can isolate one peptide from many peptides entering a mass spectrometer. A second mass analyzer then stabilizes the peptide ions while they collide with a gas, causing them to fragment by collision-induced dissociation. A third mass analyzer then sorts the fragments produced from the peptides. Tandem MS can also be done in a single mass analyzer over time, as in a quadrupole ion trap. Four main scan experiments are possible using tandem MS: (1) Precursor ion scan: The product ion is selected in the second mass analyzer, and the precursor masses are scanned in the first mass analyzer. A precursor ion scan cannot be done with time-based MS instruments. Note that “precursor ion” is synonymous with “parent ion” and “product ion” is synonymous with “daughter ion”; however, the use of these anthropomorphic terms is discouraged. (2) Product ion scan: A precursor ion is selected in the first stage, allowed to fragment, and then all resultant masses are scanned in the second mass analyzer and detected in the detector, which is positioned after the second mass analyzer. This experiment is commonly performed to identify transitions used for quantification by tandem MS. (3) Neutral loss scan: The first mass analyzer scans all the masses. The second mass analyzer also scans, but at a set offset from the first mass analyzer.

Fig. 4  Tandem mass spectrometry

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This offset corresponds to a neutral loss that is commonly observed for the class of compounds. Neutral loss scans cannot be done with time-based MS instruments either. (4) Selected reaction monitoring: Both mass analyzers are set to a selected mass. This mode is analogous to selected ion monitoring for MS experiments.

2.2.5  MS Data Analysis MS produces various types of data. The most common data representation is the mass spectrum. Certain types of MS data are best represented as a 3D contour map. In this form, m/z is on the x-axis, intensity is on the y-axis, and an additional experimental parameter, such as time, is recorded on the z-axis (Fig. 4). MS data analysis is a complicated subject that is very specific to the type of experiment producing the data. There are general subdivisions of data that are fundamental to understand any data. Many mass spectrometers work in either negative ion mode or positive ion mode. It is very important to know whether the observed ions are negatively or positively charged. This is often important in determining the neutral mass, but it also indicates something about the nature of the molecules. Different types of ion source result in different arrays of fragments produced from the original molecules. An electron ionization source produces many fragments and mostly odd-electron species with one charge, whereas an electrospray source usually produces quasimolecular even-electron species that may be multiply charged. Tandem MS purposely produces fragment ions postsource and can drastically change the sort of data obtained in an experiment. If the origin of a sample is known, certain expectations can be assumed as to the component molecules of the sample and their fragmentations. A sample from a synthesis/manufacturing process will likely contain impurities chemically related to the target component. A relatively crudely prepared biological sample will likely contain a certain amount of salt, which may form adducts with the analyte molecules in certain analyses.

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Results can also depend heavily on how the sample was prepared and how it was run/introduced. An important example is the issue of which matrix is used for MALDI spotting, since much of the energetics of the desorption/ ionization event is controlled by the matrix rather than the laser power. Sometimes samples are spiked with sodium or another ion-carrying species to produce adducts rather than a protonated species. The greatest source of trouble when non-mass spectrometrists try to conduct MS on their own or collaborate with a mass spectrometrist is inadequate definition of the research goal of the experiment. Adequate definition of the experimental goal is a prerequisite for collecting the proper data and successfully interpreting them. Among the determinations that can be achieved with MS are molecular mass, molecular structure, and sample purity. Each of these requires a different experimental procedure. Simply asking for a “mass spec” will most likely not answer the real question at hand. Since the precise structure or peptide sequence of a molecule is deciphered through the set of fragment masses, the interpretation of mass spectra requires combined use of various techniques. Usually the first strategy for identifying an unknown compound is to compare its experimental mass spectrum against a library of mass spectra. If the search comes up empty, then manual interpretation or software-assisted interpretation of mass spectra is performed. Computer simulation of ionization and fragmentation processes occurring in the mass spectrometer is the primary tool for assigning a structure or peptide sequence to a molecule. A priori structural information is fragmented in silico and the resulting pattern is compared with the observed spectrum. Such simulation is often supported by a fragmentation library that contains published patterns of known decomposition reactions. Software taking advantage of this idea has been developed for both small molecules and proteins. Another way of interpreting mass spectra involves spectra with accurate mass. A mass-to-charge ratio (m/z) with only integer precision can represent an immense number of theoretically possible ion structures. More precise mass figures significantly reduce the number of candidate molecular formulas, albeit each can still represent a large number of structurally diverse compounds. A computer algorithm called “formula generator” calculates all molecular formulas that theoretically fit a given mass with a specified tolerance.

2.2.6  P  rotein Identification by In-Gel Digestion and MS Analysis Gel plugs containing a specific protein spot are usually excised from a 2DE gel, digested with trypsin (or other proteases and chemical cleaving agent), and submitted to

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MALDI-TOF MS for PMF identification. Since trypsin cleaves at the C-terminal to basic amino acids (Arg and Lys), a number of small peptides amenable to ionization are obtained from the parental protein. The mixture of peptides depends on the primary sequence of the protein and thus is characteristic for every protein (fingerprint). The mixture of peptides is then co-crystallized with a matrix, a chemical compound able to transfer a proton to every peptide in the mixture after being bombarded by a laser beam in a MALDI source. Charged peptides are separated in the TOF analyzer, generating a list of molecular weights, also referred to as PMF. The experimental peptide mass fingerprint can be compared with theoretical ones through several search and retrieval systems freely available on the Web (see http:// www.expasy.ch). Different protein databases (TrEMBL, SwissProt, NCBI protein, etc.) were derived from genomic databases, representing a unique legacy for the proteomic era. PMF analysis can provide a fast identification of the protein in a relatively easy way. PTMs can also be indicated, but further levels of identification are needed to achieve higher confidence in both protein identification and PTM characterization. Amino acid sequence information can be retrieved by tandem MS (MS/MS) methods. Classically, ESI represents a more convenient source for tandem MS. In ESIQ-TOF devices, ionized peptides are selected by the first quadrupole analyzer and fragmented in a collision chamber downstream. In opportune kinetic energy conditions, fragmentation occurs at peptide bonds, generating a pattern of ions from which a sequence can be calculated; usually at least two or three peptides are sequenced for every protein to obtain a high level of confidence in the identification. Other MS analyzers, such as ion traps, can generate analogous information by iteratively isolating and further fragmenting ions. This feature has proven to be particularly useful for phosphorylation. A MS can also be used for protein-abundance comparison (quantitation) within different samples. At the beginning of the last century, MS was use to “weigh” atoms and thus calculate the isotopic abundance for the elements. This feature can now be applied to protein chemistry through the use of stable isotopes. Peptides from different samples can be labeled with different stable isotopes of the same element (1H/–2H, 12C–13C, 14N–15N, 16O–18O), producing a reproducible peak shift in the mass spectra. Comparison of peak height for peaks obtained by alternatively introducing a heavier isotope in the peptide chain can provide a reliable evaluation of the relative protein abundance. Labeling can be performed on intact cell cultures using different labeled media for different samples prior to protein extraction. Other labeling can be used after protein extraction or during protein digestion. Protein samples can be derived from SDSPAGE and are then subjected to some chemical modifications. Disulfide bridges in proteins are reduced and cysteine amino acids are carboxymethylated chemically or acrylamidated

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during the gel electrophoresis. Then the proteins are cut into several fragments using a proteolytic enzyme such as trypsin, chymotrypsin, or Glu-C. A typical sample-to-protease ratio is 50:1. The proteolysis is typically carried out overnight and the resulting peptides are extracted with acetonitrile and dried under a vacuum. The peptides are then dissolved in a small amount of distilled water and are ready for MS analysis.

3  Functional Proteomics Functional proteomics approaches are used to elucidate biological functions of unknown proteins and to define cellular and molecular mechanisms of biological functions at the protein level. In live cells, many proteins do not act as single players but display their biological functions through the rapid and transient association within large functional complexes whose composition, subcellular localization, and interaction determine their biological role under different conditions. The activities of protein complexes have to be regulated both in time and in space for them to be integrated within the overall cell programs. The cell orchestrates individual proteins to assemble into integrated networks fulfilling particular and superimposed functional roles. Functional proteomics studies provide insight into the properties of cellular protein complexes, their modular nature, their interaction with other complexes, and the resulting phenotypes of the proteome. In fact, the association of an unknown protein with partners belonging to a specific functional proteome or subproteome involved in a particular mechanism would be strongly suggestive of its biological function. Furthermore, the elucidation of protein–protein interactions in  vivo will also provide detailed insights into the cellular signaling pathways. The identification of the interacting protein partners, therefore, becomes very crucial for the understanding of protein functions as well as the underlying molecular mechanisms for the cellular functions [44, 45].

3.1  I dentification of Protein Complexes by Functional Proteomics Approaches The identification of interacting partners in protein complexes in vivo essentially relies on affinity-based procedures. Figure 5 illustrates an example of the functional proteomics strategies and technologies using animal models to identify specific protein complexes responsible for the genesis of a physiological or pathophysiological phenotype. To test a hypothesis for a functional protein complex, tissue samples from age-matched wild-type or genetically engineered,

Fig. 5  A hypothesis-driven functional proteomics strategy for study of cellular function in the context of health and disease. To test a hypothesis, tissue samples from age-matched wild-type (WT) or genetically engineered (GE) healthy (sham) or diseased animal models are processed. Proteins are isolated and fractionated (sarcolemmal membrane, cytosol, and mitochondria). Isolation, separation, and identification of interacting partners for protein complexes is done by recombinant protein-based affinity pull-down, immunoprecipitation, or yeast twohybrid. Further characterization of the functional subproteome can be done by western blotting, mass spectrometry, database search, etc. These putative candidate proteins are then validated by classic biochemical analysis (protein expression, kinases activity, molecular interaction), immunocytochemistry (subcellular localization), bioinformatic modeling, and physiological techniques at the cellular, tissue, organ, and whole-animal levels, which characterize the role of protein complexes responsible for the physiological or pathophysiological phenotypes

healthy (sham) or diseased animal models are processed. Proteins are isolated and fractionated (sarcolemmal membrane, cytosol, and mitochondria). Isolation, separation, and identification of candidate proteins for the functional protein complexes can be done by 1D/2D gel electrophoresis, in-gel digestion and MS, western blotting, etc. These putative candidate proteins are then validated by classic biochemical analysis (protein expression, activity of kinases, molecular interaction), subcellular localization, protein arrays, bioinformatic modeling, and physiological techniques, which characterize the role of the protein complexes responsible for the physiological or pathophysiological phenotypes. In addition to the proteomic techniques described above, many strategies such as recombinant protein-based affinity pulldown [44–46], antibody-based immunoprecipitation, and the

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Y2H system [47] have been commonly used to identify protein interacting partners in functional proteomics studies. These strategies are usually used in parallel to gain complementary information about protein complexes.

3.1.1  I dentification of Protein Interacting Partners by Recombinant Protein-Based Affinity Pull-Down The recombinant protein-based affinity pull-down assay is an in  vitro method used to determine physical interaction between two or more proteins. The strategy of this assay is to introduce a purified and tagged exogenous full-length protein, i.e. the “bait” protein, into the system, which competes with endogenous proteins to capture and “pull down” proteinbinding partners and form a new multiprotein complex (the prey). An outline of the affinity pull-down approach is shown in Fig. 6. The first step is to construct the “bait” protein. The gene encoding the protein of interest is cloned and subcloned into a vector containing a fusion protein such as glutathione S-transferase (GST), a poly-His tail, or a small peptide epitope tag (e.g. FLAG, HA, c-myc). The hybrid fused protein is expressed using a commercially available protein expression system and then isolated and purified using affinity chromatography, ion-exchange chromatography, size-exclusion chromatography, etc. The tagged bait can be immobilized onto agarose affinity beads derivatized with the appropriate antitag ligand (glutathione, antiepitope antibodies, nickel ions, streptavidin, etc.). The tagged bait can be immobilized on the Ciphergen surface-enhanced laser desorption/ionization chip and on-chip digests are possible and can be used for analysis of proteins using the LC/MS/MS system. All these affinity tag systems provide a general applicability with a large number of proteins and a minimal effect on the tertiary structure and biological activity of the bait, preventing instability of the complexes. The entire cellular extracts (lysates) and/or the extracts from specific organelles can then be incubated with the immobilized bait. The immobilized protein forms noncovalent interactions with specific partners in the cellular extract, whereas the unbound proteins will be eluted during the washing. The protein components specifically recognized by the bait and retained on the agarose beads can then be eluted and fractionated by utilizing SDS-PAGE loading buffer or a competitive analyte specific for the tag on the bait protein. SDS-PAGE loading buffer is a harsh treatment that will denature all protein in the sample, and it restricts analysis to SDSPAGE. This method may also strip excess protein off the affinity support that is nonspecifically bound to the matrix, and this material will interfere with analysis. Competitive analyte elution is much more specific for the bait–prey interaction because it does not strip proteins that are nonspecifically bound to the affinity support. This method is nondenaturing; thus, it can elute a biologically functional protein

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complex, which could be useful for subsequent studies. An alternative elution protocol allows selective elution of prey proteins while the bait remains immobilized. This is accomplished using a stepwise gradient of increasing salt concentration or a stepwise gradient of decreasing pH. A gradient elution is not necessary once the critical salt concentration or pH has been optimized for efficient elution. These elution methods are also nondenaturing and can be informative in determining the relative interaction strength. In addition, LC and other nondenaturing tools, e.g. clear native PAGE and blue native PAGE, can be used to enhance the sensitivity of the purification procedure. Protein complexes contained in eluted samples can be visualized by associated detection methods, including gel staining, western blotting detection, and 3535S radioisotopic detection. The protein bands detected on the gel are in situ enzymatically digested and the resulting peptide mixtures are analyzed by MALDI-TOF MS, leading to the identification of the proteins by a database search. Finally, the pinpointing of the subcellular locations of all proteins involved in the newly discovered “subproteome” can be done by immunofluorescence. The affinity pull-down assays can be used not only as a discovery tool for an initial screening (fishing) to identify new proteins in the endogenous environment that interact with a given bait protein, but also as a confirmatory tool for proteins that are suspected as being partners within a given interacting proteome predicted by other research techniques (e.g. co-immunoprecipitation and Y2H). The use of the pulldown technique to identify protein interacting partners depends heavily on the nature of the interaction under study. Interactions can be “stable” or “transient” and this characteristic determines the conditions for optimizing binding between bait and prey proteins. Stable interactions make up most cellular structural features but can also occur in enzymatic complexes that form identifiable structures. Since these interactions often contribute to cellular structure, the protein dissociation constant is usually low, correlating to a strong interaction. Stable protein–protein interactions are the easiest to isolate by physical methods such as pull-down assays because the protein complex does not disassemble over time. The protein complexes with strong interaction can be washed extensively with high ionic strength buffers to eliminate any falsepositive results due to nonspecific interactions. Transient interactions are defined by their temporal interaction with other proteins and are the most challenging protein–protein interactions to isolate. These interactions are more difficult to identify using physical methods because the complex may dissociate during the assay. Since transient interactions occur during transport or as part of enzymatic processes, they often require cofactors and energy via hydrolysis of nucleotide triphosphates. The transient interaction of the protein complex has a higher dissociation constant and is a weaker interaction. Optimization of the assay conditions

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Fig. 6  Outline of the strategy of fishing for interacting partners using recombinant protein-based affinity pull-down

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related to pH, salt species, and salt concentration can be used to improve the interaction strength and thus protein complex recovery. Incorporating cofactors and nonhydrolyzable nucleotide triphosphate analogues during assay optimization can serve to “trap” interacting proteins in different stages of a functional complex that is dependent on the cofactor or nucleotide triphosphate. In all pull-down assays, carefully designed control experiments are absolutely necessary for minimizing problems of nonspecific interaction and generating biologically significant results. A negative control consisting of a nontreated affinity support (minus bait protein sample, plus prey protein sample) helps identify and eliminate false positives caused by nonspecific binding of proteins to the affinity support. The immobilized bait control (plus bait protein sample, minus prey protein sample) helps identify and eliminate false positives caused by nonspecific binding of proteins to the tag of the bait protein. The immobilized bait control also serves as a positive control to verify that the affinity support is functional for capturing the tagged bait protein.

3.1.2  I dentification of Protein Interacting Partners by Antibody-Based Immunoprecipitation If a member of a larger complex of proteins is known, an antibody that specifically targets this protein can be used to precipitate a protein antigen and eventually pull the entire protein complex out of solution and thereby identify unknown members of the complex. The antibody-based “pull-down” process of capturing interacting partners of a protein complex from the solution is referred to as immunoprecipitation. The immunoprecipitation of intact protein complexes is known as co-immunoprecipitation [47]. Co-immunoprecipitation is conducted in essentially the same manner as immunoprecipitation. However, in co-immunoprecipitation the target antigen precipitated by the antibody “coprecipitates” a binding partner–protein complex from a lysate, i.e. the interacting protein is bound to the target antigen, which becomes bound by the antibody that is captured on the gel support. It is assumed that when proteins are coprecipitated these proteins are related to the function of the target antigen at the cellular level. This is only an assumption, however, and is subject to further verification. This works when the proteins involved in the complex bind to each other tightly, making it possible to pull multiple members of the complex out of solution by latching onto one member with an antibody. Traditional co-immunoprecipitation involves the following steps: (1) An antibody (monoclonal or polyclonal) against a specific target antigen is allowed to form an antigen–antibody protein complex in vivo within the cell and the cell extracts by incubation of a specific antibody with the antigen-containing sample (or cell lysate) for 1 h or more. (2) The antigen–antibody

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protein complex is captured on an immobilized agarose gel support by incubation for 0.5–2  h. (3) Any unbound protein (nonimmune complex sample components) is removed from the precipitated complex by washing the gel support with additional sample buffer. (4) The immunoprecipitated material containing the protein bait and its interacting partners can then be fractionated by SDS-PAGE. (5) Western blot analysis is performed, probing with antigen-specific antibody. (6) The individual protein components are identified by different MS methods. Identifying the members of protein complexes may require several rounds of precipitation with different antibodies for the following reasons: (1) A particular antibody often selects for a subpopulation of its target protein that has the epitope exposed; thus, it will not be able to identify any proteins in complexes that hide the epitope. This can be seen in that it is rarely possible to precipitate even half of a given protein from a sample with a single antibody, even when a large excess of antibody is used. (2) The first round of immunoprecipitation will often result in the identification of many new proteins that are putative members of the complex being studied. Antibodies that specifically target one of the newly identified proteins will then be obtained to repeat the entire immunoprecipitation experiment. This second round of immunoprecipitation may result in the recovery of additional new members of a complex that were not identified in the previous experiment. As successive rounds of targeting and immunoprecipitations have taken place, the number of identified proteins may continue to grow. The identified proteins may not ever exist in a single complex at a given time, but may instead represent a network of proteins interacting with one another at different times for different purposes. (3) Repeating the experiment by targeting different members of the protein complex allows a double-check of the results. Each round of pull-downs should result in the recovery of the original known protein and other previously identified members of the complex, as well as some new additional members. In this way, each identified member of the protein complex can be verified. If a particular protein can only be recovered by targeting one of the known members but not by targeting other known members, then that protein’s status as a member of the complex may be subject to question.

3.1.3  Protein Microarrays Protein microarrays can be used to screen binding of specific inhibitors or ligands to receptors, for evaluation of enzymatic activity, protein–protein, protein–lipid, and protein–DNA interaction profiling in answering a variety of biological questions. It is worthwhile mentioning that it is now possible to interrogate a sample with a panel of a specified set of protein targets using customized protein chips or arrays. Various

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platforms of protein microarrays are now readily accessible. A popular platform is the forward-phase protein microarrays or antibody arrays, which consist of a series of immobilized antibodies on a membrane or solid medium to provide relative protein expression levels for a specific sample. This method is particularly useful in evaluating a large number of similar samples in a high-throughput manner. However, different antibody affinities and microreaction kinetics might lead to false readings and complicate the interpretation of the results when the entire array is processed under identical conditions. The recent development of reversed-phase protein microarrays provides an alternative to classic antibody arrays. They are fabricated by depositing small volumes of a large number of protein samples or cell lysates onto a highprotein-binding substratum with a robotic precision spotter. Each protein microspot contains the full complement of the representative sample, including phosphorylated analytes, as appropriate. Because thousands of samples can be spotted in high density onto a single slide, a large number of samples can be monitored simultaneously. However, because the same antibody is being used, the comparisons between samples will be more valid. Interestingly, autoantibodies can also be detected with reverse arrays, which is particularly useful for the investigation of autoimmune-related disorders such as cardiomyopathies.

3.1.4  T  he Y2H System for Detecting Protein Interacting Partners Y2H can be used to identify novel protein–protein interactions, screen potential and confirm suspected interactions, and define interacting domains [47]. This system often utilizes a genetically engineered strain of yeast in which the biosynthesis of certain nutrients (usually amino acids or nucleic acids) is lacking. When grown on media that lack these nutrients, the yeast fails to survive. This mutant yeast strain can be made to incorporate foreign DNA in the form of plasmids. In Y2H screening, separate bait and prey plasmids are simultaneously introduced into the mutant yeast strain. Plasmids are engineered to produce a protein product in which the DNA binding domain (BD) fragment is fused onto a protein and another plasmid is engineered to produce a protein product in which the activation domain (AD) fragment is fused onto another protein. The protein fused to the BD is the bait protein and is typically a known protein that is used to identify new binding partners. The protein fused to the AD is the prey protein and can be either a single known protein or a library of protein-encoding sequences that represent all the proteins expressed in a particular organism or tissue or may be generated by synthesizing random DNA sequences. There are two broad categories of hybrid libraries: complementary DNA (cDNA)-based libraries and random libraries. The

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cDNA produced through reverse transcription of messenger RNA collected from specific cells constitutes a cDNA library. This library can be ligated into a construct so that it is attached to the BD or AD being used in the assay. A random library uses lengths of DNA of random sequence in place of these cDNA sections. A number of methods exist for the production of these random sequences, including cassette mutagenesis. Regardless of the source of the DNA library, it is ligated into the appropriate place in the relevant plasmid/ phagemid using appropriate restriction endonucleases. Regardless of the source, they are subsequently incorporated into the protein-encoding sequence of a plasmid which is then transfected into the cells chosen for the screening method. When a library is used, it is assumed that each cell is transfected with no more than a single plasmid and that, therefore, each cell ultimately expresses no more than a single member from the protein library. If the bait and prey proteins interact (i.e. bind to each other), then the AD and BD of the transcription factor are indirectly connected, bringing the AD in proximity to the transcription start site and transcription of the reporter gene or genes can occur. If the two proteins do not interact, there is no transcription of the reporter gene. In this way, a successful interaction between the fused proteins is linked to a change in the cell phenotype. A reporter gene, therefore, is provided with the upstream activation sequence (UAS) to which the BD binds, resulting in gene expression in successful cases of interaction, which can allow selection through a simple color change or through automatic death of cells in which the interaction does or does not take place. For example, the lacZ reporter gene will allow the highlighting of cells in which the UAS–BD–AD interaction is taking place. b-Galactosidase, the protein product of the lacZ gene, produces a blue coloration through the metabolism of 5-bromo-4-chloro-3-indolyl-b-dgalactoside (X-Gal), which allows the manual differentiation of cells according to color. There are a number of engineered genetic sequences which must be incorporated into the host cell in order to perform a two-hybrid analysis or one of its derivative techniques. The considerations and methods used in the construction and delivery of these sequences differ according to the needs of the assay and the organism chosen as the experimental background. One advantage of the Y2H system is the ease of acquiring the whole sequence information of the protein of interest. This system has some disadvantages: (1) The protein synthesized in yeast may lack some important posttranscription modifications and hence the circumstances in mammalian cells may not be mimicked. (2) Y2H requires the candidates to stay in the nucleus to initiate transcription. One drawback of this is that it may not reflect the real condition, since the conformation of the intracellular segment constructed for the bait may not be exposed in the native protein and thus may not be able to interact with other proteins (false

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negative). In addition, many other potential candidates binding to the bait protein may not directly bind to the protein; instead they may bind indirectly through other proteins. (3) Sticky proteins may give false-positive results. Therefore, further complementary experiments, such as affinity pull-down and co-immunoprecipitation, are required to confirm the true interactions and to discover in which manner the proteins form the complexes in vivo. (4) The Y2H experiments are time-consuming and labor-expensive owing to the manual selection of positive cells.

3.2  I dentification of Protein Phosphorylation Sites and Phosphoproteomics Reversible protein phosphorylation on serine, threonine, and tyrosine residues is well established as a key PTM that regulates protein function, subcellular localization, complex formation, degradation of proteins, and therefore, cell signaling networks. Protein kinases are one of the largest gene families in humans and mice, accounting for 1.7% of the human genome, and it is assumed that up to 30% of all proteins may be phosphorylated. Traditional biochemical and genetic analyses of phosphoproteins, and of the kinases and phosphatases that modify them, typically focus on one protein at a time and are not readily amenable to understanding the complexity of protein phosphorylation or how individual phosphoproteins function in the context of signaling networks. Phospho proteomics is a branch of functional proteomics that identifies and characterizes proteins containing a phosphate group as a PTM. Compared with the analysis of expression profiles and interactions of protein complexes, phoshoproteomics provides two additional layers of information. First, it provides clues on what protein or pathway might be activated because a change in phosphorylation status almost always reflects a change in protein activity. Second, it indicates what proteins might be involved in signaling transduction pathways responsible for a phenotype and might be potential drug targets. Although phosphoproteomics will greatly expand our knowledge of the numbers and types of phosphoproteins, its greatest promise is the rapid analysis of entire phosphorylation-based signaling networks [48]. Current methods used in phosphoproteomics rely heavily on MS and phosphospecific enrichment technologies. There are several different MS instrumentations and scanning modes that are of particular use in monitoring for phosphorylated peptides in MS/MS techniques. The most popular methods are parent ion and neutral loss scanning, both of which rely on the characteristic loss of H3PO4, HPO3, PO3−, or PO2− by phosphorylated peptides upon ionization dependent on the residue phosphorylated and the MS technology. Although MS-based approaches both allow large-scale analysis and provide the ability to discover new phosphoproteins with

D.D. Duan

important advantages of high speed, selectivity, and sensitivity over biochemical methods for the analysis of protein phosphorylation, there are several limitations that need to be taken into account when analyzing phosphorylation modifications with MS. The first problem stems from the low in vivo stoichiometry of phosphoproteins with respect to their nonphosphorylated analogue. The low abundance of phosphopeptides greatly complicates analysis. The other confounding problem in MS analysis is that the nonphosphorylated forms of the peptides tend to suppress the ionization and flight of the phosphorylated counterpart in the mass spectrometer. In addition, the energy used to ionize the peptides from the sample source can often cause the liberation and loss of the phosphate from the peptide. Therefore, a considerable amount of effort has been devoted to the development of phosphospecific enrichment methods that are compatible with, or directly coupled to, MS. Emerging technologies that are likely to have important impacts on phosphoproteomics include protein and antibody microarrays, and fluorescence-based single-cell analysis. Although these methods have the potential for high sensitivity and high throughput, they require prior knowledge of particular phosphoprotein targets. The application of gel-based and gel-free techniques in identification of phosphorylation sites will be briefly discussed.

3.2.1  Gel-Based Techniques  P Labeling

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One of the oldest biochemical methods for analysis of phosphorylation is to monitor the incorporation of radiolabeled phosphate into protein species. This technique has been most often applied using in vitro systems by incubating proteins or organelles with [32P]ATP and monitored using gel techniques combined with audioradiography.

Fluorescent Phosphoprotein Stains Fluorescent dyes that bind phosphate-containing proteins have been developed to stain gels for the identification of the presence and location of phosphoproteins. For example, ProDiamond Q (Invitrogen) has proved to be very useful in global screening of proteomes for potential phosphorylated proteins. The dye can be applied for use on 1D gels of small subproteomes or on 2DE gels of larger proteomes. ProDiamond Q can also be used along with DIGE methods to correlate protein spot quantitation on 2DE gels with the presence of phosphoprotein species as a unique method to connect PTM detection with protein quantitation. This dye is very easy to use and is convenient for laboratories that are already proficient in 2DE gel technology. It also provides a visual and easy to understand readout of the overall

41  Proteomics and Functional Proteomics

phosphorylation status of a given proteome. The main drawback of this method is that the dye will bind to proteins that contain any areas of large negative charge and can therefore produce false positives in proteins with long stretches of negatively charged amino acids. As a result, the dye can only provide a list of potentially phosphorylated proteins and its use needs to be followed up with downstream validation.

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lated at substoichiometric levels, it is very popular now to use phosphopeptide enrichment strategies prior to MS/MS analysis of phosphorylation sites. Enrichment of phosphorylated peptides away from the nonphosphorylated forms can help to overcome some of the limitations MS technology has in analyzing phosphorylation modifications by increasing the probability of seeing the phosphopeptides and by decreasing the suppression of the phosphopeptides.

Antibody-Based Recognition of Phosphoproteins Phosphospecific antibodies for phosphorylated serine, threonine, and tyrosine are commercially available and can be used for recognition on either 1D or 2D western blots. Such blots can be used along with other protein-specific antibo­ dies for correlation of protein identification. These antibodies are also useful for immunoprecipitation of phosphorylated proteins followed by downstream gel or MS analyses. Phosphorylated tyrosine antibodies are highly specific and very useful for proteomics projects such as 2D western blotting and immunoaffinity pull-downs for downstream MS/ MS analyses. Unfortunately, phosphorylated serine and phosphorylated threonine antibodies tend to be highly crossreactive to other negative charges and can produce high false-positive rates, making them much less useful in proteomic applications.

Phosphatase Dephosphorylation Assays Protein phosphatases, such as alkaline phosphatase, remove phosphate groups from modified protein species and this enzymatic dephosphorylation can be used in identifying phosphoproteins on the basis of changes in the charge of the proteins. Phosphatase treatment of samples can be coupled with 2DE to cause pI shifts and thereby identify the phosphoprotein component of a proteome or demonstrate the phosphorylation of individual proteins. For example, the phosphatase shifts can be used to label control and treated samples with different CyDye dyes in a DIGE style experiment. This technique allows for the easy visualization and confident assessment of pI shifts associated with phosphatase shifts. It also has the advantage of being highly specific because only those protein species that are modified from phosphorylated to dephosphorylated will be shifted. Mapping of the shifting proteins and thus protein identification using MS methods, however, can be challenging, especially if the proteome is complex.

3.2.2  Gel-Free Techniques Because many phosphoproteins, especially signaling intermediates, are low-abundance proteins phosphory-

Phosphopeptide Enrichment Strategies Immobilized Metal Affinity Chromatography Immobilized metal affinity chromatography (IMAC) uses a nanocolumn packed with immobilized and positively charged metal ions (usually iron) to bind tightly to negative regions of proteins or peptides (such as phosphate groups). Peptides or proteins flowed over an IMAC column will bind if they contain areas of large negative charge, whereas neutral or positively charged peptides will flow through. This allows for the enrichment and concentration of phosphorylated protein species on the IMAC column and then these species can be eluted (online with MS or offline) and run in a concentrated form in an MS/MS spectrometer for identification of the protein and site of phosphorylation. However, on the one hand, IMAC is not specific to phosphorylation and thus also pulls down peptides that have large negative charges, which can confound analysis. This nonspecific binding can be limited by conversion of peptides using O-methylesterification, which blocks the acidic residues. Esterification reduces sample complexity from nonspecific binding, but may also decrease the sensitivity of the technique. On the other hand, IMAC will not necessarily bind all the phosphopeptides, especially those peptides which also contain stretches of positively charged amino acids, and thus valuable information can be lost. Finally, IMAC preferentially binds peptides containing more than one phosphate group, which biases the analysis away from the single phosphorylated proteins.

Titanium Dioxide Affinity Titanium dioxide (TiO2) is similar to IMAC in that it is typically used as an immobilized column that is capable of binding negatively charged regions of peptides, such as phosphorylations. TiO2 columns with 2,5-dihydroxybenozic acid (DHB) in the sample buffer exhibit a higher selectivity and sensitivity for binding of phosphorylated peptides over IMAC. The TiO2/DHB combination limits the binding of negative, but nonphosphorylated, peptides and reduces sample complexity in the mass spectrometer.

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Enzymatic Treatment As with gel-based analyses, phosphate treatments can aid in MS analysis. Phosphatase treatment of proteins or peptides creates an 80-Da mass shift in a mass spectrometer as phosphate-containing peptides are converted to their dephosphorylated forms. This 80-Da shift can then be monitored in a mass spectrometer in control and phosphatase-treated samples. This is of particular use if the mass spectrometer available is a MALDI-TOF MS system for visualization of the phosphorylated peptides. Phosphatase treatment has the advantage of being specific to this modification and enzyme treatment allows for easy visualization of peptide species that contain phosphates. This has the advantage of being useful when simpler and less expensive MS instruments are used, compared with other phosphopeptide techniques that require much more complex and expensive MS/MS instrumentation. This method can only identify the peptides that contain phosphorylations and does not allow for the sitespecific information that MS/MS techniques can provide. However, site information can be deduced if there is only one phosphorylatable residue in the peptide sequence.

4  Conclusions and Perspectives Proteins are the ultimate biological determinants of physiological and diseased phenotypes. The diversity of the mammalian proteome, with one million proteins, and the dynamic nature of various types of PTMs and moment-to-moment protein interactions have necessitated a major paradigm shift of the study of the protein structure and function. The “one gene–one protein” concept and the “one single molecule at a time” strategy have yielded the way to the largescale proteomic approaches of broad analysis of the dynamic changes in proteomes under various normal and diseased conditions. We are witnessing a rapid advance in proteomics technology. Both gel-based and gel-free proteomics techniques have been widely used in combination with much improved MS technologies to gain new insights into essential issues, such as the identity, localization, and function of protein–protein interaction, PTMs, protein targeting, protein damage and repair, protein aggregation and degradation, and signaling complexes at a proteome-wide scale. In this chapter only some commonly used proteomics strategies and approaches in current practice were briefly discussed. Readers are referred to several recently published excellent books for more detailed description of experimental protocols [49, 50]. Regardless of the method or combination of methods used, one major benefit afforded by proteomic studies is the potential for unraveling the mysteries of biological functions

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and a greater understanding of the cellular processes that are responsible for the physiological phenotypes but also for identifying and developing novel diagnostic and prognostic biomarkers useful for a better understanding of many diseases. For example, the application of complementary approaches, of gel-based and LC/MS-based proteomic techniques to profile the changes in the total detectable protein pool of sputum and/or bronchoalveolar lavage in a timeand disease-dependent manner may provide a better understanding of the proteome differentially expressed by COPD patients in the course of the disease. The identification of appropriate and reliable biomarkers is suggested as an essential step for the diagnostics and treatment of these patients [51]. Recent progress in technologies as well as conceptual strategies is also resulting in the transition of proteomics from inventory mapping to functional mapping of cellular signaling pathways. Although the functional proteomic strategies and approaches described in this chapter have proven to be useful tools for the detection of interacting partners of a target protein [52], each of them has some drawbacks that might result in misleading interpretations. The use of a combination of several approaches to offset the intrinsic drawbacks of each approach has become a common practice in the identification and characterization of protein–protein interactions. It should also be kept in mind that many protein complexes might transiently assemble at the right place and the right time to fulfill a specific function. The complexes will then dissociate and individual components can participate in the formation of other complexes following the occurrence of specific signals. The transient interactions are common events in signal transduction networks. Proteomics has become more and more powerful and effective not only in capturing dynamic changes in protein expression, locali­zation, and modification but also in mapping signal transduction pathways in living cells. Therefore, the functional information from proteomic studies will certainly provide novel and decisive insights into the cellular and molecular mechanisms for disease. Acknowledgements  The research in the Laboratory of Functional Genomics and Proteomics, Center of Biomedical Research Excellence and the Department of Pharmacology, University of Nevada, School of Medicine was supported by grants from the National Institutes of Health (HL63914 and HL106256), the National Center of Research Resources (NCRR, P20RR15581), the American Diabetes Association Innovation Award (#7-08-IN-08).

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41  Proteomics and Functional Proteomics 2. O’Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007–4021 3. Scheele GA (1975) Two-dimensional gel analysis of soluble proteins. Characterization of guinea pig exocrine pancreatic proteins. J Biol Chem 250:5375–5385 4. Wasinger VC, Cordwell SJ, Cerpa-Poljak A et al (1995) Progress with gene-product mapping of the Mollicutes: Mycoplasma genitalium. Electrophoresis 16:1090–1094 5. Wilkins MR, Sanchez JC, Gooley AA et  al (1996) Progress with proteome projects: why all proteins expressed by a genome should be identified and how to do it. Biotechnol Genet Eng Rev 13:19–50 6. Pandey A, Mann M (2000) Proteomics to study genes and genomes. Nature 405:837–846 7. Kandasamy K, Keerthikumar S, Goel R et  al (2009) Human Proteinpedia: a unified discovery resource for proteomics research. Nucleic Acids Res 37:D773–D781 8. Prasad TS, Goel R, Kandasamy K et al (2009) Human Protein Reference Database – 2009 update. Nucleic Acids Res 37:D767–D772 9. Herbert B (1999) Advances in protein solubilisation for two-dimensional electrophoresis. Electrophoresis 20:660–663 10. Santoni V, Kieffer S, Desclaux D, Masson F, Rabilloud T (2000) Membrane proteomics: use of additive main effects with multiplicative interaction model to classify plasma membrane proteins according to their solubility and electrophoretic properties. Electrophoresis 21:3329–3344 11. Tastet C, Charmont S, Chevallet M, Luche S, Rabilloud T (2003) Structure-efficiency relationships of zwitterionic detergents as protein solubilizers in two-dimensional electrophoresis. Proteomics 3:111–121 12. Rothemund DL, Locke VL, Liew A, Thomas TM, Wasinger V, Rylatt DB (2003) Depletion of the highly abundant protein albumin from human plasma using the Gradiflow. Proteomics 3:279–287 13. Lopez MF, Kristal BS, Chernokalskaya E et  al (2000) Highthroughput profiling of the mitochondrial proteome using affinity fractionation and automation. Electrophoresis 21:3427–3440 14. Molloy MP, Herbert BR, Williams KL, Gooley AA (1999) Extraction of Escherichia coli proteins with organic solvents prior to two-dimensional electrophoresis. Electrophoresis 20:701–704 15. Molloy MP, Herbert BR, Walsh BJ et  al (1998) Extraction of membrane proteins by differential solubilization for separation using two-dimensional gel electrophoresis. Electrophoresis 19:837–844 16. Pieper R, Gatlin CL, Makusky AJ et al (2003) The human serum proteome: display of nearly 3700 chromatographically separated protein spots on two-dimensional electrophoresis gels and identification of 325 distinct proteins. Proteomics 3:1345–1364 17. Liotta LA, Ferrari M, Petricoin E (2003) Clinical proteomics: written in blood. Nature 425:905 18. Liotta LA, Petricoin EF (2006) Serum peptidome for cancer detection: spinning biologic trash into diagnostic gold. J Clin Invest 116:26–30 19. Luche S, Santoni V, Rabilloud T (2003) Evaluation of nonionic and zwitterionic detergents as membrane protein solubilizers in twodimensional electrophoresis. Proteomics 3:249–253 20. Rabilloud T, Blisnick T, Heller M et al (1999) Analysis of membrane proteins by two-dimensional electrophoresis: comparison of the proteins extracted from normal or Plasmodium falciparum-infected erythrocyte ghosts. Electrophoresis 20:3603–3610 21. Blonder J, Chan KC, Issaq HJ, Veenstra TD (2006) Identification of membrane proteins from mammalian cell/tissue using methanolfacilitated solubilization and tryptic digestion coupled with 2D-LC-MS/MS. Nat Protoc 1:2784–2790 22. Bjellqvist B, Pasquali C, Ravier F, Sanchez JC, Hochstrasser D (1993) A nonlinear wide-range immobilized pH gradient for twodimensional electrophoresis and its definition in a relevant pH scale. Electrophoresis 14:1357–1365

611 23. Rabilloud T, Luche S, Santoni V, Chevallet M (2007) Detergents and chaotropes for protein solubilization before two-dimensional electrophoresis. Methods Mol Biol 355:111–119 24. Westbrook JA, Wheeler JX, Wait R, Welson SY, Dunn MJ (2006) The human heart proteome: two-dimensional maps using narrowrange immobilised pH gradients. Electrophoresis 27:1547–1555 25. Kane LA, Yung CK, Agnetti G, Neverova I, Van Eyk JE (2006) Optimization of paper bridge loading for 2-DE analysis in the basic pH region: application to the mitochondrial subproteome. Proteomics 6:5683–5687 26. Rabilloud T (2008) Mitochondrial proteomics: analysis of a whole mitochondrial extract with two-dimensional electrophoresis. Methods Mol Biol 432:83–100 27. Candiano G, Bruschi M, Musante L et al (2004) Blue silver: a very sensitive colloidal Coomassie G-250 staining for proteome analysis. Electrophoresis 25:1327–1333 28. Chevallet M, Diemer H, Luche S, Van DA, Rabilloud T, LeizeWagner E (2006) Improved mass spectrometry compatibility is afforded by ammoniacal silver staining. Proteomics 6:2350–2354 29. Gharbi S, Gaffney P, Yang A et al (2002) Evaluation of two-dimensional differential gel electrophoresis for proteomic expression analysis of a model breast cancer cell system. Mol Cell Proteomics 1:91–98 30. Neverova I, Van Eyk JE (2005) Role of chromatographic techniques in proteomic analysis. J Chromatogr B Anal Technol Biomed Life Sci 815:51–63 31. Wang H, Hanash S (2003) Multi-dimensional liquid phase based separations in proteomics. J Chromatogr B Anal Technol Biomed Life Sci 787:11–18 32. Washburn MP, Wolters D, Yates JR III (2001) Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat Biotechnol 19:242–247 33. Gevaert K, Van DP, Martens L, Vandekerckhove J (2005) Diagonal reverse-phase chromatography applications in peptide-centric proteomics: ahead of catalogue-omics? Anal Biochem 345:18–29 34. Gevaert K, Goethals M, Martens L et al (2003) Exploring proteomes and analyzing protein processing by mass spectrometric identification of sorted N-terminal peptides. Nat Biotechnol 21:566–569 35. Gevaert K, Van DJ, Goethals M et  al (2002) Chromatographic isolation of methionine-containing peptides for gel-free proteome analysis: identification of more than 800 Escherichia coli proteins. Mol Cell Proteomics 1:896–903 36. Gevaert K, Impens F, Van DP, Ghesquiere B, Hanoulle X, Vandekerckhove J (2007) Applications of diagonal chromatography for proteome-wide characterization of protein modifications and activity-based analyses. FEBS J 274:6277–6289 37. Adamczyk M, Gebler JC, Wu J (2002) Identification of phosphopeptides by chemical modification with an isotopic tag and ion trap mass spectrometry. Rapid Commun Mass Spectrom 16:999–1001 38. Schwartz JC, Jardine I (1996) Quadrupole ion trap mass spectrometry. Methods Enzymol 270:552–586 39. Schwartz JC, Senko MW, Syka JE (2002) A two-dimensional quadrupole ion trap mass spectrometer. J Am Soc Mass Spectrom 13:659–669 40. Marshall AG, Hendrickson CL, Jackson GS (1998) Fourier transform ion cyclotron resonance mass spectrometry: a primer. Mass Spectrom Rev 17:1–35 41. Henzel WJ, Billeci TM, Stults JT, Wong SC, Grimley C, Watanabe C (1993) Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases. Proc Natl Acad Sci U S A 90:5011–5015 42. James P, Quadroni M, Carafoli E, Gonnet G (1993) Protein identification by mass profile fingerprinting. Biochem Biophys Res Commun 195:58–64

612 43. Pappin DJ, Hojrup P, Bleasby AJ (1993) Rapid identification of proteins by peptide-mass fingerprinting. Curr Biol 3:327–332 44. Gavin AC, Superti-Furga G (2003) Protein complexes and proteome organization from yeast to man. Curr Opin Chem Biol 7:21–27 45. Gavin AC, Aloy P, Grandi P et al (2006) Proteome survey reveals modularity of the yeast cell machinery. Nature 440:631–636 46. Ping P (2003) Identification of novel signaling complexes by functional proteomics. Circ Res 93:595–603 47. Uetz P, Giot L, Cagney G et al (2000) A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae. Nature 403:623–627 48. Macek B, Mann M, Olsen JV (2009) Global and site-specific quantitative phosphoproteomics: principles and applications. Annu Rev Pharmacol Toxicol 49:199–221

D.D. Duan 49. Walker JM (2005) The proteomics protocols handbook. Humana, Totowa 50. Westermeier R, Naven T (2002) Proteomics in practice: a laboratory manual of proteome analysis. Wiley-VCH, Weinheim 51. Casado B, Iadarola P, Luisetti M, Kussmann M (2008) Proteomicsbased diagnosis of chronic obstructive pulmonary disease: the hunt for new markers. Expert Rev Proteomics 5:693–704 52. Ping P (2009) Getting to the heart of proteomics. N Engl J Med 360:532–534 53. Yamamoto-Mizuma S, Wang GX, Liu LL et  al (2004) Altered properties of volume-sensitive osmolyte and anion channels (VSOACs) and membrane protein expression in cardiac and smooth muscle myocytes from Clcn3−/− mice. J Physiol 557:4 39–456

Chapter 42

Maintenance, Propagation, and Differentiation of Human Embryonic Stem Cells and Induced Pluripotent Stem Cells Zoë L. Vomberg, Megan Robinson, Thomas Fellner, and Karl Willert

Abstract  The methods described here for maintenance, propagation and freezing of human embryonic stem cells (hESCs) represent a combination of protocols obtained from different sources, and have been extensively tested and are routinely used to culture different hESC lines. As it has been demonstrated that hESCs and induced pluripotent stem cells (iPSCs) are very similar to one another, the same protocols are used for both cell types. In addition to standard maintenance techniques, we describe a method in this chapter to assess the pluripotency of hESCs through the formation of embryoid bodies (EBs). As is the case for culture techniques, several protocols for EB formation exist and certain EB protocols work better for Hues hESC lines than for H hESC lines, and vice versa. Although many of the methods for hESC culture are similar to standard cell culture techniques, greater care and vigilance need to be exercised to maintain the phenotypic and genotypic properties of hESCs. In particular, many established nonprimary cell lines frequently harbor bacterial contaminants, such as mycoplasma, and are able to thrive while maintained in antibiotic-containing media. In contrast, mycoplasma-contaminated hESCs grow poorly, fail to form EBs, and will eventually die. Owing to their sensitivity to any level of contamination, we highly recommended establishing a culture facility dedicated to hESC work only. In this chapter, we outline detailed procedures on how to grow and propagate hESCs (and iPSCs), include detailed protocols for the characterization of hESCs, and provide specific methods for the in vitro differentiation of hESCs via EB formation. Keywords  Stem cell culture • Embryoid body formation • Proliferation • Cell lineage • Self-renewal • Differentiation

K. Willert (*) Human Embryonic StemCell Core Facility, University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA e-mail: [email protected]

1 Introduction There are a variety of protocols in the public domain for maintenance, propagation, and freezing of human embryonic stem cells (hESCs). The methods described here represent a combination of protocols obtained from different sources. All methods have been extensively tested and are routinely used in our laboratory to culture H1/WA01, H9/WA09 [1] (WiCell and the National Stem Cell Bank, http://www.wicell.org) and Hues1, Hues7, Hues9, and Hues11 [2] (Douglas Melton, Howard Hughes Medical Institute, Harvard University, http:// www.mcb.harvard.edu/melton/hues) hESC lines. As it has been demonstrated that hESCs and induced pluripotent stem cells (iPSCs) are very similar to one another [2, 3], the same protocols are used for both cell types in our laboratory. In addition to standard maintenance techniques, we describe a method to assess the pluripotency of hESCs through the formation of embryoid bodies (EBs). As is the case for culture techniques, several protocols for EB formation exist and certain EB protocols work better for Hues lines than for H lines, and vice versa. Although many of the methods for hESC culture are similar to standard cell culture techniques, greater care and vigilance need to be exercised to maintain the phenotypic and genotypic properties of hESCs. In particular, many established nonprimary cell lines frequently harbor bacterial contaminants, such as mycoplasma, and are able to thrive while maintained in antibiotic-containing media. In contrast, mycoplasma-contaminated hESCs grow poorly, fail to form EBs, and will eventually die (personal observation). Owing to their sensitivity to any level of contamination, we highly recommended establishing a culture facility dedicated to hESC work only. Furthermore, in contrast to most other standard cell lines, hESCs require daily attention. Poor treatment of hESCs can result in karyotypically abnormal cells, which can, owing to their growth advantage, outgrow karyotypically normal cells. A reliable visual sign for abnormal hESCs is colony morphology. Whereas normal cells show some amount of variability in morphology (i.e., the majority of colonies have

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smooth edges and contain tightly packed cells), abnormal cells are strikingly uniform in their colony morphology and exhibit a very rapid growth rate. A healthy culture of hESCs is teetering at the edge of differentiation and consequently contains some, albeit a limited number of, differentiated cells. The key to successful growth and propagation of hESCs is to maintain the culture in this finely balanced state without allowing them to slip into a differentiated state or acquire chromosomal abnormalities. In the following section we will outline detailed procedures on how to grow and propagate hESCs. These methods are generally applicable to hESCs and iPSCs. Then follows a section that contains protocols for the characterization of hESCs. The final section provides methods for the in vitro differentiation of hESCs via EB formation.

2 Methods for the Propagation of hESCs 2.1 Derivation of hESCs Several publications have described the derivation of hESCs from donated human embryos [4, 5]. In all cases the earlystage preimplantation embryos, mostly blastocyst stage but occasionally morula stage [6], were obtained from in vitro fertilization clinics and under strict internal review board

Fig. 1  Human embryonic stem cells on mouse embryonic feeder cells. H9/WA09 with good (a) and poor morphology (b). Hues9 at low density (c) and high density (d)

Z.L. Vomberg et al.

guidelines and procedures. Most institutes utilize additional regulatory mechanisms, such as an embryonic stem cell research oversight committee, to ensure that hESC research is conducted under strict ethical guidelines. These checks and balances, although essential, create a logistic and regulatory morass that significantly complicates the procedures required for derivation of hESCs. Fortunately, several hESC lines are readily available, from the National Stem Cell Bank, various companies, or academic laboratories. With the recent advances in generating iPSC lines from terminally differentiated cells [1], the need for hESC derivations will likely wane in the future. However, for as long as iPSC generation involves irreversible genetic modification, iPSC lines will only be useful for nonmedical applications. Thus, for now, regenerative medical applications will require newly derived hESC lines.

2.2 Propagating hESCs Here we describe methods for the long-term culture of hESCs received from two different sources: WiCell/National Stem Cell Bank (H lines) and Douglas Melton’s laboratory (Hues lines). These cells share the characteristics that define them as hESCs. Nevertheless, there are several notable differences. First, to the trained eye the cell morphologies are strikingly different (Fig. 1), with H lines forming more tightly packed

42  Maintenance, Propagation, and Differentiation of Human Embryonic Stem Cells

colonies than Hues lines. Second, H lines are passaged as cell clumps, either by mechanical dissociation or by mild enzymatic dissociation. In contrast, Hues lines are usually passaged as single cells after enzymatic dissociation. Both cell types can be adapted to either passaging method; however, a short adjustment period may be necessary. Generally speaking, colony morphology changes significantly with different passaging techniques. Third, media compositions vary significantly and great care should be taken to closely reproduce the culture conditions (see later) recommended by the distributors.

2.3 Initiating hESC Cultures Distributors of hESC lines generally ship their cells on dry ice. Upon receipt, cells should be immediately thawed and grown or stored in an ultra-low-temperature (-140°C) or liquid nitrogen freezer. In our experience, hESCs obtained directly from distributors are very difficult to culture in the beginning and require special attention and patience. For example, if the cell density is not optimal, hESCs have the tendency to differentiate extensively. In this case, hESC colonies need to be passaged manually by picking undifferentiated colonies and growing them in a relatively small dish until a dense culture is achieved. Once a stable culture has been established, enzymatic passaging may be employed. The most robust and reliable method for growing hESCs, in particular upon thawing, is growth on mitotically inactivated feeder cells. However, for certain applications the use of feeder cells is not an option, and instead cells are grown on commercially available matrixes. In this case, survival of hESCs upon thawing can be significantly improved by adding Rho kinase (ROCK) inhibitor (Christopher Flaim, personal communication, and see [7, 8]). In the following section, we will provide a protocol for thawing hESCs in the presence of feeder cells.

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2.3.1 Preparing Feeder Dishes Feeder cells, generally irradiated mouse embryonic fibroblasts (MEFs), are commercially available from different vendors or can be generated in-house. Be aware that the quality of MEFs can vary significantly between vendors and/or batches. When using a new batch of MEFs, we recommend determining the ideal cell density as follows: coat a six-well cell culture dish with gelatin by adding sufficient sterile 0.1% gelatin solution to cover the surface of the dish and incubate the mixture for 10 min. Seed MEFs at differing densities, e.g., resuspend a vial of irradiated MEFs in 12 mL MEF medium and seed 4, 3, 2, 1, 0.5, and 0.25 mL and adjust the volume to 4 mL per well. The next day, visually determine the optimal density (Fig. 2). Once you know the number of dishes one vial of hESCs seeds, prepare a few wells of a six-well plate 1 day prior to thawing hESCs.

2.3.2 Generating MEF Conditioned Medium Seed MEFs on a 10-cm cell culture dish according to the afore-determined density. Once the MEFs are attached to the dish (minimum of 6  h) aspirate the medium and wash the dish once with phosphate-buffered saline (PBS). Add 10 mL of the appropriate hESC medium without basic fibroblast growth factor (bFGF) and incubate the mixture at 37°C and 5% CO2 for 24 h. The next day, remove the MEF conditioned medium (MCM) and use it directly to culture hESCs. Alternatively, the MCM can be filtered to remove cell debris and stored for up to 1 week at 4°C. MEF dishes can be used up to five times for collecting MCM.

2.3.3 Thawing hESCs Quickly thaw a vial of hESCs in a 37°C water bath until a small frozen pellet remains. Transfer the cell suspension from the vial to a 15-mL conical tube. Add about 10 mL prewarmed

Fig. 2  Mouse embryonic fibroblasts (MEFs) at three different densities. The right panel shows the density of MEFs recommended for human embryonic stem cell culture (hESC) co-culture

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hESC medium and centrifuge the vial at 200g for 4 min at 20°C. Aspirate the medium from one well seeded with MEFs, wash the well once with PBS, and add 2 mL MCM medium. Carefully aspirate the supernatant from the 15-mL tube and resuspend the cell pellet with 3 mL MCM using a 5-mL serological pipet and transfer the cell suspension to the prepared well. Add bFGF to a final concentration of 10–20  ng/mL. Some investigators have found that higher concentrations of bFGF (100 ng/mL) improve long-term maintenance of hESCs [9]. Incubate the cells in a humidified 37°C/5%CO2 incubator. Upon thawing, hESC colonies may be slow to appear. The number of colonies that grow out is dependent on the number of cells originally frozen down and on the quality of the storage conditions. Cells should be monitored and fed until colonies are visible; in some cases this may take as long as 10–14 days. If colonies are slow to appear, supplement each feeding with MCM. Once the colonies are large and visible to the naked eye (approximately 1,000 cells) passage them onto fresh MEFs as described later. If one to five colonies appear, passage them 1:1 onto fresh MEFs.

2.4 Feeding hESCs A healthy culture of hESCs will grow quickly and will deplete the medium of essential nutrients and growth factors and therefore should be fed daily. To feed cells, aspirate the medium and add fresh prewarmed hESC medium supplemented with bFGF. Growth of hESC cultures can be improved by supplementing the medium with MCM: 20% MCM for healthy cultures and 100% MCM for sparse or recovering cultures.

2.5 Passaging hESCs hESCs are passaged by either mechanical dissociation or enzymatic dissociation, whereby mechanical dissociation is used mostly to passage freshly thawed hESCs or hESC colonies that contain a relatively high number of differentiated cells. In the following section, we describe three methods to passage hESCs: (1) mechanical passaging, (2) enzymatic passaging with collagenese IV, and (3) enzymatic passaging with AccutaseTM. Passaging with trypsin is not recommended: several groups, including ours, have observed that use of trypsin results in accumulation of cells with an abnormal karyotype. Cells should be passaged when they reach the appropriate density (see Fig. 1d). hESC density is critical for maintaining an undifferentiated state. Once a culture is growing at a healthy rate, hESCs should be passaged when they reach 90% confluency. The passaging ratio depends on the cell line. This is especially important for the H lines from WiCell passaged using collagenase IV: passaging them before the optimal

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density is reached can lead to increased differentiation. When using AccutaseTM compared with collagenase IV, cell death is extremely low and hESCs consequently grow faster.

2.5.1 Mechanical Passaging This procedure is ideally performed under a dissecting microscope (e.g., EVOS from AMG) inside a biosafety cabinet: • Replace the medium with 100% MCM on a semiconfluent to confluent H9 dish. • Under the dissecting microscope, select morphologically superior colonies by cross-hatching with a 26-gauge needle attached to a 1-mL syringe. • By lightly “flicking” with a needle (causing a brief but rapid and localized rush of fluid across the sectioned colony), one can detach the cells from the dish. Use of a blunt-end needle is suggested to avoid cutting into the plastic. Select approximately 30–50 colonies per well of a six-well dish. • Transfer the medium containing the dissociated colonies to a MEF dish previously washed once with PBS. • Add MCM to a final volume of 3 mL and add the appropriate amount of bFGF (see earlier).

2.5.2 Enzymatic Passaging with Collagenase IV Once hESCs have fully recovered from a thaw and are growing such that they become confluent after 3 days at a 1:4–1:6 split, then enzymatic passaging can be used: • Aspirate the medium from a nearly confluent hESC dish. • For a 10-cm dish add 5 mL of 1 mg/mL collagenase IV (prepare 10 mL of a 1 mg/mL solution in hESC medium and sterile-filter through a 0.2-mm syringe filter). • Incubate the dish at 37°C for 5–7 min or until the edges of the colonies detach from feeder cells. Make sure that only the edges of the colonies detach and not the entire colony. • Aspirate the collagenase IV solution and wash the dish gently three times with hESC medium. Add about 3–5mL MCM and dislodge the colonies by scraping using a cell scraper. Triturate the cells with a 10-mL pipet to disrupt large cell clumps. • Rinse fresh 10-cm MEF plates once with PBS before seeding the cells. Transfer the appropriate amount of cell suspension to the MEF plate. If the optimal seeding density is not yet known, use a range of cell densities (e.g., seed cells at 1:2, 1:4, and 1:6). • Add hESC medium or MCM to a final volume of 10 mL and add the appropriate amount of bFGF (see earlier).

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2.5.3 Enzymatic Passaging with Accutase • • • • •

• • • • •

Aspirate the medium from a hESC cell dish. Wash the hESC dish with PBS and aspirate. Add 1–2 mL AccutaseTM per 10-cm dish. Incubate the mixture at 37°C until cells start to detach, which takes about 4 min. Pipette 10 mL hESC medium onto the cells and break up the colonies and dissociate the cells by pipetting up and down, spraying the dish without creating bubbles. Transfer the cell suspension to a 15-mL conical vial and centrifuge at 200g for 4 min. Aspirate the supernatant and resuspend the cell pellet in 10 mL hESC medium and/or MCM. Aspirate the medium from a fresh MEF dish and wash the dish once with PBS. Seed hESCs onto a MEF plate at the density previously determined to be optimal. Add hESC medium or MCM to a final volume of 10 mL and add the appropriate amount of bFGF (see earlier).

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be prepared as described in Brafman et al. [10]. Switching hESCs from feeder to feeder-free conditions may require a short “adjustment” period during which the cells require more care and supervision. For coating dishes with extracellular matrix, follow the vendors’ recommendations or see Brafman et al. [10]. The coating can be done the day of or the day before passaging hESCs (dishes coated with extracellular matrix can be stored at 4°C for several days). For passaging hESCs into MCM, use the same procedure to dissociate hESCs (either mechanically or enzymatically) as described earlier. For the first passage from feeder cells into feeder-free conditions, use a range of cell densities (e.g., transfer the cells at 1:2 or 1:3). If it is critical to eliminate all traces of MEFs, cells should be passaged at least twice in feeder-free conditions.

3 Characterization of hESCs For long-term culture of pluripotent stem cells, the following properties should be checked on a regular basis:

2.6 Freezing hESCs Once the cells have been expanded to two or more 10-cm dishes, the cells should be frozen for long-term storage. Use one of the methods described earlier to dissociate hESCs and continue as follows: • Transfer the cell suspension to a 15-mL tube and centrifuge the cells at 200g for 4 min. • Aspirate the supernatant and resuspend the cell pellet in freezing medium [60% hESC medium, 30% KnockOut serum replacement (KSR), 10% dimethyl sulfoxide, sterile-filtered]: freeze a single well of a six-well plate in one cryovial and a 10 cm dish in four cryovials. • Aliquot 1 mL cell suspension per cryovial. • Place the cryovials in a Mr. Frosty freezing container (Nalgene) containing 2-propanol and place it into a −80°C freezer overnight. • Move the cryovials to an ultra-low-temperature (−150°C) or liquid nitrogen freezer.

2.7 Feeder-Free Culture of hESCs Several vendors sell media that allow hESCs to grow in the absence of feeder cells. All feeder-free media require a suitable extracellular matrix that is commercially available from BD (MatrigelTM) and Invitrogen (CELLStartTM) or that can

Growth rate:  Embryonic stem cells grow quite rapidly, with duplication in 18–24 h. A sudden increase in the growth rate is often indicative of the cells having acquired a chromosomal abnormality, whereas a decrease in growth rate could be due to bacterial contamination, such as mycoplasma, or cell differentiation. Pluripotency marker expression:  A large collection of genes and proteins have been identified as useful markers of the pluripotent state. Some of these genes (e.g., OCT4, NANOG, SOX2) are critical for maintenance of pluripotency, whereas others (e.g., SSEA4, AP activity, TRA-1-81) are merely correlated with the pluripotent state. Expression of these markers is usually determined by immunofluorescent staining (Fig. 3a–c) or quantitative PCR. Chromosome number: In contrast to other cell types in culture, hESCs are genomically remarkably stable. However, once a single cell acquires an abnormality through chromosomal duplication or rearrangements, it has a growth advantage. The population of chromosomally normal cells will be quickly outcompeted within a few passages. The most reliable method to detect such genomic alterations and ensure the cells carry a full complement of 46 chromosomes is by karyotyping. A disadvantage of this method is that usually only 20 cells are analyzed (Figs.  3d, e). Therefore, a fluorescent in situ hybridization analysis of some of the more common chromosomal abnormalities (i.e., chromosomes 12 and 17) is suggested to detect a small subpopulation of abnormal cells, which can be missed in karyotyping.

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Fig. 3  Characterization of hESCs. Fixed H9/WA09 stained for DNA (a), SSEA4 (b), and OCT4 (c). Karyotype of a normal (d) and an abnormal (trisomy 17, e) female hESC line

Differentiation potential:  The hallmark of embryonic stem cells is their ability to differentiate into all derivatives of the three germ layers, ectoderm, endoderm, and mesoderm. For mouse embryonic stem cells and iPSCs, the gold standard test is to show that the cells can contribute to all cell types in a chimeric mouse including the germ line. Because of ethical reasons this method cannot be used to determine pluripotency of hESCs. Alternatively, the differentiation potential of hESCs can be assessed by in vitro differentiation (usually by EB formation; described later) or in  vivo differentiation (teratoma formation in a xenograft model).

4 Differentiation of hESCs via EBs Many protocols have been described to initiate differentiation via EB formation. The underlying principle of all these protocols is to culture cells in such a manner that they aggregate with

one another in suspension rather than adhere to a cell culture dish. As small clumps (EBs) form (Fig. 4a), exterior and interior cells are exposed to differing conditions and signals. In the absence of self-renewal factors, such as bFGF, pluripotency genes are downregulated, allowing cells to turn on a differentiation program resulting in cells of the three germ layers. Cell aggregation can be promoted using various methods, including self-aggregation in ultra-low-binding plastic dishes, hanging drop aggregation, and forced aggregation by centrifugation. Certain methods work better for one cell line than another. Self-aggregation is simple and quick but produces a low yield of cell clumps of variable sizes. With Hues9 cells, self-aggregation is achieved by culturing the dissociated cells in ultra-low-binding plastic dishes (about 106 cells per well of a six-well plate). In the case of H9/WA09, a dense and healthy culture of cells can be scraped off the dish, followed by mild trituration. For more uniform-sized cell clumps we recommend forced aggregation by centrifugation to produce socalled spin EBs. EB yield can be further increased by pretreating hESCs with ROCK inhibitor.

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Fig. 4  Embryoid bodies (EBs) formed from H9/WA09 human embryonic stem cells. (a) After 5–7 days in ultra-low-binding culture dishes, hESCs aggregate into tight balls of cells. (b, c) Upon transfer to adher-

ent culture dishes, EBs spread and generate cell types with variable morphologies. After long-term culture, structures resembling neuritis are readily visible (c)

The following protocol will yield derivatives of all three germ layers, assuming the starting cells are in fact pluripotent. For directed differentiation into specific cell types, the reader is referred to the primary literature and needs to develop novel methods that lead to a higher yield of the cell type of interest. Spin EB formation:

The type and length of culture is dictated by the desired outcome. If the goal is to verify the pluripotency of hESCs, derivates of the three germ layers are detectable after 2 weeks of culturing the EBs in adherent conditions (Fig. .4b, c). If more mature cell types are desired, longer culture periods are necessary. The presence of differentiated cell types can be confirmed either by immunofluorescence or by quantitative PCR.

• Pretreat hESCs with 10 mM ROCK inhibitor (5 mM stock solution) for 3 h before EB formation. • Aspirate the medium from the cells and wash the dish once with PBS. • For a 10-cm dish, add 2 mL Accutase and incubate the mixture for 4 min at 37°C or until colonies start to dissociate. • Wash off cells with 8–10 mL EB medium and transfer the cell suspension to a 50-mL conical tube. • Centrifuge the cells at 200g for 5 min at 20°C. • Aspirate the supernatant and resuspend the cell pellet in 2 mL EB medium. • Count the cells using a hemocytometer or a cell counter. • Dilute the cells with EB medium to a final concentration of 50,000 cells/mL and supplement the medium with 10 mM ROCK inhibitor. • Transfer 0.1 mL of cell suspension (equivalent to 5,000 cells) to all wells of an untreated V-shaped 96-well plate. • Centrifuge the plate at 950g for 5 min at 20°C. • Return the cells to a 37°C/5% CO2 incubator. • The following day, check the wells for compact ballshaped aggregates of cells. • Transfer about 60 cell clumps to one well of an ultra-lowbinding six-well plate using a 1-mL pipet. • Add EB medium to a final volume of 4 mL per well. • Replenish the medium every 2–3 days as described: carefully remove 2  mL per well using a 5-mL serological pipet, leaving behind all EBs. Add 2  mL fresh media resulting in a final volume of 4 mL. • After 1 week in ultra-low-binding conditions, transfer EBs to a six-well tissue culture dish coated with a suitable adhesive matrix (e.g., Matrigel). • Add fresh medium to the EBs every 2–3 days being careful not to dislodge the cell clusters.

5 Reagents hESC lines:  Several lines are presently available from various sources, including the National Stem Cell Bank (http://www. nationalstemcellbank.org/), Douglas Melton’s laboratory (Howard Hughes Medical Institute, Harvard University, http:// www.mcb.harvard.edu/melton/hues/), and certain vendors. MEFs:  Even though alternative culture methods have been developed (e.g., with human feeder cells or in feeder-free media), using mitotically inactivated MEFs as feeder cells is presently the most reliable and cost-effective method to maintain hESCs. MEFs from E13–E14 pregnant mice and expanded in cell culture to passage 3 or 4 can be purchased from various vendors. Generally it is recommended to generate MEFs from a CF-1 strain; however, a study demonstrating their superior performance has not been published. Mitotic inactivation prior to co-culture can be achieved either by irradiation (3,000–5,000 rad with a cesium-source irradiator) or mitomycin C treatment. MEF medium:  Dulbecco’s modified Eagle’s medium (DMEM), 10% fetal bovine serum supplemented with antibiotics (penicillin/streptomycin) and glutamine if desired. hESC medium: We recommend using the medium composition recommended by the supplier of the particular hESC line. Once cells have been expanded to several plates, they can be transferred into other media. Generally the medium is composed of DMEM supplemented with 20% KSR [or 10% KSR and 10% human serum albumin (e.g., Plasmanate)]. Antibiotics can be included but we do not recommend

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their use since they can mask low-level chronic bacterial contaminations, especially mycoplasma.

References

bFGF: Multiple companies provide bFGF. We recommend using recombinant human bFGF. However, some investigators have successfully used bFGF from other organisms (e.g., zebrafish).

1. Takahashi K, Yamanaka S (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126:663–676 2. Liu Y, Shin S, Zeng X et al (2006) Genome wide profiling of human embryonic stem cells (hESCs), their derivatives and embryonal carcinoma cells to develop base profiles of U.S. Federal government approved hESC lines. BMC Dev Biol 6:20 3. Muller FJ, Laurent LC, Kostka D et  al (2008) Regulatory networks define phenotypic classes of human stem cell lines. Nature 455:401–405 4. Cowan CA, Klimanskaya I, McMahon J et al (2004) Derivation of embryonic stem-cell lines from human blastocysts. N Engl J Med 350:1353–1356 5. Thomson JA, Itskovitz-Eldor J, Shapiro SS et al (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147 6. Klimanskaya I, Chung Y, Becker S, Lu SJ, Lanza R (2006) Human embryonic stem cell lines derived from single blastomeres. Nature 444:481–485 7. Damoiseaux R, Sherman SP, Alva JA, Peterson C, Pyle AD (2009) Integrated chemical genomics reveals modifiers of survival in human embryonic stem cells. Stem Cells 27:533–542 8. Watanabe K, Ueno M, Kamiya D et al (2007) A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat Biotechnol 25:681–686 9. Xu RH, Peck RM, Li DS, Feng X, Ludwig T, Thomson JA (2005) Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human ES cells. Nat Methods 2:185–190 10. Brafman D, Shah K, Fellner T, Chien S, Willert K (2009) Defining long-term maintenance conditions of human embryonic stem cells with arrayed cellular microenvironment technology. Stem Cells Dev 18:1141–1154

EB medium:  DMEM-F12 or KnockOut DMEM supplemented with 10–20% fetal bovine serum or KSR. bFGF is usually omitted. Other growth factors or small molecules (retinoic acid) can be included to push the differentiated cells into a particular lineage. Gelatin, 0.1% solution:  Autoclave 1 g gelatin from porcine skin, type A, in 1 L distilled water. Freezing medium:  60% hESC medium, 30% KSR, 10% dimethyl sulfoxide, sterile-filtered. Cell dissociation reagents:  Various reagents for cell dissociation are in use. Ezymatic dissociation can be achieved with mild trypsinization, Accutase, collagenase, or Dispase. Manual dissociation can be simplified using a passaging tool available from Invitrogen (StemPro® EZPassage™ disposable stem cell passaging tool). Acknowledgements  We are grateful to all past and present users of the UCSD Human Embryonic Stem Cell Core Facility, especially David Brafman, for useful interactions and communications that have allowed us to refine the protocols described in this chapter. We are particularly grateful to Ji-Eun Kim for her contributions to the development of a robust protocol for spin EBs and to Christopher Flaim for communicating the effectiveness of ROCK inhibitor during thawing of hESCs.

Chapter 43

Identification of Adult Stem and Progenitor Cells in the Pulmonary Vasculature Amy L. Firth, Weijuan Yao, and Jason X.-J. Yuan

Abstract  Adult stem cells retain some capacity for selfrenewal, although it is limited in comparison with embryonic stem cells, and are more restricted in their differentiation capacity. Adult stem cells are also commonly referred to as tissue specific stem cells. Some adult stem cells are still able to give rise to several specialized cell types (multipotent stem cells), whereas others are limited to a single specialized cell type (unipotent stem cells). Progenitor cells, the progeny of adult stem cells, differ from stem cells in that the potential for long-term self-renewal is lost. Scientifically, progenitor cells are more differentiated than stem cells. Primary stem cell progeny progenitor cells, usually known as multipotent adult progenitor cells, have full lineage-specific potential, whereas next-generation progenitors (oligopotent progenitors) are more lineage-restricted. This adult stem cell and progenitor cell hierarchy system exists to preserve a homeostatic repair and maintenance of the body, replenishing specialized cells and sustaining the routine cellular turnover in regenerative organs. Furthermore, the properties of these cells make them good candidates for targeted drug/gene delivery to specific organs; for example, mesenchymal stem cells (MSCs) have recently been shown to preferentially home to the lung. Adult stem and progenitor cells may be either circulating or resident in a particular tissue/organ system, including the lung and pulmonary vasculature. This chapter briefly describes the adult stem and progenitor cells currently identified in the pulmonary vasculature and introduces methodological approaches to successfully identify stem and progenitor cells. Keywords  Progenitor cells • Adult stem cells • Self-renewal • Differentiation • Multipotency • Mesenchymal stem cell • Endothelial progenitor cells

A.L. Firth (*) Laboratory of Genetics, The Salk Institute of Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA, 92037, USA e-mail: [email protected]

1 Introduction Stem cells are characterized by their ability to self-renew through mitotic cell division and differentiate into a diverse range of specialized cell types [1]. Two distinct categories of stem cells exist; embryonic stem cells and adult stem cells [2, 3]. Embryonic stem cells are pluripotent, being able to both selfrenew indefinitely and differentiate into cells of all three germ layers: endoderm, ectoderm, and mesoderm. Adult stem cells retain some capacity for self-renewal, although limited in comparison with embryonic stem cells, and are more restricted in their differentiation capacity. Adult stem cells are also commonly referred to as tissue-specific stem cells. Some adult stem cells are still able to give rise to several specialized cell types (multipotent stem cells), whereas others are limited to a single specialized cell type (unipotent stem cells) [4]. Progenitor cells, the progeny of adult stem cells, differ from stem cells in that the potential for long-term self-renewal is lost. In the current literature, stem and progenitor cell terminology is often confused; scientifically, progenitor cells are more differentiated than stem cells. A hierarchy of stem and progenitor cells exists, a system particularly well defined for hematopoietic stem cells (HSCs) [4]. Primary stem cell progeny progenitor cells, usually known as multipotent adult progenitor cells, have full lineage-specific potential, whereas next-generation progenitors (oligopotent progenitors) are more lineage-restricted. This adult stem cell and progenitor cell hierarchy system exists to preserve a homeostatic repair and maintenance of the body, replenishing specialized cells and sustaining the routine cellular turnover in regenerative organs [5]. Furthermore, the properties of these cells make them good candidates for targeted drug/gene delivery to specific organs; for example, mesenchymal stem cells (MSCs) have recently been shown to preferentially home to the lung [6]. Adult stem and progenitor cells may be either circulating or resident in a particular tissue/organ system, including the lung and pulmonary vasculature [3]. This chapter will briefly describe the adult stem and progenitor cells currently identified in the pulmonary vasculature and introduce methodological approaches to successfully identify stem and progenitor cells.

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_43, © Springer Science+Business Media, LLC 2011

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2 Stem Cells in the Pulmonary Vasculature Despite the lung having an impressive regenerative capacity, the restoration of pulmonary function rarely occurs. Endogenous lung stem cells are an attractive target for regenerative lung therapies. Recently, a subset of multipotent bone marrow cells, known as side population (SP) cells, was identified in the lung. Lung SP cells are enriched with CD45+ HSCs; CD45- cells have been shown to represent a population containing lung MSCs [7–9]. HSCs and MSCs may be circulating in the blood or resident cells with organ/tissue specificity. Such lung SP cells are postulated to be pulmonary progenitor cells with endothelial, epithelial, and mesenchymal potential [10]. Stem and progenitor cells have been implicated in a variety of pulmonary disease, including pulmonary arterial hypertension (PAH) [11–13], chronic obstructive pulmonary disease [14], and pulmonary fibrosis [15]. The precise role of these cells in disease remains uncertain; studies have shown both pathogenic and restorative roles, both of which are currently being investigated. These cells, for example, are shown to facilitate vascular repair [16]; however, they may conversely contribute to the pathogenesis of pulmonary vascular disease by enhancing vascular lesion formation [17–19].

3 Adult Stem Cells The bone marrow harbors an expansive repertoire of stem and progenitor cells which can be mobilized in response to tissue injury or disease. These cells include HSCs, MSCs, and endothelial progenitor cells (EPCs). Each is briefly described in the subsequent sections with specific reference to their roles in pulmonary vascular disease.

3.1 Hematopoietic Stem Cells HSCs are among the first stem cells that are specified during embryogenesis from the epiblast. They are able to differentiate into all blood cell types, which include (1) myeloid cells encompassing monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes/platelets, and dendritic cells, and (2) lymphoid cells, comprising T cells, B cells, and natural killer cells. Cell-surface markers commonly used in combination to select for mononuclear HSCs include CD34, CD133, CXCR4, and CD150 in the human, in addition to a lack of markers of differentiated blood lineages (Lin-). A

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distinct, and now well-defined hierarchy of HSCs exists, including cells with long-term and short-term regenerative capacity and more differentiated multipotent, oligopotent, and unipotent progenitor cells (Fig. 1). HSC are nonadherent cells, morphologically round with a high nucleus-to-cytoplasm ratio. In addition to cell-surface markers, HSCs can also be distinguished by their low ability to accumulate metabolic fluorochromes such as DNA stain Hoechst 33342, rhodamine 123, and messenger RNA marker pyronin Y. A Ficoll gradient centrifugation can be used to deplete erythrocytes and granulocytes from a blood sample [20]. Subsequent HSC purification steps include fluorescence-activated cell sorting (FACS) and/or use of paramagnetic beads (e.g., CD34+ or CD133+ paramagnetic beads). Granulocyte/macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor that stimulates myeloid cells and their precursors in vitro. Although GM-CSF is usually considered exclusively as a hematopoietic regulator, recent animals studies have established that its activity is essential for normal pulmonary homeostasis [21, 22].

3.2 Mesenchymal Stem Cells MSCs (or multipotent mesenchymal stromal cells) are thought to be located wherever mesenchymal tissue turns over, including the bone marrow, muscle, fat, skin, and cartilage. MSCs have great plasticity, being able to change from one lineage to another in the right environmental conditions, which makes characterization of multiple mesenchymal phenotypes difficult. In 2006, the International Society for Cellular Therapy issued a statement setting minimal criteria for putative MSCs to fulfill: (1) MSCs must be plastic-adherent in standard culture conditions; (2) MSCs must express CD105, CD73, and CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79a or CD19, and HLA-DR surface molecules (as shown in representative FACS images in Fig.  2); and (3) MSCs must differentiate to osteoblasts, adipocytes, and chondroblasts in  vitro [23]. MSCs have a great therapeutic potential and have been widely studied in the cardiovascular system as an autologous cell therapy [24]. Beneficial effects of MSC engraftment have been observed in rats with bleomycin-induced lung injury [25]. A schematic detailing MSC self-renewal and differentiation is shown in Fig. 3. Furthermore, MSCs are emerging as a suitable vehicle for gene therapy, MSCs have been demonstrated to preferentially home to the lung and benefits of such delivery of angiopoietin-1 have been observed in acute lung injury [6], and endothelial nitric oxide synthase delivery helped to improve PAH-related right ventricular impairment and survival [11].

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Fig. 1  Hematopoietic stem cell hierarchy. Fully self-renewing hematopoietic stem cell give rise to several multipotent progenitors [colony-forming units (CFU), common myeloid progenitor (CMP) and common lymphoid progenitors (CLP)] which, in turn, produce oligopotent progenitors, unipotent progenitors, and eventually differentiated cells. The CMP is able to

produce granulocyte-macrophage progenitors (GMP) and megakaryocyte/ erythrocyte progenitors (MEP), giving rise to monocyte/macrophages/ granulocytes and megakaryocytes/platelets/erythrocytes, respectively. Erythoid burst forming units (BFU-E) give rise to proerythroblast colonyforming unit – erythroid (CFU-E) before erythrocytes are formed

3.3 Endothelial Progenitor Cells

postcapillary venules or (2) arteriogenesis due to the de novo growth of collateral conduits from large-diameter arteries. EPCs challenged the premise that new blood vessels were formed from the proliferation, migration, and remodeling of fully differentiated endothelial cells. EPCs were first

There are two mechanisms of postnatal vasculogenesis: new blood vessels can be formed from either (1) angiogenesis resulting from the sprouting of endothelial cells from

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Fig.  2   Fluorescence-activated cell sorting (FACS) analysis of cell-surface markers typical of mesenchymal stem cells (MSC). This figure highlights a core set of marker expression patterns

used in flow cytometry to isolate putative MSC. (Reproduced with permission from the American Society for Clinical Investigation [7])

identified when a population of circulating CD34+ cells was identified in adult peripheral blood and deemed capable of in vitro differentiation and de novo vessel formation [26]. The function of EPCs in vascular homeostasis and repair is currently controversial. Studies have postulated the potential for EPCs to be used as a biomarker for cardiovascular disease and vascular function, although there is substantial ambiguity between whether a decrease or an increase in circulating EPC levels reflects the risk. EPCs are also directly linked to the restoration of the endothelial cell layer during vascular injury [27]. Vascular endothelial growth factor is known to effectively mobilize EPCs and potently induces angiogenesis. Furthermore, shear stress can promote the differentiation of EPCs into mature endothelial cells [28]. EPCs express the chemokine receptor CXCR4 and are believed to home to a site of injury owing to the chemoattractant pull of stromal-cell-derived factor-1, released from endothelial cells and platelets. In addition, high levels of b2 integrins on EPC can interact with their ligands P-selectin, E-selectin, and intracellular adhesion molecule 1, which are expressed on endothelial cells [29]. With use of a clonogenic assay, a hierarchy of EPCs has been identified primarily on the basis of the clonogenic and proliferative potential of the cells [30, 31]. The term “endothelial

progenitor cell” (EPC) is often used to refer to all cells in this “family” but should truly be restricted to those cells with minimal criteria of cell-surface markers and the ability to form vessels in vivo.

3.4 Resident Tissue Progenitor Cells Resident tissue progenitor cells are a rare and diverse group of adult stem/progenitor cells. These cells are, in general, small and have been identified in many tissues and organs, including vascular walls. As with most stem and progenitor cells, tissue-resident cells are identified by their cell-surface-marker expression, including CD133, CD44, and nestin in the lung. Furthermore, lung tissue specific (or SP) SP cells are identified by their ability to efflux Hoechst 33342 [32]. SP cells have a high expression level of the ATP binding cassette transporters (ABC), for example, ABCG2 enabling active efflux of the dye [33]. Tissue specific stem/progenitor cells present an attractive possibility to stimulate in vivo cell production and differentiation and to develop tissue specific cell based therapeutic approaches.

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Fig. 3  The mesengenic process. MSC self-renewal, proliferation, and potential lineage specific differentiation pathways are depicted. MSCs differentiate by committing, differentiating, and maturing in a lineage-specific fashion

4 Methodology Delineated The criteria for stem cells depicts an ability to self-renew and to functionally differentiate into tissue-specific cells both in vivo and in vitro [34–36]. Self-renewal is defined as having the capability to generate progeny with the same characteristics as the parent cell without undergoing senescence. The identification of adult stem and progenitor cells requires rigorous characterization: firstly, cells are characterized on the basis of the presence (or absence) of cell-surface and cytosolic markers in a particular cell population; next, cells fulfilling this complement of markers can be isolated using FACS prior to carrying our functional assays to confirm the self-renewal and differentiation potential of the isolated cell population. The methods that are currently adopted to identify

stem and progenitor cells in the pulmonary vasculature are summarized in a schematic diagram in Fig. 4.

5 Techniques for the Identification of Adult Stem and Progenitor Cells in the Pulmonary Vasculature 5.1 Cell-Surface-Marker Expression Table 1 summarizes the profiles of cell-surface and cytosolic markers known to be present (or absent) on the variety of stem and progenitor cells relevant to the pulmonary vasculature in health and disease. The expression of combinations of

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Fig. 4  The key methodology for characterization of putative progenitor cells in the pulmonary vasculature

these markers can be confirmed using an array of techniques, such as immunohistochemistry, immunofluorescence, reversetranscription (RT) polymerase chain reaction (PCR) or protein detection by western blotting. As the techniques of PCR and western blotting are common to most laboratories and are described extensively in the literature, they will not be discussed and we invite the reader to refer to Kurien and Schofield [37] and Kubista et  al. [38] for more details, if necessary. Table 1, although not exhaustive, summarizes the key markers used to identify putative stem or progenitor cells.

5.1.1 Immunohistochemistry Immunohistochemistry is the technique used to detect selected proteins within a tissue section using specific antibodies raised against the antigen in question. It is a useful technique to study the distribution and localization of the biomarkers for stem and progenitor cells in a biological tissue.

Equipment required: plastic bags, cryomold, cryostat, light microscope, fluorescence microscope. Protocol

Frozen Section Preparation 1. Lung tissue is harvested and washed quickly in chilled phosphate-buffered saline (PBS). If the sample is already frozen, the lung tissue should be placed immediately in 4% paraformaldehyde/PBS solution for 18–24 h. 2. The sample should then be placed in 30% sucrose/PBS solution at 4°C for 8–10 h prior to washing it twice with PBS (5 min each). 3. Ensure detailed labeling of all samples, plastic bags, and cryomolds. 4. Pour 100–200 mL of 2-methlylbutane into a plastic beaker and add dry ice slowly until the temperature reaches −40°C. 5. Blot the excess sucrose from the tissue with gauze.

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Table 1  Expression of key cell-surface markers defining distinct cell populations with multilineage potential present in the pulmonary vasculature (data from [7, 23, 31, 55, 56]) Marker A.k.a. HSC MSC MPC EPC CD10 Neural endopeptidase + CD11b Integrin aM CD13 Alanine aminopeptidase + CD14 Coreceptor + CD29 Integrin b1 + CD31 PECAM-1 + CD34 Mucosialin + + CD38 +LO CD44 Receptor for hyaluronic acid + +LO CD45 LCA + +/CD49A Half of a1b1 integrin duplex + CD49B Half of a2b1 integrin duplex + + CD49C Integrin a3 + CD49E Integrin a5 + CD51 Integrin aV + CD54 Intercellular adhesion molecule 1 + CD58 Lymphocyte-function-associated antigen 3 + CD59 Protectin + CD61 Integrin b3 + CD62L L-selectin + CD71 Transferrin receptor + CD73 5¢-nucleotidase, ecto + CD79a Ig-a CD90 Thy-1 + + +LO CD102 Intercellular adhesion molecule 2 + CD104 Integrin b4 + CD105 Endoglin + CD106 Vascular cell adhesion molecule 1 + CD117 c-kit + CD120A Tumor necrosis factor receptor + CD122 Interleukin-2 receptor + CD124 Interleukin-4 receptor + CD126 Interleukin-6 receptor + CD127 Interleukin 7 receptor + CD133 Promimin-1 + CD144 VE-cadherin + CD166 ALCAM + CD309 VEGFR2 + HSC hematopoietic stem cell, MSC mesenchymal stem/stromal cell, MPC mesenchymal progenitor cell, EPC endothelial progenitor cell, PECAM-1 platelet endothelial cell adhesion molecule 1, LCA leukocyte common antigen, VE-cadherin vascular endothelial cadherin, ALCAM activated leukocyte cell adhesion molecule, VEGFR2 vascular endothelial growth factor receptor 2

6. Fill a cryomold (Sakura) halfway with optimal cutting temperature (OCT) compound (Sakura). It is important that there are no bubbles in the OCT compound. 7. Orient the tissue in the mold with the desired area of analysis face down and fill in the remainder of the mold with OCT compound. The sample should be at the bottom of the mold. 8. Immerse the entire mold into the 2-methylbutane for 30–40 s, and be careful to not disturb the orientation of the tissue. Remove the mold containing the tissue, place it in a labeled plastic bag, and store it at –70°C. 9. Prior to the sectioning, the samples should be placed in a cryostat for 1  h to bring them to –20°C. Cut the tissue

block to 5–8-mm thickness and place the sections on microscope slides. Store the slides at –20°C until use.

Immunohistochemistry: Detection 1. Thaw the frozen tissue sections from the −20°C freezer at room temperature. 2. Fix the tissue sections in 4% paraformaldehyde/PBS for 5–10 min and then wash them with PBS for three times for 5 min. 3. Slides may be incubated for 5–10 min in 0.1–1% hydrogen peroxide diluted in PBS, deionized H2O, or methanol

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to quench endogenous peroxidase activity. Wash the slides twice in PBS. 4. Incubate sections for 1 h in 1.5% normal blocking serum in PBS + 2% serum + 0.1% Triton X-100. The blocking serum should be derived from the same species as the secondary antibody was raised. Remove blocking serum from the slides. 5. Incubate the blocked sections with primary antibody for 30  min at room temperature or overnight at 4°C. The optimal antibody concentration should be determined by titration; the recommended range is 0.5–5.0  mg/mL diluted in PBS with 1.5% normal blocking serum. Wash the primary antibody stained sections with PBS three times for 5 min. 6. Immunoperoxidase staining using the ABC staining system (Santa Cruz Biotechnology). Incubate the primary labelled and washed sections for 30  min with biotin-­ conjugated secondary antibody as provided, or at approximately 1  mg/mL diluted in PBS with 1.5% normal blocking serum. Wash the secondary antibody labelled sections with PBS three times for 5 min. 7. Incubate the secondary antibody labelled and washed sections for 30 min with avidin–biotin enzyme reagent. Wash these sections with PBS three times for 5 min. 8. Incubate the sections in peroxidase substrate, as provided, for 0.5–10 min, or until the desired stain intensity develops. Individual slides should be monitored to determine the best development time. Wash sections in deionized H2O for 5 min. If desired, counterstain in Gill’s formulation no. 2–hematoxylin for 5–10 s. Immediately wash the sections with several changes of deionized H2O. 9. Dehydrate the sections through alcohols and xylenes as follows: Soak the sections in 95% ethanol twice for 10 s, then in 100% ethanol twice for 10 s, then in xylene three times 10 s. Wipe off excess xylene from the sections and immediately add one or two drops of permanent mounting medium to the section on a slide (e.g., CC/Mount, Sigma), cover this with a glass coverslip, and observe the stained and fixed section by light microscopy.

5.1.2 Fluorescence-Activated Cell Sorting Putative stem and progenitor cells can be isolated from a mixed population of cells by FACS or magnetic bead separation using antibodies for specific protein markers. FACS is a specialized form of flow cytometry; cells are sorted one at a time by the light scatter of fluorescent labeled antibodies/ probes. Cells can be sorted into sterile containers ready for further analysis. Equipment required:  Falcon® no. 2052 or no. 2054 tubes, Becton Dickinson FACSCalibur machine (or equivalent), and Becton Dickinson FACSAria machine (or equivalent).

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Protocol 1. For adherent cells create a single cell suspension by using trypsin/EDTA, TrypLE (Invitrogen), or other suitable agent. 2. Wash the cells with 2 mL cold PBS by centrifuging them at 150–300g in the cold (4°C). 3. Wash the cells again (optional). 4. Resuspend the cells in buffer [PBS + 1% bovine serum albumin (BSA) + 0.1% NaN3] and aliquot 1× 105–100 × 105 cells per 100 mL (total volume once antibodies have been added) into Falcon® no. 2052 or no. 2054 tubes on ice. 5. Add 20 mL of monoclonal antibody (1–10 mg/mL final concentration) or isotype control antibody. 6. Incubate the mixture for 30 min on ice. 7. Wash the cells again with 2 mL cold azide buffer. 8. Resuspend the cells in 100  mL of secondary antibody (e.g., fluorophore-conjugated goat anti-mouse IgG). Skip to step 10 if directly conjugated monoclonal antibody is used. 9. Incubate the cells for 30 min on ice in the dark. Wash the cells again with azide buffer. 10. Resuspend the cells in 100–500 mL cold wash buffer and (a) Add an equal volume of cold buffered 3% paraformaldehyde to fix the cells and analyze the using a Becton Dickinson FACSCalibur machine and CellQuest software (BD Biosciences) or (b) Leave in the cells in wash buffer and analyze them live, add propidium iodide, 0.5 mg/mL final concentration. Live cells can be sorted by a Becton Dickinson FACSAria machine into tubes containing 100% serum. The ideal final concentration of cells should be 1 × 106/mL. NB: Using directly conjugated antibodies is a faster and more desirable approach for FACS analysis and eliminates the necessity for primary and secondary antibody incubation.

5.1.3 Immunofluorescence Imaging Like FACS, immunofluorescent labeling of cells and tissues enables the detection of a specific factor using antibodies. Direct immunofluorescence staining takes advantage of using a primary antibody directly conjugated to a fluorescent dye. Indirect immunofluorescence staining uses a fluorochrome-labeled secondary antibody to detect the specific primary antibody. Cell expression of a specific factor can then be studied in detail with a suitable microscope. Equipment required:  Fluorescence microscope Protocol 1. For frozen tissue sections, remove them from the −20°C freezer and thaw them at room temperature. For adherent cells on coverslips, wash them three times in PBS.

43  Identification of Adult Stem and Progenitor Cells in the Pulmonary Vasculature

2. Fix the tissue/cells in 4% paraformaldehyde/PBS for 5–10  min and then wash them in PBS three times for 5 min. 3. For tissue sections, clean the slide to remove water around tissue. Use a water-resistant pen to circle the tissue sections. 4. Incubate the tissue/cells in 100 mL of blocking solution (PBS + 0.1% Triton X-100 + 2% normal blocking serum) at room temperature for 1 h. 5. Incubate the tissue/cells in 50–100 mL of primary antibody diluted in blocking solution for 1 h at room temperature or 4°C overnight. Incubate a negative control in blocking solution without primary antibody. 6. Wash the slide in PBS three times for 10 min at room temperature. 7. Incubate the tissue/cells in 50–100 mL of secondary antibody conjugated with fluorophore diluted in blocking solution at room temperature for 45 min (protect from light). 8. Incubate the tissue/cells in 50–100  mL of 4¢,6-diamidino-2-phenylindole dihydrochloride (DAPI) (5  mM) diluted in PBS for 5 min at room temperature to counterstain the nucleus. 9. Wash the slide in PBS three times for 10 min at room temperature (protect from light). 10. Mount the slide/coverslip using with antifade mounting medium (e.g., Fluoromount G, Electron Microscopy Sciences), and observe the cells under the fluorescence microscope. NB:  For double immunofluorescence staining, use two antibodies raised in different species in step 6 and two secondary antibodies conjugated with different colors of fluorophores in step 8. The blocking solution should include the sera from both of the animals in which the secondary antibodies were raised.

5.1.4 Single-Cell Clonogenic Assay The most rigorous test for the clonogenic potential of a progenitor cell is to determine whether a single cell is able to divide and form a colony in the absence of other cells [30, 39]. This single-cell assay could be used to quantitate the proliferation and clonogenic potential of the marker-positive cells. This approach has been utilized to identify a hierarchy existing in EPCs which is comparable to that of the hematopoietic cell system. Equipment required:  40 mm filter; Falcon® no. 2052 or no. 2054 tubes, FACSVantage cell sorter (or equivalent), 96-well plates, fluorescence microscope, FACSVantage SE machine (or equivalent) Protocol 1. Wash 96-well collagen-coated plates with 200 mL sterile PBS per well and replace the PBS with 200 mL standard medium (e.g., Dulbecco’s modified Eagle’s medium + 10% fetal bovine serum + penicillin/streptomycin).

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2. Resuspend adherent cells using trypsin/EDTA or TrypLE (Invitrogen) and strain the cells using a 40-mm filter. 3. Transfer the cells to flow cytometry tubes containing the standard culture medium at 1 × 106 cells per tube. 4. Sort the cells with a FACSVantage sorter (BD Biosciences) at a rate of 100 events per second; gate the cells on the basis of viability and morphology. 5. Seed each phenotype in triplicate in the 96-well collagencoated plates with medium. Culture the cells at 37°C with 5% CO2/21% O2, and add 100 mL medium per well at the top of the old medium every 4 days for 12 days. 6. After colonies have started to form, remove them using trypsin/EDTA or TrypLE (Invitrogen) and expand the cells into six-well plates. Be careful to clearly label the individual clones. It is possible to generate about 20 clones from each 96-well plate utilized.

5.1.5 Telomerase Activity and Telomere Length Measurement Telomeres are portions of genetic material involved in stabilizing the chromosome ends [40, 41]. Telomeres consist of many thousands of copies of TTAGGG six-base-pair repeats in human chromosomes. Short telomeres are associated with replicative senescence and loss of stem cell proliferation capacity in  vitro, whereas long telomeres are prominently found in rapidly growing cells. There are currently several techniques that can be adopted to assess telomere length: (1) terminal restriction fragment Southern blot, (2) quantitative fluorescent in situ hybridization (Q-FISH), (3) RT-PCR, and (4) Flow-FISH. RT-PCR and Flow-FISH overcome the necessity for large amounts of genomic DNA required for Q-FISH. RT-PCR establishes the telomere to single copy gene ratio, which is proportional to the average telomere length within a cell, whereas Flow-FISH is adapted from Q-FISH and uses the median fluorescence detected by flow cytometry. The telomerase activity is an indicator of cell growth kinetics; thus, telomere length or telomerase activity measurements serve as a criterion to identify stem and progenitor cells. Equipment required: Thermocycler, ELISA plate reader (need 450- and 690-nm filters), PCR tubes, aerosol-resistant pipette tips (RNase-free), laboratory film. Protocol

Telomere Length 1 . Expand the cells to 60–80% confluence. 2. Resuspend the cells and transfer them to 1.7-mL microtubes at 1 × 106 cells per tube. 3. Following the manufacturer’s directions, suspend the cells in a hybridization solution containing a fluorescein

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i­sothiocyanate conjugated telomere probe or in a ­probe-free hybridization solution provided in, for example, a ­telomere assay kit (Dako Cytomation). 4. Hybridize each phenotype overnight at room temperature, in duplicate. 5. Wash the cells and incubate the cells with propidium iodide (0.5 mg/mL) to select the cells in G0/G1 of the cell cycle to normalize the data to equivalent genome loads. 6. Analyze the cells in a FACSVantage SE machine (BD Biosciences; or equivalent) to detect the fluorescence intensity of the samples. 7. The fluorescence intensity reflects the relative telomere length [telomere fluorescence per chromosome/genome in the sample with respect to that in control (no fluorescent probe)]. 8. Each duplicate forms one experiment; an average of three experiments is required to determine the significance. Telomerase Activity 1. Lyse 103 cells with lysis buffer provided by the Telomeric Repeat Amplification Protocol (TRAP)-eze telomerase detection kit (Chemicon). 2. After following the manufacturer’s detailed instructions, the PCR products and a six-base-pair incremental ladder are electrophoresed on a 12.5% nondenaturing polyacrylamide gel and visualized by SYBR gold staining (Molecular Probes).

6 Hematopoietic Stem Cells Pre-enrichment of mononuclear cells is an important initial step in the recovery of rare stem cell populations from blood. This is most efficiently carried out using a FicollPaque density gradient centrifugation. This technique removes erythrocytes and granulocytes in the sediment from centrifuged, anticoagulant-treated, and diluted blood.

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2. Carefully layer 30  mL of diluted blood on the FicollPaque solution. It is important not to mix the blood and Ficoll-Paque solution. 3. Centrifuge the mixture for 40 min at 400g at 20°C. 4. Collect the mononuclear cell fraction carefully using a Pasteur pipette at the interface between plasma and FicollPaque and transfer it into a clean centrifuge tube. N.B. • If erythroid cells are present in the interface, try treatment with 8% ammonium chloride or 3% diethylene glycol. −− Centrifuge cells for 10 min at 700g at 20°C. −− Add 5–20 mL of lysis solution to the pellet, mix the suspension, and incubate the mixture for 5–10 min at room temperature. −− Centrifuge the mixture for 10  min at 700g at 20°C. Discard the supernatant and proceed to step 5. • Add 40 mL PBS/EDTA to wash the mononuclear fraction and centrifuge the mixture for 10  min at 300g at 20°C. • Discard the supernatant and repeat the wash with 40 mL PBS/EDTA and centrifuge the mixture again. • Discard the supernatant and resuspend the mononuclear cells in 5–10  mL of PBS/0.5% BSA/2  mM EDTA and count the cells. • HSCs or MSCs can be further sorted by FACS using the specific cell-surface markers listed in Table 1.

7 Endothelial Progenitor Cells The isolation and characterization of circulating EPCs is described in detail in Mead et al. [42]. Table 1 details the key cell-surface markers used to identify EPCs and Table  2 provides a detailed comparison of the cellular markers and vasculogenic activity of derivatives of EPCs.

6.1 Ficoll-Paque Density Gradient Centrifugation

7.1 Uptake of 1,1¢-Dioctadecyl-3,3,3¢,3¢tetramethylindocarbocyanine Perchlorate Acetylated Low-Density It is imperative that all solutions and equipment are sterile Lipoprotein and used with proper aseptic technique [20]. Equipment required: Ficoll-Paque Plus (Amersham Biosciences) or equivalent, 500-mL Erlenmeyer flask or equivalent, 50-mL centrifuge tubes, centrifuge (preferably swinging-bucket rotor). Protocol

1. Place 15 mL Ficoll-Paque solution into a 50-mL centrifuge tube.

Acetylated low density lipoprotein (LDL) [30] is taken up by endothelial cells via the “scavenger cell pathway” of LDL metabolism [43]. By examining the fluorescent signals, uptake of 1,1¢-dioctadecyl-3,3,3¢,3¢-tetramethylindocarbocyanine perchlorate acetylated LDL (DiI-Ac-LDL) has been used to demonstrate that putative progenitor populations take on properties of functional endothelial cells [44, 45].

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Table  2  Delineation of endothelial progenitor cells (EPCs): key cellular markers and functional behavior. (Adapted from [31]. with permission. The data were collated from independent studies [31, 57]) Markers Circulating EPCs Resident EPCs Early-outgrowth EPCs (CFU-EC)

Late-outgrowth EPCs (ECFC)

Multipotent adult progenitor cells (MAPC)

Conduit-intimaderived EPCs

Conduit-vesselwall-derived EPCs(Vw-EPC)

Microcirculationderived EPCs (RMEPCs)

+ +/+ +

+ + ? +

+ + ? +

+ + ? +

+ ? +

+ + + +

+ + ?

+ + ?

+ +/-

? + ?

+ + ?

+ + -

+

-

?

-

+

-

Endothelial cell PECAM-1 VE-cadherin eNOS vWF Progenitor cell Mucosialin VEGFR2 Prominin-1 Leukocyte CD45 Functional behavior Vessel formation Morphology

No Spindle-like shape

Yes Yes Yes Yes Yes Cobble-stonelike pattern, spindle-like shape PECAM-1 platelet endothelial cell adhesion molecule, VEGFR2 vascular endothelial growth factor receptor 2, VE-cadherin vascular endothelial cadherin, eNOS endothelial nitric oxide synthase, vWF von Willebrand factor, CFU-EC colony forming unit - early outgrowth EPCs, ECFC Late outgrowth EPC, MAPC multipotent adult progenitor cells, VwEPC Conduit vessel wall derived EPCs, RMEPC resident microvascular EPCs

Equipment required: glass-bottom chamber slides, fluorescence microscope. Protocol 1 . Propagate endothelial cells on glass-bottom chamber slides. 2. Aspirate the culture medium and wash the cells with PBS. 3. Incubate the attached cells with 10 mg/mL DiI-Ac-LDL in complete endothelial growth medium (e.g., EGM, Lonza) for 4 h at 37°C. 4. Aspirate the medium and wash the cells twice in PBS to remove free DiI-Ac-LDL. 5. Fix the cells with 3% paraformaldehyde/PBS for 10 min. 6. Mount the slide with Vectashield with DAPI (Vector Lab). 7. Examine the slide with a rhodamine filter for DiI-Ac-LDL and a Hoechst filter for DAPI in a fluorescence microscope. Acquire images.

7.2 Network Formation In Vivo and Tube Formation In Vitro To assess the functional properties of putative EPCs, an important criterion is to demonstrate the formation of bona fide tubes, which is a characteristic unique to endothelial cells [46]. The in vitro tube formation assay is designed to grow putative EPCs in Matrigel and assess the formation of tube structures. Although this assay can reveal the endothelial potential of a specific cell population, the most rigorous test of the true

potential of putative progenitors is engraftment into, or de novo formation of, functional blood vessels in vivo. To do this, putative progenitors are intravenously injected into injured animals, directly injected into injured tissues, or implanted within a matrix or tumor environment. Prelabeling the cells of interest with a fluorescent dye [47] or transducing cells with a viral-driven fluorescent reporter [48] is usually carried out prior to injection to allow for precise microscopic examination of vasculature within injured or implantation sites. Furthermore, this will determine whether the cells are engrafted into the endothelium or a mural cell layer of blood vessels. Analysis of thick (50–70-mm) sections via confocal imaging, image analysis, and 3D-image reconstruction allows for the clear determination of cell engraftment in vessel structures. Transmission electron microscopy may be used to demonstrate further the ultrastructure of the tube formed in the in vitro assay and the blood vessels formed in the in vivo assay.

7.2.1 Assessment of Tube Formation In Vitro Equipment required: 48-well plates, light microscope [31]. Protocol 1. Seed putative progenitor cells at 2 × 104 cells/cm2 in Matrigel (BD Biosciences) coated (100 mL/cm2) 48-well plates, approximately 20,000 cells per well. Each well should contain 400  mL standard culture medium. Lung fibroblasts are suitable for use as a control. Each putative progenitor cell phenotype should be seeded triplicate.

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2. Incubate the plates in a humidified atmosphere at 37°C with 5% CO2/21% O2. The medium should be changed 4 days after the initial seeding. 3. Take images with a phase-contrast microscope at ×10 magnification at 8, 24, and 48 h and 1 week after seeding. 4. Determine the network formation by counting the number of branches that connect two distant cells. 5. Average counts from three wells at ×10 magnification are classed as one experiment; three separate experiments should be completed to determine a result. 7.2.2 Assessment of De Novo Vessel Formation In Vivo Equipment required: 23-gauge needle, microtome, light microscope [31]. Protocol 1. Resuspend the cells in 1.7-mL microtubes containing 250 mL standard culture medium at 4°C. The cell density should be 375,000 cells per tube. 2. Mix the cell-containing solution with 500 mL of unpolymerized Matrigel at 4°C. The cold temperature is critical to prevent polymerization of the Matrigel. 3. Mildly sedate a rat (e.g., intraperitoneal administration of ketamine 75 mg/kg). 4. Inject the 750 mL of the cell/Matrigel mix subcutaneously into the left and right lumbar abdominal regions of rats using a 23-gauge needle. This will create two plugs per rat. The injected mixture polymerizes at body temperature and forms a plug following subcutaneous contact. 5. Controls should be carried out in parallel consisting of a Matrigel mix with no cells. 6. Anesthetize the rat using sodium pentobarbital (50 mg/ kg admistered intraperitoneally). 7. Excise the Matrigel plugs from the abdominal wall of animals at 4 and 10 days after injection and fix the plugs by immersion in 4% paraformaldehyde for 18–24 h. 8. Dehydrate the fixed plugs in ethanol and embed them in paraffin. 9. Attach the tissue block to a plastic block by melting the back of the tissue block with a warm spatula and firmly pressing the two together and then use a standard microtome to cut 5-mm sections. 10. Stain the sections with hematoxylin and eosin following a standard protocol. 11. Examine the stained sections with a light microscope to count the total number of tubes containing red blood cells (i.e., blood vessels) within the gel. Blood vessels at the periphery of the gel in close proximity to the subjacent tissue or embedded within native tissue anywhere in the gel are not included for quantification. 12. The results denote the average from six different plugs obtained from different rats.

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8 Mesenchymal Stem Cells Table 1 details the key cell-surface markers used to identify MSCs and mesenchymal progenitor cells. Once isolated by FACS, putative MSCs can be confirmed by verifying their characteristic ability to differentiate into adipocytes, chondrocytes, osteocytes, and myocytes. Complete kits designed for adipocyte, chondrocyte, and osteocyte differentiation from MSCs are commercially available from Invitrogen. The method is described briefly in the following section. To preserve MSCs in culture without skewing the differentiation potential, use the MSC growth medium (Lonza) [49].

8.1 Adipocyte Differentiation Equipment required: culture vessel (12- or 96-well tissueculture plates or 16-well Culture Well slides are ideal). Protocol: The protocol is adapted from the Invitrogen adipocyte differentiation protocol and is designed for use with the StemPro® adipocyte differentiation kit: 1. Grow putative MSCs in a suitable growth medium to 60–80% confluence. Aspirate the medium and gently wash the cells in Dulbecco's PBS (DPBS). 2. Add 2–7 mL of prewarmed TrypLE™ Express (Invitrogen), sufficient to cover the culture surface and incubate the mixture until cells have detached (approximately 3–8 min at 37°C) 3. Gently triturate the detached cells to form a single cell solution using a wide-bore glass pipette and check the suspension using an inverted microscope. 4. In a centrifuge tube, pellet the cells at 100g for 5 min. 5. Cell viability and total cell density may be determined at this stage using trypan blue stain and by counting the cells using a hemocytometer (or other suitable cell counting method). 6. Resuspend the pellet in an appropriate volume of prewarmed MSC growth medium and seed the putative MSCs into the selected culture vessel at a density of 1 × 104 cells/ cm2. Culture vessels should be selected as follows: (a) For classic stain differentiation assay, use a 12-well plate. (b) For gene expression profiling, use a T-75 flask. (c) For immunocytochemistry, use a 16-well CultureWell™ chambered cover glass. 7. Incubate the cells in a humidified atmosphere at 37°C, 5% CO2, for 2 h to 4 days. 8. Replace the medium with prewarmed adipogenesis differentiation medium and continue incubation, changing the medium every 3–4 days. It should be noted that MSCs may continue to undergo limited expansion as they differentiate. 9. After determined periods of incubation, staining the cultures with Oil Red O will confirm the presence of adipocytes.

43  Identification of Adult Stem and Progenitor Cells in the Pulmonary Vasculature

8.2 Chondrocyte Differentiation The protocol is adapted from the Invitrogen chondrocyte differentiation protocol and is designed for use with the StemPro® chondrocyte differentiation kit. 1. Respect steps 1–5 from the adipogenesis differentiation protocol. 2. Resuspend the pellet in an appropriate volume of prewarmed MSC growth medium and generate a cell solution of viable cells at a density of 1.6 × 107 cells/mL. 3. Generate micromass cultures by seeding 5-mL droplets of the cell solution either: (a) In the center of multiwell plate wells for classic staining or (b) In the center of a 100-mm Petri dish for gene expression, protein detection, or immunohistochemistry. 4. After cultivating these micromass cultures for 2 h under high-humidity conditions, add warmed chondrogenesis medium and incubate the mixture at 37°C in 5% CO2. 5. Fresh medium should be added every 2–3 days. 6. After determined periods of incubation, the chondrogenic pellets can be confirmed by staining with alcian blue or safranin O (more than 14 days). • Alcian blue stain analysis: −− After differentiation for more than 14 days remove the medium, wash the cells and fix them in 4% paraformaldehyde for 10–30 min. −− Wash the fixed cells and stain them with 1% alcian blue solution prepared in 0.1 N HCl for 30 min. −− Wash the cells three times with 0.1 N HCl, add distilled water to neutralize the acidity, and visualize the cells under a light microscope. Blue staining is indicative of proteoglycan synthesis by chondrocytes.

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• Alizarin red S stain analysis: −− After more than 21  days, remove the medium and wash the cells once in DPBS. Fix the cells with 4% paraformaldehyde for 10–30 min. −− Wash the fixed cells twice with distilled water and stain them with 2% alizarin red S solution (pH 4.2) for 2–3 min. −− Wash the cells three times with distilled water and visualize them under a light microscope.

8.4 Myocyte Differentiation The myogenic differentiation potential of MSCs has been demonstrated and, most recently, human MSCs have proven to be an excellent source of smooth muscle cells for arterial engineering [49, 50]. Transforming growth factor b (TGFb) enhances this differentiation potential and progression to a smooth muscle cell contractile phenotype [51]. Gong et al. assessed the effects of culture medium and growth factors on the smooth muscle differentiation potential of MSCs and concluded that a combination of MesenPro RS™ (Invitrogen) and TGFb was the strongest inducer of a smooth muscle cell phenotype [49]. Protocol [49] 1. Repeat steps 1–8 from the adipogenesis differentiation protocol, except. (a) Seed the putative MSCs into the selected culture vessel at a density of 2.4 × 103 cells/cm2. (b) Replace the medium with prewarmed MesenPro RS™ with 1  ng/mL TGFb and change the medium twice a week. 2. After approximately 14  days, cultures can be processed for expression of smooth muscle markers, including, but not exclusively, SM-22a (transgelin), smooth muscle myosin heavy chain, smoothelin, and caldesmon.

8.3 Osteocyte Differentiation The protocol is adapted from the Invitrogen chondrocyte differentiation protocol and is designed for use with the StemPro® osteocyte differentiation kit. 1. Repeat steps 1–8 from the adipogenesis differentiation protocol, except: • Seed the putative MSCs into the selected culture vessel at a density of 5 × 103 cells/cm2. • Replace the medium with prewarmed complete osteogenesis differentiation medium. 2. After specific periods of cultivation, osteogenic cultures can be processed for alkaline phosphatase staining (7–14 days) or alizarin red S staining (more than 21 days).

9 Example Study: Identification and Characterization of EPCs Alvarez et al. published a comprehensive study which encapsulates the methodology outlined above to identify and characterize progenitor cells in the vascular endothelium [31]. The objective was to determine whether the lung microvasculature possesses EPCs and whether they are capable of rapid de novo vasculogenesis. Pulmonary arterial endothelial cells (PAECs) and pulmonary microvascular endothelial cells (PMVECs) were isolated from rats and characterized initially by FACS using a multipanel of cell-surface and cytosolic markers. A single-cell

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clonogenic assay was performed as described earlier. Owing to the highly proliferative nature of the PMVECs, the presence of resident microvascular EPCs (RMEPCs) was predicted. Using the single-cell clonogenic assay Alvarez et al. sorted individual cells into collagen I coated 96-well plates. RMEPCs were selected as the single cells which had the ability to rapidly generate colonies of more than 2,000 cells. The RMEPCs were immunophenotyped by flow cytometry and defined as expressing endothelial cell markers (CD31, CD144, enothelial nitric oxide synthase, von Willebrand factor) and progenitor cell antigens (CD34 and CD309), but not the leukocyte marker CD45. Telomere lengths were examined and compared with those of PAECs and PMVECs by

fluorescent labeling of the telomeres. RMEPCs possessed the longest telomere length among all phenotypes, suggesting RMEPCs were in a rapid proliferation state [52]. Functional assays to examine the vasculogenesis of RMEPCs were then carried out. An in vitro network formation assay was used; PMVEC and RMEPCs generated extensive networks as early as 8 and 24 h after seeding, but PAECs failed to generate extensive networks (Fig. 5). De novo vessel formation in vivo was also assessed. After 4 days, blood vessels (tubes containing red blood cells) were found in all plugs and after 10 days, all plugs were enriched in numerous, wellorganized vessels (Fig. 6). PMVECs and RMEPCs formed twofold more blood vessels than PAECs. To verify that

Fig.  5  In vitro endothelial progenitor cell (EPC) tube formation from EPCs. Resident microvascular EPCs (RMEPCs) demonstrated a rapid tube formation in the in vitro Matrigel assay. At 24 h after seeding, pulmo-

nary microvascular endothelial cells (PMVECs) and RMEPCs have interconnecting cellular extensions which were not observed in pulmonary arterial endothelial cells (PAECs). (Reproduced with permission [31])

Fig. 6  In vivo EPC de novo vessel formation in Matrigel plugs. RMEPCs exhibit rapid neovasculogenesis in in vivo Matrigel plugs. The number of de novo vessels formed by 10 days after injection was not different among

cell phenotypes. The arrows indicate the appearance of blood vessels. Notice that blood vessels also appear in the middle of collagen tracks in the control plugs injected with no cells. (Reproduced with permission [31])

43  Identification of Adult Stem and Progenitor Cells in the Pulmonary Vasculature

de  novo vessels were derived from the injected cells, RMEPCs were transduced with a retroviral construct encoding green fluorescent protein (GFP). GFP-positive cells were detected lining the vessel lumens. Ultrastructural analysis of the in vitro tube formation and de novo vessels was examined by transmission electron microscopy to determine the quality of the vessels formed. Distinguishable lumens and vessel junctions were observed. This study elegantly shows that the pulmonary microvasculature is enriched with RMEPCs that have vasculogenic competence and contribute to the maintenance of a functional endothelium. The study provides a nice representative example of isolation, identification, and characterization of a functional progenitor cell population within the pulmonary vasculature.

10 Application and Limitations The methodology described in the current chapter could be used to complete a basic identification and characterization of stem and progenitor cells in the pulmonary vasculature of both normal and diseased subjects. The major limitation currently in identifying putative stem cells is the lack of exclusively specific identifying markers for different progenitor cells. In addition, the functional tests cannot be completely conclusive. For EPCs, for example, some of the phenotypic characteristics and behaviors that are often specifically attributed to endothelial cells are, in fact, shared by other cell types, most notably myeloid cells and epithelial cells. Monocytes and macrophages also take up acylated LDL via the scavenger pathway and thus have properties similar to those of endothelial cells [53, 54]. Therefore, uptake of DiI-Ac-LDL alone cannot be used to specifically identify an endothelial cell phenotype or to demonstrate endothelial cell functional properties of putative progenitors. Similarly, in the in  vitro tube formation assay, many cell types are capable of forming cord-like structures in similar 3D systems. It is important to demonstrate the formation of bona fide tubes not just cord structures. In de novo vessel formation assay, the demonstration of residence in the endothelial cell layer alone is not indicative of “differentiation” of putative progenitors into functional endothelial cells.

11 Summary and Discussion Identification of a genuine stem or progenitor cell is not an easy task. Preliminary investigations rely upon immunophenotyping of the cell population and sorting putative stem and progenitor cells on the basis of their expression of certain cell-surface or cytosolic markers. Only then can a rigorous functional characterization take place to delineate

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the proliferative potential of the cells in question. Although this chapter is not exhaustive, it serves to provide examples of essential methodological techniques to start a basic characterization process. The field of stem and progenitor cells in pulmonary vascular disease is continually progressing and more defined lineage differentiation pathways of stem and progenitor cells will unfold, allowing for a more precise characterization of putative progenitor cells.

References 1. Becker AJ, Mc CE, Till JE (1963) Cytological demonstration of the clonal nature of spleen colonies derived from transplanted mouse marrow cells. Nature 197:452–454 2. Thomson J, Itskovitz-Eldor J, Shapiro SS et al (1998) Embryonic stem cell lines derived from human blastocysts. Science 282:1145–1147 3. Jiang Y, Jahagirdar BN, Reinhardt RL et al (2002) Pluripotency of mesenchymal stem cells derived from adult marrow. Nature 418:41–49 4. Chao MP, Seita J, Weissman IL (2008) Establishment of a normal hematopoietic and leukemia stem cell hierarchy. Cold Spring Harb Symp Quant Biol 73:439–449 5. Leblond CP (1964) Classification of cell populations on the basis of their proliferative behavior. Natl Cancer Inst Monogr 14:119–150 6. Xu J, Qu J, Cao L et al (2008) Mesenchymal stem cell-based angiopoietin-1 gene therapy for acute lung injury induced by lipopolysaccharide in mice. J Pathol 214:472–481 7. Lama V, Smith L, Badri L et al (2007) Evidence for tissue-resident mesenchymal stem cells in human adult lung from studies of transplanted allografts. J Clin Invest 117:989–996 8. Martin J, Helm K, Ruegg P, Varella-Garcia M, Burnham E, Majka S (2008) Adult lung side population cells have mesenchymal stem cell potential. Cytotherapy 10:140–151 9. Summer R, Kotton DN, Liang S, Fitzsimmons K, Sun X, Fine A (2005) Embryonic lung side population cells are hematopoietic and vascular precursors. Am J Respir Cell Mol Biol 33:32–40 10. Summer R, Fitzsimmons K, Dwyer D, Murphy J, Fine A (2007) Isolation of an adult mouse lung mesenchymal progenitor cell population. Am J Respir Cell Mol Biol 37:152–159 11. Kanki-Horimoto S, Horimoto H, Mieno S et al (2006) Implantation of mesenchymal stem cells overexpressing endothelial nitric oxide synthase improves right ventricular impairments caused by pulmonary hypertension. Circulation 114:I181–I185 12. Baber SR, Deng W, Master RG et al (2007) Intratracheal mesenchymal stem cell administration attenuates monocrotaline-induced pulmonary hypertension and endothelial dysfunction. Am J Physiol Heart Circ Physiol 292:H1120–H1128 13. Asosingh KAM, Vasanji A, Drazba J et al (2008) Circulating angiogenic precursors in idiopathic pulmonary arterial hypertension. Am J Pathol 172:615–627 14. Peinado VI, Ramírez J, Roca J, Rodriguez-Roisin R, Barberà JA (2006) Identification of vascular progenitor cells in pulmonary arteries of patients with chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol 34:257–263 15. Hashimoto N, Jin H, Liu T, Chensue SW, Phan SH (2004) Bone marrow-derived progenitor cells in pulmonary fibrosis. J Clin Invest 113:243–252 16. Walter DH, Rittig K, Bahlmann FH et  al (2002) Statin therapy accelerates reendothelialization: a novel effect involving mobilization and incorporation of bone marrow-derived endothelial progenitor cells. Circulation 105:3017–3024

636 17. Saiura A, Sata M, Hirata Y, Nagai R, Makuuchi M (2001) Circulating smooth muscle progenitor cells contribute to atherosclerosis. Nat Med 7:382–383 18. Sata M, Saiura A, Kunisato A et al (2002) Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis. Nat Med 8:403–409 19. Werner N, Priller J, Laufs U et al (2002) Bone marrow-derived progenitor cells modulate vascular reendothelialization and neointimal formation: effect of 3-hydroxy-3-methylglutaryl coenzyme a reductase inhibition. Arterioscler Thromb Vasc Biol 22:1567–1572 20. Jaatinen T, Laine J (2007) Isolation of mononuclear cells from human cord blood by Ficoll-Paque density gradient. Curr Protoc Stem Cell Biol Unit 2A:1 21. Robb L, Drinkwater CC, Metcalf D et al (1995) Hematopoietic and lung abnormalities in mice with a null mutation of the common beta subunit of the receptors for granulocyte-macrophage colony-stimulating factor and interleukins 3 and 5. Proc Natl Acad Sci U S A 92:9565–9569 22. Stanley E, Lieschke GJ, Grail D et  al (1994) Granulocyte/macrophage colony-stimulating factor-deficient mice show no major perturbation of hematopoiesis but develop a characteristic pulmonary pathology. Proc Natl Acad Sci U S A 91:5592–5596 23. Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I et  al (2006) Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8:315–317 24. Loebinger MR, Sage EK, Janes SM (2008) Mesenchymal stem cells as vectors for lung disease. Proc Am Thorac Soc 5:711–716 25. Zhao F, Zhang YF, Liu YG et al (2008) Therapeutic effects of bone marrow-derived mesenchymal stem cells engraftment on bleomycininduced lung injury in rats. Transplant Proc 40:1700–1705 26. Asahara T, Murohara T, Sullivan A et al (1997) Isolation of putative progenitor endothelial cells for angiogenesis. Science 275:964–967 27. Zhao Y, Courtman DW, Deng Y, Kugathasan L, Zhang Q, Stewart DJ (2005) Rescue of monocrotaline-induced pulmonary arterial hypertension using bone marrow-derived endothelial-like progenitor cells: efficacy of combined cell and eNOS gene therapy in established disease. Circ Res 96:442–450 28. Obi S, Yamamoto K, Shimizu N et  al (2009) Fluid shear stress induces arterial differentiation of endothelial progenitor cells. J Appl Physiol 106:203–211 29. Zampetaki A, Kirton JP, Xu Q (2008) Vascular repair by endothelial progenitor cells. Cardiovasc Res 78:413–421 30. Ingram DA, Mead LE, Tanaka H et  al (2004) Identification of a novel hierarchy of endothelial progenitor cells using human peripheral and umbilical cord blood. Blood 104:2752–2760 31. Alvarez DF, Huang L, King JA, ElZarrad MK, Yoder MC, Stevens T (2008) Lung microvascular endothelium is enriched with progenitor cells that exhibit vasculogenic capacity. Am J Physiol Lung Cell Mol Physiol 294:L419–L430 32. Asakura A, Rudnicki MA (2002) Side population cells from diverse adult tissues are capable of in vitro hematopoietic differentiation. Exp Hematol 30:1339–1345 33. Budak MT, Alpdogan OS, Zhou M, Lavker RM, Akinci MA, Wolosin JM (2005) Ocular surface epithelia contain ABCG2dependent side population cells exhibiting features associated with stem cells. J Cell Sci 118:1715–1724 34. Anderson DJ, Gage FH, Weissman IL (2001) Can stem cells cross lineage boundaries? Nat Med 7:393–395 35. Holden C, Vogel G (2002) Stem cells. Plasticity: time for a reappraisal? Science 296:2126–2129 36. Verfaillie CM (2002) Adult stem cells: assessing the case for pluripotency. Trends Cell Biol 12:502–508

A.L. Firth et al. 37. Kurien BT, Scofield RH (2006) Western blotting. Methods 38:283–293 38. Kubista M, Andrade JM, Bengtsson M et al (2006) The real-time polymerase chain reaction. Mol Aspects Med 27:95–125 39. Ingram DA, Mead LE, Moore DB, Woodard W, Fenoglio A, Yoder MC (2005) Vessel wall-derived endothelial cells rapidly proliferate because they contain a complete hierarchy of endothelial progenitor cells. Blood 105:2783–2786 40. Allsopp RC, Morin GB, Horner JW, DePinho R, Harley CB, Weissman IL (2003) Effect of TERT over-expression on the longterm transplantation capacity of hematopoietic stem cells. Nat Med 9:369–371 41. Artandi SE (2006) Telomeres, telomerase, and human disease. N Engl J Med 355:1195–1197 42. Mead LE, Prater D, Yoder MC, Ingram DA (2008) Isolation and characterization of endothelial progenitor cells from human blood. Curr Protoc Stem Cell Biol Unit 2C:1 43. Voyta JC, Via DP, Butterfield CE, Zetter BR (1984) Identification and isolation of endothelial cells based on their increased uptake of acetylated-low density lipoprotein. J Cell Biol 99:2034–2040 44. Reyes M, Dudek A, Jahagirdar B, Koodie L, Marker PH, Verfaillie CM (2002) Origin of endothelial progenitors in human postnatal bone marrow. J Clin Invest 109:337–346 45. Werner N, Junk S, Laufs U et al (2003) Intravenous transfusion of endothelial progenitor cells reduces neointima formation after vascular injury. Circ Res 93:e17–e24 46. Madri JA, Pratt BM (1986) Endothelial cell-matrix interactions: in vitro models of angiogenesis. J Histochem Cytochem 34:85–91 47. Yoon CH, Hur J, Oh IY et  al (2006) Intercellular adhesion molecule-1 is upregulated in ischemic muscle, which mediates trafficking of endothelial progenitor cells. Arterioscler Thromb Vasc Biol 26:1066–1072 48. Brunt KR, Hall SR, Ward CA, Melo LG (2007) Endothelial progenitor cell and mesenchymal stem cell isolation, characterization, viral transduction. Methods Mol Med 139:197–210 49. Gong Z, Calkins G, Cheng EC, Krause D, Niklason LE (2008) Influence of culture medium on smooth muscle cell differentiation from human bone marrow-derived mesenchymal stem cells. Tissue Eng Part A 14:1615 50. Gong Z, Niklason LE (2008) Small-diameter human vessel wall engineered from bone marrow-derived mesenchymal stem cells (hMSCs). FASEB J 22:1635–1648 51. Kinner B, Zaleskas JM, Spector M (2002) Regulation of smooth muscle actin expression and contraction in adult human mesenchymal stem cells. Exp Cell Res 278:72–83 52. Murasawa S, Llevadot J, Silver M, Isner JM, Losordo DW, Asahara T (2002) Constitutive human telomerase reverse transcriptase expression enhances regenerative properties of endothelial progenitor cells. Circulation 106:1133–1139 53. Rohde E, Malischnik C, Thaler D et  al (2006) Blood monocytes mimic endothelial progenitor cells. Stem Cells 24:357–367 54. Rafii S, Lyden D (2008) Cancer. A few to flip the angiogenic switch. Science 319:163–164 55. Reyes M, Lund T, Lenvik T, Aguiar D, Koodie L, Verfaillie CM (2001) Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells. Blood 98:2615–2625 56. Pittenger MF, Mackay AM, Beck SC et  al (1999) Multilineage potential of adult human mesenchymal stem cells. Science 284:143–147 57. Mukai N, Akahori T, Komaki M et al (2008) A comparison of the tube forming potentials of early and late endothelial progenitor cells. Exp Cell Res 314:430–440

Chapter 44

Differentiation of Embryonic Stem Cells to Vascular Cell Lineages Andriana Margariti, Lingfang Zeng, and Qingbo Xu

Abstract  In the face of extraordinary advances in the ­prevention, diagnosis, and treatment of human diseases, devastating cardiopulmonary diseases continue to deprive people of health. Research in developmental biology has led to the discovery of stem cells, which provide a platform to understand the basic mechanism of those diseases and give a hope for potential clinical applications. Three main types of stem cells have been characterized, including the embryonic stem (ES) cells, embryonic germ stem cells, and tissue-­ resident, or “adult,” stem cells, on the basis of their origin and pluripotent ability. The source of the ES cells is the inner cell mass of a blastocyst, which is a very early embryo that contains 200–250 cells and is shaped like a hollow sphere. Embryonic germ line cells come from a human fetus that is 5–9 weeks old and are derived from tissue that would have developed into the ovaries or testes. “Adult” stem cells are derived from the umbilical cord and placenta or from blood, bone marrow, skin, and other tissues. A major distinction between the ES cells and the germ line cells is the capacity of the former to develop into every type of tissue found in an adult, whereas the latter are less versatile. Similarly, progenitor cells are characterized as pluripotent cells that can replicate and differentiate into a restricted number of cell types. This chapter will focus on the methods, mechanisms, and applications of ES cells derived from both human and mouse origins that differentiate into vascular cells. Keywords  Stem cell differentiation • Pluripotency • Multipotency • Vascular progenitor cell • Smooth muscle cell • Endothelial cell

1  Introduction In the face of extraordinary advances in the prevention, diagnosis, and treatment of human diseases, devastating illnesses such as heart disease, diabetes, cancer, and Parkinson’s disease continue to deprive people of health. Research in developmental biology has led to the discovery of stem cells, which provide a platform to understand the basic mechanism of those diseases and give a hope for potential clinical applications. Three main types of stem cells have been characterized, including the embryonic stem (ES) cells, embryonic germ stem cells, and tissue-resident, or “adult,” stem cells, on the basis of their origin and pluripotent ability. The source of the ES cells is the inner cell mass of a blastocyst [1, 2], which is a very early embryo that contains 200–250 cells and is shaped like a hollow sphere. Embryonic germ line cells come from a human fetus that is 5–9 weeks old and are derived from tissue that would have developed into the ovaries or testes. “Adult” stem cells are derived from the umbilical cord and placenta or from blood, bone marrow, skin, and other tissues [3]. A major distinction between the ES cells and the germ line cells is the capacity of the former to develop into every type of tissue found in an adult, whereas the latter are less versatile. Similarly, progenitor cells are characterized as pluripotent cells that can replicate and differentiate into a restricted number of cell types. This chapter will focus on the methods, mechanisms, and applications of ES cells derived from both human and mouse origins that differentiate into vascular cells.

2  Embryonic Stem Cells

A. Margariti (*) Cardiovascular Division, King’s College London, 127 Coldharbour Lane, London SE5 9NU, UK e-mail: [email protected]

In 1967, embryonic carcinoma cell lines were established as the first pluripotent cell lines, derived from the undifferentiated compartment of murine and human germ cell tumors [4]. These cells had the capability to proliferate continuously in culture and differentiate into derivates of all three embryonic

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_44, © Springer Science+Business Media, LLC 2011

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germ layers: endoderm, ectoderm, and mesoderm [5]. Embryonic carcinoma cell lines have been proven to be a very useful model system in the laboratory. However, a number of limitations precluded their use for clinical application. In 1981, murine ES cells were isolated and grown in vitro [1, 2], providing a model system to study the basic processes of early embryonic development and cellular differentiation. Human ES cells were eventually derived 17 years later [6], making regenerative medicine and tissue engineering a possibility for the future treatment of human disease. The source of human ES cells varies, including donated blastocysts, poor-quality discarded embryos, embryos carrying genetic defects, and from unusable aneuploid zygotes. The main characteristics of ES cells are their ability to be maintained and expanded as pure populations of undifferentiated cells for extended periods of time in culture, and their unique totipotent capacity to differentiate into specific cell lineages in response to different stimuli in vitro [7–9]. In the absence of the factors that maintain ES cell pluripotency, the differentiation process is initiated, and under specific conditions ES cells undergo differentiation toward specific cell lineages consisting of derivatives of the three embryonic germ layers [8]. Here, we will discuss in detail the potential of ES cells to differentiate toward vascular cell lineages such as endothelial cells (ECs) and smooth muscle cells (SMCs).

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number of cell lineages in response to specific stimuli and the microenvironment (Fig. 1). In the last few years, an exciting goal was achieved as ES cells were reported to differentiate into vascular ECs and SMCs [21], demonstrating a potential cell source for vascular injury repair [7]. Wartenberg et  al. induced ES cells to differentiate into ECs within the three-dimensional tissue of embryoid bodies forming capillary-like structures, and expression of CD31 (platelet EC adhesion molecule 1, PECAM1) [22]. Other groups have demonstrated that mouse ES cells are able to differentiate into ECs [14, 23], and SMCs [12, 24]. In particular, cells expressing endothelial-specific cell markers were obtained after embryoid bodies had been embedded into collagen in the presence of angiogenic growth factors [vascular endothelial growth factor (VEGF), basic fibroblast growth factor (FGF), interleukin-6 ,and erythropoietin] [25, 26]. Consistent with the above-mentioned findings, a study demonstrated that human ES cells contained a population of vascular progenitor cells that had the ability to differentiate into EC-like and SMC-like cells [11]. Specifically, vascular progenitor cells were isolated from embryoid bodies and grown in suspension for 10 days; they expressed endothelial/hematopoietic marker CD34. When these cells were subsequently cultured in endothelial growth medium 2 (EGM-2) supplemented with VEGF165, they were able to give rise to endothelial-like cells characterized by a cobblestone cell morphology, and expression of endothelial

3  ES Cell Differentiation Toward Vascular Cell Lineages In vivo, stem cells reside in a special microenvironment called a niche, which maintains their self-renewal ability. Similarly, the specific microenvironment determines the “direction” of stem cell differentiation. Such microenvironmental cues include cytokines and growth factors [10, 11], extracellular matrix [12, 13], mechanical forces [14, 15], and communication with adjacent cells [16, 17]. A number of different approaches have been reported to initiate the ES cell differentiation process in vitro. For example, ES cells are cultured directly on stromal cells and differentiate when they are in contact with these cells [18], or ES cells are allowed to aggregate and form three-dimensional colonies known as embryoid bodies [8]. ES cells can also be differentiated by culturing on extracellular matrix proteins [19]. In addition to these different culture methods, ES cells are treated with growth factors and cytokines to direct the differentiation to a specific cell lineage. Selection of positive cells with specific cell markers and genetic manipulation are other remarkable approaches to direct ES cell differentiation. Importantly, parameters such as the duration in combination with culture conditions may modulate the differentiation process [20]. The data obtained provide the basic principle of stem cell differentiation that ES differentiation can be directed to a

Fig. 1  Embryonic stem (ES) cell differentiation to vascular lineages. Cytokines or growth factors, extracellular matrix, mechanical forces and communication with adjacent cells determine the “direction” of ES cell differentiation to embryoid bodies (EBs) and vascular progenitors. Selection of positive cells for specific cell markers and genetic manipulation are useful methods to direct the differentiation to specific and a pure vascular cell lineage such as endothelial cells (ECs) and smooth muscle cells (SMCs)

44  Differentiation of Embryonic Stem Cells to Vascular Cell Lineages

markers. In contrast, when CD34+ cells were cultured in EGM-2 supplemented with platelet-derived growth factor (PDGF)-BB, they gave rise to SMC-like cells characterized by spindle-shape morphology, and SMC marker expression [11]. Another report also showed that vascular progenitor cells were able to differentiate into contractile-type SMCs in the absence of VEGF, or into a synthetic type of SMCs in the presence of PDGF-BB [27]. These findings provide direct evidence that ES cells can differentiate to ECs and SMCs through a common vascular progenitor cell under the influence of the appropriate microenvironment, which activates specific pathways and directs the differentiation process. Importantly, it was shown that these cells were functional and able to integrate into endogenous heart tissue, demonstrating the ability to vascularize, while continuing to differentiate in vivo after engraftment into a mouse model [28]. In the present chapter, we will focus on the detailed description of the main protocols reported in ES cell differentiation toward vascular lineages. We will also discuss their potential clinical applications and limitations.

4  Human Versus Mouse ES Cells A number of protocols which highlight the potential of ES cells to differentiate toward vascular cell lineages have been developed in recent years. For better understanding of these protocols describing ES cell culture, it is essential for us to emphasize briefly the main differences between ES cells derived from human origin and ES cells derived mouse origin to speculate whether the same protocols can apply for both ES cell lineages. Over the last 20 years, mouse ES cells have been used in many fields of research and have made an enormous impact. Mouse ES cells were used to study the initial stages of mammalian development in vitro to dissect the basic mechanisms underlying pluripotency and cell lineage specification, also used widely to reconstitute early mouse embryos [29] and to produce “knockout” and “knock-in” transgenic animals. In contrast, the progress of human ES cell biology is less advanced. Human ES cells grow slowly compared with the murine cells, tending to form flat rather than spherical colonies and are more easily dissociated into single cells [30]. In addition, these two pluripotent stem cell lineages display several other important differences in cultures. Mouse ES cells are maintained in culture in an undifferentiated state in the presence of leukemia inhibitory factor (LIF) and/or on a feeder layer of murine embryonic fibroblasts (MEFs) [31]. Human ES cells do not respond to LIF and require culture on MEF feeder layers in the presence of bFGF [6], or on Matrigel or laminin in MEF-conditioned medium [32], suggesting that they use different signaling pathways to maintain their pluripotent status. Human ES cell differentiation is mainly induced by removing from the feeder layer and growing the cells in

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suspension. The differentiation process in suspension results in aggregation of the cells and formation of embryoid bodies [33]. In contrast, mouse ES cell differentiation is easily induced in adherent monolayer culture. Furthermore, bone morphogenic proteins (BMPs) have been shown to induce human ES cell differentiation in sharp contrast to mouse ES cells, in which BMP4 synergized with LIF to maintain selfrenewal [6, 32]. Therefore, it seems that these cells have a number of differences in terms of maintenance in undifferentiated stages and basic culture conditions. However, an interesting finding claimed that a “core molecular program” was shared between human and mouse ES cells. This was based on global transcriptional profile analysis of the state of “stemness” in human ES cells, showing several specific signaling components common with the mouse ES cell data [34]. Consequently, we may expect that the main protocols can apply in both ES cell lineages with equal success to generate vascular cell lineages.

5  Mouse ES-Derived Vascular Cells in Response to Extracellular Matrix Proteins and Growth Factors Extracellular matrix proteins consist of several types of structural proteins, such as collagens and fibronectin. Different types of extracellular matrix proteins appear to exert different effects on stem cell differentiation, leading to specific cell lineages. We have shown that collagen-IV favors stem cell differentiation toward vascular lineages, particularly SMCs [12] but also ECs [10]. In the absence of LIF, seeded on plates coated with collagen IV, ES cells spontaneously differentiated into SMCs, giving rise to a highly pure population of SMCs, which expressed a number of specific SMC markers, such as smooth muscle a-actin (SMA), SM22, smooth muscle myosin heavy chain (SMMHC), and calponin.

5.1  Protocol Mouse ES cells (ES-D3 cell line, CRL-1934; ATCC) are ­cultured in Dulbecco’s modified essential medium (DMEM, ATCC), supplemented with 10  ng/mL recombinant human LIF (Chemicon), 10% fetal bovine serum (FBS; ATCC), 2 mM l-glutamine (Sigma), 0.1  mM 2-mercaptoethanol (Sigma), 100 U/mL penicillin, and 100 mg/mL streptomycin (GIBCO) [35]. ES cells are fed every 2 days with fresh medium and passaged into gelatin-coated flasks with a 1:6 to 1:10 ratio every 2–4 days. To induce ES cell differentiation, cells are seeded on type IV mouse collagen (5  mg/mL) (Trevigen) in differentiation medium [MEM alpha medium (GIBCO) supplemented with 10% FBS (GIBCO), 0.05 mM

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2-mercaptoethanol, 100  U/mL penicillin, and 100  mg/mL streptomycin] for 3–4 days. The medium is refreshed every other day. Stem cell antigen-1-positive (Sca-1+) cells (vascular progenitors) are sorted from the cell culture by magnetic labeling cell sorting with anti-Sca-1 microbeads (Miltenyi Biotec). ES cells are detached with a 0.05% trypsin–EDTA solution (GIBCO) from flasks and incubated with the antibody-conjugated/coated microbeads at 4°C with occasional agitation for 15 min. The bead-bound cells are then selected using a magnetic cell separator (Miltenyi Biotec). Sca-1+ cells are resuspended and cultured in fresh ES cell growth medium. For SMC differentiation, Sca-1+ cells are plated on dishes or flasks coated with collagen IV, cultured in basic differentiation medium or basic differentiation medium plus 10 ng/mL PDGF-BB (Sigma) for 6 days (first passage), and then passaged every 3 days. For long-period culture of ES-cell-derived SMCs, cells grown to more than 80% confluence can be passaged into new collagen-coated flasks with a 1:2 to 1:3 ratio every 2–3 days and cultured in differentiation medium [12]. For EC differentiation, Sca-1+ cells are plated on dishes or flasks coated with collagen IV, and cultured in EC differentiation medium [basic differentiation medium plus 10  ng/mL VEGF165 (Bender MedSystems) for 21 days, passaged every 3 days, to obtain EC differentiation [10].

5.2  Application The first protocol provides a method for producing a large number of SMCs from ES cells and demonstrates that Sca-1+ progenitor cells derived from ES cells can differentiate into SMCs with high purity (more than 95%), expressing SMCspecific markers such as SMA, calponin, and SMMHC (Fig.  2). The remarkable role of the signaling pathways mediated by collagen IV and PDGF-BB important for Sca-1+ progenitor cell differentiation into SMCs is highlighted. Therefore, stem-cell-derived SMCs can be used as a source of cells for vascular tissue engineering and repair of injured vessels. Indeed, in vivo experiments have demonstrated that Sca-1+ cells can differentiate into SMCs that contribute to neointima formation [12]. Moreover, the second protocol describes an approach to select ECs using magnetic beads, which may hold the potential to be used therapeutically to treat arterial injury. Fluorescence-activated cell sorting analysis confirmed that 99% of ECs derived from ES cells were CD31-positive and 75% were von Willibrand factor positive. Almost all cells were positive for 1,1¢-dioctadecyl3,3,3¢,3¢-tetramethylindocarbocyanine perchlorate acetylated LDL uptake. Interestingly, further experiments revealed that when ES-derived ECs were mixed with Matrigel and subcutaneously implanted into mice, various vessel-like structures were observed. When ES-derived ECs were injected into

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Fig. 2  Stem cell antigen-1-positive progenitor cells derived from ES cells can differentiate into SMCs with high purity, expressing SMCspecific markers such as smooth muscle a-actin (SMA), calponin, and smooth muscle myosin heavy chain (SMMHC), as immunostaining experiments revealed. Nuclei were counterstained with 4¢,6-diamidino2-phenylindole (blue); original magnification ×400. (Reproduced from [12] with permission)

denuded femoral arteries of mice, they were found to form a neoendothelium that covered the injured areas (86 ± 13.6%), which resulted in a 73% decrease in neointimal area 2 weeks after injury [10]. Conclusively, ECs derived from ES cells are able to accelerate re-endothelialization of injured arteries and reduce neointima formation [10] (Fig. 3).

6 Human ES-Derived SMCs Human ES cells can differentiate to the SMC lineage by using a combination of cell culture medium and extracellular matrix environment [36], the detailed protocol is described next.

6.1 Protocol Human ES cells (H1 and H9 cells from WiCell, passages 32–45) are expanded on an irradiated MEF feeder layer in growth medium consisting of KnockOut Dulbecco’s modified Eagle’s medium (KO-DMEM), KnockOut serum replacement (KO-SR), l-glutamine, 2-mercaptoethanol, nonessential amino acids, and human bFGF (Invitrogen). Cells are split once a week by incubation in 1  mg/mL collagenase IV (Invitrogen) for 10 min at 37°C and seeded on freshly prepared inactivated MEFs. For formation of embryoid bodies, human ES cell colonies are digested by using 1  mg/mL

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Fig. 3  Transfer of ES-cell-derived ECs inhibited neointimal formation in injured artery. Representative photographs of normal artery (a), wireinjured artery (b), and ES-cell-derived EC-treated (c, d) femoral arteries.

Two weeks after cell transfer, mice were euthanized, and injured arteries were harvested, fixed, sectioned, and stained with H&E. Original magnification a–c ×100, d ×200. (Reproduced from [10] with permission)

c­ ollagenase type IV and transferred to an ultra-low-­attachment six-well plate (Corning) to allow aggregation in suspension. Human embryoid bodies are grown in DMEM (high glucose) supplemented with 10% FBS, l-glutamine, 2-mercaptoethanol, and nonessential amino acids. The medium is changed every day. Six-day old embryoid bodies can be transferred to six-well plates coated with 0.1% gelatin for 5–6 days, followed by digestion with TrypLE™ Express (Invitrogen) and cultured on Matrigel-coated six-well plates with 5 × 105 cells/ cm2 in smooth muscle growth medium from Lonza/Cambrex (growth conditions). Medium is then changed every day and the cells are further replated when 80–90% confluency is reached. To induce cell differentiation to SMCs, the cells should be replated in DMEM containing 5% FBS on gelatincoated plates (differentiation conditions). This cell model system provides a useful tool to study human SMC differentiation and vascular development as well as potential therapeutic targets for preventing vascular disease.

7.1  Protocol

7  Shear-Stress-Induced EC Differentiation We and others have shown that the imposition of mechanical stress on ES cells or embryonic mesenchymal progenitor cells induced differentiation into ECs and promoted the expression of angiogenic factors [14, 15].

Mouse ES cells (ES-D3 cell line, CRL-1934; ATCC, Manassas, USA) are cultured in gelatin-coated flasks in DMEM (ATCC) supplemented with 10% FBS (ATCC), 10  ng/mL LIF (Chemicon), 0.1  mM 2-mercaptoethanol, 100  U/mL penicillin, and 100  mg/mL streptomycin in a humidified incubator supplemented with 5% CO2, and split at a 1:6 ratio every other day. For differentiation, ES cells are cultured on glass slides coated with mouse collagen IV (5 mg/mL) in differentiation medium (MEM alpha medium; Invitrogen) supplemented with 10% FBS (Invitrogen), 0.05  mM 2-mecaptoethanol, 100  U/mL penicillin, and 100  mg/mL streptomycin for 3–4 days. The medium is refreshed every other day. Shear experiments are performed by exposing the predifferentiated cells to a laminar flow generated by the pressure difference between two reservoirs, with the effluent differentiation medium circulating back to the upper reservoir through a peristaltic pump. The shear stress, which was determined by the flow rate and the channel dimensions, is set at ~ 12 dyn/cm2, which is comparable to the physiological range in human major arteries. The entire flow system is placed in an incubator at 37°C and supplemented with 5% CO2 to maintain the pH. Static controls are cells cultured on slides not exposed to flow. Gentamycin (5  mg/mL) and 0.1 mg/mL doxycycline are included in the shear medium to prevent contamination [14].

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7.2  Application This protocol demonstrated that laminar flow enhanced the differentiation of stem cells into functional ECs, as sheared ES cells formed tubelike structures on Matrigel (Fig. 4). Further experiments showed that in vitro differentiated ECs could form blood vessels in  vivo, and appeared to play a crucial role in endothelial restoration and vascular repair [14]. These observations provide further support for the concept that differentiation of stem cells toward ECs is beneficial to the blood vessel.

8  ECs Derived from Mouse ES Cells Through Embryoid Body Formation Another protocol demonstrates that mouse ES-cell-derived embryoid bodies can be used as a vasculogenesis model. Embryoid bodies are formed for 3 days using cultures and plated on gelatin-coated plates in EGM-2 to promote vascular development [37].

8.1  Protocol Mouse D3 ES cells (ATCC) are co-cultured with mitomycin C treated MEF cells in high-glucose DMEM (GIBCO) containing 15% FBS (Hyclone), 1,000  U/mL of LIF/

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ESGRO (Chemicon), and basic ES medium components [50  U/mL of penicillin and 50  mg/mL streptomycin (GIBCO), 1% nonessential amino acids (GIBCO), and 0.1 mM 2-mercaptoethanol (GIBCO)]. Differentiation is induced using the hanging drop method. Twenty microliters of stem cell suspension (3.75 × 10[5]/mL) in ES cell medium [Dulbecco’s modified Eagle’s medium with 4.5 g glucose/L and 3.7 g NaHCO3/L (Biochrom) supplemented with 20% fetal calf serum (FCS) (Boehringer, Mannheim), 50  U penicillin/mL and 50  mg streptomycin/mL (Bio­ chrom), 1% NAA (Biochrom), 0.1  mM 2-mercaptoethanol (Sigma) and 1  mM glutamine (Biochrom)] is placed on the lids of Petri dishes (10-cm diameter) filled with phosphate-buffered saline (PBS). After incubation for 3 days, embryoid bodies are transferred to gelatin-coated wells of chamber slides (Nunc) or 60-mm dishes to allow attachment. To promote EC differentiation, 3-day-old embryoid bodies are placed in DMEM containing 10% FBS and medium consisting of EBM-2, 5% FBS, growth factor cocktail, and ascorbic acid (EGM2-MV Bullet Kit; Lonza/Clonetics) [37]. This protocol showed that mouse ES cells differentiated into ECs through embryoid body formation. Further experiments demonstrated that when placed in Matrigel, mouse ES-cell-derived endothelial-like cells formed networks similar to vascular structures, indicating that mouse ES cells are able to differentiate to functional ECs and can be used to study vasculogenesis.

9 Human ES Cell Differentiation into SMCs in Adherent Monolayer Culture Another study reported a direct and highly efficient SMC differentiation system by treating the monolayer-cultivated human ES cells with all-trans-retinoic acid [38].

9.1 Protocol

Fig. 4  Flow shear stress induces stem cell differentiation into ES cells. Tube-formation assay with static and sheared ES cells on Matrigel revealed that the ECs derived from mouse ES cells under shear are functional. (Reproduced from [14] with permission, Copyright Zeng et al. 14 2006. Originally published in the Journal of Cell Biology. Doi: 10.1083/jcb/200605113)

The human ES cell line HUES3 is cultured on g-irradiated MEFs at 37°C and 5% CO2 with daily change of the medium. The medium is composed of Dulbecco’s modified Eagle’s medium, 10% FCS, 10% KO-SR, 2  mM l-­glutamine, 0.1  mM 2-mercaptoethanol, 1% nonessential amino acids, penicillin, and streptomycin, 10 ng/mL bFGF (GIBCO), and 10 ng/mL human LIF (Chemicon) [38]. For SMC differentiation, an easy protocol has been designed to separate the feeder MEFs from the human ES cells, based on the differential rates of adhesion of MEFs and ES cells to the plate. The resuspended human ES cells are ­cultivated on gelatin-coated

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12-well plastic plates (Becton, Dickinson) at a density of approximately 4 × 104/cm2 at 37°C, 5% CO2 in 2 mL of differentiation medium with the presence of 10 mM all-transretinoic acid. The differentiation medium is composed of DMEM, 15% FCS, 2  mM l-glutamine, 1  mM monothioglycerol (MTG) (Sigma), 1% nonessential amino acids, penicillin, and streptomycin. The cells are cultured for 10 days with daily change of fresh medium. Starting from the 11th day, the differentiation medium is replaced by the serum-free culture medium, which is composed of KO-DMEM, 15% KO-SR, 2 mM l-glutamine, 1 mM MTG, 1% nonessential amino acids, penicillin, and streptomycin. The cultures are continued for an additional 10 days with daily change of the serum-free medium.

9.2 Application This protocol showed that more than 93% of the cells expressed SMC-marker genes along with the steady accumulation of SMC-specific proteins such as SMA and SMMHC. The fully differentiated SMCs were stable in phenotype and capable of contraction. Therefore, this inducible and highly efficient in vitro human SMC system can be an important resource to study the mechanisms of SMC phenotype determination in human [38]. However, the disadvantage of the majority of the ES cell differentiation protocols used today is that they generate heterogeneous mixtures of different cell types. This is a limitation which should be addressed before the ES cells are used for clinical application. Therefore, other protocols have been developed, which attempt to select a pure population of progenitor or more differentiated cells using a cell sorting method for a subpopulation of the cells which express specific cell markers. Such protocols have been successfully applied in both mouse and human ES cells and representative ones are described next.

10 Cell Isolation Protocols Interaction between ECs and SMCs is essential for vascular development and maintenance [39, 40]. ECs arise from Flk1-expressing (Flk1+) mesoderm cells [41], whereas SMCs possibly derive from mesoderm, neural crest, or epicardial cells and migrate to the vessel wall [42]. Therefore, on the basis of the possible common origin of these two cell lineages, a study developed a protocol showing that Flk1+ cells derived from ES cells can differentiate into both ECs and SMCs and can reproduce the vascular organization process [43].

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10.1  Protocol ES cells are maintained on mitomycin C (Kyowa-Hakko)treated embryonic fibroblast layers in DMEM (GIBCO) containing 15% FCS (Whittaker, USA), 5,000 U/mL LIF (R&D Systems) and 5 × 10–5 M 2-mercaptoethanol. Two weeks before the differentiation induction, 1,000 ES cells are transferred to gelatin (Sigma)-coated culture dishes to remove fibroblasts. ES cells (104 cells) are then transferred to each well of six-well cluster dishes (Biocoat, Becton, Dickinson) coated with type IV collagen and incubated in MEM alpha medium supplemented with 10% FCS and 5 × 10–5 M 2-mercaptoethanol, and cultured for 4 days to induce Flk1+ cells. Flk1+ cells (at a purity of greater than 95%) are obtained by flow cytometry sorting. After an additional 4 days of culturing with 10% FCS, more than 95% of the Flk1+ cells become positive for SMA. Addition of 50 ng/mL VEGF (VEGF165) to the culture results in the appearance of PECAM1+ sheets of ECs, which are also positive for vascular endothelial cadherin (VE-cadherin), CD34, endoglin, and acetyl-LDL incorporation. The remaining cells surrounding these sheets are identified as SMA+ and express other mural cell markers such as CGA7, a marker for differentiated vascular SMCs [43]. Further analysis by polymerase chain reaction with reverse transcription has confirmed that other SMC markers such as SMMHC, calponin and SM22a are upregulated in SMA+ cells, providing a strong indication that Flk1+ cells predominantly differentiate into either ECs or SMCs under these culture conditions. For culturing FLK1+VE-cadherin+ cells, a mixture of recombinant growth factors containing VEGF stem cell factor, interleukin 3, erythropoietin, and granulocyte growth factor (G-CSF) were used. Recombinant human VEGF, erythropoietin, and G-CSF were from R&D Systems (Minneapolis) [43].

10.2  Application On the basis of this protocol, ES-derived Flk1+ cells give rise to two major vascular cell types (ECs and SMCs), providing new insight into the vascular developmental pathway that originates in Flk1+ cells. Importantly, further experiments have shown that vascular cells derived from Flk1+ cells can organize into vessel-like structures consisting of endothelial tubes supported by mural cells in three-dimensional culture. Injection of Flk1+ cells into chick embryos showed that they could incorporate as ECs and SMCs to the developing vasculature in  vivo. Therefore, Flk1+ cells can act as “vascular progenitor cells” to form mature vessels and thus offer potential for tissue engineering of the vascular system [43].

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11  PECAM1+ Cell Isolation Another protocol have been developed showing that PECAM1+ cells derive from human ES cells grown in culture, displaying characteristics similar to those of vessel endothelium [23].

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cells. It was reported that VEGF and PDGF receptors were expressed on ECs and SMCs, respectively, and VEGF and PDGF stimulated the growth of the respective cell types. In this study, human ES-cell-derived vascular progenitors cells expressed both VEGF and PDGF receptors [44].

12.1  Protocol 11.1 Protocol Human ES cells are grown on MEFs (Cell Essential) in knockout medium on tissue culture plates coated with 0.1% gelatin (Sigma). Cultures are grown in 5% CO2 and routinely passaged every 5–6 days after being disaggregated with 1 mg/ mL collagenase type IV (GIBCO-BRL). To induce formation of embryoid bodies, human ES colonies are digested by using either 1 mg/mL collagenase type IV or trypsin–EDTA (0.1% of 1  mM solution) and transferred to Petri dishes to allow aggregation and prevent adherence to the plate. Human embryoid bodies are grown in the same culture medium without LIF and bFGF. For isolation of PECAM1+ cells, embryoid bodies at days 13–15 are dissociated with 0.01% trypsin– EDTA, washed with PBS containing 5% (vol/vol) FBS, and incubated with fluorescently labeled PECAM1 antibodies (30884X, PharMingen) on ice for 30  min. Fluorescently labeled cells are isolated using a flow cytometry cell sorter (Becton, Dickinson) and plated on 1% gelatin-coated plates with EC growth medium (Lonza/Clonetics) and passaged by using 0.01% trypsin–EDTA (Lonza/Clonetics) [23].

11.2  Application This protocol provides a model of human ES cell differentiation to ECs. Specifically, it has revealed that human ES cells, when induced to form embryoid bodies, could spontaneously differentiate into the endothelial lineage, ultimately forming vascular structures [23]. Moreover, the differentiated cells expressed EC markers in a pattern similar to that of human umbilical vein ECs with correctly organized cellular junctions and high metabolism of acetylated LDL. In addition, these cells were able to differentiate and form tubelike structures when cultured on Matrigel. In vivo, when transplanted into SCID mice, the cells appeared to form microvessels containing mouse blood cells [23].

12  VE-Cadherin and Cell Sorting Another study developed a protocol which highlighted the potential of human ES cells to differentiate into mature ­vascular cell components, through cell sorting of VE-cadherin+

Human ES cells are maintained on mitomycin C inactivated OP9 feeder cells. ES cells are dissociated into small colonies with the aid of 0.1% collagenase (Wako) and differentiation is induced in in  vitro two-dimensional culture on an OP9 feeder layer in a differentiation medium (minimal essential medium, GIBCO) supplemented with 5 × 10–5 M 2-mercaptoethanol with 10% FCS. VE-cadherin+ cells are sorted on day 8 and cultured on a dish coated with collagen IV without a feeder cell layer for an additional 8 days in the differentiation medium with the addition of 10% FCS, VEGF (100 ng/ mL; PeproTech EC), or PDGF-BB (10  ng/mL; PeproTech EC) [44].

12.2  Application This study provides a protocol to obtain mature vascular cells from human ES cells [44]. The authors concluded that different stages of vascular cells during the differentiation process could be derived, such as vascular progenitor cells and mature vascular cells. Further experiments showed that the ES-derived mature ECs were successfully incorporated into the host circulation and significantly accelerated improvement of local blood flow, whereas the vascular progenitor cells did not [44]. Thus, vascular progenitor cells are possibly too immature to be used directly as the source for vascular regeneration. This protocol provides useful information on the differentiation stage of the cells derived from ES cells and their potential clinical application. Consequently, we have now seen protocols that successfully direct ES cell differentiation to vascular lineages through supplementation of various cytokines and chemical factors within the culture media, or positive cell sorting of a subpopulation of cells expressing a specific marker such as VE-cadherin or Flk-1. Nevertheless, these approaches are highly inefficient and often limited by cells in different stages of cell differentiation and poor reproducibility. In addition, the fact that different cell types express a number of cell markers in a specific and nonspecific way, and the poor recovery and viability of the cell after sorting, should not be neglected when cell sorting methods are applied. Therefore, possibly, alternative approaches for ES cell differentiation to specific cell lineages may be achieved through genetic modulation with recombinant DNA.

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13  ES Cell Differentiation Through Genetic Modulation Reproducible methods for genetic manipulation of ES cells and their derivatives are highly desirable and are anticipated to be instrumental for the characterization of those candidate genes expected to regulate key processes such as cell differentiation. Several studies have reported successful transfection and stable viral transduction of genes in undifferentiated ES cells [45, 46] followed by differentiation into vascular lineages.

13.1  Human ES Differentiation into ECs by VEGF Expression A study has shown that transduction of human ES cells with an adenoviral vector expressing the human VEGF165 gene (Ad-hVEGF165) can enhance endothelial-lineage differentiation [47].

13.1.1  Protocol Undifferentiated human ES cells (passages 35–70) are propagated on a feeder layer of mitotically inactivated MEFs, plated on 0.1% gelatin-coated wells of a six-well culture dish. Each well contains approximately 2 × 105 MEF cells. The medium was composed of Dulbecco's modified Eagle’s medium/F-12 medium supplemented with 20% (v/v) KO-SR, 2 mM l-glutamine, 0.1 mM 2-mercaptoethanol, 1% nonessential amino acid stock, and 4 ng/mL bFGF. All cultures are maintained within a humidified 37°C incubator with a 5% CO2 atmosphere, and are routinely passaged every 5–7 days after disaggregation with 1  mg/mL collagenase type IV. The colonies of human ES cells are incubated with collagenase for 5–10 min at 37°C and are subsequently scraped off with a pipette tip, washed in fresh culture medium by centrifugation at 200g, and then replated onto mitomycin C inactivated MEFs. For embryoid body formation, the colonies of human ES cells are incubated with 1 mg/mL collagenase type IV for 5 min at 37°C, and dissociated into small clumps by scratching with a pipette tip, followed by placing them in suspension culture within ultra-low-attachment plates (Corning). The culture medium consists of Dulbecco's modified Eagle’s medium/F-12 medium supplemented with 20% (v/v) KO-SR, 2  mM l-glutamine, 0.1  mM 2-mercaptoethanol, and 1% nonessential amino acid solution. Within the suspension culture, the human ES cell clumps aggregate together to form compact embryoid bodies, and are maintained in culture from 5 to 21 days. The embryoid bodies are incubated in 2 mg/mL

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collagenase IV for 15 min at 37°C, followed by centrifugation at 200g for 5 min and a further 20-min incubation in a solution mixture containing cell dissociation buffer and 0.1% trypsin at a ratio of 2:1 at 37°C. The dissociated cells are washed by centrifugation at 200g for 5 min and filtered with a 40-mm cell strainer. Embryoid bodies are seeded on gelatin-coated plates in the absence of bFGF, and random differentiation occurs, resulting in the appearance of a heterogeneous population of mesodermal, endodermal, and ectodermal cell types. For transduction of human ES cells, the cells (1 × 105 cells/cm2) are exposed to either Ad-hVEGF165 or null adenoviral vector (Ad-null) at various titers, ranging from 50 plaque-forming units (pfu)/cell to 2,000 pfu/cell. The viral transduction medium is replaced after 4  h with normal 10% (v/v) Dulbecco's modified Eagle’s medium/F-12 medium for 24  h. The transduction procedure was repeated on three consecutive days to achieve optimum transduction efficiency. Ad-hVEGF165-transduced cells secreted hVEGF165 for more than 30 days after transduction. These results indicate that an adenovirus vector is an efficient tool in genetic manipulation of differentiating human ES cells [47], and is able to direct the ES cell differentiation to specific a cell lineage, which may be useful in future clinical applications.

13.2  Mouse ES Differentiation into SMCs by Histone Deacetylase 7 Overexpression Another study has shown that SMC differentiation is achieved in the mouse ES cell system by overexpression of histone deacetylase 7 (HDAC7) by adenoviral infection [48].

13.2.1  Protocol Mouse ES cells (ES-D3 cell line, CRL-1934; ATCC, Manassas, USA) are cultured in gelatin-coated flasks in DMEM (ATCC) supplemented with 10% FBS (ATCC), 10  ng/mL LIF (Chemicon), 0.1  mM 2-mercaptoethanol, 100 U/mL penicillin, and 100 mg/mL streptomycin in humidified incubator supplemented with 5% CO2, and split at a 1:6 ratio every other day. To induce SMC differentiation, ES cells are seeded on mouse collagen IV (5  mg/mL)-coated flasks or plates in differentiation medium [MEM alpha medium (GIBCO) supplemented with 10% FBS (GIBCO), 0.05  mM 2-mercaptoethanol, 100  U/mL penicillin, and 100  mg/mL streptomycin]. Three days later, the cells are infected with Ad-HDAC7 multiplicity of infection (MOI) 10 and further incubated in differentiation medium for 2 days. The empty virus Ad-tTA is used as a control virus and to compensate for the MOI. Further analysis has shown these

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cells are positive for HDAC7 and in parallel for a number of SMC markers such as SMA, calponin, and SM22 (Fig. 5). Therefore, this protocol provides a model to successfully induce SMC differentiation from mouse ES cells through adenoviral induction of the gene of interest, HDAC7 [48].

13.2.2 Application Ex vivo culture of embryonic cells from SM22-LacZ transgenic mice showed that overexpression of HDAC7 by adenoviral gene transfer significantly increased b-galactosidase+ cell numbers and enzyme activity, indicating its crucial role in SMC differentiation during embryonic development. Thus, targeting the gene of interest, such as HDAC7 in this study, will provide a new therapeutic strategy for intervention in vascular diseases [48]. Consequently, gene targeting has become an essential tool for functional genomics in ES cells [1], and provides a powerful tool to study and understand the differentiation mechanisms. Homologous recombination is also likely to be an important technique as a fundamental research tool for both basic and applied research. However, mouse and human ES cells have significant differences in both stable transfection efficiency and cloning efficiency from single cells, making it difficult to target a specific gene in human ES cells. Therefore, a feasible method able to identify and select a specific cell type undergoing differentiation needs to be developed. The introduction of a reporter/selection gene by lentivirus into ES cells, which may permit tracing of the altered cells among a heterogeneous population after ­specific

Fig. 5  SMCs were derived from ES cells after adenoviral induction of histone deacetylase 7 (HDAC7). (a) Immunostaining experiments showed that HDAC7 colocalized with the SMC marker calponin. (b) Western blot showed that overexpression of exogenous HDAC7

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cell selection, will be a useful tool in this aspect. This technique will also be applicable not only to understand better the development and spatial organization of a given cell lineage during in vitro differentiation but also to eliminate any potential contamination by undifferentiated cells in future use of the ES cell derivatives in cell-based therapies, which may result in the development of teratomas. Therefore, the development of lentiviral constructs in which the reporter/ selection gene is tightly expressed under the control of vascular-lineage-specific promoters, such as PECAM1 and VE-cadherin for ECs and SMA and SM22 for SMCs, will provide a dynamic tool to study the ES cell differentiation toward vascular lineages.

14 Discussion 14.1  Applications Today, scientific research focuses on the regenerative ability of stem/progenitor cells following organ injury, in addition to their role in the pathogenesis of disease processes. Pluripotent ES cells have the potential to differentiate into most cell types of the adult human body, including vascular cells. Vascular cells, such as ECs and SMCs, are important contributors to tissue repair and regeneration. In addition to their potential applications for treatment of vascular diseases and stimulation of ischemic tissue growth, it is also possible that ECs and SMCs derived from human ES cells may be

increased the expression of the SMC markers SMA and calponin. Zero to 20 multiplicity of infection of Ad-HDAC7 was applied to ES cells. Control Ad-tTA was used to compensate for the total amount of virus. (Reproduced from [48] with permission)

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used to engineer artificial vessels to repair damaged vessels and form vessel networks in engineered tissues. “Vascular disease” is a broad term encompassing a ­number of conditions which affect the vascular system, including ­pulmonary-hypertension-induced vascular ­remodeling, ­hyperlipidemia-induced (native) atherosclerosis, angioplasty-induced restenosis, transplant arteriosclerosis, vein bypass graft atherosclerosis, and aneurysms [49]. Accumulating evidence suggests that vascular progenitors contribute to vascular healing and remodeling under physiological and pathological conditions. The reduced number of endothelial progenitor cells are implicated in the pathogenesis of vascular diseases [50, 51]. Importantly, studies have reported that the levels of vascular progenitor cells are decreased in the bone marrow of aged apoE-deficient mice [50], indicating that progressive progenitor cell deficits and consequent delayed vascular healing may account for the pathogenesis of atherosclerosis [52]. A number of in  vivo studies have reported the contribution of vascular progenitors derived from stem cells to atherosclerosis. ECs derived from ES cells were able to accelerate re-endothelialization of injured arteries and reduce neointima formation in in vivo experiments [10]. It has been shown that the transplantation of human vascular-progenitor-cell-derived vascular cells at the proper differentiation stage successfully promoted vascular regeneration in the setting of tissue ischemia [53]. Therefore, ES-cell-derived ECs and SMCs can be used as new, promising cell sources for therapeutic vascular regeneration in patients with tissue ischemia. Pulmonary hypertension is defined as a refractory disease, which is characterized by progressive increase in pulmonary artery pressure and resistance. In reality, the cause of pulmonary hypertension appears to be heterogeneous. However, it seems that the pulmonary vasculature initially undergoes persistent vasoconstriction and structural remodeling, which results in increased medial thickness of muscular arteries, peripheral extension of arterial muscularization, and increased matrix deposition [54]. It was then speculated that endothelial dysfunction or damage possibly triggered the pathogenesis of hypoxia-induced pulmonary hypertension [55]. Therefore, transplantation of ECs derived from stem or vascular progenitor cells can be applied to restore microvascular structure and function. Such studies using EC progenitor cells have been reported in rats [56] and dogs [57]. Importantly, another study by Nagaya et al. has reported that intravenously administered endothelial progenitor cells were shown to incorporate into pulmonary arterioles. Moreover, transplantation of adrenomedullin, a target gene to transduce endothelial progenitor cells, significantly improved pulmonary hypertension [58]. Therefore, these studies revealed an important role of ECs in inhibition of pulmonary hypertension, and pulmonary arterial remodeling by accelerating

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endothelial healing of the damaged pulmonary arterioles [58]. These findings suggest that ECs derived from ES or progenitor cells have a remarkable potential for clinical application in treatment of pulmonary disease.

14.2  Limitations Scientific obstacles need to be overcome before clinical applications of human ES cells are applied, such as contamination with animal product, tumorigenicity, immunocompatibility, isolation of pure cell types, and availability of appropriate animal models to test cell function. At present, the ES cell differentiation protocols generate heterogeneous mixtures of a number of different cell types. Therefore, it will be important to elucidate why apparently homogenous populations of ES cells differentiate at different rates, or into a number of different lineages, even under simple, fully defined differentiation protocols. By obtaining highly differentiated ES cells with low proliferation potential, one will reduce the possibility of teratoma formation. Importantly, a study has shown that when ECs derived from human CD34+ progenitor cells were implanted in SCID mice under the cranial windows, stable blood conduits formed for more than 150 days without teratomas [59]. These findings suggest that human ES-cell-derived ECs are safe for potential clinical applications, when a suitable protocol is applied. Therefore, it is very important to optimize the protocols to induce and isolate the cells at the most appropriate differentiation stage. This knowledge will be essential for clinical applications using human pluripotent cells in regenerative medicine. Among the ES cell differentiation approaches used, a number of advantages and disadvantages exist. Undoubtedly, the embryoid body model provides a three-dimensional structure that induces cell–cell interactions, which are essential for certain developmental programs. However, this model generates a number of cytokines and inducing factors within these structures, increasing the possibility to complicate the signaling pathways involved in lineage commitment, and to induce specific signals able to direct the ES cell differentiation to unwanted or nonspecific cell lineages. Also undefined factors may be produced by the supportive cells, which could influence the differentiation of the ES cells to undesired lineages. On other hand, the model of differentiation in a monolayer “of extracellular matrix proteins” is a simple and well-defined model, which minimizes the influence of neighboring cells and supportive stromal cells, providing an efficient and reproducible protocol. A possible limitation of this model is that the presence of matrix proteins that are critical for the differentiation process may dramatically influence the generation and survival of derived cell types. Thus, an accumulated attempt is also focused on defining a ­serum-free

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differentiation protocol to avoid the complication of ­maintaining an additional stromal cell line. Additionally, a limitation of the mouse ES cells is their significant differences with the human ES cells, indicating differences in the mechanism of differentiation. For example, growth factor (such as bFGF, BMPs) is reported to induce endothelial differentiation in mouse but not in human embryoid bodies. These observations point out the potential differences between the two systems in the molecular mechanisms, and highlight the need to focus on developmental processes by using human systems. Therefore, understanding how extrinsic factors control human ES cell self-renewal and differentiation will give us the knowledge to culture and differentiate these pluripotent cells with higher efficiency. This emphasizes the need for establishment of an in vitro differentiation system of human vascular cell components exclusively from human ES cells to decipher cellular mechanisms in human vascular development and disease stages. A potential genetic approach which will delete or downregulate genes encoding class I major histocompatibility complex molecules may assist the derivation of “safer” human ES cell lines to be used for cellular transplantation. Furthermore, somatic cell nuclear transfer has also been used as an alternative approach to overcome transplant rejection [60–62]. However, the application of ES cells in humans is still a challenge especially owing to ethical issues and technical limitations that make the procedure very inefficient. Consequently, what we need is a solid combination of reliable and reproducible protocols to prospectively select pure and functional vascular cells which will be safe for use in clinical applications. To address this, several fundamental questions regarding the nature of ES cells in “in vitro” culture compared with their “in vivo” characteristics need to be elucidated. Further research should address the improvement of culture conditions to grow human ES cells by understanding those molecular mechanisms leading to retention of an undifferentiated and pluripotent state, and only then the development of new protocols for differentiation and genetic modulation will be facilitated.

15  Concluding Remarks ES cells have already overcome fundamental restrictions such as low frequency, difficultly to be isolated from their niche, restricted lineage potential, and poor growth in cell culture. Their features including indefinite replication, pluripotency, and genetic flexibility provide a useful model to study the molecular mechanisms of stem cell differentiation. Unequivocally, ES cell differentiation is a complicated process, and standard criteria should be strictly followed to avoid undesirable cell lineage differentiation. Lineage development from ES cells should mimic the developmental

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p­ rogram that establishes the lineage in the early embryo, and the differentiated cell populations, derived from these differentiation models in culture, must present appropriate functional properties both in  vitro and in  vivo when transplanted into suitable models. Vascular cells play a critical role in both physiological maintenance of the cardiovascular system in embryonic development, and in the pathophysiological ­processes of vascular diseases in adults [63–67]. Further investigation to elucidate the genetic changes and specific signaling pathways involved in the transition of either ECs or SMCs from their common progenitor will provide a beneficial model to study and understand the mechanisms during embryonic development and disease. Recognition of this great potential of ES cells will depend on our ability to develop feasible and reproducible protocols to maintain, prospectively differentiate, and genetically modulate ES cells and their derivatives. Since stem cell research is developing quickly, we believe that the results obtained soon could help us not only understand the mechanisms of stem cell differentiation, but also will be useful for potential application for treatment of vascular diseases. Acknowledgements  This work was supported by grants from the British Heart Foundation and the Oak Foundation.

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44  Differentiation of Embryonic Stem Cells to Vascular Cell Lineages 12. Xiao Q, Zeng L, Zhang Z, Hu Y, Xu Q (2007) Stem cell-derived Sca-1+ progenitors differentiate into smooth muscle cells, which is mediated by collagen IV-integrin a1/b1/av and PDGF receptor pathways. Am J Physiol Cell Physiol 292:C342–C352 13. Schenke-Layland K, Angelis E, Rhodes KE, Heydarkhan-Hagvall S, Mikkola HK, Maclellan WR (2007) Collagen IV induces trophoectoderm differentiation of mouse embryonic stem cells. Stem Cells 25:1529–1538 14. Zeng L, Xiao Q, Margariti A et  al (2006) HDAC3 is crucial in shear- and VEGF-induced stem cell differentiation toward endothelial cells. J Cell Biol 174:1059–1069 15. Wang H, Riha GM, Yan S et al (2005) Shear stress induces endothelial differentiation from a murine embryonic mesenchymal progenitor cell line. Arterioscler Thromb Vasc Biol 25:1817–1823 16. Vats A, Bielby RC, Tolley N et al (2006) Chondrogenic differentiation of human embryonic stem cells: the effect of the micro-environment. Tissue Eng 12:1687–1697 17. Vanikar AV, Mishra VV, Firoz A et al (2007) Successful generation of donor specific hematopoietic stem cell lines from co-cultured bone marrow with human embryonic stem cell line: a new methodology. Transplant Proc 39:658–661 18. Nakano T, Kodama H, Honjo T (1994) Generation of lymphohematopoietic cells from embryonic stem cells in culture. Science 265:1098–1101 19. Nishikawa SI, Nishikawa S, Hirashima M, Matsuyoshi N, Kodama H (1998) Progressive lineage analysis by cell sorting and culture identifies FLK1+ VE-cadherin+ cells at a diverging point of endothelial and hemopoietic lineages. Development 125:1747–1757 20. Abe K, Niwa H, Iwase K et  al (1996) Endoderm-specific gene expression in embryonic stem cells differentiated to embryoid bodies. Exp Cell Res 229:27–34 21. Owens GK (1995) Regulation of differentiation of vascular smooth muscle cells. Physiol Rev 75:487–517 22. Wartenberg M, Gunther J, Hescheler J, Sauer H (1998) The embryoid body as a novel in vitro assay system for antiangiogenic agents. Lab Invest 78:1301–1314 23. Levenberg S, Golub JS, Amit M, Itskovitz-Eldor J, Langer R (2002) Endothelial cells derived from human embryonic stem cells. Proc Natl Acad Sci U S A 99:4391–4396 24. Gerecht-Nir S, Ziskind A, Cohen S, Itskovitz-Eldor J (2003) Human embryonic stem cells as an in vitro model for human vascular development and the induction of vascular differentiation. Lab Invest 83:1811–1820 25. Li ZJ, Yang C, Tang FW et al (2005) In vitro vasculogenesis and angiogenesis of mouse embryonic stem cells. Zhongguo Yi Xue Ke Xue Yuan Xue Bao 27:62–66 26. Feraud O, Cao Y, Vittet D (2001) Embryonic stem cell-derived embryoid bodies development in collagen gels recapitulates sprouting angiogenesis. Lab Invest 81:1669–1681 27. Miyata T, Iizasa H, Sai Y, Fujii J, Terasaki T, Nakashima E (2005) Platelet-derived growth factor-BB (PDGF-BB) induces differentiation of bone marrow endothelial progenitor cell-derived cell line TR-BME2 into mural cells, and changes the phenotype. J Cell Physiol 204:948–955 28. Klug MG, Soonpaa MH, Koh GY, Field LJ (1996) Genetically selected cardiomyocytes from differentiating embryonic stem cells form stable intracardiac grafts. J Clin Invest 98:216–224 29. Bradley A, Evans M, Kaufman MH, Robertson E (1984) Formation of germ-line chimaeras from embryo-derived teratocarcinoma cell lines. Nature 309:255–256 30. Laslett AL, Filipczyk AA, Pera MF (2003) Characterization and culture of human embryonic stem cells. Trends Cardiovasc Med 13:295–301 31. Smith AG, Heath JK, Donaldson DD et  al (1988) Inhibition of pluripotential embryonic stem cell differentiation by purified polypeptides. Nature 336:688–690

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650 53. Yamahara K, Sone M, Itoh H et al (2008) Augmentation of neovascularizaiton in hindlimb ischemia by combined transplantation of human embryonic stem cells-derived endothelial and mural cells. PLoS ONE 3:e1666 54. Sata M (2006) Role of circulating vascular progenitors in angiogenesis, vascular healing, and pulmonary hypertension: lessons from animal models. Arterioscler Thromb Vasc Biol 26:1008–1014 55. Partovian C, Adnot S, Raffestin B et al (2000) Adenovirus-mediated lung vascular endothelial growth factor overexpression protects against hypoxic pulmonary hypertension in rats. Am J Respir Cell Mol Biol 23:762–771 56. Zhao YD, Courtman DW, Deng Y, Kugathasan L, Zhang Q, Stewart DJ (2005) Rescue of monocrotaline-induced pulmonary arterial hypertension using bone marrow-derived endothelial-like progenitor cells: efficacy of combined cell and eNOS gene therapy in established disease. Circ Res 96:442–450 57. Takahashi M, Nakamura T, Toba T, Kajiwara N, Kato H, Shimizu Y (2004) Transplantation of endothelial progenitor cells into the lung to alleviate pulmonary hypertension in dogs. Tissue Eng 10:771–779 58. Nagaya N, Kangawa K, Kanda M et  al (2003) Hybrid cell-gene therapy for pulmonary hypertension based on phagocytosing action of endothelial progenitor cells. Circulation 108:889–895 59. Wang ZZ, Au P, Chen T et al (2007) Endothelial cells derived from human embryonic stem cells form durable blood vessels in  vivo. Nat Biotechnol 25:317–318

A. Margariti et al. 60. Rideout WM 3rd, Hochedlinger K, Kyba M, Daley GQ, Jaenisch R (2002) Correction of a genetic defect by nuclear transplantation and combined cell and gene therapy. Cell 109:17–27 61. Wakayama T, Tabar V, Rodriguez I, Perry AC, Studer L, Mombaerts P (2001) Differentiation of embryonic stem cell lines generated from adult somatic cells by nuclear transfer. Science 292:740–743 62. Hwang WS, Ryu YJ, Park JH et al (2004) Evidence of a pluripotent human embryonic stem cell line derived from a cloned blastocyst. Science 303:1669–1674 63. Campbell JH, Campbell GR (1994) The role of smooth muscle cells in atherosclerosis. Curr Opin Lipidol 5:323–330 64. Gittenberger-de Groot AC, DeRuiter MC, Bergwerff M, Poelmann RE (1999) Smooth muscle cell origin and its relation to heterogeneity in development and disease. Arterioscler Thromb Vasc Biol 19:1589–1594 65. Hillebrands JL, Klatter FA, van den Hurk BM, Popa ER, Nieuwenhuis P, Rozing J (2001) Origin of neointimal endothelium and alpha-actin-positive smooth muscle cells in transplant arteriosclerosis. J Clin Invest 107:1411–1422 66. Hu Y, Mayr M, Metzler B, Erdel M, Davison F, Xu Q (2002) Both donor and recipient origins of smooth muscle cells in vein graft atherosclerotic lesions. Circ Res 91:e13–e20 67. Liu C, Nath KA, Katusic ZS, Caplice NM (2004) Smooth muscle progenitor cells in vascular disease. Trends Cardiovasc Med 14:288–293

Chapter 45

Statistics and Clinical Data Analysis: A Reference Guide Michael C. Donohue, Tanya Wolfson, and Anthony C. Gamst

Abstract  Statistics, which is widely used in medicine, is a branch of mathematics that focuses on collection, presentation, analysis, and interpretation of numerical and categorical data. Examples include the analysis of data from clinical trials of drugs and other interventions, modeling the progression of a disease over time, examining the diagnostic performance of medical tests, studying relationships between biomarkers and cognitive performance, and retrospective evaluation of cancer diagnosis via magnetic resonance (MR) imaging. This chapter presents an abbreviated reference guide to statistical methods in clinical research. One of the goals of this chapter is to emphasize applications to current problems and data from pulmonary vascular disease. We attempt to cover the basics of statistical thinking and the issues that arise frequently in clinical research, and provide references to the literature offering more extensive coverage of the topics considered here and pointers to work on more technical or specialized topics. The major goal is to present and discuss the statistical methods commonly used and relevant to research in pulmonary vascular disease. Keywords  Statistical analysis • Clinical trial • Bioinformatics • Epidemiology • Data analysis

1 Introduction Statistics is a branch of mathematics that focuses on collection, presentation, analysis, and interpretation of numerical and categorical data. It is widely used in any discipline where data are collected and conclusions are drawn from them.

M.C. Donohue (*) Division of Biostatistics and Bioinformatics, University of California, San Diego, 9500 Gilman Dr., MC 0717, La Jolla, CA 92093-0717, USA e-mail: [email protected]

In particular, medicine makes enormous use of statistics. Examples include the analysis of data from clinical trials of drugs and other interventions, modeling the progression of a disease over time, examining the diagnostic performance of medical tests, studying relationships between biomarkers and cognitive performance, and retrospective evaluation of cancer diagnosis via magnetic resonance (MR) imaging. This chapter presents an abbreviated reference guide to statistical methods in clinical research. Many excellent introductory textbooks on statistical methods have been written already and we will be recommending personal favorites as we proceed. One of the goals of this guide is to emphasize applications to current problems and data from pulmonary vascular disease. We cannot possibly cover all the potential topics, but we will attempt to cover the basics of statistical thinking and the issues that arise frequently in clinical research. We will also provide references to the literature offering more extensive coverage of the topics considered here and pointers to work on more technical or specialized topics. The chapter is generally organized as proceeding from the beginning to the end of a medical study. We begin with conception and design, move on to discussing the collection of data, examining, summarizing, and graphically presenting data, general data analysis techniques, and finally interpreting results and drawing conclusions. The data analysis section discusses a number of methods with guidelines for correct application; potential pitfalls and methods for avoiding them are discussed throughout. The underlying formal statistical theory, as well as stepby-step instructions on how to carry out the analyses are largely omitted: the former because it is outside the scope of this reference, the latter because it is not realistic to do these analyses “by hand” and, in general, software must be used. We will provide references to textbooks, but the main goal of this guide is to present and discuss the statistical methods commonly used and relevant to research in PVD, the main ideas behind those methods, and the dos and don’ts of applying them.

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_45, © Springer Science+Business Media, LLC 2011

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2 Experimental Design, Model Building, and Causality 2.1 Two Types of Statistical Models There are two schools of thought about statistical models: the classical (“clinical trials” or “physics”) and the more modern (“machine learning”) school [1]. In the classical school, we believe that we know all the important factors and how they interact to produce the outcome of interest and that we also understand the mechanism generating “randomness” (randomization, random sampling, or measurement error). Under these assumptions, using significance tests and a lot of caution about correctly attributing causality, we can assess whether a factor X causes the outcome Y, whether X has no role in the mechanism (model), or whether the model itself is wrong (i.e., inconsistent with the data). The modern (“machine learning”) school is not concerned with the details of the data-generating mechanism and, as a result, does not claim that any model M has a direct connection to reality. (That is, there is no claim that “the world works in such a way that outcome Y is produced through a mechanism very much like the model M.”). There is no claim that X causes Y, only that X is important in predicting Y in the model M, and X and Y are (in some unknown manner) correlated. Of interest here is simply predicting Y from interactions of variables X1, X2, … Xn. Here, significance tests are of little direct importance. To summarize, in the classical school we are interested in building and testing models of nature (of how the world really works), whereas in the modern school we are only interested in building models that allow to us predict well. An example of the classical school is a clinical trial: does a drug cause improvement in survival? An example of the modern school is diagnosing disease from a combination of many texture features on an MR image. In this guide we will be concentrating mostly on the former: modeling with the goal of establishing causality.

2.2 Design of a Study An analysis is only as good as the data on which it rests and it is the study design which largely determines the quality of collected data [2]. Two major types of study designs have been used to attribute causality: observational studies and controlled experiments. Anecdotal evidence can be (and often is) used as a starting point and a motivation for studies but is not evidence of causality by itself. An example concerning magnetic fields and cancer is given in [2]. Some children who live near power lines develop leukemia. Does this mean that magnetic fields cause leukemia? Anecdotal evidence is not

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sufficient to answer the question. Some children who do not live near the power lines also develop leukemia. Children who live near power lines may be demographically different from children who do not live near power lines; therefore, factors other than the power lines might be involved in developing leukemia. The hypothesis based on anecdotal evidence needs to be tested in a carefully designed study [3–5]. A randomized, double-blinded study design is the best option for establishing causality. The question of interest is whether a factor X (such as treatment) causes the outcome Y (such as a quantified improvement in health). The ideal design would let the subject groups differ only on the factor X, keeping all other factors equal. Randomization to treatment and control groups is the best way to balance groups in terms of all potential confounders (additional factors with the potential to influence the outcome), including those we do not or cannot measure. Keeping the study double-blind, a design where neither the investigators nor the patients know who is in the treatment or the control group, removes human biases from influencing the outcome data. (Single-blind is another type of design, where the subjects do not know which group they are in, but the investigators do. Sometimes only a single-blind design is feasible, although it gives rise to a potential for investigator bias.) A randomized, double-blind, placebo-controlled design may be ideal, but it is not always feasible. Some studies cannot be blinded: subjects in a study about the benefits of exercise will know which group they are in since there is no easy way to disguise exercise with a placebo equivalent. Some studies cannot be randomized: subjects in a study about effects of a disease cannot be randomized to disease and control groups by an investigator. It might be a possibility with disease models in animal studies, but human studies frequently have to be observational for practical or ethical reasons. A study is called “observational” when the investigators have no control over the assignment of subjects to study groups: the groups are formed “naturally” and the investigators merely observe the effects. The main difficulty with observational studies is that factors other than the factor of interest will differ between the two groups. Suppose a study is interested in comparing rates of lung cancer in long-term smokers and nonsmokers. Longterm smokers are more likely to be men than women; more likely to be older than nonsmokers; more likely to be in a different socioeconomic strata than nonsmokers; more likely to have different lifestyles. All of these factors can influence the rate of lung cancer. Thus, if a difference between smokers and nonsmokers is found, it could be due to age or gender or lifestyle, not necessarily smoking. For a causal interpretation to be justified, the two groups must be made as comparable as possible by matching them on confounding factors and by covarying for those factors in analyses (described in Sect. 7). Here the term “matching” means ensuring the groups are similar on a particular factor overall rather than attempting to

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match subjects in the groups on a one-to-one (or many-to-one) basis. Because it is impossible to foresee and account for all the potential confounding factors, randomization is preferable since, on average, it balances the groups on everything at once, including factors unknown. To summarize this section, randomized designs should be used wherever feasible. Within the limits of statistical (sampling) error it will make the groups comparable (aside from the main factor of interest, such as treatment). If randomization is not feasible and the study is observational, the groups need to be made as comparable as possible by matching or covarying for the main confounding factors expected by the investigators. A study should be double-blind, if possible, to remove human bias. Anecdotal evidence by itself is not informative (comparison with an appropriate control group is necessary), although it could be the starting point for designing a study.

3 Hypothesis Testing 3.1 Hypothesis Testing and p Values It is difficult if not impossible to find a medical research article that does not contain hypothesis tests and p values. In this section we will define some basic ideas and terminology associated with hypothesis testing. All statistical tests of significance broadly address the same question: could the observations (e.g., the difference between treatment and control groups) have occurred entirely by chance? A statistical test allows the investigator to choose between two positions formally called the null hypothesis and the alternative hypothesis (hence the term “hypothesis testing”). The null hypothesis states that the observed difference is due to chance variability alone, a fluke of sampling, or measurement error; there is no real difference between the groups. It is the argument in favor of the status quo, requiring no further action to be taken (i.e., the treatment is no better than the placebo, so there is no need to discard the status quo and begin administering the new treatment). The alternative hypothesis states that the observed difference is real and is an argument in favor of change. Hypothesis tests are performed under the assumption that the null hypothesis is true; the formal term is “under the null.” A p value is the probability, under the null (i.e., assuming that only chance variability is at play), of observing a difference as extreme or more extreme than the one observed in the data. Thus, a high p value speaks for the null hypothesis, a low p value speaks against it. If the p value is low enough, the null hypothesis is rejected, and the alternative hypothesis is accepted. Conventionally, the threshold for rejecting the null hypothesis in favor of the alternative hypothesis is 5%, or 0.05, which means that the probability

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of the observed difference being as extreme or more extreme owing to chance alone is less than 5%. The formal term is “the test is performed at a significance level of a = 0.05.” If a p value is less than 0.05, it is assumed that a factor beyond mere chance (and presumably it is the factor which separates the two groups, e.g., exposure to treatment) is generating the difference, although important caveats to that assumption are discussed in Sects. 4 and 11.

3.2 More About Hypothesis Tests Significance tests can be one-sample or two-sample; onesided or two-sided; independent-sample or paired-sample; parametric or nonparametric. In this section we will briefly discuss these concepts. The meaning of one-sample versus two-sample is intuitive. Are two groups of subjects being compared (e.g., a treatment and a control group)? If so, then the test is a twosample test. Is one group of subjects being compared for a predetermined measure? (For instance, it might be hypothesized that a group of subjects with a particular disease has a mean BMI greater than 25.) This is a one-sample test. What happens when each subject in a group receives both treatment and a placebo in random order, in what is a called a crossover trial? At the end of the study the placebo and treatment results (e.g., distance on a 6-min walk) are compared using a paired test. Clearly even though there are two sets of ­measurements they are not independent: each subject in the study serves as his or her own control and the placebo-treatment results are paired by subject. One-sided versus two-sided is a concept which refers to the alternative hypothesis. If the alternative hypothesizes a difference from the null which could go in either direction, that is a two-sided hypothesis. If it is believed that the difference from the null must go in a specific direction (e.g., the treatment group must perform better than the control group), then a one-sided alternative is hypothesized. In reality, a priori information about sidedness is usually unavailable. Twosided testing is more conservative and prudent, and generally preferable. One would, for example, be interested to know if a treatment which was thought to improve outcomes is actually harmful. The choice of parametric versus nonparametric test depends on what one knows about the data at hand. To be completely accurate, most tests are in some ways nonparametric in the sense that they rely on an approximation of the unknowable real-world data-generating mechanism. For practical purposes, however, when the study data come from one of the known probability distributions and the characteristics of that distribution are used to compute test statistics, we call such a test parametric. The two-group t test, for example, depends on the

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assumption that the sample means have a roughly Gaussian distribution. This is intuitively obvious if the data were sampled from two approximately Gaussian populations. More broadly, the central limit theorem (one of the most important theorems in statistics) states that the mean of a sample of values from an arbitrary distribution will be approximately Gaussian distributed under very mild assumptions. Even though we never actually know the distribution of the data, we can often specify an approximate distribution for them (and test for departures from that assumption). Additionally, the parametric procedures used are often robust to departures from the assumptions that were made; a robust method is one that is not strongly affected by small violations of the assumptions. Of course, robustness is not a guarantee, and caution should be exercised in the application of hypothesis tests, which rest heavily on parametric assumptions. Some distributions (e.g., the Cauchy) do not have a (finite) mean, but all distributions have a median. So, rather than testing for a difference in means, we could look for a difference in medians. The Mann–Whitney–Wilcoxon (MWW) rank-sum statistic can be thought of as a two-sample test for a difference in medians – a nonparametric competitor to the two-sample t test. This does not mean that the MWW test can be applied without making any assumptions at all about the data [6], but fewer assumptions are necessary than are made with the two-sample t test, for example. There is some cost for this flexibility: if the data are Gaussian, the t test is more efficient – that is, has more power – than the MWW (although it has been shown that the MWW is 96% efficient if the data are Gaussian and at least 86% efficient, in general, relative to the t test [7], which is not nearly so robust). Nonparametric statistics is a large and diverse subfield of statistics. One of its goals is to find estimators and test statistics which apply in situations where some of the assumptions of parametric statistics fail to hold. This branch of statistics includes nonparametric regression, also known as smoothing, and other techniques. Another goal of nonparametric statistics is to find procedures which work regardless of the distribution of the data. This branch is classical nonparametrics and research in this area deals mostly with rank-based estimators and test statistics. An example of a rank-based estimator is Spearman’s correlation coefficient, a nonparametric competitor to the parametric Pearson’s correlation coefficient, which was developed under the assumption that the data are jointly Gaussian. There is little penalty in using a nonparametric test when a parametric test would have been appropriate: the result will still be correct, although possibly less powerful. Using a parametric test, when the data come from a distribution not close to that assumed, can lead to incorrect inferences. Nonparametric tests should be used when little can be safely assumed about the distribution of the data. For more on (classical) nonparametric statistics, see the book by Lehmann [8].

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Whenever a p value is involved there is an implied (if not explicitly stated) test of significance. The p value associated with a correlation, for instance, corresponds to a test of significance; by default a one-sample, two-sided test of a difference between the true correlation and zero; i.e., the p value is computed under the null hypothesis of no correlation between two variables, and the two-sided alternative hypothesis states that the correlation coefficient is significantly different from zero in either the positive or the negative direction. Of course a different test is possible: the correlation coefficient could be compared to a hypothetical coefficient other than zero (it is sometimes of interest to test whether correlation is “high,” significantly higher than 0.5, for example). Correlation between several variables can be compared, as can the correlation between the same two variables in different groups of subjects; it is all a matter of the questions posed by the study. The p value of a regression coefficient is computed under the null hypothesis that the coefficient is zero (i.e., the predictor corresponding to that coefficient has no influence in the regression equation and is therefore not significant), and so on.

4 Power and Sample Size Determination Two types of error are associated with hypothesis testing. Type I error is the probability of rejecting the null hypothesis when it is true: a false positive. Type II error is the probability of failing to reject the null hypothesis when it is false: a false negative. Type I error is controlled by setting significance levels in the analysis of study data, as discussed in Sect. 11. Type II error is dealt with at the design stage by ensuring that the study is adequately powered. The power of a statistical test is the probability of not making a type II error, or the probability that the test will detect a departure from the null (i.e., “proclaim significance”) when the null is false. (If the null is true, a power analysis cannot help: the test should not be significant. If the null is false, it is important to have enough power a priori to distinguish deviations from the null from noise in the data.) Power depends on a trade-off between the sample size and the size of the expected deviation from the null. As the sample size grows, the error in estimating the size of the departure from the null, also called “the effect” (e.g., the difference in 6-min walk distance between two groups of subjects), grows smaller, making smaller and smaller differences detectable. Conventionally, a sample size is chosen so that the power of a test of the main hypothesis is at least 0.8 (80%). Note that power analyses are procedure-specific. A t test requires a different power computation than a test of proportions. It is not possible to estimate power without first identifying the main hypothesis on which the power analysis should be based, and the statistical analysis which will be used to test

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that hypothesis. Thus, power analysis is the last step of a study design: first the questions of interest are determined, then the outcomes and independent measures, then the statistical analyses, and finally the power and sample size are computed. It is also worth noting that the more complicated the statistical analysis, the more complicated it is to compute power precisely (mainly because too many features of the underlying model are allowed to vary at the same time). However, it is often possible to compute power for procedures simplified in such a way that the power analysis, although not entirely accurate, becomes conservative (i.e., the actual analysis will have at least as much power as the simplified analysis). To compute power it is necessary to know something about the random variation one can expect to see in the data. If two groups are being compared in terms of distance walked in 6 min, what is the standard deviation of this measure? That is, how much variability can we expect in these distances in a random sample of subjects from each group? Of course, this is still the design stage, so the actual data have not been collected. But are there pilot data? Are there published data on the 6-min walk distance of patients similar to those who will be sampled? For the purposes of a power analysis, a reasonable approximation to the standard deviation is adequate. Next, the effect size needs to be established. What difference do the researchers consider clinically meaningful? (This is an important part of the question: a large enough sample size will make a trivial difference between two groups detectable, but subjects will be difficult to recruit and expensive to evaluate, and the result may not be useful. Conversely, if an unreasonably large number of patients is needed to detect clinically meaningful effect, it is better to

Fig. 1  Power versus effect size showing the relationship between effect size and power when the sample size is held fixed

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wait until resources are available than to begin a study that is underpowered.) Finally, we need to set the significance level for the test (most commonly 0.05). Two figures are presented to demonstrate the relationship between sample size, effect size, and power. We assume an overall standard deviation of 80  meters (m) on the 6-min walk test (this is a simulated example). The significance level is set at 0.05. In Fig. 1, the sample size has been set at 100 patients per group. The curve shows the increase in power associated with increasing effect size. For this sample size the effect size (the difference between two groups in mean distance walked in 6 min) of 32 m or greater is associated with a power of at least 80%. In Fig. 2, the effect size has been fixed at 25 m. As the sample size increases, so does the power. A sample size of just over 160 subjects per group is necessary to ensure power of at least 80% to detect the proposed effect size of 25 m. What if there are no pilot data or previously published data? A useful resource for power computation based on an assumed relationship between the mean and standard deviation of a measure (without knowing either the actual mean or the standard deviation) is offered in Cohen [9]. A range of standardized effect sizes is assumed: e = d/s, where d is the raw difference, and s is the standard deviation. An effect size e = 0.2 or smaller is considered small, 0.5 is medium, 0.8 or greater is large. Power computations can be made using these guidelines and are helpful, although power based on actual data is preferable since it contains more information and the resulting study is less likely to be underpowered. Examples of power and sample size computations for a variety of statistical analyses are given in Zar [10].

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Fig. 2  Power versus sample size showing the relationship between sample size and power when the effect size is held fixed

5 Summary Statistics A sample of continuous measures is usually summarized by providing some indication of the center and spread of the sample. The center can be summarized by the arithmetic mean, median, or other measures of central tendency. The median is equivalent to the 50th percentile or second quartile (Q2): that value for which half the observations are below and the remaining half are above. The arithmetic mean is the most commonly used of the three, although outliers have less influence on the median. If associated inference is based on the parametric t test, it common to report the mean. Medians are more compatible with the nonparametric MWW ranksum test. The t test is sometimes referred to as a test of the mean, whereas the MWW rank-sum test can be thought of as a test of the median. It is also important to summarize the spread or variability of the data. Measures of variability include the range, interquartile range, and standard deviation. Range is the difference between the maximum and minimum value, which is informative but clearly driven by only the two most extreme values and can thus vary greatly from one sample to the next. The interquartile range is the difference between the 25th and 75th percentiles (or first and third quartiles), where, by definition, p percent of the sample lies below the pth percentile. Since the interquartile range depends on half of the data, it is much more stable than the range. The standard deviation is the square root of the mean squared distance to the mean, which, in a sense, provides a measure of the average deviation from the mean. Standard error is the standard deviation divided by the square root of the sample size, and depicts the uncertainty regarding the estimate of the mean. In other words, standard

error is the standard deviation of an estimate (a sample mean, for instance) about its own mean. Standard errors can be used to construct confidence intervals for an estimate. If a sample of size n is used to estimate a population parameter, such as the population mean, the estimate will be off owing to variability in the data and limitations of sample size. For example, a poll of the electorate uses a sample to estimate preferences in the population at large. “Confidence” in the estimate increases with decreasing variability and increasing sample size. For instance, using the observed variability and sample size, we can construct a 95% confidence interval about the sample mean which we believe includes the population mean at least 95% of the time and excludes the population mean less than 5% of the time. In other words, given 100 samples from a given population and the 100 associated confidence intervals, we would expect 95 of the 100 confidence intervals to include the population mean. The smaller the interval, the less confident we are that the interval contains the population mean: a 68% confidence interval is roughly two standard errors wide (estimate ± one standard error), a 95% confidence interval is roughly four standard errors wide (estimate ± two standard errors), and a 99.7% confidence interval is roughly six standard errors wide (estimate ± three standard errors). The widths are determined on the basis of approximation to the normal distribution, which becomes more accurate, in general, as the sample size increases. Simple summaries, although concise, can mask features of the data that are obvious when the data are plotted. For instance, reporting the mean and standard error might not satisfactorily summarize data from a bimodal or one-tailed distribution. Multimodality, skewness, outliers, and other features of the data may not be apparent without the use of visual displays. Even if the displays themselves are not used in the publication,

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they are a crucial step in alerting investigators to anomalies in and gaining a complete understanding of the data. If the data contain outliers, it is prudent to consider additional summaries (and analyses) excluding or downweighting the outlying points. There are no hard-and-fast rules about when to exclude outliers. Doing so largely depends on the context and cause of the extreme values. When in doubt, it always best to err on the side of full disclosure and present summaries with and without the outliers. Outliers can also be very informative, suggesting changes to experimental procedures. Categorical or qualitative data are much simpler to summarize. For instance, to summarize the sex of study participants, one presents the number of females and males. It is also helpful to include the percentage of either or each. It is important to include the denominators in such summaries, as they indicate the sample size and confidence we should have in the estimated proportions. This confidence, of course, can be formalized by providing confidence intervals for the proportions. For more information on this and other summary statistics common in medical literature, see [11].

6 Visual Displays of Data Graphical depictions of data are important at every stage of data collection and analysis. During data collection, graphics can instantly convey the progress of a study and highlight potential quality control problems such as missing or aberrant values. Visual displays can suggest the need to clean the data or transform variables before analysis. They can indicate trends and associations that can guide the nuances of a planned analysis or motivate an exploratory analysis. An elegant visual display of data concisely demonstrates effects which might otherwise be lost in an unwieldy table of numbers. Classic references in this area include [12, 13]. We provide an overview of the topic and some examples of useful graphic displays for pre- and post-operative hemodynamic data from patients undergoing pulmonary thromboendarterectomy courtesy of Dr. Kim Kerr at the University of California, San Diego. The graphics in this section were produced using the statistical software program R [14] with the Lattice Graphics package [15].

6.1 Box-and-Whisker Plots, Bar Plots, and Histograms Box-and-whisker plots (or box plots), bar plots, and histograms are commonly used to illustrate and compare univariate distributions, as opposed to scatter plots, which depict bivariate associations, and time series plots, which depict temporal dependence. Figure 3 shows a box plot and histogram

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Fig. 3  Box-and-whisker, rug, and histogram plots all depicting a leftskewed distribution of the change in pulmonary vascular resistance (PVR), posttreatment PVR minus pretreatment PVR

of the change scores, posttreatment pulmonary vascular resistance (PVR) minus the pretreatment PVR. The bottom panel of the figure gives the histogram, which shows the frequency of observations within bins of width 200 dyn s/cm5. Below and above the histogram is a “rug” which shows the actual location of the observations along the continuum. Finally, in the top panel, there is a box plot. Box plots were introduced in Tukey [16]. The ends of the box and the bold line in the middle represent the first, second (median), and third quartiles; or Q1, Q2, and Q3. Box plots use the interquartile range, or IQR = Q3 − Q1 to define outliers. The “whiskers” extend outward from Q1 and Q3 a distance of 1.5 × IQR, or to the most extreme observation, if the most extreme observation is less extreme than 1.5 × IQR. Any observations beyond Q1 − 1.5 × IQR or Q3 + 1.5 × IQR are considered outliers and are plotted separately. Box plots are slightly less intuitive, but more compact than histograms. Histograms are also sensitive to the selection of bin width and end-point location. In Fig. 3, one can see that the box hovers above the center of mass of the rug, and the peak of the histogram. The fact that box is shifted to the right with respect to the whiskers indicates the skewness, which is clearly apparent from the histogram and rug. Box plots are useful in demonstrating group or treatment differences. For example, after looking at the box plot in Fig. 3, you might conclude the mean, or median, of the change in PVR is negative. Both the t test and the Wilcoxon signed-rank test support this conclusion with highly significant p values (p < 0.001). Of course, such a post hoc observation should be

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verified with independent data and possibly a study designed to test that specific hypothesis. In other instances, multiple box plots can be used to suggest differences in the location of multiple distributions, which might be confirmed with an analysis of variance (ANOVA) or a Kruskal–Wallis test. Bar plots are also commonly used in this setting. Bar plots can be used to represent any quantitative value (e.g., counts, means, medians,) across multiple groups. In basic and clinical research settings, the bars themselves generally depict means, and standard errors or standard deviations are represented by features similar to whiskers on a box plot. Bar plots are clear and simplistic ways to show relative differences, but do not always illustrate variability, range, or skewness as well as a box plot would.

6.2 Scatter Plots The simplest and most familiar depiction of two continuous measures on a sample of data is the bivariate scatter plot. A classic example of the importance of plotting the data is provided in [13]. Anscombe describes four datasets, each with 11 observations on two measures. In each dataset, the correlation between the two measures is the same and each dataset leads to exactly the same linear regression output. However, scatter plots show that only one of the datasets is satisfactorily summarized by the correlation and linear regression (Fig. 4). The other plots reveal either a curvilinear association or the presence of a single heavily influential

Fig. 4  Anscombe’s example. The plots illustrate the importance of looking at data. Only the data at the top left are appropriately summarized by correlation and linear regression

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observation which, if ignored, would result in a very different correlation and regression line. Reporting the correlation alone ignores important information in the data and, as a result, might lead to spurious conclusions. A superimposed model fit, such as the ordinary least squares (OLS) regression used by Anscombe, or a more flexible data smoother, such as the locally weighted scatter-plot smoother (LOWESS) [17], is often used to embellish the scatter plot and suggest trends. Multiple scatter plots can be arranged into a scatter plot matrix (also often called a pairs plot, since pairs of variables are being plotted) so that clusters of associations might be assessed. Color can be used to simultaneously depict a categorical variable such as sex or provide evidence of an associated clustering within a single scatter plot. Figure  5 depicts a scatter plot matrix of pre-intervention measures from the thromboendarterectomy data. The plot includes mean systolic and diastolic pulmonary pressure (M), cardiac output (CO), pulmonary capillary wedge pressure (W), and PVR [PVR = (M − W)/CO]. The OLS regressions are represented by dashed lines, LOWESS smooths are given by solid lines, and correlations are shown in the upper-right boxes. The CO versus W plot (r = 0.13) reveals a possible outlier with one CO value (CO = 11.9) much higher than the rest. The LOWESS line is pulled down by the outlier, and the correlation excluding this value is substantially different (r = 0.21). The CO outlier affects the LOWESS curve as well in the CO versus PVR plot (r = −0.71). Coupled with a PVR outlier, the effect suggests the nonlinear trend that might be expected given the deterministic relation between PVR and

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Fig. 5  Pairs plots with smoothing splines, correlations, and histograms. Pairs plots of mean systolic and diastolic pulmonary pressure (M), cardiac output (CO), pulmonary capillary wedge pressure (W), and PVR [PVR = (M − W)/CO]. The ordinary least squares regressions are repre-

sented by dashed lines, locally weighted scatter-plot smoothing smooths are given by solid lines, and Pearson’s correlations are shown in the upper-right boxes

CO [PVR = (M − W)/CO]. Notice that in all of the other plots, the two outliers play less of a role and the LOWESS and OLS lines are very much in agreement. A traditional scatter plot is not equipped to illustrate multiple observations on the same point, so information is lost in the plot. One remedy for this is the sunflower plot, which adds a “petal” to each point for each replicated observation.

6.3  Time Series Plots One of the earliest, and still most common, uses of visual techniques is the depiction of time series data (see page 28 in Tufte [12] for a discussion of such a plot of planetary observations dating back to circa tenth century). Longitudinal plots (also known as spaghetti plots) depict the variations in an observation, usually on the vertical axis (ordinate), over

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Fig. 6  Pre- and posttreatment PVR slopes

time, usually on the horizontal axis (abscissa). The simplest example is taken from an intervention trial, in which preintervention measurements are associated with postintervention measurements on the same subject by a line connecting the two. Figure 6 depicts longitudinal PVR values before and after the thromboendarterectomy intervention, corresponding to the change scores plotted in Fig. 3. Longitudinal plots are generally more interesting and better suited to more than two time points. Although even with two time points, the spaghetti plot can be a useful quality assurance tool, calling to attention impossible trajectories. In this case, we see that most subjects experienced a decrease in PVR after intervention, but this could also be summarized with a box-and-whisker plot, a bar plot, or a histogram of the change scores.

7 Correlation, Regression, and ANOVA The most ubiquitous measures of correlation are the Pearson product moment correlation coefficient and Spearman’s rank correlation. Pearson’s r is the sample covariance of two measures divided by the product of their standard deviations. Spearman’s rank correlation, r, is Pearson’s correlation applied to the ranks. Pearson’s r characterizes how well a line describes the association between two variables and Spearman’s rank correlation characterizes how well an arbitrary monotone function describes the data. Each coefficient ranges from −1 to 1. A Pearson’s r of −1 or 1 indicates that all of the pairs can be plotted on a line with negative or positive slope, respectively. A value of 0, indicates no linear association. As discussed in Sect., one should always assess

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scatter plots for influential outliers and nonlinear associations. Bivariate data can be perfectly associated with r = 0. Another way to depict linear association is so-called ordinary least squares (OLS) regression. This assumes the response variable, y, can be modeled as a linear function of the predictor, x, and independent and identically distributed Gaussian error, e, with variance s 2. That is, for each subject i, yi = b0 + xib1 + ei. Equivalently, we assume yi has a distribution with mean mi = E( yi) = b0 + xib1 and variance s 2. The intercept, b0, and slope, b1, can be estimated by minimizing the squared distance between fitted and observed values. The OLS estimate of b1 can be shown to be equivalent to a rescaled Pearson’s r between predictors and responses. Specifically, b1 is the sample covariance between the predictors and responses divided by the standard deviation of the predictors. Choosing to report the estimated slope from OLS regression versus Pearson’s correlation is a matter of taste, although the regression setting inherently suggests “predictor” and “response” roles for each variable. The predictor is assumed to be measured without error, and overall the regression results are more informative. Learning that a one-unit increase in additives produces a b1 increase in gas mileage may be more useful than learning the correlation between additives and gas mileage. Pearson’s r makes no suggestion about predictor and response and treats each measure interchangeably. For example, consider again the thromboendarterectomy data and mean systolic and diastolic pulmonary pressure (M) versus CO. As depicted in Fig. 5, Pearson’s r is −0.30, which confirms the negative trend seen in the scatter plot. The dashed line represents the OLS regression line. The intercept, b0, is estimated to be 5.7 (standard error 0.63) and the slope, b1, is −0.04 (standard error 0.014). The intercept, of course, is the height of the dashed line as it crosses the vertical axis (M = 0), which is just left of the plot window in Fig. 5. If our null hypothesis were that there is no linear association between CO and M, we would reject the null hypothesis at the p value 0.003. Of course, OLS regression can be generalized to model multiple covariates, i.e., yi = b0 + b1x1i + … + bpxpi + ei. Estimates of the regression coefficients bi can be computed as in the simple case of one covariate: by minimizing the sum of squared distances between observed and fitted values. Furthermore, we can derive standard errors, confidence intervals, and p values for these estimates as we would with a simple mean. In OLS regression (and many other statistical procedures) a minimum of 12 data points per covariate are recommended [18]. Thus far we have only discussed continuous covariate measures, but covariates could also be “dummy” variables encoding the levels of categorical variable. For instance, assume we design a study to have a placebo group, a low-dose therapy, and a high-dose therapy. To simultaneously model the response in each of the three groups, we would create a dummy variable for each group that is 1 if the subject is in the group and 0 otherwise. The model looks like yi = bPxPi + bLxLi + bHxHi + ei.

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This is the model underlying the classic one-way ANOVA. A one-way ANOVA assesses the ratio of the variance of the group means (the estimates of bP, bL, and bH) to the mean variances within each group. This ratio is called the F statistic. If the variance of the group means is large with respect to the mean within-group variance, this suggests the groups are different. This is formalized by approximating the F statistic by its asymptotic distribution, the F distribution. From this approximation, we can derive p values associated with the F statistic for testing the null hypothesis that all three (or more) groups share the same mean. In many studies the response variable may be other than normally distributed or continuous. Generalized linear models are appropriate for binomial and count response variables, for example. They [19] use a family of “link” functions to relate linear predictors, e.g., b0 + b1x1i + … + bpxpi, to the mean of the distribution of the response, denoted E(yi) or m. That is,

E ( yi ) = m = g −1 (b 0 + b1 x1i + … + b p x pi ),, where g is the link function. The simplest case is the identity link, which yields the linear model and OLS regression. The distribution of the responses is also generalized to distributions other than the Gaussian distribution. For example, count data can be handled by assuming a Poisson-like distribution for the response and the natural log for the link function, g. Binomial distributed responses (0 or 1) can be accommodated by assuming a “logit” link function, g(m) = ln[(1 − m)/m], where m represents the probability of a 1 response. This is logistic regression. Linear and generalized linear models can be used to model nonlinear associations between predictors and responses. For instance, if we let the covariates be time and time squared (x1 = t and x2 = t2), we can model a quadratic relationship between time and the response variable. When the data contain repeated measures on the same subject, it is generally no longer safe to assume the errors are independent. In this case we might expect the errors to be correlated, as in a random effects model [20] or a model fit by generalized estimating equations [21]. These models deal with correlation structures that can arise in analysis of longitudinal or “clustered” data.

8 Survival Analysis In studies where the end point of interest is time until a particular event, survival analysis, so-called because the event under consideration is often death, is the appropriate technique. Of course, any well-defined clinically interesting event could be considered. For example, the analysis of bosentan therapy for pulmonary arterial hypertension [22] included a secondary analysis of time to clinical worsening, which was defined as “the combined end point of death, lung

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transplantation, hospitalization or discontinuation of the study treatment because of worsening pulmonary arterial hypertension.” As with most survival datasets, some event times are unobserved, or censored, i.e., when a subject survives the entire study period event-free or is lost to follow-up before any event occurs. Such censoring must be independent of the event of interest and any covariates of interest, otherwise the estimates of survival rates or covariate effects may be biased. In general, the hypothesis that censoring is independent of the event of interest is untestable, but it is considered a reasonable assumption if the subjects leave the study for reasons unrelated to health or treatment and the known relevant characteristics are similar to those of the subjects who were not lost to follow-up. The bosentan analysis includes so-called Kaplan–Meier estimates of the proportion of patients not experiencing clinical worsening over time and logrank tests comparing this proportion at various times over the course of the trial. In this section we will discuss these analyses as well as (proportional hazards) regression analyses and the analysis of clustered survival time data. An overview of survival analysis is provided in [11]. For an introduction to the detailed mathematical considerations, classic references include [23, 24]. A comprehensive overview of the subject is provided in [25]. Analyses and graphics discussed in this section can be produced in R [14] using the survival [26] package.

8.1 Kaplan–Meier Curves One of the most common graphical representations of survival data is the Kaplan–Meier curve [27]. The Kaplan–Meier curve is the nonparametric maximum likelihood estimator of the survival function, S(t), the probability of surviving eventfree to a given time t. Time is represented on the horizontal axis and the probability of survival is represented on the vertical axis. The plot is a stair-step with vertical jumps at each of the observed event times. As mentioned earlier, a key feature of survival analysis is the fact that we do not typically observe the event time for all subjects. For example, some subjects survive the entire study period without experiencing an event, others might be lost to follow-up during the trial. The Kaplan– Meier estimate of the probability of surviving the interval between two consecutive event times is simply the number observed to have survived the interval divided by the number observed to be at risk throughout the interval, thereby accounting for the censored observations by incrementally dropping them from the denominator as the risk set changes (over time). The cumulative probability of surviving to time t is then the product of all the preceding interval probabilities, which is why it is sometimes called the product-limit estimator. Table 1 and Fig. 7 depict a hypothetical example.

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M.C. Donohue et al. Table 1  Kaplan–Meier estimates

i

Time ti (days)

Number at risk at time ti

Number of events at time ti

Probability of surviving the interval ti to ti − 1

Kaplan–Meier estimate S(ti)

1 3 100 6 0.94 0.94 2 5 88 15 0.83 0.78 3 7 66 8 0.88 0.69 4 12 48 3 0.94 0.64 5 15 41 9 0.78 0.50 6 23 23 5 0.78 0.39 7 35 15 9 0.40 0.16 The Kaplan–Meier estimate of the probability of surviving the interval between two consecutive event times (column 5) is simply the number observed to have survived the interval (value in column 6 subtracted from value in column 3) divided by the number observed to be at risk throughout the interval (column 3). The cumulative probabilities (column 6) are products of the interval probabilities, and are plotted in Fig. 7.

each treatment group, one can compute the expected number of events in each group. A large difference between the observed and expected number of events is evidence against the null hypothesis. These differences are summed over all time points and a test statistic is derived with formal inference similar to the Mantel–Haenszel test [28].

8.3 Proportional Hazards Regression

Fig. 7  Kaplan–Meier estimate of survival based on hypothetical data depicted in Table 1

8.2 Logrank Test The test most typically employed to compare the rate of survival in two independent samples, placebo and active treatment, for instance, is the logrank test. In [22], for instance, the authors provide logrank test p values for 16and 28-week survival in the cases of 250  mg bosentan versus a placebo and 125  mg bosentan versus a placebo. The paper also provides superimposed Kaplan–Meier plots for the three study arms. Inference is based on the distribution of the 2 × 2 contingency table of treatment groups (e.g., active vs placebo) and survival status (e.g., died vs survived) at each of the event times. Under the null hypothesis of no treatment effect, the proportion surviving should be the same at each event time. Given the total number of events and the number at risk in

When one wishes to model the effects of covariates on the probability of remaining event-free to time t, a common model is Cox’s proportional hazards model [29]. This model assumes the instantaneous rate of failure, or “hazard,” at time t and for subjects with covariate vector x, denoted l(t;x), is proportional over time to the hazard for subjects with other covariate values. More specifically,

λ (t ; x) = λ 0 (t )exp(bx), where bx = b1x1 + b2x2 + … + bpxp, is the linear predictor and l0(t) is the so-called baseline hazard. The covariate effects, b, and the baseline hazard, l0(t), can be estimated by maximizing the semiparametric likelihood which arises from the model assumptions. This likelihood does not make use of the event times, only their ranks. For instance, the probability that an event occurs for subject 1 at time tj is

exp(b x1 ) / ∑ exp(b xi ), where the sum in the denominator is over all individuals i at risk of failing at time tj. So the likelihood only depends on t through the risk set, which is completely determined by the rank order of the event times. This simplifies estimation but makes estimates of the covariate effects sensitive to the handling of ties. Of course, ties are common in practice

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with a large dataset and discrete failure times. Available software has typically used Breslow’s method [30] for dealing with ties, which is known to be conservative. For a cautionary discussion on the handling of tied observations, see Hsieh [31]. Residual plots [32], residuals, martingale residuals, or deviance residuals [33, 34] can be used to check the assumptions of the model and assess the fit.

8.4 Competing Risks The techniques discussed thus far are predicated on there being only one event of interest and censoring being independent of the event. In practice, however, there may more than one event of interest and events, censoring included, may not be completely independent. For instance, instead of the combined end point of death, lung transplantation, hospitalization, or discontinuation of the study treatment because of worsening pulmonary arterial hypertension [22], one might consider the time to each of the specific end points separately. We cannot simply consider each event type in turn as the event of interest and the others as censored observations. This type of censoring is not independent because the risks for the different event types are dependent or competing risks [35, 36]. For example, lung transplantation and hospitalization increase the risk of death. In the case of multiple failure types, one common approach is based on cause-specific hazard rates. In [36], it is shown that, in fact, functions derived from the cause-specific hazards are the only estimable quantities possible (without making further assumptions). In [35], the authors review the available tools for summarizing data with competing risks and suggest methods including the cause-specific hazard rate, cumulative incidence curves, conditional probability curves, and bounds on the true marginal survival functions in the absence of competing risks. Inference based on the cumulative incidence curves is proposed in [37] and an accompanying R package [38] is available.

8.5 Clustered Data Classical Cox proportional hazards regression assumes the observations are independent. In some cases, we might expect greater correlation among clusters of data. For instance, in a multicenter study of a complicated treatment we might expect some sites to be better equipped to administer the study procedures. In a familial study we might expect observations on members of the same family to be more closely correlated than those on unrelated strangers owing to

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shared environmental or genetic factors. Similar correlations may be expected for matched-pair studies. Cases in which individuals may experience repeated events also fall in the category of clustered data. Ignoring such dependence can inflate the estimated error or, worse, bias the results. Clustering is commonly accounted for by including a so-called frailty in the model. Frailty terms are random variables that extend the classical Cox proportional hazards model to accommodate within-cluster correlation. More specifically, the frailty model is

l (t ; x) = gl 0 (t )exp(b x), where g is assumed to be a random variable shared by members of the same cluster. The frailty is classically assumed to have a gamma distribution [39], but may be assumed to have any of several distributions. The frailty model is akin to a mixed effects model incorporating a random intercept term on a log scale:

log l (t ; x) = log g + log l 0 (t ) + b x. More complex mixed effects models, incorporating covariate interactions, are also possible [40, 41].

9 Binary Data 9.1 Proportions This section discusses outcome data, which takes only two classes (hence binary or binomial). Examples of such binomial populations include adults and minors, having or not having a disease, surviving or not surviving over a fixed period of time. The proportion of the population belonging to one category is usually denoted by p, and the proportion of the population in the second category is q = 1 − p. Probabilities observed when sampling from these binomial populations are determined by the binomial distribution. Detailed overviews of analyses of binomial data are provided elsewhere [10, 42, 43]. Using the binomial distribution and the F distribution [44] (1 − a), one can compute confidence limits for the binomial proportions p and q as follows:

Lower limit =

X , X + (n − X + 1) F α (2),v1 ,v2

where n is the number of subjects, X is the number of subjects in one category, v1 = 2(n − X + 1),and v2 = 2 X .

Upper Limit =

( X + 1) Fα (2),v1¢ ,v2¢ n − X + ( X + 1) F α (2),v1¢ ,v2¢



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where n and X are the same as above, v1′ = 2 (X + 1) = v2 + 2

and v2′ = 2 (n − X ) = v1 − 2. Alternatively, approximate 100(1 − a)% confidence intervals can be obtained for p (or q) on the basis of the asymptotic normality of the sample proportion pˆ = X / n as follows: pˆ ± zα / 2 pq ˆ ˆ / n . (This is called the Wald confidence interval, first described in [45].) This method is very commonly used; however. caution should be exercised since this approximation is not accurate either when p is close to 0 or 1, or when npˆ or nqˆ is less than 5. In general, unless sample sizes become quite large, these confidence limits are too conservative (wider than needed). Agresti and Coull [46] introduced a modification of this approximate confidence interval by adding two successes and two failures to the data. The adjusted sample proportion is pˆ a = ( X + 2) / (n + 4) , and the confidence interval is computed using the adjusted sample proportion and (n + 4) instead of n. It has been shown that the confidence intervals so computed have better coverage properties than the Wald confidence intervals. Analysis of proportions frequently arises in medical fields: it might be of interest to assess rates of adverse events, or to compare the proportion of long-term survivors in different treatment groups. This is not the same as survival analysis, although broadly a similar question is being addressed. Survival analysis is interested in time: modeling or comparing time to a binary event. Here we are estimating or comparing proportions of binary events which took place over some fixed period of time.

9.2 Diagnostic Tests One common occurrence of proportions in medicine is in diagnostic tests for the presence of a particular condition and the evaluation of the tests’ performance. To assess how well a diagnostic test performs, the patients in the sample must be classified as having the disease or not having the disease using a reference (or “gold”) standard; the true diagnosis, or at least the reference standard diagnosis must be known. Then, each patient is classified as being healthy or having the disease on the basis of the diagnostic test, and the test’s agreement and disagreement with the true status are summarized in a table (Table  2) with four possible categories:

Table 2  Diagnostic test result categories Test positive (T+)

Test negative (T-)

Disease present (D+) Disease absent (D-)

False negative (FN) True negative (TN)

True positive (TP) False positive (FP)

• True positives (TP): the patients who test positive and have the disease • False negatives (FN): the patients who test negative but have the disease • False positives (FP): the patients who test positive but are healthy • True negatives (TN): the patients who test negative and are healthy Performance parameters are standard measures used to evaluate a diagnostic test. The discussion that follows is limited to the most commonly used subset of such parameters. Sensitivity [equivalent to TP/(TP + FN)] is the proportion of subjects testing positive for the disease among those who really have the disease; or the estimated probability of a positive result conditional on the disease being present. Sometimes it is called the true positive rate. When sensitivity is high, there are relatively few false negatives (as seen from the formula); thus, in a test with high sensitivity, a negative result can rule out the disease with high probability. Specificity [equivalent to TN/(TN + FP)] is the proportion of subjects testing negative for the disease among those who are healthy; or an estimated probability of a negative result, conditional on the subject being healthy. Sometimes this is called the true negative rate. If specificity is high, there are few false positives; thus, in a test with high specificity, a positive result rules in the disease with high probability. Different conditional probabilities are estimated by the positive predicted value (PPV) and negative predicted value (NPV). PPV [equivalent to TP/(TP + FP)] is the proportion of subjects who have the disease among all subjects who tested positive for the disease; or an estimated probability of a subject having the disease given that the test was positive. PPV is high when the test has high specificity (few false positives) and the disease is common (relatively many true positives). NPV [equivalent to TN/ (TN + FN)] is the proportion of subjects who do not have the disease among all subjects who tested negative for the disease; or an estimated probability of a subject being healthy given that the test was negative. NPV is high when the test has high sensitivity (few false negatives) and the disease is rare (relatively many true negatives). Since actual prevalence of disease affects PPV and NPV, caution must be exercised when interpreting them if the proportions of healthy and sick subjects in the study are not truly representative of the population prevalence but are artificially controlled. Finally there is total accuracy, defined as (TP + TN)/ (TP + TN + FP + FN), and its opposite, the total misclassification, defined as (FP + FN)/(TP + TN + FP + FN) or 1-total accuracy. The underlying goal is to be able to project the performance of the diagnostic test from the study sample to the population at large. Each of these performance parameters is a proportion of one category in a binomial population (e.g.,

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TPs in a population of TPs and FNs) and can be analyzed under the assumptions of the binomial distribution. One natural question is to compare selected performance parameters of a test to a chance proportion of 0.5. At the very least one wants to ensure that a diagnostic test is better than guessing at random. But in many cases that is not good enough. Both false negatives and false positives in a diagnostic test can have serious consequences. The former allows the disease to progress untreated; the latter may involve harmful effects of being aggressively treated for a misdiagnosed condition. A good diagnostic test therefore must minimize both false positives and false negatives, i.e., be both highly sensitive and highly specific. Sometimes it is of interest to compare performance parameters with predetermined proportions. More generally, a confidence interval of level 1 − a, often 95%, placed around the performance parameter allows for inferences about the performance of the test in a larger (but otherwise similar) population. Consider an example from cardiopulmonary exercise testing. These data have been published and are used with permission of Paul Phillips of San Diego’s Scripps Mercy Hospital [47]. VO2-max (the maximum capacity for utilizing oxygen during exercise) was measured in a group of 105 subjects with the goal of separating normal controls from patients with myopathy or rhabdomyolysis. Twenty-six liters per minute was considered to be the best cutoff for separating controls from affected subjects (less than 26 L/min, sick; 26  L/min or more, healthy). The diagnostic contingency table for VO2-max is shown in Table  3. The performance parameters and the exact binomial 95% confidence intervals around them are shown in Table 4.

Table 3  Summary table of myopathy/rhabdomyolysis versus control classification using dichotomized VO2-max VO2-max < 26 VO2-max ³ 26 Myopathy or rhabdomyolysis Normal control

59

18

 7

21

Table 4  Performance parameters Numerator

Denominator

9.3 Receiver Operating Characteristic Curves The previous section discussed a diagnostic test with the binary classification: the disease is present or absent. Sometimes it is also of interest to examine the ability of a continuous measure to predict the presence of disease, and to select the best possible classification threshold. One way to address this question is by use of the receiver operating characteristic (ROC) curves. A ROC curve is simply a diagram which plots sensitivity versus 1-specificity for every potential classification threshold of a numeric variable. To continue with the VO2-max example, in the group of normal and affected subjects VO2-max took on values between 6.6 and 72.9 L/min. Every value is a potential classification threshold, and for each such threshold, sensitivity versus 1-specificity can be plotted as shown in Fig. 8. The highest sensitivity/specificity combinations will be found in the top-left corner of the graph. In this example, the highest combined sensitivity and specificity (0.766 and 0.750, respectively) are indicated with an arrow and correspond to the VO2-max threshold of 26 L/min (less than 26 L/ min, affected; 26  L/min or more, healthy). The summary table for that threshold is shown in Table 3. The efficacy of a variable for diagnosis can be assessed by comparing the area under the curve (AUC) for corresponding ROC curve with the null AUC of 0.5. AUC of 0.5 occurs when the ROC is the y = 1-x diagonal shown on the plot, which means the true positive rate is equal to the false positive rate; in other words use of the variable of interest is no better than guessing at random. The statistic for evaluating the AUC of an empirical ROC curve has been shown to be equivalent to the MWW statistic for comparing distributions of values from two samples [48, 49]. In other words, to see if the variable of interest is a significant diagnostic factor, use the MWW test to compare the disease and no disease groups on the variable of interest (e.g., VO2-max of the normal controls with the VO2-max of the myopathy/rhabdomyolysis group). The p value of the test corresponds precisely to the p value associated with the AUC.

CI 95% lower bound

Estimate

CI 95% upper bound

Sensitivity 59   77 0.656 0.766 0.855 Specificity 21   28 0.551 0.750 0.893 PPV 59   66 0.794 0.894 0.956 NPV 21   39 0.372 0.538 0.699 Total accuracy 80 105 0.669 0.762 0.840 Total misclassification 25 105 0.160 0.238 0.331 The performance parameters for myopathy/rhabdomyolysis versus control classification using dichotomized VO2-max are shown. The numerator divided by the denominator give the estimate (e.g., sensitivity is 59/77 = 0.766). Lower and upper bounds of the 95% binomial confidence interval (CI) around the estimate are shown

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Fig. 8  Receiver operating characteristic (ROC) curve for the VO2-max example. The best sensitivity–specificity trade-off is shown with an arrow. Area under the curve (AUC) and the associated p value are shown on the plot

The ROC curve for the VO2-max example shows that although even in the best case it is not perfect as a classifier of myopathy/rhabdomyolysis, it is definitely informative (p < 0.0001). Which brings us to the next natural question: how to assess the joint capacity of several classifiers.

9.4 Logistic Regression A ROC curve can only be used to examine a single predictor; however, it is more common to have several simultaneous predictors of interest. To continue the previous example, suppose we are interested in separating controls from myopathy and rhabdomyolysis patients using a combination of several gas exchange predictor variables: VO2-max, respiratory exchange ratio (RER), aerobic threshold (AT), and anaerobic threshold (S1). A popular approach to this problem is called logistic regression. Logistic regression, mentioned in Sect., is a modeling technique from the class of generalized linear models which estimates the probability of an event associated with each value of a predictor (continuous, ordinal, or binary), or a combination of multiple predictors, by fitting the data to a logistic regression curve, as follows:

p(event) =

1 1+ e

− ( b0 + b1 x1 + b 2 x2 + ...b p x p )

,

where p(event) is the probability of an event, x1, x2, …xp are the predictors of interest, and b0 , b1 ,...b pare the coefficients to be estimated. The logistic distribution function is S-shaped, which involves specific assumptions about the probability (or risk) of the event: the probability is bound between 0 and 1, monotonic, and steeper in the middle than at either the high or the low ends. From the epidemiological point of view, this is a reasonable assumption about the behavior of risk probability, which is why the logistic regression approach is a popular one. Figure  9 shows an example of a logistic probability curve for the single predictor VO2-max: as VO2max decreases, the probability of myopathy/rhabdomyolysis increases. Logistic regression allows the investigator to identify factors associated with increased likelihood of an event. Another useful result of a logistic regression is the odds ratio associated with each predictor. The odds ratio for a given predictor xi can be estimated from its coefficient bi simply as e bi , and it means that one unit change bin xi when all other predictors are held fixed results in the e i change in the odds of an event. Using logistic regression to predict the probability of myopathy/rhabdomyolysis from the combination of all four gas exchange variables shows that only two of the four predictors are significant: VO2-max and RER (Table 5). Low VO2-max and high RER are associated with increased probability of myopathy/rhabdomyolysis. S1 and AT have no significant influence on the probability of myopathy/rhabdomyolysis.

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Fig. 9  Logistic regression probability curve for the VO2-max example

Table 5  Partial output from the logistic regression Coefficient Standard error

p

Intercept -19.97 6.77 0.0032 VO2-max -0.1567 0.0568 0.0058 RER 28.53 7.95 0.0003 AT -0.002 0.072 0.9776 S1 3.593 3.707 0.3323 The size, standard error, and p value of the coefficients in logistic regression predicting myopathy/rhabdomyolysis with a combination of all gas exchange variables are shown. Only VO2-max and the respiratory exchange ratio (RER) are significant.

statistical software packages include CART or similar decision tree methods. CART is described elsewhere [50]. It is worth noting that CART and multiple logistic regression are different methods but they address the same question (as in some ways do ROC and univariate logistic regression), so the results, although not identical, are generally consistent. Both ROC and univariate logistic regression confirm that VO2-max is a significant predictor of myopathy/ rhabdomyolysis. CART and multivariate logistic regression select the same two gas exchange variables as the best predictors of myopathy/rhabdomyolysis.

10 Classification and Regression Trees Another approach to describe the relationship between several predictor variables and a binary outcome is called classification and regression trees (CART). (CART can be used with continuous outcomes as well but only binary outcomes are discussed in this section.) CART is a recursive technique, which builds a decision tree from multiple predictors to classify the dependent variable. Once a splitting rule has been selected, it is applied to all subsequent nodes of the tree, which is what is meant by a recursive procedure. Figure 10 illustrates a tree for classifying controls and myopathy/ rhabdomyolisis patients using VO2-max, RER, AT, and S1 as potential predictors. The splitting rules in a tree are based on optimal classification and the algorithm selected only two out of four potential predictor variables. The advantage of CART is the production of simple flow-chart-like rules for classifying the outcome. The disadvantage is that this technique is not as accurate as more sophisticated techniques. In spite of this, CART should produce reasonable and simple-to-follow guidelines. Many

10.1 Contingency Tables, c2 Test, and Fisher’s Exact Test Tables such as Table  3 require further discussion as an example of another broad type of data summarization and analysis. Binary data (or categorical data in general) are often collected simultaneously for two variables. In the example of Table 3 the two variables are disease status and test result. A table, such as Table  3, which shows the frequency of each combination of categories is called a contingency table. Another example of a contingency table includes counting the frequency of patients with and without combinations of conditions. A simulated example of patients with and without pulmonary venous obstruction and with a PVD index above or below 1.25 is summarized in Table 6. In this example, it is of interest to examine whether the relative frequencies of a high PVD index are the same for patients with and without pulmonary venous obstruction (which is equivalent to asking whether the relative frequencies

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Fig. 10  Classification tree for the myopathy/rhabdomyolysis example. The terminal nodes are marked C for control and MR for myopathy/ rhabdomyolysis. Twenty-three subjects with VO2-max ³ 26.2 and respiratory exchange ratio (RER) less than 0.815 are classified as controls; of those, 19 are classified correctly. Eleven of 12 subjects with VO2max ³ 26.2 and RER > 8.15 are correctly classified as having myopathy/ rhabdomyolysis

Table  6  Contingency table: pulmonary venous obstruction versus pulmonary vascular disease (PVD) index PVD PVD index £ 1.25 index ³ 1.25 Total Pulmonary venous obstruction absent Pulmonary venous obstruction present Total

45

30

  75

15

60

  75

60

90

150

of pulmonary venous obstruction are the same for patients with and without a high PVD index). This question can be answered by computing and comparing row or column proportions. More generally, one is concerned with the question of whether the frequencies in the rows of a contingency table are independent of the frequencies in the columns. This question is most commonly addressed by the evaluation of a c2 statistic, a measure of how far the sample frequencies deviate from what would be expected under the null hypothesis of independence. The c2 statistic is computed as follows:

χ2 = ∑∑

( fˆij − fij )2 fij

,

where fˆij is the observed frequency for the cell in the ith row and jth column, and fij is the expected frequency for the same cell. To compute fij, the sum total of the ith row is multiplied by the sum of the jth column and divided by the total sum of the table. Using the example of Table 6, if pulmonary venous

obstruction were independent of the PVD index, then we would expect half (75/150) of all patients with a low PVD index to have pulmonary venous obstruction. Thus, 60(75/150) = 30 is the expected frequency f21. The rest of the frequencies are similarly computed, and the table’s c2 = 25, the probability of having a c2 statistic as large or larger than 25 under the assumption of independence (the p value of the c2 test), is evaluated by calibrating it against the c2 distribution with one degree of freedom. In this example, the probability is very small (p < 0.0001); the hypothesis of independence between rows and columns is rejected. In other words, we conclude that the proportions of high (or low) PVD index are not the same for patients with and without pulmonary venous obstruction. A contingency table need not be limited to only two rows and two columns. Two variables with more than two categories can summarized in a similar table (with more rows or columns), and the c2 statistic to test the hypothesis of row and column independence is computed in exactly the same way. However, this time the statistic is calibrated against the c2 distribution where the degrees of freedom equal to (rows − 1) × (columns − 1) . The 2 × 2 table is a special case of this general rule. A word of caution: if a contingency table has an expected frequency less than 1 in any of the cells, or an expected frequency less than 5 in more than one-fifth of its cells, the resulting c2 statistic will be biased, and a c2 test is not recommended [51]. Another way to analyze a contingency table is with a Fisher’s exact test, which examines the exact probabilities of cell frequencies under the null of random distribution of cell counts. In this test the margins (row and column totals of the contingency tables) are assumed to be fixed and the exact probability of a frequency of any given cell can be computed using the hypergeometric distribution. Using the example of Table .6, if we know that we have 150 patients, 75 with and 75 without pulmonary venous obstruction, 60 with a PVD index less than 1.25 and 90 with a PVD index greater than 1.25, what is the probability that purely by chance there will be 45 patients with pulmonary venous obstruction and a PVD index under 1.25? Any other cell can be selected: if the margins are fixed, knowing one cell allows all four cells to be computed and the probability is the same for all of them. This probability can be computed by hand, but only for 2 × 2 contingency tables with small cell counts; it is too complicated otherwise. Software, however, is not subject to such limitations. An advantage of Fisher’s exact test is that it is not affected by very small cell counts, unlike the c2 test. Applying Fisher’s exact test to Table 6 yields p < 0.0001, a result similar to (and consistent with) the result obtained with the c2 test. Discussions of contingency tables, c2 tests, and Fisher’s exact tests are presented elsewhere [10, 52].

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11 Controlling Type I Error 11.1 Multiple Comparisons It does not often happen that precisely one test of significance is performed in the course of a study. Much more common is the scenario where a number of measures are simultaneously collected on a group of subjects, and a number of comparisons are made. The problem with multiple comparisons is that the overall probability of finding at least one spurious significance in a collection of tests (this is type I error: the probability of a false positive) is higher than the significance level – the type I error threshold set for a single test. The overall type I error probability for a set of independent tests is easily computed. Given N tests with the null hypotheses true in all of them (nothing going on), the probability of at least one false positive finding P(fp), can be computed as follows: P(fp) = P(at least one false positive finding in a set of N tests) = 1 − P (no false positive findings in a set of N tests) = 1 − (1 − a)N This overall probability of at least one significant finding due to chance alone grows quite rapidly as the number of comparisons grows. For 45 tests it is over 90%, for 60 comparisons it is 95%, etc. (Keep in mind that these are error rates and something like 5% is desired. Granted, this calculation is strictly correct only for independent tests, whereas in practice sets of tests are usually carried out over variables, which are to some extent dependent. In that case the probability of at least one case of spurious dependence is not as high as in this computation, but the problem is still a severe one.) Suppose subjects are enrolled in a study, every conceivable piece of information about those subjects is recorded, and everything is correlated with everything else. Almost surely there will be at least one significant but spurious (and possibly absurd) correlation, for instance, between pre-SAT score and the number of onions consumed at age 35. This is pure chance: a fluke combination of two variables in that particular group of subjects only: it will not be true for another group of similar subjects. There are several methods of addressing this problem, none of them perfect. The simplest is the Bonferroni method: a single comparison is considered significant when the p value is less than 0.05/N, where N is the total number of comparisons. This correction is easy to apply and it ensures an overall significance level of 0.05, but it is very severe and overly conservative especially as the number of comparisons rises. There are several alternative corrections to the significance level that are simple to apply, similar in structure to the Bonferroni method, but somewhat “milder” [53–55]. Simes [56] modified the Bonferroni approach by ranking p values,

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whereas Hommel [57] modified the Simes procedure further with a decision strategy for individual hypotheses. With a larger number of tests, however, all of these corrections will make it more difficult to identify significant results; that is, all such corrections are power-reducing. Better use of the Bonferroni method and its cousin procedures lies in realizing that there is not necessarily one overall null hypothesis per study. Study questions can be clustered, according to type and scientific intent, and the Bonferroni method (or a similar procedure) can be applied within each cluster, each of which has its own, smaller, N. There is also the method of validation, where the hypothesis testing is done first on one randomly selected half of the data, and then on the remaining half. Only the significant results that are “confirmed” in this two-phase procedure are considered significant. Both of these approaches are especially reasonable if the study is exploratory rather than confirmatory, meant to gather information to inform further research rather than being the final statement of inference. A graphic method called p plots [58], which uses p values as data, can be used in cases of a large (say, more than 30) number of simultaneous tests of hypotheses to get an idea of how many are “truly” significant. The rub is that the p-plot procedure can only identify how many significant tests there should be; it cannot identify which ones actually are significant and it would be incorrect to pick out the set with the lowest p values. Thus, validation or confirmation is still required with p plots. Finally, a separate set of recent methods for controlling the false discovery rate have been developed for situations where the number of tests is very large both in absolute terms and relative to the number of subjects. An example where such methods are appropriate is genetics, where hundreds of genetic markers have been obtained on relatively few subjects and are tested for association with specific diseases. An overview is given in Chap. 39.

11.2 Interim Analyses Studies are designed to have a fixed final sample size, and power analyses are predicated on that assumption. Assume a hypothesis test is carried out at the conventional 0.05 level, then under the null hypothesis (i.e., under the assumption that nothing is going on) the chance of a false significant finding (type I error) should be limited to 5%. But this is only true when the hypothesis testing is done only once: at the end. If frequent (or even occasional) interim testing is done while the data are being collected and before the final sample size is reached, then there are more opportunities to find a significant result at the nominal 5% level, and the true probability under the null that there will a spurious significant finding will exceed 5%.

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Fig. 11  Simulation of unplanned interim analyses showing the progression of p values as the comparison is repeated after every new pair of “subjects.” The horizontal line indicates a p value of 0.05

Figure  11 illustrates this problem using simulated data. “Samples” were drawn at random from the standard normal distribution (so, of course, no difference between the two groups is expected except by chance; i.e., data were simulated under the null condition). As soon as two new “subjects” were acquired, they were added, one each, to the two groups, a two-sample test was applied, and a p value was obtained. The graph shows the “progress” of the p values as the sample size increases. Note that at the end of the study, with 100 “subjects” per group, the comparison is not significant, but there is an instance of the p value trending toward 0.05 and, at one point, dipping below it. The point of this discussion is not to dissuade investigators from looking at their data as they evolve, but to prevent them from stopping the experiment as soon as an interim p value of 0.05 is reached and rushing prematurely to publication. Finishing the final analysis, observing a p value slightly higher than 0.05, and then continuing to add subjects and repeating the analysis until the p value dips below the 0.05 threshold is equally incorrect, and for the same reason. Doing so turns what should have been the final analysis into an unscheduled interim analysis, and with the new final analysis the type I error probability has been increased. Unless interim analyses are properly designed, with built-in significance level penalties, interim looks should have no influence on the final hypothesis testing and inference. An overview of formal interim analysis design can be found in other sources [59–61].

12 Summary Remarks and Further Reading In this chapter we presented an overview of statistical methods to address problems we think are likely to occur in medical research practice. Some of these methods were

standard, others somewhat more advanced or outside of the mainstream. Readers interested in additional or more detailed presentation of analysis techniques are referred to other sources which can offer a detailed and practical exposition [10, 11, 52, 62]. Excellent nontechnical presentations of statistical reasoning, its common sense and its philosophy, are given in [2, 63]. For some entertainment of a statistical variety, a witty illustrated guide which covers traditional topics of a first course in statistics is offered in [64]. Of course, statistics is a large field and many methods and issues fell outside the scope of this chapter. We discussed briefly but not in detail the analyses of repeated measures data. It is intuitively clear that repeated measures from the same patient are not independent and should not be analyzed as such. Mixed effects models and generalized estimating equations, mentioned in Sect., can be used to adjust for within-patient dependence in analysis. For more information on mixed effects models and generalized estimating equations, see [19, 65]. We have not discussed resampling techniques – permutation tests, cross-validation, the jackknife, and the bootstrap. These are nonparametric techniques which can be used to construct confidence intervals for parameter estimates, estimate the accuracy of fitted models, and compare the fit of one model with that of another. See [66, 67] for further reading. We have not discussed issues of model selection, a famously difficult problem, and to this day one without a universally applicable solution. Suppose a number of predictors X are of potential interest to model outcome Y. How do we select the best model for Y from the large collection of candidate models, each corresponding to a different subset of the available covariates X? Standard stepwise regression

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techniques take a “greedy” approach, and tend to fail, selecting only locally optimal models [68]. Regularization is required to avoid this and other problems [69], but postselection inference remains difficult (and resampling techniques are often used). Machine learning techniques, such as least angle regression [70] and the least absolute shrinkage and selection operator [71], which regularize during model selection, are becoming increasingly useful and easy to apply. Information criteria such as Akaike’s information criterion [72], the Bayesian information criterion of Schwarz [73], and Mallow’s Cp [74] can also be used to good effect. In general, the Bayesian information criterion should be used when the number of potential predictors is large (p > log n, where p is the number of predictors and n is the sample size), unless some other form of regularization is used. The methods discussed so far fall under the global heading of “frequentist” statistics. A separate and growing field of statistics called Bayesian statistics is outside the scope of this chapter. However, interested readers will find a good overview in [75, 76]. Finally, statistics, like other fields, is guided by common sense. Common sense suggests, among other things, that strong conclusions based on small or unrepresentative datasets are not warranted, and a p value of 0.0499 is the same as a p value of 0.0501. Acknowledgements  The authors would like to thank Dr. Ian Abramson (Department of Mathematics, University of California, San Diego) for the discussion on controlling type I error, Dr. Kim Kerr (Division of Pulmonary and Critical Medicine, University of California, San Diego) and Dr. Paul Phillips (Scripps Mercy Hospital, San Diego) for allowing the use of their data to illustrate statistical methods, and Dr. Shu-Hong Zhu (Department of Family and Preventive Medicine, University of California, San Diego and Center for the Tobacco Cessation, University of California, San Diego) for reading, reviewing, and offering feedback.

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M.C. Donohue et al. 58. Abramson I, Wolfson T, Marcotte T, Grant I (1998) HNRC Group. Extending the p-plot: heuristics for multiple testing. J Int Neuropsychol Soc 5:510–517 59. Geller N, Pocock C (1987) Interim analyses in randomized clinical trials: ramifications and guidelines for practitioners. Biometrics 43:213–223 60. Jennison C, Turnbull B (1990) Statistical approaches to interim monitoring of medical trials: a review and commentary. Stat Sci 5:299–317 61. Siegmund D (1985) Sequential analysis: tests and confidence intervals. Springer, New York 62. Siebel S, Castellan NJ (1988) Nonparametric statistics for the behavioral sciences, 2nd edn. McGraw-Hill, New York 63. Freedman D, Pisani R, Purves R (1997) Statistics, 3rd edn. Norton, New York 64. Gonick L, Smith W (1993) Cartoon guide to statistics. HarperCollins, New York 65. Brown H, Prescott R (2006) Applied mixed models in medicine. Wiley, New York 66. Good PI (2005) Permutation, parametric and bootstrap tests of hypotheses. Springer, New York 67. Efron B (1987) The jackknife, the bootstrap, and other resampling plans. The Society for Industrial and Applied Mathematics, Philadelphia 68. Rencher AC, Pun FC (1980) Inflation of R² in best subset regression. Technometrics 22:49–54 69. Copas JB (1983) Regression, prediction and shrinkage. J R Stat Soc Ser B 45:311–354 70. Efron B, Hastie T, Johnstone I, Tibshirani R (2004) Least angle regression. Ann Stat 32:407–499 71. Tibshirani R (1996) Regression shrinkage and selection via the lasso. J R Stat Soc Ser B 58:267–288 72. Akaike H (1974) A new look at the statistical model identification. IEEE Trans Automat Control 19:716–723 73. Schwarz G (1978) Estimating the dimension of a model. Ann Stat 6:461–464 74. Mallows CL (1973) Some comments on Cp. Technometrics 15:661–675 75. Berry DA, Stangl DK (1996) Bayesian biostatistics. Statistics textbooks and monographs, v. 151. Dekker, New York 76. Gelman A, Carlin JB, Stern HS, Rubin DB (2003) Bayesian data analysis. Chapman & Hall, Boca Raton

Part III

Pathology and Pathobiology

Chapter 46

Hypoxic Pulmonary Vasoconstriction E. Kenneth Weir, Jesús A. Cabrera, Douglas A. Peterson, Saswati Mahapatra, and Zhigang Hong

Abstract  Hypoxic pulmonary vasoconstriction (HPV) is a homeostatic mechanism that, in the adult animal, diverts desaturated, mixed venous blood from poorly ventilated (hypoxic) areas of the lung to other, better ventilated (normoxic) areas of lung. Because of the relatively small amount of actively metabolizing tissue in the lungs, the proximity of the small resistance pulmonary arteries to the alveoli, and the presence of the bronchial circulation, which brings highly oxygenated blood to the lungs, the resistance pulmonary arteries are exposed to a relatively high oxygen tension, close to alveolar levels. This is particularly true in comparison with systemic resistance arteries, for instance, in the kidneys, or in skeletal or cardiac muscle, which are exposed to much lower oxygen tensions and also to the vasoactive products of metabolism. It may be useful to distinguish between the responses to hypoxia of arteries in these oxygen-consuming organs and those relating to oxygen supply, such as the pulmonary arteries and the ductus arteriosus. This chapter discusses cellular mechanism involved in hypoxia-induced constriction in pulmonary arteries and dilation in the ductus arteriosus. Keywords  Alveolar hypoxia • Vasoconstriction • Vasodilation • Pulmonary artery • Ductus arteriosus • Oxygen sensing • Redox status

1 Introduction Hypoxic pulmonary vasoconstriction (HPV) is a homeostatic mechanism that, in the adult animal, diverts desaturated, mixed venous blood from poorly ventilated (hypoxic) areas of the lung to other, better ventilated (normoxic) areas of lung. Because of the relatively small amount of actively metabolizing tissue in the lungs, the proximity of the small E.K. Weir (*) University of Minnesota, VA Medical Center, 1 Veterans Drive, 111C, Minneapolis, MN 55417, USA e-mail: [email protected]

resistance pulmonary arteries to the alveoli, and the presence of the bronchial circulation, which brings highly oxygenated blood to the lungs, the resistance pulmonary arteries are exposed to a relatively high oxygen tension, close to alveolar levels. This is particularly true in comparison with systemic resistance arteries, for instance, in the kidneys, or in skeletal or cardiac muscle, which are exposed to much lower oxygen tensions and also to the vasoactive products of metabolism. It may be useful to distinguish between the responses to hypoxia of arteries in these oxygen-consuming organs and those relating to oxygen supply, such as the pulmonary arteries and the ductus arteriosus. It is teleologically appropriate for arteries in the former to dilate in response to the metabolic consequences of hypoxia – acidosis, lactate, and an increase in the ADP-to-ATP ratio – and thus increase blood supply to the working tissue. In the case of the pulmonary arteries and the ductus arteriosus, the smooth muscle cells of these vessels respond directly to the change in oxygen tension, without the need for signaling from adjacent tissue. It is important to note that in the fetus the pulmonary arteries and ductus arteriosus experience very much the same intravascular pressure and oxygen tension. Consequently, the diametrically opposite responses of the two vessels to hypoxia (HPV and hypoxic vasodilatation of the ductus arteriosus) cannot be explained on the basis of different baseline conditions. HPV or its equivalent is old in an evolutionary sense and has been described in fish [1], amphibians [2], reptiles [3], and birds [4]. One study, using direct visualization of the small arteries (30–200 mm in diameter) in the bullfrog lung observed constriction in response to local hypoxia [5]. Another experiment used videomicrometry to observe the responses of arterioles in lung tissue transplanted to the hamster cheek pouch [6]. A low oxygen tension (between 20 and 30  mmHg) in the superfusate caused constriction of the pulmonary arterioles but vasodilatation of the systemic arterioles of the cheek pouch itself; an elegant demonstration of the opposite responses of small pulmonary and systemic arteries to hypoxia. In the adult mammal, alveolar hypoxia (less than 70  mmHg) causes constriction of the adjacent small pulmonary arteries and diverts the desaturated, mixed venous blood to better ventilated/oxygenated areas of the

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lung. Thus, in a patient with localized atelectasis, HPV matches ventilation and perfusion, and may prevent systemic hypoxemia. A case report illustrates the importance of the mechanism [7]. A 63-year-old man had lung atelectasis as a result of a car accident. Because he had a subarachnoid hemorrhage, he was treated with the calcium channel blocker nimodipine and within 20 min the systemic arterial oxygen tension fell from 114 to 33 mmHg. He was rapidly stabilized, but 3 days later a second attempt to use nimodipine caused an acute drop in arterial pO2 from 89.5 to 58.7 mmHg. Even in the absence of lung disease, HPV helps to match ventilation and perfusion [8]. The importance of acute HPV and the contribution of vasoconstriction to chronic hypoxic pulmonary hypertension [9] stimulate interest in dissecting the underlying mechanism.

2 Other Examples of Oxygen Sensing Neuroepithelial bodies (NEBs) are clusters of oxygensensitive neuroendocrine cells in the mucosa of the small airways [10]. They share a common derivation with the small cell lung carcinoma and its associated cell line H146 [11]. In NEBs and H146 cells, hypoxia inhibits an outward potassium current, leading to membrane depolarization, calcium entry through L-type calcium channels, and the exocytosis of serotonin [12–14]. The mechanism by which hypoxia is sensed appears to involve NADPH oxidase, as the potassium current is not inhibited by hypoxia in NEBs taken from mice in which the gp91phox subunit of NADPH oxidase is genetically nonfunctional [15]. Concordant with this observation, the inhibitor of NADPH oxidase diphenylene iodonium did not reduce the potassium current in the oxidase-deficient NEBs, but reduced it by approximately 30% in the wild-type NEBs. Diphenylene iodonium had no effect in the presence of hypoxia. These findings indicate that reactive oxygen species generated by NADPH oxidase during normoxia, or perhaps the related status of the NADP/NADPH redox couple, keep the potassium channels open. The fact that hydrogen peroxide reversibly increased the potassium current in both types of cells further strengthens the hypothesis [15]. Adrenal chromaffin cells and the associated PC12 cell line also demonstrate hypoxic inhibition of a potassium current, membrane depolarization, and an increase in cytosolic calcium level, which can be partially inhibited by L-type calcium channel blockers [16, 17]. Loss of oxygen sensitivity during the days after birth may be explained in part by a loss of expression of the T-type calcium channel [18]. The signaling of hypoxia appears to involve a decrease in the production of reactive oxygen species by the mitochondria because the decrease in the outward potassium current caused by hypoxia is absent in mitochondria-deficient cells, rho (0) [19, 20], is

Fig. 1  Hypoxia reduces the potassium current in neonatal adrenomedullary cells, an effect that is mimicked by N-acetylcysteine (50 mM) and reversed by hydrogen peroxide (50  mM). Current–voltage (I–V) plots are shown for eight cells. (Reprinted from [22] with permission from Elsevier)

reversed by hydrogen peroxide, and is mimicked by intracellular catalase, by the reducing agent dithiothreitol and by N-acetylcysteine [21, 22] (Fig. 1). The studies on NEBs and adrenochromaffin cells suggest that hypoxia results in a decrease in hydrogen peroxide level caused by NADPH oxidase and mitochondria, respectively, which signals the change in oxygen to the potassium channel.

3 Carotid Body Sensing of Hypoxia A fall in systemic arterial oxygen tension below 65 mmHg inhibits an outward potassium current in the type 1 cell of the carotid body. The consequent depolarization of the cell membrane leads to opening of the L-type calcium channel, calcium influx, and exocytosis of several neurotransmitters [23]. Among these, acetylcholine and ATP stimulate sensory nerve endings of the carotid sinus nerve and result in increased respiration. A variety of potassium channels (KV, KCa, and TASK) in the type 1 cell are oxygen-sensitive and are involved in the signaling cascade that is activated by hypoxic inhibition of the channel [24–26]. An interesting case report illustrates the involvement of both the carotid body and NEBs in oxygen sensing [27]. Two infants are described who had a hypoventilation syndrome. At autopsy, the carotid bodies were small and the type 1 cells few; however, there was a marked increase in the number and size of the NEBs in the lungs, suggesting a compensatory hyperplasia of these airway chemoreceptors. In the NEBs and adrenochromaffin cells discussed earlier, hypoxia represents a shift to a more reduced redox status. Recent experiments on the stimulation

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of ventilation by hypoxia suggest that the same redox control may be involved. The acute ventilatory response to hypoxia in human subjects is reduced by the carbonic anhydrase inhibitor acetazolamide and by the anesthetic isoflurane. In both cases, the normal ventilatory response is restored by the administration of the antioxidants a-tocopherol and ascorbic acid [28, 29]. Similarly, the oxygen radical scavenger N-acetylcysteine increases the normal ventilatory response to hypoxia in young adults [30].

4 Vascular Responses to Hypoxia There are considerable similarities between the mechanisms of oxygen sensing just described and those involved in HPV and in normoxic contraction of the ductus arteriosus. In HPV it is well documented that hypoxia inhibits an outward potassium current in the cell membrane of smooth muscle cells dispersed from resistance pulmonary arteries [31, 32]. This inhibition does not occur in renal or mesenteric smooth muscle cells. The decrease in outward potassium current results in membrane depolarization [33] and calcium entry through voltage-dependent calcium channels [34–36]. The inhibition of the potassium current is proportional to the severity of hypoxia [37]. Not all potassium channels are oxygen-sensitive, so considerable effort has been made to identify those involved in HPV. The use of antibodies directed against specific voltage-gated potassium channels identified KV1.5 and KV2.1 as likely candidates [38]. Subsequently, it was observed that mice lacking KV1.5 in the pulmonary arteries had markedly diminished HPV [39]. Interestingly, the lungs of rats that have been exposed to chronic hypoxia for 4–6 weeks have significantly reduced HPV [40, 41]. The pulmonary artery smooth muscle cells (PASMCs) from such rats have reduced outward potassium current and depolarization of the cell membrane, together with decreased expression of KV1.5 and KV2.1 [42–44]. The decreased expression of potassium channels and reduced current density is probably signaled by the increased activity of hypoxia-inducible factor 1a [45]. Aerosol transfection of the gene for KV1.5 reduces chronic hypoxic pulmonary hypertension in the intact rat and restores acute HPV in the isolated perfused lungs [46]. The importance of KV1.5 is further illustrated by elegant experiments using single PASMC reverse-transcription PCR [47]. In these studies hypoxia only reduced the potassium current in those cells that expressed high levels of KV1.5. It has been pointed out that KV channels may not be open at the normoxic resting potential. The role of initiating hypoxic depolarization of PASMCs may belong to the two-pore-domain potassium channels, such as TASK-1, that are voltage-independent and regulate resting potential [48].

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TASK-1 is reversibly inhibited by acute hypoxia [49, 50] and the use of small interfering RNA (siRNA) against TASK-1 precludes the inhibitory effect of hypoxia on the potassium current.

5 Sources of Calcium in HPV What is the source of calcium that triggers HPV? Most of the calcium giving rise to the hypoxic increase in cytosolic calcium level in the PASMC comes from outside the cell, but some is released from internal stores [37]. As stated earlier, membrane depolarization results in calcium entry through L-type voltagedependent calcium channels, but hypoxia also increases the voltage-independent influx of calcium through the L-type channel [51]. Interestingly, this is only true in PASMCs taken from resistance pulmonary arteries. In PASMCs from the conduit arteries, the direct effect of hypoxia on the L-type channel, independent of membrane potential, is to inhibit calcium entry. Hypoxia leads to dilatation of the conduit pulmonary arteries. In the resistance arteries, the influx of the calcium plays a significant part in causing the membrane depolarization seem with hypoxia and inhibitable by verapamil [52]. The role of calcium released from the sarcoplasmic reticulum (SR) has been emphasized by many investigators [47, 53–57]. More recently, attention has been paid to the capacitative entry of calcium that repletes the SR [58]. The calcium entering through L-type voltage-dependent channels has been discussed already but the dihydropyridine-insensitive influx of calcium is also important. The nonselective calcium entry blocker lanthanum inhibits repletion of calcium stores in PASMCs after they have been depleted by thapsigargin [58]. Both lanthanum and another nonselective calcium entry blocker, SK&F 96365, inhibit the early, phase 1, contraction of isolated rat intrapulmonary arteries (150–550  mm in internal diameter) to hypoxia. The low concentration of lanthanum used suggested that much of the calcium influx was related to capacitative calcium entry (CCE). Subsequent studies found that the dihydropyridine-insensitive portion of the rise in calcium level caused by hypoxia in PASMCs could be inhibited by SK&F 96365 and nickel, again suggesting involvement of CCE [59, 60]. Similar results were observed in the isolated perfused rat lung and the authors suggested that both CCE and calcium entry through voltage-gated calcium channels are necessary for HPV [61] (Fig. 2). The store-operated channels responsible for CCE are likely to be composed of canonical transient receptor potential (TRPC) proteins. It has been demonstrated that CCE, stimulated by cyclopiazonic acid to deplete SR calcium stores, is greater in the resistance artery PASMCs [62]. The messenger RNA and protein of several transient receptor potential isoforms is also greater in resistance than in conduit

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Fig. 2  Hypoxic pulmonary vasoconstriction (HPV) in the isolated rat lung. (a) Switching to calcium-free perfusate ablates HPV. (b) The time course of mean pulmonary artery pressure during 45 min of hypoxia in untreated control lungs and lungs exposed to antagonists of store-operated Ca2+ channels (SK&F 96365, NiCl2), voltageoperated Ca2+ channels (nifedipine), or Ca2+-free perfusate during the last 15 min of hypoxia (arrow). (Reproduced from [61] with permission)

pulmonary arteries. The absence of the early phase of HPV in mice genetically deficient in TRPC6 suggests that this particular channel protein may be involved [63]. It is likely that stromal interacting molecule 1 (STIM1) may convey the signal of calcium depletion by binding to and activating several of the TRPC channels [64, 65]. The possibility that this mechanism is involved in HPV is enhanced by the finding that siRNA directed against STIM1 markedly reduces the increase in calcium level in PASMCs from resistance rat pulmonary arteries caused by acute hypoxia [66].

6 Interaction of Membrane Potential and SR The relationship between membrane potential and the release of calcium from the SR is not clear. It has been suggested that the entry of calcium through store-operated channels actually causes depolarization of PASMCs and subsequent calcium entry through reverse-mode sodium/calcium exchange and through L-type channels [60, 61]. The recent work from the same laboratory using siRNA against STIM1 cited above [66] makes this less likely. It was observed that the siRNA did not alter the initial transient increase in calcium level caused by hypoxia, which is probably related to membrane depolarization but diminished the later, sustained calcium level elevation.

Thus, the depolarization would precede the CCE not vice versa. Recently, another mechanism has been described. The concept is that the L-type calcium channel is activated by membrane depolarization and that this activation, without the need for calcium entry, stimulates the release of calcium from the SR of basilar artery myocytes; calcium-channel-induced calcium release (CCICR) [67] (Fig. 3). The calcium release is increased by calcium channel agonists such as Bay K-8644 and FLP-64176 and is decreased by diltiazem and nifedipine. The release of calcium, initiated by L-type channel activation, in the absence of calcium entry, is markedly enhanced by low levels of ATP even after blockade of purinergic receptors and the inhibition of G proteins [68]. It is not known whether this mechanism augments HPV, but systemic vessels are relaxed by hypoxemia, so it is interesting that CCICR is diminished by hypoxia in coronary arteries [69]. The evidence supporting a role in HPV for the ryanodine receptor in the SR has been greatly strengthened by studies in mice lacking a subtype of the ryanodine receptor, either RyR3−/− or RyR1+/− [70, 71]. The calcium release stimulated by hypoxia in the PASMCs freshly isolated from these mice is significantly reduced, as is the contraction of the pulmonary arteries induced by hypoxia (Fig. 4). Related to the release of calcium from the SR caused by membrane depolarization in basilar arteries discussed earlier [67], it is interesting to note that the PASMCs of RyR1+/− mice have a smaller increase in

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Fig. 3  Effect of membrane depolarization on calcium release in basilar artery smooth muscle cells. (a) Membrane depolarization causes release of calcium in the absence of external calcium, whereas under voltage-clamp conditions 70 mM potassium does not. (b) There is a progressive increase in the calcium release in response to an increase in the amplitude of the depolarizing steps. (Reproduced from [67] with permission)

Fig.  4  In mice with decreased expression of the ryanodine receptor RyR1 (RyR1−/− or RyR1+/−), there is a smaller increase in the level of calcium in pulmonary artery smooth muscle cells in response to hypoxia and less vasoconstriction in the pulmonary arteries than in the wild type (RyR+/+). Numbers shown are the number of cells and the number of mice. (Reproduced from [71] with permission from Springer)

calcium caused by KCl-induced membrane depolarization in the absence of extracellular calcium [71]. The relationship between the L-type calcium channel and calcium release from the SR may prove to be an important mechanism in HPV.

7 Calcium Sensitization The executive mechanisms of HPV discussed up to this point involve an increase in cytosolic calcium level whether that occurs by influx of calcium through the L-type calcium channel (voltage-dependent or voltage-independent), release from the SR, or entry through a store-operated channel.

Over the last 10 years it has become apparent that, through the RhoA/Rho kinase system, hypoxia increases constriction of the small resistance pulmonary arteries beyond that caused by an increased level of calcium alone [72]. In rat PASMCs from resistance arteries, hypoxia causes an increase in Rho kinase activity and, related to the subsequent inactivation of myosin light chain phosphatase, an increase in myosin light chain phosphorylation. The use of the Rho kinase inhibitor Y-27632 reduces HPV in the isolated perfused rat lung and in pulmonary artery rings [72, 73]. At least in porcine conduit arteries, the RhoA activity induced by hypoxia depends on the neonatal age and whether the inner or outer media is being studied [74]. Hypoxia increases the level of activated (GTP-bound) RhoA in the media of fetal pulmonary arteries and in the inner media of 3-day-old neonates, whereas it decreases the level of activated RhoA in the outer media of 14-day-old piglets. Rac activity is not changed. In chronic hypoxic pulmonary hypertension, most investigators attribute the pulmonary hypertension to structural remodeling of the pulmonary vasculature; however, Y-27632 rapidly reverses the pulmonary hypertension in chronically hypoxic rats [75], indicating that hypoxiainduced vasoconstriction is still an important component of the hypertension. Similarly, acute inhibition of the Rho kinase inhibitor fasudil reduces the pulmonary hypertension that develops spontaneously in fawn-hooded rats [76]. The executive mechanisms of HPV include inhibition of several potassium channels, leading to PASMC depolarization and calcium entry through the L-type calcium channel; an increase in influx through the L-type channel which is unrelated to membrane potential; release of calcium from the SR and repletion by CCE through store-operated channels, perhaps controlled by STIM1; and calcium sensitization involving RhoA/Rho kinase. However, the calcium-dependent and calcium-independent mechanisms do not act in isolation. For instance, membrane depolarization induced by KCl increases the concentration of activated RhoA [77] and the pulmonary vasoconstriction stimulated by KCl is inhibited by Y-27632 [78]. The latter suggests the involvement of Rho kinase,

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although other mechanisms might be involved. In airway smooth muscle, Y-27632 reduces the contractions induced not only by KCl but also by cyclopiazonic acid (SR release of calcium) and by the calcium ionophore A-23187 [79]. It is also clear that membrane depolarization and activation of the L-type channel can increase the amount of Rho kinase colocalized to caveolae [80]. These studies indicate that there are several potential points of interaction between the executive mechanisms.

8 Conclusion Unfortunately, the four executive mechanisms listed earlier are exactly the same in the smooth muscle cells of the resistance pulmonary artery and of the ductus arteriosus [81–83]. Consequently, they do not tell us why the response to hypoxia in terms of vascular tone is opposite in the two vessels. For that answer we have to examine the more proximal signaling [see Chaps. 18, 19]. Any postulated mechanism has to be able to explain the different behavior of the pulmonary artery and ductus arteriosus, as well as oxygen sensing in the carotid body, adrenomedullary cells, and NEBs.

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Chapter 47

Pathogenic Roles of Ca2+ and Ion Channels in Hypoxia-Mediated Pulmonary Hypertension Jian Wang, Dandan Zhang, Carmelle V. Remillard, and Jason X.-J. Yuan

Abstract  Chronic hypoxia causes pulmonary hypertension (CHPH), which occurs in residents living in high altitude and patients with chronic obstructive pulmonary disease, interstitial lung disease, and sleep-disordered breathing. The pathogenic mechanisms of CHPH are unclear, but involve sustained vasoconstriction and excessive vascular remodeling. A rise in cytosolic Ca2+ concentration ([Ca2+]cyt) in pulmonary artery smooth muscle cells is a major trigger for pulmonary vasoconstriction and an important stimulus for vascular smooth muscle proliferation. Multiple ion channels in the plasma membrane participate in the regulation of [Ca2+]cyt, while hypoxia upregulates Ca2+ channels and downregulates K+ channels in pulmonary vascular smooth muscle cells. This chapter discusses the potential mechanism by which hypoxia causes pulmonary hypertension via modulating Ca2+ and K+ channel expression and function, and regulating intracellular Ca2+ stores in the sarcoplasmic reticulum. Keywords  Hypoxia • Voltage-gated K+ channel • Storeoperated Ca2+ channel • Receptor-operated Ca2+ channel • Membrane potential • Pulmonary vasoconstriction

1  Introduction Chronic exposure to high altitude or some lung diseases such as chronic obstructive pulmonary disease, interstitial lung disease, and sleep-disordered breathing can lead to chronic hypoxic pulmonary hypertension (CHPH). According to the Venice 2003 Revised Classification system, CHPH belongs to the third classification of pulmonary hypertension as WHO group III – pulmonary hypertension associated with lung diseases and/or hypoxemia. The mechanisms of CHPH remain unclear despite more than 50 years of investigation. Whatever J. Wang (*) Division of Pulmonary and Critical Care, First Affiliated Hospital of Guangzhou Medical University, 151 Yanjiang Rd., Guangzhou Guandong 510102, PR China e-mail: [email protected]; [email protected]

the initial cause, CHPH (WHO group III) involves the vasoconstriction or tightening of blood vessels (also known as vascular remodeling) connected to and within the lungs. This makes it harder for the heart to pump blood through the lungs, much as it is harder to make water flow through a narrow pipe as opposed to a wide one. Over time, the affected blood vessels become both stiffer and thicker, in a process known as fibrosis. This further increases the blood pressure within the lungs and impairs their blood flow. Abnormal homeostasis of intracellular Ca2+ is the converging point of a lot of misregulated processes (e.g., vasoconstriction and vascular smooth muscle proliferation) during CHPH pathogenesis and progression. The development of the patch-clamp technique in the early 1980s revolutionized cellular physiology as well as the study of ion channels in pulmonary hypertension [1, 2]. After that, studies on intracellular Ca2+, ion channels, transmembrane ion influx, and membrane potential became more and more popular, which provide us with more insight into and greatly benefited our understanding of CHPH. Although modulated by endothelial vasoactive substances, the constrictor response to hypoxia is intrinsic to the smooth muscle cell. Ion channels are important elements in two of the three components of the response. Hypoxia causes release of calcium from the sarcoplasmic reticulum (SR), with consequent repletion through store-operated Ca2+ channels (SOCCs) [3, 4]. It also inhibits several potassium channels (voltagegated and TASK), leading to membrane depolarization and calcium entry through L-type Ca2+ channels [5–8]. Finally, the effect of the rise in cytosolic calcium level is amplified by enhanced calcium sensitivity of the actin–myosin interaction, achieved by the hypoxia-induced increase in Rho kinase activity. The change in oxygen tension that stimulates these three “executive” components is signaled by a change in the redox status of the smooth muscle cell and probably by downstream changes in G proteins. Ion channels also play a critical role in the vascular remodeling that results in CHPH [3, 4], seen when all the pulmonary vascular bed is hypoxic, at high altitude, and in patients with chronic lung diseases. The same inhibition of potassium channels and influx of calcium results in high cytosolic levels

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of potassium and calcium [5, 6, 9, 10]. An increase in cytoplasmic Ca 2+ concentration is used as a key signaling messenger for regulating a host of kinetically distinct processes leading to cell growth and proliferation. This leads to inhibition of apoptosis and an increase in cellular proliferation. A better understanding of the pathophysiological mechanisms of hypoxic pulmonary vasoconstriction and vascular remodeling will enable the design of better treatments for hypoxic and other forms of pulmonary hypertension.

2  Roles of Ca2+ in Pulmonary Vasoconstriction and Proliferation In vascular smooth muscle, global increases in intracellular Ca2+ concentration trigger contraction via a signaling pathway that includes formation of Ca2+/calmodulin (CaM), activation of myosin light chain kinase (MLCK), and actin–myosin interaction. The increase in intracellular Ca2+ concentration not only triggers contraction, but also mediates proliferation of pulmonary artery smooth muscle cells (PASMCs).

2.1 Ca2+ Is a Major Trigger to Activate MLCK MLCK is a serine/threonine-specific protein kinase that phosphorylates the regulatory light chain of myosin II. Three different MLCK isoforms exist. There is a cardiac MLCK encoded by mylk3, a skeletal MLCK encoded by mylk2, and smooth muscle MLCK encoded by mylk. Smooth muscle MLCK and nonmuscle MLCK are identical and are the product of the same gene, mylk. This protein is important in the mechanism of contraction in smooth muscle. Once there is an influx of calcium into the smooth muscle, either from the SR or, more importantly, from the extracellular space, contraction of smooth muscle fibers may begin. First, the calcium will bind to CaM. This binding will activate MLCK, which will go on to phosphorylate the myosin light chain at serine residue 19. This will enable the myosin cross-bridge to bind to the actin filament and allow contraction to begin (through cross-bridge cycling). Since smooth muscle does not contain a troponin complex like striated muscle does, this mechanism is the main pathway for regulating smooth muscle contraction. CaM is a ubiquitous Ca2+ sensor protein through which a variety of the second messenger effects are mediated. CaM is a 17-kDa Ca2+-binding molecule that has been highly conserved throughout biological evolution. It is composed of an N-terminal lobe and a C-terminal lobe tethered by a highly flexible helical linker region that allows CaM to adopt a variety of conformations when bound to different targets. Each lobe

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of CaM contains a pair of EF-hand motifs allowing it to bind four Ca2+ ions, and saturation of CaM with Ca2+ induces a conformational change that permits the protein to interact with and activate a surprisingly diverse set of target enzymes. Of the many downstream targets of CaM, a family of enzymes known as the CaM-dependent kinases (CaM kinases) is one of the best characterized. As intracellular Ca2+ concentrations rise (through the opening of receptor/ion channels or release from intracellular stores), CaM becomes saturated with four Ca2+ ions and undergoes a conformational change allowing it to bind to its target site on CaM kinases. The crystal structures of the catalytic domains of two CaM kinases (CaMKI and CaMKII) have been solved, and available data support the idea that all CaM kinases share similar catalytic domain structures. CaMKII was first isolated from brain and is one of the most well studied members of the CaM kinase family. There are four isoforms of CaMKII (a, b, g, and d) in mammals and each is expressed from a separate gene [11]. CaMKII is present in vascular smooth muscle, with the d and g isoforms of CaMKII predominating. Several smooth muscle proteins have been identified as in vitro substrates for CaMKII, including MLCK [12], myosin light chains (LC20) [13], caldesmon [14], and calponin. Although the roles of CaMKII in the regulation of vascular smooth muscle contractility remain unclear, several laboratories have suggested that CaMKII does take part in the regulation of vascular smooth muscle contractility and the regulation is Ca2+-dependent. Antisense oligonucleotides or inhibitors of CaMKII decrease contractility and myosin II light chain phosphorylation [11]. Ca2+ depletion will abandon the activation of CaMKII or decrease the contractile force. It is generally accepted that Ca2+/CaM-dependent activation of MLCK and the subsequent phosphorylation of myosin light chains is a major regulatory mechanism of smooth muscle contractility. This will enable the myosin cross-bridge to bind to the actin filament and allow contraction to begin (through the cross-bridge cycle). The second possible way is CaMKII can directly phosphorylate 20-kDa myosin light chain or through a mitogen-activated protein kinase dependent manner.

2.2 Ca2+/CaM Complex Propels Progression of the Cell Cycle Abnormal or misregulated pulmonary vascular smooth muscle cell (VSMC) proliferation in response to hypoxia, hyperlipidemia, or oxidative stress greatly contributes to vascular remodeling which will lead to the pathogenesis of vascular hypertrophic disorders such as atherosclerosis and hypoxic pulmonary hypertension [12]. In vascular smooth muscle, physiological resting levels of intracellular free Ca2+ concentration in the nanomolar range are necessary to maintain basal vascular tone. Vascular smooth muscle activation is associated with an increase in

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Fig. 1  Agonist (A)–receptor (R) interaction increases the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) and Inositol 1,4,5trisphosphate (IP3) production in vascular smooth muscle. IP3 stimulates Ca2+ release from the sarcoplasmic reticulum (SR). Also, extracellular Ca2+ enters the vascular smooth muscle cell (VSMC) through Ca2+ channels. Ca2+ binds calmodulin (CaM), which could activate myosin light chain (MLC) kinase and initiate contraction, or regulate the activity of K+ channels, Ca2+-release channels, Ca2+

pumps, and CaM kinase II. During vascular injury, the VSMC transforms into an undifferentiated phenotype and enters a cell cycle which consists of growth (G1), and preparation of the chromosomes for replication, synthesis of DNA (S), preparation for mitosis (G2), and mitosis (M). Ca2+/CaM increases the activity of cyclin E/CDK2 and stimulates G1/S transition, and thereby promotes vascular smooth muscle growth and proliferation. (Reproduced from Koledova and Khalil [17] with permission)

intracellular Ca2+ concentration in the micromolar range, and large increases in vascular smooth muscle intracellular Ca2+ concentration have been identified in excessive vasoconstriction disorders such as hypertension and coronary vasospasm [13, 15, 16]. Activation of surface membrane receptors in vascular smooth muscle triggers increases in intracellular Ca2+ concentration attributable to Ca2+ release from the intracellular stores in the SR and Ca2+ entry from the extracellular space through Ca2+ channels [17] (Fig. 1). Ca2+ then activates specific protein kinases and phosphatases that are involved in vascular smooth muscle contraction and relaxation. Ca2+ may also function as a second messenger to activate other signaling pathways such as cytosolic phospholipase A2, phospholipase C (PLC), protein kinase C, and phosphodiesterase [13]. An increase in intracellular Ca2+ concentration could also modulate plasma membrane channels and pumps such as Ca2+-activated K+ channels and the plasma membrane Ca2+ATPase. Additionally, Ca2+ may affect SR channels and pumps such as the inositol 1,4,5-trisphosphate (IP3) receptor, the ryanodine-sensitive receptor and intracellular Ca2+ release channels, and the Ca2+ uptake pump (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase, SERCA) [14, 18]. Studies have shown that depletion of Ca2+ pools in VSMCs using the SERCA inhibitor thapsigargin or cyclopiazonic acid (CPA) causes inhibition of translocation of activated

ERK1/2 to the nucleus and prevents cyclin D1 expression, thus delaying the progression into the S-phase and the cell cycle [19]. Also, forced gene expression of SERCA2a in a model of balloon injury of the rat carotid artery is associated with decreased expression of cyclin D1 in VSMCs, cell cycle arrest at the G1 phase, and reduced VSMC proliferation and neointima formation [20]. Although these studies suggest possible effects of intracellular Ca2+ on cyclin expression and the CDK activity, the specific molecular interactions and targets involved have not been clarified.

3  R  egulation of Cytosolic Ca2+ Concentration in PASMCs 3.1 Calcium Homeostasis in Vascular Smooth Muscle Increases in intracellular Ca2+ concentration can be caused by (1) release of Ca2+ from internal storage sites, such as the SR, or (2) influx of Ca2+ from extracellular fluid through L-type voltage-dependent Ca2+ channels (VDCCs), receptoroperated Ca2+ channels (ROCCs), or SOCCs [21] (Fig.  2).

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evaluated by monitoring the fluorescence of Fura-2 excited at 360 nm before and after addition of MnCl2 (200 mM) to the cell perfusate. It was evaluated from the rate at which Fura-2 fluorescence was quenched by Mn2+, which entered the cell as a Ca2+ surrogate and reduced Fura-2 fluorescence upon binding to the dye. The fluorescence caused by excitation at 360 nm was the same for Ca2+-bound and Ca2+-free Fura-2; therefore, changes in fluorescence can be assumed to be caused by Mn2+ alone [23].

3.2 Voltage-Dependent Ca2+ Channels Fig. 2  Modes of regulated Ca2+ entry across the plasma membrane. Calcium can enter cells by any of several general classes of channels, including voltage-operated channels (VOC), second-messengeroperated channels (SMOC), store-operated channels (SOC), and receptor-operated channels (ROC). VOC are activated by membrane depolarization, and SMOC are activated by any of a number of small messenger molecules, the most common being inositol phosphates, cyclic nucleotides, and lipid-derived messengers (diacylglycerol and arachidonic acid and its metabolites). SOC are activated by depletion of intracellular Ca2+ stores, and ROC are activated by direct binding of a neurotransmitter or hormone agonist (Ag). In addition, under some conditions, Ca2+ can enter cells via the Na+–Ca2+ exchanger (NCX) operating in reverse mode. (Reproduced from Parekh and Putney [21] with permission)

VDCCs, the main Ca2+ channels in the VSMC membrane, are (1) activated by membrane depolarization and (2) blocked by Ca2+ channel blockers such as nifedipine and verapamil. ROCCs are present in many types of smooth muscle, and can be activated by inositol lipid signaling, one of the most widespread signal transduction cascades. This transduction cascade, mediated via G-protein-activated PLC, produces two second messengers: diacylglycerol and IP3. Store-operated calcium entry (SOCE) plays a critical role to refill Ca2+ in the SR and to maintain Ca2+ homeostasis in PASMCs. Recent reports have suggested that SOCE does occur in PASMCs [22]. Activation of this pathway can be independent of IP3 production, since various procedures that deplete internal stores (e.g., use of thapsigargin and CPA) are able to stimulate Ca2+ entry across the plasma membrane without affecting the intracellular IP3 level. SOCE in smooth muscle cell can be observed in two ways. First, SOCE is observed as a sharp rise in intracellular Ca2+ concentration occurring right after passive store depletion with CPA. In the absence of extracellular Ca2+, CPA, by blocking Ca2+ sequestration into the SR, induces a transient rise in intracellular Ca2+ concentration due to leakage of Ca2+ from the SR. The intracellular Ca2+ concentration then declines back to the original baseline level after 5–10  min as the SR Ca2+ is depleted. Under these conditions, restoration of extracellular Ca2+ induces a further rise in intracellular Ca2+ concentration due to SOCE, which is inhibited reversibly by SOCC blockers such as SK&F  96365 and Ni2+. Second, SOCE can be

VDCCs are ion channels which display selective permeability to calcium ions. In smooth muscle cells or PASMCs, the VDCC is mainly of L-type and is gated by high voltage. The individual channel is composed of multiple subunits (a1, b, a2/d, g). The L-type Ca2+ channel, compared with other VDCCs, has large Ca2+ conductance and long opening duration when it is activated by depolarization, thus it is called L-type, and is responsible for the entry of a large amount of Ca2+ into the cytoplasm. This channel was initially purified and cloned as a 1,4-dihydropyridine (DHP) binding protein. In addition to DHPs, small molecular compounds called phenylalkylamines (e.g., verapamil) and benzothiazepines (e.g., diltiazem) can selectively block the L-type Ca2+ channel, and a DHP analogue, Bay K 8644, specifically activates this channel. L-type VDCCs open in response to the membrane depolarization, then start to inactivate even during the depolarization. Ca2+ channels are regulated by two different inactivation mechanisms: voltage-dependent inactivation and Ca2+-dependent inactivation. In response to the prolonged membrane depolarization, the Ca2+ channel shifts to a nonconducting inactivated state, which is called voltage-dependent inactivation. The degree of the voltage-dependent inactivation correlates with the membrane potential. The higher the membrane potential is, the more strongly Ca2+ channels are inactivated. On the other hand, the Ca2+-dependent inactivation is induced by the elevation of intracellular Ca2+ concentration. Other cations permeable thorough the Ca2+ channel, Ba2+ and Na+, are not able to induce the Ca2+-dependent inactivation, indicating that this inactivation mechanism operates only when Ca2+ enters through the Ca2+ channels. The Ca2+-dependent inactivation is faster than the voltage-dependent inactivation; thus, is a powerful negative-feedback mechanism to strictly regulate the amount of Ca2+ influx via the channel. This pheno­menon has been shown to be mediated by CaM that is constitutively bound to L-type Ca2+ channels [24]. Before the 1990s, VDCCs were considered the primary calcium channels in PASMCs responsible for calcium influx or SOCE in response to hypoxia. However, more and more data and evidence suggest that mechanisms of pulmonary

47  Pathogenic Roles of Ca2+ and Ion Channels in Hypoxia-Mediated Pulmonary Hypertension

hypertension are more complicated, and besides VDCCs there are some other Ca2+ channels, such as SOCCs, that may also contribute to the progression of pulmonary hypertension.

3.3 Store-Operated Ca2+ Channels In 1986, on the basis of the findings of a series of experiments in parotid acinar cells investigating the relationship between Ca2+ release from internal stores, Ca2+ entry, and store refilling, the concept of SOCE was first proposed [25]. On the basis of this work, and a few eclectic observations in previous literature, it was suggested that the amount of Ca2+ in the stores controlled the extent of Ca2+ influx in non­excitable cells, a process originally called capacitative calcium entry (CCE). When stores were full, Ca2+ influx did not occur but as the stores emptied, Ca2+ entry developed. Subsequently, more straightforward evidence for capacitative or store-operated entry was obtained through the use of thapsigargin, a specific SERCA inhibitor, discussed in more detail later. Direct evidence in support of the basic tenet of this model, namely, that store depletion activates Ca2+ influx, was provided by electrophysiology studies which established that the process of emptying the stores activated a Ca2+ current in mast cells called Ca2+-release-activated Ca2+ (CRAC) current (ICRAC) [26]. ICRAC is non-voltage-activated, inwardly rectifying, and remarkably selective for Ca2+ [27, 28]. The gene or genes encoding the CRAC channel and other store-operated channels remain elusive. From research on Drosophila phototransduction, a transient receptor potential (TRP) gene encoding a subunit of a Ca2+-permeable channel which was identified in 1969 was thought to be involved in SOCE. There are seven related members of the TRP channel (TRPC) family, designated TRPC1–TRPC7 (the numbering reflects the order of their discoveries) [22, 29]. The first mammalian TRPC proteins to be cloned and expressed were TRPC1 and TRPC3. Some research data show that several members of the TRPC family such as TRPC1 and TRPC6 may be involved in hypoxic pulmonary hypertension [22, 29].

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a full electrochemical cell. If only one type of ion penetrates the membrane, the Nernst equation can be used to calculate the membrane potential with a fair degree of accuracy. The equation is based upon the idea that at equilibrium the concentration gradient forces acting on ions will be exactly balanced by opposite electrical forces. The equation states

Ei ≡ Vin − Vout =

RT [C]out ln . zF [C]in

The Nernst equation is used in physiology to find the electric potential of a cell membrane with respect to one type of ion. When the membrane is in thermodynamic equilibrium, i.e., no net flux of ions, the membrane potential must be equal to the Nernst potential. However, owing to active ion pumps, the inside and the outside of a cell are not in equilibrium. In this case the resting potential can be determined from, e.g., the Goldman–Hodgkin–Katz (GHK) voltage equation. The GHK voltage equation, more commonly known as the Goldman equation, is used in cell membrane physiology to determine the potential across a cell’s membrane taking into account all of the ions that are permanent through that membrane. It is “Nernst-like” but has a term for each permanent ion. The Nernst equation can be considered a special case of the Goldman equation for only one ion. If we take PK+ and PNa+ into account, the GHK equation can be written as

Vm =

RT  pK [K]o + pNa [Na]o + pCl [Cl]i  ln F  pK [K]i +pNa [Na]i +pCl [Cl]o 

The usefulness of the GHK equation to electrophysiologists is that it allows one to calculate the predicted membrane potential for any set of specified permeabilities. For example, if one wanted to calculate the resting potential of a cell, one would use the values of ion permeability that are present at rest (e.g., PK+ >> PNa+). If one want to calculate the peak voltage of an action potential, one would simply substitute the permeabilities that are present at that time (e.g., PK+ >> PNa+).

4.2 Resting Membrane Potential by Special K+ Channels 4  R  egulation of Membrane Potential by K+ Permeability 4.1 The Nernst and Goldman–Hodgkin–Katz Equations The Nernst equation is named after the German physical chemist who first formulated it, Walther Nernst. It can be used to determine the total voltage (electromotive force) for

The membrane potential, or better the membrane voltage, is the voltage across the plasma membrane between the inside and the outside of a cell. When the membrane voltage of a cell does not change in time, it is called the resting potential (or resting voltage), as opposed to the dynamic potential. The resting potential is mostly determined by the concentrations of the ions in the fluids on both sides of the cell membrane and the ion transport proteins that are in the cell membrane.

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For most animal cells, potassium ions are the most important for the resting potential. Owing to the active transport of potassium ions, the concentration of potassium is higher inside cells than outside. Most cells have potassium-selective ion channel proteins that remain open all the time. There will be net movement of positively charged potassium ions through these potassium channels, with a resulting accumulation of excess negative charge inside of the cell. The outward movement of positively charged potassium ions is due to random molecular motion (diffusion) and continues until enough excess negative charge accumulates inside the cell to form a membrane potential which can balance the difference in concentration of potassium inside and outside the cell. “Balance” means that the electrical force (potential) that results from the buildup of ionic charge, and which impedes outward diffusion, increases until it is equal in magnitude but opposite in direction to the tendency for outward diffusive movement of potassium. This balance point is an equilibrium potential as the net transmembrane flux (or current) of potassium ions is zero. Four main classes of K+ channels have been described in vascular smooth muscle on the basis of heir biophysical and pharmacological properties: ATP-sensitive (KATP), inward rectifier (KIR), large-conductance Ca2+-activated K+ (BKCa), and voltage-gated (KV) [30]. Of these, the KV channels are most likely to control the PASMC membrane potential and thus regulate vessel tone. The first complete nucleotide sequence encoding a KV channel was reported in 1987 with the cloning of the Drosophila Shaker channel. In addition to the four mammalian subfamilies relating to the four subfamilies found in Drosophila, five additional subfamilies (KV5–KV9) have also been described [31]. Currently, over 30 KV channels have been cloned and expressed in heterologous expression systems. These channels often display differences in voltage sensitivity, current kinetics, and steady-state activation and inactivation [32].

4.3 Molecular Composition and Structure of K+ Channels KV channels exist as tetramers formed by four six-transmembrane-spanning a-subunits combining to form a functional channel. Not only can identical a-subunits combine to form a functional channel, but distinct b-subunits can also combine to form functional heteromeric channels both in  vitro and in vivo [16, 33, 34]. These heteromeric channels have unique properties that often represent a blend of the observed properties of the corresponding homomeric channels. Furthermore, several KV a-subunits are nonfunctional when expressed alone [30]. For example, the KV9.3 subunit, the most recently

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identified member of the mammalian KV family, does not form a functional homomeric channel itself but rather functions only in heteromeric complexes, where it confers altered voltage sensitivity and activation kinetics. Accessory b-subunits can combine with KV a-subunits to add even more diversity to KV channel function. Currently, four KV a-subunit gene families have been described [35–37]. All are cytoplasmic proteins, approximately 40 kDa in mass, with a conserved core sequence and variable N-termini. KV b-subunits have been shown to confer functional effects onto a-subunits, including both fast and slow inactivation, altered voltage sensitivity, and slowed deactivation [30, 38]. Additionally, the b-subunit may play a role as a cellular redox sensor as it appears to confer O2 sensitivity on the KV4.2 channel in heterologous expression systems [38]. Thus, the roles of KV b-subunits are many, and, indeed, they may be important not only in the functional modulation of KV a-subunits but also in the PASMC response to hypoxia.

5  A  cute Hypoxic Regulation of Ion Channel Function 5.1 Cellular Mechanisms by Which Hypoxia Inhibits K+ Channels It was stated in previous sections that membrane K+ channels play an essential role in smooth muscle excitability. In VSMCs, KV channels are integral in the regulation of membrane potential and vascular tone; therefore, inhibition or closure of VSMC K + channels, which are open at the resting membrane potential, causes membrane depolarization. This change in membrane potential activates voltagegated Ca2+ channels, leading to an increase in intracellular Ca2+ concentration and vasoconstriction. VSMCs have a high input resistance; therefore, even a small change in K+ channel activity can have a significant effect on membrane potential and, consequently, on vascular tone. Indeed, in isolated PASMCs, acute hypoxia has been shown to significantly depolarize the membrane potential by approximately 15–20 mV [2], leading to contraction of individual PASMCs. It is assumed that acute hypoxia acts first to depolarize the membrane by inhibiting the K+ channels involved in setting the resting membrane potential. The membrane depolarization will then activate VDCCs and calcium influx, which will lead to increased intracellular Ca2+ concentration and vasoconstriction. It has been confirmed that the hypoxiainduced increase in intracellular Ca2+ concentration is inhibited by L-type Ca2+ channel blockers [39–41] and that hypoxia-induced constriction of small pulmonary arteries (less than 300 mm) associated with membrane depolarization

47  Pathogenic Roles of Ca2+ and Ion Channels in Hypoxia-Mediated Pulmonary Hypertension

could be inhibited by verapamil, a VDCC antagonist [42, 43]. These studies clearly illustrate the importance of Ca2+ influx through membrane VDCCs in HPV. However, because these VDCCs are generally closed at the resting membrane potential of PASMCs, it is likely that hypoxia first acts on inhibition of K+ channels and membrane depolarization. Several hypotheses have been proposed to explain the hypoxic inhibition of KV channels in PASMCs, but the mechanism by which these channels sense low levels of O2 is still unclear. KV channels could be the O2 sensors themselves through the direct interaction of O2 with the channel, or they may be an effector, responding through some indirect mechanism [44]. The indirect mechanism seems more likely, however, because cloned KV channels are O2-sensitive in some heterologous expression systems but not in others. One possible explanation is the existence of a membrane-bound heme-linked protein closely associated with the channel [45]. The binding of O2 to the sensor could affect its conformation, which, in turn, would affect the conformation of the KV channel and thus the K+ current. Alternatively, hypoxia could cause a decrease in the production of reactive oxygen species generated by a membrane-bound NADPH oxidase [43], modulating the channel through a redox mechanism. In support of a membrane-bound O2 sensor, KV channel inhibition has been clearly demonstrated in excised patches in the absence of intracellular mediators in type I cells of the carotid body as well as in heterologous expression systems [45]. Furthermore, it is thought that charged amino acids called “gating charges” move through the membrane electric field in association with pore opening, allowing membrane voltage to balance the equilibrium between closed and opened conformations [45]. All members of the voltage-dependent cation channel family contain six hydrophobic segments per subunit, S1–S6. Four subunits (most often identical in K+ channels) surround a central ion-conduction pore. S5 and S6 segments line the pore and determine ion selectivity, whereas S1–S4 form the voltage sensor [45]. Charged amino acids within the voltage sensors, particularly the first four arginine residues in S4, account for most of the gating charge. It could be possible that the conformation change of the KV channel during exposure to acute hypoxia will inhibit K+ current efflux and alter the membrane potential; therefore, it will lead to movement of gating charges and, as a result, completely close the KV channels on PASMCs.

5.2 Cellular Mechanisms by Which Hypoxia Activates SOCCs In the previous section, we discussed the potential roles and mechanisms by which K+ channels react to acute hypoxia. This evidence includes demonstrations that hypoxia reduced

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KV currents, caused depolarization, increased intracellular Ca2+ concentration, and induced contraction in isolated pulmonary arteries and PASMCs. Moreover, VDCC antagonists or removal of extracellular Ca2+ inhibited hypoxic responses in isolated pulmonary arteries and PASMCs, as well as HPV in isolated lungs and intact animals. There is also other evidence, however, suggesting that the mechanisms of HPV are more complicated and besides KV channels there should be other channels or pathways by which intracellular Ca2+ concentration is regulated. First, KV channel antagonists could not completely prevent hypoxic responses in PASMCs, pulmonary arteries, or isolated lungs [3, 46–48]. In addition, antagonists of VOCCs cannot completely block intracellular Ca2+ concentration responses to hypoxia in PASMCs or HPV in isolated lungs [47, 48]. Furthermore, contractile and intracellular Ca2+ concentration responses to hypoxia persisted in pulmonary arteries after depolarization and inhibition of VOCCs, and the increase in intracellular Ca2+ concentration caused by hypoxia in PASMCs occurred before depolarization [49]. Finally, hypoxic responses in PASMCs, pulmonary arteries, and isolated lungs were inhibited by ryanodine, which blocks Ca2+ release from the SR [42, 50–52]. These data suggest that HPV requires Ca2+ release from the SR and Ca2+ influx through pathways other than VDCCs. One such pathway could be capacitative Ca2+ entry (CCE). CCE has long been recognized as a major pathway for Ca2+ influx in nonexcitable cells; there is growing appreciation that CCE is also important in excitable cells, such as vascular smooth muscle. CCE is triggered by depletion of SR Ca2+ stores, which can result from either enhanced release of SR Ca2+ through ryanodine and IP3 receptors in the SR membrane or depressed SR Ca2+ uptake by SERCA pumps [27, 29]. The pathway for CCE is probably sarcolemmal cation channels composed of proteins homologous to the TRP proteins that provide Ca2+ entry. CCE is thought to replete SR Ca2+ stores and signal cellular responses. It has been confirmed that the intracellular Ca2+ concentration increase induced by hypoxia required activation of SOCCs [3, 47]. SK&F 96365 and NiCl2, antagonists of SOCCs, were tested and showed concentration-dependent inhibition of CCE as well as hypoxic response. It is thought that hypoxia can cause Ca2+ influx from the intracellular storage site, SR, the depletion of which will trigger a sharp extracellular Ca2+ influx into the cell through TRP-protein-composed SOCCs on the cell membrane to refill the SR store [3, 22]. Despite intense investigation, the genes that encode SOCCs remain elusive. It was suggested that TRPC might be a long-sought store-operated channel owing to the following findings: [22, 27, 29] (1) the TRPC mutant was associated with defective Ca2+ influx and (2) the TRPCs were relatively selective for Ca2+ and had a predicted topology similar to that of voltage-gated Ca2+ channels (with the notable exception that the S4 segments of Drosophila TRPC were relatively

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devoid of the voltage-sensing positively charged amino acids); this Ca2+-permeable channel was activated downstream of PLC. Because SOCE also occurs downstream of PLC and together with the other research findings, speculation as to the possible role of this channel in the SOCE pathway arose. The first mammalian TRP proteins to be cloned and expressed were TRPC1 and TRPC3. There are seven related members of the TRPC family, designated TRPC1–TRPC7 (the numbering reflects the order of their discoveries) [22, 27, 29]. TRPC2 is a pseudogene in humans (but not in most other mammalian species). The remaining six appear to fall into two groups based on structural and functional similarities: TRPC1, TRPC4, and TRPC5 and TRPC3, TRPC6, and TRPC7. Among all TRPCs, the case for TRPC1 as a store-operated channel (or subunit) is perhaps the strongest. Initial studies on functional expression of human TRPC1 in COS cells resulted in a modest increase in thapsigargin-evoked Ca2+ influx [22, 27, 29]. Other data from Zitt et al. [53] on whole-cell recordings in CHO cells expressing TRPC1 showed that dialysis with either IP3 or thapsigargin activated nonselective currents that were permeable to Ca2+, Na+, and Cs+. They concluded that TRPC1 encoded a nonselective channel that was gated by store depletion. However, whether or which TRPCs are involved in forming store-operated cation channels is worthy of further investigation and discussion owing to discordant findings that have emerged from different research groups.

6  C  hronic Hypoxic Regulation of Ion Channels Expression 6.1 Downregulation of K+ Channels in CHPH Acute hypoxia can lead to the inhibition of KV channel activities, which will greatly alter the membrane potential called depolarization and finally lead to the activation of VDCCs and increase in intracellular Ca2+ concentration. In a chronic hypoxic environment, not only the activities of K+ channels are inhibited, but most importantly the expressions of KV channels are also downregulated. Actually, the resting potential of PASMCs from chronically hypoxic animals is more depolarized, while the amplitude of KV currents is significantly lower, than cells from normoxic animals. In 1997, the first evidence for downregulation of specific KV channel a-subunits (KV1.2 and KV1.5) in cultured rat PASMCs under chronic hypoxia was reported by Wang et al. [5]. After that, decreased messenger RNA (mRNA) expression of KV1.1, KV1.5, KV2.1, KV4.3, and KV9.3 a-subunits in cultured rat PASMCs under chronic hypoxia was reported and confirmed by other research groups [9, 54–58]. The response of downregulation of KV channels is

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specific to PASMCs [9, 54, 55] and therefore selective for the pulmonary circulation since, until now, chronic hypoxia inhibition of the expression of a- or b-subunits of KV channels has not been reported in mesenteric artery smooth muscle cells. Two animal models, the chronically hypoxic animal model and the KV1.5 knockout mouse model, have been set up and studies based on these two models have emphasized the importance of KV channels in the pulmonary vascular response. Investigations of freshly isolated PASMCs from chronically hypoxic animal models showed downregulation of KV1.2 and KV1.5 and a decrease in the O2-sensitive component of the K+ current [7, 10, 41]. Studies with KV1.5 knockout mice models showed impaired HPV and reduced O2-sensitive K+ current in PASMCs. All these findings provide strong evidence for the role of KV channels in the chronic pulmonary vascular response to hypoxia [7, 9, 10, 41, 54–58]. The mechanisms by which the expression of KV channels is downregulated are not totally understood yet. Many hypotheses have been put forward to explain the chronic hypoxia induced inhibition of KV channel expression in PASMCs, which include the following: (1) upregulation or downregulation of the transcription factors and signal transduction proteins that can directly bind to the KV channel gene promoters (e.g., KV1.5 repressor element) and modulate the KV channel gene transcription [59–61] and (2) induction of transcription factors that upregulate intermediate inhibitors of the KV channel genes, such as endothelin-1 (ET-1) [59, 62]. Phosphorylation and redox modification of responsive transcription factors are the most common pathways that are involved in the transduction of hypoxic signals from the sensor to appropriate gene expression. A variety of transcription factors and signal transduction signaling proteins such as hypoxia-inducible factor 1 (HIF-1), nuclear factor kB, c-fos/c-jun, bone morphogenetic protein, p53, KRE binding factor, FixL, and FixJ can be modulated by hypoxia [54, 56, 59, 61–73], suggesting that a large number of transcriptional pathways contribute to the response under a chronic hypoxic environment. For example, it has recently been reported that overexpression of the c-jun gene can significantly decrease the whole-cell KV current, downregulate the mRNA expression of the KV1.5 channel subunit, and accelerate KV current inactivation in cultured PASMCs [36]. There is also a large amount of data indicating that the c-jun family of genes is inducible by hypoxia [74], suggesting that c-jun may indirectly downregulate transcription of the KV1.5 gene and decrease the KV current.

6.2 Upregulation of TRPCs in CHPH Development of CHPH is associated with elevated resting intracellular Ca2+ concentration in PASMCs and contraction of pulmonary vascular smooth muscle. It has been wildly accepted

47  Pathogenic Roles of Ca2+ and Ion Channels in Hypoxia-Mediated Pulmonary Hypertension

that the maintenance of increased PASMC intracellular Ca2+ concentration and tone during chronic hypoxia requires Ca2+ influx through pathways other than VDCCs. As discussed in Sect. 5.2 that SOCCs which are believed to be composed of TRPC proteins may play very important roles in response to acute hypoxia, it is likely that SOCCs also contribute to the development of CHPH. Evidence provided by several research groups has confirmed that exposure to chronic hypoxia caused enhanced activity of SOCCs [3, 4, 75], which was required for maintenance of elevated resting intracellular Ca2+ concentration and active tone and correlated with elevated expression of TRPC proteins, especially TRPC1 and TRPC6. Compared with KV channels, little is known about the roles and functions of TRPC proteins as well as the upregulation mechanisms in the development of chronic pulmonary hypertension. Yu et al. were the first to demonstrate the role for HIF-1 in mediating the pulmonary responses to chronic hypoxia. The research data showed that the development of right ventricular hypertrophy, and vascular remodeling was delayed in Hif1a +/- compared with Hif1a +/+ mice [76]. A similar study by Wang et al. provides compelling evidence that the developing stories of HIF-1 and TRPCs in the pulmonary vascular response to chronic hypoxic form part of the same narrative [3]. The authors confirmed previous observations of elevated intracellular [Ca2+]i concentrations and CCE in transiently cultured PASMCs from rats made hypoxic for 21 days. They then showed that in vivo chronic hypoxia in both rats and control mice resulted in an upregulation of the expression of protein and mRNA for TRPC1 and TRPC6, but not TRPC4. Crucially, they demonstrated that the increases in basal intracellular Ca2+ concentration and TRPC1/TRPC6 expression are absent in HIF1a+/– mice. Accordingly, overexpression of a constitutively active form of HIF-1a also increases the expression of TRPC1/TRPC6, but not TRPC4 [3].

7  M  icrodomains and Organized Signaling in Regulating Ion Channel Function 7.1 Caveolin and Caveolae Caveolae were first described in 1953 by George Palade from electron micrographs of endothelial cells [77]. In 1955, Yamada described similar invaginations in the gall bladder epithelium and created the term “caveolae” to reflect their appearance as little caves [78]. Caveolae are 50–100-nm flask-shaped invaginations of the plasma membrane. Biochemically, caveolae represent a subdomain of the plasma membrane enriched in cholesterol, sphingolipids, and a family of integral membrane proteins named caveolins.

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Caveolins are a family of 21–24-kDa integral membrane proteins with three mammalian isoforms identified as caveolin-1 (Cav-1), caveolin-2 (Cav-2), and caveolin-3 (Cav-3) [79, 80]. Cav-1 is most prominently expressed in endothelial, fibrous, and adipose tissue. The expression pattern of Cav-2 is similar to that of Cav-1, which indicates Cav-2 may be coexpressed with Cav-1. The expression of Cav-3 is restricted to striated and smooth muscle. The formation of caveolae was greatly suppressed in the VSMCs in Cav-1 knockout mice [81], whereas this formation remained present in striated muscle types, suggesting that Cav-1 is required for formation of caveolae in smooth muscle and that Cav-3 cannot compensate for the physiological function of Cav-1 in smooth muscle cells. Additionally, in Cav-3 knockout mice, skeletal and heart muscle lack caveolae, whereas smooth muscle still demonstrated formation of caveolae [81]. Therefore, it is reasonable to predict that Cav-1 is the major structural caveolin isoform in smooth muscle and that Cav-2 and Cav-3 may play roles other than simply formation of caveolae, such as modulation of Cav-1 expression, modulation of cell signaling, and modulation of metabolism.

7.2 Colocalization of Caveolins and Ion Channels in Caveolae Investigations about the ion channels and related signaling pathways localized in caveolae in PASMCs and endothelial cells will be helpful in determining their roles in the pulmonary circulation. It is suggested that the caveolae, by virtue of enrichment in signaling molecules and ion channels, would be key in excitation–contraction coupling and perhaps in O2 sensing or susceptibility to CHPH [82]. The recruitment of signaling molecules to caveolae can determine Ca2+ sensitization in smooth muscle in response to K+ depolarization and receptor agonists [82, 83]. It has been documented that the voltage-gated K+ channel KV1.5 targets to caveolae and colocalizes with caveolin [84, 85]. The latter studies demonstrated that a cholesterol-binding agent 2-hydroxypropyl-b-cyclodextrin, which disrupts caveolae, induced a hyperpolarizing shift in the voltage dependence of activation and inactivation. In 2005, Wang et  al. provided more evidence that large-conductance Ca2+-activated K+ channels were associated with Cav-1 in vascular endothelial cells by immunoprecipitation and glutathione S-transferase pull-down assays [86]. These and other data suggest that Cav-1 is a regulator of K+ channel function. Ca2+ influx across the plasma membrane following depletion of the intracellular Ca 2+ stores is known as storeoperated Ca2+ entry (SOCE). As described already, a relation between SOCE and the members of the TRPC family of

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proteins has recently emerged. TRPC1, TRPC3, and TRPC4 have been suggested to associate with caveolae via interaction with Cav-1. Bergdahl et  al. have demonstrated that TRPC1 is functionally coupled to ET-1-type receptors in intact arteries and that it is localized to caveolae [87]. Additionally, disruption of caveolae and lipid rafts by cholesterol depletion resulted in increased sensitivity of TRPC1 to SOCE and ET-1 responses. A recent report by the same group demonstrated that the plasticity of TRPC expression is enhanced by vascular injury in arterial smooth muscle, that TRPC expression correlates with cellular Ca2+ handling, and that TRPC1 is a subunit that is highly upregulated by SOCE channels [87].

8  Summary Acute hypoxia induces pulmonary vasoconstriction and chronic hypoxia causes structural changes and remodeling in pulmonary arteries, eventually leading to chronic pulmonary hypertension and right ventricular hypertrophy. In both cases, the increases in intracellular Ca2+ concentration is the key factor responsible for both transient, or reversible, changes and permanent, or irreversible, changes. The function of ion channels involved in the former process, including Kv channels, VDCCs, and SOCCs, will have been changed, either “turned on” or “turned off.” The functional changes of these ion channels are not totally separated or individual events and may have effects on one another. The effects of acute hypoxia on both the ion channels and the intracellular Ca2+ concentration are reversible once the “alarm” of the absence of oxygen has been spared and the cells are treated under regular oxygen concentration. However, chronic hypoxia has long-term and irreversible effects upon ion channels and intracellular Ca2+ concentration owing to sustained absence of oxygen. Under chronic hypoxic conditions, both mRNA and protein expression levels of the ion channels involved have been altered, providing the basis for the sustained effects. Decreased mRNA expression of KV1.1, KV1.2, KV1.5, KV2.1, KV4.3, and KV9.3 a-subunits in cultured rat PASMCs under chronic hypoxia has been confirmed. However, the mechanisms by which the expressions of KV channels are downregulated are not completely understood. It has been proposed that a variety of transcription factors and signal transduction proteins that can directly bind to the KV channel gene and modulate the KV channel gene transcription. Induction of transcription factors that upregulate intermediate inhibitors of the KV channel genes, such as ET-1, may be a common mechanism for mitogenic factormediated downregulation of KV channels. Compared with KV channels, little is known about the roles and functions of TRPC proteins or their upregulation mechanisms in the

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development of chronic pulmonary hypertension, despite the fact that the expression of TRPC proteins, especially TRPC1 and TRPC6, has been confirmed. Acknowledgements  This work was supported by an NIH Independent Scientist Award (J.W.), an American Heart Association Scientist Development Grant (J.W.), the American Lung Association of Maryland (J.W.), NIH research grants (J.X.-J.Y), the National Natural Science Foundation of China (30770953), and Chinese Central Government key research projects of the 973 grants (2009CB522107).

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Chapter 48

Roles of Endothelium-Derived Vasoactive and Mitogenic Factors in the Development of Chronic Hypoxia-Mediated Pulmonary Hypertension Rajeev Malhotra and Kenneth D. Bloch

Abstract  Chronic hypoxic exposure in humans has been linked to the development of pulmonary hypertension (PH) and pulmonary vascular remodeling. Human diseases in which alveolar hypoxia plays a direct causative role in the development of PH include chronic mountain sickness and hypoventilatory syndromes, such as obstructive sleep apnea and obesity–hypoventilation syndrome (Pickwickian syndrome). Additional diseases of the lung that result in hypoxemia-associated PH occur in conjunction with other primary abnormalities such as inflammation and abnormal immunologic responses. These disorders include chronic obstructive pulmonary disease (COPD), interstitial lung disease, cystic fibrosis, radiation fibrosis, and collagen vascular diseases. Low oxygen tension is sensed by numerous cell types of the pulmonary vasculature, including endothelial cells (ECs), smooth muscle cells (SMCs), smooth-musclelike cells termed “pericytes,” and adventitial fibroblasts. Hypoxia-mediated molecular signaling is a complex process integrating pathways from all of these cell lineages. Ultimately, these signals result in increased vasoconstriction, proliferation, and migration of SMCs and pericytes with enhanced muscularization of resistance pulmonary arterioles and the development of PH. There is a significant degree of modulation of these responses by the endothelium. The focus of this chapter is to review the processes by which vascular ECs contribute to the development of PH in chronic hypoxia. Keywords  Hypoxic pulmonary vasoconstriction • Endothelial cell • Pulmonary vascular remodeling • Extracellular matrix • Vasoconstrictor • Vasodilator • Endothelial progenitor cell

R. Malhotra (*) Massachusetts General Hospital, Harvard Medical School, Division of Cardiology, Yawkey 5700, 55 Fruit St, Boston, MA 02114, USA e-mail: [email protected]

1 Introduction Chronic hypoxic exposure in humans has been linked to the development of pulmonary hypertension (PH) and pulmonary vascular remodeling (reviewed in [1]). The World Health Organization has identified hypoxemia-associated PH, also termed “cor pulmonale,” as a distinct class of pulmonary hypertensive disorders (class III) related to hypoxemia either induced by alveolar hypoxia or from underlying lung disease. Human diseases in which alveolar hypoxia plays a direct causative role in the development of PH include chronic mountain sickness and hypoventilatory syndromes, such as obstructive sleep apnea and obesity– hypoventilation syndrome (Pickwickian syndrome). Additional diseases of the lung that result in hypoxemiaassociated PH occur in conjunction with other primary abnormalities such as inflammation and abnormal immunologic responses. These disorders include chronic obstruc­tive pulmonary disease (COPD), interstitial lung disease, cystic fibrosis, radiation fibrosis, and collagen vascular diseases [1]. It is thought that the hypoxemia observed in this latter group of pulmonary disorders is a major contributing factor to overall disease progression [2]. Hence, understanding the mechanisms by which hypoxia results in pulmonary vascular remodeling and PH may provide valuable insights into the molecular underpinnings of an important spectrum of human pulmonary diseases. Low oxygen tension is sensed by numerous cell types of the pulmonary vasculature, including endothelial cells (ECs), smooth muscle cells (SMCs), smooth-muscle-like cells termed “pericytes,” and adventitial fibroblasts [1]. Hypoxiamediated molecular signaling is a complex process integrating pathways from all of these cell lineages. Ultimately, these signals result in increased vasoconstriction, proliferation, and migration of SMCs and pericytes with enhanced muscularization of resistance pulmonary arterioles and the development of PH. There is a significant degree of modulation of these responses by the endothelium. The focus of this chapter is to review the processes by which vascular ECs

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contribute to the development of PH in chronic hypoxia. We first discuss the differences in the response of the pulmonary vasculature to chronic hypoxia from that of acute hypoxic pulmonary vasoconstriction (HPV). We subsequently review the morphologic characteristics of pulmonary vascular remodeling associated with chronic hypoxia. The endothelial modulators of hypoxic PH include vasoactive compounds, growth factors, cytokines, procoagulants, and extracellular matrix (ECM) molecules. We specifically highlight endothelium-derived vasomotor and mitogenic factors, focusing on phenotypes in animal models as well as clinical studies in humans. In addition, we explore how endothelial progenitor cells (EPCs) that are recruited to the pulmonary vasculature contribute to hypoxia-mediated PH and remodeling. The therapeutic implications derived from an understanding of these endothelial regulators in hypoxic PH will be highlighted.

2 Pulmonary Vascular Responses to Acute and Chronic Hypoxia Most animals reliably develop chronic PH when exposed to low levels of ambient oxygen resulting in alveolar hypoxia [3]. The information obtained from these animal experiments is directly applicable to hypoxic human conditions such as chronic mountain disease, high-altitude PH, and sleep apnea syndromes [4]. The contribution of hypoxemia to the development of PH in diseases such as COPD and interstitial lung disease where inflammation also plays a significant role remains incompletely elucidated. However, the importance of hypoxemia for underlying disease progression is suggested by human clinical trials demonstrating that the administration of supplemental oxygen in COPD patients provides a survival benefit [5, 6]. The same reduction in mortality has not been demonstrated with drugs that suppress inflammation, such as inhaled corticosteroids [7]. Not all patients with COPD develop PH; however, the development of PH is strongly associated with the presence of hypoxemia. Increases in pulmonary artery pressure (PAP) correlate inversely with PaO2 in patients with severe COPD [2, 8]. On the other hand, COPD patients without a significant increase in PAP do not exhibit hypoxemia [8]. In addition, the pulmonary vascular remodeling observed with PH in primary lung disorders is similar to that seen in alveolar hypoxic diseases [2]. Therefore, models of PH derived from chronic states of low ambient oxygen are likely to be relevant to the wide range of primary pulmonary diseases associated with hypoxemia.

R. Malhotra and K.D. Bloch

Evidence in humans that chronic hypoxia alone can result in the vascular changes associated with PH derives from studies of individuals living at high altitudes [1]. Highaltitude residents exhibit elevated PAP and minimal vasodilation in response to supplemental oxygen [9]. Furthermore, when humans live in a hypobaric, hypoxic environment for periods of time as little as 40 days, they develop a progressive elevation in PAP and pulmonary vascular resistance (PVR), an exaggerated increase in PAP with exercise, and an attenuated vasodilator response to supplemental oxygen [10]. These findings suggest that hypoxia can induce functional and structural changes in the pulmonary vascular bed even over relatively short periods of time. Residence at high altitudes induces changes in the pulmonary vasculature in humans that can be irreversible, with persistent elevations in PAP with exercise despite a return to sea level [11, 12]. In certain susceptible individuals, the PH from chronic hypoxia progressively worsens, leading to right ventricular failure and ultimately death. The changes associated with chronic hypoxia, however, should not be confused with the physiologic pulmonary vasoconstriction observed with acute hypoxia, a process termed “hypoxic pulmonary vasoconstriction” (HPV). A major distinction between the pulmonary circulation and the systemic circulation is the response to acute changes in oxygen tension. Pulmonary arterioles constrict to hypoxia, whereas systemic arterioles vasodilate. HPV is an adaptive response that ensures that blood is preferentially distributed to adequately ventilated regions of the lung and that maintains the appropriate ventilation–perfusion ratio [13]. In pulmonary conditions that result in ventilation– perfusion mismatch such as pneumonia and aspiration, HPV is a focal response to regions of hypoxia in the lung [14]. However, with global hypoxia, as in situations of high altitude or sleep apnea, HPV occurs throughout the pulmonary arteriolar bed and PVR increases. As reviewed by Moudgil et  al. [14], the HPV response in humans occurs within minutes and can increase the PVR by 50–300%. Studies in both humans and animal models have demonstrated that alveolar hypoxia, rather than mixed-venous hypoxemia, is the major determinant of HPV [15]. For instance, when unilateral hypoxia is delivered to patients in one lung while the other lung receives 100% oxygen, the mixed venous oxygen tension does not change significantly. However, perfusion to the alveolar hypoxic lung is reduced significantly [16]. The principal site at which HPV occurs is within the small resistance pulmonary arterioles [15]. Although the exact mechanisms underlying HPV have not been completely elucidated, one hypothesis is that O2 levels are “sensed” by the mitochondrial electron transport chain of

48  Roles of Endothelium-Derived Vasoactive and Mitogenic Factors

pulmonary artery SMCs (reviewed in [14]). The prevailing data suggest that hypoxia results in a reduced production of reactive oxygen species (ROS). These ROS are thought to modulate function of K+ and Ca2+ channels in pulmonary artery SMCs, possibly through direct oxidation–reduction reactions with key sulfhydryl groups [17]. These redox-sensitive K+ channels are inhibited under hypoxic conditions, leading to a change in cellular membrane potential and activation of L-type voltage-gated Ca2+ channels [14, 18]. Other potential mechanisms for HPV have been proposed that differ from the model described above, including an increase in ROS with hypoxia (reviewed in [19]). However, the final common pathway in HPV is an increase in the level of intracellular calcium, which signals for vasoconstriction via the actin and myosin contractile apparatus. Further support for this mechanism comes from studies in both COPD patients and in rats in which L-type calcium channel antagonists attenuate the HPV response [20, 21]. A growing body of literature reports that hypoxia-induced intracellular calcium influx also occurs through non-voltage-gated calcium channels (reviewed in [22]). Theories on the exact mechanisms of HPV remain controversial and are reviewed elsewhere in this textbook. Although HPV can be sustained for hours, it does not reflect a permanent response to global hypoxia. Persistent hypoxia ultimately results in a downregulation of the HPV response, within as little as 3  h in hypoxic rats [23, 24]. Humans that have adapted to life at high altitudes, such as native Tibetans, have substantially reduced HPV responses [25]. Whereas acute hypoxia results in an immediate increase in PAP that is reversible upon recovery of oxygen concentration, sustained hypoxia results in durable elevations in pulmonary pressures with only partially reversible vasoconstriction and long-term vascular remodeling. The key features and molecular regulators of acute HPV are different from those of chronic hypoxia-mediated PH, and it is the latter condition that we review here.

3 Pulmonary Vascular Remodeling Due to Chronic Hypoxia The pulmonary vessel wall is composed of three layers termed the adventitia, media, and intima, consisting of ­fibroblasts, SMCs, and ECs, respectively. A homeostatic balance between cell growth and proliferation, apoptosis, and migration determines in large part the vessel wall thickness. When this balance is altered in favor of prolif-

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eration as in chronic hypoxia, then thickening of the vascular wall occurs, which contributes to an increase in flow resistance [13]. Although some mammalian species such as Bos grunniens (yak) and Lama glama (llama) have adapted to high-altitude living and do not exhibit pulmonary vascular remodeling with chronic exposure to hypoxia, the vast majority of mammals do exhibit structural changes [1, 26]. Hypoxia-induced remodeling classically involves muscularization of precapillary arteriole resistance vessels with the appearance of a-smooth muscle actin expressing cells in previously nonmuscularized vessels [27]. The resultant increase in vascular resistance from inward arteriolar thickening contributes to the overall development of PH with chronic hypoxia [1]. Interestingly, the same distal muscularization observed with chronic hypoxia is also observed in other causes of PH, including pulmonary arterial hypertension (PAH) and PH secondary to lung disorders such as COPD (reviewed in [2]). Presumably each of the differing causes of PH leads to a common stressor or cell signal that initiates the process of vascular remodeling. Medial thickening extends throughout the pulmonary arterial bed from proximal elastic arteries to distal muscular arterioles in response to chronic hypoxia (reviewed in [1]). This process is dependent on both proliferation and hypertrophy of SMCs, as well as increased deposition of ECM. Reduced SMC apoptosis in lungs is also observed in some but not all models of hypoxic PH and can contribute to the overall muscularization of the vessel wall [28, 29]. Induction of SMC apoptosis is protective in animal models of PH [30]. Expression of the antiapoptotic protein Bcl-xL is increased in chronically hypoxic rat lungs [31]. Similarly in humans, pulmonary artery SMCs of patients with COPD exhibit decreased apoptotic activity compared with pulmonary artery SMCs of control individuals, and those patients with both COPD and PH have the lowest levels of apoptosis [32]. The distal muscularization of pulmonary vessels can also be attributed to the proliferation and migration of pericytes, which are smooth-muscle-like cells that exhibit contractile properties but have biochemical and morphologic signatures different from those of differentiated SMCs [1, 33]. Adventitial thickening is observed in hypoxic PH with increased proliferation of fibroblasts and deposition of ECM components such as collagens, elastin, and fibronectin [1]. The degree of remodeling in hypoxic rats is dependent on the age and sex of the animal, with infants and males exhibiting more structural changes than adult and female counterparts through unclear mechanisms [34]. Controversy currently exists over the effects of chronic hypoxia on the total number of pulmonary resistance arterioles. Earlier data indicate a loss of arteriole numbers, with

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a significant decrease in small arteries up to 200  mm in external diameter by 2 weeks of hypoxia in rat models [35, 36]. This loss of cross-sectional area is termed “rarefaction” or “pruning” and is thought to contribute to the overall increase in PVR and PH seen with chronic hypoxia. However, more recent data point toward hypoxia as a potent stimulus for pulmonary vessel angiogenesis (reviewed in [12, 37]). Stereological techniques with confocal microscopy have demonstrated an increase in overall pulmonary vessel length, volume, endothelial surface area, and EC number in rat models of chronic hypoxia [38]. However, other studies have demonstrated that expression of the potent proangiogenic factors angiopoietin, Tie2, and vascular endothelial growth factor (VEGF) is decreased in the setting of chronic hypoxic PH [39]. A potential explanation for the disparity in results is that hypoxia induces a state of ineffective angiogenesis [37]. Although the total length and surface area of the pulmonary vasculature may increase (based on stereological techniques), the effective pulmonary circulation may actually be reduced owing to abnormal breaks or terminations in the precapillary vessels [40]. The loss of distal arterioles has been demonstrated in numerous models of hypoxia-induced PH and has been attributed to the apoptosis of ECs and surrounding pericytes [37]. The ensuing neoangiogenesis results in a structurally and functionally dysregulated overgrowth of vessels primarily consisting of apoptosis-resistant ECs [41]. The topic of pulmonary arteriolar number remains controversial and the mechanisms by which ineffective angiogenesis takes place in the setting of chronic hypoxia require further investigation.

4 Hypoxia-Induced Changes in the Pulmonary Endothelium The endothelium serves many important and distinct roles in the pulmonary vasculature. The EC acts as the inner lining of blood vessels and is a critical regulator of vascular development, hemostasis, leukocyte transmigration, repair after injury, vessel permeability, and vasomotor tone (reviewed in [42]). The structure and function of ECs differ depending on anatomic location within the pulmonary vasculature. The ECs of rat pulmonary arteries are broad and short with a rectangular shape, whereas those in the pulmonary vein are large and round [43]. This structural heterogeneity correlates with differing regulatory functions. ECs in the pulmonary arterioles modulate vasomotor tone via a highly regulated and complex balance of vasoconstrictor and vasodilatory molecules such as nitric oxide (NO), prostacyclin (prostaglandin I2, PGI2), and

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endothelin-1 (ET-1). ECs in the pulmonary capillaries, on the other hand, maintain an extensive network of intercellular clefts, intracellular vesicles for transcytosis, and transendothelial channels for the purpose of gas and nutrient exchange [42]. ECs exhibit a significant degree of plasticity even during adulthood and can undergo remodeling with changes in hemodynamic shear stress [42, 44, 45].The endothelium is in a unique position to sense signals from circulating factors and blood elements and to transduce these signals to the underlying vascular smooth muscle. The oxygen tension in arterial blood is typically 150 mmHg at normal altitudes and approximately 40 mmHg in body tissues. Pulmonary ECs are able to detect and respond to arterial oxygen tensions below 70  mmHg [46]. Both EC proliferation and EC apoptosis are altered by long-term states of low oxygen tension [41, 47].Chronic hypoxia ultimately results in a threefold thickening of the intimal layer in rat pulmonary arteries, with associated EC hyperplasia and hypertrophy in addition to subendothelial deposition of microfibrils, collagen, and elastin [47, 48]. The underlying mechanisms of pulmonary intimal remodeling remain incompletely understood, and may in part be explained by an initial increase in permeability of the EC layer with hypoxia [49, 50]. The resulting extravasation of plasma proteins into the vessel wall may activate proteases that can then initiate the process of remodeling (reviewed in [1, 51]). In addition to structural remodeling, pulmonary endothelial function and metabolism are altered in states of chronic hypoxia, and these changes contribute to the development of PH (reviewed in [1]). Hypoxia alters the homeostatic balance of expression between vasodilators and vasoconstrictors in pulmonary arterioles such as NO, ET-1, PGI2, and serotonin (5-hydroxytryptamine, 5-HT). Increased signaling of endothelium-derived mitogenic factors such as fibroblast growth factor (FGF), plateletderived growth factor (PDGF), VEGF, and bone morphogenetic proteins (BMPs) enhances proliferation of vascular SMCs (VSMCs). Hypoxic ECs upregulate proinflammatory and procoagulant factors [52] such as P-selectin [53], intercellular adhesion molecule 1 [54, 55], vascular cell adhesion molecule 1 [56, 57], tissue factor [58], and plasminogen activator inhibitor 1 [59] that contribute to the inflammation and thrombosis associated with PH. The endothelium also modulates production of ECM proteins such as laminin, fibronectin, and thrombospondin-1 in states of chronic hypoxia [46, 60]. The extent of the changes depends on the degree and duration of hypoxia. In this chapter, we focus our discussion on the contribution of endothelium-derived vasoactive and mitogenic molecules to the development of chronic hypoxia-mediated PH (see Fig. 1).

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Fig. 1  Role of endothelium-derived factors in hypoxia-mediated pulmonary hypertension. Nitric oxide (NO) and prostacyclin (PGI2) are protective in hypoxia-mediated pulmonary hypertension and mediate vasorelaxation, whereas endothelin-1 (ET-1) and serotonin (5-HT) signal for vasoconstriction of smooth muscle cells. Asterisk denotes a

decrease in production with chronic hypoxia, whereas all other endothelial mediators are upregulated with hypoxia. eNOS, endothelial nitric oxide synthase; PGIS, PGI2 synthase; sGC, soluble guanylate cyclase; AC, adenylate cyclase; PKC, protein kinase C

5 Endothelium-Derived Vasoactive Mediators of Hypoxic PH

domain contains binding sites for heme, l-arginine, and the cofactor tetrahydrobiopterin and is the location of NO synthesis. Electron transport occurs at the reductase domain from NADPH to molecular oxygen bound to the heme iron [64]. This process of electron transfer allows NOS to oxygenate l-arginine, resulting in the production of l-citrulline and NO. The NOS isoforms differ in their distribution of expression and the regulation of their activities. In the respiratory system, eNOS is expressed primarily in ECs of the pulmonary vasculature. Neuronal NOS and iNOS are expressed in airway epithelium and in pulmonary VSMCs [61]. Expression of sGC primarily occurs in VSMCs and platelets. Direct binding of NO to the heme site of sGC results in a conformational change and a 200-fold increase in cGMP production from GTP (reviewed in [65]). The downstream effects of cGMP are in large part mediated by cGMP-dependent protein kinases as well as cGMP-regulated phosphodiesterases and ion channels (reviewed in [66]). cGMP relaxes pulmonary vascular smooth muscle through multiple mechanisms, including cell membrane hyperpolarization and inhibition of calcium influx. The cellular levels of cGMP are regulated both by NO-induced production and by phosphodiesterase V (PDE V)-mediated hydrolytic breakdown. Alveolar hypoxia is a potent regulator of pulmonary NO signaling (reviewed in [62]). Molecular oxygen binds to the heme group of NOS and is a direct substrate in the catalytic reaction to produce NO. A reduction in oxygen

5.1 Nitric Oxide NO, also known as endothelium-derived relaxing factor, is an intracellular signaling molecule that is essential for numerous physiologic processes (reviewed in [61]). NO is a potent pulmonary and systemic vasodilator that is produced in the endothelium. NO then diffuses to its target tissues and activates soluble guanylate cyclase (sGC) to produce guanosine 3¢,5¢-cyclic monophosphate (cGMP), with the downstream effects of SMC vasodilation and inhibition of platelet aggregation. NO can act in a cGMP-independent manner as well. NO regulates pulmonary vascular remodeling by inhibiting SMC proliferation and migration as well as ECM production. Hence, a reduction in NO signaling can result in the development of PH both by increasing vessel tone and by inducing vascular proliferation and remodeling [61, 62]. NO is a free-radical molecule that is synthesized via the oxidation of l-arginine by NO synthases (NOS). The NOS family consists of three isoenzymes: neuronal NOS, inducible NOS (iNOS), and endothelial NOS (eNOS) (reviewed in [63]). NOS is active as a homodimer, with each subunit containing a reductase domain and an oxygenase domain linked by a cal­ modulin-binding sequence (reviewed in [61]). The oxygenase

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tension ­(substrate concentration) should therefore lead to a decrease in catalytic NO production. Changes in oxygen tension within pathophysiologic ranges do in fact affect NO production [67]. Fike et  al. examined the effects of breathing 10% O2 for 10–12 days on NO production in newborn pigs and found at least a twofold reduction in exhaled NO and plasma nitrites and nitrates (stable metabolic products of NO) [68]. In addition, expression of eNOS was reduced by 40% in the lungs of hypoxic newborn pigs. However, in hypoxic adult rats, despite a decrease in pulmonary NO production, the expression levels of all three isoforms of NOS increase [69, 70], indicating that hypoxia reduces NO production through a mechanism independent of NOS expression. The contrasting observations of NOS expression in the hypoxic pig and rat highlight the differential regulation of NO production across species and at different stages of life. Exposure of rats to 3 weeks of hypoxia results in at least a twofold increase in expression of both eNOS and iNOS in the pulmonary vasculature [69, 70]. Although eNOS is normally expressed in the large pulmonary artery beds, de  novo expression of eNOS is detected in the resistance arterioles upon hypoxic exposure [69]. Congenital deficiency of eNOS [71] or the sGC subunit a1 [72] in mice enhances the pulmonary vascular remodeling, PH, and right ventricular hypertrophy observed with chronic hypoxia, thus highlighting the protective role of NO signaling in the progression of hypoxic PH. Although most humans who migrate to high altitudes for extended lengths of time will develop hypoxic PH, the Tibetans have adapted to high-altitude living conditions and develop minimal PH compared with lowlanders [73]. This adaptation to high elevation correlates with increased exhaled NO levels, allowing for enhanced pulmonary blood flow without a concomitant rise in PAP and implicating NO signaling as an important therapeutic target in humans [74]. The utility of NO as a potential treatment of hypoxic PH and other forms of PH has been investigated. Delivery of continuous inhaled NO (iNO) significantly reduces the development of pulmonary vascular smooth muscle hypertrophy and hyperplasia and right ventricular hypertrophy in hypoxic newborn rats [75]. In addition, administration of the PDE V inhibitor sildenafil to mice during chronic hypoxia attenuates the progression of PH and this is coupled with a decrease in pulmonary vascular remodeling [76]. The use of iNO has shown promise in treating humans with different forms of PH (reviewed in [77]). iNO improves oxygenation in newborns with severe hypoxemia and persistent PH of the newborn [78] and reduces PAP and PVR in patients with COPD and secondary PH [79]. Thus, further investigation of the potential benefits of iNO, PDE V inhibition [80], and sGC activation [81] in human PH is warranted.

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5.2 Endothelin-1 Exposure to chronic hypoxia results in an increase in endothelin-1 (ET-1) and endothelin-A (ETA) receptor expression in the lung and pulmonary artery [82, 83]. Human ET-1 is a 21-residue peptide hormone that is initially synthesized in a pre-pro form, posttranslationally modified, and secreted by vascular ECs (reviewed in [84]). Three different isoforms of ET exist, but ECs only produce ET-1. ET-1 acts as a potent vasoconstrictor of the pulmonary vasculature. In addition to hypoxia, numerous stimuli result in increased ET-1 production by ECs, including inflammatory molecules such as transforming growth factor-b (TGF-b) and tumor necrosis factor-a, coagulation factors such as thrombin and bradykinin, hormones such as insulin and angiotensin, and mechanical factors such as a decrease in vascular shear stress. These stimuli typically regulate ET-1 production at the transcriptional level [84]. Two primary receptors for ET-1 have been identified, ETA and endothelin-B (ETB) (reviewed in [84–87]). Both receptors belong to the family of G-protein-coupled receptors with seven transmembrane domains. The ETA receptor is primarily expressed on VSMCs and mediates vasoconstriction. The ETB receptor similarly promotes vasoconstriction when expressed in smooth muscle. However, ETB is also expressed on ECs and stimulates production of PGI2 and NO, hence creating a negative-feedback circuit for ET-1-mediated vasoconstriction. ETA receptor activation promotes vasoconstriction through protein kinase C activation [88], resulting in a sustained rise in intracellular calcium levels in SMCs. Another mechanism by which ET-1 induces sustained SMC vasoconstriction, particularly in the setting of hypoxia, is by decreasing the transcription and expression of potassium channels, thereby altering cellular membrane potential and activating calcium influx through voltage-gated calcium channels [89]. ET-1 also possesses mitogenic and differentiation effects on the pulmonary vasculature leading to remodeling [84, 85]. ET-1 increases DNA synthesis and proliferation of SMCs, ECs, and fibroblasts. An early induction of growth response genes such as c-jun and c-fos is observed with ET-1 signaling [90], correlating with the mitogenic and vascular remodeling effects [84–87]. Hypoxia is one of the most potent stimulators of ET-1 expression in ECs [82, 91–93]. Prolonged exposure of ECs to low oxygen tension stimulates the expression of hypoxiainducible factor-1 (HIF-1), a transcription factor that in turn enhances production of ET-1 [94]. The ET-1 promoter contains a binding site for HIF-1, which is essential for hypoxia-induced ET-1 expression [93, 94]. In addition to the rise in ET-1 levels, chronic hypoxia stimulates at least a twofold increase in ETA receptor messenger RNA (mRNA) expression in rat lungs [83]. However, ETB receptor mRNA expression remains unchanged under the same conditions, implicating differential regulatory mechanisms for the expression patterns of these two receptors [83]. The role of ET-1 in chronic hypoxic PH encompasses

48  Roles of Endothelium-Derived Vasoactive and Mitogenic Factors

both vasoactive and mitogenic effects primarily mediated by the ETA receptor. Specific ETA receptor antagonism in rats has been shown to decrease the PH, right ventricular hypertrophy, arteriolar muscularization, and pulmonary vascular remodeling induced by chronic hypoxia [95–97]. Murine models of ETA receptor deficiency develop severe craniofacial and cardiovascular developmental defects [98] and therefore the specific effects of ETA receptor expression deficiency in the progression of hypoxic PH are unknown. Alternatively, rats deficient in the ETB receptor specifically in the pulmonary vasculature develop exaggerated PH, diminished cardiac output, a fivefold higher expression of plasma ET-1, and reduced NO and PGI2 metabolite production when exposed to 3 weeks of hypoxia [99]. Thus, ETB receptor activity plays a protective role in hypoxia-mediated PH. Endothelin receptor antagonists represent an important class of therapy for humans with PAH [100] (reviewed in [101]). Despite the advantages in vasomotor response observed with selective ETA receptor inhibition in animals [102], human clinical trials of PAH have not identified differences in efficacy between specific ETA receptor antagonists and dual ETA–ETB receptor antagonists (reviewed in [103, 104]).

5.3 Prostacyclin PGI2 is a potent vasodilator of the pulmonary vasculature that is synthesized by ECs and acts primarily through adenosine 3¢,5¢-cyclic monophosphate (cAMP) signaling pathways (reviewed in [105]). Severe PAH in humans is often treated with continuous intravenous formulations of PGI2, making it an important molecule in the clinical arena (reviewed in [101]). PGI2 is a member of the eicosanoid family and is derived from arachidonic acid via the cyclooxygenase (COX) system [105]. Arachidonic acid is a fatty acid present in cellular membranes that is released from the lipid bilayer by a cleavage reaction catalyzed by phospholipase A2. Both isoforms of COX, including constitutive COX-1 and inducible COX-2, can convert arachidonic acid to prostaglandin H2 (PGH2), which is a common precursor of multiple prostanoids. COX-1 is the primary producer of PGH2 in blood platelets, where PGH2 is further enzymatically modified to thromboxane A2, a potent stimulator of vasoconstriction and thrombosis. COX-2 is the primary catalyst for PGH2 formation in the endothelium, where it is ultimately converted to PGI2. The production of specific prostanoids including PGI2 and thromboxane from PGH2 occurs by way of tissue-specific synthases. PGI2 is specifically synthesized from PGH2 by the membrane-bound enzyme PGI2 synthase (PGIS). Other prostanoids include prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), and prostaglandin F2, all of which are involved in the regulation of pulmonary vasomotor tone (reviewed in [105]).

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The principal mechanism of PGI2 signaling is by means of  a paracrine second messenger system (reviewed in [106, 107]). The PGI2 receptor (PGI-R) is a member of the G-protein-coupled-receptor family and is expressed primarily in platelets and the SMCs of arteries. Upon ligand binding, PGI-R activates Gs, which subsequently binds and activates adenylate cyclase, thereby increasing production of cAMP. Intracellular cAMP in turn activates protein kinase A and affects calcium mobilization via phospholipase C and inositol pathways, ultimately resulting in increased SMC vasorelaxation and inhibition of SMC proliferation [28, 106, 107]. The prostanoid ligands and receptors are unique in their tissue distribution [105–107]. In both rat main pulmonary arteries and human ECs, PGI2 expression is approximately 27-fold higher than PGD2 expression and at least 20-fold higher than PGE2 expression under normoxic conditions [108, 109]. These ratios are similar under hypoxic conditions [108, 109]. PGI-R is the predominant prostanoid relaxant receptor expressed in the pulmonary vascular bed [107]. Hence, PGI2 is a dominant mediator of prostanoid signaling in the pulmonary vasculature. The effects of chronic hypoxia on pulmonary PGI2 signaling have been studied in animal models. Seven days of hypoxic exposure in rats results in an approximately threefold increase in PGI2 synthesis in the main pulmonary arteries, both in the endothelium and in the vascular smooth muscle [109]. Pulmonary PGIS expression also increases in rats exposed to prolonged hypoxia and is a consequence primarily of the hemodynamic consequences associated with hypoxia [110]. Support of this finding comes from the observation that treatment of rats with vasodilators attenuates the PGIS response despite ongoing hypoxic exposure [110]. An increase in COX, PGIS, and PGI2 metabolite production is observed in human vascular ECs [111] and rat pulmonary arteries [112] in response to increases in shear stress, further supporting the view that vasoconstriction is a potent stimulus of PGI2 synthesis. Additionally, although ET-1 signaling increases production of PGI2 [86, 87] via the ETB receptor, treatment of hypoxic rats with a specific ETA receptor antagonist reduces PGI2 synthesis, further implicating hemodynamic influences as the dominant factor increasing PGI2 production in models of hypoxic PH [110]. Prolonged hypoxia in mice with transgenic overexpression of pulmonary PGIS results in reduced PAPs and normal pulmonary arteriolar thickness compared with wild-type hypoxic mice [113]. Moreover, mice deficient in PGI-R exhibit a greater severity of vascular medial wall thickening after chronic exposure to hypobaric hypoxia compared to control hypoxic mice [114]. Thus, endogenous PGI2 signaling plays a protective role in chronic-hypoxia-mediated PH by attenuating both vasoconstriction and the development of pulmonary vascular remodeling. The increase in PGI2 levels detected in animal models of hypoxic PH, however, is not observed in human pulmonary disease. Patients suffering from PAH or PH secondary to COPD

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exhibit a reduction in urinary PGI2 metabolite release with a concomitant rise in thromboxane A2 metabolite levels [115], implicating the prostanoid pathway as a contributor to the development or maintenance of human PH. Furthermore, immunohistochemistry and western blot experiments have demonstrated a reduction in PGIS protein expression in lung tissues obtained from humans with severe PH of differing causes [116]. Thus, in humans, a reduction in PGI2 production is observed in PH and may contribute to overall disease pathogenesis. Although animal models of hypoxic PH exhibit an increase in PGI2 synthesis, the data from both humans and animals point toward a protective role for PGI2 in PH. The benefits of PGI2 in human disease are highlighted by the widespread utilization of PGI2 infusion therapy for advanced PAH, which for many patients has obviated the need for lung transplantation (reviewed in [101]).

5.4 Serotonin Individuals with PAH have elevated levels of circulating 5-HT that may contribute to the progression of vascular remodeling (reviewed in [117]). Although primarily stored in platelets, for the most part 5-HT is produced by enterochromaffin cells of the gastrointestinal tract and neuroendocrine cells and neuroepithelial bodies of the lung (reviewed in [118]). In pathologic states, including hypoxia, these sources secrete enhanced amounts of 5-HT (reviewed in [85]). The pulmonary endothelium can also act as a source of 5-HT secretion in diseased states [119, 120]. Once released, 5-HT is taken up by cells via a transporter (5-HTT) that is expressed on platelets and pulmonary VSMCs. Intracellular 5-HT has potent downstream effects on transcription that increase the overall proliferative state of the SMC [121]. 5-HT also functions through second messenger systems by direct interactions with G-proteincoupled cell-surface receptors, of which there are multiple isoforms. These receptors significantly reduce adenylate cyclase activity and cAMP levels in target tissues, including SMCs, and ultimately activate pulmonary artery vasoconstriction as well as mitogenic pathways (reviewed in [85, 118]). There has been a long-standing association between 5-HT and PAH, dubbed the “serotonin hypothesis of PAH” ever since the 1960s, when an association between anorexigen use and PAH was discovered [122, 123]. Anorexigens, such as fenfluramine, increase 5-HT availability by inducing its release from platelets and by inhibiting monoamine oxidase activity, thereby potentially slowing 5-HT metabolism (reviewed in [85, 118]). In addition to the PH associated with anorexigens, abnormal handling and elevated plasma 5-HT concentrations may contribute to the pathogenesis of other forms of PAH [124]. Pulmonary artery SMCs from patients with PAH exhibit faster growth kinetics, in part, owing to increased expression of 5-HTT, as evidenced in studies demonstrating the attenuat-

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ing effects of 5-HTT antagonists [125]. Growth medium from cultured pulmonary microvessel ECs isolated from patients with idiopathic PH potently stimulates proliferation of pulmonary artery SMCs compared with media from control individuals [119]. 5-HT accounts for 60% of this growth effect, identifying 5-HT as a dominant modulator of endotheliumderived SMC proliferation in PH [119]. Patients with PAH are approximately twofold more likely to be homozygous for an allelic variant of the 5-HTT gene promoter corresponding to a state of overexpression [125]. A familial platelet storage pool disease that results in abnormally elevated 5-HT uptake has also been associated with PAH [126]. Therefore, 5-HT is a potent vasoconstrictor and SMC mitogen that has been tightly linked to various forms of human PAH. Hypoxic conditions modify 5-HT uptake levels, 5-HTT activity, and expression of 5-HT receptors [127]. Rats exposed to 15 days of hypoxia have a significantly increased expression of pulmonary 5-HTT levels, corresponding to a threefold increase in 5-HT uptake in pulmonary artery SMCs [128]. Inhibition of 5-HT production or transport has a beneficial effect in hypoxic PH. Mice deficient in tryptophan hydroxylase 1, an essential enzyme in the synthetic pathway for 5-HT, exhibit significantly reduced PH, right ventricular hypertrophy, and distal vessel muscularization compared with wild-type mice in states of chronic hypoxia [129]. Deficiency of 5-HTT protects mice from developing hypoxiamediated PH and vascular remodeling [130], whereas ubiquitous overexpression of 5-HTT potentiates PH and vascular remodeling with hypoxia [131]. Targeted overexpression of 5-HTT in the smooth muscle alone using a transgenic approach in mice results in spontaneous PH, and compared with wildtype mice a severer PH phenotype occurs upon exposure to hypoxic conditions [132]. Important effects of 5-HT in PH not only occur via intracellular transport but also by way of signaling initiated at the cell surface (reviewed in [85]). The mitogenic effects of 5-HT on SMCs are mediated in part by mitogen-activated protein kinase (MAPK) upregulation via the 5-HT2B receptor [133]. Chronic hypoxia in mice results in a twofold increase in 5-HT2B receptor expression in the lung vasculature [134]. Furthermore, when mice homozygous for loss-of-function mutations in 5-HT2B−/− are exposed to chronic hypoxia, no increase in right ventricular systolic pressure, right ventricular hypertrophy, or distal vessel muscularization is observed [134]. Thus, the deve­ lopment of hypoxia-mediated PH as well as other forms of PH in humans is promoted by 5-HTT activity and 5-HT receptor activity. Although no therapies directed at inhibiting 5-HT transport or signaling are currently utilized in the management of human PH, small-molecule inhibition of 5-HTT with selective 5-HT reuptake inhibitors such as citalopram and fluoxetine in animal models is protective in hypoxic PH [135] and warrants further investigation in humans (reviewed in [101]).

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6 Endothelium-Derived Mitogenic Signals from FGF, PDGF, and VEGF in Chronic-Hypoxia-Mediated PH Excessive VSMC proliferation plays a significant role in the pathogenesis of hypoxia-mediated PH [1, 2]. Although the exact mechanisms are incompletely understood, hypoxia is thought to cause metabolic disturbances or hemodynamic injury to the vessel wall, leading to an activation of multiple mitogenic pathways [46]. The mitogenic effects of hypoxia are exerted on ECs, VSMCs, and adventitial fibroblasts [1], but here we focus on the effects of hypoxic ECs on SMC hyperplasia. Under normal conditions, the SMCs of the vascular wall have abundant myofilaments, reflecting a contractile phenotype with little endoplasmic reticulum or Golgi apparatus and a minimal response to mitogens [136]. However, under certain stimuli, including chronic hypoxia, these SMCs can dedifferentiate into a proliferative and synthetic phenotype with an active response to mitogens and enhanced production of ECM proteins (reviewed in [1]). In addition to the alterations in resident medial SMCs, other cells, including adventitial fibroblasts or circulating progenitor cells, can be recruited to the media and adopt a SMC-like phenotype [1, 137]. The endothelium plays an important role in regulating the proliferation of these resident and recruited SMCs. In vitro studies have demonstrated that human ECs exposed to hypoxia produce and secrete soluble growth factors, including basic FGF (FGF2), PDGF, and VEGF, as well as ET-1 (discussed earlier), which induce SMC proliferation [13, 46, 136, 138]. The addition of medium derived from cultured hypoxic bovine pulmonary ECs to cultured pulmonary SMCs results in a 60% increase in SMC number after 24 h [139]. Hence, the endothelium exerts a significant proliferative effect on neighboring SMCs via the secretion of paracrine growth factors. Stored in the ECM, FGF2 is a known potent proangiogenic stimulator of endothelial and SMC proliferation, vascular cell migration, and synthesis of ECM proteins (reviewed in [140]). Overall there are 18 different mammalian FGFs that bind to one or more of seven different FGF receptors (FGFRs), encoded by four different FGFR genes with alternative splice variants (reviewed in [141]). The FGFRs are transmembrane receptor tyrosine kinases that signal via phospholipase Cg1 and FGFR substrate 2, with downstream activation of the MAPK and phosphoinositide 3-kinase (PI3-K) pathways. The FGFs exert broad effects on cellular signaling and developmental processes, with FGF2 playing the primary role in the vasculature [141]. Although Fgf2-deficient mice are morphologically normal, they exhibit systemic hypotension, suggesting an important role for FGF2 in the regulation of vascular tone [142]. Alveolar hypoxia increases the expression of FGF2 by cultured human ECs, the secretion of which induces proliferation

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of neighboring SMCs [136]. FGF2 also stimulates the expression of the ETA receptor on pulmonary artery SMCs, thus potentiating the effects of ET-1 in hypoxic PH [143]. The same study by Li et al. demonstrated that hypoxia alone does not stimulate ETA receptor expression in cultured pulmonary artery SMCs, suggesting that hypoxia stimulates ETA receptor expression indirectly through induction of growth factors from the endothelium. The role of FGF in hypoxia-mediated PH in humans is incompletely understood. In a study involving 117 patients with PAH, median plasma and urine levels of FGF2 were increased at least twofold when compared with those of control individuals, and higher FGF2 levels were associated with a reduced clinical functional capacity [144]. Hence, FGF2 secreted by ECs is an important mitogen for pulmonary VSMCs under hypoxic conditions, and FGF2 levels correlate with clinical severity in humans with PAH. PDGF polypeptides belong to a family of structurally and functionally related growth factors that also includes VEGF (reviewed in [145]). PDGFs are soluble dimers formed from two out of four possible peptides (A, B, C, or D) that bind and stimulate dimerization of its transmembrane receptor, PDGFR. The receptor is a tyrosine kinase that autophosphorylates upon ligand-induced dimerization and activates several signaling pathways, including PI3-K, MAPK, and phospholipase C, thereby regulating numerous cellular and developmental processes [145]. PDGF-B expression occurs primarily in the vascular endothelium and is a potent stimulator of SMC growth (reviewed in [46]). Exposure of adult rats to alveolar hypoxia results in at least a twofold increase in PDGF-B expression in the lung parenchyma [146, 147]. A concomitant rise in PDGFR expression is observed in porcine pulmonary VSMCs cultured under hypoxic conditions [148], as well as in progenitor VSMCs detected in rat models of PH [149]. Treatment of chronically hypoxic mice with the PDGFR antagonist imatinib improves survival and reverses PH and right ventricular hypertrophy 4 weeks after induction of the disease process [150]. Although imatinib is a nonspecific inhibitor of PDGFR, its other molecular targets are not known to be involved in the progression of hypoxia-mediated SMC hyperplasia and PH [150]. Thus, inhibition of PDGF signaling is a potentially attractive target in the treatment of hypoxic PH. VEGF functions as a potent mitogen and survival factor for ECs, as well as an inducer of EC migration and angiogenesis, both in the physiologic state and in disease states such as diabetic retinopathy and wound healing (reviewed by [151]). There are a total of five VEGF polypeptide chains: VEGF-A, VEGF-B, VEGF-C, VEGF-D, and placental growth factor. VEGFs, like PDGFs, consist of dimers with disulfide-linked polypeptides. VEGF-A is the prototypical member of the VEGF family, and its role in the pulmonary vasculature has been well delineated and is the focus of this discussion. VEGF-A interacts with two different tyrosine kinase receptors (reviewed in [152]), VEGFR-1 (also known

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as fms-like tyrosine kinase, flt1) and VEGFR-2 (KDR in humans, Flk-1 in mice). VEGFR-1 plays an important role during development but then exhibits weak tyrosine autophosphorylation in response to VEGF-A in adult animals, whereas VEGFR-2 is the primary receptor that mediates endothelial mitogenesis and survival in adults [153, 154]. Upon binding of a ligand, VEGFR-2 undergoes autophosphorylation and induces the phosphorylation of phospholipase Cg, PI3-K, and GTPase-activating protein among others, stimulating multiple endothelial cellular processes involving growth and survival (reviewed in [151]). VEGFR-2 knockout mice are embryonic lethal and completely lack vasculogenesis, underscoring the pivotal role of this receptor in vascular biologic processes [155]. VEGF-A is synthesized by many cell types of the lung parenchyma, including ECs, particularly in diseased states; however, other major sources of VEGF-A are airway epithelial cells and SMCs (reviewed in [1, 152, 156]). In addition to being a potent EC mitogen, VEGF-A stimulates endothelial production of NO and PGI2 and upregulates expression of eNOS, thereby indirectly regulating vascular tone [157]. Hypoxia is a strong inducer of VEGF-A expression [1]. Chronic hypoxia increases levels of mRNA encoding VEGF-A and VEGFR-2 in the rat lung, with the highest levels achieved after 3 weeks [158]. Hypoxic rats pretreated with intratracheal administration of adenovirus encoding human VEGF-A exhibit reduced PAPs, right ventricular hypertrophy, and distal vessel muscularization compared with control rats under the same hypoxic conditions [159], thus highlighting the protective role of VEGF-A in hypoxic PH. The administration of VEGF-A is associated with an increase in eNOS activity, thus attenuating the vasoconstriction associated with chronic hypoxia [159]. The combination of chronic alveolar hypoxia and administration of the small-molecule VEGFR tyrosine kinase inhibitor SU-5416 in rats results in a severe, irreversible PH with marked obliteration of the precapillary arterioles by proliferating ECs after 3 weeks of exposure [41]. After the first and second weeks of exposure to hypoxia and SU-5416, the pulmonary arteries of rats are characterized by widespread EC apoptosis, implicating the death of normal ECs and the proliferation of an apoptosis-resistant EC population as a possible cause for the severe PH [41]. When human pulmonary microvascular ECs are cultured with SU-5416, widespread apoptosis ensues, with expansion of CD34+ precursor cells that transdifferentiate into a SMC-like phenotype, thus highlighting the importance of VEGFR in maintaining proper EC survival and a normal endothelial– mesenchymal balance [160]. These striking data identify angioproliferation as a potentially important target in the treatment of PH. In humans with COPD, sputum levels of VEGF correlate with increases in PAP and PVR with exercise, underscoring a potential role for VEGF as a nonin-

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vasive marker of pulmonary vascular remodeling [161]. The prognostic value of VEGF in other forms of human PH remains to be investigated. Hypoxia is thus an important pathophysiologic stimulus for the expression of multiple mitogenic factors by the pulmonary endothelium. The exact mechanisms by which these factors are upregulated in hypoxia are not clearly understood. The induction of SMC proliferation is, however, not entirely due to an increase in expression levels of growth factors. There is evidence supporting SMCs becoming sensitized to endothelium-derived growth factors during states of hypoxia and exhibiting potentiated proliferative responses [162, 163]. The stimulation of SMC growth is, at least in part, mediated by hypoxia-induced expression of SMC transcription factors such as HIF-1a [164, 165]. Knockdown of HIF-1a expression in pulmonary artery SMCs inhibits FGF2- and PDGFinduced SMC proliferation under hypoxic conditions [163]. In addition, heterozygote deficiency of HIF-1a in mice delays the progression of PH, pulmonary vascular remodeling, and right ventricular hypertrophy that occurs with exposure to chronic hypoxia [166]. The mechanism by which HIF-1a potentiates SMC proliferation requires further elucidation. Hence, second messenger signals from endotheliumderived growth factors synergize with hypoxia-induced intracellular signals within SMCs to mediate the vascular smooth muscle remodeling seen in hypoxic PH.

7 BMPs in Hypoxia-Mediated PH BMPs are members of the TGF-b superfamily (reviewed in [167]) and are increasingly becoming recognized as potent regulators of vascular remodeling in PH. The TGF-b superfamily consists of more than 40 members, including TGF-bs, activins, BMPs, and growth and differentiation factors. TGF-b family members signal via cell-surface serine–threonine kinase type I and type II receptors, both of which are required for signaling. Upon binding a ligand, the type II receptors phosphorylate the type I receptors, which in turn activate downstream transcription factors known as Smads. These regulatory Smads are then transported into the nucleus and activate transcription of specific target genes with the help of Smad 4. BMPs signal primarily via BMPR-II, which can form complexes with four type I receptors: ALK1, ALK2, ALK3/BMPR-IA, and ALK6/BMPR-IB. Type I BMP receptors phosphorylate and activate Smads 1, 5, and 8. Smads 1, 5, and 8 are transcription factors activated by BMPs that induce expression of Id (inhibitors of DNA binding) proteins, which generally act as inhibitors of differentiation but otherwise have a broad range of cellular effects. Initially identified as stimulators of ectopic bone formation, it is now known that BMPs are critical regulators of development and cellular growth [167, 168].

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Just within the past 10 years, loss-of-function mutations in BMPR-II have been identified in both familial and approximately 25% of sporadic forms of idiopathic PAH, implicating the BMP signaling pathway as an important contributor to the pathophysiologic changes in PH [169–171]. BMPs play a critical role in lung vasculogenesis and vascular homeostasis [172]. BMPR-II under normal conditions is abundantly expressed in vascular ECs, SMCs, and adventitial fibroblasts of the lung [173] and expression is significantly reduced in various forms of PH [174]. Reductions in the BMP type I receptor ALK3 have also been observed in patients with PAH and other forms of PH [175]. The direct link between altered BMP signaling and PH has been confirmed in animal models. Whereas homozygous knockouts of BMPR-II−/− in mice result in embryonic lethality [176, 177], heterozygous BMPR-II mutant mice survive and appear to exhibit modest increases in PAP and PVR [178]. When BMPR-II is deleted specifically in the pulmonary endothelium, a subset of mice develop PH, implicating abnormal EC function as the origin for PAH [179]. An important role for BMPs in pulmonary vascular remodeling has been established. BMP signaling inhibits vascular smooth muscle proliferation in rat and murine models [180, 181], as well as the in vitro proliferation of normal human pulmonary artery SMCs [182]. However, in patients with PAH, BMPs fail to suppress SMC proliferation, reflecting altered growth signaling in the diseased state [182]. Functional BMPR-II is required for BMP-mediated growth suppression of murine pulmonary artery SMCs, highlighting the lack of functional redundancy between BMPR-II and other BMP type II receptors [181]. In contrast to their effect on SMCs, BMPs appear to promote survival of pulmonary artery ECs, and aberrant BMP signaling is associated with increased endothelial apoptosis and the loss of distal pulmonary vessels [183, 184]. The abnormal growth responses of the pulmonary vasculature to BMPs have been implicated in the vascular obliteration and pathogenesis of PH both in animal models and in human disease. Chronic hypoxia is an important regulator of BMP signaling. When rats are exposed to a hypobaric hypoxic chamber for 2 weeks, BMPR-II expression is reduced in pulmonary ECs and VSMCs and is associated with a decrease in Smad 1, 5, and 8 phosphorylation and Id-1 gene expression [173, 185]. Interestingly, hypoxia results in an increase in BMP-2 expression and EC apoptosis in rats, and use of the endogenous BMP inhibitor Noggin inhibits this apoptosis [186]. These results reflect a complex system of BMP signaling in which hypoxia-mediated downregulation of BMPR-II results in excessive BMP-2 signaling via other BMP receptors, ultimately leading to endothelial apoptosis. The exact mechanisms by which hypoxia alters BMP signaling are not understood, although the transcriptional corepressor C-terminal-binding protein 1 has been implicated in the reduction of Id-1 gene expression in hypoxic human pulmonary artery SMCs [187]. The effects of hypoxia on BMP

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expression and signaling are similar to those observed in other models of PH [188], making hypoxia-mediated PH a useful tool in the broader investigation of BMPs and human PH.

8 Endothelial Progenitor Cells Hypoxia-mediated PH is characterized by hyperplasia of all three layers of the vessel wall [1]. In addition to a heightened proliferation of resident cells in the vasculature, the increased cell number is due to recruitment from exogenous sources [189, 190]. Circulating EPCs are mobilized from the bone marrow in response to vascular injury or ischemia in adults (reviewed in [191]). During development, outer EPCs (also known as angioblasts in the developing embryo) and inner hematopoietic stem cells constitute the embryonic blood islands that ultimately create a tubular network of blood vessels with inner circulating blood cells [191, 192]. In normal adult individuals, a low basal level of vascular EC turnover occurs at all times [193]. However, acute vascular injury or stress results in a rapid accumulation of circulating EPCs [191]. EPCs that are administered to ischemic adult mice incorporate into regions of neovascularization, pointing toward the critical process of postnatal vasculogenesis [189, 192, 194]. EPCs are recruited to the pulmonary vasculature in PH owing to a variety of causes, including chronic hypoxia [195]. Although there is no consensus for the exact cellular markers that best identify EPCs, CD34, VEGFR-2, and c-kit have been utilized [191, 194]. CD34 is a cell-surface glycoprotein that functions as a cellular adhesion factor for stem cells, whereas c-kit is a transmembrane tyrosine kinase receptor that binds to stem cell factor [191]. Chronic hypoxia more than doubles the number of c-kit+ precursor cells in the peripheral blood and increases the incorporation of these cells into the adventitia of pulmonary arteries obtained from calves with hypoxia-induced PH [190]. These precursor cells differentiate into both endothelial and SMC phenotypes when cultured in  vitro, thus providing evidence for their role in hypoxic pulmonary vascular remodeling [190]. Although EPC recruitment to the pulmonary vasculature is enhanced in hypoxia-mediated PH, an unresolved question is whether EPCs are beneficial or harmful to disease progression. To address this question, Satoh et  al. [196] investigated hypoxic PH in mice deficient in the erythropoietin receptor in nonerythroid cell lineages (EpoR−/−). Epo is a hypoxia-induced hormone that, among other essential functions, stimulates mobilization of bone-marrowderived EPCs during ischemic conditions or with vascular injury (reviewed in [197]). EpoR−/− mice with hypoxiainduced PH have increased mortality and accelerated PH and pulmonary vascular remodeling compared with control mice exposed to the same degree and duration of hypoxia

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[196]. The mobilization and recruitment of EPCs to the pulmonary vessel wall is impaired in chronically hypoxic EpoR−/− mice and the severity of PH is ameliorated by reconstitution of wild-type bone marrow in the EpoR−/− mice [196]. These data suggest that EpoR expression plays a protective role in hypoxia-induced PH through mobilization and recruitment of EPCs to the ­pulmonary vasculature. The benefit of recruited EPCs in PH may be related to the secretion of paracrine factors that stimulate the proliferation of mature, native ECs [198]. However, the therapeutic benefit of EPCs recruited under hypoxic conditions remains to be proven. Marsboom et al. [195] demonstrated that although prolonged hypoxia in mice results in large numbers of EPCs mobilized to the peripheral blood, these EPCs exhibit reduced homing and adhesive capacity, decreased integrin expression, enhanced senescence, increased mitochondrial dysfunction, reduced angiogenic potential, and attenuated NO release compared with EPCs recruited under normoxic conditions. The evidence suggests that a prolonged hypoxic milieu significantly impairs the reparative capacity and function of EPCs [195]. Further investigation is needed to assess the essential determinants of EPC function and recruitment in PH.

R. Malhotra and K.D. Bloch

Fig. 2  The mechanisms of chronic hypoxia-mediated pulmonary vasoconstriction and vascular remodeling. Items within { } denote signaling that inhibits the depicted process. ET-1, endothelin-1; 5-HT, serotonin; NO, nitric oxide; PGI2, prostacyclin; FGF, fibroblast growth factor; PDGF, platelet-derived growth factor; BMPs, bone morphogenetic proteins; VEGF, vascular endothelial growth factor; BMPR-II, BMP Type II Receptor; Epo, erythropoietin; EpoR, Epo Receptor; EC, endothelial cell; EPC, endothelial progenitor cell

insights of the key molecular mechanisms of PH expand, so too will our ability to guide therapeutic decision-making.

References 9 Summary Hypoxia is a potent stimulator of pulmonary vasoconstriction in humans that, depending on the degree and duration of exposure, ultimately results in pathologic remodeling of the vasculature with endothelial hyperplasia and SMC proliferation (Fig. 2). Individuals with sleep apnea or those who reside at high altitudes can develop PH from the direct effects of chronic alveolar hypoxia. Those individuals with underlying pulmonary disease such as COPD suffer from hypoxemia, which is a contributor to the overall progression of PH. The endothelium is an important biologic sensor of oxygen tension and is induced by hypoxia to produce potent vasoactive molecules and mitogens that exert paracrine effects on the smooth muscle. This induction is primarily regulated at the transcriptional level by direct oxygen sensors such as HIF-1. Clinically, PH is a disease of vasoconstriction and vessel wall thickening that begins with symptoms of exertional dyspnea and can progress to end-stage right ventricular failure. Current therapies for symptomatic PH target endothelium-derived pathways with potent vasoactive properties such as NO, ET-1, and PGI2 signaling. A growing understanding of the underlying mechanisms of hypoxic PH points to new vasoactive mediators and mitogens as potential therapeutic targets. EC-based therapies represent an exciting and promising new avenue of treatment and highlight the essential role of abnormal endothelial function in the development of hypoxia-mediated PH. As our

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Chapter 49

Oxygen-Sensitive Transcription Factors and Hypoxia-Mediated Pulmonary Hypertension Louise Østergaard, Vinzenz H. Schmid, and Max Gassmann

Abstract  Dynamic changes in oxygen partial pressure (pO2) constitute a potential signaling mechanism for the regulation of expression and activation of oxygen-responsive transcription factors. Hypoxia might affect tissues or even the whole body: defective oxygen uptake in the lung, impaired oxygen transport capacity in the blood, or lower pO2 in the environment, as experienced at high altitude. The reaction to this potentially life-threatening situation requires hypoxia-dependent gene regulation that induces a variety of specific adaptation mechanisms that ensure survival at cellular and systemic levels alike. Once exposed to hypoxia, the pulmonary and systemic vasculature, and potentially also the airway chemoreceptors, enables a corresponding response within seconds; changes in gene expression on the other hand require minutes to hours. To mediate these adaptive effects a range of oxygen-sensitive transcription factors play important roles. An adaptive response of the pulmonary circulation to low oxygen tension is the increase in pulmonary pressure. This is a self-regulatory mechanism for maintaining the optimal balance between ventilation and perfusion. During acute hypoxia, the vasoconstriction acts to reduce the perfusion of underventilated parts of the lungs and instead divert blood to well-ventilated regions, meaning that the acute response is mainly associated with a change in vascular tone. Chronic hypoxia and the resulting endothelial dysfunction, on the other hand, are associated with sustained pulmonary hypertension. This is a serious condition characterized by elevation in pulmonary arterial pressure. A constant elevation in pulmonary arterial pressure will lead to right ventricular hypertrophy and ultimately right ventricular failure and possibly death. Keywords  Hypoxia • Transcription factor • Hypoxia-inducible factor • Gene expression • Hypoxia-mediated pulmonary hypertension M. Gassmann (*) Institute of Veterinary Physiology, Vetsuisse-Faculty and Zurich Center for Integrative Human Physiology (ZIHP), University of Zurich, Winterthrurerstrasse 260, 8057 Zurich, Switzerland e-mail: [email protected]

1 Introduction Oxygen is essential for the survival of aerobic organisms; from prokaryotes to mammals, all have adapted to maintain a supply-and-demand homeostasis. However, the level of oxygen, low or high, can be a source of stress depending on the biological context. Dynamic changes in oxygen partial pressure (pO2) constitute a potential signaling mechanism for the regulation of expression and activation of oxygen-responsive transcription factors. Hypoxia (e.g., reduced oxygenation) might affect tissues or even the whole body: defective oxygen uptake in the lung, impaired oxygen transport capacity in the blood, or lower pO2 in the environment, as experienced at high altitude. The reaction to this potentially life-threatening situation requires hypoxia-dependent gene regulation that induces a variety of specific adaptation mechanisms that ensure survival at cellular and systemic levels alike. Once exposed to hypoxia, the pulmonary and systemic vasculature, and potentially also the airway chemoreceptors, enables a corresponding response within seconds; changes in gene expression on the other hand require minutes to hours. To mediate these adaptive effects a range of oxygen-sensitive transcription factors play important roles. The main transcription factor mediating these molecular responses is hypoxia-inducible factor (HIF)-1, but other transcription factors such as nuclear factor kappa B (NF-kB), peroxisome proliferator-activated receptors (PPARs), and early growth response-1 (Egr-1) are also involved. The common perception is that a putative oxygen sensor that responds to changes in pO2, will relay signals for the regulation of gene expression through activation of specific transcription factors. An adaptive response of the pulmonary circulation to low oxygen tension is the increase in pulmonary pressure. This is a self-regulatory mechanism for maintaining the optimal balance between ventilation and perfusion. During acute hypoxia, the vasoconstriction acts to reduce the perfusion of underventilated parts of the lungs and instead divert blood to wellventilated regions, meaning that the acute response is mainly associated with a change in vascular tone. Chronic hypoxia and the resulting endothelial dysfunction, on the other hand,

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are associated with sustained pulmonary hypertension. This is a serious condition characterized by elevation in mean pulmonary arterial pressure (mPAP). A constant elevation in mPAP will lead to right ventricular hypertrophy and ultimately right ventricular failure and possibly death.

L. Østergaard et al.

tive variations. For the transcription factor to be capable of promoting transcription, it must translocate from the cytoplasm to the nucleus. This can be archived by a signaling event, such as a change of the state of phosphorylation. Therefore, the gene regulation as a result of oxygen perturbations involves complex interactions of multiple cellular pathways.

2 Pulmonary Hypertension The pulmonary circulation is a low-pressure, high-flow system in humans. The normal mPAP is below 25  mmHg, and the resistance is approximately one tenth of that in the systemic circulation (average 67 ± 30 dyn s cm-5) [1]. The system is capable of adjusting to large increases in blood flow, e.g., during exercise, with only a minor rise in pressure. But if a constant elevation in mPAP is present, this will lead to right ventricular hypertrophy and ultimately to right ventricular failure. Pulmonary hypertension is defined as a mPAP above 25 mmHg at rest or 30 mmHg during exercise, the normal mPAP being between 12 and 16 mmHg at rest. A typical feature is luminal vascular obstruction caused by cell proliferation of endothelial cells and smooth muscle cells or by thrombosis and also to a varying degree vasoconstriction. This can reflect a homeostatic imbalance manifested by a disproportion in secreted vasodilators and constrictors and mediators that affect growth and thrombosis. A possible cause of this imbalance could be endothelial dysfunction. The role of the endothelium in hypoxia-induced pulmonary vasoconstriction is controversial, but it seems that the sustained constriction is, at least partly, dependent on an intact endothelium [2]. The effects of hypoxia on the endothelium may include a change in the secretion of, for instance, vasodilators and constrictors such as nitric oxide, prostacyclin, endothelin-1, angiotensin II, and serotonin [3]. Generally, constrictors and/or their receptors are upregulated, whereas the release of dilators is decreased, but there is evidence that differences in the response exist, depending on whether the hypoxia is acute or chronic. Several other endothelial factors involved in cellular functions such as migration, growth, and survival are important too.

3 Oxygen-Sensitive Transcription Factors The level of oxygen can regulate the cellular signaling response, which is governed at the genetic level by transcription factors that bind to control regions of target genes and alter their expression. Transcription factors can modulate the initiation, stimulation, or termination of the transcriptional process. Changes in the composition of gene expression through regulatory transcription factors are fundamental components in determining the cellular response to oxida-

4 Hypoxia-Inducible Factors In the past, expression of erythropoietin (EPO) was used as a prototype system to study hypoxia-responsive genes and transcription factors regulated by oxygen levels [4]. Soon the existence of a hypoxia-inducible enhancer 3¢ to the human EPO gene was discovered (5¢-RCGTG-3¢), containing a hypoxia-responsive element (HRE), that was required for hypoxia-mediated transcription [5]. This observation subsequently led to the isolation and cloning of a factor binding to the HRE upon hypoxia, namely, HIF-1 [6–8]. HIF-1 is a ubiquitously expressed heterodimeric basic helix–loop– helix/PER/aryl hydrocarbon receptor nuclear translocator (ARNT)/SIM (PAS) domain transcription factor composed of an oxygen-labile a subunit and a constitutively expressed b subunit, the latter being shared with other transcription factors (for reviews, see [9, 10]). The b subunit was earlier identified as the heterodimerization partner of the dioxin receptor/ aryl hydrocarbon receptor and was hence called aryl hydrocarbon receptor nuclear translocator (ARNT; see [11]). Later studies revealed, however, that the heterodimerization of the a subunit with ARNT is required for DNA binding and transactivation of target genes [12–14], but not for translocation into the nucleus [15, 16]. The oxygen-labile HIF a subunit provides the ability to rapidly switch on and off the activity of the transcription factor according to changes in pO2. This very efficient mode of control is illustrated by a half-life of HIF-1a of under 5 min upon normoxia [17], and accumulation of HIF-1a in the nucleus of HeLa cells already after 2 min of hypoxia [18].

4.1 HIF Isoforms HIF comprises a family of three transcription factors: HIF-1, HIF-2, and HIF-3, all consisting of an a subunit and a b subunit. HIF-1a and HIF-2a share 48% overall amino acid identity [19]. HIF-1a is the most widely distributed and is believed to be the most important concerning adaptations and/or pathophysiological processes, but specialized roles for HIF-2a have also been suggested [20]. Whereas HIF-1a is ubiquitously distributed, HIF-2a expression is mainly found in vascular endothelium, kidney, and heart [19–21].

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Furthermore, the two isoforms regulate both shared and unique target genes, indicating that they are not functionally redundant. HIF-3a is expressed in a number of tissues [22, 23], and, remarkably, also gives rise to a HIF antagonist. This oxygendependent-degradation-domain (ODD)-lacking (see later), thus stable, splice variant of HIF-3a, termed “inhibitory PAS protein,” functions as a hypoxia-induced dominant negative repressor of HIF targets, by, for example, capping the angiogenic stimulus exerted via HIF-1 in tissues where too much vascularization would only lead to functional impairment (e.g., human cornea) [22].

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HREs and recruits transcriptional coactivators such as p300/ cyclic AMP response element binding protein (CREB)binding protein (CBP) for the transactivation of genes. Several genes have been found to carry regulatory sequences similar to the EPO 3¢ HRE, and now more than 70 validated HIF target genes such as glycolytic enzymes, proteins involved in angiogenesis or cell proliferation, transcriptional regulation and others have been identified (for reviews, see [33–35]). Recent studies even suggest that HIF-1 regulates the expression of hundreds of genes in human cells [36, 37]. Notable is the fact that HIF-1 and HIF-2 rarely upregulate just one target in a pathway, but rather coordinate the response of all necessary steps to hypoxia. Table 1 shows a selection of validated HIF-1a targets and their function.

4.2 Control of HIF Activity: ODD The ODD is responsible for normoxic HIF-1a ubiquitination and subsequent degradation via the proteasome pathway [24, 25]. This is initiated by hydroxylation of two proline residues (Pro-402 and Pro-564 in human HIF-1a) in the ODD [26, 27]. The HIF-prolyl hydroxylases responsible have been named PHD (prolyl hydroxylase domain containing) 1, 2, and 3, respectively [27, 28]. All require oxygen and 2-oxoglutarate as cosubstrates and contain iron in coordination with two histidines and one aspartic acid residue. To retain iron in its enzymatically active ferrous state, ascorbate is also needed. These findings explain how hypoxia-mimicking agents such as iron chelators (e.g., desferrioxamine) or transition metals such as cobalt can either deplete (desferrioxamine) or substitute (cobalt) the catalytic iron of PHDs, resulting in the suppressed hydroxylation of the ODD prolyl residues and, ultimately, the stabilization of the HIF a subunit under normoxic conditions [4]. Once the ODD prolyl residues have been hydroxylated, the degradation pathway is triggered by the immediate binding of the von Hippel–Lindau tumor suppressor protein (pVHL) to the a subunit [26, 27]. pVHL is the recognition component of an E3 ubiquitin-protein ligase, and its binding, in turn, recruits the other proteins involved; elongin C, elongin B, cullin 2, and RBX-1 [29]. The complete ligase complex can then interact with E1 ubiquitin-activating and E2 ubiquitin-conjugating enzymes, thus mediating ubiquitin tagging of the target protein (in this case HIF-a) and initiating its subsequent degradation in the 26S proteasome [30–32] (Fig. 1).

4.3  HIF Target Genes Upon stabilization, the HIF-1a/HIF-2a protein heterodimerizes with HIF-b/ARNT, forming the now active transcription factor HIF-1/HIF-2. This factor then binds target DNA at the

4.4  HIF and Pulmonary Hypertension As mentioned already, HIFs are the main regulators of oxygensensitive transcription in the development of pulmonary hypertension. The involvement of HIF was first described by the finding that HIF-1a+/− mice in contrast to wild-type mice did not develop polycythemia, right ventricular hypertrophy, pulmonary hypertension, pulmonary vascular remodeling, and weight loss when exposed to hypoxia [38]. Furthermore, HIF-2a heterozygous mice exposed to chronic hypoxia do not develop pulmonary hypertension and right ventricular hypertrophy at all [39], indicating a very important role for HIF-2 in the pulmonary response to hypoxia. EPO contributes to the development of pulmonary hypertension by increasing the production of red blood cells during hypoxia [40, 41] and possibly by stimulating the release of endothelin-1 by endothelial cells [42]. Another prominent target of HIF, vascular endothelial growth factor (VEGF), is implicated in vascular remodeling [43], a well-known characteristic of pulmonary hypertension. Other HIF-regulated factors implicated in hypoxia-induced pulmonary remodeling are proliferative and vasoactive factors such as endothelin-1, platelet-derived growth factor, and angiotensin-converting enzyme [44]. In addition, the serotonin transporter is regulated by HIF and is linked to augmented smooth muscle cell proliferation [45]. The increase in RhoA expression and concomitantly downregulation of Rho kinase, at least in fibroblasts, can be mediated by HIF, but a clear role in pulmonary hypertension has not yet been clarified [46]. The vasodilator nitric oxide is produced by endothelial nitric oxide synthase and inducible nitric oxide synthase, which are reported to be induced by HIF [47, 48]. This vasodilator is very important for regulating pulmonary vascular tone and is therefore also implicated in pulmonary hypertension [49]. Furthermore, nitric oxide can also reduce vascular

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Fig.  1  Hypoxia-inducible factor (HIF) pathway. In the presence of oxygen, prolyl hydroxylase domains (PHDs) catalyze the posttranslational modification of HIF-a, causing the immediate binding of the von Hippel–Lindau tumor-suppressor protein (pVHL). pVHL binding, in turn, recruits binding of elongin B, elongin C, cullin 2 [29], and RBX-1. Upon hydroxylation of Pro-402 and Pro-564 within the oxygen-dependent-degradation domain of HIF-1a, the pVHL/elongin complex, in concert with ubiquitin-activating (E1) and ubiquitin-conjugating (E2)

enzymes, mediates the ubiquitination of HIF-1a, leading to subsequent degradation in the proteasome. Under hypoxia, the O2-requiring prolyl hydroxylases cannot modify HIF-a, and the protein remains stable. Stabilized HIF-a translocates into the nucleus, where it dimerizes with HIF-1b and cofactors such as cyclic AMP response element binding protein binding protein/p300 and the DNA polymerase II complex to bind to hypoxia-responsive element and activate gene transcription. (Modified from Fandrey et al. [112])

remodeling [50], thereby further influencing the chronic change in pulmonary tone. Other downstream effectors of HIF can involve, for instance, modulation of ion channels such as the downregulation of KV+ channels and subsequent membrane depolarization in pulmonary artery smooth muscle cells [51]. This mechanism has been postulated to lead not only to enhanced vasoconstriction, but also to conferral of resistance to cell death via hyperpolarization of mitochondria and enhanced survivin expression [52]. Cell growth, and in addition increased cellular pH, can also be influenced by the Na+/H+

exchanger, the expression and activity of which are increased by HIF upon chronic hypoxia [53]. The expression of canonical transient receptor potential channels 1 and 6 was also observed to be augmented by HIF [54]. These channels act to increase intracellular calcium concentrations in pulmonary artery smooth muscle cells, which contribute to vascular remodeling and reactivity. As described, HIF is involved in numerous pathways that are essential for the progression of pulmonary hypertension, and is therefore fundamental for the advancement of the disease.

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Table 1  Hypoxia-inducible factor (HIF)-1a targets Gene Product Function

Gene product

Function

a1B-Adrenergic receptor

Vascular tone

IGF-2

Adenylate cyclase Adrenomedullin Aldolase-A Aldolase-C Aminopeptidase-A Angiopoietin-2 Angiopoietin-4

Nucleotide metabolism Vascular tone, cell survival Glucose metabolism Glucose metabolism Metabolism Angiogenesis Angiogenesis

Keratin-14 Keratin-18 Keratin-19 Lactate dehydrogenase A LDL-receptor-related protein-1 Leptin Matrix metalloproteinase-2

Autocrine motility factor Carbonic anhydrase-9 Carbonic anhydrase-12 Cathepsin D

Motility pH regulation pH regulation Extracellular-matrix metabolism Iron metabolism Extracellular-matrix metabolism Cell proliferation Motility

MIC2/CD99 NIP3 Nitric oxide synthase-2 NIX

Cell proliferation and survival Cytoskeletal structure Cytoskeletal structure Cytoskeletal structure Glucose metabolism Motility Energy metabolism Extracellular-matrix metabolism Cell adhesion Apoptosis Vascular tone, cell survival Apoptosis

p21 p35srj

Cell proliferation Regulation of HIF-1 activity

Phosphofructokinase L 6-Phosphofructokinase-2 kinase/ fructose 2,6-bisphosphatase Phosphoglycerate kinase-1 Placental growth factor Plasminogen activator inhibitor-1 Prolyl-4-hydoxylase a (I) Pyruvate kinase M RTP801 Stanniocalcin-1

Glucose metabolism Glucose metabolism

Erythropoiesis, cell survival Extracellular-matrix metabolism Glucose metabolism Glucose metabolism

Transferrin Transferrin receptor

Iron metabolism Iron metabolism

Transforming growth factor-a Transforming growth factor-b3

Glucose metabolism

Transglutaminase-2

Cell proliferation Angiogenesis, cell proliferation Amino-acid metabolism

Vascular tone, cell survival Glucose metabolism

Triosephosphate isomerase Urokinase plasminogen activator receptor VEGF VEGF receptor-1 (flt-1) Vimentin

Glucose metabolism Extracellular-matrix metabolism Angiogenesis/cell survival Angiogenesis Cytoskeletal structure

Ceruloplasmin Collagen type V Cyclin G2 CXCR4 DEC1/Stra13 DEC2 Endocrine gland VEGF Endoglin Endothelin-1 Enolase-1 Erythroblastosis virus transforming sequence ETS-1 Erythropoietin Fibronectin-1 Glucose transporter-1 Glucose transporter-3 Glyceraldehyde 3-phosphate dehydrogenase Heme oxygenase-1 Hexokinase-1

Transcriptional regulation Transcriptional regulation Angiogenesis Angiogenesis Vascular tone Glucose metabolism Angiogenesis, cancer invasion

Hexokinase-2 Glucose metabolism IGF-binding protein-1 Cell proliferation and survival IGF-binding protein-2 Cell proliferation and survival IGF-binding protein-3 Cell proliferation and survival Data from [36, 37, 109, 110] IGF insuline-like growth factor, VEGF vascular endothelial growth factor

5 Nuclear Factor Kappa B The NF-kB family of transcription factors comprises proteins with a highly conserved Rel homology domain. This domain is responsible for DNA binding, dimerization, and interactions with IkB, the intracellular inhibitor for NF-kB. Five members of this family have been identified: NF-kB1 (p50/p105), NF-kB2 (p52/p100), p65 (RelA), cRel, and RelB. The NF-kB family of proteins can form either homodi-

Glucose metabolism Cell proliferation Angiogenesis Collagen metabolism Glucose metabolism Apoptosis/cell survival Cellular respiration

meric or heterodimeric complexes. These different complexes recognize slightly different binding motifs. For example, p50/p65 preferably binds the sequence 5¢-GGGRNNYYCC-3¢, whereas the RelA/c–Rel complex binds 5¢-HGGARNYYCC-3¢ (where H is A, C, or T; R is a purine; and Y is a pyrimidine) [55]. These sequences are found in genes that code for cytokines, chemokines, growth factors, and cell adhesion molecules as well as some acute-phase proteins [56]. NF-kB is found in the cytoplasm in numerous cell types bound to an

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inhibitory protein known as IkB. Upon cellular activation, IkB is phosphorylated and proteolytically degraded or processed by proteasomes. This exposes the nuclear localization signal and NF-kB then translocates into the nucleus [56]. The activation can occur in response to a wide range of inducers besides hypoxia [57], such as ultraviolet irradiation, cytokines, inhaled occupational particles, and bacterial or viral products. Several mechanisms as to how NF-kB is activated by hypoxia have been suggested, such as the tyrosine phosphorylation of IkB by the Ras/Raf kinases downstream from Src, or the involvement of mitochondria-derived reactive oxygen species. Furthermore, the activation can be mediated by p42/44 and phosphatidylinositol 3-kinase. Target genes for hypoxia-induced NF-kB include cyclooxygenase-2, tumor necrosis factor (TNF)-a, interleukin-6, and macrophage inflammatory protein-2 [58]. NF-kB has been implicated in the development of pulmonary hypertension in relation to enhanced thrombogenicity in the pulmonary vascular wall and the prothrombotic state, which is frequently associated with pulmonary hypertension. BelAiba et  al. [59] have proposed that NF-kB is activated through the serum- and glucocorticoid-inducible kinase-1 and then translocates to the nucleus, where it facilitates the transcription of tissue factor, which in turn increases the formation of thrombin and thereby may contribute to the prothrombotic state and vascular remodeling of pulmonary arteries. Pulmonary artery endothelial cells isolated from piglets with hypoxia-induced pulmonary hypertension exhibited increased activity of NF-kB, most likely due to RhoGTPases [60]. This may be involved in the regulation of endothelial permeability. Another important aspect is that under hypoxic conditions, NF-kB is able to bind to an element in the proximal promoter of the HIF-1a gene and thereby upregulate the transcription of HIF-1a [61, 62]. There is now also evidence for the regulation of NF-kB by HIF-1 [63], suggesting a coupling between the two essential stress responses: innate immunity and the hypoxic response. In addition, NF-kB has been associated with the development of monocrotalineinduced PH in rats [64] and bleomycin-induced pulmonary hypertension in mice [65].

6 PPAR Gamma The PPARg transcription factor belongs to the superfamily of nuclear receptors that also includes the two other PPAR isoforms: PPARa and PPARb. PPARg forms a heterodimer complex with retinoid X receptor-a (RXRa), which in turn binds to peroxisome proliferator response elements (PPREs) within the promoters of PPARg-targeted genes [66]. The

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PPARs all consist of a structure with functionally distinct domains called A/B (ligand-independent activation domain), C (DNA binding domain), D (hinge domain), and E/F (ligand-binding domain) [67]. This is shared by all nuclear receptors. The gene transcription mechanism is similar to that of other members of the nuclear receptor family; the C-terminal A/F domain of the receptor heterodimerizes with RXRa and the PPAR/RXRa complex binds to the PPRE sequence of the target gene promoters [67]. The activity of PPARg can be modulated by posttranslational modifications such as phosphorylations. For instance mitogen-activated protein kinase can phosphorylate the Ser-112 residue, which will inhibit PPARg activity [68]. Several factors can modulate PPARg transcriptional activity, including the HIF downstream target DEC1/Stra13, which can inhibit the gene expression of PPARg [69]. Recently, it was also found that a hypoxia-activated signal, other than the HIF-1 pathway, could reduce the expression of PPARg [70]. PPARg is mainly known for its role in controlling energy balance and can modulate both lipid and glucose homeostasis [71], but the transcription factor also controls expression of genes that are involved in cellular differentiation, induction of apoptosis [72], and inhibition of angiogenesis [73] and can be found in, besides adipose tissue, lung alveolar type II cells [74]. The PPARg agonist rosiglitazone can greatly attenuate hypoxia-induced pulmonary artery remodeling in chronic hypoxic rats, whereas there is only a small effect on pulmonary artery pressure and a moderate effect on right ventricular hypertrophy [75]. It was also suggested that PPARg could be involved in the recruitment of c-Kit-positive cells, progenitor cells which may contribute to adventitial neovascularization, in the pulmonary artery wall of animals exposed to chronic hypoxia [75]. Furthermore, the PPARg transcription factor is downregulated in lungs from patients with severe pulmonary hypertension and in addition a complete loss in the complex vascular lesions is reported [76]. It was not completely clear, though, whether the regulation of PPARg was directly due to hypoxia or if it was shear-stress-induced. In a chronic hypoxic mouse model, pulmonary hypertension and right ventricular hypertrophy were attenuated by treatment with rosiglitazone, and it is proposed that PPARg by reducing the amount of reactive oxygen species can improve the bioavailability of nitric oxide [77].

7 Early Growth Response-1 The zinc finger transcription factor Egr-1 is activated in responses to a variety of stimuli, including growth factors, hormones, neurotransmitters, and hypoxia [78]. It is an early

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response gene and can therefore act as a convergence point for many signaling cascades. The hypoxia-induced activation of Egr-1 involves members of the protein kinase C (PKC) family. This was demonstrated by the fact that PKCb null mice exhibit markedly decreased Egr-1 levels in response to hypoxia [79]. Furthermore, in hypoxic endothelial cells, PKCa and its downstream targets Ras/Raf/extracellularsignal-regulated kinase (ERK) 1/2 are important for Egr-1mediated gene transcription [80]. Egr-1 preferentially binds with high affinity to the Egr-1 binding site: 5¢GCGGGGGCG-3¢ [81]. This consensus site is also found in the Egr-1 gene, resulting in the Egr-1 protein binding to its own gene and, as a result, can prevent further Egr-1 transcription [82]. The activity of Egr-1 can also reduce binding of two transcriptional cofactors: NGFI-A binding proteins 1 and 2 (NAB1, NAB2) [83]. Moreover, the transcription of NAB2 is controlled by Egr-1 [84], suggesting another negative-feedback loop. This indicates a very tight regulation of Egr-1 activity. The target genes of Egr-1 are involved in many physiological functions, ranging from synaptic plasticity to neurite outgrowth, wound repair, female reproductive capacity, extracellular matrix remodeling, thrombosis, as well as growth control and apoptosis [78]. Several growth factor genes are targeted by Egr-1, including insulin-like growth factor-II, platelet-derived growth factors A and B, and transforming growth factor-1 [78], pointing in the direction of a growth stimulatory autocrine loop, since platelet-derived growth factor enhances Egr-1 synthesis via the ERK or alternative signaling cascades [78]. Besides the role as a growth-inducing transcription factor, Egr-1 is also proposed to be a proapoptotic protein. This is mainly due to the fact that Egr-1 induces synthesis of the tumor suppressor protein p53, it can bind to the transcription factor c-Jun and augments its proapoptotic activity, and in addition it can transactivate PTEN [85]. The proapoptotic activity of Egr-1 holds true under some circumstances, but it seems to depend on the cell type and the character of the cytotoxic stimulus. There are several reports of the increased expression of Egr-1 in the lung and in pulmonary vascular cells after hypoxic exposure [86, 87]. Furthermore, as Egr-1 can activate several downstream targets involved in vascular remodeling, it is plausible that this transcription factor is involved in hypoxia-induced pulmonary hypertension. For example, upon hypoxia, Egr-1-mediated production of tissue factor is observed in monocytes, in bronchial and vascular smooth muscle, and in alveolar macrophages, which leads to hypoxia-induced fibrin deposition in the pulmonary vasculature [88]. Indirect evidence for the involvement of Egr-1 in hypoxiainduced pulmonary hypertension was recently demonstrated in extracellular superoxide dismutase (EC-SOD) overexpressing

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mice [89]. These mice did not exhibit increased Egr-1 protein levels upon hypoxia, nor the downstream targets tissue factor and NAB2. In addition, the hypoxia-induced increase in right ventricular systolic pressure was attenuated, indicating a role for Egr-1 and tissue factor.

8 Activating Protein 1 Activating protein 1 (AP-1) is a pleiotropic dimeric transcription factor that comprises members of Fos, Jun, activating transcription factor(ATF) and musculoaponeurotic fibrosarcoma protein families. These proteins can homodimerize or heterodimerize to form the active AP-1 complex that modulates gene expression. Target genes are involved in diverse cellular functions related to apoptosis, cell proliferation, cell differentiation, catecholamine biosynthesis, inflammation, xenobiotic metabolism, tumor invasion, and angiogenesis [90]. A range of stimuli, including growth factors, proinflammatory cytokines, UV radiation and hypoxia, activate AP-1. Hypoxic genes in whose regulation AP-1 is involved include tyrosine hydroxylase [91], VEGF [92], and endothelial nitric oxide [93]. These regulations can be solely by AP-1 or in cooperation with other transcription factors such as HIF-1, GATA-2, NF-1, and NF-kB [91, 92, 94]. The hypoxia-induced activation of AP-1 has not been fully clarified, but may involve a Jun N-terminal kinase dependent pathway [95], a change in the redox environment [93], or the hypoxia-induced change in intracellular calcium levels [91, 92]. In human pulmonary artery endothelial cells, AP-1 binding activity was increased in response to hypoxia. This was mediated by enhanced Ca2+ influx [96]. Furthermore, doubleTNF-receptor-deficient mice did not activate NF-kB or AP-1 and as a result did not develop bleomycin-induced pulmonary hypertension. Pharmacological blocking of these two transcription factors in wild-type mice also attenuated the bleomycin-induced pulmonary hypertension [65].

9 Nuclear Factor of Activated T Cells This family of transcription factors was first characterized in T lymphocytes as inducers of cytokine expression, but since then functions in the cardiovascular system have also been described. The family consists of four members – NFATc1, NFATc2, NFATc3, and NFATc4 – that are Ca2+/calcineurindependent for nuclear translocation and a fifth member – NFAT5 – that is Ca2+-independent [97]. Nuclear factor of activated T cells (NFAT) proteins are found in the cytoplasm in resting cells, where they are phosphorylated. When the

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cells are stimulated and intracellular calcium is increased, NFATs are dephosphorylated by calcineurin. This activates the transcription factors and they translocate to the nucleus, where gene expression is then initiated. Modulating NFAT kinases and nuclear partner proteins such as AP-1 can further adjust NFAT activity [97]. NFAT is mainly known for regulating genes involved in the inflammatory response, but it also appears to regulate the expression of smooth muscle myosin heavy chain, smooth muscle a-actin, integrins, and caldesmon genes [98]. An increase in intracellular calcium level is mainly responsible for NFAT activation but other stimuli can also be involved. For instance, hypoxia can activate NFAT-mediated gene expression of resistin in rat aortic smooth muscle cells [99] and it has been shown that reactive oxygen species are required for NFAT activation mediated by angiotensin II [100]. Furthermore, also extracellular glucose and lipids can induce nuclear accumulation of NFAT [101]. In both human and experimental pulmonary hypertension, an activation of NFAT has been observed, which is suggested to downregulate the Kv1.5 channel, increase bcl-2 expression, and modulate mitochondrial membrane potential. All these contribute to the survival and proliferation of vascular smooth muscle cells and thereby the vascular remodeling [102], which is a prominent feature of pulmonary hypertension. In addition, NFAT3c is found expressed in the smooth muscle cells of murine pulmonary arteries, where its activity is highly increased upon hypoxic exposures. This is followed by an amplification of a-actin [103]. In a mouse model of pulmonary arterial hypertension where the gene for vasoactive intestinal peptide is knocked out, NFAT has been proposed to play a role both in the inflammatory response and for the development of remodeling [104]. Also in the mouse model of hypoxia-induced pulmonary hypertension, NFAT was activated and connected to increased a-actin levels [103]. In the hypoxic rat, NFAT is furthermore suggested to play a role in the development of right ventricular hypertrophy [105].

10 Cyclic AMP Response Element Binding Protein The CREB family of leucine zipper transcription factors consists of, besides CREB, the cAMP response element modulator and ATF 1. These transcription factors are activated after phosphorylation at a conserved serine residue in response to cAMP, which alone can, at least in some instances, lead to gene binding of the cAMP response element, TGACGTCA, and subsequent cellular gene expression. Additional promoter-bound factors, such as the coactivator

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CBP/p300, can further modulate the activity and thereby discriminate between different inputs [106]. CREB is involved in the transcription of genes involved in, for instance, growth, survival, inflammation, metabolism, and long-term memory [106]. In addition, CREB can in some cases act as a repressor of gene expression [107]. The gene transcription induced by CREB can be influenced by hypoxia. Interestingly, it seems that the degree and duration of hypoxia is highly important for how the activity of CREB is modulated. It has been shown that acute mild hypoxia can increase CREB activity through increased phosphorylation, whereas a severer oxygen deprivation can target the transcription factor for ubiquitin-mediated degradation. Long exposures to severe hypoxia, on the other hand, results in CREB stabilization [58]. With use of tissue samples from neonatal calves with hypoxia-induced pulmonary hypertension, CREB binding was found to be reduced in the smooth muscle cells surrounding remodeled, hypertensive pulmonary arteries. In addition, in cultured cells CREB could inhibit smooth muscle cell proliferation and migration [108]. In mice exposed to 10% oxygen, phosphorylation of CREB and ATF 1 was observed in the lungs but not in liver, kidney, or spleen. Furthermore, CREB-responsive genes were upregulated in the lung in response to hypoxia, whereas their expression was unchanged in five systemic organs [109]. This suggests a key role in the lung-specific responses to hypoxia.

11 Summary For the development of strategies for the prevention and/or treatment of hypoxia-mediated pulmonary hypertension, an understanding of the molecular pathophysiological processes is imperative. This must ultimately be in terms of changes in gene expression, which are mediated by transcription factors. Within the lung and its vasculature, hypoxia can activate a wide range of transcription factors and in that way modulate several signaling pathways and cellular responses. Although HIF is considered the most significant factor in influencing the cellular response to hypoxia [111], other oxygen-sensitive transcription factors are, as described, also important in this regard, emphasizing that most likely a number of signaling pathways are involved in the hypoxic response of a cell or a tissue. Several of the transcription factors can also induce the expression or stabilization of each other and some can cooperate in the modulation of certain genes. This underlines the complicated interplay of the oxygen-sensitive transcription factors, which is important for fine-tuning the exact response.

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Chapter 50

Developmental Regulation of Pulmonary Vascular Oxygen Sensing David N. Cornfield

Abstract  The in utero environment of the pulmonary circulation is unique. During fetal life, the lung is fluid filled, oxygen tension low, pulmonary blood flow limited, and pulmonary vascular resistance exceeds systemic vascular resistance. Blood is shunted away from the lungs at the level of the ductus arteriosus. At birth, pulmonary blood flow increases eightfold to tenfold and pulmonary artery pressure decreases to 50% of systemic levels within 24 h after birth concomitant with an increase in oxygen tension. The critical physiologic stimuli that account for perinatal pulmonary vasodilation include rhythmic distention of the lung and an increase in both shear stress and oxygen tension. Data accumulated over the past several decades implicate distinct and complementary roles for both pulmonary artery endothelial cells (PAECs) and pulmonary artery smooth cells (PASMCs) in promoting fetal lung growth and development and in mediating the postnatal adaptation of the pulmonary circulation. Keywords  Lung development • Neonatal and perinatal • Endothelial cell • Smooth muscle • Fetal lung growth

1 Introduction The in utero environment of the pulmonary circulation is unique. During fetal life, the lung is fluid filled, oxygen tension low, pulmonary blood flow limited, and pulmonary vascular resistance exceeds systemic vascular resistance. Blood is shunted away from the lungs at the level of the ductus arteriosus. At birth, pulmonary blood flow increases eightfold to tenfold [1] and pulmonary artery pressure decreases to 50% of systemic levels within 24  h after birth [2] concomitant with an increase in oxygen tension. The critical physiologic

D.N. Cornfield (*) Department of Pediatrics – Pulmonary Medicine, Lucile Salter Packard Children’s Hospital at Stanford, 770 Welch Rd., Ste. 350, Stanford, CA 94304, USA e-mail: [email protected]

stimuli that account for perinatal pulmonary vasodilation include rhythmic distention of the lung [3] and an increase in both shear stress [4] and oxygen tension [5]. Data accumulated over the past several decades implicate distinct and complementary roles for both pulmonary artery endothelial cells (PAECs) and pulmonary artery smooth cells (PASMCs) in promoting fetal lung growth and development and in mediating the postnatal adaptation of the pulmonary circulation.

2 Pulmonary Artery Endothelial Cell The PAEC plays a critically important role in the developing pulmonary circulation, both during and after fetal life. Vasoactive products produced by the PAECs simultaneously preserve and constrain fetal pulmonary blood flow. During fetal life, inhibition of endothelin, a vasoconstrictive protein produced by the endothelium [6], causes marked pulmonary vasodilation [7]. Conversely, pharmacologic inhibition of nitric oxide decreases pulmonary blood flow and increases pulmonary artery blood pressure [4, 8]. These two observations suggest that carefully circumscribed pulmonary blood flow is of critical importance in normal pulmonary vascular development. In response to key perinatal physiologic stimuli, the pulmonary endothelium elaborates vasoactive mediators such as nitric oxide [9], endothelin [6], bradykinin, and prostaglandins [10, 11]. PAEC nitric oxide production is necessary [8], though not always sufficient, for the transition of the pulmonary circulation from a high-resistance to a low-resistance circuit. Among the most important properties of the pulmonary circulation is the ability to sense and respond to alterations in oxygen tension. During air-breathing life, the pulmonary circulation is adapted to respond to decreases in oxygen tension to match ventilation and perfusion and thereby mitigate hypoxemia. At birth, it is biologically imperative for the pulmonary circulation to respond to an acute increase in oxygen tension. Interestingly, the capacity of the pulmonary circulation to sense and respond to an acute increase in oxygen tension is developmentally regulated. Not until gestation is

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_50, © Springer Science+Business Media, LLC 2011

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approximately 85% complete is the fetal pulmonary circulation able to respond to an increase in oxygen tension [12]. Vasodilator agents that act through endothelial-cell-mediated mechanisms produce only transient pulmonary vasodilation [5, 13]. The relatively narrow developmental window wherein the fetal pulmonary circulation is able to respond to an increase in oxygen tension may serve a protective purpose as either too much [14, 15] or too little pulmonary blood flow compromises pulmonary vascular development. The capacity of PAECs to produce nitric oxide [16] underlies, to a significant degree, the ability of the pulmonary circulation to dilate in response to increasing oxygen tension with maturation. By the end of gestation, the pulmonary circulation is exquisitely sensitive to alterations in oxygen tension, as an increase of only 8 Torr results in a threefold increase in pulmonary blood flow [17]. Several investigators have demonstrated that production of nitric oxide by the endothelial cells mediates oxygen-induced pulmonary vasodilation [4, 18] (Fig. 1). Recent data have provided further

Fig.  1  Hemodynamic effects of pharmacologic inhibition of nitric oxide (nitro-l-arginine) on left pulmonary arterial blood flow (LPA flow; top panel) and mean pulmonary arterial pressure (MPAP; bottom panel) during sequential ventilation with low and high concentrations of oxygen. In comparison with control animals (closed circles; n = 6 animals), inhibition of nitric oxide (open circles; n = 7 animals) markedly attenuated the rise in left pulmonary arterial blood flow during ventilation with a low and high fraction of inspired oxygen concentration as well as the decline in mean pulmonary artery pressure during ventilation with a high fractional concentration of inspired oxygen. (Reprinted from [4] with permission)

D.N. Cornfield

detail of the mechanism, however, how fetal PAECs sense and respond to an acute increase in oxygen tension remains incompletely understood. In a confluent monolayer of fetal PAECs, an acute increase in oxygen tension directly increases the level of cytosolic calcium. In the PAEC, an acute increase in oxygen tension results in membrane depolarization and an increased rate of calcium entry into the PAEC. The sustained and progressive increase in PAEC cytosolic Ca2+ concentration entails calcium release from intracellular stores. The observation that acetylcholine causes a greater increase in the level of PAEC cytosolic calcium under hypoxic, compared with normoxic, conditions provides inferential evidence that the low oxygen tension environment of the normal fetus may ready the perinatal pulmonary circulation to respond to an acute increase in oxygen tension by augmented loading of intracellular stores. In PAECs and lung tissue derived from fetal lambs with chronic uterine pulmonary hypertension, the response of the pulmonary circulation to vasodilator stimuli is attenuated. Endothelial-derived nitric oxide expression is decreased. PAECs derived from animal models of perinatal pulmonary hypertension of the newborn do not respond to an acute increase in oxygen tension with an increase in either cytosolic Ca2+ concentration or nitric oxide production [19] (Fig. 2). These observations support the proposition that the intrauterine environment informs the immediate cellular response of endothelial cells [20].

Fig. 2  Effect of an acute increase in oxygen tension on nitric oxide production in fetal pulmonary artery endothelial cells. An acute increase in oxygen tension caused an increase in DAF-FM membrane fluorescence. In pulmonary artery endothelial cells (PAEC) derived from fetal lambs with chronic intrauterine pulmonary hypertension, an acute increase in oxygen tension did not increase nitric oxide production. With an acute increase in oxygen tension, DAF-FM membrane fluorescence did not increase in pulmonary artery endothelial cell treated with the competitive antagonist of nitric oxide production nitro-l-arginine. (Reprinted from [19] with permission)

50  Developmental Regulation of Pulmonary Vascular Oxygen Sensing

The response of the PAEC to birth-related physiologic stimuli is critically important for postnatal adaptation of the pulmonary circulation. PAECs must contract, increase production of nitric oxide [8, 21] and prostacyclin [10, 22], as well as decrease endothelin production [23] in a remarkably short time frame to facilitate the transition of the neonatal pulmonary circulation from high to low tone and thereby enable effective gas exchange to occur in the lungs. The consequences of an attenuated response of endothelial cells to an acute increase in oxygen tension are significant, with blood shunting away from the lungs resulting in severe central hypoxemia that is relatively unresponsive to high concentrations of inspired oxygen [24]. Recent data provide clear evidence that inadequate production of nitric oxide and relatively increased production of endothelin play an etiologic role in persistent pulmonary hypertension of the newborn (PPHN) [25]. The observation that endothelial cells from an animal model of PPHN demonstrate no increase in either cytosolic Ca2+ concentration or only a minimal increase in nitric oxide production in response to an acute increase in oxygen tension provides the first direct evidence that the chronic intrauterine pulmonary hypertension compromises endothelial cell oxygen sensing and nitric oxide production [19]. Moreover, the observation that acetylcholine caused a greater increase in cytosolic Ca2+ concentration in PAECs from hypoxic, compared with normoxic, lambs suggests that the normally low oxygen tension environment of the developing pulmonary circulation prepares the lung for extrauterine, airbreathing life, providing a mechanistic link between endothelial cell cytosolic calcium and an acute increase in oxygen tension, one of the central perinatal physiologic stimuli [1]. The observation that fetal PAECs maintained in culture respond to an acute increase in oxygen tension with an increase in cytosolic Ca2+ concentration, in direct contrast to the response of PAECs derived from the adult pulmonary vasculature [26, 27], lends weight to the proposition that maturation-related differences in the pulmonary circulation extend not only to the molecular expression, but also to the function of individual cellular constituents. Further, the observation that PAECs derived from fetal lambs with chronic intrauterine pulmonary hypertension do not respond to an acute increase in oxygen tension with either an increase in cytosolic Ca2+ concentration or increased nitric oxide production [19] provides still further evidence of a link, albeit an inferential one, between an acute increase in oxygen tension and nitric oxide production in fetal PAECs. Calcium-sensitive (KCa) K+ channels also play an integral role in the regulation of fetal PAEC cytosolic Ca2+ concentration and in modulating both nitric oxide production and the response to changes in O2 tension. Basal fetal PAEC cytosolic Ca2+ concentration is determined by KCa channel activity, as pharmacologic blockade of the KCa, but not the voltage-gated (KV), channel increases PAEC cytosolic Ca2+ concentration.

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Similarly, the decrease in PAEC cytosolic Ca2+ concentration that occurs in response to acute hypoxia is blocked by KCa channel antagonists, implying that O2 sensing is mediated by the KCa channel. Moreover, PAEC nitric oxide production is attenuated by KCa but not KV or ATP-sensitive K+ channel antagonists. Thus, the KCa channel plays an essential role in enabling both PAECs and PASMCs to sense and respond to physiologic stimuli that lead to the postnatal adaptation of the pulmonary circulation [28]. Thus, in the perinatal pulmonary circulation, PAECs are able to sense and respond to an acute increase in oxygen tension with an increase in cytosolic Ca2+ concentration. The acute increase in oxygen tension leads to membrane depolarization and an increase in the rate of extracellular calcium entry. Augmented entry of extracellular calcium entry leads to calcium release from inositol trisphosphate sensitive intracellular calcium stores. The sustained elevation of PAEC Ca2+ concentration results, in part, from release of calcium from ryanodine-sensitive stores. From a teleologic perspective, the response of the PAEC to an acute increase in oxygen tension is biologically imperative. Compromised PAEC oxygen sensing may underlie the attenuated response of the pulmonary circulation to perinatal vasodilator stimuli, thereby leading to PPHN.

3 Role of the KCa Channel in the Perinatal Lung Although physical factors and vasoactive products elaborated by the pulmonary vascular endothelium are involved in the regulation of perinatal pulmonary vascular tone [8], sustained and progressive perinatal pulmonary vasodilation requires pulmonary vascular K+ channel activation [29] (Fig.  3). PASMC K+ activation causes membrane hyperpolarization, closure of voltage-operated calcium channels, a decrease in cytosolic Ca2+ concentration, and vasodilation [30]. Data demonstrate that activation of KCa channels in the pulmonary vasculature plays a critical role in perinatal pulmonary vasodilation. The first definitive evidence that KCa channel activation mediates O2-induced fetal pulmonary vasodilation came from studies in acutely instrumented lategestation ovine fetuses [31]. O2-induced fetal pulmonary vasodilation was blocked by either tetraethylammonium (TEA), a K+ channel blocker [32, 33], or iberiotoxin, a specific KCa channel antagonist [34], but was unaffected by glibenclamide, a blocker of the KATP channel [35, 36] (Fig. 3). The data suggested that O2 causes fetal pulmonary vasodilation through KCa channel activation. Inhibitors of either guanylate cyclase or cyclic nucleotide dependent kinases also attenuated O2-dependent fetal pulmonary vasodilation, implying that an elevated level of fetal O2 acts to increase

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Fig. 3  Effect of K+ channel inhibition with either tetraethylammonium (TEA) or glibenclamide (GLI) on perinatal pulmonary vasodilation. In response to sequential ventilation, left pulmonary artery (LPA) blood flow in response to sequential ventilation with low and high levels of O2. Left pulmonary artery flow in the tetraethylammonium group was attenuated compared with that in the control (CTRL) in response to both low and high levels of O2. There was no difference in left pulmonary artery flow between conrol and glibenclamide groups. *p < 0.01 all groups compared with the baseline value. •p < 0.01 control group versus tetraethylammonium group [29]

guanylate cyclase activity, cyclic GMP concentration, and activate cyclic nucleotide dependent kinases, causing KCa channel activation and vasodilation [31]. K+ channel inhibition with TEA, a preferential KCa channel antagonist in millimolar concentrations [37, 38], blocks the perinatal pulmonary vasodilation caused by ventilation [29] (Fig. 4). This observation implies that ventilation causes sustained and progressive perinatal pulmonary vasodilation through activation of TEA-sensitive K+ channels. Nitric oxide causes perinatal pulmonary vasodilation, in part, through activation of the KCa channel [39]. Even shear-stress-induced fetal pulmonary vasodilation, induced by compression of the ductus arteriosus during fetal life, requires KCa and apaminsensitive KCa channel activation [40].

4 Ontogeny of the Pulmonary Vascular K+ Channel Although the KCa channel is crucially important in the perinatal pulmonary circulation, its importance seems to decrease with maturation. The observation that the K+ channel setting resting membrane potential in the pulmonary circulation changes following birth from a KCa to a KV channel suggests developmental regulation of K+ channels in the pulmonary circulation [41]. In coordination with a maturation-related decrease in KCa channel expression (Fig. 5) and activation, KV expression increases with maturation. There is relatively more KV2.1 channel protein and message in the adult than in the fetal and neonatal pulmonary circulation [42]. The increase in KV2.1 channel parallels the increasing capacity of PASMCs to sense and respond to acute decreases in O2 tension, as in response to acute hypoxia, cytosolic Ca2+ concentration

D.N. Cornfield

Fig. 4  The effect of K+ channel inhibition on oxygen-induced fetal pulmonary vasodilation. In instrumented fetal lambs, delivery of supplemental oxygen to the maternal ewe increased fetal oxygen tension and in the control periods caused a marked decrease in pulmonary vascular resistance (PVR) (p < 0.05), compared with the baseline. K+ channel inhibition had no effect on basal pulmonary vascular resistance. Following administration of tetraethylammonium (TEA) (p < 0.05) and iberiotoxin, a specific KCa channel blocker, to the pulmonary circulation, oxygen-induced fetal pulmonary vasodilation was blocked. Glibenclamide, an ATP-sensitive K+ channel antagonist, had no effect on attenuated oxygen-induced fetal pulmonary vasodilation (p < 0.05). (From [31]. Copyright National Academy of Sciences)

increases more rapidly and to a greater degree in adult, as compared with fetal, PASMCs [42]. The relatively greater abundance of the KV2.1 channel in the adult may represent an adaptation that allows the pulmonary circulation to respond to a specific physiologic signal that is relevant to a particular developmental stage. Reports that the O2 sensor in the adult PASMC is a KV channel are consistent with this construct [43, 44], since the KV channel is inactivated by acute hypoxia, causing PASMC depolarization, opening of voltage-operated calcium channels, increase in cytosolic Ca2+ concentration, and vasoconstriction. Thus, the maturationrelated increase in hypoxic pulmonary vasoconstriction that has been previously reported [45, 46] might derive from the parallel increases in KV2.1 channel activity, protein, and message with maturation. In contrast to pulmonary vascular KV channel expression and activity, pulmonary vascular KCa activity and expression decreases with maturation. Data from both pulmonary arteries and PASMCs in primary culture indicate that KCa channel expression is greatest in the fetal lung and decreases with maturation [47]. Indeed, the functional changes of ion channels in pulmonary vascular smooth muscle cell are consistent with the transcriptional and translational changes of the corresponding channel proteins molecular biology. In fetal PASMCs, the resting membrane potential is determined by the KCa channel, whereas in adult PASMCs KV channel activity determines the resting membrane potential [41]. In fetal PASMCs, an acute increase in oxygen tension results in membrane hyperpolarization and a decrease in PASMC cytosolic Ca2+ concentration and has no effect on adult pulmonar artery membrane potential

50  Developmental Regulation of Pulmonary Vascular Oxygen Sensing

Fig. 5  (a) Western blot of protein extract from the fetal and adult pulmonary vasculature. More BKCa channel protein is present in the fetus than in the adult. Mouse brain served as the positive control. Co-incubation of the antigen and antibody did not produce a band, indicating specificity of the antibody. (b) BKCa channel a subunit messenger RNA (mRNA) levels in the distal pulmonary vasculature decrease with maturation. The BKCa channel band was normalized to the 18S band intensity. mRNA was isolated from fetal, newborn, and adult animal pulmonary arteries. (c) Representative gel of a reverse-transcription PCR analysis of BKCa channel mRNA expression during development. The BKCa band was compared with the control 18S band in fetal and adult samples. The BKCa channel band intensity was determined by densitometry and normalized to that of the 18S band [47]

or cytosolic Ca2+ concentration [47]. Taken together, these observations indicate that the developmental regulation of KCa channel expression allows for the fetal PASMC to be uniquely well adapted to respond to an acute increase in oxygen tension with a decrease in cytosolic Ca2+ concentration and vasorelaxation. The molecular mechanisms whereby a maturationrelated decrease in KCa and increase in KV channel activity occurs remain unknown. Although there is precedent for the concept that hypoxia modulates K+ channel activity [48, 49], no studies have demonstrated a definitive relationship between hypoxia and KCa channel expression.

5 Ductus Arteriosus Closure of the ductus arteriosus at birth is a necessary step for the normal transition from fetal to air breathing life [50]. Previous studies demonstrated that an acute increase in PO2 causes an increase in tension of ductus arteriosus rings. In rabbit ductus arteriosus smooth muscle cells acute normoxia

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inhibits K+ current, leading to membrane depolarization [51–53]. Pharmacologic blockade of the voltage-gated K+ channels mimics the effect of acute normoxia. Consistent with such data, 4-aminopyridine mimics an acute increase in oxygen tension in ductus arteriosus smooth muscle cells [54]. Moreover, preventing extracellular Ca2+ entry accentuates the initial increase in cytosolic Ca2+ concentration in ductus arteriosus smooth muscle cells exposed to acute normoxia. An acute increase in oxygen tension directly increases cytosolic Ca2+ concentration in a subconfluent monolayer of ductus arteriosus smooth muscle cells. Pharmacologic inactivation of voltage-sensitive, but not ATP- or calcium-sensitive, K+ channels mimics the effect of an acute increase in oxygen tension, providing support for the proposition that KV channel inactivation mediates the O2-induced increase in the cytosolic Ca2+ concentration of ductus arteriosus smooth muscle cells [54]. Alternative reports have demonstrated that an acute increase in oxygen tension initially decreases the rate of calcium entry into ductus arteriosus smooth muscle cells [55]. Data demonstrate that entry of extracellular calcium is not necessary for the initial increase in ductus arteriosus smooth muscle cell cytosolic Ca2+ concentration. Rather, release of calcium from inositol trisphosphate sensitive stores accounts for the initial increase in ductus arteriosus smooth muscle cell cytosolic Ca2+ concentration. The sustained and progressive increase in ductus arteriosus smooth muscle cell cytosolic Ca2+ concentration entails subsequent extracellular calcium entry [54]. The initial release of Ca2+ from intracellular stores is necessary to trigger the second phase of extracellular Ca2+ entry, in a process termed “calcium-induced calcium entry.” The observation that blockade of calcium release from ryanodine-sensitive stores has no effect on the normoxia-induced increase in ductus arteriosus smooth muscle cell cytosolic Ca2+ concentration offers further support for the notion that release of calcium from an inositol trisphosphate sensitive store plays a pivotal role in the normoxia-induced increase in ductus arteriosus smooth muscle cell cytosolic Ca2+ concentration [54]. Thus, pathological patency of the ductus arteriosus may result from defects in oxygen sensing.

6 Hypoxia-Inducible Factor 1a Modulates KCa Channel Expression The KCa channel is critically important for the transition of the pulmonary circulation at birth and diminishes relative to its physiologic significance and expression with maturation. Although the physiologic underpinnings of the developmental regulation of the pulmonary vascular KCa channel remains unknown, recent data indicate that the low oxygen tension environment of the fetal pulmonary circulation might motivate KCa channel expression.

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Hypoxia-inducible factor 1a (HIF-1a), a key transcription factor that is central to oxygen homeostasis, enables the cell to respond to changes in O2 availability, an essential response in many developmental, physiological and pathological processes. Semenza and others have shown that HIF-1a regulates the transcription of many genes involved in the cellular and systemic response to oxygen availability, including genes involved in angiogenesis (i.e., vascular endothelial growth factor), oxygen transport (i.e., erythropoietin), and energy metabolism (i.e., glycolytic enzymes) [56]. HIF-1a is also a key player in pathophysiological processes such as cancer, as hypoxic microenvironments in a tumor trigger expression of angiogenic genes that promote the growth of the newly vascularized tumor [57, 58]. From a developmental perspective, the transition from the relatively low oxygen tension intrauterine environment to air-breathing life entails an unprecedented and absolutely required response to an increase in oxygen availability. The physiologically low oxygen environment of the fetus might be essential for fetal vascular growth and lung morphogenesis [59], as well as other early embryo developmental programs [60]. As a master regulator of the oxygen response, it follows that the molecular mechanisms responsible for fetal life and the transition to a newborn might involve HIF-1a. Under normoxic conditions, the a chain of HIF-1a is eliminated by degradation, rendering HIF-1a inactive. In lowoxygen environments, HIF-1a forms a dimer, is stabilized, and can translocate to the cell nucleus, thereby promoting transcription of hypoxia-sensitive genes [61, 62]. Deferoxamine mesylate (DFX), a known hypoxia mimic, is an iron chelator capable of inducing HIF-1a protein stability and hypoxia-dependent gene expression, probably by inhibiting the action of the prolyl hydroxylase enzyme responsible for hypoxia-inducible factor 1ab. Normoxic fetal PASMCs supplemented with 200 mM DFX demonstrated increased expression of the b subunit of the KCa channel. DFX-treated cells showed an increased KCa a subunit messenger RNA expression, at levels comparable to those observed under hypoxia [63] (Fig. 6). As DFX prevents HIF-1a degradation, augmented expression of the KCa channel implicates a role for HIF-1a in the regulation of the BKCa channel. Consistent with the notion that HIF-1a regulates BKCa expression is the presence of putative hypoxia-response elements on the 5¢ genomic sequences of the human KCa channel gene b1 subunit [64], two elements that closely matched the consensus sequence for the HIF-1a binding site, 5'-RCGTG-3¢ [65]. One of the sequences perfectly matches the “extended” consensus (5¢-GCACGTA-3¢), as found in many hypoxia-related genes [66–70]. The position of these sites proximal to the start of transcription and their precise match to elements previously shown to contain the HIF-1a binding site strongly suggest the presence of hypoxia-response elements on the KCa channel. More definitive evidence for the sensitivity of the KCa chanel to hypoxia derives from the fetal PASMC transfected

D.N. Cornfield

Fig. 6  Desferoxamine mimics the effect of hypoxia on the calciumsensitive K+ channel a subunit gene expression, suggesting a possible involvement of hypoxia-inducible factor 1. The aggregate reversetranscription PCR data (n = 3) of the relative expression of the KCa channel a subunit in normal and desferoxamine-treated cells is shown. Hypoxic cells were used as controls. The b-actin band was used to normalize the results for total mRNA content [63]

Fig.  7  Ovine pulmonary artery smooth muscle cells in primary cell cultures were transfected with 0.5–1  mg of KCa:luc+ plasmids and a plasmid control for 1–2 h. Cell cultures were placed under normoxic (pO2 = 120 Torr) or hypoxic (pO2 = 30 Torr) conditions. After 24–36 h in culture, cells were washed and lysed. Protein extracts were assayed for luciferase activity and normalized for transfection efficiency and protein concentration. Whereas the b1 subunit of the KCa channel showed a significant threefold induction under hypoxic conditions, the b2 subunit showed only a 38% increase in luciferase activity. (Reprinted from [63] with permission)

with 0.5–1 mg of KCa:luc+ plasmids and a plasmid control (to assess transfection efficiency) for 1–2 h. Cell cultures were placed under normoxic (pO2 = 120  Torr) or hypoxic (pO2 = 30  Torr) conditions. Interestingly, although the b1 subunit of the KCa channel showed a significant threefold induction under hypoxic conditions, the b2 subunit showed only a 38% increase in luciferase activity [63] (Fig. 7).

50  Developmental Regulation of Pulmonary Vascular Oxygen Sensing

Since changes in oxygen tension at birth might signal an important transcriptional adaptation of the fetus to the air-breathing life of the newborn infant, Resnik et  al. investigated the role of HIF-1a in that transition and found that expression of HIF-1a is developmentally regulated, as is the expression of the prolyl and asparagyl hydroxylases and other competing factors that control HIF-1a expression and activity [71]. The well-coordinated developmental regulation of the components of the HIF-1a pathway in the fetus suggests that the fetus represents a particular transcriptional paradigm that, in response to differential oxygen availability, produces a specific physiological and developmental response.

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Chapter 51

Pulmonary Vascular Remodeling by High Oxygen Rosemary C. Jones and Diane E. Capen

Abstract  As an organ of gas exchange, the lung and its ­vasculature function optimally at the level of oxygen in air. This chapter reviews the effect of breathing a higher than normal atmospheric level of oxygen on the vasculature of the lung. The earth’s atmosphere contains 20.99% oxygen, and at this concentration efficiently sustains life (cell respiration and metabolism). Studies have demonstrated the toxic effects of breathing a high oxygen tension on lung tissue, mainly in response to normobaric hyperoxia, as differences began to emerge between the response of an organ, or tissue, and how the oxygen is administered. Higher than normal partial pressures of oxygen can be administered in an atmosphere of reduced total barometric pressure (hypobaric hyperoxia), at sea level (normobaric hyperoxia), or at a total partial pressure of oxygen greater than the total barometric pressure at sea level (hyperbaric hyperoxia). Studies show that breathing high oxygen injures the adult lung’s vasculature, while prolonged exposure to hyperbaric hyperoxia induces vasculitis, with endothelial cell proliferation and hyalinization of the walls of large and small pulmonary arteries. This chapter discusses the potential mechanisms involved in pulmonary vascular remodeling in response to cycles of breathing high oxygen, or high oxygen and then air, discusses the observations that would improve understanding of the pathobiology of vascular changes that characterize the adult lung in pulmonary hypertension, and highlights the striking degree of plasticity of the lung’s vascular bed in matching vascular density to the ambient alveolar oxygen tension. Keywords  Hyperoxia • Normobaric and hyperbaric hyperoxia • Pulmonary vascular remodeling • Pulmonary vascular density • Plasticity

R.C. Jones (*) Harvard Medical School, Boston, MA, USA e-mail: [email protected]

1  Introduction Powerful in opposite directions, Janus (the Roman god of doors and archways) symbolizes the opposing roles of oxygen as both a life-giving and a deadly agent; hence, normal atmospheric levels of oxygen are essential to all life forms, whereas higher ones are toxic. As an organ of gas exchange, the lung and its vasculature function optimally at the level of oxygen in air. For a description of normal lung structure and function in relation to oxygen exchange, the excellent monograph by Weibel [1] is recommended. This chapter reviews the effect of breathing a higher than normal atmospheric level of oxygen on the vasculature of the lung. The earth’s atmosphere contains 20.99% oxygen, and at this concentration efficiently sustains life (cell respiration and metabolism). The rise in oxygen to this level is estimated to have begun with photosynthesis over two billion years ago, and a fascinating relationship subsequently developed between fluctuations in the atmosphere and stages of biological evolution, culminating in mammals with air-breathing lungs [1–3]. Photosynthesis by green plants produces some 4.3 × 1011 tons of atmospheric oxygen per year, and 99.99% of annual production is utilized by earth and its inhabitants, a fortuitous balance between generation and utilization being maintained [3]. The generation of atmospheric oxygen levels has been paralleled by the development of protective cellular antioxidant mechanisms [4, 5], without which life could not be sustained [3]. In his historical review of the discovery of oxygen, Balentine [3] credits Priestley in 1775 and Scheele in 1778 [3] with independent discoveries of oxygen as an atmospheric gas, and Lavoisier between 1773 and 1785 [3] as the first to demonstrate the clinical and pathological effects of oxygen toxicity – although he attributed his findings to an accumulation of carbon dioxide rather than the direct effect of oxygen. In 1874, Bert demonstrated the toxicity of oxygen but did not show its effects on the lung since he studied the response to hyperbaric hyperoxia, which induces mainly neurological changes [3]. Later studies demonstrated the toxic effects of breathing a high oxygen tension on lung tissue, mainly in

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_51, © Springer Science+Business Media, LLC 2011

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response to normobaric hyperoxia, as differences began to emerge between the response of an organ, or tissue, and how the oxygen was administered. Higher than normal partial pressures of oxygen can be administered in an atmosphere of reduced total barometric pressure (hypobaric hyperoxia), at sea level (normobaric hyperoxia), or at a total partial pressure of oxygen greater than the total barometric pressure at sea level (hyperbaric hyperoxia). In the late twentieth and early twenty-first centuries, studies of hypobaric hyperoxia related most to aviation and aerospace research and, although hyperbaric hyperoxia became the tool of choice in early studies of the therapeutic effects of oxygen, the use of normobaric hyperoxia has since become an integral part of respiratory care therapy [3]. In the early 1930s, Smith et  al. [6] demonstrated in an in vivo model system that breathing high oxygen injures the adult lung’s vasculature. Prolonged exposure to hyperbaric hyperoxia (PAO2 3,040 mmHg = 4 ATA = 83% O2) induced vasculitis, with endothelial cell proliferation and hyalinization of the walls of large and small pulmonary arteries. Increase in right ventricular weight [i.e. right ventricular hypertrophy (RVH)] suggested mild pulmonary hypertension (PH) [6]. Similar vascular changes were reported after breathing pure (100%) oxygen under hypobaric (PAO2 550 mmHg) and normobaric (PAO2 760 mmHg) conditions [7, 8]. These studies were followed by classic accounts of the effect of normobaric hyperoxia (85–99% O2) for relatively short periods (days to 1 week) on lung capillaries. Morphometric analyses of alveolar–capillary membrane cells demonstrated a striking (50%) fall in the number of endothelial cells, hypertrophy of ones remaining, and increased numbers of interstitial cells [9–12]. Studies with germ-free rodents (rats) confirmed that the high alveolar oxygen level is the direct cause of injury [13]. These early reports, and additional quantitative studies of pulmonary artery [14, 15] and venous [16] changes, first demonstrated that breathing very high oxygen tensions decreases the number and size of lung capillary networks and, consistent with the development of PH, affects small pulmonary vessels. The models discussed in detail later were developed in the 1980s [14, 15] as a tool to better understand vascular remodeling in response to cycles of breathing high oxygen, or high oxygen and then air (Fig. 1a); and to improve understanding of the pathobiologic process of vascular changes that characterize the adult lung in PH (Fig. 1b, c). These changes significantly restrict the distal vascular segments of the lung (arteries, capillaries, and veins) that form its microcirculation. Here the wall structure of the vessel population is heterogeneous in terms of the distribution of smooth muscle cells (SMCs), i.e. some of the vessels have a nonmuscular wall, whereas others have a muscular or partially muscular wall (see later). The nonmuscular vessel population normally predominates, but as wall muscle increasingly develops in

R.C. Jones and D.E. Capen

PH, the vessel ­population shifts to one that is more muscular. In addition, some distal vascular segments are occluded or lost. Section  2 reviews these changes in small vessels and capillaries of the normal lung after breathing high oxygen: these exemplify microvascular changes in the clinical PHs [14–20] (Fig. 1b). Section 3 addresses vascular changes that occur in response to air breathing after high oxygen has remodeled the lung’s vasculature: these highlight exquisite readjustments to lung vascular structures, triggered by a fall in the alveolar oxygen tension [15, 21] (Fig. 1c). Section 4 outlines the role of the alveolar oxygen tension as a regulator of vascular density, and Section 5 links to vascular remodeling in human disease and the potential for therapeutic intervention. Overall, these studies highlight the striking degree of plasticity of the lung’s vascular bed in matching vascular density to the ambient alveolar oxygen tension.

2 Lung Vascular Remodeling in Hyperoxia-Induced PH In vivo model systems using tensions close to that of pure oxygen (99–100%) at normobaric pressure provide critical data for the acute stages of lung injury by high oxygen, but these tensions are fatal within 48–72 h. To induce the vascular changes of PH, slightly lower (sublethal) tensions (approximately 80–90%) breathed for prolonged periods (of 1–4 weeks) are optimal. The studies reviewed here are based on an in vivo (Sprague-Dawley) rat model [14, 15, 22]. In the rat, normal vessel wall structures [23, 24], including the presence of conventional and supernumerary vessels (see Chapter 3), closely resemble ones in the human lung, as do the vascular changes in PH [14, 15, 21, 25, 26]. Typically, during the first days of breathing 80–90% oxygen, large areas of the alveolar–capillary membrane are disrupted: swollen, necrotic, and apoptotic capillary and vessel endothelial, and epithelial (type 1 and type 2), cells are evident in these areas and scattered throughout still intact regions of the membrane. On the vascular side, interstitial edema follows loss of endothelial cell junctional complexes, and gaps, as capillary endothelial cells retract by the rearrangement of their actin filaments, and by loss of adhesion molecules present in focal adhesion plaques and along their membrane surface [27]. Protein-rich fluid tracking between the basal surfaces of capillary endothelial and epithelial cells further widens the interstitium throughout the membrane. Gradually, in the face of a consistently high oxygen tension, the membrane starts to reform as endothelial (and epithelial) cells adapt via intracellular antioxidant mechanisms [4, 5, 28]. By days 4–7, as the attenuated, dendritic-like, processes of adjacent capillary endothelial cells again form contacts, continuity is restored to even the most disrupted regions of the membrane. As the membrane

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Fig. 1  In vivo models of high oxygen-induced pulmonary hypertension (PH) and pulmonary vascular remodeling. (a) Rodent (rat) lungs are studied after breathing 87% O2 at normobaric pressure (normoxia– hyperoxia cycle, for up to 4 weeks) or after similarly breathing 87% O2 for 4 weeks, followed by lower oxygen levels over 1 week (weaning), and then air for up to 4 weeks (hyperoxia–normoxia cycle, for up to 9 weeks). Additional lungs are studied after 8 weeks of air breathing (not shown). Changes in distal lung vessels and capillaries are assessed quantitatively and qualitatively by brightfield and by transmission microscopy. Similar capillary changes occur in the mouse lung in (b, i) and (c, ii) but in this species perivascular cell development and rise in pulmonary artery pressure (PAP, see b) are not as robust as in the rat. Note also that in the mouse there are important inter-strain variations in susceptibility to high oxygen [159, 160] and the ability to mount an angiogenic response [161]. (b) High oxygen induces vascular changes leading to PH that exemplify the vascular and hemodynamic changes in clinical PH. Consistent with its function as a high flow-low pressure system, the normal adult human lung has a complex and highly dense arrangement of distal (intra-acinar/pre-capillary) arteries and (intra-acinar/post-capillary) veins: in clinical PH, as in the model described, wall overgrowth by perivascular cells results in lumen restriction in these segments. Both artery and venous segments narrow but particularly arterial segments draining blood to the capillary bed: when widespread, veno-occlusive changes prevent capillary outflow. Loss of functional (i.e. perfused) vascular segments by

injury and obliteration further restricts distal lung vessels. As these structural changes impede blood flow through small arterial segments (less than 1 mm in length), pulmonary vascular resistance (PVR) and PAP rise, and, working against the increase in PAP, the right ventricle hypertrophies (RVH). As hemodynamic and structural changes increase blood flow and shear stress, i.e. the tangential pressure exerted on the vessel wall, this promotes flow-related injury to the walls of still patent vessels, and contributes to the process of vascular restriction. Whereas in normal lung vascular function (at rest) can be maintained when vascular volume is significantly reduced (as by resection), in the injured lung the loss of vascular volume created by diffuse restriction of small vessels severely reduces the lung’s ability to maintain adequate function. (c) High oxygen followed by air induces divergent patterns of vessel and capillary remodeling. Wall overgrowth by developing perivascular cells initially continues, and PAP and RVH further increase (week 6, i.e. after 4 weeks in hyperoxia, 1 week of weaning, and 1 week in air). Capillary growth is marked throughout the period in air (weeks 6–9, i.e. after 4 weeks in hyperoxia, 1 week of weaning, and up to 4 weeks in air), and the number of patent (perfused) vessels increases (the term “density” here denotes a qualitative assessment of distribution rather than number of capillaries/vessels per unit area of lung). As the vascular density approaches normal, perivascular cell loss (weeks 7–9, see above) results in vessel wall thinning, and with restored patency comes a decrease in high flow/ shear stress, PAP and RVH

re-forms, however, perivascular cells develop in the walls of small intra-acinar arteries and veins (less than 100 mm in external diameter, ED), and the overall number of small arteries, veins, and capillary networks falls. In response to this restriction, and to the associated hemodynamic changes that lead to increasing pulmonary artery pressure (PAP), perivascular cells also develop in the large (preacinar) arteries [14, 15, 17]. The extent of restriction of the distal lung vascular networks relates directly to the oxygen tension breathed and the duration of the exposure [14, 15]. Hemodynamic measurements (assessed via indwelling pulmonary and femoral artery catheters) confirm that the above-mentioned vascular changes increase pulmonary vascular resistance (PVR) and PAP [14, 21]. Raised PVR in the lung after high oxygen (to almost twice normal) [14] is a mechanical factor to which the upstream vascular bed and right ventricle must adapt: at maximum, PAP increases from approximately 19 mmHg in the normal lung to ­approximately

64  mmHg after 4 weeks in high oxygen [21]. RVH – as judged by the weight of the right ventricle to that of the left ventricle and septum – provides additional evidence of increased peripheral resistance [15, 16]. Angiograms (arteriograms and venograms) provide a broad overview of the degree of vascular loss in relation to the high oxygen tension breathed [14, 15, 21]. By the late stage, major conducting arteries and veins have narrowed, and there is widespread loss of small arteries and veins (less than 160 mm in diameter) when compared with normal lung (Fig. 2). Analysis of vessel profiles confirms the contribution of vessel wall thickening, and vessel loss, to the reduced filling patterns evident by angiography (Figs. 3, 4). Hence, breathing high oxygen for several weeks remodels the normal vascular bed from the hilum to the periphery, particularly those vascular segments that feed and drain the capillary bed. Of note, although in larger arterial segments (axial and lateral preacinar branches 1–6) the remodeled

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Fig.  2  Pulmonary arteriograms and venograms of normal lung (a, b and e) and of the lung after high oxygen for 28 days (c, d, and f). Note the extent of filled distal arteries in the normal lung (a, b) and loss of distal filled arteries after breathing high oxygen (c, d). Similarly, when compared with the dense filling of veins in the normal lung (e), the lateral pathways of veins are narrow after high oxygen, and the filling of distal veins is markedly reduced (f). Angiograms are prepared after the pulmonary artery (or pulmonary venous) bed is distended with a barium sulfate– gelatin mixture, and the airways are distended with fixative – each

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infused under standard pressures via catheters in the main pulmonary artery (or vein) and trachea [14, 15, 22]. The barium mixture fills large and small arteries (or veins): vessels more than 160 mm in diameter are identified as separate lines on the angiograms; smaller vessels form a fine background haze [14]. Original magnification: ×1.8 (a, c); ×3.6 (b, d); ×2.2 (e, f). Note for these and subsequent images, magnifications indicate the original image size by brightfield or transmission microscopy. (a–d Reprinted from [21] with permission. (f) Reprinted from [16] with permission from the American Society for Investigative Pathology)

vasoactive mediators, the basis of this likely being an increase in thickness and amount of wall muscle in preacinar vessels, and in intra-acinar pre- and post-capillary vessels, rather than alterations in vascular geometry, such as reduced vessel number or narrowed lumen diameter [30]. Since narrowing and occlusion of distal vessel segments (by the development of new perivascular cells), and vessel and capillary loss, form the structural basis of distal vascular restriction and the development of hyperoxia-induced PH, the cellular basis of these changes is described in detail.

Fig. 3  Photomicrographs of distal pulmonary arteries in normal lung and after high oxygen for 28 days. Thin-walled artery (external diameter 72 mm) with a single elastic lamina in normal lung (a) and a thick-walled artery (external diameter 53 mm) with a newly developed medial layer of smooth muscle cells (SMCs) between an internal and an external lamina, in the lung after high oxygen (b). In both arteries the lumen is distended with barium sulfate–gelatin. Paraffin sections (4 mm-thick) stained with Miller-elastic van Gieson stain. Original magnification: ×640 (a, b). (Reprinted from [15] with permission from the American Society for Investigative Pathology)

wall is more muscular, it fails to generate more force; it is also stiffer, independent of the degree of collagen present [29]. The vascular bed of the isolated (and perfused) high oxygen-injured lung is also more reactive to challenge by

2.1 Perivascular Cell Development in Distal Pulmonary Vessels and Capillaries Depending on their normal location within a vessel segment, new perivascular cells develop [18, 21, 32–35] in the high oxygen lung from existing vascular cells (intermediate cells and rare SMCs; see later) and/or by the recruitment of nonvascular cells (interstitial fibroblasts present throughout the alveolar–capillary membrane) to vessel walls. As these cell types transition to a contractile phenotype, circumferential layers of SMCs appear in normally “nonmuscular” lung vessel segments (see later). The new cells result in

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Fig. 4  Photomicrographs of distal pulmonary veins in the normal lung and after high oxygen for 28 days. Thin-walled vein (external diameter 23 mm) in normal lung (a) and thick-walled veins (b–d) after high oxygen (external diameters 27, 28, and 34 mm, respectively). The pulmonary venous bed is perfused with barium sulfate. Note the presence of well-developed elastic laminae and lumen narrowing by new cell layers (b–d). Paraffin sections (4 mm-thick) stained with Verhoeff–elastic van Gieson stain. Original magnification: ×1,375 (a, b); ×1,594 (c, d) (c Reprinted from [16] with permission from the American Society for Investigative Pathology)

wall overgrowth (with up to a tenfold increase in wall thickness). In the normal lung the distribution and type of perivascular cells matches vessel size and location, i.e. few ­capillaries (7–14 mm diameter) have these cells, adjacent vessels (18–30 mm diameter) have more, and their number and organization then increase with vessel size. SMCs and adventitial fibroblasts form the perivascular cell layers ­supporting the endothelium until, at the level of the respiratory bronchiole (at the entrance to the acinus), or at a more distal point within the acinus (in alveolar ducts or in the alveolar wall), most of the SMCs change pitch to form a spiral before they, and the adventitial fibroblasts, disappear from the vessel wall (see Chapter 3). Hence, only rarely do distal segments in the normal lung retain a thin but full SMC layer: most distal arterial and venous segments have a nonmuscular wall or a partially muscular wall (with a nonmuscular region in those segments having a SMC spiral). Throughout the normal vascular bed, the thickness of a vessel wall typically decreases as lumen diameter falls. An exception is found in the small (so-called) resistance vessels located near the entrance of the acinus, where wall thickness is normally high for lumen size (see Chapter 3). Penetrating their basal laminae, SMC processes form lateral homeotypic (SMC–SMC) contacts; and, penetrating the basal lamina of adjacent endothelial cells, heterotypic ones (SMC–endothelial cell). In some segments (particularly along the axial arterial pathway – which splits into two at the entrance to the acinus [31]), intermediate cells form an additional layer beneath the endothelium. These cells [18, 24, 26, 31] fall between a SMC and a pericyte in phenotype and in their location in the vascular branching pattern. Typically, they are encased in a separate basement membrane and have some filaments, but lack the dense ­bodies and hemi-desmosomes of mature SMCs. Approaching

the capillary bed, endothelial contacts increase in number, and their processes penetrate the basal lamina to contact the surrounding perivascular cells, i.e. pericytes. Pericytes share the capillary endothelial basement membrane and typically have few if any contractile filaments.

2.1.1 Cell Proliferation Early in the response to high oxygen, new perivascular cells develop by proliferation [17, 36]. Specific in the timing of the response, a marked burst of proliferating endothelial cells (day 7) follows one of intermediate cells and interstitial fibroblasts (day 4) (Fig. 5a). Some of the small number of SMCs present also proliferate (day 7). Each cell type thus contributes to the new perivascular cell layers developing at this stage [17, 36]. Although an occasional proliferating cell is evident later, activity then falls (days 7–28) [17]. Although increased numbers of proliferating fibroblasts and capillary endothelial cells are also present early (day 4) within the alveolar–capillary membrane (Fig.  5b), later (days 7–28) proliferating endothelial cells alone increase in number [36].

2.1.2 Cell Recruitment The fibroblasts recruited as perivascular cells (Fig. 6) develop a (polarized) leading cell edge, migrate toward the organizing vessel wall, and, by extending lamellapodia along their adluminal edge, align circumferentially around a vessel. The formation of an external elastic lamina incorporates the aligned cells into the vessel wall [18, 19, 21]. In the absence or presence of low numbers of proliferating cells, perivascular cells

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Fig. 5  Early development of new vascular wall cells by proliferation. Photomicrographs of autoradiographs illustrating (3H)-thymidine labeled fibroblasts in a distal lung vessel (a) and in an endothelial cell after high oxygen (a, inset). Images are inverted to highlight silver grains identifying labeled cells. The fibroblasts lie close to an alveolar wall vessel (external diameter approximately 65 mm): these cells are interstitial fibroblasts that become vascular (SMC) precursors. Quantitative analysis, i.e. the number of labeled cells per 1,000 cells of the same type (entering the S phase of the mitotic cycle) in vessels (external diameter less than 100 mm), demonstrates peak labeling of fibroblasts at this time (data not shown). In similar vessels, peak labeling of endothelial cells (a, inset) occurs later. Similar quantitative analysis of alveolar–capillary membrane cells shows increased numbers of proliferating fibroblasts and endothelial cells early (day 4) in high oxygen (b); later (days 7–28), proliferating endothelial cells alone increase in number [36]. To overcome the change in the distribution of cell types as vessel walls remodel in high oxygen,

the number of labeled and unlabeled nuclei of endothelial cells, subintimal precursor SMCs, medial SMCs, and fibroblasts were noted, related to unit length of vessel wall, and were expressed as the labeling index for each cell type (number of labeled cells/total number of cells of the same type × 1,000). In the alveolar–capillary membrane, the main cell types were counted as endothelial cell (EC), interstitial fibroblast (FB), epithelial cell (EP), or infiltrating cells in the alveolar space, i.e. monocyte (MONO) or pulmonary alveolar macrophage (PAM), and the number of labeled cells expressed per square millimeter of the alveolar region. These cell populations were analyzed in sections from lungs prepared following perfusion of the pulmonary circulation and airways with fixative (after ligating the main pulmonary vein to retain fixative in the lung); in these sections, small vessels are not classed as an artery or vein [18, 22]. Resin section (2 mm-thick) stained with toluidine blue (a). Original magnification: ×1,000. (b Reprinted from [36] with permission. Official journal of the American Thoracic Society copyright American Thoracic Society)

Fig.  6  Perivascular cell development in distal lung vessels in high ­oxygen after 4 or 7 days. In high resolution images intermediate cells are identified from recruited fibroblasts. In most alveolar vessels of normal lung (day 0), endothelial cells (EC) and intermediate cells (IC) alone form the vessel wall (a, external diameter 20 mm). Both intermediate cells and fibroblasts form perivascular cell layers from day 4, and (as shown) at day 7, fibroblasts (asterisk) are seen enveloping vessels (b, external diameter 12 mm), and aligning (c–e, external diameter 24 mm). The processes of an

aligning cell (c, see box) are shown at higher magnification (d, e). Several ­fibroblasts (asterisks) are aligning around endothelial cells in the vessel shown (f, external diameter 20 mm). IS interstitial space, IFB interstitial fibroblast, Alv alveolus, Cap capillary. Resin sections (80 nm-thick) stained with uranyl acetate and lead citrate (a–f). Original magnification: ×2,618 (a); ×2,880 (b); ×1,000 (c); ×3,541 (d); ×15,120 (e); ×6,570 (f) (a Reprinted from [35] with permission. d, f Reprinted from [18] with permission from the American Society for Investigative Pathology)

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Fig.  7  Perivascular cell development in distal lung vessels in high oxygen after 28 days. Note the absence of perivascular cells in the vessel profile from normal lung illustrated by brightfield microscopy (a, external diameter 27  mm), and the number of perivascular cells narrowing the lumen in the vessel after high oxygen (b, external diameter 32  mm, arrowheads). By high resolution microscopy the single perivascular cell (c, open arrow) present in the nonmuscular alveolar wall vessel of a normal lung is identified as an intermediate cell (c, external diameter 25  mm). In an alveolar wall vessel (d, external diameter 16 mm) after high oxygen the processes of perivascular cells (asterisks) lie adlu­minal to a well-defined electron-dense elastic lamina

(el) and abluminal to endothelial cells (EC). Microfilaments are heavily distributed throughout cell processes within the wall. Numerous vesicles line the edge of a sub-endothelial cell process, adluminal to the forming elastic lamina, in another alveolar wall vessel of similar size (e, external diameter 23 mm, arrows). Resin sections (2 mm-thick) stained with toluidine blue (a, b). Resin sections (80 nm-thick) stained with uranyl acetate and lead citrate (c–e). EC endothelial cell, IFB interstitial fibroblast, Ep2 epithelial type 2 cell, Alv alveolus. Original magnification: ×400 (a, b); ×1,412 (c); ×2,919 (d); ×15,428 (e) (c–e Reprinted from [18] with permission from the American Society for Investigative Pathology)

continue to develop (days 7–28, Fig.  7a–d): quantitative ­analysis confirms their low number in vessel profiles of the normal lung (less than 5%) and increasing number in high oxygen, i.e. in approximately 40–80% of profiles in the early stage (days 4–7) and in 100% later (day 28) [18]. Intermediate cells disappear as they transition to SMCs (approximately days 14–28), and recruited fibroblasts continue as the major source of perivascular cells [18, 34, 35]. Perivascular cells also develop in capillary walls after high oxygen. Interstitial fibroblasts (distinct from capillary pericytes by their lack of basement membrane) are seen enveloping capillary structures (Fig. 8), indicating a role for these cells as a pericyte precursor in the lung, as in mesenteric capillaries [37].

2.1.3 Neomuscularization (Precursor Cell Transition to SMC) The intermediate cell and recruited fibroblast transition to a SMC phenotype by synthesizing proteins and the organelles characteristic of contractile cells, i.e. extensive a-smooth muscle actin and smooth muscle myosin heavy chain filament networks, and dense bodies and hemi-desmosomes (Fig. 9c–g). The intermediate cell rapidly acquires more filaments and SMC organelles. The fibroblast begins this transition when aligning and continues to acquire the organelles of a SMC as it integrates into the vessel wall [18, 19]. A new internal elastic lamina forms to separate the cells from endothelium, and the original lamina (now external) marks the outer margin of

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Fig. 8  Development of perivascular cells in pulmonary capillary walls after high oxygen for 28 days. Note that in the relatively large pulmonary capillary shown (external diameter 14 mm, a, b) the perivascular cell (a, boxed area) is a pericyte (asterisk) which shares the basal lamina of the endothelial cell (EC) and possesses few microfilaments (b). The peg and socket contact between the two cells is characteristic (b, white arrow). Endothelial cell junctions (arrows) are clearly delineated, as is the elastic lamina (el) which lies adluminal to interstitial fibroblast (IFB) processes. In a smaller pulmonary capillary (external diameter 11 mm, c and inset in c) endothelial cells (EC) and a pericyte (Pc) form

the wall and the extended processes of an interstitial fibroblast (IFB) are seen enveloping the capillary wall. Unlike the pericyte, which shares the endothelial basal lamina (small double arrows), a lamina is absent from the fibroblast processes. Ep epithelium, col collagen. Resin sections (80 nm-thick) stained with uranyl acetate and lead citrate (a–d). Original magnification: ×3,042 (a); ×15,050 (b); ×10,183 (c, d); ×3,643 (inset) (a, b Reprinted from [18] with permission from the American Society for Investigative Pathology. (c) and inset in (c) Reprinted from [19] with permission from the European Respiratory Society Journals Ltd)

the vessel wall [18]. Other fibroblasts that continue to align as perivascular cells after an external lamina has developed become adventitial cells [19] and synthesize collagen (types I/III). Hence by phenotypic transition, intermediate cells and fibroblasts give rise to SMCs, forming a medial layer between an internal and an external lamina (Fig. 9a, b). The development of SMCs in distal vessel segments, where these cells normally are rare or absent from the wall, is illustrated by the analysis of artery profiles, high oxygen increasing the number of arteries with a muscular or partially muscular wall at the expense of nonmuscular ones [14, 15] (Fig.  10). Similarly, more small veins (less than 150 mm ED) are narrow and have a muscular or partially muscular wall, whereas the larger veins are spared. For example, the proportion of the vein population with SMCs in their wall increases thirteen-fold in the smallest group (less than 25–50 mm ED), threefold in a

larger group (50–100 mm ED), and less than onefold in the largest veins (less than 100–150 mm ED) [16]. In many veins, SMCs develop adjacent to endothelium (without the formation of a separating elastic lamina) – at the level where the overall restriction in cross-sectional area of the vascular bed in high oxygen has likely increased the velocity of venous blood flow. The restriction of small veins may so impede distal blood flow that it contributes to the increase in peripheral resistance.

2.2 Loss of Distal Pulmonary Vessels and Capillaries As wall overgrowth occludes the lumen of artery and venous segments in high oxygen, vessel remnants appear within the

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Fig.  9  Development of SMCs in distal lung vessels after high ­oxygen for 28 days. Alveolar wall vessel with SMCs forming a medial cell layer after high oxygen (a, b, external diameter 20 mm): the medial cells and cell processes (asterisks) lie adluminal to a thick external electron-lucent elastic lamina (a, large arrowheads) and abluminal to endothelial cells (EC). An electron-lucent internal lamina (a, small double arrows) separates the cells from endothelium. At higher magnification (b), SMCs forming the vessel wall have a ­well-developed arrangement of intracellular filaments (asterisks), subplasmalemmal densities (i.e. attachment plaques, arrows), and basal lamina (arrowheads). P platelet, le leukocyte. Inverted images of cells in alveolar vessels (c–g), highlighting (10 nm) protein A (pA)gold-labeled antigenic sites for a-smooth muscle actin (a-SMA) (c–e) and smooth muscle myosin heavy chain (SM-MHC) (f, g). In normal lung (c, external diameter 25  mm, see inset), fine arrays of a-SMA filaments decorate a sub-endothelial cell (asterisk), whereas in the associated endothelial cell (EC) a-SMA localizes to the

nucleus. After high oxygen (a, e), dense a-SMA filaments now decorate a sub-endothelial cell (d, external diameter 31  mm, see arrow in inset) and a SMC (e, external diameter 20 mm, see arrow in inset). Similarly, in normal lung (f, external diameter 29  mm, see inset), SM-MHC (SM-B isoform) decorates a perivascular cell (asterisk) of a similar alveolar wall vessel (external diameter 41 mm); after high oxygen (g, external diameter 33  mm, see inset) the cell illustrated is heavily labeled with antigenic sites (asterisks, and see arrow in inset) for SM-MHC (SM-B isoform). Resin sections (80 nm-thick) stained with uranyl acetate and lead citrate (a–g). Original magnification: ×8,757 (a); ×28,673 (b); ×49,041 and ×3,541 (c); ×,877 and ×1,416 (d); ×74,057 and ×1,416 (e); ×9,818 and ×1,167 (f); ×19,636 and ×2,090 (g) (a, b: Reprinted from [18] with permission from the American Society for Investigative Pathology. c–e Reprinted from [32] with permission. Official journal of the American Thoracic Society copyright American Thoracic Society. f, g Reprinted from [33] with permission)

alveolar–capillary membrane [14–16] (Fig.  11). These increase in number in relation to the length of time breathing high oxygen and to the degree of fibrosis (Fig.  11a). Quantitative data (expressed as the ratio of the number of arteries to the number of alveoli) highlights, in particular, the loss of intra-acinar arteries from the distal lung [14, 15]. Capillary networks also decrease in number at this time (Fig. 12a, b). Avascular alveolar structures (not seen in normal lung) are present, indicating where capillary segments have been effaced from the membrane (Fig.  12f, g). These are formed by the thin opposing processes of epithelial type 1 cells with or without an interstitial fibroblast (or single endothelial cell), and/or fine collagen deposits, between [21]. As capillary loss proceeds, the alveolar–capillary membrane includes a mixture of patent residual capillaries, some close to

normal in size (Fig. 12c) and others short and only one fourth of the normal diameter. Trapped erythrocytes are evident in residual segments partially occluded by hypertrophied endothelial cell processes (Fig. 12d). Despite a striking degree of lumen narrowing (Fig.  12e), the absence of cell necrosis indicates that flow of plasma through these ­segments is still sufficient to maintain viable endothelial cells [38].

3 Lung Vascular Remodeling in Air After High Oxygen Return to air breathing after weeks of breathing high oxygen requires gradual weaning through lower oxygen tensions to prevent dyspnea, cyanosis, and asphyxia [15]. The ­immediate

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the number of filled segments then continues to increase with time. Because of the wall remodeling occurring in high oxygen, at the start of weaning and air breathing, almost all distal vascular segments already have either a partially muscular wall or a muscular wall. Divergent patterns then occur as vessel walls thicken further by cell acquisition and then thin by cell loss. The pulmonary capillary networks effaced in high oxygen undergo a marked growth response. The veins, less affected than pulmonary arteries in this model, have not been analyzed after air breathing.

3.1 Perivascular Cell Development in Distal Pulmonary Vessels and Capillaries Fig.  10  Quantitative analysis of SMC development in distal artery profiles (classed by location and wall structure) after high oxygen for 28 days. Comparison of vessel profiles by size and wall structure in the normal lung and after high oxygen is possible after distension of pulmonary arteries (or veins) with barium sulfate–gelatin. As shown, artery profiles within the acinus are associated with respiratory bronchioli (RB), alveolar ducts (AD1–3) or lie within the alveolar wall (AW). When classed by wall structure, i.e. the presence or absence of SMCs, artery (or vein) profiles are muscular (M), partially muscular (PM), or nonmuscular (NM). Such analyses demonstrate an increase in the number, i.e. relative percent of the population of muscular and partially muscular artery profiles at the expense of nonmuscular ones, in the lung after high oxygen (day 28) when compared with normal lung (day 0)

response in the high oxygen-adapted lung (with its greatly restricted distal vascular bed) is vasoconstriction [15]. The vascular and hemodynamic response and ­worsening RVH, which follows during weaning and early air breathing (weeks 1 and 2), indicate that the lower tension is sensed as “relative hypoxia” [15, 21]. This triggers pulmonary artery and capillary remodeling in ways that mirror the response of the normal vascular bed to hypoxia (Fig.  13). Of note, whereas structural vessel loss occurs in hyperoxia, any loss appears functional in hypoxia – since (as judged by the absence of vessel remnants) there is little evidence of obliterated vessels in the hypoxic lung [20, 39, 40]. Hemodynamic measurements show that PAP (increased to +∆45 mmHg at the end of high oxygen) falls steadily in air (being –∆20  mmHg after 4 weeks). However, it persists above normal [21]. Weaning accentuates the RVH caused by high oxygen. After 2 and 4 weeks in air, the ratio improves to match the degree of RVH after high oxygen; and later, by 4–8 weeks, the ratio approaches, but does not reach, the normal value [21]. Arteriograms detect an increase in the number of barium-filled vessels (more than 160 mm ED) during air breathing (Fig. 14). Increasing after only 1 week in air,

The early pattern of perivascular cell development during air breathing recapitulates, in part, that seen after breathing high oxygen, in that recruited fibroblasts are their source.

3.1.1 Cell Proliferation Fewer proliferating cells are present in the walls of distal lung vessels during weaning and in the early stage of air breathing than early in high oxygen. After 2 weeks in air, relatively higher numbers of proliferating endothelial cells, fibroblasts, and SMCs are present in distal vessel walls and in large pulmonary arteries [17]. As in the lung after high oxygen, however, the low proliferative activity does not support the large increase in perivascular cells developing in the distal vessel population [21].

3.1.2 Cell Recruitment The elastic laminae (assembled in high oxygen) initially disappear from vessel walls during air breathing [21]. After 1 week in air, elastin fragments are evident and large numbers of interstitial fibroblasts are aligning as vascular cells. In the absence of elastic laminae, they abut endothelial cells, or form additional layers around the SMCs that developed in high oxygen. These additional cells result in vessel walls that are thicker at this time than after high oxygen alone (Fig. 15a, b) After 2 weeks, fibroblast and SMC cell processes form complex interdigitating networks within vessel walls, and dense plaques indicate an increase in the number of cell attachment sites to the surrounding extracellular matrix. Although elastic laminae have now re-formed, in most vessels the cell layers remain disorganized, indicating loss of

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Fig. 11  Vessel occlusion and loss in the distal lung after high oxygen for 28 days. Occluded vessels, and vessel remnants, are present within the alveolar–capillary membrane after high oxygen (a–d). In the same strain but in different groups of Sprague-Dawley rats studied (with slight variation in their genetic background), the degree of fibrosis varies. Occluded and obliterated vessel remnants develop when (interstitial) fibrosis is absent or minimal; their number, however, closely follows the degree of fibrosis elicited in the lung. Large numbers of occluded vessels and vessel remnants develop when (interstitial and intra-alveolar) fibrosis is moderate to severe (as may occur in the lung breathing air after high oxygen, see Fig. 15a). Perivascular cells narrow (a, c) and occlude (b, d) the vessel lumen, the extent of lumen occlusion being evident along the segments shown in b. High

resolution images identify a remnant in which the lumen is still patent although the wall is greatly disrupted (c): endothelial cells form a discontinuous lining (arrows), necrotic SMCs and proteinaceous material lie between the remains of both an external elastic lamina (double arrowheads) and a fragmented internal elastic lamina (white arrow). In a further remnant (d), the vessel outline is barely detectable (see dotted outline) and disorganized cell layers occlude the original lumen and blur the outline of cell layers. Alv alveolus. Resin sections (2 mm-thick) stained with toluidine blue (a, b). Resin sections (80 nm-thick) stained with uranyl acetate and lead citrate (c, d). Original magnification: ×200 (a, b); ×4,200 (c, d). (Reprinted from [15] with permission from the American Society for Investigative Pathology)

cell–cell contacts during ongoing wall reorganization [21] (Fig. 15c, d).

i­ndicating a slow process of SMC de-differentiation and loss in these segments [15] (Fig. 16, compare with Fig. 10).

3.1.3 Neomuscularization (Precursor Cell Transition to SMC)

3.2 Loss of Perivascular Cells in Distal Pulmonary Vessels and Capillaries

The proportion of muscularized arteries after weaning remains constant, but after 2 weeks in air more distal vessels are muscular and partially muscular than after high oxygen (Fig. 16, compare with Fig. 10). By 8 weeks, nonmuscular arteries increase in number (as a proportion of the population) as SMCs disappear from vessel walls: still more are muscularized at this time than in the normal lung, however, and many have more muscle than after high ­oxygen,

Perivascular cell development ceases as the vascular threshold of the lung is reset, and vessel walls undergo a process of thinning by cell loss (Fig. 17a–d). As cells are lost, elastin fragments (the remnants of laminae) increase in number. After 4 weeks, some vessels still have hypertrophied walls but relatively thin walled vessels now predominate, shifting the vessel population toward the distribution in normal lung.

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Fig. 13  The response of the lung to breathing high oxygen (hyperoxia) versus low oxygen (hypoxia). The pulmonary response to air breathing after high oxygen (relative hypoxia) mirrors the response of the normal lung to hypoxia. Following early edema, an extended period in high oxygen (28 days) leads to pulmonary vessel and capillary remodeling. Subsequently, in the high oxygen-adapted lung, a lower oxygen level represents “relative hypoxia” and causes (pulmonary artery) acute vasoconstriction. In a reverse pattern, acute vasoconstriction typically occurs in the normal lung breathing a lower than normal oxygen level, i.e. hypoxia, and return to breathing air represents “relative hyperoxia” and causes edema

The walls of the thinnest vessels [21] consist only of ­endothelial cells and an occasional SMC that expresses a synthetic rather than a contractile phenotype.

3.3 Restoration of Distal Pulmonary Vessel and Capillary Networks

Fig. 12  Pulmonary capillaries are narrowed or lost after high oxygen for 28 days. Illustration of a normal capillary bed with patent capillaries (a, arrows). After high oxygen, many avascular zones that lack a capillary structure (b, double arrows) are present. These structures later act as templates for re-forming capillaries (see Fig. 18a), ensuring that the structure of the restored alveolar–capillary membrane is close to normal (see Fig. 18b). The newly forming networks are thus very different from less structured arrangements of capillaries that form as part of an inflammatory response, in bronchiectasis, in capillary overgrowth, and in capillary malformations in hereditary hemorrhagic talengiectasia. High resolution images show that the open lacy capillary networks of normal lung are lost after high oxygen (c–g, asterisks denote endothelial cell ­processes). Some patent segments, identified by the presence of circulating ­erythrocytes, persist (c); the lumen of other segments is narrowed by the apical processes of opposing endothelial cells (d, asterisks), is occluded (e), or is replaced by collagen deposits (f). Regionally, large areas of the ­membrane consist of the processes of back-to-back

Although some vessels occluded in high oxygen re-open during air breathing, vessel remnants persist (Figs.  15a–g, 17f). Those vessels in which a lumen is re-established contribute to the increasing number evident on arteriograms after 2 and 4 weeks (Fig. 14). Most are considerably larger (more than 160 mm ED) than ones assessed in the alveolar– artery ratio (less than 100 mm ED), which confirms that the number of smallest arteries is not restored during air breathing [15]. Since arteriograms reveal the gross number of filled vessels through the full thickness (three-dimensional volume) of the lung, their presence in its thinnest region, at the lung edge, may obscure the small vessel loss detected in tissue sections by the ratio. Thus, although air breathing restores lumen patency to a significant number of preacinar Fig. 12  (continued) epithelial type 1 cells with collagen and fibroblast processes between and no residual capillary segment evident (g). Alv alveolus, Ep1 epithelial type 1 cell, p platelet, FB fibroblast, col collagen. Resin sections (2 mm-thick) stained with toluidine blue (a, b). Resin sections (80 nm-thick) stained with uranyl acetate and lead citrate (c–g). Original magnification: ×200 (a, b); ×4,075 (c, e); ×5,268 (d); ×12,000 (f, g). (c–e Reprinted from [21] with permission)

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Fig. 14  Pulmonary arteriograms in the lung breathing air after high oxygen for 28 days show the degree of vessel patency restored: high oxygen plus 7 days of weaning (a, b), high oxygen plus 7 days of weaning plus 2 weeks in air (c, d), and high oxygen plus 7 days of weaning plus 4 weeks in air (e, f). More small filled arteries are seen along the lateral branches 2 and 4 weeks after air breathing than at

the end of weaning. Quantitative analysis demonstrates, however, that even after 4 weeks the number of patent vessels remains below the normal density. Original magnification: ×1.8 (a, c, e); ×3.6 (b, d, f). (a Reprinted from [15] with permission from the American Society for Investigative Pathology. c–f Reprinted from [21] with permission)

Fig. 15  Remodeling of distal pulmonary vessels in the lung breathing air after high oxygen for 28 days. Occluded alveolar vessel (a, external diameter 47  mm) and remnant (b) after high oxygen plus 7 days of weaning. Residual regions of the lumen lined by endothelial cells persist. Occluded alveolar vessel after high oxygen plus 7 days of weaning (c, external diameter 114  mm). A concentric arrangement of cells is present around an almost completely occluded lumen – the internal lamina and traces of erythrocytes mark the residual lumen. In a still patent alveolar wall vessel (d, external diameter 56 mm) after high oxygen plus 7 days of weaning plus 1 week in air, the lumen is largely narrowed by eccentric cell layers. High resolution image of a similar vessel after

high oxygen plus 7 days of weaning plus 1 week in air (e,  external diameter 20  mm). The lumen is narrowed by swollen (nucleated) endothelial cell (EC) processes, and fibroblasts are aligning as vascular cells (asterisks). After 2 weeks in air, the walls of some patent alveolar vessels reorganize by thinning (f, external diameter 31 mm, arrowheads indicate external elastic lamina) or remain occluded (g, external diameter 36 mm). mc mast cell, Alv alveolus. Resin sections (2 mm-thick) stained with toluidine blue (a–d, f, g). Resin section (80 nm-thick) stained with uranyl acetate and lead citrate (e). Original magnification: ×200 (a, b); ×400 (c, d); ×3,456 (e); ×630 (f, g). (f, g Reprinted from [21] with permission)

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Fig.  16  Quantitative analysis of SMC development in distal artery profiles (classed by location and wall structure) in the lung breathing air after high oxygen for 28 days. Artery profiles are quantified as in Fig. 10. Analysis confirms the progression of wall thickening by SMCs. Relatively more alveolar muscular and partially muscular artery profiles are present in the lung after high oxygen plus 7 days of weaning plus 2 or 8 weeks of recovery in air than after high oxygen alone (see Fig. 10). (Reprinted from [15] with permission)

vessels, and to some large intra-acinar vessels (more than 160 mm ED), the smallest lung vessels lost in high oxygen are not restored in a significant number. Residual capillary structures in the alveolar–capillary membrane increase in length and diameter as their lumen re-opens (Figs.  18a, b). Endothelial cells contact the processes of adjacent endothelial cells to form new capillary structures and single cells form seamless capillaries. With time, as capillaries increase in density, the presence of erythrocytes indicates functional networks. These networks continue to form until (by week 4 in air) no avascular alveolar structures are evident. The first capillary segments forming are simple endothelial tubules and these increase in complexity with time until they closely resemble the lacy networks of normal lung [15, 21] (Fig. 18c–e).

4 Alveolar Oxygen Tension as a Vascular Regulator The previously described studies demonstrate that the adult lung responds to excess iatrogenic oxygen by trimming its vascular networks, and to insufficient oxygen (exchange) by expanding them. Concurrently, perivascular cells develop in large numbers as the vascular density falls below normal, and are deleted on increase in the vascular density to a more normal distribution. Weibel [41] defines the link between lung structure and function by the principle of symmorpho-

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sis, i.e. “a state of structural design commensurate to ­functional needs resulting from regulated morphogenesis,” and further, if this principle holds “that the vascular supply of an organ is commensurate with the needs for blood flow imposed by the metabolic activity of its cells” [42]. This principle appears to apply to the vascular bed remodeling in the adult (and developing) lung. In the embryonic, fetal, and postnatal lung, the programmed patterning of vascular development is regulated by transcription and growth factors, with regression signals removing redundant (unperfused) segments. Vascular networks in the adult lung are also vulnerable to growth and regression signals leading to wall cell gain and loss but are not subjected to the same developmental controls; thus, they refashion in a less ordered response to the interaction of growth factors [43]. Through complex patterns that include cell loss and adaptive responses for cell survival and growth, the vascular networks of the adult lung remodel in response to the alveolar oxygen tension, with vascular density likely regulated to match tissue needs – governed by the principle of symmorphosis. Increasingly it appears that cellular and molecular mechanisms of vascular reorganization in the mature (and developing) lung should be studied in relation to the alveolar oxygen tension, including normoxia, hyperoxia, and hypoxia [44, 45]. As shown by “gain and loss of function” studies in mice, growth factors are essential regulators of blood vessel ­formation [46–50], e.g. platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), placental growth factor, angiopoietin, transforming growth factor-b, fibroblast growth factor, and their cognate receptors, as well as cytokines, chemokines, adhesion molecules, and extracellular matrix factors (see also Chapter 3). Perturbation of these signaling systems leads to malformed vascular structures. These signaling pathways are proving relevant to ones for vascular wall assembly and network formation in the adult lung. Each has a multifunctional rather than a singular role in vessel and capillary wall assembly, and is modulated in a coordinate manner to construct functional networks [51–56].

4.1 Cellular and Molecular Mechanisms of Perivascular Cell Development and Loss The PDGFs and their receptors regulate perivascular cell development [57–59]. Each ligand (i.e. PDGF-A, PDGF-B, PDGF-C, and PDGF-D) signals through two receptor tyrosine kinases (RTKs, i.e. PDGF-Ra and PDGF-Rb). PDGF-B/PDGF-Rb forms an important signaling system for mesenchymal cell proliferation and migration, and for SMC differentiation [46–48] (see Chapter 3). Furthermore, PDGF signaling drives mesenchymal cell responses in inflammation, and in clinical vascular disorders such as atherosclerosis,

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Fig. 17  Vessel lumen restoration and perivascular cell loss in the lung breathing air after high oxygen for 28 days. After high oxygen plus 7 days of weaning plus 4 weeks in air, cell loss leaves some vessels with walls that are still thick (a, external diameter 33 mm) relative to vessels in normal lung but many now have a wall closer to normal (b, external diameter 38 mm). High resolution images of alveolar wall vessels (c, d), at the same time point, show that in thicker-walled vessels (c,  external diameter 20 mm) the endothelium (EC) is relatively thin but SMCs, now characterized by a synthetic rather than a contractile phenotype, remain hypertrophied. Their separation by an internal elastic lamina is minimal (c, arrowhead),

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with endothelial cell and SMC processes focally apposed (white arrow). In a thinner-walled vessel (d, external diameter 20 mm), where the wall is close to normal in structure, endothelium (EC) and a subintimal cell process (white asterisk), with fibroblasts and elastin deposits (double asterisks), form the wall. Thick-walled vessel segments in the process of reopening at this time are illustrated (e, see arrows) and occluded segments persist (f). Alv alveolus. Resin sections (2 mm-thick) stained with toluidine blue (a, b, e, f). Resin sections (80 nm-thick) stained with uranyl acetate and lead citrate (c, d). Original magnification: ×630 (a, b); ×9,818 (c, d); ×200 (e, f). (a–d Reprinted from [21] with permission)

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Fig. 18  Pulmonary capillaries re-form in the lung breathing air after high oxygen for 28 days. Gradual restoration of patent capillary networks is evident: high oxygen plus 7 days of weaning plus 1 week in air (a), and high oxygen plus 7 days of weaning plus 4 weeks in air (b). After 4 weeks in air, more segments are patent (b, arrows) and the more complex (lacelike) structure of a normal alveolar–capillary membrane (see Fig. 12a) is restored. High resolution images demonstrate increased numbers of extended tubular capillary structures after high oxygen plus 7 days of weaning plus 1 week in air (c), and after high oxygen plus 7 of days weaning plus 2 weeks in air (d). After 4 weeks in air, the re-forming membrane resembles that of normal lung (e). Alv alveolus. Resin sections (2 mm-thick) stained with toluidine blue (a, b). Resin sections (80 nm-thick) stained with uranyl acetate and lead citrate (c–e). Original magnification: ×200 (a, b); ×1,167 (c); ×2,090 (d); ×2,880 (e). (c–e Reprinted from [21] with permission)

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restenosis, PH, and retinal and fibrotic diseases [60]. Endothelial cells direct the migration of mesenchymal cells to become perivascular in a process mediated by PDGF-B [61]. As a corollary to the failed development of perivascular cells in the absence of this signaling, PDGF-B/PDGF-Rb interactions can be expected to promote their development. As a source of perivascular cells in vessel and capillary wall remodeling in high oxygen, interstitial fibroblasts serve a function in the adult lung similar to that of mesenchymal cells in the developing lung. To establish how the PDGF-B/PDGF-Rb signaling pathway is modulated in distal vessels in the high oxygen cycle, and if reciprocal signaling occurs between cells, ligand and receptor levels were assessed in endothelial cells and perivascular cells in the early (days 1–4) and late (days 7–28) stages [34]. To link these findings to the other major PDGF signaling system for mesenchymal cells, PDGF-A and PDGFRa levels were analyzed [35]. Using quantitative high-resolution immunogold labeling approaches, constitutive levels of PDGF-Rb were approximately tenfold higher than those of PDGF-BB in the cells targeted. In high oxygen, PDGF-BB levels did not then change, whereas PDGF-Rb levels greatly increased. Initially, cells preferentially expressed both PDGF-AA and PDGF-Rb molecules, but with time PDGF-AA levels fell by half and PDGF-Rb levels increased approximately sixfold [34, 35] (Fig. 19). Hence, in contrast to their constitutive pattern, relatively low PDGF-AA and high PDGFRb levels characterize the cells in high-oxygen-induced PH. Since all cells expressed the ligands and their RTKs, no reciprocal signaling pathways were detected. Tight regulatory control of these molecules was evident in a cell population of a single vessel (e.g. endothelial cells: all expressed either a high ligand or RTK level, or a low one). At the same time, expression levels could be opposite for the same cell type in adjacent vessels within the branching pattern. Increasing the stability of vessel walls by matrix ­deposition, and inhibiting endothelial cell proliferation and ­migration [62], perivascular cells normally develop in response to transmural pressure [63]. Mechanical signals (transmitted via the cell cytosol) in response to increasing pressure may promote their development to stabilize the vessel wall in response to rising PAP in high oxygen. The response may be initiated and sustained by signaling within the vessel wall, i.e. fibroblasts migrating toward endothelial cells in response to their expression of PDGF-B. Continued ligand expression by endothelial cells is then required for their retention as perivascular cells. As vessel walls remodel, endothelial cells and SMC precursors (intermediate cells and  cells derived from interstitial fibroblasts) each express these molecules, holding ligand ­levels stable and increasing ­receptor levels exponentially [34]. It is not known if air breathing after high oxygen (relative hypoxia) stimulates

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Fig.  19  Platelet-derived growth factor (PDGF) ligand and receptor expression by cells in distal lung vessels: inverted images highlighting (10 nm) pA gold-labeled antigenic sites for PDGF-AA and PDGF-Rb in endothelial cells and fibroblast-derived vascular cells of normal lung (a, c, e, g) and in the lung after high oxygen for 28 days (b, d, f, h). Mean values for the density of antigenic sites (for each illustrated cell population) are shown. Endothelial cell (EC) expression of PDGF-AA, high in the normal lung (a, 1,200 sites, vessel external diameter 47 mm), falls in high oxygen (b, 720 sites, vessel external diameter 29 mm); and in a reverse pattern the low PDGF-Rb expression level present in normal lung (c, 440 sites, vessel external diameter 30 mm) increases in high

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oxygen (d, 2,760 sites, vessel external dia­meter 29 mm). The response is similar for interstitial fibroblasts (IFB) expressing PDGF-AA (e, 1,120 sites, vessel external diameter 47  mm) and PDGF-Rb (g, 480 sites, vessel external diameter 37  mm) in normal lung; and for these cells expressing PDGF-AA (f, 680 sites, vessel external diameter 29 mm) and PDGF-Rb (h, 2,880 sites, vessel external diameter 42 mm) when recruited as perivascular cells (PFBs) in high oxygen. Resin sections (80 nm-thick) stained with uranyl acetate and lead citrate. Original magnification: ×19,636 (a–h). (a, b, e, f Reprinted from [35] with permission. c, d, g Reprinted from [34] with permission from Taylor & Francis)

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a  second wave of perivascular cells via vasoconstriction (via ­hypoxia-inducible factor 1a) and increased PAP and transmural pressure [64]. A change in ­oxidant stress levels at this time may occur by a decrease in the antioxidant capacity of cells, or by increased production of reactive oxygen species (ROS), i.e. superoxide anion, hydrogen peroxide, hypochlorous acid, and the hydroxyl ­radical [65]. A burst in ROS generation can lead to (hypoxic) ­pulmonary vasoconstriction [66]; alternatively, as previously closed vascular segments begin to open throughout the alveolar–capillary membrane, higher blood flow can increase ROS generation by vascular cells, leading to endothelial dysfunction and impaired endothelium-dependent pulmonary relaxation [67]. Perivascular cells are also a local source of the endothelial survival factor VEGF, which serves to retain endothelial channels by protecting against regression signals and/or the loss of survival signals. In the face of decreasing VEGF levels in high oxygen, the preferential development of PDGFRb+ cells may promote vessel and capillary survival (via VEGF or via VEGF–PDGF interactions) [68–70].

4.2 Cellular and Molecular Mechanisms of Vessel and Capillary Loss Unlike the normal lung, where all segments are continually (although not continuously) perfused, blood flow is impeded as the distal lung’s vessel and capillary segments remodel. In the absence of inflammatory cells, remnants of small arteries (and veins), evident by wall rarefaction and by intimal and medial cell degeneration (lysis), persist after the cycles of high oxygen and of high oxygen and air: lumen occlusion by swollen endothelial cells further indicates unperfused or underperfused capillary networks, whereas disorganized endothelial cell clusters identify capillary remnants, and avascular alveolar regions highlight capillary loss. Under conditions of no flow, imposed by an upstream (or downstream) block, vessel and capillary collapse and loss of lumen space will lead to occlusion, either fully or in part; and any resulting unperfused vascular segments are effaced. Concurrent disturbance in blood flow will lead to the collapse of adjoining segments. One aspect of the loss of small vessels and capillaries in high oxygen likely reflects overlapping processes of cell death and loss by apoptosis, oncosis, and/or necrosis [65, 71–75]. The importance of ROS as mediators of lung cell injury, and the detrimental effects of an imbalance in lung oxidant and antioxidant levels, is well established [4]. Hyperoxia-mediated cell injury occurs by lipid peroxidation, protein sulfhydryl oxidation, enzyme activation, and/or DNA damage and depletion of cellular reducing agents [65]. These pathways, counterbalanced by changes in cellular levels of

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antioxidants, e.g. superoxide dismutase (SOD), glutathione peroxidase, and catalase [65], and lung levels of extracellular SOD [76], determine the number of viable vascular cells in the high oxygen-breathing lung. Endothelial cell loss, in particular, is likely promoted by ROS triggering patterns of cell death – apoptosis via caspase-dependent and caspaseindependent pathways [75]. Regression of vascular networks in response to oxidative stress may also occur from the loss of endothelial mitogen and survival factors (e.g. VEGF and survivin), triggered by overwhelming ROS generation during prolonged high oxygen breathing [77]. For example, levels of VEGF (VEGF-A) and its receptors (VEGF-R1 and VEGF-R2) increase and decrease in response to oxygen tensions: ligand and receptor levels fall in the lung breathing high oxygen [78] and increase in the lung breathing air after high oxygen [79, 80] – likely via hypoxia-dependent induction of VEGF [81]. In a ­complex pattern, VEGF/VEGF-R2 signaling results in endothelial cell apoptosis, whereas VEGF/ VEGF-R1 signaling promotes endothelial survival [82]. The balance toward regression may be favored in high oxygen as VEGF levels fall below the level needed to maintain existing networks, or as survivin levels fall, and the protection offered by PDGF (against ­caspase-activated apoptosis) is lost [83, 84]. Vessels of the adult lung also receive endothelial survival signals from their surrounding matrix, and (although less dependent on short-term withdrawal of VEGF-related signaling than nascent/immature vessels) are unable to survive a prolonged period of VEGF withdrawal [85]. VEGF is normally derived via paracrine signals from alveolar epithelial cells – an important source in normal lung [86, 87]. In the remodeled alveolar–capillary membrane, this signaling pathway may be altered by the presence of epithelial cells in which VEGF expression is low or lost. Since endothelial cells, however, also require autocrine VEGF to maintain viability and vascular homeostasis [88], this signaling being different from events initiated by paracrine VEGF, their loss may result from a decrease in autocrine and/or paracrine VEGF signaling.

4.3 Cellular and Molecular Mechanisms of Vessel and Capillary Restoration As capillary restoration proceeds during air breathing there is little evidence of new capillary formation by sprouting angiogenesis. Rather, residual networks re-form as the apposing apical membranes of endothelial cells retract to enlarge the lumen and establish a patent channel; and as local endothelial cells extend the diameter and length of existing capillary structures by thinning and extension of their processes, i.e. organelle

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streaming. This occurs over relatively long ­distances within the matrix of the alveolar–capillary ­membrane. It is not currently known if capillaries re-form (in  vivo) by the “guided migration” of endothelial cells (proposed as an alternative mechanism to sprouting in vitro), the cells sliding over each other (in three dimension) to form extended structures with a lumen [89]. Where present, remnants of basement membrane will provide ready templates for endothelial cell migration: fragments rich in basic fibroblast growth factor within the membrane [89, 90] will enhance this response, by acting as a molecular track and inducing capillary endothelial cells to invade collagen matrix and form tubules [91]. Where present, fibroblast-derived soluble factors and fibroblast–endothelial cell contacts will further promote the response [92]. Where endothelial cells fail to contact other endothelial cells, they form simple seamless capillary structures (consisting of a single endothelial cell profile), the cells forming a junction at the point of self-contact. These capillaries form [91, 93] mostly at the terminal end of capillary networks and connect directly to adjacent functional segments [93]. Gradually the structure of the alveolar–capillary membrane is restored as re-forming capillary networks invade the “avascular” regions left in the lung after high oxygen. These regions promote ordered capillary growth by restricting the caliber (diameter) of the developing endothelial tubes, and by guiding the regenerating branches of capillary networks. Consistent with this response at a time when the lung is challenged by a lower oxygen tension, in  vitro studies have demonstrated that pulmonary endothelial cells spontaneously form into channels in hypoxia [94]. In addition to resident endothelial cells, progenitor/precursor cells circulating through the adult lung may contribute to the restoration of capillary structures, i.e. circulating “angiogenic” progenitor/precursor cells, bone-marrowderived (BMD) cells, and cells in peripheral blood [95–99]. Each may home to sites of neovascularization, and have the potential to give rise to endothelial cells that expand the vasculature of distal organs. However, they also have the potential to contribute to the pathobiology of vascular changes (e.g. SMC development) [100, 101]. How such cells contribute to vascular remodeling in response to the alveolar oxygen tension is currently being analyzed. To date, in terms of phenotype and their potential role during the time of capillary expansion in the model, the data identify a novel population of circulating myeloid precursor (CD11b+ VEGF-R2+ PDGF-Rb+) cells [102] interacting with the apical plasmalemmal membrane of capillary endothelial cells (e.g. by CXCR4/stromal-cellderived factor 1a signaling) [103]. Further investigation is under way to determine whether the precursor cells are retained in this location to promote endothelial cell proliferation, or as a prelude to their (temporary or permanent) integration into endothelial surfaces. Balasubramaniam et  al. have elegantly demonstrated the requirement for circulating BMD cells to maintain vascular density in the developing and adult lung

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challenged by hyperoxia [104]. Although progress in this field has been made, much is still unknown of the cellular and molecular basis of the patterns of vascular remodeling in the adult lung in response to events triggered by the alveolar oxygen tension.

5 Lung Vascular Restriction in Human Disease and Future Directions In addition to documenting the vascular response to cycles of breathing high oxygen and air, the studies discussed here provide a useful tool to dissect the pathobiology of some of the important vascular changes in the clinical PHs and to highlight potential therapeutic strategies. Although only briefly mentioned here, important studies of the effects of breathing a higher than normal oxygen tension have focused on the cellular and molecular basis of impaired vascular development in the human neonatal lung, and assessment of strategies to address the outcome.

5.1 Vascular Restriction in Adult Respiratory Distress Syndrome and Acquired/ Idiopathic PH Pulmonary arterial hypertension (PAH) is defined clinically as a sustained increase in the mean PAP (more than 25 mmHg at rest or more than 35 mmHg on exercise) in the presence of a low or normal pulmonary capillary wedge pressure (less than 15 mmHg). The restrictive vascular changes leading to PH occur in patients with severe forms of adult respiratory distress syndrome (ARDS), and in other groups of patients who develop PH in an acquired or idiopathic forms. Although treatment approaches are making steady progress, dependable strategies to prevent/ameliorate the vascular changes are less advanced. The following outlines human vascular disease, where appropriate the relationship of vascular changes to breathing high oxygen, and the direction of potential therapeutic strategies to ameliorate vascular remodeling in PH. In patients with respiratory failure, i.e. acute lung injury (ALI), and with compromised gas exchange, iatrogenic oxygen is administered at higher than normal levels to maintain adequate oxygenation to the brain and peripheral tissues. This supportive treatment raises the question of hyperoxia’s effect on an already injured lung, particularly since many of the morphological changes in in vivo models of oxygen toxicity resemble ones in these patients, and in patients with its severer form – ARDS. Currently, ALI is defined as hypoxemic respiratory failure with bilateral lung infiltrates not attributable to left atrial hypertension [105] and ARDS as a subtype of ALI

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with a severer form of hypoxemia [106, 107], resulting from the complex response of the lung to a combination of direct and indirect insult [108]. ARDS affects nearly 200,000 patients per year in the USA [107] and, despite improvements in supportive care, ALI/ARDS has a high mortality rate. ARDS after non-thoracic trauma gradually became a recognized syndrome by the mid-1960s [109, 110] – a finding evident in soldiers saved on the battlefield (in Vietnam) by improved survival approaches, who after successful resuscitation from shock later died of lung complications from their injuries [111]. Also known as “shock lung” at that time, ARDS gradually became widely recognized in civilian patients who developed severe airway and vascular changes after an injury other than to the lung, such as abdominal sepsis or long bone trauma with fat embolism; a further link was made between lung changes and the prolonged use of high oxygen tensions in inspired gas mixtures and mechanical ventilation [112, 113]. By the mid-1970s and early 1980s, it was demonstrated that patients with the severest forms of ARDS also developed vascular changes and PH [114–117]. The site and nature of vascular injury in the lungs of patients dying from severe ARDS included extensive pulmonary thromboemboli and small vessel occlusion and obliteration [115, 116]. These patients also ­generally required long periods of supplemental oxygen treatment and, although it was difficult to determine its effect on an already injured lung, the consensus was that high-pressure mechanical ventilation, and the generation of oxygen metabolites, could amplify existing changes. Thus, although the primary injury of ARDS often resulted from a single event, a complex response leading to secondary injury of the lung could continue throughout the course of the illness [118]. As treatment approaches improved, and as lower oxygen levels became effective with the introduction of lower mechanical ventilation and positive endexpiratory pressures, fewer of these patients developed PH. Currently, ALI/ARDS patients needing supplemental oxygen treatment for long periods continue to receive the lowest fraction of inspired oxygen (FiO2) consistent with adequate gas exchange [119]. Thus, although experimental studies implicate hyperoxia as a cause of many lung changes, it is not proven as a clinically important cause of lung injury in humans; rather its combined use with high mechanical ventilator pressures continues to be recognized as a potential cause of injury [120]. To prevent this outcome, ongoing studies seek to understand the signaling pathways responsible [121–124]. Associated (acquired) PAH (APAH) occurs in conditions that include collagen vascular diseases (scleroderma), congenital right-to-left cardiac shunts, portal hypertension (associated with liver disease), infection (HIV), and drugs such as fenfluramine. In patients with PAH and no associated disease (idiopathic PAH, IPAH), the cause is unknown and these patients are currently the focus of genetic analysis studies [125, 126]. A variety of vascular changes develop in  both APAH and IPAH: the principal ones include

R.C. Jones and D.E. Capen

t­ hromboemboli, medial hypertrophy, intimal hyperplasia, and lumen ­occlusion, together with focal changes considered important markers of the severity or rapid progression of the disease, i.e. plexiform lesions, dilatation lesions, and arteritis [127]. The proliferation of endothelial cells forming plexiform lesions, which occurs in IPAH in particular, is thought to mark an abnormality similar to that of neoplastic cells [127]. Venous changes are also reported in IPAH [128]. Medial and adventitial thickening and occlusive and obliterative lesions also form the structural basis of veno-occlusive disease, i.e. pulmonary venous hypertension [129–131]. All clinical forms are vascular diseases with high morbidity and mortality. To prevent or control the structural changes in human PH, the predominant cellular events need to be further understood, including vascular loss and the unregulated development of endothelial and perivascular cells throughout the vascular branching pattern, but particularly in the distal vessels. Studying the prevention of lumen occlusion by proliferating endothelial cells, and the development of plexiform lesions (endothelial channels forming luminal nests) in distal vessels in clinical PH [132–134], requires an appropriate experimental model such as IL-6 overexpressing mice breathing high oxygen [135]. In this mouse model, the development of occluded vessels and PH results from an imbalance in endothelial (and perivascular) cell proliferation and apoptosis. In the rat models highlighted here, however, a similar process of lumen occlusion by endothelial cells is not seen; rather, an endothelial monolayer is preserved as perivascular cells encroach on the lumen. By their physical restriction of distal vessels, both by lumen encroachment and by wall thickening, and by promoting wall contractility and reactivity, the unrestricted growth of these cells represents a major problem in clinical PH: it would be clearly advantageous to modulate/abrogate their development. Experimental data highlighting a potential therapeutic approach come from blocking studies of PDGF signaling. Blocking PDGF-Rb, for example, modulates SMC proliferation in a rat model of endothelin-mediated pulmonary vascular remodeling [136], and in sheep prevents the additional development of SMCs triggered in hypoxia-driven PH [137]. Furthermore, PDGF-Rb blockade is reported to reverse experimentally induced PH in rodent (mouse) models of monocrotaline or hypoxia PH after the induction of lung vascular changes, including SMC development in distal vessels [138]. The finding of higher than normal PDGF-Rb levels in patients with PH highlights the potential benefit of a receptor blocking agent as a therapeutic approach to ameliorate the cycle of vessel wall occlusion and neomuscularization in the human lung [138, 139]. Although fibroblasts are shown to undergo phenotypic transition to SMCs in the studies described here, the development of SMCs from endothelial cells in PH offers an alternative cell population as their source, and approaches to prevent development of these cells have been discussed by Arciniegas et  al. [140]. The

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potential role for BMD cells in addressing lung vascular changes is also of high interest [141–143]. Although severely damaged regions of the distal vascular bed may not be salvageable, therapies directed toward re-endothelialization of less injured regions (where residual structures persist to offer a template for repair) may lead to vascular restoration. To address the inherent concerns, and possible consequences, of reintroducing expanded vascular progenitor/precursor cell populations into the lung [101, 144] further data are needed to identify which cells give rise to appropriate “endothelial” and perivascular cells to repair vascular surfaces or form new vascular channels. Avenues to prevent vascular regression and/or promote appropriate vascular growth and network formation (based on the administration of these cells) are currently being explored [145]. Further studies are needed to assess optimal approaches to promote the remodeling of vascular networks to more normal patterns by targeting relevant cellular and molecular pathways.

5.2 Vascular Restriction (Failed Vascular Development) in Neonatal Respiratory Distress

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that follows ventilator and oxygen therapy for acute respiratory failure after premature birth [150], BPD was initially characterized by airway injury and severe fibrosis: the use of lower oxygen tensions, however, has changed its pathological features to forms of pulmonary hypoplasia with fewer blood vessels and alveoli [151–157]. An important question is the ability of the lung with BPD to achieve catch-up growth in the postnatal period and to accomplish the developmental patterns that normally occur in childhood and in the young adult (see Chapter 3). Hence, although breathing higher than normal oxygen levels remodels the lung’s vascular bed at all ages, its effects on the developing lung are especially important in that the response is superimposed on the critical periods of airway and vascular growth that immediately precede and follow birth [45] (see Chapter 3). Impairment of the vascular template at this time has severe consequences for future periods of growth and maturation [44, 45]. To promote normal lung development and improve the clinical outcome of respiratory distress and BPD, approaches to facilitate vascular and epithelial interactions [44], and the use of BMD cells [158], offer encouraging therapeutic strategies.

6 Summary of Lung Vascular Remodeling in PH The effects of breathing high oxygen tensions on the neonatal and early postnatal lung have long been the subject of intense study [11, 146–148]. In this context, current in vivo models are increasing understanding of the basis, and long-term effects, of impaired vascular development and growth. The normal transition to air breathing at the time of birth (from the hypoxic environment in utero to the hyperoxic environment of air) challenges the developmental program of both the distal airway and vascular compartments of the still immature lung. The newborn lung at term is normally well shielded by relatively high antioxidant levels, and other protective mechanisms during this transition, and so can withstand this challenge to its cells and tissue structures. The clinical ability to support premature infants born at early developmental times, goes hand in hand with vascular and airway structures that are underdeveloped. It may be that the low oxygen (hypoxic) tension present in utero is needed to drive angiogenesis and the normal vascular–epithelial interactions for vascular development that occur before birth [149]. Premature infants that have not developed their epithelial surfaces and surfactant systems sufficient for lung expansion develop respiratory distress, receive surfactant replacement therapy, and typically also receive supplemental oxygen (at levels above 21%), for adequate oxygen exchange. This can result in injury to small airways and vessels and lead to bronchopulmonary dysplasia (BPD). In vivo model systems documenting the effects of hyperoxia on the neonatal lung clearly highlight the extent of impaired vascular and epithelial growth and the changes leading to respiratory distress and BPD. First recognized as a (chronic) lung disease of infancy

6.1 Microvascular Remodeling in the Lung Breathing High Oxygen 1. The distal (intra-acinar) vascular bed of the normal adult lung is characterized by extensive networks of small vessels and capillaries that reorganize in response to a change (increase) in the ambient alveolar oxygen tension. 2. Breathing a high (but sublethal) oxygen tension for several weeks greatly restricts the lung’s distal vascular bed, including both vessel and capillary networks. 3. Distal vessel and capillary networks are restricted by lumen occlusion, obliteration (evident by vessel remnants), and/or regression (evident by capillary loss giving rise to “avascular” zones). 4. Distal vessel restriction increases PVR, PAP, and RVH, and leads to wall remodeling (thickening) in large vessels. 5. In vessels, lumen occlusion results from an early burst of endothelial cell and perivascular cell proliferation, and from the persistent development of (subintimal) perivascular cells. 6. Perivascular cells develop in vessels from intermediate cells and (recruited) interstitial fibroblasts; in capillaries they develop from the latter. 7. Intermediate cells and recruited fibroblasts transition to contractile cells by expressing organelles and proteins typical of a SMC phenotype, converting nonmuscular vessel segments to ones with a muscular or partially muscular wall.

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6.2 Microvascular Remodeling in the Lung Breathing Air After High Oxygen Early phase (weeks 1 and 2 in air): 1. The hyperoxia-adapted lung is characterized by “pruned” networks of small vessels and capillaries throughout the alveolar–capillary membrane which then reorganize in response to a change (decrease) in the ambient alveolar oxygen tension. 2. Air (relative hypoxia) triggers pulmonary vasoconstriction in the hyperoxia-adapted lung, leading to further increase in PAP and RVH. 3. Additional perivascular cells develop from interstitial fibroblasts recruited to vessel walls. 4. Functional capillary networks increase in number throughout the alveolar–capillary membrane as endothelial cell retraction restores lumen patency to occluded segments, and as segments increase in size (length and diameter) and form connections. 5. Vessel remnants persist. Late phase (weeks 3 and 4 in air): 6. Vessel remnants persist. 7. Perivascular cells are deleted from occluded and patent vessel walls and the number of nonmuscular vessels increases at the expense of muscular and partially muscular ones. 8. Lumen patency is restored to occluded vessels throughout the alveolar–capillary membrane by perivascular cell deletion, and by endothelial cell reorganization as vessel walls expand in diameter. 9. Capillary networks continue to expand in number throughout the alveolar–capillary membrane (by growth processes other than sprouting angiogenesis), and remodel from simple tubes into complex structures. 10. Excluding the presence of vessel remnants, the overall structure of the alveolar–capillary membrane is restored close to normal as the avascular zones (formed in hyperoxia) are invested with functioning capillary and vessel networks, and vessel walls thin by perivascular cell deletion. Acknowledgement  Studies reported in this chapter are based on published work by the authors and were supported by grants (to RJ) from the National Institutes of Health. The writing of this chapter by the authors (RJ and DC) was also supported by a grant from the National Institutes of Health (NIH HL 089252).

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758 147. Roberts RJ, Weesner KM, Bucher JR (1983) Oxygen-induced alterations in lung vascular development in the newborn rat. Pediatr Res 17(5):368–375 148. Pappas CT, Obara H, Bensch KG, Northway WH Jr (1983) Effect of prolonged exposure to 80% oxygen on the lung of the newborn mouse. Lab Invest 48(6):735–748 149. van Tuyl M, Liu J, Wang J, Kuliszewski M, Tibboel D, Post M (2005) Role of oxygen and vascular development in epithelial branching morphogenesis of the developing mouse lung. Am J Physiol Lung Cell Mol Physiol 288(1):L167–L178 150. Northway WH Jr, Rosan RC, Porter DY (1967) Pulmonary disease following respirator therapy of hyaline-membrane disease. Bronchopulmonary dysplasia. N Engl J Med 276(7):357–368 151. Jobe AJ (1999) The new BPD: an arrest of lung development. Pediatr Res 46(6):641–643 152. Jobe AH, Bancalari E (2001) Bronchopulmonary dysplasia. Am J Respir Crit Care Med 163(7):1723–1729 153. Abman SH (2001) Bronchopulmonary dysplasia: “a vascular hypothesis”. Am J Respir Crit Care Med 164(10 Pt 1):1755–1756 154. Bhatt AJ, Pryhuber GS, Huyck H, Watkins RH, Metlay LA, Maniscalco WM (2001) Disrupted pulmonary vasculature and decreased vascular endothelial growth factor, Flt-1, and TIE-2 in human infants dying with bronchopulmonary dysplasia. Am J Respir Crit Care Med 164(10 Pt 1):1971–1980

R.C. Jones and D.E. Capen 155. Thebaud B, Abman SH (2007) Bronchopulmonary dysplasia: where have all the vessels gone? Roles of angiogenic growth ­factors in chronic lung disease. Am J Respir Crit Care Med 175(10):978–985 156. Alejandre-Alcazar MA, Kwapiszewska G, Reiss I et  al (2007) Hyperoxia modulates TGF-beta/BMP signaling in a mouse model of bronchopulmonary dysplasia. Am J Physiol Lung Cell Mol Physiol 292(2):L537–L549 157. Abman SH (2008) The dysmorphic pulmonary circulation in bronchopulmonary dysplasia: a growing story. Am J Respir Crit Care Med 178(2):114–115 158. van Haaften T, Thebaud B (2006) Adult bone marrow-derived stem cells for the lung: implications for pediatric lung diseases. Pediatr Res 59(4 Pt 2):94R–99R 159. Hudak BB, Zhang LY, Kleeberger SR (1993) Inter-strain variation in susceptibility to hyperoxic injury of murine airways. Pharmacogenetics 3(3):135–143 1 60. J ohnston CJ, Stripp BR, Piedbeouf B et  al (1998) Inflammatory and epithelial responses in mouse strains that differ in sensitivity to hyperoxic injury. Exp Lung Res 24(2):189–202 161. Rohan RM, Fernandez A, Udagawa T, Yuan J, D’Amato RJ (2000) Genetic heterogeneity of angiogenesis in mice. FASEB J 14(7):871–876

Chapter 52

Pulmonary Vascular Remodeling: Cellular and Molecular Mechanisms Kurt R. Stenmark and Maria G. Frid

Abstract  The common pathologic feature of all forms of chronic pulmonary hypertension (PH) is the increase in pulmonary artery (PA) wall thickness. Although most attention has been focused on the increase in thickening in the walls of small PAs, it is evident that there are changes in the thickness of vessels along the entire longitudinal axis of the pulmonary circulation, from the most proximal elastic vessels to the most distal muscular and even nonmuscular PAs. The arteries are thickened by (a) an increase in endothelial cell numbers and matrix protein deposition in the intima (in concentric, eccentric, and plexiform lesions), which may account for most of the reduction in the luminal area of small PAs in human PH (but only in a few animal models of PH); (b) an increase in the size and number of smooth muscle cells (SMCs) and even smooth muscle (SM)-like cells not fully differentiated (e.g., myofibroblasts), and the accumulation of extracellular matrix proteins in the arterial media; and (c) a marked increase in fibroblast and myofibroblast numbers and extracellular matrix protein deposition in the adventitia. In addition, there is also increasing evidence that inflammatory and progenitor cells accumulate both within and around the remodeled vessels and contribute significantly to the overall remodeling process. The goal of this chapter is to describe the changes in the structure of the pulmonary arterial wall that occur at different locations along the longitudinal axis of the pulmonary circulation and to provide insight into the contributing cellular and molecular mechanisms. Further, this chapter emphasizes the changes that occur in different compartments (intimal, medial, and adventitial) of the pulmonary arterial wall and discusses the phenotypic changes of the endothelial cells, SMCs, and fibroblastic cells which contribute directly to the pathologic changes observed. Lastly, the chapter discusses the impact of the recruited inflammatory and progenitor cells on the structure and function of resident PA wall cells. K.R. Stenmark (*) Department of Pediatrics, University of Colorado, Denver, 12700 E. 19th Ave., Mail Stop B131, Aurora, CO 80045, USA e-mail: [email protected]

Keywords  Pulmonary vascular wall thickening • Intima • Media • Adventitia • Extracellular matrix • Myofibroblast • Inflammatory cell • Progenitor cell

1  Introduction The common pathologic feature of all forms of chronic ­pulmonary hypertension (PH) is the increase in pulmonary artery (PA) wall thickness. Although most attention has been focused on the increase in thickening in the walls of small PAs, it is evident that there are changes in the thickness of vessels along the entire longitudinal axis of the pulmonary circulation, from the most proximal elastic vessels to the most distal muscular and even nonmuscular PAs [1–8] (Fig.  1). The arteries are thickened by (a) an increase in endothelial cell numbers and matrix protein deposition in the intima (in concentric, eccentric, and plexiform lesions), which may account for most of the reduction in the luminal area of small PAs in human PH (but only in a few animal models of PH); (b) an increase in the size and number of smooth muscle cells (SMCs) and even smooth muscle (SM)-like cells not fully differentiated (e.g., myofibroblasts), and the accumulation of extracellular matrix proteins in the arterial media; and (c) a marked increase in fibroblast and myofibroblast numbers and extracellular matrix protein deposition in the adventitia. In addition, there is also increasing evidence that inflammatory and progenitor cells accumulate both within and around the remodeled vessels and contribute significantly to the overall remodeling process. The goal of this review is to describe the changes in the structure of the pulmonary arterial wall that occur at different locations along the longitudinal axis of the pulmonary circulation and to provide insight into the contributing cellular and molecular mechanisms. Further, this review will emphasize the changes that occur in different compartments (intimal, medial, and adventitial) of the pulmonary arterial wall and will discuss the phenotypic changes of the endothelial cells, SMCs, and fibroblastic

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_52, © Springer Science+Business Media, LLC 2011

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2 Vascular Remodeling at Different Locations Along the Longitudinal Axis of the Pulmonary Circulation 2.1 Remodeling in Conductance Vessels

Fig. 1  Significant increases in the thickness of all three vascular compartments (intima, media, and adventitia) are observed for pulmonary arteries of all sizes in patients with idiopathic pulmonary arterial hypertension compared with normal controls. (Adapted from [1] with permission of the American Association of Pathologists and Bacteriologists)

cells which contribute directly to the pathologic changes observed. Lastly, it will consider the impact of the recruited inflammatory and progenitor cells on the structure and function of resident PA wall cells.

In the large PAs (extrapulmonary and intrapulmonary elastic), increases in medial and adventitial thickness are commonly observed in humans and in animal models [3, 5, 7, 8]. In addition, variable degrees of intimal thickening can be seen in these vessels. The cellular mechanisms contributing to these changes appear to be dependent on the species examined and on the resident cellular composition of the vessel wall. For instance, in the hypoxic rat model of PH, adventitial thickening occurs early and is dramatic, whereas thickening of the media lags behind [2, 6]. Adventitial fibroblasts demonstrate earlier and more significant increases in DNA synthesis compared with SMCs. In fact, medial SMCs did not demonstrate an increase in DNA synthesis until at least day 3 and, at this point in time, the labeling index increased from less than 0.5 to only 1–1.5 [4]. The findings in the mouse are similar, with a brief rise in the medial SMC ­labeling index in the main PA at days 4–6, followed by a rapid decline in medial SMC labeling and total SMC number compared with controls at 3 weeks [9]. The observations suggest that the medial thickening in the large vessels of rodents is largely due to SMC hypertrophy and increased matrix (elastin and collagen) deposition. The significant adventitial thickening in mice and rats is due to an ­accumulation of fibroblasts/myofibroblasts and matrix ­proteins ­(predominately collagen, fibronectin, and tenascin) [10–12]. In large animals (e.g., calf and pig) the responses are different, with early and dramatic medial thickening predominating [7, 13, 14] (Fig. 2). The differences may be explained by the fact that the cellular composition of the proximal ­pulmonary and systemic vessels of larger ­mammalian species (including the cow, lamb, pig, and human) is more complex than that of rodent species [3, 7, 15–19]. The media of large conducting pulmonary and systemic vessels in various large mammalian species, including the human, is composed of multiple phenotypically distinct SMC populations [3, 7, 15–17, 19] (Fig. 3). On the basis of observations of distinct proliferative and matrix-producing capabilities and ion channel expression in response to many stimuli, including hypoxia, these different SMC populations appear to serve different functions [20–26]. There is evidence to support the argument that these cells are derived from distinct lineages and are not simply a common cell exhibiting different states of differentiation [15, 16, 19, 27]. Although it is evident that these phenotypically distinct cell populations serve different functions in health and disease, presently many questions remain to be determined, such as the embryonic origin of distinct cell

52  Pulmonary Vascular Remodeling: Cellular and Molecular Mechanisms

Fig. 2  Marked changes in the structure of the main pulmonary artery are observed in animal models of hypoxia-induced pulmonary hypertension. Whereas adventitial thickening predominates in the mouse (a) and rat (b) models, marked thickening of the media (in addition to adventitial changes) is observed in the neonatal calf (c). (a Adapted from [11] with permission. (b) Adapted from [28]. (c) Adapted from [38] with permission from the American Society for Clinical Investigation)

populations, the ratio of different cells at a given vascular site in a given species, and the mechanisms ­directing the developmental assembly of these cells types into a ­functioning, mature blood vessel wall. Furthermore, the ­possibility that cells with progenitor-like capabilities are contained within specific sites in the large vessels needs to be considered [7]. It has been shown, in larger mammalian species, that the medial thickening of proximal PAs in response to chronic hypoxia can be accounted for, in part, by the proliferation of a distinct SM-like subpopulation residing within the media [15, 28] (Fig.  4). This SM-like population exists in a relatively “undifferentiated state” (as assessed by SM-specific cytoskeletal and contractile protein marker expression) in

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comparison with differentiated SMCs within the vessel wall. These in situ observations are consistent with the findings of cell culture studies demonstrating that only less differentiated SM-like cells demonstrated increases in proliferation in response to hypoxic exposure [22, 23, 29]. SMCs that ­maintain a more differentiated phenotype in culture exhibited growth inhibition under hypoxic conditions [22]. The ­observation that the adventitia of the large PA in calves underwent less thickening than in rats or in mice is perhaps due to the fact that the PA media in larger mammals contains cells capable of rapidly responding to hypoxia and increases in wall tension by proliferating and secreting matrix proteins. Since thickening serves to decrease wall stress, the existence of such a cell population within the media of large vessels may provide the vascular wall with a mechanism to adapt rapidly to increased wall tension. Thus, highly differentiated SMCs are “spared” from dedifferentiating and proliferating and may continue to maintain their normal homeostatic functions such as contraction in these larger vessels. Little is known regarding the mechanisms that confer unique proliferative characteristics to specific cell populations that exist in the large PAs. It has been demonstrated that less differentiated and more proliferation-prone medial cells are characterized by exuberant responses to G-protein-coupled receptor (GPCR) agonists, in contrast with the differentiated medial SMCs, which do not exhibit proliferative responses to hypoxia [15, 22, 23, 29]. These observations suggest that there may be differences in receptor expression and/or intracellular signaling among various cell types within the vessel wall media. Differences in endothelin-1 (ET-1) production, receptor expression, and responses have been described in distinct cell populations derived from the ovine PA [30, 31]. As mentioned above, marked increases in collagen and elastin accumulation are observed in proximal pulmonary vessels of humans and animals with PH. Less is known of the pathways stimulating matrix protein production in PH than of those responsible for cell proliferation. There is reason to believe the former are distinctly different and perhaps even more complex than those regulating proliferation. One cytokine likely involved in controlling both responses is transforming growth factor b (TGF-b). TGF-b is an important regulator of collagen and elastin synthesis in SMCs and fibroblasts, both in  vivo and in  vitro [7, 11, 14, 32–34]. Increases in TGF-b expression are observed in the large (and small) vessels in animal models of PH. The importance of TGF-b in hypoxia-induced collagen production and vascular remodeling was demonstrated in recent studies where a dominant negative mutation in the TGF-b receptor blocked chronic hypoxia-induced PH in rats [35]. In addition, administration of recombinant human relaxin, a hormone with direct effects on collagen and fibronectin production by fibroblasts, as well as administration of other antifibrotic agents reduce hypoxia-induced PH, findings which again

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Fig. 3  Bovine large elastic vessels are composed of a mosaic of phenotypically distinct smooth mucle cells which exhibit stable differences in contractile and cytoskeletal protein expression both in vivo and in cell culture. (Adapted from [22] with permission from Wolters Kluwer)

Fig. 4  Hypoxia-induced cell proliferation in the media of large elastic pulmonary arteries of hypertensive neonatal calves occurs almost exclusively in a specific cell subpopulation. Double immunofluorescence staining shows that differentiated medial smooth muscle cells expressing the smooth-muscle-specific cytoskeletal protein meta-vinculin

(green, asterisk, “mVN-positive”) do not replicate, whereas less differentiated (meta-vinculin-negative) medial cells actively replicate (express nuclear proliferation-associated Ki-67 antigen, red, arrowheads). (Modified from [28] with permission from the American Society for Clinical Investigation)

emphasize the importance of matrix accumulation in large vessels [36]. Significant increases in elastin production and accumulation are also observed in large pulmonary hypertensive vessels [34, 37]. In addition, recent studies also show that elastin undergoes significant decreases in its capacitive characteristics in the hypertensive vessel wall [37]. The changes in cell and matrix composition in the large vessels lead to marked increases in their stiffness [37, 38] (Fig. 5). This may significantly contribute to the increases in impedance and right ventricular afterload observed in PH, changes that are important predictors of outcome in patients with PH [7, 10, 11, 39–42]. In fact, increases in wall stiffness in large vessels contribute as much as 30–40% of the increase in load to which the right ventricle is exposed [37, 41, 43]. In addition, this increase in stiffness in proximal vessels leads to an inability of conductance vessels to store and deliver the entire stroke volume of the right ventricle and ultimately results in a loss of pulmonary flow during diastole. This disturbance of flow not only increases the load on the right ventricle but also may contribute to a decrease in nitric oxide

(NO) production in distal PA endothelial cells, which require steady pulsatile flow for optimal NO production [44]. Additionally, stiffening of the capacitance vessels appears to increase pulse wave velocity and magnitude in the distal circulation. In the systemic circulation this leads to activation of endothelial cells, resulting in a proinflammatory and prothrombotic state [45]. Similar studies are needed in the pulmonary circulation.

2.2 Remodeling in Distal Muscular PAs The distal pulmonary circulation, which is the primary site of pulmonary vasoconstriction, also undergoes significant structural changes in all forms of chronic PH. The changes include medial and adventitial thickening and often marked intimal thickening and fibrosis, findings which are common in human PH but are lacking in most animal models of PH (Fig. 6). In fact, it is presently thought that intimal lesions

52  Pulmonary Vascular Remodeling: Cellular and Molecular Mechanisms

Fig. 5  Significant increases in the stiffness of the neonatal calf main pulmonary artery occur in the presence of pulmonary hypertension. In vivo increases in the impedance modulus (a) as well as ex vivo

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increases in overall tissue stiffness (b) and in elastin stiffness (c) are observed in pulmonary hypertension

Fig. 6  Hypoxia-induced perivascular remodeling (adventitial tickening, accumulation of myofibroblasts and inflammatory cells) varies significantly among species, ranging from minimal perivascular changes in mice to marked changes in neonatal calves. Note the strong correlation between remodeling and inflammation. SD rat Sprague–Dawley rat strain, WKY rat Wistar–Kyoto rat strain

account for most of the reduction of luminal area of small PAs and thus contribute significantly to the fixed increases in vascular resistance that characterize human chronic PH. With regard to the medial thickening observed in distal vessels, an important question is whether the cellular mechanisms which act to cause structural changes in the media of distal vessels are the same as those utilized in the proximal arteries. Studies among various species have raised the possibility that they are different. Significant differences in the electrophysiological properties between proximal and distal SMCs exist and contribute to the unique nature of the distal

circulation [20, 21, 46]. Distal PA SMCs cultured from normal calves demonstrate a resistance to traditional growthpromoting stimuli, including platelet-derived growth factor (PDGF)-BB, angiotensin II, and TGF-b1, as well as to serotonin (5-HT) and endothelin. Importantly, growth of distal PA SCMs was also consistently inhibited by hypoxia in either the presence or the absence of serum [47]. Thus, it appears that the medial SMCs in the distal pulmonary circulation, at least in some species, constitute a uniform population of well-differentiated and relatively growth resistant cells. In contrast, in the far more commonly utilized bovine

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vascular SMCs derived from the proximal media of the main PA, responses to growth factors, cytokines, and environmental stress and PDGF, 5-HT, endothelin, and angiotensin stimulate cell proliferation rather than cell hypertrophy [48–50]. Similarly, it has been reported that SMCs derived from large PAs of rats or mice exhibit proliferative responses to these factors as well as to hypoxia. Studies in the human pulmonary circulation also suggest differences in proliferative capacity between SMCs derived from proximal and distal vessels although presently these differences are not entirely consistent with those in animal studies [51]. In some reports, cells described as “SM-like” and derived from the distal vessels exhibit greater ­proliferative responses to mitogens than cells from the proximal ­vessels [52] although it is unclear whether the cells obtained were only from normal patients or if pulmonary hypertensive patients were also included. Further, cells were characterized as SMCs only on the basis of expression of a-SM actin (no other SM-specific marker was tested), raising the possibility that nonmedial cells were cultured and studied (e.g., myofibroblasts, also a-SM actin+). Intriguingly, Yang et al. reported that serum-stimulated proliferation of cells harvested from the main or lobar PA tends to be inhibited by TGF-b and bone morphogenetic proteins (BMPs) 2, 4, and 7. In contrast, in PA SMCs isolated from small distal PAs (1–2 mm), BMPs 2 and 4 stimulated proliferation [53]. Observations of a phenotypically uniform population of growth-resistant differentiated SMCs in the distal circulation raise a question as to what, if not the hyperplasia of the resident medial SMC, could explain the marked medial thickening observed in most forms of PH [47]. Most evidence would suggest that SMC hypertrophy together with accumulation of extracellular matrix account for most of the medial thickening in PH [8, 54]. SMC hyperplasia probably plays a minor role in the distal PA medial thickening since it has been experimentally shown in hypoxic rats that whereas the proliferation index of SMC doubled in large PAs, cells of the intra-acinar vessels did not show evidence of [3H]thymidine uptake [4]. Similar findings were observed in PH caused by monocrotaline in rats [55]. The apparent predominant role of SMC hypertrophy over hyperplasia in medial thickening is also supported by human PH studies [56–58]. On the other hand, in PH studies where cell proliferation has been assessed, early and significant increases in fibroblast proliferation were reported [4, 6, 13]. Further, many stimuli and cytokines known to participate in the pulmonary hypertensive process (including endothelin, TGF-b, angiotensin, and hypoxia) have been demonstrated to increase myofibroblast numbers in the PA adventitia and to induce differentiation of fibroblasts into myofibroblasts [59]. A myofibroblast is a highly metabolically active cell that secretes matrix proteins, including collagen, fibronectin, tenascin, and osteopontin. Thus, these cells directly contribute to the adventitial thickening observed. In the systemic

K.R. Stenmark and M.G. Frid

circulation, the processes of early activation, differentiation, and subsequent migration of fibroblasts/myofibroblasts into the media have been suggested as mechanisms contributing to medial and even intimal thickening [60]. The possibility that these or similar mechanisms operate to affect medial thickening in small PAs was suggested over 20 years ago by Sobin et  al. and more recently by Jones et  al. [2, 61, 62]. Both sets of investigators have presented evidence in support of the idea that either an adventitial fibroblast or an interstitial lung fibroblast is activated by hypoxia, recruited, and differentiates into a myofibroblast or even a SM-like cell in the PA wall. It seems logical that cells with a larger repertoire of functional capabilities, such as adventitial fibroblasts or even recently described resident adventitial stem-like cells, may be more capable of proliferating, migrating, and secreting matrix proteins than highly differentiated contractile medial SMCs whose function is to control vascular tone and blood flow within the distal circulation [63–66]. Recent in vivo and in vitro studies evaluated the cellular composition of the distal PA media of normoxic and chronically hypoxic neonatal calves. The distal PA media of control calves comprised a phenotypically uniform population of differentiated SMC, whereas at least two phenotypically distinct cell populations were detected in PAs of chronically hypoxic calves. One population comprised differentiated and significantly hypertrophied SMCs, and another represented an undifferentiated cell population with high proliferative and migratory capabilities. Experimental data suggested that this latter cell population was of a nonresident, nonmedial origin (be it adventitial, interstitial, or circulating) and that these cells had migrated into the media. This possibility was supported by recent studies showing that hypoxia induces recruitment of mesenchymal precursors, including fibrocytes, into the vessel wall [12, 67]. Fibrocytes constitute a small subpopulation of circulating leukocytes (CD45+, CD11b+) that, at the site of tissue injury, can assume a mesenchymal phenotype ­(type-I collagen+, a-SM actin+) and the functions of resident ­fibroblasts or even myofibroblasts. Fibrocytes have been described as significantly contributing to fibrosis in a number of other fibroproliferative conditions in the lung [68–70]. Intimal lesions account for most of the reduction of ­luminal area of small PAs (in human PH) and potentially largely influence the overall increase in pulmonary vascular resistance. Intimal lesions are often observed in the distal vasculature of pulmonary hypertensive patients, and animals, and may consist of eccentric intima thickening or, in more advanced PH, fibrotic, concentric, plexiform, and dilation/angiomatoid lesions. Fibrotic lesions are characterized by accumulation of fibroblasts and matrix (usually mucopolysaccharides and collagen) as has been shown in lungs of patients with PH [71]. The organization of myofibroblasts and/or endothelial cells in “onion-skin” layers is characteristic of concentric lesions. These lesions are characteristic of severe PH. Concentric

52  Pulmonary Vascular Remodeling: Cellular and Molecular Mechanisms

lesions composed of SMCs have also been described [72]. It is unclear whether endothelial- or SMC-based lesions have a similar impact on the course of PH. However, it is conceivable that these lesions may arise from similar progenitors, as endothelial cells can transdifferentiate into SMCs when stimulated with PDGF-BB or TGF-b1 [73, 74]. PAs may also be nearly occluded by the accumulation of acellular matrix, suggestive of a terminal scarring process. Approximately 20–25% of PAs ranging between 25–200 mm in diameter are compromised by intimal occlusion or concentric lesions [1]. Plexiform lesions, typically located at branching points of muscular arteries, consist of a network of vascular channels lined by endothelial cells and a core of myofibroblastic or less differentiated cells [75–77]. These lesions are characteristically found in cases of severe PH, including idiopathic pulmonary arterial hypertension (IPAH), and PH associated with HIV infection, liver cirrhosis, CREST, congenital heart malformations, and schistosomiasis. Recently, dilation lesions have been suggested to represent another potential endothelial cell lesion, with the formation of a rosary of dilated channels (also known as angiomatoid lesions), often distal to plexiform lesions [78]. It has also been proposed that plexiform lesions represent a process of misguided angiogenesis on the basis of the findings of expression of vascular endothelial growth factor (VEGF), its receptors VEGF-R1 (Flt) and VEGF-R2 (KDR), and hypoxia-inducible factors (HIF)-1a and HIF-1b [79]. The findings of monoclonality of endothelial cells in plexiform lesions in IPAH, but not in similar lesions in PH associated with congenital heart malformation, suggest that these lesions might arise from mutations in tumor suppressor genes [80]. Somatic loss of expression of TGF-b receptor 2 and proapoptotic Bax, potentially due to microsatellite instability, is also documented in IPAH plexiform lesions [81]. Heterozygous germline mutations of BMP receptor 2 (BMPR2) (a component of the TGF-b-related family) is found to underlie up to 60% of cases of familial IPAH [25, 26]. BMPR2 expression is documented in plexiform lesions [72] and in remodeled PAs in IPAH lungs, predominantly in endothelial cells. However, studies have revealed that the cells in the central core of plexiform lesions lack the expression of TGF-b receptor 1 and their signaling Smad(s). The absence of expression of the phosphorylated Smads is the best indication that there is no TGF-b or BMP signaling in these cells [82].

2.3 Structural Changes in Nonmuscular Alveolar Wall Vessels Perhaps the most characteristic, yet least understood, change in the pulmonary vasculature observed in PH is the “muscularization” of the vascular segments that normally lack a

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muscular coat, thus converting the precapillary segment into a resistance structure. In hypoxic models of PH, this is the first cellular event to occur in response to hypoxic exposure, as well as the first to disappear upon withdrawal of the hypoxic stimulus [2]. Mural cells are reported to increase in size and number, and both media and adventitia are formed to produce a new structural vessel profile. Several mechanisms have been invoked to explain the distal muscularization process. Certain vessels, termed “partially muscular,” contain pericytes and/or “intermediate cells.” These cells exhibit SM-like characteristics, although they can be distinguished morphologically and biochemically from a differentiated SMC [2, 6, 83]. In vessels where either pericytes or intermediate cells are present, it has been suggested that environmental stimuli such as hypoxia act directly to induce differentiation and proliferation of these cells, and so they directly contribute to the observed muscularization. Additionally, factors secreted by the endothelium in response to injury or to abnormal changes in blood flow such as PDGF, 5-HT, epidermal growth factor, and even ATP or monocyte chemoattractant protein 1 (MCP-1) could stimulate growth of SM-like cells. Other mechanisms, including those discussed below, may also operate in conjunction to contribute to “distal muscularization.” In the smallest alveolar wall vessels, which normally lack any elastic lamina and possess a capillary-like wall structure, the mechanisms of muscularization may involve more than simple migration of pericytes down the vessel. One suggested mechanism is the recruitment of interstitial fibroblasts via migration, alignment, and incorporation into the vessel wall [62, 84]. It is also possible that in these vessels, especially so-called corner vessels in the alveolus, there is extravasation of mesenchymal precursor cells [12, 85]. Similar to their role in muscularized vessels discussed above, these cells could differentiate into SM-like cells and contribute to hypoxia-induced distal muscularization. The possibility that endothelial cells transition/transdifferentiate into SM-like cells and contribute to distal muscularization also needs to be considered, as discussed further below. Endothelial–mesenchymal transition (EnMT) plays an important role in vascular development of the pulmonary and systemic circulations [86]. Morphology studies in human embryos have suggested that endothelial-like cells may give rise to SMCs during the maturation of both PAs and veins [87, 88]. Findings in experimental wound repair have suggested that EnMT may also take place in the adult, reporting that capillary endothelial cells could undergo conversion into interstitial connective tissue mesenchymal-like cells in granulation tissue. Others have observed that microvascular endothelial cells transition into mesenchymal cells in response to chronic inflammatory stimuli [89]. A possible role for EnMT in the neointimal thickening observed in transplant atherosclerosis and restenosis has also been suggested [90, 91].

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A number of in  vitro studies have demonstrated that endothelial cells from a variety of vascular beds retain the capability of transitioning into mesenchymal or even SM-like cells under a variety of culture conditions [74, 86, 92]. Endothelial cells derived from the adult bovine aorta convert to spindle-shaped a-SM-actin-expressing cells when treated with TGF-b1 [93]. Of relevance and importance to the discussions regarding inflammation in PH are recent studies demonstrating that human dermal microvascular endothelial cells can be induced to transform into myofibroblasts in vitro, following long-term exposure to inflammatory cytokines [94]. Recent studies have demonstrated that hypoxia is also capable of inducing the transdifferentiation of PA endothelial cells into myofibroblasts or SM-like cells in a process regulated by myocardin [95]. Hypoxic exposure of porcine PA endothelial cells induced changes from the typical cobblestone morphology to the spindle-shaped morphology associated with the mesenchymal, or fibroblast-like phenotype. Transfection of endothelial cells with small interfering RNA against myocardin prevented this transdifferentiation process. Another intriguing possibility, although never explored to our knowledge in pulmonary vascular disease, is that lung epithelial cells may transdifferentiate into mesenchymal cells. Epithelial–mesenchymal transition (EMT) is critical during normal embryonic development and is increasingly appreciated to contribute to fibrosis and cancer development and progression [96, 97]. TGF-b and Wnt signaling are important transducers of the EMT process. These systems are known to be involved in the pulmonary hypertensive ­process, thus supporting the idea that EMT could also ­contribute to the distal PA muscularization process. A better understanding of distal muscularization could provide new opportunities for therapy.

3 Changes in Vascular Cell Phenotype, and Their Contribution to Vascular Remodeling 3.1 Changes in Endothelial Cell Phenotype in PH Endothelial cells are recognized as major regulators of vascular function and homeostasis. Disturbance in endothelial cell function, often referred to as “endothelial dysfunction,” is universally observed in PH. Increases in endothelial cell permeability, as well as imbalance in endothelial cell production of vasoconstrictors versus vasodilators, activators versus inhibitors of SMC growth and migration, prothrombotic mediators, and proinflammatory versus anti-inflam-

K.R. Stenmark and M.G. Frid

matory signals all contribute to vascular remodeling in chronic PH. Significant changes in the physical state and composition of endothelial cell plasma membrane lipids, phospholipids, and fatty acid composition, as well as in membrane fluidity have been described in animal models of PH [98–104]. The mechanisms causing these alterations in plasma membranes in the pulmonary circulation are not clear. Increased contents of malondialdehyde and conjugated dienes suggest that lipid peroxidation occurs and plays a role [99]. Another potential mechanism is the activation of membrane phospholipases, which would account for the reduction in the content of plasma membrane phospholipids and the concomitant increase in the release of fatty acids [99]. Increases in phospholipase A and C and diacylglycerol lipase activity have been demonstrated [105]. Membrane changes observed in endothelial cells contribute directly to the alterations in growth factor receptor function, as well as in the transport and processing of biogenic amines, extracellular nucleotides, and adenosine, and thus could have significant effects on pulmonary vascular tone and structure [102, 103, 106]. An increase in endothelial cell permeability to plasma factors may be an important component of the pathogenesis of PH. The GTPases RhoA and Rac1 play opposing roles in the regulation of endothelial cell barrier function. Stimuli such as thrombin activate RhoA/Rho kinase (ROCK), which increases formation of F-actin stress fibers, cell contraction, and permeability. On the other hand, barrier-enhancing mediators such as sphingosine 1-phosphate and prostacyclin (PGI2) stimulate Rac1/PAK-1, which counteracts the effects of RhoA/ROCK and promotes cortical F-actin ring ­formation and barrier integrity [107]. PA endothelial cells cultured from chronically hypoxic piglets have an abnormal phenotype with low Rac1 and high RhoA activities, which correlates with increased stress fiber formation and permeability [108]. Activation of Rac1/PAK-1, as well as inhibition of RhoA, reverses the abnormal phenotype and acts to decrease permeability of the hypertensive endothelial cells. Alterations in the surface coagulant properties of endothelial cells, characterized by suppressed thrombomodulin production and increased procoagulant activity, largely through the increased expression of tissue factor, are observed in PH models. These alterations are important because in situ thrombosis of small peripheral PAs is not uncommon in human PH and is known to contribute to the severity of the disease [109]. In addition, in response to cytokines, hypoxia, and/or shear stress, endothelial cells display proadhesive, proinflammatory, and prothrombotic phenotypes and release soluble vascular cell adhesion molecule (VCAM-1), von Willebrand actor, plasminogen activator inhibitor 1, and endothelium-derived microparticles [110–114]. For instance, hypoxia increases interleukin-1 (IL-1) and interleukin-6 (IL-6) messenger RNA (mRNA) levels and IL-1a production in

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cultured endothelial cells, probably through nuclear factor kb or nuclear factor IL-6 [113–115]. Hypoxia (and/or IL-1) can also increase expression of endothelial leukocyte adhesion molecule 1, intracellular adhesion molecule 1, and VCAM-1 on endothelial cells. Enhanced expression of RANTES, mainly originating from endothelial cells, has been observed in lungs of patients with severe PH [116]. These observations support the idea that activation of the endothelial cells, leading to prothrombotic and proinflammatory interactions with circulating cells, participates in the pulmonary hypertensive response. This proinflammatory response could also facilitate recruitment and migration of progenitor cells into the vessel wall [117, 118]. A derangement in the endothelial production and release of substances that modulate vascular tone and the proliferative and synthetic state of SMCs is well documented [119–123]. Decreased production and/or activity of PGI2 and NO have been reported [119–121, 124–126]. Increases in the production of platelet-activating factor, leukotrienes, hydroxyeicosatetraenoic acids, endothelin, 5-HT, and PDGF have been observed [127–130]. Reduced NO bioavailability in PH can be due to decreased expression of endothelial NO synthase (eNOS), inhibition of eNOS enzymatic activity, and inactivation of NO by superoxide anion. Activation of endothelial RhoA/ROCK signaling can be involved in at least the first two processes. For example, RhoA/ROCK activation mediates hypoxia- and thrombin-induced inhibition of both eNOS expression and activity in cultured endothelial cells [122, 123]. The activity of arginase II, which reduces NO synthesis by competing with eNOS for the substrate l-arginine, is increased in PH ­endothelial cells [131], and RhoA/ROCK signaling mediates thrombinand TNF-a/lipopolysaccharide-induced activation of the enzyme in human endothelial cells [132]. Patients with IPAH have increased plasma levels of the endogenous inhibitor of eNOS asymmetric dimethylarginine (ADMA) [133], and the levels of ADMA and the enzyme that degrades it, dimethylarginine dimethyaminohydrolase, are, respectively, increased and decreased in the PA endothelium of IPAH patients [133]. In addition, recent reports have suggested that there is a release of extracellular ATP by PA endothelial cells under hypoxic conditions [134]. This is interesting because ATP is also released under conditions such as osmotic swelling, which, as discussed above, has been described in the setting of hypoxic exposure [135]. Extracellular ATP has direct effects not only on the endothelial cells, where, acting through purinergic receptors and AMP-activated protein kinase, it participates in controlling NO release as well as other endothelial cell functions [136]. Released nucleotides also directly activate SMC and fibroblast proliferation and migration [134, 137, 138]. The direct relevance of the release (or lack thereof) of many of these molecules to the hypertensive process has been confirmed in whole-animal studies. Rats treated with plateletactivating factor inhibitors and 5-lipoxygenase inhibitors, as

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well as mice lacking 5-lipoxygenase, demonstrate less PH and vascular remodeling in response to hypoxia [139, 140]. Endothelin receptor antagonists inhibit hypoxia-induced PH [129]. Germline deletions in eNOS are associated with increased right ventricular pressure and distal muscularization in both normoxia and hypoxia [141, 142]. However, one group of investigators found a decrease in pulmonary arterial muscularization early in the course of hypoxic exposure [143]. Increased production of the eNOS cofactor tetrahydrobiopterin is associated with attenuated development of hypoxic PH and vascular remodeling, whereas eNOS deficiency results in enhanced PH and remodeling [144]. Deficiency in other NOS isoforms has not been clearly associated with increased pulmonary vascular responses to hypoxia [145]. A complementary observation is that PGI2overexpressing mice are protected against hypoxia-induced PH [146]. Similarly, gene transfer of human PGI2 synthase ameliorates monocrotaline-induced PH [147]. Decreases in other vasodilator genes, such as atrial natriuretic peptide, adrenomedullin, heme oxygenase-1, and PGI2, are all also associated with enhanced hypoxia-induced PH and/or remodeling [145, 148–151]. Conversely, increased expression of the 5-HT transporter (5HTT) gene is associated with enhanced hypoxic PH [152]. Decreased expression of the 5HT-1B receptor is associated with decreased hypoxia responses [153]. Deficiency in the ET-1B receptor in rats is associated with increased hypoxic PH and remodeling. Interestingly, germline overexpression of ET-1 in a mouse did not result in significant PH but was associated with pulmonary inflammation and fibrosis [154]. The collective body of evidence described above ­demonstrates that “endothelial dysfunction” contributes significantly to vascular remodeling. Marked changes in endothelial permeability, coagulant, inflammatory, and protein synthetic capabilities are observed in chronic PH. These changes can affect directly or indirectly underlying SMC and adventitial fibroblasts, thus contributing to chronic abnormalities in vascular tone and structure.

3.2 Changes in SMC Phenotype in PH The severity of chronic hypoxic PH is determined, at least in part, by the extent of structural changes in the medial compartment of the PA wall. As mentioned already, these changes include SMC hypertrophy, proliferation, and matrix protein production/accumulation as well as recruitment of adventitial and/or circulating cells. Hypoxia, blood borne cytokines and those locally produced by endothelial cells, SMCs, fibroblasts, and inflammatory cells, as well as mechanical stress, collectively act to drive the cellular responses within the media. These stimuli activate a cascade of intracellular

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s­ ignaling mechanisms, including tyrosine kinases, mitogenactivated protein kinases, protein kinase C (PKC), phosphoinositidyl 3-kinase (PI3K), Smad phosphorylation, calcium (Ca2+) entry and ROCKs. All these collectively act to control SMC contractility, growth, differentiation, and matrix protein synthesis. Synergy between different stimuli and the resulting “cross talk” between signal transduction pathways augment the extent of vascular changes within the media. Susceptibility to these stimuli is enhanced when inhibiting mechanisms such as endothelial barrier function, local production of heparan sulfates and PGI2- and NO-induced increases in cyclic nucleotides are impaired. Intrinsic (genetic, developmental, acquired, or epigenetic) differences in growth and matrix synthetic capacity, as well as local and regional phenotypic heterogeneity of pulmonary SMCs, also regulate the pattern of remodeling of the tunica media in response to chronic hypoxia. Changes in receptor expression or function as well as in Ca2+ handling have been observed in SMCs from hypertensive PAs. Examples of these changes are discussed.

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5-HT1B receptor and that 5-HT1BR knockout mice develop less pulmonary vascular remodeling and PH than do wildtype mice [153]. However, Marcos et al. reported that chronic hypoxia-induced PH and increased vessel muscularization were not reduced by the 5-HT1B/1D receptor antagonist GR127935 [162]. The 5-HT2B receptor has also been implicated in hypoxic PH, as it has been shown to regulate 5-HTT activity [163]. 5-HT2BR null mice fail to develop hypoxic PH despite maintaining an acute response to hypoxia [163]. Moreover, 5-HT1BR, 5-HT2BR, and 5-HTT are colocalized in PA SMCs, and the 5-HT2BR functionally interacts with 5-HT1BR. This cross talk between the Gq-coupled 5-HT2BR and the Gi-coupled 5-HT1BR has been suggested to facilitate the development of PH [158]. It is possible that 5-HT receptors act by producing reactive oxygen species via the NADPH oxidase like enzyme to induce SMC proliferation [164]. Collectively, these observations suggest that the mechanisms of action of 5-HT, its transporter, and its receptors are rather complex and may be species- and cell-type-dependent. Further studies are needed to delineate how hypoxia effects 5-HT-induced changes in SMC phenotype and the relation of these changes to hypoxia-induced vascular remodeling.

3.3 Changes in 5-HTT and 5-HT Receptor Function in SMCs

3.4 K+ and Ca2+ Homeostasis in SMCs: Effects on Tone and Proliferation Many studies support a role for 5-HT in the pathogenesis of the SMC abnormalities that characterize PH, particularly as they relate to contraction and growth of SMCs [152, 155–158]. 5-HT is a vasoactive and, in some SMCs, a mitogenic ­molecule released from platelets, pulmonary neuroendocrine cells, and endothelial cells. Moreover, increases in plasma 5-HT levels have been observed under hypoxic conditions and in patients with PH [159, 160]. Exposure of PA SMCs to elevated levels of 5-HT results in increased SMC contraction and proliferation [160]. These observations have generated great interest in elucidating the mechanisms through which 5-HT exerts its effects and contributes to the remodeling process. An active transporter that internalizes 5-HT (5-HTT) and multiple cell-surface 5-HT receptors (5-HT2A, 5-HT1B/1D, and 5-HT2B) have been described on SMCs, and it appears that a balance of transporter and receptor expression and function determines the cellular responses to 5-HT [158]. Overexp‑ ression of the 5-HTT gene in rats exposed to chronic hypoxia augmented vascular remodeling, as did continuous infusion of 5-HT [152]. Administration of the 5-HTT inhibitors citalopram and fluoxetine partially reduced increased pulmonary vascular resistance in chronically hypoxic mice, a finding supported by other experiments in which hypoxic PH was attenuated in knockout mice with disruption of 5-HTT [161, 162]. It has also been demonstrated that the increased contractile response to 5-HT in PAs isolated from chronically hypoxic wild-type mice is mediated through the

In PA SMCs the free Ca2+ concentration in the cytosol is an important determinant of contraction, migration, and proliferation. As cytosolic Ca2+ concentration increases, Ca2+ is bound by calmodulin, which activates myosin light chain kinase (MLCK), which, in turn, phosphorylates the myosin light chain (MLC). The phosphorylated MLC initiates cycling of the myosin cross-bridges with actin filaments and causes PA SMC contraction and vasoconstriction. In addition, an elevated concentration of nuclear Ca2+ propels quiescent cells into the cell cycle and through mitosis, promoting cellular proliferation. Cytosolic Ca2+ concentration in PA SMCs can be increased by (a) Ca2+ influx through voltagedependent Ca2+ channels, receptor-operated Ca2+ channels, and store-operated Ca2+ channels [store-operated Ca2+ channels in vascular SMCs include the transient receptor potential (TRP) channels], (b) Ca2+ release from intracellular stores (e.g., sarcoplasmic reticulum) via Ca2+-release channels (e.g., inositol 1,4,5-trisphosphate receptors and ryanodine receptors), and (c) inward transportation of Ca2+ via Ca2+ transporters in the plasma membrane, such as the reverse mode of the Na+/Ca2+ exchanger. PA SMC proliferation is associated with a significant increase in mRNA and protein expression of TRP channels such as TRPCs 1, 3, and 6 [165, 166]. In addition, cytosolic Ca2+ concentration is significantly greater in proliferating PA SMCs than in quiescent cells. Inhibition

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of TRP channel expression with antisense oligonucleotides significantly inhibits PA SMC proliferation. Thus, upregulation of TRP channels may contribute to heightened PA SMC proliferation in chronic PH. Resting Em in vascular SMCs normally ranges between –70 and –50 mV. Decreased expression and/or function of K+ channels leads to sustained membrane depolarization and contributes to sustained elevation of cytosolic Ca2+ concentration by (a) activating voltage-dependent Ca2+ channels, (b) facilitating the production of inositol 1,4,5-trisphosphate, which stimulates the release of sarcoplasmic reticulum Ca2+ into the cytoplasm, and (c) promoting Ca2+ entry via the reverse mode of Na+/Ca2+ exchange. In PA SMCs from IPAH patients, the amplitude of whole-cell IK(V) and mRNA/protein expression levels of Kv channel subunits (e.g., Kv1.2 and Kv1.5) are both significantly decreased in comparison with those in cells from controls or patients with secondary PH [167]. The downregulated Kv channels and decreased IK(V) are associated with a more depolarized Em in IPAH PA SMCs and the resting cytosolic Ca2+ concentration is much higher than in PA SMCs from controls, again leading to PA SMC proliferation. Further, in PA SMCs from IPAH patients, apoptosis induced by BMP and staurosporine is inhibited in comparison with cells isolated from secondary PH [168]. Decreased Kv expression is an emerging hallmark of the PH PA SMCs occurring in human PH and all known experimental models (including those due to BMPR2 dysfunction or excess 5-HT activity) [169, 170]. Interestingly, both Kv channels involved in hypoxic pulmonary vasoconstriction (Kv1.5 and Kv2.1) are inhibited by anorexigens, 5-HT and ET-1, which significantly and reversibly reduces the Kv1.5 currents [171, 172]. As would be predicted, restoring Kv1.5 expression reduces chronic hypoxic PH and restores hypoxic pulmonary vasoconstriction.

3.5 RhoA/ROCK Signaling in SMCs Intracellular signaling, via GTP-RhoA and its downstream effector ROCK (Rho/ROCK signaling) is also increasingly appreciated as an important signaling pathway in the pathogenesis of PH because of its potential to cause sustained vasoconstriction and its effects on the proliferative and differentiation state of PA wall cells [173, 174]. The contractile state of vascular SMCs is regulated by phosphorylation (causing contraction) and dephosphorylation (causing relaxation) of the 20-kDa regulatory MLC. Phosphorylation of MLC is catalyzed by Ca2+/calmodulin-dependent MLCK and dephosphorylation is catalyzed by calcium-independent MLC phosphatase (MLCP), which is targeted to myosin by its regulatory myosin binding subunit. Thus, the balance and activity of MLCK and MLCP regulate contraction. At a

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given level of cytosolic Ca2+, the activity of both enzymes can be modulated by second-messenger-mediated pathways to change MLC phosphorylation and thus the force of contraction; in other words, the activity of these pathways will change the calcium sensitivity of contraction [173, 174]. There is indirect evidence that hypoxia activates RhoA in SMCs [174, 175]. Further, several vasoconstrictors speculated to participate in the sustained vasoconstriction implicated in PH (including ET-1, 5-HT, and thromboxane) activate RhoA in vascular SMCs through GPCR-mediated signaling pathways [173, 174]. Interestingly, GPCRindependent signaling to RhoA also occurs and can be mediated by receptor and nonreceptor tyrosine kinases and by recruitment of other GTPases [173, 174]. In addition, RhoA/ROCK signaling is differentially regulated among subsets of SMCs within a given segment of the PA [176]. The importance of ROCK signaling in chronic hypoxic PH is highlighted by the experiments of McMurtry et al., in which the ROCK inhibitor Y27632, but not nifedipine, caused marked vasodilation in the hypertensive animals [177]. The severity of chronic hypoxic PH has also been shown to be reduced by chronic blockade of the Rho/ROCK signaling [177, 178]. ROCK inhibition has also recently been reported to significantly decrease PA pressures in a model of severe PH characterized by occlusive intimal lesions [179].

3.6 Changes in Adventitial Fibroblast Phenotype and Function in PH A rapidly emerging concept suggests that the vascular adventitia acts as a biological processing center for the retrieval, integration, storage, and release of key regulators of vessel wall function [66] (Fig.  7). In fact, under certain conditions, the adventitial compartment may be considered the principal injury-sensing tissue of the vessel wall [66]. In animal models of PH, early and often dramatic increases in fibroblast proliferation and adventitial remodeling have been reported [4, 6, 66]. Accumulating experimental data suggest that, in response to vascular stresses including hypoxia and overdistention, the adventitial fibroblast is the first cell to be activated, to proliferate, to upregulate contractile and extracellular matrix proteins, and to release factors that can directly affect medial SMC tone and growth, as well as stimulate the recruitment of inflammatory and progenitor cells [4, 6, 12, 13, 60, 66]. Each of these changes in fibroblast phenotype modulates either directly or indirectly overall vascular function and structure. In vitro experimental studies support the notion that adventitial fibroblasts have greater propensity to proliferate under hypoxic conditions than do resident PA SMCs

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K.R. Stenmark and M.G. Frid

Fig. 7  Adventitial fibroblasts play a pivotal role in control of vascular tone and structure. Adventitial fibroblasts are rapidly activated in response to a variety of pathophysiologic stimuli and release potent

mediators capable of affecting the function of resident vessel wall cells, and also facilitate recruitment of inflammatory and progenitor cells. (Adapted with permission [66])

[180–182]. It has even been suggested that hypoxiainduced proliferation is a unique property of the PA fibroblast [181]. Current evidence implies that activation of Gai and Gq family members, perhaps in a ligand-independent fashion, with subsequent stimulation of PKC and mitogenactivated PKC family members, is an important regulator of hypoxia-induced fibroblast proliferation [134, 138, 180]. Activation of PI3K and synergistic interaction with Akt, mammalian target of rapamycin, and p70 ribosomal protein S6 kinase have also recently been demonstrated to be essential for the proliferative response of PA adventitial fibroblast to hypoxia [134, 138]. Transcriptional targets of hypoxia and/or the aforementioned signaling pathways include early growth response-1 (Egr-1), HIF-1a, and HIF-2a. Knockdown experiments have shown that both Egr-1 and HIF-2a participate in the proliferative response [183, 184]. It has also been shown that downstream targets of HIF-1a, angiotensin-converting enzyme (ACE) and angiotensin II receptor type I (AT-I), are upregulated by hypoxia and that inhibitors of ACE and AT-I can block hypoxia-induced, HIF-dependent, adventitial fibroblast proliferation [185–187]. In addition, mice partially deficient in either HIF-1a or HIF-2a have attenuated hypoxiainduced PH and remodeling [188, 189]. It should be stressed that the arterial adventitia, like the lung interstitium, is composed of heterogeneous fibroblast populations, and that only specific subsets of fibroblasts appear capable of proliferating in response to hypoxia [66, 190]. Experimental evidence suggests that a selective expansion of these “hypoxia-sensitive” fibroblast subpopulations accounts for the marked accumulation of fibroblasts in the adventitia of chronically hypoxic animals, also consistent with observations that fibrogenic foci in the lung comprise selective fibroblast populations [66, 190].

PA adventitial fibroblasts derived from chronically hypoxic animals exhibit heightened growth responses to a number of growth-promoting stimuli, including hypoxia, as well as alterations of proliferation-associated signaling pathways, compared with fibroblasts derived from normoxic animals [181, 182, 191–193]. For instance, the atypical PKCz isozyme acts as a pro-proliferative kinase in fibroblasts from chronically hypoxic animals, whereas it exhibits antiproliferative actions in fibroblasts from normoxic animals [192]. These observations raise the possibility that chronic hypoxia leads to the emergence of fibroblasts in the adventitia that have lost their ability to limit stimulus-induced proliferation [193]. Activated PA adventitial fibroblasts may exert significant effects on other cell types within the vessel wall through the production of paracrine factors (by HIF-1a-dependent mechanisms), which have potent stimulatory effects on SMC proliferation or production of reactive oxygen species (generated in large part through a distinct NADPH oxidase system [194, 195]). Fibroblast-produced reactive oxygen species can exert potent effects on vascular tone directly by stimulating contraction of SMCs and/or by acting as a sink and destroying endothelial-derived NO before its optimal effects on SMC relaxation have been achieved [195]. In support of the important role of NADPH-derived radicals in hypoxia-induced PH and vascular remodeling is the fact that both responses are completely abolished in NADPH (GP91FOX) knockout mice [196]. Hypoxia-induced PH and numerous other vasculopathies are associated with early and dramatic increases in the differentiation of fibroblasts toward a myofibroblast (a-SM actin+) phenotype [12, 59, 62, 197–200]. Activated fibroblasts and myofibroblasts contribute to the structural remodeling of the vessel through increased production of collagen and other extracellular matrix proteins, including fibronectin,

52  Pulmonary Vascular Remodeling: Cellular and Molecular Mechanisms

tenascin, and elastin [12, 14, 66, 197, 198]. Myofibroblast accumulation can also contribute to the abnormalities of tone observed in chronic hypoxic PH. The contractile properties of myofibroblasts appear to be different from those of SMCs, with a slower onset of contraction in response to vasoactive stimuli and a failure to relax in response to vasodilating ­stimuli [197]. Significant changes in the production of chemokines and cytokines by the adventitial fibroblast (including MCP-1, stromal cell-derived factor-1, thrombin, and VEGF) can be induced in response to hypoxia [66, 67]. These chemokines can serve to recruit inflammatory circulating progenitor cells (discussed later). In addition, activated adventitial fibroblasts are known to secrete a variety of proangiogenic factors [60, 66, 67]. In this context, it has been reported that hypoxia induces a rapid expansion of the PA adventitial vasa vasorum [12, 67]. Fibroblasts were recently shown to directly regulate vasa vasorum endothelial cell proliferation and assembly into networks [12]. Expansion of the vasa vasorum appears important in contributing to the progression of many vascular diseases, possibly by providing a conduit for delivery of inflammatory and progenitor cells to the vessel wall [66, 201].

4 Role of Inflammation, Progenitor Cells, and Vasa Vasorum in Pulmonary Vascular Remodeling Despite strong evidence that inflammation is heavily involved in PH, this is the least studied aspect of the disease. There is evidence of global immunologic alterations in IPAH patients and PH occurs in the setting of profound immune deregulation and underlying HIV infection and collagen vascular ­disease [202, 203]. It is important to note that patients in whom inflammation is present are also the subgroups of PH with the worst prognosis. Several types of inflammatory cells, including activated T cells, B cells, monocytes, and macrophages, have been documented to infiltrate the PA wall in IPAH [8, 77]. Concordantly, PAs of IPAH patients express IL-1, IL-6, PDGF-BB, as well as the chemokine RANTES, (an important chemoattractant for monocytes and T cells) and macrophage inflammatory protein (MIP)-1a [116, 202, 204]. Lungs of IPAH patients also have increased expression of fractalkine, a chemokine involved in T-cell trafficking and monocyte recruitment, and these patients have higher levels of the fractalkine receptor CX3CR-1 on T cells as compared with controls [205]. The important role of inflammation is further highlighted in cases of PH where a viral cause can be identified. Viral proteins can affect vascular remodeling, with direct effects on vascular wall cells and extracellular matrix,

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or indirectly through the dysregulation of inflammation and immunity [206]. The HIV Nef (negative factor) was recently found in endothelial cells from patients with HIV PH [207]. Nef can affect several signaling pathways, including survival, proapoptotic, and proangiogenic pathways [208]. Human herpes virus 8 (the Kaposi-associated herpes virus), which carries several inflammatory, proangiogenic, and proproliferative genes, has also been found in human plexigenic lesions [209]. Similar findings have been reported in inflammatory cells, which show activation of nuclear factor of activated T cells (NFAT), the transcription factor that regulates the activation of T cells and the expression of many inflammatory mediator and cytokine genes. PH patients with scleroderma, in fact, have very high levels of NFAT in their circulating lymphocytes [210]. CD3+ cells with activated NFAT were also observed in the PA wall of patients with PH but not in normal subjects [210]. Intriguingly, in both humans and animal models of PH, activated NFAT has been observed in PA SMCs, again supporting a role for this proinflammatory transcription factor in the disease process. Collectively, these observations point out that inflammation plays a critical, as of yet unrecognized role in many forms of human PH. Several recent studies have also documented the enhanced expression of inflammatory mediators (MCP-1, MIP-2, IL-1b, IL-6) and increased numbers of macrophages and lymphocytes in the lungs of animals with experimental PH [12, 150, 211–213]. Chronic hypoxic exposure results in the robust and persistent appearance of mononuclear cells in the PA adventitia and media of both weanling rats and neonatal calves [12]. Interestingly, at least in these two animal models, hypoxia-induced accumulation of mononuclear cells appeared specific to the pulmonary circulation since no mononuclear cell recruitment was noted in systemic vessels (aorta). No neutrophils were identified in the pulmonary perivascular areas at any of the time points evaluated (24 h or more) [12]. Further, the accumulation of monocytes appeared to correlate with the degree of perivascular remodeling observed in different species (Fig.  5). These observations appear to substantiate a critical role for inflammatory cell accumulation in the vascular remodeling process. A substantial proportion of the mononuclear cells that accumulated around PAs comprised fibrocytes (cells characterized by dual expression of leukocytic and mesenchymal markers) [68]. As mentioned earlier, fibrocytes may play a particularly important role in the hypoxia- or injury-induced vascular remodeling process. They have been described as important contributors to the fibrosis observed in lung injury, wound healing, and asthma [68–70, 214–216]. The recruited monocytes and fibrocytes release a variety of factors, which can act to increase the proliferation and/or differentiation of resident fibroblasts and SMCs. In addition, fibrocytes are potent producers of extracellular matrix proteins, especially collagen, raising the possibility that nonresident, recruited

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mesenchymal cells contribute to vascular fibrosis [12, 68–70]. Fibrocytes also produce angiogenic factors and could contribute to the neovascularization of the vasa vasorum described above [68, 70]. Other investigators have also suggested a role for circulating bone-marrow-derived cells in chronic-hypoxia-induced remodeling [217]. The contribution of circulating mononuclear cells/fibrocytes to the hypoxia-induced vascular remodeling process was confirmed by depletion studies, in which the number of these cells was reduced in the circulation of hypoxic rats using intravenous injections of liposome-encapsulated clondronate or gadolinium chloride [12]. A striking reduction in adventitial thickening as well as a near-complete inhibition of the accumulation of collagen, fibronectin, and tenascin-C were documented [12]. In the monocrotaline model, it was shown that interleukin-10 delivery by an adeno-associated virus to monocrotaline-treated rats decreased PA pressure and improved survival while dramatically reducing macrophage infiltration and cell proliferation in the vessel wall [213]. Collectively, these observations in humans and in animal models of PH demonstrate a critical role for inflammation in the vascular remodeling process. Questions arise as to how leukocytes and precursor cells traffic into the injured/diseased vascular wall, particularly the adventitia. In the systemic circulation, the adventitial vasa vasorum microcirculation undergoes marked neovascularization in a number of vasculopathies and is thought to serve as a conduit for continued delivery of inflammatory and progenitor cells to the vessel wall [201, 218, 219]. Similar changes have been documented in the PAs of chronically hypoxic calves [67]. Further, experiments have shown that inhibition of plaque neovascularization reduced macrophage accumulation and progression of advanced ­atherosclerosis [201]. Other experiments have shown that hypoxic fibroblasts support the growth of vasa vasorum endothelial cells [220]. Thus, local fibroblasts and recruited inflammatory cells play a role in driving the neovascularization of the vasa vasorum and contribute to a feed-forward loop of vascular ­remodeling. Future experiments need to be directed at determining the factors which recruit and retain inflammatory and progenitor cells in the vessel wall so as to ultimately design specific therapies to turn the process off.

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201. Moulton KS, Vakili K, Zurakowski D et al (2003) Inhibition of plaque neovascularization reduces macrophage accumulation and progression of advanced atherosclerosis. Proc Natl Acad Sci USA 100:4736–4741 202. Dorfmüller P, Perros F, Balabanian K, Humbert M (2003) Inflammation in pulmonary arterial hypertension. Eur Respir J 22:358–363 203. Nicolls MR, Taraseviciene-Stewart L, Rai PR, Badesch DB, Voelkel NF (2005) Autoimmunity and pulmonary hypertension: a perspective. Eur Respir J 26:1110–1118 204. Humbert M, Monti G, Fartoukh M et  al (1998) Platelet-derived growth factor expression in primary pulmonary hypertension: comparison of HIV seropositive and HIV seronegative patients. Eur Respir J 11:554–559 205. Perros F, Dorfmüller P, Souza R et al (2007) Fractalkine-induced smooth muscle cell proliferation in pulmonary hypertension. Eur Respir J 29:937–943 206. Spiekerkoetter E, Alvira CM, Kim YM et al (2008) Reactivation of gammaHV68 induces neointimal lesions in pulmonary arteries of S100A4/Mts1-overexpressing mice in association with degradation of elastin. Am J Physiol Lung Cell Mol Physiol 294:L276–L289 207. Marecki JC, Cool CD, Parr JE et al (2006) HIV-1 Nef is associated with complex pulmonary vascular lesions in SHIV-nef-infected macaques. Am J Respir Crit Care Med 174:437–445 208. Fackler OT, Baur AS (2002) Live and let die: Nef functions beyond HIV replication. Immunity 16:493–497 209. Cool CD, Rai PR, Yeager ME et al (2003) Expression of human herpesvirus 8 in primary pulmonary hypertension. N Engl J Med 349:1113–1122 210. Bonnet S, Rochefort G, Sutendra G et al (2007) The nuclear factor of activated T cells in pulmonary arterial hypertension can be therapeutically targeted. Proc Natl Acad Sci USA 104:11418–11423 211. Madjdpour C, Jewell UR, Kneller S et al (2003) Decreased alveolar oxygen induces lung inflammation. Am J Physiol Lung Cell Mol Physiol 284:L360–L367 212. Zampetaki A, Minamino T, Mitsialis SA, Kourembanas S (2003) Effect of heme oxygenase-1 overexpression in two models of lung inflammation. Exp Biol Med (Maywood) 228:442–446 213. Ito T, Okada T, Miyashita H et al (2007) Interleukin-10 expression mediated by an adeno-associated virus vector prevents monocrotaline-induced pulmonary arterial hypertension in rats. Circ Res 101:734–741 214. Bayes-Genis A, Campbell JH, Carlson PJ, Holmes DR Jr, Schwartz RS (2002) Macrophages, myofibroblasts and neointimal hyperplasia after coronary artery injury and repair. Atherosclerosis 163:89–98 215. Quan TE, Cowper S, Wu SP, Bockenstedt LK, Bucala R (2004) Circulating fibrocytes: collagen-secreting cells of the peripheral blood. Int J Biochem Cell Biol 36:598–606 216. Schmidt M, Sun G, Stacey MA, Mori L, Mattoli S (2003) Identification of circulating fibrocytes as precursors of bronchial myofibroblasts in asthma. J Immunol 171:380–389 217. Hayashida K, Fujita J, Miyake Y et al (2005) Bone marrow-derived cells contribute to pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension. Chest 127:1793–1798 218. Barger AC, Beeuwkes R 3rd, Lainey LL, Silverman KJ (1984) Hypothesis: vasa vasorum and neovascularization of human coronary arteries. A possible role in the pathophysiology of atherosclerosis. N Engl J Med 310:175–177 219. Hayden MR, Tyagi SC (2004) Vasa vasorum in plaque angiogenesis, metabolic syndrome, type 2 diabetes mellitus, and atheroscleropathy: a malignant transformation. Cardiovasc Diabetol 3:1 220. Davie NJ, Gerasimovskaya EV, Hofmeister SE et  al (2006) Pulmonary artery adventitial fibroblasts cooperate with vasa vasorum endothelial cells to regulate vasa vasorum neovascularization: a process mediated by hypoxia and endothelin-1. Am J Pathol 168:1793–1807

Chapter 53

Carbon Monoxide and Heme Oxygenase in the Regulation of Pulmonary Vascular Function and Structure Stella Kourembanas

Abstract  Accumulating evidence indicates that antioxidant and/or stress response genes play a critical, cytoprotective role in the systemic and pulmonary vasculature. One of these genes is heme oxygenase-1 (HO-1). HO-1 is a microsomal enzyme that is ubiquitously distributed and strongly induced by several stressors, including oxidative, environmental, and hemodynamic stresses. HO-1 catalyzes the oxidative degradation of free heme to biliverdin (BV), free iron, and carbon monoxide (CO). BV is reduced to bilirubin (BR), a potent endogenous antioxidant with potential anti-inflammatory properties, whereas iron is sequestered by ferritin, leading to additional antioxidant and antiapoptotic effects. CO is a gaseous molecule with increasingly recognized biological properties, including anti-inflammatory effects, vasodilation, and neurotransmission in the central and peripheral nervous systems. It shares many similarities with nitric oxide (NO) as an activator of soluble guanylyl cyclase, leading to an increase in cyclic GMP (cGMP) levels and causing vasodilation. Like NO, CO is an inhibitor of platelet aggregation and smooth muscle cell proliferation. These properties of HO-1 and its enzymatic products make it a suitable candidate to orchestrate regulatory functions in the lung and to potentially be a key mediator of the pathways underlying the control of lung vascular homeostasis. This chapter focuses on the protective effects of HO-1 and its products, CO and BR, in pulmonary hypertension and in the maintenance of cardiovascular function. Keywords  Hemo oxygenase-1 • Nitric oxide • Heme • Oxidative stress • Anti-inflammatory effect • Redox status

1 Introduction Accumulating evidence indicates that antioxidant and/or stress response genes play a critical, cytoprotective role in the systemic and pulmonary vasculature. One of these genes is heme oxygenase-1 (HO-1). HO-1 is a microsomal enzyme that is ubiquitously distributed and strongly induced by several stressors, including oxidative, environmental, and hemodynamic stresses [1–3]. HO-1 catalyzes the oxidative degradation of free heme to biliverdin (BV), free iron, and carbon monoxide (CO) (Fig. 1). BV is reduced to bilirubin (BR), a potent endogenous antioxidant with potential antiinflammatory properties, whereas iron is sequestered by ferritin, leading to additional antioxidant and antiapoptotic effects [4, 5]. CO is a gaseous molecule with increasingly recognized biological properties, including anti-inflammatory effects, vasodilation, and neurotransmission in the central and peripheral nervous systems. It shares many similarities with nitric oxide (NO) as an activator of soluble guanylyl cyclase, leading to an increase in cyclic GMP (cGMP) levels and causing vasodilation. Like NO, CO is an inhibitor of platelet aggregation and smooth muscle cell proliferation. These properties of HO-1 and its enzymatic products make it a suitable candidate to orchestrate regulatory functions in the lung and to potentially be a key mediator of the pathways underlying the control of lung vascular homeostasis. This review will focus on the protective effects of HO-1 and its products, CO and BR, in pulmonary hypertension and in the maintenance of cardiovascular function.

2 Molecular Mechanisms of Pulmonary Arterial Hypertension S. Kourembanas (*) Department of Pediatrics, Division of Newborn Medicine, Children’s Hospital, Harvard Medical School, 300 Longwood Ave, Enders 961, Boston, MA 02115, USA e-mail: [email protected]

Pulmonary arterial hypertension (PAH) is a serious lung disease with multifactorial etiology as described elsewhere in this book. Vasoconstriction, remodeling of the pulmonary vessel wall, and thrombosis contribute to increased pulmonary vascular resistance in PAH and lead to right ventricular

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_53, © Springer Science+Business Media, LLC 2011

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Fig. 1  Enzymatic activity of heme oxygenase-1 (HO-1). HO-1 catalyzes the degradation of heme to biliverdin, carbon monoxide (CO), and iron. Biliverdin is reduced to bilirubin, iron is sequestered by ferritin, and CO activates guanylyl cyclase to form the second messenger cyclic GMP

(RV) hypertrophy and ultimately RV failure and death. The process of pulmonary vascular remodeling involves all layers of the vessel wall and is complicated by cellular heterogeneity within each compartment of the pulmonary arterial wall. Several cell types including endothelial cells, smooth muscle cells, and fibroblasts, as well as inflammatory cells and platelets, may play a significant role in PAH. It has been proposed that the initial insult may be endothelial cell dysfunction from hypoxia, shear stress, toxins, or inflammation. This can lead to endothelial cell apoptosis and clonal expansion of apoptosis-resistant endothelial cells resulting in intimal cell hyperplasia and plexiform lesions with lumen occlusion [6]. In addition, injury to the endothelium causes the release of vasoconstrictors and mitogens that stimulate smooth muscle cell proliferation, migration, as well as matrix deposition [7–9]. As the vessel wall thickens, there is a concomitant expansion of the adventitial vasa vasorum, possibly in response to proangiogenic factors secreted by the adventitial fibroblasts [10]. Circulating endothelial progenitor cells or inflammatory cells can gain access to the vessel wall and contribute to arteriolar remodeling through these newly formed vessels [11, 12]. Transdifferentiation of endothelial cells to vascular smooth muscle cells may also contribute to vascular remodeling [13]. The progressive thickening of the vessel wall results in narrow or occluded vessels and interferes with normal blood flow, causing RV pressure overload. The response of the right ventricle to the increased afterload is an important determinant of patient outcome. Although several studies have addressed the molecular and cellular mechanisms underlying lung vascular remodeling, there is a gap in our understanding of the molecular mechanisms that underlie the transition from compensated hypertrophy to dilation and failure of the right ventricle that occurs during the course of the disease. The increase in afterload is the first trigger for RV adaptation in PAH; however, oxidative stress, inflammation, ischemia, and cell death may contribute to the eventual development of RV dilation and failure.

The identification of heterozygous germline mutations in the gene encoding the bone morphogenetic protein (BMP) type II receptor in patients with familial PAH has highlighted the important role of the BMP/transforming growth factor-b pathway in lung vascular biology [14, 15]. BMP-2, BMP-4, and BMP-7 inhibit the proliferation of smooth muscle cells derived from normal pulmonary arteries and from PAH patients with congenital heart diseases, but they fail to suppress proliferation of cells from patients with idiopathic or familial PAH [16]. An attractive hypothesis is that a failure of the growth-inhibitory effects of BMPs in idiopathic or familial PAH cells could contribute to the vascular obliteration and remodeling that characterize the condition. The failure to suppress growth of idiopathic or familial PAH cells was observed in all cases, whether or not specific BMPR2 mutations were identified, suggesting that defective BMP-mediated signaling may be a common factor in idiopathic or familial PAH. In pulmonary artery smooth muscle cells (PASMCs), Smad 1/5 signaling is coupled to growth inhibition [17, 18] and apoptosis [19]. Mutations in the BMPR2 gene favor activation of p38 mitogen-activated protein kinase (MAPK)-dependent pro-proliferative pathways, while inhibiting Smad-dependent signaling in a mutationspecific manner. Thus, a feature common to all mutants is a gain of function involving p38 MAPK activation. In a recent study, HO-1 was found to be a BMP-4 inducible gene in human PASMCs [20]. This study demonstrated that the induction of HO-1 messenger RNA was dependent on p38 MAPK and extracellular-signal-regulated kinase (ERK) 1/2 and was Smad-independent. Inhibition of HO-1 signaling in BMPR2 mutant cells by a second prohypertensive stimulus would interfere with the body’s adaptive, cytoprotective mechanisms and lead to manifestation of disease. The specific cell–cell interaction and signaling pathways underlying PAH result from an imbalance of vasoconstrictors and vasodilators, increased vascular inflammation, differential vascular cell apoptosis, KV channel inhibition and dysfunction, and enhanced proteolysis of extracellular

53  Carbon Monoxide and Heme Oxygenase in the Regulation of Pulmonary Vascular Function and Structure

matrix. There is downregulation of vasodilators, including reduced endothelial NO production [21, 22], reduction of vasointestinal peptide [23], and inhibition of KV channels; [24], an increase in smooth muscle cell mitogens and vasoconstrictors such as endothelin-1 (ET-1) [8], platelet-derived growth factor (PDGF)-B [7], and serotonin; [25], as well as increased elastase activity [26] and, importantly, haplotype insufficiency of the BMPR2 gene [14, 15]. HO-1 may play a key cytoprotective role in the lung vasculature to modulate the above-described molecular/cellular pathways via its antimitogenic, antiapoptotic, and anti-inflammatory effects, described later.

3 Heme Oxygenase-1 and Cytoprotection in the Lung In the lung, HO-1 is expressed in various cell types, including type II pneumocytes, alveolar macrophages, fibroblasts, and vascular cells. It is induced by heme, hyperoxia, hypoxia, endotoxin, heavy metal exposure, allergen exposure, and proinflammatory cytokines such as IL-6 and TNF-a [27, 28]. The molecular regulation of HO-1 occurs primarily at the transcriptional level. There are two major upstream enhancers at −4 kb and at −10 kb relative to the transcription initiation site that are responsible for the inducible expression of the murine HO-1 gene in response to diverse stimuli, including hypoxia, oxidant stress, heavy metal response, and lipopolysaccharide (LPS) treatment [29, 30]. Among the many transcription factors involved in HO-1 regulation, NF-E2-related factor-2 (Nrf2) binds to the stress-response elements which bear homology to the antioxidant responsive element consensus sequence and mediate the response to many inducers, including LPS, heme, and heavy metals [30]. Other transcriptional regulators of HO-1 include hypoxia-inducible factor-1, heat shock factor-1, activator protein-1, early growth-response factor-1, and nuclear factor k-B. To date, there has been a single reported case of a patient with HO-1 deficiency who died in childhood from chronic oxidative stress and vascular injury [31]. There has been increasing recognition, however, that polymorphisms of the HO-1 promoter that lead to reduced HO-1 expression may be associated with increased risk of cardiovascular and respiratory diseases. A dinucleotide repeat polymorphism, [GT]n, within the proximal promoter of the human HO-1 gene at approximately −200 was reported to modulate the transcriptional activity of the gene [32]. The length of the dinucleotide repeat ranges from [GT]10 to [GT]40, and this represents a continuum of alleles, with [GT]25 often used as a cutoff. Repeat lengths less than 26 are classified as short (S) alleles and those of 26 or more are classified as long (L) alleles. Studies suggest that patients with the S-class polymorphisms

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exhibit better outcomes in cardiopulmonary disease, compared with patients with the L-class allele. In pulmonary disease, the L-class genotype has been associated with increased susceptibility to smoking-induced emphysema [33]. In addition, the L-class genotype may also be associated with lung adenocarcinoma in male smokers [34] and with susceptibility to pneumonia in elderly Japanese [35]. In cardiovascular disease, the S-class genotype has been associated with attenuated restenosis after angioplasty or stent implantation and a decreased inflammatory response [32]. Several other reports link the S-class genotype with improved protection and outcomes in vascular disease. The induction of HO-1 in various pathological conditions in animal models of disease has been shown to confer protection from injury. Rats receiving Ad5-HO-1 before exposure to hyperoxia manifested reduced lung injury such as edema, hemorrhage, and inflammation relative to controls [36]. Ad-HO-1 treatment resulting in increased lung HO-1 expression protected against bleomycin-induced pulmonary fibrosis [37] and cardiac-specific expression of HO-1 protected from ischemia–reperfusion (I/R) injury in transgenic mice [38]. The adenoviral-mediated transduction of HO-1 into ApoE-deficient mice inhibited atherosclerotic plaque lesions [39] and Ad-HO-1 delivery in pigs inhibited vascular arterial injury, whereas HO-1 null mice displayed hyperplastic arterial injury and intimal hyperplasia compared with wild-type controls [40]. Additionally, homozygous HO-1 null mice displayed increased mortality in a model of lung I/R injury that was partially rescued by inhaled CO [41], and HO-1 was critical in determining cardiac xenograft survival in mice [42]. There is increased recognition that HO-1 may mediate the cytoprotective actions of other therapeutic agents. For example, the cytoprotective effects of vascular endothelial growth factor (VEGF) in hyperoxic lung injury were shown to be mediated by HO-1, since when VEGF-overexpressing mice were treated with short interfering RNA (siRNA) targeting HO-1, all markers of injury increased [43]. In a separate model, HO-1 was shown to mediate the cytoprotective, anti-inflammatory actions of interleukin-10 (IL-10) [44], inhibiting the neointimal proliferation in aortic allografts in mice [45].

3.1 Actions of Carbon Monoxide CO is a ubiquitous air pollutant and is classified as a toxic molecule. Indeed, high levels of CO displace oxygen from hemoglobin, resulting in tissue hypoxia. Heme oxygenasederived CO was formerly considered a catabolic elimination product with no physiological significance [46]. Human blood contains CO, which originates in part from the degradation of the oxygen carrier hemoglobin. Heme metabolism

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accounts for greater than 85% of the endogenous CO production in man [47] and the remainder is derived from other metabolic pathways, including the cytochrome P-450 dependent oxidation of xenobiotics and lipid substrates [48]. Increased exhaled CO levels have been reported in several pulmonary pathological conditions, including asthma exacerbation, chronic obstructive pulmonary disease (COPD), and lung infections. The increased CO levels may result from increased activity of endogenous lung HO-1 in response to stress as well as from an additional systemic inflammatory response that is accompanied by a corresponding increase in carboxyhemoglobin (COHb) levels [49, 50]. Basal levels of COHb reach 1–3% depending on the contribution of environmental CO in nonsmokers and may be as high as 10–15% in smokers [51]. The binding of CO to soluble guanylyl cyclase stimulates its enzymatic activity and increases the production of cGMP. cGMP is an important, widely recognized second messenger molecule that mediates vascular relaxation, neuronal transmission, bronchodilation, inhibition of platelet aggregation, and inhibition of smooth muscle cell proliferation. CO-induced increases in cGMP production are 30–100 times lower than those of its cognate gas, NO [52]. We and others have proposed that NO may be the critical molecule that regulates baseline cGMP levels and under conditions of stress associated with endothelial cell injury and an underlying NO-deficient state, it is CO that substitutes for NO in the maintenance of vascular homeostasis. Indeed, the vasodilatory effects of CO may be important for maintaining tissue perfusion in lungs during hypoxic states to compensate for reduced NO levels [53, 54]. A role of CO in vasodilation has been reported in numerous studies. Exogenous CO relaxed the aortic rings in a cGMP-dependent fashion [55], whereas overexpression of HO-1 by Ad-HO-1 infection of the vessels inhibited phenylephrine-dependent vasoconstriction in isolated aortic rings by inducing cGMP production. The effects of CO are not limited to the vascular smooth muscle. Airway smooth muscle also relaxes with exogenously administered CO via a cGMP-dependent pathway as demonstrated in guinea pig tracheas [56]. cGMP-independent mechanisms of vasodilation by CO have also been proposed. Just as with NO, CO may dilate blood vessels by directly activating calciumdependent potassium channels [57, 58]. At low concentrations, CO exerts a potent antiproliferative effect on vascular cells, including endothelial and smooth muscle cells, and thus has the potential to prevent or reverse tissue remodeling. CO was reported to inhibit smooth muscle cell proliferation in culture by activation of cGC and upregulation of p21Waf1/Cip1 as well as by inhibition of the cell-cycle-specific transcription factor E2F1, preventing cell cycle progression [40, 59]. In addition, CO has indirect smooth muscle inhibitory effects in vivo, by inhibiting the expression of the endothelial derived

S. Kourembanas

Fig. 2  Cytoprotective actions of CO in pulmonary arterial hypertension (PAH). CO enhances vasodilation and inhibits prohypertensive pathways in the vasculature of the lung: it inhibits inflammation, endothelial cell (EC) inactivation and apoptosis, and smooth muscle cell (SMC) proliferation and migration

mitogens ET-1 and PDGF-B [53] (Fig. 2). The antiproliferative effects of CO were associated with protection in animal models of vascular injury [60]. More recently, inhaled CO rescued photochemical-induced endothelial injury consisting of endothelial cell loss and vessel denudation that was associated with platelet-rich microthrombi in the subendothelium of HO-1 deficient mice [61].

3.2 Actions of Bilirubin/Biliverdin The bile pigments BV and BR originate primarily from the turnover of hemoglobin heme and have long been regarded as toxic metabolites of heme degradation. BR is rapidly formed in cells by the enzymatic activity of biliverdin reductase. In neonates, the excessive accumulation of BR can cause neurologic complications including hearing deficits and, in severe cases, kernicterus. Despite these adverse consequences of severe elevations in BR levels, it is increasingly recognized that moderate levels of BR may have important homeostatic effects and serve to protect the newborn infant in its transition from the in utero, low-oxygen environment, to the high oxidative stress it encounters at birth. Specifically, in 1989, Stocker et al. demonstrated that BV and BR have potent antioxidant properties and could confer antioxidative protection to cells and tissues [62]. Several in  vitro and in vivo studies have since supported this protective role of BR conferring resistance to oxidative stress. For example, jaundiced Gunn rats displayed reduced plasma biomarkers of oxidative stress after exposure to hyperoxia [63] and recent clinical studies demonstrated correlations between increased circulating BR levels and decreased risk for cardiovascular disease. In healthy subjects, serum BR levels were inversely correlated with two indicators of atherosclerosis [64]. Therapeutic application of BR was shown to preserve myocardial function during cardiac I/R injury in an isolated perfused heart model [65] and in organ transplantation models of I/R, including the kidney, liver, and heart

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[66–68]. In addition to reduced markers of oxidative injury, the protection afforded by BR was associated with decreased neutrophil infiltration and reduced levels of proinflammatory markers. To date, very few studies have examined the protective role of BR on the lung. The few that have include models of LPS- and bleomycin-induced injury demonstrating decreased inflammation, lung edema and fibrosis, and increased survival [69, 70]. In addition to possible antioxidative effects, BR was shown to inhibit smooth muscle cell proliferation and suppress neointimal hyperplasia in a rat model of balloon injury [71]. We found BR to inhibit PASMC proliferation but compared with CO, its antimitogenic effects were far less potent [72].

3.3 Protective Effects of Heme Oxygenase-1 in Pulmonary Hypertension A number of studies have demonstrated that HO-1 or CO, or both, can prevent and reverse the development of PAH. With use of the hypoxic mouse model of PAH, HO-1 null mice were shown to have lung vessel remodeling and elevations in RV systolic pressure (RVSP) comparable to those of wildtype controls; however, the HO-1 deficient mice manifested protracted lung inflammation and two thirds died after 5 weeks of hypoxia owing to RV dilation and RV thrombus [73]. What was interesting in this model is the rapid progression to RV dilation and failure as opposed to the RV hypertrophy and compensation evident in the wild-type controls, all of whom survived the hypoxia exposure. Chemical inducing agents of HO-1 such as heme and NiCl2 inhibited the development of PAH in the hypoxic rat model [74]. In monocrotaline-induced PAH, treatment with rapamycin inhibited PAH via the induction of HO-1 in the lung [75]. Exposure of wild-type mice to hypoxia resulted in a dramatic increase in lung neutrophil and macrophage accumulation, peaking at 48 h of hypoxia and resolving over the subsequent week [76]. This inflammatory cell infiltrate was accompanied by a significant rise in the proinflammatory cytokines interleukin-1b, interleukin-6 (IL-6), monocyte chemoattractant protein-1, and macrophage inflammatory protein-2. Transgenic mice with targeted overexpression of HO-1 in the lung did not develop inflammation or cytokine induction during this time period and manifested normal RV pressures with absence of lung vascular remodeling, demonstrating that sustained elevations in the level of HO-1 ameliorate or prevent the features of PAH. In the rat monocrotaline model of PAH, IL-10 was delivered via intramuscular injection with an adeno-associated viral vector and was shown to inhibit the development of PAH, improving survival rates [77]. IL-10 reduced perivascular macrophage infiltration and smooth muscle cell proliferation in the arteriolar walls and decreased

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IL-6 levels. Interestingly, the actions of IL-10 were mediated, at least in part, by HO-1. These studies did not investigate the specific enzymatic product of HO-1 that mediated the lung cytoprotective, vascular effects. In another report, inhaled CO was shown to reverse established PAH and RV hypertrophy in rodent models of monocrotaline-induced and hypoxic PAH [78]. In this work, the ability of CO to reverse PAH required intact endothelial production of NO as mice with nitric oxide synthase-3 deficiency were not protected by inhaled CO. In the mouse model of hypoxia-induced PAH, Vitali et al. [72] demonstrated that HO-1 null mice did not develop elevations in PAP nor lung vessel wall remodeling when they were treated with inhaled CO intermittently or continuously throughout the hypoxic exposure. On the basis of the findings of in vitro and in vivo work, the protective actions of HO-1 are likely the result of the action of its enzymatic product CO. In addition to the study by Zuckerbraun et al. on the ability of inhaled CO to reverse PAH [78], CO was found to be protective in several animal models of inflammatory conditions such as sepsis, hyperoxic lung injury, ventilator-induced lung injury, and transplant rejection [79, 80]. In the heart, CO delivered by a CO-releasing molecule has been shown to reduce infarct size in mouse and rat models of cardiac I/R [81, 82]. Increasing evidence suggests that BR and BV may have protective effects that rival those of the better-studied HO-1 product CO. Injected BV has been shown to be protective in the models of cardiac transplant rejection already described, yet no report has demonstrated a protective effect of BR on lung vascular remodeling. When BR was administered to HO-1 deficient mice exposed to 5–7 weeks of hypoxia, the animals still manifested profound PAH with elevations in RVSP and vessel wall remodeling. However, unlike most of the untreated animals, which developed RV dilation and RV thrombus between 5 and 7 weeks of hypoxia exposure, none of the HO-1 null mice that received BR developed RV thrombus/infarct [72]. In contrast, HO-1 null mice treated with continuous or intermittent inhaled CO were protected from pulmonary vascular remodeling; however, they still developed RV failure and thrombus with significant mortality, despite normal pulmonary artery pressures. The mechanisms behind the myocardial protective effects of BR include the antioxidant and antiapoptotic actions of BV on the cardiomyocyte as demonstrated in studies of cultured cardiomyocytes exposed to anoxia– reoxygenation injury. Apoptosis was prevented and cardiomyocyte survival was significantly increased with BR [72, 83] but not with CO [72]. Redout and others recently reported that progression from RV hypertrophy to failure in a rat monocrotaline pulmonary hypertension model is associated with increased reactive oxygen species production by mitochondria and NADPH oxidase [84]. These failing right ventricles showed evidence of reduced antioxidant

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capacity and increased oxidative stress. That the antioxidant property of BR is important for cardioprotection is supported by recent data showing that HO-1 overexpression protected cardiomyocytes from anoxia–reoxygenation-induced oxidative injury and apoptosis [85]. Inhaled CO concurrent with chronic hypoxia completely prevented hypoxia-induced pulmonary vascular remodeling and elevated RVSP in both the rat and the mouse models. The actions of CO are due, at least in part, to the prevention of hypoxia-induced PASMC proliferation as has been previously reported and summarized above. However, CO was recently also found to significantly inhibit smooth muscle cell migration. In cultured PASMCs, exposure to CO prevented hypoxia-induced smooth muscle migration in response to the chemoattractant PDGF [72]. The mechanism for hypoxia-induced smooth muscle cell migration has not been studied. PDGF-induced PASMC migration was reported to be dependent on ERK MAPK phosphorylation [86] and, therefore, it is plausible that CO inhibits migration via dephosphorylation of ERK 1/2. BR had no inhibitory effect on PASMC migration under hypoxia and this may explain its inability to ameliorate lung vessel wall remodeling and hypoxia-induced vasoconstriction. The divergent effects of the two enzymatic products of HO-1 in the same disease model highlight the complexity of HO-1’s protective actions in the cardiovascular system. Moreover, the finding that CO provided protection from pulmonary hypertension but failed to provide protection from RV injury indicates that hypoxia has a direct effect on the right ventricle that is not mediated by pulmonary vascular constriction or remodeling and may explain why treatment with vasorelaxant agents has not reduced mortality in patients with PAH where hypoxia is an underlying or associated underlying contributor to disease. When chronic hypoxia is the cause of PAH, the right ventricle is subjected not only to elevated pulmonary vascular resistance from vascular remodeling, but also to systemic hypoxia. Both pressure overload [87, 88] and chronic hypoxia [73] are known to independently increase HO-1 expression in the heart, pointing to a possible independent role of these two stressful stimuli. As patients with PAH secondary to hypoxia (e.g., secondary to COPD) make up a continuously increasing percentage of patients with PAH, it is important to investigate this independent effect of systemic hypoxia on myocardial performance and progression to failure. In general, therapeutic strategies for PAH should not be solely targeted at reducing pulmonary arterial pressure as they will ultimately not improve long-term survival in PAH patients as historical experience has shown. The combined lungspecific as well as cardioprotective effects of each of the enzymatic products of HO-1 may provide new opportunities to investigate therapeutic approaches to PAH that target both organ systems.

S. Kourembanas

4 Heme Oxygenase-1 and Therapeutic Implications for Pulmonary Arterial Hypertension Animal studies have suggested that overexpression of the HO-1 gene is beneficial in PAH. Possible approaches to modulate HO-1 expression in patients include gene therapy, pharmacological inducers of HO-1, and HO-1 targeted siRNA. Significant challenges regarding safe and tissuespecific HO-1 gene transfer to humans preclude this approach at present. Genetically modified bone-marrow-derived stem cells overexpressing HO-1 may be a potential strategy to achieve HO-1 gene delivery as is being currently tested for the NOS gene in human trials of PAH. Pharmacologic inducers of HO-1 such as cobalt protoporphyrin and hemin are toxic and further development of nontoxic and specific agonists is required. Interestingly, currently tested therapies such as statins may confer protective effects via the induction of endogenous HO-1 [89, 90]. The demonstrated efficacy of low-dose CO as a therapeutic agent in multiple models of vascular disease, including PAH, is a potential new therapeutic approach. The recently developed CO-releasing molecules (i.e., transition metal carbonyls) provide an alternative vehicle for pharmacological delivery of CO [91]. However, it must be noted that CO by itself may not alter long-term outcomes of PAH if it cannot protect against RV failure as demonstrated in the hypoxia mouse model of disease [72]. Combinations of antioxidant and anti-inflammatory approaches may be necessary to address the lung vascular and cardiac-specific pathophysio­ logy of PAH to decrease mortality rates and long-term morbidity.

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S. Kourembanas 72. Vitali SH, Mitsialis SA, Liang OD et al (2009) Divergent cardiopulmonary actions of heme oxygenase enzymatic products in chronic hypoxia. PLoS One 4:e5978 73. Yet S-F, Perrella MA, Layne MD et  al (1999) Hypoxia induces severe right ventricular dilatation and infarction in heme oxyg­ enase-1 null mice. J Clin Invest 103:R23–R29 74. Christou H, Morita T, Hsieh CM et al (2000) Prevention of hypoxiainduced pulmonary hypertension by enhancement of endogenous heme oxygenase-1 in the rat. Circ Res 86:1224–1229 75. Zhou H, Liu H, Porvasnik SL et al (2006) Heme oxygenase-1 mediates the protective effects of rapamycin in monocrotaline-induced pulmonary hypertension. Lab Invest 86:62–71 76. Minamino T, Christou H, Hsieh C-M et al (2001) Targeted expression of heme oxygenase-1 prevents the pulmonary inflammatory and vascular responses to hypoxia. Proc Natl Acad Sci USA 98:8798–8803 77. Ito T, Okada T, Miyashita H et al (2007) Interleukin-10 expression mediated by an adeno-associated virus vector prevents monocrotaline-induced pulmonary arterial hypertension in rats. Circ Res 101:734–741 78. Zuckerbraun BS, Chin BY, Wegiel B et al (2006) Carbon monoxide reverses established pulmonary hypertension. J Exp Med 203: 2109–2119 79. Dolinay T, Szilasi M, Liu M et al (2004) Inhaled carbon monoxide confers antiinflammatory effects against ventilator-induced lung injury. Am J Respir Crit Care Med 170:613–620 80. Sato K, Balla J, Otterbein L et al (2001) Carbon monoxide generated by heme oxygenase-1 suppresses the rejection of mouse-to-rat cardiac transplants. J Immunol 166:4185–4194 81. Guo Y, Stein AB, Wu WJ et  al (2004) Administration of a CO-releasing molecule at the time of reperfusion reduces infarct size in vivo. Am J Physiol Heart Circ Physiol 286:H1649–H1653 82. Stein AB, Guo Y, Tan W et  al (2005) Administration of a CO-releasing molecule induces late preconditioning against myocardial infarction. J Mol Cell Cardiol 38:127–134 83. Foresti R, Goatly H, Green CJ et  al (2001) Role of heme oxyg­ enase-1 in hypoxia-reoxygenation: requirement of substrate heme to promote cardioprotection. Am J Physiol Heart Circ Physiol 281: H1976–H1984 84. Redout EM, Wagner MJ, Zuidwijk MJ et al (2007) Right-ventricular failure is associated with increased mitochondrial complex II activity and production of reactive oxygen species. Cardiovasc Res 75:770–781 85. Pachori AS, Smith A, McDonald P et al (2007) Heme-oxygenase-1induced protection against hypoxia/reoxygenation is dependent on biliverdin reductase and its interaction with PI3K/Akt pathway. J Mol Cell Cardiol 43:580–592 86. Yamboliev IA, Gerthoffer WT (2001) Modulatory role of ERK MAPK-caldesmon pathway in PDGF-stimulated migration of cultured pulmonary artery SMCs. Am J Physiol Cell Physiol 280:C1680–C1688 87. Katayose D, Isoyama S, Fujita H et al (1993) Separate regulation of heme oxygenase and heat shock protein 70 mRNA expression in the rat heart by hemodynamic stress. Biochem Biophys Res Commun 191:587–594 88. Delcayre C, Samuel JL, Marotte F et al (1988) Synthesis of stress proteins in rat cardiac myocytes 2-4 days after imposition of hemodynamic overload. J Clin Invest 82:460–468 89. Hsu HH, Ko WJ, Hsu JY et al (2009) Simvastatin ameliorates established pulmonary hypertension through a heme oxygenase-1 dependent pathway in rats. Respir Res 10:32 90. Grosser N, Hemmerle A, Berndt G et  al (2004) The antioxidant defense protein heme oxygenase 1 is a novel target for statins in endothelial cells. Free Radic Biol Med 37:2064–2071 91. Motterlini R, Mann BE, Foresti R (2005) Therapeutic applications of carbon monoxide-releasing molecules. Expert Opin Investig Drugs 14:1305–1318

Chapter 54

Shear Stress, Cell Signaling, and Pulmonary Vascular Remodeling Shampa Chatterjee and Aron B. Fisher

Abstract  Mechanotransduction is defined as the sensing of physical stimuli such as flow and the mechanisms by which cells convert those signals into a biochemical response. Mechanical forces important to pulmonary structure and function can be produced by gradients in gravity, motion, osmotic forces, and by the interactions between adjacent cells and between cells and the adjacent matrix. Because of the complexity of the pulmonary structure, the variety of cell types, and the variety of physical forces to which cells in the lung are exposed, there are many variations and possible mechanisms for mechanical stimulation and the cellular responses that may be induced. The lung endothelial cells lining the blood vessels serve as the interface between blood and tissue and are in intimate contact with the blood. As a result, these cells are directly exposed to the physical forces associated with pulmonary perfusion. The endothelial cells transduce these hemodynamic forces into biochemical signals which play an important role in physiological and pathophysiological responses of the pulmonary endothelium. One of the important forces is the tangential shear that serves to distort the endothelial cell in the direction of flow. Shear is transmitted through the endothelium by a complex process that involves the interplay between cytoskeletal and biochemical elements and results in changes in structure, metabolism, and gene expression. Whereas physiological shear modulates the pulmonary vascular properties and function, chronic and abrupt changes in shear may cause vascular injury, dysfunction, and remodeling. This chapter focuses on the molecular mechanisms associated with shear signaling in the pulmonary vasculature, and how the changes in the hemodynamic environment affect physiological signaling and lead to the development of pulmonary diseases.

S. Chatterjee (*) Institute for Environmental Medicine, University of Pennsylvania Medical Center, 1 John Morgan, 3620 Hamilton Walk, Philadelphia, PA 19104, USA e-mail: [email protected]

Keywords  Mechanotransduction • Mechanical force • Endothelial cell • Blood flow • Shear signaling • Hemodynamic environment

1 Introduction Mechanotransduction, defined as the sensing of physical stimuli such as flow and the mechanisms by which cells convert those signals into a biochemical response, is a relatively recent subject of study. Mechanical forces important to pulmonary structure and function can be produced by gradients in gravity, motion, osmotic forces, and by the interactions between adjacent cells and between cells and the adjacent matrix. Because of the complexity of the pulmonary structure, the variety of cell types, and the variety of physical forces to which cells in the lung are exposed, there are many variations and possible mechanisms for mechanical stimulation and the cellular responses that may be induced. The lung endothelial cells lining the blood vessels serve as the interface between blood and tissue and are in intimate contact with the blood. As a result, these cells are directly exposed to the physical forces associated with pulmonary perfusion. The endothelial cells transduce these hemodynamic forces into biochemical signals which play an important role in physiological and pathophysiological responses of the pulmonary endothelium. One of the important forces is the tangential shear that serves to distort the endothelial cell in the direction of flow. Shear is transmitted through the endothelium by a complex process that involves the interplay between cytoskeletal and biochemical elements and results in changes in structure, metabolism, and gene expression. Whereas physiological shear modulates the pulmonary vascular properties and function, chronic and abrupt changes in shear may cause vascular injury, dysfunction, and remodeling. This chapter will focus on the molecular mechanisms associated with shear-dependent signaling in the pulmonary vasculature, and how the changes in the hemodynamic environment affect physiological signaling and lead to the development of pulmonary diseases.

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_54, © Springer Science+Business Media, LLC 2011

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2 Shear Stress in the Pulmonary Vasculature 2.1 Pulmonary Blood Flow and Forces Acting on the Endothelial Cells The pulmonary vasculature, like its systemic counterpart, exists in a complex environment where mechanical forces arising from shear stress and blood pressure interact to shape the vascular wall. The forces associated with blood flow in the vasculature can be resolved into two vectors: shear stress is the frictional force acting tangentially at the blood–endothelial interface; the hydrostatic pressure is the perpendicular force on the vessel wall that imposes circumferential stretch. It is now well established that physio­ logical shear acts through the endothelium to regulate vessel tone and restructuring of blood vessels [1, 2]. However, acute and chronic changes in shear can alter shear-signaling mechanisms and cause endothelial damage. As integrity of the pulmonary endothelial cell monolayer is a critical requirement for preservation of pulmonary function, this sets the stage for pulmonary vascular remodeling and disease. 2.1.1 Laminar, Pulsatile, and Disturbed Flow Shear stress in the vascular endothelium is due to the force that blood flow exerts on the vessel wall. It is defined as force per unit area and has units of dynes per square centimeter or newtons per square meter. The latter is also defined as a pascal; 1 Pa = 10  dyn/cm2. Some older texts refer to shear rate, which is the ratio of the velocity of a particle (e.g., centimeters per second) to the distance traveled (e.g., centimeters) and is expressed as a dimensionless value per second. Depending on the flow conditions, shear stress may be steady (laminar), pulsatile, or disturbed (turbulent or oscillatory) (Fig.  1). Laminar or steady shear stress arises from an orderly flow (i.e., constant velocity over time) of the fluid which moves parallel to the vessel wall. With laminar flow, all elements of the fluid move in streamlines that are parallel to the long axis of the tube, with the central streamlines having the greatest velocity, resulting in a paraboloid

Fig. 1  Profiles of laminar (a) and turbulent flow (b). Laminar flow is streamlined and occurs in parallel layers. The flow is greatest at the center and decreases toward the periphery, making a parabolic profile. Turbulent flow is random with vortices and swirling (eddies) of the fluid

S. Chatterjee and A.B. Fisher

flow pattern. Regions of the arterial tree with uniform geometry are exposed to unidirectional, undisturbed laminar flow. Pulsatile flow is the intermittent flow of fluid or blood through a vessel due to the rhythmic property of the cardiac cycle; it is otherwise similar to laminar flow in terms of the calculated forces. Disturbed flow occurs when the flow patterns within the blood vessel are random and unpredictable. It is either turbulent, in which case the flow forms vortices, eddies, and wakes, or oscillatory, where blood moves back and forth in response to an oscillating pressure gradient. Laminar shear is directly proportional to the fluid flow rate and the viscosity of the medium as calculated (in dynes per square centimeter) by the following equation:

Shear stress = 4h Q / p r 2 l , where h is the viscosity (dyn�s/cm2), Q is the flow rate (mL/s), r is the radius of the vessel (cm), and l is the length of the vessel (cm). For a given bulk flow rate, the shear stress varies inversely with the vessel radius (squared) and therefore increases significantly with decreasing vessel radius. Turbulence, in part, reflects conditions of high flow and also occurs in association with areas of narrowing or partial obstruction. Turbulent flow results in variable and inconsistent levels of shear. The point at which laminar flow becomes turbulent has been estimated by calculation of a dimensionless quantity called the Reynolds number (Re):

Re = vDr / h, where n is the velocity of a particle (cm/s), D is the vessel diameter (cm), r is the blood density (g/cm3), and h is the blood viscosity (dyn�s/cm2). Turbulence usually develops with Re > 2,000, but local conditions are very important. If a constant ratio of blood density to viscosity is assumed, turbulence is encouraged by increasing the velocity of flow or the diameter of the vessel. Regions of the arterial tree with arches, extensive branching, or partial obstruction are more likely to experience disturbed flow.

2.1.2 Shear Values in the Vessels of the Pulmonary and Systemic Vasculature The vasculature, both pulmonary and systemic, consists of large conduit vessels (macrocirculation) that provide convective transport over large distances and smaller vessels (microcirculation) that permit diffusive exchange between blood and tissue. Shear stress in these vessels depends on the flow rate and vessel diameter. Vascular shear stress of large conduit arteries typically varies between 5 and 20 dyn/cm2; however, significant instantaneous values range from near zero up to 40  dyn/cm2 during states of increased cardiac output. Table 1 shows typical wall shear stress values for vessels of various size in the human

54  Shear Stress, Cell Signaling, and Pulmonary Vascular Remodeling Table 1  Shear stress in selected human blood vessels (data from [3, 4]) Vessel Shear stressa(dyn/cm22) Aorta Large arteriole Small arteriole Capillary Small vein Large vein Pulmonary artery a Approximate normal values

5 8 10 24 11 5 2

system circulatory system [3, 4]. Because the lung has a highly compliant, high-capacity vascular bed, the particle flow is relatively slow (although bulk flow is equal to the flow of the systemic circulation) and the wall shear stress is correspondingly lower compared with that in systemic vessels.

3 Physiological Response to Shear It is now well established that hemodynamic forces such as shear play an important role in physiological responses of endothelial cells. Shear resulting from blood flow leads to immediate responses in the form of altered ion channel activity and activation of signaling molecules and subsequent later responses manifested by changes in structure, metabolism, and gene expression [1, 5–8]. Overall these changes occur as a result of complex interactions between the mechanical component of shear and the cytoskeletal and biochemical components of the cell. In vivo studies have demonstrated that arteries adapt to chronic changes in blood flow by altering their structure to accommodate the new condition [1, 8]. However, in vivo studies are not suitable for accurately monitoring changes in cellular phenotype or gene expression. Thus, the use of endothelial cells in culture or isolated intact vessels in vitro has filled the gap. These models can be subjected to controlled shear stresses, and monitored for endothelial responses and properties, including release of mediators, gene expression, and protein production [6, 7, 9, 10]. Most of the published studies utilized cells cultured under static conditions and subjected to the onset of shear. This relatively unphysiologic preparation can give information regarding the cellular machinery that is involved in the response to shear but might not accurately reflect the changes that occur with altered shear in  vivo. A more physiologic preparation is isolated cells that have been flow adapted in vitro and then subjected to altered shear. This section will present three aspects of mechanotransduction. Firstly, candidate flow sensors will be discussed from the perspective of molecules at the luminal cell surface that by virtue of being in direct contact with the flowing blood are activated directly by physical changes (conformational

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change) or indirectly by ligand–receptor interactions. Secondly, the physiological changes with immediate and long-term shear will be presented with emphasis on ion channel activation and the endothelial gene expression involved in adaptation to shear. The third aspect will be the signaling pathway associated with change of shear.

3.1 Flow Sensors and Mechanosensing Although the identity of the endothelial cell component that is responsible for sensing shear has not been precisely identified, a number of candidate cell membrane shear sensors have been suggested for endothelial cells. A cell membrane protein would be the ideal candidate, but an internal sensor based on a change in the internal stresses also is possible. This latter condition has been termed “cellular tensegrity” [11]. Tensegrity as defined by Buckminster Fuller [12] is the mechanism for stabilization of shape of a structure by continuous tension or “tensional integrity.” The cellular tensegrity model proposes that the whole cell is a prestressed structure where tensional forces are borne by cytoskeletal microfilaments and intermediate filaments, and these forces are balanced by interconnected structural elements (extracellular matrix adhesion and microtubules) that resist compression. The tensional prestress (pre-existing tensile stress) that stabilizes the whole cell is generated by the contractile machinery of the cell (actomyosin apparatus). Changes in shear can then be sensed by changes in the internal cell “tension.” The alternative sensing mechanism is that the change in the local environment of a cell membrane protein results in a “biochemical” response.

3.1.1 Ion Channels A change in membrane potential has been demonstrated as one of the earliest responses to a change in flow. Patch clamp studies have shown that an initial K+ current is activated with onset of flow and is followed by a Cl- current. This presumably reflects a change in activity of endothelial plasma membrane ion channels. Two types of ion channels have been reported to be flow-sensitive: (1) an inwardly rectifying K+ channel that with activation hyperpolarizes the cell membrane, whereas channel inactivation results in membrane depolarization [13], and (2) an outwardly rectifying Cl- channel that when activated depolarizes the endothelial membrane [14, 15]. These channels are activated independently of each other and exhibit different sensitivities to applied shear stress [16]. Inwardly rectified K+ channels (KIR) represent a relatively large family of proteins that are divided into six types. The KATP channel is included in this family and is classified as KIR type 6. KATP channels (specifically KIR6.2) are expressed at low levels in

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isolated, cultured endothelial cells, but are induced during long-term (48–72-h) exposure to shear [17]. Induction of these channels during flow adaptation indicates that some other cellular protein serves as a flow sensor to mediate KATP channel gene expression. The KATP channels appear to be maintained in an active state by flow and are inactivated (decreased open probability) by loss of shear [18]. Another inwardly rectified K+ channel, KIR2.1, has also been reported to be flow-sensitive but its presence and physiological role in endothelial cells have not been confirmed [19]. Other ion channels that may be involved in the response to flow include members of the transient receptor potential (TRP) superfamily. TRP channels in the endothelium belong to the subfamily TRPC, including TRPC1 and TRPC6, which display physiological responses in a pure bilayer [20, 21]. The TRPC channels are important in regulating Ca2+ entry into cells. At least one of these channels, TRPC1, is located in caveolae, thought to be a site of mechanosensing (see later). TRPC4 has been reported to be activated by cell swelling, which represents in part a mechanical stimulus [22, 23].

3.1.2 Integrins Integrins are transmembrane receptors that link cytoskeletal proteins with the proteins in the extracellular matrix so as to facilitate cell–matrix communication. Integrins connect to the cytoskeleton through focal adhesions that contain multiple actin-associated proteins, including talin, vinculin, paxillin, and zylin [24]. The cytoskeleton responds mechanically to forces transferred via the extracellular matrix and channeled through integrins by rearranging its interlinked actin microfilaments, microtubules, and intermediate filaments. Focal adhesions undergo constant remodeling on the abluminal side of endothelial cells and integrins are suggested to exert their influence through the activation of focal adhesion kinase. Integrins have two subunits, a and b. One of the major integrins in vascular endothelium is avb3, which interacts with the matrix protein fibronectin. Shear-induced signaling has been shown to decrease in the presence of anti-avb3 antibodies [25]. 3.1.3 Caveolae Caveolae are lipid-rich invaginations on the cell surface that serve as a platform for clustering of many cell-membraneassociated proteins. They participate in endocytosis and also are reported to serve as mechanotransduction sites within the plasma membrane. A role for caveolae in flow sensing in the pulmonary endothelium has been proposed [26–29]. Chronic exposure to shear stress altered caveolin expression and distribution, increased the density of caveolae, and led to enhanced mechanosensitivity to subsequent changes in hemodynamic forces by endothelial cells in culture [26]. Depletion of caveolae prevented the endothelial

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response to stop of flow [28, 29]. Caveolae associate with b1 integrins during flow and have been reported to be functionally linked in their flow-sensing properties [26, 30]. Cells with disrupted caveolar structural domains show attenuated integrin-dependent caveolin-1 phosphorylation and activation of Src (a tyrosine kinase) in response to alterations in shear.

3.1.4 Cytoskeleton The cytoskeleton and other structural components can transmit shear-induced deformation of the cell surface throughout the cell via changes in cytoskeletal tension. The proteins involved include focal adhesion sites, integrins, cellular junctions, and extracellular matrix [5]. The cytoskeleton is composed of three major types of protein filaments: microtubules, microfilaments, and intermediate filaments. These form a cytoskeletal network that provides a scaffold for organizing several signaling molecules whose activation appears to be mediated through the cytoskeleton. The tensional forces that are generated within contractile microfilaments and transmitted throughout the cell are balanced by internal microtubules and by adhesions to extracellular matrix and to other cells [5]. This allows cells to shift compressive forces between microtubules and extracellular matrix adhesions. Thus, extracellular matrix bears most of the shear stress in cells spread on adhesive substrates, whereas microtubules bear the mechanical forces in cells with fewer anchoring points.

3.1.5 Mechanosensory Complexes In addition to individual mechanosensors, a multicomponent complex has been reported to mediate shear response in endothelium. This complex is composed of the platelet endothelial cell adhesion molecule (PECAM), vascular endothelial growth factor receptor 2, and vascular endothelial cadherin [31]. Together these receptors confer shear responsiveness to endothelial cells. This is further supported by observations in vivo where PECAM null lungs do not show the endothelial response to disturbed flow, i.e., activation of nuclear factor kB (NF-kB) and inflammatory genes [31].

3.2 Immediate Response to Shear Responses to either acute or chronic alterations in blood flow in the vasculature are endothelium-dependent [32]. The earliest change that is detected in endothelial cells in culture upon onset or cessation of flow, respectively, is activation [13] or deactivation of ion channels [18]. This is followed by

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other signaling events, such as increases in cytosolic Ca2+ concentration, activation of intracellular kinases, and generation of nitric oxide (NO) and reactive oxygen species (ROS). Cells that are initially cultured under static conditions and then exposed to flow undergo adaptive changes that result in a flow-adapted state. The vascular endothelium in  vivo is assumed to be flow-adapted and the demonstration of physiological responses to flow cessation in  vitro requires prior adaptation of cells to flow.

3.3 Adaptation to Flow and Development of a Stable Phenotype Endothelial cells in conducting vessels of the arterial vascular tree exhibit an ellipsoidal shape and are aligned with the direction of blood flow. On the other hand, the shape of individual cells near vessel branch points fails to show any orientation, consistent with local temporal and spatial fluctuations in flow [8, 33]. Exposure of endothelial cells in vitro to unidirectional laminar shear induces a time- and forcedependent change in cell shape and alignment that appears to reflect the in vivo condition. The changes are reversible upon cessation of flow [8]. These changes in cell shape and alignment are seen with endothelial cell cultures that have been derived from large conducting vessels, but to date have not been reported with endothelial cells derived from the microcirculation. The shape changes in endothelial cells require reorganization of the cytoskeleton [1]. Phenotypic changes may extend to the level of the individual endothelial cells that compose the continuous vascular lining. Prolonged exposure of endothelial cells to flow in  vitro also causes changes in gene expression. These changes represent the response of shear-sensitive elements that trigger a biochemical cascade. This cascade activates protein kinases that in turn lead to activation of cytosolic transcription factors and regulation of gene transcription in the nucleus. Persistent changes in blood flow, such as those resulting from diseaserelated or experimentally induced vascular narrowing or shunts, also can cause structural remodeling involving cell proliferation and apoptosis and hypertrophy of extracellular matrix. These longer-term structural modifications are discussed in section 5.

3.3.1 Shear-Dependent Endothelial Cell Gene Regulation Flow-dependent regulation of endothelial cell gene expression has been demonstrated in vitro by measuring messenger RNA and protein levels as a function of time after the application of continuous laminar shear stress [7, 34, 35]. With

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use of gene array methods, shear stress was found to significantly affect the expression of about 3,000 endothelial cell genes, of which 232 genes were affected by the frequency of shear and about 3,000 genes were affected by shear magnitude. Of the 232 genes sensitive to differences in frequency, 185 were also sensitive to mean shear [36]. Several patterns of response of genes to shear have been described: 1. Rapid induction under shear followed by rapid return to the baseline [37, 38]. An example of this pattern is shown by c-fos, a proto-oncogene that encodes a DNA binding protein of the activator protein (AP-1) family. c-fos has low transcript levels in cells cultured under static conditions, but rapid induction to a high level of expression (20-fold over the basal level) in response to shear followed by a rapid return to the baseline [37, 38]. 2. Initially high transcript levels under basal conditions that increase further upon exposure to shear. Examples include platelet-derived growth factor (PDGF)-B chain, endothelial nitric oxide synthase (eNOS), c-jun, PDGF-A, monocyte chemotactic protein-1 (MCP-1), transforming growth factor- b (TGF-b), basic fibroblast growth factor, tissue plasminogen activator and intercellular adhesion molecule (ICAM)-1 [39–42]. 3. A third response is biphasic with a relatively slight increase associated with onset of flow followed by a decrease in expression to levels below basal. This response has been described for endothelin-1 [43]. 4. A fourth response requires a cofactor for induction. Human umbilical vascular endothelial cells exposed to shear exhibit relatively little change in vascular cell adhesion molecule (VCAM)-1 messenger RNA expression, but show significant induction if they are pre-exposed to endotoxin or various cytokines [41].

3.3.2 Shear Stress Response Element The observation that gene transcription in endothelial cells can be modulated by laminar shear stress led to the search for conserved cis elements in the promoter sequences of shear-inducible genes. Several such cis elements have now been identified, indicating that there may be various sequences that regulate transcription upon shear. The most common transcriptional regulatory unit is a six-base-pair sequence, GAGACC, that was identified on the basis of the effect of shear on the promoter of PDGF-B [44]. Several endothelial genes unrelated to PDGF have been reported to be responsive to laminar shear stress and encode the shear stress response element (SSRE) or its complementary sequence (GGTCTC) in their promoter regions. Examples of genes that have an SSRE in their respective promoters include eNOS, MCP-1, and TGF-b [44].

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3.4 Signaling Associated with Altered Shear Experimental models to study the effects of altered shear have evaluated either the onset of shear in cells cultured under static conditions or the cessation of shear in flowadapted cells. Cellular responses with these two approaches have many similarities, leading to the generalization that cells respond to a change from the steady-state condition. The model of abrupt cessation of shear in flow-adapted cells mimics the expected response to acute arterial obstruction such as that associated with an embolus. The in vivo counterpart of the increased laminar shear model in cells cultured under static conditions is less clear; a more physiological in vitro model would be the study of increased laminar flow in cells that have been flow-adapted (shear-adapted), but those studies have not been done. The effects of disturbed flow (oscillatory or turbulent) have been interpreted as an indication of the changes in a vessel with partial luminal occlusion, such as the change associated with atherosclerosis, or with vasoconstriction. Here again, flow-adapted cells may represent a better basal (control) condition. Although the procedure to “flow adapt” endothelial cells in vitro is in principal straightforward, it is time-consuming and requires close attention to maintain sterility [18, 29, 45]. Nevertheless, use of this procedure can be expected to result in a significant increase in physiological relevance. A schematic of a commercially available chamber that has been used for this purpose is shown in Fig.  2. The physiological changes with altered shear range from virtually instantaneous electrochemical responses to more delayed changes in gene expression and cell phenotype.

3.4.1 Onset of Flow Candidate flow sensors as mentioned already can directly or indirectly sense flow and generate a biochemical signaling

Fig. 3  Time course of endothelial response to onset of laminar shear stress in cells cultured under static conditions. See the text for details

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cascade in response. The timescale for shear-induced responses by endothelial cells can be grouped into three categories (Fig.  3). Immediate responses generated subsequent to the acute sensing of shear are rapid, on the order of seconds, and may be initiated by changes in ion permeability due to the activation of flow-sensitive K+ and Cl− channels [13, 14]; these changes play an important role in regulating overall endothelial responsiveness to flow. Subsequent responses include an increase in intracellular free Ca2+ concentration, increased adenylate cyclase activity, inositol trisphosphate generation [1, 5, 13, 46], and the phosphorylation of the serine/threonine kinase Akt [15]. Intermediate

Fig. 2  A chamber that is used for adapting endothelial cells in vitro to flow. The artificial capillary system (FiberCell Systems) is made up of semipermeable polypropylene hollow fibers approximately 200 mm in diameter into which cells are seeded. The fibers are encased in a cartridge, with the ends forming the inlet and outlet ports to allow for perfusion. Before the seeding, the fibers are coated with a fibronectin matrix. After the seeding, the chamber is perfused via the side ports to provide substrate while allowing the cells to attach. After 24 h, perfusion is re-routed to the main ports and cells are subjected to laminar shear stress for an additional 24–72  h to obtain a flow-adapted state. Experimentally, re-routing the perfusate to the side ports simulates ischemia (loss of shear) while maintaining delivery of O2 and substrates. This chamber can be used to obtain cells that show a programmed response to ischemia [17, 28, 29, 45]. (Adapted from [45] with permission)

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responses that develop over minutes include activation of intracellular signaling molecules, release of mediators such as ROS and TGF-b [42], and the redistribution of cytoskeletal elements. Late responses (hours) are cellular adaptations and involve regulation of protein synthesis, changes in the cell shape, and alignment of cells in the direction of flow [6, 47].

3.4.2 Cessation of Flow Endothelial cells also show an immediate response to an abrupt decrease in shear. On the basis of a limited number of studies, there is a threshold for this effect, with indications that the shear stress must be decreased by more than 80% from the steady-state value for a response to be elicited [48]. Decreased shear can occur with various diseases owing to vascular obstruction associated with embolism/thrombosis, tumor growth, or (rarely in the lung) atherosclerosis. From a research standpoint, the lung permits study of the effects of the mechanical component of flow (i.e., decreased shear stress) without the attendant tissue anoxia that accompanies stop of blood flow in the vascular beds of other organs. Thus, ischemia/reperfusion in the lung can be differentiated from anoxia/reoxygenation. Decreased shear (i.e., ischemia) in the pulmonary vasculature has been studied using both the intact lung (isolated perfused rat or mouse lung) and in  vitro flow-adapted pulmonary endothelial cells. In these systems, the control condition is the continuously perfused lung or cells; ischemia is achieved by stopping the perfusate flow. The use of realtime high-resolution digital imaging of the subpleural microvasculature in the intact lung by either confocal or epifluorescence microscopy in the presence of fluorophores has enabled the measurement of the rapid changes that occur in endothelial cells with the abrupt stop of flow. These events include changes in plasma membrane polarity, in the generation of ROS and NO, and increase in the intracellular Ca2+ concentration [18, 49–52]. The use of cells and lungs from gene-targeted mice has allowed the determination of the role of various mediators and pathways in the lung response. These responses are described in greater detail in the next section.

4 Cellular Response to Altered Shear 4.1 Membrane Depolarization Membrane-potential-sensitive dyes, such as bisoxonol and di-8-ANEPPS, localize in the membrane lipid bilayer and show increased fluorescence when the membrane is in a depolarized state. On the basis of changes in fluorescence, cell membrane potential decreases within several seconds after stop of flow [50]. Pretreatment of the lungs with a KATP

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channel agonist to maintain the open state blocked membrane depolarization, indicating that inactivation (closure) of these channels could be responsible for the change in membrane potential with ischemia. Lungs from KATP (KIR6.2) null mice showed a greatly diminished response to the abrupt decrease in shear [51]. Patch clamp studies with isolated flow-adapted endothelial cells confirmed the presence of a K+ current that was responsive to modulators of KATP channel activity and that decreased with abrupt flow cessation [18]. These studies led to the conclusion that inactivation of a cell membrane KATP channel resulting in membrane depolarization is one of the initial events following cessation of flow. The change in cell membrane potential with ischemia is not seen in cells from caveolin null mice (that lack caveolae), indicating that these organelles may serve as a flow sensor that is coupled to the subsequent inactivation of KATP channels.

4.2 Generation of ROS Various fluorophores, including dihydrochlorofluorescein, hydroethidine, and Amplex red, are readily oxidized, resulting in increased cellular fluorescence. These fluorophores have been used with intact organ and cell systems to image the cellular production of ROS. In the presence of these dyes, there is a rapid increase in endothelial cell fluorescence in the intact lung in the immediate period (minutes) subsequent to stop of flow [49–51]. A similar finding has been observed with endothelial cells in  vitro provided they have been flowadapted prior to the experiment. Thus, ROS formation appears to be an early event associated with altered shear stress.

4.2.1 Role of NADPH Oxidase in ROS Generation Investigation into the pathway for endothelial generation of ROS with ischemia revealed that diphenyleneiodonium (a flavoprotein inhibitor that inhibits NADPH oxidase) effectively prevented ischemia-induced ROS generation. It is now known that endothelial cells possess a plasma membrane NADPH oxidase enzyme system that is similar to that described for phagocytic cells and is called “NOX2” [53, 54]. The generation of a functional NADPH oxidase necessitates the translocation of three cytosolic protein components (p47phox, p67phox, p21rac1) to the plasma membrane, where they associate with the cytochrome b-558 heterodimer (gp91phox and p22phox) [54, 55]; this assembly is required for ROS generation. Mice with “knockout” of gp91phox, the flavoprotein of the multicomponent NADPH oxidase enzyme complex, fail to generate ROS with lung ischemia, providing a strong indication that NOX2 is the enzyme responsible [49, 52]. NOX2 is a transmembrane protein that transfers electrons

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across the plasma membrane to generate superoxide anion (O2•−) in the extracellular (intravascular) space. O2•− in turn dismutes to H2O2, either spontaneously or through catalysis by extracellular superoxide dismutase (SOD). With the in vitro flow-adapted endothelial cell system, generation of ROS was demonstrated by the SOD-dependent reduction of cytochrome c added to the medium. These results indicate that the ROS produced with ischemia is O2•−, as expected for NADPH oxidase. Further, reduction of extracellular cytochrome c indicates that O2•− is generated on the extracellular side of the plasma membrane with ischemia, again consistent with the known activity of NOX2. The activation of NOX2 with altered shear is linked to cell membrane depolarization as shown by the absence of ROS generation in the presence of agents that prevent the closure of KATP channels [52]. A linkage between membrane potential and activation of NADPH oxidase has also been demonstrated for phagocytic cells [56, 57] although the mechanism is still undefined.

4.3 Intracellular Ca2+ Cessation of flow in the pulmonary endothelium in situ or in flow-adapted endothelial cells in vitro results in a rapid rise in the intracellular Ca2+ concentration [50, 58–60]. This effect is also dependent on the change in membrane potential (depolarization) of the lung endothelium in flow-adapted bovine pulmonary artery endothelial cells. The concentration of intracellular Ca2+ (as monitored by Fura-2) was estimated at approximately 135  nM under control conditions and began to increase within 30 s after flow cessation, reaching a plateau within 10  min at approximately double the basal concentration. The increase was inhibited by removal of Ca2+ from the perfusate, indicating that at least some of the increase in the internal Ca2+ concentration came by translocation from the extracellular medium across the cell membrane. It is generally accepted that voltage-gated Ca2+ channels mediate Ca2+ influx in response to cell membrane depolarization [61]. Ca2+ influx in endothelial cells in response to ischemia was not altered by inhibitors of L-type Ca2+ channels, which led to a search for T-type channels in these cells. These channels have indeed been detected in freshly isolated microvascular endothelial cells, but they are poorly expressed in endothelial cells cultured under static conditions [62]. However, the expression of the two subunits of this channel, the a1G and b3 proteins, is significantly increased during flow adaptation, suggesting that these channels are present and function in cells in vivo [60]. Ca2+ influx is blocked if cells are pretreated with the semispecific T-type channel blocker mibefradil. T-type Ca2+ channels open after a relatively small depolarization (10–20  mV),

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unlike L-type Ca2+ channels which require a significantly larger depolarization [63]. Calculations based on fluorescence imaging and using the Nernst equation show that membrane potential in the in situ lung decreases by approximately 15–20 mV with ischemia, compatible with the window for T-type channel activation [58, 60]. In addition to influx of Ca2+ through the cell membrane channels, there is some release of Ca2+ from its intracellular stores into the cell cytoplasm [58].

4.4 NO Generation Increased NO production has been detected in the pulmonary endothelium with cessation of flow in the intact lung as well as in flow-adapted endothelial cells in vitro. NO generation was observed through use of the fluorophore diaminofluorescein, and temporally appeared to follow the increase in intracellular Ca2+ concentration. Inhibition by Nw-nitro-l-arginine methyl ester indicates that NO generation with ischemia is due to activation of nitric oxide synthase. Further, inhibition by chelation of Ca2+ or treatment with a calmodulin inhibitor suggests that eNOS is the isoform responsible for the major fraction of NO production [28].

4.5 Phosphorylation Signaling Cascades Mitogen-activated protein kinases appear to be important mediators of mechanosignal transduction. Ischemia in the flow-adapted endothelial cell model leads to an increase in phosphorylation of extracellular-signal-regulated kinase (ERK) 1 and 2 during the first 10 min of stop of flow and reaches a plateau at 20–30  min. ERK phosphorylation (activation) is suppressed when ROS generation is inhibited. On the other hand, inhibitors of ERK phosphorylation suppressed NO generation but had no effect on ROS generation. These results indicate that ERK phosphorylation is downstream of (dependent on) ROS but upstream of eNOS [28].

4.6 Activation of Transcription Factors The initial responses to ischemia, viz., increased levels of ROS, Ca2+, and NO, are compatible with cell signaling and led to a search for activation of transcription factors under these conditions. Analysis of nuclear extracts from flow-adapted endothelial cells by electrophoretic mobility shift assay at

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1 h of ischemia showed increased NF-kB components (p65 and p50), indicating activation of this transcription factor. AP-1 also showed activation at 1 h of ischemia, with increases of the c-Jun/c-fos subunits of AP-1 [45]. Activation of NF-kB as well as AP-1 was abolished by pretreatment of cells with inhibitors of ROS generation, indicating that their activation is linked to oxidative events.

4.7 Cell Proliferation ROS generation is known to mediate Ras-induced cell cycle progression and mammalian cell proliferation [64]. Further, activation of transcription factors, NF-kB and AP-1, reportedly is coupled to increased cell division. On the basis of these observations, the proliferative response of flow-adapted endothelial cells subjected to ischemia was evaluated. Pulmonary artery endothelial cells demonstrated twice as much 3H-thymidine incorporation as control cells at 24  h after stop of flow, indicating an increase in DNA synthesis. Further, pulmonary microvascular endothelial cells studied by flow cytometry showed a 2.5-fold increase in the cellular proliferation index with ischemia, whereas an increase in the number of cells in the S and G2/M phases was observed with cell cycle analysis [29, 45]. Cell proliferation is blocked by inhibition of ROS generation, thus linking the ROS produced with ischemia to DNA synthesis, activation of transcription factors, and cell division [65, 66].

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of eNOS and increased synthesis of NO. The latter represents another mechanism for the restoration of blood flow, that is, via NO-mediated vasodilation. Of course, production of ROS and NO can result in cellular manifestations of oxidative/nitrosative stress such as lipid peroxidation and protein nitration under conditions where the rate of oxidant generation exceeds the oxidant scavenging ability of the cells. The signaling cascade in response to acute ischemia is shown in Fig. 4.

5 Manifestations of Disease The role of hemodynamic factors in diseases of the systemic circulation has long been established with the association between regions of disturbed flow and atherosclerotic lesions in vivo [67, 68]. Studies with endothelial cells in vitro have also shown that changing shear forces or hemodynamics alters endothelial cell structure, function, and gene expression and activates endothelial cell intracellular signaling pathways as described in previous sections [1, 9]. The changes accompanying altered shear in the pulmonary circulation have been less intensively studied, but clearly alteration of lung structure/function is seen with increased shear such as that accompanying left-to-right blood shunt or the decreased shear associated with thromboembolic disease. This section will present the pathophysiological alteration in the lung associated with altered shear. An important, unresolved issue

4.8 Summary of the Endothelial Signaling Cascade with Altered Shear The studies described in the previous sections are consistent with a defined endothelial cell signaling cascade that is initiated by the abrupt cessation of flow. A comparable cascade may be initiated by increased shear, although the precise details in this latter circumstance are not as well described. After sensing in the cell membrane, the initial response is cell membrane depolarization due to inactivation of KATP channels; this results in activation of NADPH oxidase and generation of ROS. The consequences of increased ROS production include mitogen-activated protein kinase (ERK) activation, activation of transcription factors (NF-kB, AP-1), and endothelial cell proliferation. Cell proliferation with ischemia may be an attempt to generate new capillaries to restore impeded blood flow. Cell membrane depolarization also leads to opening of T-type voltage-dependent Ca2+ channels in endothelium, with consequent Ca2+ influx and elevated intracellular Ca2+ concentration, which in turn leads to activation

Fig.  4  Endothelial cell response to abrupt loss of shear stress (simulated ischemia). See the text for details. (Modified from [87] with permission from Springer)

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regarding the contribution of altered flow to maintaining and advancing vascular disease is the relative contribution of altered shear versus the continuing contributions of the underlying disease process. In other words, it is important to differentiate the lung disease caused by altered shear from the manifestations of the underlying disease process.

5.1 Disturbed Flow and Cell Proliferation The normal structure and function of the vasculature is dependent upon the normal shear stresses produced by laminar blood flow at physiological levels. Laminar flow maintains vascular homeostasis and is considered to be antiapoptotic antiatherosclerotic, and antiproliferative [5, 6, 8]. Under normal laminar flow, the production of NO, TGF-b, PDGF, and other factors by the endothelium which regulate smooth muscle cell growth is maintained in a basal state [1]. This balance is altered in the presence of disturbed (turbulent or oscillatory) shear. Disturbed flow promotes endothelial dysfunction in the form of endothelial cell damage, increased macromolecular permeability, lipoprotein accumulation, and leukocyte adhesion [33, 69], and leads to structural modifications of the vessel wall. The response includes abnormal proliferation of both endothelial and smooth muscle cells and increased synthesis of matrix proteins. These changes associated with shear can amplify lung alterations owing to progression of the underlying disease process or can incite new disease processes. The production of ROS through activation of vascular NADPH oxidase appears to play a central role in cell proliferation [70] as demonstrated experimentally with smooth muscle proliferation induced by the NOX2 activator angiotensin II [71, 72].

5.2 Inflammatory Responses to Shear Disturbed flow also has been associated with induction of vascular wall inflammation. A physiological level of laminar shear stress is considered to be anti-inflammatory and maintains mediators of inflammation in a basal state [73]. This anti-inflammatory role of laminar shear stress may be mediated partly by NO since NO synthesis limits the production of adhesion molecules, cytokines, and chemokines. Endothelial cells exposed to disturbed flow in vitro over prolonged periods develop a proinflammatory phenotype with increased expression of adhesion molecules (ICAM, VCAM, P-selectin, and E-selectin), chemokines (monocyte chemotactic protein) and cytokines (tumor necrosis factor, interleukins) [35, 74, 75]. Like the mechanisms for cell proliferation, the effect on inflammations occurs at least in part through endothelial

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ROS production mediated via activation of vascular NADPH oxidase [35, 75].

5.3 Rarefaction of Pulmonary Vasculature and Increased Pulmonary Vascular Resistance Increased or disturbed shear can result in an increased pulmonary vascular resistance that is attributed, in part, to remodeling of the walls of resistance vessels. Thus, intimal, medial, and adventitial hypertrophy leads to reduction of the diameter of the vascular lumen and increased vascular resistance. The results of vasculature remodeling in the lung are a reduction of the total number of pulmonary arterioles with consequent altered perfusion of the pulmonary alveoli and abnormal ventilation–perfusion relationships [76]. This effect of shear in the pulmonary vasculature contrasts with its effects in the systemic vasculature, where vascular disease promotes angiogenesis. In a rat model of pulmonary hypertension, extensive new vessel formation was observed in the peribronchial region and on the pleural surface arising from systemic (bronchial and intercostal) vessels, but there was no evidence of new vessel formation in the alveolar walls [77]. A similar response has been seen with decreased shear associated with experimental pulmonary artery ligation [78]. These results imply that unlike in the systemic vasculature, angiogenesis does not occur in the adult pulmonary circulation. The mechanisms for the differential responses of the pulmonary and systemic vascular beds are not clear.

5.4 Pulmonary Hypertension Pulmonary hypertension has multiple causes with a range of underlying mechanisms resulting in areas of the vasculature that are subjected to either excessive or decreased shear [79, 80]. Thus, increased shear results from increased blood flow, disturbed (turbulent) shear results from partial intravascular obstruction, and decreased shear is associated with vascular occlusion. Clinical examples include left-to-right shunt (increased shear), vasculitis (intravascular obstruction), and thromboembolic disease (occlusion). Pulmonary vasoconstriction would be expected to result in increased shear, but turbulent shear or decreased shear could also result depending on the pulmonary blood flow. In most cases of pulmonary hypertension (e.g., caused by lung damage), areas of increased shear, turbulent shear, and decreased shear coexist within the lung circulation. Persistent changes in pulmonary shear stress result in structural alterations of the vessel wall, indicated by changes

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in wall diameter and thickness, with eventual modification of vessel diameter. This vascular remodeling is characterized by thickening of all three layers of the blood vessel wall, viz., the adventitia, the media, and the intima, due to hypertrophy and/or proliferation (hyperplasia) of smooth muscle, endothelial, and fibroblastic cells as well as increased deposition of extracellular matrix components such as collagen, elastin, and fibronectin. The effects of altered shear stress on vascular remodeling have been demonstrated in animal models of increased pulmonary blood flow due to experimental left-toright shunts [81, 82]. Another manifestation of altered shear is the formation of neointimal lesions that are hyperplastic areas composed of smooth muscle cells and extracellular matrix [83]. These lesions are seen with severe pulmonary endothelial injury. Experimentally, the pulmonary vascular injury associated with the monocrotaline model of pulmonary hypertension is characterized by the development of the neointimal pattern. The intimal changes can include fibrotic or cellular thickening and acute and organizing thrombi, all of which can result from or lead to disordered flow. Finally, a hallmark of obstructive intimal remodeling is the pathological structure called the “plexiform lesion” that is associated with severe pulmonary artery hypertension [84]. This lesion is manifested by granulation tissue that forms to repair transmural destruction of the vessel wall. The lesions generally develop at the branch point of a muscular artery, compatible with an important role for shear stress in their pathogenesis. These lesions are most commonly observed in primary pulmonary hypertension, but can occur in other forms of pulmonary hypertension as well.

5.5 Atherosclerosis Shear stress appears to play a contributing role in development of atherosclerotic lesions since atheroma (plaque) are rarely seen in straight sections of the arterial tree, where flow is laminar, but develop in regions associated with disturbed or oscillatory flows near arterial bifurcations, branch ostia, and curvatures. Arteries such as the coronary, carotid, cerebral, iliac, and splenic arteries are selectively involved in comparison with other branches. Although pulmonary artery atherosclerosis is rare, there are reports of macrophage-rich lesions in patients with severe pulmonary hypertension [85]. Inflammation is thought to play an important role in the pathogenesis and progression of atherosclerosis [86]. As plaque forms, monocytes present in the plaque proliferate, oxidize LDL, and generate multiple cytokines that act as chemoattractants for other inflammatory cells [86]. These cells induce neointimal lesions with increased extracellular matrix production, thereby contributing to the progression of atherosclerotic lesions.

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6 Conclusions Shear stress plays an important role in lung (and other vascular) disease and disease processes. Laminar shear is sensed by the endothelium and perhaps other sections of the vascular wall and helps to maintain those cells in a quiescent state, consistent with their functions in pulmonary perfusion and gas exchange. Disturbed or abruptly decreased flow can be associated with inflammation and alterations of the vascular wall, including neointimal and plexiform lesions. Thus, the pattern of shear represents a fundamental mechanism that is crucial to the health and viability of the lung as the organ of gas exchange.

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54  Shear Stress, Cell Signaling, and Pulmonary Vascular Remodeling 59. Manevich Y, Al-Mehdi A, Muzykantov V et  al (2001) Oxidative burst and NO generation as initial response to ischemia in flowadapted endothelial cells. Am J Physiol Heart Circ Physiol 280:H2126–H2135 60. Wei Z, Manevich Y, Al-Mehdi AB et al (2004) Ca2+ flux through voltage-gated channels with flow cessation in pulmonary microvascular endothelial cells. Microcirculation 11:517–526 61. Catterall WA (2000) Structure and regulation of voltage-gated Ca2+ channels. Annu Rev Cell Dev Biol 16:521–555 62. Wu S, Haynes J Jr, Taylor JT et al (2003) Cav3.1 a1G T-type Ca2+ channels mediate vaso-occlusion of sickled erythrocytes in lung microcirculation. Circ Res 93:346–353 63. Perez-Reyes E (1999) Three for T: molecular analysis of the low voltage-activated calcium channel family. Cell Mol Life Sci 56:660–669 64. Boonstra J, Post JA (2004) Molecular events associated with reactive oxygen species and cell cycle progression in mammalian cells. Gene 337:1–13 65. Milovanova T, Manevich Y, Haddad A et al (2004) Endothelial cell proliferation associated with abrupt reduction in shear stress is dependent on reactive oxygen species. Antioxid Redox Signal 6:245–258 66. Milovanova T, Chatterjee S, Manevich Y et al (2006) Lung endothelial cell proliferation with decreased shear stress is mediated by reactive oxygen species. Am J Physiol Cell Physiol 290:C66–C76 67. Kohler TR, Jawien A (1992) Flow affects development of intimal hyperplasia after arterial injury in rats. Arterioscler Thromb 12: 963–971 68. Kimura BJ, Russo RJ, Bhargava V et al (1996) Atheroma morphology and distribution in proximal left anterior descending coronary artery: in vivo observations. J Am Coll Cardiol 27:825–831 69. Bussolari SR, Dewey CF Jr, Gimbrone MA Jr (1982) Apparatus for subjecting living cells to fluid shear stress. Rev Sci Instrum 53:1851–1854 70. Weber DS, Rocic P, Mellis AM et al (2005) Angiotensin II-induced hypertrophy is potentiated in mice overexpressing p22phox in vascular smooth muscle. Am J Physiol Heart Circ Physiol 288:H37–H42 71. Ushio-Fukai M, Zafari AM, Fukui T et al (1996) p22phox is a critical component of the superoxide-generating NADH/NADPH oxidase system and regulates angiotensin II-induced hypertrophy in vascular smooth muscle cells. J Biol Chem 271:23317–23321 72. Griendling KK, Sorescu D, Lassegue B et al (2000) Modulation of protein kinase activity and gene expression by reactive oxygen species and their role in vascular physiology and pathophysiology. Arterioscl Thromb Vasc Biol 20:2175–2183

799 73. Sampath R, Kukielka GL, Smith CW et  al (1995) Shear stressmediated changes in the expression of leukocyte adhesion receptors on human umbilical vein endothelial cells in  vitro. Ann Biomed Eng 23:247–256 74. Chappell DC, Varner SE, Nerem RM et al (1998) Oscillatory shear stress stimulates adhesion molecule expression in cultured human endothelium. Circ Res 82:532–539 75. Hwang J, Saha A, Boo YC et  al (2003) Oscillatory shear stress stimulates endothelial production of O2- from p47phox-dependent NAD(P)H oxidases, leading to monocyte adhesion. J Biol Chem 278:47291–47298 76. Partovian C, Adnot S, Raffestin B et al (2000) Adenovirus-mediated lung vascular endothelial growth factor overexpression protects against hypoxic pulmonary hypertension in rats. Am J Respir Cell Mol Biol 23:762–771 77. Schraufnagel DE (1990) Monocrotaline-induced angiogenesis. Differences in the bronchial and pulmonary vasculature. Am J Pathol 137:1083–1090 78. Mitzner W, Lee W, Georgakopoulos D et al (2000) Angiogenesis in the mouse lung. Am J Pathol 157:93–101 79. Voelkel NF, Tuder RM (1995) Cellular and molecular mechanisms in the pathogenesis of severe pulmonary hypertension. Eur Respir J 8:2129–2138 80. Rubin LJ (1997) Primary pulmonary hypertension. N Engl J Med 336:111–117 81. Bousamra M 2nd, Rossi R, Jacobs E et  al (2000) Systemic lobar shunting induces advanced pulmonary vasculopathy. J Thorac Cardiovasc Surg 120:88–98 82. Friedli B, Kent G, Kidd BS (1975) The effect of increased pulmonary blood flow on the pulmonary vascular bed in pigs. Pediatr Res 9:547–553 83. Tanaka Y, Schuster DP, Davis EC et al (1996) The role of vascular injury and hemodynamics in rat pulmonary artery remodeling. J Clin Invest 98:434–442 84. Yi ES, Kim H, Ahn H et al (2000) Distribution of obstructive intimal lesions and their cellular phenotypes in chronic pulmonary hypertension. A morphometric and immunohistochemical study. Am J Respir Crit Care Med 162:1577–1586 85. Botney MD (1999) Role of hemodynamics in pulmonary vascular remodeling: implications for primary pulmonary hypertension. Am J Respir Crit Care Med 159:361–364 86. Ross R (1999) Atherosclerosis-an inflammatory disease.[see comment]. N Engl J Med 340:115–126 87. Chatterjee S, Chapman KE, Fisher AB (2008) Lung ischemia: a model for endothelial mechanotransduction. Cell Biochem Biophys 52:125–138

Chapter 55

Pulmonary Hypertension and the Extracellular Matrix Marlene Rabinovitch

Abstract  The extracellular matrix (ECM) plays a pivotal role in regulating cell shape and cell signaling, in maintaining cell–cell communication, and in controlling cell differentiation and dedifferentiation. The stiffness of a vessel is directly related to the proportion of the various ECM components. It is not surprising then that even haploinsufficiency of a single component of the ECM such as elastin or fibrillin can lead to profound developmental abnormalities and propensity to diseases of blood vessels. For example, haploinsufficiency of elastin (Williams syndrome) is associated with pulmonary and systemic stenoses and haploinsufficiency of fibrillin (Marfan syndrome) leads to aneurismal dilatation of the aorta. A variety of growth factors and cytokines regulate the production of the various components of the ECM and are in fact bound by different components of the ECM. Turnover of the ECM and release of growth factors is controlled by the balance between proteolytic enzymes such as serine elastases and matrix metalloproteinases (MMPs) and their endogenous inhibitors. So it is not surprising that abnormalities in the regulation of the ECM would contribute in a fundamental way to the pathobiology of pulmonary vascular disease leading to pulmonary arterial hypertension (PAH). Keywords  Elastase • Elastin • Fibrillin • Vascular wall stiffness • Growth factors • Pulmonary vascular remodeling

1 Introduction The extracellular matrix (ECM) plays a pivotal role in regulating cell shape and cell signaling, in maintaining cell– cell communication, and in controlling cell differentiation and dedifferentiation. The stiffness of a vessel is directly related to the proportion of the various ECM components. It M. Rabinovitch (*) Department of Pediatrics, Wall Center for Pulmonary Vascular Diseases, Stanford University School of Medicine, CCSR-Room 2245B, 269 Campus Drive, Stanford, CA 94305-5162, USA e-mail: [email protected]

is not surprising then that even haploinsufficiency of a single component of the ECM such as elastin or fibrillin can lead to profound developmental abnormalities and propensity to diseases of blood vessels. For example, haploinsufficiency of elastin (Williams syndrome) is associated with pulmonary and systemic stenoses and haploinsufficiency of fibrillin (Marfan syndrome) leads to aneurismal dilatation of the aorta. A variety of growth factors and cytokines regulate the production of the various components of the ECM and are in fact bound by different components of the ECM. Turnover of the ECM and release of growth factors is controlled by the balance between proteolytic enzymes such as serine elastases and matrix metalloproteinases (MMPs) and their endogenous inhibitors. So it is not surprising that abnormalities in the regulation of the ECM would contribute in a fundamental way to the pathogenic mechanisms of pulmonary vascular remodeling leading to pulmonary arterial hypertension (PAH).

2 Pulmonary Hypertension and Elastase Activity Ultrastructural assessment of pulmonary arteries (PA) in lung biopsy tissue from patients with congenital heart defects and PAH [1] first suggested that changes in the ECM could contribute to the pathobiology of PAH. In association with alterations in endothelial structure and function, we noted fragmentation of elastic laminae (Fig. 1). These early features, apparent by electron microscopy, preceded the later development of occlusive lesions. Gaps in the internal elastic laminae were also reported in patients with PAH and neointimal lesions [2]. Fragmentation of elastin occurred in experimental rats in which PAH was induced in association with endothelial injury [3] following injection of the toxin monocrotaline [4]. Consistent with the ultrastructural changes reflecting degradation of PA elastin was heightened activity of a serine elastase in the PA wall [4]. The increased activity of this endogenous vascular elastase (EVE) preceded the development of PAH and the accompanying vascular lesions. Those vascular changes consisted of abnormal muscularization and

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_55, © Springer Science+Business Media, LLC 2011

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Fig. 1  (a) A section of pulmonary artery 92 mm in diameter in a patient with normal pulmonary arterial pressure shows an intact elastic lamina. (b) In a section from a pulmonary artery 108 mm in diameter in a patient with increased pulmonary blood flow and pressure, microfibrillar material is present in the subendothelium but there is no true internal elastic lamina. The endothelial cells (EC) and smooth muscle cells (SMC) are separated by only a thick basement membrane (bm). Bars 1  mm. (Modified from [1] with permission)

loss of peripheral alveolar duct and wall PAs and medial hypertrophy of the more proximal intra-acinar PAs at the level of the terminal and respiratory bronchioli [5]. Moreover, inhibition of EVE resulted in attenuation of the PAH as well as the associated vascular changes in a variety of experimental rodent models [5]. More recently in a mouse that overexpresses S100A4 and that develops neointimal lesions after injection of the murine gamma herpes virus 68 (MHV-68) [6], we showed that serine elastase activity is increased after infection of the virus and with reactivation. In an effort to understand how EVE activity was regulated in the vessel wall, we cultured PA smooth muscle cells (SMC). We were able to show that EVE could be induced by serum and endothelial factors, to which SMC would be exposed under conditions of endothelial injury [7]. It appeared that these factors signaled through the mitogen-activated protein kinase pathway, causing induction and nuclear translocation of phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2) that was inhibited by NO. We showed that pERK1/2 nuclear translocation was necessary for the activity of the transcription factor for elastase, AML1 [8]. Then we documented that EVE could release SMC mitogens such as basic fibroblast growth factor that are normally stored in the ECM in an inactive form [9]. In addition, we showed that elastin peptides [10], produced in response to EVE, could mediate both SMC proliferation and fibronectindependent SMC motility [11] (Fig.  2). As well, elastin peptides are chemoattractants for inflammatory cells [12], and can induce proliferation and migration of SMC [13, 14]. Further studies revealed that EVE, either directly or through activation of MMPs [15], could, by degrading collagen and exposing cryptic RGD sites, cause heightened signaling through avb3 integrins [16]. This signaling was responsible for production and secretion of tenascin-C, a glycoprotein that binds to and clusters avb3 integrins [16]. In so doing,

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tenascin-C alters the actin cytoskeleton and this causes clustering and activation of growth factor receptors [16]. These receptors transduce both pro-proliferative and cell survival signals in PA SMCs. So when we suppressed EVE or MMP activity in proliferating PA SMCs, we not only prevented their proliferation but also induced apoptosis [16]. We then documented that we could induce regression of hypertrophied PAs in the intact animal by inhibiting EVE and by inducing PA SMC apoptosis [17] (Fig. 3). Interestingly, inhibition of EVE also reversed the loss of small precapillary vessels (alveolar duct and wall arteries), suggesting that this therapy may also regenerate the lost microcirculation associated with PAH. Although heightened MMP activity is associated with the development of PAH, we could not reverse experimentally induced PAH in the monocrotaline-treated rat with MMP inhibition, although the length of survival was improved [18]. Others have shown that inhibition of MMPs may actually worsen hypoxia-induced PAH [19]. In fact, we have shown that MMPs are activated in SMCs downstream of bone morphogenetic protein receptor II (BMPRII) signaling [20, 21] and are necessary for normal migratory and proliferative behavior of SMCs and pericytes in development, and potentially in response to injury. An extensive body of work has shown that metalloproteinase activity is required for the regression of chronic-hypoxia-induced PAH [22].

3 Elafin, an EVE Inhibitor To further address the importance of EVE in the pathology of cardiovascular disease, we made a mouse that overexpressed human elafin, a molecule that inhibits leukocyte elastase but that we had shown was also an endogenous inhibitor of EVE [23]. Elafin was initially isolated from bronchial secretions [24] and from psoriatic skin [25] as a high-affinity serine elastase inhibitor (ki for neutrophil elastase 4.2 ×10−9 M). It is similar functionally, structurally, and by chromosomal location to secreted leukocyte protease inhibitor [26] but with limited targets of inhibitory activity. These include neutrophil elastase, pancreatic elastase, and proteinase 3 [25]. Elafin is produced as a 12-kDa pro-form called trappin and in this form consists of a signal peptide, a transglutaminase domain that binds to a variety of ECM components and an elastase inhibitory domain. Upon cleavage of the signal peptide and the transglutaminase domain, the 6-kDa elafin is released. In designing the transgenic mouse, we overexpressed elafin under the regulation of the preproendothelin ­promoter. Thus, it was induced under conditions of endothelial injury when endothelin transcription is activated. The elafinoverexpressing mouse was not only protected against PAH [27], but also against a variety of other cardiovascular diseases

55  Pulmonary Hypertension and the Extracellular Matrix

Fig. 2  (a) We speculate, on the basis of our studies in cultured cells and in experimental animals, as to how a stimulus could induce activity of an elastolytic enzyme and how this might stimulate the remodeling process. In response to an injurious stimulus, the first casualty is the endothelial cell. As a result of structural and functional alterations in endothelial cells, some of the barrier function would be lost, allowing a leak into the subendothelium of a serum factor normally excluded from this region. The serum factor could induce activity of an endogenous vascular elastase (EVE). This enzyme released from precursor or mature smooth muscle cells (SMCs) would activate growth factors normally stored in the extracellular matrix in an inactive form, which are known to induce smooth muscle hypertrophy and proliferation and increases in connective tissue (CT) protein (e.g., collagen and elastin) synthesis. The growth factors could also result in the differentiation of precursor cells to mature smooth muscle

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related to the muscularization of normally nonmuscular small peripheral arteries. Elastase activity could also contribute to the loss of small arteries. (b) As a result of elastase activity, a proteolytic cascade could be ­amplified through the activation of matrix metalloproteinases. We have shown that proteolysis of the matrix leads to the induction of tenascin-C, which in clustering to b3 integrins results in the clustering and activation of growth factor receptors which send important survival as well as growth signals to the cells. Continued elastase activity would cause migration of smooth muscle cells in several ways. The elastin peptides or degradation products of elastin can stimulate fibronectin, a glycoprotein that is pivotal in altering smooth muscle cell shape and in switching smooth muscle cells to the motile phenotype. Inhibition of elastase activity could lead to smooth muscle cell apoptosis and regression of pulmonary vascular disease (PVD). (Reproduced from [11] with permission)

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Fig.  3  Cellular mechanism responsible for reversal of pulmonary artery muscularity. Elastase inhibition arrests tenascin-C accumulation and proliferation and induces apoptosis and loss of extracellular matrix (such as elastin). (a–p) Days refer to time after injection of monocrotaline: (a, e, j, m) day 21; (b, f, j, n) day 28; (c, g, k, o) day 28; (d, h, l, p) day 28. (a–d) Saline-perfused pulmonary arteries stained with Movat pentachrome stain. (e–h) Pulmonary arteries after tenascin-C

immunohistochemistry. Arrows indicate positive brown peroxidase staining. (i–l) In situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assays identifying apoptosis. Arrows indicate TUNEL-positive vascular cells. (m–p) Proliferating vascular cells, shown by immunohistochemistry for proliferating cell nuclear antigen (PCNA); dark nuclei are PCNA-positive cells. Scale bars 5 mm. (Reproduced from [17] with permission)

such as myocardial infarction [28], myocarditis [23], and carotid artery stenosis [29]. Gene transfer of elafin attenuated vein graft atherosclerosis [30] and infusion of elafin in rabbits attenuated both posttransplant coronary artery disease and rejection [31].

occlusion. Haploinsufficiency of elastin causes PAH and replacement of the null allele with the human tropoelastin gene to generate 35% of normal tropoelastin [32] further increases the severity of PAH and vascular stiffness. Structural changes in the distal PA vasculature are not, however, very severe [33] (Fig. 4). A variety of mice have been created with loss of elastin assembly genes such as fibrillin-1 and fibulin-5, and deletions of some of these genes (fibulin-4) cause lethality in embryonic or early postnatal life, so the presence of PAH has not been assessed. In the newborn calf that is made hypoxic, there is evidence for marked stimulation of tropoelastin synthesis in the media and adventitia of the PAs [34] (Fig.  5). We observed that in rats treated with monocrotaline, there is a twofold increase in net production of both elastin and collagen,

4 Elastin, Elastin Assembly Genes, and Pulmonary Hypertension Intact well-assembled elastin plays a critical role in vascular resilience and in preventing stiffening of the vasculature and in controlling SMC proliferation. Deletion of elastin results in neonatal lethality owing to proliferation of SMCs and vascular

55  Pulmonary Hypertension and the Extracellular Matrix Fig. 4  (a) Right ventricular (RV) pressure was measured using a microcatheter introduced through the internal jugular vein; six animals per genotype. Bars represent mean systolic (solid bars) and diastolic (shaded bars) pressures ± the standard error. *P < 0.00001, P < 0.0001, ¶P = 0.96, and P = 0.41 versus the wild type (WT). (b) Pressure tracings from wild-type and human elastin transgene (ELN +/+) expressed in a bacterial artificial chromosome and a mouse null for the elastin gene (ELN -/-), (hBAC-mNULL) mice demonstrating measured systolic and diastolic pressure differences. (Reproduced from [33] with permission)

Fig. 5  In situ hybridization localization of tropoelastin messenger RNA (mRNA) in control and hypertensive vessels from neonatal calves. White staining over areas indicates tropoelastin mRNA labeling. In normotensive vessels (top), labeled cells (35S-labeled T66-T7) were confined to the inner media. Minimal signal is noted in the outer vessel wall. In vessels from hypertensive animals (14 days of hypoxia) (bottom), an intense autoradiographic signal was observed throughout the media, albeit in a patchy distribution. (Reproduced from [34] with permission)

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but the turnover of elastin is high, so the ratio of synthesis to accumulation is about 5:1 [4]. The newly synthesized elastin is not distributed as thicker elastic laminae but rather as scattered interlamellar elastin fragments [4, 35]. In a transgenic mouse that overexpresses the calcium binding protein S100A4, PAH is observed under room-air conditions and is aggravated by exposure to chronic hypoxia. In this mouse, we documented thick elastic laminae and increased deposition of elastin in association with a twofold increase in the production and incorporation of the elastin assembly protein fibulin-5 [36]. Further studies showed that these S100A4-overexpressing mice had less distal remodeling for their severity of PAH in response to chronic hypoxia [37]. However, other studies showed that the S100A4 mice (but not their littermate controls) developed neointimal lesions at times spontaneously [38] but more reproducibly following inoculation with MHV-68. We established that this was associated with heightened activity of a serine elastase in the S100A4 lung and also with greater susceptibility of the thick but poorly assembled fibulin-5 rich elastic laminae to elastase-mediated degrada-

Fig. 6  Prx1 and tenascin-C expression in isolated normal, F14, and F28 pulmonary artery (PA) smooth muscle cells (SMCs). (a) Semi­ quantitative reverse-transcription (RT) PCR for Prx1a, Prx1b, and tenascin-C mRNA expression in normal (Nor), F14, and F28 pulmonary artery smooth muscle cells. 18S was used as an input control; 28, 30, and 32 PCR cycles were used for each complementary DNA amplification. (b) Quantification of RT-PCR data (n = 3) for Prx1a (top) and tenascin-C (bottom) relative to 18S mRNA. (c) Western immunoblotting for tenascin-C using total cell lysates derived from normal, F14, and

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tion [6]. It is of further interest that another elastin assembly protein (fibrillin-1) may be implicated in PAH since antifibrillin-1 antibodies were identified in patients with idiopathic PAH [39].

5 Tenascin-C, Matrix Metalloproteinases, and Pulmonary Hypertension Tenascin-C, in binding to avb3 integrins results in clustering and activation of growth factor receptors in PA SMCs [16]. Recently, it has been shown that mutations in BMPRII or in its downstream signaling pathway, specifically in Smad8, result in upregulation of tenascin-C and its transcription factor, Prx1 [40] (Fig. 6). It also has been shown that alterations in the SMC or fibroblast cytoskeleton can, through focal adhesion kinase, promote Prx1 production and transcriptional activity, resulting in the production of tenascin-C [41]. Heightened activity of MMPs induced Prx1 and tenascin-C production by activating avb3 integrins [42]. Not only was

F28 smooth muscle cells. b-Actin was used as a loading control. Familial pulmonary arterial hypertension smooth muscle cells expressed a higher-molecular-mass tenascin-C isoform of 220  kDa, whereas normal smooth muscle cells expressed an 180-kDa isoform. (d) Representative immunofluorescence photomicrograph showing Prx1 (in red) and tenascin-C (in green) expression in normal, F14, and F28 pulmonary artery smooth muscle cells. Nuclei are labeled with 4,6-diamidino-2-phenylindole (DAPI, in blue). Scale bars 10  mM. (Reproduced from [40] with permission)

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Fig. 7  Representative photomicrographs showing immunoperoxidase staining for tenascin-C (TN) (a, d, g), PCNA (b, e, h), and epidermal growth factor (EGF) (c, f, i) in graded lung biopsy tissue sections. (a–c) Vessel showing a typical grade IA lesion. (d–f) Vessel showing a typical grade IC lesion. (g–i) Vessel showing a typical grade IIIC lesion. In low-grade lesions (a), modest tenascin-C immunostaining was evident in the adventitia. With medial hypertrophy, tenascin-C immunoreactivity became more prominent in the periendothelium (d), with the most intense immunostaining being apparent within the neointima of high-

grade lesions showing occlusive neointimal formation (g). In the lowest grade of lesion, PCNA was negative (b), despite foci of EGF in the media. With medial hypertrophy, PCNA was expressed in the media (e), together with foci of EGF (f). With the development of higher-grade occlusive lesions, tenascin-C (g), PCNA (h), and EGF (i) colocalized to the neointimal cell layers. Note that tenascin-C and PCNA staining was performed on serial sections, whereas EGF detection was carried out on similar vessels within the same biopsy. Magnification ×40. (Reproduced from [43] with permission)

heightened expression of tenascin-C associated with progression of clinical pulmonary vascular disease [43] (Fig. 7), but regression of hypertrophied PAs was also accompanied by suppression of tenascin-C. It is interesting that when we suppressed tenascin-C alone using antisense RNA, the cells upregulated another matrix component and avb3 ligand, osteopontin [44], and in this way limited the amount of regression of pulmonary vascular disease.

[45] so its deposition in the subendothelium provided a gradient that could facilitate that development of a neointima. Fibronectin peptides also facilitate perivascular inflammation [46].

6 Fibronectin and Pulmonary Hypertension We observed in clinical pulmonary vascular disease associated with PAH that along with the increase in the expression of tenascin-C there was an increase in the level of fibronectin, particularly in the subendothelial region [43]. Our previous studies had related fibronectin to SMC migration

7 Collagen and Pulmonary Hypertension Advanced pulmonary vascular lesions were previously considered to be stable fibrotic scars, but a study showing deposition of the pro-form of collagen suggested that there was active turnover of the matrix and that this might be important to target in reversing the lesions [47]. The cells that produce the collagen are believed to be myofibroblasts and these cells could be derived from fibrocytes (i.e., cells with characteristics of both fibroblasts as well as monocytes) that migrate from the adventitia [48].

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8 Hyaluronic Acid, Other Glycosaminogycans, and Proteoglycans in PAH An increase in the levels of hyaluronic acid and hyaluronan synthase has been demonstrated in PAs in patients with PAH. Hyaluronan facilitates SMC migration in response to a fibro­ nectin gradient [45]. Other proteoglycans and glycosaminoglycans implicated in systemic vascular pathobiology are also likely to play an important role in the pathobiology of PAH. On the other hand, gene transfer of biglycan limits neointimal formation by enhancing the synthesis and ­assembly of elastic fibers [49].

9 Summary Abnormalities in the expression of a single ECM component can have profound effects on the assembly of other ECM ­components and on the signals that the cells receive. These signals result from altered contact of cells with ECM components and from alterations in the stiffness of the ECM. The signals in turn influence cell behavior, related to survival versus apoptosis, proliferation versus differentiation or migration. Genetic factors alter production of the various matrix components as do growth factors and proteolytic enzymes such as elastases. Elastin and elastase have been best characterized in relation to the development of PAH.

References 1. Rabinovitch M, Bothwell T, Hayakawa BN et al (1986) Pulmonary artery endothelial abnormalities in patients with congenital heart defects and pulmonary hypertension: a correlation of light with scanning electron microscopy and transmission electron microscopy. Lab Invest 55:632–653 2. Aiello VD, Gutierrez PS, Chaves MJ, Lopes AA, Higuchi ML, Ramires JA (2003) Morphology of the internal elastic lamina in arteries from pulmonary hypertensive patients: a confocal laser microscopy study. Mod Pathol 16:411–416 3. Rosenberg HC, Rabinovitch M (1988) Endothelial injury and vascular reactivity in monocrotaline pulmonary hypertension. Am J Physiol 255:H1484–H1491 4. Todorovich-Hunter L, Dodo H, Ye C, McCready L, Keeley FW, Rabinovitch M (1992) Increased pulmonary artery elastolytic activity in adult rats with monocrotaline-induced progressive hypertensive pulmonary vascular disease compared with infant rats with nonprogressive disease. Am Rev Respir Dis 146:213–223 5. Ye C, Rabinovitch M (1991) Inhibition of elastolysis by SC-37698 reduces development and progression of monocrotaline pulmonary hypertension. Am J Physiol 261:H1255–H1267 6. Spiekerkoetter E, Alvira CM, Kim YM et al (2008) Reactivation of gHV68 induces neointimal lesions in pulmonary arteries of S100A4/ Mts1 over-expressing mice in association with degradation of elastin. Am J Physiol Lung Cell Mol Physiol 294:L276–L289

M. Rabinovitch 7. Thompson K, Kobayashi J, Childs T, Wigle D, Rabinovitch M (1998) Endothelial and serum factors which include apolipoprotein A1 tether elastin to smooth muscle cells inducing serine elastase activity via tyrosine kinase-mediated transcription and translation. J Cell Physiol 174:78–89 8. Mitani Y, Zaidi SHE, Dufourcq P, Thompson K, Rabinovitch M (2000) Nitric oxide reduces vascular smooth muscle cell elastase activity through cGMPmediated suppression of ERK phosphorylation and AML1B nuclear partitioning. FASEB J 14:805–814 9. Thompson K, Rabinovitch M (1996) Exogenous leukocyte and endogenous elastases can mediate mitogenic activity in pulmonary artery smooth muscle cells by release of extracellular-matrix bound basic fibroblast growth factor. J Cell Physiol 166:495–505 10. Hinek A, Molossi S, Rabinovitch M (1996) Exploration of the molecular mechanisms leading to upregulation of fibronectin production by the arterial smooth muscle cells. Exp Cell Res 225:111–120 11. Rabinovitch M (2007) Pathobiology of pulmonary hypertension. Annu Rev Pathol 2:369–399 12. Houghton AM, Quintero PA, Perkins DL et al (2006) Elastin fragments drive disease progression in a murine model of emphysema. J Clin Invest 116:753–759 13. Hinek A, Rabinovitch M (1993) The ductus arteriosus migratory smooth muscle cell phenotype processes tropoelastin to a 52-kDa product associated with impaired assembly of elastic laminae. J Biol Chem 268:1405–1413 14. Karnik SK, Brooke BS, Bayes-Genis A et al (2003) A critical role for elastin signaling in vascular morphogenesis and disease. Development 130:411–423 15. Nagase H, Enghild J, Suzuki K, Salvesen G (1990) Stepwise activation mechanisms of the precursor of matrix metalloproteinase 3 (stromelysin) by proteinases and (4-aminophenyl) mercuric acetate. Biochemistry 29:5783–5789 16. Jones P, Crack J, Rabinovitch M (1997) Regulation of tenascin-C, a vascular smooth muscle cell survival factor that interacts with the avb3 integrin to promote epidermal growth factor receptor phosphorylation and growth. J Cell Biol 139:279–293 17. Cowan KN, Heilbut A, Humpl T, Lam C, Ito S, Rabinovitch M (2000) Complete reversal of fatal pulmonary hypertension in rats by a serine elastase inhibitor. Nat Med 6:698–702 18. Merklinger SL, Jones PL, Martinez EC, Rabinovitch M (2005) Epidermal growth factor receptor blockade mediates smooth muscle cell apoptosis and improves survival in rats with pulmonary hypertension. Circulation 112:423–431 19. Vieillard-Baron A, Frisdal E, Eddahibi S et al (2000) Inhibition of matrix metalloproteinases by lung TIMP-1 gene transfer or doxycycline aggravates pulmonary hypertension in rats. Circ Res 87:418–425 20. El-Bizri N, Guignabert C, Wang L et  al (2008) SM22a-targeted deletion of bone morphogenetic protein receptor 1A in mice impairs cardiac and vascular development, and influences organogenesis. Development 135:2981–2991 21. El-Bizri N, Wang L, Merklinger SL et  al (2008) Smooth muscle protein 22a-mediated patchy deletion of Bmpr1a impairs cardiac contractility but protects against pulmonary vascular remodeling. Circ Res 102:380–388 22. Tozzi C, Thakker-Varia S, Yu S et al (1998) Mast cell collagenase correlates with regression of pulmonary vascular remodeling in the rat. Am J Respir Cell Mol Biol 18:497–510 23. Zaidi SHE, Hui C-C, Cheah AYL, You X-M, Husain M, Rabinovitch M (1999) Targeted overexpression of elafin protects mice against cardiac dysfunction and mortality following viral myocarditis. J Clin Invest 103:1211–1219 24. Sallenave J-M, Ryle AP (1991) Purification and characterization of elastase-specific inhibitor: Sequence homology with mucous proteinase inhibitor. Biol Chem Hoppe Seyler 372:13–21

55  Pulmonary Hypertension and the Extracellular Matrix 25. Wiedow O, Luademann J, Utecht B (1991) Elafin is a potent inhibitor of proteinase 3. Biochem Biophys Res Commun 174:6–10 26. Moreau T, Baranger K, Dade S, Dallet-Choisy S, Guyot N, Zani ML (2008) Multifaceted roles of human elafin and secretory leukocyte proteinase inhibitor (SLPI), two serine protease inhibitors of the chelonianin family. Biochimie 90:284–295 27. Zaidi SHE, You X-M, Ciura S, Husain M, Rabinovitch M (2002) Overexpression of the serine elastase inhibitor elafin protects transgenic mice from hypoxic pulmonary hypertension. Circulation 105:516–521 28. Ohta K, Nakajima T, Cheah AY et al (2004) Elafin-overexpressing mice have improved cardiac function after myocardial infarction. Am J Physiol Heart Circ Physiol 287:H286–H292 29. Zaidi SH, You XM, Ciura S, O’Blenes S, Husain M, Rabinovitch M (2000) Suppressed smooth muscle proliferation and inflammatory cell invasion after arterial injury in elafin-overexpressing mice. J Clin Invest 105:1687–1695 30. O’Blenes SB, Zaidi SHE, Cheah AYL, McIntyre B, Kaneda Y, Rabinovitch M (2000) Gene transfer of the serine elastase inhibitor elafin protects against vein graft degeneration. Circulation 102: III289–III295 31. Cowan B, Baron O, Crack J, Coulber C, Wilson GJ, Rabinovitch M (1996) Elafin, a serine elastase inhibitor, attenuates post-cardiac transplant coronary arteriopathy and reduces myocardial necrosis in rabbits after heterotopic cardiac transplantation. J Clin Invest 97:2452–2468 32. Hirano E, Knutsen RH, Sugitani H, Ciliberto CH, Mecham RP (2007) Functional rescue of elastin insufficiency in mice by the human elastin gene: implications for mouse models of human disease. Circ Res 101:523–531 33. Shifren A, Durmowicz AG, Knutsen RH, Faury G, Mecham RP (2008) Elastin insufficiency predisposes to elevated pulmonary circulatory pressures through changes in elastic artery structure. J Appl Physiol 105:1610–1619 34. Prosser IW, Stenmark KR, Suthar M, Crouch EC, Mecham RP, Parks WC (1989) Regional heterogeneity of elastin and collagen gene expression in intralobar arteries in response to hypoxic pulmonary hypertension as demonstrated by in situ hybridization. Am J Pathol 135:1073–1088 35. Todorovich-Hunter L, Ranger P, Johnson D, Keeley F, Rabinovitch M (1988) Altered elastin and collagen synthesis associated with progressive pulmonary hypertensin induced by monocrotaline. A biochemical and ultrastructural study. Lab Invest 58:184–195 36. Yanagisawa H, Davis EC, Starcher BC et al (2002) Fibulin-5 is an elastin-binding protein essential for elastic fibre development in vivo. Nature 415:168–171

809 37. Merklinger SL, Wagner RA, Spiekerkoetter E et al (2005) Increased fibulin-5 and elastin in S100A4/Mts1 mice with pulmonary hypertension. Circ Res 97:596–604 38. Greenway S, van Suylen RJ (2004) Du Marchie Sarvaas G, et al. S100A4/Mts1 produces murine pulmonary artery changes resembling plexogenic arteriopathy and is increased in human plexogenic arteriopathy. Am J Pathol 164:253–262 39. Morse JH, Antohi S, Kasturi K et al (2000) Fine specificity of antifibrillin-1 autoantibodies in primary pulmonary hypertension syndrome. Scand J Immunol 51:607–611 40. Ihida-Stansbury K, McKean DM, Lane KB et al (2006) Tenascin-C is induced by mutated BMP type II receptors in familial forms of pulmonary arterial hypertension. Am J Physiol Lung Cell Mol Physiol 291:L694–L702 41. McKean DM, Sisbarro L, Ilic D et al (2003) FAK induces expression of Prx1 to promote tenascin-C-dependent fibroblast migration. J Cell Biol 161:393–402 42. Jones FS, Meech R, Edelman DB, Oakey RJ, Jones PL (2001) Prx1 controls vascular smooth muscle cell proliferation and tenascin-C expression and is upregulated with Prx2 in pulmonary vascular disease. Circ Res 89:131–138 43. Jones PL, Cowan KN, Rabinovitch M (1997) Tenascin-C, proliferation and subendothelial fibronectin in progressive pulmonary vascular disease. Am J Pathol 150:1349–1360 44. Cowan KN, Jones PL, Rabinovitch M (2000) Elastase and matrix metalloproteinase inhibitors induce regression, and tenascin-C antisense prevents progression of vascular disease. J Clin Invest 105:21–34 45. Boudreau N, Turley E, Rabinovitch M (1991) Fibronectin, hyaluronan, and a hyaluronan binding protein contribute to increased ductus arteriosus smooth muscle cell migration. Dev Biol 143:235–247 46. Pozzetto U, Aguzzi MS, Maggiano N et al (2005) RGDS peptide inhibits activation of lymphocytes and adhesion of activated lymphocytes to human umbilical vein endothelial cells in vitro. Immunol Cell Biol 83:25–32 47. Botney MD, Liptay MJ, Kaiser LR, Cooper JD, Parks WC, Mecham RP (1993) Active collagen synthesis by pulmonary arteries in human primary pulmonary hypertension. Am J Pathol 143:121–129 48. Frid MG, Brunetti JA, Burke DL et  al (2006) Hypoxia-induced ­pulmonary vascular remodeling requires recruitment of circulating mesenchymal precursors of a monocyte/macrophage lineage. Am J Pathol 168:659–669 49. Hwang JY, Johnson PY, Braun KR et al (2008) Retrovirally mediated overexpression of glycosaminoglycan-deficient biglycan in arterial smooth muscle cells induces tropoelastin synthesis and elastic fiber formation in  vitro and in neointimae after vascular injury. Am J Pathol 173:1919–1928

Chapter 56

Role of Progenitor Cells in Pulmonary Vascular Remodeling Kurt R. Stenmark, Susan M. Majka, and Maria G. Frid

Abstract  All forms of chronic pulmonary hypertension are characterized by cellular and structural changes in the walls of pulmonary arteries. Intimal thickening and fibrosis, medial hypertrophy, and fibroproliferative changes in the adventitia are common. In addition, extension of smooth muscle (SM) into previously nonmuscularized vessels is a consistent feature. Virtually all of these changes are characterized, to a greater or lesser degree, by increased numbers of cells expressing a-smooth muscle actin (a-SMA) and the accumulation of inflammatory cells. In addition, increased numbers of endothelial cells, particularly in the intima (plexiform lesions) but also in the perivascular regions, with distinct functional characteristics have been described. However, neither the origin(s) of these functionally distinct cells nor the molecular mechanisms operating to cause their accumulation in any of the three compartments of the vessel wall have been fully elucidated. The purpose of this chapter is to review the data supporting a contributory role for vascular progenitors, including endothelial progenitor cells, smooth muscle progenitor cells and fibrocytes, in systemic and pulmonary vascular remodeling. Keywords  Adult stem cell • Fibrocyte • Endothelial ­progenitor cell • Smooth muscle progenitor cell • Pulmonary vascular wall thickening • Plexiform lesion

1 Introduction All forms of chronic pulmonary hypertension are characterized by cellular and structural changes in the walls of pulmonary arteries. Intimal thickening and fibrosis, medial hypertrophy, and fibroproliferative changes in the adventitia are common. K.R. Stenmark (*) Department of Pediatrics, University of Colorado, Denver, 12700 E. 19th Ave., Mail Stop B131, Aurora, CO 80045, USA e-mail: [email protected]

In addition, extension of smooth muscle (SM) into previously nonmuscularized vessels is a consistent feature. Virtually all of these changes are characterized, to a greater or lesser degree, by increased numbers of cells expressing a-smooth muscle actin (a-SMA) and the accumulation of inflammatory cells [1–4]. In addition, increased numbers of endothelial cells, particularly in the intima (plexiform lesions) but also in the perivascular regions, with distinct functional characteristics have been described [5, 6]. However, neither the origin(s) of these functionally distinct cells nor the molecular mechanisms operating to cause their accumulation in any of the three compartments of the vessel wall have been fully elucidated. Traditionally, it has been thought that the a-SMA-­ expressing cells, accumulating in vascular lesions (both ­systemic and pulmonary), were exclusively derived from the resident vascular SM cells (SMCs) and/or from adventitial fibroblasts through the processes of “dedifferentiation” of the former or “differentiation” of the latter (Fig. 1). Over the past decade, however, this concept has been expanded by new experimental data suggesting many alternative sources of a-SMA-expressing cells (SM-like cells or myofibroblasts) in various vascular diseases, including pulmonary hypertension. For instance, the possibility that both epithelial and endothelial cells have the capability of transitioning into a mesenchymal, SM-like phenotype has been raised [7–9] (Fig. 1). Resident vascular progenitor cells have been demonstrated to exist within the vessel wall and, in response to certain stimuli, to expand and express myofibroblastic, endothelial, or even hematopoietic markers [10–12]. Multipotent resident lung vascular progenitors have also been demonstrated to reside within the Hoechstlow lung side population (SP) of cells [13, 14]. Bone-marrow-derived or circulating progenitor cells have also been shown to be recruited to sites of vascular injury and to assume both endothelial and SM-like phenotypes [15–17] (Fig.  1). The purpose of this chapter is to review the data supporting a contributory role for vascular progenitors [including endothelial progenitor cells (EPCs), SM progenitor cells (SPCs), and fibrocytes] in systemic and pulmonary vascular remodeling.

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Fig.  1  Potential origins of fibroblasts/myofibroblasts in the vessel wall. Several potential origins for tissue myofibroblast have been ­proposed: (1) in several organs, a-smooth muscle actin (a-SMA)expressing myofibroblasts are believed to originate from tissue resident fibroblasts; (2) in the vasculature, myofibroblasts may arise through dedifferentiation of resident smooth muscle cells (SMC); (3) epithelial cells can give rise to fibroblasts/myofibroblasts in the lung and other organs through a process of endothelial–mesenchymal transdifferentiation (EMT); (4) in the lung, endothelial-to-mesenchymal (EnMT) transition may provide another mechanism to generate myofibroblasts; (5) in various fibrotic lesions in tissue injury/repair processes, bone-marrowderived circulating progenitor cells are proposed to contribute to the pool of myofibroblasts. (From Hinz et al. [8]. Adapted with permission from the American Society of Pathologists and Bacteriologists)

2 Origin and Phenotypic Characterization of Endothelial, Smooth Muscle, and Fibroblast/Myofibroblast (i.e., Fibrocyte) Progenitors Currently, EPCs represent the most widely studied adult human progenitor cell subpopulation. Their discovery in 1997 by Asahara et al. marks a milestone in our ­understanding of the development of blood vessels in the adult [18]. These cells can be derived from the bone marrow and peripheral blood and have recently been reported to reside also in the vascular adventitia [19, 20]. Interestingly, the endothelium lining blood vessels may be composed of cells with progenitorlike characteristics [12, 21–23]. Presently, however, the definition of EPC remains controversial and is not yet consistent. Most commonly, marker combinations for ­identifying the putative circulating EPC comprise certain hematopoietic lineage markers such as CD133, CD34, vascular endothelial growth factor receptor (VEGFR)-2, Tie-2, and UEA-1 lectin [22–25]. However, recent studies have provided growing evidence supporting the inclusion of some circulating myeloid cells as functional EPCs, which can contribute to endothelial regeneration and ischemic or tumor angiogenesis. These cells are characterized by expression of CD14low; subpopulations

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include CD14+/CD34low, CD14+/VEGFR-2+, CD14+/VEGFR-2+/ CXCR2+, and CD14low/CD16+/Tie-2+ [26, 27]. Thus, it seems that circulating pools of functional EPCs correspond to a rather heterogeneous cell population of multiple origins, phenotype, and tissue distribution. The common features of these cells, arguably, encompasses the expression of progenitor and endothelial markers (i.e., CD34, VEGFR-2, CD31, vascular endothelial cadherin, von Willebrand factor), colony-forming capacity, and differentiation toward the endothelial lineage with formation of angiogenic structures in  vitro as well as in vivo [23, 26]. The function of these EPCs in vivo is not known and may depend on the interplay with inflammation and the tissue microenvironment. Investigators have also explored the growth properties and colony-forming-unit potential of these cells, which has been found to be useful as a biomarker for outcomes in acute lung injury, as well as in cardiovascular diseases, including pulmonary hypertension [28–30]. Another vascular progenitor subtype, the SPC, has not been studied as intensively as the EPC. It has been shown that, similar to the EPCs, these progenitor cells can reside in the bone marrow, can circulate, or can be found in peripheral tissues. Circulating SPCs can be distinguished by expressing markers of mesenchymal/SM lineage such as endoglin (CD105), a-SMA, calponin, SM myosin heavy chain, SM22, and platelet-derived growth factor (PDGF) receptor-b (PDGFR-b) [31–33]. Bone-marrow-derived cells expressing SM markers have been observed in the remodeled intima of patients who have undergone sex-mismatched bone marrow transplants [16, 34]. Further, in a murine model, a subpopulation of sorted c-Kit-/Sca-1+/Lin-/PDGFR-b+ cells was reported to acquire the phenotype of mature SMCs in the presence of PDGF-BB [33]. Interestingly, similar to EPCs, human myeloid CD14+ SPCs have also been recently identified as a circulating CD14+/CD105+ subpopulation [32]. Another cell type that has received much attention as a potential vascular progenitor is the fibrocyte. Fibrocytes are bone-marrow-derived mesenchymal progenitors that coexpress hematopoietic stem cell (HSC) antigens, markers of the monocyte lineage and fibroblast products [35–38]. These cells produce extracellular matrix (ECM) components as well as ECM-modifying enzymes and can further differentiate into myofibroblasts both in  vitro and in  vivo under permissive microenvironmental conditions [36]. Fibrocytes express the common leukocyte antigen CD45 as well as monocyte markers CD11a and CD11b, and are variably reported to express CD14 and CD34. Upon stimulation, these cells express type 1 collagen, fibronectin, vimentin, and matrix metalloproteinase-9. This expression pattern has led several investigators to hypothesize that fibrocytes, like some dendritic cell subsets, derive from precursors of the monocyte lineage. In fact, these cells express the major histocompatability complex class I and class II and the costimulatory molecules CD80 and

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CD86. Fibrocytes also exhibit antigen-presenting activity and activate both CD4 and CD8 T lymphocytes [36, 39]. The combination of collagen production and expression of CD45 (or one of the hematopoietic or myeloid antigens, CD11b, CD13, or CD34) is considered as a sufficient criterion to discriminate fibrocytes from leukocytes, dendritic cells, endothelial cells, and tissue resident fibroblasts in vivo and in vitro. Fibrocytes can be distinguished from circulating or tissue resident mesenchymal stem/stromal cells, because the latter cells express fibroblast products but do not express CD34 or CD45, or the monocyte markers [40]. Several factors affect the differentiation of fibrocytes from the CD14+ monocyte precursors into mature mesenchymal cells. PDGF, interleukin (IL)-4, and IL-13 promote differentiation of CD14+ mononuclear cells into fibrocytes [41]. By contrast, serum amyloid-P inhibits the differentiation of CD14+ mononuclear cells into fibrocytes [41, 42]. Recent studies also suggest that TH-1 derived cytokines such as IL-12 and interferon-g may inhibit fibrocyte differentiation. On the other hand, TH-2 products, such as IL-4 and IL-13, stimulate fibrocyte differentiation as does transforming growth factor b (TGF-b). Further differentiation into cells ultrastructurally, phenotypically, and functionally similar to mature fibroblasts and/or myofibroblasts is promoted by stimulation with TGF-b or endothelin [41]. The resulting cell population produces more collagen and fibronectin than the relatively immature fibrocyte. In certain circumstances, the fibrocyte begins to express a-SMA, while ­downregulating leukocyte marker expression, and assumes a myofibroblast phenotype.

In addition to a circulating source of EPCs and mesenchymal cells, there is increasingly good evidence suggesting that cells with progenitor characteristics exist within the vessel wall itself. Following the discovery of circulating EPCs, it was demonstrated that EPCs exist in the wall of human embryonic aorta, which can differentiate into mature endothelial cells and form vascular-like structures under in vitro conditions [43]. Further studies revealed the presence of a complete hierarchy of EPCs in the wall of human adult blood vessels as well as umbilical cord [44]. Recently, it was shown that large and medium-sized human arteries and veins in several organs contain EPCs in a distinct zone of the vascular wall (termed “vascular-wall-resident EPCs,” VW-EPCs), and that region was named the “vasculogenic zone” (Fig. 2). This zone is located between the outer media and the adventitial layers [12]. Interestingly, the VW-EPCs have been reported to differentiate not only into mature endothelial cells but also into hematopoietic and local immune cells such as macrophages. In the vessel wall these cells are CD34+ but CD31-, they also express VEGFR-2 and Tie-2. Only a few cells in this zone of the vascular wall are positive for the leukocytic antigen CD45. Other investigators have reported that non-EPCs reside in the adventitia of the vessel wall and are capable of giving rise to vascular wall cells including SMCs. Hu et al. reported that stem cell antigen-1 (Sca-1)+ cells with a potential to differentiate into SMCs reside in the aortic adventitia of adult mice [45]. Subsequent studies by Passman et al. confirmed the presence of Sca-1+ progenitor cells at the medial–adventitial border. In vivo these adventitial Sca-1+ cells do not express SMC marker

Fig. 2  Hypothetical scheme illustrating the concept of the “vasculogenic zone.” This vascular mural zone at the border between the media and adventitia contains endothelial progenitor cells (EPCs) and probably also multipotent mesodermal stem cells. EPCs present in this zone are

p­ roposed to differentiate into endothelial cells and form capillary-like sprouts from the vascular wall, whereas the multipotent mesodermal stem cells in this zone may serve as precursors of macrophages, fibroblasts, and SMCs. (Adapted from Zengin et al. [12] with permission)

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Fig. 3  The lung side population (SP) is characterized by CD45+ and CD4− subpopulations. Hoechst red versus Hoechst blue dot plots defined an SP. Lung SP cells were identified and represented less than 1% of the total viable lung cells on the basis of propidium iodide exclusion. Flow-cytometric analysis indicated that lung SP cells express CD45

proteins. They do express, however, transcription factors thought to be required for SMC differentiation, including serum response factor and myocardin family members, and in vitro they readily differentiate into SM-like cells. It is possible that these cells, when activated in response to injury, contribute to the accumulation of SM-like cells in the vessel wall [46]. Another source of resident lung vascular progenitors which may play a role in the pathogenesis of pulmonary arterial hypertension (PAH) is the Hoechstlow CD45- SP of cells. The term “side population” (SP) is based on the cytometric profile in which there is a side arm of cells protruding from the main Hoechst-stained population and is referred to as Hoechstlow (Fig. 3). Hoechst vital dye is taken up by live cells and fluoresces in red and blue when excited by a UV laser. An ABCG2 multidrug resistance (MDR) transporter mechanism allows the cells to pump out the dye, thus leading to a lower fluorescence intensity [47, 48]. ABCG2 as well as other MDR transporters are believed to be a hallmark of primitive cell types, HSCs, and cancer cells [47–52]. The

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murine lung SP (Hoechstlow CD45-) has been isolated and characterized in vitro as an enriched tissue-specific source of organ-specific pulmonary precursors having mesenchymal stem cell properties as well as both endothelial and epithelial lineage potential [13, 14, 53–56]. The origin of the lung SP was examined by bone marrow transplantation analyses and it was found that the CD45+ fraction was derived from bone marrow, whereas the origin of the CD45- population is undefined [56]. These studies form the basis for the identification of the SP, or ABCG2, as a resident adult-tissue-­specific stem cell marker. The vascular potential of the lung SP is defined in vitro by the ability of the cells to express vascular endothelial cadherin, bind isolectin B4, take up diLDL, and to form angiogenic tubes in Matrigel [13]. More recent studies defined the mesenchymal stem cell potential of this Hoechstlow CD45- SP in both a mixed population of cells as well as with single cell clones. These cells express high levels of telomerase relative to liver tissue, which does not decrease significantly over time [14]. They also express characteristic mesenchymal markers (CD44, CD90, CD105, CD106, CD73, and Sca-I) and lack any hematopoietic markers (CD45, c-Kit, CD11b, CD34, and CD14). The clonal analyses demonstrated that a single lung SP could assume the phenotype of mesenchymal lineages such as bone, cartilage, and fat [14]. Although the true role of these cells in vivo is yet to be determined, because this cell population has both vascular endothelial and mesenchymal stem cell characteristics, it is likely that the cells differentiate to endothelial, SM, and myofibroblast lineages depending on the microenvironment. Thus the lung SP may play a role in remodeling associated with pulmonary hypertension.

3 Contribution of Circulating Vascular Progenitors to Systemic Vascular Remodeling The contribution of circulating progenitor cells to vascular remodeling has been studied more extensively in systemic vascular disease than in pulmonary vascular disease. Many of these studies serve as the rationale for investigations in the pulmonary circulation and thus a brief review of this work is critical for a better understanding of progenitor cells in pulmonary vascular remodeling. As mentioned already, in contrast to the initial conventional assumption that damaged organs are repaired only by migration and proliferation of adjacent resident cells, accumulating evidence supports the idea that multifunctional progenitor cells are mobilized into the circulation and recruited specifically into sites of tissue regeneration. Many reports have demonstrated that bone-marrow-derived EPCs significantly contribute to neovascularization and re-endothelialization

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after acute vascular injury [18, 57]. Further, several animal and clinical studies have demonstrated that transplantation of autologous EPCs or unfractionated bone marrow cells is effective for the treatment of ischemic cardiovascular disease [17, 58]. On the other hand, there is increasingly good evidence demonstrating that bone marrow cells or circulating progenitor cells can participate not only in the maintenance or restoration of vascular homeostasis but also in the pathogenesis of various vascular diseases. The role of EPCs in vascular healing has been well demonstrated and recently reviewed and will not be discussed further [59, 60]. However, the role of progenitor cells in pathologic angiogenesis and intimal lesion formation is briefly reviewed here because of its relevance to the mechanisms involved in vascular remodeling in pulmonary hypertension. Angiogenesis has been implicated in the pathogenesis of a variety of systemic disorders, including diabetic retinopathies, tumors, rheumatoid arthritis, and cirrhosis [61, 62]. EPCs have been shown to contribute to the pathologic angiogenesis observed in these conditions [10, 63, 64]. Tumor angiogenesis, in particular, is associated with the recruitment of hematopoietic and circulating EPCs [65]. In fact, experimental evidence has demonstrated that hematopoietic progenitors expressing VEGFR-1 are required for regulation of tumor metastasis [66]. Along these lines, injection of bone marrow cells promoted injury-associated retinal angiogenesis [67]. Further, endothelial cells, that accumulate in neointimal lesions in allografts and originate from circulating and bone-marrow-derived progenitors are responsible for the formation of microvessels in the setting of transplant atherosclerosis [10]. These observations raise the possibility that transplantation of EPCs or bone marrow cells may, under certain circumstances, promote tumor formation, diabetic retinopathy, and atherosclerosis by augmenting ­disease-associated pathologic angiogenesis. However, some clinical studies have demonstrated that the number of circulating EPCs correlates inversely with risk factors for coronary artery disease [29]. In addition, the levels of circulating EPCs have been reported to predict the occurrence of cardiovascular events [68]. These observations prompted some investigators to suggest that physiologic levels of circulating EPCs in fact function to prevent atherosclerosis without promoting unfavorable angiogenesis. The observations that most cells in neointimal lesions express certain SM markers (typically a-SMA [69]) led to the general assumption that SMCs in the adjacent medial layer migrated into the subendothelial space, proliferated, and synthesized ECM, thereby contributing to neointimal formation [70]. However, many recent pathology studies have demonstrated that neointimal SM-like cells are phenotypically distinct from medial SMCs. For example, unlike SMCs in the normal vascular media that express a differentiated contractile phenotype, neointimal SM-like cells are

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characterized by a large number of synthetic and secretory organelles. These “synthetic” SM-like cells secrete ECM components and express lower levels of SM-specific contractile proteins [71]. These synthetic SM-like cells proliferate and migrate significantly in response to various growth factors present in the injured vessel wall [72]. In fact, much effort has been devoted to understanding the regulators of vascular SM phenotype that control the transition from a quiescent differentiated SMC, under normal conditions, to a proliferative dedifferentiated SM-like cell in the presence of pathologic stimuli. Intriguingly, however, it was reported that some neointimal SM-like cells express a number of hematopoietic lineage makers along with certain SM markers [73]. Macrophage-like SM-like cells with phagocytic activity have been obtained from human arteries [74]. Although it has been assumed that resident medial SMCs “transdifferentiate” into macrophage-like cells, recent studies have demonstrated that some “synthetic” SM-like cells with characteristics of macrophages are derived from circulating blood cells rather than from medial SMCs [75, 76]. Taking advantage of both genetically modified mice as well as the use of a chimeric mouse model of parabiosis, recent studies demonstrate convincingly that, in a variety of systemic vascular injuries, bone-marrow-derived cells home to the damaged vessels and contribute to both vascular repair and pathologic remodeling by differentiating into cells expressing mesenchymal or SMC characteristics [20, 77]. In addition to EPCs and SPCs, fibrocytes have also been described as being present in the diseased systemic vessel wall. Fibrocytes (procollagen-1+/CD34+) have recently been identified in the fibrous cap of human atherosclerotic lesions [78, 79]. Moreover, subendothelial a-SMA+ myofibroblasts coexpressing CD68 or CD34 have been found in lipid-rich areas of the atherosclerotic intima in human aorta and in the fibrous cap of human carotid arteries, suggesting that these cells are ­fibrocytes [78, 80]. Further, one of the subsets of monocytes that are preferentially recruited into the arterial wall during experimentally induced arteriosclerosis in mice represents the murine counterpart of the human inflammatory subset of CD14+/CD16- mononuclear cells that express the monocyte chemoattractant protein 1 (MCP-1) receptor CCR2 [81]. These observations are consistent with elegant new studies by Varco et al. demonstrating that the intimal hyperplasia in an ovine carotid artery patch graft model is partially due to hematopoietic circulating progenitor cells, fibrocytes, that acquire mesenchymal features as they mature at the site of injury [82]. Thus, a CD14+/CD16-/CCR2+ subpopulation may be involved in atherogenesis and may contain fibrocyte precursors that contribute to lesion formation. Collectively, these observations are supported by epidemiologic data showing that MCP-1 plays a major role in the pathogenesis of atherosclerosis and disease progression and may promote recruitment of fibrocytes to the vessel wall [83].

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4 Contribution of Circulating Vascular Progenitors to Pulmonary Vascular Remodeling 4.1 Animal Studies On the basis of observations in systemic vascular disease, Davie et al. were among the first to examine the possibility that circulating progenitor cells contribute to pulmonary vascular remodeling [84]. They tested the hypothesis that hypoxia would stimulate mobilization of bone-marrow-derived c-Kit cells (c-Kit is a transmembrane tyrosine kinase receptor for stem cell factor and a generally accepted marker for bone-marrowderived HSCs) into the circulation, and create an environment, specifically in the pulmonary artery, that facilitates circulating c-Kit cell adhesion and thus an increase in the number of c-Kit cells within the remodeled pulmonary artery. The study demonstrated that chronic hypoxia significantly increased the number of c-Kit cells in the circulation and decreased their numbers in the bone marrow, thus supporting the previously reported idea that ischemic stimuli lead to the mobilization of bonemarrow-derived stem cells [85]. Of relevance is the fact that genes regulated by hypoxia, including erythropoietin and vascular endothelial growth factor (VEGF), have been implicated in the generation and differentiation of hemangioblasts, the precursors of both HSCs and primitive endothelial cells [86]. Moreover, VEGF has been shown to mediate mobilization of bone-marrow-derived HSCs and EPCs, which promotes tumor blood vessel growth [65]. Davie et al. also identified a greater ­number of c-Kit cells in the remodeled adventitia as well as in the expanding vasa vasorum of hypoxic pulmonary arteries compared with the relatively acellular pulmonary adventitia of normoxic animals (Fig. 4). Further, it was shown that the hypoxic pulmonary artery wall exhibits elevated expression of proteins that serve critical roles in recruitment, retention, and differentiation of progenitor cells (such as VEGF, fibronectin, and thrombin osteopontin). Each of these molecules has been implicated in inflammatory cell responses, including adhesion,

Fig.  4  Identification of c-Kit cells in the hypoxic pulmonary artery. Immunohistochemistry (brown peroxidase signal) revealed a greater number of c-Kit cells (arrows) in the vessel wall of distal pulmonary artery from hypoxic animals compared with the control. The c-Kit cells were localized contiguous to, and within, the vasa vasorum in the adventitia. (Adapted from [84] with permission)

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migration, division, and differentiation of multiple cell types. Indirect evidence that the c-Kit cells might contribute to the expansion of the vasa vasorum and accumulation of fibroblasts/ myofibroblasts in the vessel wall was provided by in  vitro observations that peripheral blood mononuclear cells derived from hypoxic calves exhibited the potential to differentiate into endothelial as well as SM-like cell phenotypes, depending on the culture conditions. Shortly after publication of these data came other reports supporting the idea that circulating progenitor cells contribute to remodeling in various models of pulmonary hypertension. Hayashida et  al. used green fluorescent protein (GFP)–bone marrow transplant chimeric mice to investigate the possible role of bone-marrow-derived cells in hypoxiainduced pulmonary vascular remodeling [87]. These investigators demonstrated that hypoxia induced the recruitment of significant numbers of GFP+ bone-marrow-derived cells into the pulmonary artery wall, including the adventitia. Very few cells were observed in the control mice. The investigators quantified the number of GFP+ cells that coexpressed a-SMA in the lung tissues of both control and pulmonary hypertensive mice and found significant increases in the numbers of GFP+/a-SMA+ cells in the pulmonary media and adventitia. They found that the number of GFP+/a-SMA+ cells increased with time in parallel with the progression of pulmonary hypertension. Collectively, the Davie et  al. and Hayashida et  al. studies provide evidence that bone-marrow-derived cells, which are mobilized and accumulate in the pulmonary arteries, contribute to the pulmonary vascular remodeling in hypoxia-induced pulmonary hypertension. Subsequently, additional reports have emerged ­supporting a role for specific progenitor cells, including fibrocytes and bonemarrow-derived mesenchymal progenitors, in the pulmonary vascular remodeling process. Circulating fibrocytes have been implicated in the pathogenesis of lung fibrosis in several mouse models, including irradiation- and bleomycin-induced lung injury [88, 89]. Fibrocytes have also been shown to contribute to the subepithelial fibrosis observed in the airways of mice with experimentally induced asthma [90]. In experimental animal models (rat and neonatal calf) of pulmonary hypertension, Frid et al. reported that exposure of these animals to chronic hypoxia led to robust accumulation of fibrocytes in the adventitia of pulmonary but not systemic vessels [15]. Using confocal microscopy, the investigators found that at least 40% of the monocyte-like cells accumulating in the pulmonary artery adventitia coexpressed the leukocytic marker CD45 and the mesenchymal marker type 1 procollagen, consistent with a fibrocyte phenotype (Fig. 5). A significant, but lesser proportion of the cells coexpressed CD45 and a-SMA, thus indicating the transition of the recruited circulating fibrocytes toward the myofibroblast phenotype (Fig. 6). In vivo labeling of cir­c ulating monocytic cells with liposome-encapsulated 1,1¢dioctadecyl-3,3,3¢,3¢-tetramethylindocarbocyanine perchlorate

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Fig.  5  Hypoxia-induced pulmonary remodeling is characterized by monocyte/fibrocyte accumulation in the adventitia and media. (a) Sustained hypoxia induces a robust appearance of mononuclear CD45+ cells (indirectly labeled with red fluorochrome) in the pulmonary arterial adventitia (these lesions are marked by triple large arrowheads in the “hypoxia” columns). (b) A large proportion (around 40%) of

CD45+ leukocytic cells (red fluorescence) accumulating in the remodeled pulmonary arterial adventitia of hypoxic hypertensive calves coexpress type 1 procollagen (green fluorescence), suggestive of a fibrocyte phenotype. Scale bars 5 mm. (a From Frid et al. [15]. Adapted with permission from the American Society of Pathologists and Bacteriologists)

(a red fluorochrome) provided additional evidence that, in response to chronic hypoxic exposure, circulating cells were recruited specifically to pulmonary but not to systemic vessels, and consequently expressed fibroblast antigens (type 1 procollagen) in the remodeled pulmonary vessel wall. Most importantly, the investigators demonstrated a causal link between fibrocyte accumulation and vascular wall remodeling by showing that depletion of circulating monocytes/fibrocytes using two different strategies (gadolinium chloride encapsulated and chlodronate-encapsulated liposomes) abrogated hypoxiainduced perivascular remodeling in the rat model of hypoxic

pulmonary hypertension. These results are consistent with studies in other organ systems where inhibition of fibrocyte accumulation resulted in reduced collagen deposition and reduced accumulation of myofibroblasts [82, 91–93]. However, it should be noted that fibrocytes themselves are proinflammatory cells and produce a number of cytokines and growth factors that can induce angiogenesis, fibroblast hyperplasia, and release of ECM molecules from the resident tissue fibroblasts [94]. Therefore, the correlations between fibrocyte accumulation and tissue remodeling may also reflect the paracrine effects of these cells on resident vascular wall cells.

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Fig. 6  Hypoxia promotes the emergence of cells coexpressing leukocytic marker CD11b and the myofibroblast marker a-SMA. (a) A robust influx of CD11b+ cells (red) in the pulmonary arterial adventitia of chronically hypoxic rats correlates with a significant number of a-SMA+ myofibroblasts in that region. A adventitia, M media. (b) Double-label immunofluorescent staining for CD11b (red) and a-SMA (green), followed by deconvolution confocal microscopy analysis of the boxed area in a, demonstrates cells that coexpress both markers (arrowheads), or express only one of the markers (a-SMA, single arrow, or CD11b, triple arrows).

(c) Significant numbers of a-SMA+ myofibroblasts (brown staining in the adventitia, labeled A are present in distal pulmonary arteries of the chronically hypoxic calves. (d) Double-label immunofluorescent staining, followed by deconvolution confocal microscopy, demonstrate that some of the a-SMA+ myofibroblasts (green) coexpress the macrophage marker CD68 (red) (arrowheads). 4¢,6-Diamidino-2-phenylindole (DAPI) (blue) labels cell nuclei. M media, A adventitia. Scale bars 50 mm (a, c); 5 mm (b, d) (From Frid et al. [15]. Adapted with permission from the American Society of Pathologists and Bacteriologists)

Recent studies by Spees et  al. examined the potential of bone-marrow-derived progenitor cells to contribute to the repair and remodeling observed in the lung and heart during monocrotaline (MCT)-induced progressive pulmonary hypertension [95]. Female rats transplanted with GFP+ male (Y-chromosome+) bone marrow cells were used as a model. In control untreated GFP+–bone marrow transplant chimeric rats, approximately 15% of the cells in the lung were GFP+, whereas in the MCT-treated chimeric rats, the number of GFP+ cells increased to 35–45%. The pulmonary GFP+ cells in both cases were a mix of hematopoietic inflammatory and nonhematopoietic cells. A large number of the bone-marrow-derived GFP+ cells in the lung were fibroblasts and myofibroblasts and thus may have contributed to lung fibrosis following irradiation and MCT treatment. The possibility that cell fusion could have played a role in the differentiation of bone-marrowderived cells into mesenchymal cells was addressed. However, as indicted by fluorescent in situ hybridization

assays, the incidence of cell fusion in the lung was very low, ruling out this as a major contributing mechanism. Importantly, the investigators also showed the presence of bone-marrow-derived cells in the right ventricle of MCTtreated chimeric rats. Therefore, as in the case of many fibrotic diseases, these results identify the bone-marrow-derived progenitors as an important player in vascular disease and suggest that strategies aimed at the inhibition of the recruitment of these cells may constitute important ­therapeutic options in the treatment of pulmonary vascular disease.

4.2 Human Studies Newly emerging data in humans with pulmonary hypertension support a role for progenitor cells in the vascular remodeling that characterizes these patients. Patients with COPD often

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exhibit striking vascular remodeling in pulmonary muscular arteries in precapillary vessels. The hallmark of remodeling in this group of patients is the thickening of the intima due to the accumulation of SM-like cells. Peinado et al. presented evidence that CD133+ EPCs participate in this process [96]. They demonstrated increased numbers of CD133+ cells infiltrating the hyperplastic intima of pulmonary arteries very close to areas of denuded endothelium. The number of progenitor cells recruited correlated with the thickness of the arterial wall, suggesting a potential association with the severity of the remodeling process [96]. Subsequently, in vitro studies by the same group demonstrated that CD133+ cells may acquire the morphology and phenotype of not only endothelial cells but also of SM-like cells. CD133+ cells coincubated with isolated human pulmonary artery were shown to migrate into the intima and differentiate into SMCs. Progenitor cell differentiation was produced without fusion with mature SMCs. Thus, CD133+ cells exhibit a certain degree of plasticity with an ability to differentiate into both endothelial and SM-like cells, which re-enforces the idea regarding their potential role in pulmonary hypertension in COPD. It has also been reported recently that the numbers of apoptosis-resistant, highly proliferative circulating CD133+ cells increase in human idiopathic PAH (IPAH) [5, 97]. The investigators raised the possibility that these cells contributed to the intimal lesions observed in these patients. Importantly, a recent study by Majka et al. demonstrated increased numbers of CD133+ cells in and around the pulmonary vascular lesions in both IPAH and familial PAH (FPAH) [2] (Fig. 7). The increases in the numbers of CD133+ and CD45+ cells raised questions as to how these cells affect resident cell function and the remodeling process in general. One possibility is that cell–cell fusion events are present and contribute to the remodeling process. Cell fusion can result in genetic changes, which confer selective growth advantages as well as microsatellite instability, point mutations, allelic imbalance, and loss of heterozygosity [98–100]. The result of such changes is uncoupling from normal checkpoint apoptosis, germline mutation or hypermethylation, which could, in part, explain the presence of apoptosis-resistant cells in PAH. Majka et al. therefore analyzed ploidy in cells composing vascular lesions, since any cell fusion event would result in at least twice the chromosomal content (4N). The investigators did not detect any chromosomal content amplification in either IPAH or FPAH specimens, thus suggesting that genetic instability and proliferative advantage of cells in the hypertensive tissues were not the result of aneuploidy. Thus, it was speculated by the authors that if a neoplastic “switch” has occurred in cells that characterize the vascular lesions in PAH, it does not involve genes typical of those usually observed in lung cancer [101, 102]. The possibility that fibrocytes also were present in the remodeled vessel wall of patients with severe PAH was

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Fig. 7  CD133+ cells accumulate and proliferate in the pulmonary artery intima of pulmonary arterial hypertension patients. (a) Immunostaining with antibodies to a-smooth muscle actin (SMA) demonstrates accumulation of mesenchymal cells in the intima. (b) DAPI staining demonstrates total cell accumulation. (c) CD133+ cells compose a portion of the cells accumulating in the intima. (d) Ki67 immunostaining on the same section rephotographed demonstrated that the majority of proliferating cells were CD133+. High-magnification views of (c) and (d) are shown in (e) and (f), respectively

raised though not demonstrated as conclusively as was their presence and contribution to airway remodeling in patients with asthma [90].

5 Chemokine Signaling Involved in Progenitor Cell Recruitment Presently, the molecular mechanisms controlling the recruitment, retention, and differentiation of circulating progenitor cells into the vessel wall remain largely undefined. It is clear, however, that chemokines play an important role in the vascular remodeling process through guidance of circulating mononuclear cells to the injury site and activation of resident vascular cells [103, 104]. Considering the cell-type-specific

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expression of chemokine receptors and the substantial overlap in ligand–receptor specificity, an interactive network of chemokines and chemokine receptors has emerged which exhibits enormous plasticity in different types of injury/ repair processes. A growing number of chemokine/chemokine receptor pairs with confined effects in vascular diseases have been described [105]. For instance, in the systemic circulation, stromal-cell-derived factor-1 (SDF-1) has been shown to regulate vascular repair by CXCR4-dependent SPC recruitment, which contributes to the maladaptive response to injury [106]. Further, three distinct chemokine/chemokine receptor pairs (MCP-1/CCR2, RANTES/CCR5, and fractalkine/CX3CR1) have been shown to direct lesional leukocyte infiltration [107]. In addition, MCP-1/CCR2 and fractalkine/CX3CR1 increase expansion of neointimal SM-like cells [108]. Thus, to ultimately pharmacologically modulate maladaptative responses in arterial remodeling, it will be essential to identify specific chemokine/chemokine receptor pairs that play specific roles in the remodeling process. It is possible that these will be vessel- and diseasespecific and may, in fact, vary over time. Recent studies have begun to address specific chemokine/ chemokine receptor pairs involved in inflammatory/progenitor cell recruitment to the pulmonary arteries in models of pulmonary hypertension. An advancement in this area has come about through the use of laser capture microdissection (LCM) to evaluate vessel-specific changes, since it is becoming increasingly clear that analysis of the whole lung tissue may not accurately reflect specific changes in gene and protein expression in the vessel wall itself [109–111]. With use of the LCM technique, it was recently demonstrated that sustained hypoxia induced upregulation of a wide range of inflammatory mediators and growth-, differentiation-, adhesion-, and fibrosis-associated molecules in a pulmonary artery-specific fashion [112]. The study demonstrated upregulation of a number of chemokines and receptors, as well as adhesion molecules, which preceded the influx of ­inflammatory cells and the onset of perivascular fibrosis. These included CXCL12 (SDF-1)/CXCR4, vascular cell adhesion molecule 1, intercellular adhesion molecule 1, TGF-b, and 5-lipoxygenase. Interestingly, over time, the pulmonary artery inflammatory microenvironment became more complex, with the appearance of other cytokine, adhesion, growth and differentiation mediators (such as IL-6, MCP-1, and PDGF-BB), which was consistent with the increasing numbers and persistent presence of monocytes and other inflammatory cells in the perivascular regions. Importantly, upon removal of the hypoxic stimulus, expression of specific chemokine/ chemokine receptors and adhesion molecules rapidly returned to control levels, which preceded the disappearance of inflammatory cells and ultimately reversal of vascular remodeling and resolution of pulmonary hypertension. Similarly, in patients with IPAH increases in fractalkine, RANTES, and

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PDGF expression specifically within the pulmonary arterial wall have been observed [111, 113, 114]. Further work is essential to define more precisely the chemokine/chemokine receptor pairs involved in the pulmonary vascular remodeling process. This information will be critical to the development of new therapeutic interventions.

6 Summary In summary, substantial experimental evidence points to a role for circulating progenitor cells in the vascular pathology that characterizes chronic pulmonary hypertension. More work is needed to determine the types of progenitor cells involved in the vascular remodeling and their specific functions once they have taken up residence in the vessel wall. Much work is needed to determine the factors involved in their recruitment and retention. Additionally, work is needed to determine factors involved in controlling their proliferative and differentiation state. Work also needs to be done to address the role of resident lung and vascular progenitor cells in the remodeling process. Collectively, these experiments may yield opportunities for critical new treatment strategies.

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K.R. Stenmark et al. 74. Matsumoto K, Hirano K, Nozaki S et  al (2000) Expression of macrophage (Mj) scavenger receptor, CD36, in cultured human aortic smooth muscle cells in association with expression of peroxisome proliferator activated receptor-g, which regulates gain of Mj-like phenotype in vitro, and its implication in atherogenesis. Arterioscler Thromb Vasc Biol 20:1027–1032 75. Tanaka K, Sata M, Hirata Y, Nagai R (2003) Diverse contribution of bone marrow cells to neointimal hyperplasia after mechanical vascular injuries. Circ Res 93:783–790 76. Tanaka K, Sata M, Natori T et  al (2008) Circulating progenitor cells contribute to neointimal formation in nonirradiated chimeric mice. FASEB J 22:428–436 77. Iwata H, Sata M (2007) Potential contribution of bone marrowderived precursors to vascular repair and lesion formation: lessons from animal models of vascular diseases. Front Biosci 12:4157–4167 78. Medbury H (2007) Role of fibrocytes in atherogenesis. World Scientific, Singapore 79. Medbury HJ, Tarran SL, Guiffre AK et al (2008) Monocytes contribute to the atherosclerotic cap by transformation into fibrocytes. Int Angiol 27:114–123 80. Andreeva ER, Pugach IM, Orekhov AN (1997) Subendothelial smooth muscle cells of human aorta express macrophage antigen in situ and in vitro. Atherosclerosis 135:19–27 81. Tacke F, Randolph GJ (2006) Migratory fate and differentiation of blood monocyte subsets. Immunobiology 211:609–618 82. Varcoe RL, Mikhail M, Guiffre AK et al (2006) The role of the fibrocyte in intimal hyperplasia. J Thromb Haemost 4:1125–1133 83. McDermott DH, Yang Q, Kathiresan S et al (2005) CCL2 polymorphisms are associated with serum monocyte chemoattractant protein-1 levels and myocardial infarction in the Framingham Heart Study. Circulation 112:1113–1120 84. Davie NJ, Crossno JT Jr, Frid MG et al (2004) Hypoxia-induced pulmonary artery adventitial remodeling and neovascularization: contribution of progenitor cells. Am J Physiol Lung Cell Mol Physiol 286:L668–L678 85. Takahashi T, Kalka C, Masuda H et  al (1999) Ischemia- and cytokine-induced mobilization of bone marrow-derived endothelial progenitor cells for neovascularization. Nat Med 5:434–438 86. Ramirez-Bergeron DL, Simon MC (2001) Hypoxia-inducible factor and the development of stem cells of the cardiovascular system. Stem Cells 19:279–286 87. Hayashida K, Fujita J, Miyake Y et al (2005) Bone marrow-derived cells contribute to pulmonary vascular remodeling in hypoxiainduced pulmonary hypertension. Chest 127:1793–1798 88. Gomperts BN, Strieter RM (2007) Fibrocytes in lung disease. J Leukoc Biol 82:449–456 89. Lama VN, Phan SH (2006) The extrapulmonary origin of fibroblasts: stem/progenitor cells and beyond. Proc Am Thorac Soc 3:373–376 90. Schmidt M, Sun G, Stacey MA, Mori L, Mattoli S (2003) Identification of circulating fibrocytes as precursors of bronchial myofibroblasts in asthma. J Immunol 171:380–389 91. Haudek SB, Xia Y, Huebener P et al (2006) Bone marrow-derived fibroblast precursors mediate ischemic cardiomyopathy in mice. Proc Natl Acad Sci USA 103:18284–18289 92. Ishida Y, Kimura A, Kondo T et al (2007) Essential roles of the CC chemokine ligand 3-CC chemokine receptor 5 axis in bleomycininduced pulmonary fibrosis through regulation of macrophage and fibrocyte infiltration. Am J Pathol 170:843–854 93. Phillips RJ, Burdick MD, Hong K et al (2004) Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate fibrosis. J Clin Invest 114:438–446 94. Wang JF, Jiao H, Stewart TL, Shankowsky HA, Scott PG, Tredget EE (2007) Fibrocytes from burn patients regulate the activities of fibroblasts. Wound Repair Regen 15:113–121

56  Role of Progenitor Cells in Pulmonary Vascular Remodeling 95. Spees JL, Whitney MJ, Sullivan DE et  al (2008) Bone marrow progenitor cells contribute to repair and remodeling of the lung and heart in a rat model of progressive pulmonary hypertension. FASEB J 22:1226–1236 96. Peinado VI, Ramírez J, Roca J, Rodriguez-Roisin R, Barberà JA (2006) Identification of vascular progenitor cells in pulmonary arteries of patients with chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol 34:257–263 97. Asosingh K, Aldred MA, Vasanji A et al (2008) Circulating angiogenic precursors in idiopathic pulmonary arterial hypertension. Am J Pathol 172:615–627 98. Gao Q, Reynolds GE, Innes L et al (2007) Telomeric transgenes are silenced in adult mouse tissues and embryo fibroblasts but are expressed in embryonic stem cells. Stem Cells 25: 3085–3092 99. Niv Y (2007) Microsatellite instability and MLH1 promoter hypermethylation in colorectal cancer. World J Gastroenterol 13: 1767–1769 100. Yeager ME, Golpon HA, Voelkel NF, Tuder RM (2002) Microsatellite mutational analysis of endothelial cells within plexiform lesions from patients with familial, pediatric, and sporadic pulmonary hypertension. Chest 121:61S 101. Jacobsen BM, Harrell JC, Jedlicka P, Borges VF, Varella-Garcia M, Horwitz KB (2006) Spontaneous fusion with, and transformation of mouse stroma by, malignant human breast cancer epithelium. Cancer Res 66:8274–8279 102. Varella-Garcia M (2006) Stratification of non-small cell lung cancer patients for therapy with epidermal growth factor receptor inhibitors: the EGFR fluorescence in situ hybridization assay. Diagn Pathol 1:19 103. Charo IF, Ransohoff RM (2006) The many roles of chemokines and chemokine receptors in inflammation. New Engl J Med 354:610–621 104. Schober A (2008) Chemokines in vascular dysfunction and remodeling. Arterioscler Thromb Vasc Biol 28:1950–1959

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Chapter 57

Receptor Signaling in Pulmonary Arterial Hypertension Patricia A. Thistlethwaite, Robin N. Leathers, Xioadong Li, and Xiaoxue Zhang

Abstract  Research on the molecular basis of pulmonary arterial hypertension (PAH) caused by perturbations in receptor signaling in vascular endothelial and smooth muscle cells is beginning to yield novel approaches to future therapies for this disease. This chapter focuses on recent findings of the role of two different receptors – NOTCH3 and bone morphogenetic protein (BMP) receptor (BMPR) type 2 (BMPR-2) – in regulating vascular smooth muscle cell and endothelial cell behavior and phenotype, and discusses the potential role of ligand–receptor signaling in the genesis of PAH. Changes in the structure, function, and integrity of blood vessels are necessary for the pathogenesis of many diseases, including PAH. This disease is characterized by structural remodeling of small pulmonary arteries and arterioles, due to vessel thickening and luminal occlusion by vascular smooth muscle cell and endothelial proliferation. The vasculopathy seen in PAH is progressive, diffuse, and eventually results in obliteration of the distal pulmonary arterial tree. From a clinical point of view, PAH manifests itself as sustained elevation in pulmonary arterial pressures and pulmonary vascular resistance, leading to right-sided heart failure and death. Although several stimuli and conditions, such as hypoxia, fenfluoramine ingestion, collagen vascular disease, portal hypertension, and intracardiac left-to-right shunting, are associated with this disease [3], the exact mechanism of how the lung remodels its vascular architecture in the pulmonary artery bed in PAH is not known. Increasing evidence suggests that two types of receptors, NOTCH and BMPR, may each play an important role in the genesis of this disease. This emerging body of literature lays the groundwork for designing therapy based on the basic biology of the pulmonary vascular wall. Keywords  Notch signaling • Bone morphogenetic protein receptor • Signal transduction • Tyrosin kinase receptor • Smooth muscle cell recruitment • Endothelial cell

P.A. Thistlethwaite (*) Division of Cardiothoracic Surgery, University of California, San Diego, 200 West Arbor Drive, San Diego, CA 92103-8892, USA e-mail: [email protected]

1 Introduction Pulmonary arterial hypertension (PAH) constitutes a major and growing health burden around the world. In the USA alone, PAH afflicts approximately 100,000 people each year, and is the cause of death in 20,000 individuals [1]. Despite considerable treatment advances over the past several decades, it is a disease that continues to carry a poor prognosis, with impaired quality of life and premature death. The development of novel treatments for patients with PAH therefore remains a major priority. Research on the molecular basis of PAH caused by perturbations in receptor signaling in vascular endothelial and smooth muscle cells is beginning to yield novel approaches to future therapies for this disease. This chapter will focus on recent findings of the role of two different receptors – NOTCH3 and bone morphogenetic protein (BMP) receptor (BMPR) type 2 (BMPR-2) – in regulating vascular smooth muscle cell and endothelial cell behavior and phenotype, and discuss the potential role of ligand–receptor signaling in the genesis of PAH. Changes in the structure, function, and integrity of blood vessels are necessary for the pathogenesis of many diseases, including PAH. This disease is characterized by structural remodeling of small pulmonary arteries and arterioles, due to vessel thickening and luminal occlusion by vascular smooth muscle cell and endothelial proliferation [2]. The vasculopathy seen in PAH is progressive, diffuse, and eventually results in obliteration of the distal pulmonary arterial tree. From a clinical point of view, PAH manifests itself as sustained elevation in pulmonary arterial pressures and pulmonary vascular resistance, leading to right-sided heart failure and death. Although several stimuli and conditions, such as hypoxia, fenfluoramine ingestion, collagen vascular disease, portal hypertension, and intracardiac left-to-right shunting, are associated with this disease [3], the exact mechanism of how the lung remodels its vascular architecture in the pulmonary artery bed in PAH is not known. Increasing evidence ­suggests that two types of receptors, NOTCH and BMPR, may each play an important role in the genesis of this disease. This emerging body of literature lays

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the groundwork for designing therapy based on the basic biology of the pulmonary ­vascular wall.

2 Notch Signaling in the Pulmonary Vasculature 2.1 Overview of Notch Signaling Genes of the Notch family encode single-pass transmembrane receptors that transduce signals through cell–cell interactions (Fig. 1). In mammals, four Notch family receptors have been described: Notch1–Notch4 [4]. The extracellular domain of Notch family proteins contains up to 36 tandemly repeated copies of an epidermal growth factor (EGF)-like motif, involved in ligand interaction, and three juxtamembrane

Fig. 1  Structure of the Notch receptor. The Notch receptor is located at the cell surface as a heterodimer following protease cleavage (S1 cleavage) during protein maturation. The extracellular domain associates noncovalently with a membrane-tethered intracellular domain (ICD) and is composed of up to 36 epidermal growth factor (EGF)-like repeats and three cysteine-rich Lin-12 repeats. EGF repeats 11 and 12 are sufficient to mediate the interaction between Notch and its ligands. In the ICD, Notch has a region called “RAM” (RBPJ-associated module) followed by repeated structural motifs named “ankyrin repeats” (these mediate the action between Notch and CBF1), a transactivation domain (TAD) and a proline-, glutamine-, serine-, threonine-rich (PEST) domain. The PEST domain is involved in the degradation of Notch

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repeats known as Lin-12-Notch repeats which modulate ­interactions between the extracellular and the membranetethered domains [5]. The intracellular region of Notch includes seven ankyrin repeats flanked by nuclear localization signals, a proline-, glutamine-, serine-, threonine-rich (PEST) domain and a tranactivation domain [6, 7]. Notch receptors interact with single-pass transmembrane ligands on adjacent cells (paracrine) or the same cell (autocrine). This restricts the Notch pathway to regulating short-range intercellular interactions. Notch cell-bound ligands are encoded by the Jagged (Jag1, Jag2) and Delta-like (Dl1, Dl3, and Dl4) gene families [8, 9]. To date, in adult mammals, including humans, NOTCH ligands have been found to be principally expressed on vascular endothelium, although recent reports suggest that these ligands may also have ­limited expression on vascular smooth muscle cells [10, 11]. An important feature of Notch is that it acts, at the same time, as a transmembrane receptor and a transcription factor (2). After ligand binding, Notch receptors undergo a series of proteolytic events, including a final cleavage by a g-secretase enzyme, that lead to release of the intracellular domain (ICD) of these receptors. The Notch ICD fragment translocates to the nucleus because of the presence of nuclear localization signals located within it [12]. Once in the nucleus, the Notch ICD forms an active transcriptional complex with the DNA binding protein, Rbpj recombination signal binding protein for immunoglobulin kappa J region (Fig.  2) [13]. In the absence of the Notch ICD, Rbpj binds to specific DNA sequences in the regulatory elements of various target genes and represses transcription of these genes by recruiting histone deacetylases to form a corepressor complex. The Notch ICD displaces the histone deacetylase–corepressor complex from the Rbpj protein. Subsequently, the Notch ICD–Rbpj complex recruits the protein mastermind-like 1 (Maml1) and histone acetyltransferases (HAC), leading to the transcriptional activation of Notch target genes [14]. The key downstream genes of Notch signaling are the Hes (Hairy/Enhancers of Split) and Hrt (Hairy/Enhancers of Split-related) gene families, which, when activated by Notch, reduce expression of downstream transcriptional effectors such as Mash [15], MyoD1 [16], and myocardin [17], as well as cell-cycle regulatory proteins, p27kip1 and p21waf/cip1 [18, 19]. Notch signaling has also been found to directly modulate the expression of the platelet-derived growth factor-b receptor [20].

2.2 Notch3 Regulates Specification of Arterial Smooth Muscle Cells The Notch3 gene is expressed solely in vascular smooth muscle cells of arteries and arterioles, and is not seen in vascular smooth muscle cells of veins or venules. Clues as to the

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cells in Notch 3-/- mice were abnormally aggregated within the vessel wall. It is of interest that in arteries of Notch3-/mice which did not express arterial markers for smooth muscle cells (i.e., Smtn), normal expression of several endothelial cell arterial markers occurred, including that of ephrin-B2, connexin-40, Hes1, Hey1 (also known as Hrt1), and Hey2 (also known as Hrt2) [23]. These results suggest that in the systemic circulation the arterial identity of endothelial cells and vascular smooth muscle cells surrounding them is independently specified.

2.3 Studies Supporting a Role of Notch3 in Vascular Smooth Muscle Cell Proliferation and Dedifferentiation Fig. 2  The Notch signaling pathway. Ligands of the Jagged (Jag1 and Jag2) and Delta-like (Dll1, Dll3, and Dll4) families interact with Notch receptors (Notch1–Notch4) on an adjacent cell. The Notch receptor exists at the cell surface as a proteolytically cleaved heterodimer consisting of a large extracellular domain and a membrane-tethered intracellular domain. The receptor–ligand interaction induces two proteolytic cleavages – S2 cleavage is mediated by the ADAM metalloproteinase family protein TNFa-converting enzyme, and the subsequent S3 cleavage within the transmembrane domain is mediated by g-secretase – that free the Notch ICD from the cell membrane. The Notch ICD translocates to the nucleus, where it forms a complex with the Rbpj protein, displacing a histone deacetylase (HDAc)–corepressor (CoR) complex from the Rbpj protein. Components of an activation complex, such as mastermind-like 1 and histone acetyltransferases (HAc), are recruited to the ICD–Rbpj complex, leading to the transcriptional activation of Notch target genes (Hes and Hrt)

function of Notch3 have been revealed through the study of Notch3-null mice, which are viable and fertile [21, 22]. In the mouse, maturation of arterial vessels occurs from birth to postnatal day 28, with vascular smooth muscle undergoing changes both in morphology and orientation. This ­remodeling process is strongly impaired in Notch3-/- mice. Arteries of Notch3-/- mice are enlarged with a thinner vascular smooth muscle cell coat than is found in wild-type arteries [23]. Arterial defects were observed in all organs analyzed in homozygous Notch3 knockout mice, but were milder or absent in the major elastic arteries of the trunk, suggesting an important role of Notch3 in small resistance arteries. Morphologically, vascular smooth muscle cells of Notch3-/mice resembled smooth muscle cells surrounding veins in wild-type mice, with elongated cell shape and poorly oriented clusters of smooth muscle cells around lumens. Arterial smooth muscle cells from Notch3-/- mice were found to lack the arterial smooth muscle cell marker smoothelin (Smtn) [24], yet had generalized smooth muscle cells markers, such as myosin heavy chain (Myh11) and a-smooth muscle actin (Acta2) at birth. By the seventh postnatal day, smooth muscle

Several lines of evidence suggest that Notch3 signaling is crucial for maintenance of arterial vascular smooth muscle cell proliferative capacity. First, Notch3 expression is restricted to vascular smooth muscle cells of arteries, and is not seen in veins or other tissues [25]. Second, expression of this gene has been linked to modulation of vascular smooth muscle cells into an undifferentiated, proliferative state. Campos et al. [26]. have described the growth behavior of a constitutively expressing Notch3 ICD stable cell line generated from rat embryonic vascular smooth muscle cells in which the growth rate failed to decelerate at postconfluence. Notch3 ICD-induced proliferation was associated with decreased expression of the cell-cycle inhibitor p27kip. Overexpression of p27kip1 has been shown to result in a dosedependent rescue of the Notch-induced proliferative phenotype and exit from the cell cycle [18]. Similarly, when smooth muscle cells were forced to express Hrt1, a downstream effector of Notch3 signaling, they displayed postconfluence growth coincident with diminished levels of p21waf1/cip1 [19]. Sweeney et  al. [13, 27] found that NOTCH3 promotion of vascular smooth muscle cell proliferation was RBPJdependent. Although serum is known to stimulate vascular smooth muscle cell proliferation, it also upregulates expression of NOTCH target genes, HRT1 and HRT2. In addition, cultured vascular smooth muscle cells isolated from Hrt2 knockout mice have been shown to have a decreased proliferative capacity and diminished migratory response to platelet-derived growth factor [28]. Third, Notch3 signaling has been shown to repress terminal smooth muscle cell differentiation and maintenance of a contractile smooth muscle cell phenotype [17]. Forced expression of either the Notch ICD or Hrt2 in C3H10T1/2 fibroblasts can inhibit myocardindependent transcription of smooth-muscle-cell-restricted genes and activity of multiple smooth muscle cell transcriptional regulatory elements. In addition, expression of the constitutively active form of NOTCH3 ICD has been found

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to downregulate ACTA2, MYH11, calponin (CNN1), and SMTN in human aortic smooth muscle cells [29]. This effect was CBF1/RBPJ-dependent and was mediated by the NOTCH target genes HRT1, HRT2, and HRT3. Taken together, these data support a critical role of Notch signaling in repressing expression of genes encoding contractile proteins and promoting cell proliferation. The importance of NOTCH3 signaling in arterial vascular disease is further highlighted by the fact that its impairment is responsible for two congenital diseases that affect the vasculature, the cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) [30], and Alagille syndrome (AGS) [31]. CADASIL is a hereditary vascular degenerative disorder caused by mutations in the human NOTCH3 gene. This syndrome is characterized by arteriopathy that affects the small cerebral arteries and arterioles, leading to stroke and dementia in humans. In fact, CADASIL is the most common single gene disorder associated with ischemic stroke, but because it is underdiagnosed, its true prevalence is not known. The genetic defects in CADASIL are typically missense mutations, most of which translate into amino acid exchanges within the first five EGF-like repeats of the NOTCH3 ectodomain. Although the disease is characterized by the progressive degeneration of vascular smooth muscle cells, the downstream consequences of NOTCH3 mutation (whether mutations lead to gain or loss of NOTCH signaling) are not known. AGS is a congenital disorder caused by mutations in the human gene for Jagged 1 (JAG1). Although this syndrome is associated with abnormalities in the liver, heart, eye, and skeleton, the most frequent vascular anomalies in AGS patients are segmental and subsegemental pulmonary artery stenoses, contributing to the development of severe pulmonary hypertension. AGS is caused by JAG1 haploinsufficiency. Both CADASIL and AGS, which affect the NOTCH signaling pathway, manifest disease in mid- to small-sized arteries and arterioles in different target organs (brain and lung). To date, the causes of CADASIL and AGS remain insufficiently understood, so recent advancements toward understanding the role of NOTCH3 in the vasculature may allow new insight into the pathogenesis of these diseases.

2.4 Role of NOTCH3 in the Development of Pulmonary Arterial Hypertension Recently, NOTCH3 signaling has been studied in the context of PAH in humans as well as pulmonary hypertension models in rodents [32]. Biopsies from patients with end-stage PAH undergoing lung transplantation have been found to have higher levels of NOTCH3 messenger RNA (mRNA) and NOTCH 3 ICD protein compared with biopsies from

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patients undergoing lung resection for benign conditions. In a similar fashion, the lungs of mice with hypoxia-induced pulmonary hypertension and the lungs of rats with monocrotaline-induced pulmonary hypertension have been demonstrated to have elevated levels of Notch3 mRNA and Notch3 ICD protein compared with species- and strain-specific nonpulmonary hypertensive lungs. Notch3 mRNA levels were found to be higher in the lung, brain, heart, and kidney in animals exposed to 10% oxygen for 6  weeks (pulmonary hypertensive animals) compared with normoxic animals (nonpulmonary hypertensive animals). However, Notch3 ICD protein was only minimally detected in most organs, whereas lung levels of Notch3 ICD protein more than tripled in animals with hypoxia-induced pulmonary hypertension. A link has been found between NOTCH3 signaling and the magnitude of PAH in humans and pulmonary hypertension in animals [32]. NOTCH3 expression has been found to correlate with disease progression as measured by pulmonary vascular resistance in humans and pulmonary arterial pressures in rodents, making it a sensitive molecular marker for the severity of pulmonary hypertension. No difference in the expression of the other NOTCH proteins, NOTCH1, NOTCH2, or NOTCH4, has been found in the lungs of normotensive versus pulmonary hypertensive humans, rats, and mice. The level of HES5, a known downstream effector of NOTCH3 signaling, has also been shown to be increased in the lungs of humans with PAH and rodents with pulmonary hypertension. High levels of HES5 in lung tissue correlated with worsening disease severity. Studies using immunofluorescent staining have shown that NOTCH3 expression and HES5 expression are confined to vascular smooth muscle cells in pulmonary arteries and arterioles in the lung. These results established a link between NOTCH3 signaling and the magnitude of pulmonary arterial pressures in human PAH and in two animal models of pulmonary hypertension, and suggested that NOTCH3 and HES5 are sensitive ­markers for the severity of pulmonary hypertension. Recently, constitutive Notch3 ICD expression has been found to induce small pulmonary artery smooth muscle cell (sPASMC; isolated from vessels less than 1,500 uM in diameter) proliferation. Using an adenoviral transfection system, Li et al. [32] have shown that overexpression of Notch3 in human sPASMCs was associated with significantly increased growth rates at preconfluence as measured by cell count and 3[H] leucine incorporation. Knockdown of HES5 abolished the proliferative effect of Notch3 ICD in human subcultured sPASMCs. These results suggested that the upregulated NOTCH3–HES5 signaling pathway may play a role in the proliferation of human sPASMCs, and that inhibition of HES5 can attenuate this process. Recent observations suggest that enhanced NOTCH signaling through HES5 in PAH sPASMCs contributes to the ability of these cells to selectively proliferate and lose

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e­ xpression of markers of differentiated smooth muscle cells [32]. First, sPASMCs from patients with PAH have been shown to have lower levels of differentiated smooth muscle cell markers (SMTN and MYH11) compared with pulmonary vascular smooth muscle cells from nonpulmonary hypertensive patients. Second, sPASMCs from human lungs with PAH have been shown to have significantly shorter doubling times and higher rates of 3[H]leucine incorporation compared with vascular smooth muscle cells from non-PAH lungs. Inhibition of endogenous HES5 expression using small interfering (siRNA) has been shown to markedly attenuate PAH sPASMC proliferation and 3[H]leucine incorporation, suggesting that NOTCH signaling through HES5 may play a role in the development of pulmonary medial hypertrophy. Similarly, siRNA knockdown of endogenous HES5 in human pulmonary sPASMCs has also been shown to induce increased expression of smooth muscle cell markers, MYH11 and SMTN, over that seen in scramble-treated PAH sPASMCs. “Proof of concept” that Notch3 signaling is a requisite for the development of pulmonary hypertension has been the demonstration that mice with homozygous knockout mutation in Notch3 do not develop pulmonary hypertension in response to hypoxic stimulation [32]. Mice with homozygous deletion of the Notch3 allele lacking 2.5  kb of genomic sequence encoding EGF-like repeats 8–12 in the extracellular domain [21] have been found to have minimal expression of Hes5 in the lung in conditions of hypoxia and normoxia. In contrast, Notch3+/+ mouse lungs were found to have elevated levels of Notch3 ICD and Hes5 protein when the animals were subjected to 6 weeks of hypoxia compared to levels of these proteins seen in the lungs of normoxic mice. Notch3-/- mice were resistant to the development of pulmonary hypertension and did not develop elevated right ventricular pressures, right ventricular hypertrophy, muscularization of small pulmonary arteries, or small vessel pruning on pulmonary angiography when subjected to 6 weeks of hypoxia. In contrast, wild-type littermates manifested histological and clinical signs of ­pulmonary hypertension under the same conditions. These results suggested that Notch3 is a requisite for the development of hypoxic pulmonary hypertension in rodents. Recently, reversal of pulmonary hypertension in a rodent model has been demonstrated by treatment with an inhibitor of Notch3 cleavage/activation [32]. The g-secretase inhibitor N-[N-(3,5-diflurophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) has been shown to block the in vitro and in  vivo cleavage of Notch proteins to ICD peptides [33]. Chemical blockage of Notch3 cleavage (and thus activation) to Notch3 ICD using this drug has been shown to effectively treat and reverse hypoxic pulmonary hypertension in mice. In these experiments, mice were placed in 10% oxygen for 2 weeks and they started to develop pulmonary hypertension as expected. Subsequent to this, the animals were treated with a daily subcutaneous dose of DAPT from weeks 3 to 6 while they were in

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10% oxygen. Verification of inhibition of Notch3 cleavage into its active form in lung tissue was done by western blotting at weekly intervals during DAPT administration. Administration of DAPT reversed pulmonary hypertension induced by chronic hypoxia, as measured by right ventricular systolic pressures, muscularization and morphology of small pulmonary arteries, and pulmonary angiography. Sham-treated animals developed progressive medial thickening of small pulmonary arteries consistent with the usual pattern of pulmonary hypertension development in hypoxic animals, whereas DAPT-treated animals did not. Mice treated with DAPT had minimal elevations in right ventricular systolic pressures and had regression of right ventricular hypertrophy, further indicating that they were effectively treated for pulmonary hypertension.

2.5 Notch3: Conclusions The study of Notch signaling as it relates to smooth muscle cell growth and differentiation is in its infancy. Several key findings that suggest that NOTCH signaling may play a role in the pathogenesis of PAH: (1) high steady-state levels of NOTCH3 and HES5 are seen in the lungs of patients with PAH, (2) the levels of NOTCH3 and HES5 protein in the lung are predictive of the severity of PAH in humans and pulmonary hypertension in two rodent models of the disease, (3) Notch3 signaling is a requisite for the development of hypoxic pulmonary hypertension in mice, (4) Notch3 signaling induces sPASMCs into a proliferative phenotype, and (5) pulmonary hypertensive vascular disease in vivo can be prevented by treatment with a drug that blocks Notch signaling. These results suggest that inhibition and molecular targeting of the NOTCH3–HES5 axis in pulmonary vascular smooth muscle cells may be novel strategies for the treatment of PAH in the future.

3 BMP Signaling in the Pulmonary Vasculature 3.1  Overview of BMPR Signaling BMPs are the largest group of cytokines within the ­transforming growth factor-b (TGF-b) superfamily [34]. They were first identified as cellular products found in normal bone that promote ectopic bone formation and healing of fractures [35]. BMPs are known to regulate proliferation, apoptosis, and differentiation in multiple different cell types in vitro, as well as contribute to the embryological development of the mammalian cardiopulmonary system [36–38]. TGF-b superfamily type II receptors are active serine/threonine kinases. BMPs exert their effect through activation of BMPR type 1 (BMPR-1) and BMPR-2, which are

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located adjacent to each other on the cell surface and transduce intracellular signals through a Smad-dependent pathway. The affinity of BMPs (BMP-2, BMP-4, BMP-6, BMP-7, GDF-5, and GDF-6) for type 2 receptors is much weaker than for type 1 receptors [39]. BMP-6 demonstrates specificity for BMPR-2. BMPs bind with high affinity to a heterotetrameric type 1/type 2 receptor complex, including type 1 receptors, BMPR-1A (also called ALK-3), BMPR-1B (also called ALK-6), and ALK-1 [40]. Ligand specificity for different components of the BMP receptor complex may have functional significance in specific tissues where BMPR signaling occurs [41]. One of the paradigms of BMPR signaling is the observation that the biologic response to receptor activation is dependent on dosage as well as cellular and organ context. Whereas BMPR2 mutation is associated with heritable PAH, BMPR1A mutation causes familial juvenile colonic polyposis [42], and BMPR1B causes hereditary brachydactyly [43]. After ligand binding, BMPR-2 phosphorylates a glycine/serine-rich domain on the proximal intracellular portion of BMPR-1 [44]. Activated BMPR-1 initiate intracellular signaling by phosphorylating specific receptor-regulated R-Smad proteins (Smad1, Smad5, and/or Smad8) which assemble into heteromeric complexes with the common partner, Co-Smad: Smad4 [45]. These heteromeric complexes translocate efficiently to the nucleus, where they regulate the transcription of target genes (both in a positive and in a negative fashion) with nuclear binding factors [46]. BMPs can also signal via mitogen-activated protein kinase (MAPK) pathways [p38MAPK, p42/44MAPK (extracellular signal-regulated kinases 1/2, ERK1/2), c-Jun NH2-terinal kinase/stress-activated protein kinase, JNK/SAPK in a Smad-independent fashion [47, 48]. A major transcriptional target of Bmp signaling is the inhibitor of DNA binding (Id) family of proteins (Fig.  3) [49, 50]. Id proteins are thought to be involved in the proliferation–differentiation transition because they have the ability to control specific genes which regulate proliferation and cell-cycle progression in a variety of mature and embryonic lineages, including vascular smooth muscle and endothelium [51, 52]. Id proteins exert control of differentiation and proliferation by preventing transcription factors from binding DNA by direct physical interaction. Primary targets for Id proteins are the basic helix–loop–helix (bHLH) transcription factors which regulate cell-type-specific gene expression and the expression of cell-cycle regulatory genes [53]. Id acts primarily by sequestering the ubiquitously expressed bHLH proteins known as E proteins, preventing them from binding DNA alone or as heterodimers with tissue-restricted bHLH proteins [54]. As Id proteins lack a basic DNA-binding domain, heterodimers between Id and bHLH proteins cannot bind DNA, and Id proteins exert their effect through a dominant negative mode of regulation. Specific proliferative genes associated with Ids include the cyclin-dependent kinase (Cdk) 1 genes, p15, p16, and p21, as well as the Cdk ­inhibitor p21waf/cip1 [55, 56]. In addition to blocking the function of

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Fig. 3  Bone morphogenetic protein (BMPs) strongly induce Id1–Id3 expression in a Smad-dependent manner. Id proteins act as dominant negative inhibitors of basic helix–loop–helix (bHLH) transcription factors. Class A bHLH transcription factors are ubiquitously expressed; class B bHLh proteins show a tissue-restricted pattern of expression. bHLH proteins bind to specific DNA sequences called E-boxes. Dimerization of class A and class B bHLH proteins is essential for DNA binding and transcriptional activity. Id proteins bind to both class A and class B bHLH proteins and inhibit their ability to bind DNA, resulting in changes in cellular proliferation

bHLH proteins, Id proteins have also been shown to modulate the function of several non-bHLH proteins, including (1) the retinoblastoma tumor suppressor protein [57], (2) mouse Id-associated protein 1 (MIDA-1), a protein necessary for proliferation of erythroleukemia cells [58], and (3) ADD1/ SREBP-1, a leucine-zipper transcription factor controlling expression of liopgenesis genes [59], and Pax-2, Pax-5, and Pax-8 transcription factors [55]. BMP signaling has been shown to be regulated at multiple levels. These include (1) inhibitors of BMP binding (ligand traps – sequestration of the extracellular ligand) and/or signaling by proteins such as noggin, chordin, follistatin, and BAMBI, (2) modulation of the levels of expression of specific BMPs and availability of BMPR-1 on the cell surface for dimerizations with BMPR-2, (3) modulation of the levels of expression and activation of signaling of Smads and inhibitory Smads (Smad6 and Smad7), (4) degradation of Smads via

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Smad ubiquitination and Smurf regulatory ­factors, as well as by Smad phosphatases, and (5) extensive cross talk with other signaling pathways, such as Notch, serotonin, angiopoietin, p38MAPK, c-src, Rack-1, and the cytoskeletal-associated proteins Tctex-1 and Limk-1. Such diverse levels of regulation may be responsible for the pleiotropic effects of BMP signaling in different tissues, and may account for the diverse spectrum of human disease associated with BMPR mutations.

3.2 BMPR2 Mutations in Heritable Pulmonary Arterial Hypertension The field of pulmonary hypertension research was greatly expanded in 2000, when several groups found that mutations in BMPR2 were associated with the development of heritable PAH [60]. Heritable PAH is a rare (1:105–1:108) autosomal dominant disorder that has reduced penetrance of 10–20%, has a female-to-male ratio of 1.7:1, and demonstrates genetic anticipation with a worsening of disease in younger generations [61]. Subsequent to the discovery of BMPR2 mutations in heritable PAH patients, approximately 25% of sporadic idiopathic cases of PAH have been found to be associated with haploid mutations in BMPR2 [62]. A proportion of these are examples of heritable PAH in which the condition has not manifested itself in relatives because of low penetrance, whereas others are examples of new mutation. A recent study has found that patients with PAH and an identifiable mutation in BMPR2, regardless of family history, had a severer form of the disease, with clinical presentation at an earlier age with faster acceleration of declining hemodynamics. Patients with mutation in BMPR2 have been found to be less likely to respond to acute vasodilator testing in the setting of right-sided heart catheterization [63]. The BMPR2 gene comprises 13 exons and generates a polypeptide of 1,038 amino acids. BMPR-2 comprises four discrete functional domains including the extracellular ligand binding domain, a single-pass transmembrane domain, a serine/threonine kinase domain, and a large cytoplasmic tail (not seen in other BMPRs) (Fig. 4). Mutations in heritable PAH have been reported in all exons of the gene except for exon 13 [64]. More than 140 BMPR2 mutations have been identified in patients with heritable PAH. The mechanism by which mutations in BMPR2 affect signaling is heterogeneous and mutation-specific [65, 66]. Most (approximately 70%) BMPR2 mutations lead to haploinsufficiency through a process of nonsense-mediated RNA decay. The remaining missense mutations in functional domains of the receptor cause loss of receptor function, such as reduced trafficking of the mutant protein to the cell surface, inability to phosphorylate BMPR-1, and a dominant negative effect on wild-type receptor function in terms of Smad signaling [67]. Haploinsufficiency is thought to lead to a loss or attenuation of signaling via the

Fig.  4  Structure of BMP receptor type 2 (BMPR-2). BMPR-2 is a polypeptide of 1,038 amino acids, with four function domains including the extracellular ligand binding domain, a single-pass transmembrane domain, a serine/threonine kinase domain, and a large cytoplasmic tail. Substitution of cysteine residues in the ligand binding or kinase domain of BMPR-2 leads to reduced trafficking of the mutant to the cell surface. Non-cysteine mutations within the kinase domain of the BMPR-2 gene make the receptor unable to phosphorylate BMPR-1A or BMPR-1B. Missense mutations in the cytoplasmic tail region of the BMPR-2 gene render the receptor unable to transduce signals via Smads

Smad1/Smad5 pathway. In addition, genetic analysis has recently shown that an increasing number of families have BMPR2 mutation secondary to gene deletions and rearrangements [68, 69].

3.3 Role of BMPR2 Mutation in the Development of Pulmonary Hypertension: Animal Studies The BMP pathway plays a critical role in embryonic vascular development and atrioventricular valve development. Mouse embryos with homozygous mutation in Bmpr2 die in early gastrulation [70], whereas mouse embryos with homozygous mutation in Bmpr1a die at approximately 8 days of gestation during formation of the atrioventricular cushions [71]. Although heterozygous Bmpr2+/− mice are genotypically similar to humans with heritable PAH, these mice survive to adulthood with no apparent phenotypic deficit. Bmpr2+/− mice have been found to have no resting elevation in pulmonary arterial pressure under normal conditions [72]. However, more recent studies using the same mouse strain have shown some significance in right ventricular systolic pressure between

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Bmpr2+/− mice and wild-type littermates [73]. It is unknown why the lung histology of Bmpr2+/− mice containing a Bmpr2 mutation essentially the same as in humans does not recapitulate the histological characteristics seen in human PAH patients. Although hypoxia is commonly used as a mouse model system for pulmonary hypertension, Bmpr2+/− mice showed only mild elevations in right ventricular systolic pressures compared to wildtype littermates when exposed to 10% oxygen [72]. In contrast, when Bmpr2+/− mice were infused with serotonin [72] or induced to constitutively express IL-1b [74] in the lung, they developed elevated pulmonary arterial pressures compared with wild-type mice. These experiments suggested that Bmpr2 haploinsufficiency predisposes animals to develop elevated pulmonary arterial pressures when challenged with a second stimulating agent. The low penetrance of human heritable PAH also suggests that “two hits,” one genetic and one environmental, may be necessary for the development of this disease. Bmpr2 knockout mice studies have revealed conflicting results, and no genetically altered mouse line to date recapitulates a phenotype identical to human PAH. Bmpr2 knockdown mice, made with an siRNA technology that showed up to a 90% reduction in Bmpr-2 expression compared with normal mice, had no significant elevation in pulmonary arterial pressure [75]. Unfortunately, further reduction in Bmpr-2 levels as could be obtained with Bmpr2 hypomorphs or homozygous null mice leads to embryonic lethality [76]. Early embryonic lethality limits further Bmpr-2 dose reduction experiments to test phenotype in the adult lung. Recently, conditional deletion of Bmpr2 in pulmonary endothelial cells has been shown to cause pulmonary hypertension in subsets of mice by 7 months of age [77]. In addition, targeted gene delivery of Bmpr2 to pulmonary vascular endothelium was found to significantly reduce pulmonary arterial pressure and right ventricular hypertrophy in chronically hypoxic rats [78]. Conditional overexpression of a dominant negative kinase domain mutant Bmpr2 in vascular smooth muscle cells of adult mice has been found to increase pulmonary vascular remodeling and pulmonary hypertension [79]. In general, further studies are needed to examine the role of Bmpr signaling in pulmonary vascular endothelium versus pulmonary vascular smooth muscle. An animal model based on genetic mutation which mimics the human phenotype would clearly be of great benefit to the study of this disease.

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smooth muscle cells, vascular endothelial cells, or vascular adventitial cells, or combinations of these cell types. In the lung, BMPR-2 is highly expressed on vascular endothelium of pulmonary arteries [80], with lower expression seen in pulmonary artery smooth muscle cells and fibroblasts. The expression of BMPR-2 in the pulmonary arterial tree is markedly diminished in patients with BMPR2 mutations and in patients with PAH who do not harbor BMPR2 mutations. Similarly, phosphorylation of Smad1/Samd5 is also reduced in the pulmonary arterial wall of individuals with heritable and idiopathic PAH [81]. Reduced levels of BMPR-1A have also been found in the lungs of patients with different forms of nonheritable PAH [82]. In heritable PAH cases, the level of BMPR-2 in the lung has been found to be substantially lower than that predicted for haploinsufficiency, suggesting that additional factors may be necessary to reduce BMPR-2 levels below a threshold that triggers pulmonary vascular disease [83]. At a cellular level, BMP signaling produces different physiologic results in pulmonary vascular smooth muscle cells depending upon anatomic locations. For example, BMP2, BMP-4, and BMP-7 have been shown to inhibit serumstimulated proliferation [81] and induce apoptosis [84] of main or lobar pulmonary arterial smooth muscle cells in a Smad1-dependent fashion. However, in smooth muscle cells isolated from 1–2-mm-diameter pulmonary arteries, BMP-2, BMP-4, and BMP-7 were found to stimulate proliferation [81]. The proliferative effect of BMPs on smooth muscle cells in small pulmonary vessels was ERK1/2- and p38MAPK-dependent. These conflicting results suggest that the physiological effect of BMP signaling differs depending on smooth muscle cell location within the pulmonary vascular tree. Studies of how BMP stimulation affects pulmonary vascular endothelium suggest that it plays a role in endothelial proliferation and resistance to apoptosis. Endothelial cells in vitro proliferate and form tubular structures in response to Bmp-4 and Bmp-6 stimulation [85]. This phenomenon is dependent on Smad1/Smad5 phosphorylation and the subsequent transcriptional activation of Id genes. In addition, siRNA attenuation of BMPR2 increases the susceptibility of pulmonary artery endothelial cells to apoptosis, whereas BMP stimulation protects endothelial cells from apoptosis [86].

3.5 New Directions with BMPR-2 Signaling 3.4 Role of BMPR2 Mutation in the Development of Pulmonary Arterial Hypertension: Cellular Studies To date, it is unknown whether mutations in BMPR2 cause the development of PAH through its effect on vascular

Although most studies addressing BMPR-2 in the pulmonary vasculature have focused on (1) the effect of BMP signaling through downstream pathways including Smad/ID, ERK, and MAPK, (2) its physiologic effect on vascular endothelial and smooth muscle cells, and (3) its role when mutated in human and rodent disease, there is new evidence to suggest

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that BMPR mutation may influence other pathways and work on other cell types. Recently, Long et  al. have argued that one possible consequence of reduction in BMPR-2 signaling is a deleterious increase in TGF-b signaling [87]. This may occur as a consequence of cross talk between BMP- and TGF-b-activated Smad signaling pathways. BMP/Smad1 signaling and TGF-b/Smad3 signaling have been shown to exert opposite and antagonistic effects on cell function in endothelial cells and in the process of epithelial to mesenchymal transition [88]. In support of this view that decreased BMP signaling modulates TGF-b signaling are three observations. First, pulmonary artery smooth muscle cells isolated from patients with PAH demonstrate an abnormal proliferative response to TGF-b [89]. Second, increased TGF-b/ Smad2 signaling has been noted in lung tissue from individuals with PAH [90]. Third, inhibition of TGF-b signaling via activin-receptor-like kinase-5 prevents development and progression of pulmonary hypertension in a rodent monocrotaline model [87]. Further studies will be needed to clarify the reciprocal relationship between reduction in BMP signaling and a gain in TGF-b signaling in human and rodent studies of pulmonary hypertension.

4 Linking BMPR and Notch Signaling Pathways An emerging body of literature now suggests that there is extensive cross talk, reciprocal regulation, and signal integration between Bmpr and Notch signaling pathways (reviewed in [91, 92]). Notch and TGF-b/Bmpr signaling converge in the regulation of a number of developmental processes, including myogenic, endothelial, pancreatic, and neuronal differentiation. Both synergy and antagonism have been found between the Bmp and Notch signaling pathways. Activation of Bmp signaling has been found to lead to enhanced activation of the Notch target gene Herp2 (Hrt1) through interactions between the intracellular Bmp mediator Smad1 with Notch ICD [93]. Smad1 was found to bind to Notch ICD and act as a coenhancer to stimulate transcription of the Notch downstream gene Hrt1. Interestingly, Hrt1 was then found to bind to and efficiently induce degradation of Id1, a downstream effector of Bmp signaling. The physical interaction between Notch ICD and Smad1 has also been demonstrated by coimmunoprecipitation and glutathione aid S-transferase (GST) pull-down experiments [94]. Protein– protein interactions between Id2 (in Bmpr/Id signaling) and Hes1 (in Notch signaling) have also been demonstrated in the chicken hindbrain to be key regulators of expression of genes involved in neurogenesis [95]. Dahlqvist et  al. have found that Bmp-4-induced inhibition of myogenic differentiation is abolished by use of either a dominant negative Rbpj

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protein or inhibition of Notch receptor cleavage, and have provided evidence that Bmp mediation requires functional Notch signaling [96]. Of note, Kennard et al. [97] have shown that Notch3 blocks TGF-b1-dependent differentiation of mouse fibroblasts to a smooth muscle cell phenotype; whereas in a reciprocal fashion, TGF-b1 causes a decrease in Notch3 expression which is dependent on both Smad4 and p38MAPK. Finally, functional interaction between TGF-b and Notch signaling has been demonstrated in the regulation of Hes1 in myogenic cells [97]. These complex feedback loops provide evidence that Notch signaling, in the context of different cell types and different organs, may modulate downstream Bmpr signaling or vice versa. Further research will be needed to address the possible levels of regulation between these two signaling systems in the pulmonary vasculature in the context of pulmonary hypertensive vasculopathy.

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57  Receptor Signaling in Pulmonary Arterial Hypertension 56. Peng Y, Kang Q, Luo Q, Jiang W, Si W, Liu BA et al (2004) Inhibitor of DNA binding/differentiation helix-loop-helix proteins mediate bone morphogenetic protein-induced osteoblast differentiation of mesenchymal stem cells. J Biol Chem 279:32941–32949 57. Lasorella A, Noseda M, Beyna M, Iavarone A (2000) Id2 is a retinoblastoma protein target and mediates signalling by Myc oncoproteins. Nature 407:592–598 58. Inoue T, Shoji W, Obinata M (1999) MIDA1, an ID-associating protein, has two distinct DNA binding activities that are converted by the association with Id1: a novel function of Id protein. Biochem Biophys Res Commun 266:147–151 59. Moldes M, Boizard M, Liepvre XL, Feve B, Dugail I, Pairault J (1999) Function antagonism between inhibitor of DNA binding (Id) and adipocyte determination and differentiation factor 1/sterol regulatory element-binding protein-1c (ADD1/SREBP-1c) transfactors for the regulation of fatty acid synthase promoter in adipocytes. Biochem J 344:873–880 60. Lane KB, Machado RD, Pauciulo MW, Thomson JR, Phillips JA, Loyd JE et al (2000) Heterozygous germline mutations in BMPR2, encoding a TGF-b receptor, cause familial primary pulmonary hypertension. The International PPH Consortium. Nat Genet 26:81–84 61. Loyd JE, Butler MG, Foroud TM, Conneally PM, Phillips JA, Newman JH (1995) Genetic anticipation and abnormal gender ratio at birth in familial primary pulmonary hypertension. Am J Respir Crit Care Med 152:93–97 62. Thomson J, Machado R, Pauciulo M, Morgan N, Humbert M, Elliot G et al (2000) Sporadic primary pulmonary hypertension is associated with germline mutations of the gene encoding BMPR-II, a receptor member of the TGF-b family. J Med Genet 37:741–745 63. Rosenweig EB, Morse JH, Knowles JA, Chada KK, Khan AM, Roberts KE et al (2008) Clinical implications of determining BMPR2 mutation status in a large cohort of children and adults with pulmonary arterial hypertension. J Heart Lung Transplant 27:668–674 64. Machado RD, Eickelberg O, Elliott CG, Geraci MW, Hanaoka M, Loyd JE et al (2009) Genetics and genomics of pulmonary arterial hypertension. J Am Coll Cardiol 54:S32–S42 65. Rudarakanchana N, Flanagan JA, Chen H, Upton PD, Machado RD, Patel D et al (2002) Functional analysis of bone morphogenetic protein type II receptor mutations underlying primary pulmonary hypertension. Hum Mol Genet 11:1517–1525 66. Nishihara A, Watabe T, Imamura T, Miyazono K (2002) Functional heterogeneity of bone morphogenetic protein receptor-II mutants found in patients with primary pulmonary hypertension. Mol Biol Cell 13:3055–3063 67. Machado RD, Aldred MA, James V, Harrison RE, Patel B, Schwalbe EC et al (2006) Mutations of the TGF-b type II receptor BMPR2 in pulmonary arterial hypertension. Hum Mutat 27:121–132 68. Aldred M, Vijayakrishnan J, James V, Soubrier F, Gomez-Sanchez M, Martensson G et al (2006) BMPR2 gene rearrangements account for a significant proportion of mutations in familial and idiopathic pulmonary arterial hypertension. Hum Mutat 27:212–213 69. Cogan JD, Vnenak-Jones CL, Phillips JA, Land KB, Wheeler L, Robbins IM et al (2005) Gross BMPR2 gene rearrangements constitute a new cause for primary pulmonary hypertension. Genet Med 7:169–174 70. Beppu H, Kawabata M, Hamamoto T, Chytil A, Minowa O, Noda T et al (2000) BMP type II receptor is required for gastrulation and early development of mouse embryos. Dev Biol 221:249–258 71. Kaneko K, Li X, Zhang X, Lamberti JJ, Jamieson SW, Thistlethwaite PA (2008) Endothelial expression of bone morphogenetic protein receptor type 1a is required for atrioventricular value formation. Ann Thorac Surg 85:2090–2098 72. Long L, MacLean MR, Jeffery TK, Morecroft I, Yang X, Rudarakanchana N et al (2006) Serotonin increases susceptibility to pulmonary hypertension in BMPR2-deficient mice. Circ Res 98:818–827

835 73. Beppu H, Ichinose F, Kawai N, Jones RC, Yu PB, Zapol WM et al (2004) BMPR-II heterozygous mice have mild pulmonary hypertension and an impaired pulmonary vascular remodeling response to prolonged hypoxia. Am J Physiol Lung Cell Mol Physiol 287:L1241–L1247 74. Song Y, Jones JE, Beppu H, Keaney JF, Loscalzo J, Zhang YY (2005) Increased susceptibility to pulmonary hypertension in heterozygous BMPR2-mutant mice. Circulation 112:553–562 75. Liu D, Wang J, Kinzel B, Mueller M, Mao X, Valdez R et al (2007) Dosage-dependent requirement of BMP type II receptor for maintenance of vascular integrity. Blood 110:1502–1510 76. Delot EC, Bahamonde ME, Zhao M, Lyons KM (2003) BMP signaling is required for septation of the outflow tract of the mammalian heart. Development 130:209–220 77. Hong K, Lee JL, Lee E, Park SO, Beppu CH, Li E et  al (2008) Genetic ablation of the Bmpr2 gene in pulmonary endothelium is sufficient to predispose to pulmonary arterial hypertension. Circulation 118:722–730 78. Reynolds AM, Xia W, Holmes MD, Hodge SJ, Danilov S, Curiel DT et al (2007) Bone morphogenetic protein type 2 receptor therapy attenuates hypoxic pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 292:L1182–1192 79. West J, Fagan K, Steudel W, Fouty B, Lane K, Harral J et al (2004) Pulmonary hypertension in transgenic mice expressing a dominant-negative BMPRII gene in smooth muscle. Circ Res 94:1109–1114 80. Atkinson C, Stewart S, Upton PD, Machado R, Thomson JR, Trembath RC et  al (2002) Primary pulmonary hypertension is associated with reduced pulmonary vascular expression of type II bone morphogenetic protein receptor. Circulation 105:1672–1678 81. Yang X, Long L, Southwood M, Rudarakanchana N, Upton PD, Jeffery TK et al (2005) Dysfunctional Smad signaling contributes to abnormal smooth muscle cell proliferation in familial pulmonary arterial hypertension. Circ Res 96:1053–1063 82. Du L, Sullivan CC, Chu D, Cho AJ, Kido M, Wolf PL et al (2003) Signaling molecules in nonfamilial pulmonary hypertension. N Engl J Med 348:500–509 83. Morrell N, Adnot S, Archer SL, Dupuis J, Jones PL, MacLean MR et  al (2009) Cellular and molecular basis of pulmonary arterial hypertension. J Am Coll Cardiol 54:S20–S31 84. Zhang S, Fantozzi I, Tigno DD, Yi ES, Platoshyn O, Thistlethwaite PA et al (2003) Bone morphogenetic proteins induce apoptosis in human pulmonary vascular smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 285:L740–L754 85. Valdimarsdottir G, Goumans MJ, Rosendahl A, Brugman M, Itoh S, Lebrin F et al (2002) Stimulation of Id1 expression by bone morphogenetic protein is sufficient and necessary for bone morphogenetic protein-induced activation of endothelial cells. Circulation 106:2263–2270 86. Teichert-Kuliszewska K, Kutryk MJ, Kuliszewski MA, Karoubi G, Courtman DW, Zucco L et al (2006) Bone morphogenetic protein receptor-2 signaling promotes pulmonary arterial endothelial cell survival: implications for loss-of-function mutations in the pathogenesis of pulmonary hypertension. Circ Res 98:209–217 87. Long L, Crosby A, Yang X, Southwood M, Upton PD, Kim D et al (2009) Altered bone morphogenetic protein and transforming growth factor-b signaling in rat models of pulmonary hypertension: potential for activin receptor-like kinase-5 inhibition in prevention and progression of disease. Circulation 119:566–576 88. Zeisberg M, Hanai J, Sugimoto H, Mammoto T, Charytan D, Strutz F et  al (2003) BMP-7 counteracts TGF-b1-induced epithelial-tomesenchymal transition and reverses chronic renal injury. Nat Med 9:964–968 89. Morrell NW, Yang X, Upton PD, Jourdan KB, Morgan N, Sheares KK et  al (2001) Altered growth responses of pulmonary artery smooth muscle cells from patients with primary pulmonary

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Chapter 58

Role of Endothelium in the Development of Pulmonary Hypertension Bryan Ross and Adel Giaid

Abstract  Pulmonary hypertension (PH) primarily affects the small pulmonary resistance arteries, whereby marked pulmonary vascular wall thickening is observed. Disordered and uncontrolled proliferation of the endothelial layer often occurs. This, as well as inflammation, apoptosis and many other factors, can lead to endothelial dysfunction and an imbalance of key endothelium-derived factors (EDFs). The balance between these factors is crucial to overall control of vascular function. The pathological imbalance of endothelial factors observed in PH contributes to abnormal endothelial and smooth muscle cell (SMC) proliferation and a loss of control over pulmonary vascular tone. The resulting narrowed lumen and increased pulmonary vascular resistance often lead to secondary pathological conditions such as an increased stress and preload in the right side of the heart, which is why right ventricular failure often accompanies PH. Regardless of the general classifications and conditions promoting the disease, little is known about the underlying physiological and molecular initiating events that trigger PH. It is becoming increasingly clear that the endothelium plays a central role in the cause and progression of PH. The role of the endothelium in the maintenance of proper pulmonary blood flow and function has only come to realization in the last three decades, from the first discovery that the endothelium secretes a vasodilatory substance termed “endotheliumderived relaxing factor”. This compound, subsequently discovered to be nitric oxide (NO), is pivotal in regulating physiological vascular function and will be discussed in detail in this chapter along with many other important EDFs. Many factors that interact with or affect normal endothelial function will also be discussed. It becomes increasingly clear that there is a great deal of cross-regulation across the several endothelial functional pathways. This critical observation illustrates the importance of the fine-tuning and control that must occur, as the disruption of even one EDF can have B. Ross (*) Department of Cardiology, McGill University Health Centre, Montreal QC, Canada e-mail: [email protected]

significant effects on a number of pathways. The healthy, functional pulmonary endothelium is responsible for this control. An understanding of endothelium-related components and their interactions with one another is critical in understanding PH: the role of the endothelium is surely a central topic in present and future PH research and treatment. Keywords  Endothelium-derived factor • Nitric oxide • Endothelin • Pulmonary vascular function • Vascular remodeling

1 Pulmonary Hypertension and the Endothelium The pulmonary vasculature is a low-pressure, thin-walled conduit. Pulmonary hypertension (PH) is characterized by a pulmonary arterial pressure of greater than 25 mmHg at rest [1]. PH primarily affects the small pulmonary resistance arteries [2], whereby marked pulmonary vascular wall thickening is observed [3]. Disordered and uncontrolled proliferation of the endothelial layer often occurs [4]. This, as well as inflammation, apoptosis [5], and many other factors can lead to endothelial dysfunction. Importantly, an imbalance of key endothelium-derived factors (EDFs) results. The balance between these factors is crucial to overall control of vascular function. The pathological imbalance of endothelial factors observed in PH contributes to abnormal endothelial and smooth muscle cell (SMC) proliferation and a loss of control over pulmonary vascular tone [4]. The resulting narrowed lumen and increased pulmonary vascular resistance often lead to secondary pathological conditions such as an increased stress and preload in the right side of the heart, which is why right ventricular failure often accompanies PH [6]. There are two very broad categories of PH, primary (or idiopathic) PH and secondary PH [7]. Patients are essentially divided into a given category on the basis of whether the cause of disease is known (as in secondary PH) or unknown (primary PH) [7]. PH can be induced by a variety

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_58, © Springer Science+Business Media, LLC 2011

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of conditions and stresses. Some specific conditions are used to induce PH in experimental models. Examples include chronic hypoxia [8] and exposure to monocrotaline [8, 9]. Regardless of the general classifications and conditions promoting the disease, little is known about the underlying physiological and molecular initiating events that trigger PH. Further research is also needed to assess the underlying factors that drive the progression of PH. Despite the fact that PH research is still evolving, it is becoming increasingly clear that the endothelium plays a central role in the cause and progression of PH [3]. The role of the endothelium in the maintenance of proper pulmonary blood flow and function has only come to realization in the last three decades, from the first discovery that the endothelium secretes a vasodilatory substance termed “endotheliumderived relaxing factor” [10]. This compound, subsequently discovered to be nitric oxide (NO), is pivotal in regulating physiological vascular function and will be discussed in detail in this chapter along with many other important EDFs. Many factors that interact with or affect normal endothelial function will also be discussed. It becomes increasingly clear that there is a great deal of cross-regulation across the several endothelial functional pathways. This critical observation illustrates the importance of the fine-tuning and control that must occur, as the disruption of even one EDF can have significant effects on a number of pathways. The healthy, functional pulmonary endothelium is responsible for this control [11]. An understanding of endothelium-related components and their interactions with one another is critical in understanding PH: the role of the endothelium is surely a central topic in present and future PH research and treatment.

2 Endothelium-Derived Vasoactive Mediators The pulmonary endothelium of a healthy lung closely regulates many components of the pulmonary vasculature to allow for proper hemodynamic blood flow to occur [11]. Endothelial control over vascular tone is a primary example. The endothelium is also a key mediator of inflammation and coagulation [11]. This thin layer releases a variety of factors that regulate the tone and proliferative potential of the SMC layer [11]. Many endothelium-derived vasoactive mediators are discussed in this chapter. Each factor will be briefly introduced and their interrelations with one another discussed. As we will see, the fine balance between these many components is what maintains proper vascular tone and homeostasis; an imbalance in the secretion level of any of these opposing substances has indeed been theorized to be the currently unknown precipitating factor in the progression of PH [12]. Three predominant endothelium-derived vasoactive mediators are depicted in Fig. 1.

B. Ross and A. Giaid

2.1 The Principal Vasodilator, Nitric Oxide Nitric oxide (NO) is, in essence, the principal endotheliumderived vasodilator. Its effects span many processes and pathways, as NO mediates both the basal smooth muscle layer tone as well as a variety of other EDFs. Its antiproliferative [13], antithrombogenic [13], and potent vasodilative [3] properties help to maintain a healthy pulmonary vasculature. In addition to these important roles, it should be noted that NO also minimizes the risks associated with the most prevalent pathological condition in cardiovascular disease, atherosclerosis [14]. This is accomplished by the prevention of adhesion and migration of monocytes into the intimal layer and by opposing plaque rupture, thereby preventing deadly thrombogenic occlusion [15]. NO is formed from the conversion of l-arginine to l-citrulline at the endothelial layer [3]. This reaction is catalyzed by the endothelial nitric oxide synthase (eNOS) enzyme [16]. To carry out the conversion, eNOS requires the presence of a variety of compounds, which include substrates L-arginine and O2, the cofactor tetrahydrobiopterin (BH4), the electron donor NAPDH, and the electron carriers flavin adenine mononucleotide (FAM) and flavin adenine dinucleotide (FAD) [17]. The actual production of NO occurs in two steps. The first step requires the consumption of O2 and two electrons from NAPDH via FAM and FAD to yield N-hydroxy-l-arginine [17]. The second step also requires O2 plus an additional electron to yield NO [17]. NO levels are typically proportional to the amount of eNOS expression in the endothelium [16], and as this chapter discusses, NO levels also depend on the presence of a variety of factors that either activate or inhibit eNOS or NO itself. The potent vasoconstrictor endothelin-1 (ET-1) can inhibit NO production by directly increasing the presence of compounds such as reactive oxygen species (ROS) [18]. Upon production, NO diffuses to the smooth muscle layer of the pulmonary vasculature. There, it will promote a decrease in intracellular Ca2+ availability and thus vasodilation in SMCs [3]. This occurs through the direct stimulation of soluble guanylate cyclase, which subsequently increases the intracellular levels of cyclic GMP (cGMP), causing a marked decrease in intracellular Ca2+ levels [19]. A variety of stimulants can induce NO release. Compounds such as acetylcholine, thrombin, bradykinin, ATP, histamine, and vascular endothelial growth factor (VEGF) all induce endothelial-dependent NO production [20]. Certain states such as hypoxia and, in particular, increased shear stress are also very potent stimulators of NO release [21]. It is important to note at this point that although its actions are short-lived, NO is constitutively expressed and is the primary regulator of basal vascular tone [14]. NO degrades easily and further can even be transformed and deactivated through the binding of ROS [5]. The latter fate of NO is an important consideration when treating patients with PH; NO

58  Role of Endothelium in the Development of Pulmonary Hypertension

839

Fig. 1  Precursors and targets of three predominant endotheliumderived factors: the vasoconstrictive endothelin-1, and the vasodilatory nitric oxide and prostacyclin. Endothelial dysfunction impairs these and many other critical endothelial mechanisms that act on the smooth muscle layer. Blue endothelial cells, red smooth muscle cells, cAMP cyclic adenosine monophosphate, cGMP cyclic guanosine monophosphate, ET endothelin, ETA endothelin receptor A, ETB endothelin receptor B, PDE5 phosphodiesterase type 5. (Reproduced from [2] with permission. Copyright Elsevier, 2004)

reduction is detrimental to the pulmonary vasculature; however, overexposure to NO as observed in acute inhalant treatment can also be disruptive [22].

2.2 The Principal Vasoconstrictor, Endothelin-1 Along with the discovery of an “endothelium-derived relaxing factor” came the discovery of an “endothelium-derived constricting factor.” [23] This factor is now known to be one of the three endothelin subtypes found in the body, ET-1. ET-1 is a 21 amino acid peptide that is produced from the endothelial multiple-step conversion of arachidonic acid to prostacyclin (also known as prostaglandin I2) [24]. ET-1 originally exists in an inactive, 212-residue pre-pro-form [25]. It is then cleaved to become the 39-residue “big ET-1,” or pro-ET1, by the action of endopeptidases [25]. Finally, it is once again cleaved into its 21-residue, mature form by endothelin-converting enzyme 1 (ECE-1) [25]. It elicits a number of effects on the body and alters an innumerable number of pathways. ET-1 displays potent vasoconstrictive and mitogenic effects on the smooth muscle layer [3]. In addition, it plays a great number of regulator functions in neighboring endothelial processes and pathways. Examples relevant to this chapter include cytokine and chemokine pathways, superoxide formation, angiogenic pathways, and, of particular importance, regulation over NO production. Chronically elevated ET-1 expression has severe implications for certain diseased states. Its stimulation occurs from pathological conditions such as the aforementioned experimental inducers of PH, hypoxia and monocrotaline [25]. When viewed collectively, the potentially harmful effects of ET-1 overactivity help to illustrate the central pathological role that this EDF plays in PH.

ET-1 binds to ETA receptors (found mainly in the smooth muscle layer) as well as to ETB receptors (found mainly in the endothelial layer and scattered in the smooth muscle layer); both of these belong to the G-protein-coupled receptor (GPCR) superfamily [25]. Through the binding of ETA receptors, ET-1 elicits vasoconstriction and SMC proliferation [26]. These effects are mediated in part by the negative feedback from ET-1 binding to ETB receptors, which aids in ET-1 clearance and induces elevated NO and prostacyclin synthesis and release from the endothelium [24, 27]. It is important to note that ET-1 is one of the most potent physiological vasoconstrictors in the body [28]. As such, the endogenous ET-1 levels have been suggested as a marker in diagnosing endothelial dysfunction [29]. In essence, increased ET-1 expression is detrimental to the pulmonary vasculature and is in itself a chief mediator of the detrimental pathways involved in PH [26]. There is a clear, well-characterized, and often inhibitory regulation between EDFs. Of particular importance is the interaction between NO and ET-1 [15]. The effects of these opposing compounds, both on the pulmonary vasculature as well as on one another, are pivotal in the progression of PH. One can see that a disruption of such a delicate and complicated balance can be detrimental to the pulmonary vasculature and may help to explain the uncontrollable snowball effect observed during the course of PH progression. Chronically decreased levels of NO [16] and chronically increased levels of ET-1 [30] both appear to be particularly detrimental to the pulmonary vasculature. This is well evidenced from those experiments performed on patients with PH. When compared with control subjects, patients with PH have significantly greater levels of ET-1 in the pulmonary vasculature [30]. Secondly, the degree of pulmonary vascular resistance and arterial pressure in these patients is proportional to the local concentrations of ET-1 [30]. Lastly, ET-1

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expression appears to be particularly high in the plexiform lesions [30], pathological formations described later in this chapter. Chronically reduced expression of NO is observed in these patients as well [31]. Importantly, the expressions of the key endothelial enzymes eNOS and ECE-1 are altered in PH; there is an observed lack of eNOS and an overabundance of ECE-1 [31]. The presence of NO appears to affect the release of ET-1 from the endothelium and vice versa, both in pathological and in physiological states [3]. ET-1 inhibits normal eNOS function [15]. Conversely, endogenous NO modifies endothelial ET-1 levels by downregulating its expression in both hypoxia-induced and in physiological conditions [24]. ET-1 binding to ETB results in ET-1 clearance and NO release [26]. These examples all illustrate the cross-regulation that occurs between these chief antagonists. The mechanism of progression of PH is still largely unknown. However, ET-1 and NO levels are, respectively, increased and decreased in the pulmonary vasculature of patients with PH [31]. A dysfunctional pulmonary endothelium cannot regulate the respective levels of vasoconstrictors and vasodilators or proliferative and antiproliferative factors [3]. This is a key concept in understanding how a damaged endothelium can result in the progression of PH.

2.3 Prostacyclin and Thromboxane Prostacyclin is another important endogenous vasodilator. It also plays a predominant role in the inhibition of platelet aggregation. Prostacyclin and analogues have emerged as a gold standard therapy in prolonging the life of PH patients [26]. Endogenously, prostacyclin is synthesized by cyclooxygenase (COX) and ultimately causes an increase in cyclic AMP (cAMP) expression in the SMC layer [32]. It is a downstream product of arachidonic acid [33]. It is noteworthy that thromboxane A2 (TxA2) is also a product of a separate arachidonic acid pathway which utilizes the same COX enzyme [32, 33]. However, COX-1 appears to predominantly synthesize TxA2, whereas COX-2 predominantly synthesizes prostacyclin [34]. Additionally, prostacyclin is produced mainly in endothelial cells, whereas TxA2 is mainly produced by COX in circulating platelets [32]. Prostacyclin and TxA2 are, by nature, antagonists of mirroring pathways. Whereas prostacyclin causes antiplatelet aggregation, vasodilation, and opposes inflammation, the effects of TxA2 are the exact opposite [33]. Classic symptoms that accompany PH include vasoconstriction, inflammation, proliferation, and thrombosis [3]. These symptoms can be countered by adequate production of endothelial prostacyclin. As the endothelium becomes compromised in a variety of pathological conditions, prostacyclin and prostacyclin analogue administration becomes an ideal clinical pharmacological intervention [26].

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3 Caveolae, Caveolin-1, and eNOS Caveolae are functional invaginations in the plasma membrane that typically span 50–100 nm [35]. The presence of caveolae is necessary and critical; just as a functional endothelium is central in the inhibition of PH, functional caveolae are central in the inhibition of endothelial dysfunction. In essence, caveolae are responsible for maintaining the proper function of cells. And, as we will see, caveolin-1 is essential to the presence of a functional caveola [36]. Endothelial cells, SMCs, epithelial cells, and adipocytes all rely on caveolae [35]. They compose a particularly large portion of the surface area on the membranes of endothelial and SMCs [13]. These invaginations are extremely populated with structures that are essential to proper endothelial and ultimately pulmonary function [13]. eNOS, for example, is primarily located in caveolae. Caveolae also contain the cationic amino acid transporter which supplies eNOS with its l-arginine substrate [37]. Transport, signaling, transcytosis, tumor suppression, cholesterol transport [35], homeostasis [35], and recently angiogenesis [13] have also been implicated as caveolardependent cellular functions [35]. Endothelial abnormalities that result in the absence of these highly functional rafts result in the imbalance of EDFs that would result in PH. The caveolins are a group of integral membrane proteins necessary for both the formation of and for structurally maintaining caveolae [13]. Caveolae are formed as the cholesteroland sphyngolipid-sequestering caveolins stabilize an area of the plasma membrane [13]. This creates a foundation on which a variety of enzymes and receptors congregate [13]. Three caveolins, caveolin-1, caveolin-2, and caveolin-3, have been identified [13] and a variety of experiments have been conducted on these isoforms. The presence of caveolin-1 in particular appears to be crucial in maintaining caveolar structure and function [20]. The central hydrophobic region of caveolin-1 is located in the plasma membrane, whereas both its NH2 and COOH terminal ends face the cytoplasm [13]. Forty residues on caveolin-1 form important homo-oligomerization interactions with other caveolins [13]. Caveolin-1 also contains the crucial caveolin scaffolding domain (SCD) [13]. Many proteins found within caveolae contain caveolin binding motifs [13], allowing regulatory caveolin-1 binding and conferring stability to many proteins located within a given caveola [13]. eNOS is an exception to this phenomenon, as it does not express a caveolin binding motif [13]. Instead, caveolin-1 binds to the calmodulin found on eNOS, thereby inhibiting eNOS function [20]. Caveolin-1 is thought to interfere primarily with the NAPDH-dependent electron transfer [37]. Intracellular increases in endothelial calcium concentration allow increased Ca2+ binding to calmodulin [20]. This binding dissociates the inhibitory caveolin-1–calmodulin interaction, enabling the freed eNOS to produce and release NO [20]. The overall result is increased vascular vasodilation (see Fig. 2).

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Fig.  2  Calmodulin-dependent activation of endothelial nitric oxide synthase on caveolae and subsequent endothelial release of nitric oxide. eNOS endothelial nitric oxide synthase, CaM calmodulin, Hsp90 heat shock protein 90, Arg arginine, NO nitric oxide, P phosphate

Several factors, including shear stress, VEGF, thrombin, histamine, and bradykinin, can induce intracellular increases in Ca2+ concentration to promote this vasodilatory pathway [20]. Recent evidence indicates that heat-shock protein 90 is instrumental in facilitating this caveolin-1–eNOS dissociation [38]. In addition, a very recent experiment elucidated that caveolar internalization may in fact be even more important in eNOS activity than Ca2+ levels [39], once again underlining the significance of caveolae in overall endothelial function. Supporting results have been documented for experiments whereby caveolin-1 was ablated in mice. In the absence of caveolin-1, there is a complete absence of caveolae on the pulmonary endothelial cell surface [35]. Caveolin-1-deficient mice also lacked the ability to downregulate NO release, corresponding to a roughly threefold higher cGMP concentration in SMCs and marked vasodilation [35]. This chronically elevated production of NO in endothelial cells may spark the notion that caveolin-1 targeting could be helpful in treating PH. However, it is important to recall that the loss of caveolin-1 also results in a loss of regulation over NO release and thus an absence of basal control over resting vascular tone [35]. A paradoxical concept thus emerges: caveolin-1, in creating the potential for caveolar formation, allows for maximal eNOS localization and activation [13]. At the same time, caveolin-1 directly binds to eNOS, thereby inhibiting its function [13]. Thus, caveolin-1, although inhibitory to eNOS

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function, is nonetheless instrumental in preventing endothelial dysfunction and subsequent PH by creating ideal conditions and localization of eNOS on caveolae. Although the eNOS activation–inactivation pathway seems simple, such an important process is in actuality modulated and fine-tuned by a great variety of factors, scaffolds, proteins chaperones, and kinases [21]. In addition, eNOS and NO production can be hampered by excess levels of circulating LDLs in the vasculature, as this results in an upregulation of caveolin-1; [13] oxidized LDL has even been shown to remove eNOS from caveolae altogether [13]. In addition, hypercholesterolemia has been demonstrated to reduce the stability of eNOS messenger RNA (mRNA) [21] and decrease the available amounts of essential cofactors and substrates such as BH4 and l-arginine [38] while allowing for the accumulation of inhibitory caveolin-1. Conversely, the aforementioned factors including VEGF and thrombin increase intracellular Ca2+ concentrations, thereby dissociating eNOS from caveolin-1 [21]. In conclusion, the presence of caveolae is absolutely essential to pulmonary vascular function, as it allows for an ideal localization, activation, and even regulation via caveolin-1 and other factors. A disruption in these structures leads to endothelial dysfunction, uncontrolled vascular tone, and eventual PH [4].

4 Tetrahydrobiopterin As mentioned, a variety of substrates and cofactors are present during the synthesis of NO by eNOS. One cofactor gaining acceptance as a major protector from the progression of PH is BH4. Its presence is in fact mandatory for the endothelial production of NO [40]. BH4 plays the essential role of coupling eNOS to form homodimers and thus also reduces the alternative pathway of eNOS in which superoxide production is catalyzed by uncoupled eNOS [41]. BH4 is produced in the endothelium by GTP cyclohydrolase I [40]. This rate-limiting enzyme catalyzes the conversion of GTP to dihydroneopterin triphosphate, upon which 6-pyruvoyltetrahydropterin synthase and sepiapterin reductase complete the conversion to BH4 [41]. In addition to eNOS, all other NOS isoforms require the formation of homodimers to function [17]. It is only in this conformation that NO synthases can use the electrons donated from NADPH via FMN and FAD that are needed to carry out the NO-producing reaction [17]. The key concept surrounding BH4 is that it binds to the heme that is found at the interface of the eNOS dimer; this heme is in fact the theorized catalysis site for eNOS uncoupling [41, 42]. Thus, BH4 stabilizes and activates eNOS by binding to and altering this heme [17, 41]. At increased levels BH4 is especially beneficial in that it increases NO production, minimizes the formation of superoxide production, opposes endothelial dysfunction and thus prevents PH [17, 40].

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5 Vascular Endothelial Growth Factor

6 Serotonin

In severe forms of PH, prominent structures known as plexiform lesions are present [16]. Plexiform lesions are a mass of concentric endothelial cell layers whose form resembles that of glomeruloid structures [3] and “onion bulbs” [43]. Plexiform lesions are thought to arise from an uncontrolled, disorderly state of pathological pulmonary angiogenesis [44]. Inflammation also plays a key role in the formation of plexiform lesions [43]. Physiologically, angiogenesis is considered to be a positive and reparative mechanism [3]. However, the formation of plexiform lesions appears to be a pathological, misguided attempt at revascularization [3]. They are typically found in proximity to bifurcations along the pulmonary precapillary vasculature, in the small resistance vessels [44]. They contribute to the increased pulmonary vascular resistance and therefore contribute to secondary right ventricular failure [8]. The presence of these lesions in PH patients has been linked to the effects of increased VEGF expression [3, 4]. Just as angiogenesis is typically physiologically reparative, VEGF is typically a positive growth factor [8]. In fact, its blockade during development induces lifelong PH [45]. This is a prime example demonstrating the importance of moderate expression of EDFs. VEGF is the principal endothelium-derived angiogenic growth factor and at elevated concentration it has been implicated as the primary inducer of uncontrolled angiogenesis and thus of plexiform lesions [3]. VEGF is also a potent mitogen for endothelial cells, which physiologically allows for new tube formation but pathologically enhances plexiform lesions [46]. As a promoter of angiogenesis, VEGF is significantly upregulated in response to chronic hypoxia [8]. This is achieved partly through the effects of the transcription factor hypoxia-inducible factor (HIF) [44]. Circulating levels of VEGF [47], expression of VEGF receptors 1 and 2 on endothelial cells [47], and expression of both HIF subunits a and b [44] have all been shown to be elevated in the cells that compose plexiform lesions in patients with severe PH [44, 47]. Thus, it appears that the endothelium itself undergoes a state of autocrine and paracrine angiogenesis and proliferation that results in the observed plexiform lesions [44]. An interesting phenomenon is the observation of elevated levels of HIF in these endothelial cells [44]. This may be explained by the fact that plexiform lesions receive a significantly reduced blood flow. Thus, a continuous cycle of hypoxia, HIF upregulation, and chronic VEGF expression ensues in the diseased resistance vessels. VEGF also regulates the release of other endothelial factors. For example, VEGF blockade results in decreased eNOS expression [48]. In addition, VEGF binding to the endothelial VEGF receptor induces a vasodilatory effect via NO [48] and prostacyclin [44] synthesis and release [3]. As we now know, VEGF accomplishes these endothelial effects by increasing intracellular Ca2+ levels to free eNOS from caveolin-1 [21].

Serotonin (5-HT) is a potent growth factor released from the endothelium [49]. It has a paracrine effect on neighboring endothelial cells and also diffuses to the vascular SMC layer [49]. In the normal vasculature, 5-HT is largely stored in circulating platelets in the bloodstream [2]. This sequestration helps to maintain low normal plasma levels of 5-HT [2]. Increased 5-HT levels in the plasma and low platelet storage are observed in idiopathic PH [50] as well as in pulmonary arterial hypertension (PAH) [51]. PH appears to be dependent on 5-HT owing to its effect on pulmonary SMCs. Firstly, 5-HT is mitogenic [52]. Secondly, the 5-HT pathway elicits endothelial-dependent vasoconstriction [53]. 5-HT primarily affects SMC tone through a G-protein-dependent increase in intracellular SMC Ca2+ concentrations, constitutively increasing Ca2+ permeability and increasing L-type Ca2+ channel activity [53]. It should be noted that the mechanisms by which 5-HT affects the progression of PH are still somewhat inconclusive [54]; however, the 5-HT-induced growth and proliferative effects appear to be dependent on the mitogen activated protein kinase- and Rho pathways [55, 56]. Regardless, it is known that 5-HT transporter (5-HTT) overexpression as well as 5-HT receptor defects both contribute to SMC hyperplasia and vascular remodeling [54, 57]. In recent years the receptors and transporter of 5-HT have been studied in regard to their interactions with other cellular processes. 5HTT and 5-HT1B blockade decreases the expression of the S100 calcium-binding protein (S100A4/Mtsl), reducing the contraction of SMCs [52, 58]. The 5-HT2B receptor has also recently been studied, specifically in mice. This particular receptor appears to be instrumental in conducting hypoxiadependent PH progression [2]. Heightened expression of the 5-HT2A receptor appears to inhibit the voltage-gated potassium (Kv) channels in SMCs [59]. This finding is particularly significant. In physiological conditions there is a delicate balance between SMC depolarization and hyperpolarization, between voltagegated Ca2+ channel and K+ channel activity, and between relaxation and contraction [2]. Patients with PAH are found to have downregulated Kv channel expression [2]. The finding that 5-HT mediates Ca2+ channel influx while inhibiting K+ channel efflux underlines 5-HT’s potent effects on vasoconstriction and PH pathogenesis. To summarize, increased S100A4/Mtsl levels,l increased intracellular calcium concentration [2], and reduced Kv channel activity [59] are all vasoconstrictive phenomena, and all are observed in PH progression [59]. 5-HTT overexpression allows for 5-HT influx and intracellular action; the presence of this overexpression is a particularly strong indicator of hyperplasia [54]. 5-HTT may further contribute to PH through its 5-HT-mediated phosphorylation of the platelet-derived growth factor (PDGF)

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b receptor [60]. This transactivation is important, as the chemokine PDGF is directly associated with PH progression [60]. 5-HTT is expressed by a single gene [2, 54]. Of the long (L) and short (S) gene polymorphisms, the L variant is homogyzously present in 65% of idiopathic PAH patients [2]. This also contributes to the notion that some forms of PH may in fact have a genetic predisposition [54].

7 Reactive Oxygen Species ROS such as superoxide (O2·−) and hydrogen peroxide (H2O2) can have a very significant impact on overall vascular health as they affect both the endothelium as well as the SMC layer [61]. Superoxide can be produced by a variety of enzymes such as NADP(H) oxidases and xanthine oxidases [5]. Of particular relevance, ROS are also closely tied to NOSs in a variety of ways. An elaboration of this latter point will clearly illustrate the implications of ROS in PH. Production of NO via eNOS is dependent on the presence of a variety of compounds such as the substrate l-arginine and the cofactor BH4 [17]. The presence of substrate, as well as eNOS conformation, affects the release of NO [40]. Decreased levels of either l-arginine or unoxidized BH4 promote an alternative eNOS pathway in which superoxide is produced; the uncoupling of eNOS dimers also promotes the alternative production of ROS [40]. In addition, ROS can affect eNOS function by directly interacting with its product, NO [61]. NO is a short-lived compound [22], but particularly so in the presence of superoxide, which binds and inactivates it [61]. Other peroxides have also been implicated in this inhibitory alteration of NO. NO–ROS interaction produces the compound peroxynitrite (ONOO·) [62]. In addition to itself being a dangerous compound to the vasculature, peroxynitrite can also induce the eNOS-dependent production of more harmful ROS. Upon its formation, peroxynitrite oxidizes the essential eNOS cofactor BH4, transforming it into BH2 and thereby inactivating it [62]. This results in the uncoupling of eNOS, which in turn results in even more production of ROS [61]. Thus, ROS are harmful species that can impair the vasculature while at the same time directly and indirectly inhibiting the vasodilatory function of the endothelium. At increased quantities in the pulmonary vasculature, ROS indeed induce chronic SMC vasoconstriction [63]. The process leading to endothelial dysfunction is highly ROS dependent [40]. Inflammatory and structural changes brought about by ROS contribute to a decreased output of essential EDFs that regulate vascular tone. ROS play a direct role in endothelial cell apoptosis by interacting with and activating the proapoptotic caspase-3 enzyme [61]. Also, the many cytokines and growth factors that stimulate adhesion molecule expression appear to do so in ROS-dependent pathways [61]. Vascular adhesion molecule 1 (VCAM-1) expression

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and intercellular adhesion molecule 1 (ICAM-1) expression are both upregulated by ROS, which allows for the attachment of detrimental inflammatory cells [64]. Lastly, ROS can alter inherent structural properties of the endothelial barrier. Cadherin is responsible for endothelial cell-to-cell binding and thus for structural integrity of the endothelial layer; ROS regulate many cadherin-dependent properties [65]. These numerous mechanisms illustrate how ROS can impair the endothelium to the extent that endothelial regulation over vascular tone becomes deregulated and the integrity of the endothelium becomes compromised. Thus, ROS act directly on the endothelium in a variety of ways to optimize the conditions for subsequent PH progression. ROS clearly interfere with normal endothelial layer structure and function. They can also diffuse to the SMC layer to alter a variety of SMC processes that lead to PH [18]. For example, ROS can promote SMC contraction and migration [18]. H2O2, for example, is important in both vasoconstriction and vascular remodeling [5]. In a hypoxic state, mitochondrial- and electron-transport-chain-dependent H2O2 is released, which directly increases intracellular calcium levels to induce vasoconstriction [62]. Also, H2O2 is thought to be the ROS that is most instrumental in the vascular remodeling process [61], however the presence of the aforementioned peroxynitrite also appears to be important in the late remodeling stages [61]. ROS activate a variety of mitogenic cytokines and chemokines including tumor necrosis factor a (TNF-a) and monocyte chemoattractant protein 1 (MCP-1), and in doing so further contribute to vascular remodeling [61]. In addition, ROS are closely involved in 5-HT-dependent effects on SMCs, and increased levels of 5-HT also directly increase SMC production of harmful ROS and subsequent vasoconstriction [66]. The presence of ROS in the pulmonary vasculature is evident in the late stages of PH. ET-1 [18] as well as VEGF [5] appear to induce ROS production; both are increased in PH and both induce the formation of pathological plexiform lesions [5]. Endothelial cell migration and proliferation are essential angiogenic events; H2O2 indeed induces both of these [5]. Lastly, VEGF is the pivotal growth factor in angiogenesis and plexiform lesion formation [5]; NADP(H) oxidases produce ROS which directly mediate VEGF expression as well as VEGF-dependent angiogenesis [5].

8 Adrenomedullin Discovered in a pheocromocytoma in 1993, adrenomedullin (AM) is a multifunctional peptide consisting of 52 amino acids [67]. AM is produced in response to a variety of stimulants, including hypoxia, shear stress and cytokines, of which the hypoxia/HIF stimulation appears to be most effective [12]. It is produced in a variety of organs and tissues, but mainly in

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vascular endothelial cells and SMCs [12]. The importance of AM in the pulmonary vasculature is illustrated by the finding that the levels of AM and AM-receptor mRNA are both physiologically elevated in the lung as compared with the systemic circulation [68]. In fact, AM preferentially vasodilates the pulmonary vasculature [12]. In addition, AM regulates the healthy growth and survival of endothelial cells [12], an essential function for those PH patients with a compromised endothelial layer. AM plays an antiproliferative role in plexiform lesions and an antioxidant role in countering ROS [12]. It is thus clear why intravenous and intratracheal artery infusion of AM is emerging as a potential treatment for PH [65]. Like NO and prostacyclin, AM is a potent vasodilator [33]. However, unlike NO, AM is stable and thus exerts a longer-lasting hypotensive effect in the pulmonary vasculature [12, 67]; unlike prostacyclin, AM is also a natural diuretic and natriuretic [67]. Interestingly, AM plasma concentrations are elevated in patients with PH and in proportion to individual severity [69]. In addition, higher AM levels are accompanied by greater mean pulmonary arterial pressure, resistance, and right arterial pressure [33]. Owing to the many beneficial effects of AM, this observed increase is thought to be a compensatory mechanism against the disease rather than having a causative role in PH [33]. AM interacts with its specific GPCR to exert its effects via two main pathways. One pathway is cAMP-dependent, whereas the other is NO-dependent [12, 67]. In the former pathway, cAMP accumulation results in protein kinase A activation and a subsequent intracellular SMC Ca2+ concentration decrease [12, 67]. In the latter pathway, AM specifically activates eNOS through either calmodulin/Ca2+- or phosphatidylinositol 3-kinase/Akt-dependent functions [12, 67].

9 Thrombin Thrombin is a proteinase that is worth consideration in PH progression. It plays a central role in promoting thrombosis, blood coagulation, and hemodynamic flow [70]. Prothrombin is membrane-bound and self-activates to a-thrombin, a two-chain complex that is released into the vascular lumen [70]. Thrombin has several important implications owing to the nature of PH [71]. As the upstream small arterial vasculature becomes increasingly compromised from lesions and vasodilatory defects, the pulmonary vasculature and flow of blood becomes disrupted [72]. The resulting minimized flow and pooling downstream of the capillaries tends to encourage venous coagulation and thromboembolism [26]. Circulating prothrombin becomes activated to thrombin from the binding and activation by coagulation factors X and V at the surface of endothelial and platelet cells [71]. From there, thrombin converts fibrininogen into its active fibrin form, in addition to releasing fibrinopeptide A [71]. There is evidence showing continuous levels of thrombin

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and blood coagulation in all forms of PAH as indexed by the elevated levels of fibrino­peptide A in PAH [72]. Blood coagulation is an important physiological function and is normally present, nonetheless downregulated, when the endothelium is intact [72]. In fact, the endothelium itself produces low basal levels of clotting factors as well as binding sites for additional clotting factors [72]. Endothelial dysfunction is agreed to be the precipitating event that results in pathological thrombin activity [3, 71, 72]. In particular, NO and prostacyclin both play significant roles in the inhibition of the coagulation pathway [71]. This is yet another piece of evidence that illustrates the delicate balances that exist between endothelial factors, enzymes, and physiological functions. A decrease in NO or prostacyclin release as observed in endothelial dysfunction can trigger further pathological conditions of thrombosis and occlusion [71]. NO and prostacyclin inhibition thus result in direct chronic vasoconstriction as well as an indirect pathological increase in prothrombotic pathways [71]. In addition to its coagulant and thrombotic properties, thrombin is also an endothelial-barrier-disrupting agonist [73, 74] (Fig.  3). Thrombin induces almost immediate structural impairment of the integrity of the endothelial membrane [73], acting along with ROS and TNF-a [74]. In fact, thrombin indirectly and additionally disrupts the endothelium by causing an increase in ROS production via NADPH oxidase, which itself also stimulates TNF-a release [61].

10 Cytokines There is a clear role for inflammation in the pathological states of many forms of PH, as evidenced by secondary PH from sclerodoma and lupus and connective tissue diseases [43]. The role of inflammatory cytokines in PH is also clearly demonstrated by the heightened levels of cytokine gene mutations in primary PH patients and increased expression of many cytokines in various other forms of PH [2]. For example, increased levels of IL-1 and IL-6 are found in a subset of PH patients [3]. A particularly relevant group of cytokines are those belonging to the transforming growth factor b (TGF-b) superfamily (Fig. 4). There are three isoforms of TGF-b, TGF-b1, TGF-b2, and TGF-b3; their functions range from cell proliferation to an increased extracellular matrix and elastin production [2]. These isoforms appear to have differential effects on the pulmonary vasculature in healthy versus pathological lungs and thus their roles are highly context specific [2]. For example, TGF-b1 is an antiproliferative agent in normal lungs, but is a proliferative agent in the lungs of primary PH patients [2]. Importantly, TGF-b1 has also been found to increase ET-1 expression in the human pulmonary vasculature [2]. Lastly, the TGF-b receptors ALK-1 and endoglin appear to have a higher incidence of mutation in PH [75, 76].

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Fig. 3  Pulmonary endothelial inflammation, leading to paracellular gap formations along the vascular endothelium and eventual endothelial dysfunction. Entry of inflammatory cells becomes facilitated. Thrombin acts directly on the endothelial layer, as well as indirectly through the stimulation of tumor necrosis factor and reactive oxygen species. TNF tumor necrosis factor. (Reproduced from [89] with permission of Macmillan Publishers Ltd. Copyright 2000)

Fig. 4  The importance of the transforming growth factor b superfamily in altering endothelial processes, as illustrated by its vast and numerous functions. TGF transforming growth factor, BMP bone morphogenetic protein, GDF growth and differentiation factor. (Reproduced from [2] with permission. Copyright Elsevier, 2004)

Many researchers believe that PH is caused, regardless of origin, by a disruption of the normal intracellular trafficking and extracellular signaling pathways [77]. The pathways regulated by bone morphogenetic proteins (BMPs) (Fig. 4) and BMP receptor 2 (BMPR2) are particularly good examples of this hypothesis in action. BMPR2 is a serine/threonine kinase receptor that will bind a variety of TGF-b members, including BMP2, BMP4, and BMP7 [33], and growth and differentiation factor (GDF) 5 and GDF6 [2]. A variety of phosphorylations occur on kinases downstream of BMPR2 to ultimately induce the receptor-activated members of the Smad family of proteins, the R-Smads (specifically Smads 1, 5, and 8) [2]. Cytoplasmic Smad complexes eventually translocate to the nucleus to affect

transcription [2], ultimately inducing proliferation and vascular remodeling [33]. Thus, activating BMPR2 affects transcription levels of a variety of pivotal cellular processes. BMPR2 is found on the endothelium of both systemic and pulmonary vasculatures [2]. Interestingly, BMPR2 expression defects affect only the pulmonary vasculature, to the extent that a decrease of BMPR2 expression is indicative of PAH [78]. Cytokine expression is yet another example of the potential genetic component involved in the onset of PH: a pronounced overlap has been observed between mutations in the TGF-b superfamily and in patients with PH [33]. For example, mutations in TGF-b receptor 2, and particularly in BMPR2, are commonly observed in PH [2].

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11 Chemokines A special subclass of cytokines particularly relevant to endothelial inflammation is the chemokine family. Chemokines are essentially involved in attracting leukocytes to sites of inflammation [79]. This cellular migration toward an increasing gradient is known as chemotaxis (chemotactic cytokines are chemokines) [79]. Chemokines are considerably small proteins with four conserved cysteine residues that form two disulfide bonds. The arrangement of cysteine residues in different chemokines, whether adjacent or separated by an amino acid, introduces the classification of chemokines into the two broad CC and CXC categories, respectively. Endothelial cells primarily express cell-surface CXCR receptors [77]. There appears to be a great homogeneity among chemokines, both in general structure as well as in nonspecific binding [80]. Chemokines exert their effects upon binding to GPCRs on leukocytes [80]. The resulting actin filament rearrangements and lamellipodia alterations confer movement to the activated leukocyte [80]. Notably, integrin expression on the leukocyte surface is also increased by chemokines [80]. Integrin expression is essential to binding immunoglobulin endothelial adhesion molecules such as VCAM-1 and ICAM-1 [81]. Increases in the levels of intracellular Ca2+ and free oxygen radicals are also observed in these leukocytes [80]. The endothelium synthesizes a variety of chemokines, which include TGF-b1, PDGF, MCP-1, and macrophage inflammatory protein 1a [82]. Although PDGF is not produced by the endothelium, it is nonetheless another important mitogen and chemokine in the progression of PAH [83]. PDGF blockade indeed aids in reversing vascular remodeling in experimentally induced PH [83]. Increased levels of the chemokine fractalkine (FKN) are observed in severe primary PH cases [43]. In fact, the FKN/ CX3CL1 complex acts as an adhesion molecule for leukocytes that express the CX3CR1 receptor [43]. FKN specifically appears to play a significant role in attracting monocyte/T cell recruitment [43]. “Regulated upon activation, normal T-cell expressed, and secreted” (RANTES) is another important chemokine [84]. RANTES has been shown to increase ET-1 production by stimulating ECE-1 in the endothelium [84]. Lastly, the level of RANTES mRNA is increased in patients with PAH [43]. Increased expression of both RANTES and FKN is observed in severe PH [2].

12 Adhesion Molecules Adhesion molecules, particularly on endothelial cells, play an essential role in inflammation and endothelial dysfunction. Indeed, these structures interact with the leukocytes

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attracted to the endothelium by chemokines. Adhesion molecules are transmembrane glycoproteins with three domains: an external binding domain, a hydrophobic transmembrane domain, and a cytoplasmic domain responsible for communicating with intracellular signaling pathways [81]. Localization and recruitment of leukocytes and other inflammatory cells are dependent upon the transient expression of adhesion molecules, as their expression on the endothelial cell surface is critical to binding [81]. A particular importance of those adhesion molecules belonging to the selectin and immunoglobulin superfamilies is implicated in vascular inflammation [64]. Most notable are the selectins E-selectin and P-selectin, and the immunoglobulins VCAM-1, ICAM-1, and platelet–endothelial adhesion molecule 1 (PECAM-1) [81]. It is these immunoglobulin adhesion molecules that are expressed on the endothelial surface [85]. VCAM-1 and ICAM-1 bind integrins on leukocytes, whereas PECAM-1 binds itself between the endothelium [85]. Thus, VCAM-1 and ICAM-1 are heterophylic binders, whereas PECAM-1 is a homophilic binder [85]. The selectins are involved in the beginning steps of leukocyte recruitment to vascular inflammatory lesions along the endothelium [81]. Briefly, the leukocyte must roll along the pulmonary endothelium to tether; this occurrence is selectindependent [81]. Upon selectin-dependent rolling along the endothelial surface, the immunoglobulin adhesion molecules VCAM-1 and ICAM-1 tether these leukocytes [81]. They also play an essential role in leukocyte transmigration across the endothelial barrier [81]. This is a potentially harmful inflammatory process which can lead to gaps in the endothelium and damage to the vasculature [74]. Adhesion molecules can be constitutively expressed (as is the case for PECAM-1), expressed in response to specific stimulants, or both. P-selectin is always present in the endothelium and stored in Wiebel–Palade bodies to be expressed on the cell surface in response to stimulants such as thrombin [81]. P-selectin transcription is increased in the presence of cytokines (TNF-a [64], IL-1b via the nuclear factor kB dependent pathway [81]). Conversely, VCAM-1 and ICAM-1 are both transiently upregulated, particularly in response to these same inflammatory cytokines [81]. Interestingly, ET-1 directly stimulates the expression of adhesion molecules [26]. Additionally, adhesion molecule expression is strongly regulated by NO to the extent that the presence of NO greatly decreases the cytokine-dependent surface expression of both VCAM-1 and ICAM-1 [86]. This inhibition occurs possibly via inhibition of the nuclear factor kB pathway [86]. Once again, the question of cause and effect is raised in the development of PH, as the level of NO is decreased, the level of ET-1 is increased and inflammatory lesions arise during the course of PH development [81]. An important aspect

58  Role of Endothelium in the Development of Pulmonary Hypertension

of future PH research will surely be distinguishing those events that are triggers from those that are effects of PH.

13 Interventions and Treatments for Pulmonary Hypertension PH is a spiraling condition in which the accumulation of endothelial deficiencies imposes a real and potentially irreversible stress. This creates a situation of uncontrolled, increased tone and vascular remodeling. This chapter has demonstrated the complicated, interrelated effects that many components of the endothelium have on one another and on their ultimate targets. As a summary to the chapter, the gold standard therapies for treating PH are discussed (Fig. 5). It is interesting to note that most PH treatments in use today attempt to mimic normal endothelial function. One can conclude that an understanding of the endothelium and its various regulatory roles is central in comprehending the progression of PH. Figure 5 represents an important reference and summary for the current approaches to treating PH. It outlines the three primary pathways regulating pulmonary arterial tone. The overlap between Figs. 5 and 1 once again underscores that understanding the role of the endothelium is central in comprehending and treating PH. One of the most widely employed, standard forms of treating PH is the administration of prostacyclin and prostacyclin analogues. As observed in the Fig. 5, these analogues stimulate cAMP production in SMCs in the same way that endogenous prostacyclin does. This line of treatment began in the early 1980s with the administration of epoprostenol [87]. Despite the potent vasodilatory effects, its short 3-min half-life necessitates an intravenous administration of the drug [88]. Although a large number of cohort analyses and prospective studies have supported the benefits of epoprostenol, it is not universal in its beneficial effects on the large variety of PH patients [26]. In addition, the risks and cost that accompany chronic intravenous administration can both be severe [26]. Risks such as catheter-related sepsis have led to the invention of prostacyclin analogues that do not require intravenous administration [26]. One such example is treprostinil, which is instead given subcutaneously owing to its greater stability. This analogue appears to be especially effective for those patients able to tolerate maximal doses. Another alternative to intravenously administered epoprostenol is beraprost [88]. This analogue, unlike epoprostenol and treprostinil, is given orally and as such is biologically stable [26]. Studies have shown marked improvement during exercise tests of patients receiving beraprost [88]. Lastly, iloprost is delivered by inhalation [88]. This chemically stable analogue, although relatively short acting, also shows promising short-term results in treating the symptoms of PH [88].

847

As opposed to stimulating a major vasodilatory pathway, another line of treatment aims at inhibiting the major vasoconstrictive pathway; the ET-1 pathway. In doing so, secondary antiproliferative and anti-inflammatory effects are also minimized. This line of treatment consists of a variety of endothelin receptor antagonists. Bosentan blocks both ETA and ETB receptors and is metabolized in the liver [26]. Even though it is an extremely effective drug that significantly ameliorates a vast number of detrimental PH symptoms, its effects on hepatic function require careful administration and observation [26]. There are also ETA-specific antagonists. From the content of this chapter one would reasonably theorize this to be an ideal therapy: ETA inhibitors would block the vasoconstrictive ETA effects, while at the same time allowing ETB to carry out its negative-feedback actions of ET-1 clearance and NO-mediated vasodilatation. However, the long-term benefit as well as the unwanted hepatic side effects of these drugs are under current investigation [26, 88]. As already mentioned, prostacyclin and prostacyclin analogue administration remains one of the primary treatments to positively target a vasodilatory pathway. The NO pathway, unfortunately, is more difficult to target. Shortterm inhalation of NO is an effective and potent vasodilatory treatment [88]. However, chronic administration of inhaled NO is impractical and may even be harmful [22]. NO sources such as nitroglycerin have also been used. Nitroglycerin is an effective endothelial-independent source of NO and thus a useful vasodilator [22]. However, patients tend to develop tolerance quickly to the therapy, rendering it of little use as a long-term PH treatment. It has been demonstrated that inhibition of cGMP breakdown in pulmonary vascular smooth muscle cells by sildenafil (an inhibitor of phosphodiesterase) is potentially a promising long-term treatment for PH [26].

14 Summary PH is a life-threatening disease of which the progression and cause is still under investigation. As we learn more about this disease, it becomes increasingly clear that the endothelium, its many secreted factors, and its innumerable associated pathways are monumental; current clinical treatment of PH indeed lies in ameliorating or mimicking endothelial function. The endothelial structures and pathways are vast and intertwined. In conclusion, the endothelium maintains a delicate and complicated control over the pulmonary vasculature, and further investigation into its many roles is a major component in the future of PH research. Acknowledgements  A.G. is supported by the Canadian Institute of Health Research and The Heart and Stroke Foundation of Quebec.

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B. Ross and A. Giaid

Fig. 5  Primary targets in the pharmacological treatment of pulmonary hypertension. The red arrows indicate the sites of therapeutic inhibition, whereas the green arrows represent therapeutic activation. cGMP

cyclic guanosine monophosphate. (Reproduced from [26] with permission. Copyright 2004 Massachusetts Medical Society. All rights reserved)

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Chapter 59

Coagulation and the Vessel Wall in Pulmonary Embolism Irene M. Lang

Abstract  Abnormal thrombus formation and resolution underlie many vascular disorders, including venous thromboembolism (VTE), stroke, hypertension, and myocardial infarction. Conditions that account for both venous and arterial thrombosis such as the antiphospholipid antibody syndrome, hyperhomocysteinemia, malignancies, infections, and hormone treatment provide a pathophysiological link between arterial and venous thrombosis. Furthermore, recent trials have demonstrated that patients with VTE are at increased risk of arterial thrombosis. The scope of this chapter is to define the characteristics of venous thrombosis and VTE, the role of soluble factors, and to emphasize the role of the vascular wall and of circulating cells, thus creating a new view of thrombosis, beyond classic coagulation and fibrinolysis. Keywords  Thrombosis • Pulmonary embolism • Venous thromboembolism • Embolus • Thromboembolic pulmonary hypertension

1 Introduction Abnormal thrombus formation and resolution underlie many vascular disorders, including venous thromboembolism (VTE), stroke, hypertension, and myocardial infarction. Conditions that account for both venous and arterial thrombosis such as the antiphospholipid antibody syndrome, hyperhomocysteinemia, malignancies, infections, and hormone treatment provide a pathophysiological link between arterial and venous thrombosis [1]. Furthermore, recent trials have demonstrated that patients with VTE are at increased risk of arterial thrombosis [2]. The scope of this chapter is to define the characteristics of venous thrombosis and VTE, the role of

I.M. Lang (*) Department of Internal Medicine, Division of Cardiology, Medical University of Vienna, Währinger Gürtel 18–20, 1090 Vienna, Austria e-mail: [email protected]

soluble factors, and to emphasize the role of the vascular wall and of circulating cells, thus creating a new view of thrombosis, beyond classic coagulation/fibrinolysis.

2 The Spectrum of VTE 2.1 Epidemiology of VTE VTE is understood as a continuum extending from deep vein thrombosis, thrombus in transit, acute pulmonary embolism (PE), to the rare sequela of VTE that is labeled “chronic thromboembolic pulmonary hypertension” (CTEPH) (Fig. 1) VTE occurs frequently (approximately 500–800 cases per million individuals per year) and carries a high morbidity and mortality, leading to sudden death in about 10% of patients, accounting for approximately 300,000 yearly clinical episodes and 50,000 deaths in the USA [3, 4]. The prevalence of PE among hospitalized patients in the USA according to data collected between 1979 and 1998 was 0.4% [5]. Corresponding figures for Europe are not available. About 50% of patients with proximal deep vein thrombosis have an associated, usually clinically asymptomatic, PE at lung scan [6]. The death rate from PE increases with age, and is higher in African-American than in white patients [7].

2.2 Varicose Veins and Deep Vein Thrombosis In humans, VTE originates in the deep veins. The most common anatomic alterations in the venous system that are risk factors for thrombosis are varicose veins. Increased venous pressure, hemodynamic consequences of arteriovenous communications, descending valvular incompetence, and primary connective tissue abnormalities have emerged as the

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_59, © Springer Science+Business Media, LLC 2011

851

852

Fig. 1  The spectrum of venous thromboembolism. Note that the timelines between an initial thromboembolic event and pulmonary embolism may be as long as 6 weeks, whereas the interval between an initial thromboembolic event and chronic thromboembolic pulmonary hypertension (CTEPH) may be decades

principal pathogenic theories of primary varicose vein formation. Endothelial nitric oxide synthase (eNOS) is involved in the regulation of resting and stimulated arterial tone by producing NO from the guanidino nitrogen of l-arginine. To investigate endothelial dysfunction in veins, the expression of eNOS and inducible nitric oxide synthase (iNOS) was analyzed in veins from 24 patients undergoing elective vein resection (mean age 56.1 ± 4 years, 14 females/10 males) and was compared with the expression in segments of greater saphenous veins from controls (mean age 56.4 ± 3.5 years, six females/six males, unpublished data). In addition, endothelial cell (EC) lysates freshly scraped from the vessel walls were used to determine the nitric oxide synthase activity by the conversion of l-[14C]arginine to l-[14C]citrulline. Analysis of the specimens revealed severe intimal and medial thickenings in all patients undergoing elective vein resections, and in two of nine veins harvested from controls. eNOS expression in the endothelium of varicose veins was dramatically reduced compared with that in controls as determined by protein measurement (1.5 ± 0.4 versus 6.1 ± 1.1, p = 0.0001) and on a messenger RNA (mRNA) level (unpublished data). In contrast, iNOS expression was not different between the groups. Analysis of EC scrapings demonstrated a decreased eNOS activity in varicose compared with control veins. These data illustrate that the endothelium of normal leg veins expresses high basal levels of eNOS. In contrast, in varicose veins eNOS disappears from the luminal endothelium. These alterations favor thrombosis.

I.M. Lang

hypercoagulable states such as deficiencies in protein S, protein C, and antithrombin III, mutations in factor V (e.g. “factor V Leiden”), the single point mutation in the 3¢ untranslated region of prothrombin (G20210A), acquired phospholipid antibodies, lupus anticoagulans, and a history of previous VTE, active cancer, and neurologic disease with extremity paresis. Setting-related factors are fracture (hip or leg), hip or knee replacement, major general surgery, major trauma, spinal cord injury, arthroscopic knee surgery, central venous lines, chemotherapy, immobility due to sitting (e.g. prolonged car or air travel), medical disorders causing bed rest for more than 3 days such as heart or acute respiratory failure, and congenital or acquired thrombophilia, hormone replacement therapy, and treatment with oral contraceptives. Apart from known alterations of the coagulation system, such as antithrombin 3 deficiency, factor V Leiden, and the G20210A variant of prothrombin [10], polymorphisms in ADRB2, a b2adrenergic receptor that functions as an inflammatory mediator, and lipoprotein lipase, a protein with a key role in lipid metabolism, have recently been independently associated with idiopathic venous thrombosis in large population-based studies [11].

3 The Balance Between Thrombosis and Thrombus Resolution: A New View of Thrombosis The generation of thrombin from its precursor prothrombin is the central event of blood coagulation, which is essential for hemostasis and the culprit in vascular thrombosis. It represents a highly regulated, dynamic, and rapid process. By contrast, the timely removal of thrombus results from the concerted action of plasmatic fibrinolysis and a complex vascular remodeling process [12], which is time-consuming (Fig. 2), and involves circulating cells [13] and cells within the vessel wall [12]. Thrombosis itself may be considered the first event in this timeline, which takes between seconds and several hours.

2.3 Risk Factors of VTE

3.1 Plasmatic Thrombosis and the Importance of D-Dimer, Endogenous Thrombin Although PE can occur in patients without any identifiable Potential and Phospholipid Antibodies predisposing factors, one or more of these factors are usually identified (secondary PE). The proportion of patients with idiopathic or unprovoked PE is estimated at 20% [8]. VTE is currently regarded as the result of the interaction between patient-related and setting-related risk factors [9]. Patientrelated predisposing factors are usually permanent, whereas setting-related predisposing factors are more often transient. Patient-related predisposing factors include age, primary

Thrombophilia can be identified in about half of all patients presenting with venous thrombosis. In association with venous and arterial thrombosis as well as pregnancy complications, the most commonly tested thrombophilic abnormalities have been protein C, protein S, and antithrombin deficiencies, the F5 R506Q (factor V Leiden) and F2 G20210A (prothrombin

59  Coagulation and the Vessel Wall in Pulmonary Embolism

Fig.  2  The balance between thrombosis and thrombus resolution. Although plasmatic thrombosis may occur within seconds, and is intrinsically disturbed in only a few patients, the process of resolution is time-consuming and is subjected to numerous disturbances, e.g. infection, inflammation, and autoimmunity

G20210A) mutations, and elevated levels of coagulation factor VIII [14]. Testing for hereditary thrombophilia generally does not alter the clinical management of patients with venous or arterial thrombosis or pregnancy complications and should not be performed on a routine basis. It appears that clinical factors are more important than laboratory abnormalities in predicting complications and recurrence. However, the critical question remains of whether coagulation/fibrinolytic parameters may be employed as biomarkers to predict recurrence and optimize the duration of anticoagulation. Fibrin degradation products (FDPs) are formed whenever fibrin is cleaved by enzymes (e.g. plasmin). The determination of FDPs is not useful as FDPs may derive from thrombus or as part of an unspecific inflammation. D-dimers are unique in that they are the breakdown products of a fibrin mesh that has been stabilized by factor XIII cross-linking the E element to two D elements as the final step in the generation of a thrombus. Plasmin cannot break down the bonds between one E unit and two D units. The protein fragment thus left over is a D-dimer. In the PREVENT trial (a randomized, double-blind, placebo-controlled trial comparing the combination of usual care and targeted low-dose warfarin with usual care plus a placebo), patients with idiopathic unprovoked VTE in the placebo group received 6 months of anticoagulation therapy. D-dimer was measured 7 weeks after discontinuation of oral anticoagulation therapy with warfarin. The subsequent recurrence rate was 12% per year in those with elevated D-dimer levels compared with 5.6% in those with normal D-dimer levels. The data suggest that D-dimer may be useful to prognosticate VTE recurrence after 6 months of oral anticoagulation therapy [15]. According to in  vitro experiments, thrombin generation occurs in two phases. Initially, small amounts of thrombin are produced after the activation of factor X by tissue factor/ factor VIIa. The initial thrombin propagates coagulation by activating platelets, factor V, and factor VIII. Factors VIIIa and IXa, which are generated by tissue factor/factor VIIa, combine on the surface of activated platelets, leading to

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factor X activation. This results in the generation of large amounts of thrombin, fibrin formation, and, ultimately, clot formation [16]. Thrombin activity can be defined by continuously measuring cleavage of a chromogenic or fluorescent substrate, resulting in a thrombin generation curve. From this curve, various parameters, including time until thrombin burst, peak amount of thrombin generation, velocity of thrombin generation, and total amount of thrombin generated (area under the thrombin generation curve), can be calculated. Peak thrombin generation may be utilized as another marker for the risk of recurrence [17], with a low degree of thrombin generation predicting a low likelihood of recurrence. These observations suggest that a major mechanism of recurrence may be persistent thrombosis. Residual (semi)organized thrombus in the vessel wall may represent an ongoing stimulus to coagulation. Antiphospholipid antibodies are a family of autoantibodies that exhibit a broad range of target specificities and affinities, all recognizing various combinations of phospholipids, phospholipid-binding proteins, or both. It is generally accepted that the major autoantigen for antiphospholipid antibodies is b2-glycoprotein I, which mediates the binding of antiphospholipid antibodies to target cells (i.e. ECs, monocytes, platelets, trophoblasts, etc.), leading to thrombosis and fetal loss. Antiphospholipid antibodies exert their mainly procoagulant effect by various mechanisms, e.g. inhibition of the activated protein C pathway, upregulation of the tissue factor pathway, inhibition of antithrombin III activity, disruption of the annexin V shield on membranes, inhibition of fibrinolysis and the action of b2-glycoprotein by increasing its membrane fixation, enhanced expression of adhesion molecules on ECs with increased adherence of leukocytes and in particular neutrophils, subsequent activation and degranulation of neutrophils, activation/degranulation of platelets, and activation of the complement cascade. In addition, antiphospholipid antibodies are a risk factor for recurrent pregnancy loss and obstetric complications in patients with the antiphospholipid syndrome [18].

3.2 Thrombus Resolution Is a Complex Vascular Remodeling Process The vascular wall is also an important contributor to the pathophysiologis processes of thrombosis, in addition to the velocity of blood flow and plasmatic coagulation (also addressed as Virchow’s triad). However, in contrast with the scientific interest in the mechanisms of thrombus formation that has primarily focused on the plasmatic components of thrombus formation [19, 20], a new view of thrombosis will address mechanisms of vascular remodeling in response to thrombosis as illustrated in Fig. 3.

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Fig. 3  A new view of thrombosis. (a) A hematoxylin–eosin stain of a histological section of the pulmonary artery from a human transplant donor. (b) A histological section contrasted with a trichrome stain of a freshly thrombosed pulmonary artery harvested from a patient who had succumbed to acute pulmonary embolism. (c) A subacute partially

organized occlusion of a pulmonary artery from another area of the same lung tissue as in (b), stained with hematoxylin–eosin. (d) A trichrome stain of a completely occluded pulmonary artery. The vascular lumen is filled with collagenous tissue that has replaced a previous thrombotic occlusion

A crucial physiologic function of the endothelium is to facilitate blood flow by providing an antithrombotic surface that inhibits platelet adhesion and clotting [21]. The intact endothelium represents a barrier separating platelets from adhesive substrates in the subendothelial connective tissue matrix. Disruption of the integrity of the vessel wall by mechanical or functional trauma allows circulating platelets to come in contact with and adhere to the thrombogenic subendothelial matrix. After disturbance, ECs undergo programmatic biochemical changes that result in their transformation to a prothrombotic surface [21]. Thrombus formation ensues once a critical mass of fibrinogen is cleaved, and serves to protect organ integrity by limiting vascular damage. Once the procoagulant stimulus has disappeared, the injured endothelium can often return to its unperturbed state. Overall, thrombus organization is very similar to the formation of granulation tissue [22]. Initially, on top of erythrocytes, platelets, fibrin, and eosinophils, neutrophils and subsequently monocytes are recruited into organizing thrombi (Fig. 2). Neutrophils are likely important for early thrombus retraction and lysis [13]. A primary function of neutrophils is the secretion of cytokines, proteases, and reactive oxygen species. Experimental neutropenia compromised

venous thrombus resolution in the rat [23]. Increased collagen and profibrotic growth factors were found in the venous wall of neutropenic rats [24]. Moreover, vein wall and intrathrombus urokinase and intrathrombus matrix metalloproteinase-9 activities were significantly diminished in neutropenic rats compared with controls, but no difference in thrombus neovascularization was observed. These data suggest a critical early role for neutrophils in postthrombotic vein wall remodeling.

3.2.1 The Endogenous Plasmatic and Tissular Fibrinolytic System Cell invasion and migration depend upon the action of highly regulated and specific proteases that can locally digest tissue proteins without the widespread damage associated with, for example, chronic inflammatory disease. This “targeted” proteolysis is accomplished in part by the plasminogen activation system, a versatile, temporally controlled enzymatic cascade that, when activated, generates locally expressed extracellular proteolytic activity [25]. In circulating blood, the same enzyme system functions to maintain vessel patency

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by specifically removing fibrin deposits from the vasculature (fibrinolysis) [26]. This diversity of function indicates that the plasminogen activation system is broadly used in human physiologis function, and that plasminogen activator (PA) activity must be precisely regulated. This regulation is achieved primarily at the level of PA inhibitors (PAIs). Four molecules inhibit PAs in vitro, including PAI-1, PAI-2, PAI3, and protease nexin 1, with PAI-1 being the primary physiological inhibitor of tissue-type PA (tPA) and urokinase-like (uPA) in  vivo. Activation of this system is initiated by the release of PAs (tPA and uPA) from cells in response to signals (e.g. growth factors, cytokines, hormones) liberated during tissue remodeling, inflammation, and thrombosis, and it leads to the formation of the broadly acting, trypsin-like protease plasmin. In addition, binding of tPA to ECs promotes its fibrinolytic activity and stimulates cell proliferation [27, 28]. ECs produce abundant PAI-1 that is associated primarily with the extracellular matrix, resulting in stabilization of its activity [29]. PAI-1 is a serine protease inhibitor and its main function is to inhibit tPA and uPA. The liver is the major source of plasma PAI-1 and its synthesis is stimulated by thrombin, endotoxin, various cytokines, lipoprotein (a), oxidized LDL, and other mediators [30]. Quiescent ECs express little or no PAI-1, but after exposure to inflammatory stimuli, the expression of PAI-1 is highly upregulated, which results in impaired fibrinolytic function [31]. Because of these findings, the plasminogen activation system was a primary target for research to understand thrombosis and thrombus resolution as it might provide a link between plasmatic fibrinolysis and vascular remodeling. Because pulmonary emboli do generally resolve within 1 year, it has been assumed that the pulmonary circulation harbors remarkable fibrinolytic capacity [32]. In accord with this paradigm, tPA is expressed in ECs and smooth muscle cells of human main pulmonary artery. These cells may become a significant source of luminal PA activity. In combination with a relative decrease of whole vessel wall PAI-1 expression, an increased net fibrinolytic activity is the consequence. Within hours after pulmonary thromboembolism, a sequential upregulation of fibrinolytic genes occurs in the wall of mainstem pulmonary artery [12]. One may speculate that the capacity to efficiently lyse pulmonary emboli may be developmentally required in humans to compensate for the increased rate of thromboembolic episodes due to erect posture. An elevated tPA-to-PAI-1 ratio reflects decreased steady-state expression of PAI-1, at the mRNA and protein levels, with free tPA rapidly accessible for thrombolysis in the pulmonary artery vessel wall [33]. The adventitia is an important source for PA activity, but does not account for differential fibrinolytic gene expression [33]. Although shear stress was shown to increase EC tPA mRNA and tPA protein release [34], tPA steady-state expression is not physiologically different between aorta and

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pulmonary artery [33]. However, more efficient macrophage recruitment in this vascular compartment [35] could contribute to differential fibrinolytic gene expression and enhanced spontaneous thrombus degradation.

3.2.2 Vascular Remodeling in Acute PE Pulmonary thromboemboli resolve in the majority of cases with restoration of normal hemodynamic conditions [36–38]. Resolution occurs through fibrinolysis, but also by mechanical fragmentation in the course of embolization [39], and through organization of the thromboembolus by invasion of capillary buds and fibroblasts with recanalization. Only in a small percentage of cases do venous thromboemboli fail to lyse, thereby resulting in CTEPH, or in occlusion of the deep veins. Immediately after clot formation, ECs proliferate and migrate to the site of endothelial damage. On the basis of the knowledge of a key role of the fibrinolytic system in thrombus organization, human tissues from patients who had died from acute PE were investigated. Several important observations were made. First, evidence was found for a staged process of thrombosis. Different stages of thrombus organization were found in a single individual specimen. Local expression of PAs and PAI-1 was observed in distinct patterns. tPA antigen was primarily detected in regions containing an intact endothelial lining, whereas uPA expression was initially restricted to monocytic cells within thromboemboli and subsequently in cells that appeared to be migrating from the vessel wall toward the thrombus. The distribution of uPA was in accord with published data on uPA expression in monocytes/macrophages [40] and with the proposed role of uPA in supporting cell migration [41]. High PAI-1 concentrations were detected in ECs directly in contact with fibrin, which is in good agreement with data showing PAI-1 induction by thrombin [42] and by transforming growth factor-b (TGF-b), a polypeptide growth factor released from platelets [43]. PAI-1 expression induced by picomolar concentrations of TGF-b occurs within 2–4  h [44]. In another model system, PAI-1 expression was observed within neoendothelial cells overgrowing vascular thrombi, as soon as 1 week after thrombosis [45]. An embolizing thrombus may cause local EC injury by impinging on the EC surface. Direct contact of ECs with fibrin has been shown to modulate a number of phenotypic properties, including loss of organization, severing of cell–cell contacts, and cell retraction [46]. In addition to enhancing the production of PAI-1 in the single layer of ECs lining the vessel wall, the deposition of a thromboembolus within a pulmonary vessel results in the generation of a new interface, which is subsequently penetrated by cells that are involved in the organization process. These cells are characterized by the concomitant production of proteases and protease inhibitors, initiating the entry of macrophages,

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and facilitating the degradation of fibrin and capillary sprouting [41]. Furthermore, local hypoxia, which has been shown to be a trigger for PAI-1 upregulation [47], could be a factor responsible for the elevation of PAI-1 level in the remodeling/ organization regions within the thrombi, and for the induction of connective tissue growth factor (CTGF) via a hypoxiaresponsive element in the CTGF promoter [48].

4 Vena Cava Stenosis Model To Study Thrombus Resolution To tease out mechanisms involved in thrombus resolution and organization, we have started to utilize a murine model of inferior vena cava (IVC) thrombosis [49]. In this model, thrombosis occurs within several hours after the ligation, and thereafter, because of continuous slow flow rather than no reflow, processes of resolution can be studied. In brief, adult female BALB/c mice (age 8 weeks, weight 18–20  g, Fig.  4a) are

Fig. 4  Vena cava stenosis model to study thrombus resolution. (a) The experiments were performed in BALB/c mice. (b) Induction of stagnant flow venous thrombosis as described in the text. (c) Representative histological sections are shown by day. The sizing bar represents 100 mm

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anesthetized with 100 mg/kg ketamine (Ketavet) and 5 mg/kg xylacine (Rompun) intraperitoneally. The IVC is exposed below the renal veins through a midline laparotomy incision. The intestines are retracted, and retroperitoneal blunt dissection of the infrarenal vena cava is performed to mobilize a 5-mm segment distal to the left renal vein. In contrast to the original description [49], endothelial denudation with a neurosurgical vascular clip is not performed because of a high rate of rupture of the IVC wall with this method. The injury to the endothelial layer occurring as a consequence of the manipulation is a sufficient prothrombotic stimulus. A 5-0 Prolene suture is placed alongside the vena cava (Fig. 4b). A stenosis is produced in the vein by tying a 4-0 silk suture around the IVC to include the Prolene suture. The Prolene suture is then pulled to allow blood to continue to pass up the vein. The intestines are replaced, and the abdominal wall is sutured. The mice are allowed to recover from anesthesia. Pain is treated with 0.05  mg/kg buprenorphine (Temgesic) subcutaneously every 8 h. Mice have access to water ad libitum and conventional commercial chow.

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In this model, thrombi resolve to approximately one sixth of their original size by 28 days after IVC ligation (Fig. 4c). Histologically and by gene array analyses (unpublished data), genes involved in matrix accumulation predominate by day 7 after ligation, followed by the appearance of genes associated with ingrowth of smooth muscle cells. By day 14 after the ligation, genes involved in inflammation are strongly expressed, and then disappear. Beyond day 14, genes mediating fibrosis, collagen synthesis, and scarring appear. In addition to understanding the natural history of thrombus organization, the model can be modified by manipulating mice, or employing genetically modified animals.

4.1 Mouse Thrombosis Model with Staphylococcal Infection Because of the clinical observation that infected intravenous leads may cause nonresolving pulmonary emboli, a model of thrombus infection was established. For infection, at day 1 after ligation 20  mg/kg of Staphylococcus aureus (ATCC 12600, S. aureus subsp. aureus Rosenbach) dissolved in 200 mL saline was injected into mice (eight per time point) via the tail vein. Control mice received an equivalent volume of saline alone. Staphylococcal infection delayed thrombus resolution in parallel with upregulation of TGF-b and CTGF. The data suggest that infection with Staphylococcus sp. enhances fibrotic vascular remodeling after thrombosis, resulting in misguided thrombus resolution [50].

4.2 Mouse Thrombosis Model of Conditional Fetal Liver Kinase-1 Deletion Currently, there exists no evidence that different rules underlie thrombus organization in the pulmonary circulation and that in the deep veins. Thrombosis of the veins of the lower extremities usually occurs without the prerequisite of a primary inflammatory stimulus [51]. However, histological evidence suggests that inflammatory cells immediately appear at the sites of attachment of the thrombus to the endothelium, in parallel with the appearance of capillary buds [12]. Angiogenesis is the growth of new capillary blood vessels (neovascularization). Angiogenesis is a key histologic feature of thrombus growth and organization. Previous animal studies demonstrated that recanalization of the vein may occur within 24  h of thrombus formation [13]. During the early phase of resolution, thrombi begin to contract and retract from the vein wall, which results in the formation of cell-lined pockets between the body of the thrombus and the intima of the vein wall. Over time, these channels coalesce and enlarge, and blood

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flow is restored through and around them. New vascular channels are also observed within the body of thrombi and contribute to restoration of a patent lumen [13]. The appearance of these channels is associated with the expression of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF), which are thought to drive neovascularization within the thrombus [52]. Recently, Modarai et al. showed that bonemarrow-derived, Tie-2-expressing cells are recruited into resolving thrombi. Many of these cells expressed a macrophage phenotype. However, these cells did not line the newly formed vessels either between the thrombus and the vein wall or within the thrombus itself. This led to the speculation that the revascularization process during thrombus resolution involves mature ECs derived from the vena venora (microvessels that supply the vein wall), and that the stimulus for revascularization may be provided by bone-marrow-derived progenitors within the thrombus, which may represent a population of plastic stem cells that orchestrate thrombus angiogenesis [13]. Two growth factors, bFGF and VEGF, are required for the initiation of vascular development [53]. VEGF interacts with specific tyrosine kinase receptors and stimulates receptor autophosphorylation, EC replication, and migration. In the mouse, fetal liver kinase-1 (flk-1) protein (corresponding to human VEGF receptor 2; KDR) is upregulated during thrombus organization. Contact of angioblasts and ECs is essential for the expansion of the vascular bed. Enhanced revascularization of thrombi has been reported following treatment with the proangiogenic factors IL-8 and VEGF [54, 55]. In an occlusive rodent model of thrombosis, daily intravenous application of recombinant human IL-8 led to increased recruitment of neutrophils, monocytes, and spindle-shaped cells (fibroblasts and ECs), and improved blood flow through the thrombus [54]. In rats, a single bolus injection of recombinant VEGF protein into newly formed venous thrombi under reduced flow conditions resulted in enhanced recanalization and organization, and stimulated the monocyte recruitment in a dose-dependent manner [55]. Although treatment with bFGF improved revascularization and blood flow in experimental thrombi, no effect was observed on thrombus weight, indicating no effect on thrombus resolution [56]. The proangiogenic factor epithelial neutrophil activating protein (ENA-78) and the angiostatic compound interferon inducible protein (IP-10) were also tested in the same study, and no effect was observed on thrombus neovascularization or resolution. Recently, adenovirus-mediated VEGF gene therapy enhanced venous thrombus recanalization and resolution in both rats and mice. The authors proposed that this effect was due to macrophage recruitment into the thrombi [57]. Distinct cell surface receptors, including platelet EC adhesion molecule 1 (PECAM-1, CD31) and vascular endothelial (VE)-cadherin, mediate important cell–cell adhesions [58]. As another example for these important biological

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domains, the soluble form of the cell adhesion molecule P-selectin was recently identified as risk factor for VTE in cancer patients [59]. In our system, we were able to employ mice with conditional cell-specific deletion of angiogenic molecules (tie-2/cre, VEGF receptor 2/flk-1/flx) as well as mice with constitutive deletions of PECAM-1 (CD31-/-). We were able to demonstrate a downregulation of the angiogenic molecules VEGF and VE-cadherin, and of leukocyte-associated genes CD45 and CD68. These observations paralleled a loss of isolectin B4-positive microvessels within organizing thrombus. The data are in good accord with the concept that angiogenesis is required for thrombus resolution, and that it functions in concert with macrophage recruitment.

5 Models for Nonresolving Thrombosis 5.1 Vascular Remodeling in CTEPH CTEPH is characterized by predominantly major-vessel obstructions [60] resulting in increased pulmonary vascular resistance. The molecular mechanisms underlying thrombus persistence are unknown [61], but the investigation of CTEPH has revealed new concepts of thrombosis. The natural history of acute pulmonary thromboemboli is for them to undergo almost complete resolution within 6 months [62]. However, in 0.1–5% of survived acute events [63–66] thromboemboli undergo an organization process leading to permanent fibrotic obstruction of the pulmonary vascular bed. CTEPH is largely understood as of thromboembolic origin. However, patients with CTEPH lack classic plasmatic thromboembolic risk factors [67], such as antithrombin III deficiency, alterations in proteins C and S, and factor V Leiden [68]. Furthermore, neither systemic [69] nor local [70] imbalances of fibrinolytic proteins in the pulmonary arterial wall have been detected. In addition, it is not possible to induce the disease in animal models by repeated embolizations [71], suggesting alternative nonthromboembolic hypotheses [72]. The difficulty to induce CTEPH by repeated release of preformed clots from the IVC of mongrel dogs [71] was resolved by a thorough biochemical dissection of factors contributing to increased vascular fibrinolytic activity in these animals [73]. It was found that high plasma levels of uPA activity are present in this species. Furthermore, uPA is associated with canine platelets and mediates rapid clot lysis. We investigated the EC-associated fibrinolytic system in major pulmonary vessels free of thrombus of patients with CTEPH. To dissect the endothelial fibrinolytic system in nonresolving pulmonary thromboemboli, conditions were established to culture ECs from unthrombosed main pulmonary arteries of patients during surgical pulmonary endarterectomies. The levels of tPA antigen and PAI-1 activity in

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media conditioned by primary ECs harvested from areas free of thrombus were not significantly different between patients with chronic thromboemboli and organ donors. Cultured patient pulmonary arterial ECs increased the secretion of tPA and PAI-1 in response to thrombin similar to donor pulmonary artery ECs [70]. In recent years, it has been recognized that major-vessel remodeling and classic small-vessel pulmonary arteriopathy coexist in CTEPH [74], suggesting a complex remodeling process involving factors beyond traditional thrombosis [75]. As a first step, we analyzed the pulmonary vascular obstruction of CTEPH by histology, in situ hybridization, and quantitative gene expression. Red, fibrin-rich thrombi within thromboendarterectomy specimens lined with a single layer of ECs exhibited high levels of PAI-1 antigen and in situ hybridization signal, in comparison with the signal present in the ECs from noninvolved areas of patients’ pulmonary arteries (p < 0.001) [76]. Yellowish-white thrombi were composed of smooth muscle cells and ECs in numerous vessels that stained prominently for PAI-1 antigen. Both types of cells within the highly organized tissues also exhibited elevated PAI-1 mRNA levels in comparison with patient pulmonary artery specimens that were free of thrombus (p < 0.02). The prevalence of PAI-1 expression within pulmonary thromboemboli suggests that this inhibitor may play a role in the stabilization of vascular thrombi, and provide grounds for in situ thrombosis. CTEPH serves as a model disease, yielding clinical data and surgical thrombus specimens, thus permitting translational insights into the mechanisms underlying an extreme variant of thrombus nonresolution. For example, the observation that splenectomy is a condition increasing the risk of CTEPH at least tenfold above the background risk [75] has led to the concept that phospholipids from recycling cell membranes that cannot be deposited in the spleen may alter the propensity of vascular thrombosis. Furthermore, the observation that infected intravenous lines increase the risk for CTEPH has led to the discovery of S. aureus promoting thrombosis and thrombus persistence [50]. In situ thrombosis is triggered by abnormal gene expression, i.e. increased PAI-1 expression [76], increased von Willibrand factor expression (unpublished data), inadequate expression of antagonists of thrombosis [77], abnormal TGF-b receptor expression [78], and slow flow. Key mechanisms that have to be addressed in the future concern angiogenesis, leukocyte–EC interactions, immune mechanisms, EC regeneration, and the effects of endothelial shear stress in the context of thrombosis/ thrombus organization. Research that is able to define the mechanisms underlying the vascular remodeling processes following thrombosis will provide tools to modify vascular patency after thrombosis (Fig. 5).

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Fig. 5  Clinical models for nonresolving thrombosis

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860 37. Paraskos JA, Adelstein SJ, Smith RE et al (1973) Late prognosis of acute pulmonary embolism. N Engl J Med 289:55–58 38. Murphy ML, Bulloch RT (1968) Factors influencing the restoration of blood flow following pulmonary embolization as determined by angiography and scanning. Circulation 38:1116–1126 39. Wagenvoort CA (1995) Pathology of pulmonary thromboembolism. Chest 107:10S–17S 40. Samad F, Schneiderman J, Loskutoff D (1997) Expression of fibrinolytic genes in tissues from human atherosclerotic aneurysms and from obese mice. Ann N Y Acad Sci 811:350–358 41. Pepper MS, Montesano R, Mandriota SJ, Orci L, Vassalli JD (1996) Angiogenesis: a paradigm for balanced extracellular proteolysis during cell migration and morphogenesis. Enzyme Protein 49:138–162 42. Fearns C, Samad F, Loskutoff DJ (1995) Synthesis and localization of PAI-1 in the vessel wall. Harwood, Amsterdam 43. Fujii S, Hopkins WE, Sobel BE (1991) Mechanisms contributing to increased synthesis of plasminogen activator inhibitor type 1 in endothelial cells by constituents of platelets and their implications for thrombolysis. Circulation 83:645–651 44. Sawdey M, Podor TJ, Loskutoff DJ (1989) Regulation of type 1 plasminogen activator inhibitor gene expression in cultured bovine aortic endothelial cells. Induction by transforming growth factorbeta, lipopolysaccharide, and tumor necrosis factor-alpha. J Biol Chem 264:10396–10401 45. Sawa H, Fujii S, Sobel BE (1992) Augmented arterial wall expression of type-1 plasminogen activator inhibitor induced by thrombosis. Arterioscler Thromb 12:1507–1515 46. Kadish JL, Butterfield CE, Folkman J (1979) The effect of fibrin on cultured vascular endothelial cells. Tissue Cell 11:99–108 47. Gertler JP, Perry L, L’Italien G et al (1993) Ambient oxygen tension modulates endothelial fibrinolysis. J Vasc Surg 18:939–946 48. Hong KH, Yoo SA, Kang SS, Choi JJ, Kim WU, Cho CS (2006) Hypoxia induces expression of connective tissue growth factor in scleroderma skin fibroblasts. Clin Exp Immunol 146:362–370 49. Singh I, Burnand KG, Collins M et al (2003) Failure of thrombus to resolve in urokinase-type plasminogen activator gene-knockout mice: rescue by normal bone marrow-derived cells. Circulation 107:869–875 50. Bonderman D, Jakowitsch J, Redwan B et al (2008) Role for staphylococci in misguided thrombus resolution of chronic thromboembolic pulmonary hypertension. Arterioscler Thromb Vasc Biol 28:678–684 51. Stein PD, Evans H (1967) An autopsy study of leg vein thrombosis. Circulation 35:671–681 52. Waltham M, Burnand KG, Collins M, Smith A (2000) Vascular endothelial growth factor and basic fibroblast growth factor are found in resolving venous thrombi. J Vasc Surg 32:988–996 53. Beck L Jr, D’Amore PA (1997) Vascular development: cellular and molecular regulation. FASEB J 11:365–373 54. Wakefield TW, Linn MJ, Henke PK et al (1999) Neovascularization during venous thrombosis organization: a preliminary study. J Vasc Surg 30:885–892 55. Waltham M, Burnand KG, Collins M, McGuinness CL, Singh I, Smith A (2003) Vascular endothelial growth factor enhances venous thrombus recanalisation and organisation. Thromb Haemost 89:169–176 56. Varma MR, Moaveni DM, Dewyer NA et  al (2004) Deep vein thrombosis resolution is not accelerated with increased neovascularization. J Vasc Surg 40:536–542 57. Modarai B, Humphries J, Gossage JA et  al (2008) Adenovirusmediated VEGF gene therapy enhances venous thrombus recanalization and resolution. Arterioscler Thromb Vasc Biol 28: 1753–1759

I.M. Lang 58. Woodfin A, Voisin MB, Nourshargh S (2007) PECAM-1: a multi-functional molecule in inflammation and vascular biology. Arterioscler Thromb Vasc Biol 27:2514–2523 59. Ay C, Simanek R, Vormittag R et al (2008) High plasma levels of soluble P-selectin are predictive of venous thromboembolism in cancer patients: results from the Vienna Cancer and Thrombosis Study (CATS). Blood 112:2703–2708 60. Moser KM, Auger WR, Fedullo PF (1990) Chronic major-vessel thromboembolic pulmonary hypertension. Circulation 81:1735–1743 61. Lang IM (2004) Chronic thromboembolic pulmonary hypertension – not so rare after all. N Engl J Med 350:2236–2238 62. Menendez R, Nauffal D, Cremades MJ (1998) Prognostic factors in restoration of pulmonary flow after submassive pulmonary embolism: a multiple regression analysis. Eur Respir J 11:560–564 63. Becattini C, Agnelli G, Pesavento R et  al (2006) Incidence of chronic thromboembolic pulmonary hypertension after a first episode of pulmonary embolism. Chest 130:172–175 64. Pengo V, Lensing AW, Prins MH et al (2004) Incidence of chronic thromboembolic pulmonary hypertension after pulmonary embolism. N Engl J Med 350:2257–2264 65. Ribeiro A, Lindmarker P, Johnsson H, Juhlin-Dannfelt A, Jorfeldt L (1999) Pulmonary embolism: one-year follow-up with echocardiography Doppler and five-year survival analysis. Circulation 99:1325–1330 66. Fedullo PF, Auger WR, Kerr KM, Rubin LJ (2001) Chronic thromboembolic pulmonary hypertension. N Engl J Med 345: 1465–1472 67. Wolf M, Boyer-Neumann C, Parent F et al (2000) Thrombotic risk factors in pulmonary hypertension. Eur Respir J 15:395–399 68. Lang IM, Klepetko W, Pabinger I (1996) No increased prevalence of the factor V Leiden mutation in chronic major vessel thromboembolic pulmonary hypertension (CTEPH). Thromb Haemost 76:476–477 69. Olman MA, Marsh JJ, Lang IM, Moser KM, Binder BR, Schleef RR (1992) Endogenous fibrinolytic system in chronic large-vessel thromboembolic pulmonary hypertension. Circulation 86:1241–1248 70. Lang IM, Marsh JJ, Olman MA, Moser KM, Schleef RR (1994) Parallel analysis of tissue-type plasminogen activator and type 1 plasminogen activator inhibitor in plasma and endothelial cells derived from patients with chronic pulmonary thromboemboli. Circulation 90:706–712 71. Marsh JJ, Konopka RG, Lang IM et al (1994) Suppression of thrombolysis in a canine model of pulmonary embolism. Circulation 90:3091–3097 72. Egermayer P, Peacock AJ (2000) Is pulmonary embolism a common cause of chronic pulmonary hypertension? Limitations of the embolic hypothesis. Eur Respir J 15:440–448 73. Lang IM, Marsh JJ, Konopka RG et al (1993) Factors contributing to increased vascular fibrinolytic activity in mongrel dogs. Circulation 87:1990–2000 74. Hoeper MM, Mayer E, Simonneau G, Rubin LJ (2006) Chronic thromboembolic pulmonary hypertension. Circulation 113:2011–2020 75. Bonderman D, Wilkens H, Wakounig S et al (2009) Risk factors for chronic thromboembolic pulmonary hypertension. Eur Respir J 33:325–331 76. Lang IM, Marsh JJ, Olman MA, Moser KM, Loskutoff DJ, Schleef RR (1994) Expression of type 1 plasminogen activator inhibitor in chronic pulmonary thromboemboli. Circulation 89:2715–2721 77. Lang IM, Moser KM, Schleef RR (1996) Expression of Kunitz protease inhibitor-containing forms of amyloid b-protein precursor within vascular thrombi. Circulation 94:2728–2734 78. Hong KH, Lee YJ, Lee E et al (2008) Genetic ablation of the Bmpr2 gene in pulmonary endothelium is sufficient to predispose to pulmonary arterial hypertension. Circulation 118:722–730

Chapter 60

Alveolar Epithelial Fluid Transport in Lung Injury Hans G. Folkesson

Abstract  This chapter discusses the regulation of lung fluid transport by lung epithelial active ion transport mechanisms. Ion transport occurs across both the alveolar and the distal airway epithelium. Both experimental models of pulmonary edema and clinical studies are considered to demonstrate how active Na+ and active Cl- transport participate and regulate alveolar edema resolution. Some of the material in this chapter has been discussed in recent reviews. For decades, it was believed that Starling forces, i.e. differences in hydrostatic and protein osmotic pressures, accounted for removal of excess air space fluid. This misconception remained partly because experiments measuring solute fluxes across the lung epithelial and endothelial barriers were done at room temperature and the animal species used was the dog, a species that later was demonstrated to have a very low rate of active ion and fluid transport. Also, until the late 1970s and early 1980s, there were few good animal models to study the resolution of pulmonary edema. Furthermore, isolation and culture of alveolar epithelial type II cells was just evolving to become a useful experimental technique. Although removal of interstitial pulmonary edema by lung lymphatics and lung microcirculation had been discussed by Staub in his review of pulmonary edema, there was still no information on how pulmonary edema was removed from the distal air spaces of the mature lung. Keywords  Lung fluid transport • Ion transportation • Airway epithelium • Pulmonary edema • Vascular permeability

1 Introduction This chapter discusses the regulation of lung fluid transport by lung epithelial active ion transport mechanisms. Ion transport occurs across both the alveolar and the distal airway epiH.G. Folkesson (*) Department of Physiology and Pharmacology, Northeastern Ohio Universities College of Medicine, 4209 Street Route 44, Rootstown, OH 44272-00, USA e-mail: [email protected]

thelium. Both experimental models of pulmonary edema and clinical studies are considered to demonstrate how active Na+ and active Cl− transport participate and regulate alveolar edema resolution. Some of the material in this chapter has been discussed in recent reviews [1, 2]. For decades, it was believed that Starling forces, i.e. differences in hydrostatic and protein osmotic pressures, accounted for removal of excess air space fluid. This misconception remained partly because experiments measuring solute fluxes across the lung epithelial and endothelial barriers were done at room temperature [3] and the animal species used was the dog, a species that later was demonstrated to have a very low rate of active ion and fluid transport [4]. Also, until the late 1970s and early 1980s, there were few good animal models to study the resolution of pulmonary edema. Furthermore, isolation and culture of alveolar epithelial type II cells was just evolving to become a useful experimental technique. Although removal of interstitial pulmonary edema by lung lymphatics and lung microcirculation had been discussed by Staub in his review of pulmonary edema [5], there was still no information on how pulmonary edema was removed from the distal air spaces of the mature lung.

2 Lung Fluid Absorption The human lung consists of a series of highly branched hollow tubes that end blindly in alveoli, with conducting airways (cartilaginous trachea, bronchi, and membranous bronchioles) occupying the first 16 airway generations [6]. Gas exchange occurs in the branches making up the last seven generations, which include respiratory bronchioles, alveolar ducts, alveolar sacs, and alveoli [7]. The airways, approximately 1.4 m2, and alveoli, approximately 143 m2, constitute the interface between the lung internal environment and the external environment and are lined by a continuous epithelium [8]. Distally, the airway epithelium is composed of polarized epithelial cells having the ability to transport Na+ and Cl−, including ciliated Clara cells and nonciliated cuboidal cells. The alveoli themselves are composed of a thin alveolar epithelium (0.1-0.2 mm)

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that covers 99% of the air space surface area in the lung and contains thin, squamous type I cells and cuboidal type II cells [8]. The alveolar type I cell covers 95% of the alveolar surface [8]. The close apposition between the alveolar epithelium and the vascular endothelium facilitates the efficient exchange of gases, but forms a tight barrier to movement of liquid and proteins from the interstitial and vascular spaces, thus ­assisting in maintaining relatively dry alveoli [9]. The large alveolar surface area favors the idea that most fluid ­absorption occurs at the alveolar level, although active fluid ­absorption may occur across all segments of the pulmonary epithelia. The contribution of each anatomic segment of the lung to fluid absorption remains to be established. The tight junction is another critical structure for the barrier function of the alveolar epithelium. This junction connects adjacent epithelial cells near their apical surfaces, thereby maintaining apical and basolateral cell polarity [10]. Ion transporters are asymmetrically distributed on opposing cell surfaces, conferring vectorial transport properties to the epithelium. It was originally thought that tight junctions were rigid structures, physically restricting passage of larger molecules. However, the permeability of tight junctions is dynamic and regulated by cytoskeletal proteins and intracellular Ca2+ concentration [10]. Studies of tight junctions in the alveolar epithelium indicate that diffusion of water-soluble solutes between alveolar epithelial cells is much slower than through the intercellular junctions of the adjacent lung capillary endothelium [5, 11, 12]. In addition, studies of protein flux across the endothelial–epithelial barrier of the sheep lung suggested that 92% of the resistance to albumin flux resides in the epithelium [13]. Removal of large quantities of soluble protein from the air spaces appears to occur primarily by restricted diffusion [14, 15], although there is evidence for endocytosis and transcytosis of albumin across alveolar epithelium [14–18]. The general model for transepithelial fluid movement is that active salt transport drives osmotic water transport. This paradigm has proven to be true for fluid absorption from the distal airs paces on the basis of several animal and human studies [19]. The results of in vivo studies demonstrated that changes in hydrostatic or protein osmotic pressures cannot account for removal of excess fluid from the distal air spaces [20–23]. Pharmacologic inhibitors of Na+ transport also reduce the rate of fluid absorption in the lungs [4, 24–26]. Additionally, there is good evidence that isolated alveolar epithelial cells actively transport Na+ and other ions [27–29]. Multiple preparations have been used to study fluid and protein transport from the distal air spaces, including isolated perfused lungs, in situ lung perfusions, surface fluorescence, and in  vivo lung preparations (30  min to 144  h). The advantages and disadvantages of these preparations have been reviewed in some detail [1, 19]. The initial in vivo evidence of active ion transport that may account for pulmonary edema fluid removal in mature lungs came from anesthetized, ventilated sheep [21, 22]. In those

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studies, the key discovery was that isosmolar fluid absorption occurred in spite of a rising alveolar protein concentration and protein oncotic pressure over 4-24 h. After 4 and 24 h, air space protein concentration had risen from approximately 6.5–8.4 g/dL, respectively, to 12.9 g/dL. Plasma protein concentration never changed. This rise in protein concentration was equivalent to an increase in air space protein osmotic pressure (oncotic pressure) from 25 to 65 cmH2O. As the epithelial barrier was intact and prevented protein leak and no rapid protein absorption or secretion occured in the lung, the only way that the protein concentration could have increased was if water had been absorbed. This process has since been confirmed in multiple animal species and humans [24–26]. Elimination of ventilation to one lung did not change the rate of alveolar fluid clearance, thus ruling out changes in transpulmonary airway pressure as a major determinant of alveolar fluid clearance [20]. Furthermore, if active ion transport was responsible for alveolar fluid clearance, then alveolar fluid clearance should be temperature-dependent. In fact, in an in situ rat lung preparation, alveolar fluid clearance was inhibited by low temperature [30]. This was also true in an in situ perfused goat lung preparation [31] and in ex vivo human lung studies [26]. Additional evidence for active ion transport was obtained with the use of pharmacologic blockade of Na+ uptake by amiloride, an inhibitor of Na+ uptake by the apical membrane of alveolar and distal airway epithelia. Amiloride inhibits between 40 and 70% of basal alveolar fluid clearance in sheep, rabbit, rat, guinea pig, mouse, and human lungs [1]. To further explore the role of active Na+ transport, experiments were designed to inhibit the Na+/K+ATPase. This turned out to be more challenging in vivo since ouabain, the commonly used Na+/K+-ATPase blocker, is cardiotoxic. However, in isolated rat lungs, ouabain inhibits more than 90% of alveolar fluid clearance [32]. Following the development of an in situ sheep preparation for ­measuring alveolar fluid clearance, it was reported that ouabain inhibited more than 90% of alveolar fluid clearance [20].

3 Ion Transport in Alveolar and Distal Airway Epithelia The success in isolating and culturing alveolar epithelial type II cells was pivotal to the researchers’ success in studying the transport properties of these cells. Initial studies showed that when alveolar epithelial type II cells were grown on a nonporous surface such as plastic, they formed a continuous confluent layer of polarized cells [33, 34]. Then, after 3–5 days, small domes of fluid were observed where the substratum was detached. These domes were later shown to form from active ion transport from the apical to the basal surface, because they were inhibited by both the replacement of Na+ by another ­cation and pharmacologic inhibitors of Na+ transport, such as amiloride

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Fig. 1  Na+ absorption occurs from the apical surface of both alveolar epithelial type I and alveolar epithelial type II cells via the epithelial Na+ channel (ENaC) and via cyclic-nucleotide-gated (CNG) channels (seen only in alveolar epithelial type I cells). Cl− transport occurs through the cystic fibrosis transmembrane conductance regulator in alveolar epithelial type I and type II cells. K+ may be transported from the alveolar epithelial cells via basolateral K+ channels in alveolar epithelial type I and type II cells. Aquaporin 5 is expressed at the apical cell membrane of the alveolar epithelial type I cell. The basolaterally expressed Na+/ K+-ATPase provides the driving force for Na+ absorption through the ENaC and CNG channels in the apical membrane

and ouabain. More information on the nature of ion transport across alveolar epithelial type II cells was gained by culturing these cells on porous supports and mounting them in Using chambers and measuring short-circuit current (Isc) and ion flux under voltage-clamp conditions [1, 33]. Na+ entering the epithelial cells passively through specialized pathways apically is pumped out of the cells basolaterally by the Na+/K+-ATPase (Fig. 1). Because of its continuous pumping, the Na+ electrochemical potential is lower inside the cell, Na+ flows down its electrochemical gradient, and is extruded basolaterally. Owing to the pump activity, K+ electrochemical potential is larger inside the cell, and K+ leaks through the basolateral cell membrane and is then recycled by the Na+/K+-ATPase. Na+ entry pathways into alveolar epithelial type II cells are numerous (Fig.  1). Amiloride inhibited dome formation [33, 34] and decreased Isc in in vitro studies [35], an observation that supported the critical importance of Na+ uptake through an amiloride-sensitive pathway present in the apical membrane of the alveolar epithelial cells, e.g. the epithelial Na+ channel (ENaC). A detailed discussion of pharmacologic, biophysical, and molecular bases for fluid clearance across the alveolar and distal airway epithelium is available in other reviews [1, 36]. The role of alveolar epithelial type I cells in vectorial fluid transport in the lung is becoming clearer as these cells have now been successfully isolated. On the basis of studies in freshly isolated alveolar epithelial type I cells, it has been shown that these cells have a high osmotic permeability to water with apical surface expression of aquaporin 5 [37] (Fig. 1). Also, studies have reported that the Na+/K+-ATPase is expressed in the alveolar epithelial type I cells [28, 29, 38] (Fig. 1). The presence of the Na+/K+-ATPase could be

consistent with a role for the alveolar epithelial type I cell in vectorial fluid transport, although this is not conclusive, because the Na+/K+-ATPase can also be used to maintain cell volume. Recent data demonstrate that alveolar epithelial type I cells express a multitude of Na+ transporting proteins, such as the amiloride-sensitive ENaC and amiloride-insensitive cyclic-nucleotide-gated Na+ channels [29] (Fig. 1). There are also new data confirming the importance of ENaC for alveolar fluid clearance using RNA interference (RNAi) against the aENaC subunit [39]. In those studies, inhibition of aENaC expression by specific small interfering RNA against aENaC attenuated the amiloride sensitivity of both normal and stimulated alveolar fluid clearance. In addition, the alveolar epithelial type I cells express Cl− transporters, i.e. the cystic fibrosis transmembrane regulator (CFTR), and thus have the ion transporters needed to participate in vectorial ion transport and clearance of alveolar fluid. The large surface area that the alveolar epithelium covers in the lung suggests that removal of edema fluid from the lung may primarily occur across the alveolar epithelium. However, it has been demonstrated that the distal airway epithelium also actively transports Na+, a process that depends on amilorideinhibitable uptake of Na+, likely by ENaC, on the apical surface and extrusion of Na+ through a basolateral Na+/K+-ATPase [1]. Clara cells actively absorb and transport Na+ from the apical to the basal surface [40]. This information provides support for a possible role of distal airway epithelia in fluid clearance. Thus, even though their surface area is limited, a contribution from distal airway epithelia to the overall fluid transport is likely, especially as cells from distal airway epithelia primarily transport salt from the apical to the basolateral surface.

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4 Regulation of Lung Fluid Absorption This section considers how the rate of vectorial fluid transport across the distal pulmonary epithelium is regulated by catecholamine-dependent [cyclic AMP (cAMP)-dependent] or catecholamine-independent mechanisms. Studies in newborn animals indicated that endogenous catecholamine release, particularly epinephrine, is critical for stimulation of reabsorption of fetal lung fluid from the newborn lung air spaces [41, 42]. In most adult mammalian species, b2-adrenoceptor stimulation increases alveolar fluid clearance [43, 44]. The stimulatory effect occurs rapidly after administration of epinephrine or terbutaline, and is completely prevented by a nonspecific b-receptor antagonist (propranolol).

4.1 Catecholamine Regulation The increased alveolar fluid clearance by b2-adrenoceptor agonists can be prevented by amiloride or RNAi against aENaC, indicating that stimulation is related to increased transepithelial Na+ transport [39]. In anesthetized ventilated sheep, terbutaline-induced stimulation of alveolar fluid clearance was also associated with an increase in lung lymph flow, a finding that reflected increased removal of the alveolar fluid volume to the lung interstitium [43]. Although terbutaline increased pulmonary blood flow, this is not important, because control studies with nitroprusside, an agent that increased pulmonary blood flow, did not increase alveolar fluid clearance. Other studies have demonstrated that b-adrenoceptor agonists increased alveolar fluid clearance in rats, dogs, guinea pigs, mice, and humans [1] (Table 1). The existence of b1- and b2-adrenoceptors on alveolar epithelial type II cells has been shown in  vivo by autoradiographic and immunochemistry techniques [1]. It has been difficult to quantify the effect of catecholamines on the rate of alveolar fluid clearance and edema resolution in humans [45]. However, studies of alveolar fluid clearance in isolated human lungs have shown that b-adrenoceptor agonist therapy increases alveolar fluid clearance, and this increased alveolar fluid clearance can be inhibited by propranolol or amiloride [26]. The magnitude of the stimulatory effect is similar to that observed in other species, with a b-adrenoceptor-agonist-dependent doubling of alveolar fluid clearance over baseline levels. These data are particularly important because aerosolized b-adrenoceptor agonist treatment in some patients with pulmonary edema might be used to accelerate the resolution of alveolar edema. On the basis of in  vitro studies, it was proposed that increased intracellular cAMP levels, such as those observed after b-adrenoceptor stimulation, resulted in increased

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Na+ transport across alveolar epithelial type II cells by an independent upregulation of apical Na+ conductive pathways and basolateral Na+/K+-ATPases. Proposed mechanisms for upregulation of Na+ transport proteins by cAMP include augmented Na+ channel open probability, increased Na+/K+ATPase a-subunit phosphorylation, and increased delivery of ENaCs to the apical membrane and of Na+/K+-ATPases to the basolateral cell membrane [1, 46] (Table 1). Most experimental studies have attributed a role for active Na+ transport in the apical-to-basolateral transport of salt and water across the alveolar epithelium of the lung. The potential role of Cl−, especially in mediating the cAMP-mediated upregulation of fluid clearance across distal lung epithelium, has also been the subject of a few recent studies. A study in cultured alveolar epithelial cells suggested that vectorial Cl− transport across the alveolar epithelium occurs paracellularly under basal conditions and perhaps transcellularly in the presence of cAMP stimulation [47]. A second study in cultured alveolar epithelial type II cells suggested that cAMP-mediated apical Na+ uptake may depend on an initial Cl− uptake [48]. A third study of cultured alveolar epithelial type II cells under apical surface–air interface conditions reported that b-adrenoceptor agonists produced acute activation of apical Cl− channels with enhanced Na+ absorption [49] (Table 1). However, the results of these studies are considered inconclusive [50] because the data depend on cultured cells of an uncertain phenotype. Furthermore, studies of isolated alveolar epithelial type II cells do not address the possibility that vectorial fluid transport may be mediated by other epithelial cells, including alveolar epithelial type I cells as well as distal airway epithelial cells. To better define the role of Cl− transport for active salt and water transport across the distal pulmonary epithelia, in  vivo lung studies were designed to study mechanisms regulating Cl− transport [51]. This approach is important because studies in several species have indicated that both distal airway and air-space epithelia are capable of ion transport and both ENaC and CFTR are expressed in alveolar and distal airway epithelia. Inhibition and ion substitution studies ­demonstrated that Cl− transport was necessary for basal alveolar fluid clearance. The role of CFTR under basal and cAMP-stimulated ­conditions was tested using conditions in which CFTR was not functional because of failure in CFTR trafficking to the cell membrane, the most common human mutation in cystic fibrosis (D1F508). The results suggested that CFTR was essential for cAMP-mediated upregulation of fluid clearance from the distal air spaces, because alveolar fluid clearance could not be increased in D1F508 mice either with b-adrenoceptor agonists or with forskolin [51] (Table 1). Studies using pharmacologic CFTR inhibition in both mouse and human lungs with glibenclamide or CFTRInh-172 supported the results [51, 52]. Although CFTR absence in upper airways results in

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Table 1  Overview of factors that regulate transepithelial ion transport across alveolar and airway epithelia and modulate lung fluid balance Factor Effect on transepithelial ion transport or lung fluid transport b-Adrenergic agonists Dopamine Cyclic AMP Cl− transport (CFTR) Glucocorticoids Mineralocorticoids Thyroid hormones Growth factors (KGF, EGF, TGFa) Cytokines (TNFa, IL-1, TGF-b1) Serine proteases Hyperoxia

Hypoxia Nitric oxide and nitrate products Anesthetics

Increase Na+ transport and fluid clearance [24, 33, 35, 43, 56, 86, 92, 93] Down-regulate fluid clearance with prolonged delivery [94, 95] Increase fluid clearance [67, 68] No effect [96] Increase fluid clearance [97, 98] Increase Na+ transport [48, 49] Increase Na+ transport and fluid clearance [48, 49, 51, 52] Increase fluid clearance [56, 99], ENaC, and Na+/K+-ATPase expression [100, 101] No effect in alveolar cells [102] or in upper airways [103] Increase fluid clearance [57, 58] Increase fluid clearance [56] and Na+/K+-ATPase expression [59] Increase fluid clearance [60–64] and Na+/K+-ATPase activity in alveolar epithelial type II cells [63] Increase fluid clearance [66, 104] Decrease fluid clearance [83, 84, 105] Increase Na+ transport and fluid clearance [70, 71] Increase Na+ transport [32, 106, 107] and Na+/K+-ATPase expression [108] No effect [109] Decrease Na+ transport and fluid clearance [110] Decrease Na+ transport [75, 111] and fluid clearance [72] Decrease Na+ absorption [81, 82, 112, 113] Decrease fluid clearance [77, 79] No effect [114]

enhanced Na+ absorption, the data in these studies provide evidence that CFTR absence prevents cAMP-upregulated distal air space fluid clearance, a finding that is similar to the results from studies on the importance of CFTR in mediating cAMP-stimulated Na+ absorption in human sweat ducts [53]. Additional studies suggested that the lack of CFTR results in greater accumulation of pulmonary edema in hydrostatic stress, thus demonstrating the potential physiologic importance of CFTR in upregulating fluid transport from the distal air spaces [51]. There is new evidence that functional CFTR Cl− channels are present in both adult rat alveolar epithelial type I and adult rat alveolar epithelial type II cells on the basis of the findings of whole-cell patch-clamp experiments [29]. In conjunction with progress in experimental studies of lung fluid balance under clinically relevant pathologic conditions, further studies should be done to test the potential role of catecholamine-dependent therapies that may enhance the resolution of clinical pulmonary edema. Recent experimental data in a rat model of acute lung injury suggest that b2adrenoceptor agonist therapy can decrease lung endothelial permeability, increase alveolar fluid clearance, and decrease pulmonary edema even when given after lung injury has developed [54]. The feasibility of delivering therapeutic concentrations of aerosolized b-adrenoceptor agonist therapy to the distal air spaces of ventilated patients has also been demonstrated [55]. Therefore, clinical trials could be carried out to test the potential value of this therapy for enhancing the resolution of pulmonary edema and improving clinical outcomes.

4.2 Non-catecholamine Regulation Several interesting catecholamine-independent mechanisms have been identified that can upregulate fluid transport in the distal air spaces. Hormonal factors such as glucocorticoids and mineralocorticoids upregulate Na+ transport by transcriptional mechanisms [56–58] (Table 1), whereas thyroid hormones work by a posttranslational mechanism [59] (Table 1). Growth factors such as epidermal growth factor, transforming growth factor-a, and keratinocyte growth factor (KGF) work by either transcriptional or direct membrane effects, or by enhancing the number of alveolar epithelial type II cells [60–65] (Table 1). For example, KGF is a potent mitogen for alveolar epithelial type II cells and distal air space administration of KGF increased alveolar fluid clearance by 66% [62]. KGF can also enhance Na+ and fluid transport in injured rat lungs [63, 64]. KGF also increases Na+ transport protein expression [65]. There is also evidence that a proinflammatory cytokine, tumor necrosis factor-a (TNFa), can rapidly upregulate Na+ uptake and fluid transport [66] (Table  1). The effect of TNFa is amiloride-inhibitable in both rats and isolated A549 human cells [66]. Another catecholamine-independent mechanism is represented by dopamine. Dopamine is a vasoactive agonist that has been described to impair Na+ reabsorption in renal tubules. In rat lungs, the effect of dopamine is opposite to that in the renal tubular epithelium, as dopamine increased fluid clearance in isolated perfused rat lungs [67, 68] (Table 1). This increase depended on Na+ transport and was mediated by D1 receptors in

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alveolar epithelial type II cells and not via the b-adrenoceptor. In alveolar type II cells, dopamine increased expression of basolateral Na+/K +-ATPase a1-subunits [69]. Finally, serine proteases can regulate ENaC activity and potentially increase alveolar fluid clearance [70, 71] (Table  1). These catecholamine-independent mechanisms are also explored in more detail in a recent review [1].

5 Mechanisms Impairing Alveolar Edema Resolution Several mechanisms have also been identified that can impair fluid transport from the distal air spaces. This section considers three conditions that have relevance to human disease: hypoxia, the use of anesthetics, and the presence of reactive oxygen and nitrogen species. In addition, this section reviews mechanisms that impair fluid transport under pathologic conditions.

5.1 Hypoxia Hypoxia is a condition that may occur during residence or recreation at high altitudes and under a variety of pathologic conditions associated with acute and chronic respiratory disease. Therefore, it is important to understand the effect of hypoxia on ion and fluid transport across the lung epithelia. In anesthetized rats, hypoxia decreased alveolar fluid clearance by inhibition of the amiloride-sensitive ENaC [72] (Table 1). The effect of hypoxia could not be explained by transcriptional effects on ENaC or Na+/K+-ATPase. Instead, the results suggested a posttranslational mechanism, i.e. a change of ENaC activity or ENaC transport to the plasma membrane. This hypothesis was supported by the normalization of alveolar fluid clearance by a b-adrenoceptor agonist (terbutaline), which increased trafficking of Na+ transporter proteins from the cytoplasm to the membrane [73, 74]. Direct evidence for this mechanism in hypoxic alveolar epithelial type II cells was demonstrated for ENaC [75] as well as for an inhibitory effect of hypoxia on Na+/K+-ATPase activity [76].

5.2 Anesthesia Anesthetics represent another group of agents that have the potential to inhibit Na+ transport across the air space epithelia and thereby reduce alveolar fluid clearance (Table 1). In alveolar epithelial cells, halogenated anesthetics affect Na+ transport. In the rat, halothane and isoflurane decreased alveolar fluid clearance by inhibition of the amiloride-sensitive

H.G. Folkesson

ENaC. This effect was rapidly reversible after cessation of halothane exposure [77]. In vitro, exposure to a low halothane concentration (1%) for a short time (30 min) induced a reversible decrease in Na+/K+-ATPase activity and amiloridesensitive [22] Na influx via ENaC in rat alveolar epithelial type II cells [78]. The mechanisms whereby halothane induced this decrease in Na+ transport protein activity have not been elucidated, but they are not related to a decrease in intracellular adenosine triphosphate content or to a change in cytosolic free calcium concentration. Taken together, these observations suggest that halogenated anesthetics may interfere with the clearance of alveolar edema. Lidocaine is a local anesthetic that is widely used in patients with acute cardiac disorders and has been recently implicated as a possible cause of pulmonary edema following liposuction. In experimental rat studies, both intravenous and intra-alveolar administration of lidocaine reduced alveolar fluid clearance by 50% [79]. Because lidocaine did not inhibit ENaC when expressed in oocytes, the inhibitory effect on vectorial ion and fluid transport was likely through an effect on the Na+/K+-ATPase activity or through an indirect effect via blockade of K+ channels, a wellknown property of lidocaine. The effect of lidocaine was also completely reversible with b2-adrenoceptor agonist therapy, further excluding direct effects on the Na+ transport machinery [79].

5.3 Reactive Oxygen Species During many pathologic conditions, in response to proinflammatory cytokines activated neutrophils and macrophages may localize in the lungs and migrate into the air spaces. There they release reactive oxygen species by the membranebound enzyme complex nicotinamide adenine dinucleotide phosphate oxidase and nitric oxide (NO). NO has been shown to decrease Isc across cultured rat alveolar epithelial type II cells without affecting transepithelial resistance and to inhibit 60% of the amiloride-sensitive Isc across alveolar epithelial type II cell monolayers [80] (Table 1). NO reacts with superoxide (·O2) to form peroxynitrite (ONOO–), which is a potent oxidant and nitrating species that directly oxidizes a wide spectrum of biologic molecules, such as DNA constituents, lipids, and proteins [36]. Boluses of peroxynitrite delivered into suspensions of freshly isolated rabbit alveolar epithelial type II cells decreased the amiloride-inhibitable Na+ uptake by approximately 65% without affecting cell viability (Table  1). Some investigators also have reported that macrophage products, including NO, downregulate Na+ transport in endotoxin-stimulated fetal distal lung epithelium [81]. These data suggest that oxidation of critical amino acid residues in ENaC might be responsible. This matches well with other studies that have shown that protein nitration and oxidation

60  Alveolar Epithelial Fluid Transport in Lung Injury

by reactive oxygen and nitrogen species are associated with diminished function of important proteins in the alveolar spaces, including surfactant protein A [82].

5.4 Cytokines Specific factors may also decrease ENaC function and thereby reduce alveolar fluid clearance. As an example of this, there is new evidence that transforming smooth factorb1 decreases expression of ENaC and alveolar epithelial Na+ and fluid transport by an extracellular signal-regulated kinase 1 and 2 (ERK1/2)-dependent mechanism in both primary rat and human alveolar epithelial type II cells [83] (Table  1). Interleukin-1b is a proinflammatory cytokine that has been associated with membrane dysfunction and a reduced fluid absorptive capacity in the lungs [84] (Table 1).

5.5 Pathologic Conditions In the clinical setting, fluid clearance from the distal air spaces has been measured in mechanically ventilated patients having acute respiratory failure from pulmonary edema. In addition, there have been numerous studies with animal models of relevant pathologic conditions [85–88]. Studies of alveolar fluid clearance in humans have been done in intubated, ventilated patients by measuring the concentration of total protein in sequential samples of undiluted pulmonary edema fluid aspirated from the distal air spaces with a standard suction catheter passed through the endotracheal tube into a wedged position in the distal airways [23, 45, 89]. This method for measuring alveolar fluid clearance in patients was adapted from the method for aspirating fluid from the distal air spaces in experimental studies in small and large animals [19, 43]. The clinical procedure has been validated in patients by demonstrating that there is a relationship between alveolar fluid clearance and improvement in oxygenation and chest radiographs [45, 89]. In patients with severe hydrostatic pulmonary edema, net alveolar fluid clearance existed in most of them during the first 4  h after endotracheal intubation and onset of positivepressure ventilation [23]. Upon a more detailed investigation, it was found that the rate of alveolar fluid clearance in these patients varied between maximal (more than 14%/h) in 38% of the patients and submaximal (3–14%/h) in 37% of the patients. Overall, 75% of the patients had intact alveolar fluid clearance. There was no significant correlation between the levels of alveolar fluid clearance and endogenous plasma epinephrine levels, although twice as many of the patients with intact alveolar fluid clearance received aerosolized b-adrenoceptor therapy as did those with impaired alveolar fluid clearance; this difference did not reach statistical significance, perhaps because the total number of patients studied was modest.

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Most patients with increased permeability edema and acute lung injury have impaired alveolar epithelial fluid transport, a finding that is associated with more prolonged respiratory failure and a higher mortality. In contrast, a few patients can remove alveolar edema fluid rapidly, and these patients have a higher survival rate [45, 89]. These results indicate that a functional, intact distal lung epithelium is associated with a better outcome in patients with acute lung injury, thus supporting the hypothesis that the degree of injury to the distal lung epithelium is an important determinant of the outcome in patients with increased permeability pulmonary edema from acute lung injury. What are the mechanisms that impair alveolar fluid clearance in the patients with acute lung injury? As already explained, alveolar hypoxia can depress alveolar epithelial fluid transport. In addition to hypoxia, viral or bacterial lung infection can depress alveolar fluid transport, by interfering with normal ion transport, by inducing apoptosis or necrosis of the distal lung epithelium, and/or by affecting the sensitivity of the alveolar epithelium to b-adrenoceptor stimulation [90, 91]. There are also some clinical data indicating that a decrease in alveolar fluid clearance may be associated with higher levels of nitrate and nitrite in pulmonary edema fluid, a finding that supports the hypothesis that nitration and oxidation of proteins essential to epithelial fluid transport may occur in some patients with lung injury, further depressing their ability to remove alveolar edema fluid [82].

6 Summary Remarkable progress has been made in understanding the basic mechanisms that regulate salt and water transport across the distal airway and alveolar epithelia. Removal of excess air space fluid, particularly alveolar edema resolution, is driven by active ion transport. The rate of alveolar fluid clearance can be upregulated by cAMP-dependent stimulation, including endogenous catecholamines or exogenous, aerosolized b2-adrenoceptor agonists. Impaired alveolar ion and fluid transport may contribute to the severity of lung edema in multiple pathologic conditions, including clinical acute lung injury. Therapies hastening the repair of the injured alveolar epithelium or upregulating alveolar epithelial ion and fluid transport may have clinical value in reducing morbidity and mortality in patients with acute pulmonary edema.

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H.G. Folkesson 98. Berthiaume Y (1991) Effect of exogenous cAMP and aminophylline on alveolar and lung liquid clearance in anesthetized sheep. J Appl Physiol 70:2490–2497 99. Norlin A, Baines DL, Folkesson HG (1999) Role of endogenous cortisol in basal liquid clearance from distal air spaces in adult guinea-pigs. J Physiol 519:261–272 100. Barquin N, Ciccolella DE, Ridge KM, Sznajder JI (1997) Dexamethasone upregulates the Na-K-ATPase in rat alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 273:L825–L830 101. Dagenais A, Denis C, Vives M-F et  al (2001) Modulation of a-ENaC and a1-Na+-K+-ATPase by cAMP and dexamethasone in alveolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 281:L217–L230 102. O’Brodovich H, Rafii B, Perlon P (1992) Arginine vasopressin and atrial natriuretic peptide do not alter ion transport by cultured fetal distal lung epithelium. Pediatr Res 31:318–322 103. Grubb BR, Boucher RC (1998) Effect of in vivo corticosteroids on Na+ transport across airway epithelia. Am J Physiol Cell Physiol 275:C303–C308 104. Rezaiguia S, Garat C, Delclaux C et  al (1997) Acute bacterial pneumonia in rats increases alveolar epithelial fluid clearance by a tumor necrosis factor-a-dependent mechanism. J Clin Invest 99:325–335 105. Laffon M, Lu LN, Modelska K, Matthay MA, Pittet JF (1999) a-Adrenergic blockade restores normal fluid transport capacity of alveolar epithelium after hemorrhagic shock. Am J Physiol Lung Cell Mol Physiol 277:L760–L768 106. Nici L, Dowin R, Gilmore-Hebert M, Jamieson JD, Ingbar DH (1991) Upregulation of rat lung Na-K-ATPase during hyperoxic injury. Am J Physiol Lung Cell Mol Physiol 261:L307–L314 107. Yue G, Russell WJ, Benos DJ, Jackson RM, Olman MA, Matalon S (1995) Increased expression and activity of sodium channels in alveolar type II cells of hyperoxic rats. Proc Natl Acad Sci USA 92:8418–8422 108. Johnson CR, Guo Y, Helton ES, Matalon S, Jackson RM (1998) Modulation of rat lung Na+,K+-ATPase gene expression by hyperoxia. Exp Lung Res 24:173–188 109. Carter EP, Wangensteen OD, O’Grady SM, Ingbar DH (1997) Effects of hyperoxia on type II cell Na-K-ATPase function and expression. Am J Physiol Lung Cell Mol Physiol 272:L542–L551 110. Olivera WG, Ridge KM, Sznajder JI (1995) Lung liquid clearance and Na,K-ATPase during acute hyperoxia and recovery in rats. Am J Respir Crit Care Med 152:1229–1234 111. Tomlinson LA, Carpenter TC, Baker EH, Bridges JB, Weil JV (1999) Hypoxia reduces airway epithelial sodium transport in rats. Am J Physiol Lung Cell Mol Physiol 277:L881–L886 112. Ding JW, Dickie J, O’Brodovich H et  al (1998) Inhibition of amiloride-sensitive sodium-channel activity in distal lung epithelial cells by nitric oxide. Am J Physiol Lung Cell Mol Physiol 274:L378–L387 113. DuVall MD, Zhu S, Fuller CM, Matalon S (1998) Peroxynitrite inhibits amiloride-sensitive Na+ currents in Xenopus oocytes expressing abg-rENaC. Am J Physiol Cell Physiol 274: C1417–C1423 114. Nielsen VG, Baird MS, Geary BT, Matalon S (2000) Halothane does not decrease amiloride-sensitive alveolar fluid clearance in rabbits. Anesth Analg 90:1445–1449

Chapter 61

High-Altitude Pulmonary Edema Erik R. Swenson

Abstract  High-altitude pulmonary edema (HAPE) is an uncommon form of pulmonary edema that occurs in healthy individuals within a few days of arrival at altitudes above 2,500–3,000 m. The crucial pathophysiology is an excessive hypoxia-mediated rise in pulmonary vascular resistance (PVR) or hypoxic pulmonary vasoconstriction (HPV) leading to increased microvascular hydrostatic pressures despite normal left atrial pressure. The resultant hydrostatic stress can cause both dynamic changes in the permeability of the alveolar capillary barrier and mechanical damage leading to leakage of large proteins and erythrocytes into the alveolar space in the absence of inflammation. Bronchoalveolar lavage (BAL) and pulmonary artery (PA) and microvascular pressure measurements in humans confirm that high capillary pressure induces a high-permeability non-inflammatorytype lung edema; a concept termed “capillary stress failure.” Measurements of endothelin and nitric oxide (NO) in exhaled air, NO metabolites in BAL fluid, and NO-dependent endothelial function in the systemic circulation all point to reduced NO availability and increased endothelin in hypoxia as a major cause of the excessive hypoxic PA pressure rise in HAPE-susceptible individuals. Other hypoxia-dependent differences in ventilatory control, sympathetic nervous system activation, endothelial function, and alveolar epithelial sodium and water reabsorption likely contribute additionally to the phenotype of HAPE susceptibility. Recent studies using magnetic resonance imaging in humans strongly suggest nonuniform regional hypoxic arteriolar vasoconstriction as an explanation for how HPV occurring predominantly at the arteriolar level can cause leakage. This compelling but not yet fully proven mechanism predicts that in areas of high blood flow due to lesser vasoconstriction edema will develop owing to pressures that exceed the structural and dynamic capacity of the alveolar capillary barrier to maintain normal

E.R. Swenson () Pulmonary and Critical Care Medicine, Veterans Affairs Puget Sound Health Care System, 1660 South Columbian Way, Seattle, WA 98108, USA e-mail: [email protected]

alveolar fluid balance. Numerous strategies aimed at lowering HPV and possibly enhancing active alveolar fluid reabsorption are effective in preventing and treating HAPE. Much has been learned about HAPE in the past four decades such that what was once a mysterious alpine malady is now a wellcharacterized and preventable lung disease. This chapter will relate the history, pathophysiology, and treatment of HAPE, using it not only to illuminate the condition, but also for the broader lessons it offers in understanding pulmonary vascular regulation and lung fluid balance. Keywords  High altitude pulmonary edema • Hypoxia • Alveolar fluid balance • Alveolar capillary barrier • Microvascular hydrostatic pressure • Capillary stress failure

1  Introduction High-altitude pulmonary edema (HAPE) is an uncommon form of pulmonary edema that occurs in healthy individuals within a few days of arrival at altitudes above 2,500–3,000 m. It can be life-threatening, but with recognition, descent, and/ or treatment complete recovery is the rule. Indeed, a previous bout of HAPE does not preclude future successful ascents to even higher altitudes or sojourns in the mountains, especially if the rate of ascent is slowed allowing for acclimatization. Effective drug prophylaxis of HAPE and treatment are possible if slow ascent rates are not feasible and descent is not possible. The crucial pathophysiology is an excessive hypoxia-mediated rise in pulmonary vascular resistance (PVR) or hypoxic pulmonary vasoconstriction (HPV) leading to increased microvascular hydrostatic pressures despite normal left atrial pressure. The resultant hydrostatic stress can cause both dynamic changes in the permeability of the alveolar capillary barrier and mechanical damage leading to leakage of large proteins and erythrocytes into the alveolar space in the absence of inflammation. Bronchoalveolar lavage (BAL) and pulmonary artery (PA) and microvascular pressure measurements in humans confirm that high capillary pressure induces a high-permeability non-inflammatory-type

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_61, © Springer Science+Business Media, LLC 2011

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lung edema; a concept termed “capillary stress failure.” Measurements of endothelin and nitric oxide (NO) in exhaled air, NO metabolites in BAL fluid, and NO-dependent endothelial function in the systemic circulation all point to reduced NO availability and increased endothelin in hypoxia as a major cause of the excessive hypoxic PA pressure rise in HAPE-susceptible individuals. Other hypoxia-dependent differences in ventilatory control, sympathetic nervous system activation, endothelial function, and alveolar epithelial sodium and water reabsorption likely contribute additionally to the phenotype of HAPE susceptibility. Recent studies using magnetic resonance imaging in humans strongly ­suggest ­nonuniform regional hypoxic arteriolar vasoconstriction as an explanation for how HPV occurring predominantly at the arteriolar level can cause leakage. This compelling but not yet fully proven mechanism predicts that in areas of high blood flow due to lesser vasoconstriction edema will develop owing to pressures that exceed the structural and dynamic capacity of the alveolar capillary barrier to maintain normal alveolar fluid balance. Numerous strategies aimed at lowering HPV and possibly enhancing active alveolar fluid reabsorption are effective in preventing and treating HAPE. Much has been learned about HAPE in the past four decades such that what was once a mysterious alpine malady is now a well-characterized and preventable lung disease. This chapter will relate the history, pathophysiology, and treatment of HAPE, using it not only to illuminate the condition, but also for the broader lessons it offers in ­understanding pulmonary vascular regulation and lung fluid balance.

2 Clinical Presentation 2.1 Epidemiology Two distinct populations are affected by HAPE. The first involves well-acclimatized high-altitude residents returning from low-altitude stays (re-entry HAPE) and the second involves rapid ascent of unacclimatized lowlanders. Although good clinical descriptions of HAPE date back over 100 years, it was variably diagnosed as bronchitis, pneumonia, or heart failure. The first medical description of HAPE as a distinct entity was recognized in Peru as re-entry HAPE [1]. The first cases of HAPE in unacclimatized lowlanders going to high altitude were reported from the Rocky Mountains [2]. Most likely, both forms share the same pathophysiology; that of a pulmonary vasculature and lung parenchyma ill prepared to handle a sudden rise in vascular resistance induced by a low inspired partial pressure of oxygen, but most of our knowledge has been gained in studying lowlanders. Altitude, ascent rate, and, importantly, individual susceptibility are the major determinants of HAPE in mountaineers and trekkers and in

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those with re-entry HAPE. The prevalence of HAPE ranges from less than 0.2% in a general mountaineering population when climbing in 3 days or more to altitudes between 4,000 and 5,000 m to 7% if the same altitudes are reached within 1 day. A similar increase in HAPE incidence of 2.5 versus 15.5% occurs when an altitude of 5,500 m is reached by airlift as opposed to trekking over 4–6 days. In those with a history of radiographically documented HAPE at 4,559  m, the likelihood of developing HAPE is 60% with a 1–2-day ascent to the same altitude [3]. Much has been learned about the pathophysiology, treatment, and prevention of HAPE from studies of these individuals, who have generously and enthusiastically volunteered for a variety of investigations, often of an invasive nature in the laboratory and at high altitude. They have a variety of physiological characteristics and responses to hypoxia, which appear to be quite disadvantageous at high altitude, as discussed below. Although HAPE is a risk in mountaineers it also occurs at lower altitudes between 2,500 and 3,000  m in the ski and mountain resorts of Asia, Europe, South America, and North America. The estimated incidence in visitors to the Colorado Rockies is 0.01–0.1% [3]. Women may be slightly less susceptible to HAPE than men [4]. Re-entry HAPE has been also reported from the Rocky Mountains and appears to run in susceptible families and affect predominantly children, findings similar to those reported from the Andes [5, 6]. Most recently, a case series of HAPE at altitudes between 1,400 and 2,400 m in ski resort areas with facilities for skiing up to 3,200 m [7] has been reported, but no incidence figures were given. Susceptibility for HAPE in the tourists may have been increased by preceding or unrecognized underlying illness as discussed later in this review, such as viral infections, pulmonary embolism, or diastolic heart dysfunction. Subclinical or very mild HAPE likely occurs and causes either no problems or minimal symptoms that are ignored or attributed to other factors. The true incidence of subclinical HAPE is unknown, although several studies at altitudes between 4,000 and 5,000 m have suggested that as many as 50% of persons may have subclinical fluid accumulation in the lung consistent with occult edema that resolves spontaneously despite remaining at high altitude [8–11]. This incidence is as high as that for acute mountain sickness (AMS), which has been associated with mild gas exchange impairment [12, 13]. The incidence may be overestimated because these studies did not use chest radiographs, but rather used very indirect measures of lung water changes such as spirometry, closing volume, and/or transthoracic impedance, all of which can vary for other reasons related to the high-altitude environment, including intense exercise and increased cardiac output, cold/dry-air-induced mild bronchoconstriction, and hypocapnia [14]. Recently a high incidence of subclinical HAPE could not be confirmed at 4,559 m in a study of climbers using an array of pulmonary function measurements including body plethysmography, chest radiography, arterial blood

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gases, and compliance measurements [15]. The same measurements made in a smaller group of subjects diagnosed with mild HAPE by detectable radiographic interstitial edema showed only very mild abnormalities, suggesting that many pulmonary function parameters may not be very sensitive to small changes in interstitial fluid volume.

2.2 Diagnosis and Evaluation HAPE in lowlanders occurs within 2–5 days after arrival at high altitude [3]. It is rarely observed below altitudes of 2,500–3,000 m and after 1 week of acclimatization. In highaltitude residents retuning home after a stay at low altitude the onset is similar. In most cases, it is preceded by symptoms (headache, nausea, and lassitude) of AMS, which afflict upward of 50% of newcomers to high altitude. The clinical presentation of HAPE does not differ much from that of any other form of pulmonary edema as it evolves from its mild to its severe form. Early symptoms of HAPE include exertional dyspnea different from past experience at that altitude or in relation to that of one’s companions, cough, chest tightness, and reduced exercise performance. As edema progresses, cough worsens, breathlessness is present at rest, and orthopnea develops. Gurgling in the chest and pink frothy sputum indicate advanced cases. The physical examination reveals cyanosis, tachypnea, tachycardia, and elevated body temperature generally not exceeding 38.5°C. Crackles are discrete at the onset, typically located over the middle lung fields. Often, auscultation underestimates the more widespread disease on the chest radiograph. In advanced cases, as arterial oxygen saturation plummets, signs of concomitant high-altitude cerebral edema such as ataxia and decreased consciousness are frequent findings and consequences. There are no unequivocal characteristic diagnostic laboratory findings with HAPE [3]. Abnormal results may be due to accompanying dehydration, stress, and preceding exercise. Arterial blood gas and oxygen saturation measurements in advanced HAPE at 4,559  m demonstrate its severity; mean arterial PO2 in the mid 20-mmHg range versus 40–45 mmHg in healthy controls, and arterial oxygen saturations below 50% versus 70–85% [3]. Chest radiographs and CT scans of early symptomatic HAPE show a patchy and sometimes peripheral distribution of edema. The radiographic appearance of advanced HAPE becomes more homogenous and diffuse [16, 17]. Surprisingly, in patients with two episodes of HAPE, the radiographic appearance is not always similar, suggesting that intrinsic structural vascular and parenchymal regional differences do not necessarily dictate where edema may first develop [17] except in the rare cases of unilateral absence of a PA, in which the edema always occurs in the contralateral lung receiving the entire cardiac output [18]. BAL findings show a protein-rich exudate and mild alveolar hemorrhage (Fig.  1)

Fig.  1  Chest radiograph with bronchoalveolar lavage fluid aliquots (first and fifth) from a representative subject with early high-altitude pulmonary edema. The radiograph shows interstitial and alveolar infiltrates and the lavage performed after the X-ray was taken shows mild alveolar hemorrhage. (Reproduced from [20] with permission. Copyright 2002 American Medical Society. All rights reserved)

which initially is noninflammatory, but can progress to a more inflammatory picture as discussed below [19–21]. In all cases echocardiographic and PA catheterization studies at high altitude have always shown marked pulmonary hypertension. Autopsies reveal distended PAs with thrombi and infarction, diffuse bloodstained alveolar edema with bloody foamy fluid present in the airways, hyaline membranes, but little to no evidence of left ventricular failure or leukocytic infiltration [22].

3 Pathophysiology Since the recognition of HAPE as a noncardiogenic pulmonary edema in the 1960s, a number of pathophysiological mechanisms have been postulated to explain the sudden appearance of pulmonary edema in otherwise young, healthy, and physically fit individuals. From the first modern descriptions it was clear that pulmonary hypertension and HAPE were inextricably linked, suggesting a primary hemodynamic basis, but the story became more complicated with subsequent studies in both human and animals suggestive of an inflammatory pathogenesis. A final chapter in our understanding of HAPE is evolving with the growing appreciation that active alveolar epithelial sodium and water reabsorption, and its sensitivity to hypoxia, must now be taken into account, as is true for other forms of pulmonary edema [23].

3.1 Hemodynamics Right-sided heart catheterization studies in untreated cases of HAPE at high altitude [24, 25] revealed mean PA pressures of 60 mmHg (range 35–115 mmHg), but normal PA wedge pressures. These pioneering studies were crucial in putting to

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rest the idea that HAPE was acute left-sided heart failure at high altitude. Subsequently, less invasive estimations of systolic PA pressure by echocardiography have substantiated the catheterization data with values between 50 and 100 mmHg for subjects with HAPE and 30–50  mmHg for healthy controls [26, 27]. It is well established that excessive PA pressure precedes the development of HAPE and is not a consequence [26]. The critical role of high PA pressure is further confirmed by the fact that any intervention (descent, oxygen, or drugs) which lowers PA pressure can improve gas exchange in HAPE [27, 28] and in the case of drugs is effective for treatment [29] and prevention [26]. Individuals susceptible to HAPE, as mentioned already, have many physiological characteristics (Table 1) that place them at risk for high-altitude problems. The most important among these is a strong HPV response. Although they have resting PA pressures within the normal range at low altitude, their exaggerated PA pressure responses with exercise and even during sleep in normoxia [30–32] point to a constitutional hyperreactivity of the pulmonary circulation to stress (Fig. 2). Studies in relatives of HAPE-susceptible individuals have not been undertaken as yet, but in a recent study of young children and their fathers at high altitude (3,450  m) it was shown that the increase in PA pressure from low to high altitude of each child correlated with that in the father [33], ­suggesting that HPV is in part genetically determined, as already established for the hypoxic ventilatory response [34]. Recently, right-sided heart catheterization studies by Maggiorini et al. [35] using the single occlusion technique at the Capanna Margherita in the Swiss–Italian Alps (4,559 m) in controls and HAPE-susceptible climbers showed that the exaggerated rise in PA pressure in individuals susceptible to HAPE is accompanied by increased microvascular (vessels less than 100 mm in diameter) pressure above 20 mmHg in those developing HAPE (Fig. 3). This threshold value accords with experimental observations in dogs of a ­PO2-independent critical microvascular pressure of 17–24 mmHg, at which the lungs continuously leak fluid and gain weight [36]. These results suggest that increased microvascular pressures with Table 1  HAPE-susceptibility characteristics Hemodynamic • Exaggerated hypoxic pulmonary vasoconstriction (HPV) • Greater normoxic exercise-induced PA pressure elevation • Augmented sympathetic tone with hypoxia • Reduced vascular endothelial nitric oxide production • Increased vascular endothelial endothelin production Pulmonary • Smaller lung volumes • Reduced recruitment of diffusing capacity with hypoxia and exercise • Possibly reduced alveolar epithelial Na+/H2O reabsorptive capacity Ventilatory and Renal • Lower hypoxic ventilatory responsiveness (HVR) • Possibly reduced natriuretic response to acute hypoxia

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pulmonary arterial hypertension rather than upstream arterial pressure elevation itself are the crucial hemodynamic factor in the pathophysiology of HAPE, since as this study showed some susceptible subjects with equivalently high PA pressures but lower microvascular pressures did not develop ­clinical or radiological evidence of edema. The basis for high hypoxic PA pressures in HAPEsusceptible subjects is not fully known and likely is multifactorial. PVR is the sum of many influences including those intrinsic to the vascular smooth muscle, but also those related to vascular endothelium, lung volume, ventilatory control, left ventricular end-diastolic pressure, and neurohumoral responses. As noted in Table  1, HAPE-susceptible subjects have lower hypoxic ventilatory responsiveness (HVR) (set largely by the peripheral chemoreceptors) [37–39], which results in a lower alveolar PO2 at the same altitude (or FIO2) than in a HAPE-resistant subject, and thus a stronger stimulus for HPV. In addition to a lower alveolar PO2, a lower HVR may lead to a smaller fall in alveolar PCO2 and less hypocapnic inhibition of HPV [40]. To what extent the greater HPV of HAPE-susceptible subjects is due to lower HVR has never been established by testing HAPE-susceptible and HAPEresistant control subjects over a range of inspired PO2, so that the influence of differences in HVR can be eliminated by comparing the two groups at equivalent alveolar PO2s (the predominant stimulus of HPV) and arterial oxygen saturation. Animal studies have revealed that HVR and HPV may be linked in two other ways. The first is the influence of arterial PO2 itself because the bronchial arterial circulation perfuses the vaso vasorum of the pulmonary vasculature. Isolated perfusion of the bronchial artery in sheep with deoxygenated blood, when alveolar PO2 and systemic PO2 are held constant, increases PA pressure [41]. The second is via the peripheral

Fig.  2  Pulmonary artery pressure (PAP) in high-altitude pulmonary edema (HAPE)-susceptible (HAPE-s) individuals (continuous lines and filled symbols) and in nonsusceptible controls (dashed lines and open symbols) during exposure to normobaric hypoxia (left) and before and during exercise on a bicycle ergometer (right). The highest PAP recordings during exercise (75–150 W) are shown. (Reprinted from [32] with permission from Elsevier)

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Fig. 3  Mean PAP (Ppa) and pulmonary capillary pressure (Pcap) in 14 controls and in 16 HAPE-s subjects at high altitude. HAPE-s is further divided into those who developed HAPE (HAPE) and those who did not develop

HAPE (non-HAPE). Bars indicate the mean values in each group. *p < 0.05, **p < 0.01 versus control, †p < 0.01 versus non-HAPE. (Reproduced from [35] with permission from Lippincott Williams & Wilkins)

chemoreceptors independent of the alveolar PO2. In anesthetized animals with fixed minute ventilation, vagotomy of the lungs [42] or manipulation of the carotid body [43], both of which alter a neural afferent–efferent pathway, results in greater HPV for an equivalent alveolar PO2. These findings suggest that the peripheral chemoreceptors when stimulated help to blunt HPV by two mechanisms: by increasing ventilation to reduce the stimulus itself and by diminishing the vascular responsiveness to that stimulus. There is another peripheral chemoreceptor-mediated response to hypoxia that may have relevance in HAPE susceptibility. In animal and human studies, acute hypoxia of moderate intensity (FIO2 of 0.12–0.14) causes diuresis and natriuresis; this in humans is greater in those with higher HVR [44]. Because HAPEsusceptible subjects have low HVR, they may be ­disadvantaged by a limited hypoxic diuretic response as suggested by a field study in which several HAPE-susceptible subjects had no diuresis in contrast to HAPE-resistant subjects [45], in part by greater activation of the renin–angiotensin system [46, 47] and higher sympathetic activity [48], but also less ­chemoreceptor-mediated natriuresis. Increased sympathetic tone may also contribute to stronger HPV. Microneurography in HAPE-susceptible subjects demonstrates increased skeletal muscle sympathetic tone during hypoxia at low altitude and prior to HAPE at high altitude [47], although this study, similar to the HPV studies, suffers from a lack of control on the strength of the hypoxic stimulus due to differences in HVR. In accordance with these findings, increased plasma and/or urinary levels of norepinephrine compared with the levels for controls precede and accompany HAPE [48]. Some studies in adult animals and man have found that stimulation of the sympathetic innervation of the lung augments HPV [49] via a-receptor stimulation [50] and that it is reduced it with autonomic blockade [51, 52]. In an in  vivo canine model of isolated cerebral hypoxia by selective perfusion of the brain with hypoxic

venous blood, there was intense sympathetic activation, increased PA pressure, and considerable pulmonary edema [53]. Thus, it is conceivable that increased hypoxia-mediated sympathetic activity contributes to brisk HPV and HAPE development and a-adrenergic antagonists may be useful [28]. The importance of heightened sympathetic-mediated pulmonary hypertension is well established in neurogenic pulmonary edema [54], an acute pulmonary edema that may share similarities with HAPE. A difference in lung volumes is a very consistent finding in studies of lung function in HAPE-susceptible and HAPEresistant subjects. HAPE-susceptible individuals have 10–15% lower total lung capacity (TLC) and vital capacity than HAPE-resistant controls [39, 55–57] and in the only study to measure functional residual capacity (FRC) a 30% lower value was found [56]. The possible greater importance of differences in FRC than in the maximal lung volumes is that FRC is the operating lung volume around which normal breathing and perfusion occurs and itself is a determinant of PVR. Studies in isolated dog lungs have shown that PVR is minimal at 50% of TLC and rises greatly with a fall in volume [58]. Interestingly, the HAPE-resistant subjects had a FRCto-TLC ratio of 0.52, whereas the ratio was 0.42 in the HAPE-susceptible group [56]. HAPE-susceptible subjects show greater steady-state arterial desaturation in the supine position than HAPE-resistant individuals at a simulated altitude of 4,000 m that resolves with 2 min of stimulated breathing [57], indicative of a lower FRC close to the point of airway closure in dependent lung regions typically seen when breathing at lower lung volumes. Pointing to a slight but potentially important restrictive defect is the higher ratio of the forced expiratory volume in 1 s to the forced vital capacity in HAPE-susceptible subjects and their associated reduced maximal diffusing capacity [56] that indicate a smaller capillary bed and less vascular recruitability. Because of their lower lung volumes, HAPE-susceptible subjects may need to

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recruit a larger fraction of their total alveolar surface area as they breathe and thus subject their lungs to greater stretch. In vitro studies have shown that alveolar epithelial permeability increases with both greater frequency and peak stretch magnitude [59]. It must be noted that lacking any pre-HAPE lung function measurements and longitudinal follow-up data, one cannot distinguish whether HAPE-susceptible persons have an intrinsically different lung structure or whether an episode of HAPE heals with a small loss in lung volume and capillary bed, leading to an apparent slight restrictive defect. In addition to the aforementioned extrinsic influences on the pulmonary vasculature, growing evidence points to possible differences of the vasculature itself. Presently, we lack any evidence for or against differences in vascular smooth muscle responsiveness to hypoxia, since studies in isolated smooth muscle from such subjects and controls have never been performed. The intrinsic responsiveness of the pulmonary vascular endothelium to hypoxia may likely be greater in HAPE-susceptible subjects; however, again owing to the problems arising from differences in HVR in awake and spontaneously breathing subjects, the true magnitude of pulmonary endothelial response differences may be overestimated. Nonetheless, compelling evidence is accumulating that the susceptibility to HAPE resides at the level of the vascular endothelium. NO and endothelin 1 (ET-1) are important endothelialderived vasodilator and vasoconstrictor mediators, respectively, in the pulmonary circulation and have received considerable attention in many forms of pulmonary hypertension. Lung NO production is reduced in HAPE-susceptible individuals, since they have lower exhaled NO concentrations during acute [60] and prolonged [61] exposure to hypoxia (Fig. 4) than HAPE-resistant individuals, and they have lower concentrations of the NO metabolites nitrate and

nitrite in BAL fluid [20]. Such measurements of lung NO metabolism do not perfectly reflect vascular production differences because NO is produced both by the vascular endothelium and by the alveolar and bronchial epithelia [62]. It is very likely that both vascular and bronchial sources of bioactive NO contribute to low pulmonary vascular tone and reduced HPV [62–64]. To better and more directly investigate the role of vascular endothelial NO differences, Berger et al. [65] assessed plasma nitrite concentration and showed that systemic vascular endothelial NO generation is reduced more in hypoxic HAPE-susceptible subjects than in controls within 90 min of hypoxic exposure and as a possible consequence there is less endothelial-dependent vasodilation to acetylcholine in hypoxia. Likely this is the case for the ­pulmonary circulation as the above-cited measurements of exhaled NO and metabolites of NO in BAL fluid suggest, however this has not been formally tested, or that of bronchial NO production. Tibetans, the population best adapted to high altitude, interestingly have greater pulmonary NO production than lowlanders and have minimal HPV [66]. The observation that inhaled NO fails to normalize PA pressure in hypoxic HAPE-susceptible individuals, in contrast to those resistant to HAPE [67], indicates impaired NO synthesis cannot fully account for the excessive pulmonary vascular reactivity in HAPE-prone subjects. Circulating ET-1 concentrations are elevated almost threefold at high altitude after 1–2 days and to a greater degree in HAPE-susceptible subjects [65, 68–70] and correlate with the rise in PA pressure. However, when ET-1 concentrations are measured with more acute hypoxia ranging from 5 to 90 min, there is no change at 5 min, when HPV is already evident, in either control or HAPE-susceptible subjects [71] and there is an equal rise of roughly 50% at 90 min in the two groups [65]. In humans, ET-1 infusion sufficient to raise

Fig. 4  (a) Exhaled NO after 40 h at 4,559 m in individuals developing HAPE (left) and in individuals not developing HAPE (HAPE-R) despite identical exposure to high altitude. (b) Exhaled NO in individuals with susceptibility (HAPE-S) and without

susceptibility (HAPE-R) to HAPE after 4 h of exposure to hypoxia (FIO 2 = 0.12) at low altitude (elevation 100  m). (a) Reproduced from [61] with permission; (b) reproduced from [60] with permission)

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c­ irculating concentrations tenfold has no effect on HPV after 2 h [72]. These data suggest that the early immediate rise in PA pressure with hypoxia may not be related to changes in circulating ET-1 levels, but that with longer hypoxic duration differences in ET-1 synthesis and/or clearance may become more important. When HAPE-susceptible subjects and HAPE-resistant subjects were studied, no important differences in either endothelial-derived vasodilator (prostacyclin and/or prostaglandin E) or vasoconstrictor (thromboxane A2 and/or prostaglandin F2a) prostaglandins and atrial natriuretic peptide were found with hypoxia of 30-min duration [30]. Longer periods of hypoxia or high-altitude sojourns have not been studied, despite the major role of these endogenous ­pulmonary vasoactive mediators in other forms of pulmonary hypertension. Other potential circulating pulmonary vasodilators and vasoconstrictors such as brain natriuretic peptide, adrenomedullin, and epoxyeicosatrienoic acids have not been studied, nor have differences in PA smooth muscle ion transporters or calcium signaling or responsiveness. The heightened responsiveness of the pulmonary circulation in HAPE-susceptible persons raises several ­intriguing questions. What is the incidence of this in the general population? A tentative answer may be 9–10% as recently shown in a random group of 86 young healthy subjects that served as a control group in a study of primary pulmonary hypertension (PPH) [73]. Is this behavior a “forme frusta” of PPH and thus a risk for these individuals later in life to develop fixed PA hypertension with other pulmonary vascular stresses? Only prospective studies will provide the answer. Preliminary studies have ruled out differences in bone morphogenic protein receptor II, a leading candidate for familial PPH and some cases of sporadic PPH [74]. Clinically serious pulmonary hypertension does develop in a small but significant fraction of patients with sleep apnea, left ventricular failure, and chronic hypoxic lung disease [75], although why it occurs in some and not others with equally severe disease remains a mystery. Perhaps these individuals constitute the unknown percentage of persons with pulmonary vascular hyperreactivity, which in their earlier and healthier lives at low altitude never caused problems. Since capillary pressure is elevated and likely the important site of excessive pressure in HAPE (Fig. 2), the question arises how hypoxic constriction of arterioles leads to edema. Three mechanisms have been suggested: transarteriolar leakage, irregular regional vasoconstriction with overperfusion in certain areas, and hypoxic venoconstriction. The evidence in hypoxic animals from the double occlusion technique [76, 77] reveals that the small arterioles are exposed to high ­pressure and that they are a site of transvascular leakage in the presence of markedly increased PA pressure in hypoxia [78]. Recently Parker et  al. [79] showed that pulmonary microvascular endothelial cells grown in culture have a

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20-fold lower hydraulic conductance than macrovascular arterial endothelial cells. Pulmonary veins also constrict in response to hypoxia [80], thus increasing the resistance downstream of the fluid filtration region. Both mechanisms, alone or in combination, may contribute to edema formation; however, they cannot explain the patchy radiographic appearance of early HAPE as it appears on chest radiographs or CT scans, unless one postulates, in addition, that there is regional heterogeneity of hypoxic pulmonary arterial vasoconstriction. If vasoconstriction in hypoxia is inhomogeneous, HAPE could be the consequence of uneven regional distribution of perfusion, with high flow in those areas with lesser vasoconstriction leading to increased microvascular pressure. The elevated microvascular pressures would be due to a longitudinal pressure drop across the upstream vessels insufficient to lower the tension below the threshold of 17–24 mmHg at the point of entry into the alveolar microvasculature [81], possibly aggravated further by increased venous resistance [36]. The concept of increased capillary pressure by a high blood flow was first postulated by Visscher [82] and was adapted to HAPE by Hultgren [83]. Recent investigations which demonstrate that HPV is uneven at rest provide substantial support to the concept of regional overperfusion in some areas. Intravascular microspheres used to map regional perfusion reveal that the spatial heterogeneity of the pulmonary perfusion in rats, pigs, and dogs increases with hypoxia [84]. This has now been corroborated in humans in studies using magnetic resonance imaging which found greater regional blood flow heterogeneity in hypoxic HAPE-susceptible individuals than in nonsusceptible controls [85, 86]. The basis of uneven regional HPV is not known, but may involve inhomogeneous localization of arterial smooth muscle, both in radial thickness and in distribution longitudinally along the arterial tree. As well, it may have its origin in intrinsic differences in local endothelial vasoactive mediator production or expression and heterogeneity of membrane ion channels and receptors involved in HPV, such as have been shown for endothelial-derived NO in the horse between dorsal and ventral lung regions [87]. If unevenness of regional HPV is responsible for HAPE, it would appear that it might decrease with time at altitude since slow ascent prevents HAPE even in susceptible individuals and HAPE rarely occurs after the first 5 days at a given altitude. One hypothesis accounting for these observations is that there may be rapid remodeling and generalized muscular hypertrophy of all pulmonary arterioles, which lead to a more even distribution of blood flow, but may also contribute to persistent excessive pulmonary hypertension and lead over some weeks to months to congestive right-sided heart failure termed “subacute mountain sickness” [88]. Although the idea that acute hypoxia-induced arteriolar muscularization and more

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regional homogeneity in arteriolar resistance protects the microvasculature is attractive, it remains unproven and possibly refuted by recent data in children with pulmonary hypertension, who already have well-developed extensive PA smooth muscularization. These children are at considerable risk for HAPE with even slight gains in elevation [89]. What may be more important in acute acclimatization of the lung against the hydrostatic stress of high HPV is upregulated gene expression for collagen and other extracellular matrix proteins that help to strengthen the alveolar capillary barrier [90] as has been demonstrated in animal models of acute hypoxia and pulmonary vascular and parenchymal stress. Perhaps these changes in the parenchyma explain the smaller lung volumes noted earlier in HAPE-susceptible subjects. In summary, these most recent investigations provide evidence for the concept of an overperfusion edema in HAPE occurring in some lung regions as a result of high blood flow under such large driving gradients that the increased microvascular and capillary pressures exceed the capacity of the alveolar barrier to maintain a fluid-free air space. The final piece in the puzzle will be to show with a single imaging modality capable of resolving flow and change in extravascular fluid that it is indeed these areas of high flow that do leak. The relevance of overperfusion injury extends to other clinically encountered forms of pulmonary edema, including alveolar fluid accumulation in nonoccluded areas in pulmonary embolism [91], following pulmonary thromboendarterectomy [92] and lung transplantation [93], and in the pulmonary edema during vigorous exercise and high cardiac outputs in highly trained athletes [94] or racehorses [95]. Increasing cardiac output manyfold over the baseline level with exercise will contribute to edema formation by increasing capillary pressure in overperfused areas owing to the rise in PA pressure as well as some further increase in the regional heterogeneity of blood flow [96]. Furthermore, it has been postulated that the greater increase of PA pressure in HAPEsusceptible individuals may slightly impair left ventricular filling because of leftward ventricular septal shift [97] and possible mild left ventricular stiffness [97] arising from decreased cardiac lymph clearance to the right side of the heart [98]. Mild diastolic dysfunction due to pulmonary hypertension could explain the significantly greater wedge pressure increase in HAPE-susceptible individuals compared with nonsusceptible controls during exercise in hypoxia [31], whereas at rest wedge pressure is normal in untreated HAPE [24, 25, 35]. A more recent echocardiographic study in HAPE-susceptible subjects exercising at 4,559 m before and after vasodilator therapy, however, found no evidence for left ventricular diastolic dysfunction [99]. Nonetheless, in many cases of HAPE, particularly at lower elevations, exercise may be the essential component of the setting that leads to pulmonary edema.

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3.2  Inflammation The first measurements of alveolar lavage fluid in ­mountaineers with and without HAPE were performed on Mt. McKinley in Alaska by Schoene et al. [19] and were then followed up by studies in hospitalized patients with HAPE in Japan [100]. In these reports the onset of HAPE could not be established with certainty, but particularly in the Japanese studies, the duration of HAPE may have been greater than 3 days. In addition to the high protein concentrations and mild alveolar hemorrhage, some but not all patients had significant neutrophilia and elevations of the levels of proinflammatory cytokines and neutrophil chemotactic factors, such as tumor necrosis factor (TNF)-a, interleukin (IL)-1, IL-6, and leukotriene B4. These observations strongly suggested that inflammation might be a causal factor in HAPE, consistent with an altered permeability leading to protein and fluid leak. Support for the concept was further marshaled in studies of animals with viral priming [101] or endotoxin administration [102] showing increased lung edema with hypoxic exposure compared with controls and prevention of lung edema with corticosteroid pretreatment, a classic anti-inflammatory therapy [103]. These studies lacked either histological or lavage measurements to show the proposed inflammation. Gonzalez and Wood pursued the hypoxia-mediated inflammation paradigm in a series of studies in rats exposed to 10% oxygen for upward of 4 h and found in various systemic vascular beds (brain, skeletal muscle, and brain) that hypoxia stimulated leukocyte adhesion to capillaries and increased their permeability via increased levels of reactive oxygen species and depletion of endogenous NO [104]. The lung microvasculature was not studied to determine whether the same leukocyte– endothelial adherence and increased permeability was taking place in the pulmonary capillaries. Intriguingly, they determined that the systemic capillary changes were the result of mast cell degranulation triggered in some way by hypoxic alveolar macrophages, because the effect of systemic hypoxia could be abrogated by cromolyn pretreatment and/or more than 90% depletion of alveolar macrophages and was not linked to the level of hypoxia in the systemic vascular bed [105]. Numerous in vitro studies have ­established that very severe (and likely lethal in vivo hypoxia – 7–20 mmHg) can cause spontaneous release of many proinflammatory and chemotactic mediators by leukocytes (reviewed in [20]) and vascular endothelial cells. These findings taken together continued to sustain the idea that HAPE might have a critical inflammatory pathogenesis. The findings, however, that not all cases of HAPE had evidence of inflammation in alveolar lavage fluid [19], and the lack of evidence of in vivo thrombin and fibrin formation except in very advanced HAPE [106], indicated that in humans inflammation is possibly a secondary response to alveolar–capillary barrier disruption or edema. Furthermore,

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prospective studies provided no evidence that inflammation and increased systemic vascular permeability preceded HAPE [107]. BAL in HAPE-susceptible and HAPE-resistant climbers within 1 day after ascent to 4,559 m from low altitude showed mild alveolar hemorrhage and increased serumderived protein concentrations in the air space (Table  2) both in subjects ill with HAPE at the time of bronchoscopy and in those who developed HAPE within the next 24 h [20]. In fact, fully supporting the central role of hemodynamics in HAPE, we demonstrated a very strong correlation between the magnitude of pulmonary hypertension by echocardiography and the degree of hemorrhage and protein level elevation in the alveolar space (Fig. 5). In contrast, there were no increases in the levels of alveolar macrophages, neutrophils, and proinflammatory mediators (TNF-a, IL-1, and IL-8) at high altitude early in course of HAPE, despite the very high PA pressures recorded in these subjects. The only cytokine whose level was elevated both in the circulation and in the alveolar space was IL-6 and its level was only elevated after

the onset of HAPE. For ethical and practical reasons we could not perform serial lavages to document the time course of secondary inflammation. Alveolar macrophages harvested from the lavage fluids at sea level and at high altitude showed no differences in TNF, IL-8, IL-6, and IL-1 production between the HAPE-resistant and HAPE-susceptible subjects when stimulated under normoxic or hypoxic conditions, before and after endotoxin stimulation [108]. Thus, with regard to the work of Gonzalez and Wood discussed above, if alveolar macrophages sense hypoxia and elicit systemic permeability changes, altered permeability does not appear to involve signaling by these cytokines. In numerous studies of humans at high altitude, increases in circulating IL-6 concentration are observed [105, 109–111] and have been taken as prime facie evidence for an inflammatory effect of hypoxia that might be critical to HAPE development [109]. The problem with this idea is that exercising muscle releases IL-6 in proportion to the intensity and duration of work both in normoxia and in hypoxia and is

Table 2  Bronchoalveolar lavage (BAL) characteristics in early HAPE 550 m CONT (n = 8)

4,559 m HAPE-S (n = 9)

CONT (n = 6)

HAPE-S (pre-HAPE) (n = 6)

HAPE-S (ill) (n = 3)

Leukocyte count (×10 /mL) 8.1 6.3 9.5 8.1 9.8 Macrophages (%) 94 95 83 82 85 Neutrophils (%) 1 0  0  1 1 Red cell count (×103/mL) 3 4 10 26* 623* Total protein (mg/dL) 1.8 1.5 14* 34* 163* Systolic PAP (mmHg) 22 26 37* 61*   81* BAL at 550 m and on the second day at 4,559 m in eight control subjects (CONT) and in nine HAPE-susceptible subjects (HAPE-S), of whom three had pulmonary edema at the time of BAL. Of those six HAPE-S without pulmonary edema at the time of BAL (pre-HAPE), four developed HAPE within 18 h after BAL *p < 0.05 versus 550 m 3

Fig. 5  Individual bronchoalveolar lavage (BAL) red blood cell count and albumin concentrations versus pulmonary artery systolic pressures at high altitude (4,559 m). The vertical lines denote a threshold systolic PAP (more than 60 mmHg) above which red blood cells (a) appear in the BAL fluid, in contrast to the lower pressure (35 mmHg) at which

albumin leakage occurs (b). The open circles at the lower left of (a) and (b) show the normal values for these at low altitude. The correlation coefficients are given for the best-fit curves of the values at high altitude (p < 0.05 for both curves). (Reproduced from [20] with permission. Copyright 2002 American Medical Society. All rights reserved)

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driven to some extent by sympathetic nervous system activation with exertion [111, 112] and that with passive ascent to high altitude there is little to no increase in IL-6 concentration, even in HAPE-susceptible persons [113, 114]. Although IL-6 is considered a classic proinflammatory cytokine, there is considerable evidence that it may have equally important anti-inflammatory and endothelial permeability protective actions [112] and thus it might be equally plausible that IL-6 is released with severe hypoxia and after injury in the lung in an effort to limit inflammatory damage and capillary permeability. Studies in a recently developed IL-6 knockout mouse would be interesting in determining the role of IL-6 in high-altitude pathophysiology. What initiates the secondary inflammation is not clear. It may be that sustained and increasing high pressures in untreated HAPE of sufficient duration can trigger inflammation [115] or the inflammation may represent part of the healing process of a markedly disrupted alveolar–capillary barrier that occurs in the severest cases of HAPE, especially with alveolar hemorrhage. Interestingly, it has been shown recently that heme and other breakdown products of red cell hemoglobin are chemotactic for neutrophils [116]. The surprise is that despite the intensity of apparent inflammation found in some subjects [100], the resolution of HAPE even at this stage is rapid and without apparent sequelae as opposed to the acute respiratory distress syndrome (ARDS), in which the concentration of alveolar neutrophilia and inflammatory cytokines is of magnitude equal to that seen in the later stages of HAPE [19, 100]. Despite overwhelming evidence against a primary inflammatory alteration of the alveolar–capillary barrier in HAPE, it is nevertheless conceivable that any concurrent process altering the permeability of the alveolar–capillary barrier will lower the pressure required for edema formation. Indeed, increased fluid accumulation during hypoxic exposure after priming by endotoxin or virus in animals [101, 102] and the association of preceding respiratory viral infections with HAPE in children [89, 117] support this concept. In conditions of increased permeability, HAPE may occur in individuals with more normal moderate hypoxic pulmonary vascular responsiveness. Thus, upper respiratory tract infections shortly before a sojourn in the mountains and vigorous exercise at altitudes between 2,000 and 3,000  m may explain in some cases why HAPE develops at a modestly low altitude [7].

3.3 Alveolar Fluid Clearance Disruption of the alveolar capillary barrier and leak are the proximal cause of HAPE, but recent work in cell cultures and animal models has highlighted that alveolar fluid clearance mechanisms dependent upon active alveolar epithelial sodium and water reabsorption by both type II and type I

E.R. Swenson

pneumocytes may contribute to the pathophysiology of HAPE. Active water and sodium transport from the alveolar space into the lung interstitium is important in normal lung fluid balance. Hypoxia decreases transepithelial sodium transport by reducing the expression and activity of the epithelial sodium channel (ENaC) and Na+/K+ ATPase proteins [118] in cultured alveolar epithelial cells possibly by an impairment of b2 adrenergic receptor signaling [119]. In vivo hypoxia depresses fluid clearance from the alveoli of hypoxic rats and rabbits [120, 121]. Mice partially deficient in the ENaC develop greater accumulation of lung water in hypoxia [122] as well as in other forms of lung injury [123]. In an attempt to determine whether differences in active alveolar salt and water transport lead to HAPE susceptibility, Sartori et al. [124, 125] measured lower transepithelial nasal potentials in normoxia in HAPE-susceptible individuals than in nonsusceptible controls. The easily measured potential difference across the nasal mucosa is considered to be a good, but not prefect, surrogate for the alveolar epithelium. The reduced nasal potential difference in HAPE-susceptible individuals was attributed to lower sodium transport by the epithelial apical sodium channel (ENaC), pointing to a constitutional, possibly genetically, determined reduction of sodium transport across the respiratory epithelium. This has been questioned with a recent investigation that confirmed the difference in nasal potential between HAPE-susceptible individuals and controls in normoxia, but that found that this difference could not be attributed to differences in ENaC activity [126, 127], but rather to differences in chloride secretion, which contributes a large fraction of the potential difference in the nasal mucosa but not in the alveolar epithelium. Another approach to study the relevance of alveolar transepithelial fluid reabsorption in HAPE has involved the use of b2-receptor agonists and glucocorticoids, both of which are known to upregulate ENaC and Na+/K+ ATPase (reviewed in [23]). Two recent field studies reported successful prevention of HAPE in HAPE-susceptible climbers with inhalation of salmeterol, a long-acting b2 agonist [124] and orally administered dexamethasone [128] begun 1 day before ascent to 4,559 m. Owing to multiple actions of salmeterol and other b2 adrenergic agonists, such as inhibition of HPV, increased HVR and ventilation, tightening of cell-to-cell contacts, and upregulation of NO production [129–131], the contribution of enhanced alveolar fluid clearance to the positive outcome of the study remains uncertain. Indeed, the protective effect of dexamethasone from a putative enhancement of alveolar fluid reabsorption could not be correlated to indirect measures of enhanced active alveolar sodium and fluid reabsorption (transepithelial nasal potential difference and expression of leukocyte messenger RNA for the sodiumtransporting proteins), but rather to a surprising reduction of PA pressure at high altitude to be discussed below with respect to prevention of HAPE. Thus, what are needed are

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selective and specific drugs to better evaluate the role of active alveolar fluid clearance in HAPE pathophysiology. Interestingly, a putative link between virus infection and HAPE may rest in part upon infection-related downregulation of ENaC activity and diminished fluid clearance as recently shown in lungs of rats [132] infected with a nonreplicating influenza virus. In addition to the deleterious effect of reduced NO production in HAPE-susceptible subjects on PVR, NO may have a permissive and stimulatory effect on alveolar Na+ reabsorption as indirectly shown in rabbit lungs, which gain more weight at constant vascular pressure with inhibition of all nitric oxide synthase (NOS) activity [133] and more directly in cell culture studies showing depressed ENaC activity and fluid reabsorption in mice with loss of inducible NOS (iNOS), either by genetic knockout or by pharmacological inhibition [134]. In mice and man, a constitutive form of iNOS is the isoenzyme responsible for airway epithelial NO production [135]. ET-1 expression, as discussed earlier, increases with hypoxia, and plays a role in the elevated vascular pressures and lung water accumulation in HAPE. Two recent studies [136, 137] add another face to ET-1; that of inhibiting alveolar fluid clearance by activation of endothelial cell ETB receptors. ETB receptor binding by ET-1 generates NO, which as one of its effects includes downregulation of alveolar epithelial Na+/K+ ATPase by a non-cyclic GMP (GMP)-mediated mechanism [137]. The clinical importance of this possible effect of ET-1 on alveolar fluid clearance and HAPE awaits human studies with selective ETB receptor antagonists, because two studies at high altitude with bosentan (a nonselective ETA/ETB receptor antagonist) have yielded conflicting results on PA pressure, exercise capacity, and gas exchange [70, 138].

4 Mechanisms of Increased Capillary Permeability Although it is clear that microvascular pressures in HAPE are high, the reasons why the pulmonary vasculature leaks under high pressure are not completely resolved and remain an area of active investigation. Traditionally, pulmonary edema has been categorized as either noncardiogenic (increased permeability with exudative characteristics: high protein concentrations and markers of inflammation in the setting of normal or only modestly elevated intravascular pressures) or cardiogenic (elevated hydrostatic pressures leading to a noninflammatory protein-poor transudative leak). As alluded to already and discussed further later, BAL findings in nascent HAPE [20] reveal characteristics of a hydrostatic, but noncardiogenic noninflammatory edema suggesting pressure-induced alterations to the normal

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p­ ermeability of the alveolar–capillary barrier or frank ­traumatic injury. This novel constellation of features in HAPE does not fit the conventional dichotomy of pulmonary edema, particularly in violating the dogma that high hydrostatic pressures alone should not lead to changes in microvascular permeability to high molecular weight substances.

4.1 Stress Failure Early work by Fishman et al. [139] and amplified by Bachofen et al. [140] established that cardiogenic edema might, in fact, lead to alveolar protein accumulation. The concept was further advanced by West for HAPE (reviewed in [141]) after the publication of the first lavage findings in HAPE by Schoene et al. [19]. West et al. showed histological evidence obtained by electron microscopy for discrete ultrastructural disruptions in the alveolar capillary barrier in in situ perfused rabbit lungs with very high transmural pressures typical of severe HAPE, within cell membranes, between cells, and in the basement membranes of both the capillary endothelium and the alveolar epithelium, which could be responsible for the characteristic leak in HAPE. These changes, which they also showed in rats exposed to rapid simulated hypobaric “ascents” to 8,800 m [142], were termed “stress failure” of the pulmonary capillaries. They ascribed them to stretch and deformation of the extracellular collagen matrix in excess of its load-bearing capacity to maintain normal structural and permeability characteristics. Despite their traumatic-like appearance, even allowing red cell egress, these discontinuities can quickly close with reductions in pressure. Recently, it was proposed that hydrostatic disruptions of the alveolar– capillary barrier permit the leak of vascular endothelial growth factor (VEGF) from the alveolar air space (where it is in high concentration) to the capillary endothelium, where VEGF receptors are expressed and when activated promote vascular leakiness [143].

4.2 Dynamic Alterations in Permeability Others have more recently suggested that the hydrostatic-induced permeability changes with hypoxia may have more than a mechanical basis. These may be dynamic noninjurious cellular responses because b adrenergic agonists [144] and gadolinium [145] reduce hydrostatic edema at constant vascular pressure. Perhaps dynamic changes in transcellular leakage via vesicle formation and fusion to create pathways that traverse the cell [146, 147] and paracellular pathways via alterations in gap junction assembly [148] arise from signals initiated when cells are deformed by pressure or stretch.

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These responses may represent a pre-emptive attempt of the alveolar–capillary barrier to lower stress forces temporarily and prevent damage to the basement membranes as has been suggested in the systemic vasculature [149]. It is conceivable that these dynamic changes occur with moderate pressure elevations and precede the more profound “stress failure” changes observed in the studies by West et  al. In the only study to examine in vivo permeability and histological disruptions, there was no correlation between the number of ultrastructural lesions and the extent of leak [150], suggesting that some of the permeability changes are not due to stress failure disruptions. Our lavage data at high altitude support this in showing a very mild protein leak even in HAPE-resistant subjects, in whom lavage protein concentrations were increased several-fold (Table 2) over those at low altitude [20]. Additionally, we found in nascent HAPE [20] that although there was considerable movement of serumderived proteins into the air space, no equivalent air space to blood movement of two alveolar lining fluid proteins (surfactant protein A and Clara cell protein) was detectable. This unidirectional noninjurious selective leak is in marked contrast to the profound injury of the alveolar–capillary barrier in ARDS, in which air space proteins are detected in blood and reflect the severity of injury [151]. We did not measure VEGF back leak, but our findings with surfactant and Clara cell proteins do not support the idea [20] that VEGF is an amplifying factor. One, however, cannot rule out back leak of an alveolar space substance sufficient to interact with the endothelium but not to raise circulating levels. Studies with selective VEGF blockade are ultimately needed. If hydrostatic forces persist, then gene upregulation and transcription of collagen and other extracellular matrix ­proteins are initiated to strengthen the alveolar–capillary barrier [90] and ultimately reduce stress failure and leak. These observations offer an explanation for the rapid recovery from HAPE and the protection from recurrence when reascending only several days after recovery from HAPE. The same changes in alveolar–capillary barrier strength occur in chronic heart failure and explain the lack of pulmonary edema in some patients despite chronically elevated pulmonary venous pressures, but at the price of mild restrictive changes and reduction in diffusing capacity.

5 Prevention and Treatment 5.1 Ascent Rates and Activity Level Slow ascent is the most effective form of prevention even in susceptible individuals when the average daily ascent rate above 2,000  m does not exceed 350–400  m/day or with

E.R. Swenson

staged ascent [3]. To avoid life-threatening illness, persons should not ascend further with any symptoms of altitude ­illness and should descend when mild symptoms do not improve after 1 day of rest. Because exercise-induced circulatory changes may enhance or cause pulmonary edema, vigorous exercise should be avoided during the first days of altitude exposure by individuals with a history of HAPE, and by those with symptoms of altitude illness or after a rapid ascent to altitudes above 3,500–4,000 m. As pointed out earlier, susceptibility to HAPE may be increased during and shortly after any infection.

5.2 Prediction of Susceptibility: Phenotypic and Genotypic Characteristics Although as a group HAPE-susceptible individuals have a number of physiological characteristics and responses to hypoxia that arguably set them at risk (Table  1), these responses are not easily tested except in specialized laboratories. In a retrospective study, measurement of systolic PA pressure in hypoxia (2 h at an FIO2 of 0.12) identified HAPEsusceptible individuals with a specificity of 93% and a sensitivity 77% [152]. Measurements of lung volumes and HVR did not improve the identification. Given the low prevalence of HAPE between 0.2 and 15% (depending on the setting), such testing will always result in a considerable overestimation of HAPE-prone individuals. In fact, certain individuals with exaggerated acute HPV, perhaps as a result of perinatal hypoxia, do not develop HAPE in rapid ascents to 4,559 m [153]. Recently it was reported that subjects with a detectable patent formamen ovale (PFO) at sea level were at a fourfold greater risk for HAPE [154]. This study could not determine whether shunting across a PFO, which theoretically would act to unload the pulmonary circulation at the expense of greater arterial hypoxemia, was causative or simply a marker of susceptibility. If a PFO despite its potential to lower PA pressures by flow diversion were to predispose to HAPE, it could be explained by the greater stimulus of more arterial hypoxemia acting to enhance HPV by bronchial arterial hypoxemia [41] and greater sympathetic activation [49, 50, 53]. In the last analysis, general screening of trekkers or mountaineers for susceptibility to HAPE is not necessary, since this illness can clearly be avoided by slower ascent rates that permit adaptation of the pulmonary microvasculature to increasing pressures by remodeling [90]. Since accurate prediction of HAPE susceptibility by physiological characteristics has not been proven, there has been considerable interest in identifying a genetic marker(s) that might more readily serve the purpose. Numerous candidates have been sought on the basis of reported and hypothesized possible differences in vasoactive mediator production.

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Although the pathogenesis of reduced NO production in HAPE susceptibility is unknown, a leading candidate is ­vascular endothelial NOS (eNOS). This has received some ­support in a Japanese and Asian Indian population [155] but these studies only found a weak segregation of eNOS polymorphisms between HAPE-resistant and HAPE-susceptible subjects, which was not borne out in an investigation of Caucasians with equal power [156]. Ethnic or environmental differences or different linkage disequilibrium in Asians and Caucasians could account for the differing results, or numerous other factors that determine eNOS activity and expression may be more important, such as differences in expression of caveolin 1 or the cationic amino acid transporter responsible for l-arginine uptake into endothelial cells. There is no association of HAPE susceptibility with the well-characterized angiotensin converting enzyme deletion polymorphism [157], which is associated with pulmonary hypertension in COPD [158] and in chronic highaltitude pulmonary hypertension [159]. Polymorphisms in the human VEGF gene were not found to be correlated to HAPE susceptibility [160]. Other gene polymorphisms, including those for the angiotensin receptor, aldosterone synthase, and serotonin transporter, have had very limited study [161]. Lastly, given the exciting results with dexamethasone prophylaxis described in the next section, it may be fruitful to explore whether HAPE susceptibility has any link to polymorphisms in the glucocorticoid receptor, which influence numerous aspects of cardiovascular and metabolic control [162].

5.3 Pharmacological Prophylaxis The decision to use drug prophylaxis must be based upon an individual’s past history at high altitude and a risk–benefit discussion of prophylaxis versus no prophylaxis. Nifedipine and other Ca2+ channel blockers that inhibit HPV are recommended for individuals with a history of unquestionable HAPE when slow ascent is not possible. Sixty milligrams daily of a slow-release formulation should be started with the ascent and end on the third to fourth day after arrival at the final altitude. Given the uncertainties mentioned already with regard to the preventive effect of high-dose inhaled b2-receptor agonists, nifedipine remains the drug of choice for HAPE prophylaxis. Other drug prophylaxis options in animals and man have been recently studied. Not only do these other agents offer more flexibility and options, but also the surprising results obtained are providing new insights into HAPE pathophysiology and pulmonary vascular regulation. Interestingly, acetazolamide, long used for AMS prevention, blunts or abolishes HPV in animals and man at doses relevant to its clinical use [163, 164] and it was successful in an animal model of mild HAPE in reducing alveolar edema and hemorrhage

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[165]. Studies in isolated PA smooth muscle cells suggest that acetazolamide prevents the increase in intracellular Ca2+ concentration with hypoxia that initiates smooth muscle contraction and that it does so by a mechanism not involving carbonic anhydrase inhibition [166]. Acetazolamide may also act to minimize HAPE by stimulating alveolar fluid clearance as recently shown in rats [167]. Maggiorini et al. [128] have recently reported that dexamethasone and tadalafil (a long-acting phosphodiesterase type 5 inhibitor) were as equally effective (reduction of recurrence rate to less than 10%) as nifedipine in HAPEsusceptible subjects. The effectiveness of tadalafil was expected given the prominent role of NO in modulating HPV, as shown with acute hypoxia or at high altitude with sildenafil [168], but the efficacy of dexamethasone was not so certain. Dexamethasone was chosen, on the basis of much animal work [23], as it might be a more selective agent in upregulating alveolar epithelial sodium and water reabsorption than salmeterol and so interpretation of its results would not be confounded by any potential ventilatory stimulation or HPV inhibition known for b2 adrenergic agonists. The potent prophylactic effect of dexamethasone, however, was mediated by an equally striking reduction in PA pressures as in the tadalafil group, and even better arterial oxygenation. What appears to be a leading explanation for the efficacy of dexamethasone is its ability to upregulate pulmonary vascular eNOS and NO production, which was recently shown in isolated PA endothelial cells [169] and indirectly suggested by higher urinary cGMP excretion in patients treated with dexamethasone for HAPE prophylaxis [128]. Evidence in this same study for a sympatholytic effect of dexamethasone (lower heart rates) may have been another contributing factor to the reduced PA pressures. Another relevant effect of dexamethasone is increased surfactant production and secretion, which is used to enhance lung function in premature infants, but is also demonstrable in adult lungs [170]. Increased surfactant production would have possibly two salutary actions. By increasing the production of alveolar lining fluid surfactant, dexamethasone reduces surface tension, which in turn reduces negative forces at the air–liquid interface and thus lowers the alveolar–capillary transmural pressure difference [171]. This mechanism may explain the reduction in vascular permeability in hypoxic mice treated with dexamethasone in which no changes in PA pressure were reported [103]. Thus, dexamethasone may act in numerous ways to prevent HAPE; via its enhancement of NO production, decreased pulmonary vascular sympathetic tone, upregulation of alveolar sodium and water reabsorption mediated by corticosteroid-induced upregulation of alveolar epithelial apical membrane ENaC activity and basolateral membrane Na+/K+ ATPase activity [23], and enhanced surfactant secretion. All mechanisms

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could lower PVR, either directly or by improving ventilation and raising alveolar and arterial PO2.

5.4 Treatment Immediate improvement of oxygenation is the treatment of choice. How to attain this depends on where HAPE occurs. In a remote area without medical care descent has the first priority. The tourist with HAPE in a skiing resort may stay at altitude if the arterial oxygen saturation can be kept above 90% with low-flow oxygen (2–4 L/min), monitoring by family or friends is guaranteed, and access to clinical care is close at hand. Relief of symptoms is achieved within hours and complete clinical recovery usually occurs within 2–3 days. Individuals with severe, advanced cases need to be evacuated to low altitude, where they may require prolonged hospitalization, especially if they have coexisting high-altitude cerebral edema. Mortality is estimated to be around 50% when neither descent nor other treatment is possible. Without either oxygen or descent, portable hyperbaric chambers [172] or treatment with nifedipine (20 mg slow-release formulation every 6 h) should be initiated until descent can be accomplished. In a monitored clinical setting diuretics may be given, but this is not recommended in the field since the victims are often volume-depleted. In mountaineers with HAPE at 4,559  m persistent relief of symptoms, improvement of gas exchange, and radiographic appearance were documented over 34  h with 20 mg nifedipine every 6 h [29]. Nifedipine causes no significant side effects when administered either prophylactically or therapeutically in slow-release formulations. Inhaled NO, although presently technically difficult to provide, is effective [27], suggesting that other inhaled NO donors such as nitroglycerine, nitroprusside, and nitrite may be more practical. Whether inhalation of b2-receptor agonists might be used in addition to nifedipine needs study, as does the use of any of the lesser studied prophylactic therapies, particularly the phosphodiesterase type 5 inhibitors.

6 Summary HAPE is now well established as a consequence of HPV and sufficient transmission of high PA pressure and blood flow to portions of the pulmonary capillary bed, most likely due to regional unevenness in HPV with a possible contribution by venoconstriction. Although strong HPV is a characteristic shared by most individuals who develop HAPE, there probably can be no absolute resistance to HAPE, even in “nonsusceptible” individuals, if the altitude and the ascent rate are

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high enough or if other factors such as a concurrent respiratory infection transiently increase susceptibility. The fluid leak in humans with HAPE (and in animal models) affirms the concept proposed by West et  al. that increased pulmonary capillary pressure can lead to a permeability-type edema in the absence of inflammation and challenges the classical paradigm that hydrostatic stress can only lead to ultrafiltration of protein-poor fluid. The same pathophysiology of capillary leak at high pressure and flow (overperfusion edema) exceeding local mechanisms of fluid absorption and clearance that is central to HAPE is also relevant to other forms of pulmonary edema at low altitude. The study of HAPE has taught us much about the lung and pulmonary vasculature and it is hoped that successful strategies for HAPE prevention and treatment may find broader clinical application in pulmonary edema of other origins.

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887 122. Egli M, Cook S, Hugli O et al (2001) Delayed resolution of pulmonary edema in mice with defective sodium transport-dependent alveolar fluid clearance. FASEB J 15:860 123. Egli M, Duplain H, Lepori M et  al (2004) Defective respiratory amiloride-sensitive sodium transport predisposes to pulmonary oedema and delays its resolution in mice. J Physiol 560:857–865 124. Sartori C, Allemann Y, Duplain H et al (2002) Salmeterol for the prevention of high-altitude pulmonary edema. N Engl J Med 346:1631–1636 125. Sartori C, Duplain H, Lepori M et al (2004) High altitude impairs nasal trans-epithelial sodium transport in HAPE-prone subjects. Eur Resp J 23:916–920 126. Mairbäurl H, Weymann J, Möhrlein A et al (2003) Nasal epithelium potential difference at high altitude (4,559 m): evidence for secretion. Am J Respir Crit Care Med 167:862–867 127. Mairbäurl H, Schwobel F, Hoschele S et al (2003) Altered ion transporter expression in bronchial epithelium in mountaineers with high-altitude pulmonary edema. J Appl Physiol 95:1843–1850 128. Maggiorini M, Brunner-La Rocca HP, Peth S et al (2006) Both tadalafil and dexamethasone may reduce the incidence of high-altitude pulmonary edema: a randomized trial. Ann Intern Med 145:497–506 129. Bärtsch P, Mairbäurl H (2002) Salmeterol for the prevention of high-altitude pulmonary edema. N Engl J Med 347:1283 130. Easton PA, Katagiri M, Johnson MW, Rothwell BC, Holroyde MC, Kusuhara N (2008) Effect of salbutamol on respiratory muscle function and ventilation in awake canines. Respir Physiol Neurobiol 161:253–260 131. Adding LC, Agvald P, Artlich A, Persson MG (1999) Gustafsson. Beta-adrenoceptor agonist stimulation of pulmonary nitric oxide production in rabbit. Br J Pharmacol 126:833–839 132. Chen XJ, Seth S, Yue G et al (2004) Influenza virus inhibits ENaC and lung fluid clearance. Am J Physiol Lung Cell Mol Physiol 287:L366–L373 133. Mundy AL, Dorrington KL (2000) Inhibition of nitric oxide synthesis augments pulmonary edema in isolated perfused rabbit lung. Br J Anaesth 85:570–576 134. Hardiman KM, McNicholas-Bevensee CM, Fortenberry J et  al (2004) Regulation of amiloride-sensitive Na+ transport by basal nitric oxide. Am J Respir Cell Mol Biol 30:720–728 135. Ricciardolo FL, Sterk PJ, Gaston B, Folkerts G (2004) Nitric oxide in health and disease of the respiratory system. Physiol Rev 84:731–765 136. Berger MM, Rozendal CS, Schieber C et al (2009) The effect of endothelin-1 on alveolar fluid clearance and pulmonary edema formation in the rat. Anesth Analg 108:225–231 137. Comellas AP, Briva A, Dada LA et al (2009) Endothelin-1 impairs alveolar epithelial function via endothelial ETB receptor. Am J Respir Crit Care Med 179:113–122 138. Scheult RD, Ruh K, Foster GP, Anholm JD (2008) Prophylactic bosentan does not improve exercise capacity or lower pulmonary artery systolic pressure at high altitude. Respir Physiol Neurobiol 165:123–130 139. Szidon JP, Pietra GG, Fishman AP (1972) The alveolar-capillary membrane and pulmonary edema. N Engl J Med 286:1200–1204 140. Bachofen H, Schürch S, Weibel ER (1993) Experimental hydrostatic pulmonary edema in rabbit lungs: barrier lesions. Am Rev Respir Dis 147:997–1004 141. West JB (2003) Thoughts on the pulmonary blood-gas barrier. Am J Physiol Lung Cell Mol Physiol 285:L501–L513 142. West JB, Colice GL, Lee YJ et  al (1995) Pathogenesis of highaltitude pulmonary oedema: direct evidence of stress failure of pulmonary capillaries. Eur Respir J 8:523–529 143. Kaner RJ, Crystal RG (2004) Pathogenesis of high altitude pulmonary edema: does alveolar epithelial lining fluid vascular endothelial growth factor exacerbate capillary leak? High Alt Med Biol 5:399–409

888 144. Parker JC, Ivey CL (1997) Isoproterenol attenuates high vascular pressure-induced permeability increases in isolated rat lungs. J Appl Physiol 8:1962–1967 145. Parker JC, Ivey CL, Tucker JA (1998) Gadolinium prevents high airway pressure-induced permeability increases in isolated rat lungs. J Appl Physiol 84:1113–1118 146. Dvorak A, Feng D (2001) Vesiculo-vacuolar organelle: a new endothelial cell permeability organelle. J Histochem Cytochem 49:419–431 147. DeFouw DO (1983) Ultrastructural freatures of alveolar epithelial transport. Am Rev Respir Dis 127:S9–S13 148. Cavanaugh KJ, Oswari J, Margulies SS (2001) Role of stretch on tight junction structure in alveolar epithelial cells. Am J Respir Cell Mol Biol 25:584–591 149. Curry FE, Clough GH (2002) Flow-dependent changes in microvascular permeability -an important adaptive phenomenon. J Physiol 543:729–732 150. Maron MB, Fu Z, Mathieu-Costello O, West JB (2001) Effect of high transcapillary pressures on capillary ultrastructure and permeability coefficients in dog lung. J Appl Physiol 90:638–648 151. Greene K, Wright J, Steinberg K et  al (1999) Serial changes in surfactant-associated proteins in lung and serum before and after onset of ARDS. Am J Respir Crit Care Med 160:1843–1850 152. Dehnert C, Grunig E, Mereles D, von Lennep N, Bärtsch P (2005) Identification of individuals susceptible to high-altitude pulmonary oedema at low altitude. Eur Respir J 25:1–7 153. Sartori C, Allemann Y, Trueb L et al (2000) Exaggerated pulmonary hypertension is not sufficient to trigger high-altitude pulmonary oedema in humans. Schweiz Med Wochenschr 130:385–389 154. Allemann Y, Hutter D, Lipp E et al (2006) Patent foramen ovale and high-altitude pulmonary edema. J Am Med Assoc 296:2954–2958 155. Ahsan A, Charu R, Pasha MAQ et al (2004) ENOS allelic variants at the same locus associate with HAPE and adaptation. Thorax 59:1000–1002 156. Weiss J, Haefeli WE, Gasse C et al (2003) Lack of evidence for association of high altitude pulmonary edema and polymorphisms of the NO pathway. High Alt Med Biol 4:355–366 157. Dehnert C, Weymann J, Montgomery HE et al (2002) No association between high-altitude tolerance and the ACE I/D gene polymorphism. Med Sci Sports Exerc 34:1928–1933 158. Kanazawa H, Okamoto T, Hirata K, Yoshikawa J (2000) Deletion polymorphisms in the angiotensin converting enzyme gene are associated with pulmonary hypertension evoked by exercise challenge in patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 162:1235–1238 159. Aldashev AA, Sarybaev AS, Sydykov AS et al (2002) Characterization of high-altitude pulmonary hypertension in the Kyrgyz: associa-

E.R. Swenson tion with angiotensin-converting enzyme genotype. Am J Respir Crit Care Med 166:1396–1402 160. Hanaoka M, Droma Y, Ota M, Ito M, Katsuyama Y, Kubo K (2009) Polymorphisms of human vascular endothelial growth factor gene in high altitude pulmonary edema susceptible subjects. Respirology 14:46–52 161. Mortimer H, Patel S, Peacock AJ (2004) The genetic basis of highaltitude pulmonary oedema. Pharmacol Ther 101:183–192 162. DeRijk R, Schaaf M, de Kloet ER (2002) Glucocorticoid receptor variants: clinical implications. J Steroid Biochem Mol Biol 81:103–122 163. Höhne C, Pickerodt PA, Francis RC, Boemke W, Swenson ER (2007) Pulmonary vasodilation by acetazolamide during hypoxia is unrelated to carbonic anhydrase inhibition. Am J Physiol Lung Cell Mol Physiol 292:L178–L184 164. Teppema LJ, Balanos GM, Steinback CD et al (2007) Effects of acetazolamide on ventilatory, cerebrovascular, and pulmonary vascular responses to hypoxia. Am J Respir Crit Care Med 175:277–281 165. Berg JT, Ramanathan S, Swenson ER (2004) Inhibitors of hypoxic pulmonary vasoconstriction prevent high-altitude pulmonary edema in rats. Wilderness Environ Med 15:32–37 166. Shimoda LA, Luke T, Sylvester JT, Shih HW, Jain A, Swenson ER (2007) Inhibition of hypoxia-induced calcium responses in ­pulmonary arterial smooth muscle by acetazolamide is ­independent of carbonic anhydrase inhibition. Am J Physiol Lung Cell Mol Physiol 292:L1002–L1012 167. Runyon M, Bhargava M, Wangensteen D et al (2005) Acetazolamide stimulates alveolar fluid clearance in ventilated adult rats. Am J Respir Crit Care Med 171:A561 168. Ghofrani HA, Reichenberger F, Kohstall MG et al (2004) Sildenafil increased exercise capacity during hypoxia at low altitudes and at Mount Everest Base Camp. Ann Intern Med 141:169–177 169. Murata T, Hori M, Sakamoto K, Karaki H, Ozaki H (2004) Dexamethasone blocks hypoxia-induced endothelial dysfunction in organ-cultured pulmonary arteries. Am J Respir Crit Care Med 170:647–655 170. Young SL, Ho YS, Silbajoris RA (1991) Surfactant apoprotein in adult rat lung compartments is increased by dexamethasone. Am J Physiol 260:L161–L167 171. Albert RK, Lakshminarayan S, Hildebrandt J, Kirk W, Butler J (1979) Increased surface tension favors pulmonary edema formation in anesthetized dogs’ lungs. J Clin Invest 63:1015–1018 172. Taber RL (1990) Protocols for use of portable hyperbaric ­chambers for treatment of high altitude disorders. J Wilderness Med 1:181–192

Chapter 62

Statins and Acute Lung Injury Amit K. Mahajan and Jeffrey R. Jacobson

Abstractâ•… The development of acute lung injury (ALI), with progression to acute respiratory distress syndrome (ARDS), is associated with an in-hospital mortality as high as 38%. The ageadjusted incidence of ALI and ARDS is reported to be as high as 86.2 per 100,000 person-years, with approximately 74,500 deaths in the USA each year. Although the ultimate cause of death in patients with ALI is often secondary to an underlying systemic process, the occurrence of ALI€can lead to refractory hypoxemia and devastating parenchymal lung damage. Although the application of low-tidalvolume ventilator strategies has improved outcomes in patients with ALI, further strides toward effective treatment have been few and far between. However, investigations into the pathophysiology of ALI have elucidated mechanisms of vascular incompetence secondary to inflammation and endothelial dysfunction, offering direction for physicians to tailor novel treatment approaches. In this regard, extending beyond their utility as highly effective lipid-lowering drugs, statins serve as a highly promising treatment for pulmonary vascular diseases characterized by inflammation and endothelial dysfunction, including ALI. Pathologic findings in bronchoalveolar lavage (BAL) fluid from patients with ALI include abundant neutrophils, macrophages, and protein-rich edema fluid, consistent with capillary injury and endothelial incompetence. These characteristic vascular changes serve as potential targets for statin therapy. Through complex interactions involving endothelial proteins, cytoskeletal structures, and vascular gene expression, statins directly effect endothelial activation, resulting in improved vascular integrity. In addition, statins exert direct anti-inflammatory effects on macrophages and neutrophils which may independently provide clinical benefits in ALI. Keywordsâ•… Lung vascular permeability • Barrier function • Inflammation • Endothelial dysfunction • Acute respiratory distress syndrome J.R. Jacobsonâ•›(*) University of Illinois at Chicago, 840 South Wood Street, 920-N CSB, Illinois 60612, Chicago e-mail: [email protected]

1╇Introduction The development of acute lung injury (ALI), with progression to acute respiratory distress syndrome (ARDS), is associated with an in-hospital mortality as high as 38% [1]. The ageadjusted incidence of ALI and ARDS is reported to be as high as 86.2 per 100,000 person-years, with approximately 74,500 deaths in the USA each year [1, 2]. Although the ultimate cause of death in patients with ALI is often secondary to an underlying systemic process, the occurrence of ALI can lead to refractory hypoxemia and devastating parenchymal lung damage. While the application of low-tidal-volume ventilator strategies has improved outcomes in patients with ALI, further strides toward effective treatment have been few and far between. However, investigations into the pathologic process of ALI have elucidated mechanisms of vascular incompetence secondary to inflammation and endothelial dysfunction, offering direction for physicians to tailor novel treatment approaches. In this regard, extending beyond their utility as highly effective lipid-lowering drugs, statins serve as a highly promising treatment for pulmonary vascular diseases characterized by inflammation and endothelial dysfunction, including ALI. Pathologic findings in bronchoalveolar lavage (BAL) fluid from patients with ALI include abundant neutrophils, macrophages, and protein-rich edema fluid, consistent with capillary injury and endothelial incompetence. These characteristic vascular changes serve as potential targets for statin therapy. Through complex interactions involving endothelial proteins, cytoskeletal structures, and vascular gene expression, statins directly effect endothelial activation, resulting in improved vascular integrity. In addition, statins exert direct antiinflammatory effects on macrophages and neutrophils which may independently provide clinical benefits in ALI.

2╇ Overview of Statins and Their Mechanism of Action The prospect of statins as a treatment for ALI reflects the pleiotropic properties of this class of drugs observed clinically.

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The benefits of statins in patients with cardiovascular disease are well established. For example, aggressive statin therapy reduces the levels of low-density lipoproteins and decreases the incidence of ischemic cardiac events as effectively as angioplasty in low-risk patients with stable coronary artery disease [3]. Statin treatment has also been associated with a decreased incidence of death following acute coronary syndrome [4]. However, evidence suggests that statins may confer benefits in a wide range of inflammatory diseases, including asthma [5], rheumatoid arthritis [6], and multiple sclerosis [7]. The diverse properties of statins in these contexts, independent of their effects on serum cholesterol levels, underlie the basis for their consideration as a novel treatment for ALI. The statins are a class of 3-hydroxy-3-methylglutarylcoenzymeA (HMG-CoA) reductase inhibitors that block the rate-limiting step in the de novo synthesis of cholesterol. The first HMG-CoA reductase inhibitor, discovered in 1976 by Akira Endo, was named “compactin” and was initially utilized in the treatment of patients with heterozygous familial hypercholesterolemia [8]. Inhibition of HMG-CoA reductase by statins prevents protein prenylation, a posttranslational modification culminating in either farnesylation or geranylgeranylation, the addition of a 15-carbon side chain or two 20-carbon side chains, respectively. Although one downstream product of farnesylation is cholesterol, this pathway also regulates the function of a number of proteins, including Ras GTPases. Similarly, geranygeranylation is requisite for membrane translocation and localized activation of the Rho family of small GTPases, including Rho and Rac. The ability of statins to modulate Rho GTPase signaling is a critical determinant of their vascular protective properties as differential effects on RhoA and Rac1 by statins induce endothelial cell (EC) cytoskeletal rearrangement, resulting in augmented vascular integrity. Reduced reactive oxygen species (ROS) production via the inhibition of NAPDH oxidase in concert with increased nitric oxide (NO) production, effects also linked to Rho GTPase modulation, further contribute to enhanced endothelial function by statins. Finally, the influence of statins on differential EC gene expression, including those of cytoskeletal proteins and inflammatory mediators, confers vascular protection as well. Cumulatively, these effects provide compelling justification for the investigation of statins as a therapeutic option in the context of increased vascular leak and inflammation, characteristic of ALI.

3 Endothelial Cytoskeletal Rearrangement by Statins The vascular endothelium serves as a metabolically active and dynamic semiselective barrier between the interstitium and circulating blood. The endothelium is also responsible for maintaining intravascular homeostasis and vascular tone.

A.K. Mahajan and J.R. Jacobson

The luminal vascular lining is composed of ECs that are directly involved in lung inflammatory responses as fluid and leukocytes traverse the vessel wall into the surrounding interstitium and alveoli. In addition to modulating vascular tone, the endothelium also guards against damage from toxic metabolites and adapts to stresses threatening vascular integrity. Hazards to the vessel wall such as lipopolysaccharide (LPS), a component of the cell wall of gram negative bacteria, lead to local inflammation with resulting endothelial dysfunction and increased vascular permeability. The EC cytoskeleton is a complex network of actin microfilaments, intermediate filaments, and microtubules that provides structural stability and shape to individual cells. The cytoskeleton regulates the dynamic balance between competing adhesive, barrier-promoting tethering forces and contractile, tension-producing forces that drive EC barrier dysfunction [9]. Whereas EC adherens junctions regulate macromolecule leakage, rearrangements of actin microfilaments in response to external stimuli may precipitate alternations in EC shape, with a resultant increase in vascular permeability. For example, ECs exposed to the inflammatory agonist thrombin undergo a cytoskeletal structural transformation due to myosin light chain (MLC) phosphorylation with subsequent actomyosin contraction and actin stress fiber formation, resulting in the formation of paracellular gaps via cellular contraction, thereby facilitating leukocyte trafficking and leakage of fluid into the interstitium. The reorganization of the actin cytoskeleton in response to external stimuli, such as thrombin, is a complex process featuring the myosin light chain kinase (MLCK) and small GTPases, RhoA and Rac1. The MLCK isoform expressed in human ECs is a 1,914 amino acid high molecular weight (214-kDa) protein, derived from a single gene on chromosome 3 [10]. MLC phosphorylation is catalyzed by Ca2+/camodulin-dependent MLCK at Ser-19 and Thr-18. Separately, RhoA can also drive MLC phosphorylation. The conversion of inactive RhoA-GDP to active RhoAGTP via Rho kinase results in the inhibition of MLC phosphatase, thereby favoring MLC phosphorylation. The functional significance of these events is suggested by marked reductions of both thrombin-induced barrier dysfunction and MLC phosphorylation upon the inhibition of RhoA by C3 transferase toxin of Clostridium botulinum [11]. Rac1, however, serves an opposing role. Rac1 activation is associated with the translocation of cortactin, an actin-binding protein, to the EC periphery and the subsequent polymerization of cortical actin, key elements involved in the augmentation of EC barrier function. Moreover, inhibition of Rac1 is associated with an attenuation of EC barrier enhancement induced by a variety of agonists [12, 13]. Statin inhibition of Rho GTPase geranygeranylation would predict competing effects on RhoA and Rac1 with respect to cytoskeletal rearrangement and EC barrier function. Nonetheless, inhibition of RhoA appears to be the overriding effect in this context as EC monolayers treated with statins

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demonstrate marked reductions in transcellular actin stress fibers in association with enhanced monolayer integrity as well as augmented cortical actin rings despite the inhibition of Rac1 geranylgeranylation [14] (Fig. 1). In addition, the inhibition of RhoA alone via specific silencing RNA replicates

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statin-mediated EC barrier protection to a significant degree [15]. Accordingly, although Rho GTPase modulation by statins induces EC signaling with numerous effects beyond cytoskeletal rearrangement, these changes likely represent the major determinants of their ability to confer protection of the vasculature (Fig. 2). Interestingly, increased levels of GTP-bound Rho GTPases are found in the cytosol of statin-treated ECs [14]. However, as these proteins are unable to be geranylgeranylated and engage in signaling events initiated at the cell membrane, the functional significance of these cytosolic GTP-bound Rho GTPases is unknown.

4 Statins and Endothelial NO

Fig. 1  Endothelial cytoskeletal rearrangement by statins. Monolayers of human lung microvascular endothelial cells (ECs) grown to confluence exhibit transcellular actin stress fibers under basal conditions (small arrows). In response to treatment with simvastatin (5 mM), cytoskeletal rearrangement occurs in a time-dependent manner characterized by an attenuation of transcellular stress fibers and augmentation of cortical actin rings (large arrows). These changes have been shown to correspond to the time course of statin-mediated EC barrier protection

Aside from their effects on the EC cytoskeleton, the ability of statins to augment NO bioavailability also offers strong support for their use as a treatment for ALI. NO is the end product of the conversion of l-arginine to l-citrulline by nitric oxide synthase (NOS), which is present throughout the body in three isoforms: endothelial NOS (eNOS), neuronal NOS, and inducible NOS (iNOS). NO is responsible for cyclic-GMP-mediated vasodilation but also functions to inhibit platelet aggregation, prevent leukocyte adhesion, and attenuate vascular smooth muscle cell proliferation. A potential therapeutic role for NO in ALI is suggested by its vasodilatory properties which would predict favorable effects on pulmonary hemodynamics and ventilation–perfusion matching. A therapeutic role for NO in ALI is also suggested by its anticoagulant effects and by its ability to attenuate neutrophil chemotaxis and activation [16]. Chemotaxis of neutrophils is a complicated process involving initial attachment, rolling, and transmigration across the vasculature. Localized neutrophil activation is associated with lung parenchymal damage due the release of ROS and production of multiple cytokines [17]. The degree of neutrophil activation corresponds to both the extent of lung injury and the levels of a number of inflammatory cytokines, including tumor necrosis factor a (TNF-a), interleukin 6 (IL-6), and IL-8 [18]. Whereas eNOS is expressed constitutively by ECs, production of NO is modulated by alterations in vascular homeostasis. Physiologic stressors such as hypoxia [19], low-density lipoproteins [20], and TNF-a [21] lead to the upregulation of eNOS with a subsequent increase in NO production. ECs exposed to 13-hydroperoxyoctadecadienoic acid, a biologically activated component of oxidized low-density lipoproteins, also generate elevated levels of eNOS via both transcriptional and posttranscriptional mechanisms [22]. Moreover, conditions associated with prolonged endothelial injury, such as smoking, may lead to the upregulation of eNOS, although synthesized proteins tend to be dysfunctional owing to direct oxidative injury resulting from cigarette smoke [23]. Additionally, superoxide anions

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Fig.  2  Mechanisms of endothelial cytoskeletal regulation by statins. Endothelial barrier protection by statins is largely attributable to the inhibition of RhoA geranylgeranylation. At the cell periphery, RhoA inhibition promotes polymerization of cortical actin, which strengthens cell–cell interactions and barrier integrity. At the same time, barrier

disruption induced by agonists such as thrombin is also attenuated as a result of RhoA inhibition as RhoA drives transcellular actin stress fiber formation through Rho kinase, resulting in increased myosin light chain (MLC) phosphorylation via the inhibition of MLC phosphatase. MLCK myosin light chain kinase

(O2−) produced by reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidases react directly with circulating NO to create peroxynitirite (ONOO−), a metabolite directly injurious to ECs. The promise of NO as a therapeutic target for ALI is provided by encouraging results in animal models although trials in humans have proven largely indeterminate. Induction of ALI using LPS and formyl-norleucyl-phenylalanine in mice has revealed that NO generated as a result of iNOS activation blocks the full expression of tissue damage in the lungs [24]. Moreover, inhaled NO attenuates alveolar–capillary membrane injury in a porcine model of Gram-negative sepsis without adverse systemic hemodynamic effects [25]. Although clinical trials in patients with ARDS have demonstrated selective pulmonary arterial vasodilation, decreased venous admixture, and improved arterial oxygenation induced by inhaled NO, significant differences in clinical outcomes have not been reported [26, 27]. Statins have been shown to increase NO bioavailability by upregulating eNOS in several animal models. These studies include rat models of myocardial ischemia [28] and cirrhotic portal hypertension [29], in addition to diabetic microangiopathic hindlimb ischemia [30] and ischemic stroke [31] in mice. Augmentation of functional eNOS with resulting elevations in NO levels confers vascular protection through inhibition of neutrophil transmigration, reduction in leukocyte trafficking, and improved endothelial function. Additionally, messenger RNA (mRNA) expression of eNOS is significantly increased in the aortas of mice treated with atorvastatin [32]. This increase in eNOS mRNA expression is due to enhanced

mRNA stability rather than increased gene transcription and is directly attributable to the inhibition of RhoA geranylgeranylation, a negative regulator of eNOS [33, 34]. In addition to the upregulation of eNOS, statins also effect increased NO bioavailability through increased eNOS phosphorylation and activity. This occurs as a result of two distinct mechanisms, namely, the activation of the serine/threonine protein kinase Akt and the downregulation of caveolin-1 (Fig. 3). Statin treatment of EC induces tyrosine phosphorylation of phosphatidylinositol 3-kinase, a signaling molecule upstream of Akt [35]. Subsequent Akt phosphorylation and translocation to the cell membrane then induces phosphorylation of eNOS [36]. Separately, caveolin is an abundant component of cholesterol-rich EC plasmalemmal microdomains which serves as a negative regulator of eNOS activation. Statin inhibition of cholesterol synthesis results in decreased caveolin-1 expression, which in turns results in the disinhibition of eNOS activation [37]. Thus, the cumulative effects of statins on ECs including increased eNOS abundance through augmented mRNA stability and increased eNOS activity via effects on Akt and caveolin result in increased NO production.

5 Statins and Endothelial NADPH Oxidase Another important mechanism of vascular protection by statins is via inhibition of EC NADPH oxidase. The NADPH oxidase complex is a superoxide-generating enzyme composed

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Fig. 3  Statin effects on nitric oxide and reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Vascular protection by statins is mediated in part via increased of EC nitric oxide (NO) production and inhibition of NADPH oxidase activity, both of which occur through several distinct mechanisms. The inhibition of RhoA geranylgeranylation results in increased expression of endothelial nitric oxide synthase (eNOS) messenger RNA, whereas increased eNOS activity results from both the

downregulation of caveolin-1 (Cav-1) and the activation of the of phosphatidylinositol 3-kinase (PI3K)–Akt pathway. Increased eNOS expression and activity in turn leads to increased NO production. Separately, statins inhibit NADPH oxidase both directly via the inhibition of Rac1 geranygeranylation and indirectly via the downregulation of the angiotensin II (Ang-II) receptor AT-1, which activates NADPH oxidase through separate pathways involving Rac1 and protein kinase C (PKC)

of multiple subunits, including membrane-bound p22phox and Nox2 (also known as gp91phox) as well as the cytosolic subunits p47phox, p67phox, and Rac1. In unstimulated, nonphagocytic cells, including ECs, NADPH oxidase is constitutively active at low levels, yet the enzyme can be further stimulated acutely by specific agonists such as angiotensin II (Ang-II) as well as various cytokines. Formation of the NADPH oxidase complex occurs in response to a variety of stimuli effecting vascular homeostasis, including hypoxia– reoxygenation [38] and ischemia [39]. Complex assembly then facilitates electron transfer from NADPH to molecular oxygen, generating superoxide that then reacts with NO to form peroxynitrite. Moreover, the reaction to form peroxynitrite depletes available NO, a factor that independently contributes to augmented vascular injury. The significance of these events with respect to the pathophysiology of ALI is supported by evidence of lung microvascular injury and neutrophil infiltration mediated by NADPH oxidase activity in animal models of ALI [40, 41]. Statins bolster endothelial integrity through the inhibition NADPH oxidase activation via two distinct mechanisms involving Rac1 (Fig. 3). As the translocation of Rac1 is requisite for NADPH oxidase complex assembly and production of ROS, the direct inhibition of geranylgeranylation by statins prevents the translocation of Rac1 to the EC membrane, thereby preventing NADPH oxidase complex formation and subsequent activity [15]. Separately, the binding of Ang-II to the AT-1 receptor independently activates Rac1, with

subsequent NADPH oxidase activation. AT-1 receptor ligation also activates NADPH oxidase through a distinct pathway mediated by protein kinase C [42]. However, the AT-1 receptor mRNA half-life is significantly reduced by statins [43]. Consistent with these effects, EC signaling by Ang-II via AT-1, including downstream ROS generation, is demonstrably inhibited by statins [43, 44]. Thus, statin inhibition of the geranylgeranyl-dependent translocation of Rac1 and the destabilization of AT-1 receptor mRNA reduces NADPH oxidase activity and superoxide generation. Although these effects of statins are certainly important with respect to their potential therapeutic role in ALI, the lack of evidence to support the use of antioxidants in ALI patients suggests that they may ultimately only prove to be beneficial in combination with the other vascular protective and anti-inflammatory properties of statins [45].

6 Anti-inflammatory Properties of Statins Independent of their complex effects on ECs, statins have demonstrated a number of anti-inflammatory properties. As they relate to the pathophysiology of ALI, these effects are attributable in part to peroxisome-proliferator-activated receptor a (PPARa) signaling. PPARa, a nuclear receptor that regulates gene expression, is found in a variety of human cells, including ECs, smooth muscle cells, neutrophils, and macrophages. Through the inhibition of the transcription factors nuclear factor (NF)-kB and activating protein 1 (AP-1), PPARa ligation effects

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Fig. 4  Endothelial genes differentially regulated by statins. Among the many genes differentially regulated by statins, several are highly relevant to their vascular-protective effects. These include mediators of coagulation and fibrinolysis such as thrombomodulin, tissue factor (TF), plasminogen activator inhibitor-1 (PAI-1), and the thrombin receptor protease-activated receptor 1. Statins also induce the

differential regulation of several Rho GTPase modulators as well as the upregulation of eNOS and the downregulation of the angiotensinII (Ang-II) receptor AT-1 and caldesmon, an actin-binding protein that regulates contraction. Integrin b4 has been identified as a EC gene dramatically upregulated by statins which may also be relevant to vascular protection

the downregulation of inflammatory genes, including IL-6 [46]. Notably, in a relevant murine model, PPARa has been identified as a significant modulator of ALI as treatment of animals with a fibrate, known PPARa agonists, was associated with an attenuation of LPS-induced lung vascular injury. Moreover, these effects were absent in PPARa-deficient mice [47]. Early evidence of the anti-inflammatory properties of statins including the downregulation of proinflammatory genes highly reminiscent of that effected by PPARa led to the investigation of PPARa as a key regulator of these effects. Subsequently, in mouse models of acute inflammatory injury, the protective properties of statins were found to be absent in PPARa-deficient animals [48]. Moreover, statin treatment was associated with the inhibition of protein kinase C in macrophages and neutrophils, resulting in the PPARadependent attenuation of LPS-induced inflammatory gene expression. Accordingly, PPARa-mediated vascular protection in response to inflammatory stimuli represents yet another distinct attribute of statins relevant to the treatment of ALI.

that of genes highly relevant to inflammatory responses and vascular function. Among these genes are mediators of Rho GTPase activation and the thrombin regulatory elements thrombomodulin, plasminogen activator inhibitor-1 (PAI-1), tissue factor, and protease-activated receptor 1 (PAR-1). Marked alterations in these genes and others contribute significantly to vascular protection by statins (Fig. 4). In mouse aortas and cultured ECs, RhoA expression is upregulated by statins and RhoA accumulates in the cytosol owing to the inhibition of geranygeranylation, which prevents translocation to the cell membrane [32]. The increase in Rho gene transcription by statins is regulated by a negativefeedback mechanism mediated by the inhibition of actin stress fiber formation. Separately, a modest increase in the level of Rac1 and differential expression of Rho GTPase modifiers, including specific GTPase activating proteins and guanine nucleotide exchange factors, has been reported in statin-treated ECs [14]. These effects further implicate the Rho GTPases as key regulators of statin-mediated signaling and likely account for the increased levels of GTP-loaded Rho GTPases induced by statins within the cytosol of ECs [14, 49]. Thrombomodulin is an endothelial cell-surface thrombinbinding glycoprotein with anticoagulant properties. The binding of thrombin to thrombomodulin, with subsequent protein C activation, enables the complex to function as an anticoagulant and the amplification of thrombomodulin expression

7 Differential Endothelial Gene Expression by Statins In addition to their direct effects on EC signaling, statins are also able to induce differential EC gene expression, including

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serves to mitigate EC inflammatory responses. Statin treatment of ECs results in the upregulation of thrombomodulin [14] as well as the downregulation of tissue factor, a cell-surface receptor involved in the generation of thrombin through the extrinsic coagulation pathway, and PAI-1, a serine protease inhibitor that specifically inhibits fibrinolysis. Notably, the inhibition of Rho GTPase geranygeranylation by statins has been implicated as the mechanism underlying the differential expression of both of these genes [50, 51]. Furthermore, statins also downregulate PAR-1, the EC thrombin receptor [14]. The combined effects of increased thrombomodulin expression and decreased expression of tissue factor, PAI-1, and PAR-1 results in statinmediated EC thrombin resistance and improved endothelial integrity [14]. Among the EC genes that are most dramatically altered by statin treatment, two are particularly intriguing with respect to potential mechanisms of statin protection in ALI. Expression of caldesmon, a cytoskeletal protein involved in the actin rearrangement and contractility associated with agonist-induced EC barrier disruption, is decreased more than twofold at both the gene and the protein level in response to statins [14]. Evidence of the important role of cytoskeletal rearrangement in statin-mediated EC barrier protection suggests that this marked downregulation of caldesmon may be a significant contributing factor independent of statin modulation of Rho GTPase signaling. Separately, the EC gene which is most significantly modulated by statins is integrin b4, found to be upregulated more than sevenfold at both the gene and the protein level [14]. The integrins are heterodimeric transmembrane proteins with a and b subunits that mediate both inside-out and outside-in signaling pathways. Among the eight b subunits, integrin b4 is uniquely characterized by its long cytoplasmic tail which comprises over 1,000 amino acids. As the literature on the role of integrin b4 in EC signaling is sparse, the functional significance of increased levels of integrin b4 in statin-treated ECs is unclear. However, integrin b4 has been implicated in activation of the Rho GTPases [52] as well as mitogen-activated protein kinase and NF-kB [53] signaling, pathways highly relevant to inflammation and injury. In addition, several studies have implicated other b integrins as mediators of lung vascular permeability and EC barrier function. In particular, integrin b5 has been characterized as a mediator of ALI in distinct models of rat ischemia–reperfusion lung injury and murine ALI [54]. Moreover, integrin b5 inhibition in vitro was found to attenuate agonist-induced EC actin stress fiber formation and monolayer permeability. Additionally, in separate reports, integrin b2 [55] and integrin b6 [56] have also been implicated as mediators of increased lung vascular permeability associated with ALI, although the precise mechanisms of integrin-mediated effects in these contexts remain to be fully characterized. Accordingly, the dramatic upregulation of EC integrin b4 by statins may ultimately prove

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to be among the most important mechanisms of statin-mediated vascular protection.

8 Statins in Animal Models of ALI The well-established beneficial role of statins in endothelial function in  vitro paved the way for investigations into the utility of these drugs in preclinical studies of inflammatory lung injury. Abundant evidence for the beneficial effects of statins on eNOS upregulation, NAPDH oxidase inhibition, and cytoskeleton stabilization has translated into wellfounded optimism regarding the use of these drugs in relevant clinical settings. Subsequently, animal models of LPS-induced ALI and ischemia–reperfusion lung injury have provided further evidence to support the use of statins as a therapeutic option in these settings. In one report, mice pretreated with simvastatin prior to the intratracheal administration of LPS exhibited a marked decrease in indices of lung vascular leak and inflammation at 24 h, including significant reductions in BAL neutrophils and albumin content [57]. Additionally, statin treatment decreased Evans blue albumin dye extravasation into the lung tissue consistent with an attenuation of LPS-induced vascular leakage. These findings were corroborated by decreased infiltration of inflammatory cells and tissue edema evident by histology of lungs from animals pretreated with simvastatin prior to LPS compared with controls (Fig.  5). LPS-mediated lung gene expression was also significantly modulated in statin-treated mice within a number of relevant gene ontologies, including inflammation and immune responses, along with individual genes implicated in the development or severity of ALI [57]. Additional studies utilizing similar murine models of lung injury induced by LPS or the intratracheal administration of Klebsiella pneumoniae have further confirmed the protective of effects of statin pretreatment with respect to microvascular injury, tissue influx of neutrophils, and release of cytokines [58]. These studies provide compelling in vivo evidence for the role of statins in the treatment of ALI through the attenuation of vascular permeability and inflammation. Ischemia–reperfusion represents a distinct mechanism of ALI that may be encountered in patients immediately after lung transplantation. The cause of ischemia–reperfusion lung injury is attributable to ROS generated during periods of both tissue ischemia and subsequent reperfusion after transplantation. This production of ROS has been linked to increased NADPH oxidase activity with concurrent decreases in the level of bioavailable NO and is associated with an increase in vascular permeability. In a relevant animal model, statin pretreatment prior to the induction of lung ischemia for 90 min followed by reperfusion for up to 4  h was associated with a significant reduction in lung vascular permeability as measured by

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In a relevant animal model, pretreatment with cerivastatin in mice rendered septic by the intraperitoneal injection of LPS, Staphylococcus aureus, or Salmonella typhimurium was associated with improved survival, enhanced bacterial clearance, and significantly reduced expression of serum cytokines, TNF-a and IL-6 [61]. Additionally, in another model of sepsis induced by cecal ligation and perforation in mice, statin administration initiated after sepsis was also found to confer significant survival benefits [62].

9 Looking Forward: Clinical Trials of Statins in ALI

Fig. 5  Statin protection in a murine model of acute lung injury (ALI). Lung histological sections from a mouse model of lipopolysaccharide (LPS)-induced ALI is shown. Compared with vehicle controls, lungs from animals administered LPS intratracheally (2 mg/g, 24 h) exhibit significant infiltration of inflammatory cells (small arrows) and interstitial edema (large arrows). Pretreatment of mice with simvastatin (20 mg/ kg, 24 h prior to LPS administration and again concomitant with LPS administration, via intraperitoneal injection) confers significant protection with lung histological structure in these mice comparable to those of uninjured vehicle controls. (Reproduced with permission [57])

I-bovine serum albumin extravasation [59]. Additionally, treatment with a statin resulted in a significant reduction in lung tissue myeloperoxidase content and marked reductions in the number of BAL inflammatory cells. These findings are supported by a report of improved outcomes in human lung transplant recipients who had coincidentally been prescribed statins for hyperlipidemia. Compared with lung transplant patients who had not been prescribed a statin, patients taking statins had significantly better lung function by spirometry and improvement in 6-year survival (91% vs. 54%) [60]. Notably, as the available animal studies of ALI demonstrate the protective effects of statin pretreatment, it is unclear if statin treatment after the development of ALI in these models would provide the same degree of protection. Accordingly, the most relevant extension of these studies to humans applies to patients at risk for developing ALI rather than to patients with previously established ALI. For this reason, as sepsis represents the most common precipitating factor for the development of ALI, the role of statins as a novel therapy for sepsis has garnered significant interest. Sepsis is defined by a systemic inflammatory response occurring in response to an infection. In this context, the resulting immune response may be injurious to multiple organs throughout the body and, in the severest cases, may culminate in multiorgan dysfunction syndrome (MODS). Lung involvement is typically characterized by vascular dysfunction and increased vascular permeability which may progress to ALI.

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A preponderance of in vitro and animal data now firmly support the investigation of statins as a novel therapy for patients either with or at risk for ALI. Additional supportive evidence is available through reports of the association of statins on improved outcomes in a number of relevant patient populations. For example, a populationbased cohort analysis of 141,487 patients over the age of 65 years hospitalized for acute coronary syndrome, ischemic stroke, or revascularization between 1997 and 2002 who survived at least 3 months after discharge investigated the incidence of sepsis in those prescribed a statin after leaving the hospital. This study found that the incidence of sepsis was lower in patients receiving a statin compared with controls (71.2 vs. 88.0 events per 10,000 person-years; hazard ratio 0.81) [63]. Among hospitalized patients with acute bacterial infections, statin use has been reported to be associated with a dramatic decrease in the incidence of both severe sepsis (2.4% vs. 19%) and admissions to the intensive care unit (4% vs. 12%) [64]. Separately, at least two published studies have reported significantly reduced hospital mortality in bacteremic patients who were taking a statin compared with patients who were not (6–8% vs. 23–28%) [65, 66]. At the other end of the clinical spectrum, statin therapy has been reported to be associated with significantly reduced mortality (72% vs. 35%) in the setting of MODS secondary to advanced sepsis, ALI, or other causes [67]. Most recently, a meta-analysis identified 20 studies in which the effect of statins in infections and sepsis was studied [68]. Of these, 11 studies had data regarding mortality as the main outcome and eight of these reported evidence of decreased mortality associated with statin use (one study reported increased mortality, whereas the other two reported no association). Taken together, these studies provide both compelling data to support the potential efficacy of statin therapy in patients with sepsis and ALI as well as evidence consistent with a highly favorable safety profile of statin use in infected patients, including those who are critically ill.

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10 Summary The promise of statins for the treatment of ALI highlights the pleiotropic properties of these drugs. Through the upregulation of eNOS and inhibition of NADPH oxidase, statins serve as potentially powerful modulators of endothelial inflammatory injury. Additionally, by stabilizing the EC cytoskeleton and altering EC gene expression, statins target processes central to ALI, namely, endothelial permeability and the activation of coagulation, with subsequent microthrombi formation. Investigations of statin effects on these various pathways have yielded impressive in vitro results that have been translated into success in relevant animal models. Finally, compelling observational data related to the use of statins in various patient populations has now set the stage for definitive randomized, placebo-controlled clinical trials. The potential use of statins in the treatment of ALI signifies the progression of the application of these drugs to a vastly diverse field of clinical medicine, and ultimately may point the way for other clinical applications related to diseases characterized by inflammation and vascular dysfunction.

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Chapter 63

Genomics of Acute Lung Injury and Vascular Barrier Dysfunction Roberto F. Machado and Joe G.N. Garcia

Abstract  Acute lung injury (ALI) is a devastating ­syndrome of diffuse alveolar damage that develops via a variety of local and systemic insults such as sepsis, trauma, ­pneumonia, and aspiration. It is interestingly to note that only a subset of individuals exposed to potential ALI-inciting insults develop the disorder and the severity of the disease varies from complete resolution to death. In addition, ALI susceptibility and severity are also affected by ethnicity as evidenced by the higher mortality rates observed in African-American ALI patients compared with other ethnic groups in the USA. Moreover, marked differences in strain-specific ALI responses to inflammatory and injurious agents are observed in preclinical animal models. Together, these observations strongly indicate genetic components to be involved in the pathogenesis of ALI. The identification of genes contributing to ALI would potentially provide a better understanding of ALI pathobiology, yield novel biomarkers, identify individuals or populations at risk, and prove useful for the development of novel and individualized therapies. Genome-wide searches in animal models have identified a number of quantitative trait loci that associate with ALI susceptibility. In this chapter, we utilize a systems biology approach combining cellular signaling pathway analysis with population- based association studies to review established and suspected candidate genes that contribute to dysfunction of endothelial cell barrier integrity and ALI susceptibility. Keywords  Alveolar capillary permeability • Lung injury • Genomics • Genetics • Bioinformatics • Polymorphism

1 Introduction  

Acute lung injury (ALI) is a devastating syndrome of diffuse alveolar damage that develops via a variety of local and ­systemic insults such as sepsis, trauma, pneumonia, and J.G.N. Garcia (*) University of Illinois at Chicago, 1737 West Polk Street, Suite 310, Chicago, IL 60612, USA e-mail: [email protected]

a­ spiration [1]. Deranged alveolar capillary permeability, ­profound inflammation, and extravasation of edematous fluids into the alveolar spaces are critical elements of ALI, reflecting the substantial surface area of the pulmonary vasculature needed for alveolar gas exchange. ALI, together with its severest form, acute respiratory distress syndrome (ARDS), afflicts approximately 190,000 patients per year in the USA and has a mortality rate of 35–50% [2, 3]. It is interestingly to note, however, that only a subset of individuals exposed to potential ALI-inciting insults develop the disorder and the severity of the disease varies from complete resolution to death. In addition, ALI susceptibility and severity are also affected by ethnicity as evidenced by the higher mortality rates observed in African-American ALI patients compared with other ethnic groups in the USA [4]. Moreover, marked differences in strain-specific ALI responses to inflammatory and injurious agents are observed in preclinical animal models [5]. Together, these observations strongly indicate genetic components to be involved in the pathogenesis of ALI. The role that genetics plays in determining ALI risk or the subsequent severity of the outcome is one of the many unanswered questions regarding ALI pathogenesis and epidemiology. The identification of genes contributing to ALI would potentially provide a better understanding of the pathogenic mechanisms of ALI, yield novel biomarkers, identify individuals or populations at risk, and prove useful for the development of novel and individualized therapies. However, a traditional genetic approach to studies using family linkage mapping is not feasible given the sporadic nature of ALI and the necessity of an extreme environmental insult. Further, genetic studies of ALI are challenging owing to the substantial ­phenotypic variance in critically ill patients, diversity in the lung injury evoking stimuli, presence of varied comorbid illnesses common in the critically ill patient, complex gene–environment interactions, and potentially incomplete gene penetrance [6, 7]. Despite these inherent challenges, the unrivaled progress made in the post-human genome era combined with the utilization of sophisticated bioinformatics and high-throughput methods have allowed significant advances to be made. For example, these tools are now linked to escalating knowledge of the

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_63, © Springer Science+Business Media, LLC 2011

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­ olecular mechanisms of lung endothelial permeability, a hallm mark of ALI and an attractive target for the design of novel therapies, to identify candidate genes whose variants are potentially involved in ALI susceptibility. Genome-wide searches in animal models have identified a number of quantitative trait loci that associate with ALI susceptibility [8]. In this chapter, we utilize a systems biology approach combining cellular signaling pathway analysis with population-based association studies to review established and suspected candidate genes that contribute to dysfunction of endothelial cell barrier integrity and ALI susceptibility. The integration of high-throughput gene expression profiling in preclinical models of ALI with bioinformatics has led to the identification of differentially expressed genes in response to ALI whose variants are potentially involved in ALI susceptibility and severity. This approach confirmed long-suspected ALI-associated candidate genes, but more importantly, identified novel genes not previously implicated in ALI. Increasing knowledge of the molecular mechanisms of endothelial-barrier-regulatory pathways has also enhanced the ability to find novel ALI candidate genes. The analysis of the molecular pathways involving the cytoskeletal scaffolding and the dynamic cytoskeletal changes driving cell shape alterations, a key feature of vascular permeability, has identified additional genes contributing to the development and severity of ALI, thereby providing novel therapeutic targets in this devastating illness. Genes encoding proinflammatory cytokines, growth factors and mediators, receptors for barrier-regulatory agonists, and mechanical-stress-sensitive genes expressed in endothelium which regulate inflammatory responses also serve as attractive ALI candidate genes and are representative of the diverse but fertile areas of exploration for candidate SNPs affecting ALI susceptibility and severity.

2 Candidate Genes in Acute Lung Injury: Vascular Barrier Regulatory Cytokines, Growth Factors, and Mediators

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via type I and type II angiotensin II receptors that ­subsequently activate the nuclear factor kB (NF-kB) pathway [13, 14]. The RAS is also involved in the fibrotic response to ALI via induction of transforming growth factor expression [15]. The most compelling evidence for RAS involvement in ALI has come from the effective attenuation of ALI pathobiology by ACE inhibitors or angiotensin receptor blocking drugs [16, 17] and ACE knockout mice in preclinical models of ALI [18]. An intronic insertion (I) or deletion (D) of a 287-bp Alu repeat sequence in the human ACE gene, located on chromosome 17q35, has been associated with ACE levels and activity in serum [19, 20]. The D allele possesses a higher enzyme activity which parallels the higher gene expression in individuals with DD genotype [21]. The initial association of the DD genotype in the ACE gene with increased ALI mortality provided the impetus for subsequent studies to more firmly establish a genetic basis of ALI and to identify ALI candidate genes [22]. Caucasian patients with ARDS show significantly higher frequencies of the DD genotype and the D allele as compared with ventilated intensive care unit (ICU) patients without ARDS, patients after coronary artery bypass surgery, or healthy controls. Moreover, ARDS patients with DD genotype show markedly higher mortality (54%) in comparison with the II genotype (11%) or strike ‘‘4’’ ID genotype (28%) [22]. The higher mortality rate in ARDS patients with DD or ID genotype as compared with II genotype was subsequently confirmed in Han Chinese patients in Taiwan, although the frequency of the D allele is significantly lower in the Chinese population as compared with Western populations [23]. Compared with Caucasians, a higher frequency of D allele has been reported among Africans (Nigerian and African-American populations) [24, 25], potentially contributing to the observed disparity in ALI-associated higher mortality rates in African-Americans [4]. However, to date, no association study of ACE polymorphisms and lung injury has been performed in African-Americans. In contrast, Mexican and Amerindian populations have slightly lower allelic frequencies of the D allele [25]. Thus, ACE represents a highly viable endothelial candidate gene and attractive target in acute inflammatory lung disease.

2.1 Angiotensin-Converting Enzyme Angiotensin-converting enzyme (ACE) is a member of the rennin–angiotensin system (RAS), balancing the levels of angiotensin I and angiotensin II, with significant expression in lung vascular endothelium as compared with other vascular beds [9]. The RAS is considered to be an important regulator of inflammation that contributes to ALI by altering vascular permeability, vascular tone, fibroblast activation, and endothelial–epithelial cell survival [10–12]. For example, angiotensin II activates inflammatory processes by upregulating proinflammatory cytokines and chemokines

2.2  Tumor Necrosis Factor Tumor necrosis factor (TNF) a, an early mediator of ALI development, is a potent proinflammatory cytokine which dramatically increases endothelial cell permeability, cytokine production, and a variety of cytotoxic and proinflammatory compounds which lead to subsequent vascular leakage and disturbed lung water balance. Both TNFa and TNFb subtypes appear in the circulation, in bronchoalveolar lavage (BAL) fluid and in pulmonary edema fluid during the onset of

63  Genomics of Acute Lung Injury and Vascular Barrier Dysfunction

lung injury. As such, the elevated levels of TNF and its ­soluble receptors are commonly used as markers of inflammation and are associated with morbidity and mortality in ALI patients [26]. Both the TNFa and TNFb genes lie in close proximity within the major histocompatibility complex, with several polymorphisms described in this region. The -308G/A promoter polymorphism in the TNFa gene and the NcoI restriction fragment length polymorphism in the TNFb gene appear to influence the expression of TNFa. The carriers of the -308A allele and homozygotes for the TNFb2 allele exhibit increased TNFa expression and have increased susceptibility and mortality to sepsis [27, 28]. In patients with ARDS, the -308A allele is also associated with increased 60-day mortality, with the strongest association found among younger individuals [29]. However, in ARDS patients with direct or indirect pulmonary injury, these SNPs are associated with alterations in ALI susceptibility (TNFa -308 G/A SNP only in the direct pulmonary injury group, and TNFb NcoI only in the indirect pulmonary injury group). Owing to the extent of linkage disequilibrium in the region, it remains unclear as to whether these are regulatory SNPs or if the TNF protein level is modulated by a third locus or a haplotype [30]. Promoter SNPs within the TNFa gene (-238 G/A, -857 C/T) have been associated with inflammatory bowel disease along with the -308 G/A SNP [31]. Thus, the role of TNF variants in inflammatory disorders is apparent and indicates a need for further study of other TNF variants in association with ALI.

2.3 Interleukin-6 Interleukin-6 (IL-6) is an acute-phase response cytokine that plays a key role in the activation of B and T cells. Inflammatory cytokines, including IL-6, are essential for the immune system homeostasis; however, when IL-6 production is exaggerated as observed in inflammatory lung disorders including ALI [22, 32], clearly detrimental outcomes are observed. ALI-related increased levels of IL-6 have been established in the BAL fluid of critically ill patients with ARDS, sepsis, and trauma [33, 34] in association with ALI adverse outcome [35] and development of multisystem organ failure [36]. In prior reports, we observed significantly higher expression of IL-6 and the IL-6 receptor genes across multiple-species ALI models and in human lung endothelium exposed to ventilator-induced mechanical stress as well as in differential region-specific expression in lungs of the canine ALI model [37–39]. On the basis of these data, the IL-6 gene constitutes an excellent ­candidate gene to understand the genetic basis underlying ALI. A functional polymorphism in the IL-6 gene promoter region at the -174 position (G-174C) has been associated with ­alterations in both gene expression and IL-6 levels and lower circulating IL-6 concentrations and lower mortality rates in

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patients with acute respiratory failure admitted to the ICU [22]. The ­contrasting correlation between G-174C alleles and circulating IL-6 levels has also been reported [32]. The haplotype involving -174 G/C, 1753C/G, and 2954G/C is associated with higher mortality (and other secondary clinical outcomes) in a cohort of septic patients of European descent [40]. We further evaluated 14 IL-6 gene tagging SNPs covering the entire gene for potential association in sepsis and ALI patients of European descent [32]. No single SNP was identified as significantly associated with ALI; however, a  common haplotype (comprising -1363G/-572G/-174G/ 1208A/1305A/4835C) with a frequency of 63% in cases and 49% in controls showed a significant association with ALI susceptibility. In addition, homozygote carriers of the risk haplotype are twice as frequent in ALI cases (44.8%) than in controls (22.9%), yielding a highly significantly increased odds ratio for developing ALI (odds ratio 2.73; 95% confidence interval, 1.39–5.37; p = 0.003). This haplotype spans the entire IL-6 gene including the G allele at position -174, i.e. the risk allele for susceptibility to ALI noted above. These data support the association of the IL-6 gene with ALI susceptibility and illustrate the value of haplotype analysis as a robust approach in association studies.

2.4 Vascular Endothelial Growth Factor Vascular endothelial growth factor (VEGF) is an endothelialcell-specific mitogen that regulates angiogenesis, migration, and cell permeability [41]. VEGF plays an important role in several organs by directly regulating vascular permeability to water and proteins. Lung overexpression of VEGF induces increased pulmonary vascular permeability, resulting in marked pulmonary edema [42], and plasma VEGF levels are significantly elevated in ALI patients [43]. Several studies have reported the association of low levels of VEGF with the severity of ARDS and elevated levels with the recovery from ARDS, indicating a role for VEGF in the repair process of lung injury [44]. Several polymorphisms have been described in the VEGF gene, primarily in association with cancer susceptibility and severity. The C/T SNP at position 936 of the 3¢ untranslated region (UTR) of the gene has been associated with higher VEGF plasma levels in healthy subjects [45]. Recently, the C936T SNP in the VEGF gene has been associated with ARDS susceptibility and severity (increased mortality) in subjects of European descent [46, 47]. The haplotype TCT at position C-460 T, C + 405G, and C + 936 T was significantly associated with a higher rate of mortality in ARDS patients and higher plasma levels of VEGF [47]. These studies highlight the VEGF gene as an attractive barrier-regulatory ALI candidate gene and molecular target in ALI therapeutic strategies.

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Table 1  Genes with significant differential expression in multispecies models of acute lung injury and number of PubMatrix citations PubMatrix (tool for multiplex literature mining, http://pubmatrix.grc.nia. nih.gov), April 2009 Fold Acute lung Endothelial Lung change Gene Endothelium permeability Sepsis Inflammation diseases (p < 0.05) injury Genes symbol Interleukin-1b Interleukin-6 Tissue factor/thromboplastin Plasminogen activator inhibitor type I Cyclooxygenase II Interleukin-13 Aquaporin 1 Plasminogen activator urokinase receptor Interleukin-1 receptor antagonist Pre-B cell colony enhancing factor Chemokine receptor 4 Growth arrest DNA damage inducible a

IL-1b IL-6 F3 PAI-1 COX2 IL-13 AQP-1 PLAUR

1.53 1.84 1.52 1.47 1.79 1.3 1.3 1.47

168 459 39 54 2 16 11 10

829 1,571 971 1270 278 85 74 285

66 168 34 23 11 12 52 8

523 1,906 452 189 60 52 1 21

4,219 14,178 792 888 717 1,467 18 165

649 2,446 530 303 107 1,070 19 122

IL-1RA PBEF CXCR4 GADD45a

2 2.82 1.62 1.71

24 16 24 0

83 16 316 1

4 5 8 1

208 10 90 0

1,200 59 1,265 11

166 17 394 7

2.5 Chemokine Receptor 4 Chemokine receptor 4 (CXCR4) is an a-chemokine receptor specific for stromal-derived factor 1 (SDF-1; also known as CXCL12) that plays an important role in cell migration, inflammation, B lymphocyte development, angiogenesis, and human immunodeficiency virus (HIV) infection (HIV coreceptor) [48–50]. Chemokine receptors are G-protein-coupled receptors, which trigger diverse signaling cascades including activation of G proteins and the phosphatidylinositol 3-kinase, Janus kinase/signal transducer and activator of transcription, Rhop160 Rho kinase, and mitogen-activated protein kinase signaling pathways [51]. The activation of these signaling pathways is often accompanied by the internalization of chemokine receptors and their trafficking back to the plasma membrane. This intracellular turnover determines the leukocyte responsiveness to chemokines [52]. Nonmuscle myosin II A is a molecular motor that binds with the cytoplasmic tail of CXCR4 and CCR552 and participates in the SDF-1-dependent endocytosis of CXCR4 via dynamic interaction with a-arrestin, a key component of the CXCR4 internalization pathway [50]. The CXCR4 gene was identified as a novel candidate gene in ALI as it survived two filtering strategies dedicated to identifying ALI-susceptibility genes associated with elevated levels of mechanical stress as observed in mechanical ventilator-associated lung injury (VALI). Our orthologous gene approach determined ALI-specific gene ontologies – coagulation, inflammation, chemotaxis/cell motility, and immune response [38] – involving recognized genes likely to participate in ALI pathogenesis [IL-6, aquaporin 1 (AQP-1), plasminogen activator inhibitor type I (PAI-1)], as well as novel genes not previously known to be mechanistically involved in ALI, including CXCR4 [38] (Table  1). We subsequently utilized a consomic rodent approach with introgression of rat chromosomes 2, 13, 16, and 17, which contained the highest density of VALI-responsive genes [39]. Introgression of the VALI-sensitive Brown Norway

(BN) rat chromosome 13, containing several genes, including CXCR-4, into the VALI-resistant Dahl salt-sensitive (SS) rat resulted in conversion of the SS consomic rats to a VALIsensitive phenotype [39]. Surface expression of CXCR4 is downregulated by interleukin-4, interleukin-13, and granulocyte–macrophage colony-stimulating factor and upregulated by interleukin-10 and transforming growth factor-b (TGFb) [53], suggesting that CXCR4 may also play a role in the fibrotic response to ALI via TGFb signaling. Polymorphisms in the CXCR4 gene have not yet been reported; however, a SNP in the 3¢ UTR of the SDF-1 gene (G801A), is associated with susceptibility to AIDS and type 1 diabetes [54, 55]. We are currently exploring CXCR4 as a potential ALI-associated candidate gene as suggested by the density of PubMatrix citations relating CXCR4 to inflammation (1,151 published papers), endothelium (297 published papers), ALI (28 published papers) and endothelial permeability (eleven published papers). PubMatrix is a Web-based tool that allows simple text-based mining of the NCBI literature search service PubMed using any two lists of keywords terms, resulting in a frequency matrix of term co-occurrence.

3 Strategies to Identify New Genes and Biomarkers in Acute Lung Injury The advent of high-throughput gene sequencing and expression technologies, and complete genome sequencing of model organisms, now provides the tools to perform largescale analyses of the genome in complex disorders such as ALI. Whole genome scans, in silico approaches, utilization of consomic rats, and a candidate gene approach involving expression profiling and pathway analysis are proving exceptionally useful in identifying novel candidate genes and genetic variations (Fig. 1).

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Fig.  1  Novel approaches to identify acute lung injury (ALI) genes. High-throughput gene sequencing and expression technologies, and complete genome sequencing of model organisms, now provide the tools to perform large-scale analyses of the genome in complex disorders such as ALI. Genome-wide association study (GWAS) platforms are effective and have been successfully used in diverse disorders, but although this approach has yet to be employed in either sepsis or ALI, the application of GWAS to the disease is clearly imminent. The differential gene expression between lung apex/base regions as well as between gravitationally dependent/nondependent regions of the lung base in a canine model of ventilator-associated lung injury (VALI) identified ALIimplicated lung genes in response to local mechanical stress within the lung. This approach identified the already established ALI gene macrophage migration inhibitory factor and novel genes such as growth

arrest DNA damage inducible (GADD45) and pre-B cell colony enhancing factor (PBEF). Our multispecies orthologous gene approach in human (endothelial cells), rat, mouse, and canine models of VALI exhibits expression of common ALI-implicated evolutionarily conserved genes (orthologues) across the species. The genes with a unidirectional 1.3-fold change (p > 0.05) are found to reside in high density on rat chromosomes 13 and 16, the chromosomal loci used to develop the consomic rodent model. Together, these approaches identified novel ALI genes such as PBEF, chemokine receptor (CXCR-4) GADD45. Interrogating the prospective pathways involved in endothelial permeability and correlation with these differentially expressed genes in VALI models identified the most putative ALI genes such as myosin light chain kinase (MYLK), sphingosine 1-phosphate receptor 1, cMet, and vascular endothelial growth factor (VEGF)

High-throughput whole genome scanning technology has recently emerged as a powerful tool, particularly in detecting disease-susceptibility genes with modest effects. The Haplotype Mapping Project [56], which identified blocks of SNPs associated with each other, has allowed selection of the most informative SNPs for further disease association studies [57]. Currently, the most commonly used high-throughput SNP platforms involve assessment of over one million SNPs spanning the genome, i.e. genome-wide association studies (GWAS). GWAS platforms are effective and have been successfully used in diverse disorders such as agerelated macular degeneration [58], inflammatory bowel

d­ isease [59], type 2 diabetes [60], and stroke [61]. Although this approach has yet to be employed in either sepsis or ALI, the application of GWAS to the disease is clearly imminent. Another method to identify ALI candidate genes is an orthologue gene in silico approach. The basis of this approach is the hypothesis that patients with ALI and preclinical animal models of ALI would exhibit commonality in expression of evolutionarily conserved genes across ­species. For example, profiling results from more than 50 Affymetrix microarray chips obtained from ventilator-associated ALI models (human, rat, mouse, canine) identified 3,077 genes whose expression was altered across all four species in response to ventilator-associated

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mechanical stress [37, 38]. Filtering these results for a ­unidirectional change in gene expression with greater than 1.3fold change in expression refined the list to 69 genes, reflecting specific ALI-associated gene module/ontology categories: coagulation, inflammation, chemotaxis/cell motility, and immune response. This approach identified multiple genes already recognized as ALI genes (such as IL-6, AQP-1, and PAI-1), but also identified several novel genes that were not previously known to be mechanistically involved in ALI [38]. Complementing the in silico approach described above, a consomic rat approach can also be utilized to identify novel ALI gene candidates. In an experimental study, two strains of inbred rodents were determined to have differing susceptibility to VALI (20 mL/kg, 4 h): VALI-sensitive BN rats and the VALIresistant Dahl SS rats. Using microarray analysis and a bioinformatic-intense ­candidate gene approach, we identified 245 differentially expressed potential VALI genes with ontologies such as transcription, chemotaxis, and inflammation. Because chromosomes 2, 13, 16, and 17 were found to contain the highest number of VALI-response genes, consomic SS rats containing substituted BN chromosome 13 were exposed to VALI mechanical stress, resulting in conversion of the resistant SS rat to VALI sensitivity [39]. Extensive expression profiling across preclinical ALI models can extend the identification of ALI gene candidates to determination of allelic frequencies of gene polymorphisms (SNPs) that may confer ALI risk or severity. This “candidate gene approach” has identified several candidates with hypothesized significant mechanistic roles in lung injury, inflammation, or repair in the setting of ALI and VALI [62]. Further, given the availability of sophisticated bioinformatic methods and increasing knowledge of the molecular and cellular mechanisms of lung injury, candidate genes can also be identified via analysis of cellular pathways involved in ALI pathogenesis [63, 67].

4 Novel Acute Lung Injury Candidate Genes and Biomarkers The application of the novel techniques described in the previous section is proving to be exceptionally useful in identifying novel candidate genes and genetic variations in the study of the pathobiology of ALI. These novel gene and biomarkers are discussed in this section.

4.1 Myosin Light Chain Kinase Myosin light chain kinase (MLCK) is an enzyme that phosphorylates regulatory myosin light chains, which allows myosin cross-bridging interactions with F-actin. In endothelial

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cells, the contraction of the actomyosin complex generates a stronger centripetal force that overcomes the force keeping the adjacent endothelial cell tethered, leading to endothelial retraction, decreased intercellular adhesion, and increased vascular permeability [68, 69]. This phenomenon is physiologically relevant as evidenced by nonmuscle MLCK (nmMLCK) isoform knockout mice [which retain the smooth muscle MLCK (smMLCK) isoform] that are less susceptible to lipopolysaccharide (LPS)- and ventilator-induced ALI [70, 71]. Further, treatment with a MLCK inhibitor prior to LPS exposure in the wild-type mice attenuates endothelial cell barrier dysfunction and inflammation [70]. Thus, the myosin light chain kinase gene (MYLK), which encodes for MLCK, is an excellent ALI candidate gene. Since initial cloning of the highly expressed nmMLCK in endothelium in our laboratory [72], we have identified substantial roles of nmMLCK in cytoskeleton rearrangement of endothelial cells regulating vascular barrier function [64, 68], angiogenesis, and leukocyte diapedesis [73], consistent with a potential mechanistic role for MLCK in the genesis of ALI. The human MYLK gene is located on chromosome 3q21 and encodes three proteins, including nmMLCK, smMLCK, and telokin. We sequenced exons, exon–intron boundaries, and 2 kb of the 5¢ UTR of MYLK in healthy individuals, patients with sepsis alone, and patients with sepsis-associated ALI, all of European and African-American descent [66], and identified 51 SNPs (ten exonic, 31 intronic, nine in the 5¢ UTR, and one in the noncoding exon 1), of which 28 were chosen for further linkage disequilibrium studies. Five of the ten coding MYLK SNPs confer an amino acid change (Pro21His, Pro147Ser, Val261Ala, Ser1341Pro, and Arg1450Gln) in MLCK. Subsequently, association analysis of both single SNPs and haplotypes demonstrated very strong associations in both ­ethnic groups [66]. In European Americans, the rs3845915A/ MYLK_037C haplotype was associated with more than a fivefold increase in the risk of developing ALI and sepsis. In  contrast, the haplotype MYLK_021G/MYLK_022G/ MYLK_011T conferred specific risk for ALI but not sepsis [66]. The 5¢ haplotype of the MYLK gene also conferred ALIspecific risk in both European- and African-descent subjects; however, the 3¢ region haplotype was associated with ALI only in African-descent subjects. In African-Americans, the haplotype hcv1602689C/MYLK_037A/rs11707609G is substantially more prevalent in ALI (11%) as compared with sepsis (1%). This CAG haplotype is not found in European Americans, suggesting a potential genetic contribution to the observed ethnicity-specific differences in ALI/ARDS prevalence and susceptibility [4]. We noted similar findings in association studies involving a cohort with trauma-induced ALI [74]. We have also evaluated the association of 17 MYLK genetic variants with severe asthma in both European American and African-American populations and identified  a  SNP highly associated with severe asthma in

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­ frican-Americans [75] consistent with data linking this A chromosomal locus (MYLK, 3q21.1) to asthma and asthmarelated phenotypes [75]. Taken together, these data strongly implicate MYLK genetic variants as risk variants in inflammatory lung disorders, such as ALI and asthma.

4.2 Macrophage Migration Inhibitory Factor Macrophage migration inhibitory factor (MIF) is an ALI candidate gene and recognized biomarker, initially discovered as a soluble product of activated T cells and named for its role in inhibiting random macrophage migration [76]. MIF is a proinflammatory cytokine which binds to CD44 and CD74 and is produced by many cell types, including monocytes/macrophages, pituitary cells, vascular endothelium, and respiratory epithelium [77, 78]. MIF may serve as a delicate regulator of the cytokine balance between immunity and inflammation as MIF counterregulates the immunosuppressive effects of glucocorticoids [79]. The role of MIF as an endogenous prosurvival factor has been demonstrated in vitro. LPS-mediated induction of Flice-like inhibitory protein (FLIP) by MIF confers resistance to LPS-mediated endothelial cell death [80]. Suppression of MIF by RNA interference induces cell death and sensitivity to apoptotic stimuli [80]. In addition, MIF interacts with the multidimensional nmMLCK [81] isoform which regulates TNF-mediated apoptosis in addition to its potent effects on endothelial cell barrier dysfunction as discussed earlier [68, 69]. Together, these findings implicate the role of MIF in regulation of nonmuscle cytoskeletal dynamics and vascular pathophysiology, which is evident from the enhanced MIF levels in the serum, BAL fluid, and alveolar endothelium of patients with ARDS as compared with other critically ill patients [76, 78, 82]. We found significant increases in MIF transcript and protein ­levels in murine and canine models of ventilator-induced lung injury (VILI) (using high mechanical ventilation and endotoxin exposure, respectively) [82] and in human lung endothelium cells exposed to 48 h of cyclic stretch [83]. MIF deficiency or immunoneutralization appears to protect mice or rats from fatal endotoxic shock or other inflammatory diseases [84] although these results are not without controversy [85] and our own studies in 8–12-week-old mice failed to demonstrate a VILI/ALI-related phenotype which was different from controls (data not shown). MIF also upregulates the expression of AQP-1, the water channels expressed in alveolar endothelial and epithelial cells, and a candidate gene we identified in models of VILI-associated mechanical stress [38]. MIF may serve to modulate fluid movement into alveolar spaces, a cardinal feature of ALI [86]. To extend the likelihood that MIF serves as a putative candidate gene in ALI and sepsis, we studied the association of eight MIF polymorphisms, including the most studied MIF

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promoter G/C SNP at position -173, in a sepsis-induced ALI cohort (n = 506) of African- and European-descent cases [82]. No individual SNP showed a significant association with either ALI or sepsis; however, the carriers of the CC genotype (rs755622) and the carriers of the TT genotype (rs2070767) showed more than twofold increased risk of developing sepsis and ALI, respectively. This association was lost, however, after age and gender adjustment in a logistic regression model. In contrast, MIF haplotypes at the 3¢ region of the gene display strong association with ALI and sepsis, conferring both protection as well as susceptibility to ALI, in European and African populations [82]. Furthermore, the haplotype at the 5¢ promoter region of the gene involving a short tandem repeat at position -794 (CATT)5 and the -173 G allele show significant association with both ALI and trauma [82]; however, no association was found between promoter region haplotypes and MIF levels. Rheumatoid arthritis patients with the -173C allele have higher levels of MIF in the serum and synovial fluid than the carriers of the G allele and have a higher probability of developing idiopathic arthritis [87]. Thus, given these diverse MIF functions, MIF remains an attractive target in inflammatory diseases including the lung.

4.3 Sphingosine 1-Phosphate Receptor 1 The bioactive sphingolipid metabolite sphingosine 1-phosphate (S1P) is an important lipid mediator that enhances endothelilal cell barrier function in vivo and in vitro by ligating S1P receptor 1 (S1P1), which is encoded by an endothelial differentiation gene (EDG1 or S1P1) [88, 89]. S1P1 is a pertussis-toxin-sensitive, Gi-coupled receptor which induces Rac GTPase-dependent substantial increases in cortical actin polymerization critical to endothelial cell barrier enhancement [88, 90]. S1P1 activation enhances the organization and redistribution of vascular endothelial cadherin and b-catenin in junctional complexes in endothelium by phosphorylation of cadherin as well as p120catenin and inducing the formation of cadherin/catenin/actin complexes [91]. Understanding the role of S1P in enhancing endothelial cell barrier function underscores its importance as a therapeutic target in reversing loss of endothelial cell barrier integrity. In vivo administration of selective S1P1 competitive antagonists induces a dose-dependent disruption of barrier integrity in pulmonary endothelium [92, 93], whereas S1P1 agonists, SEW2871 and FTY720, promote vascular endothelial barrier function [94–96]. A compelling argument for S1P1 as an attractive ALI candidate gene is not only its ability to transduce signals which restore barrier integrity but also that S1P1 is the target for transactivation by receptors for other potent barrier-­protective agonists. These include EPCR (receptor for activated protein C) [65], c-Met [receptor for hepatocyte growth factor (HGF)] [97], CD44 (receptor for high molecular

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weight hyaluronan) [67], and the ATP receptor [98]. We recently resequenced the S1P1 gene (14 African-Americans and 13 European Americans) to search for common variations in the EDG1 gene and identified 39 SNPs in the EDG1 gene, with several promoter SNPs associated with asthma, another inflammatory lung syndrome [99].

4.4 c-Met (Hepatocyte Growth Factor Receptor) The role of HGF and its tyrosine kinase receptor c-Met has been investigated in lung development, inflammation, and repair [100] as well as in neoplastic processes such as cellular transformation, neoplastic invasion, and metastasis [101, 102]. SNPs causing underexpression of c-Met have been associated with autism and c-Met SNPs/mutations appear to be linked to lung cancer disparities in different ethnic groups. These include an N375S mutation in the HGFbinding domain of c-Met, an R988C SNP/mutation in the juxtamembrane domain, and an activating M1268T mutation in the tyrosine kinase domain (exon 19), all linked to development of solid tumors such as lung cancer, renal cancer, gastric cancer, and hepatocellular carcinoma [102]. HGF influences morphogenesis in epithelial cells from a variety of organs, including lungs, where HGF antisense oligonucleotides block alveolar and branching morphogenesis [103]. HGF expression and activity increase after 3–6 h of lung injury with intratracheally administered hydrochloric acid, suggesting that HGF plays a role in reparative responses to lung injury [104]. c-Met expression on type II pneumocytes is likely involved in increased type II pneumocyte proliferation and restoration of an intact alveolar epithelium [105]. c-Met is composed of a 50-kDa extracellular a subunit and a 145-kDa transmembrane b subunit [106] which contains tyrosine kinase domains, tyrosine phosphorylation sites, and tyrosine docking sites [107]. We demonstrated that HGF-mediated c-Met phosphorylation and c-Met recruitment to caveolin-enriched microdomains (CEMs) protects against the LPS-induced pulmonary vascular hyperpermeability that is regulated by high molecular weight hyaluronan (CD44 ligand) [108]. Our novel findings indicate that HGF/c-Met-mediated, CD44-regulated CEM signaling promotes Tiam1 (a Rac1 exchange factor)/dynamin 2 dependent Rac1 activation, and peripheral recruitment of cortactin (an actin cytoskeletal regulator), processes essential for endothelial cell barrier integrity. Understanding the mechanism(s) by which HGF/c-Met promotes increased endothelial cell barrier function may lead to novel treatments for diseases involving vascular barrier disruption, including inflammation, tumor angiogenesis, atherosclerosis, and ALI. However, on the contrary, the higher mortality rate in ALI patients with increased levels of HGF in BAL

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fluids [109] and in pulmonary edematous fluids [110] ­indicates severer injury and inflammation in response to increased HGF levels. It has now become increasingly clear that HGF plays an important role in normal and injured lung and may have a therapeutic potential in lung diseases.

4.5 Pre-B Cell Colony Enhancing Factor Pre-B cell colony enhancing factor (PBEF), was first identified by Samal and colleagues in 1994 as a protein secreted by activated lymphocytes in bone marrow stromal cells that stimulate early stage B cell formation in conjugation with stem cell factor and interleukin-7. A large body of work has now highlighted the power of a systems biology approach in the search for novel disease-susceptibility genes and potentially novel biomarkers, with PBEF serving as an excellent example of this approach (Fig.  2). We first identified marked upregulation of PBEF via microarray analyses of murine and canine models of VILI/ALI with increased gene/ protein expression in BAL fluid and serum samples from critically ill ICU patients with ALI and sepsis [111]. With only a total of eight papers in PubMed at that time, we next directly sequenced the PBEF gene in 36 subjects with ALI, sepsis, and healthy controls and conducted a PBEF SNP-based association study in ALI subjects of European and African-American descent [111]. We identified 11 SNPs in the PBEF gene with two promoter SNPs, T-1001G and C-1543  T, associated with ALI and sepsis. Genotyping of PBEF C-1543  T and T-1001G SNPs revealed significant associations of sepsis and ALI, with the strongest association found with the -1543C/-1001G haplotype. Univariate analysis found carriers of the G allele (T1001G) to have 2.75-fold higher risk of developing ALI as compared with controls (p = 0.002) [111]. These results were subsequently confirmed in a comparable but distinct replicate ALI population [112]. Interestingly, the -1543G/-1001C haplotype was also associated with increased ICU parient mortality, whereas the -1543 T/-1001 T haplotype was associated with fewer ventilator days and decreased ICU patient mortality [112]. A key challenge in genomic explorations is the ability to confirm the contribution of a SNP to a dysfunctional-geneinvolved disease process. Additional reports have highlighted the capacity for the PBEF gene to have an influence far beyond any B-cell regulatory function, with a key role in regulating vascular permeability [113] as well as inhibiting neutrophil apoptosis [114]. To further explore mechanistic participation of PBEF in ALI and VILI, we focused on the contribution of PBEF to endothelial function. Our prior immunohistochemical staining of canine-injured lung tissues localized PBEF expression to vascular endothelial cells, in addition to infiltrating neutrophils and type 2 alveolar epithelial cells [111]. Our in vitro studies showed that expression

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Fig. 2  Systems biology approach in defining the role of PBEF in ALI and ventilator-induced lung injury (VILI). Each systems biology compartment has been utilized to address PBEF involvement across the entire systems biology spectrum. This includes genes (microarrays, SNP discovery, small interfering RNAs, microRNAs, promoter luciferase assays), proteins (bacterial two hybrid studies, recombinant

human PBEF (rhPBEF), cell signaling, site-directed mutagenesis), organelles (nuclear events, mitochondrial function, cytoskeleton), cells (endothelial cells, neutrophils), organs (lung), preclinical animal models (genetically engineered PBEF+/− mice, VILI, and sepsis models), and populations [bronchoalveolar lavage (BAL) studies, biomarker studies in intensive care unit (ICU) subjects, SNP association studies]

of  PBEF in pulmonary artery endothelial cells increases ­thrombin-mediated vascular permeability [111], suggesting that enhanced PBEF expression may mediate the early increase in vascular permeability that is characteristic of ALI. Neutrophils harvested from the circulation of septic and ALI patients show marked inhibition of the apoptotic process in association with evidence of enhanced respiratory burst capacity [115, 116], with both activities largely restored with administration of PBEF antisense oligonucleotides. Our initial in vitro studies further demonstrated recombinant human PBEF (rhPBEF) as a direct rat neutrophil chemotactic factor, with in vivo studies demonstrating marked increases in BAL fluid leukocytes (polymorphonuclear leukocytes, PMNs) following intratracheal injection in C57BL/6 J mice [117]. These changes were accompanied by increased BAL fluid levels of PMN chemoattractants (KC and MIP2) and modest increases in lung vascular and alveolar permeability. We also noted synergism between rhPBEF challenge and a model of limited VILI and observed dramatic increases in BAL fluid PMNs, BAL protein, and cytokine levels (IL-6, TNFa, KC) compared with either challenge alone. Gene expression profiling identified induction of ALI- and VILI-associated gene modules (NF-kB, leukocyte extravasation, apoptosis, toll-receptor pathways). Heterozygous PBEF+/− mice were significantly protected (reduced BAL fluid protein levels, BAL fluid IL-6 levels, peak inspiratory pressures) when exposed to a model of severe VILI (4 h, 40 mL/kg tidal volume) and exhibited significantly reduced gene expression of VILI-associated modules. Finally, strategies to reduce PBEF availability (neutralizing antibody) resulted in significant protection from VILI [117]. PBEF is now recognized as associated with modestly increased risk of type 2 diabetes and elevated levels of acute-phase proteins [118] and a C-948G SNP has been associated with an increased diastolic blood pressure in obese

children [119]. These studies implicate PBEF, now associated with a number of inflammatory disorders such as inflammatory bowel disease, multiple sclerosis, cystic fibrosis, and asthma [120–122], as a key inflammatory mediator intimately involved in both the development and the severity of ventilator-induced ALI.

4.6 Growth Arrest DNA Damage Inducible a (GADD45a) Growth arrest DNA damage inducible a (GADD45a), a member of an evolutionarily conserved gene family, is implicated as a stress sensor that modulates the response of mammalian cells to genotoxic or physiological stress [123, 124]. GADD45a is a small 21-kDa predominantly nuclear protein that interacts with other proteins implicated in stress responses, including proliferating cell nuclear antigen, p21, Cdc2/cyclin B1, MEKK4, and p38 kinase [125, 126]. GADD45 induces cell cycle arrest and apoptosis in most of cells as well as promoting DNA repair functions and survival [126]. Growth Arrest and DNA Damage gene (GADD45) also maintains genomic stability in a p53-responsive manner [127]. Despite the multiple known functions of GADD45, its role ALI, endothelial/epithelial barrier dysfunction, or repair of injured lung is unknown [38]. GADD45 exhibited differential expression in orthologous global gene expression profiling, in multispecies ALI models [38], in region-specific lung tissue expression profiling [62], and was markedly upregulated in response to the VILI [128]. We explored the mechanistic involvement of GADD45a in endotoxin (LPS)and ventilator-induced inflammatory lung injury (VILI) by comparing multiple biochemical and genomic parameters of

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inflammatory lung injury in wild-type C57Bl/6 and GADD45a−/− knockout mice exposed to high tidal volume ventilation (VILI) or intratracheally administered LPS [129]. GADD45a−/− mice were modestly susceptible to LPSinduced injury but were profoundly susceptible to VILI, demonstrating increased inflammation and increased microvascular permeability. VILI-exposed GADD45a−/− mice manifested striking neutrophilic alveolitis with increased BAL fluid levels of protein, IgG, and inflammatory cytokines. Expression profiling of lung homogenates revealed strong dysregulation in the B cell receptor signaling pathway in GADD45a−/− mice, ­suggesting the involvement of phosphatidylinositol 3-kinase/Akt signaling components. Western blots confirmed a threefold reduction in Akt protein and phosphorylated Akt levels observed in GADD45a−/− lungs. Electrical resistance measurements across human lung endothelial cell monolayers transfected with small interfering RNAs to reduce GADD45a or Akt expression revealed significant potentiation of LPS-induced endothelial barrier dysfunction which was attenuated by overexpression of a constitutively active Akt1 transgene. Whereas other lung injury studies failed to demonstrate a role for GADD45 in hyperoxic lung injury [130, 131], our studies validate GADD45a as a novel inflammatory lung injury candidate gene and a significant participant in vascular barrier regulation via effects on Akt-mediated endothelial signaling [132]. Thus, both Akt and GADD45 are extremely attractive ALI candidate genes. The human GADD45a gene contains 25 validated SNPs (National Center for Biotechnology Information SNP database) whose role in ALI pathogenesis is completely unknown [123]. We are currently pursuing further characterization of the role of GADD45a and its association of genetic variants with sepsis and ALI.

5 Novel Therapeutics The identification of novel pathways involved in the pathobiology of ALI also opens doors for the exploration of new therapeutic targets for the disease. As such, the use of agents that attenuate the endothelial barrier dysfunction and the inflammatory response characteristic of ALI have shown promise in preclinical studies which will hopefully lead to their use in trials of human ALI (Fig. 3).

5.1 Sphingosine 1-Phosphate S1P, an important lipid mediator generated by the phosphorylation of sphingosine by sphingosine kinase, decreases endothelial permeability to both water and solute via cytoskeletal reorganization and adherens junction assembly

R.F. Machado and J.G.N. Garcia

Fig. 3  Mechanism-based novel therapies for ALI. The identification of novel pathways involved in the pathobiology of ALI also facilitates the exploration of new therapeutic targets. Sphingosine 1-phosphate (S1P1) attenuates the endothelial barrier dysfunction associated with ALI, whereas blocking of PBEF attenuates VALI

[88, 89]. S1P-induced barrier protective effects could serve to attenuate the increased pulmonary vascular permeability essential factor in the development of ALI. The S1P analogue FTY720 (0.1 mg/kg),when administered to C57BL/6 mice with endotoxin-induced lung injury, decreases lung edema formation, solute transport across the alveolar capillary endothelium, and inflammatory cell infiltration into lung parenchyma [94]. Similarly, the prophylactic administration of S1P attenuates both alveolar and vascular barrier dysfunction while significantly reducing shunt formation associated with lung injury in rodent and canine models of ALI induced by combined intrabronchial endotoxin administration and high-tidal-volume mechanical ventilation [133]. In a recent study of a canine model of ALI, we demonstrated that when bacterial endotoxin was instilled intratracheally followed in 1 h by intravenous administration of S1P (85 mg/kg) or vehicle and 8 h of high-tidal-volume mechanical ventilation [134], S1P treatment attenuated the severity of ALI-induced increases in shunt fraction and the presence of both protein and neutrophils in BAL fluid compared with vehicle ­controls. Interestingly, BAL fluid cytokine production was not altered

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significantly by intravenous administration of S1P and S1P potentiated the endotoxin-induced systemic production of the inflammatory cytokines TNFa, C-X-C chemokine ligand-1, and IL-6, without resulting in end-organ dysfunction. These data suggest that S1P may represent a viable therapy for the prevention and treatment of ALI.

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gene–environment interactions, which add complexity to our understanding of the genome. These novel genetic approaches may prove exceptionally useful in ushering in the era of ­personalized medicine for critically ill individuals.

References 5.2 Pre-B Cell Colony Enhancing Factor Blockage As previously described in this chapter, PBEF appears to play a central role in the promotion of several pathogenetic aspects of ALI and VALI. Therefore, interventions aimed at attenuating the effects of PBEF could have a potential therapeutic effect in these disorders. To begin to address the potential for PBEF to serve as a therapeutic target in ameliorating VILI, we assessed the effect of PBEF neutralizing antibody on rhPBEF-stimulated lung inflammation [117]. Simultaneous instillation of rhPBEF and PBEF neutralizing antibody produced dramatic reductions in rhPBEF-induced neutrophil recruitment. Further, the intratracheal delivery of PBEF neutralizing antibody (30 min before high-tidal-volume mechanical ventilation) abolished VILI-induced increases in total BAL fluid cell counts and significantly decreased neutrophil influx into the alveolar space as well as VILI-mediated increases in the level of lung tissue albumin.

6 Summary ALI is a major cause of morbidity and mortality in critically ill patients. Given the unacceptably high mortality rate observed in ALI and the paucity of novel therapies and biomarkers, it is essential to recognize molecular targets associated with ALI to identify individuals at risk and to develop novel therapeutic targets and biomarkers. It is clear that derangements in endothelial cell barrier regulation play a major role in the pathobiology of ALI and genetic variants regulate endothelial cell barrier function, thereby determining ALI risk or subsequent severity of outcome. High-throughput gene sequencing and expression technologies, and complete genome sequencing of model organisms, have allowed for the performance of large-scale analyses of the genome in ALI. In this chapter, we have highlighted how global gene expression profiling in multispecies ALI models served to broaden our net knowledge of ALI-implicated genes and provide a basis for hope that increased insights and therapies may be forthcoming. As genotyping becomes more rapid and easily accessed, combining advanced bioinformatics techniques with ­high-throughput methods will be the future practice of personalizing treatment strategies. Continued challenges will be the gene–gene and

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912 is mediated by the small GTPase Rac and cortactin. Am J Physiol Lung Cell Mol Physiol 291:L289–L295 99. Sun X, Ma SF, Wade MS et  al (2009) Sphingosine-1-phosphate receptor 1 variant increases promoter activity and decreases ­susceptibility to human asthma. Am J Respir Crit Care Med 175:A5420 100. Brinkmann V, Foroutan H, Sachs M, Weidner KM, Birchmeier W (1995) Hepatocyte growth factor/scatter factor induces a variety of tissue-specific morphogenic programs in epithelial cells. J Cell Biol 131:1573–1586 101. Lesko E, Majka M (2008) The biological role of HGF-MET axis in tumor growth and development of metastasis. Front Biosci 13:1271–1280 102. Ma PC, Kijima T, Maulik G et al (2003) c-MET mutational analysis in small cell lung cancer: novel juxtamembrane domain mutations regulating cytoskeletal functions. Cancer Res 63:6272–6281 103. Itakura A, Kurauchi O, Morikawa S, Okamura M, Furugori K, Mizutani S (1997) Involvement of hepatocyte growth factor in formation of bronchoalveolar structures in embryonic rat lung in primary culture. Biochem Biophys Res Commun 241:98–103 104. Yanagita K, Matsumoto K, Sekiguchi K, Ishibashi H, Niho Y, Nakamura T (1993) Hepatocyte growth factor may act as a pulmotrophic factor on lung regeneration after acute lung injury. J Biol Chem 268:21212–21217 105. Panos RJ, Patel R, Bak PM (1996) Intratracheal administration of hepatocyte growth factor/scatter factor stimulates rat alveolar type II cell proliferation in vivo. Am J Respir Cell Mol Biol 15:574–581 106. Hammond DE, Carter S, Clague MJ (2004) Met receptor dynamics and signalling. Curr Top Microbiol Immunol 286:21–44 107. Kermorgant S, Parker PJ (2005) c-Met signalling: spatio-temporal decisions. Cell Cycle 4:352–355 108. Singleton PA, Salgia R, Moreno-Vinasco L et  al (2007) CD44 regulates hepatocyte growth factor-mediated vascular integrity. Role of c-Met, Tiam1/Rac1, dynamin 2, and cortactin. J Biol Chem 282:30643–30657 109. Stern JB, Fierobe L, Paugam C et al (2000) Keratinocyte growth factor and hepatocyte growth factor in bronchoalveolar lavage fluid in acute respiratory distress syndrome patients. Crit Care Med 28:2326–2333 110. Verghese GM, McCormick-Shannon K, Mason RJ, Matthay MA (1998) Hepatocyte growth factor and keratinocyte growth factor in the pulmonary edema fluid of patients with acute lung injury. Biologic and clinical significance. Am J Respir Crit Care Med 158:386–394 111. Ye SQ, Simon BA, Maloney JP et  al (2005) Pre-B-cell colonyenhancing factor as a potential novel biomarker in acute lung injury. Am J Respir Crit Care Med 171:361–370 112. Bajwa EK, Yu CL, Gong MN, Thompson BT, Christiani DC (2007) Pre-B-cell colony-enhancing factor gene polymorphisms and risk of acute respiratory distress syndrome. Crit Care Med 35:1290–1295 113. Ye SQ, Zhang LQ, Adyshev D et  al (2005) Pre-B-cell-colonyenhancing factor is critically involved in thrombin-induced lung endothelial cell barrier dysregulation. Microvasc Res 70:142–151 114. Jia SH, Li Y, Parodo J et al (2004) Pre-B cell colony-enhancing factor inhibits neutrophil apoptosis in experimental inflammation and clinical sepsis. J Clin Invest 113:1318–1327 115. Jimenez MF, Watson RW, Parodo J et  al (1997) Dysregulated expression of neutrophil apoptosis in the systemic inflammatory response syndrome. Arch Surg 132:12639, discussion 9–70 116. Taneja R, Parodo J, Jia SH, Kapus A, Rotstein OD, Marshall JC (2004) Delayed neutrophil apoptosis in sepsis is associated with maintenance of mitochondrial transmembrane potential and reduced caspase-9 activity. Crit Care Med 32:1460–1469

R.F. Machado and J.G.N. Garcia 117. Hong SB, Huang Y, Moreno-Vinasco L et al (2008) Essential role of pre-B-cell colony enhancing factor in ventilator-induced lung injury. Am J Respir Crit Care Med 178:605–617 118. Zhang YY, Gottardo L, Thompson R et  al (2006) A visfatin promoter polymorphism is associated with low-grade inflammation and type 2 diabetes. Obesity (Silver Spring) 14:2119–2126 119. Körner A, Böttcher Y, Enigk B, Kiess W, Stumvoll M, Kovacs P (2007) Effects of genetic variation in the visfatin gene (PBEF1) on obesity, glucose metabolism, and blood pressure in children. Metabolism 56:772–777 120. Koppelman GH, Stine OC, Xu J et al (2002) Genome-wide search for atopy susceptibility genes in Dutch families with asthma. J Allergy Clin Immunol 109:498–506 121. Satsangi J, Parkes M, Louis E et al (1996) Two stage genome-wide search in inflammatory bowel disease provides evidence for susceptibility loci on chromosomes 3, 7 and 12. Nat Genet 14:199–202 122. Vandenbroeck K, Fiten P, Heggarty S et al (2002) Chromosome 7q21-22 and multiple sclerosis: evidence for a genetic susceptibility effect in vicinity to the protachykinin-1 gene. J Neuroimmunol 125:141–148 123. Lal A, Gorospe M (2006) Egad, more forms of gene regulation: the gadd45a story. Cell Cycle 5:1422–1425 124. Liebermann DA, Hoffman B (2007) Gadd45 in the response of hematopoietic cells to genotoxic stress. Blood Cells Mol Dis 39:329–335 125. Fornace AJ Jr, Jackman J, Hollander MC, Hoffman-Liebermann B (1992) Liebermann DA Genotoxic-stress-response genes and growth-arrest genes gadd, MyD, and other genes induced by treatments eliciting growth arrest. Ann N Y Acad Sci 663:139–153 126. Liebermann DA, Hoffman B (2002) Myeloid differentiation (MyD)/growth arrest DNA damage (GADD) genes in tumor suppression, immunity and inflammation. Leukemia 16:527–541 127. Hollander MC, Philburn RT, Patterson AD, Wyatt MA, Fornace AJ Jr (2005) Genomic instability in Gadd45a−/− cells is coupled with S-phase checkpoint defects. Cell Cycle 4:704–709 128. Dolinay T, Szilasi M, Liu M, Choi AM (2004) Inhaled carbon monoxide confers antiinflammatory effects against ventilatorinduced lung injury. Am J Respir Crit Care Med 170:613–620 129. Meyer NJ, Huang Y, Singleton PA et  al (2009) GADD45a is a novel candidate gene in inflammatory lung injury via influences on Akt signaling. FASEB J 23(5):1325–1337 130. O’Reilly MA, Staversky RJ, Watkins RH, Maniscalco WM, Keng PC (2000) p53-independent induction of GADD45 and GADD153 in mouse lungs exposed to hyperoxia. Am J Physiol Lung Cell Mol Physiol 278:L552–L559 131. Roper JM, Gehen SC, Staversky RJ, Hollander MC, Fornace AJ Jr, O’Reilly MA (2005) Loss of Gadd45a does not modify the pulmonary response to oxidative stress. Am J Physiol Lung Cell Mol Physiol 288:L663–L671 132. Altemeier WA, Matute-Bello G, Gharib SA, Glenny RW, Martin TR, Liles WC (2005) Modulation of lipopolysaccharide-induced gene transcription and promotion of lung injury by mechanical ventilation. J Immunol 175:3369–3376 133. McVerry BJ, Peng X, Hassoun PM, Sammani S, Simon BA, Garcia JGN (2004) Sphingosine 1-phosphate reduces vascular leak in murine and canine models of acute lung injury. Am J Respir Crit Care Med 170:987–993 134. Szczepaniak WS, Zhang Y, Hagerty S et  al (2008) Sphingosine 1-phosphate rescues canine LPS-induced acute lung injury and alters systemic inflammatory cytokine production in vivo. Transl Res 152:213–224

Chapter 64

Ventilator-Induced Mechanical Stress and Lung Vascular Dysfunction Konstantin G. Birukov

Abstract  By virtue of their design as a gas-exchange organ, lungs are constantly exposed to mechanical stimulation associated with inflation–deflation cycles. Pulmonary vasculature experiences mechanical forces resulting from blood circulation and respiratory cycles. These forces are transduced onto pulmonary vessels in the form of shear stress, intravascular hydrostatic pressure, and tensile stress. Normally, mechanical loads on pulmonary vasculature are well tolerated even during intense exercise, several-fold elevation of inhaled air pressure during scuba diving, or ascension to high altitude. However, extreme loads experienced by elite athletes elevate the pulmonary capillary pressure beyond physiological limits and may cause transient pulmonary vascular leak. In the clinical setting, mechanical ventilation of patients at high tidal volumes leads to pulmonary edema and a severer complication, acute respiratory distress syndrome (ARDS). Chronic elevation of pulmonary artery pressure due to cardiogenic or primary pulmonary hypertension leads to vascular remodeling, vascular occlusion, and severe lung dysfunction. Relatively rare cases of pulmonary vascular leak and edema have been described when a lung collapsed for several days as a result of pneumothorax is rapidly re-expanded. In such cases, the protein concentration in sputum is very high during the onset of so-called pulmonary re-expansion edema (RPE), suggesting that RPE belongs to a type of permeability pulmonary edema caused by pulmonary microvascular dysfunction. These examples demonstrate the significance of the physiologic biomechanical milieu in the pulmonary vascular barrier regulation and proper functioning of the lung as a gasexchange organ. Keywords  Lung injury • Mechanical stimulation • Vascular permeability • Barrier dysfunction • Pulmonary microcirculation

K.G. Birukov (*) Section of Pulmonary and Critical Medicine, Department of Medicine, University of Chicago, GCIS Bldg., W410, 929 East 57th Street, Chicago, IL 60637, USA e-mail: [email protected]

1  Biology of Mechanical Stress 1.1  Mechanical Forces and the Lung Function By virtue of their design as a gas-exchange organ, lungs are constantly exposed to mechanical stimulation associated with inflation–deflation cycles. Pulmonary vasculature experiences mechanical forces resulting from blood circulation and respiratory cycles. These forces are transduced onto pulmonary vessels in the form of shear stress, intravascular hydrostatic pressure, and tensile stress. Normally, mechanical loads on pulmonary vasculature are well tolerated even during intense exercise, several-fold elevation of inhaled air pressure during scuba diving, or ascension to high altitude. However, extreme loads experienced by elite athletes elevate the pulmonary capillary pressure beyond physiological limits and may cause transient pulmonary vascular leak. In the clinical setting, mechanical ventilation of patients at high tidal volumes leads to pulmonary edema and a severer complication, acute respiratory distress syndrome (ARDS). Chronic elevation of pulmonary artery pressure due to cardiogenic or primary pulmonary hypertension leads to vascular remodeling, vascular occlusion, and severe lung dysfunction. Relatively rare cases of pulmonary vascular leak and edema have been described when a lung collapsed for several days as a result of pneumothorax is rapidly re-expanded. In such cases, the protein concentration in sputum is very high during the onset of so-called pulmonary re-expansion edema (RPE), suggesting that RPE belongs to a type of permeability pulmonary edema caused by pulmonary microvascular dysfunction. These examples demonstrate the significance of the physiologic biomechanical milieu in the pulmonary vascular barrier regulation and proper functioning of the lung as a gas-exchange organ.

1.2 Mechanical Forces and the Vascular Wall Pulmonary vascular cells experience four principal mechanical forces: (1) shear stress resulting from blood fluid flow and

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local stresses resulting from blood cells passing through the terminal capillary system; (2) vessel wall strain from intraluminal hydrostatic pressure; (3) cyclic strain caused by heart propulsions; and (4) distension of lung microvasculature caused by respiratory cycles. These additional mechanical forces form a unique mechanical environment experienced by pulmonary vasculature.

1.2.1  Shear Stress Although both pulmonary and systemic circulations are constantly exposed to shear stress, lung endothelial cells (ECs) experience variable flow patterns in vivo under physiological and pathological conditions that are dictated by unique features of the pulmonary circulation. Owing to the highly branched structure of the pulmonary circulation that ensures efficient gas exchange, large- and small-caliber pulmonary vessels experience reduced resistance and intraluminal pressure and comparable levels of shear stress (5–10 dyn/cm2 compared with 15–40  dyn/cm2 in the systemic circulation) [1, 2]. The regional distribution of blood flow throughout the human pulmonary vasculature is nonuniform and decreases from the base to the apex of the lung. In pathological situations, such as severe hypovolemia and mechanical ventilation at excessive airway pressure, apical pulmonary artery pressures may fall below alveolar pressures, resulting in capillary collapse and cessation of blood flow. Cessation of ­pulmonary flow through defined segments may be caused by thromboembolism, during lung infections and inflammation in ARDS patients, and in response to hypoxic vasoconstriction, a condition unique to the pulmonary circulation, which may result in capillary collapse. Thus, the differences in ­systemic and pulmonary flow patterns may dictate different mechanisms of flow-induced regulation of endothelial functions in these systems.

1.2.2 Mechanical Strain Mechanical stretch of vascular cells is a superposition of ­pulsatile and tonic components. Tensile stress is imposed by hydrostatic pressure counteracted by tonic contraction of vascular smooth muscle cells (VSMCs) and elastic components of extracellular matrix in the vessel wall. Cyclic stretch is imposed by heart propulsions. Pulsatile distension of the arterial wall in the systemic circulation normally does not exceed 10–12%, but various vasomotor reactions may change the diameter of smaller-caliber “resistance” arteries to 60% of the initial diameter or more and may last minutes or hours [3]. The pulmonary circulation is characterized by lower hydrostatic pressure, but is subjected to additional mechanical stretch due to respiratory cycles or mechanical ventilation. Thus, cyclic stretch is a more prominent factor in the

K.G. Birukov

lung alveolar capillaries, whereas shear forces may have differential effects on various potions of the pulmonary vascular bed. The examples of pathological conditions associated with increased mechanical stress in the lung are briefly discussed herein.

2 Mechanical Stresses in the Pulmonary Circulation 2.1  Vascular Wall and Hypertension Increased vascular wall stress has been implicated in the pathogenesis of hypertension. Whereas physiological cyclic stretch causes cell cycle arrest in the VSMCs [4], a chronic increase in blood pressure activates vascular cell proliferation and collagen and fibronectin synthesis, which results in thickening of the vascular wall as a feature of hypertensioninduced vascular remodeling [5–7]. These changes lead to the vessel wall thickening, increased media-to-lumen ratio, and decreased luminal diameter. Such remodeling of resistance vessels alters arterial compliance and further propagates arterial hypertension [8]. Similar to systemic hypertension, endothelial dysfunction and vascular remodeling are hallmarks of pulmonary hypertension, reviewed in detail in Chaps. 52, 57, and 58. This chapter discuss the vascular dysfunction induced by ventilator-associated mechanical stretch with focus on cellular responses by pulmonary vascular ECs and VSMCs.

2.2 Pulmonary Capillary Stress Failure The total area of the blood–gas barrier (BGB) in the human lung is 50–100  m2 and for over more than half of this ­enormous area, its thickness is only 0.2–0.3 mm to ensure efficient and fast gas exchange. At the same time, the BGB must be immensely strong, because the hydrostatic pressure in the pulmonary capillaries may rise to high levels during heavy exercise as result of the large filling pressures demanded by the left ventricle to develop the necessary cardiac output. Studies by West’s group determined that elevations in capillary pressure up to approximately 50 cmH2O in rabbit, approximately 100 cmH2O in dog, and approximately 145 cmH2O in horse lung capillaries would be required to cause ultrastructural changes in the capillary walls [9, 10]. Pulmonary artery pressure in human subjects can rise up to 30 mmHg during intense exercise, with a mean pulmonary artery pressure of as much as 37 mmHg. Theoretical calculations suggest that at these hydrostatic pressure levels the walls of human pulmonary capillaries have little strength in

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reserve at high levels of exercise. Increased capillary pressure leads to breaks in the capillary endothelial layer referred to as “stress failure.” Interestingly, these breaks appear to be within the cells themselves [10]. Brakes in the pulmonary capillaries were found in lung preparations of thoroughbred racehorses shortly after they were galloping on a treadmill. At these loads, capillary pressure is raised up to approximately 100 mmHg. Similar findings were reported in elite human athletes. For example, hemoptysis and hemorrhagic pulmonary edema were seen in two athletes participating in the Comrade’s Marathon in South Africa. This race is over a distance of 90 km and the competitors run for 8–11 h.

2.3 Pathological Conditions Associated with Lung Capillary Stress Failure Similar to elevation of capillary pressure during extreme exercise, development of high-altitude pulmonary edema is also associated with pulmonary vasoconstriction leading to increased capillary pressure. Increased vascular permeability in high-altitude pulmonary edema was confirmed by high concentrations of high molecular weight proteins and large numbers of leukocytes detected in bronchoalveolar lavage (BAL) fluid [11]. Other pathological conditions associated with stress failure of pulmonary capillaries include neurogenic pulmonary edema characterized by very high pulmonary vascular pressures [12], and accumulation of high molecular weight proteins and red blood cells in the alveolar edema fluid. Experimental neurogenic pulmonary edema induced disruptions of both capillary EC and alveolar epithelial cell layers [13]. Pulmonary capillary stress failure may also occur during left ventricular failure and mitral stenosis. Weakening of the collagen-containing basal membrane has been found in Goodpasture’s syndrome, in which type IV collagen is abnormal because of autoimmune attack of the NC1 globular domain and leads to BGB disruption and bleeding into the alveolar spaces even under normal ­capillary pressures.

2.4 Mechanical Ventilation and Capillary Stress Failure A particularly important pathological condition involving stress failure of pulmonary capillaries leading to increased pulmonary permeability is lung injury induced by excessive mechanical ventilation. Mechanical ventilation is a lifesaving intervention for patients with respiratory failure from various injuries, including pneumonia, sepsis, and trauma. However, pathological lung overdistension caused by

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Fig. 1  Capillary endothelial stress induced by alveolar wall distension associated with mechanical ventilation at high tidal volumes

mechanical ventilation at high tidal volumes transmits pathologic mechanical stress to alveolar epithelium and pulmonary vasculature, leading to barrier dysfunction (Fig. 1). Studies of animal preparations have shown that increasing lung volume from normal to high levels at constant capillary transmural pressure values markedly increases the number of disruptions in both capillary endothelial and alveolar ­epithelial layers [14]. In these conditions, the alveolar wall can be regarded as a string of pulmonary capillaries, with the result that some of the increased tension in the alveolar wall caused by high states of lung inflation is transmitted to the capillary wall [15].

2.5 Evaluation of Physiologic and Pathologic Lung Stretch Regimen Direct measurements of interstitial/vascular distension in the mechanically ventilated lung remain a challenging task because of the complexity of local distension patterns in the lung parenchyma, which become further complicated owing to uneven regional lung distension observed in inflamed and injured lung. However, studies of alveolar epithelial cell cultures exposed to mechanical strain in vitro suggest that a 25% increase in cell surface area, corresponding to 8–12% linear distension, likely correlates with physiological levels of mechanical strain experienced by alveolar epithelium, whereas cyclic stretch resulting in 37–50% increase in cell surface area corresponding to 17–22% linear distension is relevant to pathophysiological conditions produced by mechanical ventilation and causes progressive cell death [16]. Furthermore, calculations based on animal models ­suggest that if the lung volume increases from 40% to 100% of the total lung capacity, the alveolar epithelial cell basal surface area increases by 34–35% [17, 18]. Indeed, pulmonary ECs exposed to cyclic stretch at 5% or 18% linear elongation show different patterns of activated signaling and respond differently to edemagenic agents [19–21]. These regimens represent conditions of low and high tidal volume mechanical ventilation in vivo.

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3 Plasma Membrane Reparation Mechanisms Is there a basement membrane strain threshold beyond which the plasma membrane breaks and, if this happens, what could be the mechanisms of membrane repair? When the basement membrane in the overinflated lung is strained, adherent alveolar epithelial cells as well as capillary ECs must change shape. Provided that the cells remain attached to the basal membrane and maintain constant cell volume and continuous cell–cell junctions, their surface (plasma membrane)-to-volume ratio must increase during such a deformation. The review by Vlahakis and Hubmayr [22] summarizes four hypothetical plasma membrane deformation responses applicable for ventilation-induced lung cell overdistension, all of which have been observed in experimental systems: (1) unfolding of excess plasma membrane; (2) elastic deformation (i.e. stretch) of the plasma membrane: (3) translocation of lipids from intracellular stores to the plasma membrane; and (4) plasma membrane stress failure. Approaches for monitoring the plasma membrane disruptions include laser confocal microscopy, electron microscopy, and the uptake of high molecular weight fluorescent dextran (dextran labeled with fluorescein isothiocyanate, FITC-Dx) [23–25]. When the plasma membrane is disrupted, FITC-Dx molecules are able to enter the cell down a concentration gradient and thus homogenously label the cell interior [25], which is different from the granular intracellular pattern of dextran uptake by pinocytosis. Cells that reseal their membranes retain FITC-Dx, remain attached to the substratum, and are viable in culture. Cells that do not reseal their membranes will not retain FITC-Dx and will lift off the cell substratum and lyse. With the use of FITC-Dx of differing molecular size, this approach allows analysis of deformation type and ­estimations of amplitudedependent effects on plasma membrane break size [26, 27].

4 Barotrauma and Biotrauma in Lung Injury Lung diseases associated with excessive mechanical stress on lung capillaries or alveoli may directly cause capillary stress failure and resulting BGB compromise leading to ­pulmonary edema and alveolar flooding. These findings emphasize a role of cell membrane stress failure and direct cell injury, or barotrauma, underlying some forms of hydrostatic pulmonary edema. May the barotrauma paradigm entirely explain the lung injury induced by overdistension? Exemplary studies by Berg et al. used asymmetric ventilation of left and right lungs in anesthetized open-chest rabbits, where inflation of one lung was greatly increased, whereas the other lung was ventilated at a normal volume [28].

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High-magnitude inflation of one lung while keeping the second lung ventilated normally over 4 h resulted in increased expression of genes encoding growth factors and extracellular matrix proteins [28]. An unexpected finding was that these changes in messenger RNA were identical in both the overinflated lung (9-cmH2O positive end-expiratory pressure) and in the normally inflated lung (1-cmH2O positive end-expiratory pressure) in the same preparation. These studies illustrate a generalized organ-specific response after the localized (unilateral) application of a mechanical force, suggesting other mechanisms than just direct mechanical cell injury (or barotrauma) in development of ventilatorinduced lung dysfunction. The clinical observations and further experimental studies further showed that besides direct mechanical effects on epithelial and endothelial integrity, mechanical ventilation at high tidal volumes causes release of inflammatory cytokines, which further exacerbate ventilatorinduced lung injury (VILI), lipopolysaccharide (LPS)induced lung injury, ventilator-induced vascular leak, and LPS-induced vascular leak [29–31]. The term “biotrauma” has been introduced to describe the process by which stress produced by mechanical ventilation leads to the upregulation of an inflammatory response. For mechanical ventilation to exert its deleterious effect, cells are required to sense mechanical forces and activate intracellular signaling pathways able to communicate the information to its interior. This information must then be integrated in the nucleus, and an appropriate response must be generated to implement and/or modulate its response and that of neighboring cells. How do vascular cells sense mechanical forces? How does mechanical stimulation translate to a physiological response by pulmonary vasculature? These important questions will be discussed next.

5 Cellular Mechanisms of Mechanosensing 5.1 Mechanosensors Because cell membranes, cell attachment sites, and the cytoskeletal network directly experience hemodynamic forces, they are considered as primary mechanosensors [1]. Cells adhere to neighboring cells and to the extracellular matrix via transmembrane receptors of cadherin (cell-tocell) and integrin (cell-to-substrate) families. Thus, mechanical perturbation of the cell monolayer would immediately trigger intracellular signaling because cytoplasmic domains of cell contact receptors are coupled to cytoskeleton and protein complexes (Fig. 2), which mediate mechanical signal transduction via activation of tyrosine kinases [focal adhesion kinase (FAK), p60Src], serine/threonine protein kinases [protein kinase A (PKA), protein kinase C (PKC),

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6 Mechanosensing and Mechanotransduction in the Pulmonary Vasculature 6.1  Cell–Matrix Contacts Fig. 2  Putative mechanosensors activated in pulmonary vascular and alveolar cells by mechanical strain

extracellular-signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase (MAPK)], inositol lipid kinases (phospholipase C), and some growth factor receptors [vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) ­receptors] [1, 2, 7, 32].

5.2 Ion Channels Stretch-activated ion channels represent another example of such mechanosensors [33]. These channels are tethered to cytoskeletal and external anchors via intracellular and extracellular linkers. The mechanical forces transduced via these linkers to the channel can affect ion conductivity and activate intracellular signaling in an amplitude-dependent fashion. Two different mechanosensitive channels have been described in vascular cells: shear-activated potassium channels and stretch-activated channels [34]. Stretch-activated ion channels are cation-specific and have an electric activity mainly detectable at the time of their opening. The activation of these channels leads to calcium (Ca2+) influx followed by membrane depolarization. Stretch-activated ion channel activities have been found in lung cells. A 20% single static stretch of rat pulmonary artery smooth muscle cells increased both Ca2+ influx and Ca2+ efflux [35]. Thus, discovery of the involvement of stretch-activated ion channels in Ca2+ influx and physiological responses in ECs [36] suggests a possibility of amplitude-dependent regulation of cellular functions by mechanical strain. In addition, mechanical stress may be sensed differently by vascular cells in a normal or a pathologic state. For example, stretch activation of Na+ and Ca2+ channels was greater in VSMCs isolated from spontaneously hypertensive rats compared with those from normotensive Wistar Kyoto rats [37]. These findings illustrate two major paradigms of mechanotransduction that may be applied in pathologic states: (1) amplitude-dependent effects of mechanical stress on vascular cells; (2) different responses of healthy and diseased vascular cells to the same levels of mechanical stress.

Mechanical stretch from underlying capillary wall is transmitted to ECs through their adhesive contacts. Engagement of integrin-containing focal adhesions and application of an external force to cells may be achieved experimentally by twisting of cell-attached magnetic beads coated with integrin ligand RGD. Mechanically challenged cells increase their resistance to applied deformation and exhibit a “stiffening response” [38]. Stretch-induced activation of integrins in cell experiments may be judged by their association with the adaptor protein Shc. This interaction triggers binding of focal-adhesion-associated structural proteins and signaling complex to Shc, which converts the mechanical signal to biochemical cascades. Focal adhesions are multimolecular complexes consisting of more than 50 different proteins [39]. Focal adhesions form a bidirectional linkage between the actin cytoskeleton and the cell–extracellular matrix interface and provide additional tethering forces that help maintain EC barrier integrity. Binding to the extracellular matrix induces the attachment of integrins to intracellular actin fibers, a ­process in ECs that stimulates phosphorylation of multiple proteins such as paxillin, GIT-2, p21-activated kinase 1 (PAK1), and FAK [39]. Because integrins not only physically connect the cytoskeleton to the extracellular matrix but also function as signaling receptors, they are recognized as ­important transmitters of physical forces into chemical ­signals, ­examples of which include c-Src-dependent tyrosine ­phosphorylation of paxillin, FAK, and p130Cas, and ­activation of MAPK signaling [40–42]. Mechanical strain or centripetal pulling of the cell by a micropipette causes redistribution of focal adhesions, their elongation, and increases in size [43, 44]. This process triggers Rho signaling leading to activation of a Rho-associated kinase (Rho kinase)-dependent increase in actomyosin contraction [43] and signaling in the focal adhesions [41, 45]. Another Rho target, formin homology protein mDia1, is specifically involved in force-induced focal contact formation [43]. Studies of vascular cells exposed to cyclic stretch show a critical role of integrin signaling in cell responses. Uniaxial cyclic stretch upregulates the expression of integrin b3 in ECs, which further enhances the cell adhesiveness and resistance of the EC monolayer to hemodynamic forces or excessive vessel distension [46]. Besides different cyclic stretch effects on ECs and smooth muscle cells, different profiles of mechanical strain may have contrasting effects. Application of 10% cyclic stretch in pressurized and perfused aorta

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preparations ex vivo did not induce significant FAK phosphorylation relative to nonpulsatile controls. Although ERK1/2 was activated in vessels exposed to pulsatility, it was not blocked by PP2 or RGD peptide treatment. This study demonstrates that steady and cyclic modes of stretch trigger different patterns of intracellular signaling which may be differentially regulated by FAK, Src, and integrins [47]. Thus, besides the cell-type-specific differences in mechanotransduction by cell-extracellular matrix contacts discussed above, cellular responses to mechanical stress also depend on the nature and magnitude of the applied mechanical stimulation, and the exact mechanisms of stretch-induced signaling by focal adhesions await further investigation.

6.2 Cell–Cell Adhesions Vascular endothelial-specific cadherin (VE-cadherin) is a transmembrane domain that forms homotypic interactions (adherens junctions) between adjacent ECs and links them with cell cytoskeleton via the catenin family of proteins. VE-cadherin has been associated with endothelial responses to shear stress [48]; however, its role in mechanical signaling is less obvious. VE-cadherin appears to be involved in stretchinduced endothelial proliferation. ECs require cell–cell contact and VE-cadherin engagement to transduce stretch into proliferative signals. In contrast, smooth muscle cells respond to stretch without contact with neighboring cells [49]. VE-cadherin may also serve as an adaptor in endothelial orientation and gene expression response to flow, where platelet endothelial cell adhesion molecule-1 (PECAM-1) served as a force transducer leading to ­activation of signaling by VEGF receptor-2 and phosphatidylinositol 3-kinase (PI3K) [50]. This and other studies consider PECAM-1 as a mechanosensor located within endothelial cell–cell adhesions. When ECs are stimulated by fluid shear stress, PECAM-1 is tyrosinephosphorylated and activates the ERK1/2 MAPK signaling cascade. The same ERK1/2 activation occurs if pulling force is applied directly on PECAM-1 on the EC surface using magnetic beads coated with antibodies against the external domain of PECAM-1. These findings suggest that PECAM-1 may sense mechanical forces generated by both flow-induced shear stress and mechanical stretch applied on the vascular endothelial monolayer by external mechanical stress [51].

6.3 Receptor Tyrosine Kinases Interestingly, growth factor receptors also referred as receptor tyrosine kinases appear to be involved in mechanotransduction and may become transactivated by cell contact

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receptors. Stretching of VSMCs induces a rapid phosphorylation of PDGF receptor a (PDGFRa) in a magnitudedependent fashion [5]. This stretch-induced PDGFRa phosphorylation is not affected by PDGF blocking antibody, and conditioned medium from the stretched cells does not cause PDGFRa phosphorylation in static VSMCs [5]. These results indicate that the stretch-induced PDGFRa activation is not the result of the paracrine or autocrine release of its ligand PDGF. Similar to PDGFRa, stretch also induces the phosphorylation of epithelial growth factor receptor (EGFR) and its recruitment of adaptor proteins Shc and Grb2, which in turn lead to ERK1/2 activation [52]. The mechanisms of such growth factor receptor transactivation by mechanical forces are not completely clear, but may involve formation of molecular scaffolds containing cell–cell or cell–substrate receptors linked to receptor tyrosine kinases via adaptor proteins such as Shc, which is an adaptor protein containing a C-terminal Src-homology-2 (SH2) domain. Tyrosinephosphorylated Shc becomes associated with the cognate receptor tyrosine kinases through SH2 binding and mediates the integrin-induced signal transduction caused by mechanical strain. Thus, transactivation of receptor tyrosine kinases by mechanical forces may not only mediate stretch-induced mechanotransduction and immediate cell responses such as permeability, contraction, and secretion, but may also control vascular remodeling, cell proliferation, and cell survival. These processes are critical for pulmonary vascular reparation during recovery after acute lung injury (ALI). Observed upregulation of the key tyrosine kinase receptors Flk-1, Tie2, and Tie-1 in cyclic-stretch-stimulated vascular ECs [53] further increases the EC sensitivity to growth factors and, therefore, facilitates angiogenesis and tissue repair.

6.4 Heterotrimeric G Proteins Heterotrimeric G proteins consist of three subunits, a, b, and g, which couple membrane receptors with intracellular ­signaling cascades. Receptor-mediated activation of the ­heterotrimeric G-protein complex leads to dissociation of b and g subunits from a subunits and activation of downstream effectors by G-protein b and g subunits. Participation of ­heterotrimeric G proteins in stretch-induced mechanotransduction was shown in ECs, alveolar epithelial cells, and nonpulmonary cells: myoblasts and osteoblast-like cells. Direct screening of stretch-activated G proteins was performed by Clark et al. [54] by loading cells with a radioactively labeled nonhydrolyzable photoreactive GTP analog prior to cyclic stretch stimulation. This study identified small GTPase rab5 and the G ai subunit of heterotrimeric G proteins as novel stretch-activated signal proteins. The g subunit of heterodimeric G proteins has been localized to integrin-rich focal

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adhesion sites and adjacent to F-actin stress fibers [55], suggesting a potential mechanism of stretch-induced regulation of its activity via signaling from focal adhesions and cytoskeleton. However, precise mechanisms of stretch-induced G-protein activation remain elusive.

6.5 Caveolae Many cell types, including ECs, contain specialized plasma membrane microdomains also referred to as lipid rafts or caveolae. These cholesterol-enriched plasma microdomains also contain sphingomyelin, gangliosides, phosphatidylinositol 4,5-bisphosphate, diacylglycerol, and a specific ­scaffolding protein, caveolin-1, and are important sites for concentrating transmembrane receptors, and signaling proteins that transduce chemical and mechanical signals into the cell [56, 57]. In VSMCs, caveolin mediates stretch-induced ERK activation through an association with b1 integrins/Fyn/ Shc [58]. Serine/threonine signaling kinase Akt is essential for many cell functions, including survival, migration, and permeability regulation. In ECs Akt activation is insensitive to stretch, but is activated by shear stress in a caveolin-1-dependent fashion. However, in mesenchymal cells, actin cytoskeleton and focal adhesions are not involved in stretchinduced activation of Akt, which, however, depends upon EGFR transactivation. EGFR transactivation requires intact caveolae and the Src-mediated phosphorylation of caveolin-1 [59]. These studies define a novel function for caveolae in the stretch-induced signal transduction via EGFR transactivation and Akt stimulation. In VSMCs, stretch-induced activation of FAK, ERK1/2, and Rho does not require caveolin-1 [60]. In contrast, ­exposure of mesangial cells to 10% equibiaxial cyclic stretch activated Rho in a caveolin-1-dependent fashion and was abrogated by chemical disruption of caveolae [61]. Stretchinduced activation of RhoA and Rac1 through caveolae was reported in cardiomyocytes [62]. It was suggested that in these cell types activation of RhoA and Rac1, localized in a caveolar compartment, was essential for sensing an externally applied force and transducing this signal to the actin cytoskeleton and ERK1/2 translocation. Thus, available data suggest that caveola-mediated signaling induced by mechanical stress may be very different in different cell types and should be analyzed in particular cellular context.

6.6 Stress-Activated Kinases MAPKs are a family of serine/threonine kinases that are activated through a cascade of dual-specificity MAPK kinases in response to distinct extracellular stimuli. Many

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activities stimulated by growth factors and other mitogens are mediated through so-called ERKs belonging to the MAPK family. Parallel to the ERK pathway, two MAPK pathways, namely, the p38 MAPK and JNK pathways, become activated in response to many cellular stress stimuli, including heat shock, ultraviolet radiation, endotoxin, and inflammatory cytokines. JNK is also called “stress-activated protein kinase.” Stretch-induced activation of ERK, p38, and JNK MAPK cascades is a common cellular response to mechanical strain or flow-induced shear stress and has been ­demonstrated in many cell types [63, 64]. Several upstream mechanisms mediate mechanical-stress-induced MAPK activation. For example, shear stress activates FAK and its association with adaptor protein Grb2 suggesting signaling by cell–matrix contacts. This rapid and transient interaction then leads to the shear-stress-induced ERK2 and JNK1 activation [65]. Several review articles summarize basic aspects of MAPK signaling and regulation by mechanical forces [51, 63, 64, 66]. The complexity of signaling pathways activated by mechanical stress suggests potential involvement of multiple mechanosensors in MAPK activation. For example, stretch-induced activation of MAPK requires tyrosine kinase, PKC activities, and elevation of the level of intracellular Ca2+ [67]. On the other hand, stretch-induced JNK activity in rat cardiac myocytes is not dependent on secreted angiotensin II, PKC, or Ca2+ [68]. Shear-stress-induced ERK activation in ECs depends on Gi-2 protein, Ras, and protein tyrosine kinase activities [69]. As mentioned earlier, cholesterol-sensitive microdomains in the plasma membrane, such as caveolae-like domains, play a critical role in differential activation of ERK and JNK by shear stress [70], implicating a role for caveolae as mechanosensors. The role of VE-cadherin in stretch-induced proliferative signals implies cell–cell junctions in MAPK mechanoregulation [49]. Some effects of mechanical stress on MAPK activation are indirect and involve paracrine mechanisms. For example, mechanical-stretch-induced ERK activation in VSMCs is mediated via angiotensin and endothelin systems [71]. MAPK activation by mechanical stress associated with extensive lung mechanical ventilation plays a critical role in the pathogenesis of pulmonary edema associated with VILI. The following examples support this point. Inhibition of stretch-induced production of the inflammatory cytokine IL-8 by bronchial epithelial cells is achieved by pharmacological blockade of p38 MAPK [72]. Pharmacological inhibition of JNK, p38 MAPK, or apoptosis-signal-related kinase, a member of the MAPK kinase kinase family, attenuates high tidal volume ventilation-induced cytokine production, neutrophil migration into the lung, and vascular leak [73]. Activation of p38 and ERK MAPKs in pulmonary ECs by mechanical stress increases xanthine

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oxidoreductase activity and exacerbates oxidative stress involved in VILI-associated pulmonary edema [74]. The role of mechanical stress in vascular dysfunction associated with VILI will be discussed in more detail in the following sections. In summary, mechanical stretch activates multiple signaling pathways to affect different molecules in the MAPK family, leading to the activation of various transcription factors, e.g., c-myc, c-fos, and c-jun, to modulate VSMC gene expression. Available data indicate that that the particular cell type and the amplitude and frequency of the applied mechanical stimulation dictate which particular member MAPK family will be activated and whether this activation will be sustained or transient. These parameters eventually determine the specificity of cellular response to a particular mechanical stimulus.

6.7 Small GTPases Rho GTPases are members of the Ras superfamily of monomeric 20–30-kDa GTP-binding proteins. The most extensively characterized members are Rho, Rac, and Cdc42, which have distinct effects on actin cytoskeleton, cell adhesions, and cell motility [75–79]. Among 30 potential Rho GTPase effectors identified to date [80], mDia and Rho kinase appear to be required for Rho-induced assembly of stress fibers, myosin light chain (MLC) phosphorylation and actomyosin-driven cell contraction [42, 81–83]. Rac initiates membrane ruffling, lamellipodia extension, and formation of new cell–substrate and cell–cell adhesions. Rac effectors, WAVE, WASP, interact with the Arp2/3 complex and stimulate actin polymerization required for lamellipodia formation and cell motility [84–86]. Thus, the differential effects of Rac and Rho on endothelial cytoskeleton and permeability suggest that the balance between Rho- and Rac-mediated signaling may be a critical component of pulmonary endothelial barrier regulation in pulmonary endothelium exposed to mechanical and chemical stimulation. Cyclic stretch in the 10–20% range activates Rho in pulmonary smooth muscle cells [87], epithelium [88], and endothelium [19, 20, 44, 45]. In contrast to 10–20% stretch, 5% stretch did not cause marked Rho activation or reduction of Rac activity in ECs [20, 89]. In addition to stimulation of small GTPase Rho without affecting Rac, exposure of pulmonary ECs to high-magnitude cyclic stretch (18% elongation) in vitro also potentiates stress fiber formation and barrier dysfunction induced by edemagenic agonists [19, 20]. In nonpulmonary cells, 10–15% cyclic stretch also caused activation of Rac [90, 91]. These findings indicate that stretch-induced small GTPase signaling

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and cell responses may be differentially regulated by cyclic stretch amplitudes and may also be cell-type-specific.

6.8 Nonreceptor Protein Tyrosine Kinases Stimulation of vascular cells by mechanical stretch or shear stress results in tyrosine phosphorylation of multiple cellular protein targets. Stretch-induced activation of tyrosine phosphorylation is required for the formation of focal adhesions, which contain integrin clusters, focal-adhesion-associated proteins (paxillin, talin, vinculin, GIT, PAK1, bPIX, etc.), and cytoskeletal proteins, as well as various tyrosine kinases, including focal-adhesion-associated tyrosine kinase FAK. c-Src, a tyrosine kinase associated with the membrane, plays a role in the stretch-mediated signal transduction. Following activation by stretch, c-Src translocates to the focal contacts [92], where it interacts with an autophosphorylation site on FAK and creates an acceptor for the SH2 domain of Grb2 and thus supports association of FAK with the paxillin–Src complex. Pharmacological inhibition of Src abolishes the stretchinduced cell orientation response. Stretch-induced activation of FAK may also activate RhoA; however, the precise mechanism is not well understood. Although several candidate proteins associated with focal adhesions (including paxillin) may also be involved in mechanotransduction, the role of FAK in this context has been best studied. FAK is activated in stretched pulmonary vessels, in particular in the endothelium [93], and in cultured ECs exposed to shear stress [94]. The recruitment of integrins to focal adhesion sites is mediated by their cytoplasmic domains, which bind proteins of the cytoskeleton. The proteins present at focal adhesions become phosphorylated on tyrosine when the cells are stimulated. In a proposed mechanism of stretch-induced signal transduction leading to cell remodeling [95], ­activation of stretch-activated ion channels leads to elevation of the intracellular Ca2+ level that stimulates Src activity, leading to protein tyrosine phosphorylation, rearrangement of cytoskeletons and focal adhesions, and ultimately cell remodeling. Another mechanism of stretch-induced FAK tyrosine phosphorylation is via stretch-induced mitochondrial reactive oxygen species (ROS) signaling [96]. Studies of pulmonary ECs isolated from lungs ventilated at low or high tidal ­volumes have shown that high tidal volumes enhanced tyrosine phosphorylation of focal adhesion protein paxillin, increased focal adhesion formation, and increased surface expression of PECAM-1 in isolated ECs. These results show amplitudedependent, stretch-induced regulation of tyrosine phosphorylation of cytoskeletal and cell contact proteins in the vascular cells, which may reflect enhancement of cell mechanical and adhesive properties to withstand increased mechanical load.

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6.9 Phosphatidylinositol 3-Kinase/Akt Signaling PI3K and its downstream target Akt participate in cellular signaling in response to growth factors directed to cell survival, proliferation, and migration responses. However, PI3K appears to be also involved in stretch-induced signal transduction. Stretching of VSMCs leads to a rapid PI3K/Akt activation, which can be inhibited by pretreatment with N-acetylcysteine, a scavenger of ROS [97], suggesting that free radicals also play a role in mechanotransduction in VSMCs. Interestingly, cyclic-stretch-induced activation of PI3K and Akt in mesangial cells does not require signaling by integrins or cytoskeleton, but is triggered by transactivation of EGFR and Src-mediated signaling in caveolae. The downstream effect of such PI3K/Akt activation is increased type 1 collagen production [59]. In ECs, stretch-activated PI3K/Akt signaling leads to upregulation of endothelialspecific nitric oxide synthase (eNOS) [98], which is important for regulation of local hemodynamics.

6.10 Reactive Oxygen Species Mechanical stretch of pulmonary vasculature affects both the endothelium and vascular smooth muscle. Increased production of ROS in response to cyclic mechanical stretch has been described in ECs [99–102], VSMCs [103–105], and fibroblasts [106]. Superoxide appears to be the initial species generated in these cell types. Potential sources for increased superoxide production in response to mechanical stress include the NADPH oxidase system [100, 103, 107, 108], mitochondrial production [96, 99, 109], and the xanthine oxidase system [74, 108]. ROS production by nitric oxide synthase has been implicated in the pathogenesis of pulmonary hypertension [110]. Whether these potential sources are activated directly or indirectly by mechanical stress is unclear. However, increased ROS production in response to elevated stretch may contribute to the onset of VILI [111–113]. Stretch-induced increase in ROS production by vascular cells results in activation of nuclear factor kB (NFkB), activation of matrix metalloproteinases, MAPK activation, angiogenesis, LDL oxidation, and altered vasomotion. Studies of isolated mouse arteries have shown that highpressure-induced activation of the NFkB pathway is critically dependent on NADPH-mediated increased ROS production [114, 115]. In cardiac myocytes, high-amplitude cyclic strain induced an increase in ROS production, associated with activation of JNK and ERK1/2 and induction of an apoptotic phenotype [116]. In VSMCs, cyclic stretch induced

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ROS production by rapid activation of NADPH oxidase, leading to activation of MAPK signaling, including p38, JNK, and ERK1/2, and inhibition of NADPH or p38 prevented strain-induced cell alignment [116]. These studies delineated the role of ROS-sensitive p38 activation in vascular remodeling by cyclic stretch. Similar results were obtained using an organ culture model of rabbit aorta. It was shown that pulsatile stretch induces ERK1/2 activation via ROS production [117]. Other studies suggested additional mechanisms and demonstrated that in a cell culture model of arterial hypertension, cyclic stretch induced ROS production via NADPH oxidase and led to vascular remodeling via matrix metalloproteinase activation [103]. In pulmonary artery smooth muscle cells, cyclic stretch contributed to vascular remodeling via an increase in VEGF expression, which was mediated by cyclic-stretch-induced activation of NADPH oxidase and elevation of ROS production [118]. Therefore, increased ­oxidative stress in response to stretch contributes to activation of proinflammatory transcription factors, activation of growth-promoting MAPKs, upregulation of profibrogenic mediators, and altered vascular tone, the important mechanisms ­contributing to the lung vascular dysfunction ­associated with high tidal volume mechanical ventilation or pulmonary hypertension. Stretch-induced ROS production in endothelium upregulates expression of cell adhesion molecules and chemokines [102, 119]. Several mechanisms of ROS production in ECs have been described. Cyclic stretch stimulated ROS production via increased expression of ROS-generating enzymes: NADPH oxidase and eNOS [120–122]. Kuebler et al. reported that circumferential stretch activates nitric oxide production in pulmonary ECs via activation of signaling cascade involving PI3K, Akt and eNOS [123]. Utilization of pulmonary ECs exposed to high-magnitude cyclic stretch in vitro and in animal models of high tidal volume mechanical ventilation revealed stretch-induced activation of endothelial xanthine oxidoreductase that occurred in a p38 and ERK1/2 MAPKdependent fashion. These mechanisms may contribute to the development of VILI [74]. The other part of stretch-induced modulation of ROS signaling is feedback regulation of antioxidant systems. Although the mechanisms by which cyclic stretch regulates the antioxidant enzyme expression are not well understood, the transcription factor Nrf2 appears to be involved in this process [124]. Nrf2, via the antioxidant response element (ARE), alleviates pulmonary toxicant- and oxidant-induced oxidative stress by upregulating the expression of several antioxidant enzymes [125]. Cyclic stretch exposure stimulates ARE-driven transcriptional responses and subsequent expression of antioxidant enzymes such as glutathione peroxidase 2, heme oxygenase-1, and glutamate cysteine ligase in stretched pulmonary ECs [124]. Cyclic stretch then transactivates

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EGFR, and the PI3K/Akt pathway acts as the downstream effector of EGFR and regulates cyclic-stretch-induced ARE activation in an oxidative-stress-dependent manner. Thus, EGFR-activated signaling and actin remodeling act in concert to regulate the stretch-induced Nrf2–ARE transcriptional response and subsequent expression of antioxidant enzymes. It is important to note that NADPH oxidase inhibitor, which inhibits the generation of ROS, N-acetylcysteine, and other flavoproteins that regulate ROS production, blocked cyclic-stretch-induced EGFR phosphorylation, suggesting that ROS-mediated signaling regulates EGFR activation in response to cyclic stretch. These mechanisms again reflect tight relations between cyclic stretch and ROS in mechanochemical regulation of vascular function.

7 Effects of Cyclic Stretch on Pulmonary Vascular Pathophysiological Responses 7.1  Endothelial Permeability Pulmonary endothelium has long been considered a passive barrier for water and macromolecules. However, a growing body of literature has demonstrate sophisticated molecular mechanisms involved in EC barrier regulation under normal and pathological conditions. The two major pathways of endothelial permeability: transcellular and paracellular permeability, are now well recognized. In the transcellular pathway, albumin and other macromolecules are actively transported through cells by an elaborate system of vesicles. Water can also permeate ECs via endothelial water channels called aquaporins. Increased paracellular permeability is a result of the formation of gaps between adjacent ECs, leading to extravasation of water and macromolecules in the lung tissue. A working model of paracellular EC barrier regulation [126, 127] suggests that paracellular gap formation is regulated by the balance of competing contractile forces imposed by actomyosin cytoskeleton, which generate centripetal tension, and adhesive cell–cell and cell–matrix tethering forces imposed by focal adhesions and adherens junctions, which together regulate cell shape changes. Increased EC permeability in response to agonist stimulation is associated with activation of myosin light chain kinase (MLCK), RhoA GTPase, MAPKs, and tyrosine kinases, which trigger actomyosin cytoskeletal rearrangement, phosphorylation of regulatory MLCs, activation of EC contraction, destabilization of intercellular (adherens) junctions, and gap formation [127]. Although involvement of mechanical forces in vascular permeability is apparent, imperfections in existing models limit the development of this area. Several groups have characterized stretch-induced orientation of vascular cells

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induced by cyclic stretch in  vitro and defined signaling pathways that control stretch-induced cell orientation response [19, 41, 116, 128–131]. Fewer studies have addressed the mechanisms of endothelial barrier regulation by VILI-relevant levels of mechanical stress or combination of mechanical stress and stimulation by edemagenic agonists in vitro. Cyclic stretch induces time- and magnitude-dependent changes in pulmonary endothelial signaling and specific protein expression, which may contribute to the altered responsiveness to edemagenic agents and exacerbation of lung vascular barrier dysfunction associated with mechanical ventilation at high tidal volumes [19, 132, 133]. The effects of cyclic stretch on agonist-induced MLC phosphorylation may be observed within minutes of cyclic stretch stimulation. Several mechanisms may be involved in synergistic effects of pathologic cyclic stretch on the agonist-induced EC contractility and barrier dysfunction. First, stretch-induced Ca2+ influx may cause additional MLC phosphorylation by Ca2+/ calmodulin-dependent MLCK [134]. Second, cyclic-stretchinduced activation of signaling serine/threonine- and tyrosine-specific protein kinases [52, 96, 135, 136] may lead to activation of Rho-specific guanine nucleotide exchange factors and trigger the Rho pathway of barrier dysfunction. Third, pathologic cyclic stretch triggers generation of ROS, which may function as second messengers in the signal transduction cascades, including the Rho pathway, that activate cellular responses to strain [96]. Stretch-induced ROS production is dependent on the integrity of cytoskeletal and focal adhesion elements [96, 137].

7.2 Inflammation Mechanical ventilation, an indispensable therapeutic ­modality for the treatment of respiratory failure, can lead to a ­number of serious complications, including initiation or exacerbation of underlying lung injury. Inflammatory response is one of the major lung reactions to overinflation. Injurious ventilation increases cytokine levels in lavage fluid, including those of tumor necrosis factor a (TNF-a), IL-1b, IL-6, IL-10, macrophage inflammatory protein-2, and interferon-g [138], which may contribute to ALI and the development of multiple organ dysfunction syndrome (Fig. 3). The role of stress kinases in cyclic-stretch-induced gene expression was discussed in Sects. VI.F and VI.J. These responses to excessive mechanical strain may also be reproduced in cultures of lung cells exposed to high-magnitude cyclic stretch in  vitro. For example, exposure of human macrophages to 8- to 32-h stretch at 20 cycles per minute stimulated production of IL-8, and the same stretch regimen also enhanced LPS-induced TNF-a and IL-6 production

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Fig. 3  Positive-feedback mechanism of stretch-induced cytokine production involved in the pathogenesis of ventilator-induced lung injury. Mechanical stress induces production of inflammatory cytokines and edemagenic mediators, which further potentiate cyclic-stretch-induced

activation of intracellular signaling. In cells exposed to pathologic mechanical stretch activation of Rho GTPase, myosin light chain kinase and stress-activated protein kinases enhance endothelial contraction and the disruption of cellular contacts, leading to increased vascular permeability

from macrophages [139]. Co-culture of alveolar type II cells and macrophages exposed to pathologic cyclic stretch levels increased intracellular TNF-a levels [140]. Biologically active eicosanoids contribute to the lung inflammatory reaction in VILI. Isoprostanes are the products of lipid oxidation resulting form ALI-associated oxidative stress. Isoprostanes resemble prostanoid structures and have been identified in the alveolar fluid and exhaled air of VILI/ARDS patients. These compounds may further promote lung inflammation and are now used as valuable biomarkers reflecting the severity of VILI/ARDS [31, 141].

pulmonary vasculature leading to activation of apoptosis that may contribute to vascular dysfunction in VILI.

7.3 Apoptosis Apoptosis, or programmed cell death, plays an important role in vascular dysfunction and hyperpermeability caused by VILI-related inflammatory mediators such as TNF-a [142, 143]. However, pathologic mechanical forces may directly stimulate apoptosis in vascular cells. Cyclic stretch with an area change of 25% increased apoptosis in VSMCs, which was accompanied by sustained activation of JNKs and the MAPK p38. In contrast, cyclic stretch with an area change of 7% had no such effect [144]. Stretch-induced apoptosis of VSMCs is possibly regulated by GADD153, a growth arrest and DNA damage-inducible gene [145]. Interestingly, involvement of the b1 integrin signaling pathway in the mechanical-stretch-induced apoptosis in smooth muscle cells [146] suggests a role of mechanosensing via cell–cell contacts in the amplitude-dependent control of vascular cell apoptosis by cyclic stretch. In ECs, physiological levels of cyclic stretch (6–10% at 1 Hz) inhibit EC apoptosis; however, a higher potentially pathologic level of cyclic stretch (20% at 1 Hz) stimulated EC apoptosis [147]. These examples illustrate pathologic effects of high-magnitude cyclic stretch on

8 Pulmonary Vasculature in Ventilator-Induced Lung Injury 8.1  Two-Hit Model of Ventilator-Induced Lung Injury Pathological lung overdistension and circulation disturbances associated with mechanical ventilation at high tidal volumes compromise the BGB, increase lung permeability, and may culminate in VILI or pulmonary edema [148, 149]. Clinical studies suggest that vascular leak observed in VILI patients is associated with increased levels of edemagenic agents and inflammatory cytokines such as thrombin, histamine, TNF-a, IL-8, and IL-1 [139, 148, 150, 151]. Increasing evidence suggests that changes in the patterns of flow and cyclic stretch may directly contribute to the increased EC permeability resulting in pulmonary edema as a complication of mechanical ventilation at high tidal volumes [148, 150, 152]. However, the significance of the interactions between the edemagenic agents and pathological mechanical distension of the lung tissue in the progression of VILI-associated vascular leak and pulmonary edema has been recognized only recently.

8.2 Experimental Models of Ventilator-Induced Lung Injury Because complications of mechanical ventilation develop in patients with pre-existing lung inflammation, infection, or trauma, a National Heart, Lung, and Blood Institute (NHLBI)

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working group emphasized the importance of two-hit animal models, which combine experimentally induced lung inflammation and mechanical ventilation at high tidal volumes to more appropriately reflect common comorbidities and risk factors present in patients with ALI [153]. Risk factors for human ARDS were reproduced in animal models of lung injury. In humans with clinical risk factors for ARDS, mechanical ventilation is superimposed on an ongoing inflammatory process in the lungs. Thus, two major groups of animal models are currently used: a model of VILI, in which mechanical ventilation is the sole method used to generate injury, and a model of ventilator-associated lung injury, in which mechanical ventilation modifies lung injury due to a clinically relevant cause, such as pulmonary infection, instillation of LPS, acid aspiration, or lung injury secondary to other pathological conditions. Remarkably, noninjurious mechanical ventilation combined with small doses of LPS or bacteria that cause minimal inflammation results in the development of a form of lung injury characterized by cytokine release, neutrophilic alveolitis, and protein leakage into the air spaces. These studies show that mechanical ventilation and bacterial products are synergistic in causing lung injury [154, 155]. Animal studies in isolated perfused ventilated lungs have shown direct effects of lung overinflation caused by high tidal volume mechanical ventilation and decreased vascular flow rates on vascular filtration coefficient [156–158]. The rapid onset of the increase in vascular permeability represents direct effects of mechanical strain on lung barrier function and activation of rapid intracellular signaling events. Indeed, exposure of isolated perfused mouse lungs to high peak inflation pressure (PIP) ventilation (35-cmH2O PIP) increased the lung filtration coefficient, perivascular cuff, and alveolar fluid volumes, which was caused by ventilatorinduced rapid activation of Ca2+-selective TRPV4 channels [159]. The second phase is characterized by a slower increase in cytokine levels and mobilization of inflammatory cell infiltrates observed a few hours later. High tidal volume mechanical ventilation triggers several pathogenic mechanisms: cellular plasma membrane disruption, increased cytokine production, increased concentration of ROS leading to the oxidative stress, compromised alveolar reabsorption, and increased endothelial permeability [139, 148, 160]. Studies of cellular responses to VILI have indicated that several cell types (alveolar epithelium, neutrophils, alveolar macrophages, and lung endothelium) are involved in this process (for a review, see [148]). Several in  vitro models of cultured ECs, alveolar epithelial cells, lung fibroblasts, or macrophages have been established to reproduce the effects of pathologic cyclic stretch and inflammatory agents on lung cells. Consistent with clinical observations, these models showed cytokine

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production and exacerbation of agonist-induced endothelial barrier dysfunction by high-amplitude cyclic stretch observed in specific lung cell types and explored the molecular and biochemical mechanisms of mechanical stress responses in particular cells [16, 19, 139, 148, 151]. These models are now intensively utilized in the studies of pathophysiological mechanotransduction, genomics, and proteomics applications.

8.3 Magnitude-Dependent Effects of Stretch on Lung Cells In Vitro Physiological levels of cyclic stretch and intraluminal pressure are essential for the maintenance of VSMC contractile phenotype, control of vascular tone, regulation of mass transport across the vessel wall, and expression of native constituents of vascular wall extracellular matrix [161]. However, physiologically and pathologically relevant mechanical stresses may exhibit opposite effects on certain cell functions. For example, whereas physiologic cyclic stretch inhibits apoptosis in vascular endothelium, pathologic cyclic stretch promotes it and causes mitotic arrest of VSMCs [4, 147, 162]. Primary alveolar epithelial cells tolerate physiologically relevant mechanical strain well, resulting in a 25% increase in cell surface area, whereas a 37–50% increase in cell surface area, corresponding to 90–100% of total lung capacity, causes progressive cell death [16]. Analysis of the effects of different stretch regimens on IL-8 production from A549 cells shows that 30% (but not 20%) stretch at 20 or 40 cycles per minute increased IL-8 production after 12–48 h [151]. VSMC stretching at 14% and 33% elongation, but not at 5% elongation, induces release of fibroblast growth ­factor-2, a growth factor involved in cellular reparation after injury [163]. EC exposure to cyclic stretch at 15% elongation increases production of the inflammatory cytokine IL-8, whereas 6% cyclic stretch was without effect [164]. Exposure of EC cultures to 15–20% cyclic stretch in  vitro reproduces activation of VEGF expression also observed in VILI patients [165]. Low and high mechanical strains alternatively regulate matrix metalloproteinase-1 expression, suggesting amplitude-dependent mechanosensitive regulation of vascular remodeling [166]. Long-term preconditioning at 18% cyclic stretch enhances thrombin-induced permeability in pulmonary EC monolayers even after cessation of cyclic stretch stimulation [19]. In contrast, stretch preconditioning of pulmonary ECs at physiological levels decreases thrombin-induced barrier dysfunction and accelerates barrier recovery. These differences are associated with magnitude-dependent regulation of intracellular signaling by yet unknown mechanisms and

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long-term cyclic stretch effects on EC permeability associated with stretch-induced sustained EC phenotypic changes [132, 133].

[174] considers edemagenic effects of acute increases in VEGF levels in the early stage of lung injury induced by inflammatory stimuli or mechanical stretch.

9 Interactions Between Mechanical Stress and Bioactive Molecules in Ventilator-Induced Lung Injury

9.2 Hepatocyte Growth Factor

Interactions between cyclic stretch and agonist stimulation may represent a fundamental mechanism of mechanochemical control of vascular remodeling and barrier function.

9.1  Vascular Endothelial Growth Factor VEGF is a potent angiogenic factor, and its presence at threshold concentrations in essential for EC survival. VEGF production induced by physiologic cyclic stretch described in VSMCs [167] may provide an arterial stimulus for ­maintenance of steady-state levels of VEGF essential for EC survival. On the other hand, VEGF, originally called “vascular permeability factor,” also controls lung vascular permeability to water and proteins. VEGFinduced endothelial permeability is mediated by MAPKs and Rho-dependent signaling [21, 168, 169]. VEGF overexpression in the lungs or injection of purified VEGF increases endothelial permeability in vivo [170, 171]. The role of VEGF in lung disease is ­controversial. VEGF is primarily produced by type II alveolar epithelial cells and is a survival factor for the lung microvascular ECs [172]. VEGF production induced by ­physiologic cyclic stretch described in VSMCs [167] may also provide an arterial stimulus for maintenance of steady-state levels of VEGF essential for EC survival. In healthy human subjects, VEGF is highly compartmentalized to the lung with alveolar VEGF protein levels 500 times higher than in plasma [173]. During excessive lung mechanical stress or injury such as in ALI or VILI, because of the anatomic proximity between alveolar epithelial and microvascular ECs, VEGF may literally spill pulmonary edema [173, 174]. VEGF level increases in the lung have been reported in several lung diseases, including hydrostatic edema, ARDS, and LPS-induced lung injury [175, 176]. High tidal volume ventilation and cyclic stretch of vascular ECs and VSMCs in vitro also stimulates VEGF and VEGF receptor expression [53, 118, 177]. In turn, VEGF-induced endothelial monolayer barrier disruption is enhanced by high-magnitude cyclic stretch in  vitro [21]. A model describing the role of VEGF in ALI recently proposed by Mura et  al.

Hepatocyte growth factor (HGF) is a multifunctional mesenchyme-derived pleiotropic factor secreted by several cell types. Clinical studies have shown dramatic (up to 25-fold) elevation of HGF levels in plasma and BAL fluid in patients with ALI/ARDS [178–180]. This elevation may be directly induced by pathologic mechanical stretch associated with mechanical ventilation, since it was demonstrated that exposure of human alveolar epithelial cells (A549) cultured on a silicoelastic membrane to high-magnitude cyclic stretch in  vitro induces HGF expression and its release [181]. This study also reported cyclic-stretch-induced expression of the inflammatory cytokine IL-8 by the same cells. HGF stimulates alveolar epithelial proliferation and is likely involved in alveolar epithelial repair processes in the lung [182]. In addition, HGF promotes cell survival and enhancement of lung endothelial barrier properties critical for restoration of lung vascular barrier function [183–185]. These biological activities suggest a role for increased HGF levels induced by pathologic mechanical stress in the lung reparation and vascular barrier recovery. Thus, in contrast to “vicious circles,” the signaling loops resulting in escalation of a pathogenic process such as stretchinduced production of inflammatory agents induced by pathologic stretch, or potentiation of ­barrier-disruptive Rho signaling described earlier, stretch-induced HGF production in VILI represents the autoregulatory mechanism directed at resolution of the pathological ­condition. These clinical and experimental findings also ­emphasize the importance of interactions between protective and disruptive bioactive molecules in control of vascular ­permeability and lung inflammation in VILI/ ARDS. Potential therapeutic role of HGF in the treatment of VILI/ADRS will be discussed later.

9.3 Coagulation: Role of Thrombin Dysregulation of coagulation and fibrinolysis are frequent complications of systemic infection. Clinical investigations and experimental models of ALI/ARDS, VILI, and pneumonia have clearly demonstrated beneficial effects of anticoagulant therapy [186–189]. The important role of pulmonary coagulation has been recognized in the development of VILI [187, 188]. Because thrombin activates Rho both in vivo and in vitro, increased thrombin levels may become a considerable

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factor contributing to the Rho-mediated vascular endothelial barrier dysfunction caused by high tidal volume mechanical ventilation. In vivo, the thrombin-derived nonthrombogenic protease-activated receptor 1 ligand TRAP-6 exacerbates high-tidal-volume-induced lung dysfunction [190].

9.4 GTPases in Regulation of Vascular Permeability: Testing the Two-Hit Ventilator-Induced Lung Injury Model As discussed already, the Rho pathway is directly involved in the lung EC barrier disruption induced by pathologically relevant levels of cyclic stretch and inflammatory agonists. Experimental models strongly suggest that attenuation of Rho activity and stimulation of the Rac pathway reduces lung vascular leak and promotes lung barrier recovery in the in vitro and in vivo models of ALI/VILI [20, 190–192]. In vitro studies have shown synergistic effects of pathologic cyclic stretch amplitudes (18% cyclic stretch) and thrombin in the pulmonary endothelial barrier disruption. Remarkably, pathologic cyclic stretch alone does not cause considerable barrier disruption in this model. These data strongly support the two-hit model of VILI currently considered by ARDS Network [193]. A hypothesis considering a shift in the Rac– Rho balance toward Rac activation as the pulmonary vascular protective mechanism (Fig. 4) has received further support from other in  vitro and animal studies that have utilized structurally unrelated agents with Rac-activating potential.

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Their properties as potential protective agents in the treatment of VILI-associated vascular dysfunction will be discussed next in more detail.

10 Candidate Molecules for Treatment of Ventilator-Induced Vascular Dysfunction The mortality from ALI/ARDS remains high. A critical role of ventilatory strategies in reduction of ALI/ARDS mortality has been emphasized in a large prospective NHLBI-supported study which showed that mortality was reduced from 40% to 31% if mechanical ventilation was performed at low tidal volume (6  mL/kg) and limited plateau pressure (less than 30 cmH2O) [193]. However, despite two major advances in treatment, low tidal volume ventilation for ALI/ARDS and activated protein C for severe sepsis (the leading cause of ALI/ARDS), additional research is needed to develop specific treatments and improve understanding of the pathogenesis of these syndromes [153]. Recent studies used in vitro models of endothelial barrier dysfunction to test a number of molecules with proven barrier-protective effects in EC cultures.

10.1  Sphingosine 1-Phosphate Sphingosine 1-phosphate (S1P) is a biologically active sphingolipid produced in many cells by the phosphorylation of sphingosine, a molecule derived from the degradation of the plasma membrane component sphingomyelin [194]. Activated platelets represent an important and primary source of secreted S1P [195]. Being a potent angiogenic factor, S1P also exhibits prominent barrier-protective effects on pulmonary endothelium via ligation of the EC surface receptor Edg-1 [196], which leads to stimulation of Rac GTPasedependent cytoskeletal rearrangement, adherens junction formation and stabilization, and focal adhesion assembly [197, 198]. A single dose of S1P markedly attenuated pulmonary vascular leak in rodent and canine models of ALI induced by combined intrabronchial endotoxin administration and high tidal volume mechanical ventilation [29] and significantly reduced microvascular permeability and inflammation in a murine model of LPS-mediated lung injury [199].

10.2 FTY720

Fig. 4  Interaction between mechanical strain and agonists in ­regulation of Rho–Rac balance, cytoskeletal remodeling, and pulmonary endothelial permeability

FTY720 [2-amino-2-(2-(4-octylphenyl)ethyl)-1,3-propanediol], is a synthetic compound with structural similarity to sphingosine and is currently in phase III clinical trials as a immunosuppressive agent for the prevention of solid organ

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transplant rejection [200]. The proposed FTY720 action invokes phosphorylation of FTY720 in situ by cellular sphingosine kinases, thereby greatly increasing its homology to S1P and the affinity for the S1P family of receptors, particularly S1P1 and S1P3 [201]. The mechanism of FTY720mediated immunosuppression involves binding of endogenously phosphorylated FTY720 to S1P1 receptor on lymphocytes, leading to internalization of the receptor [202]. FTY720 improves survival and upregulation of cytokine production in the transient ischemia–reperfusion model [203] and exhibits potent barrier-protective effects in pulmonary endothelial cultures [204] and a murine ALI model [199]. Surprisingly, pulmonary EC barrier enhancement by FTY720 neither requires the S1P1 receptor nor causes the characteristic enhancement of cortical actin cytoskeleton observed in S1P-stimulated endothelium [204]. Both S1P and FTY720 diminish acute pulmonary injury in rats with acute necrotizing pancreatitis [205], reflected by marked attenuation of lung histological changes, significant reduction of pulmonary vascular leak, BAL fluid protein concentration, total cell count, polymorphonuclear leukocyte percentage, and production of proinflammatory cytokines. It is important to note that although they ameliorated pulmonary injury, S1P and FTY720 did not protect against acute necrotizing pancreatitis induced by the injection of 5% sodium taurocholate. These data support a potential therapeutic role for S1P and FTY720 for the targeted treatment of lung injury associated with acute necrotizing pancreatitis and possibly other diseases.

10.3 Rho Kinase Inhibitors Increased Rho kinase activity results in inappropriate coronary artery vasoconstriction in the vascular system and is likely involved in the pathogenesis of exercise-induced myocardial ischemia, spontaneous coronary artery spasm, and hypertension. In clinical trials, the Rho kinase inhibitors fasudil and Y-27632 have demonstrated anti-ischemic, antivasospastic, and antihypertensive effects [206]. Inhibition of Rho kinase using fasudil has also been considered for treatment of pulmonary hypertension [207]. Besides regulation of smooth muscle tone, Rho signaling plays an important role in regulation of pulmonary endothelial permeability by mechanical stretch, and inflammatory mediators [20, 190]. Inhibition of Rho kinase by Y-27632 abolished pulmonary endothelia barrier disruption induced by pathologically relevant amplitudes of cyclic stretch and thrombin [19]. The Rho kinase inhibitor Y-27632 attenuates endotoxin-induced lung edema and neutrophil emigration into the lung parenchyma in mice [192] and reverses oleic acid induced lung damage [208]. These findings strongly suggest that Rho kinase inhibitors may be a promising strategy for the treatment of VILI and ARDS.

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10.4 Oxidized Synthetic 1-Palmitoyl-2Arachidonoyl-sn-Glycero-3Phosphorylcholine The role of lipid oxidation products in the pathogenesis of VILI is actively debated. For example, lipid oxidation products such as isoprostanes serve as a marker of the severity of ALI/ARDS, and oxidation of surfactant phospholipids [209, 210]. However, isoprostanes appear to be more than inert markers, and may also act as signal transduction molecules. As isomers of prostaglandins, they can exert powerful biological effects on many lung cell types through actions on prostanoid receptors and promote both beneficial and deleterious effects on lung function [211]. Specific products resulting from oxidation of phospholipids containing arachidonic acid exhibit potent barrier-protective properties. Oxygenation of nonsaturated bonds at the sn-2 position in the arachidonic acid moiety in 1-palmitoyl2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) produces cyclopenthenone-containing compounds such as 1-palmitoyl-2-(5,6-epoxyisoprostane E2)-sn-glycero-3-phos phocholine and 1-palmitoyl-2-(5,6-epoxycyclopentenone)sn-glycero-3-phosphocholine that possess barrier-protective activities toward pulmonary endothelium [212]. Other products of phospholipid oxidation belonging to a group of fragmented phospholipids such as 1-palmitoyl-2-(5-oxovaleroyl) -sn-glycerophosphatidyl choline, 1-palmitoyl-2-glutaroyl-snglycerophosphatidyl choline, and lyso­phosphatidylcholine induce endothelial permeability via activation of the Rho pathway [212–214]. A preparation of oxidized synthetic PAPC (OxPAPC) with a predominant content of oxygenated barrier-protective compounds, as verified by mass spectrometry analysis, has been extensively characterized in vitro and in vivo [190, 212, 215–221]. A single dose of OxPAPC significantly reduces lung vascular leak, neutrophil accumulation, and inflammatory cytokine production in the rat and murine models of ventilator-induced and LPS-induced ALI [190, 221]. Barrierprotective effects of OxPAPC are mediated by activation of Rac signaling and Rac-dependent inhibition of Rho-activated vascular permeability [190, 216, 217]. Whereas cyclic stretch of pulmonary ECs in vitro at pathologically relevant amplitudes (18% elongation) stimulates Rho activity on its own and further potentiates Rho activation induced by thrombin [20], OxPAPC potently inhibits activation of the Rho pathway caused by pathologic cyclic stretch amplitudes in both pulmonary EC cultures and in mice exposed to mechanical ventilation at high tidal volume [190]. OxPAPC is also effective in reduction of vascular leak in the two-hit model of ALI caused by administration of thrombin-derived activating signaling peptide (TRAP-6) combined with high tidal volume mechanical ventilation [190]. OxPAPC exhibits potent vasoprotective and anti-inflammatory effects in the lung when

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injected intravenously [191, 220, 222, 223] or intratracheally [220]. However, very high OxPAPC concentrations may exert deleterious effects on endothelial barrier properties [212] and other parameters of lung function [224]. These results suggest that OxPAPC may be considered as a therapeutic agent for future protective strategies, however, its dosage and composition requires further characterization.

10.5 Hepatocyte Growth Factor Similar to S1P, HGF is a multifunctional pleiotropic factor secreted by several cell types. Novel therapeutic strategies using HGF to ameliorate cardiovascular disease have been suggested [183, 225, 226]. HGF protects against endothelial barrier dysfunction via stimulation of PI3K and its downstream effector glycogen synthase kinase 3b (GSK-3b) leading to activation of Rac and GSK-3b-dependent stabilization of peripheral actin cytoskeleton [184, 185]. Furthermore, HGF attenuates VEGF-induced hyperpermeability in pulmonary ECs, and combination of HGF and EC exposure to physiologically relevant cyclic stretch amplitudes abolishes VEGF-induced EC barrier disruption [21]. Thus, HGF administration in the acute phase of VILI may reduce lung vascular leak and injury in the acute phase and accelerate resolution of ALI.

10.6  b  -Adrenergic Agonists and Elevation of Cyclic AMP Levels Several reports suggest beneficial effects of endogenous b-adrenergic tone in the models of ALI. One component of such protection is activation of alveolar fluid clearance by cAMP-dependent mechanisms [227], whereas the other modality is attenuation of ALI-associated vascular hyperpermeability associated with b-agonist-induced elevation of intracellular cyclic AMP (cAMP) levels in pulmonary endothelium. What are the effects of the elevation of cAMP levels on pulmonary vasculature? The first mechanism is relaxation of actomyosin cytoskeleton via cAMP-dependent signaling. cAMP-mediated relaxation and barrier protection has been generally associated with PKA-mediated inhibition of MLCK enzymatic activity [228–230] leading to decreased levels of phosphorylated MLC essential for activation of actomyosin contraction in smooth muscle and nonmuscle (endothelial) cells. As described earlier, activated Rho GTPase engages Rho kinase, which inactivates myosin light chain phosphatase activity and thus increases the levels of phosphorylated MLC [231]. RhoGDP dissociation inhibitor

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(Rho-GDI) is a negative regulator of Rho. PKA-mediated phosphorylation of Rho-GDI leads to inactivation of Rho [232]. Thus, PKA-dependent attenuation of MLCK and Rho activities directly reduces endothelial actomyosin contraction and alleviates increased vascular permeability. Studies of animal models have shown that inhibition of MLCK and elevation of intracellular cAMP levels reduces capillary fluid leak after a modest high-PIP injury, suggesting protective effects in VILI [152]. Recent studies described a novel cAMP-activated signaling cascade, Epac–Rap1 pathway, which works in parallel to PKA signaling and contributes to endothelial barrier protection [233–235]. A small-moleculespecific activator of Epac has been also described and shown to reproduce barrier-protective effects on pulmonary endothelium elicited by cAMP [234, 236]. The role of Epac–Rap1 signaling in protection against ventilator-induced vascular leak has not been tested yet. However, these signaling molecules may be important targets for drug design.

11  Concluding Remarks It now becomes clear that excessive mechanical strain associated with mechanical ventilation at high tidal volumes triggers multiple pathologic mechanisms leading to pulmonary vascular dysfunction, inflammation, and pulmonary edema. Mechanical strain may (a) permanently or reversibly damage vascular and alveolar cells leading to cell death or reversible cell membrane failure, (b) stimulate signaling pathways leading to endothelial barrier compromise or exacerbation of vascular cell responses to edemagenic and inflammatory agonists, and (c) induce release and production of inflammatory cytokines (IL-8, IL-6, TNF-a, etc.) that induce further vascular leak, lung inflammation, and alveolar flooding, which are the hallmarks of ALI and ARDS. This review discussed involvement of MLCK, Rho GTPase, and serine/threonine and tyrosine kinase signaling in endothelial vascular leak induced by excessive mechanical strain. The complexity of the pathologic mechanisms triggered by high tidal volume mechanical ventilation clearly indicates that the treatment of this devastating syndrome should focus on targeting several major pathologic mechanisms: reduction of oxidative stress, control of actomyosin contractility in vascular ECs and possible alveolar epithelial cells with MLCK and Rho GTPase as major targets, improvement of alveolar fluid reabsorption, and stimulation of Rac signaling in vascular endothelium that leads to enhancement or restoration of endothelial barrier properties. A number of bioactive molecules with barrier-protective properties targeting small GTPases, signaling kinases, receptors, and the coagulation system have been described and tested in cell culture and animal models of VILI. Most

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recent findings have described novel signaling pathways of endothelial barrier protection that revealed potential novel targets for drug therapy. Further testing of already described and emerging candidate molecules in the models of ventilator-induced vascular leak is an area of active research directed at the mechanism-based strategies for treatment of lung vascular dysfunction associated with injurious mechanical ventilation.

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Part IV

Pulmonary Vascular Diseases

Chapter 65

Classification of Pulmonary Hypertension: History and Perspectives David B. Badesch

Abstract  A well-organized, systematic classification scheme is important in any disease process; this is especially true in pulmonary hypertension. Classification serves as a framework for clinical studies, as well as patient care. It provides structure for inclusion and exclusion criteria for clinical trials, as well as the development of screening, diagnostic, and treatment algorithms. There are several distinct classification schemes used in pulmonary hypertension: diagnostic category, functional, and pathologic. This chapter will briefly review each of these classification schemes, providing a historical perspective, and illustrating the utility of each. Keywords  Pulmonary arterial hypertension • Pulmonary venous hypertension • Genetics • Associated pulmonary arterial hypertension

1  Introduction A well-organized, systematic classification scheme is ­important in any disease process; this is especially true in pulmonary hypertension (PH). Classification serves as a framework for clinical studies, as well as patient care. It provides structure for inclusion and exclusion criteria for clinical trials, as well as the development of screening, diagnostic, and treatment algorithms. There are several distinct classification schemes used in PH: diagnostic category, functional, and pathologic. This chapter will briefly review each of these classification schemes, providing a historical perspective, and illustrating the utility of each.

2 Diagnostic Classification The diagnostic category classification scheme has evolved from a very simple primary (unexplained) versus secondary (due to an underlying disease process) format to the more detailed Evian, Venice, and finally, Dana Point systems. The latter classification systems were each developed as consensus statements from world symposia on pulmonary hypertension held at these three venues. These meetings have occurred every 5 years, beginning with the second world symposium held in Evian, France, in 1998, followed by the third world symposium in Venice, Italy, in 2003, and the fourth world symposium in Dana Point, California, USA, in 2008. Each meeting has resulted in an update to the diagnostic classification scheme.

2.1 The Evian Classification The Evian classification scheme was designed to create ­diagnostic categories based on similarities in pathological and pathophysiological characteristics, clinical presentation, and treatment options (Table  1) [1, 2]. This scheme was well accepted, and widely used in clinical practice, also being used by the US Food and Drug Administration (FDA) and the European Agency for Drug Evaluation (EMEA) for the labeling of newly approved medications [3–6]. The Evian classification used five major categories: (1) pulmonary arterial hypertension (PAH), (2) pulmonary venous hypertension, (3) PH associated with disorders of the respiratory system, (4) PH associated with thrombotic or embolic diseases, and (5) PH associated with diseases affecting the pulmonary vasculature. 2.1.1   Pulmonary Arterial Hypertension

D.B. Badesch (*) University of Colorado, Denver, Leprino Bldg., Rm 536, Box 957, 12401 E. 17th Ave., Aurora, CO 80045, USA e-mail: [email protected]

PAH included disease processes largely localized to the small pulmonary arterioles; precapillary in location. This category included a subgroup without an identifiable cause,

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_65, © Springer Science+Business Media, LLC 2011

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938 Table 1  The Evian clinical classification (reprinted from [16] with permission from Elsevier) 1. Pulmonary arterial hypertension 1.1. Primary PH (a) Sporadic (b) Familial 1.2. Related to (a) Collagen vascular disease (b) Congenital systemic to pulmonary shunts (c) Portal hypertension (d) Human immunodeficiency virus infection (e) Drugs/toxins: anorexigens, other (f) Persistent PH of the newborn (g) Other 2. Pulmonary venous hypertension 2.1. Left-sided atrial or ventricular heart disease 2.2. Left-sided valvular heart disease 2.3. Extrinsic compression of central pulmonary veins (a) Fibrosing mediastinitis (b) Adenopathy/tumors 2.4. Pulmonary veno-occlusive disease 2.5. Other 3. PH associated with disorders of the respiratory system or hypoxemia 3.1. Chronic obstructive pulmonary disease 3.2. Interstitial lung disease 3.3. Sleep-disordered breathing 3.4. Alveolar hypoventilation disorders 3.5. Chronic exposure to high altitude 3.6. Neonatal lung disease 3.7. Alveolar-capillary dysplasia 3.8. Other 4. PH caused by chronic thrombotic or embolic disease 4.1. Thromboembolic obstruction of proximal pulmonary arteries 4.2. Obstruction of distal pulmonary arteries (a) Pulmonary embolism (thrombus, tumor, ova, or parasites, foreign material) (b) In situ thrombosis (c) Sickle-cell disease 5. PH caused by disorders directly affecting the pulmonary vasculature 5.1. Inflammatory (a) Schistosomiasis (b) Sarcoidosis (c) Other 5.2. Pulmonary capillary hemangiomatosis

“primary PH” (PPH). This incorporated both sporadic and familial forms of the disease. A second subgroup included PAH occurring in relationship to a variety of underlying disease processes, including collagen vascular disease (now often referred to as connective tissue disease), congenital systemic-to-pulmonary shunts (such as atrial septal defects, ­ventricular septal defects, patent ductus arteriosis, etc.), portopulmonary hypertension (PAH occurring in relationship to portal hypertension or late-stage liver disease), human immunodeficiency virus infection, drug or toxin exposure (including anorexigens and others), and persistent PH of the

D.B. Badesch

newborn. Although these conditions may involve somewhat different pathophysiologic mechanisms leading to remodeling of the pulmonary arterioles, it was appreciated that they shared similar clinical presentations, and responsiveness to therapy (particularly to epoprostenol [7, 8]).

2.1.2  Pulmonary Venous Hypertension This category focused largely on left-sided cardiac disease, including left ventricular dysfunction, and mitral and aortic valvular disease. It also included extrinsic compression of the central pulmonary veins (as can be seen with fibrosing mediastinitis, adenopathy, or tumors), and pulmonary venoocclusive disease (PVOD). These diseases may share a common lack of response to pulmonary vasodilator therapy, such as epoprostenol, and sometimes demonstrate a distinctly adverse response to such agents [9].

2.1.3  P  H Associated with Disorders of the Respiratory System or Hypoxemia Included in this category is PH occurring in association with chronic obstructive pulmonary disease, interstitial lung ­disease, hypoventilation, chronic exposure to high altitude, etc. Treatment is often focused on the underlying disease process, and the correction of hypoxemia. There is ­increasing interest in patients who have PH that seems out of ­proportion to the severity of these underlying processes, and whether they might be candidates for PH-specific therapy. Further study is needed in such situations.

2.1.4  PH Caused by Thrombotic or Embolic Diseases Chronic thromboembolic PH due to proximal organized clot in major pulmonary arteries may benefit from pulmonary thromboendarterectomy [10, 11]. More peripheral emboli or thrombi similar to the thrombotic lesions observed in PPH may sometimes be treated with medical therapy [12], although further studies are needed. In most cases, long-term anticoagulation is indicated.

2.1.5  P  H Caused by Disorders Directly Affecting the Pulmonary Vasculature Included in this category were diseases associated with inflammatory processes or mechanical obstruction (such as schistosomiasis [13] and sarcoidosis [14]), and pulmonary capillary hemangiomatosis (PCH) [15].

65  Classification of Pulmonary Hypertension: History and Perspectives

2.2  The Venice Classification The classification of PH was updated at the third world ­symposium held in Venice, Italy, in 2003 (Table  2) [16]. The structure of the Evian classification scheme was maintained, with major changes including the addition of a genetic

Table  2  Revised clinical classification of pulmonary hypertension (Venice 2003) (reprinted from [16] with permission from Elsevier) 1. Pulmonary arterial hypertension (PAH) 1.1. Idiopathic (IPAH) 1.2. Familial (FPAH) 1.3. Associated with (APAH) (1) Collagen vascular disease (2) Congenital systemic-to-pulmonary shunts (**below) (3) Portal hypertension (4) HIV infection (5) Drugs and toxins (6) Other (thyroid disorders, glycogen storage disease, Gaucher disease, hereditary hemorrhagic telangiectasia, hemoglobinopathies, myeloproliferative disorders, splenectomy) 1.4. Associated with significant venous or capillary involvement (1) Pulmonary veno-occlusive disease (PVOD) (2) Pulmonary capillary hemangiomatosis (PCH) 1.5. Persistent pulmonary hypertension of the newborn 2. Pulmonary hypertension with left heart disease 2.1. Left-sided atrial or ventricular heart disease 2.2. Left-sided valvular heart disease 3. Pulmonary hypertension associated with lung diseases or hypoxemia 3.1. Chronic obstructive pulmonary disease 3.2. Interstitial lung disease 3.3. Sleep-disordered breathing 3.4. Alveolar hypoventilation disorders 3.5. Chronic exposure to high altitude 3.6. Developmental abnormalities 4. Pulmonary hypertension caused by chronic thrombotic and/or embolic disease 4.1. Thromboembolic obstruction of proximal pulmonary arteries 4.2. Thromboembolic obstruction of distal pulmonary arteries 4.3. Non-thrombotic pulmonary embolism (tumor, parasites, foreign material) 5. Miscellaneous Sarcoidosis, histiocytosis X, lymphangiomatosis, compression of pulmonary vessels (adenopathy, tumor, fibrosing mediastinitis ** Guidelines for classification of congenital systemic-to-pulmonary shunts 1.  Type: simple, atrial septal defect (ASD), ventricular septal defect (VSD) patent ductus arteriosus, total or partial unobstructed anomalous pulmonary venous return, combined (describe combination and define prevalent defect if any), complex, truncus arteriosus, single ventricle with unobstructed pulmonary blood flow, atrioventricular septal defects. 2.  Dimensions: small (ASD 2.0 cm and VSD 1.0 cm), large (ASD >2.0 cm and VSD >1.0 cm) 3.  Associated extracardiac abnormalities 4.  Correction status: noncorrected, partially corrected (age), corrected [spontaneously or surgically] (age)

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classification; discontinuation of the use of the term “primary pulmonary hypertension” (PPH), and substitution of the term “idiopathic pulmonary arterial hypertension” (IPAH); reclassification of PVOD and PCH; an update to the risk factors for PAH; and reassessment of congenital systemic-to-pulmonary shunts. In this revised classification scheme, the first category, “pulmonary arterial hypertension” (PAH), came to consist of IPAH, familial PAH, associated PAH, PAH associated with significant venous or capillary involvement (including PVOD and PCH), and persistent PH of the newborn. A new category of “miscellaneous” was added, including sarcoidosis, histiocytosis X, lymphangiomatosis, and compression of pulmonary vessels (adenopathy, tumor, fibrosing mediastinitis). The revised classification also provided guidelines for the classification of congenital systemic-topulmonary shunts.

2.3  The Dana Point Classification Classification of PH was again updated at the fourth world symposium held in Dana Point, California, USA, in 2008 (Table 3) [17]. Again, the general structure of the classification scheme was preserved, but several important changes were made. Highlighted below are several of the more important changes to group 1 PAH in the Dana Point scheme.

2.3.1  Heritable PAH “Familial PAH” was dropped in favor of the term “heritable PAH.” This was due to the realization that mutations in the bone morphogenetic protein receptor 2 (BMPR2) gene have been detected in 11–40% of apparently idiopathic cases with no family history [18, 19], the distinction between idiopathic and familial BMPR2 mutations is artificial, and in 20% or fewer families with PAH, no BMPR2 mutation has been identified. Heritable forms of PAH therefore include IPAH with germline mutations (largely BMPR2, but also activinreceptor-like kinase-1 or endoglin) and familial cases with or without germline mutations [20, 21].

2.3.2  Risk Factors and Drug- and Toxin-Induced PAH An updated list of risk factors and associated conditions for PAH was developed at the Dana Point symposium (Table 4) [17]. Definite risk factors are thought to include exposure to aminorex, fenfluramine, dexfenfluramine, and toxic rapeseed oil. Likely risk factors are thought to include amphetamines, l-tryptophan, and methamphetamines [22].

940 Table  3  Updated clinical classification of pulmonary hypertension (Dana Point classification, 2008) (reprinted from [17] with permission from Elsevier) 1. Pulmonary arterial hypertension 1.1. Idiopathic PAH 1.2. Heritable (1) BMPR2 (2) ALK1, endoglin (with or without hereditary hemorrhagic telangiectasia) (3) Unknown 1.3. Drug- and toxin-induced 1.4. Associated with (1) Connective tissue disease (2) HIV infection (3) Portal hypertension (4) Congenital heart diseases (5) Schistosomiasis (6) Chronic hemolytic anemia 1.5. Persistent pulmonary hypertension of the newborn 1.6. Pulmonary veno-occlusive disease (PVOD) and or pulmonary capillary hemangiomatosis (PCH) 2. Pulmonary hypertension due to left heart disease 2.1. Systolic dysfunction 2.2. Diastolic dysfunction 2.3. Valvular disease 3. Pulmonary hypertension due to lung diseases and/or hypoxia 3.1. Chronic obstructive pulmonary disease 3.2. Interstitial lung disease 3.3. Other pulmonary diseases with mixed restrictive and obstructive pattern 3.4. Sleep-disordered breathing 3.5. Alveolar hypoventilation disorders 3.6. Chronic exposure to high altitude 3.7. Developmental abnormalities 4. Chronic thromboembolic pulmonary hypertension (CTEPH) 5. PH with unclear multifactorial mechanisms 5.1. Hematologic disorders: myeloproliferative disorders, splenectomy 5.2. Systemic disorders: sarcoidosis, pulmonary Langerhans cell histiocytosis, lymphangioleiomyomatosis, neurofibromatosis, vasculitis 5.3. Metabolic disorders: glycogen storage disease, Gaucher disease, thyroid disorders 5.4. Others: tumoral obstruction, fibrosing mediastinitis, chronic renal failure (on dialysis)

2.3.3  Congenital Heart Diseases Following the Dana Point symposium, the pathologic and pathophysiologic classification of congenital heart disease with systemic-to-pulmonary shunts was updated to provide a more detailed description of each condition (Table 5) [17]. 2.3.4  Schistosomiasis Schistosomiasis has been added to group 1 in the Dana Point classification (Table 3) [17]. This is a frequently occurring form of PAH in areas where the infection is endemic.

D.B. Badesch Table 4  Updated risk factors and associated conditions for pulmonary arterial hypertension (reprinted from [17] with permission from Elsevier) Possible Definite Cocaine and Phenylpropanolamine Aminorex St. John’s wort Fenfluramine Chemotherapeutic agents Dexfenfluramine SSRIs Toxic rapeseed oil Likely Amphetamines l-tryptophan Methanmphetamines

Unlikely Oral contraceptives Estrogen Cigarette smoking

Table 5  Anatomic-pathophysiologic classification of congenital systemic-to-pulmonary shunts associated with pulmonary arterial hypertension (modified from the Venice 2003 classification; reprinted from [17] with permission from Elsevier) 1. Type 1.1. Simple pre-tricuspid shunts (1) Atrial septal defect (ASD) −  Ostium secundum −  Sinus venosus −  Ostium primum (2) Total or partial unobstructed anomalous pulmonary venous return 1.2. Simple post-tricuspid shunts (1) Ventricular septal defect (VSD) (2) Patent ductus arteriosus 1.3. Combined shunts (describe combination and define ­predominant defect) 1.4. Complex CHD (1) Complete atrioventricular septal defect (2) Truncus arteriosus (3) Single ventricle physiology with unobstructed pulmonary blood flow (4) Transposition of the great arteries with VSD (without pulmonary stenosis) and/or patent ductus arteriosus (5) Other 2. Dimension (specify for each defect if more than one congenital heart defect) 2.1. Hemodynamic (specify Qp/Qs)a (1) Restrictive (pressure gradient across the defect) (2) Nonrestrictive 2.2. Anatomic (1) Small to moderate (ASD £ 2.0 cm and VSD £ 1.0 cm) (2) Large (ASD >2.0 cm and VSD > 1.0 cm) 3. Direction of shunt 3.1. Predominantly systemic-to-pulmonary 3.2. Predominantly pulmonary-to-systemic 3.3. Bidirectional 4. Associated cardiac and extracardiac abnormalities 5. Repair status 5.1. Unoperated 5.2. Palliated (specify type of operation(s), age at surgery) 5.3. Repaired (specify type of operation(s), age at surgery) a Ratio of pulmonary (Qp) to systemic (Qs) blood flow

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Table 6  Functional assessment (reprinted from [31] with permission of the American College of Chest Physicians) A. New York Heart Association functional classification Class 1 No symptoms with ordinary physical activity Class 2 Symptoms with ordinary activity. Slight limitation of activity Class 3 Symptoms with less than ordinary activity. Marked limitation of activity Class 4 Symptoms with any activity or even at rest B. World Health Organization functional assessment classification Class I Patients with PH but without resulting limitation of physical activity. Ordinary physical activity does not cause undue dyspnea or fatigue, chest pain, or near syncope Class II Patients with PH resulting in slight limitation of physical activity. They are comfortable at rest. Ordinary physical activity causes undue dyspnea or fatigue, chest pain, or near syncope Class III Patients with PH resulting in marked limitation of physical activity. They are comfortable at rest. Less than ordinary activity causes undue dyspnea or fatigue, chest pain, or near syncope Class IV Patients with PH with inability to carry out any physical activity without symptoms. These patients manifest signs of right-heart failure. Dyspnea and/or fatigue may even be present at rest. Discomfort is increased by any physical activity

2.3.5  Chronic Hemolytic Anemia A new subcategory was added to group 1 PAH in the Dana Point classification scheme for the chronic hemolytic anemias. These conditions include sickle cell disease [23, 24], thalassemia [25], hereditary spherocytosis [26], stomatocytosis, and microangiopathic hemolytic anemia.

2.3.6  PVOD and/or PCH PVOD and PCH have been placed into group 1¢ in the Dana Point classification system [17]. Fig. 1  Plexiform lesion

3  Classification by Functional Class PH can also be classified by functional class, a measure of the limits placed on a patient by the disease. It is predictive of mortality [27], and plays an important role in the selection of PAH therapy according to the treatment algorithm published by the American College of Chest Physicians Guidelines Panel [28, 29], as well as the Task Force on Diagnosis and Treatment of Pulmonary Arterial Hypertension of the European Society of Cardiology [30]. There are two widely used functional classification systems for assessing patients with PH, the New York Heart Association (NYHA) system (Table  6), and the World Health Organization (WHO) system (Table  6) [31]. The WHO system acknowledges the importance of near syncope and syncope in PAH. Syncope is generally thought to be associated with a worse prognosis, and for this reason patients with a history of syncope are generally assigned to WHO functional class IV.

4  “Classification” by Pathologic Findings A focus on the pathologic changes occurring in PH ­dominated much of the early work in the field [32]. It is now recognized that PH of differing origin may share some of the same, or similar, pathologic findings in the pulmonary arterial wall. The so-called plexiform lesion can be seen in IPAH (Fig. 1), as well as in PAH occurring in association with congenital heart disease. Concentric lesions composed of smooth muscle cells have also been described. Recognition of the potential importance of cellular proliferation and structural changes, in addition to vasoconstriction, has led to recent exploration of antiproliferative therapy for the disease.

5  Summary The classification of PH is an evolving area, adapting to advances in our understanding of the pathogenesis of the disease, as well as response to therapy. As we learn more about

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the differences in pathobiology and therapeutic ­responsiveness between various forms of PH, it is increasingly important to utilize a well-organized and structured approach to diagnosis and treatment.

References 1. Rich S, Rubin LJ, Abenhail L et al (1998) Executive summary from the World Symposium on Primary Pulmonary Hypertension (Evian, France, September 6–10, 1998). The World Health Organization, Geneva. http://www.who.int/ncd/cvd/pph.html 2. Fishman A (2001) Clinical classification of pulmonary hypertension. Clin Chest Med 22:385–391 3. Channick RN, Simonneau G, Sitbon O et al (2001) Effects of the dual endothelin-receptor antagonist bosentan in patients with ­pulmonary hypertension: a randomised placebo-controlled study. Lancet 358:1119–1123 4. Rubin LJ, Badesch DB, Barst RJ et al (2002) Bosentan therapy for pulmonary arterial hypertension. N Engl J Med 346:896–903 5. Simonneau G, Barst RJ, Galie N et al (2002) Continuous subcutaneous infusion of treprostinil, a prostacyclin analogue, in patients with pulmonary arterial hypertension: a double-blind, randomized, placebo-controlled trial. Am J Respir Crit Care Med 165:800–804 6. Olschewski H, Simonneau G, Galie N et al (2002) Inhaled iloprost for severe pulmonary hypertension. N Engl J Med 347:322–329 7. Barst RJ, Rubin LJ, Long WA et  al (1996) A comparison of ­continuous intravenous epoprostenol (prostacyclin) with conventional therapy for primary pulmonary hypertension. The Primary Pulmonary Hypertension Study Group. N Engl J Med 334:296–302 8. Badesch DB, Tapson VF, McGoon MD et  al (2000) Continuous intravenous epoprostenol for pulmonary hypertension due to the scleroderma spectrum of disease. A randomized, controlled trial. Ann Intern Med 132:425–434 9. Palmer SM, Robinson LJ, Wang A et al (1998) Massive pulmonary edema and death after prostacyclin infusion in a patient with pulmonary veno-occlusive disease. Chest 113:237–240 10. Jamieson SW, Kapelanski DP, Sakakibara N et al (2003) Pulmonary endarterectomy: experience and lessons learned in 1,500 cases. Ann Thorac Surg 76:1457–1462, discussion 1462–1454 11. Auger WR, Kim NH, Kerr KM et al (2007) Chronic thromboembolic pulmonary hypertension. Clin Chest Med 28:255–269, x 12. Hoeper MM, Kramm T, Wilkens H et al (2005) Bosentan therapy for inoperable chronic thromboembolic pulmonary hypertension. Chest 128:2363–2367 13. Lapa M, Dias B, Jardim C et al (2009) Cardiopulmonary manifestations of hepatosplenic schistosomiasis. Circulation 119:1518–1523 14. Barnett CF, Bonura EJ, Nathan SD et al (2009) Treatment of sarcoidosis-associated pulmonary hypertension: a two-center experience. Chest 135:1455–1461 15. Ito K, Ichiki T, Ohi K et al (2003) Pulmonary capillary hemangiomatosis with severe pulmonary hypertension. Circ J 67:793–795

D.B. Badesch 16. Simonneau G, Galie N, Rubin LJ et al (2004) Clinical classification of pulmonary hypertension. J Am Coll Cardiol 43:5S–12S 17. Simonneau G, Robbins IM, Beghetti M et al (2009) Updated clinical classification of pulmonary hypertension. J Am Coll Cardiol 54(Suppl 1):S43–S54 18. Machado RD, Aldred MA, James V et al (2006) Mutations of the TGF-beta type II receptor BMPR2 in pulmonary arterial hypertension. Hum Mutat 27:121–132 19. Thomson JR, Machado RD, Pauciulo MW et  al (2000) Sporadic primary pulmonary hypertension is associated with germline mutations of the gene encoding BMPR-II, a receptor member of the TGF-beta family. J Med Genet 37:741–745 20. Chaouat A, Coulet F, Favre C et al (2004) Endoglin germline mutation in a patient with hereditary haemorrhagic telangiectasia and dexfenfluramine associated pulmonary arterial hypertension. Thorax 59:446–448 21. Trembath RC, Thomson JR, Machado RD et al (2001) Clinical and molecular genetic features of pulmonary hypertension in patients with hereditary hemorrhagic telangiectasia. N Engl J Med 345:325–334 22. Chin KM, Channick RN, Rubin LJ (2006) Is methamphetamine use associated with idiopathic pulmonary arterial hypertension? Chest 130:1657–1663 23. Castro O, Hoque M, Brown BD (2003) Pulmonary hypertension in sickle cell disease: cardiac catheterization results and survival. Blood 101:1257–1261 24. Gladwin MT, Sachdev V, Jison ML et al (2004) Pulmonary hypertension as a risk factor for death in patients with sickle cell disease. N Engl J Med 350:886–895 25. Aessopos A, Stamatelos G, Skoumas V et  al (1995) Pulmonary hypertension and right heart failure in patients with beta-thalassemia intermedia. Chest 107:50–53 26. Smedema JP, Louw VJ (2007) Pulmonary arterial hypertension after splenectomy for hereditary spherocytosis. Cardiovasc J Afr 18:84–89 27. McLaughlin VV, Presberg KW, Doyle RL et al (2004) Prognosis of pulmonary arterial hypertension: ACCP evidence-based clinical practice guidelines. Chest 126:78S–92S 28. Badesch DB, Abman SH, Ahearn GS et al (2004) Medical therapy for pulmonary arterial hypertension: ACCP evidence-based clinical practice guidelines. Chest 126:35S–62S 29. Badesch DB, Abman SH, Simonneau G et al (2007) Medical therapy for pulmonary arterial hypertension: updated ACCP evidencebased clinical practice guidelines. Chest 131:1917–1928 30. Galie N, Torbicki A, Barst R et al (2004) Guidelines on diagnosis and treatment of pulmonary arterial hypertension. The task force on diagnosis and treatment of pulmonary arterial hypertension of the European society of cardiology. Eur Heart J 25:2243–2278 31. Rubin LJ (2004) Diagnosis and management of pulmonary arterial hypertension: ACCP evidence-based clinical practice guidelines. Chest 126(Suppl 1):7s–10s 32. Tuder RM, Marecki JC, Richter A, Fijalkowska I, Flores S (2007) Pathology of pulmonary hypertension. Clin Chest Med 28:23–42 33. Rich S (1998) Executive summary from the World Symposium on primary pulmonary hypertension. World Health Organization, Geneva. http://www.who.int/ncd/cvd/pph.html

Chapter 66

Epidemiology of Pulmonary Arterial Hypertension Jess Mandel and Darren B. Taichman

Abstract  The past two decades has seen remarkable advances in the understanding of the basic biology of various forms of pulmonary hypertension as well as the development of much more effective therapies. These advances have also impacted the prognosis of many patients with pulmonary hypertension; whereas pursuing an evaluation to determine the presence and cause of pulmonary hypertension was once considered a pointless endeavor with little, if any, impact on an immutably dismal outcome, a comprehensive evaluation is now the standard of care as patients have a number of therapeutic options available to them. Keywords  Incidence of pulmonary hypertension • Disease outcome • Clinical management of pulmonary hypertension

1  Introduction The past two decades has seen remarkable advances in the understanding of the basic biologic processes of various forms of pulmonary hypertension as well as the development of much more effective therapies. These advances have also impacted the prognosis of many patients with pulmonary hypertension; whereas pursuing an evaluation to determine the presence and cause of pulmonary hypertension was once considered a pointless endeavor with little, if any, impact on an immutably dismal outcome, a comprehensive evaluation is now the standard of care as patients have a number of therapeutic options available to them.

J. Mandel (*) Division of Pulmonary and Critical Care Medicine, University of California, San Diego, 9500 Gilman Drive, #0729, San Diego, CA 92093, USA e-mail: [email protected]

2  C  hanges in the Classification of the Pulmonary Hypertensive Diseases A “sclerosis of the pulmonary arteries” without apparent explanation was first documented in 1891 by Ernst von Romberg, and was again described as “cardiacos negros” in 1901 by Abel Ayerza of Argentina [1]. Thereafter, the entity was referred to as “Ayerza’s disease,” and was believed to be a consequence of luetic vasculitis. The belief in syphilis as the cause of pulmonary hypertension persisted until the 1940s, when Oscar Brenner described the histopathologic changes in the arteries of 100 patients with pulmonary hypertension, and notably did not uncover any features suggestive of syphilis as a cause. During the 1950s, when cardiac catheterization permitted elucidation of the disease’s hemodynamic abnormalities [2], Dresdale et  al. described a hypertensive vasculopathy of the pulmonary circulation. It was characterized by vasoconstriction, an elevation in pulmonary artery pressures, and a response to the injection of tolazoline, a vasodilator with both pulmonary and systemic effects [3]. As no underlying cause could be identified for the disease, it was termed “primary pulmonary hypertension.” Cases of pulmonary hypertension for which a cause could be established were thereafter labeled “secondary” pulmonary hypertension (e.g. pulmonary hypertension secondary to left ventricular failure, chronic pulmonary diseases, hypoxemia, etc.) [4]. Later, using acetylcholine, a vasodilator that is cleared exclusively within the pulmonary circulation, Paul Wood demonstrated a transient pulmonary-specific hemodynamic improvement in patients with primary pulmonary hypertension [2]. The underlying histopathologic changes were investigated by Wagenvoort and Wagenvoort, who described extensive vascular injury and remodeling which they termed “plexogenic pulmonary arteriopathy,” and believed to be pathognomonic of the disease [5]. Although the terms “primary,” “secondary,” and “plexogenic” have become a part of the familiar lexicon, an appreciation of important similarities and differences in the pathologic and clinical characteristics of differing patient groups has prompted the adoption of more precise terminology.

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In part to reach consensus on clinically useful classification schemes for the pulmonary hypertensive disorders, there have been a number of international working groups under the sponsorship of the World Health Organization (WHO) since 1973. These classification schemes have evolved as new information has emerged regarding both pathophysiologic mechanisms and clinical characteristics. Pathologic findings are no longer the key element in classification, as few of the pathologic patterns observed are truly disease specific and biopsies are now only rarely performed. The current (2008 Dana Point) approach to classification (Table 1) is based upon a hemodynamic definition of pulmonary hypertension, coupled with clinical characteristics and associated risk factors [6]. Pulmonary hypertension is deemed present when the mean pulmonary artery pressure (mPAP) exceeds 25 mmHg at rest. The presence of pulmonary arterial hypertension (PAH) further requires a normal left ventricular end-diastolic pressure directly measured or indirectly approximated by a pulmonary artery occlusion pressure (PAOP) less than 15 mmHg. Classification as PAH further requires the absence of significant left-sided heart disease, chronic respiratory disease, or thromboembolic disease, and the presence of an elevated pulmonary vascular resistance (PVR; more than 3 Wood units) [7]. It is important to distinguish between the spectrum of PAH and other causes of pulmonary hypertension (e.g. due to chronic left-sided heart or chronic respiratory disease). This distinction arises from recognition of similarities in the pathologic and clinical features of patients with various forms of PAH. This category (now termed WHO “group 1” diseases) includes patients with identifiable genetic causes of PAH (i.e. those with heritable PAH), and those with collagen vascular diseases or other conditions known to be associated with PAH (associated PAH). Patients with PAH in whom no known associated disease entity or genetic cause can be found are classified as having idiopathic PAH (IPAH), in place of the previously used term “primary pulmonary hypertension.” Abandonment of the name “primary pulmonary hypertension” is important as a means of discouraging the confusing and clinically inappropriate term “secondary pulmonary hypertension.” Use of such “primary” and “secondary” groupings inappropriately suggests similarities in the pathophysiologic mechanisms and treatment of patients with many, very different diseases. For example, patients with chronic obstructive pulmonary disease (COPD), chronic thromboembolic disease, and congenital heart disease might each be loosely labeled as having “secondary pulmonary hypertension,” although the pathogenesis is distinct in each category and the appropriate therapy is extremely different. Conceptualizing patients as having either “primary” or “secondary” disease may also obscure important clinical similarities (including appropriate treatment) between what was previously called

J. Mandel and D.B. Taichman Table 1  2008 Dana Point clinical classification of pulmonary 1 Pulmonary arterial hypertension (PAH)   1.1  Idiopathic PAH   1.2  Heritable     1.2.1  BMPR2     1.2.2 ALK1, endoglin (with or without hereditary hemorrhagic telangiectasia)     1.2.3  Unknown   1.3  Drug- and toxin-induced   1.4  Associated with     1.4.1  Connective tissue diseases     1.4.2  HIV infection     1.4.3  Portal hypertension     1.4.4  Congenital heart diseases     1.4.5  Schistosomiasis     1.4.6  Chronic hemolytic anemia   1.5  Persistent pulmonary hypertension of the newborn 1’ Pulmonary veno-occlusive disease (PVOD) and/or pulmonary capillary hemangiomatosis (PCH) 2 Pulmonary hypertension owing to left heart disease   2.1  Systolic dysfunction   2.2  Diastolic dysfunction   2.3  Valvular disease 3 Pulmonary hypertension owing to lung diseases and/or hypoxia   3.1  Chronic obstructive pulmonary disease   3.2  Interstitial lung disease   3.3 Other pulmonary diseases with mixed restrictive and obstructive pattern   3.4  Sleep-disordered breathing   3.5  Alveolar hypoventilation disorders   3.6  Chronic exposure to high altitude   3.7  Developmental abnormalities 4 Chronic thromboembolic pulmonary hypertension (CTEPH) 5 Pulmonary hypertension with unclear multifactorial mechanisms   5.1 Hematologic disorders: myeloproliferative disorders, splenectomy   5.2 Systemic disorders: sarcoidosis, pulmonary Langerhans cell histiocytosis: lymphangioleiomyomatosis, neurofibromatosis, vasculitis   5.3 Metabolic disorders: glycogen storage disease, Gaucher disease, thyroid disorders   5.4 Others: tumoral obstruction, fibrosing mediastinitis, chronic renal failure on dialysis ALK1 activin receptor-like kinase type 1; BMPR2 bone morphogenetic protein receptor type 2; HIV human immunodeficiency virus

“primary pulmonary hypertension” and other entities labeled “secondary” [e.g. patients with congenital heart disease or human immunodeficiency virus (HIV) infection]. Although understanding of the basic mechanisms which produce pulmonary hypertension remains incomplete, a number of risk factors for the development of PAH are recognized. The causal relationship of some risk factors has been firmly established by controlled epidemiology studies (e.g. exposure to fenfluramine-derived anorectic agents).

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Table 2  Risk factors for the development of pulmonary arterial hypertension [6] Definite

Possible

Aminorex Fenfluramine Dexfenfluramine Toxic rapeseed oil  

Cocaine Phenylpropanolamine St. John’s Wort Chemotherapeutic agents SSRI

Likely

Unlikely

Amphetamines Oral contraceptives l-tryptophan Estrogen Methamphetamines Cigarette smoking PAH pulmonary arterial hypertension; SSRI selective serotonin reuptake inhibitor

Others (e.g. thyroid disease) are suspected but not definitively established [8–10] and emerging information suggests additional potential risks in need of further study (e.g. observational studies suggest that the maternal use of serotonin receptor reuptake inhibitors during pregnancy may increase the risk of persistent pulmonary hypertension in newborns; these same inhibitors, however, may even be protective against the development of PAH and may even be therapeutic in adults) [11–13] (Table 2).

3  Idiopathic PAH IPAH is a rare disease with an estimated incidence of one to 3.5 cases per million individuals in industrialized countries [14–19]. The paucity of patients with IPAH and the likelihood that diverse causes might produce similar clinical syndromes have complicated descriptions of the natural history of the disease. To overcome the limitations of scattered case reports, the National Institutes of Health (NIH) established the prospective National Registry for the Characterization of Primary Pulmonary Hypertension in the early 1980s. Overall, 187 patients from 32 centers were enrolled between 1981 and 1985 [20]. The mean age of patients in the NIH registry was 36.4 years, and was similar for women and men. Women were affected more frequently, however, with a female-to-male ratio of 1.7:1 (Fig. 1). Nine percent of patients were older than 60 years. Race and ethnicity of the cohort were similar to those of the general population. Similar demographic trends have been reported in series from France, Israel, Japan, and Mexico [16, 21–23]. Dyspnea was the most common presenting symptom, and the mean time from the onset of symptoms to diagnosis among patients in the NIH registry was approximately 2 years. In two more recently reported series, 674 patients referred for treatment of PAH were enrolled in a French national

Fig.  1  Distribution of patients with idiopathic pulmonary arterial hypertension entered into the National Institutes of Health National Registry for the Characterization of Primary Pulmonary Hypertension. The patients were most often in their third and fourth decades of life, with a similar ratio of women to men (1.7:1) in all decades. Open bars represent women; shaded bars represent men. (Adapted from Rich et  al. [20] with permission from the American College of Chest Physicians)

registry between 2002 and 2003, and 578 patients were enrolled at a single referral center in the USA between 1982 and 2006 [17, 24]. Both prevalent and incident patients were evaluated, making up 18% and 82% of the study population in France and 14% and 86% in the USA, respectively. IPAH patients accounted for 39–48% of these registries and, as in prior studies, disease was seen more commonly in women. The mean age of patients with IPAH was 45 and 52 years in the USA and France, respectively, older than that seen in prior series. Although data specific to IPAH were not available, 20–25% of patients with PAH of any form in these registries were older than 60 years, and some patients were diagnosed in their eighties. Unfortunately, despite the significant advances in awareness and therapy that have occurred since the NIH registry, a significant delay in the diagnosis of IPAH was still noted. Indeed, 80% of IPAH patients had WHO functional class III or IV symptoms (Table 3) at the time of diagnosis in both registries. Exercise capacity was severely impaired in the French patients, with a mean 6-min walk distance of only 328  m, and hemodynamic values nearly identical to those of the NIH registry population. Only very small improvements in hemodynamics were seen in the US registry in comparing baseline values of patients diagnosed more recently (e.g. from 2002 to 2006) with those diagnosed before 1996; exercise capacity continued to be severely impaired. In a preliminary report from a large, multicenter US PAH registry [25], 46% of 1,226 patients had IPAH. The mean age of all patients was 53 years, and at the time of enrollment into the registry most patients had either WHO functional class III (49%) or WHO functional class IV (9%) symptoms [26].

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3.1  Prognostic Factors in IPAH The prognosis of IPAH is exceedingly poor in the absence of effective therapy. This was documented in the NIH registry, where the median survival of patients was 2.8 years, with survival rates of 68% at 1 year, 48% at 3 years, and 34% at 5 years [20]. Similar dismal outcomes have been reported in other series from various countries [27, 28], with the majority of patients dying of right-sided heart failure. Neither age nor the duration of symptoms at the time of diagnosis has been found to predict survival. Survival has been similar between men and women in studies both before and since the availability of effective therapies [29–32]. Demographic factors do not appear to be important predictors of survival. However, in a single retrospective study, nonwhite race was associated with an increased risk of death despite the use of similar medications [33]. The prognosis is generally worse as symptoms progress. In the NIH registry, the median survival of patients with milder symptoms (WHO functional classes I or II; see Table  3) was 58.6 months as compared with only 31.5 months for patients with severer symptoms (WHO functional class III). Patients with the severest functional impairment (WHO functional class IV) had a median survival of less than 6 months [20, 29]. Although the outlook has improved with the availability of effective therapy, functional status remains a highly significant prognostic variable [16, 30–34]. Indeed, in one study, the persistence of WHO functional class III or IV symptoms despite a mean of 17 months of epoprostenol therapy was associated with a markedly reduced survival [35].

Table 3  World Health Organization functional classification Class 1 Patients with pulmonary hypertension but without resulting limitation of physical activity. Ordinary physical activity does not cause undue dyspnea or fatigue, chest pain, or near syncope Class II Patients with pulmonary hypertension resulting in slight limitation of physical activity. They are comfortable at rest. Ordinary physical activity causes undue dyspnea or fatigue, chest pain, or near syncope Class III Patients with pulmonary hypertension resulting in marked limitation of physical activity. They are comfortable at rest. Less than ordinary activity causes undue dyspnea or fatigue, chest pain, or near syncope Class IV Patients with pulmonary hypertension with inability to carry out any physical activity without symptoms. These patients manifest signs of right-sided heart failure. Dyspnea and/or fatigue may even be present at rest. Discomfort is increased by any physical activity Data from [4]

J. Mandel and D.B. Taichman

Objective measurements of exercise capacity are also predictive of survival. Among 43 patients treated for the most part with parenteral or oral prostanoids, the pretreatment 6-min walk distance was independently associated with survival, which was significantly better for those who could walk farther than 332 m [36] (Fig. 2). Similarly, in randomized trials of epoprostenol therapy, patients with a lower baseline 6-min walk distance had poorer survival [32, 37]. Maximal oxygen consumption during cardiopulmonary exercise testing also correlates with survival [38]. Echocardiographic findings, including right atrial enlargement and the presence and size of a pericardial effusion, can connote a worse prognosis [39–41]. An index of right ventricular function derived by dividing the combined isovolumetric contraction and relaxation times by the right ventricular ejection time (the Tei index) is also predictive of survival, with a higher index associated with a poorer prognosis [42]. More recently, the degree of tricuspid annular displacement during systole was shown to be associated with right ­ventricular function, hemodynamic measures, as well as survival in a cohort of patients with various forms of PAH, as well as others with pulmonary hypertension associated with chronic respiratory or thromboembolic disease [43] (Fig. 3). Assessments of right ventricular size and function made with cardiac magnetic resonance imaging also have been shown to be independent predictors of outcome [44, 45]. Hemodynamic measurements have been found to predict survival in both observational and clinical trials. Despite some differences, taken in total these studies suggest that values reflecting a declining right ventricular function (e.g. an increased right atrial pressure and a decreased cardiac

Fig. 2  Survival in idiopathic pulmonary arterial hypertension according to the 6-min walk test distance. Kaplan–Meier survival curves according to the median value of distance walked in meters during a 6-min walk test. Patients unable to walk more than 332 m had a lower survival (p < 0.001). (Reprinted from Miyamoto et al. [36] with permission. The official journal of the American Thoracic Society. Copyright American Thoracic Society)

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Fig. 3  Survival according to an echocardiographic assessment of right ventricular function. The displacement from end-diastole to end-systole of the lateral tricuspid annulus (tricuspid annular plane excursion,) is shown in M-mode views from two patients, one with relatively ­preserved tricuspid excursion (and right ventricular function) (a) as compared with a patient with greater impairment (b). Kaplan–Meier

estimates of survival in a cohort of patients with either pulmonary ­arterial hypertension (idiopathic or associated with collagen vascular disease) or chronic respiratory or thromboembolic pulmonary hypertension (c, d). (Adapted from Forfia et  al. [43] with permission. The official journal of the American Thoracic Society. Copyright American Thoracic Society)

index) are associated with poorer survival [16, 22, 23, 30]. Interestingly, survival is less consistently linked with mPAPs, and both increasing and decreasing values have been associated with worsened outcomes. This reflects the natural history of right-sided heart failure in PAH: mPAP increases progressively as the vascular derangements worsen, only to decline later as the right side of the heart progressively fails and is no longer able to generate an increased pressure (Fig. 4). The levels of many serum markers are elevated in patients with IPAH and are associated with a poorer prognosis. Increasing levels of serum creatinine and decreasing levels of sodium are each associated with worsening hemodynamics and survival in patients with PAH (including those with IPAH, although not studied separately) [46, 47]. Uric acid levels are increased in hypoxic states, and the degree of increase correlates with hemodynamic and functional decline in patients with IPAH [48, 49]. Levels of B-type natriuretic peptide are also increased in patients with IPAH, reflective of

Fig. 4  Hemodynamic changes with progression of pulmonary arterial hypertension. The changes in cardiac output (CO), mean pulmonary artery pressure (PAP), and pulmonary vascular resistance in the absence of effective therapy. Pulmonary vascular resistance rises progressively as the vascular derangements progress, eventually leading to the development of right-sided heart dysfunction. Pulmonary artery pressures initially rise as the right ventricle remains capable of generating the increased pressures required to maintain a given degree of cardiac output. Note, however, that with more advanced disease cardiac output decreases and the pulmonary artery pressure falls owing to the inability of the failing right ventricle to generate increased pressures (Reproduced from Voelkel et al. [48] with permission)

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right-sided heart failure analogous to that seen in patients with left-sided heart dysfunction. In one series of 60 patients with IPAH, serum brain natriuretic peptide (BNP) concentrations were increased versus controls, and inversely correlated with both functional status and survival [50]. BNP levels declined as hemodynamic measures improved with therapy, and a persistently elevated BNP level (more than 180 pg/mL) despite therapy predicted a poorer prognosis. Elevated serum levels of N-terminal BNP similarly are associated with a poor prognosis [51]. Serum concentrations of endothelin-1, catecholamines, and atrial natriuretic peptide have also been correlated with disease severity, although none are commonly measured clinically. Increases in the levels of von Willebrand factor, D-dimer, and troponin-T or a decrease in the serum albumin level have been individually associated with poorer survival in patients with IPAH [33, 50–56]. None of these putative prognostic markers, however, are routinely incorporated into clinical decision making. Not surprisingly, the prognosis of patients with IPAH who have suffered cardiac arrest is dismal even when resuscitative efforts are initiated promptly. In one retrospective review of records from over 3,000 patients, 132 episodes of attempted cardiopulmonary resuscitation following cardiac arrest were identified. Survival at 90 days following cardiopulmonary resuscitation was only 6% [57].

J. Mandel and D.B. Taichman

forms of PAH about the expected outcomes of treatment. It is also important to acknowledge the limits in understanding the relative efficacy of available agents, as data from direct comparisons are lacking. Most often, patients treated with intravenously administered epoprostenol have been more severely ill than those treated with oral therapies, making comparisons between studies problematic. Limitations in currently employed clinical trial enrollment criteria and end points further restrict the ability to compare results across studies [58–60]. A regression equation predicting survival on the basis of hemodynamic values was derived using data from the NIH registry [22, 29] and has been used extensively to assess the efficacy of new therapies. As the prognosis in the absence of treatment is dismal, it would be unethical to include longterm, untreated control groups in clinical trials [61]. Instead, survival with new therapies is frequently compared with “expected” outcomes predicted by the NIH registry equation. Such comparisons have indicated improved survival with the use of prostanoid therapies, calcium channel antagonists, or endothelin receptor antagonists. It must be recognized, however, that the NIH registry was conducted in an era lacking effective therapy. As available therapies have since improved dramatically and specialized centers for the care of persons with pulmonary vascular disease have emerged, the NIH registry equation may no longer be an appropriate metric for predicting survival [33].

3.2  I mportant Caveats in Assessing the Impact of Therapy on Prognosis

3.3  The Impact of Therapy on Prognosis

Whereas in the past a diagnosis of IPAH was associated with an invariably dismal prognosis, therapies developed in the last 15 years have significantly improved the prognosis of the condition and yielded many long-term survivors. However, no currently available medical treatment is considered curative and lung transplantation fails to yield truly long-term survival for most patients. Although multiple studies have evaluated the survival of IPAH patients treated with newer therapies, and studies have frequently included PAH patients within the scleroderma spectrum of collagen vascular disease, information on patients with other forms of PAH is sparse. Most subjects in controlled clinical trials of treatment with calcium channel antagonists, prostanoids, endothelin receptor antagonists, or phosphodiesterase inhibitors have had IPAH. Fewer patients with either familial PAH or various forms of associated PAH have been studied; patients with PAH associated with portal hypertension have been routinely excluded from these clinical trials. This paucity of data must be kept in mind when informing patients with some

In the French national registry population from 2002 to 2003, the 1-year survival of PAH patients with IPAH, ­familial PAH, or anorexigen-associated PAH was 89.3%. Although information regarding the specific treatments that were employed was not reported, the expected survival of these patients on the basis of the NIH registry equation would have been only 71.8% [17]. Although specific rates for patients with IPAH were not available, the 1-, 3-, and 5-year survival of all PAH patients in the single-center US study was 84, 67, and 58%, respectively [24]. The long-term prognosis is much better for the small group of IPAH patients who respond acutely to the administration of short-acting pulmonary vasodilators. Testing of responsiveness is performed at the time of right-sided heart catheterization [62–68]. Although the definition of an acute response has varied in the past, currently the consensus definition of responsiveness is a decrease in the mPAP of at least 10 mmHg to a final value of less than 40 mmHg, with either an increased or an unchanged cardiac output [7, 69, 70]. In one study performed prior to the availability of other effective treatments,

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the survival rate of acutely responsive patients treated chronically with oral calcium channel antagonists was 94% at 5 years, compared with only 38% among nonresponders [71]. Unfortunately, only approximately 12% of patients in a recent series demonstrated acute vasoreactivity, and of these only about half experienced a sustained clinical response to oral calcium channel antagonist therapy. Only 4.5% of 48 patients with IPAH or familial PAH from another recent retrospective series were vasoreactive [24]. For the few acutely vasoreactive patients who do experience a sustained response to oral calcium channel antagonists, the prognosis is excellent. In one retrospective study, Sitbon et  al. found that among 38 patients with sustained clinical response (defined as being in WHO functional class I or II after 1 year of treatment), survival was 97% in up to 7 years of follow-up [72]. However, oral calcium channel antagonists are of no benefit and indeed can cause considerable harm when administered to patients who do not display acute pulmonary vasoreactivity. The prognostic significance of acute pulmonary vasoreactivity is less clear in patients treated with therapies other than oral calcium channel antagonists. The magnitude of improvements in mPAP and cardiac index at the time of acute vasodilator testing correlated with survival in one series of patients treated with long-term epoprostenol infusion; such a correlation was not seen in another group of similarly treated patients [31, 32]. One retrospective evaluation of survival among patients treated with calcium channel antagonists, prostanoids, bosentan, warfarin, or a combination of these agents found no correlation between survival and acute vasoreactivity at the baseline [33]. Little is known regarding the prognostic significance of acute vasoreactivity in nonidiopathic forms of PAH; available data suggest that few ­non-IPAH patients are acutely vasoresponsive [73–75]. Several studies have demonstrated that continuously infused epoprostenol improves survival in patients with IPAH. In the single randomized study performed, 81 IPAH patients were randomized to treatment with intravenous epoprostenol infusion or “conventional therapy” (which at the time included oral vasodilators, diuretics, cardiac glycosides, and anticoagulants). After 12 weeks, only one of the 41 epoprostenoltreated patients had required lung transplantation, whereas two of the 40 patients in the “conventional treatment” group had undergone transplantation and eight had died [37]. Additional studies have confirmed these favorable findings with epoprostenol treatment. In a cohort of 162 IPAH patients, McLaughlin et al. observed 1- and 3-year survival rates of 88% and 62% with epoprostenol infusion, compared with rates of 59% and 35% predicted by the NIH registry equation [31]. Sitbon et  al. observed remarkably similar results in 178 patients at 1 and 3 years, whereas somewhat lower impacts were documented by Kuhn et al. In each study, survival with epoprostenol therapy was improved compared

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with that predicted by the NIH registry equation [32, 35]. Indeed, although originally conceived as a “bridge” to lung transplantation, the long-term use of epoprostenol and other treatments have resulted in long-term responses that have resulted in a decrease in the demand for lung transplantation for patients with IPAH [76]. In 1998, Robbins et al. reported that more than two thirds of their patients treated with epoprostenol improved sufficiently to be removed from the waiting list for lung transplantation [77]. Despite these encouraging findings, an analysis by McLaughlin et  al. showed that approximately one third of patients from several series died within 3 years notwithstanding treatment with epoprostenol, and nearly half had died within 5 years [27]. Comparison with survival predicted by the NIH registry equation has also be used to assess the efficacy of oral therapy with the endothelin receptor antagonist bosentan. In an openlabel extension of placebo-controlled trials, survival of 169 patients with IPAH initially treated with bosentan was 96% at 1 year and 89% after 2 years [78]. In a noninferiority study comparing this same group with historical control patients treated with epoprostenol infusion, outcomes were similar at 1 and 2 years [79]. It must again be noted that, in general, patients treated with epoprostenol in clinical trials have been more ill than those enrolled into studies of oral therapies. Given the inconvenience and significant risks of continuously infused therapies, however, randomized trials comparing intravenous with oral treatments have not been performed. It is thus difficult to draw firm conclusions regarding their ­relative efficacy. Autopsy studies have revealed in situ thrombosis within venous and arterial vessels in a significant proportion of patients with PAH, suggesting a potential therapeutic role for systemic anticoagulation [76, 79, 80]. Warfarin therapy for PAH has not been studied in randomized trials, although observational reports have demonstrated an association between nonrandomized warfarin use and increased survival. As an example, in a prospective study of 64 IPAH patients treated with or without calcium channel antagonists, survival after 5 years was greater among those in either group who had received warfarin at their physician’s discretion [71]. Likewise, a retrospective cohort study of IPAH and aminorex-associated PAH patients reported that treatment with warfarin was associated with improved survival at 5 years [82]. On the basis of these data, anticoagulation with warfarin is generally recommended for patients with significant PAH in the absence of contraindications. Lung transplantation remains an important option for suitable patients with IPAH whose disease fails to respond adequately to medical therapy. Single-lung, double-lung and heart-lung transplantations have been performed, although most centers favor double-lung transplantation procedures for this indication when feasible [83]. Unfortunately, the

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reported survival of patients who undergo lung transplantation for IPAH is 66% at 1 year and 47% at 5 years [84]. Of note, advances in medical therapies have markedly reduced the need for lung transplantation [85]. Whereas 10% of all lung transplant recipients in 1990 had IPAH, more recently these patients account for only 4% of lung transplantation procedures [86]. The survival of patients undergoing lung transplantation for IPAH is comparable to that of those ­transplanted for other indications [87].

4  Familial PAH A genetic basis for the development of PAH has been suspected since a family of patients with IPAH was described by Dresdale et al. [3] in 1951. In the NIH registry, 6% of patients reported one or more affected family members [20]. Loyd and Newman showed that familial PAH has an autosomal dominant pattern of inheritance, an increased tendency for female carriers to manifest clinical disease, and an earlier onset in successive generations (a phenomenon known as genetic anticipation) [88, 89]. Mutations in the gene in the 2q33 region for a member of the transforming growth factor b (TGF-b) superfamily of receptors, bone morphogenetic protein receptor type II (BMPR2 gene), have been identified as the major cause of familial PAH [90–93]. More than 140 distinct BMPR2 mutations have been identified to date, and disease is believed to occur when haploinsufficiency results in inadequate quantities of functional protein being ­produced [94]. A penetrance of only 20% has been observed in familial PAH, suggesting that environmental factors or additional genetic factors likely contribute to disease development in persons with BMPR2 abnormalities [95–97]. Mutations in another member of the TGF-b superfamily, activin-receptor-like kinase-1, predisposes patients with hereditary hemorrhagic telangiectasia to develop PAH [98–101]. Germline mutations in BMPR2 have been identified in up to 60% of patients with familial PAH. Some patients with IPAH (nonfamilial) and other associated forms of PAH also have BMPR2 mutations [92, 93, 102–107] and common ancestries have been identified in up to 20% of patients with PAH previously assumed to be sporadic [96, 102]. Familial cases of PAH might not be recognized on account of incomplete family history taking or reporting, as well as low disease penetrance in smaller families [103, 108]. Carriers of gene mutations may be asymptomatic despite mild pulmonary hypertension documented by echocardiography [109]. However, among patients with clinically apparent PAH, the presence of BMPR2 abnormalities is associated with disease onset at an earlier age, and with severer hemodynamic derangements at the time of diagnosis [110].

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Genetic testing of family members can help assess the risk of developing PAH. There is a roughly one in five chance of PAH developing in a first-order relative who carries a disease-causing BMPR2 mutation. In the absence of genetic testing results, the risk of disease developing in the first-order relative of a patient with known familial PAH can be approximated as one in ten. When testing demonstrates an absence of disease-causing BMPR2 mutations, the risk is the same as in the general population (estimated at one to four per million) [95]. Genetic testing should only be performed in conjunction with qualified genetic counseling because of the potential interpersonal, psychological, and economic ­implications of identifying an at-risk genotype.

5  PAH Associated with a Specific Condition 5.1  Collagen Vascular Diseases Among the identified risk factors for the development of PAH, the presence of systemic sclerosis is the most commonly reported. In the French national registry, 76% of PAH patients with collagen vascular disease had scleroderma, accounting for 11.6% of all patients enrolled [17]. A Scottish registry estimated the incidence of PAH associated with collagen vascular diseases to be 2.8 annual cases per million population, making it slightly more common than IPAH [18]. Patients with PAH associated with collagen vascular disease tend to be older and have a lower cardiac output at the time of diagnosis than patients with IPAH [111]. PAH occurs most often in patients with limited disease or the CREST syndrome (calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasia). Estimates have varied significantly, but when assessed by right-sided heart catheterization, PAH has been documented in between 7 and 29% of patients [112]. When screening of symptomatic patients was performed by echocardiogram, approximately 13% of patients with systemic sclerosis were found to have pulmonary hypertension in each of two large series [113, 114]. In a cross-sectional analysis of 654 patients with systemic sclerosis evaluated at multiple centers in the Netherlands, approximately 5% had PAH [115]. PAH appears to occur within 5 years of the diagnosis of systemic sclerosis in approximately half of patients [116]. The prognosis of patients with scleroderma and PAH is worse than even that of those scleroderma patients who develop severe pulmonary fibrosis. Median survival of patients with scleroderma and PAH is approximately 1 year, versus 3 years among those with scleroderma and pulmonary fibrosis alone [117, 118]. Even when similar therapies are used (including epoprostenol, bosentan, sildenafil, calcium channel antagonists, and warfarin) survival of patients with PAH associated

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Fig.  5  Survival in pulmonary arterial hypertension associated with systemic sclerosis as compared with survival in idiopathic pulmonary arterial hypertension (IPAH). Kaplan–Meier estimates of survival in patients with pulmonary arterial hypertension associated with the systemic sclerosis spectrum of diseases (SS-PAH) and idiopathic pulmonary arterial hypertension. (Reproduced from Kuwat et al. [117] with permission)

with systemic sclerosis is less favorable than for patients with IPAH [117, 119] (Fig. 5). As an example, whereas hemodynamic values and exercise capacity improve when patients with IPAH are treated with bosentan, the benefit observed among similarly treated patients with scleroderma appears to be primarily a slowing in the rate of deterioration [120]. PAH also occurs in patients with other forms of collagen vascular disease, including systemic lupus erythematosis, rheumatoid arthritis, Takayasu’s arteritis, Sjögren’s ­syndrome, polymyositis, and dermatomyositis. In most studies, pulmonary artery pressures have been estimated by echocardiogram, and thus the true prevalence of PAH is not known. Pulmonary hypertension has been identified by echocardiography in approximately 10% of patients with systemic lupus erythematosis, and in up to 43% of patients who are followed prospectively [121–125]. Estimates are similarly broad among patients with mixed connective tissue disease, and again lack confirmation by catheterization in most studies. Regardless of the exact frequency, however, the presence of pulmonary hypertension appears to be a significant cause of death in these patients. Accordingly, the possibility of pulmonary hypertension should be considered in all collagen vascular disease patients with exertional dyspnea or other symptoms, and early evaluation is warranted.

5.2  Human Immunodeficiency Virus Infection with HIV increases the risk of developing PAH by unclear mechanisms and cannot be explained by hepatitis C or human herpesvirus 8 co-infection, or a history of stimulant abuse [126, 127]. The estimated prevalence of PAH among HIV-infected patients is 0.5% [126].

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In one large Swiss cohort of HIV-positive patients, the annual incidence of pulmonary hypertension appears to be declining, having peaked at 0.24% in 1993 and decreased to 0.02% in 2001. This reduction has coincided with the introduction of highly active antiretroviral therapies and it has been postulated that better control of HIV infection might decrease the risk of developing PAH [128]. In contrast, a prospective study of over 7,000 HIV-positive adults in France found no change in the prevalence of PAH (approximately 0.5%) between the early 1990s and 2008, when the antiretroviral management of patients changed profoundly [129]. Whether therapy for HIV infection in patients with established PAH alters the course of their vascular disease remains unknown, although some cases of regression on therapy have been reported [130]. The symptoms, hemodynamic findings, and survival of patients with PAH associated with HIV are similar to those of patients with IPAH [131]. Like the prognosis of patients with IPAH, the prognosis of patients with PAH associated with HIV infection is worse when symptoms are more advanced (e.g. WHO functional class III or IV versus either WHO functional class I or WHO functional class II). A CD4 lymphocyte count below 212/mm3 is also associated with a poorer prognosis [132]. Mortality is more often directly attributable to PAH and right-sided heart failure than to infectious complications [131, 132].

5.3  Portal Hypertension Patients with chronic liver disease are at risk for the ­development of pulmonary complications. When portal hypertension and PAH are present, the combination is referred to as “portopulmonary hypertension” (POPH) [133]. POPH involves vascular derangements that increase PVR to produce an increased mPAP, as opposed to increased pulmonary pressures due solely to the elevated cardiac output that frequently accompanies chronic liver disease. The cause of pulmonary hypertension in these patients may be difficult to unravel as the high cardiac output state may precede, or accompany, the development of POPH. The severity of underlying liver disease or portal hypertension does not appear to correlate with the severity of PAH [134, 135]. Despite similar degrees of clinical impairment, most patients with POPH may manifest numerically smaller increases in PVR or decreases in cardiac output than those with IPAH. The frequency of PAH in patients with liver disease has not been established. Estimates have ranged from 0.73% in 1,241 autopsies from patients with cirrhosis, to 16% of 62 patients undergoing right-sided heart catheterization during evaluation for transjugular intrahepatic portosystemic shunting

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[136, 137]. In one series of patients with advanced liver disease and symptoms evaluated for liver transplantation, the prevalence of POPH was 8.5% [138]. The risk factors for the development of POPH among patients with liver disease have not been established. In a case-control study of patients evaluated at tertiary care centers, women and patients with autoimmune hepatitis appeared to be at increased risk for the development of POPH [135]. Genetic evaluation of patients from this same cohort identified an increased risk of POPH associated with several polymorphisms for genes involved in estrogen signaling [139]. The prognosis of POPH is poor; in one retrospective series of 78 patients, the mean survival was only 15 months [140]. Little is known regarding the impact of current therapies on survival in POPH, as patients with POPH have been excluded from almost all of the major prospective clinical treatment trials. However, even with the use of such therapies, survival appears to be worse for patients with POPH than for those with IPAH. In a retrospective cohort of 13 patients with POPH, survival at 1 and 3 years was 85% and 38% as compared with 82% and 72% in 33 patients with IPAH [141] (Fig. 6). Many patients with POPH and advanced hepatic dysfunction require liver transplantation. The presence of PAH, however, increases the perioperative mortality, and a mPAP above 50 mmHg is considered a contraindication to liver transplantation at most centers [142, 143]. Although therapy to lower mPAP has permitted successful orthotopic liver transplantation in some patients [144–149], the pulmonary vascular abnormalities of PAH are not consistently reversed by liver transplantation. Although some instances of reversal have been reported, in other patients POPH has progressed despite transplantation [150].

Fig. 6  Survival in patients with pulmonary arterial hypertension associated with chronic liver disease and portal hypertension. Kaplan–Meier estimates of survival in patients with portopulmonary hypertension as compared with those of patients with idiopathic pulmonary arterial hypertension. (Reproduced from Kawut et al. [141] with permission)

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5.4  Anorectic and Stimulant Agents The ingestion of certain anorexigens can lead to the ­development of PAH, possibly by increasing blood levels of the vasoconstrictor serotonin. An epidemic of PAH erupted in Switzerland, Austria, and Germany between 1966 and 1968, when the incidence increased 20-fold following the introduction in those countries of the appetite-depressant aminorex fumarate [151]. Although only 2% of those exposed to the drug developed PAH, the relative risk compared with unexposed individuals was 52:1 [152]. The use of fenfluramine derivatives was similarly associated with the development of PAH both in Europe and in North America [153]. In a registry of 95 PAH patients in Europe, the odds ratio of PAH was 6.3 after any anorectic agent use, and 23.8 when anorectic agents were taken for longer than 3 months [14]. Likewise, a North American registry of 579 patients reported that the use of fenfluramine was strongly associated with the development of PAH (odds ratio of 7.5 when taken for more than 6 months). The study also identified a high frequency of anorectic agent use among patients with other associated forms of PAH (e.g. collagen vascular disease), suggesting these drugs might ­precipitate disease when combined with other risks [154]. A different series identified BMPR2 mutations among 22% of 40 patients with fenfluramine-related PAH, suggesting the importance of genetic predisposition to this disorder [155]. In a series of 62 patients evaluated over a 10-year period at a single center in France, the interval between the initial exposure to fenfluramine and the development of dyspnea was approximately 4 years [74]. In the multicenter French national registry from 2002 to 2003, anorexigen-associated PAH was diagnosed within 2 years of drug exposure in 24% of cases, between 2 and 5 years in 32% of cases, and after more than 5 years following drug exposure in 44% of cases [17]. In both series, the baseline hemodynamic values were similar to those of patients with IPAH, although patients exposed to anorectic agents were even less likely to demonstrate acute vasoreactivity. The prognosis of anorectic-agent-associated PAH has not been well established. Data comparing survival with survival of IPAH patients are conflicting. Survival of aminorex-­associated PAH patients was better than that of those with IPAH in a retrospective study of 104 patients treated with anticoagulation [82]. With additional therapies, including epoprostenol, survival in fenfluramine-exposed patients with PAH appeared to be similar to that of IPAH patients in one study [74] yet poorer in another series in which patients were matched according to treatments and disease severity [156]. Although there are fewer data regarding an association between illicit stimulant use than between anorectic drug use

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and PAH, cocaine, amphetamine, and methamphetamine are generally considered to be risk factors for the development of PAH as well. Most of the data supporting an association are in the form of case reports and case series, although one case-control study found that patients with IPAH were over 10 times as likely to have a history of illicit stimulant use than PAH patients with known risk factors [157, 158]. The mechanism by which these agents predispose to PAH is not known, but may relate to direct endothelial injury or ­dysregulation of the mediators of vascular tone.

5.5  Hemoglobinopathies The risk of developing pulmonary hypertension is increased in patients with certain chronic hemolytic disorders [160]. Hemolytic states might lead to PAH by releasing both free hemoglobin and arginase into the plasma, where they can reduce the bioavailability of both nitric oxide and its substrate, l-arginine [159–161]. Prevalence estimates of pulmonary hypertension in patients with either sickle cell disease or thalassemia have varied significantly, according to the age of the population studied, the severity of symptoms, and the method of pulmonary artery pressure assessment used [162–165]. Echocardiographic estimates in 195 adults patients with sickle cell disease reported pulmonary hypertension in 32%; however, the tricuspid regurgitation gradient used to define pulmonary hypertension was 2.5  m/s, less than the tricuspid regurgitation gradient of more than 3.0 m/s frequently used to suggest the presence of pulmonary hypertension [166]. Pathologic findings consistent with PAH were noted in 15 of 20 autopsies performed on patients with sickle cell disease [167]. Isolated cases of pulmonary hypertension with other chronic hemolytic disorders have been reported, including patients with hereditary spherocytosis and paroxysmal nocturnal hemoglobinuria [168, 169]. Factors which predispose patients with sickle cell disease to PAH, other than the magnitude of hemolysis, have not been well defined [170]. One study suggested that single nucleotide polymorphisms in genes encoding for the activin A receptor, type II-like 1 (ACVRL1), bone morphogenetic protein 6 (BMP6), and the b1 adrenergic receptor (ADRB1) are associated with the development of PAH in this population [171]. Elevated platelet counts and lower oxygen saturation have also been implicated as risk factors [172]. Patients with PAH associated with sickle cell disease tend to have lower mPAPs and higher cardiac outputs than patients with IPAH or other forms of associated PAH. Furthermore, patients with PAH associated with hemoglobinopathies also have hemodynamic findings consistent with both intrinsic

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pulmonary vascular disease (i.e. an increased PVR) and increased PAOPs, suggesting the possibility that left ventricular diastolic dysfunction also plays a role. As an example, in one study of 20 patients with pulmonary hypertension associated with sickle cell anemia, the mPAP was 36 mmHg, cardiac output was 8.6 L/min, and PAOP was 16 mmHg; half of the patients had PAOP values greater than 15 mmHg and most had PVR less than 3 Wood units [173]. The presence of pulmonary hypertension portends a worse prognosis in patients with sickle cell disease. Mortality within 2–2.5 years of almost 50% of sickle cell patients with pulmonary hypertension has been reported in several series [173–175]. In a prospective NIH registry of 195 adult patients with sickle cell anemia followed for a mean of 18 months, Gladwin et al. found mortality increased when the tricuspid regurgitant velocity was greater than 2.5  m/s (relative risk 10.1), versus when the pressures were lower [166] (Fig. 7). Markers of hemolysis, such as plasma arginase, correlated not only with the presence of pulmonary hypertension, but also with an increased risk of death [160]. The impact of therapy for pulmonary hypertension on patients with sickle cell disease (or other forms of hemolytic anemia) has not been studied in a systematic fashion. As ongoing hemolysis appears to contribute to the increased mortality associated with pulmonary hypertension in these patients, therapy to minimize hemolysis is believed to be important in improving survival [160, 166]. Although the use of PAH-directed therapies has been reported in patients with hemolytic anemias [173, 175, 177], their impact upon survival has not been adequately studied.

Fig.  7  Survival in patients with sickle cell anemia and pulmonary hypertension. Kaplan–Meier estimates of survival as affected by the presence of pulmonary hypertension as assessed by echocardiogram (defined as a tricuspid regurgitant velocity greater than 2.5 m/s. Values below lines represent the number of patients at risk at the time of each death for both groups. (Reprinted with permission from Gladwin et al. [166]. Copyright 2004 Massachusetts Medical Society. All rights reserved)

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5.6  Pulmonary Veno-Occlusive Disease Pulmonary veno-occlusive disease (PVOD) is a rare form of pulmonary hypertension that is characterized by extensive and diffuse obliteration of small pulmonary veins or venules by cellular proliferation, in situ thrombosis, and fibrous tissue [178, 179]. In most patients, pulmonary arteriolar changes accompany the venous changes, although it is unclear if arteriolar remodeling develops simultaneously with or as a consequence of progressive venous destruction [180]. Capillary proliferation (findings used to diagnose capillary hemangiomatosis) has also been seen frequently in pathologic samples containing changes consistent with PVOD, raising the possibility that these are not distinct clinical entities [181]. PVOD usually presents with dyspnea on exertion and ­findings of right ventricular failure in a manner similar to IPAH, although subtle differences from IPAH may be present, such as digital clubbing, basilar rales, pleural effusions, and radiographic evidence of pulmonary lymphatic engorgement (e.g. Kerley B lines visible on chest radiographs and septal thickening on chest CT) [178, 182, 183]. Differentiation from other forms of PAH is important because treatment with prostanoids or other agents that are effective for IPAH may produce pulmonary edema and death in a subset of PVOD patients [184]. Partially because of these difficulties in treatment, the prognosis of PVOD is very poor, with most patients dying within 2 years of diagnosis unless long-term treatment with prostanoids is tolerated [179, 182, 185]. Immediate consideration of lung transplantation is thus encouraged when the diagnosis of PVOD is established, although recurrence of the condition after transplantation has been reported [186]. The epidemiologic factors of PVOD are not clearly defined. Unlike IPAH, men and women appear equally at risk for PVOD, and the age at diagnosis has ranged from the first weeks through the seventh decade of life [179, 187–189]. Because PVOD is approximately one tenth as common as IPAH, the annual incidence of PVOD in the general population has been estimated at 0.1–0.4 cases per million persons per year [179]. However, this may represent an underestimate because many cases are probably misclassified as IPAH, heart failure, or interstitial lung disease. No well-designed studies have adequately examined the magnitude of this likely misclassification phenomenon. The cause of PVOD remains unknown and likely represents a final common clinicopathologic pathway that develops in a susceptible host following exposure to one of a number of different triggers. BMPR2 mutations have been described in a small number of patients [107, 189]. A number of factors associated with PVOD have been proposed but none of these epidemiologic hypotheses have been tested by rigorous methods. Conditions theorized to be associated

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with an increased risk of PVOD include the development of the condition in a sibling, exposure to antineoplastic chemotherapy, infection with HIV or other agents, and the presence of thrombophilia or autoimmune diseases [80, 178, 190–197]. Tobacco exposure has been reported to be significantly more common in patients with PVOD than in patients with IPAH [189].

5.7  Schistosomiasis Schistosomiasis is estimated to affect more than 200 million persons worldwide, ranking it as the third most common endemic parasitic disease [198]. The percentage of patients with the disease who develop PAH is unclear. One study of 65 Brazilian patients with hepatosplenic schistosomiasis documented PAH by invasive hemodynamic measurements in five individuals (7.7%) [199]. Given the enormous population potentially at risk for PAH due to schistosomiasis, it is vitally important to understand in greater detail the strength of this risk factor, the chronology of PAH in this setting, and the optimal management of the condition after eradication of the parasite in affected individuals.

6  E  pidemiologic Features of Certain NonPAH Forms of Pulmonary Hypertension 6.1  Chronic Respiratory Disease Parenchymal lung disease and hypoxemia are relatively common conditions and thus constitute a large proportion of conditions of all patients with pulmonary hypertension [200]. In most cases, hypoxia is thought to produce pulmonary hypertension via hypoxic vasoconstrictive mechanisms, as well as indirectly by stimulating vascular remodeling by changing the balance of endogenous vasoregulators [201]. Patients with abnormalities in BMPR2 or serotonin reuptake transporter proteins may be at particular risk for developing pulmonary hypertension in the setting of hypoxia [96]. COPD is epidemiologically the most important cause of pulmonary hypertension associated with respiratory disease. Between 12 million and 24 million individuals in the USA suffer from COPD, and it is estimated that some degree of pulmonary hypertension is present in 20–35% of all patients [202–204]. In most patients with COPD, the magnitude of pulmonary hypertension is mild (mPAP 35–40 mmHg), and correlates with the degree of hypoxemia, and to a lesser degree, airflow obstruction [205, 206]. However, a very small

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proportion of COPD patients develop pulmonary hypertension that is severer and is out of proportion with their airflow and blood gas abnormalities [207, 208]. Progression of pulmonary hypertension in patients with COPD at a rate of approximately 0.5  mmHg in mPAP per year has been described [204, 209]. Pulmonary hypertension is associated with decreased survival in COPD. As an dramatic example, a 1971 study reported the 5-year survival rate in patients with COPD was less than 10% when the mPAP exceeded 45 mmHg but was 90% when the mPAP was less than 25 mmHg [210]. Longterm oxygen therapy is the mainstay of treatment, with benefits in hemodynamics and mortality described in COPD patients in whom PaO2 is maintained above 55  mmHg (or greater than 60 mmHg if evidence of pulmonary hypertension is present.) [211–213]. Clinical trials are ongoing, but at present, there is no established role for prostanoids, endothelin receptor antagonists, or phosphodiesterase inhibitors in the treatment of COPD-associated pulmonary hypertension [200]. In a small randomized controlled trial of patients with mild to moderate pulmonary hypertension as estimated by echocardiogram, treatment with bosentan did not improve exercise capacity, but was associated with worsened oxygenation and health-related quality of life [214]. Obstructive sleep apnea (OSA) is another common condition and results in disordered sleep, hypoventilation, and chronic hypoxemia [215]. It is estimated that in the USA approximately three million men and 1.5 million women suffer from the disorder, and that approximately 20% of these individuals have pulmonary hypertension [200, 216]. In patients with concomitant lung disease or extreme obesity, the prevalence of pulmonary hypertension is increased further. In most cases, pulmonary hypertension associated with OSA is mild, with mPAP remaining below 35 mmHg [217]. Therapy is based upon relieving sleep disordered breathing and hypoxia, usually by employing continuous positive airway pressure and supplemental oxygen as necessary. Improvements in pulmonary hemodynamics following the treatment of OSA have been observed in a number of studies [218, 219]. Interstitial lung disease also may result in pulmonary hypertension, likely via both hypoxic mechanisms and ­compromise of the pulmonary vascular bed by interstitial fibrosis. Pulmonary endothelial cell dysfunction also has been implicated in the pathogenesis of pulmonary hypertension in this setting [220]. Unlike COPD, moderate to severe pulmonary hypertension is not unusual, and may occur in most patients with advanced interstitial lung disease, regardless of its pathologic subtype [200, 220]. The contribution of pulmonary hypertension to the patient’s impaired functional status may be difficult to differentiate from that of restrictive ventilatory physiology, anemia, or muscle weakness related to glucocorticoid therapy. Interstitial lung disease and pulmonary hypertension coexist in up to 30% of patients with progressive

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systemic sclerosis; the incidence and prevalence of pulmonary hypertension in other types of interstitial lung disease are not well characterized [221]. A negative association of pulmonary hypertension with survival has been documented most thoroughly in patients with the scleroderma spectrum of diseases, in sarcoidosis, and in idiopathic pulmonary fibrosis [222–224]. In treating pulmonary hypertension associated with interstitial lung disease, correction of hypoxemia is essential. Clinical trials of prostanoids, endothelin receptor antagonists, or phosphodiesterase inhibitors in patients with significant interstitial lung disease are ongoing.

6.2  C  hronic Thromboembolic Pulmonary Hypertension Chronic thromboembolic pulmonary hypertension (CTEPH) is a clinicopathologic entity characterized by progressive pulmonary hypertension and right ventricular failure in the presence of large pulmonary arteries occluded by intraluminal fibrous material that is consistent with organized thromboemboli. The magnitude of hemodynamic abnormalities frequently exceeds the observed degree of large vessel obstruction, suggesting that diffuse small vessel pulmonary vascular remodeling plays a major role in disease progression. It is generally accepted that the condition results from single or multiple episodes of acute pulmonary thromboembolism that fail to resolve normally via endogenous fibrinolytic mechanisms [225]. Treatment ideally involves surgical pulmonary thromboendarterectomy, with medical therapy playing an adjunctive role in patients who are not surgical candidates, or in patients who fail to improve optimally following the procedure [226]. Between 0.01% and 3.1% of survivors of an episode of acute pulmonary thromboembolism appear to develop CTEPH [227, 228]. Among patients with massive pulmonary embolism, the rate of progression to CTEPH appears significantly higher. As an example, one study of 227 patients with acute pulmonary embolism and obstruction of at least 50% of the pulmonary vascular bed found that 20% developed CTEPH during a 4–8-year follow-up period [229]. It is unclear what mechanisms are the culprit in the minority of patients with acute pulmonary embolism who develop CTEPH. The only thrombophilic abnormality that has consistently been overrepresented among CTEPH patients is the presence of antiphospholipid antibodies, reported in 10–20% of CTEPH patients [226, 230, 231]. Most studies have not found abnormalities of coagulation or fibrinolysis, such as factor V Leiden or deficiencies of protein C, protein S, or antithrombin III, to be more common in patients with CTEPH than in the general population, although more recently, potential roles for elevated factor VIII or homocysteine levels have been suggested in small series [232–234].

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7  Conclusion The epidemiology of PAH is changing. Remarkable advances in understanding the pathobiology and clinical care in PAH have resulted in improved quality of life and survival. Despite such important progress, however, neither exercise capacity nor survival is normal. Controlled studies have only just begun to evaluate the role of combinations of therapies, as well as the utility of genetic and other biomarkers to predict a given patient’s response. Such studies will hopefully allow for better targeting of therapy to individual patients with PAH. They may also lead to improvements in the care of patients with other forms of pulmonary hypertension. Expanding therapeutic options might also allow consideration of end points beyond physical function and survival, such as health-related quality of life, when planning care [235–241].

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66  Epidemiology of Pulmonary Arterial Hypertension 198. Butrous G, Ghofrani HA, Grimminger F (2008) Pulmonary vascular disease in the developing world. Circulation 118:1758–1766 199. Lapa M, Dias B, Jardim C et al (2009) Cardiopulmonary manifestations of hepatosplenic schistosomiasis. Circulation 119:1518–1523 200. Gerke A, Mandel J (2006) Pulmonary hypertension secondary to chronic respiratory disease. In: Mandel J, Taichman DB (eds) Pulmonary vascular disease. Elsevier, Philadelphia 201. Shimoda L, Sham J, Sylvester J (2000) Altered pulmonary vasoreactivity in the chronically hypoxic lung. Physiol Res 49:549–560 202. Burrows B, Kettel LJ, Niden AH, Rabinowitz M, Diener CF (1972) Patterns of cardiovascular dysfunction in chronic obstructive lung disease. N Engl J Med 286:912–918 203. Weitzenblum E, Hirth C, Ducolone A, Mirhom R, Rasaholinjanahary J, Ehrhart M (1981) Prognostic value of pulmonary artery pressure in chronic obstructive pulmonary disease. Thorax 36:752–758 204. Weitzenblum E, Sautegeau A, Ehrhart M, Mammosser M, Hirth C, Roegel E (1984) Long-term course of pulmonary arterial pressure in chronic obstructive pulmonary disease. Am Rev Respir Dis 130:993–998 205. Doi M, Nakano K, Hiramoto T, Kohno N (2003) Significance of pulmonary artery pressure in emphysema patients with mild-tomoderate hypoxemia. Respir Med 97:915–920 206. Chin KM, Rubin LJ (2008) Pulmonary arterial hypertension. J Am Coll Cardiol 51:1527–1538 207. Chaouat A, Naeije R, Weitzenblum E (2008) Pulmonary hypertension in COPD. Eur Respir J 32:1371–1385 208. Thabut G, Dauriat G, Stern JB, Logeart D, Lévy A, MarrashChahla R, Mal H (2005) Pulmonary hemodynamics in advanced COPD candidates for lung volume reduction surgery or lung transplantation. Chest 127:1531–1536 209. Kessler R, Faller M, Weitzenblum E et al (2001) "Natural history" of pulmonary hypertension in a series of 131 patients with chronic obstructive lung disease. Am J Respir Crit Care Med 164:219–224 210. Bishop J (1971) Role of hypoxia in the pulmonary hypertension of chronic bronchitis and emphysema. Scand J Respir Dis Suppl 77:61–65 211. Nocturnal Oxygen Therapy Trial Group (1980) Continuous or nocturnal oxygen therapy in hypoxemic chronic obstructive lung disease: a clinical trial. Ann Intern Med 93:391–398 212. (1981) Long term domiciliary oxygen therapy in chronic hypoxic cor pulmonale complicating chronic bronchitis and emphysema. Report of the Medical Research Council Working Party. Lancet 1:681–686 213. Zielinski J, Tobiasz M, Hawrylkiewicz I, Sliwinski P, Palasiewicz G (1998) Effects of long-term oxygen therapy on pulmonary hemodynamics in COPD patients: a 6-year prospective study. Chest 113:65–70 214. Stolz D, Rasch H, Linka A, Di Valentino M, Meyer A, Brutsche M, Tamm M (2008) A randomised, controlled trial of bosentan in severe COPD. Eur Resp J 32:619–628 215. White D (2006) Sleep apnea. Proc Am Thorac Soc 3:124–128 216. Namen A, Dunagan D, Fleischer A et al (2002) Increased physician-reported sleep apnea: the National Ambulatory Medical Care Survey. Chest 121:1741–1747 217. Laks L, Lehrhaft B, Grunstein RR, Sullivan CE (1995) Pulmonary hypertension in obstructive sleep apnoea. Eur Respir J 8:537–541 218. Somers VK, White DP, Amin R et al (2008) Sleep apnea and cardiovascular disease: an American Heart Association/American College of Cardiology Foundation Scientific Statement from the American Heart Association Council for High Blood Pressure Research Professional Education Committee, Council on Clinical Cardiology, Stroke Council, and Council on Cardiovascular Nursing. J Am Coll Cardiol 52:686–717 219. Atwood CW Jr, McCrory D, Garcia JG, Abman SH, Ahearn GS; American College of Chest Physicians (2004) Pulmonary artery hypertension and sleep-disordered breathing: ACCP evidencebased clinical practice guidelines. Chest 126:72S–77S

961 220. Ryu JH, Krowka MJ, Pellikka PA, Swanson KL, McGoon MD (2007) Pulmonary hypertension in patients with interstitial lung diseases. Mayo Clin Proc 82:342–350 221. Ungerer R, Tashkin D, Furst D et al (1983) Prevalence and clinical correlates of pulmonary arterial hypertension in progressive systemic sclerosis. Am J Med 75:65–74 222. Shorr A, Davies D, Nathan S (2003) Predicting mortality in patients with sarcoidosis awaiting lung transplantation. Chest 124:922–928 223. Trad S, Amoura Z, Beigelman C et  al (2006) Pulmonary arterial hypertension is a major mortality factor in diffuse systemic sclerosis, independent of interstitial lung disease. Arthritis Rheum 54:184–191 224. Lettieri CJ, Nathan SD, Barnett SD, Ahmad S, Shorr AF (2006) Prevalence and outcomes of pulmonary arterial hypertension in advanced idiopathic pulmonary fibrosis. Chest 129:746–752 225. Fedullo P, Rubin L, Kerr K, Auger W, Channick R (2000) The natural history of acute and chronic thromboembolic disease: the search for the missing link. Eur Respir J 15:435–437 226. Chin K, Fedullo PF (2006) Chronic thromboembolic pulmonary hypertension. In: Mandel J, Taichman DB (eds) Pulmonary vascular disease. Elsevier, Philadelphia 227. Moser K, Auger W, Fedullo P (1990) Chronic major-vessel thromboembolic pulmonary hypertension. Circulation 81:1735–1743 228. Pengo V, Lensing AW, Prins MH et al (2004) Incidence of chronic thromboembolic pulmonary hypertension after pulmonary embolism. N Engl J Med 350:2257–2264 229. Liu P, Meneveau N, Schiele F, Bassan JP (2003) Predictors of long-term clinical outcome of patients with acute massive pulmonary embolism after thrombolytic therapy. Chin Med J (Engl) 116:503–509 230. Auger W, Permpikul P, Moser K (1995) Lupus anticoagulant, heparin use, and thrombocytopenia in patients with chronic thromboembolic pulmonary hypertension: a preliminary report. Am J Med 99:392–396 231. Wolf M, Boyer-Neumann C, Parent F et al (2000) Thrombotic risk factors in pulmonary hypertension. Eur Respir J 15:395–399 232. Bonderman D, Turecek P, Jakowitsch J et al (2003) High prevalence of elevated clotting factor VIII in chronic thromboembolic pulmonary hypertension. Thromb Haemost 90:372–376 233. Colorio CC, Martinuzzo ME, Forastiero RR, Pombo GA, Adamczuk Y, Carreras LO (2001) Thrombophilic factors in chronic thromboembolic pulmonary hypertension. Blood Coagul Fibrinolysis 12:427–432 234. Highland KB, Strange C, Mazur J, Simpson KN (2003) Treatment of pulmonary arterial hypertension: a preliminary decision analysis. Chest 124:2087–2092 235. Hoeper MM, Oudiz RJ, Peacock A et al (2004) End points and clinical trial designs in pulmonary arterial hypertension: clinical and regulatory perspectives. J Am Coll Cardiol 43:48S–55S 236. Rubin LJ, Galie N (2004) Pulmonary arterial hypertension: a look to the future. J Am Coll Cardiol 43:89S–90S 237. Taichman DB, Christie J, Biester R et  al (2005) Validation of a brief telephone battery for neurocognitive assessment of patients with pulmonary arterial hypertension. Respir Res 6:39 238. Taichman DB, Shin J, Hud L et al (2005) Health-related quality of life in patients with pulmonary arterial hypertension. Respir Res 6:92 239. White J, Hopkins RO, Glissmeyer EW, Kitterman N, Elliott CG (2006) Cognitive, emotional, and quality of life outcomes in patients with pulmonary arterial hypertension. Respir Res 7:55 240. Roberts K, Preston I, Hill NS (2006) Pulmonary hypertension trials: current end points are flawed, but what are the alternatives? Chest 130:934–936 241. Zlupko M, Harhay MO, Gallop R et  al (2008) Evaluation of disease-specific health-related quality of life in patients with pulmonary arterial hypertension. Resp Med 102:1431–1438

Chapter 67

Air Pollution and the Pulmonary Vasculature Melissa L. Bates and Rebecca Bascom

Abstract  Particulate air pollution is associated with ­ ortality of many cardiorespiratory disease. This implaum sible association, is now well supported by epidemiology studies, as is discussed in this chapter. There is now additional evidence for morbidity from asthma, COPD, ischemic heart disease, and congestive heart failure. We now know that ultrafine particles escape detection by the macrophage defense system and translocate to the circulation. We know that, in vitro, air pollution activates endothelin-1 (ET-1), a key regulator of the pulmonary circulation. The recommended chapter outline provided by the editors points to gaps in our knowledge: Sections on clinical manifestations, complications, diagnosis, treatment and prevention, and prognosis are meager because no coherent body of information yet exists. The fragmentary evidence presented herein is intriguing in its suggestion that common air pollutants target the pulmonary circulation and the right side of the heart. The data are by no means robust. A key purpose of this chapter is to challenge pulmonary vascular biologists and clinicians to reconsider the hypothesis that ambient air pollution causes or exacerbates pulmonary vascular disease. This chapter will discuss the effects of reactive gas and particulate pollution on the pulmonary circulation, creating a link between epidemiological findings in humans and experimental findings from controlled exposure studies in human and animal models. It will discuss the pulmonary circulation as a system directly affected by pollutants, as a point of entry into the systemic circulation, and as a biochemically active entity whose mediators exert both local and systemic effects after exposure. Keywords  Epidemiology • Pollutants • Pulmonary vessels • Pulmonary circulation • Reactive gas

M.L. Bates (*) Department of Pediatrics, University of Wisconsin, 2000 Observatory Drive, #2039, Madison, WI 53706, USA e-mail: [email protected]

1  Introduction In 1987, Lewis Rubin cheerfully informed me (Rebecca Bascom) that air pollution was irrelevant to diseases of the pulmonary circulation, and I concurred. Rubin was then Pulmonary Division Chief at the University of Maryland and was recruiting me to study air pollution health effects at University of Maryland’s Environmental Research Facility. At the time, our areas of scientific interest seemed worlds apart. He was trying to save the lives of anorexigen users with end-stage idiopathic pulmonary arterial hypertension at a time when prostacyclin trials were just beginning. I was inviting healthy young adults to take a virtual trip to southern California (ozone), or a smoke-filled tavern (environmental tobacco smoke) to induce and measure subtle changes in respiratory physiology and airway inflammatory markers. The two decades later perspective shows that both Rubin and I were wrong on several counts. Particulate air pollution is associated with mortality from cardiorespiratory disease. This implausible association, first published in the early 1990s, is now well supported by epidemiology studies, as will be discussed in this chapter. There is now additional evidence for morbidity from asthma, COPD, ischemic heart disease, and congestive heart failure. We now know that ultrafine particles escape detection by the macrophage defense system and translocate to the circulation. We know that, in  vitro, air pollution activates endothelin-1 (ET-1), a key regulator of the pulmonary circulation. The recommended chapter outline provided by the editors points to gaps in our knowledge: Sections on clinical manifestations, complications, diagnosis, treatment and prevention, and prognosis are meager because no coherent body of information yet exists. The fragmentary evidence presented herein is intriguing in its suggestion that common air pollutants target the pulmonary circulation and the right side of the heart. The data are by no means robust. A key purpose of this chapter is to challenge pulmonary vascular biologists and clinicians to reconsider the hypothesis that ambient air pollution causes or exacerbates pulmonary vascular disease. Joining me as coauthor is Melissa Bates, at whose comprehensive examination in physi­ology in the Department of

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_67, © Springer Science+Business Media, LLC 2011

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Chemical Engineering at Pennsylvania State University I first seriously considered the role of the pulmonary circulation in air pollution health effects. This chapter will discuss the effects of reactive gas and particulate pollution on the pulmonary circulation, creating a link between epidemiological findings in humans and experimental findings from controlled exposure studies in human and animal models. It will discuss the pulmonary circulation as a system directly affected by pollutants, as a point of entry into the systemic circulation, and as a biochemically active entity whose mediators exert both local and systemic effects after exposure. Here, we define the pulmonary circulation as the vascular bed that perfuses the gas-exchanging regions of the lung and lies in series with the systemic circulation. This is distinct from the bronchial circulation, which perfuses the conducting airways. This chapter will not discuss foodborne or absorptive exposures, infectious or immunological sensitization, or cigarette smoking.

2  Etiology and Pathogenetic Mechanisms The lung is the only organ, other than the skin, that is in constant contact with the ambient environment. Its large cross-sectional area (the size of a tennis court!), robust immunological milieu, and thin respiratory membrane necessary for gas exchange make it especially susceptible to damage caused by inhaled pollutants. The literature with respect to the effects of pollutants on airway mechanics, lung inflammation, and pulmonary function within a population is rich. The effects of pollutants on the pulmonary circulation are more poorly characterized yet may have important health consequences. Two consequences of pollutant exposure, myocardial infarction and stroke, are probably related to or modulated by effects on the pulmonary circulation.

2.1  Methodological Constraints Environmental health effects research has two complementary goals – to understand the health effects induced by a pollutant and the related mechanisms, and to understand the threshold dose necessary to induce the effect. There is no single study design that can determine air pollution health effects. Data are contributed to this field both by epidemiology studies and controlled inhalation studies in humans and animals, and by mechanistic studies using in vitro methods. Each discipline has strengths and limitations. Epidemiology studies have the advantage of real-world exposures, real-world populations, and outcomes of evident importance (mortality, hospitalization, medication use, etc.).

M.L. Bates and R. Bascom

Limitations include concurrent exposures other that the pollutant of interest that could confound the exposure– response relationship. Controlled inhalation studies in humans provide a precise evaluation of a specific pollutant exposure under highly controlled conditions, but are limited to relatively brief exposure periods, sometimes super-physiological exposure doses, and relatively noninvasive end points. Controlled animal studies allow precise pollutant generation and exposure and a full range of end points, but are limited by cross-species extrapolation. In vitro mechanistic studies investigate in detail one aspect of a larger complex exposure response process.

2.2  Regulatory Framework In the USA, The Clean Air Act mandates the Environmental Protection Agency (EPA) to set air quality standards for six major pollutants – ozone, particulate matter, nitrogen dioxide, sulfur dioxide, carbon monoxide, and lead. These pollutants are important because of their widespread distribution and recognized adverse health effects. They are also indicator pollutants, meaning that they represent more complex exposures from anthropogenic sources including transportation, manufacturing, and energy-generation processes. Congress intended that the primary air quality standards be based on evidence of human health risk and provide a reasonable margin of safety for susceptible populations. This congressional intent resulted in a scientific focus on understanding the health impact of outdoor air pollutants on individuals with chronic illnesses. The EPA regulatory framework categorizes sources into stationary, mobile, and area sources for outdoor air pollution inventory and control purposes. These categories are useful to inventory pollutants on a regional or nationwide scale, but have notable gaps when looking at local effects. Most air pollution monitors are sited so as to identify regional pollutant patterns. Human exposures immediately adjacent to or downwind of sources may be much higher, and are typically neither measured nor subject to local control. In an effort to fill this gap, the California Air Resources Board recommends a horizontal buffer of 1,000 ft between high combustion generating areas (e.g. roadways, truck drop lots, or warehouses) and residential areas, including schools. This practical guideline can be implemented by local planning ordinances. To synthesize health effects information from diverse disciplines, the US EPA periodically prepares “Criteria Documents” to summarize the scientific health effects data of the six pollutants. Recent updates to the Criteria Docu­ ments for particle pollution and ozone are given in [1, 2]. Because they are the best characterized, these two pollutants will be the major focus of this chapter.

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Because diesel and gasoline combustion engines are a primary source of fine and ultrafine particles [8], urban areas experience a greater pollution burden than rural areas [9–11]. More recent studies have emphasized the health risks for individuals in close proximity to high-density traffic flow or to truck idling areas (e.g. warehouses or drop lots). As automobile emissions age and are transported from the emission source, their character changes. Particles nucleate and condense to form larger particles [12]. These particles may then settle to the ground. This is encouraged by moderately windy conditions [13], humidity [14], and precipitation [15]. Sunlight encourages the reaction of particles with volatile gas and liquid-phase species such as sulfuric acid [16]. Thus, morbidity and mortality related to particulate pollution is greatest during a period of warm, dry, sunny weather. Fig. 1  Idealized size distribution of the components of ambient atmospheric particles and their mechanisms of formation and growth. Mechanically generated particles are typically alkaline and result from disruption of crustal material. Submicron particles are typically acidic, rich in metals, and form as products of combustion. (From Ref. [2])

2.3  Particulate Air Pollution Particulate air pollution comprises aerosolized solid and liquid species of various sizes. Particles generally fall into two classes – coarse mode and fine mode (Fig.  1). Coarse mode particles are larger than 2.5 mm in diameter, are generated by disruption of the Earth’s crust, and are composed of heavy metals, silica, and salts. Fine mode particles are less than 2.5 mm in diameter and are the product of fuel combustion and nanomanufacturing. They are largely carbon based, but may also contain lead, bromine, and vanadium, which are components of petroleum-based fuels [3]. These particles can complex with gas-phase organic species and nitrogen and sulfur oxide species, altering the surface chemistry of the particle [4, 5]. The surface chemistry of the particle influences interparticle interactions, the length of time the particle remains aerosolized, and potentially the interaction of the particle with the respiratory mucosa. The EPA’s National Ambient Air Quality Standards group particulate matter into two classes: PM10 particles are smaller than 10 mm but larger than 2.5 mm; PM2.5 particles are smaller than 2.5  mm. To be compliant with the EPA’s standards, the concentration of PM10 must not exceed 150 mg/m3 and the concentration of PM2.5 must not exceed 35 mg/m3 more than once per year over a 3-year period. Additionally, the annual average of PM2.5 must not exceed 15.0 mg/m3 over 3 years. Of particular recent interest is the subset of fine mode particles less than 0.1 mm in diameter, described as ultrafine particles, because of both their abundance and their enhanced potential for adverse health effects. In the USA and Europe, 82–89% of aerosolized particles by number are ­ultrafine [6, 7].

2.4  Ozone Tropospheric or ground-level ozone (O3) is an oxidizing component of urban smog formed from nitrogen oxide (NOx) and volatile organic compounds (VOC) via a reaction that is catalyzed by sunlight [17]. Major sources of NOx and VOC include automobile emissions, factory exhaust, and agricultural processes. Reflective of the role of sunlight in the production of O3, ambient ground-level O3 concentrations tend to be higher both during the daytime and during the summer months compared with nighttime or the winter months [18]. Like particulate matter, because emissions are the primary source of O3, the pollutant burden is higher in urban and interurban areas than in rural areas. The Northeast Ozone Transport Region in the USA extends from northern Virginia to Maine: in the summertime, prevailing winds send precursor pollutants in a northeast direction, with ozone generation occurring outside the large cities. Ozone is a highly reactive gas and has the ability to react to generate free-radical compounds. The EPA’s National Ambient Air Quality Standards set a maximum 1-h average exposure limit of 0.12 ppm and a maximum 8-h average exposure limit of 0.08 ppm. Between 2003 and 2005, 104 of 639 monitored US counties violated the primary 8-h standard. An additional 398 of 639 US counties registered ambient O3 concentrations greater than 0.075 ppm.

2.5  Dosimetry Concepts and Terminology Medieval physician Phillipus Paracelsus wrote in the sixteenth century, “All things are poison and nothing is without poison, only the dose permits something not to be poisonous.” Although environmental standards are typically based on

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effects related to specific, whole-organism exposure doses, studies investigating the effect of pollutants on pulmonary function and lung mechanics have demonstrated that there is substantial intersubject variability in response [19, 20]. The variability in response has been best characterized for ozone, with animal studies identifying the genetic loci for specific responses, and human studies showing longitudinal intrasubject stability in lung function response phenotypes in the presence of large intersubject variation. The variability in whole-organism exposure–response relationships may be the result of differences in the dose delivered to the target organ compared with the wholeorganism dose, differences in the interorgan distribution of the pollutant, or differences in reactivity at the effector site [21, 22] (Fig. 2).

2.6  Computational Models for Ozone Computational fluid dynamics has been a valuable tool in predicting the regional delivered lung dose of a pollutant [23–25]. Although it has been largely unapplied to modeling pollutant deposition and response in the pulmonary vasculature, it may be valuable in understanding sources of intersubject variability in this area as well. Here, we provide a simple model of how ozone, a highly reactive gas, is distributed in the lung as an example of how mathematical modeling has been applied to understanding airway exposure of pollutants. The intent is to indicate to the reader the major determinants of the dose distribution and suggest how these tools could be applied to the vasculature. The total dose retained by the lung is the difference between the inhaled and the exhaled dose. The inhaled dose (mg/min) is the product of the exposure concentration (mg/L), the minute ventilation, and the duration of the exposure (min). The dose received by a lung region is dependent on

Fig. 2  Primary determinants of the pulmonary dose of inhaled pollutants

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the local airway anatomy, tidal volume, biochemistry, and the composition of the pollutant. Although more comprehensive and thus more complicated models of lung exposure are available [26, 27], here we will discuss the distribution of O3 using a simple two-region Bohr model. This model is ­demonstrated schematically in Fig. 3. The Bohr model subdivides the respiratory system into an anatomical dead space compartment corresponding to the conducting airways (VD) and a gas-exchanging compartment corresponding to the peripheral air spaces. During a single inspiration of a tidal volume VT , the alveolar compartment is ventilated with a volume of air given by VA:

VT = VA + VD .

(1)

Multiplying each side of this equation by the frequency of breathing (f ) and rearranging provides an equation for the alveolar ventilation rate:

f VT = f (VA + VD ).

(2)

The Bohr model is sufficient to predict the distribution of an inert gas to the conducting airways and peripheral air spaces. For a reactive gas, a model must also consider its uptake and reaction with the lung during the course of inspiration. The rate at which O3 reaches the alveolar compartment (DA) is the product of the concentration of O3 reaching the compartment ([O3]A), f, and VA:

Fig. 3  Bohr model of the distribution of ozone in the lung with a model containing dead space (VD) and gas-exchanging (VA) compartments. The inhaled tidal volume (VT) is distributed to VD, leaving a volume VA = VT − VD to ventilate the alveolar compartment. The dose reaching the alveolar component is equal to the dose entering at the mouth ([O3]TVT) minus the dose retained in VD ([O3]TVD) and the uptake at the conducting airway wall (U) (shown in the insert). The insert shows the composition of the airway wall with the local concentration of ozone (O3) shown as a dotted line. Ozone is taken up in the epithelial lining fluid (ELF). Because of it high reaction rate and the abundance of ELF substrates, the concentration of O3 reaches nearly zero within the conducting airway ELF

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(3)

DA = f VA [O3 ]A .

The dose reaching the alveolar compartment ([O3]A) can also be described in terms of the volume of O3 that would ventilate the alveolar region were it an inert gas minus its uptake in the conducting airways (U):

DA = f VA [O3 ]T − U .

(4)

Here, [O3]T represents the concentration of O3 in inhaled air. Substituting (2) into (4) yields (5):

DA = [O3 ]T f (VT − VD ) − U

(5)

The uptake rate of O3 in the conducting airways, measured in mass (e.g., grams or micrograms) per minute, is determined by the concentration gradient between the lumen of the airway and the airway epithelial lining fluid (ELF). Because O3 reacts very quickly with the abundant antioxidant substrates in the ELF, the assumption can be made that the concentration of O3 in the ELF is zero and that the concentration gradient is entirely determined by the conducting airway concentration, approximated as [O3]T. Uptake is also determined by the surface area (S) and a mass transfer coefficient (K) that describes the rate of transport of O3 to the ELF [28]:

U = KS[O3 ]T .

(6)

The value of K, expressed as mass per minute per surface area per concentration, is determined by the anatomy of the airways, the air velocity, and the diffusion coefficient of O3 in air and can be measured experimentally using in vitro techniques [29]. Multiplying the right-hand side of the equation by VD/VD allows uptake to be described in terms of K, a surfaceto-volume ratio (a), VD, and the concentration of O3 (7). Ka has been measured experimentally in humans [28].

U = KaVD [O3 ]T , where

a≡

will penetrate deeper into the lungs of women, who have a smaller surface-to-volume ratio and a smaller amount of dead space than men. Finally, diseases or factors that depress the normal antioxidant content of the conducting airways (such as cigarette smoking, previous pollutant exposure, and respiratory disease) will increase the depth of delivery of O3.

S . VD

(7)

Substituting (7) into (3) and dividing both sides by VT and rearranging yields (8), which describes the fraction of inspired O3 reaching the peripheral air spaces:

 DA Ka  VD . = FO3 = 1 − 1 +   f  VT f VT [O3 ]T

(8)

The model in (67.8) predicts that activities that increase breathing frequency and tidal volume, such as exercise, will increase the delivery of O3 to the gas-exchanging regions of the lung. It has been shown experimentally that increasing inspired flow increases the depth of delivery of O3 [30]. Conditions which lower the anatomic dead space, such as asthma, will increase the depth of delivery. A similarly sized inhaled volume

2.7  Computational Models for Particles Part of the simplicity of the previously described model comes from the fact that gas-phase pollutants are freely diffusive and their penetration into the lung is not dependent on their size or mass. Unlike gas-phase pollution, however, a major determinant of the distribution of particles is their diameter. Generally, the diffusivity and inhalability of a particle is inversely proportional to its diameter. Inhalability refers to the likelihood that a particle, when inhaled, will enter the respiratory tract from the ambient environment. The probability of a particles less than 10 mm in diameter penetrating past the mouth or nose opening is greater than 0.96. Large particles greater than 2.5 mm are partially removed in the oronasal and upper conducting airways by inertial impaction and sedimentation, whereas particles smaller than 2.5 mm are more likely to deposit in deeper lung regions. These are dependent on the velocity of the particle. During exercise, when humans breathe primarily orally, the nose no longer plays a role in the removal of pollutants and the lung dose increases. Thus, any model that describes particle deposition must account for the particle’s diameter [31]. Although this results in a more complicated model than the model described previously for O3 distribution, the same general approach can be taken and the same factors that determine O3 distribution also impact particle distribution.

3  Systemic and Molecular Pathophysiology With pollutant exposure, the pulmonary vasculature plays three potential roles in determining the health effects of a pollutant. First, pulmonary vasculature tone and function may be directly affected. Second, the pulmonary vasculature may produce and distribute inflammatory and vasoactive mediators with local and distal effects to pulmonary and systemic vasculature and other organs. Finally, the pulmonary vasculature may be a point of entry for ultrafine and nanoparticles. These particles can then pass to and affect the systemic circulation or end organs. This section will describe some of the pathophysiological consequences of exposure to the lung vascular to inhaled pollutants.

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3.1  P  ulmonary Hypertension and Effects on the Right Side of the Heart Animal studies using isolated vessel preparations and histology have provided compelling, but incomplete, data indicating that transient exposure to pollutants affects pulmonary vessel tone and function. These data, combined with limited human exposure data, suggest that pollutants cause direct vasculopathy that may result in cardiovascular dysfunction and rightsided heart failure in at-risk populations. Key studies supporting this link are highlighted in this section.

3.1.1  Histology and Vessel Morphometry The major studies describing the morphological effects of particulate matter exposure have been completed via collaboration between John Godleski of Harvard University and Paolo Saldiva of the University of São Paulo. Batalha et al. induced chronic bronchitis in male Sprague–Dawley rats by exposing them to 276 ± 9  ppm sulfur dioxide for 5  h/day, 5 days/week, over a 6-week period, with an appropriate control population exposed to filtered air [32]. They then exposed the rats to either concentrated ambient particulate matter or filtered air for 5 h/day over 3 days. They collected and sectioned the lungs, and imaged the pulmonary vasculature. The investigators found that, compared with control animals exposed only to filtered air, short-term exposure to particulate matters caused vascular hypertrophy similar to that seen in animals with chronic bronchitis (Fig. 4). There was not an overall additive effect of chronic bronchitis and exposure to particulate matter on vessel hypertorphy. However, the degree of vessel wall thickening observed in the control rats and the chronic bronchitis rats exposed to particulate matter was related to the amount of silicon in the particles. This indicates that an underlying pulmonary airway disease is not necessary for the development of vascular disease with pollutant exposure and that the composition of the particle, not simply its size, is important in determining the degree of disease developed. Multiday exposure is not necessary to induce morphometric changes in the pulmonary vasculature. In a subsequent experiment performed by the same group, Rivero et  al. instilled 1 mL water containing either 100 or 500 mg of 2.5-mm particles or particle-free water into the lungs of anesthetized male Wistar rats. Twenty-four hours later, the rats were killed and the lungs sectioned and imaged, and the wet-to-dry weight ratio was assessed. The investigators found evidence of leukocyte infiltration and changes in the wall vessel morphometry consistent with vasoconstriction in the particle-exposed lungs. They also found increases in the wet-to-dry weight ratio of the lung consistent with pulmonary edema.

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Finally, Lemos et  al. exposed conscious, freely moving mice for 24  h/day, 7 days/week over a 4-month period in exposure chambers to the ambient air in downtown São Paulo or filtered air. As in the previous two studies, the investigators killed the animals and sectioned and imaged the lungs. They also sectioned the heart and made measures of coronary artery morphometry. As in the first study, the investigators found that exposure to particulate matter induced pulmonary vessel hypertrophy. They also found hypertrophy of the coronary vessels. These data, taken with the data from the previous two experiments, demonstrate that ambient particles, concentrated particles, and particles instilled in solution are all sufficient to induce pulmonary vascular alterations. There appears to be a time course of events in the development of disease whereby vessel constriction and pulmonary edema occur early in the exposure period, with vessel wall hypertrophy developing later. Controlled, multistaged exposure experiments in intact, instrumented animals are still needed to determine whether this progression of events occurs in the individual animal and whether they result in altered pulmonary vascular pressure and right-sided heart mechanics.

Fig.  4  Histological sections of rat lung tissue sham rats (a), rats exposed to concentrated ambient particles (b), rats with sulfur dioxide induced chronic bronchitis (c), and rats with chronic bronchitis exposed to concentrated ambient particles (d). Black arrows note arterial walls. Note the thickening of the arterial walls in rats exposed to concentrated ambient particles. This is enhanced in concentrated ambient particle exposed rats with chronic bronchitis (From Batalha et al. [32])

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3.1.2  Isolated Vessel Function Li et al. investigated the mechanism by which pollutant exposure induces pulmonary vasoconstriction and vessel wall hypertrophy [33]. They exposed pulmonary artery rings isolated from Sprague–Dawley rats to particulate matter collected in St Louis over a 12-month period and measured the change in ring tension caused by exposure. The authors found that particulate exposure caused constriction in the rings of up to 30% of the maximum constriction induced by phenylephrine in a dose-dependent manner (Fig.  5). This was abated with the administration of losartan, an angiotensin type 1 receptor antagonist, indicating a role for angiotensin in pollutant-induced pulmonary vasoconstriction. Particulate matter caused activation of the cell-growthand ­cell-proliferation-promoting mitogen-activated protein kinase (MAPK)–extracellular-signal-regulated kinase–p38 pathway. This activation was blocked with losartan administration and with administration of captopril, an angiotensin-converting-enzyme antagonist. Combined, these in  vitro data indicate that particulate matter causes angiotensin-mediated pulmonary vessel constriction and potentially vascular remodeling as a result of MAPK pathway activation. An important caveat is that these experiments are conducted in the absence of the lung’s inflammatory milieu. Pollutant exposure causes neutrophil infiltration of the lung. Adherence of neutrophils to pulmonary vascular endothelium is maximal within 2 h after ozone exposure and returns to control levels by 12  h after exposure [34]. Because inflammatory mediators can alter vascular function [35], it is important to conduct these experiments also in vivo and in the context of relevant inflammatory mediators.

3.1.3  Right-Sided Heart Function Despite several compelling studies indicating that pollutant exposure has direct effects on the structure and function of the pulmonary vasculature, controlled human exposure experiments investigating the implication of this for the right side of the heart are unavailable. The most convincing evidence that these vascular changes may impact rightsided heart function comes from work reported by Rich et  al. Here, the investigators analyzed data from the Chronicle Offers Management to Patients with Advanced Signs and Symptoms of Heart Failure (COMPASS-HF) trial. Patients were instrumented with indwelling pressure monitors in the right side of the heart. As a secondary end point of the study Rich et  al. regressed right-sided heart pressure as a function of same-day PM2.5 concentration and found that for each 11.6  mg/m3 increase in par­ ticulate concentration, pulmonary artery diastolic pressure increased 0.19 mmHg and right ventricle diastolic pressure increased 0.23 mmHg. Although not a controlled exposure

Fig.  5  (a) Change in tension in isolated rat pulmonary artery rings exposed to urban particles. Ring tension increases with increased particle concentration. (b) Mean estimated pulmonary artery diastolic (ePAD) and right ventricular (RV) diastolic pressures in patients with right-sided heart failure as a function of the relative ambient concentration of particulate matter (PM) smaller than 2.5  mm (PM2.5). Increases in PM2.5 concentration are related to increases in ePAD and RV diastolic pressures in these patients. (a From Li et al. [67]; b from Rich et al. [68])

study, this small observational study provides evidence in support of the hypothesis that acute exposure to particles elevates right-sided heart pressure, which may contribute to morbidity in individuals at risk for heart failure. Longterm, repeated exposures, resulting in elevations in rightsided heart pressure, could result in right ventricular remodeling. However, more study in this area is needed to define the link between pollutant exposure and right-sided heart effects. The studies described in this section support a causative link between exposure of the pulmonary vasculature to pollutants and heart failure, suggesting that pollutant exposure on a single day causes acute vasoconstriction and increases in right ventricular pressure. Long term, these exposures result in vessel hypertrophy and, possibly, pulmonary hypertension

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and remodeling of the right side of the heart. However, what is severely missing from this area of research is controlled animal and human exposure studies to investigate the mechanism by which pollutants cause hemodynamic changes and the basic dose-response characteristics of these changes. There are intact animal preparations available to study the impact of pulmonary vessel changes on remodeling of the right side of the heart both with short-term and with long-term exposures [36]. Finally, these changes have only been studied in the context of particulate matter and the effects of concomitant or separate gas pollutant exposure are unknown.

3.2  Transcription of Vasoactive Mediators There is substantial evidence that pollutant exposure causes the transcription of vasoactive mediators within the pulmonary vasculature of humans and animals. The following section describes what is known about the effect of pollution on the production of the potent vasoconstrictor ET-1. The lung is a major site for both production and removal of circulating ET-1, and humans living in polluted environments have higher circulating blood ET-1 concentrations compared with individuals living in nonpolluted environments [37]. A number of animal studies offer insight into the effect of pollutant exposure on ET-1 handling by the lung. Thomson et al. described the effects of a 4 h, combined 0.8 ppm O3 and 49 mg/m3 particulate matter (mean diameter 0.5 mm) nasal exposure on transcription of endothelin-related genes in the lungs of Fischer-344 rats [38]. Exposure to O3 caused an upregulation of the transcription of preproET-1 and endothelin-converting enzyme-1 receptors 2  h after exposure. Although the authors did not distinguish between transcription uniformly throughout the lung and transcription occurring regionally, their results demonstrate that pollutant exposure has the ability to upregulate endothelin-related genes in a short period of time. Human epithelial cells exposed to particulate matter in vitro also upregulate genes related to the production of ET-1 [39]. In a subsequent study, Thomson et al. showed that O3 and particulate matter each increased circulating ET-1 levels individually immediately after exposure, but not when administered together. In contrast, Bouthillier et  al. demonstrated under similar exposure conditions that the concentration of ET-1 in plasma is increased 1 and 3 days after exposure with particulate matter exposure or a combination of particulate matter and O3, but not with O3 alone [40]. These data suggest that there could be interacting time-dependent and pollutantdependent factors that affect the production and/or metabolism of ET-1 that impacts its appearance in the plasma. This upregulation of ET-1 in the background of a pollutant exposure could contribute to myocardial infarction, pulmo-

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nary vascular disease, and stroke in at-risk populations. Campen et al. exposed isolated coronary vessels from mice deficient in apolipoprotein E, a model of atherosclerosis, to diesel exhaust. They found that the pollutant exposure enhanced the vasoconstrictive effects of ET-1 and reduced the vasodilatory effects of sodium nitroprusside [41]. Calderon-Garcidueñas et al. compared 59 children in Mexico City, a city with ambient particulate matter and O3 concentrations in excess of the USA’s air quality standards, with 22 children living in an unpolluted area. They found that the children in Mexico City had elevated pulmonary artery pressures and circulating plasma ET-1 levels compared with control children. The circulating ET-1 level was correlated with the cumulative concentration of particulate matter less than 2.5 mm in diameter over the preceding 7 days [42]. The precise systemic consequence of upregulating vasoactive mediators in the lung is unknown, but whole-organism exposure to ambient pollutants alters systemic cardiovascular function. Short-term exposure causes systemic vasoconstriction and increases blood pressure [43]. Long term, pollutant exposure alters systemic vascular responsiveness in humans apparently by uncoupling endothelial nitric oxide synthase signaling [44–46]. This could be caused by long-term exposure to the vasoactive mediators produced by the lung. Pollutants also may modulate the autonomic nervous system, leading to altered vasoresponsiveness. The mechanism by which gas-phase and particle pollutants modulates the autonomic nervous system, whether via the activation of local lung reflex pathways or via action directly on the central nervous, is not well understood [47]. Rodents exposed to particulate matter and O3 demonstrate strain-dependent reductions in heart rate and heart rate variability [48]. Whereas rats adapted to repeated particulate matter exposure, there was no apparent adaptation to repeated O3 exposure. This indicates that the autonomic modulations by particulate matter and O3 do not necessarily share a common mechanism. Ozone reacts locally with the mucous lining and epithelial layers of the lung, whereas particulate matter may cross the respiratory membrane and enter the bloodstream. Here, the pulmonary vasculature may be serving as a point of entry by which particulate matter can enter the systemic circulation and modulate the central nervous system, whereas O3 may be having more local effects. In the pulmonary function literature, activation of local, parasympathetic and nociceptive pulmonary reflex pathways is not sufficient to completely explain alterations in forced expired volume in 1 s (FEV1) with acute pollutant exposure [49, 50]. A combination of undescribed neural mechanisms and neural-independent mechanisms may explain alterations in function not explained by activation of local afferents alone. Subsequent studies interested in defining the mechanism by which pollutants affect the systemic vasculature will

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need to consider both the effects of neurally mediated and biochemically mediated alterations in function, and the fact that different pollutants may have different mechanisms of action.

3.3  V  entilation/Perfusion Mismatch and Impaired Gas Exchange The primary purpose of the lung is gas exchange. Surprisingly, very few studies have investigated the effect of pollutant exposure on the lung’s ability to exchange carbon dioxide and oxygen. Folinsbee et  al. measured maximal exercise performance after 2  h of continuous chamber exposure to 0.75 ppm O3 in human research participants [51]. The authors found that maximal achieved work (expressed as metabolic equivalents) and VO2max were both reduced approximately 10% with O3 exposure compared with filtered air exposure. The authors concluded that this exercise limitation was related to the participants’ self-limitation as a result of O3-induced lung irritation and discomfort. At that time, there were no data indicating that O3 affects cardiovascular function, and diffusion limitation was thought to be minimal. Two additional human exposure studies demonstrate that O3-exposure limits exercise performance [52, 53], but both studies concluded that exercise intolerance is related to selflimitation as a result of discomfort from the exposure. Later, Gong et al. exposed ten hypertensive and six normotensive adult men to 0.3 ppm O3 for 3 h with intermittent exercise with the intent of investigating the effect of O3 on cardiac function in these two groups [54]. Although the authors found no differences in heart rate, blood pressure, or pulmonary artery wedge pressure with exposure, the major finding was that 13 of the 16 participants experienced an  increase in the alveolar–arterial oxygen difference (Da-APO2), with the mean change greater than 10  mmHg and a range of changes between 1 and 25 mmHg. The mean Da-APO2 with filtered air exposure was less than 1 mmHg. This indicates that the inhalation of O3 alters gas exchange and that the exercise intolerance seen in other studies may be due to factors more complex than simple discomfort. Although, in both populations, the increase in Da-APO2 was not sufficient to alter cardiovascular function or exercise performance, the additional impairment of gas exchange in persons with preexisting pulmonary or cardiovascular disease may cause changes in cardiovascular function. Although Gong et  al.’s data uncovered a tremendously important effect of O3 on the lung, there are two factors that limit their interpretation. Gong et  al.’s participants completed less intense exercise under a single exposure condition that was less than half the concentration of Folinsbee et al.’s 0.75 ppm. In Gong et al.’s study, where the exercise

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intensity was such that individuals did not widen their Da-APO2 with room air exposure, the additional Da-APO2 with O3 exposure was not limiting. However, during intense exercise in room air, individuals may widen their Da-APO2 upwards of 30  mmHg [55]. An additional Da-APO2 of 25 mmHg caused by O3 exposure would certainly be limiting. Whether this gas exchange abnormality would have been present and limiting with maximal exercise or a higher exposure concentration is unknown. Second, Gong et  al.’s hypertensive participants were largely medicated with antihypertensive medications. Whether they would have responded similarly to normotensive individuals if they had been unmedicated is unknown. Pursuing the question is important considering the large number of undiagnosed and unmedicated hypertensive individuals living in polluted areas. The cause of this gas-exchange abnormality, its relationship to the inhaled and local doses, its impact on disease, and its dependence on exercise intensity cannot be inferred from these data. However, a potential cause for the increase in Da-APO2 is ventilation/perfusion (V/Q) inequality [56]. If areas of the lung become poorly ventilated or unventilated, but remain perfused, arterial hypoxemia will result (see Fig. 3.20 in [57] for a more detailed explanation). In human participants, O3 exposure changes the normal distribution of ventilation [58]. Foster et  al. exposed nine healthy male volunteers to either filtered air or 0.33 ppm O3 for 2 h with intermittent exercise. Exposure was followed by imaging using 133Xe gas. Following air exposure, 133Xe was distributed in a gradient such that the basal lobes of the lungs received a higher portion of the inhaled 133Xe bolus per unit volume compared with the middle and upper lobes. In seven of nine subjects, O3 inhalation caused a redistribution of the 133 Xe bolus such that the 133Xe signal was higher in the upper and middle lobes compared with the lower lobes. Thus, the inhalation of O3 causes a redistribution of ventilation such that ventilation is shunted from the normally highly ventilated basal lung toward the less well ventilated apical lung. Alterations in inhaled gas patterns occur in patients with COPD, often with increased central deposition of inhaled tracer. The effect of O3 exposure on ventilation distribution in this group is unknown, but would be of interest as a possible pathway for O3-associated morbidity and mortality seen in epidemiology studies. The fact that that O3 causes ventilation redistribution and that arterial hypoxemia results suggests that V/Q matching is uncoupled and that perfusion is not redistributed to match ventilation. Ozone may have a direct impact on pulmonary vascular function, resulting in increased regions of V/Q inequality. However, a single unifying study has not been completed to test this hypothesis. The potential warrants further investigation of V/Q matching with O3 exposure.

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4  Clinical Effects 4.1  Particulate Air Pollution Epidemiology studies have demonstrated an increased mortality associated with PM10 pollution. This finding, initially reported in the early 1990s, was hotly debated for several reasons. Earlier epidemiology studies suggested that current regulations were protective against particulate adverse health effects, so this was a paradigm shift for scientists and regulators. A telling editorial published at the time was entitled “The Environment and the Lung–Changing Perspectives” [59]. Responding to these concerns, the Health Effects Institute, a research institute cofunded by the EPA and the automobile manufacturers, commissioned a reanalysis of the  datasets that underlie the initial particulate–mortality association. Methodological issues were addressed in the analytic approach. The findings confirmed the increased risk of mortality with particulate exposure. Figure 6a shows the 0.2% increase in all-cause mortality per 10 mg/m3 increase in PM10. Subgroup analysis shows that this increased mortality is predominantly due to an increased mortality in cardiovascular and respiratory diseases. Models that evaluated the contribution of other ambient pollutants to the mortality effect caused small shifts or broadening of the effect curve, but did not change the main effect. The finding that PM10 pollution was associated with increased mortality prompted several lines of investigation. Investigators sought to determine which components of particulate pollution were responsible for the mortality effect. A search for susceptible subgroups within the mortality signal also began. Finally, a search began for intermediate biomarkers of effect and experimental models to understand the mechanisms of injury. The following paragraphs highlight the results of these investigations. The first task was to determine which component of PM10 air pollution was responsible for the mortality effect. As shown in Fig. 1, particulate matter under 10 mm in diameter comprises four distinct modes, and has a complex and dynamic chemistry. Broadly speaking, the larger particles form by physical disruption of surface materials (e.g. quarrying), whereas smaller particles form by the process of combustion. The ultrafine and fine particles begin as fumes or vapors, then increase in size through coagulation, nucleation, and condensation. These particles form primarily by combustion, with their chemical composition determined by the fuel source. The ultrafine particles bypass the macrophage defense system and translocate to the pulmonary vasculature, with subsequent dissemination throughout the body. Not all particulate air pollution reaches the pulmonary vasculature. The mechanically generated, coarse mode particles deposit by impaction in the nasopharynx and upper tracheobronchial

Fig. 6  (a) National average effects of PM10 for total mortality, cardiovascular–respiratory mortality, and other causes mortality for 90 US cities. (b) National average estimates of PM10 on total mortality from nonexternal causes with and without control for other pollutants for 90 US cities. Shown are 1-day-lag marginal posterior distributions, with the top-right boxes showing the posterior probabilities that the overall effects are greater than 0. (From Dominici et al. [69])

tree and are removed by the mucociliary escalator and expelled by sneezing, swallowing, or coughing. Figure 7 summarizes studies that evaluated the effects of three size fractions of particulate pollution: PM10 (all particles less than 10 mm), PM2.5 (all particles less than 2.5 mm, and PM2.5–10, particles between 2.5 and 10  mm. Figure 7 shows the consistency of the mortality and morbidity effects of particulate matter in epidemiology studies in the USA and Canada. As with the mortality study shown in Fig. 6b, the particulate matter effects are remarkably stable when evaluated as single pollutant effects or in combination with the vapor-phase pollutants ozone, nitrogen oxides, sulfur

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Fig. 7  (a) Excess risk estimates for cardiovascular-related effects, including mortality, hospital admissions, and changes in biomarkers in singlepollutant (PM only) and multipollutant models. IH ischemic heart disease, HF heart failure, HR heart rate, HRV heart rate variability. PM increments were 50 mg/m3 for PM10 and 25 mg/m3 for PM2.5 and PM between 2.5 and

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10 mm (PM10-2.5). (b) Excess risk estimates for respiratory-related effects, including mortality, hospital admissions, and medical visits in single-pollutant (PM only) and multipollutant models. Mort mortality, Pneu pneumonia, COPD chronic obstructive pulmonary disease. PM increments were 50 mg/m3 for PM10 and 25 mg/m3 for PM2.5 and PM10-2.5. (From [2])

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dioxide, and carbon monoxide. Part of the explanation for this is that pollutant sources are from mobile (transportation), stationery (e.g. power plants, factories), and area sources. The products of combustion result in cogeneration of particle and vapor-phase pollutants. The second task was to determine susceptible subgroups that were vulnerable to the effects of particulate pollution. Figure  7 summarizes some of the studies that identified patients with ischemic heart disease and heart failure showing morbidity related to particulate pollution. Other studies have since shown that patients with diabetes and cystic fibrosis are also at risk. Respiratory morbidity and mortality likewise showed associations with outcomes ranging from emergency room visits to hospitalization for pneumonia, and COPD. The effect for pneumonia seemed to lie in the  PM2.5 fraction, whereas the effect for COPD was in the PM10-2.5 fraction. This makes some sense, as COPD is a disease of the airways, and would in theory be affected by particles deposited in the airways, whereas pneumonia might be susceptible to more distally deposited pollutants. The third task was to seek intermediate biomarkers of particulate effects that might explain the pathogenesis of the mortality and morbidity response. Activation of the coagulation system has been associated with particulate pollution, specifically increased levels of fibrinogen and platelets, and autonomic dysregulation with effects on resting heart rate and heart rate variability [60]. A notable omission in the epidemiology studies of air pollution health effects is the assessment of the effect of particulate air pollution on pulmonary vascular diseases. The morbidity and mortality among patients with COPD could occur via effects on the pulmonary vessels and right side of the heart, but this has not been evaluated directly. Similarly, activation of the clotting cascade could result in increased numbers of thromboembolic events.

4.2  Tropospheric Ozone The previously described computational model and experimental data both show the importance of outdoor work at increased levels of exertion (and increased minute ventilation) to ozone health effects. The primary end points studied thus far have been changes in lung function decrements, including changes in FEV1, respiratory tract inflammation, and airway hyperreactivity. Ozone exposure is associated with increased mortality in older adults (more than 65 years). This age group, by contrast, shows a diminished FEV1 responsiveness to controlled ozone exposure [61], suggesting that the mortality effect may occur by a nonrespiratory mechanism. Genetic polymorphisms in antioxidant enzymes may contribute to the variability in the pulmonary function and inflammatory response to ozone

M.L. Bates and R. Bascom

exposure. The GSTM1 null genotype was associated with increased responsiveness to ambient ozone in asthmatic children in Mexico City [62].

5  Complications Experience with air pollution health effects in the airways leads to concern about possible complications of air pollution exposure for individuals with chronic pulmonary vascular disease. The clinical impact of an adverse effect may be greater when added to preexisting impaired function. Specific data for patients with pulmonary vascular diseases are lacking.

6  Diagnosis (and Distinguishing Diagnosis) Disease caused or exacerbated by air pollution bears no unique stamp. There are no distinguishing diagnostic studies that can be recommended to look for pollutant-associated effects.

7  Treatment and Prevention Antioxidant supplementation with vitamins C and E attenuated the ozone-associated lung function decrements in outdoor workers in Mexico City [63]. These data, although not directly related to pulmonary vascular disease, support the hypothesis that understanding mechanisms and dosimetry may lead to mitigation strategies. Outdoor air pollution is monitored throughout the USA with data reduction and aggregation performed by the departments of the environment at the state level and the EPA at the national level. Clinicians interested in learning local patterns can consult these sources. Air quality alerts are broadcast, particularly during the summer months.

8  Prognosis Outdoor air pollution is widespread in the USA and worldwide, with large segments of the population living in areas that violate current health-based standards. Exposure is thus likely to continue, creating the need for better understanding of pathogenetic mechanisms and strategies to mitigate adverse effects.

67  Air Pollution and the Pulmonary Vasculature

9  Summary and Gaps in the Knowledge Environmental air pollution is commonly defined as a composite mixture of reactive gases and aerosolized liquids and particles [64]. Government legislation and regulation promulgate health-based standards, using indicator pollutants including ozone and particulate matter. Epidemiology studies demonstrating morbidity and mortality from particulate (and to a lesser extent O3) pollution mandate a reassessment of the adequacy of the current body of experimental research. Careful dose–response studies demonstrate the effects of O3 exposure on pulmonary and ventilatory function. In contrast, there are only single-dose studies designed to investigate the mechanism of O3 exposure on the pulmonary vasculature. Carefully controlled dose–response studies in this field, using well-defined experimental end points, and a combination of human and animal exposure models, are clearly warranted. These data will guide the definition of air quality standards and guide clinicians in the evaluation and mitigation of adverse effects. Because pollutants may act through different mechanisms, these studies should be done for each of the major classes of pollutants. The data investigating the mechanism by which pollutants cause vascular disease are intriguing, but suffer from a lack of consistency in methods and pollutants used between laboratory groups. To be easily translated to air quality standards, consistent exposure protocols and experimental models will need to develop as they have in the pulmonary function literature. Despite the fact that the major function of the lung is gas exchange, gas-exchange abnormalities caused by pollutant exposure have not been well studied and the role of alterations in pulmonary vascular function in pollutant-induced gas-exchange abnormalities have not been considered. Computation fluid dynamics models have been used extensively in the pulmonary function literature, but have not been applied to the pulmonary vasculature and have not been used to predict gas-exchange abnormalities. These models could be immensely valuable in predicting gas-exchange and vascular abnormalities in normal individuals and in individuals with pulmonary diseases such as asthma, COPD, and the many forms of pulmonary hypertension. Although outdoor air pollution and domestic biomass combustion continue as important sources of indoor air pollution, the human indoor pollutant load has drastically increased and diversified compared with what is was 50 years ago. Factors driving these changes include technological advances in building design and energy efficiency, an increased reliance on air conditioning and heating, and the development of new building and cleaning products [65]. In manufacturing, the development of nanotube and nanoparticle technologies presents a potential risk to factory workers

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manufacturing the products who might be occupationally exposed to the particles [66]. Indoor air pollution and exposure to nanoparticles are potentially major sources of morbidity and mortality, but the health effects have not been well described in controlled exposure studies.

References 1. Environmental Protection Agency (2006) Air quality criteria for ozone and related photochemical oxidants (EPA/600/R-05/004aF) 2. Environmental Protection Agency (2004) Final particulate matter criteria document 3. Committee of the Environmental and Occupational Health Assembly of the American Thoracic Society (1996) Health effects of outdoor air pollution. Am J Respir Crit Care Med 153:3–50 4. Prather KA, Hatch CD, Grassian VH (2008) Analysis of atmospheric aerosols. Annu Rev Anal Chem 1:485–514 5. Kuhn T, Krudysz M, Zhu Y et  al (2005) Volatility of indoor and outdoor ultrafine particulate matter near a freeway. J Aerosol Sci 36:291–302 6. Zhang KM, Wexler AS (2004) Evolution of particle number distribution near roadways – part I: analysis of aerosol dynamics and its implications for engine emission measurement. Atmos Environ 38:6643–6653 7. Junker M, Kasper M, Röösli M et al (2000) Airborne particle number profiles, particle mass distributions and particle-bound PAH concentrations within the city environment of Basel: an assessment as part of the BRISKA Project. Atmos Environ 34:3171–3181 8. Kirchstetter TW, Harley RA, Kreisberg NM, Stolzenburg MR, Hering SV (1999) On-road measurement of fine particle and nitrogen oxide emissions from light- and heavy-duty motor vehicles. Atmos Environ 33:2955–2968 9. Hussein T, Hämeri K, Aalto PP, Paatero P, Kulmala M (2005) Modal structure and spatial-temporal variations of urban and suburban aerosols in Helsinki – Finland. Atmos Environ 39:1655–1668 10. Lin C-C, Chen S-J, Huang K-L et al (2007) Water-soluble ions in nano/ultrafine/fine/coarse particles collected near a busy road and at a rural site. Environ Pollut 145:562–570 11. Wiedensohler A, Wehner B, Birmili W (2001) Aerosol number concentrations and size distributions at mountain-rural, urban-influenced rural, and urban-background sites in Germany. In: 13th International Congress of the International Society for Aerosols in Medicine (ISAM). Liebert, Interlaken 12. Zhu YF, Hinds WC, Kim S, Sioutas C (2002) Concentration and size distribution of ultrafine particles near a major highway. J Air Waste Manag Assoc 52:1032–1042 13. Charron A, Harrison RM (2003) Primary particle formation from vehicle emissions during exhaust dilution in the roadside atmosphere. Atmos Environ 37:4109–4119 14. Jamriska M, Morawska L, Mergersen K (2008) The effect of temperature and humidity on size segregated traffic exhaust particle emissions. Atmos Environ 42:2369–2382 15. Garcia PJ, Garcia BA, Fernandez Diaz JM, Rodriguez Brana MA (1994) Parametric study of selective removal of atmospheric aerosol by below-cloud scavenging. Atmos Environ 28:2335–2342 16. Sardar SB, Fine PM, Mayo PR, Sioutas C (2005) Size-fractionated measurements of ambient ultrafine particle chemical composition in Los Angeles using the NanoMOUDI. Environ Sci Technol 39:932–944 17. Sillman S, He DY (2002) Some theoretical results concerning O-3NOx-VOC chemistry and NOx-VOC indicators. J Geophys Res Atmos 107:15

976 18. Kasibhatla P, Chameides WL (2000) Seasonal modeling of regional ozone pollution in the eastern United States. Geophys Res Lett 27:1415–1418 19. Hattis D, Russ A, Goble R, Banati P, Chu M (2001) Human interindividual variability in susceptibility to airborne particles. Risk Anal 21:585–599 20. McDonnell WF, Stewart PW, Andreoni S et al (1997) Prediction of ozone-induced FEV1 changes: effects of concentration, duration, and ventilation. Am J Respir Crit Care Med 156:715–722 21. Bates ML, Brenza TM, Ben-Jebria A, Bascom R, Ultman JS (2009) Longitudinal distribution of ozone absorption in the lung: Comparison of cigarette smokers and nonsmokers. Toxicol Appl Pharmacol 236:270–275 22. Reeser WH, Lee GM, Taylor A et  al (2005) Uptake of ozone in human lungs and its relationship to local physiological response. Inhal Toxicol 17:699–707 23. Ginsberg GL, Asgharian B, Kimbell JS, Ultman JS, Jarabek AM (2008) Modeling approaches for estimating the dosimetry of inhaled toxicants in children. J Toxicol Environ Health 71:166–195 24. Taylor AB, Borhan A, Ultman JS (2007) Three-dimensional simulations of reactive gas uptake in single airway bifurcations. Ann Biomed Eng 35:235–249 25. Bush ML, Zhang W, Ben-Jebria A, Ultman JR (2001) Longitudinal distribution of ozone and chlorine in the human respiratory tract: simulation of nasal and oral breathing with the single-path diffusion model. Toxicol Appl Pharmacol 173:137–145 26. Miller FJ, Overton JH Jr, Jaskot RH, Menzel DB (1985) A model of the regional uptake of gaseous-pollutants in the lung. 1. The sensitivity of the uptake of ozone in the human lung to lower respiratorytract secretions and exercise. Toxicol Appl Pharmacol 79:11–27 27. Overton JH, Graham RC, Miller FJ (1987) A model of the regional uptake of gaseous-pollutants in the lung. 2. The sensitivity of ozone uptake in laboratory-animal lungs to anatomical and ventilatory parameters. Toxicol Appl Pharmacol 88:418–432 28. Bush ML, Raybold T, Abeles S, Hu SC, Ben-Jebria A, Ultman JS (1996) Longitudinal distribution of ozone absorption in the lung: simulation with a single-path model. Toxicol Appl Pharmacol 140:219–226 29. Kermani S, Ben-Jebria A, Ultman JS (2006) Kinetics of ozone reaction with uric acid, ascorbic acid, and glutathione at physiologically relevant conditions. Arch Biochem Biophys 451:8–16 30. Hu SC, Benjebria A, Ultman JS (1994) Longitudinal distribution of ozone absorption in the lung – effects of respiratory flow. J Appl Physiol 77:574–583 31. Miller F (2000) Dosimetry of particles: critical factors having risk assessment implications. Inhal Toxicol 12(Suppl 3):389–395 32. Batalha JRF, Saldiva PH, Clarke RW et  al (2002) Concentrated ambient air particles induce vasoconstriction of small pulmonary arteries in rats. Environ Health Perspect 110:1191–1197 33. Blanc A, Pandey NR, Srivastava AK (2003) Synchronous activation of ERK 1/2, p38mapk and PKB/Akt signaling by H2O2 in vascular smooth muscle cells: potential involvement in vascular disease. Int J Mol Med 11:229–234 34. Lavnikova N, Prokhorova S, Lakhotia AV, Gordon R, Laskin DL (1998) Distinct inflammatory responses of adherent vascular lung neutrophils to pulmonary irritants. J Inflamm 48:56–66 35. Garrison RN, Spain DA, Wilson MA, Keelen PA, Harris PD (1998) Microvascular changes explain the “two-hit” theory of multiple organ failure. Ann Surg 227:851–860 36. Tuchscherer HA, Vanderpool RR, Chesler NC (2007) Pulmonary vascular remodeling in isolated mouse lungs: effects on pulsatile pressure-flow relationships. J Biomech 40:993–1001 37. Calderon-Garciduenas L et  al (2007) Systemic inflammation, endothelial dysfunction, and activation in clinically healthy ­children exposed to air pollutants. In: 103rd International

M.L. Bates and R. Bascom Conference of the American ThoracicSociety. Taylor & Francis, San Francisco 38. Thomson E, Goegan P, Kumarathasan P, Vincent R (2004) Air pollutants increase gene expression of the vasoconstrictor endothelin-1 in the lungs. Biochim Biophys Acta 1689:75–82 39. Chauhan V, Breznan D, Thomson E, Karthikeyan S, Vincent R (2005) Effects of ambient air particles on the endothelin system in human pulmonary epithelial cells (A549). Cell Biol Toxicol 21:191–205 40. Bouthillier L, Vincent R, Goegan P et al (1998) Acute effects of inhaled urban particles and ozone – lung morphology, macrophage activity, and plasma endothelin-1. Am J Pathol 153:1873–1884 41. Campen MJ, Babu NS, Helms GA et al (2005) Nonparticulate components of diesel exhaust promote constriction in coronary arteries from ApoE−/− mice. Toxicol Sci 88:95–102 42. Calderón-Garcidueñas L, Vincent R, Mora-Tiscareño A et al (2007) Elevated plasma endothelin-1 and pulmonary arterial pressure in children exposed to air pollution. Environ Health Perspect 115:1248–1253 43. Peretz A, Sullivan JH, Leotta DF et  al (2008) Diesel exhaust inhalation elicits acute vasoconstriction in  vivo. Environ Health Perspect 116:937–942 44. Briet M, Collin C, Laurent S et al (2007) Endothelial function and chronic exposure to air pollution in normal male subjects. Hypertension 50:970–976 45. Knuckles TL, Lund AK, Lucas SN, Campen MJ (2008) Diesel exhaust exposure enhances venoconstriction via uncoupling of eNOS. Toxicol Appl Pharmacol 230:346–351 46. O’Neill MS, Veves A, Zanobetti A et al (2005) Diabetes enhances vulnerability to particulate air pollution – associated impairment in  vascular reactivity and endothelial function. Circulation 111: 2913–2920 47. Bartoli CR, Wellenius GA, Diaz EA et  al (2009) Mechanisms of inhaled fine particulate air pollution-induced arterial blood pressure changes. Environ Health Perspect 117:361–366 48. Hamade AK, Tankersley CG (2009) Interstrain variation in cardiac  and respiratory adaptation to repeated ozone and particulate matter exposures. Am J Physiol Regul Integr Comp Physiol 296: R1202–R1215 49. Hazucha MJ, Bates DV, Bromberg PA (1989) Mechanism of action of ozone on the human lung. J Appl Physiol 67:1535–1541 50. Beckett WS, McDonnell WF, Horstman DH, House DE (1985) Role of the parasympathetic nervous system in acute lung response to ozone. J Appl Physiol 59:1879–1885 51. Folinsbee LJ, Silverman F, Shephard RJ (1977) Decrease of maximum work performance following ozone exposure. J Appl Physiol 42:531–536 52. Gong H Jr, Bradley PW, Simmons MS, Tashkin DP (1986) Impaired exercise performance and pulmonary function in elite cyclists during low-level ozone exposure in a hot environment. Am Rev Respir Dis 134:726–733 53. Foxcroft WJ, Adams WC (1986) Effects of ozone exposure on four consecutive days on work performance and VO2max. J Appl Physiol 61:960–966 54. Gong H Jr, Wong R, Sarma RJ et al (1998) Cardiovascular effects of ozone exposure in human volunteers. Am J Respir Crit Care Med 158:538–546 55. Dempsey JA, Wagner PD (1999) Exercise-induced arterial ­hypoxemia. J Appl Physiol 87:1997–2006 56. West JB (2008) Respiratory physiology: the essentials, 8th edn. Lippincott Williams & Wilkins, Baltimore 57. West JB (2001) Pulmonary physiology and pathophysiology: an integrated, case-based approach. Lippincott Williams & Wilkins, Baltimore

67  Air Pollution and the Pulmonary Vasculature 58. Foster WM, Silver JA, Groth ML (1993) Exposure to ozone alters regional function and particle dosimetry in the human lung. J Appl Physiol 75:1938–1945 59. Samet JM, Utell MJ (1991) The environment and the lung – changing perspectives. J Am Med Assoc 266:670–675 60. Rudež G, Meijer P, Spronk HM et al (2009) Effects of ambient air pollution on hemostasis and inflammation. Environ Health Perspect 117:995–1001 61. Hazucha MJ, Folinsbee LJ, Bromberg PA (2003) Distribution and reproducibility of spirometric response to ozone by gender and age. J Appl Physiol 95:1917–1925 62. Romieu I, Ramirez-Aguilar M, Sienra-Monge JJ et  al (2006) GSTM1 and GSTP1 and respiratory health in asthmatic children exposed to ozone. Eur Respir J 28:953–959 63. Romieu I, Sienra-Monge JJ, Ramírez-Aguilar M et  al (1998) Antioxidant supplementation and respiratory functions among workers exposed to high levels of ozone. Am J Respir Crit Care Med 158:226–232

977 64. Brook RD (2008) Cardiovascular effects of air pollution. Clin Sci 115:175–187 65. Weschler CJ (2009) Changes in indoor pollutants since the 1950s. Atmos Environ 43:153–169 66. Maynard AD (2006) Nanotechnology: assessing the risks. Nano Today 1:22–33 67. Li ZW, Carter JD, Dailey LA, Huang YCT (2005) Pollutant particles produce vasoconstriction and enhance MAPK signaling via  angiotensin type 1 receptor. Environ Health Perspect 113: 1009–1014 68. Rich DQ, Freudenberger RS, Ohman-Strickland P, Cho Y, Kipen HM (2008) Right heart pressure increases after acute increases in ambient particulate concentration. Environ Health Perspect 116: 1167–1171 69. Dominici F, McDermott A, Daniels M, Zeger SL, Samet JM (2003) Revised analyses of the national morbidity, mortality, and air pollution study, part II: mortality among residents of 90 cities. Health Effects Institute, Cambridge, pp 12–13

Chapter 68

Idiopathic Pulmonary Arterial Hypertension Jane E. Lewis and Richard N. Channick

Abstract  Idiopathic pulmonary arterial hypertension (IPAH), formerly referred to as primary pulmonary hypertension, is a disease in which there is a persistent elevation of pulmonary artery pressure without demonstrable cause. There is expert agreement that the definition of pulmonary hypertension, as was required for entry into the Primary Pulmonary Hypertension Registry of the US National Institutes of Health (NIH), and for inclusion in all recent clinical trials, is a resting mean pulmonary artery pressure greater than or equal to 25 mmHg. A pulmonary artery occlusion pressure, or left ventricular end-diastolic pressure less than 16 mmHg, in the setting of pulmonary hypertension, is compatible with a diagnosis of pulmonary arterial hypertension (PAH). This chapters will discuss the pathogenic mechanisms, pathological changes, clinical management and potential therapeutic approaches of idiopathic and heritable pulmonary arterial hypertension. Keywords  Primary pulmonary hypertension • Diagnosis • Treatment • Clinical management • Pathogenic mechanisms

1 Background Idiopathic pulmonary arterial hypertension (IPAH; formerly primary pulmonary hypertension) is a disease in which there is a persistent elevation of pulmonary artery pressure without demonstrable cause. There is expert agreement that the definition of pulmonary hypertension, as was required for entry into the Primary Pulmonary Hypertension Registry of the US National Institutes of Health (NIH) [1], and for inclusion in all recent clinical trials [2], is a resting mean pulmonary artery pressure greater than or equal to 25  mmHg. A pulmonary artery occlusion pressure, or left ventricular end-diastolic pressure less than 16  mmHg, in the setting of pulmonary

J.E. Lewis () The Transplant Unit, Papworth Hospital NHS Foundation Trust, Papworth Everard, Cambridge CB3 8RE, UK e-mail: [email protected]

hypertension, is compatible with a diagnosis of pulmonary arterial hypertension (PAH).

1.1 Epidemiology IPAH first was described at autopsy by Romberg in 1891 [3]. Patients with pulmonary hypertension of unknown cause were reported infrequently over the next 60 years, with only two cases identified from 10,000 consecutive autopsies at Massachusetts General Hospital [4]. However, the condition was recognized more commonly after the advent of cardiac catheterization. Wood [5] in 1950 detected six cases of what he called idiopathic pulmonary hypertension among 233 unselected British patients with presumed congenital heart disease, 152 of whom were catheterized. One year later, the term “primary pulmonary hypertension” was introduced by Dresdale et  al. [6] in a detailed clinical and hemodynamic study of similar patients. Since these original studies, primary pulmonary ­hypertension has been reported with increased frequency. Shepherd et al. [7] and Chapman et al. [8] described 14 patients with the condition in 1957; in 1958, Yu [9] reviewed the findings in 55 persons older than 12 years of age reported previously in the literature. Walcott et al. [10] selected 23 cases from among Mayo Clinic patients seen from 1946 to 1965. A review of 17,901 autopsies performed on persons older than 1 year of age from 1944 to 1981 at the Johns Hopkins Hospital yielded 24 cases of primary pulmonary hypertension; this amounted to a prevalence of 0.13% of all patients [11]. In 1981, a US national registry was established to determine epidemiology and follow the natural history of IPAH (known at that time as primary pulmonary hypertension). One hundred and eighty-seven patients were enrolled and followed prospectively [11]. The average age at enrollment was 36.4 ± 15 years, with a female predominance of 1.7:1. It should be noted that, although in the NIH registry 7% of “primary pulmonary hypertension” patients were over age 65, it is unclear how rigorously left-sided heart disease was excluded. Given the high frequency of left ventricular

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_68, © Springer Science+Business Media, LLC 2011

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d­ iastolic dysfunction, for example, one wonders how many of these older “primary pulmonary hypertension” patients actually were closer to diagnostic group 2. In 2002, a national registry in France enrolled 674 patients with PAH, of which 264 (39%) had IPAH. In the IPAH subgroup, the average age at diagnosis was 52 ± 15 years, with a female predominance of 1.6:1. Of all patients, 80.5% had New York Heart Association (NYHA) functional class III or IV symptoms with a mean 6-min walk test (6MWT) distance of 328 ± 115 m and 10.3% of IPAH patients had an acute vasodilator response on initial right-sided heart catheterization (RHC). The low estimate for IPAH incidence in this population was 5.9 cases per million individuals.

1.2 Pathology and Pathophysiology Wood [12] in 1958 divided pulmonary hypertension into six types: passive, as seen with increased pulmonary venous pressure due to raised left atrial or ventricular pressure; hyperkinetic, caused by increased pulmonary blood flow; obstructive, resulting from pulmonary embolism or thrombosis; ­obliterative, manifested by a reduction of pulmonary vascular capacity; vasoconstrictive, brought about by functional and presumably reversible vasospasm; and polygenic, arising in two or more of the preceding ways. Vasoconstrictive pulmonary hypertension occurred most characteristically in response to alveolar hypoxia and usually responded to the inhalation of oxygen or the injection of acetylcholine into the pulmonary artery. Reversible vasoconstriction also was seen as an added component of the passive hypertension of mitral stenosis and the hyperkinetic hypertension caused by congenital left-toright intracardiac shunts before the onset of Eisenmenger syndrome. The histologic lesions observed in these reversible kinds of pulmonary hypertension consisted of medial hypertrophy of the pulmonary artery and scant intimal hyperplasia. Also in 1958, Heath and Edwards [13] documented the pathology of hypertensive pulmonary vascular disease in a study of 67 patients with congenital heart disease and two patients with primary pulmonary hypertension. These investigators argued that the progression of lesions in these patients was so stereotyped as to allow division of the structural effects of pulmonary hypertension into six grades as follows: grade 1, medial hypertrophy of the pulmonary arteries and arterioles without intimal changes; grade 2, medial hypertrophy with cellular intimal proliferation; grade 3, medial hypertrophy, intimal proliferation, and intimal fibrosis; grade 4, progressive generalized vascular dilation and occlusion by intimal fibrosis and fibroelastosis; grade 5, appearance of dilation lesions, including veinlike branches of occluded pulmonary arteries, plexiform lesions, angiomatoid lesions, and cavernous lesions; and grade 6, necrotizing arteritis.

J.E. Lewis and R.N. Channick

Heath and Edwards’s grading system focused on the muscular pulmonary arteries 100–1,000 mm in size. Grades 1 and 2, which were characterized by medial thickening, were comparable to the reversible vasoconstrictive phase of Wood's scheme. In grades 3 and 4, intimal changes led to progressive obstruction of the vessels, which then dilated in the presence of high pressure. Some of the dilated areas evolved into microaneurysms in which there was endothelial proliferation and the formation of in situ thrombosis; these made up the characteristic plexiform lesions. In grade 5, the dilation became rigid, and hemosiderin was extruded from vessels that either burst or allowed the diapedesis of erythrocytes. Grade 6 was seen rarely and involved perivascular neutrophilic inflammation and fibrinoid necrosis. Heath and Edwards lumped the pathology of congenital heart disease and primary pulmonary hypertension together, and a large series of patients with the latter disorder was not available until 1970. In that year, Wagenvoort and Wagenvoort [14] described the morphology of the pulmonary vessels of 150 persons in whom the diagnosis of primary pulmonary hypertension had been made on clinical grounds. Despite their common diagnosis, the cases could be divided into ­several groups on histologic analysis: chronic pulmonary thromboembolism, chronic pulmonary venous hypertension, pulmonary veno-occlusive disease (PVOD), sarcoidosis, chronic bronchitis and pulmonary emphysema, pulmonary schistosomiasis, and vasoconstrictive primary pulmonary hypertension. The patients with the last disorder were the largest group, consisting of 36 men and 74 women with a mean age of 23 years and an age range of 4 days to 69 years. The lesions that Wagenvoort and Wagenvoort attributed to vasoconstrictive primary pulmonary hypertension were strikingly similar to those described earlier by Heath and Edwards and Wood. The earliest abnormalities were medial hypertrophy of the muscular pulmonary arteries and muscularization of the arterioles. These findings were especially prominent in children and were the only irregularities found in young infants. Although the medial thickening might be interpreted as a persistence of the high-resistance fetal circulation, it was considerably greater than that observed in infants of the same age. Less marked in children but apparent in all adults studied were intimal proliferation and laminar intimal fibrosis that gave an onion-skin appearance to the pulmonary arteries. This pattern was not seen in any patients with initially unsuspected chronic pulmonary thromboembolism and also was absent from an additional 20 patients with known thromboembolic disease. Dilation lesions, including the plexiform variety, were observed in 77 patients with vasoconstrictive primary pulmonary hypertension and in one patient with schistosomiasis (who also had Schistosoma mansoni ova) but was not evident in persons with chronic pulmonary thromboembolism or other disorders. Necrotizing arteritis occurred in 34 patients

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Despite the distinct pathologic findings in primary pulmonary hypertension, there is more recent evidence that the pathologic arteriopathic changes are in fact not unique to this disease and have been reported in congenital heart disease, connective tissue disease (CTD) [16], and chronic thromboembolic disease [17]. For this reason, in addition to the risk of lung biopsy in patients with pulmonary hypertension, one rarely obtains pathologic tissue prior to lung transplant or death.

2 Pathogenesis

Fig. 1  Typical advanced pulmonary arteriopathy in idiopathic pulmonary arterial hypertension (IPAH). Note proliferative changes in all three vessel layers, including intimal hyperplasia, plexiform lesions, medial hypertrophy, and adventitial fibrosis. (Images of plexiform lesions, intimal hypertrophy, and medial hypertrophy are courtesy of Jason Yuan and Grace Lin)

with chronic pulmonary thromboembolism; it never involved the veins as it does in PVOD. Wagenvoort and Wagenvoort noted that vasculitis occurred only in persons with advanced disease and probably was a result rather than a cause of pulmonary hypertension, in contrast to the true pulmonary vasculitides. They believed that the progression of lesions in primary pulmonary hypertension was entirely consistent with a vasoconstrictive mechanism. Subsequent investigators [15, 16] confirmed the findings of Wagenvoort and Wagenvoort, including the histologic distinctions between primary pulmonary hypertension, chronic pulmonary thromboembolism, and PVOD. Typical arteriopathic muscular arteries are shown in Fig. 1.

A dramatic change has taken place over the last several years in our thinking regarding the pathogenesis of IPAH. The “paradigm” has shifted from one of vasoconstriction to one of growth and proliferation. There are several lines of evidence suggesting that primary pulmonary hypertension develops as a result of abnormal proliferation of vascular smooth muscle cells affecting all three layers of the vessel wall and leading to intimal hyperplasia, medial hypertrophy, and adventitial proliferation. What initiates this abnormal growth is not entirely known, but there are several clues. The concept of genetic predisposition toward growth and proliferation has recently emerged. Mutations in the bone morphogenetic protein (BMP) receptor 2 (BMPR2) gene have been reported in patients with the familial form of primary pulmonary hypertension [18]. This gene contributes to the apoptotic process through a complex series of messenger proteins, as part of the transforming growth factor b family of genes. Thus, emerges the possibility that pulmonary arteriopathy is a failure of normal apoptosis. Some investigators have even referred to primary pulmonary hypertension as “cancer of the pulmonary artery.” Although this is an attractive hypothesis, the story is not that simple. For one, the presence of a BMPR2 mutation is not always associated with the development of primary pulmonary hypertension. It is likely that another genetic or acquired insult is required to initiate the arteriopathic process. Defects in a specific voltagegated potassium ion channel, the Kv1.5 channel, have been found in the pulmonary artery smooth muscle cells (PASMCs) from patients with primary pulmonary hypertension [19]. A defect or deficiency of this channel allows excess calcium to enter the cell and thus promotes both cell contraction and cell growth. Overexpression of the serotonin transporter has recently been described in patients with primary pulmonary hypertension [20]. This genetic defect might lead to increased internalization of serotonin and subsequent smooth muscle cell growth. Recent interest has focused on the role of impaired apoptosis in the development of proliferative arteriopathy in IPAH. It is clear that functional mutations seen in the BMPR2 gene with resultant abnormal downstream signaling result in impaired apoptosis [21–23].

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J.E. Lewis and R.N. Channick

In addition to potential genetic defects, several abnormalities in endothelial cell function have been found, many of which are likely a result of some vascular insult. The basis for currently approved therapeutics is the deficiency in prostacyclin and nitric oxide release and the excess of endothelin-1 and its receptor expression in patients with pulmonary hypertension [24–27]. The complex cellular and molecular processes contributing to IPAH are shown in Fig.  2: a rise in cytosolic Ca2+ concentration in PASMCs due to (a) decreased

Kv channel activity (Å in Fig. 2) and membrane depolarization which opens voltage-dependent Ca2+ channels, (b)  upregulated transient receptor potential channels that participate in forming receptor-operated and store-operated Ca2+ channels (Ç in Fig. 2), and (c) upregulated membrane receptors (e.g. serotonin, endothelin, or platelet-derived growth factor receptors) (É in Fig. 2) and their downstream signaling cascades causing pulmonary vasoconstriction, stimulating PASMC proliferation, and inhibiting the

Fig. 2  Possible pathogenic factors in IPAH (see the text for an explanation [78]). AVD apoptotic volume decrease, CaM calmodulin, DAG diacylglycerol Em, membrane potential, EGF epidermal growth factor, GPCR G-protein-coupled receptor, HHV juman herpesvirus, IP3 ­inositol

1,4,5-trisphosphate, MLC myosin light chain, MLCK myosin light chain kinase, PDGF platelet-derived growth factor, PKC protein kinase C, PLC phospholipase C, ROS reactive oxygen species, RTK receptor tyrosine kinase, SR sarcoplasmic reticulum

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BMP-signaling pathway that leads to antiproliferative and proapoptotic effects on PASMCs. Dysfunction of BMP signaling due to BMPR2 mutation and BMP receptor II/BMP receptor Ia downregulation (Ñin Fig. 2), and inhibition of Kv channel function and expression (Åin Fig. 2), attenuate PASMC ­apoptosis and promote PASMC proliferation. Increased angiopoietin-1 synthesis and release (Öin Fig.  2) from PASMCs enhance serotonin production and downregulate BMP receptor Ia in pulmonary artery endothelial cells and further enhance PASMC contraction and proliferation, whereas inhibited NO and prostacyclin synthesis (Üin Fig. 2) in pulmonary artery endothelial cells would attenuate the endothelium-derived relaxing effect on pulmonary arteries and promote sustained vasoconstriction and PASMC proliferation. Increased activity and expression of the serotonin transporter (áin Fig.  2) serve as an additional pathway to stimulate PASMC growth via the mitogen-activated kinase pathway. Activation of G-protein-coupled receptors also increases RhoA (Rho) activity, activates Rho kinase, inactivates Ca2+-independent myosin light chain phosphatase, and causes smooth muscle contraction (àin Fig. 2). Furthermore, exogenous viral (and/or bacterial) infection and inflammation (â in Fig.  2) may contribute to vasoconstriction and vascular medial hypertrophy by increasing elastase activity in extracellular matrix, which subsequently increases growth factor production and stimulates epidermal growth factor receptor activity. In addition, a variety of splicing factors, transcription factors, protein kinases, extracellular metalloproteinases, and circulating growth factors would all contribute to mediating the phenotypical transition of normal cells to contractive or hypertrophied cells and to maintain the progression of PAH (Fig. 2).

3 Diagnosis PAH is defined as a mean pulmonary artery pressure of 25 mmHg or greater at rest or 30 mmHg or greater with exercise, with a pulmonary capillary wedge pressure of 15 mmHg or lower during RHC [28]. IPAH is a diagnosis of exclusion. To conclude that a patient has IPAH, all other diagnoses within the classification system must be excluded, including associated conditions or exposures, significant left-sided heart disease, significant lung or respiratory disease, and thromboembolic disease.

3.1 Symptoms In the NIH national registry, in patients with IPAH enrolled between 1981 and 1985, dyspnea was the most frequent presenting symptom (60%) [1]. At the time of enrollment into the

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registry, 98% of patients complained of dyspnea. Other common symptoms at initial presentation included fatigue (19%), chest pain (7%), near syncope (5%), syncope (8%), leg edema (3%), and palpitations (5%). The mean time from symptom onset to diagnosis was 2.03 ± 4.9 years. In the most recent evaluation of the data from the REVEAL registry, dyspnea on exertion was the most common presenting symptom (83%), with other common symptoms including fatigue (29%), chest pain and/or discomfort (20%), presyncope and syncope (20%), dizziness and/or light-headedness (14%), cough (13%), dyspnea at rest (11%), and other symptoms (27%) [29]. The median time from symptom onset to RHC was 14 months.

3.2 Signs Common physical exam findings in IPAH include an increased second heart tone (P2) present in 93% of patients, a right-sided S3 (23%), and a right-sided S4 (38%). Other signs include an early systolic ejection click, midsystolic ejection murmur, a palpable parasternal lift, and a prominent “a” wave on jugular venous wave form examination. Signs ­consistent with more advanced disease include diastolic ­pulmonary regurgitation murmur, holosystolic tricuspid regurgitation murmur, signs of right ventricular failure, hypotension, and cool extremities, which indicate a significantly reduced cardiac output and peripheral vasoconstriction. Rich et al. reported an S3 was ­significantly associated with increased right atrial pressure (13 compared with 9 mmHg) and decreased cardiac output (1.8 vs. 2.4 L/min/ m2). Tricuspid regurgitation was evident on physical exam in 40% of patients and was also associated with increased right atrial pressure and decreased cardiac ­output. Cyanosis was present in 20% of patients and peripheral edema in 32%.

3.3 Diagnostic Testing When a clinical suspicion for pulmonary hypertension exists, further diagnostic testing is indicated. Routine chest radiography and electrocardiography are often performed. However, the sensitivity and specificity of the chest X-ray to detect IPAH are unknown. Prominent main pulmonary artery and hilar vessels with decreased peripheral vessels (“pruning”) are common findings in patients with IPAH (Fig. 3). However, Rich et al. reported that 6% of chest X-rays were reported as normal [1]. The absence of pruning should not be misinterpreted as excluding IPAH. However, chest radiographapy is useful in ruling out coexisting lung disease. Similarly, the electrocardiogram lacks sensitivity as a screening tool. In a study of 61 patients with IPAH or PAH associated with CTD and a mean pulmonary artery pressure

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Fig. 3  Typical chest radiograph in IPAH. Note clear lung fields, cardiomegaly, and decrease in retrosternal air space on lateral film, indicating right ventricular enlargement

of more than 50 mmHg, eight patients (13%) had a completely normal electrocardiogram. The sensitivity of right-axis ­deviation (defined as QRS axis greater than 100°) and right ventricular hypertrophy was 73 and 55%, respectively [30]. In patients suspected of having PAH, Doppler echocardiography is the most common screening tool. In patients without pulmonary outflow tract obstruction, right ventricular systolic pressure (RVSP) is equivalent to pulmonary artery systolic pressure. The tricuspid regurgitation jet velocity (v) is necessary to calculate RVSP. Using the modified Bernoulli equation, one calculates RVSP by CVP + 4v2, where CVP is central venous pressure [31]. Tricuspid regurgitation jets can be evaluated in between 39% [32] and 86% [33] of patients undergoing echocardiography and had a sensitivity of 90% and a specificity of 75% in one study evaluating PAH associated with systemic sclerosis. Echocardiography can provide alternative explanations for elevated pulmonary artery pressure by assessing left atrial size, left ventricular size and function, and valvular abnormalities. The accuracy of echocardiography in quantifying pulmonary artery systolic pressure is questionable. The diagnosis of PAH requires RHC to determine the pulmonary artery pressures, while providing data on disease severity and excluding other causes of pulmonary hypertension, including intracardiac shunts and left-sided heart disease. In 1958, Wood defined pulmonary hypertension as a pulmonary artery pressure greater than 30/15 mmHg, which was the upper limit of normal in 60 control patients [13]. The current definition of PAH is a mean pulmonary artery pressure of more than 25 mmHg at rest, with a pulmonary capillary wedge pressure (15 mmHg or lower) [28]. Virtually identical hemodynamic and clinical findings may be encountered in the rare form of PAH termed “pulmonary veno-occlusive disease” (PVOD). Like IPAH, PVOD appears to be a morphologic rather than an etiologic entity. In addition, PVOD tends to afflict infants, children, and

young adults, although patients older than 60 years of age have been reported. However, in the adults with PVOD who have been reported, there is a slight male preponderance. Also, a familial occurrence of PVOD has been noted. PVOD has been associated with viral syndromes, toxin exposure, and chemotherapy. The characteristic histologic feature of PVOD is obstruction of pulmonary venules and veins by intimal fibrosis; intravascular fibrous septa, which usually are considered pathognomonic of recanalized thrombi, are nearly always present [34]. These findings have led to the speculation that thrombosis is an essential pathogenetic mechanism in most cases, but the factors causing or contributing to thrombus formation are completely unknown. Of considerable interest is the observation that narrowing or obliteration of pulmonary arteries is found in approximately half the cases of PVOD [25]. The morphologic appearance of the arterial lesions indicates that they also were produced by organization of intravascular thrombi and that the abnormalities were not merely secondary to pulmonary venous obstruction with attendant medial hypertrophy and arterial hypertension. These observations raise the ­question whether there are two variations of the same basic ­disorder: the classic form of PVOD, in which the arteries are spared, and a generalized form, in which both arteries and veins are involved. PVOD cannot be distinguished with ­certainty from IPAH by RHC unless the pulmonary capillary wedge pressure is elevated. Apart from the difficulties of successfully wedging a catheter in patients with pulmonary hypertension, whether the wedge pressure is elevated depends on whether the venous obstruction is in the venules or the large veins. This is because the pressure actually measured at the tip of a catheter positioned in an obstructed ­pulmonary artery, by either wedging or inflating a balloon, is the pressure in the next freely communicating downstream vessel. Accordingly, if venules are occluded, as is often the case in

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PVOD, the next freely communicating vessel may be a vein in which the pressure is normal. In contrast, if (large) veins are the sites of occlusion, then the pulmonary venous pressure is likely to be elevated proximally and to be detectable at catheterization. Indeed, the wedge pressure may vary in different sites within the lung in patients with PVOD. The chest radiograph may show signs of pulmonary hypertension with prominent Kerley B lines in the absence of other signs of left-sided heart failure, and this is often the most useful clue to the diagnosis in a patient with unexplained severe pulmonary hypertension. CT scanning may reveal interlobular septal thickening, pleural effusions, and enlarged pulmonary arteries. The perfusion lung scan may show defects suggestive of thromboembolism but with unremarkable pulmonary angiograms [35]. In addition, patients with PVOD may develop acute pulmonary edema in response to pharmacologic agents that reduce upstream pulmonary vascular resistance and increase cardiac output, such as prostacyclin. This phenomenon probably is caused by an increase in pulmonary blood volume in the face of ­downstream ­vascular obstruction. A recent analysis of PVOD cases ­suggested that bronchoalveolar lavage could be useful, as patients with PVOD almost always had increased hemosiderin-laden macrophages in bronchoalveolar lavage fluid [36].

3.4 Acute Vasodilator Testing The purpose of acute pulmonary vasodilator testing is to identify the rare IPAH patient who is highly vasoreactive and might achieve a long-term benefit from calcium channel antagonists (CCBs). In one study evaluating the long-term outcome of IPAH, 12.3% of patients were acute responders, but on long-term follow-up only 6.3% of the total population had a long-term favorable response to CCBs [37]. Patients in  the favorable group generally had demonstrated nearnormalization of pulmonary artery pressure during the acute vasodilator test (see details later). Therefore, in current guidelines, a significant response (i.e. one that might indicate favorable CCB response in the long term), 10  mmHg or greater decrease in mean pulmonary artery pressure to 40 mmHg or less with an unchanged or increased cardiac output [38]. Accepted medications used to assess for a vasodilator response include inhaled nitric oxide, adenosine, and prostacyclin.

4 Treatment and Prognosis IPAH is now a treatable disease. Clear-cut short-term and long-term benefits are seen with currently available therapies. To optimize a patient’s outcome, a comprehensive

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­ edical approach is essential. Once the diagnostic process in m a patient with pulmonary hypertension is complete, and the patient is characterized as having IPAH (WHO group 1), therapy should be initiated. But many questions arise regarding IPAH therapy. What are the current goals and expected outcomes of IPAH therapy? Which drug should be used first? When should another therapy be added? What are the data supporting use of different therapies? Should more than one therapy be used and, if so, in what order? When should transplantation be considered? Are there published guidelines in which experts have evaluated the quality of evidence and issued a “grade” for the therapy? Therapy for IPAH may be subdivided into “supportive” or “conventional” therapies, defined as empiric treatments or recommendations for which there are no prospective, randomized controlled data, and “specific” or “targeted” therapies, which have been tested and approved by regulatory authorities for the treatment of PAH.

4.1 Supportive Therapies 4.1.1 Exercise and Physical Activity There is no evidence-based guidance regarding physical activity or exercise in PAH. Patients should avoid activities which lead to undue symptoms such as severe dyspnea, chest pain, light-headedness, and syncope. However, low to moderate levels of exercise to prevent deconditioning and improve mental outlook are appropriate. One recent study demonstrated that exercise and respiratory training is safe and results in measurable improvements in subjective and objective parameters [39]. Mereles et al. randomized 30 patients (23 PAH, 7 chronic thromboembolic pulmonary hypertension) to exercise training versus no change in treatment. In addition to being safe, patients in the exercise-training group had improved quality-of-life scores, functional class, maximal oxygen consumption, and increased exercise capacity (assessed by 6MWT) of 111 m (placebo-corrected).

4.1.2 Avoidance of Altitude Hypobaric hypoxia causes pulmonary vasoconstriction and thus can worsen the pulmonary hypertension in PAH patients. Patients with PAH not infrequently report symptomatic worsening when traveling to elevations above 3,000 ft. It is therefore generally recommended that patients flying on commercial airliners (pressurized to approximately 5,000–7,500 ft) or traveling to elevations above 5,000 ft use supplemental oxygen. Not surprisingly, patients with severe PAH residing at high elevations often improve if they move to sea level.

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4.1.3 Avoidance of Pregnancy Pregnancy, and especially delivery, are extremely risky in patients with PAH [40]. Although there are rare case reports of patients managed with epoprostenol and undergoing successful pregnancies and deliveries [41, 42], it is strongly recommended that women of childbearing potential use appropriate methods of birth control to avoid pregnancy. In terms of which birth control method is preferable, no consensus exists. Some experts recommend avoiding estrogencontaining hormonal contraceptives. Depo-Provera is an effective, convenient agent commonly used in PAH patients. It should be noted that in patients on an endothelin receptor antagonist such as bosentan or ambrisentan, an additional barrier method of birth control should be used.

4.1.4 Warfarin There is strong rationale for the use of anticoagulants in PAH. Many of the endothelial cell abnormalities that predispose patients to pulmonary arteriopathy also increase thrombosis. The presence of heart failure and/or the presence of an indwelling central venous catheter are independent risk factors for thromboembolic events, which are poorly tolerated by patients with an already marginal pulmonary vascular reserve. In addition, microscopic thrombotic lesions in the pulmonary vasculature are well appreciated in PAH patients. Warfarin is the anticoagulant most frequently used in patients with PAH. In clinical trials with PAH drugs, approximately 50–85% of patients are on anticoagulants at study entry. However, anticoagulation has risks (i.e. bleeding) as well as the need for frequent monitoring. What are the data to justify the use of warfarin in PAH? Three studies have examined the effects of warfarin in IPAH or anorexigen-induced PAH [43–45]. All three were retrospective analyses with some patients on warfarin. All three demonstrated better survival in patients using ­warfarin than those not anticoagulated. In an early study by Fuster et al. 120 patients with a clinical diagnosis of IPAH were studied [43]; anticoagulation improved survival at 3 years (49% vs. 21%). However, of note, the overall 5-year survival for the entire cohort was only 21%. In addition, 57% of the patients in whom autopsy was performed had predominantly thrombotic lesions, raising the question of whether some of these “IPAH” patients may have been misdiagnosed and actually had chronic thromboembolic pulmonary hypertension. In the Rich et al. [44] study, which primarily looked at the long-term benefit of CCBs in IPAH, a survival benefit with warfarin was noted predominantly in patients who were “nonresponders” to CCBs (Fig.  4). The third retrospective study, by Frank et al. compared survival in aminorex-induced

Fig.  4  Survival in patients with IPAH based on acute vasoreactivity and treatment with warfarin. In patients who were not vasoreactive, warfarin was associated with a modest survival advantage versus nonreactive patients who were not on warfarin

PAH and IPAH with and without anticoagulation [45]. In both groups, the use of oral anticoagulant therapy was ­associated with better survival, with the longest mean ­survival observed in anticoagulated aminorex-PAH patients (8.3 years vs. 6.1 years for nonanticoagulated aminorex-induced PAH patients). Although these three retrospective studies all suggest a survival benefit with warfarin in PAH, it is important to note that none of the studies were conducted during the “modern” era of effective, targeted PAH therapies, which are already associated with better survival than that seen in these “historical” studies. Whether warfarin further improves outcome in patients on these therapies (e.g. endothelin receptor antagonists, phosphodiesterase-5 inhibitors, prostanoids) remains unclear. Despite the serious limitations in the existing data, published guidelines recommend, with a “B” grade, that patients with IPAH be treated with warfarin [38]. There is less guidance regarding the use of anticoagulation in other forms of PAH, such as that associated with congenital systemic-topulmonary shunts or associated with CTDs. There may be specific cases where warfarin would be indicated, such as in patients with advanced heart failure, indwelling central venous catheters, or erythrocytosis. On the other hand, if there are patients at increased risk for bleeding (thrombocytopenia, history of hemoptysis, or gastrointestinal bleeding) withholding anticoagulation, given the limited data, may be prudent.

4.1.5 Supplemental Oxygen The benefits of supplemental oxygen in PAH patients, unlike patients with pulmonary hypertension associated

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with lung diseases such as COPD [46, 47], are not clear. In fact, most PAH patients are not hypoxemic, at least at rest. Mild hypoxemia, when present, is likely on the basis of reduced mixed venous oxygen saturation caused by low cardiac output with mild ventilation/perfusion inequality. The presence of more profound hypoxemia in a PAH patient should raise suspicion for underlying parenchymal lung disease, systemic-to-pulmonary shunting, PVOD, pulmonary capillary hemangiomatosis, or pulmonary arterial–venous malformations as seen in patients with PAH associated with Osler–Weber–Rendu disease. Although oxygen is a pulmonary vasodilator, there have been no long-term studies supporting its efficacy. However, the consensus is that if systemic arterial oxygen pressure is less than 60 mmHg or systemic arterial oxygen saturation is less than 90% at rest, supplemental oxygen is indicated. One exception to this approach is in patients with Eisenmenger syndrome, with hypoxemia due to right-to-left shunting. In this group, the use of supplemental oxygen remains controversial [48, 49]. There is no general agreement about whether exercise-only systemic arterial oxygen desaturation warrants oxygen supplementation. In addition, the “stigma” of nasal cannulae for a PAH patient often limits compliance outside the home.

4.1.6 Diuretics Diuretics have long been a mainstay of therapy for heart failure, including right ventricular failure. Both total body and intravascular volume overload are common in PAH patients. In pivotal trials of PAH drugs, most patients entered the studies on chronic diuretic therapy [50, 51]. In addition to causing symptomatic peripheral edema, volume overload of the right ventricle can cause compression of the left ventricle, and can contribute to decreased cardiac output and prerenal azotemia. Thus, it is a common observation that, in decompensated PAH patients, aggressive diuresis leads to clinical and physiologic improvements. Despite the benefits of diuretics in PAH patients, there have been no controlled studies to guide the clinician in using these agents. A loop diuretic is frequently employed first. Although furosemide is often the loop diuretic of choice, there is some evidence that torsemide might be more efficacious without increased side effects [52]. In addition, in patients with marked extravascular fluid accumulation and poor intestinal absorption, intravenous diuretic therapy is frequently needed. Anti-aldosterone drugs (e.g. spironalactone) are commonly combined with loop diuretics in PAH patients. Whether data suggesting a morbidity and mortality benefit of spironolactone in left-sided heart failure [53] can be extrapolated to right-sided heart failure is not known.

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In some cases, addition of a thiazide diuretic to the regimen is appropriate. The combination of metolazone and furosemide has been found by the authors to be effective in effecting a brisk diuresis. However, marked hypokalemia can occur with this regimen. Although it is possible that, in some PAH patients, the right ventricle is preload-dependent and therefore overdiuresis can be detrimental, this has not been the authors’ experience. More often, aggressive diuresis (1–3 L negative fluid balance per day) leads to improvement in renal function and blood pressure, consistent with improved cardiac output.

4.1.7 Calcium Channel Antagonists In 1958, Paul Wood first defined the clinical entity of pulmonary hypertension with reference to the “vasoconstrictive factor” [13]. It is not surprising, then, that a search for pulmonary vasodilators as effective therapies ensued. Agents including phentolamine [54], tolazine [55], captopril [56], and hydralazine [57] were evaluated in uncontrolled reports. The results, although variable, were not overwhelmingly favorable. No systematic studies of these medications were carried out. Out of the myriad of oral antihypertensive agents CCBs emerged. Ostensibly, this class of agents “made sense” for treating pulmonary hypertension. CCBs have acceptable side-effect profiles and are potent pulmonary vasodilator agents as well as systemic vasodilator agents. The role of intracellular cytosolic calcium in the vasoconstriction of PASMCs was well established. Thus, blocking influx of calcium into the cells seemed desirable. In a highly quoted paper, Rich et al. described favorable survival in a subgroup of IPAH patients treated with either diltiazem or nifedipine [44]. In that study, patients manifesting acute pulmonary vasoreactivity with CCBs, defined as an acute decrease in mean pulmonary artery pressure and pulmonary vascular resistance of at least 20%, had a 5-year survival of 94%; in contrast, patients who did not have an acute response had only a 55% 5-year survival. In addition, predicted survival for the “acute responders,” using an equation based on hemodynamics at the time of diagnosis, was significantly worse than the observed survival. Although it was not a placebo-controlled study, these data suggested a benefit with CCBs in some IPAH patients. The Rich et al. study, although seminal, likely led to overuse of CCBs, not only for IPAH, but also for other forms of PAH. CCBs are not selective pulmonary vasodilators. In addition, CCBs have potential negative inotropic effects. Thus, in patients with minimal or no acute pulmonary vasoreactivity, the negative effects of CCBs can become predominant, with potential for catastrophic consequences.

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A subsequent, large retrospective study by Sitbon et al. has further narrowed the role of CCBs in IPAH patients [37]. In that study, 557 IPAH patients were tested for acute pulmonary vasoreactivity using either intravenously administered prostacyclin (e.g. epoprostenol) or inhaled nitric oxide. Seventy patients (approximately 13%) had an acute response (at least 20% fall in mean pulmonary artery pressure and pulmonary vascular resistance), and were treated with CCBs. However, only half of those “acute responders” (approximately 7% of the total) did “well” long term on CCBs, defined as being alive and in functional class I or II at 5-year follow-up. The long-term survivors had less severe disease as assessed by hemodynamics at the baseline and had reached a lower mean pulmonary artery pressure with acute vasodilator challenge than the long-term CCB failures (33 vs. 46 mmHg). In other words, rather than the percent drop in mean pulmonary artery pressure during an acute test, the absolute mean pulmonary artery pressure reached appeared to be better in defining the patients that would benefit long term with CCBs. These data have been codified into evidence-based guidelines [38], which define potential CCB candidates as IPAH patients in whom, during an acute ­pulmonary vasoreactivity test, the mean pulmonary artery ­pressure decreases by at least 10  mmHg to a level below 40  mmHg, with no decrease in cardiac output. The method for performing acute pulmonary vasoreactivity testing varies among pulmonary hypertension centers. Most frequently, one of three short-acting pulmonary vasodilators is used in the cardiac catheterization laboratory: inhaled nitric oxide, intravenously administered adenosine, or ­intravenously administered epoprostenol. Using a shortacting agent prevents refractory systemic hypotension that could result when a PAH patient with minimal pulmonary vasoreactivity is given a systemic vasodilator. A distinct advantage of inhaled nitric oxide is the absence of systemic

Fig. 5  Three abnormal endothelial mediator pathways, which are targeted by current therapies. AA arachidonic acid, ET endothelin, ETR endothelin receptor, NO nitric oxide, PC prostacyclin, PDE5 phosphodiesterase-5, PGI2 prostaglandin I2

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hemodynamic effects, the very rapid “on/off” properties of the drug (i.e. half-life 20 s), and the absence of side effects. With inhaled nitric oxide, an acute pulmonary vasoreactivity test with repeat hemodynamic measurements can be accomplished in less than 20 min.

4.2 Targeted Therapies PAH-specific therapies have been available since 1995, when intravenously administered epoprostenol was approved by the Food and Drug Administration (FDA), on the basis of the first prospective randomized controlled trial done in PAH. Since then, an additional five therapies have been approved for PAH: subcutaneously and intravenously administered treprostinil, inhaled iloprost, the oral endothelin receptor antagonists bosentan and ambrisentan, and the phosphodiesterase-5 inhibitor sildenafil. These therapies were developed to offset the imbalance in endothelium-derived mediators seen in PAH: excessive endothelin-1 production, deficient prostacyclin, and abnormal nitric oxide production (Fig. 5).

4.2.1 Prostacyclin Analogues Epoprostenol (Flolan®) was first approved by the FDA on September 20, 1995 for the indication of “long-term intravenous treatment of primary pulmonary hypertension in NYHA Class III and Class IV patients.” Continuous intravenous infusions of prostacyclin (prostaglandin I2, epoprostenol) have been used, either as a primary mode of treatment or as a bridge to transplantation, and produce sustained improvement in hemodynamics and exercise tolerance as well as prolonged survival. In this first randomized,

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p­ rospective study [50], 81 patients with NYHA class III or class IV symptoms despite treatment with conventional therapy, which included oral vasodilators, digoxin, diuretics, and supplemental oxygen, were randomized to either continuous intravenous infusion of epoprostenol plus conventional therapy or conventional therapy alone for 12 weeks. The primary outcome was the change in exercise capacity, as measured by the 6MWT. Secondary outcomes included change in hemodynamics, quality of life, NYHA functional class, and survival. At 12 weeks, the 6MWT distance improved in the continuous intravenous infusion of epoprostenol group by 32 m and decreased by 15 m in the conventional therapy group. Mean pulmonary artery pressure, cardiac index, and pulmonary vascular resistance significantly improved in the epoprostenol group. There were also significant improvements in the epoprostenol group when measuring quality of life and NYHA functional class. Eight patients died during the 12-week study and all were in the conventional treatment arm. Notably, this is the only PAH trial ever to show, compared with a control group, a beneficial effect on survival. Follow-up studies have confirmed the long-term benefits of epoprostenol on survival compared with historical controls, with 5-year survival estimates of greater than 60% in treated patients [58], a great improvement from the median survival of less than 3 years in the “pre-epoprostenol” era. Interestingly, the benefits of epoprostenol may be progressive. In one report of four patients [59], chronic epoprostenol infusions resulted in normalization or near-normalization of pulmonary hemodynamics, allowing transitioning to oral medications Despite its benefits, chronic use of epoprostenol is complex and requires an indwelling central venous catheter, continuous infusion pump, and daily preparation of the medication. In addition, numerous side effects, including jaw claudication, leg and foot pain, diarrhea, rash, and weight loss occasionally with ascites, occur. The complexities of dosing prostacyclin and assessing response to the therapy obligates a dedicated team, typically present only at large pulmonary hypertension centers. Treprostinil sodium (Remodulin®) is a tricyclic benzindene analogue of prostacyclin that is chemically stable at room temperature and has a half-life of approximately 4-h. In a double-blind randomized controlled trial, 470 patients with IPAH (58%), PAH-associated with CTD, or PAHassociated with congenital systemic-to-pulmonary shunts with WHO functional class II, III, or IV were randomized to continuous subcutaneous administration of treprostinil or a placebo for 12 weeks [60]. As with other PAH trials, the primary outcome measure was exercise capacity as defined as placebo-corrected improvement in 6MWT distance. In addition, “reinforcing” principal end points included a composite score assessing signs and symptoms of pulmonary hypertension, dyspnea fatigue rating, and clinical worsening

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(number of patients dying, undergoing lung transplantation, or discontinuation of the study drug). The placebo-corrected improvement in 6MWT distance was 16 m (p = 0.006). Thirteen patients in the treprostinil group and 16 patients in the placebo group died, required lung transplantation, or discontinued therapy secondary to clinical worsening. Borg dysnpea scores and quality-of-life measures were significantly improved in the treprostinil group compared with the group receiving the placebo. Patients treated with treprostinil had improved pulmonary hemodynamics, including significant improvement in mean right atrial pressure, mean pulmonary artery pressure, cardiac index, pulmonary vascular resistance, and mixed venous oxygen saturation. A review of the treprostinil subset analysis suggested that significantly higher doses than with epoprostenol were needed to achieve efficacy. Infusion-site difficulties were the major adverse event, with 200 patients (85%) in the treprostinil group versus 62 patients (27%) in the placebo arm reporting site pain. Subsequent to approval of subcutaneously administered treprostinil, the intravenous route was approved on the basis of bioequivalence. There has not been a prospective randomized controlled trial of intravenously administered ­treprostinil, but two uncontrolled reports have been published [61, 62]. Iloprost is a stable prostacyclin analogue approved by the FDA in the inhaled formulation. Inhaled iloprost is delivered via an ultrasonic nebulizer directly to the lungs. It received FDA approval in December 2004 on the basis of the ­aerosolized iloprost randomized study (AIR) trial, a doubleblind, placebo-controlled trial that randomized 203 patients with NYHA class III or class IV IPAH (50%) or PAH associated with anorexigens, scleroderma, or chronic thromboembolic pulmonary hypertension to receive 2.5 or 5 mg inhaled iloprost versus a placebo six or nine times per day for 12 weeks [63]. The primary outcome was a combined end point: At least 10% improvement in 6MWT distance in the treatment group and improvement in at least one NYHA functional class was found. Secondary outcome measures included a change in the 6MWT distance and NYHA class, dyspnea index scores, pulmonary hemodynamics, and quality-of-life and clinical worsening (defined as deterioration, death, and/or need for transplantation). Seventeen patients in the iloprost group versus five in the placebo arm met the combined primary end point, which was statistically significant with an estimated odds ratio of 3.97. Cardiac output, systemic arterial oxygen saturation, and mixed venous oxygen saturation as measured at “peak” following an inhalation increased significantly and the pulmonary vascular resistance and right atrial pressure decreased significantly in the iloprost group at 12 weeks as compared with the placebo group. Adverse events included syncope, flushing, and jaw pain in the iloprost group.

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4.2.2 Endothelin Receptor Antagonists In humans, two endothelin-1 receptor subtypes are found: ETA and ETB. Antagonism of one or both receptors has been shown to be an effective treatment strategy for IPAH, with two FDA-approved therapies, bosentan and ambrisentan. A third agent, sitaxsentan, was not approved in the USA but is available in Europe and in other countries. Bosentan is a potent, nonpeptide, oral ETA and ETB receptor antagonist, with higher affinity for the ETA subtype ­receptor. Bosentan received FDA approval in November 2001 for patients with WHO functional class III or IV PAH. The first double-blind, placebo-controlled trial randomized 32 patients with IPAH (84%) or pulmonary hypertension associated with scleroderma with NYHA class III to bosentan or a placebo for 12 weeks [64]. The primary end point was the placebo-corrected change in 6MWT distance, with secondary end points including a change in pulmonary hemodynamics, WHO functional class, Borg dyspnea index, and clinical worsening. The placebo-corrected improvement in 6MWT distance was 76 m in favor of the bosentan group. In ­addition, pulmonary hemodynamics, especially cardiac index and pulmonary vascular resistance, were favorably affected by bosentan (Fig.  6). There were asymptomatic increases in the levels of liver aminotransferases in two patients on bosentan, but these returned to the baseline without discontinuing or changing the dose. The larger BREATHE-1 (bosentan randomized trial of endothelin antagonist therapy) trial, which randomized 213 patients with IPAH (70%) and pulmonary hypertension associated with CTD with WHO functional classes III and IV to a placebo or bosentan at 125 or 250 mg twice daily, confirmed the efficacy and safety of bosentan over 16 weeks [51]. At 16 weeks, a placebo-corrected 6MWT ­distance improvement of 44  m was noted (p < 0.001) (Fig.  7a). There were also improvements in the Borg

Fig.  6  Hemodynamic effects of bosentan in international society of heart and lung transplantations (PAH) patients. Bosentan-treated patients had significantly improved cardiac output, pulmonary vascular resistance, and pulmonary arterial pressure compared with patients receiving a placebo. (Data from Galiè et al. [67])

J.E. Lewis and R.N. Channick

d­ yspnea score and the time to clinical worsening in both bosentan groups. Increases in the level of liver aminotransferases greater than 8 times the upper limit of normal were again noted in the bosentan group and were dose-dependent with two patients in the 125-mg group and five patients in the 250-mg group. In addition to the above “pivotal” trials of bosentan, a recently published randomized controlled trial of bosentan in PAH patients with less functional impairment (WHO class II), the EARLY trial, demonstrated a benefit in reducing pulmonary vascular resistance and preventing clinical worsening at 6 months. No statistically significant effect on the 6MWT was seen, although the baseline walk distance was greater than 400  m, confirming that this cohort had better baseline exercise capacity [65]. Ambrisentan is a specific ETA receptor antagonist approved for PAH, functional classes II and III at doses of 5 or 10 mg once daily. Following a phase 2 dosing study showing favorable pulmonary hemodynamic effects [66], two ­randomized controlled trials (ARIES 1 and ARIES 2) of ambrisentan (ARIES 1: 5  mg, 10  mg, placebo; ARIES 2: 2.5 mg, 5 mg, placebo) were performed, enrolling a total of 394 patients [67]. Both trials achieved the primary end point of placebo-corrected improvement in 6MWT distance (Fig. 7b). In ARIES 2, there was a significant improvement in the time to clinical worsening in the treatment group as compared with the placebo group. There was a trend toward improvement in the time to clinical worsening in the ARIES 1 study, but it was not statistically significant (p = 0.307). WHO functional class improvement was significant in ARIES 1 and there was a trend toward improvement in ARIES 2 but it did not reach statistical significance (p = 0.117); 298 patients were enrolled and followed in the long-term extension study over 48 weeks. Eighteen patients required additional therapies (prostanoids or phosphodiesterase-5 inhibitors). Of the 280 patients who continued on ambrisentan monotherapy, the improvement in 6MWT distance at 12 weeks was 40  m and was maintained at 39  m. Although there were no patients with elevations in the levels of serum aminotransferases greater than 3  times the upper limit of normal while on ambrisentan, in the trials, long-term follow-up revealed cases of transaminase level elevations which resolve upon discontinuation of ambrisentan. Both approved ETA and ETB receptor antagonists require monthly liver function test monitoring and have teratogenic properties, necessitating monthly pregnancy tests in women of childbearing age (see package inserts).

4.2.3 Phosphodiesterase-5 Inhibitors Sildenafil (Revatio) was approved by the FDA in June 2005 for the indication of symptomatic IPAH. The pivotal trial of

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Fig. 7  Six-minute walk effects of the three currently approved oral therapies for PAH. Bosentan (a), ambrisentan (b), and sildenafil (c) demonstrated significant placebo-corrected improvements in 6-min walk distance, which was the primary end point in each pivotal trial.

(a, c Reprinted with permission from Rubin et al. [50] copyright 2002 and Galiè et al. [67] copyright 2002 Massachusetts Medical Society. All rights reserved. (b) Reprinted with permission from Galiè et al. [66])

sildenafil, SUPER-1 (Sildenafil Use in Pulmonary Arterial Hypertension) trial, was a 12-week randomized, doubleblind, placebo-controlled trial comparing a placebo, and 20, 40, and 80 mg twice daily [68]. The study design was similar to that for other trials. In the sildenafil 20-mg group at 12 weeks, the placebo-corrected 6MWT distance was 45  m (Fig.  68.7c). In the 40-mg group, the improvement was 46 m and in the 80-mg group, it was 50 m. There was no significant effect of sildenafil on Borg dyspnea score or the time to clinical worsening. There was an improvement in the percentage of patients with improved functional class, particularly with the higher doses of sildenafil (7% in the placebo group, 28% in the 20-mg group, 36% in the 40-mg group, and 42% in the 80-mg group). However, despite the ­dose-related improvements in hemodynamics and WHO functional class in patients on higher dosages of sildenafil, the FDA approved only the 20-mg dose of sildenafil in the treatment of IPAH. This medication appeared to be well tolerated and no laboratory abnormalities were noted. Common adverse events included flushing, epistaxis, and gastritis. Combination with nitrates is contraindicated.

4.3 Combination Therapy There is strong rationale for combining drugs to treat IPAH; however, there are very few controlled data. The PACES trial randomized 267 patients with IPAH (79%) or PAH associated with CTD, anorexigen use, or corrected congenital-systemic shunts on stable epoprostenol therapy to either additional sildenafil or a placebo for 16 weeks [69]. Patients were on continuous infusion of epoprostenol as the baseline therapy for an average of 2.91 and 2.75 years, in the treatment and placebo arms, respectively. They were randomized to either sildenafil 20 mg three times a day or a placebo for 4 weeks and uptitrated to 80 mg three times a day or placebo uptitration. The primary outcome measurement was exercise capacity as measured by the 6MWT. The placebo-corrected 6MWT distance improvement in the sildenafil/epoprostenol group was 28.8  m. Hemodynamic improvements in the sildenafil/epoprostenol group compared with the placebo/epoprostenol were noted. Although mortality was not a prespecified end point, during the 16-week trial there were seven deaths, all of which occurred in the placebo/epoprostenol group.

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The STEP trial randomized 67 patients with IPAH (55%) and PAH associated with CTD, repaired congenital heart disease, HIV, or anorexigen use on stable therapy with bosentan for 4 months or more to inhaled iloprost or a placebo for 12 weeks [70]. The study was designed primarily as a safety study, but efficacy end points were also evaluated, including the 6MWT distance, with secondary end points including NYHA functional class, Borg dyspnea score, pulmonary hemodynamics, and the time to clinical worsening (defined as PAH-related death, hospitalization, study discontinuation from worsening PAH, initiation of new PAH-specific therapy, lung transplantation, or atrial septostomy). At 12 weeks, there was a borderline statistically significant placebo-corrected improvement in 6MWT distance of 26  m in the bosentan/inhaled iloprost group (p = 0.051). In addition, there was a significant improvement in both the NYHA functional class and the time to clinical worsening in the iloprost/bosentan group compared with the placebo/bosentan group. The addition of inhaled treprostinil (currently under FDA review) to oral PAH therapy has been evaluated and shown to be beneficial, first in a pilot study [71] and then in the pivotal TRIUMPH-1 study [72], a 12-week study that compared the addition of inhaled treprostinil versus a placebo to either bosentan or sildenafil in 235 patients with NYHA functional classes III and IV. The primary outcome was a change in 6MWT distance with secondary end points including a change in NYHA functional class, Borg dyspnea score, signs and symptoms of PAH, and clinical worsening. The 6MWT distance improved significantly by 20  m in the treprostinil arm as compared with the placebo arm. There were no significant changes in the secondary end points. Inhaled treprostinil appeared to be safe, with the most frequent adverse events reported as cough, headache, and nausea. Clearly, further study is needed to elucidate the role of add-on or combination therapy. Importantly, the development of longer-term, event-driven trials will be necessary, as add-on therapy may provide less incremental improvement, as evidenced by the relatively modest effects on 6MWT distance seen in the STEP and TRIUMPH studies.

4.4 Lung Transplantation Lung transplantation is a viable option when all therapies for IPAH have been tried without success. According to recent United Network for Organ Sharing data, the minority of lung transplants are done for IPAH and are usually double-lung transplants. The primary goal of lung transplantation is to provide a survival benefit; however, in certain situations (e.g. COPD) a significant improvement in quality of life is also deemed as important as an end point.

J.E. Lewis and R.N. Channick

In patients failing maximal medical therapy, lung transplantation is the “ultimate” option. According to the International Society for Heart and Lung Transplantation, current recommendations for consideration of transplantation include persistent NYHA class III or IV on maximal medical therapy, low or declining 6MWT distance, failing therapy with intravenously administered epoprostenol, cardiac failure with cardiac index less than 2, and elevated right atrial pressure (above 15  mmHg) [73]. Considerations for referral to a transplant center include any patient with rapidly progressive disease or NYHA functional class III or IV, regardless of the therapeutic options. The lung allocation scoring system, although not perfect, has enabled more appropriate listing of IPAH patients. Patients who are declining will, hopefully, receive higher scores, allowing shorter waiting times and, presumably, better outcomes. In patients doing well on oral therapies alone, evaluation for transplant is no longer necessary. One-year survival after lung transplantation for pulmonary hypertension was estimated at 80% in one series [74]. In a recent article by a group from Pittsburgh, the actuarial survival of all lung transplants for IPAH from 1994 to 2006 was 86% at 1 year, 75% at 5 years, and 66% at 10  years [75]. However, despite these recommendations, the minority of patients undergo lung transplantation for IPAH. This is likely related to the surge of treatments now available to treat IPAH.

4.5 Overall Therapeutic Strategy: Challenges With several effective medical therapies and lung transplantation available for treatment of IPAH, how does one decide what therapy to use, when to reassess, when to add or change the therapy, and what to add or change? Several consensus committees have attempted to evaluate the evidence for approved therapies and grade recommendations for these treatments. An algorithm recently published from the Fourth World Symposium on Pulmonary Hypertension is shown in Fig. 8 [76]. Supportive therapies including diuretics, warfarin, and oxygen are considered. An acute vasoreactivity test is recommended for IPAH patients and if a positive response is seen, calcium channel blocker treatment is recommended. For patients without acute vasoreactivity, recommendations are given regarding specific therapies, based on the initial functional class. Oral therapies have been given “A” recommendations for less severely limited patients, while intravenously administered epoprostenol for functional class IV patients. Less specific guidance is given regarding combination therapy or the timing of lung transplantation, given fewer data on these interventions. In practice, when a patient either

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Fig. 8  Proposed treatment algorithm. Strengths of recommendations are defined as A strong recommendation, B moderate recommendation, C weak recommendation, E/B moderate recommendation on the basis of expert opinion only, E/C weak recommendation on the basis

of expert opinion only. APAH associated PAH, ER endothelin receptor, HPAH heritable PAH, IV intravenous, Inh inhalation, PDE-5 phosphodiesterase-5, SC subcutaneous, WHO World Health Organization

fails to improve adequately or worsens on initial therapy, additional medications are often utilized. However, if such patients can be enrolled in a clinical trial evaluating “add-on” therapy, this route is probably preferable.

acknowledged that there are no prospective controlled ­survival data with the newer agents, given obvious ethical concerns about such trials in the era of existing therapy.

4.7 Experimental Therapies 4.6 Survival Long-term survival data are emerging in the “modern era” of IPAH treatment. In patients treated with epoprostenol, two papers demonstrated survival estimates of approximately 60% at 3 years [58, 77]. In these retrospective studies, favorable survival was more predicted by response to therapy (improvement in 6MWT distance, functional class, and pulmonary vascular resistance). Survival data are also available for other therapies. For example, in 169 IPAH patients enrolled in the two pivotal bosentan trials, ­estimated survival at 1 and 2 years was 96 and 89%, respectively, as compared with the predicted survival of 69 and 57% [78] (based on a validated NIH equation calculating predicted survival from baseline hemodynamics). It should be

The clinical therapeutics development landscape in PAH continues to evolve. Several new, novel agents are being evaluated, including a new-generation, tissue-targeted ETA and ETB receptor antagonist, inhaled vasoactive intestinal peptide, a guanylate cyclase inhibitor, and a nitric oxide synthase coupler. In addition, antineoplastic agents such as imatinib and sorafinib which, through kinase inhibition, attack the excessive growth of cells are being evaluated in IPAH.

5  Summary IPAH is a severely debilitating illness that too often is diagnosed only when it is far advanced. Mortality is related to

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impaired right ventricular function and is best predicted on the basis of relationships between stroke volume index and right atrial and pulmonary artery pressures. Improvements in understanding of the pathogenesis, cause, diagnosis, and treatment of this previously deadly disease have been striking over the last several years. Since it is now clear that IPAH is a disorder of excessive growth and proliferation of the pulmonary vascular wall and not typically a vasoconstrictive disease, therapy is now aimed at inhibiting or reversing the proliferative changes in the vasculature and improving the ability of the right ventricle to function. These new treatments have dramatically affected, in a positive way, the lives of patients with IPAH. Continued insight into the basic pathogenic mechanisms of primary pulmonary hypertension should lead to even more refined therapies.

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995 59. Simonneau G, Barst RJ, Galiè N et al (2002) Continuous subcutaneous infusion of treprostinil, a prostacyclin analogue, in patients with pulmonary arterial hypertension: a double-blind, randomized, placebo-controlled trial. Am J Respir Crit Care Med 165:800–804 60. Tapson VF, Gomberg-Maitland M, McLaughlin VV et  al (2006) Safety and efficacy of IV treprostinil for pulmonary arterial hypertension: a prospective, multicenter, open-label, 12-week trial. Chest 129:683–688 61. Gomberg-Maitland M, Tapson VF, Benza RL et al (2005) Transition from intravenous epoprostenol to intravenous treprostinil in pulmonary hypertension. Am J Respir Crit Care Med 172:1586–1589 62. Olschewski H, Simonneau G, Galiè N et al (2002) Inhaled iloprost for severe pulmonary hypertension. N Engl J Med 347:322–329 63. Channick RN, Simonneau G, Sitbon O et al (2001) Effects of the dual endothelin-receptor antagonist bosentan in patients with ­pulmonary hypertension: a randomised placebo-controlled study. Lancet 358:1119–1123 64. Galiè N, Rubin LJ, Hoeper M et  al (2008) Treatment of patients with mildly symptomatic pulmonary arterial hypertension with bosentan (EARLY study): a double-blind, randomized controlled trial. Lancet 37:2093–2100 65. Galiè N, Badesch D, Oudiz R et al (2005) Ambrisentan therapy for pulmonary arterial hypertension. J Am Coll Cardiol 46:529–535 66. Galiè N, Olschewki H, Oudiz RJ et  al (2008) Ambrisentan in Pulmonary Arterial Hypertension, Randomized, Double-Blind, Placebo-Controlled, Multicenter, Efficacy Studies (ARIES) Group. Circulation 117:3010–3019 67. Galiè N, Ghofrani HA, Torbicki A et  al (2005) Sildenafil citrate therapy for pulmonary arterial hypertension. N Engl J Med 353:2148–2157 68. Simonneau G, Rubin LJ, Galiè N et al (2008) Addition of sildenafil to long-term epoprostenol therapy in patients with pulmonary ­arterial hypertension: a randomized trial. Ann Intern Med 149:521–530 69. McLaughlin VV, Oudiz RJ, Frost A et al (2006) Randomized study of adding inhaled iloprost to existing bosentan in pulmonary arterial hypertension. Am J Respir Crit Care Med 174:1257–1263 70. Channick RN, Olschewski H, Seeger W, Staub T, Voswinckel R, Rubin LJ (2006) Safety and efficacy of inhaled treprostinil as addon therapy to bosentan in pulmonary arterial hypertension. J Am Coll Cardiol 48:1433–1437 71. McLaughlin VV, Rubin LJ, Benza R et al (2008) Triumph-1. Efficacy and safety of inhaled treprostinil sodium in patients with pulmonary arterial hypertension (PAH). ATS 2008, Toronto, Canada 72. Orens JB, Estenne M, Arcasoy S et al (2006) International guidelines for the selection of lung transplant candidates: 2006 update – a consensus report from the pulmonary scientific council of the international society for heart and lung transplantation. J Heart Lung Transplant 25:745–755 73. Charman SC, Sharples LD, McNeil KD, Wallwork J (2002) Assessment of survival benefit after lung transplantation by patient diagnosis. J Heart Lung Transplant 21:226–232 74. Toyoda Y, Thacker J, Santos R et al (2008) Long-term outcome of lung and heart-lung transplantation for idiopathic pulmonary arterial hypertension. Ann Thorac Surg 86:1116–1122 75. Barst RJ, Gibbs JS, Ghofrani HA et al (2009) Updated evidencebased treatment algorithm in pulmonary arterial hypertension. J Am Coll Cardiol 54:S78–S84 76. Sitbon O, Humbert M, Nunes H et al (2002) Long-term intravenous epoprostenol infusion in primary pulmonary hypertension: prognostic factors and survival. J Am Coll Cardiol 40:780–788 77. McLaughlin VV, Sitbon O, Badesch DB et al (2005) Survival with first-line bosentan in patients with primary pulmonary hypertension. Eur Respir J 25:244–249 78. Morrell NW, Adnot S, Archer SL et al (2009) Cellular and molecular basis of pulmonary arterial hypertension. J Am Coll Cardiol 54:S20–S31

Chapter 69

Genetics of Familial and Idiopathic Pulmonary Arterial Hypertension Eric D. Austin, James E. Loyd, and John A. Phillips III

Abstract  Pulmonary arterial hypertension (PAH), formerly known as primary pulmonary hypertension (PPH), is further classified into a sporadic or idiopathic form (IPAH) and a familial or heritable form (HPAH). Multiple individual reports described 13 different families in the USA with PAH among family members. In 1984, we conducted a followup analysis of these original 13 families, including interval description of eight new cases in nine of the families since the original reports, by contacting each author and each family. In addition, we reported an additional family, the 14th family with PAH in the literature. At that time, this family contained the largest number of affected family members described to date, with six deaths due to pulmonary hypertension during two generations. That publication described characteristics of the inheritance patterns including vertical transmission through mothers or fathers to children of both sexes, which suggested an autosomal dominant mode of inheritance indicative of a single gene defect. The report also introduced the speculation that cases of pulmonary hypertension which appeared to occur in isolation may in fact have a familial basis that is not obvious owing to incomplete penetrance causing skipped generations. Two separate teams of investigators independently mapped the locus for the gene, named PPH1, for HPAH to chromosome 2q31-32 in 1997. A few years later, both teams also demonstrated that mutations in the gene encoding bone morphogenetic protein (BMP) receptor type-2 (BMPR2), a receptor which is a member of the transforming growth factor b (TGF-b) superfamily, was the gene responsible for the majority of cases (80–85%) of the autosomal dominant disease HPAH. A small percentage of HPAH families (15–20%) have multiple affected individuals but do not have mutations identified in BMPR2 despite comprehensive testing. It is likely that mutations in one or more other genes are responsible.

E.D. Austin (*) Department of Pediatrics, Vanderbilt University Medical Center, Nashville, TN 37232, USA e-mail: [email protected]

Keywords  Bone morphogenetic protein receptor type 2 • Herigable • Genetics • Gene mutation • Inheritance

1  Background and Synopsis of History Pulmonary arterial hypertension (PAH), formerly known as primary pulmonary hypertension (PPH), was first described by Dresdale et  al. in 1951 as a sporadic disease entity [1]. That report of what is now known as idiopathic PAH (IPAH) was followed by a second publication in 1954, in which Dresdale et al. described the occurrence of pulmonary hypertension among several members of the same family, including a mother as well as her sister and son [2]. These are the first known reports of IPAH and familial PAH (FPAH), which has been recently reclassified as heritable PAH (HPAH) [3]. Over the next 30 years following Dresdale et al.’s initial description of one family with HPAH, multiple individual reports described 13 different families in the USA with PAH among family members. In 1984, we conducted a follow-up analysis of these original 13 families, including interval description of eight new cases in nine of the families since the original reports, by contacting each author and each family. In addition, we reported an additional family, the 14th family with PAH in the literature. At that time, this family contained the largest number of affected family members described to date, with six deaths due to pulmonary hypertension during two generations [4]. That publication described characteristics of the inheritance patterns including vertical transmission through mothers or fathers to children of both sexes, which suggested an autosomal dominant mode of inheritance indicative of a single gene defect. The report also introduced the speculation that cases of pulmonary hypertension which appeared to occur in isolation may in fact have a familial basis that is not obvious owing to incomplete penetrance causing skipped generations. In fact, the latest classification scheme now replaces the term “familial PAH” (FPAH) with “heritable PAH” (HPAH) at least in part to recognize the fact that 10–20% of patients previously thought to have IPAH harbor

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identifiable mutations and therefore pose a hereditary risk to other ­family members [5]. The National Institutes of Health (NIH) prospective registry of PAH in the mid-1980s provided the benchmark for the clinical definition of PAH, and facilitated interaction of participating investigators to collect and organize sufficient numbers of families to provide robust statistical power for a genome-wide search for HPAH loci [6]. Two separate teams of investigators, at Columbia University and Vanderbilt University, independently mapped the locus for the gene, named PPH1, for HPAH to chromosome 2q31-32 in 1997 [7, 8]. A few years later, both teams also demonstrated that mutations in the gene encoding bone morphogenetic protein (BMP) receptor type-2 (BMPR2), a receptor which is a member of the transforming growth factor b (TGF-b) superfamily [9, 10], was the gene responsible for the majority of cases (80–85%) of the autosomal dominant disease HPAH. A small percentage of HPAH families (15–20%) have multiple affected individuals but do not have mutations identified in BMPR2 despite comprehensive testing. It is likely that mutations in one or more other genes are responsible.

2 Epidemiology of PAH The frequency of IPAH and HPAH, has been estimated at one to two cases per year per million in the general population, with a female-to-male prevalence of approximately 2:1, but with similar disease severity and outcome [11, 12]. Comprehensive clinical information about the natural history of PAH derives largely from the prospective NIH study of patients followed in the USA from 1981 to 1985 with PAH. This report meticulously described and followed 187 patients with PAH at 32 US centers, was conducted prior to development of effective therapies, and contained an ethnic background of patients comparable to that of the US ­population at that time. Consistent with previous reports, the registry confirmed that PAH can develop at any age, with a mean age at diagnosis of 36 years. In general, women tended to present in the third decade of life, and men in the fourth. The median survival following diagnosis was 2.8 years. The mean time from symptom onset to diagnosis was 2 years for the entire cohort, and not surprisingly this interval was shorter for patients with a family history of PAH. The NIH registry reported that the prevalence of heritable disease was 6% on the basis of the finding that 12 of the 187 patients reported one or more immediate family members affected with PAH. These 12 HPAH patients did not differ from the 175 other patients in terms of hemodynamic data or symptoms, although several recent reports do not fully support this conclusion [6, 13]. Similar findings were confirmed and extended in a registry of PAH patients in a national prospective registry in France established in 2000, and recently reported with 674 enrolled

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PAH cases. The investigators reported a PAH prevalence of 15 cases per million adult French inhabitants. They identified a female-to-male ratio of 1.9:1, consistent with the NIH registry data noted above. The distribution of causes of PAH in the 674 cases showed 39.2% IPAH, 3.9% HPAH, 15.3% associated with connective tissue diseases (systemic sclerosis was leading cause), 11.3% related to congenital heart diseases, 10.4% related to portal hypertension, 9.5% associated with anorexigen use, 6.2% related to HIV, and 4.3% other causes. Importantly, the design of this registry did not include children [14].

3 Clinical Transmission Patterns and Expression of PAH IPAH and HPAH are clinically and histopathologically indistinguishable, and the true incidence of HPAH is unknown for several reasons. As noted already, PAH due to BMPR2 gene mutations in families transmits in an autosomal dominant manner with incomplete penetrance, which can make establishing a role for familial transmission difficult since generations of mutation-carrying individuals may not express disease [4, 15] (Fig.  1). Owing to incomplete penetrance, approximately 20% of individuals with a known genetic mutation in BMPR2 will develop detectable PAH during their lifetime, and phenotypic expression can occur at any age (Fig. 2); untreated, death follows diagnosis within 1–5 years. Furthermore, as a highly mobile society, full family genealogical history is often impaired [16]. In addition to incomplete penetrance, several other interesting findings characterize HPAH, including female predominance (approximately 2:1 female-to-male ratio), variable age at onset, and genetic anticipation (Figs. 1, 2).

3.1 Incomplete Penetrance Initial pedigree studies of families with PAH revealed ­phenotypic expression in fewer individuals than expected for an autosomal dominant disease. In fact, individuals known to be at risk of disease by pedigree analysis do not express PAH in a consistent manner [4] (Fig. 1). It is now known that mutant BMPR2 alleles display reduced penetrance among HPAH kindreds, indicating that heterozygous mutation of the gene is required but not sufficient to precipitate clinical expression of disease [11]. To date, the mechanisms of incomplete penetrance are unknown, and suggest a role for additional genetic and/or environmental modifiers of disease expression [17]. Currently, at Vanderbilt University, we are following a research cohort of 120 families with HPAH, and each has

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Fig.  1  Pedigree of an extended kindred with heritable pulmonary arterial hypertension (HPAH) due to a BMPR2 mutation with reduced penetrance. This pedigree now links six families who previously were labeled as independent, and 28 total patients with HPAH, as well as 40 known heterozygotes (obligate carriers). Solid symbols represent individuals with disease. Circles represent women, squares represent men.

incomplete BMPR2 penetrance. Of those families with comprehensive genetic testing completed, 52 have a detectable BMPR2 mutation, and 12 have no detectable mutation. The families contain 364 known PAH patients (256 females and 108 males), whose age at death spreads across the entire age spectrum, although a greater percentage of male deaths occur in childhood compared with the percentage of female deaths in childhood (Fig. 2). There are another 4,085 bloodline family members, and half of all these individuals are predicted to inherit the mutation, putting them at risk to develop HPAH. Approximately 18% of subjects within family bloodlines have been diagnosed with PAH. Pedigree analysis of the six most heavily affected families revealed that 26% of firstdegree relatives of patients with PAH have also been diagnosed with the disease (November 2008, unpublished data). We are continuing to follow the 14th family described in our 1984 report [4]. That family has been a substantial contributor to the understanding of the genetics of HPAH, and remains the largest known family with the disease. To date, 28 patients have been diagnosed with HPAH in the extended family, including 24 females and four males diagnosed at various ages between 9 and 70 years (Fig. 1). Consistent with the incomplete genetic penetrance of HPAH, this family ­contains numerous unaffected but at-risk individuals with

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A line through a symbol represents death. A dot inside a symbol represents an obligate carrier of the BMPR2 mutation. Numbers below symbols represent age at death or current living age. Numbers inside symbols represent the numbers of unaffected siblings of each gender. S inside a symbol represents stillbirth. A diamond represents sex unknown

Fig. 2  Cumulative mortality curve for all patients with HPAH in the Vanderbilt University research cohort. The x-axis demonstrates the age in years, whereas the y-axis lists the percentage of each group deceased at each age. The female group is connoted by darkened triangles. The male group is connoted by squares. Note that a larger percentage of males are deceased by age 20 years than the percentage of females by the age of 20 years

both parents and progeny diagnosed with HPAH. Such individuals are considered obligate carriers of the autosomal dominant mutation responsible for this disease, and they are at risk to develop the disease themselves. A research cohort of similar size recruited at Columbia University by Robyn Barst and Jane Morse over the past

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15 years is currently directed by Wendy Chung and includes an additional 106 families with 278 patients, and the characteristic incomplete penetrance (personal communication). Together, the Vanderbilt University and Columbia University cohorts suggest that there are at least 226 families who have had 642 patients, along with several thousand family ­members at risk who have an estimated chance of about 1 in 10 to develop disease during their lifetime (50% chance they possess an autosomal dominant gene mutation, and 20% chance that mutation will be penetrant).

3.2 Skewed Gender Ratio For both IPAH and HPAH, females outnumber males by a ratio of approximately 2:1. In fact, female gender is the most powerful determinant of the penetrance of a BMPR2 mutation. Interestingly, a lower percentage of males are born to BMPR2 mutation carriers than would normally be expected, which suggests increased male fetal losses perhaps due to abnormalities of embryologic development [15]. Suggestions of some hormonal influence or effect by the presence or absence of sex chromosomes have yet to be substantiated. The mechanism by which gender modulates disease has yet to be uncovered and may represent a critical piece of information for the improved diagnosis and therapy of HPAH.

3.3 Variable Age of Onset IPAH and HPAH can affect individuals at any age from childhood to late adulthood [12] (Fig. 2). Whereas the NIH registry study did not identify a difference between types of what was then called PAH, investigators in France recently determined that subjects with a BMPR2 mutation are diagnosed approximately 10 years younger than those without a mutation, despite the fact that most subjects with a mutation (59%) had no known family history that would bias the age at diagnosis. Furthermore, subjects with a mutation also died at a younger age than mutation-negative subjects, which further supports the conclusion that subjects with a BMPR2 mutation have severer disease [18].

3.4 Genetic Anticipation Several studies have shown a progressively earlier age of onset in subsequent generations, a finding known as genetic anticipation [15, 19]. Although striking in some families, it remains unknown whether this finding is due to an ­ascertainment artifact or true biologic causality. In other

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d­ isease processes, two different causes of genetic anticipation are known to have a molecular basis. Trinucleotide repeat expansion was discovered for fragile X syndrome in the early 1990s, and this mechanism is known to be the basis for more than 40 different neurologic diseases, most of which exhibit genetic anticipation [20]. The other known biologic explanation for genetic anticipation is progressive telomere ­shortening in dyskeratosis congenita, which is due to mutation in telomerase reverse transcriptase [21]. Neither of these known molecular explanations has been shown to explain genetic anticipation in HPAH. Since there are numerous statistical biases that can confound anticipation, it will remain uncertain, unless a biologic mechanism is discovered, whether the earlier age of onset in successive generations in HPAH has a biologic basis or is related to a statistical artifact.

4 Mutations in BMPR2 Cause Most HPAH Cases Starting in the 1980s, DNA was collected and stored from subjects within families affected by HPAH, such that by the mid1990s the statistical power was sufficient to locate the genetic cause of PAH. The discovery of the BMPR2 mutations as the genetic basis of HPAH in most cases was made possible by the collaborative development of family cohorts with banked DNA, advances in molecular genetics, and the availability of information from the Human Genome Project [22]. Two separate groups, at Vanderbilt University and Columbia University, utilized microsatellite markers and linkage analysis simultaneously to focus the search on chromosome 2q33. Morse et al. identified the locus of an HPAH gene to chromosome 2q31-32 [23, 24]. Using DNA collected from 19 affected and 58 unaffected family members of six families with PAH that had pedigrees suggestive of an autosomal dominant pattern of gene transmission, Nichols et al. independently utilized a microsatellite marker search to establish linkage to a 30 million base pair region on chromosome 2q33 [7]. Because the segment on chromosome 2 was too large to be sequenced in that era, the investigators employed a positional candidate gene approach to identify the gene responsible. By the targeting and testing of several genes in this region known to produce products of biological relevance, the correct gene was ultimately identified [16]. BMPR2, which is a member of the TGF-b superfamily of receptors, was chosen as a biologically plausible functional candidate gene. Of important relevance to PAH, the BMPR2 protein is a constitutively active serine/threonine receptor kinase whose downstream signaling has profound effects on ­developmental processes, including vasculogenesis. In 2000, the two ­independent teams at Vanderbilt University and Columbia University characterized multiple heterozygous germline mutations in BMPR2. This large gene (approximately 192 kb)

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resides on chromosome 2q33 and spans 13 exons that include multiple mutations shown to cause HPAH in several affected kindreds [9, 10]. A 4-kb transcript is produced that generates a polypeptide, composed of four distinct functional domains, of 1,038 amino acids in humans [25].

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The discovery that BMPR2 mutations are responsible for PAH in several families in 2000 prompted investigators to search to identify mutations in other families, as well as in those with PAH without family history. Since that time, the results of molecular testing for BMPR2 abnormalities have revealed mutations among many different ethnic groups [26–28]. A variety of testing methods have been used, including sequencing of genomic DNA, Southern blot analysis, highperformance liquid chromatography, and melting curve analysis. By 2005, approximately 65% of known HPAH families had BMPR2 mutations identifiable by gene sequencing. A summary report described 144 distinct mutations among

210 subjects, and most of these coded frameshift, nonsense, or splice site donor/acceptance site mutations [29]. In 2005, investigators demonstrated that 48% of the 21 mutations discovered in 30 unrelated affected families were deletions or duplications of exons by using reverse transcriptase PCR and a new method of analysis known as multiplex ligationdependent probe amplification (MLPA) analysis of genomic DNA with confirmation by real-time PCR. They concluded that such techniques would improve the identification of genetic abnormalities in BMPR2 in HPAH, such that 70% or more of such families have identifiable BMPR2 mutations [30]. The use of MLPA analysis of genomic DNA was also recently reported by a different group, who discovered mutations in 28% of families previously reported to lack BMPR2 mutations [31]. It is now generally accepted that approximately 70% of families with documented PAH in one or more members have a detectable mutation in BMPR2 in affected individuals [3]. This agreement further supports the primary role of BMPR2 in the central pathogenesis of ­disease in HPAH. Figure 3, from a report by Machado et al. displays the distribution of most recurrent mutations in the BMPR2 gene, including their domain location [25].

Fig.  3  Distribution of mutations across the BMPR2 gene. (a) The distribution and frequency of recurrent mutations. The x-axis represents the position of mutations relative to the BMPR2 complementary DNA (cDNA) below, and the y-axis defines the number of unrelated probands observed to harbor each mutation. The contiguous black and dotted line identifies the two amino acid substitutions recurrent at position 491. (b) Proportional representation of the BMPR2 cDNA, with exons 1–13 indicated. The boxed regions delineate

the extracellular (ECD), transmembrane (TM), kinase (KD), and cytoplasmic (CD) domains of the receptor depicted below. Large gene rearrangements (deletions and duplications) identified in pulmonary arterial hypertension (PAH) cases are arrayed below the cDNA. (c) Summary of the total number of PAH-causing mutations observed in each functional domain and nondomain encoding sequence. (From Machado et al [25]. Reproduced with permission of John Wiley & Sons)

4.1  Prevalence of BMPR2 Mutations in HPAH

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4.2 BMPR2 Mutations in IPAH Given the clinical and pathologic similarity to HPAH, it was immediately suspected that mutations in BMPR2 might also play a role in IPAH for some patients. Although further ­studies are ongoing, it is already clear that a significant proportion of IPAH patients harbor mutations in the BMPR2 gene. The detection rates vary between centers and countries, ­ranging from 10% to 40% of IPAH patients with a detectable BMPR2 mutation [25, 31–33]. Mutations may occur in the setting of low-penetrance germline mutations, in which other family members transmit the mutation without disease expression, or the mutations may develop de novo [25]. This is not surprising, given that low penetrance and genetic anticipation are both hallmarks of HPAH. Of note, the rate of BMPR2 mutation in the general population is not known, but appears to be extremely low given that no mutations have been detected among a few hundred control samples [16]. Data concerning mutation detection in IPAH depend in part on which detection techniques were employed, as these techniques continue to improve. As with HPAH, when newer molecular techniques were applied to IPAH cohorts, more mutations were detected. Aldred et al. noted six of 106 (5.7%) IPAH patients previously labeled BMPR2 mutation negative had detectable gene rearrangements. Interestingly, detailed pedigree evaluation in three such patients showed two of the mutations were de novo, whereas one was inherited from an unaffected mother [31]. At Vanderbilt University, 44 patients for which comprehensive pedigree analysis found no evidence of familial disease were tested for a BMPR2 mutation by direct sequencing and MLPA; a mutation has been detected in three of 44 patients (6.8%; unpublished data).

4.3 BMPR2 Mutations Cause PAH Haploinsufficiency is generally accepted as the predominant molecular mechanism by which a BMPR2 mutation predisposes to PAH [26, 34]. Haploinsufficiency describes the condition in which heterozygosity for a gene mutation leads to insufficient protein product, with decreased protein function leading to phenotypic expression of disease. In 2001, Machado et al. examined the role of BMPR2 mutation in terms of genotype–phenotype relationships, proposing that a model of haploinsufficiency explains the development of PAH from a molecular perspective. They suggested that 60% of the reported pathogenic mutations should result in premature truncation codons of the BMPR2 transcript, which would lead to nonsense-mediated decay [26]. Recent studies have supported this by demonstrating deletions within exons 2–13, which lead to nonfunctional peptide [30, 31]. Although these studies do suggest that

Fig.  4  Model of the potential impact of nonsense-mediated decay (NMD) upon BMPR2 expression. Subjects with no BMPR2 mutation will have a normal level of BMPR2 expression, and presumably normal BMPR2 pathway signaling. However, subjects who are heterozygotes for a NMD+ BMPR2 mutation will presumably only have activity via the normal wild-type BMPR2 allele, making them haploinsufficient. In contrast, subjects with a NMD- BMPR2 mutation behave in a manner that is potentially dominant negative. That is, the mutant BMPR2 allele may produce a protein that has deleterious effects in that it not only functions incorrectly, but may also reduce the appropriate function of the normal wild-type allele. It should be noted that an unknown proportion of NMD- BMPR2 mutations will not function in a dominant negative manner

haploinsufficiency contributes to the molecular mechanisms underlying PAH through reduction in the available level of BMPR2 protein in many cases, it is unclear what percentage of BMPR2 mutations result in disease due to a dominant negative impact on molecular signaling [35, 36]. That is, specific disease-causing mutations in the ligand binding and kinase domains of BMPR2 appear to exert dominant negative effects on BMP signaling. Disruption of BMP signaling may lead to absence of critical mechanisms in the antiproliferative and differentiation mechanisms in the pulmonary vasculature [29]. In fact, functional studies have demonstrated dominant negative effects on BMP signaling in vitro in the setting of point mutations or truncations in the kinase domain of BMPR2 [37]. Although it is unclear whether phenotypic differences will emerge in comparing haploinsufficient and dominant negative mutations, we recently demonstrated that presumably dominant negative mutations are more penetrant than haploinsufficient mutations [38] (Fig. 4).

4.4 PAH, BMPR2 Mutations, and the Concept of “Multiple Hits” Incomplete penetrance implies that mutation of the BMPR2 gene is required but is not sufficient alone for phenotypic expression. It is likely that genetic and environmental ­modifiers mediate the clinical expression of disease [11]. BMPR2 mutation carriers, and others, may express disease in

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the setting of a “multiple-hit” model, analogous to models of sequential mutations in the genesis of neoplasia. It has been suggested that the characteristic plexiform lesions in the lungs of patients with PAH represent a neoplastic-like proliferative process, with resultant endothelial and smooth muscle cell proliferation and diminished apoptosis [39]. In fact, there is evidence of monoclonal proliferation of local endothelial cells as well as the migration and local proliferation of smooth muscle cells in the regions of these lesions [40]. Interestingly, BMPR2 signaling may impact the monoclonal pulmonary arterial endothelial cells that contribute to these events [41]. In contrast, the plexiform lesions in patients with secondary pulmonary hypertension contain endothelial cells that are polyclonal [42]. Recently, investigators have examined the role of circulating endothelial precursor cells (EPCs) as a cause or reparative consequence in PAH, suggesting that they migrate to the pulmonary vasculature in PAH [43]. Diller et al. recently demonstrated reduced numbers of circulating EPCs in PAH (BMPR2 status not addressed), and a correlation with disease severity. Furthermore, treatment with sildenafil resulted in a dosedependent rise in the levels of circulating EPCs, and an improvement in cardiovascular parameters [44]. In contrast, Asosingh et  al. found high levels of proangiogenic endothelial progenitor cells circulating in patients with IPAH, and concluded these cells participate in the pulmonary vascular remodeling process [45]. Further studies are needed to better understand this process in both HPAH and IPAH. Unexpectedly, immunohistochemical staining of lung tissue from HPAH patients demonstrates nearly complete absence of BMPR2 protein in those with heterozygosity by genetic testing [46]. Given the presence of one mutated germline allele and one normal allele, one would expect evidence of protein production by the wild-type allele unless the mutation were a dominant negative. This raised the question of lung-specific alteration of the wild-type BMPR2 allele by somatic mutation, similar to the loss of a second tumor suppressor gene in cancer development. However, a search for evidence of microsatellite instability with somatic loss of function in the remaining wild-type BMPR2 allele did not reveal this “two-hit process” to be a likely cause [17]. In contrast, Hamid et  al. recently demonstrated lower levels of wild-type BMPR2 transcripts in patients compared with unaffected carriers of the same haploinsufficient BMPR2 mutation, suggesting that alterations in the level of expression of a BMPR2 mutation heterozygote’s normal BMPR2 allele may modify disease expression. That is, perhaps the “second hit” is the decreased activity of a BMPR2 heterozygote’s hypomorphic wild-type allele, which interacts with the haploinsufficiency of a BMPR2 mutation heterozygote to cause further reduction in BMPR2 pathway activity similar to the situation of a dominant negative mutation. This would

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create a molecular environment suitable to trigger phenotypic expression of disease [47, 48] (Fig. 4).

5 Genetic Modifiers of PAH The inconsistent phenotypic expression of PAH, such as the variable age at disease onset between and within families, and marked gender disparity imply the importance of endogenous and exogenous factors that modify disease susceptibility. The most obvious endogenous factors are genetic in origin, with a focus upon biologically relevant candidate genes. A number of such genes have been investigated and implicated, but no finding has been convincingly replicated [38, 49–54]. It is unknown whether these might be genes whose action promotes clinical expression of a BMPR2 mutation in those who develop PAH, or whether these are protective modifiers that prevent disease in the mutation carriers who never develop disease. For example, common genetic variations (polymorphisms) in the serotonin (5-hydroxytryptamine) pathway or polymorphisms in the serotonin transporter (SERT) gene may play a role in a manner either independent of or related to the presence of a BMPR2 mutation. It has been shown that serotonin is a cellular mitogen, and stimulates pulmonary artery smooth muscle cell (PASMC) proliferation through a signaling pathway mediated via SERT [55]. Internalization of serotonin by SERT leads to downstream activation of the mitogen-activated protein kinase (MAPK) cascade, in part via the production of reactive oxygen species, which ultimately acts to stimulate transcription of genes to drive cellular proliferation. PASMCs respond strongly to these stimulatory effects in  vitro [56, 57]. Thus, upregulation of the MAPK cascade as an effect of abnormal serotonin signaling has garnered attention as a potential antagonist to the growth-inhibitory effects of BMP signaling pathways, and possibly confers susceptibility to PAH expression. Initial studies implicated this pathway in humans by identifying increased plasma serotonin levels in 16 patients with PAH [58]. Subsequently, Eddahibi et  al. found increased growth of PASMCs in culture from patients with PAH compared with controls when stimulated by serotonin or serum, and attributed these mitogenic effects to increased expression of SERT [59, 60]. Further, they proposed a clinical link to the molecular cause for SERT overexpression in the form of variations in the SERT gene promoter by comparing 89 people with severe PAH and 84 control subjects. Homozygosity for a long promoter variant (L-allelic variant) was present in approximately 65% of patients (LL genotype individuals) with PAH but in only 27% of control subjects [60]. This suggested that serotonin polymorphisms may modulate disease penetrance.

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Machado et al. recently examined the role of polymorphic variation within the SERT gene to modify PAH in 528 affected patients, building upon the work described above. They studied 133 HPAH or IPAH patients with known BMPR2 mutation, 259 IPAH patients, 136 associated-PAH patients, and 353 control subjects. The results demonstrated no difference in the frequency of SERT gene alleles among any of the groups, suggesting that the SERT mutations evaluated are not likely to contribute to phenotypic expression of PAH in subjects with or without BMPR2 mutation. Likewise, genetic variation of the SERT promoter gene locus did not correlate with differences in the age of onset of disease. Nor did such variation differ by gender, the only known risk factor for development of PAH [52]. In a separate study, Willers et al. investigated the role of polymorphisms in the SERT gene, specifically the L allele, in a cohort of patients with IPAH and HPAH. This cohort included 83 patients with IPAH, 99 patients with HPAH, 67 unaffected carriers of a known BMPR2 mutation, and 125 control subjects. Overall, results comparable to those in the study by Machado et al. [52] were found, including no difference in SERT genotype distribution between groups. Again, a lack of difference between affected patients and unaffected BMPR2 mutation carriers suggested that the polymorphisms studied do not affect disease penetrance. However, subset analysis did determine that HPAH patients homozygous for the L allele (LL genotype) demonstrated an earlier age at diagnosis than other HPAH patients without this genotype. This finding may reflect unappreciated interactions between BMPR2 mutations and polymorphisms in the SERT gene that affect disease expression [53]. Various investigators are studying the balance of signaling among the various members of the TGF-b pathway of receptors, including BMPR2 and TGF-b receptor signaling [61]. It has been suggested that a reciprocal relationship exists between pathways comprising members of this superfamily of receptors, and the perturbation of BMPR2 signaling by a genetic mutation disrupts this balance, facilitating cellular proliferation and subsequent vessel obliteration by PASMCs, and perhaps endothelial cells [62]. With this in mind, Phillips et al. investigated the contribution of common genetic variants which might promote disease expression among susceptible individuals owing to a BMPR2 mutation. In a cohort of individuals with varying types of BMPR2 mutations, those subjects with genetic polymorphism in TGF-b1 predicted to increase pathway activity were more likely to be penetrant, and to express disease at a younger age [38]. Given the biologic plausibility of this argument, the detection of an effect was notable but in need of replication. Finally, although most efforts have focused upon candidate genes involved in pathways suspected to interact with the BMP pathway, it is possible that a genetic modifier of disease is the wild-type BMPR2 allele. That is, those subjects

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heterozygous for a mutated BMPR2 gene also possess one allele (wild type) with presumably normal function. As discussed previously, Hamid et  al. recently presented data to suggest that not all wild-type alleles are equal in their generation of transcript, and those with low production of transcript are more likely to have disease expression. Presumably, variations in wild-type allele activity raise or lower a heterozygote’s likelihood of disease expression [47].

6 The BMP Signaling Pathway BMPR2 is ubiquitously expressed and highly conserved throughout nature. It is a receptor for a family of cytokines known as BMPs. BMPs are members of the TGF-b superfamily of proteins. Like BMPR2, the TGF-b family of proteins and their receptors are highly conserved throughout nature. BMPs play a crucial role in the regulation of mammalian development, such as embryonic lung morphogenesis as well as cartilage and bone development [63]. Although genetic studies have strongly implicated the TGF-b superfamily in the regulation of pulmonary vascular cell growth and differentiation, investigations have failed to reveal the precise molecular mechanisms involved. Nonetheless, progress has been made in evaluating their contribution to lung vascular development. Heterodimerization of the serine/threonine transmembrane kinases, BMPR1, and BMPR2 is critical to BMPR2 signaling. Four functional domains compose BMPR2: ligand binding, kinase, transmembrane, and cytoplasmic domains. Activation of the BMPR2/BMPR1 complex leads to phosphorylation of a series of cytoplasmic mediators, which include the Smad family. Specifically, Smad proteins 1, 5, and 8 are phosphorylated, and subsequently complex with Smad 4 for translocation into the nucleus to regulate target gene transcription in concert with specific nuclear repressors and cofactors [64]. The Smad signaling pathway appears to participate in the inhibition of cell growth and the induction of apoptosis, although its exact mechanisms have yet to be fully elucidated [65]. It is postulated that BMPR2 mutation eliminates a critical growth regulatory function in pulmonary vascular cells [66]. Why other types of vasculature in the body have not been found to be abnormal in PAH is unclear. It is clear that other substrates are affected by BMPR2/BMPR1 activation, including MAPKs and NH2terminal kinase [64]. Recent studies have implicated not only Smad signaling, but also unopposed MAPK function in association with BMPR2 mutations. Yang et  al. demonstrated kinase domain mutations of BMPR2 found in PAH patients result in downregulation of Smad signaling in PASMCs, with resultant loss of the antiproliferative effect. The resulting imbalance between Smad and MAPK function may lead to

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proproliferative and antiapoptotic effects that promote the development of PAH [37]. The BMPs and TGF-b superfamily play critical roles in a diverse array of cellular processes beyond the BMPR2 pathway. Additional growth factors that have been implicated in the pathogenesis of PAH, including vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF), interact with the BMPs and likely the BMPR2 pathway. The exact function of VEGF in the lung is unknown, but studies of plexiform lesions in PAH demonstrated elevated levels of VEGF expression. Whether VEGF acts to promote or inhibit apoptosis and remodeling in a beneficial manner in the setting of PAH is unknown [67, 68]. It does appear, however, that BMP-9, and perhaps other BMPs, can block VEGF activity [69]. PDGF is a potent mitogen and pulmonary vascular smooth muscle cell chemoattractant, which may contribute to pulmonary vasculature remodeling in PAH [70]. Elevated levels of PDGF have been demonstrated in the lungs of individuals with PAH, whereas BMPR2 activity has been shown to dampen PDGF-associated PASMC proliferation. It may be that insufficient BMPR2 activity results in a loss of PDGF restraint, which may be rescued by peroxisome-proliferatoractivated receptor l activation [71].

7 BMPR2 Mutations and Other Causes of Pulmonary Hypertension In general, mutations in BMPR2 have not been consistently found in other causes of pulmonary hypertension. The connection between BMPR2 among HPAH and IPAH suggests a relationship between these two disease entities, which may in fact exist together along a continuum. One exception to the lack of BMPR2 mutations in other forms of pulmonary hypertension is pulmonary venoocclusive disease (PVOD), a rare form of pulmonary hypertension in which the vascular changes also affect small pulmonary veins and venules. Runo et  al. documented a novel mutation in exon 1 of the BMPR2 gene, resulting in a truncated protein predicted to have no function, in a 36-year-old female with biopsy-confirmed PVOD. Further investigation revealed that the proband’s mother had died of pulmonary hypertension, and laboratory evaluation confirmed an identical BMPR2 mutation in the proband’s healthy sister [72]. Similarly, Aldred et  al. discovered a genetic mutation in exon 2 of BMPR2 in a patient with PVOD. This patient had three first-degree relatives who had died with the diagnosis of pulmonary hypertension. Interestingly, this mutation of BMPR2 had previously been described in a family with HPAH with no evidence of PVOD [31]. Furthermore, Montani et al. recently described

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24 patients with PVOD, of whom two patients had a ­detectable BMPR2 mutation [73]. The detection of BMPR2 mutations in PVOD suggests that the molecular alterations associated with BMPR2 mutations may exhibit clinical (phenotypic) heterogeneity, that is, the ability of different allelic mutations at a single locus to produce more than one disease phenotype. Further, PVOD and PAH may well represent different spectra of the same disease, with phenotype influenced by genetic and/or environmental modifiers [72]. Another cause of PAH is that which is associated with drug exposure. Specifically, PAH has been reported in conjunction with exposure to appetite suppressants, such as fenfluramine and dexfenfluramine [74, 75]. The exact mechanism of PAH promotion has not been elucidated, but these drugs may serve as an environmental mediator to facilitate disease expression, possibly in genetically susceptible individuals. Individual factors of susceptibility are particularly plausible given the low numbers of PAH in those exposed to fenfluramine (approximately one case per 10,000 people exposed) [76]. Humbert et al. found distinct BMPR2 mutations, each predicted to result in diminished receptor function, in 9% of unrelated patients with PAH associated with fenfluramine exposure. They found no BMPR2 mutations in 130 normal control subjects. Of note, when compared with other patients with PAH associated with fenfluramine, patients heterozygous for a BMPR2 mutation expressed disease following a shorter interval of exposure to fenfluramine, which was also statistically significant [34]. This information, although in need of further study, again emphasizes the importance of additional genetic and environmental factors required to produce disease expression. Although BMP pathway activity is known to be important in the embryologic development of the cardiovascular system, insufficient data exist concerning congenital heart disease and BMPR2 mutations [77]. A report by Roberts et al. noted 6% of 106 children and adults with congenital heart disease possessed mutations of the BMPR2 gene. This finding was in agreement with reports in mouse models of vasculogenesis and cardiac anomalies, which implicate abnormalities of the TGF-b pathway.

8 Hereditary Hemorrhagic Telangiectasia (Rendu–Osler–Weber Syndrome) and PAH Hereditary hemorrhagic telangiectasia (HHT) is a vascular dysplasia characterized by mucocutaneous telangiectasias, recurrent epistaxis, and gastrointestinal bleeding, as well as arteriovenous malformations of the pulmonary, hepatic, and cerebral circulations. Mutations in additional components of the TGF-b signaling superfamily receptor complex,

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a­ ctivin-receptor-like kinase 1 (ALK1) located on ­chromosome 12 and endoglin on chromosome 9, contribute to the pathogenesis of HHT [78, 79]. Pulmonary hypertension is also known to occur in HHT, typically a secondary form related to a high cardiac output state. However, PAH that is clinically and histologically identical to HPAH and IPAH has recently been described in multiple unique kindreds with HTT associated with detectable mutations in ALK1, implicating ALK1 in HPAH [80, 81]. Interestingly, an endoglin germline mutation was recently described in a patient with HHT and PAH that was associated with dexfenfluramine exposure [82]. Investigators also recently reported a child with a mutation resulting in abnormal splicing of the endoglin transcript causing markedly reduced levels, suggesting haploinsufficiency leading to disease expression. The child’s asymptomatic father was a carrier of the same mutation [83]. Identifiable abnormalities of the TGF-b signaling pathway in patients with HHT and PAH suggests that a common molecular pathway precipitates pulmonary vascular disease. No direct interaction between the gene products of the BMPR2 gene and ALK1 or endoglin genes is known, and these receptors do not appear to share activating ligands [64, 83]. However, each receptor mediates intracellular signaling via the Smad family of coactivators [84]. PAH in HHT demonstrates that TGF-b signaling pathway defects are likely to be diverse in type, interaction, and phenotypic expression.

9 Genetic Implications for Clinical Evaluation and Therapy There is paucity of data convincingly outlining the impact of BMPR2 genotype upon clinical outcomes and therapeutic response in PAH, although this is beginning to change. For instance, a small proportion of PAH patients have good longterm response to calcium channel blockers [85, 86] which is predicted by acute vasoreactivity. Elliott et al. retrospectively compared the results of vasoreactivity testing in PAH patients to the presence of known BMPR2 gene changes that code for different amino acid products (nonsynonymous BMPR2 sequence variations). They found that patients with HPAH and IPAH with nonsynonymous BMPR2 sequence variations were less likely to demonstrate vasoreactivity by standard testing [87]. Rosenzweig et al. found similar results, as well as worse hemodynamic parameters, in a cohort of adult and pediatric patients with PAH and a BMPR2 mutation [88]. An important study by Sztrymf et al. provides evidence that having certain BMPR2 mutations is associated with a more aggressive form of PAH. By examining their large registry of individuals with PAH in France, they found that 20% of patients without a family history of PAH had a detectable mutation in BMPR2, suggesting that heritable disease may

E.D. Austin et al.

be more common than currently appreciated. They also found that subjects with BMPR2 mutation, regardless of family history, had a severer form of the disease, on the basis of an earlier age at diagnosis and severer hemodynamic abnormalities than for those lacking the mutation. Although survival was comparable in both groups, patients with BMPR2 mutation were more likely to be treated with parenteral prostacyclin therapy or undergo lung transplantation [18, 89]. As Rubin and others have pointed out, studies such as these correlating genotype and phenotype are critical to refine the molecular basis of PAH variability. In addition, they provide clinical support for the growing recognition that not all BMPR2 mutations result in equal effects. For example, as noted earlier, haploinsufficient mutations may result in less severe disease than those resulting in a dominant negative effect. Also, kindred individuals with the same BMPR2 mutation may have different SERT or TGFb polymorphic alleles that modify the penetrance of their shared BMPR2 mutations. One day, patients and their physicians may be able to implement a management strategy based in part upon one’s genotype [89].

10 Screening and Counseling The issue of genetic testing on a clinical basis is a complicated one. Independent of genetic testing, the siblings or children of patients with HPAH, or of known heterozygotes for a BMPR2 mutation, have an overall risk of 50% to inherit the abnormal gene. With reduced penetrance that is on average about 20%, this yields an estimated risk of 10% to express disease [90]. Thus, most relatives will be asymptomatic even though they are heterozygotes for a BMPR2 mutation. A negative test result for the BMPR2 mutation should substantially alleviate fears for family members of PAH patients. If the test is negative, an individual PAH family member’s risk of disease falls from 1 in 10 to that of the population, which is approximately 1 in 1,000,000 (100,000fold risk reduction). However, if an asymptomatic individual at risk is found to carry the known familial BMPR2 mutation, the risk of expressing clinical disease throughout the individual’s lifetime increases only modestly, to 1 in 5 (twofold risk increase). Genetic testing on a clinical basis for HPAH is currently available in both North America and Europe. It is most commonly and efficiently used for individuals with a known family history of PAH, or with PAH themselves. Laboratory evaluation for a BMPR2 mutation should start with full sequencing of all 13 exons, including splice junctions. Further methods may be needed to identify some mutations, such as MLPA. Current costs range from US $1,000 to US $3,000 for

69  Genetics of Familial and Idiopathic Pulmonary Arterial Hypertension

testing new individuals in a family, with subsequent cost to additional family members reduced once a mutation has been detected [3]. Genetic testing should only be provided in concert with antecedent and posttesting professional genetic counseling by experienced counselors [16, 90]. It should not be overlooked that a positive test for a BMPR2 mutation in a subject with IPAH converts the concept of disease from one of a “sporadic” finding to that of a heritable family disease. Thus, the parents, siblings, and children of that individual are at 50% risk to have the same mutation. This can be terrifying for patient and family members alike. Given the vast number of potential mutations in the large BMPR2 gene, screening for a mutation is most efficient in an affected patient with IPAH or HPAH, so that the specific mutation in their family can be identified, if present. There is no rationale for clinical genetic testing of relatives of a PAH patient unless a mutation is identified in the patient sample. The detection of a BMPR2 mutation in a patient with IPAH is often surprising and alarming, converting the concept of disease from one of a “sporadic” finding to that of a potentially familial disease. As such, family members are at increased risk and should be offered counseling about their risk and about testing for the known mutation. Current recommendations for asymptomatic family members of individuals with PAH advise a screening echocardiogram at 3–5-year intervals [90]. Investigators in Germany have proposed a screening process utilizing estimation of systolic pulmonary artery pressure at rest and during exercise by echocardiography, on the basis of studies of families with HPAH. Using a systematic clinical assessment of pulmonary artery pressure at rest and during exercise in members of families with PAH, Grunig et al. reported an abnormal pulmonary artery pressure response to hypoxia during echocardiographic evaluation in individuals deemed at risk to develop PAH based upon a known BMPR2 mutation or a possible “risk haplotype” [91]. These efforts, and others such as the use of advanced imaging modalities, are critical to improve the earlier detection of PAH in at-risk individuals. However, the use of stress echocardiography as a screening tool for PAH has not been widely accepted, and requires more extensive evaluation and independent confirmation. As such, it is not currently recommended for general use.

11 Conclusion and Future Directions Investigators have made tremendous progress in the evaluation and management of PAH in relation to the genetics and pathogenic mechanisms of this devastating disease. The discovery that mutations in the BMPR2 gene underlie most cases of HPAH, and of an important subset of cases of IPAH, has energized the field and prompted significant

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p­ rogress in understanding the molecular basis of PAH. Efforts continue to reveal the full genetic basis of HPAH and IPAH. Work is still needed to elucidate the cause(s) of the reduced penetrance of BMPR2 mutations, including the identity and role of various modifiers of disease expression. Such modifiers may also be the basis of genetic anticipation in HPAH. In addition, studies of genotype–phenotype interactions should include an exploration of the role of gender, the major known risk modifier. Investigations are under way to identify novel genes and proteins involved in disease pathogenesis and progression, as well as the interaction of genes in need of replication studies, such as those involved in the serotonin and TGF-b signaling pathways. The molecular mechanisms that converge to develop a milieu of cellular proliferation and vascular remodeling require further investigation. Ultimately, a greater understanding of the genetic and molecular mechanisms of PAH should lead to earlier diagnosis, advanced pharmacogenetics, and perhaps one day in the future, disease prevention. Acknowledgements  The authors thank the many patients and families who graciously contributed to this work, and Lisa Wheeler, whose service is invaluable as coordinator of the Vanderbilt Familial Primary Pulmonary Hypertension Study. Support  This work was supported by NIH NHLBI PO1 HL72058, and the Vanderbilt GCRC grant M01 RR 00095 NCRR/NIH.

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72. Runo JR, Vnencak-Jones CL, Prince M et al (2003) Pulmonary venoocclusive disease caused by an inherited mutation in bone morphogenetic protein receptor II. Am J Respir Crit Care Med 167:889–894 73. Montani D, Achouh L, Dorfmüller P et al (2008) Pulmonary venoocclusive disease: clinical, functional, radiologic, and hemodynamic characteristics and outcome of 24 cases confirmed by histology. Medicine (Baltimore) 87:220–233 74. Simonneau G, Galiè N, Rubin LJ et al (2004) Clinical classification of pulmonary hypertension. J Am Coll Cardiol 43:5S–12S 75. Abenhaim L, Moride Y, Brenot F et al (1996) Appetite-suppressant drugs and the risk of primary pulmonary hypertension. International Primary Pulmonary Hypertension Study Group. N Engl J Med 335:609–616 76. Sztrymf B, Yaici A, Jais X, Sitbon O, Simonneau G, Humbert M (2005) Idiopathic pulmonary hypertension: what did we learn from genes? Sarcoidosis Vasc Diffuse Lung Dis 22:S91–S100 77. Roberts KE, McElroy JJ, Wong WP et al (2004) BMPR2 mutations in pulmonary arterial hypertension with congenital heart disease. Eur Respir J 24:371–374 78. McAllister KA, Grogg KM, Johnson DW et al (1994) Endoglin, a TGF-b binding protein of endothelial cells, is the gene for hereditary haemorrhagic telangiectasia type 1. Nat Genet 8:345–351 79. Johnson DW, Berg JN, Baldwin MA et al (1996) Mutations in the activin receptor-like kinase 1 gene in hereditary haemorrhagic telangiectasia type 2. Nat Genet 13:189–195 80. Trembath RC, Thomson JR, Machado RD et al (2001) Clinical and molecular genetic features of pulmonary hypertension in patients with hereditary hemorrhagic telangiectasia. N Engl J Med 345: 325–334 81. Harrison RE, Flanagan JA, Sankelo M et al (2003) Molecular and functional analysis identifies ALK-1 as the predominant cause of pulmonary hypertension related to hereditary haemorrhagic telangiectasia. J Med Genet 40:865–871 82. Chaouat A, Coulet F, Favre C et al (2004) Endoglin germline mutation in a patient with hereditary haemorrhagic telangiectasia and dexfenfluramine associated pulmonary arterial hypertension. Thorax 59:446–448 83. Harrison RE, Berger R, Haworth SG et  al (2005) Transforming growth factor-b receptor mutations and pulmonary arterial hypertension in childhood. Circulation 111:435–441 84. Fernández-L A, Sanz-Rodriguez F, Blanco FJ, Bernabéu C, Botella LM (2006) Hereditary hemorrhagic telangiectasia, a vascular dysplasia affecting the TGF-b signaling pathway. Clin Med Res 4:66–78 85. Montani D, Marcelin AG, Sitbon O, Calvez V, Simonneau G, Humbert M (2005) Human herpes virus 8 in HIV and non-HIV infected patients with pulmonary arterial hypertension in France. Aids 19:1239–1240 86. Archer SL, Michelakis ED (2006) An evidence-based approach to the management of pulmonary arterial hypertension. Curr Opin Cardiol 21:385–392 87. Elliott CG, Glissmeyer EW, Havlena GT et al (2006) Relationship of BMPR2 mutations to vasoreactivity in pulmonary arterial hypertension. Circulation 113:2509–2515 88. Rosenzweig EB, Morse JH, Knowles JA et al (2008) Clinical implications of determining BMPR2 mutation status in a large cohort of children and adults with pulmonary arterial hypertension. J Heart Lung Transplant 27:668–674 89. Rubin LJ (2008) BMPR2 mutation and outcome in pulmonary arterial hypertension: clinical relevance to physicians and patients. Am J Respir Crit Care Med 177:1300–1301 90. McGoon M, Gutterman D, Steen V et  al (2004) Screening, early detection, and diagnosis of pulmonary arterial hypertension: ACCP evidence-based clinical practice guidelines. Chest 126:14S–34S 91. Grunig E, Dehnert C, Mereles D et  al (2005) Enhanced hypoxic pulmonary vasoconstriction in families of adults or children with idiopathic pulmonary arterial hypertension. Chest 128:630S–633S

Chapter 70

Pulmonary Arterial Hypertension Related to Scleroderma and Collagen Vascular Diseases Paul M. Hassoun

Abstract  Pulmonary arterial hypertension (PAH), defined as a mean pulmonary arterial pressure of greater than 25 mmHg in the absence of elevation of the pulmonary capillary wedge pressure, is a cause of significant morbidity and mortality. PAH is characterized by remodeling and occlusion of the small pulmonary arterioles leading to a progressive increase in pulmonary vascular resistance. Left untreated, this syndrome leads irremediably to right ventricular (RV) hypertrophy, pressure overload, and dilation resulting in death within 2–3 years. PAH encompasses a heterogeneous group of clinical entities, discussed in separate sections of this book, which share similar pathological changes. PAH can also be associated with various collagen vascular diseases (CVD) such as systemic sclerosis (SSc), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and mixed connective tissue disease (MCTD), which are the focus of this chapter. The mechanisms involved in the pathogenesis of PAH are unknown; however, several lines of evidence implicate a role for autoimmunity in the development of the pulmonary vascular changes, including the presence of circulating autoantibodies, proinflammatory cytokines (e.g. IL-1 and IL-6), and in particular the association of PAH with CVD. However, despite the presence of serologic and pathologic features suggestive of inflammation in both idiopathic PAH (IPAH) and SSc-related PAH (SSc-PAH), it is likely that inflammatory pathways and autoimmunity are more pronounced in SSc-PAH and may explain the survival discrepancies between the two syndromes and a differential response to current modern therapy. As such, SSc-PAH may be considered an ideal prototypic example to study inflammatory processes potentially operative in the pathogenesis of PAH. Other connective tissue diseases such as SLE, MCTD, and to a lesser extent RA, dermatomyositis, and Sjogren’s syndrome will be discussed separately in this chapter.

P.M. Hassoun (*) Division of Pulmonary and Critical Care Medicine, Johns Hopkins University, 18 East Monument St., Baltimore, MD 28721, USA e-mail: [email protected]

Keywords  Systemic sclerosis • Autoimmunity • Systemic lupus erythematosus • Rheumatois arthritis • Inflammation

1  Introduction Pulmonary arterial hypertension (PAH), defined as a mean pulmonary arterial pressure of greater than 25 mmHg in the absence of elevation of the pulmonary capillary wedge pressure, is a cause of significant morbidity and mortality [1–3]. PAH is characterized by remodeling and occlusion of the small pulmonary arterioles leading to a progressive increase in pulmonary vascular resistance. Left untreated, this syndrome leads irremediably to right ventricular (RV) hypertrophy, pressure overload, and dilation resulting in death within 2–3 years [1]. PAH encompasses a heterogeneous group of clinical entities [4], discussed in separate sections of this book, which share similar pathological changes. PAH can also be associated with various collagen vascular diseases (CVD) such as systemic sclerosis (SSc), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and mixed connective tissue disease (MCTD), which are the focus of this chapter. The mechanisms involved in the pathogenesis of PAH are unknown; however, several lines of evidence implicate a role for autoimmunity in the development of the pulmonary vascular changes, including the presence of circulating autoantibodies [5], proinflammatory cytokines (e.g. IL-1 and IL-6) [6], and in particular the association of PAH with CVD. However, despite the presence of serologic and pathologic features suggestive of inflammation in both idiopathic PAH (IPAH) and SSc-related PAH (SSc-PAH), it is likely that inflammatory pathways and autoimmunity are more pronounced in SSc-PAH and may explain the survival discrepancies between the two syndromes and a differential response to current modern therapy [7, 8]. As such, SSc-PAH may be considered an ideal prototypic example to study inflammatory processes potentially operative in the pathogenesis of PAH. Other connective tissue diseases such as SLE, MCTD, and to a lesser extent RA, dermatomyositis, and Sjogren’s syndrome will be discussed separately in this chapter.

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_70, © Springer Science+Business Media, LLC 2011

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2  Etiology and Pathogenic Mechanisms in Scleroderma-Related PAH

P.M. Hassoun

underdiagnosed as suggested by the lower than expected prevalence of the disease in the few registries available [23, 24].

2.1  Systemic Sclerosis 2.2.1  Pathophysiology SSc is a heterogeneous disorder characterized by dysfunction of the endothelium, dysregulation of fibroblasts ­resulting in excessive production of collagen, and abnormalities of the immune system [9]. These processes lead to progressive fibrosis of the skin and internal organs resulting in organ failure and death. Although the cause of SSc is unknown, genetic and environmental factors are thought to contribute to host susceptibility [10] in the context of autoimmune dysregulation. SSc, whether presenting in the limited or in the diffuse form, is a systemic disease with the potential for multiple organ system involvement, including the gastrointestinal, cardiac, renal, and pulmonary systems [11]. Pulmonary manifestations include PAH, interstitial fibrosis, and increased susceptibility to lung neoplasms. Estimates of the incidence and prevalence of SSc have varied widely depending on the period of observation, disease definition, and population studied [12]. Although the incidence seemed to be increasing over the period from 1940 to 1970, the rate of occurrence has stabilized since the widespread use of a standard classification system [13]. However, for reasons that remain poorly understood, there continues to be marked geographic variation in the occurrence of the disease, perhaps supporting a role for environmental factors in disease pathogenesis. For example, the prevalence of SSc ranges from 30 to 70 cases per million individuals in Europe and Japan [14–16] to approximately 240 cases per million individuals in the USA [13]. The incidence varies similarly by geographic area, with the highest rates found in the USA (approximately 19 persons per ­million individuals per year) [12].

Vascular changes occur at an early stage in SSc [25], and suggest that autoimmune mechanisms are central to the vascular remodeling process, whether involving the systemic or the pulmonary vascular beds. These changes include gaps between endothelial cells [26], apoptosis [27], endothelial activation with expression of cell adhesion molecules, inflammatory cell recruitment, a procoagulant state [28], and intimal proliferation and adventitial fibrosis leading to vessel obliteration. To a large extent, prognosis and outcome of patients with SSc depends on the extent and severity of the vascular lesions [29]. Endothelial injury is reflected by increased levels of soluble vascular cell adhesion molecule 1 (VCAM-1) [30], disturbances in angiogenesis as indicated by increased levels of circulating vascular endothelial growth factor (VEGF) [31, 32], and the presence of angiostatic ­factors [31, 33]. Increased levels of VEGF, a glycoprotein with potent angiogenic and vascular permeability-enhancing properties, may be a consequence of increased angiogenesis or profound disturbances in signaling in SSc. From a histologic standpoint, the pulmonary vascular lesions in SSc-PAH are indistinguishable from those present in IPAH, with the exception of a higher rate of veno-occlusive disease in ­SSc-PAH [34]. The role of dysregulated angiogenesis in SSc-PAH, whether driven by the inflammatory process or other mechanisms, appears to be a predominant feature of the disease and should be the focus of future studies.

2.2.2  Autoantibodies in SSc-PAH

2.2  SSc-Related PAH Estimates of the prevalence of PAH in patients with SSc have varied widely depending on the definition of pulmonary hypertension and the method of obtaining the measurements (i.e. echocardiography or cardiac catheterization). In patients with limited SSc (formerly called CREST syndrome for calcinosis cutis, Raynaud’s phenomenon, esophageal dysfunction, sclerodactily, and telangiectasias), the prevalence of elevated RV systolic pressure may be as high as 60% [17–20] when based on echocardiographic criteria. However, when using strict cardiac catheterization criteria for diagnosis, the prevalence of pulmonary hypertension is 8–12% [21, 22]. However, it is likely that SSc-PAH remains

A role for an autoimmune process in the pathogenesis of SSc-PAH is further supported by the presence of various ­circulating antibodies. Antifibrillarin antibodies (anti-U3RNP) are ­frequently found in SSc-PAH patients [35], and the poorly characterized antiendothelial antibodies (aECA) correlate with digital infarcts [36]. IgG antibodies directed against endothelial cells and obtained from patients with SSc-PAH, but also from patients with IPAH, display distinct reactivity profiles against antigens from the microvascular and macrovascular beds [37]. Antibodies to fibrin-bound tissue plasminogen activator in CREST patients [38] and in IPAH patients with HLA-DQ7 antigen [39], and antitopoisomerase IIa antibodies, particularly in association with HLA-B35 antigen [40], have been reported in SSc-PAH. Nicolls et al. suggested that aECA which can

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activate endothelial cells and induce the expression of ­adhesion molecules, as well as trigger apoptosis, can potentially play a role in the pathogenesis of PAH [41]. In vitro experiments using autoantibodies from patients with connective tissue diseases (anti-U1-RNP and anti-­doublestranded DNA) can upregulate adhesion molecules (e.g. endothelial leukocyte adhesion molecule 1) and histocompatibility complex class II molecules on human pulmonary artery endothelial cells [42], suggesting that such an inflammatory process could lead to proliferative and inflammatory pulmonary vasculopathy. Fibroblasts are essential components of the pulmonary vascular wall remodeling in PAH and can be found in the remodeled neointimal layer in both SSc-PAH and IPAH. The detection of antifibroblast antibodies in the serum of SSc and IPAH patients [37, 43] has significant pathogenic importance since these antibodies can activate fibroblasts and induce collagen synthesis, thus contributing potentially directly to the remodeling process. Antibodies from sera of patients with SSc have been shown to induce a proadhesive and proinflammatory response in normal fibroblasts [43]. IgG antifibroblast antibodies are present in sera of patients with IPAH and SSc-PAH and have distinct reactivity profiles in these two conditions, as assessed by one-dimensional immunoblotting [44]. Several fibroblast antigens recognized by serum IgG from IPAH and SSc-PAH patients have been identified, including proteins involved in regulation of cytoskeletal function, cell contraction, cell and oxidative stress, cell energy metabolism, and different key cellular pathways [45]. Although the specific membrane antigen targeted by these autoantibodies remains to be determined, it is likely that they react with membrane components since they typically bind to unpermeabilized fibroblasts, and may mediate the release of cytokines and growth factors which in turn might contribute to the pathogenesis of vascular remodeling in PAH [44]. Taken together, particularly in light of the positive response to immunosuppressive therapy for some patients with PAH associated with SLE and MCTD [46, 47], these studies suggest that inflammation and autoimmunity could play a major role in the pathogenesis of PAH. Therefore, a search for specific biomarkers of inflammation should be a focus of future studies in SSc-PAH and other autoimmune conditions associated with PAH.

2.2.3 Genetic Factors in SSc-PAH and SclerodermaRelated PAH Recent advances in molecular genetics have helped define a genetic contribution to the development of PAH. To date, linkage studies have been limited to the subphenotype IPAH and have mostly been performed with modest datasets of

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multiplex families, i.e. those with two or more affected ­individuals [48]. Polymorphisms involving the bone morphogenetic protein receptor-2 (BMPRII) [49, 50] are now found to be present in about 80% of familial IPAH cases and up to 20% of sporadic cases [51, 52] of IPAH. Additional candidate genes have been proposed to influence the pathogenesis of PAH [53]. Polymorphisms of the activinreceptor-like kinase 1 (ALK1) gene, which encodes the transforming growth factor b (TGF-b) receptor, have been reported in patients with hereditary hemorrhagic telangiectasia and PAH [54]. In contrast, little is known about the genetic involvement in SSc-PAH, although an increasing number of candidate genes and single-nucleotide polymorphisms involving inflammatory genes such as monocyte chemo­ attractant protein 1 (MCP-1), TNF-a, and IL-1a, have been described in different SSc populations [55–58]. In particular, BMPR2 mutations have not been identified in two small cohorts of SSc-PAH patients [59, 60]. Recently, however, an association between an endoglin gene (ENG) polymorphism and SSc-related PAH was identified. Wipf et  al. demonstrated a significant lower frequency of the 6bINS allele in SSc-PAH patients as compared with controls or patients with SSc but no PAH [61]. Endoglin, a homodimeric membrane glycoprotein primarily present on human vascular endothelium, is part of the TGF-b receptor complex. The functional significance of the ENG polymorphism in SSc patients remains to be determined. Aside from the few examples cited above, the genes relevant to the pathogenesis and generally poor outcome associated with SSc-PAH have not been identified, and their definition will require robust, well-characterized patient populations to provide adequate power for analysis.

2.2.4  Clinical Featmures Several risk factors for the development of PAH in SSc patients have been identified and include late-onset disease [62] and long duration of the disease [63], a postmenopausal condition [64], an isolated reduction in the diffusing capacity of the lung for carbon monoxide (DLCO), a forced vital capacity to DLCO ratio of less than 1.6 [65, 66], or a combined decreased DLCO/alveolar volume with elevation of serum N-terminal pro-brain natriuretic peptide (N-TproBNP) levels [67] (Table 1). Typically, patients with SSc-PAH are predominantly women, have limited sclerosis, are older, and have seemingly less severe hemodynamic impairment compared with IPAH patients [8]. Like in IPAH, clinical symptoms are generally nonspecific, including dyspnea and functional limitation, which, in this patient population, may be related to pulmonary disease but also involvement of the musculoskeletal system.

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P.M. Hassoun Table 1  Risk factors for development of pulmonary arterial hypertension in systemic sclerosis Risk factors

Reference

Limited systemic sclerosis Long duration of disease Late age of onset Postmenopausal condition Raynaud’s phenomenon Decreased DLCO FVC/DLCO < 1.6 Increased N-T-proBNP serum level Antibodies (e.g. anti-U3 RNP)

[66] [63] [62] [64] [66] [66, 67] [66] [67] [35]

2.2.5  Other Organ Involvement SSc-PAH patients also tend to have other organ involvement such as renal dysfunction and intrinsic heart disease. Mortality from kidney disease has been declining over the past few decades [68], most likely due to the almost universal therapeutic use of antagonists of the angiotensin system. However, cardiac involvement, such as myocardial fibrosis and inflammation, conduction disturbances, and pericardial abnormalities, is common in SSc [69]. SSc patients (even in the absence of PAH) tend to have depressed RV function [70, 71] and left ventricular systolic as well as diastolic dysfunction [72]. Like IPAH patients, SSc-PAH patients have severe RV dysfunction at time of presentation but have more severely depressed RV contractility compared with IPAH patients [73]. In addition, SSc-PAH patients tend to have more commonly left ventricular diastolic dysfunction and a high prevalence of pericardial effusion (34% compared with 13% for IPAH) [8]. In both groups, pericardial effusion portends a particularly poor prognosis [8]. SSc-PAH patients also tend to have severer hormonal and metabolic dysfunction such as high levels of N-TproBNP [74] and hyponatremia [75]. Both N-TproBNP and hyponatremia have been shown, at the baseline and with serial changes (for N-TproBNP [74]), to correlate with survival in PAH [74, 75].

Fig.  1  Algorithm for detection of pulmonary arterial hypertension (PAH) in patients with systemic sclerosis (SSc). A proposed algorithm for performance of routine clinical tests in patients with systemic sclerosis which may allow early detection of pulmonary arterial hypertension or other causes of cardiac dysfunction (e.g. left ventricular dysfunction). PFTs pulmonary function tests, DLCO single breath diffusing capacity of the lung for carbon monoxide, FVC forced vital capacity, RV right ventricle, TRV tricuspid regurgitation jet, RHC rightsided heart catheterization

with known disease (prevalent cases). Therefore, unexplained dyspnea should prompt a search for PAH in these patients, in particular in the setting of a low single breath DLCO or declining DLCO over time [66], echocardiographic findings suggestive of the disease (elevated TRV jet or dilated right ventricle or atrium), or elevated levels of N-TproBNP, which can reflect cardiac dysfunction and have been found to predict the presence of SSc-PAH [74]. In fact, systematic screening should allow detection of early disease and prompt therapy which, according to some experts, may be beneficial from a prognostic standpoint [76].

2.2.6  Diagnosis of SSc-PAH

2.2.7  Prognosis

An algorithm for detection of PAH in patients with SSc may be helpful if it is based on a combination of symptoms and ­screening echocardiography (Fig. 1). In a large French registry, patients with SSc with tricuspid regurgitation velocity (TRV) jet by transthoracic echocardiography greater than 3  m/s or between 2.5 and 3  m/s if accompanied by unexplained dyspnea were systematically referred for right-sided heart catheterization [21]. This approach allowed the detection of incident cases of SSc-PAH with less severe disease (as judged by hemodynamic data) compared with patients

Pulmonary hypertension is one of the leading causes of mortality in patients with SSc [68]. In general, SSc-PAH patients tend to have a worse prognosis than patients with other forms of PAH such as IPAH (Fig.  2) as now indicated by many studies [7, 8, 77]. Indeed, 1-year survival rates for SSc-PAH patients range from 50 to 81% [7, 20, 22, 77], considerably lower than the estimated 88% 1-year survival for IPAH patients [78]. In all patients with SSc, PAH significantly worsens survival and is one of the leading causes of mortality in these patients [13, 68, 77].

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Fig. 2  Survival differences between SSc-related PAH (SSc-PAH) and idiopathic PAH (IPAH) patients treated with modern therapy: a singlecenter experience. One- and 3-year survival rates are around 87 and 48%, respectively, in patients with SSc-PAH and 95 and 83%, respectively, in IPAH patients. SSc-PAH patients are 3 times more likely to die than IPAH patients despite having similar treatment (endothelin receptor antagonists, prostaglandins, or phosphodiesterase inhibitors). (Reprinted from [8] with permission from John Wiley & Sons)

3  PAH Associated with Other CVDs PAH can complicate any CVD, most frequently SSc as discussed already, but also SLE, MCTD, RA, or other diseases such as Sjogren’s syndrome and dermatomysositis.

3.1  Systemic Lupus Erythematosus There are many reasons for pulmonary vascular involvement in SLE. Like in SSc, there is evidence of endothelial dysfunction in SLE with a potential consequent imbalance between vasodilators and vasoconstrictors. Endothelin activation is suggested by a significant increase in serum levels of endothelin-1 (ET-1), and soluble E-selectin and thrombomodulin in SLE patients [79]. Endothelin levels are high in these patients, in particular in the presence of systemic organ dysfunction [79]. Release of ET-1 from cultured endothelial cells in response to exposure to serum from SLE patients correlates with levels of aECA and immune complexes [80]. Other causes of pulmonary vascular disease that may lead to pulmonary hypertension in SLE include recurrent thromboembolic disease, in particular in patients with increased coagulability related to the presence of antiphospholipid antibodies (such as anticardiolipin antibody present in up to 10% of patients with SLE) [81], pulmonary vasculitis, and parenchymal disease (including interstitial lung disease, and the shrinking lung syndrome from myositis of the diaphragm). Combined

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vasculitis and chronic hypoxia are frequent contributing offenders in these syndromes. In addition, pulmonary venous hypertension can be a consequence of left ventricular dysfunction, myocarditis, or Libman–Sacks endocarditis. The prevalence of PAH in SLE is unclear but is likely less than in SSc, affecting about 0.5–14% patients with SLE in a large review of the literature encompassing over 100 patients [82]. The patients are predominantly female (90%), young (average age of 33 at the time of diagnosis), and often suffer from Raynaud’s phenomenon (underscoring a generalized endothelial dysfunction in these patients with SLE and pulmonary hypertension). The pathological lesions are often indistinguishable from IPAH or SSc-PAH lesions, with intimal hyperplasia, smooth muscle cell hypertrophy, and medial thickening. Survival, which was thought to be quite poor (25–50% at 2 years) even compared with that in SSc-PAH in studies antedating specific pulmonary hypertension treatment, is now estimated at 75% [24].

3.2  Mixed Connective Tissue Disease Patients with MCTD have clinical features which overlap those of SSc, SLE, RA, and polymyositis. The exact prevalence of PAH in MCTD is unknown and has been reported as high as 50% [83]. PAH in these patients may occasionally respond to immunosuppressive drugs [46, 47].

3.3  Rheumatoid Arthritis Both the prevalence and the impact of pulmonary hypertension in patients with RA are not well known. PAH is, ­however, a rather unusual complication of RA.

3.4  Primary Sjogren’s Syndrome Although primary Sjogren’s syndrome (pSS) is a relatively common autoimmune disease with glandular and extraglandular manifestations, it is very rarely complicated by PAH. In a recent review by Launay et al. of 28 well-characterized patients with pSS and PAH [84], the mean age at diagnosis of PAH of these almost exclusively female patients (27of 28 patients) was 50 years. Patients had severe functional (i.e. functional classes III and IV) and hemodynamic impairment. Standard therapy (with endothelin receptor antagonists, phosphodiesterase inhibitors, or prostanoids) was typically ineffective despite an initial improvement. Some patients were reported to respond to immunosuppressive treatment.

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However, a conclusion regarding treatment is limited by the small size of this case report. The survival rate was low (66% at 3 years).

4  Therapy for PAH Related to CVD Evidence of chronically impaired endothelial function [85–87], affecting vascular tone and remodeling, has been the basis for current therapy of PAH. Vasodilator therapy using high-dose calcium channel blockers is an effective long-term therapy [88], but only for a minority of patients (less than 7% [89] of IPAH patients) who demonstrate acute vasodilation (e.g. to nitric oxide or adenosine) during hemodynamic testing, and an even smaller number of patients with PAH related to CVD who typically fail to show a vasodilator response to acute testing (only about 2.6% of responders in that group in one large study [90]). Therefore, high-dose calcium channel therapy is usually not indicated for patients with PAH associated with CVD such as SSc-PAH although most patients often receive these drugs at low dosage, typically for Raynaud’s syndrome.

4.1  Prostaglandins Prostacyclin (epoprostenol) has potent pulmonary ­vasodilator but also antiplatelet aggregating and antiproliferative properties [91], and has proven effective in improving the exercise capacity, cardiopulmonary hemodynamics, New York Heart Association (NYHA) functional class, symptoms, as well as survival in patients with PAH when given by continuous infusion [92–94]. In SSc-PAH, continuous intravenous infusion of epoprostenol improves exercise capacity and hemodynamics [95], compared with conventional therapy; however, there has been no demonstrable effect on survival. Treprostinil, a prostacyclin analog suitable for continuous subcutaneous administration, has been shown to have modest effects on symptoms and hemodynamics in PAH [96]. In a small study of 16 patients (among whom six had connective tissue disease related PAH), recently FDA-approved intravenously administered treprostinil was shown to improve hemodynamics 6-min walk distance, functional class, and hemodynamics after 12 weeks of therapy [97]. Although the safety profile of this drug is similar to that of intravenously administered epoprostenol, required maintenance doses are usually twice as high as for epoprostenol. However, for patients with SSc-PAH, the lack of requirement of ice packing and less frequent mixing of the drug offer significant advantages considering these patients’ poor tolerance to cold exposure.

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Several reports of pulmonary edema in SSc-PAH patients treated with prostaglandin derivatives, both in acute and in chronic settings, have raised the suspicion of increased prevalence of veno-occlusive disease in these patients [98, 99], and concern about the usefulness of these drugs for this entity. Nevertheless, prostaglandin therapy remains an option for patients with SSc-PAH with NYHA class IV. Considering the frequent digital problems and disabilities that these patients often experience, this form of therapy can be quite challenging and may increase the already heavy burden of disease in these patients. In summary, both epoprostenol and treprostinil are FDA-approved for PAH, but are cumbersome therapies requiring continuous parenteral administration with the attendant numerous adverse effects (e.g. infection and possibility of pump failure [100]), which make these drugs less than ideal.

4.2  Endothelin Receptor Antagonists Randomized, placebo-controlled trials of 12–16 weeks’ duration demonstrated a beneficial effect of bosentan therapy on functional class, 6-min walk distance, time to clinical worsening, and hemodynamics in PAH [101, 102]. In these studies, roughly one fifth of the population consisted of SScPAH patients, whereas a large majority had a diagnosis of IPAH. A subgroup analysis, performed by Rubin et  al. reported a nonsignificant trend toward a positive treatment effect on the 6-min walk distance among the SSc-PAH patients treated with bosentan compared with a placebo [102]. At most, bosentan therapy prevented deterioration in these patients (as assessed by an increase of 3 m in the 6-min walk distance in the treated group compared with a decrease of 40 m in the placebo group). This less than optimal effect of therapy in patients with SSc-PAH is unclear but may be related to the severity of PAH at the time of presentation, as well as other factors, such as hypothetically, severer RV and pulmonary vascular dysfunction, as compared with patients with other forms of PAH (e.g. IPAH). In a recent analysis of patients with PAH related to CVD (e.g. patients with SLE, overlap syndrome, and other rheumatological disorders) included in randomized clinical trials of bosentan, there was a trend toward improvement in the 6-min walk distance and improved survival compared with historical cohorts [103]. Single-center experience suggests that the long-term outcome of first-line bosentan ­monotherapy is inferior in SSc-PAH patients compared with IPAH patients, with no change in functional class and worse survival in the former group [104]. Since ET-1 appears to play an important pathogenic role in the development of SSc-PAH, ­contributing to vascular damage and fibrosis, inhibiting ET-1 remains a rational and viable therapeutic strategy in these patients. In fact, in a small study of 35 patients with SSc (ten of whom

70  Pulmonary Arterial Hypertension Related to Scleroderma and Collagen Vascular Diseases

had SSc-PAH), bosentan treatment appeared to reduce endothelial cell (as determined by endothelial soluble serum factors such as intercellular adhesion molecule 1, VCAM-1, P-selectin, and platelet/endothelial cell adhesion molecule 1) and T cell subset (assessed by expression of lymphocytefunction-associated antigen-1, very late antigen-4, and l-selectin on CD3 T cells) activation [105]. Aside from improving pulmonary hypertension, ET-1 receptor antagonists (specifically bosentan) cause significant reductions in the occurrence of new digital ulcerations without, however, healing preexisting ulcers [106]. In an effort to target the vasoconstrictive effects of endothelin while preserving its vasodilatory action, selective endothelin-A receptor antagonists have been developed. Sitaxsentan, which is only approved in Europe for treatment of PAH, improved exercise capacity (i.e. change in peak VO2 at week 12, which was the main end point of the study) [107]. Elevation in liver enzyme levels were noted in 10% of patients at the higher dosage tested (300  mg orally once daily). Patients with PAH associated with CVD represented less than a quarter of the study group. A post hoc analysis of 42 patients (33 patients who received the drug and nine patients who received a placebo) with PAH associated with CVD demonstrated improved exercise capacity, quality of life, and hemodynamics with sitaxsentan although elevated liver enzyme levels were reported in two patients [108]. A large placebo-controlled, randomized trial of ambrisentan, the only currently FDA-approved selective endothelin receptor antagonist, improved the 6-min walk distance in PAH patients at week 12 of treatment; however, the effect was larger in patients with IPAH than in patients with PAH associated with CVD (range of 50–60  m vs. 15–23  m, respectively) [109]. Ambrisentan is generally well tolerated, although peripheral edema (in up to 20% of patients [109]) and congestive heart failure have been reported.

4.3  Phosphodiesterase Inhibitors Sildenafil, a phosphodiesterase type V inhibitor that reduces the catabolism of cyclic GMP, thereby enhancing the cellular effects mediated by nitric oxide, has become a widely used and highly efficacious therapy for PAH. A recent clinical trial showed that sildenafil therapy led to an improvement in the 6-min walk distance in patients with IPAH and PAH related to CVD or repaired congenital heart disease (patients were predominantly ­functional class II or III) at all three dosages tested (20, 40, and 80  mg, given three times a day) [110]. Since there were no significant differences in clinical effects and the time to clinical worsening at week 12 between the dosages, the FDA recommended a dosage of 20 mg three times a day. In a post hoc subgroup analysis of 84 patients

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with PAH related to CVD (45% of whom had SSc-PAH), data from the SUPER study suggest that sildenafil at a dose of 20 mg improved exercise capacity (6-min walk distance), hemodynamic measures, and functional class after 12 weeks of therapy [111]. However, for reasons that remain unclear (but in part related to the limitations of that study such as post hoc subgroup analysis), there was no effect for the dosage of 80  mg three times a day on hemodynamics in this ­subgroup of patients with CVD-related PAH [111]. For this reason and because of the potential of increased side effects (such as bleeding from arteriovenous malformations) at high doses, a sildenafil dosage of 20 mg three times a day is recommended for SSc-PAH patients (and perhaps for patients with PAH associated with other forms of CVD) as standard therapy. Higher doses are occasionally attempted in the case of limited response. The impact of long-term sildenafil therapy on survival in these patients remains to be determined. Finally, the results for ­tadalafil, another promising phosphodiesterase antagonist for PAH therapy which has the advantage over sildenafil of single daily dosage, have not yet been published.

4.4  Combination Therapy It is now common practice to add drugs when patients fail to improve on monotherapy. Adding inhaled iloprost to patients receiving bosentan has been shown to be beneficial in a small, randomized trial. Combining inhaled iloprost with sildenafil is mechanistically appealing and anecdotally efficacious [112, 113] as these drugs target separate, potentially synergistic pathways. Several multicenter trials are now exploring the efficacy of various combinations of two oral drugs or one oral and one inhaled drug. The recently published results of the PACES trial demonstrate that adding sildenafil (at a dosage of 80 mg three times a day) to intravenously administered epoprostenol improves exercise ­capacity, hemodynamic measurements, time to clinical worsening, and quality of life [114]. About 21% of these patients had CVD, including 11% with SSc-PAH. Although no specific subgroup analysis was provided, improvement was apparently mainly in patients with IPAH. In a smaller one-center clinical trial, adding sildenafil for patients with IPAH or SScrelated PAH after they had failed initial monotherapy with bosentan demonstrated that combination therapy improved the 6-min walk distance and functional class in IPAH patients. The outcome in patients with SSc-PAH was less favorable, although combination therapy may have halted clinical ­deterioration. In addition, more side effects were reported in the SSc-PAH patients compared with the IPAH patients, including hepatotoxicity that developed after addition of sildenafil to bosentan monotherapy [115].

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4.5  Anticoagulation The rationale for the use of anticoagulation in severe PAH is based on pathologic evidence of pulmonary thromboembolic arterial disease and thrombosis in situ in patients with IPAH [116], and two clinical studies (a retrospective analysis) [117] and a small, nonrandomized prospective study [88] demonstrating a significant beneficial effect of anticoagulation on survival in IPAH. Based essentially on these findings, anticoagulation is routinely recommended in the treatment of IPAH patients. The role of anticoagulation in other forms of PAH, in particular in SSc-PAH or other forms of CVD is much less clear. Theoretically, there is potential for increased bleeding in patients with CVD, particularly with SSc, where intestinal telangiectasias may be common. An unpublished review of our experience with anticoagulation in over 100 patients with SSc-PAH suggests that less than 50% of these patients remain on long-term anticoagulation therapy. The reason for discontinuing anticoagulation in these patients is often related to occult bleeding in the gastrointestinal tract. In our experience, the source of bleeding is often difficult to diagnose.

4.6  Anti-inflammatory Drugs As discussed already, it has been increasingly recognized that inflammation may play a significant role in various types of pulmonary hypertension, including IPAH and PAH associated with CVD. Interestingly, some patients with severe PAH associated with some forms of CVD (such as SLE, pSS, and MCTDs) have had dramatic improvement of their pulmonary vascular disease with corticosteroids and/or immunosuppressive therapy [46], emphasizing the relevance of inflammation in these subsets of patients. However, this has not been the case for patients with SSc-PAH whose PAH is usually refractory to immunosuppressive drugs [46].

4.7  Tyrosine Kinase Inhibitors The finding that there is pathologically aberrant proliferation of endothelial and smooth muscle cells in PAH, as well increased expression of secreted growth factors such as VEGF and basic fibroblast growth factor, has caused a shift in paradigm in treatment strategies for this disease as some investigators have likened this condition to a neoplastic process reminiscent of advanced solid tumors [118]. As a result, antineoplastic drugs have been tested in experimental ­models

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[119, 120] and some patients [121, 122]. Two strategies are currently being tested for treatment of PAH: disruption of platelet-derived growth factor signaling and disruption of the VEGF signaling pathway. Whether these new antineoplastic drugs with anti-tyrosine kinase activity will have a role in PAH associated with CVD such as SSc-PAH (where there is evidence for both dysregulated proliferation and increased expression of growth factors such as VEGF) remains to be determined.

4.8  Lung Transplantation Lung transplantation is typically offered as a last resort to patients with PAH who fail medical therapy. Although CVD is not an absolute contraindication to lung transplantation, patients with CVD often have associated morbidity and organ dysfunction other than that of the lung that place them at a specifically high risk for lung transplantation. The involvement of the esophagus with severe motility disorder and gastroesophageal reflux in patients with SSc is an example of specific risk that surgeons do not take lightly because of the enhanced postoperative potential of aspiration and damage to the recipient lung. For these reasons, patients with SSc-PAH are often denied the lung transplantation option. However, if properly screened and approved for lung transplantation, patients with SSc experience similar rates of survival 2 years after the procedure as patients who receive lung transplantation for pulmonary fibrosis or IPAH [123].

5  Survival of Patients with CVD and PAH Despite the recent increase in the number of drugs targeting specific pathways in PAH, survival of patients with CVD and PAH remains unacceptably low, although there is evidence of mild survival improvement in patients with SSc-PAH compared with similar historical controls [76]. The reason for a discrepancy in survival between IPAH patients and patients with CVD and PAH, particularly SSc-PAH, remains poorly understood and may involved more complex disturbances affecting not only both proximal and distal pulmonary vessels but also the heart (such as inflammatory myocarditis) in CVD. Thus, a better understanding of the underlying pathophysiological processes as well as the RV-pulmonary vascular interaction and uncoupling in this disease is needed for better targeted therapy. Whether ­specific anti-inflammatory agents or drugs targeting tyrosine kinase activity hold any promise of a better response is unclear at this time but needs to be explored.

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6  Summary and Conclusion Pulmonary hypertension is a common complication of CVD, particularly SSc, where it portends a significantly worse outcome compared with other diseases (such as IPAH) within group 1 of the WHO classification. In addition, modern therapy for PAH appears to be of limited value in SSc-PAH (Fig. 2). Similarly, currently available markers of disease severity or response to therapy in SSc-PAH and other CVDs are either limited or lacking. Therefore, there is an urgent need to identify potential genetic causes and novel physiologic, molecular, and imaging biomarkers that will allow a better understanding of the underlying pathogenesis and serve as reliable tools to monitor therapy in this devastating syndrome.

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1020 37. Tamby MC, Chanseaud Y, Humbert M et al (2005) Anti-endothelial cell antibodies in idiopathic and systemic sclerosis associated pulmonary arterial hypertension. Thorax 60:765–772 38. Fritzler MJ, Hart DA, Wilson D et al (1995) Antibodies to fibrin bound tissue type plasminogen activator in systemic sclerosis. J Rheumatol 22:1688–1693 39. Morse JH, Barst RJ, Fotino M et  al (1997) Primary pulmonary hypertension, tissue plasminogen activator antibodies, and HLA-DQ7. Am J Respir Crit Care Med 155:274–278 40. Grigolo B, Mazzetti I, Meliconi R et al (2000) Anti-topoisomerase II alpha autoantibodies in systemic sclerosis-association with pulmonary hypertension and HLA-B35. Clin Exp Immunol 121:539–543 41. Nicolls MR, Taraseviciene-Stewart L, Rai PR, Badesch DB, Voelkel NF (2005) Autoimmunity and pulmonary hypertension: a perspective. Eur Respir J 26:1110–1118 42. Okawa-Takatsuji M, Aotsuka S, Fujinami M, Uwatoko S, Kinoshita M, Sumiya M (1999) Up-regulation of intercellular adhesion molecule-1 (ICAM-1), endothelial leucocyte adhesion molecule-1 (ELAM-1) and class II MHC molecules on pulmonary artery endothelial cells by antibodies against U1-ribonucleoprotein. Clin Exp Immunol 116:174–180 43. Chizzolini C, Raschi E, Rezzonico R et al (2002) Autoantibodies to fibroblasts induce a proadhesive and proinflammatory fibroblast phenotype in patients with systemic sclerosis. Arthritis Rheum 46:1602–1613 44. Tamby MC, Humbert M, Guilpain P et  al (2006) Antibodies to fibroblasts in idiopathic and scleroderma-associated pulmonary hypertension. Eur Respir J 28:799–807 45. Terrier B, Tamby MC, Camoin L et al (2008) Identification of target antigens of antifibroblast antibodies in pulmonary arterial hypertension. Am J Respir Crit Care Med 177:1128–1134 46. Sanchez O, Sitbon O, Jais X, Simonneau G, Humbert M (2006) Immunosuppressive therapy in connective tissue diseases-­ associated pulmonary arterial hypertension. Chest 130:182–189 47. Jais X, Launay D, Yaici A et al (2008) Immunosuppressive therapy in lupus- and mixed connective tissue disease-associated pulmonary arterial hypertension: a retrospective analysis of twenty-three cases. Arthritis Rheum 58:521–531 48. Nichols WC, Koller DL, Slovis B et al (1997) Localization of the gene for familial primary pulmonary hypertension to chromosome 2q31-32. Nat Genet 15:277–280 49. Deng Z, Morse JH, Slager SL et al (2000) Familial primary pulmonary hypertension (gene PPH1) is caused by mutations in the bone morphogenetic protein receptor-II gene. Am J Hum Genet 67:737–744 50. Lane KB, Machado RD, Pauciulo MW et al (2000) Heterozygous germline mutations in BMPR2, encoding a TGF-b receptor, cause familial primary pulmonary hypertension. The International PPH Consortium. Nat Genet 26:81–84 51. Newman JH, Trembath RC, Morse JA et al (2004) Genetic basis of pulmonary arterial hypertension: current understanding and future directions. J Am Coll Cardiol 43:33S–39S 52. Koehler R, Grunig E, Pauciulo MW et al (2004) Low frequency of BMPR2 mutations in a German cohort of patients with sporadic idiopathic pulmonary arterial hypertension. J Med Genet 41:e127 53. Morse JH, Deng Z, Knowles JA (2001) Genetic aspects of pulmonary arterial hypertension. Ann Med 33:596–603 54. Trembath RC, Thomson JR, Machado RD et  al (2001) Clinical and molecular genetic features of pulmonary hypertension in patients with hereditary hemorrhagic telangiectasia. N Engl J Med 345:325–334 55. Karrer S, Bosserhoff AK, Weiderer P et al (2005) The -2518 promotor polymorphism in the MCP-1 gene is associated with systemic sclerosis. J Investig Dermatol 124:92–98 56. Tolusso B, Fabris M, Caporali R et al (2005) 238 and +489 TNF-a along with TNF-RII gene polymorphisms associate with the dif-

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70  Pulmonary Arterial Hypertension Related to Scleroderma and Collagen Vascular Diseases 76. Williams MH, Das C, Handler CE et al (2006) Systemic sclerosis associated pulmonary hypertension: improved survival in the current era. Heart 92:926–932 77. Koh ET, Lee P, Gladman DD, Abu-Shakra M (1996) Pulmonary hypertension in systemic sclerosis: an analysis of 17 patients. Br J Rheumatol 35:989–993 78. McLaughlin VV, Shillington A, Rich S (2002) Survival in primary pulmonary hypertension: the impact of epoprostenol therapy. Circulation 106:1477–1482 79. Kuryliszyn-Moskal A, Klimiuk PA, Ciolkiewicz M, Sierakowski S (2008) Clinical significance of selected endothelial activation markers in patients with systemic lupus erythematosus. J Rheumatol 35:1307–1313 80. Yoshio T, Masuyama J, Mimori A, Takeda A, Minota S, Kano S (1995) Endothelin-1 release from cultured endothelial cells induced by sera from patients with systemic lupus erythematosus. Ann Rheum Dis 54:361–365 81. Pope J (2008) An update in pulmonary hypertension in systemic lupus erythematosus – do we need to know about it? Lupus 17:274–277 82. Haas C (2004) Pulmonary hypertension associated with systemic lupus erythematosus. Bull Acad Natl Med 188:985–997 83. Sullivan WD, Hurst DJ, Harmon CE et  al (1984) A prospective evaluation emphasizing pulmonary involvement in patients with mixed connective tissue disease. Medicine (Baltimore) 63:92–107 84. Launay D, Hachulla E, Hatron PY, Jais X, Simonneau G, Humbert M (2007) Pulmonary arterial hypertension: a rare complication of primary Sjogren syndrome: report of 9 new cases and review of the literature. Medicine (Baltimore) 86:299–315 85. Giaid A, Saleh D (1995) Reduced expression of endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension. N Engl J Med 333:214–221 86. Tuder RM, Cool CD, Geraci MW et al (1999) Prostacyclin synthase expression is decreased in lungs from patients with severe pulmonary hypertension. Am J Respir Crit Care Med 159:1925–1932 87. Giaid A, Yanagisawa M, Langleben D et al (1993) Expression of endothelin-1 in the lungs of patients with pulmonary hypertension. N Engl J Med 328:1732–1739 88. Rich S, Kaufmann E, Levy PS (1992) The effect of high doses of calcium-channel blockers on survival in primary pulmonary hypertension. N Engl J Med 327:76–81 89. Sitbon O, Humbert M, Jais X et al (2005) Long-term response to calcium channel blockers in idiopathic pulmonary arterial hypertension. Circulation 111:3105–3111 90. Humbert M, Sitbon O, Chaouat A et al (2006) Pulmonary arterial hypertension in France: results from a national registry. Am J Respir Crit Care Med 173:1023–1030 91. Vane JR, Anggard EE, Botting RM (1990) Regulatory functions of the vascular endothelium. N Engl J Med 323:27–36 92. Barst RJ, Rubin LJ, Long WA et  al (1996) A comparison of ­continuous intravenous epoprostenol (prostacyclin) with conventional therapy for primary pulmonary hypertension. The Primary Pulmonary Hypertension Study Group. N Engl J Med 334:296–302 93. McLaughlin VV, Genthner DE, Panella MM, Rich S (1998) Reduction in pulmonary vascular resistance with long-term epoprostenol (prostacyclin) therapy in primary pulmonary hypertension. N Engl J Med 338:273–277 94. Rubin LJ, Mendoza J, Hood M et al (1990) Treatment of primary pulmonary hypertension with continuous intravenous prostacyclin (epoprostenol). Results of a randomized trial. Ann Intern Med 112:485–491 95. Badesch DB, Tapson VF, McGoon MD et al (2000) Continuous intravenous epoprostenol for pulmonary hypertension due to the

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1022 sildenafil in idiopathic pulmonary arterial hypertension. Eur Respir J 24:1007–1010 114. Simonneau G, Rubin LJ, Galie N et al (2008) Addition of sildenafil to long-term intravenous epoprostenol therapy in patients with pulmonary arterial hypertension: a randomized trial. Ann Intern Med 149:521–530 115. Mathai SC, Girgis RE, Fisher MR et al (2007) Addition of sildenafil to bosentan monotherapy in pulmonary arterial hypertension. Eur Respir J 29:469–475 116. Pietra GG (1994) Histopathology of primary pulmonary hypertension. Chest 105:2S–6S 117. Fuster V, Steele PM, Edwards WD, Gersh BJ, McGoon MD, Frye RL (1984) Primary pulmonary hypertension: natural history and the importance of thrombosis. Circulation 70:580–587 118. Adnot S (2005) Lessons learned from cancer may help in the treatment of pulmonary hypertension. J Clin Invest 115:1461–1463

P.M. Hassoun 119. Schermuly RT, Dony E, Ghofrani HA et  al (2005) Reversal of experimental pulmonary hypertension by PDGF inhibition. J Clin Invest 115:2811–2821 120. Moreno-Vinasco L, Gomberg-Maitland M, Maitland ML et al (2008) Genomic assessment of a multikinase inhibitor, sorafenib, in a rodent model of pulmonary hypertension. Physiol Genomics 33:278–291 121. Ghofrani HA, Seeger W, Grimminger F (2005) Imatinib for the treatment of pulmonary arterial hypertension. N Engl J Med 353:1412–1413 122. Patterson KC, Weissmann A, Ahmadi T, Farber HW (2006) Imatinib mesylate in the treatment of refractory idiopathic pulmonary arterial hypertension. Ann Intern Med 145:152–153 123. Schachna L, Medsger TA Jr, Dauber JH et al (2006) Lung transplantation in scleroderma compared with idiopathic pulmonary fibrosis and idiopathic pulmonary arterial hypertension. Arthritis Rheum 54:3954–3961

Chapter 71

Pathology and Management of Portopulmonary Hypertension Michael J. Krowka

Abstract  Portopulmonary hypertension (POPH) is the well-recognized relationship of pulmonary artery hypertension that evolves as a consequence of portal hypertension. POPH was first reported by Mantz and Craige in a 53-year-old female patient who presented with sudden hoarseness due to a large spontaneous portocaval shunt (1.5–3 cm in diameter) that lay in intimate relationship with the left recurrent laryngeal nerve as it coursed through the mediastinum. Following death from rightsided heart failure 4 days after an exploratory thoracotomy (expecting a mediastinal tumor), the postmortem study demonstrated pulmonary arterial disease that included endothelial proliferation, recanalization, old clot (in situ versus embolic) and completed vascular obliteration due to scarring. The portal vein was “strikingly narrowed” and associated with splenomegaly and normal hepatic parenchyma. Since the 1980s enhanced recognition and renewed importance of POPH has evolved with the evolution of liver transplantation (LT). Such clinical interest has been spurred by remarkable resolution or improvement of POPH following LT in highly selected, aggressively managed cases, but has been tempered by the well-documented complications of increased intraoperative and transplant hospitalization mortality due to right-sided heart failure caused by severe pulmonary artery hypertension. Specific screening recommendations and diagnostic criteria now exist for POPH. Such specific criteria are important in view of the spectrum of pulmonary hemodynamics well documented in advanced lived disease and therapeutic decisions facing the clinician. Ironically, the exclusion of POPH patients from all randomized, controlled pulmonary artery hypertension trials to date has hampered the development of therapeutic practice guidelines with reference to pulmonary vasomodulating drugs.

M.J. Krowka (*) Pulmonary and Critical Care Medicine, Mayo Clinic, College of Medicine, 200 First Street, Rochester, MN 55905, USA e-mail: [email protected]

Keywords  Liver disease • Portal hypertension • Pulmonary arterial hypertension • Endothelial proliferation • Recanalization

1 Introduction Portopulmonary hypertension (POPH) is the well-recognized relationship of pulmonary artery hypertension that evolves as a consequence of portal hypertension. In the 2003 Venice and 2008 Dana Point clinical classifications of pulmonary artery hypertension, POPH was recognized within the group 1 category owing, in part, to its identical pathologic resemblance to idiopathic pulmonary artery hypertension [1]. POPH was first reported by Mantz and Craige in a 53-year-old female patient who presented with sudden hoarseness due to a large spontaneous portocaval shunt (1.5–3  cm in diameter) that lay in intimate relationship with the left recurrent laryngeal nerve as it coursed through the mediastinum. Following death from right-sided heart failure 4 days after an exploratory thoracotomy (expecting a mediastinal tumor), the postmortem study demonstrated pulmonary arterial disease that included endothelial proliferation, recanalization, old clot (in  situ versus embolic) and completed vascular obliteration due to scarring [2]. The portal vein was “strikingly narrowed” and associated with splenomegaly and normal hepatic parenchyma. Since the 1980s enhanced recognition and renewed importance of POPH has evolved with the evolution of liver transplantation (LT). Such clinical interest has been spurred by remarkable resolution or improvement of POPH following LT in highly selected, aggressively managed cases, but has been tempered by the well-documented complications of increased intraoperative and transplant hospitalization mortality due to right-sided heart failure caused by severe pulmonary artery hypertension [3, 4]. Specific screening recommendations and diagnostic ­criteria now exist for POPH [5, 6] (Table 1). Such specific criteria are important in view of the spectrum of ­pulmonary ­hemodynamics well documented in advanced lived disease

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_71, © Springer Science+Business Media, LLC 2011

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and ­therapeutic decisions facing the clinician (Fig.  1). Ironically, the exclusion of POPH patients from all ­randomized, ­controlled pulmonary artery hypertension ­trials to date has hampered the development of therapeutic practice guidelines with reference to pulmonary ­vasomodulating drugs.

Table 1  Portopulmonary hypertension (POPH) diagnostic criteria 1. Portal hypertension (with or without cirrhosis) 2. Abnormal pulmonary hemodynamics (a) MPAP > 25 mmHg (b) PVR > 240 dyn s cm–5 (c) TPG > 12 mmHga MPAP mean pulmonary artery pressure, PVR pulmonary vascular resistance, TPG transpulmonary gradient [MPAP minus pulmonary artery occlusion pressure (PAOP)] a  This parameter replaces the PAOP (also known as pulmonary capillary wedge pressure) criteria proposed by the ERS Task Force [5]

M.J. Krowka

2 Etiology and Pathogenetic Mechanisms It is hypothesized that in genetically susceptible patients, the existence of portal hypertension allows circulating factors to bypass normal hepatic metabolism and adversely affect the pulmonary vascular bed [7–9]. Specifically, obstruction to arterial flow is caused by proliferation of pulmonary arterial endothelium and smooth muscle, in situ thrombosis, and vasoconstriction (Fig. 2). The presence of classic plexogenic arteriopathy has been well documented [10, 11]. Abnormal platelet aggregations have been documented at the small vessel and capillary level in POPH patients who have died of right-sided heart failure during LT attempts [12]. Consistent with this “bypass” hypothesis is the increased frequency of POPH seen after portocaval shunt surgery [13, 14]. Interestingly, the microscopic pulmonary pathologic changes seen in POPH are identical to those seen in idiopathic pulmonary artery hypertension [15]. Furthermore, a deficiency in endothelial prostacyclin synthase and increase in circulating endothelin-1 levels have been reported in POPH [16, 17]. Increased measured gradients of endothelin-1 between the pulmonary artery and pulmonary veins, but not between the hepatic vein and inferior vena cava have been documented (K.L. Swanson, Mayo Clinic, personal communication).

3 Prevalence and Clinical Manifestations

Fig. 1  Pulmonary hemodynamics associated with portal hypertension can be categorized into three main groups on the basis of measured mean pulmonary artery pressure (MPAP), cardiac output (CO), and pulmonary arterial occlusion pressure (PAOP). Pulmonary vascular resistance (PVR) is a calculated value [(MPAP-PAOP)/CO]

Fig. 2  Autopsy specimen from patient with mild portopulmonary hypertension (POPH) (MPAP 34 mmHg; PVR 160 dyn s cm–5, CO 9.4 L/min following treatment with sildenafil 20 mg three times daily for 3 months). Sudden cardiac arrest 3 days after liver transplantion. Distinction between in situ clot and embolic clot may be difficult (patient had no radiographic findings to suggest pulmonary emboli). Proliferation of endothelium and smooth muscle noted with plexogenic change caused vascular obstruction to flow

Although earlier autopsy studies suggested a 0.25–0.73% frequency of pulmonary artery hypertension complicating portal hypertension or cirrhosis, the clinical diagnosis of POPH in the era of pulmonary artery hypertension ­therapeutic advances and LT is more prevalent [18–20]. Data from the REVEAL registry (Registry to Evaluate Early and LongTerm Pulmonary Artery Hypertension Management),

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p­ rovided by pulmonary hypertension clinics throughout the USA since 2006 (n = 3,250), suggest that the diagnosis of POPH comprises nearly 5% of all data submissions [21]. A  recent French study has described POPH as the fourth ­leading cause of pulmonary artery hypertension seen in the 17-member hospital consortium and with a frequency of 10.4% of 674 patients seen in the time span from 2002 to 2003 [22]. Two of the largest LT centers in the USA reported a 5.3–8.5% frequency of POPH in LT candidates [23, 24]. POPH can complicate any hepatic disorder that results in  portal hypertension and usually manifests 4–7 years ­following a given hepatic diagnosis [25, 26]. Not infrequently, patients referred to pulmonary hypertension clinics with a diagnosis of idiopathic pulmonary hypertension are subsequently found to have POPH, as the only presenting clue may be a marked reduction in the serum platelet counts (without changes in the levels of serum bilirubin or liver enzymes) due to splenomegaly caused by portal hypertension. An increased frequency of POPH is suggested in autoimmune hepatic problems and the female gender [27]. Rarely, POPH occurs in the pediatric age group [28]. No relationship exists between the severity of POPH and the severity of liver disease as characterized by the Child–Pugh–Turcotte scores (ascites, encephalopathy, bilirubin, albumin and prothrombin time), model for end-stage liver disease scores (bilirubin, creatinine, and international normalized ratio) or the degree of the hepatic vein–portal gradient [4, 5, 26, 27]. As obstruction to pulmonary arterial flow worsens, progressive exertional dyspnea is usually the first patient complaint. Late

symptoms may include chest pain and syncope, caused by right-sided heart strain and reduced cardiac output, respectively [4]. The physical examination may reveal an increased intensity and wide splitting of the second heart sound, elevated jugular pressures, a systolic murmur of tricuspid regurgitation, and normal breath sounds. A hyperdynamic precordium is usually present and a right ventricular heave may be palpable. Cyanosis is not a usual sign in POPH [4]. Abnormalities in the posterolateral chest radiograph include cardiomegaly and enlarged central pulmonary arteries, but these findings are associated with at least moderate to severe pulmonary artery hypertension [4]. Further changes in chest radiographs may demonstrate other complications of advanced liver disease that occur independent of the POPH and reflect the dual nature of the concomitant pulmonary and hepatic problems (Fig. 3). Electrocardiogram changes suggest right ventricular hypertrophy, right atrial enlargement, a rightward QRS axis, and strain on the right ventricle as manifested by T-wave inversions in the precordial V1 to V4 leads (a sign of moderate to severe POPH). Pulmonary function testing is not particularly helpful and frequently reveals a nonspecific decrease in the single breath diffusing capacity for carbon monoxide; lung volumes and expiratory airflows are normal. Arterial blood gases may show a slight abnormality in oxygenation and marked reduction in carbon dioxide with a respiratory alkalosis pattern, but again these are nonspecific findings [4, 5].

Fig. 3  Chest radiograph demonstrating cardiomegaly and enlargement of central pulmonary arteries; patient receiving intravenous administration of prostacyclin. Four years later the patient presented with metastatic hepatocellular carcinoma and pericardial effusion in the setting of stable POPH

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4 Screening and Diagnostic Criteria Specific diagnostic criteria for the diagnosis of POPH have been formulated via a Pulmonary-Hepatic Vascular Disorder Task Force convened in 2004 [5]. Increased mean pulmonary artery pressure (MPAP) and pulmonary vascular resistance (PVR), accompanied by a normal pulmonary artery occlusion pressure (PAOP), form the key elements of the criteria (Table  1). The latter criterion (normal PAOP) has recently been modified and replaced by an increased transpulmonary gradient by some investigators (see later) [23]. With diagnostic criteria now recognized, practice guidelines have been proposed by the American Association for the Study of Liver Disease that recommend screening transthoracic echocardiography for all LT candidates [6]. The goal of such screening is to noninvasively identify patients with any form of pulmonary hypertension and select those that should undergo diagnostic right-sided heart catheterization. As to the clinical relevance for such screening, the presence of moderate to severe POPH (MPAP greater than 35 mmHg) in LT candidates has been associated with several intraoperative deaths and an approximate 35% frequency of posttransplant hospitalization mortality. This degree of mortality was documented in both a 2000 literature review (n = 48 cases of POPH with attempted LT) and a 2004 report from a multicenter, prospective data collection effort of LT candidates (n = 66) with POPH [29, 30]. Thus, screening and documentation of POPH has significant clinical management implications. Two key features can be elicited from echocardiography to guide the clinician toward further study: an estimate of the pulmonary artery systolic pressure and cardiac chamber size

Fig. 4  Frequency of pulmonary hemodynamic patterns documented by right-sided heart catheterization (RHC) that followed screening transthoracic echocardiography; RHC was accomplished if right ventricular systolic pressure was more than 50 mmHg. (Reproduced from [23] with permission)

M.J. Krowka

and function [31–33]. Patients with advanced liver disease commonly have a hyperdynamic circulatory state that causes high flow in the pulmonary vascular bed. Cardiac and/or renal dysfunction may complicate liver diseases, leading to excess central volume. Less commonly, obstruction to pulmonary artery flow due to endothelial/smooth muscle proliferation and/or in situ thrombosis that characterizes POPH may occur. Importantly, any or all of these mechanisms may result in increased pulmonary artery systolic pressure in a given patient that can be estimated by the right ventricular systolic pressure (as long as the pulmonic valve is normal) using the modified Bernoulli equation. In addition, measurement of the left atrial size may help discern volume status and left-sided heart function [32]. Finally, qualitative assessment of right ventricular size and function can be made, as well as documentation of abnormal shape of the left ventricle (“D” shape) in the setting of severe pulmonary artery hypertension [33]. In the largest prospective screening and diagnostic study reported to date from 1,235 patients seen at the Mayo Clinic Liver Transplant Program, 101 (8.2%) underwent right-sided heart catheterization because the right ventricular systolic pressure was found to be greater than 50 mmHg [23] (Fig. 4). Of all patients screened, 66 of 1,235 (5.3%) had both increased MPAP and increased PVR, thus fulfilling the POPH diagnostic criteria (Table  2). Selected pulmonary hemodynamic relationships observed in POPH patients are shown graphically in Fig. 5. A high flow state with normal PVR was documented in 35 patients (35%) with right ventricular systolic pressure greater than 50 mmHg. Clinically, normal PVR patients would not need pulmonary vasomodulating therapy, nor would they

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Table 2  Pulmonary hemodynamic patterns associated with advanced liver disease (mean right-sided heart catheterization data; n = 101) MPAP (mmHg) CO (L/min) PVR (dyn s cm–5) PAOP (mmHg) TPG (mmHg) 31 ± 9 8.6 ± 2.6* 142 ± 58 16 ± 6* High flow (decreased PVR) (n = 35) 28 ± 8 8.2 ± 2.3 154 ± 60 12 ± 2 Normal volume (n = 20) 34 ± 10 9.1 ± 3.0 125 ± 52 21 ± 4 Increased volume (n = 15) POPH (increased PVR) (n = 66) 49 ± 11* 6.1 ± 2.0 533 ± 247* 12 ± 6 Normal volume (n = 50) 48 ± 11 5.9 ± 2.0 571 ± 257 10 ± 3 Increased volume (n = 16) 53 ± 9 6.8 ± 2.0 407 ± 171 21 ± 5 CO cardiac output, *p < 0.001 All patients had right ventricular systolic pressure of 50 mmHg or more by echocardiography (normal is less than 35 mmHg)

16 ± 7 17 ± 7 14 ± 7 37 ± 11* 38 ± 11 34 ± 10

Fig.  5  Pressure–flow relationships via RHC in patients with POPH. (a) PVR versus CO for all patents (n = 101). As the resistance to pulmonary arterial flow increases, a reduction in blood flow as measured by CO is observed. (b) Increasing the resistance to pulmonary arterial flow is associated with increasing MPAP. However, above a PVR of 800 dyn s cm–5, a few patients appear to show reduction in MPAP, suggesting a failure of the

right ventricle as PVR further increases. (c) MPAP versus CO suggesting a nonlinear relationship as MPAP increases. All patients to the left of and above the PVR of 240 dyn s cm–5 line had increased PVR. Patients below the PVR of 240 dyn s cm–5 line had normal PVR. The group of patients between 120 and 240 dyn s cm–5 merit close follow-up. MPAP versus CO in all patients (n = 101). (Reproduced from [23] with permission)

have higher risk for LT surgery. Finally, a small number of patients (16 of 66) with portal hypertension were noted to have increased MPAP, PVR, and PAOP. Although the increased PAOP was not consistent with ERS Task Force recommendations, these patients are now considered by several investigators to have an abnormal pulmonary vascular bed with increased resistance to flow as characterized by an increased transpulmonary gradient and, hence, are a distinct subgroup of POPH.

5 Treatments There have been no controlled, randomized trials that have exclusively enrolled patients with POPH. However, small series and numerous case reports have documented the hemodynamic improvement using prostacyclins [34–45], endothelin receptor antagonists [46–55], and phosphodiesterase inhibitors [56–58]. These studies have included some patients treated both before and after LT, as well as those who did not

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undergo transplantation. The largest collective experience to date has been with the use of intravenous 24-h continuous infusion of epoprostenol. Analyses of data published from five series have documented a significant decrease in MPAP (28%) and PVR (52%), as well as an increase in cardiac output (25%) from the baseline. Epoprostenol was given for a minimum of 3 months with variable time end points (Table 3). Several single case reports have reported hemodynamic improvement using bosentan in Childs A patients with liver disease and normal or minimal abnormalities in liver enzymes. In the largest single series from Germany, investigators reported 1- and 3-year survivals of 89% and 79%, respectively, in 18 POPH patients given bosentan 125  mg twice daily [54]. Mean changes in MPAP (53 to 45 mmHg), PVR (925 to 579 dyn·s·cm–5), and cardiac output (4.4 to 5.7 L/min) were clinically significant. These same investigators reported their uncontrolled experience using inhaled iloprost (13 patients) and noted no significant hemodynamic improvements in MPAP, PVR, or cardiac output. Sildenafil has resulted in successful short-term hemodynamic improvement in uncontrolled studies of POPH patients, but the best 12-month outcomes occurred when a prostacyclin was coadministered [56–58]. Despite pulmonary hemodynamic improvement, concern has been raised about the potential deleterious effect on portal hypertension with sildenafil [59]. The use of beta-blockers in the setting of moderate to severe POPH (MPAP of 52 mmHg in ten patients) has been reported. Withdrawal of beta-blockers was associated with increased 6-min walk distance (79 m), cardiac output (28% owing to increased heart rate), with no change in MPAP. PVR decreased by 19% owing to increased cardiac output. No patient experienced a major hepatic complication (such as variceal bleed) by 23 months of follow-up [60]. At this time POPH is not considered an indication for LT, whereas the other pulmonary vascular consequence of portal hypertension–hepatopulmonary syndrome (hypoxemia due to pulmonary vascular dilatation) is an indication and receives higher priority if PaO2 is less than 60 mmHg [5]. Early single-center experience (15 out of 362 transplants) suggested that patients with MPAP between 25 and 43 mmHg and PVR less than 250 dyn·s·cm–5 were not at increased mortality risk for LT [61]. Increased pulmonary artery pressures with ­normal PVR (owing to increased cardiac outputs) were Table  3  Pulmonary hemodynamic effect of intravenously administered epoprostenol in POPH Baseline Follow-up MPAP (mmHg) 48 (35–76) 36 (24–72)* CO (L/min) 6.3 (1.9–10.5) 8.7 (4.4–14.0)* PVR (dyn s cm–5) 550 (250–1,811) 262 (120–800)* n = 48 patients reported from five studies [34, 36, 42–44]. Mean values (range) are shown *p < 0.01 at follow-up that ranged from 3 to 36 months

M.J. Krowka

d­ ocumented in 20% of transplant candidates [61]. Such ­common hemodynamic patterns, distinct from POPH, have been confirmed in subsequent studies and are important observations when decisions are made about pulmonary vasomodulating therapy and LT candidacy [23, 62, 63]. Outcomes associated with LT, as summarized in Table 4, have been variable and unpredictable [64–82]. Mortality following LT attempts in the setting of POPH is well documented, with right-sided heart failure being the direct cause of death [12, 24, 30, 65, 70, 73, 76, 81]. Hence, the existence of moderate to severe POPH (MPAP greater than 35 mmHg has been considered a relative contraindication for LT owing to the increased intraoperative and posttransplant hospitalization mortality without pulmonary vasomodulating therapy [30]. Small series, however, have demonstrated the effectiveness of treating POPH aggressively prior to LT, documenting improved hemodynamics and then proceeding to LT surgery. In approximately 50% of cases in which transplantation was successful, the pretransplant prostacyclin regimen could be discontinued and pulmonary dynamics were found to be ­normal; other patients were successfully maintained on oral regimens of either bosentan or sildenafil [38, 39, 42–44]. In short, there are selected patients who may be “cured” of POPH, at least from a hemodynamic perspective, and others may be well controlled with oral pulmonary vasomodulating drugs. Controlled studies using such medications as a bridge to LT are in the planning phases. The scope of the POPH– transplant problem is not trivial, with approximately 18,000 patients on the United Network for Organ Sharing waiting for LT (nearly 6,500 transplants accomplished annually in the USA) and an estimated POPH frequency of 5%, suggesting about 300 new LT candidates annually found to have POPH (http.//www.UNOS.com). The current POPH–LT candidate-treatment clinical algorithm followed at the Mayo Clinic since 1996 is shown in Fig. 6. Table 4  Outcomes of liver transplantation in the setting of POPH Outcome reported References Denied transplant owing to POPH Transplant surgery; but intraoperative death Transplant surgery accomplished with Transplant hospitalization mortality Subsequent nontransplant h­ ospitalization mortality Survival, worsening or no improvement Improvement/normalization of pulmonary hemodynamics with continual pulmonary ­vasomodulating therapy Resolution of POPH (normal ­hemodynamics) and no pulmonary vasomodulating therapy Multiorgan allografts (heart–lung–liver) Living donor allograft

[30] [12, 29, 30, 32, 65, 69, 72] [24, 30, 44, 65, 66, 72, 74, 77, 79] [24, 29, 63] [24, 43, 45, 71] [35, 37, 38, 56, 58, 60, 67, 68, 70, 74, 81, 85] [39–42, 48, 69, 73, 75, 76, 85] [64, 78, 82] [80]

71  Pathology and Management of Portopulmonary Hypertension

Fig. 6  Algorithm followed at the Mayo Clinic for screening, categorization via RHC and treatment decisions based upon catheterization results

6 Prognosis In the pre-LT, pre-prostacyclin eras (before 1985), scant data suggested a 5-year survival of 30% [25]. In the LT era, recent data confirm a poor outcome in advanced liver disease (Childs B and C) if POPH coexists [83, 84]. In the largest single institution experience to date (n = 75), investigators form the Mayo Clinic have documented a 14% 5-year survival in POPH patients who did not receive a pulmonary vasomodulating therapy [85] (Fig. 7). However in this uncontrolled study, those who did receive prostacyclin therapy (with or without added orally administered bosentan or sildenafil) had a 45% 5-year survival. A smaller group that received prostacyclin therapy and went on to have LT had a 70% 5-year survival. These initial data appear to suggest that pulmonary artery hypertension treatments for POPH (with or without LT) are beneficial in highly selected patients. POPH cure versus control remains problematic. Whether patients with POPH should have a higher priority for LT if pulmonary hemodynamics can be improved remains a potential practice guideline that awaits further outcome data [86]. Finally, the development of pulmonary artery hypertension de novo has been reported [87–89]. In some cases this problem developed several months after the resolution of hepatopulmonary syndrome, suggesting the possible coexistence of both vascular lesions prior to transplant and resolution of the vascular dilatation process, but not the vasoproliferative, obstructive process [87]. In other cases, the hemodynamic history suggests a posttransplant process that evolved after “normal” pulmonary hemodynamics was well documented during the transplant surgery [88–89]. The mechanism of this process remains an enigma, but suggests a complex pulmonary vascular pathogenic process that can occur in the setting of portal hypertension that may only be

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Fig. 7  Long-term survival in patients with POPH. Significant differences in 5-year survival are noted between those treated and those not treated with pulmonary vasomodulating drugs. (Reproduced from [85] with permission)

partially characterized by usual pulmonary hemodynamic measurements.

7 Summary POPH remains a challenging subgroup of pulmonary artery hypertension. The pulmonary vasculopathy that occurs as a consequence of portal hypertension appears complex, yet very similar histopathologically to that seen in idiopathic pulmonary artery hypertension. The diagnosis must be confirmed by right-sided heart catheterization owing to the spectrum of pulmonary hemodynamic abnormalities that exist in the setting portal hypertension. Long-term survival can be affected by either the unpredictable, direct consequences of liver disease or right-sided heart failure. Experience in treating this disorder with pulmonary vasomodulators is evolving, with distinct degrees of success. Limited experience suggests that LT, in combination with medical therapy, may offer a unique “cure” for this type of pulmonary artery hypertension in highly selected cases.

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47. Hinterhuber L, Graziadei IW, Kähler C, Jaschke W, Vogel W (2004) Endothelin-receptor antagonist treatment of POPH. Clin Gastroenterol Hepatol 2:1039–1042 48. Clift PF, Bramhall S, Isaac JL (2004) Successful treatment of severe portopulmonary hypertension after liver transplantation by bosentan. Transplantation 77:1174–1176 49. Kuntzen C, Gülberg V, Gerbes AL (2005) Use of a mixed endothelin receptor antagonist in POPH: a safe and effective therapy? Gastroenterology 128:164–168 50. Barth F, Gerber PJ, Reichen J, Dufour JF, Nicod LP (2006) Efficiency and safety of bosentan in child C cirrhosis with portopulmonary hypertension and renal insufficiency. Eur J Gastroenterol Hepatol 18:1117–1119 51. Stähler G, von Hunnis P (2006) Successful treatment or portopulmonary hypertension with bosentan. Eur J Clin Invest 36:62–66 52. Grander W, Eller P, Fuschelberger R, Tilg H (2006) Bosentan treatment or portopulmonary hypertension related to liver cirrhosis and hepatitis C. Eur J Clin Invest 36:67–70 53. Hoeper MM, Seyfarth HJ, Hoeffken G et al (2007) Experience with inhaled iloprost and bosentan in portopulmonary hypertension. Eur Respir J 30:1096–1102 54. Humbert M, Segal ES, Kiely DG, Carlsen J, Schwierin B, Hoeper MM (2007) Results of European post-marketing surveillance of bosentan in pulmonary hypertension. Eur Respir J 30:338–344 55. Makisalo H, Koivusalo A, Vakkuri A, Hockerstedt K (2004) Sildenafil for portopulmonary hypertension in patients undergoing liver transplantation. Liver Transpl 10:945–950 56. Reichenberger F, Voswinckel R, Steveling E et  al (2006) Sildenafil treatment for portopulmonary hypertension. Eur Respir J 28:563–567 57. Hemmes AR, Robbins IM (2009) Sildenafil monotherapy in portopulmonary hypertension can facilitate liver transplantation. Liver Transpl 15:15–19 58. Wang YW, Lin HC, Tyang YY, Hou MC, Lee SD (2006) Sildenafil decreased pulmonary artery pressure but may have exacerbated ­portal hypertension in a patient with portopulmonary hypertension. J Gastroenterol 41:593–597 59. Provencher S, Herve P, Jais X et  al (2006) Deleterious effects of b-blockers on exercise capacity and hemodynamics in patients with portopulmonary hypertension. Gastroenterology 130:120–126 60. Castro M, Krowka MJ, Schoeder DR et  al (1996) Frequency and clinical implications of increased pulmonary artery pressures in liver transplant candidates. Mayo Clin Proc 71:543–551 61. Kuo PC, Plotkin JS, Johnson LB et  al (1997) Distinctive clinical features of portopulmonary hypertension. Chest 112:980–986 62. Colle IO, Moreau R, Godinho E et al (2003) Diagnosis of portopulmonary hypertension in candidates for liver transplantation: a prospective study. Hepatology 37:401–409 63. Wallwork J, Williams R, Calne RY (1987) Transplantation of liver, heart and lungs for primary biliary cirrhosis and primary pulmonary hypertension. Lancet 2:182–185 64. DeWolf AM, Gasior T, Kang Y (1991) Pulmonary hypertension in a patient undergoing liver transplantation. Trans Proc 23:2000–2001 65. Cheng EY, Woehlck HJ (1992) Pulmonary artery hypertension complicating anesthesia for liver transplantation. Anesthesiology 77:389–392 66. Prager MC, Cauldwell CA, Ascher NL, Roberts JP, Wolfe CL (1992) Pulmonary hypertension associated with liver disease is not reversible after liver transplantation. Anesthesiology 77: 375–378 67. Yoshida EM, Erb SR, Pflugfelder PW et al (1993) Single lung versus liver transplantation for the treatment of portopulmonary hypertension – a comparison of two patients. Transplantation 53: 688–690 68. De Wolf AM, Scott VL, Gasior T, Kang Y (1994) Pulmonary hypertension and liver transplantation. Anesthesiology 78:213–214

69. Koneru B, Ahmed S, Weisse AB, Grant GP, McKim KA (1994) Resolution of pulmonary hypertension of cirrhosis after liver transplantation. Transplantation 58:1133–1135 70. Mandell MS, Duke J (1994) Nitric oxide reduces pulmonary hypertension during hepatic transplantation. Anesthesiology 81:1538–1542 71. Gillies BS, Perkins JD, Cheney FW (1996) Abdominal aortic compression to treat circulatory collapse caused by severe pulmonary hypertension during liver transplantation. Anesthesiology 85:420–422 72. Levy MT, Torzillo P, Bookallil M, Sheil AG, McCaughan GW (1996) Case report: delayed resolution of severe pulmonary hypertension after isolated liver transplantation in a patient with cirrhosis. J Gastroenterol Hepatol 11:734–737 73. Losay J, Piot D, Bougaran J et al (1998) Early liver transplantation is crucial in children with liver disease and pulmonary artery hypertension. J Hepatol 28:337–342 74. Tarquino M, Geggel RL, Strauss RS, Rhodes J, Wunderlich B, Rohrer RJ (1998) Treatment of pulmonary hypertension with inhaled nitric oxide during hepatic transplantation in an adolescent: reversibility of pulmonary hypertension after transplantation. Clin Pediatr 37:505–510 75. Schott R, Chaout A, Jaunoy A, Pottecher T, Weitzenblum E (1999) Improvement of pulmonary hypertension after liver transplantation. Chest 115:1748–1749 76. Starkel P, Vera A, Gunson B, Multimer D (2002) Outcome of liver transplantation for patients with pulmonary hypertension. Liver Transpl 8:382–388 77. Pirenne J, Verleden G, Nevens F et al (2002) Combined liver and heart-lung transplantation in liver transplant candidates with refractory portopulmonary hypertension. Transplantation 73:140–142 78. Pilatis N, Jacobs LE, Rerkpattanapipat P et al (2000) Clinical predictors of pulmonary hypertension in patients undergoing liver transplantation. Liver Transpl 6:85–91 79. Uchiyama H, Soejima Y, Taketomi A et  al (2006) Successful ­adult-to-adult living donor liver transplantation in a patient with moderate to severe portopulmonary hypertension. Liver Transpl 112:481–484 80. Austin MJ, McDougall NI, Wendon JA et al (2008) Safety and efficacy of combined use of sildenafil, bosentan, and iloprost before and after liver transplantation in severe portopulmonary hypertension. Liver Transpl 14:287–291 81. Grannas G, Neipp M, Hoeper M et al (2008) Indications for and outcomes after combined lung and liver transplantation: a single-center experience on 13 consecutive cases. Transplantation 85:524–531 82. Le Pavec J, Souza R, Herve P et al (2008) Portopulmonary hypertension: survival and prognostic factors. Am J Resp Crit Care Med 178:637–643 83. Kawut S, Taichman DB, Ahya VN et al (2005) Hemodynamics and survival in patients with portopulmonary hypertension. Liver Transpl 11:1107–1111 84. Swanson KL, Wiesner RH, Nyberg SL, Rosen CB, Krowka MJ (2008) Survival in portopulmonary hypertension: Mayo Clinic experience categorized by treatment groups. Am J Transpl 8:2445–2453 85. Krowka MJ, Fallon MB, Mulligan DC, Gish RG (2006) Model for end-stage liver disease (MELD) exception for portopulmonary hypertension. Liver Transpl 12:S114–S116 86. Aucejo F, Miller C, Vogt D, Eghtesad B, Nakagawa S, Stoller JK (2006) Pulmonary hypertension after liver transplantation in patients with antecedent hepatopulmonary syndrome: report of 2 cases and review of the literature. Liver Transpl 12:1278–1282 87. Koch D, Caplan M, Reuben A (in press) Pulmonary hypertension after liver transplantation: case presentation and literature review. Liver Transpl 88. Shirouzu Y, Kasahara M, Takada Y et al (2006) Development of pulmonary hypertension in 5 patients after pediatric living donor liver transplantation: de novo or secondary? Liver Transpl 12:870–875

Chapter 72

Pathobiology and Treatment of Pulmonary Hypertension in HIV Disease Michael H. Ieong and Harrison W. Farber

Abstract  Among HIV-infected people, the primary pulmonary complications among adults remain infectious. However, for those HIV-infected persons who receive highly active antiviral therapy, the spectrum of pulmonary disease is changing as a decrease in the incidence of opportunistic infections of the lung yields an increase of noninfectious complications. HIV-associated pulmonary arterial hypertension (HIV-PAH) is among these noninfectious complications. The prevalence of HIV-PAH (determined in different cohorts) is approximately 0.5%. The prevalence in HIV-PAH has dramatically changed during the last ten years, which is in stark contrast to another pulmonary complication of HIV infection, Pneumocystis pneumonia, in which the total number of cases declined fivefold between 1995 and 2003. The stable prevalence of HIV-PAH leads to three key observations. First, the prevalence of PAH among HIV infected patients is much greater than that of IPAH in the general population (6–12-fold greater). Second, because 40 million people in the world are estimated to be infected with HIV and these individuals are surviving much longer, the number of PAH cases identified is certain to increase. Finally, the observation that the prevalence of PAH in HIV-infected people is essentially unchanged in spite of reduced viral load suggests that the pathogenesis of HIV-PAH is only partly dependent on the degree of HIV viremia. The clinical implication of these observations is that current and investigative therapies used in PAH should significantly reduce the symptoms and improve the quality of life in HIV-PAH. The major limiting factor for use of any PAH-modulating medication is the complicated pharmacokinetics related to concurrent antiretroviral use and the higher prevalence of hepatic impairment among HIV-infected patients. Despite these limitations, it seems reasonable that, as is the case with other types of PAH, early intervention has the potential of significantly delaying disease progression.

M.H. Ieong (*) Department of Allergy, Pulmonary, and Critical Care Medicine, Boston University Medical Center, 72 East Concord St., R-304, Boston, MA 02118, USA e-mail: [email protected]

Keywords  Human immunodeficiency virus • Acquired immunodeficiency syndrome • Infection

1 Introduction Among the 40 million people infected with HIV, the primary pulmonary complications among adults remain infectious. However, for those HIV-infected persons who receive highly active antiviral therapy, the spectrum of pulmonary disease is changing as a decrease in the incidence of opportunistic infections of the lung yields an increase of noninfectious complications [1]. HIV-associated pulmonary arterial hypertension (HIV-PAH) is among these noninfectious complications. First described in 1987 by Kim and Factor, HIV-PAH is a pulmonary arteriopathy similar to that seen with idiopathic pulmonary arterial hypertension (IPAH), although differences in disease incidence and rate of progression suggest different causative mechanisms [2–4]. In 1991 Speich et al. reported the prevalence of HIV-PAH in their cohort of 1,200 patients to be 0.5% [5]. As these patients were all characterized in 1990, it was speculated that, with the advent of highly active antiretroviral therapy (HAART), there would be a change in the prevalence with improved viral control. Sitbon et al. addressed this issue in their report on the prevalence of pulmonary arterial hypertension (PAH) in a large, multicenter, French cohort of 7,648 HIV-infected patients [6]. Between 2004 and 2005 these investigators used a screening algorithm that identified HIV-positive patients with unexplained dyspnea and abnormal findings on echocardiograms. These patients were then studied by catheterization of the right side of the heart, leading to the identification of 35 patients with PAH by hemodynamic measurement. The resulting prevalence of HIV-PAH was thus found to be 0.46%, essentially unchanged from that of 1990 [6]. The unchanged prevalence in HIV-PAH is in stark contrast to another pulmonary complication of HIV infection, Pneumocystis pneumonia, in which the total number of cases declined fivefold between 1995 and 2003 [7].

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_72, © Springer Science+Business Media, LLC 2011

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The stable prevalence of HIV-PAH leads to three key observations. First, the prevalence of PAH among HIVinfected patients is much greater than that of IPAH in the general population (6–12-fold greater). Second, because 40 million people in the world are estimated to be infected with HIV and these individuals are surviving much longer, the number of PAH cases identified is certain to increase. Finally, the observation that the prevalence of PAH in HIV-infected people is essentially unchanged in spite of reduced viral load suggests that the pathogenesis of HIV-PAH is only partly dependent on the degree of HIV viremia. The clinical implication of these observations is that current and investigative therapies used in PAH should significantly reduce the symptoms and improve the quality of life in HIV-PAH. The major limiting factor for use of any PAH-modulating medication is the complicated pharmacokinetics related to concurrent antiretroviral use and the higher prevalence of hepatic impairment among HIV-infected patients [8]. Despite these limitations, it seems reasonable that, as is the case with other types of PAH, early intervention has the potential of significantly delaying disease progression.

2 Pathology HIV-PAH is grouped with IPAH (WHO group 1) because the histological characteristics of the pulmonary arterial lesions are indistinguishable [9]. The original 1987 observation identified precapillary pulmonary arteries with lumen filling endothelial cell clusters in a 40-year-old HIV-infected man who died of Pneumocystis pneumonia [3]. This initial observation was subsequently confirmed in reports of other HIV-infected patients with PAH [5, 10–14]. Interestingly, in a retrospective report of 131 published HIV-PAH cases, most (78%) patients had plexiform lesions; however, there were other histological findings, including thrombotic pulmonary arteriopathy, pulmonary medial hypertrophy with intimal fibrosis, and pulmonary veno-occlusive disease [15]. More recently, there have been HIV-PAH cases with evidence of perivascular inflammatory cells, suggesting yet another potential mechanism for the development of HIVPAH [16, 17].

3 Pathobiology The cause of HIV-PAH is likely multifactorial, resulting from a combination of viral and host factors. A number of putative mechanisms by which HIV infection might directly cause or elicit a cellular environment that would lead to the development of the hallmark plexogenic arteriopathy and

M.H. Ieong and H.W. Farber

hemodynamic disorder have been proposed. These ­mechanisms of disease development are speculated to relate directly to the virus, to the host’s response to HIV infection, and to the consequent infections or diseases that result from HIV-induced immune deficiency.

3.1 Direct Effects of HIV The proposed mechanisms that invoke a direct effect of HIV infection have generally focused on viral proteins as mediators. These HIV proteins are generated during viral replication, have been demonstrated to be bioactive, and are integral to HIV-induced pathogenesis [18–23]. The HIV proteins that have been studied for their potential to contribute to the development of HIV-PAH include gp120, Tat, Nef, and Env. Tat is an HIV protein that, in addition to being required for viral replication, interacts with a number of cytoplasmic and nuclear proteins. It is involved in mediating HIV pathogenesis and can cross cell membranes [23, 24]. Thus, uninfected cells and cells that are not infected with HIV, often referred to as “bystander” cells, can be affected. Bone morphogenetic protein receptor-2 (BMPR2) is a member of the transforming growth factor b superfamily of receptors which transduces extracellular signals through the kinases on their intracytoplasmic tail. Heritable pulmonary arterial hypertension (HPAH) is indistinguishable pathologically from IPAH and HIV-PAH. Mutations of the BMPR2 gene are associated with increased risk of HPAH in many, but not all, individuals in these families, suggesting that compromised BMPR2 signaling can contribute to development of PAH [25]. In a cellculture model using the monocyte cell line U937, Caldwell et al. demonstrated that exposure to HIV Tat protein resulted in decreased BMPR2 promoter activity and transcript copy number. Thus, HIV infection and the resulting exposure to Tat may contribute to the development of PAH by compromising BMPR2 signaling in a manner similar to that in HPAH [26]; however, small studies have not found BMPR2 mutations in patients with HIV-PAH. Larger population studies may be necessary to address this issue completely, or, more likely, other mechanisms are more important in most individuals with HIV-PAH. Gp120 is another HIV protein that mediates viral pathogenesis and can be measured in the sera of HIV-infected patients. It has been shown to recruit T cells and monocytes, impair dendritic cell maturation and antigen-presenting capacity, and induce the secretion of a number of cytokines. Endothelin (ET)-1 is a peptide that, similar to cytokines, is secreted from cells and has potent bioactivity. In patients with severe PAH, ET-1 serum and pulmonary parenchymal levels are markedly elevated and ET receptor antagonists are clinically used to mitigate PAH. It has potent smooth muscle

72  Pathobiology and Treatment of Pulmonary Hypertension in HIV Disease

p­ roliferative and vasoconstrictive effects as well as ­antiproliferative and vasodilatory effects, via nitric oxide and prostacyclin, depending on to which of its two G-proteincoupled receptors, ETA or ETB, it binds. ET-1 is produced primarily by endothelial cells, but Ehrenreich et al. also found it to be secreted by human macrophages [27]. On the basis of this observation, Ehrenreich et  al. found that exposure of human monocytes to gp120 for 24  h resulted in elevated supernatant levels of ET-1. To assess the in vivo relevance of this observation, the investigators compared monocytes isolated from HIV-infected patients at various stages of the disease with those from healthy HIV-negative donors by reverse-transcription PCR. In eight of the ten HIV-infected individuals an ET-1 PCR product was clearly visible, whereas it was undetectable in the HIV-negative controls. Monocytes from other inflammatory or virus-mediated diseases where also studied as disease controls, and none had an identifiable ET-1 PCR product. Finally, the investigators compared immunohistochemically stained macrophages in brain tissue obtained from HIV-infected patients with encephalopathy with those from HIV-uninfected controls. They again found strong staining for ET-1 in CD68-positive cells, consistent with brain microglia [27]. On the basis of the evidence that interrupting ET-1 signaling can mitigate PAH, elevation of the level of ET-1 in the presence of gp120 and in vivo HIV infection is suggestive of a role for gp120 in HIV-PAH. These studies, however, only demonstrate association. Further studies of this HIV protein are needed to clarify its role in the pathogenesis of HIV-PAH. The viral proteins Nef and Env are two other HIV proteins that have recently been implicated in the pathogenesis of HIV-PAH. In simian immunodeficiency virus (SIV)-infected nonhuman primates, HIV proteins can be studied by inserting the gene sequence for a specific HIV protein in place of the analogous SIV gene. Rhesus monkeys infected with SHIV-nef develop a rapidly progressive immunodeficiency that replicates much of the pathological characteristics of HIV-infection in humans, with most of the monkeys dying within 1 year [28]. In a histology study by Marecki et al. the lungs of five of six rhesus monkeys infected with SHIV-nef chimeric viruses had evidence of vascular remodeling with endothelial cell proliferation when compared with the lungs of monkeys infected with SIV, in which no HIV Nef was present [29]. These lesions stained positive for factor VIII and muscle-specific actin. In contrast, in SIV-infected monkeys, there was no evidence of abnormal pulmonary artery histological findings, suggesting the presence of HIV Nef was associated with the vascular remodeling found in the SHIV-nef-infected monkeys. These investigators then examined lung tissue from two patients with HIV-PAH for the presence of Nef protein. In both the HIV-PAH patients and the SHIV-nef-infected monkeys, the pulmonary artery lesions demonstrated the presence of Nef [29]. Although this provides

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some supportive evidence that Nef may contribute to HIV-PAH, examining lung tissue from HIV-infected patients without PAH was not done and is an important control needed to bolster this hypothesis. More recently, a report on the proviral DNA sequences of nef from 12 HIV-infected patients with PAH has added to this theory. In this study these signature sequences were compared with the Los Alamos/NCBI database of HIV-1 sequences; certain residues were more frequently found among patients with HIV-PAH compared with historical controls. Further, when compared with the nef sequence in the SHIV-nef monkeys, there was commonality in three out of five amino acid substitutions [30]. These data further support the hypothesis that HIV-infected patients who develop PAH may be infected with a particular HIV-1 mutant strain that produces a distinct and disease-inducing Nef protein. However, against Nef as the sole mediator of PAH, another group of investigators have found pulmonary arteriopathy in 11 of 13 SHIV-env-infected macaques. Further, and as a notable difference from the prior SHIV-nef report, three of the 11 SIV-infected control monkeys also demonstrated pulmonary vascular lesions, arguing against an absolute requirement for an HIV protein [31]. Although a number of studies have implicated HIV proteins in the development of HIV-PAH, substantial research is still required to link a specific viral protein conclusively to the development of this complication of HIV infection. These investigations do strongly suggest, however, that a viral factor is contributing to the pathogenesis of PAH. Whether it is only one viral factor, the combination of many different viral factors, or viral factors in combination with other variables is still unknown.

3.2 Host Response and Factors On the basis of our understanding of IPAH and other diseaseassociated PAH, it is very likely that in HIV infection the host genome of the host exerts a significant influence in determining the predisposition to develop PAH. Two such genomic host factors are the BMPR2 gene and the major histocompatability complex (MHC) human leukocyte antigen (HLA) class II allele. As has been discussed, there is a clear association between BMPR2 gene mutations and HPAH. In some patients with IPAH, an association with MHC HLA class II alleles of the DR and the DQ loci has been reported [32]. Both of these associations have been tested in HIV-infected patients. From a French cohort of 82 HIV-infected patients with PAH, Nunes et  al. screened 19 patients and found no BMPR2 mutations [33]. In regard to HLA type, Morse et al. examined ten racially diverse patients with HIV-PAH and defined their HLA class II alleles. They identified an increased frequency of HLA-DR6 HLA-DR52, and the linked alleles HLA-DRB1*1301/2,

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DRB3*0301, and DQB1*0603/4 ­compared with uninfected Caucasian control subjects [34]. These genotypes also differed in frequency from reports of patients with IPAH [32]. Thus, although BMPR2 does not appear to contribute to HIV-PAH, there may be HLA genotypes that are associated with individuals who are inclined to develop PAH if they are infected with HIV. These observations, however, are significantly limited by the number of patients included in each study, and are therefore far from definitive. Beyond genomic variables, a number of other host variables that could contribute to PAH pathogenesis have been characterized. The relevance of many of these pathways in the pathogenesis of HIV-PAH is not yet clear; however, on the basis of the clinical response to accepted PAH therapeutics, it is clear that certain host genome is integral. Primarily this is related to the molecules of vasomotor tone, such as ET-1, phosphodiesterase-5, prostanoids, and nitric oxide. Although it is clear that pulmonary arterial tone is of great relevance, the principal pathological finding in PAH is the obliterating foci of endothelial cells that define the plexiform lesions, and therefore endogenous mediators of proliferation have been examined in patients with PAH. A growth factor that has been studied in both PAH and HIV-PAH is platelet-derived growth factor (PDGF). PDGF is a polypeptide composed of homo- or heterodimers that is secreted by many cell types, induces proliferation, and has been identified in both inflammatory and myeloproliferative disorders [35–38]. Elevated levels of PDGF isoforms and receptor have been found in lung tissue samples from patients with PAH compared with lung tissue from individuals without PAH [17]. Lung tissue from patients with HIV-PAH has also been examined; despite a smaller number of samples, increased expression of PDGF has been found [17]. Further, there are a small number of reports describing the use of the tyrosine kinase inhibitor imatinib mesylate, a selective antagonist of the PDGF receptor, for severe pulmonary hypertension refractory to conventional medications. These case reports described significant improvement in pulmonary hemodynamics and functional class after initiating imatinib use [39, 40]. Thus, it is likely that the dysregulation of host PDGF and possibly other growth factors plays a role in the development of HIV-PAH.

M.H. Ieong and H.W. Farber

the clinical characteristics are most applicable to cohorts with limited access to antiretrovirals, and the utility of these clinical findings must be tempered in populations where HAART is in wide use. Most symptoms reported in HIV-PAH are related to right ventricular dysfunction and include dyspnea on exertion (85%), pedal edema (20–30%), nonproductive cough (19%), fatigue (13%), syncope or near syncope (12–30%), and chest pain (7–20%). The mean age of patients with HIV-PAH is 33 ± 7 years, with a female-to-male ratio of 1:1.6; this differs from IPAH, in which females predominate (female-to-male ratio between 1.7:1 and 3:1) [4, 43, 44] (Table 1). The symptoms of HIV-PAH are similar to those of IPAH; however, the time between symptom development and diagnosis is shorter in the HIV-PAH population (6 months vs. 2.5 years) [43]. This may be due to the increased medical surveillance of HIV-infected patients or is possibly a reflection of a more rapid progression of the HIV-related disease. Overall survival in patients with HIV-PAH at 1, 2, and 3 years after diagnosis of PAH has been reported as 73%, 60%, and 47%, respectively, with lower survival in patients with worse New York Heart Association (NYHA) functional class. In those with functional class III and IV, the survival has been reported as 60, 45, and 28%, respectively [33]. A more recent report of 89.7% overall 1-year survival suggests there has been improvement in the HAART era [45]; whether this is related to HAART or improved therapy for the underlying HIV-PAH is unknown. The most frequently identified risk factors for HIV infection in patients with HIV-PAH are intravenous drug use (58%), homosexual contact (22%), hemophilia (9%), and heterosexual contact (9.2%) [15]. There is no correlation between the CD4 count, HIV viral load, or history of opportunistic infections and the occurrence of HIV-PAH [6, 15]. However, many investigators have found that patients with lower CD4 counts have severer PAH [6, 33]. More recently, two large European studies of HIV-PAH patients treated during the HAART era have been reported. Table  1  Patient comparison between HIV-associated pulmonary arterial hypertension (HIV-PAH) and idiopathic pulmonary arterial hypertension (IPAH) HIV-PAH IPAH Incidence

4 Clinical Presentation and Diagnostic Evaluation The clinical findings and diagnostic approach to PAH are well described; however, distinctive features of HIV-PAH have been reported [41, 42]. In reviewing these reports, it is important to remember that most of the case reports and series include patients from the pre-HAART era. Thus,

1 in 200 HIV1–2/million persons/ infected persons year Age at diagnosis 32 ± 5 42 ± 7 Male-to-female ratio 1.6:1 1.3–4 Survival – 1 year 51–58% 68–77% Survival – 2 years 32–45% 58–63% Survival – 3 years 20–25% 40–56% Survival – median 1.3 years 2.6 years Histology findings 85% plexogenic 28–80% plexogenic lesions lesions Adapted from Farber [44] with permission. Copyright Massachusetts Medical Society. All rights reserved

72  Pathobiology and Treatment of Pulmonary Hypertension in HIV Disease

The characteristics of a French cohort of 35 patients did not diverge significantly from those described in prior series; compared with HIV patients with dyspnea but no PAH, HIV-PAH patients were predominantly male (71% vs. 53%), intravenous drug users (51% vs. 22%), and more patients had a CD4 count below 200 cells/mL (37% vs. 18%). Overall, CD4 counts did not differ significantly between the two populations. All of these patients had PAH confirmed by right-sided heart catheterization; it was noted that the five newly diagnosed patients had milder disease than the previously diagnosed patients (mean pulmonary artery pressure 30 ± 9 vs. 46 ± 13 mmHg; and pulmonary vascular resistance of 4 ± 3 vs. 10 ± 4 Wood units) [6]. However, given the small number of patients in this study, these observations should be confirmed. The Swiss HIV Cohort Study has been actively collecting data since 1988; it recently reported on a cohort of 19 patients diagnosed after 2001 [45]. In this cohort, compared with individuals diagnosed before 2001, the patients were older (mean age 43 vs. 34 years), and had a higher median CD4 cell count (215 vs. 169  cells/mL). However, by other measures, this cohort still resembled pre-HAART HIV-PAH patients in that more were male and intravenous drug users. Further characterization of HIV-PAH patients in the HAART era is clearly warranted. Once the diagnosis of PAH is suspected in an HIV-infected person, it is essential to confirm pulmonary hypertension by measuring hemodynamics directly via a right-sided heart catheterization. Once PAH is confirmed, other comorbidities contributing to the development of PAH should be considered; for example, contaminants in injection drugs such as methylcellulose induce extensive pulmonary arteriothrombosis that can lead to pulmonary hypertension; likewise, chronic liver disease and portal hypertension associated with hepatitis B or C infection, commonly seen in the HIV-infected population, can lead to pulmonary hypertension. However, by convention, HIV-infected patients with pulmonary hypertension, even if they have a significant history of liver dysfunction or intravenous drug use, are considered to have HIV-PAH because it is impossible to distinguish which disease process contributed most to the development of the PAH. Also, the treatment approach is the same as for PAH.

5 Treatment Treatment of HIV-PAH is based on symptoms, NYHA/ WHO functional class, the severity of pulmonary hypertension, and right ventricular dysfunction. Treatment goals are to reduce pulmonary vascular resistance and increase ­cardiac output through selective pulmonary vasodilatation and to improve long-term vascular remodeling. Although ­calcium

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Table 2  Treatment of HIV-PAH: a clinical breakdown Petipretz et al. [2] Nunes et al. [33] Degano et al. [61] Type of study Recruitment period Number of patients included Medication available during the study Number of patients on medication

Retrospective 1987–1992

Retrospective 1986–2000

Retrospective 2002–2007

20

82

59

Calcium channel Epoprostenol blocker

Bosentan

Unknown

All

20 received drug for 17 ± 13 months 48%

83% Percentage of 0% patients on PI plus 2 other ARVs Survival 1 year NR 73% 93% 2 years 46% 60% 86% 3 years NR 47% 66% PI protease inhibitor, ARVs antiretrovirals, NR not reported

channel blockers, oral anticoagulation, and continuous intravenous prostacyclin infusion all increase survival in patients with IPAH, the impact these therapies have on survival in the HIV-seropositive individual is not known [16, 46]. Moreover, there have been no randomized, controlled studies of any of these agents in patients with HIV-PAH. Table 2 provides an overall synopsis of three key studies in which HIV-PAH patients were treated with conventional PAH-approved compounds. The studies are described further in the following.

5.1 Calcium Channel Blockers The hemodynamic improvements with calcium channel blockers seen in IPAH have not been reproduced in HIVPAH despite the pathologic similarities of the diseases [47– 50]. Only a small subset of all IPAH patients will have sufficient vasoreactivity to be considered for calcium channel blocker therapy; perhaps this subset is even smaller in HIV-PAH patients because of a later stage of presentation. Yet, those HIV-PAH patients in NYHA/WHO class I, II, or III who have a positive vasodilator response to nitric oxide, adenosine, or prostacyclin should be considered for calcium channel blocker therapy. Oral anticoagulation to decrease in situ thrombosis has not been evaluated in HIVPAH, but its use in patients with IPAH has been extrapolated to those HIV-PAH patients without a contraindication for anticoagulation (goal international normalized ratio 1.5–2.0).

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5.2 Epoprostenol Continuous intravenous infusion of epoprostenol (prostacyclin) improves hemodynamics, 6-min walk distance, functional class, quality of life, and survival in NYHA/WHO class III and IV patients with IPAH [51–56]. In a study of 19 patients with HIV-PAH, Petitpritz et al. found that acute infusion of epoprostenol resulted in a 20% decrease in the pulmonary vascular resistance index [57]. A subsequent study of six patients with HIV-PAH also noted a significant improvement in hemodynamics with acute infusion. Five of the six patients underwent repeat cardiac catheterization at 12 months (the sixth died of nucleoside-analog-induced lactic acidosis), with four of them having additional improvement in pulmonary artery pressure, pulmonary vascular resistance, and cardiac output with chronic epoprostenol infusion. Three of these patients had continued benefit or further improvement in their PAH at catheterization at 24 months [47]. In the largest series to date, Nunes et al. described a singlecenter, retrospective case series of 82 HIV-PAH patients, which included 20 NYHA/WHO class III or IV patients treated with epoprostenol once it became available [33]. Owing to the rapidly changing therapeutic standards over the 14-year study period, only a subset of patients were treated with a protease inhibitor or HAART. Although the beneficial effects of epoprostenol as defined by improved 6-min walk distance and improved hemodynamic parameters were durable at 17 months, on multivariate analysis, only the CD4 count was independently associated with improved survival [33]. In addition to the well-described adverse effects of epoprostenol, such as headache, flushing, jaw pain, nausea, and thrombocytopenia, infection related to the need for chronic central venous access is a significant concern in the immunocompromised host [58]. Despite this increased theoretical risk, there has been no documentation of increased incidence or severity of catheter-associated sepsis in HIV-PAH patients receiving epoprostenol. Although a history of intravenous drug use may be a relative contraindication to placement of a central venous catheter, we recommend epoprostenol, if appropriate, after a 6-month period of documented drug abstinence.

M.H. Ieong and H.W. Farber

these studies suggest that it may be beneficial. Barbaro et al. compared 18 patients treated with HAART with 18 patients treated with HAART and bosentan. At 24 weeks after initiating treatment, they found that the bosentan-treated group had significantly increased average 6-min walk distance, and decreased mean pulmonary artery pressure, pulmonary vascular resistance, and right atrial pressure [59]. Sitbon et al. reported similar findings in a cohort of 16 HIV-PAH patients treated with bosentan for 16 weeks; increase in 6-min walk distance and improvement in hemodynamic parameters [60]. Degano et al. extended these observations in a retrospective review of 59 patients with HIV-PAH that were treated with bosentan for an extended period of time [61]. In this cohort, which included 12 of the 16 patients in the Sitbon et  al. study, 56 patients were evaluated after 4 months of bosentan treatment, and 38 were evaluated for a mean of 29 ± 15 months. In this study, survival at 1, 2, and 3 years was reported as 93, 86, and 66%, respectively. This was in contrast to the study of Nunes et al. who in 2003 reported 73%, 60%, and 40% survival at 1, 2, and 3 years, respectively, and the study of Petitpretz et  al. who in 1994 reported a 46% 2-year survival rate [2, 33]. It is important to note, however, that in comparing these cohorts, the prevailing treatments available at the time for HIV infection. For example, the percentage of patients receiving combination antiretroviral therapy including a protease inhibitor was 83% in the Degano et  al. study, 48% in the Nunes et  al. study, and 0% in the Petitpretz et al. study. A key limiting variable with bosentan is any active liver disease, since one of its potential side effects is transaminitis and liver injury. In Degano et al.’s cohort, more than half of the patients (n = 30) had concurrent hepatitis C (n = 24) or hepatitis B (n = 6) infection, of which ten had mild cirrhosis (Child– Pugh A class). In a univariate analysis of the data, the authors found that neither hepatitis C nor hepatitis B co-infection was associated with a poorer outcome. Three patients discontinued bosentan treatment owing to elevated levels of transaminases, but it is not clear whether any of these patients were infected with hepatitis C or hepatitis B. However, the frequency of elevated levels of transaminases was no higher in HIV-PAH than in a recently reported cohort of 4,994 PAH patients treated with bosentan, in which the incidence of elevated levels of transaminases was 7.6% over 30 months [62].

5.3 Bosentan 5.4 Sildenafil A number of newer agents have been used for IPAH and have demonstrated improvement in short-term measures such as hemodynamics and exertional capacity. Although survival data are lacking, many of these agents are now being used in HIV-PAH. The oral ET receptor antagonist bosentan has been studied in some small cohorts of HIV-PAH patients;

Other medications for the treatment of PAH are now or may soon be available and have potential use in HIV-PAH. These include more selective ET receptor antagonists, subcutaneously administered prostacyclin, and phosphodiesterase-5 inhibitors, some of which have been shown to improve

72  Pathobiology and Treatment of Pulmonary Hypertension in HIV Disease

hemodynamics, exercise capacity, and NYHA class in patients with IPAH [63–68]. Unfortunately, there have only been limited studies of these agents in the treatment of HIVPAH. Sildenafil, a selective phosphodiesterase-5 inhibitor, has shown efficacy as monotherapy and in combination treatment in several small uncontrolled studies of patients with a variety of PAH causes [69]. Case reports of patients with HIV-PAH treated with sildenafil have demonstrated decreased pulmonary artery pressures and improved clinical status [70– 72]. However, close consideration of potential interaction with concurrent antiretroviral medications is warranted since protease inhibitors can cause a significant increase in the levels of sildenafil, and sildenafil may significantly decrease levels of protease inhibitors [72].

5.5 Antiretrovirals Although medical treatment of HIV-PAH has many similarities to that of IPAH, it differs significantly in that one postulated therapeutic intervention is the use of antiretrovirals which directly target what is hypothesized to be at least one of the factors driving the pathogenesis of PAH in these patients. The impact of antiretroviral therapy on the clinical course of HIV-PAH, however, is controversial. Pugliese et al. reported a decreased incidence of HIV-PAH with nucleoside reverse transcriptase inhibitors (0.7%) compared with combination HAART therapy (2%) [73]. In a retrospective study of a Swiss cohort of 47 patients with HIV-PAH, those treated with HAART had a significantly increased median duration of survival when compared with patients who received no treatment or nonnucleoside reverse transcriptase inhibitors alone [74]. Small studies have suggested the use of zidovudine or didansosine may have a beneficial effect on rightsided heart function and HAART combinations including reverse transcriptase and protease inhibitors appear to decrease the mortality for HIV-related congestive heart failure [50, 75]. There are also case reports of the beneficial effects of protease inhibitor treatment of HIV infection on right-sided heart function and hemodynamics [76]. However, the actual effect of HAART on HIV-PAH is currently unknown; moreover, HIV-PAH is not considered an indication for initiation of HAART.

6 Summary Although there have been significant reductions in the infectious complications of HIV disease with the advent of HAART, the prevalence of HIV-PAH has not changed. HIVPAH can occur at any point during the disease and no

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d­ efinitive association with CD4 count or viral load has been observed. The cause and pathogenesis of HIV-PAH remain poorly characterized, although a number of observations in the SIV model and in vitro studies support a pathogenic role for HIV proteins. The genetics of the host and the response to HIV infection is also likely to be integral in pathogenesis. Inflammatory and cell proliferation pathways are likely key candidate networks. The most common presenting symptom in HIV-PAH is dyspnea and studies suggest that evaluation of patients without a clear reason for shortness of breath will yield new HIV-PAH diagnoses. Despite limited studies of therapy (no randomized controlled trials) in HIV-PAH, the treatment options are currently similar to those employed for PAH. The major limitations are potential interactions between the PAH medications and antiretrovirals and the potential effect on hepatic function. Nevertheless, limited series of patients treated with epoprostenol and bosentan suggest the benefit of employing standard therapeutics in these patients. As HIV-infected patients live longer, HIV-PAH is likely to become a more frequent complication despite increased access to HAART. Longitudinal studies of existing patient cohorts and more inclusion of HIV-infected patients in drug protocols are important steps toward the goal of earlier recognition and treatment of this debilitating and potentially fatal complication of HIV infection.

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72  Pathobiology and Treatment of Pulmonary Hypertension in HIV Disease 53. Shapiro SM, Oudiz RJ, Cao T et  al (1997) Primary pulmonary hypertension: improved long-term effects and survival with continuous intravenous epoprostenol infusion. J Am Coll Cardiol 30:343–349 54. Hinderliter AL, Willis PW, Barst RJ et al (1997) Effects of longterm infusion of prostacyclin (epoprostenol) on echocardiographic measures of right ventricular structure and function in primary pulmonary hypertension. Primary Pulmonary Hypertension Study Group. Circulation 95:1479–1486 55. Klings ES, Hill NS, Ieong MH, Simms RW, Korn JH, Farber HW (1999) Systemic sclerosis-associated pulmonary hypertension: s. Arthritis Rheum 42:2638–2645 56. Badesch DB, Tapson VF, McGoon MD et  al (2000) Continuous intravenous epoprostenol for pulmonary hypertension due to the scleroderma spectrum of disease. A randomized, controlled trial. Ann Intern Med 132:425–434 57. Petitpretz P, Brenot F, Azarian R et al (1994) Pulmonary hypertension in patients with human immunodeficiency virus infection. Comparison with primary pulmonary hypertension. Circulation 89: 2722–2727 58. Recusani F, Di Matteo A, Gambarin F, D’Armini A, Klersy C, Campana C (2003) Clinical and therapeutical follow-up of HIVassociated pulmonary hypertension: prospective study of 10 patients. AIDS 17:S88–S95 59. Barbaro G, Lucchini A, Pellicelli AM, Grisorio B, Giancaspro G, Barbarini G (2006) Highly active antiretroviral therapy compared with HAART and bosentan in combination in patients with HIVassociated pulmonary hypertension. Heart 92:1164–1166 60. Sitbon O, Gressin V, Speich R et al (2004) Bosentan for the treatment of human immunodeficiency virus-associated pulmonary arterial hypertension. Am J Respir Crit Care Med 170:1212–1217 61. Degano B, Yaici A, Le Pavec J et al (2009) Long-term effects of bosentan in patients with HIV-associated pulmonary arterial hypertension. Eur Respir J 33:92–98 62. Humbert M, Segal ES, Kiely DG, Carlsen J, Schwierin B, Hoeper MM (2007) Results of European post-marketing surveillance of bosentan in pulmonary hypertension. Eur Respir J 30:338–344 63. Simonneau G, Barst RJ, Galiè N et al (2002) Continuous subcutaneous infusion of treprostinil, a prostacyclin analogue, in patients with pulmonary arterial hypertension: a double-blind, randomized, placebo-controlled trial. Am J Respir Crit Care Med 165:800–804

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64. Barst RJ, Galiè N, Naeije R et  al (2006) Long-term outcome in pulmonary arterial hypertension patients treated with subcutaneous treprostinil. Eur Respir J 28:1195–1203 65. Galiè N, Olschewski H, Oudiz RJ et al (2008) Ambrisentan for the treatment of pulmonary arterial hypertension: results of the ambrisentan in pulmonary arterial hypertension, randomized, double-blind, placebo-controlled, multicenter, efficacy (ARIES) study 1 and 2. Circulation 117:3010–3019 66. Benza RL, Barst RJ, Galiè N et al (2008) Sitaxsentan for the treatment of pulmonary arterial hypertension: a 1-year, prospective, openlabel observation of outcome and survival. Chest 134:775–782 67. Barst RJ, Langleben D, Badesch D et al (2006) Treatment of pulmonary arterial hypertension with the selective endothelin-A receptor antagonist sitaxsentan. J Am Coll Cardiol 47: 2049–2056 68. Wittbrodt ET, Abubakar A (2007) Sitaxsentan for treatment of pulmonary hypertension. Ann Pharmacother 41:100–105 69. Leuchte HH, Schwaiblmair M, Baumgartner RA, Neurohr CF, Kolbe T, Behr J (2004) Hemodynamic response to sildenafil, nitric oxide, and iloprost in primary pulmonary hypertension. Chest 125:580–586 70. Prasad S, Wilkinson J, Gatzoulis MA (2000) Sildenafil in primary pulmonary hypertension. N Engl J Med 343:1342 71. Schumacher YO, Zdebik A, Huonker M, Kreisel W (2001) Sildenafil in HIV-related pulmonary hypertension. AIDS 15:1747–1748 72. Alp S, Schlottmann R, Bauer TT, Schmidt WE, Bastian A (2003) Long-time survival with HIV-related pulmonary arterial hypertension: a case report. AIDS 17:1714–1715 73. Pugliese A, Isnardi D, Saini A, Scarabelli T, Raddino R, Torre D (2000) Impact of highly active antiretroviral therapy in HIV-positive patients with cardiac involvement. J Infect 40:282–284 74. Zuber JP, Calmy A, Evison JM, Hasse B et al (2004) Pulmonary arterial hypertension related to HIV infection: improved hemodynamics and survival associated with antiretroviral therapy. Clin Infect Dis 38:1178–1185 75. Murphy RL, Barbaro G (2003) Clinical and biological insights in HIV-associated cardiovascular disease in the era of highly active antiretroviral therapy. AIDS 17:S1–S3 76. Speich R, Jenni R, Opravil M, Jaccard R (2001) Regression of HIVassociated pulmonary arterial hypertension and long-term survival during antiretroviral therapy. Swiss Med Wkly 131:663–665

Chapter 73

Pulmonary Arterial Hypertension Secondary to Anorexigens and Other Drugs and Toxins Kim Bouillon, Yola Moride, Lamiae Bensouda-Grimaldi, and Lucien Abenhaim

Abstract  Several drugs and toxins have been shown to be associated with the development of pulmonary vascular hypertension. Pulmonary hypertension (PH) related to these factors has been classified as primary PH (PPH), currently referred to as idiopathic pulmonary arterial hypertension, because its morphological findings, clinical manifestations, hemodynamic measures, and pathological changes were reported to be similar to those of PPH. In 1998, a clinical classification of PH was proposed, the “Evian classification,” which was updated in 2003. According to the revised clinical classification of PH, this disease with identifiable risk factors is called “pulmonary arterial hypertension (PAH) associated with drugs and toxins”. However, some drugs can be associated with pulmonary veno-occlusive disease (PVOD), persistent PH of the newborn (PPHN), and newly recognized systemic disease, in which the histopathological findings are characterized by pulmonary arteriopathy with medial hypertrophy of muscular and elastic arteries, dilation and intimal atheromas of elastic pulmonary arteries, and right ventricular hypertrophy. Plexogenic arteriopathy can be seen with the exception of PAH of persistent fetal circulation. The identification of drugs and toxins as risk factors for PH poses a great challenge to both the physician and the epidemiologist. Drugs and toxins are categorized according to the strength of their association with PH and the probability of causal role. Table 1 summarizes the drugs and toxins considered in this chapter. Keywords  Anorexic agents • Toxins • Diet pills • Pulmonary arteriopathy • Fenfluramine

1  Introduction Several drugs and toxins have been shown to be associated with the development of pulmonary vascular hypertension. Pulmonary hypertension (PH) related to these factors has been classified as primary PH (PPH) because its ­morphological K. Bouillon (*) Paris Santé Cochin, La-ser, 26 rue d’Ulm, 75005 Paris, France e-mail: [email protected]

findings, clinical manifestations, hemodynamic measures, and pathological changes were reported to be similar to those of PPH. In 1998, a clinical classification of PH was proposed, the “Evian classification,” which was updated in 2003. According to the revised clinical classification of PH, this disease with identifiable risk factors is called “pulmonary arterial hypertension (PAH) associated with drugs and toxins” [1]. However, some drugs can be associated with pulmonary veno-occlusive disease (PVOD), persistent PH of the newborn (PPHN), and newly recognized systemic disease, in which the histopathological findings are characterized by pulmonary arteriopathy with medial hypertrophy of muscular and elastic arteries, dilation and intimal atheromas of elastic pulmonary arteries, and right ventricular hypertrophy. Plexogenic arteriopathy can be seen with the exception of PAH of persistent fetal circulation. The identification of drugs and toxins as risk factors for PH poses a great challenge to both the physician and the epidemiologist. Drugs and toxins are categorized according to the strength of their association with PH and the probability of causal role [1]. Table 1 summarizes the drugs and toxins considered in this chapter.

2  Drugs 2.1  Anorectic Agents 2.1.1  Aminorex Aminorex (2-amino-5-phenyl-2-oxazoline) resembles adrenaline and ephedrine in its chemical structure and acts as a potent appetite-depressing agent and as a central stimulant (Fig. 1). It has an adrenergic effect. In the 1960s, there was an outbreak of chronic PH in Europe. In 1967, a sudden 20-fold increase in the referral of patients with PPH was observed in a Swiss medical clinic. The disease showed a rapid progression; the interval between the onset of symptoms and ­diagnosis was 10 months on the average. The onset of the symptoms followed the

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_73, © Springer Science+Business Media, LLC 2011

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K. Bouillon et al. Table 1  Drugs and toxins and pulmonary arterial hypertension (PAH) Name Evian classification

Evian consensusa

Best evidenceb

Drugs Anorectic agents Aminorex 4-Methylaminorex Fenfluramine Dexfenfluramine Propylhexedrine Phendimetrazine Mazindol Phenylpropanolamine Diethylpropion Benfluorex Pergolide Phenformin Oral contraceptives

Definite Not studied Definite Definite Not studied Not studied Not studied Not studied Not studied Not studied Not studied Not studied Unlikely

1 2 1 1 3 3 3 1 3 2 3 3 3 3 3 3 3 3 2 3 3 3 3 3 3 3 3

Estrogen therapy Leflunomide Interferon-a 2 Aglucerase Chemotherapeutic agents Thalidomide Mitomycin C containing regimen Carmustine Cyclophosphamide-containing regimen without BMT Cyclophosphamide-containing regimen with autologous BMT Cyclophosphamide-containing regimen with allogeneic BMT Bleomycin-containing regimen MOPP/COPP Nonsteroidal anti-inflammatory drugs Indomethacin Naproxen Ibuprofen Diclofenac Nimesulide Fluoxetine Substance abuse Methamphetamines Cocaine

APAH APAH APAH APAH APAH APAH APAH APAH APAH APAH APAH APAH APAH PVOD FPAH APAH APAH APAH

Unlikely Not studied Not studied Not studied Possible

APAH PVOD PVOD APAH PVOD PVOD PVOD PVOD PPHN PPHN PPHN PPHN PPHN PPHN

Not studied Not studied Not studied Not studied Not studied Not studied

2 3 3 3 3 1

APAH APAH PPHN APAH

Possible Possible

1 3

Glue (toluene) Not studied 3 Toxins and other Monocrotaline, fulvine, seneciphylline – – – Toxic rapeseed oil Systemic disease Definite 1 l-Tryptophan Systemic disease Very likely 1 APAH associated pulmonary arterial hypertension, PVOD pulmonary veno-occlusive disease, FPAH familial pulmonary arterial hypertension, PPHN persistent pulmonary hypertension of the newborn, BMT bone marrow transplantation, MOPP mechlorethamine, vincristine, procarbazine, and prednisone, COPP cyclophosphamide, vincristine, procarbazine, and prednisone a According to a consensus on drugs and toxins as risk factors for pulmonary arterial hypertension made during the Evian meeting (1998). “Definite” indicates an association based on several concordant observations including a major controlled study or an unequivocal epidemic. “Very likely” indicates several concordant observations (including large case series and studies) that are not attributable to identified bases. “Possible” indicates an association based on case series, registries, or expert opinions. “Unlikely” indicates risk factors that were suspected but for which controlled studies failed to demonstrate any association b According to strength of association found in the literature: 1 definite (epidemiology studies), 2 suspected (case series), 3 case report

73  Pulmonary Arterial Hypertension Secondary to Anorexigensand Other Drugs and Toxins

Fig. 1  Chemical structures of appetite suppressants

beginning of the anorectic treatment by an ­average of 8.6 months (6–12 months). There were no apparent changes in either the size or the composition of the population, nor of diagnostic procedures. About half of the patients were more or less overweight, with a female preponderance. It was noticed that a considerable number of these patients had taken aminorex to reduce weight [2]. A similar increase in the number of PPH cases observed was reported in other clinics in Switzerland [3], and also in Austria and Germany, where aminorex was available. Subsequently, a cooperative retrospective study was conducted with participation of 23 centers in these countries. A total of 582 cases of chronic PH were identified, among which 68% of patients claimed they had used aminorex, either alone or in combination with other anorectic agents [4]. Aminorex was introduced on the Swiss market in November 1965, and was withdrawn in October 1968, a few months after the communication of these findings. A decline in number of PPH cases observed was noted after its removal. The overall incidence of developing PPH in patients who used aminorex was reported to be about 0.2% [5]. The number of cases varied geographically, consistent with the marketing of the product, and temporally with the intake of aminorex. The clinical manifestations, hemodynamic measures, and pathological changes were reported to be indistinguishable from those of PPH. In patients with PAH, the survival rate after diagnosis was better among the aminorex-positive groups than among

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aminorex-free groups (at 10 years 53% vs. 34% [2] and 63% vs. 31% [6]). The prognosis in patients with vascular PH due to anorectic drug intake has showed to be critically dependent on the interruption of the drug intake. If the aminorex had been discontinued sufficiently early, a regression of PH and right ventricular hypertrophy could be observed [6]. Although one study documented that the acute administration of aminorex increased the pulmonary vascular resistance in anesthetized dogs in a dose-dependent way, attempts to develop a chronic aminorex–PH animal model failed [7]. Despite a dose–response effect between aminorex intake and the severity of PH not having been established and the evidence not having been substantiated by an animal model, the epidemiologic evidence suggesting a cause-and-effect relationship between this drug and the disease appears strong. Even though aminorex was withdrawn from the market in 1968, PH has been associated with ingestion of its analog 4-methylaminorex [street names “U-4-E-uh” (pronounced euphoria) or “ice”] in the USA in the context of illicit use as an alternative to methamphetamine [8]. In 1993, three members of a family (a man, his wife, and a nephew) were diagnosed with PH, potentially associated with this drug use. This anecdotal case suggests that substance abuse should be excluded in all cases of idiopathic PH.

2.1.2 Fenfluramines Fenfluramines (dl-fenfluramine and its analog dexfenfluramine) are phenylethylamine derivatives, serotonin-uptake inhibitors, and have been widely used for the treatment of obesity. There were several anecdotal reports associating the use of fenfluramine or dexfenfluramine with PPH in Europe between 1981 and 1992 [9–14]. All reported patients of PPH with a history of fenfluramine or dexfenfluramine intake were women, from 20 to 58 years old. The time between exposure to fenfluramine or dexfenfluramine until the ­presentation of early symptoms of PH, such as dyspnea on exertion, or hospital admission, varied from 3  months to 8  years [9–15]. In 1993, investigators at a French referral center performed a 5-year retrospective review of their experience with 73 PPH patients, and found that about 20% of them had been exposed to fenfluramine or dexfenfluramine [15]. There had been a large increase in the sales of fenfluramine or dexfenfluramine in France after the introduction of dexfenfluramine to the market (1985–1992). The role of fenfluramines in the development of PH was established by an international case-­control study conducted in 35 specialized centers in France, Belgium, the Netherlands, and the UK from September 1992 through September 1994 [16]. The International Primary Pulmonary Hypertension Study (IPPHS) found a strong association between appetite suppressants and PPH, with an odds ratio

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(relative risk estimates) of 6.3 [95% confidence interval (CI) 3.0–13.2]; 90% of patients for whom a defined product could be traced had used fenfluramine or dexfenfluramine. The risk increased markedly with the duration of use (odds ratio of 23.1 for more than 3 months; 95% CI 6.9–77.7) and decreased after cessation of use. Despite the IPPHS results, dexfenfluramine was approved for prescription use by the Food and Drug Administration (FDA) for the long-term treatment of obesity. The first cases for fenfluramine-induced PAH were reported in the USA in 1997 [17–19]. Several patients took the off-label combination of fenfluramine and phentermine, commonly called “Fen-Phen.” Fenfluramine and phentermine were approved as individual agents for appetite suppression, with phentermine approved for use in the USA in 1957 and fenfluramine approved in 1973. The total number of prescriptions in the USA exceeded 18 million [20]. Following the discovery of another cardiac disease [20], fenfluramine and dexfenfluramine were removed from the worldwide market. After their removal, several North American – Surveillance of North American Pulmonary Hypertension (SNAP) [21] and Surveillance of Pulmonary Hypertension in America (SOPHIA) [22] – and European [23, 24] epidemiology studies showed the association between the use of fenfluramine or dexfenfluramine and PAH and other results consistent with those of the IPPHS [16] and provided better understanding of PAH associated with the use of fenfluramine and dexfenfluramine. The possible interaction of fenfluramine and dexfenfluramine with female gender was suggested after the observation of a high female-to-male sex ratio in series studying these drugs [24]. This interaction has, however, not been confirmed, in view of the usual sex ratio in PPH at large (1.4:1) and the higher use of appetite suppressants by women. In the French national registry which included 17 university hospitals, 674 patients with PAH seen between 2002 and 2003 were enrolled. It showed that 9.5% of patients had a history of anorexigen exposure mainly with fenfluramine derivatives (77%). Two thirds of them (65%) were exposed for more than 6 months. The delay between the last appetitesuppressant intake and the first symptoms of PH was more than 2  years in 76% of them, and in 44% was more than 5 years [23]. The median duration of fenfluramine exposure was 6 months, with a median of 4.5 years between exposure and onset of symptoms among 109 patients followed up in a French medical center specializing in the management of PPH [24]. The long delay between exposure to fenfluramine derivatives and the onset of symptoms highlights the importance of a cardiological and pneumological surveillance among patients who have been exposed to fenfluramine derivatives for several years after their discontinuation. Regarding the survival, Rich et  al. evidenced a poorer ­survival among patients with fenfluramine-induced PAH

K. Bouillon et al.

c­ ompared with patients with idiopathic PAH or familial PAH (FPAH) (median 1.2 vs. 4.1 years) [25]. Analyzing a larger sample of patients, Souza et  al. did not find a significant ­difference between patients with fenfluramine-induced PAH and patients with idiopathic PAH or FPAH, with a median survival of 6.4  years [24]. In both studies, patients were exposed to similar PAH treatment regimens regardless of their exposure status.

Pathogenic Mechanisms The pathogenic mechanisms of PH induced by appetite suppressants are still unknown. Investigators have hypothesized that the anorexigens elevate the levels of serotonin (5-hydroxytryptamine) normally stored in platelets (“serotonin hypothesis of PPH”). Serotonin is a direct pulmonary artery vasoconstrictor, and an active mitogen for smooth ­muscle. Its levels have been found to be increased in patients with PPH, even some of those who had taken anorexigens [26]. Anorectic phenethylamine derivatives inhibit the uptake and provoke the release of serotonin from platelets [27]. Although aminorex has been shown to release noradrenaline, it also releases serotonin [27, 28]. However, several data show that administration of fenfluramines in animals and humans actually lowers blood levels of serotonin and does not increase the plasma level of serotonin [29–31]. Rothman et al. tested the hypothesis that the fenfluramines and other appetite suppressants might increase the risk of PAH through interactions with serotonin transporters (SERTs) in the lung  [32]. They showed that aminorex and fenfluramine are SERT substrates and increase the level of extracellular serotonin at therapeutically relevant doses. The concentrations of serotonin in blood and plasma may not be indicative of ­serotonin concentrations in local microenvironments ­surrounding ­pulmonary epithelial cells and arterial smooth muscle cells. To explain the hyperplasia of pulmonary artery, Rothman et al. hypothesized that SERT substrates other than serotonin might also be mitogenic [32]. They suggested that the accumulation of anorexigens in smooth muscle cells after their translocation could trigger mitogenesis through inhibition of K+ channels. They also suggested that the role of SERT in the pathogenesis of PAH is to serve as a “gateway” for the accumulation and concentration of medications in pulmonary cells [32]. Recent findings support the important role of SERT in the pathogenesis of PAH in the presence of anorexigens [33–35]. Anorexigens may have other important actions on the lung circulation. Aminorex, fenfluramine, and dexfenfluramine inhibit potassium current in rat pulmonary vascular smooth muscle cells, lowering the membrane potential and leading to contraction [36]. Because only a few patients exposed to fenfluramines develop PH, genetic susceptibility has been incriminated. Until now, several genes have been identified whose

73  Pulmonary Arterial Hypertension Secondary to Anorexigensand Other Drugs and Toxins

mutations could be associated with anorexigen-related PAH. Mutations within the bone morphogenetic protein receptor type II gene (BMPR2), coding for a receptor of the transforming growth factor b (TGF-b) superfamily, have been shown to underlie 50% (23 of 47 FPPH families) of cases of patients with familial PH [37] and 26% (13 of 50 patients) of cases of patients with sporadic PH [38]. BMPR2 mutations were found among 9% (three of 33) [39] and 18% (seven ­of 38) of patients with PAH associated with fenfluramines [24]. Recently, in a patient with hereditary hemorrhagic telangiectasia and dexfenfluramine associated PAH, an endoglin germline mutation was observed [40]. Endoglin gene encodes an endothelial cell transmembrane protein that can be defined as a component of the receptor complexes for growth factors of the TGF-b superfamily. These data are interpreted as in favor of a role of the TGF-b signaling pathway in anorexigeninduced PAH [40]. Regarding the role of SERTs in anorexigen-induced PAH, a mutation causing loss of function of the serotonin 5-HT2B receptor was found in one of ten patients with fenfluramine-induced PAH, whereas no mutations were found among 18 patients with idiopathic PAH [34]. These findings stress the complexity of the pathophysiologic mechanisms by which appetite suppressants may cause PH. Much work has been done and is ongoing to understand them. Weir stated: “we are beginning to understand the hieroglyphics on our Rosetta stone” [41].

2.1.3  Other Appetite Suppressants Phentermine Phentermine, a contraction for “phenyl tertiary butylamine,” is an appetite suppressant of the amphetamine and phenethylamine class. It has often been used in combination with fenfluramine as an appetite suppressant (see earlier). Until now, no PAH induced by phentermine alone has been reported. Propylhexedrine Propylhexedrine is an amphetamine-like appetite suppressant. Until now, only one case of PAH, which developed over a period of 8 years of propylhexedrine use, has been reported; this was in 1984, in a 39-year-old woman [42]. Propylhexedrine is also commonly found in over-the-counter cold, allergy, and allergic rhinitis remedies for the temporary symptomatic relief of nasal congestion. Phendimetrazine Phendimetrazine is an appetite suppressant as potent as amphetamines. To date, only one case of PAH associated

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with phendimetrazine has been reported; this was in 1991, in a 41-year-old woman, 6 months after the onset of her symptoms. Her symptoms, electrocardiographic abnormalities, and pulmonary hemodynamics improved several months after discontinuation of the drug [43].

Mazindol Mazindol is a sympathomimetic amine, similar to amphetamine. We found only a single case report of PH diagnosed 12 months after discontinuation of mazindol that had been taken for 10 weeks [44].

Phenylpropanolamine Phenylpropanolamine is a synthetic sympathomimetic amine commonly used as an appetite suppressant. A case-control study found that phenylpropanolamine increased the risk of hemorrhagic stroke. Therefore, since 2000, it has no longer been marketed in the USA as an appetite suppressant. Between 1998 and 2001, in the SOPHIA study, Walker et al. found an association between PAH and over-the-counter diet pills containing phenylpropanolamine [22]. In addition, Barst et  al. reported a fatal case of idiopathic PAH in a 7.5-year-old boy who had a very high exposure to phenylpropanolamine, which is commonly found in cough or cold remedies [45]. These data strengthen the hypothesis that phenylpropanolamine is a risk factor for the development of PAH. Physicians and the population should be aware of the risks of PAH and of hemorrhagic stroke when a substantial dose of phenylpropanolamine contained in over-the-counter diet pills and cough or cold remedies has been used, especially among children.

Diethylpropion Diethylpropion (amfepramone) is structurally related to amphetamines and is a sympathomimetic stimulant. The implication of diethylpropion use is not clear because of the small number of patients exclusively exposed to this drug. Between 1995 and 2003, only two cases of diethylpropionassociated PAH were reported [46, 47]. The first case was observed in the UK. A 22-year-old woman was prescribed diethylpropion for 3–5  months. Breathlessness on exertion developed when she was 27, and the diagnosis of PAH was made 4  years later [46]. The second case was observed in Belgium. A 27-year-old woman took diethylpropion during two cures of 4 and 5 weeks, respectively, 3.5 and 1.5 years before the onset of symptoms [47]. Interestingly, a mutation of BMPR2 was found in the second case of PAH associated with diethylpropion.

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2.2  Benfluorex Benfluorex is marketed as an adjunctive drug for patients with hypertriglyceridemia or diabetes with overweight. This drug, structurally related to fenfluramine derivatives, is classified by the World Health Organization (WHO) as an appetite suppressant, and is sometimes used off-license as a slimming aid [48]. In France, where benfluorex has been on the market since 1976, Boutet et al. reported the first five cases of PAH related to benfluorex consumption [49]. All patients were overweight diabetic females, aged from 50 to 57 years. The duration of benfluorex exposure ranged from 3 months to 10 years. One patient underwent lung transplantation. Pathological examination of her lungs revealed lesions characteristic of PAH. The symptoms and hemodynamics determined by right-sided heart symptoms disappeared after discontinuation of benfluorex use in some of patients. This small case series does not demonstrate any causal relationship between benfluorex use and PAH. However, the pharmacological background and clinical findings support the hypothesis that benfluorex may play a role in this association [49]. In Spain, benfluorex was removed from the market in 2003 because cases of valvular heart disease were observed after its use [50]. One case of this disease was also reported by Boutet et al. [49]. Well-designed studies similar to the IPPHS [16] and SOPHIA [22] are needed to further investigate fenfluramine cardiovascular-like effects of benfluorex use.

2.3  Pergolide Pergolide is an ergot alkaloid-derivated dopamine agonist prescribed in Parkinson disease. It alleviates Parkinson disease by stimulating the dopamine D1 and D2 receptors. It has been prescribed since the late 1980s. Pergolide is known to induce valvulopathies similar to those seen with the fenfluramines. Interestingly, a recent publication reported a case of reversible PAH in a patient after 4 years of pergolide use [51]. Like fenfluramines [36], pergolide has been shown to inhibits voltage-gated K+ channels in pulmonary arterial smooth ­muscle cells, which led to a hypoxic pulmonary vasoconstriction [52] (Fig. 2).

2.4  Phenformin Phenformin is a biguanide, and has been used as an antidiabetic drug. It was reported to be associated with severe or fatal lactic acidosis, which could induce pulmonary ­vasoconstriction in animal experiments. In 1973, two cases of PH, believed to be associated with treatment with ­phenformin, were reported [53]. This drug was withdrawn

Fig. 2  Chemical structures of drugs other than appetite suppressants

from the US market in 1977. To date, there has been no other report on its use and PAH.

2.5 Oral Contraceptives and Hormone Replacement Therapy Kleiger et al. [54] reported six cases of PH in women with a history of oral contraceptive use. Three of these women had known predisposing causes of PH. For the other three women, PAH was diagnosed after they had been exposed to oral contraceptives for about 5  years. There was no evidence of thromboembolism in the lungs or the veins of the leg. In the IPPHS, oral contraceptive use was not associated with a significantly increased risk [16]. One case report suggested the association between a fatal histologically proven PVOD and oral contraceptives use in a 24-year-old woman [55]. Morse et al. [56] described the case of a 64-year-old woman known to be an obligate carrier of FPAH who, after taking hormone replacement therapy for 3 months, had PAH. She had normal pulmonary function before this treatment.

2.6 Leflunomide Leflunomide is a pyrimidine synthesis inhibitor. It is a disease-modifying antirheumatic drug that reduces the signs and symptoms of rheumatoid arthritis and slows down disease progression. A 70-year-old woman has been reported to have PAH after 1.5 years of treatment with leflunomide [57]. The symptoms and echocardiogram signs improved after use of the drug has been stopped.

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2.7 Interferon-a2 Interferon-a2 is an immunomodulator widely used in hepatitis and high-risk melanoma. Jochmann et al. described a 40-year-old woman who developed severe PAH 30 months after initiation of interferon-a2 use for adjuvant treatment of spreading melanoma [58]. The symptoms and echocardiogram improved after discontinuation of its use. This association had been previously observed by Al-Zahrani et al. [59].

2.8 Aglucerase Gaucher disease is an autosomal-recessive disorder that is caused by a deficiency of the enzyme glucocerebrosidase. PAH has been seen in patients with type 1 Gaucher disease because of perivascular infiltration by Gaucher cells with vascular obliteration and plugging of the capillaries with Gaucher cells. However, three cases of PAH were reported to be associated with the use of alglucerase after eliminating the possible involvement of Gaucher cells [60, 61].

2.9 Chemotherapy-Related Drugs Chemotherapy is considered to be one of the possible risk factors for patients developing PAH (Table 1). In the literature, chemotherapy is reported to be associated with both PAH and PVOD (Fig. 3).

2.9.1 Thalidomide and PAH Thalidomide is an immunosuppressive agent with antiangiogenic and anti-inflammatory activities. Before 2006, it was given both as a single agent and in combination therapy against refractory or relapsing multiple myeloma, and since then thalidomide plus dexamethasone has been approved as frontline treatment of multiple myeloma. Several case reports suggested a possible association between the use of thalidomide for treatment of multiple myeloma and PAH, beginning in 2003 [62–64]. As thalidomide is known to be associated with an increased risk of thromboembolic PH, a pilot study designed to detect clinical and subclinical ­nonthromboembolic PH in multiple myeloma patients after thalidomide treatment was conducted among 82 patients [65]. Clinical and echocardiographic evaluation revealed four out of 82 (5%) patients with PAH between 1 and

Fig. 3  Chemical structures of chemotherapy drugs

6  months after thalidomide administration, eliminating a thromboembolic nature of PH. After the use of thalidomide had been discontinued, pulmonary pressure decreased but without recovering to the baseline value, possibly because of an irreversible drug effect on endothelial and muscle vessel cells. Echocardiography may be an interesting tool in monitoring patients treated with thalidomide to detect the preclinical stage of the disease. 2.9.2 Combination of Chemotherapy Used for Malignant Tumors and PAH Other combinations of chemotherapy have been reported to be associated with PAH. To our knowledge, two cases of histologically confirmed PAH have been observed [66, 67]. In both cases, a cyclophosphamide-containing regimen was used to treat neuroblasma and myelomonocytic leukemia. Table  2 describes the combination of chemotherapy drugs taken by these patients. 2.9.3 Mitomycin C Containing Chemotherapy for Solid Malignant Tumor and PVOD Mitomycin C is used in chemotherapy for malignant tumors combined with other chemotherapy. Anecdotal reports showed that histologically confirmed PVOD occurred in patients treated for metastatic cervical carcinoma [68, 69], metastatic gastric adenocarcinoma [70], and non-small-cell lung cancer [71, 72]. Table 3 shows the patients’ characteristics and a description of the combination of chemotherapy.

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Table 2  PAH related to chemotherapy treating malignant neoplasms

Publication date

Malignant neoplasm

Gender

Age at time of report

1991 [66]

Neuroblasma

Male

9 months

Female

8 years

Chemotherapy received before onset of PH Cyclophosphamide Doxorubicin Cisplatin Teniposide (VM-26)

Cytarabine (L1, L2) Mitoxantrone (L1) Etoposide (L1) Daunorubicin (L1) Busulfan (L2) Cyclophosphamide (L2) Cyclosporine Methotrexate PH pulmonary hypertension, LX X number of line of chemotherapy a Delays were estimated from the publications 2002 [67]

Myelomonocytic leukemia (allogeneic BMT)

Radiation therapy

Interval between treatment (first line) and onset of PHa

Interval between symptoms and deatha

Lung histological finding

–

5 months

2 months

PAH

–

9 months

Alive

PAH

Table 3  PVOD related to chemotherapy treating solid malignant neoplasms

Publication date Malignant neoplasm

Age at time of Gender report

1983 [68]

Female 41 years

Cervical carcinoma

Chemotherapy received before Radiation onset of PH therapy

Mitomycin Pelvis Bleomycin Cisplatin 1985 [69] Cervical carcinoma Female 36 years Mitomycin Pelvis and mediastinum Bleomycin Cisplatin Vinblastine 1984 [70] Gastric adenocarcinoma Male 68 years Mitomycin – 5-Fluorouracil Doxorubicin 2001 [71] Lung adenocarcinoma Male 57 years Docetaxel – Cisplatin Mitomycin Vindesine Gemcitamine 2002 [72] Lung adenocarcinoma Male 64 years Mitomycin – Ifosfamide Cisplatin 2002 [72] Bronchial large-cell Male 63 years Mitomycin – carcinoma Ifosfamide Cisplatin 1987 [73] Glioma Female 17 years Carmustine Brain 1987 [73] Glioma Female 21 years Carmustine Brain 2003 [74] Neuroblastoma Male 19 months Vincristine (L1) Skull base, (autologous BMT) abdomen Doxorubicin (L1) Cyclophosphamide (L1) Cisplatin (L1) Etoposide (L1) Dexamethasone (L1) Carboplatin (L2) Etoposide (L2) Melphalan (L2) PH pulmonary hypertension, BMT bone marrow transplantation, LX X number of line of chemotherapy a Delays were estimated from the publications

Interval between treatment (first line) and onset of PHa

Interval between symptoms and deatha

Lung histological findings

6 months

3 months

PVOD

?

6 months

PVOD

17 months

5 months

PVOD

2.5 years

8 months

PVOD

4 months

6 months

–

4 months

4 months

PVOD

10 months 6 months 6 months

3.5 months PVOD 2 months PVOD 45 days PVOD

73  Pulmonary Arterial Hypertension Secondary to Anorexigensand Other Drugs and Toxins

2.9.4 Other Chemotherapy for Solid Malignant Tumors and PVOD A chemotherapy regimen other than that associated with mitomycin C has been shown to be related to histologically confirmed PVOD: carmustine alone given for treatment of glioma [73]; a cyclophosphamide- and etoposide-containing regimen with autologous bone marrow transplantation for neuroblastoma [74] (Table 3); a bleomycin-containing regimen used for lymphocytic lymphoma [75]; and MOPP (mechlorethamine, vincristine, procarbazine, and prednisone) and COPP (cyclophosphamide, vincristine, procarbazine, and prednisone) or MOPP alone regimens for Hodgkin

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disease [73, 76] (Table 4). Lymphocytic lymphoma has been treated like acute lymphoblastic leukemia, i.e. with a cyclosphosphamide-containing regimen associated with bone marrow transplantation [77] (Table 5). This is also the case for T-cell lymphoma management [78].

2.9.5 Combination of Chemotherapy Used for Acute Lymphoblastic Leukemia/ Myelofibrosis and PVOD Several physicians noticed that a few patients treated for acute lymphoblastic leukemia had histologically confirmed

Table 4  PVOD related to chemotherapy treating lymphoma

Publication Malignant date neoplasm

Age at time Chemotherapy received Gender of report before onset of PH

1983 [75]

Male

Lymphocytic lymphoma Lymphocytic lymphoma

60 years

Radiation therapy

Interval between treatment (first line) and onset of PHa

Bleomycin – 3.5 months Vincristine 1983 [75] Male 32 years Chlorambucil (L1) – > 9 months Prednisone (L1, L2) Bleomycin (L2) Vincristine (L2) Male 39 years Cyclosphosphamide 1996 [77] Lymphocytic – 52 days after (L1, L3) lymphoma BMT (autologous Doxorubicin (L1) BMT) Vincristine (L1) Prednisone (L1) Etoposide (L2, L3) Cytosine arabinoside (L2) Solumedrol (L2) Cisplatin (L2) Carmustine (L3) Dacarbazine (L3) Total body 2.5 months 20 years Cyclophosphamide (L1) 2000 [78] T-cell lymphoma Male irradiation (allogeneic Adriamycine (L1) BMT) Vincristine (L1) Prednisolone (L1, L2) Carboplatin (L2) Cytosine arabinoside (L2, L3) Mitoxantrone (L3) Cyclosporine (L4) Methotrexate (L4) Mantle and 10 years 1987 [73] Hodgkin Male 23 years MOPP then COPP inverted Y lymphoma Mustargen (L1) V incristine (L1, L2) Procarbazine (L1, L2) Prednisone (L1, L2) Cyclophosphamide (L2) 6 years Inverted Y, 1993 [76] Hodgkin Male 34 years Cyclophosphamide (L1) supraclaviclymphoma Prednisolone (L1) ular fossa MOPP (L2) PH pulmonary hypertension, BMT bone marrow transplantation, LX X number of line of chemotherapy a Delays were estimated from the publications

Interval between symptoms and deatha

Lung histological findings

1 week

PVOD

?

PVOD

8 days

PVOD

6 months

PVOD

12 months

PVOD

13 months

PVOD

Table 5  PVOD related to chemotherapy treating acute leukemia/myelofibrosis

Publication Malignant date neoplasm 1984 [79]

1989 [80]

1989 [80]

1996 [81]

1996 [82]

2004 [83]

Age at time of Gender report

Male Acute lymphoblastic leukemia (allogeneic BMT)

12 years

Vincristine (L1, L2) Cyclophosphamide (L1, L3) Daunorubicin (L1) Cytarabine (L1) Prenisone (L2, L3) Mithramycin Methotrexate (L3) Female 4 years Vincristine (L1, L2) Acute lymphoblasPrednisone (L1, L2) tic leukemia Asparaginase (L1, L2) (allogeneic Daunomycin (L1) BMT) Busulfan Methotrexate (IT) Arabinosylcytosine (L2) Cyclophosphamide (L2, L3) Carmustine (L3) Etoposide (VP-16, L3) Male 7 years Prednisone (L1, L3, L4, L5, L6) Acute lymphoblasVincristine (L1, L3, L4, L5) tic leukemia Asparaginase (L1, L3, L5) (allogeneic Methotrexate, IT (L1, L5) BMT) 6-Mercaptopurine (L2, L4) Methotrexate (L2, L3, L4) Teneposide (VM-26, L3, L4) Arabinosylcytosine (L3, L4, L6) Etoposide (VP-16, L4, L7) Cyclophosphamide (L4, L7) Daunomycin (L5) Carmustine (L7) Female 19 years Cyclophosphamide (L1) Acute lymphoblasMethotrexate (L2) tic leukemia Cyclosporine (L2) (allogeneic Mitoxantrone (L3) BMT) Arabinosylcytosine (L3, L4) Busulfan (L4) Etoposide (L4) Melphalan (L4) Male 24 years Cyclophosphamide (L1) Acute lymphoblasDoxorubicin (L1) tic leukemia Methotrexate, IV + IT (L1, L3) (allogeneic Arabinosylcytosine (L1) BMT) Vincristine (L1, L2) Prednisone (L2) Cyclosporine (L2) Dactinomycin (L2) Carmustine (L2) Granulocyte colony-stimulating factor g-Immunoglobulin Mercaptopurine (L3) Asparagine (L4) Male 4 months Arabinosylcytosine (L1) Myelofibrosis (allogeneic Busulfan (L2) BMT) Cyclophosphamide (L2) Cyclosporine

IV intravenous, IT intrathecal Delays were estimated from the publications

a

Chemotherapy received before onset of PH

Interval between treatment (first line) and onset of PHa

Interval between symptoms and deatha

Total body irradiation

5.5 years

A few days

PVOD

Total body irradiation

4.5 years

2 months

PVOD

Brain, spine

3 years

Alive

–

Total body irradiation

1.5 years

5 months

PVOD

?

18 months

?

PVOD

Spleen

Symptoms existed 32 days after at time of BMT treatment owing to hepatosplenomealy

Radiation therapy

Lung histological findngs

PVOD

73  Pulmonary Arterial Hypertension Secondary to Anorexigensand Other Drugs and Toxins

PVOD following chemotherapy, described in Table 5 [79–82]. PVOD occurred in a particular context. First, all patients used in total, before the appearance of PVOD, at least seven drugs. Second, all of the patients had used cyclophosphamide. Finally, all had an allogeneic bone marrow transplantation after total body irradiation. PVOD has also been noted after treating myelofibrosis with chemotherapy and bone marrow transplantation [83]. A chemotherapy regimen has been shown to be associated with both PAH and PVOD, in particular thalidomide for PAH, and mitomycin C and bleomycin for PVOD. After examination of these cases, it is very difficult to determine whether chemotherapy is a causal agent for PAH and PVOD for several reasons. First, we cannot exclude the role of malignant neoplasms in inducing PAH or PVOD. For example, Capewell et al. described a patient with Hodgkin lymphoma who developed PVOD at the time of the diagnosis of lymphoma prior to any therapy [84]. Second, we do not know how radiation therapy interacts with chemotherapy to provoke these pulmonary diseases. Kramer et al. observed a patient who developed PVOD 6 years after receiving radiation therapy in a mantle distribution for Hodgkin disease, without use of any chemotherapy [85]. In addition, an association between PVOD and bone marrow transplantation, either allogeneic or autologous, has been noted. In most cases, conditioning was carried out with cytotoxic chemotherapy as well as total body irradiation. Finally, some chemotherapy is known to be lungtoxic, in particular mitomycin, which can also provoke interstitial pneumonitis. Therefore, lung biopsy needs to be performed to classify PH. At this time, we are very far from understanding the pathophysiologic mechanisms of chemotherapy in inducing PAH and PVOD. The cause is likely to be multifactorial. All the cases of PH associated with drugs described above concerned young persons or adults who had been exposed directly to drugs. Some of them, such as nonsteroidal antiinflammatory drugs and selective serotonin-reuptake inhibitors (SSRIs) may be associated with PH in the fetus or in the newborn after antenatal (these drugs are known to cross the placenta) or postnatal exposure. Among newborns, these drug classes have been reported to be associated with PAH and PPHN. The prevalence of PPHN is estimated at two in 1,000 live births [86]. PPHN is suspected of leading to neonatal hypoxemia, which is often refractory, and associated with a high mortality (11%) [86].

2.10 Nonsteroidal Anti-inflammatory Drugs Indomethacin is a nonselective inhibitor of cyclooxygenase which inhibits prostaglandin synthesis. It is used in obstetrics for its effect of tocolytic effect for pregnant women with

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premature labor, and for prophylaxis of patent ductus arteriosus in premature infants. Utilization of indomethacin for premature labor has been associated with PAH of newborns. The first cases of PAH in two newborns in relation to prenatal exposure to indomethacin were reported in 1976 [87]. Dalens et al. [88] found that five premature newborns whose mothers had received a prolonged indomethacin therapy (at least 9 days) at 25 or 32 weeks of gestation had clinical and biological symptoms similar to those of PH with persistence of the fetal circulation with a patent ductus arteriosus; two of them died. Their autopsies showed an important reduction of the lumen of pulmonary arterioles due to a thickening of the tunica media. Similarly, in a randomized clinical trial, Besigner et al. [89] found three cases of PAH in newborns whose mothers received indomethacin in the long-term treatment (more than 16 days) for preterm labor at 23–34 weeks of gestation. PH has been observed also among newborn pups from parturient rats which received daily injections of indomethacin [90]. The newborns pups had significantly increased numbers of muscularized peripheral pulmonary vessels. PH, however, did not persist to adulthood. However, in a randomized trial that compared short-term (48 h) indomethacin use with ritodrine use for preterm labor less than or equal to 32  weeks of gestation, among the indomethacin group composed of 52 patients no cases of premature closure of the ductus arteriosus or PH were observed [89]. According to these findings, the risk of PAH in the newborn may be significantly increased with prolonged use of indomethacin, and not with short-term (up to 48 h) courses of therapy. These results should be interpreted with caution, as the diagnosis of PAH in the newborn is difficult to make because anatomical abnormalities have to be considered. As a prostaglandin synthetase inhibitor, another well-known adverse effect of indomethacin is constriction of the fetal ductus arteriosus, which can lead to permanent PH in a premature newborn. Both Levin et al. [91] and Tarcan et al. [92] reported a case of permanent PH in a premature newborn after short-term use of antenatal indomethacin exposure. At autopsy, an increased pulmonary arterial medial thickness due to increased smooth muscle cell proliferation and growth was observed [91]. A few case reports described among preterm or term newborns persistent PH with a constricted ductus arteriosus with the use of naproxen, ibuprofen diclofenac, and nimesulide, the same as what was seen with indomethacin [92]. Patient education regarding over-the-counter pain self-medication during pregnancy should be emphasized.

2.11 Fluoxetine Fluoxetine is an SSRI (as are aminorex and fenfluramines). However, there has been no report of a relationship between

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the chronic intake of SSRI antidepressants such as fluorexine and PAH among adults. SSRIs are very widely used antidepressants especially among pregnant women, with a usage level of 77.5% among 2.6% of pregnant women who received an antidepressant prescription [93]. In 1996, Chambers et al. observed in a prospective small cohort an increased risk for perinatal complications among women who took fluoxetine in the third trimester [92]. They suggested a possible association between maternal use of fluoxetine late in the third trimester of pregnancy and the risk of PPHN in the infant. Recent data from a retrospective case-control study conducted between 1998 and 2003 by Chambers et al. supported the association between the maternal use of SSRIs after the completion of the 20th week of gestation and PPHN in the offspring: Fourteen infants with PPHN had been exposed to an SSRI compared with six in the control group (adjusted odds ratio of 6.1; 95% CI 2.2–16.8) [92]. Another cohort study based on the Swedish Medical Birth Register was in favor of this association [94]. In addition, Källén et  al. observed a risk of PPHN when women took an SSRI at an early stage of pregnancy. Experimental studies in rats support this association, showing that fluoxetine exposure in utero induced PH in the fetal rat as a result of a developmentally regulated increase in pulmonary vascular smooth muscle proliferation [95]. Other studies such as the SOPHIA [22] and the IPPHS [16] should be carried out to monitor temporal trends in the number of reported cases of PPHN to detect if an epidemic of SSRI antidepressant related PPHN occurs, and to identify which SSRI is or SSRIs are at risk of causing PPHN. The benefits/risks of the SSRI antidepressants may be evaluated by health authorities. Physicians may be aware of this potential adverse effect of SSRI, and pregnant women should be informed about it.

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Fig. 4  Chemical structures of illicit substances

3.2 Cocaine Cocaine (benzoyl methylecgonine) is derived as an alkaloid from the leaves of Erythroxylon coca. Like amphetaminelike appetite suppressants, it blocks the release and reuptake of catecholamines, including dopamine, and blocks serotonin release and reuptake. Two forms of cocaine can be found: cocaine hydrochloride is water-soluble, and “crack” cocaine (alkaloidal cocaine) is lipid-soluble and resists thermal degradation, so it can be smoked. It has been shown that cocaine or crack induced PAH among individuals who had abused this substance for more than 3 years [99, 100]. Lung biopsy specimens revealed medial hypertrophy of muscular pulmonary arteries. In the IPPHS, use of illicit drugs (cocaine or intravenous drugs) was associated with an increased point estimate of the relative risk (odds ratio of 2.8; 95% CI 0.5– 15.7), but the numbers were too small draw a conclusion [16]. In most studies, it is difficult to disentangle the role of the product used (cocaine, heroine) from that of emboli associated with intravenous drug use (in which case the PH should not be considered to be “primary”). Cocaine abuse by a mother also produced signs of persistent PH in a newborn [101] (Fig. 4).

3 Substance Abuse

3.3 Other Substances

Amphetamines, cocaine, and other stimulants have been considered “possible” or “very likely” risk factors.

In the early 1980s, one small series reported that among 18 boys with a history of chronic glue (toluene) abuse for 6 months or more, obvious PH was found on clinical examination in five of them [102]. No lung biopsy was done.

3.1 Methamphetamine As seen already for amphetamine-like appetite suppressants, amphetamine and methamphetamine, also known as “crank,” are stimulants of the central nervous system. A case report and an epidemiology study have established the relationship between PAH and methamphetamine abuse [96, 97]. Rothman et  al. [32, 98] suggested that the underlying mechanism is interactions with SERTs in the lung, as seen for aminorex and fenfluramine derivatives.

4 Toxins and Others 4.1 Herb Toxins The pyrrolizine alkaloids are amino alcohols derived from the pyrrolizidine nucleus. They are plant toxins that cause injury to the vasculature of the lungs and PH and right-sided

73  Pulmonary Arterial Hypertension Secondary to Anorexigensand Other Drugs and Toxins

Fig. 5  Chemical structures of pyrrolizine alkaloids

heart failure in animals (Fig. 5). They are named differently according to species of plants from the genera Crotalaria and Senecio. Monocrotaline is found in Crotalaria spectabilis and fulvine is found in C. fulva [103]. In Senecio jacobaea, several alkaloids are found, in particular seneciphylline [103]. The leaves and seeds of these plants are used for the preparation of bush teas, which are consumed by the indigenous population for medicinal and other purposes. Until now, no human case of PH has been reported following ingestion of these pyrrolizine alkaloids, but they are known to cause hepatic veno-occlusive disease. It is not clear why human pulmonary arteries and veins are not affected by the ­pyrrolizidine alkaloids when they exert such toxic effects on the small hepatic veins. The monocrotaline-induced PH rat is an animal model for the study of human PH. These data suggested that in the case of unexplained PH in a man, a history of ingestion of herbal remedies should be sought. PH seen in toxic oil syndrome (TOS) induced by toxic rapeseed oil and in eosinophilia–myalgia syndrome (EMS) related to l-tryptophan are not classified as PAH related to drugs and toxins but they are interesting examples of the relations between exposure to chemicals and PH.

4.2 Toxic Rapeseed Oil In May 1981, a large foodborne epidemic occurred in central and northwestern Spain. The epidemic was characterized in the initial phase by atypical pneumonia and pronounced eosinophilia in the next few days after exposure. From May to October 1981, it affected over 20,000 people. Among them over 300 died of this new multisystemic disorder now known as toxic oil syndrome (TOS) within 8  months following the onset of the outbreak. Criteria for TOS diagnosis were defined by the Spanish Clinical Commission in August 1981 [104]. Epidemiological investigations have established that the cause was consumption of adulterated food oil, which was sold principally in unlabeled, 5-L containers by traveling salesmen. This oil was in fact a mixture containing rapeseed oil originating from France. It was denatured with 2% aniline

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Fig.  6  Chemical structures of 3-phenylamino-1,2-propanediol and l-tryptophan

before being imported to Spain, theoretically for industrial use. In Spain, it was denatured and illegally refined to remove aniline, and finally mixed with other edible oils. Toxicoepidemiology studies have suggested that 3-phenylamino-1,2-propanediol or related compounds found in toxic oil may be the causal agent of TOS (Fig. 6). However, to date, none of these oil contaminants have produced results in animals or in vitro suggestive of TOS in man. In the natural history of the disease, most authors agree on the existence of three phases. The acute phase (first and second months after onset of the disease) was characterized by noncardiogenic pulmonary edema with dyspnea, skin rash, eosinophilia, and myalgia. The intermediate phase (third and fourth months) was marked by severe myalgia, subcutaneous edema, hepatic disease, arthritis, and PH. PH was found in about 20% of hospitalized patients from the second to fourth months since the onset of the disease [105]. The early chronic phase (from the fourth month to the second year) was marked by scleroderma, polyneuropathy, sicca syndrome, and joint contractures. The late chronic phase (from the second year to the end of the follow-up) was characterized by muscle cramps, chronic musculoskeletal pain, Raynaud phenomenon, carpal tunnel syndrome, and chronic lung disease [106]. Alonso-Ruiz et  al. [106] followed 332 cases of TOS for up to 8 years and found that 8.1% of patients developed PH; the condition regressed in 74% of them, and only 2.1% developed a malignant form of PH at the end of the follow-up period of 8 years. Abaitua Borda et al. [107] showed that the mortality during the first 13  years following TOS diagnosis was 5  times higher among the TOS cohort compared with the Spanish population as a whole [standardized mortality ratio (SMR) of 4.9; 95% CI 4.4–5.5]. No specific cause of death was shown to be more frequent in the TOS cohort than in the Spanish population, with the exceptions of TOS and PH. PH represented an 18-fold (SMR of 18.2; 95% CI 3.7–53.0) higher risk of death in this cohort than in the general population during this study period. Pathological changes at a later stage (at least 3 years after the onset of the disease) in the evolution of the patients with TOS were characterized by medial hypertrophy, intimal fibrosis, and plexiform lesions in pulmonary arteries [108].

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4.3 l-Tryptophan l-Tryptophan is an essential amino acid that is normally ingested as a constituent of dietary protein. l-Tryptophan supplements are used by some people for disorders such as premenstrual syndrome, insomnia, and depression (Fig. 6). In October 1989, three patients in New Mexico had a syndrome in which eosinophilia was accompanied by severe myalgias. This syndrome was associated with l-tryptophan ingestion [109]. It became a newly recognized disease named eosinophilia–myalgia syndrome (EMS) with the following Centers for Disease Control definition: peripheral blood eosinophilia (number superior to 109 [9]/L) and generalized, disabling myalgias without other recognized causes. The apparent median latency period of this disease was 127 days [109]. This is a multisystemic disease; other clinical manifestations include pulmonary involvement (interstitial infiltrates and pleural effusions), skin rash and edema, axonal polyneuropathy, perimyositis, and possible adverse neurocognitive effects [109]. Pulmonary vascular involvement rarely led to PH. It has gradually resolved except in a few catastrophic cases. Case-controls studies conducted by state health departments confirmed the association of l-tryptophan use with EMS [109]. In November 1989, the FDA recalled all dietary supplements that contained l-tryptophan. After that, the number of new cases dropped sharply. In August 1990, over 1,500 cases of EMS were reported in the USA. Epidemiology studies clearly linked illness to the ingestion of tryptophan produced by a single manufacturer in Japan, and the time course of the epidemic was most consistent with its being caused by a product contaminant [109]. However, until now, no etiologic agent has been identified. Lung biopsy specimens have been collected among female patients taking l-tryptophan for 1–9 months prior to the rapidly progressive shortness of breath. The specimens showed both interstitial inflammatory infiltrate with lymphocytes and small numbers of eosinophils; and vascular changes with arterial and arteriolar medial hypertrophy accompanied by mild PH [110].

5 Summary In the literature, several drugs and toxins have been suggested to be associated with PAH. These can be categorized according to the strength of evidence. Up to now, only anorectic agents (aminorex and fenfluramine) and toxic rapeseed oil can be considered to be definite (causal) risk factors for PAH. After reviewing the literature on association between drugs and toxins, we have six findings. First, drugs and toxins are related to PAH through heterogeneous pathways: in a direct way in the case of their being associated with pulmonary arterial hypertension; and in several

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indirect ways as seen in PVOD, PPHN, FPAH, and systemic disease. Second, except for aminorex, fenfluramine derivatives, and toxic rapeseed oil, the importance of the role attributable to drugs or toxins is difficult to evaluate in the occurrence of PAH because we do not know the role played by individual susceptibility, initial disease, and other treatment, especially in oncological treatment, where several drugs are given in one line, patients can take several lines of chemotherapy during the follow-up, and radiotherapy and bone marrow transplantation are frequent. Third, it is worth conducting a careful investigation in all patients presenting with unexplained PH to study the possibility that the disease might be related to the ingestion of a drug or toxin in both individual and public levels: when its use is discontinued sufficiently early, a regression of PH and right ventricular hypertrophy can be observed; and when the drug responsible for PAH is removed from the market, the incidence of cases with PAH decreases. This investigation may be carried out by teams responsible for pharmacovigilance to highlight a possible association between the drug and PH. To establish firmly this association, welldesigned epidemiology studies should be performed. Fourth, other epidemics of PAH due to a drug or a toxin are still possible because drugs chemically similar to fenfluramine derivatives are still massively used, in particular benfluorex, certain amphetamine-like appetite suppressants such as propylhexedrine and phenylpropanolamine are commonly found in over-the-counter cold and allergy remedies, and because of unidentified drugs and toxins. Fifth, careful attention may be addressed toward newborns that are particularly susceptible for PH when they are exposed in utero to some widely used drugs among pregnant women. Finally, it is important to understand that the list of drugs and toxins is not complete. It will be added to with time according to our current knowledge. There is a need to review and update the strength of evidence of risk factors for PAH on a regular basis. Acknowledgement  The authors thank M. Humbert for his helpful comments and advice.

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73  Pulmonary Arterial Hypertension Secondary to Anorexigensand Other Drugs and Toxins 89. Loe SM, Sanchez-Ramos L, Kaunitz AM (2005) Assessing the neonatal safety of indomethacin tocolysis: a systematic review with meta-analysis. Obstet Gynecol 106:173–179 90. Herget J, Hampl V, Povýsilová V, Slavík Z (1995) Long-term effects of prenatal indomethacin administration on the pulmonary circulation in rats. Eur Respir J 8:209–215 91. Levin DL, Fixler DE, Morriss FC, Tyson J (1978) Morphologic analysis of the pulmonary vascular bed in infants exposed in utero to prostaglandin synthetase inhibitors. J Pediatr 92:478–483 92. Silvani P, Camporesi A (2007) Drug-induced pulmonary hypertension in newborns: a review. Curr Vasc Pharmacol 5:129–133 93. Reefhuis J, Rasmussen SA, Friedman JM (2006) Selective serotoninreuptake inhibitors and persistent pulmonary hypertension of the newborn. N Engl J Med 354:2188–2190 94. Källén B, Olausson PO (2008) Maternal use of selective serotonin re-uptake inhibitors and persistent pulmonary hypertension of the newborn. Pharmacoepidemiol Drug Saf 17:801–806 95. Fornaro E, Li D, Pan J, Belik J (2007) Prenatal exposure to fluoxetine induces fetal pulmonary hypertension in the rat. Am J Respir Crit Care Med 176:1035–1040 96. Schaiberger PH, Kennedy TC, Miller FC, Gal J, Petty TL (1993) Pulmonary hypertension associated with long-term inhalation of “crank” methamphetamine. Chest 104:614–616 97. Chin KM, Channick RN, Rubin LJ (2006) Is methamphetamine use associated with idiopathic pulmonary arterial hypertension? Chest 130:1657–1663 98. Rothman RB, Baumann MH (2007) Methamphetamine and idiopathic pulmonary arterial hypertension: role of the serotonin transporter. Chest 132:1412–1413 99. Murray RJ, Smialek JE, Golle M, Albin RJ (1989) Pulmonary artery medial hypertrophy in cocaine users without foreign particle microembolization. Chest 96:1050–1053

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100. Russell LA, Spehlmann JC, Clarke M et al (1992) Pulmonary hypertension in female crack users. Am Rev Respir Dis 145:A717 101. Collins E, Hardwick RJ, Jeffery H (1989) Perinatal cocaine intoxication. Med J Aust 150(331–332):334 102. Devathasan G, Low D, Teoh PC, Wan SH, Wong PK (1984) Complications of chronic glue (toluene) abuse in adolescents. Aust N Z J Med 14:39–43 103. Kay JM (1994) Dietary pulmonary hypertension. Thorax 49:S33–S38 104. Posada de la Paz M, Philen RM, Borda AI (2001) Toxic oil syndrome: the perspective after 20  years. Epidemiol Rev 23:231–247 105. Castro García M, Posada de la Paz M, Díaz de Rojas F, Abaitua Borda I, Aonso Gordo JM, Tabuenca Oliver JM (1984) Pulmonary hypertension after toxic rapeseed oil ingestion. J Am Coll Cardiol 4:443 106. Alonso-Ruiz A, Calabozo M, Perez-Ruiz F, Mancebo L (1993) Toxic oil syndrome: a long-term follow-up of a cohort of 332 patients. Medicine 72:285–295 107. Abaitua Borda I, Philen RM, Posada de la Paz M et al (1998) Toxic oil syndrome mortality: the first 13  years. Int J Epidemiol 27:1057–1063 108. Gómez-Sánchez MA, Mestre de Juan MJ, Gómez-Pajuelo C, López JI, Díaz de Atauri MJ, Martínez-Tello FJ (1989) Pulmonary hypertension due to toxic oil syndrome. A clinicopathologic study. Chest 95:325–331 109. Kilbourne EM (1992) Eosinophilia-myalgia syndrome: coming to grips with a new illness. Epidemiol Rev 14:16–36 110. Tazelaar HD, Myers JL, Drage CW, King TE Jr, Aguayo S, Colby TV (1990) Pulmonary disease associated with L-tryptophaninduced eosinophilic myalgia syndrome. Clinical and pathologic features. Chest 97:1032–1036

Chapter 74

Pulmonary Arterial Hypertension Related to Gaucher’s Disease, Sarcoidosis, and Other Disorders Judd W. Landsberg and Jess Mandel

Abstract  The disease process underlying pulmonary ­arterial hypertension (PAH) involves a classic pattern of pulmonary vascular remodeling, consisting of muscularization (medial hypertrophy) of small and medium-sized pulmonary arteries, with endothelial abnormalities, intimal fibrosis, and occasionally thrombus or plexiform lesions. The current classification divides PAH into five major categories: IPAH, familial PAH, “associated with” PAH (APAH), “associated with pulmonary venous involvement” PAH, and persistent PAH of the newborn. The category APAH is further subdivided on the basis of individual disease association ending (inauspiciously) with “other.” Here are found rare disorders, often with well defined, systemic pathophysiologic derangements as in glycogen storage diseases. There are two important reasons to study the pulmonary vascular remodeling that occurs in the setting of such rare diseases. First, regardless of the disease association, PAH is a potentially treatable cause of increased morbidity and mortality. Second, when a rare disease is complicated by PAH, it is reasonable to assume a mechanistic link between the underlying disease process and the development of pulmonary vascular remodeling. Thus, insight into the pathophysiologic processes of IPAH may be gleaned from the study of IPAH-like changes that occur in the setting of a systemic single enzyme deficiency such as seen in Gaucher’s disease type I. This approach may help identify important pulmonary vulnerabilities to vascular remodeling, since the underlying disease is systemic, but the vascular injury is confined to the lungs. Many of these diseases have the potential to cause pulmonary hypertension by mechanisms other than pulmonary vascular remodeling, such as parenchymal lung destruction, left ventricular dysfunction, venous thromboembolism, vasculitis, and obliteration of the pulmonary microvasculature, underscoring the importance of careful study and characterization. To that end, we have decided to include both sarcoidosis and Langerhans

J.W. Landsberg (*) Division of Pulmonary and Critical Care Medicine, University of California, San Diego, 200 West Arbor Drive, MC 8383, San Diego, CA 92103, USA e-mail: [email protected]

cell histiocytosis in this category (usually ­classified under miscellaneous pulmonary hypertension), based on the belief that each has the potential to cause a variant of PAH. Keywords  Associated pulmonary arterial hypertension • Rare disease • Parenchymal lung destruction • Vasculitis • Langerhans cell histiocytosis

1  Introduction The 2003 Venice classification of pulmonary hypertension reserves the term “pulmonary arterial hypertension” (PAH) for diseases that are pathologically and clinically similar to idiopathic PAH (IPAH). In practice, this includes diseases, disorders, or exposures that lead to severe progressive pulmonary hypertension mandating specific therapy and or consideration for lung transplantation. The disease process underlying PAH involves a classic pattern of pulmonary vascular remodeling, consisting of muscularization (medial hypertrophy) of small and medium-sized pulmonary arteries, with endothelial abnormalities, intimal fibrosis, and occasionally thrombus or plexiform changes. The current classification divides PAH into five major categories: IPAH, familial PAH, “associated with” PAH (APAH), “associated with pulmonary venous involvement” PAH, and persistent PAH of the newborn. The category APAH is further subdivided on the basis of individual disease association ending (inauspiciously) with “other.” Here are found rare disorders, often with welldefined, systemic pathophysiologic derangements as in glycogen storage diseases. There are two important reasons to study the pulmonary vascular remodeling that occurs in the setting of such rare diseases. First, regardless of the disease association, PAH is a potentially treatable cause of increased morbidity and mortality. Second, when a rare disease is complicated by PAH, it is reasonable to assume a mechanistic link between the underlying disease process and the development of pulmonary vascular remodeling. Thus, insight into the pathogenic mechanisms of IPAH may be gleaned from

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the study of IPAH-like changes that occur in the setting of a systemic single enzyme deficiency such as seen in Gaucher’s disease type I. This approach may help identify important pulmonary vulnerabilities to vascular remodeling, since the underlying disease is systemic, but the vascular injury is confined to the lungs. Many of these diseases have the potential to cause pulmonary hypertension by mechanisms other than pulmonary vascular remodeling, such as parenchymal lung destruction, left ventricular dysfunction, venous thromboembolism, vasculitis, and obliteration of the pulmonary microvasculature, underscoring the importance of careful study and characterization. To that end, we have decided to include both sarcoidosis and Langerhans cell histiocytosis in this category (usually classified under miscellaneous pulmonary hypertension), based on the belief that each has the potential to cause a variant of PAH. Hereditary hemorrhagic telangiectasia and hemoglobinopathies are discussed separately in individual chapters. Lymphangiomatosis was not included in the discussion because of a paucity of available research and data.

2  PAH in Glycogen Storage Disease Type I 2.1  Background Glycogenosis or glycogen storage disorders are a group of inherited conditions characterized by impaired glycogen metabolism. Several case reports of severe, progressive, fatal pulmonary hypertension occurring in patients with glycogen storage disease type I (GSD I), von Gierke’s disease, have suggested an association between these independently rare disorders. GSD I (subsets a–d) is an autosomal recessive disorder, involving an inborn enzyme deficiency affecting glucose 6-phosphatase (Ia), glucose 6-phosphate transporter (Ib), or glucose 6-phosphate translocase (Ic and Id). The incidence of GSD I is approximately one in 100,000 live births, with more than 80% of those afflicted suffering from GSD Ia. The presence of glucose 6-phosphatase deficiency (primarily located in the liver and the kidney) leads to excessive hepatic and renal glycogen accumulation. Patients typically present in infancy with failure to thrive, growth retardation, obesity, and hepatomegaly [1]. Metabolically, GSD I patients suffer from hypoglycemia, lactic acidosis, hyperlipidemia and hyperuricemia. The natural history of the disease is quite variable, ranging from death in infancy to survival into adulthood. Overall, the prognosis has improved with the introduction of nocturnal nasogastric feeding and uncooked corn starch therapy.

J.W. Landsberg and J. Mandel

However, as survival from the metabolic abnormality increases, long-standing complications such as renal failure and ­hepatocellular carcinoma have been reported more ­commonly [2]. Pulmonary parenchymal involvement, leftsided heart failure, and portal hypertension are not features of GSD I, making its association with severe pulmonary hypertension all the more interesting.

2.2  The Prevalence of GSD I APAH Severe pulmonary hypertension complicating GSD I is an uncommon, classically fatal, late complication of the disease, with a total of seven cases reported in the literature. Of the patients involved, 70% were female, with a median age at onset of 16 years (19 years if a 4-year-old outlier with an atrial septal defect is excluded). Mild, asymptomatic elevations in pulmonary artery pressure may be more common. Kishnani et al. performed a small prospective study screening 22 GSD I patients (11 female, 11 male) for pulmonary hypertension with echocardiography. Although no subject had an estimated pulmonary artery pressure (ePAP) above 40  mmHg, 27% had an ePAP above 30 mmHg (two females and four males). Although the ­sample size was low, there were no significant differences noted in the age, sex, or metabolic disease control between GSD I patients with a normal or elevated pulmonary artery pressure [3].

2.3  Pathophysiology Three of the seven patients with GSD I APAH reported in the literature underwent autopsies. All three demonstrated the classic pulmonary vascular remodeling seen in IPAH, specifically medial hypertrophy, intimal fibrosis, and plexiform lesions. None had evidence of excessive thrombosis [1, 4] (Fig.  1). In their report of the seventh GSD I patient with severe PAH, Humbert et al. noted that she had a dramatically elevated serum serotonin level. This prompted them to prospectively measure serotonin levels in 13 patients with GSD 1 but no evidence of PAH, 16 patients with severe pulmonary hypertension (IPAH/anorexigen exposed) without GSD I, and the index patient with both GSD I and severe pulmonary hypertension. Serotonin levels were comparably elevated (approximately 37  nmol/L) in all 13 of the patients with GSD I (and no PAH) and all 16 of the severe pulmonary hypertension patients without GSD I. None of the 26 healthy controls had elevated serotonin levels (approximately 9 nmol/L). Interestingly, the index patient with both GSD I and severe pulmonary hypertension had the highest level (114 nmol/L) [5].

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six patients died from right-sided heart failure. The median time from PAH diagnosis to death was only 60 days, although there was a significant range (1 week to 4 years.) [5]. Because most of these cases predate the modern era of IPAH management, therapy consisted primarily of diuretics, warfarin, and digoxin. Presenting symptoms were those of end-stage right-sided heart failure (i.e. profound exercise limitation, volume overload, and exercise-induced syncope). In all cases, clinical suspicion and physical examination led to echocardiography, which was followed up with confirmatory right-sided heart catheterization (RHC). Interestingly, both longer-term survivors were male; one was alive 4 years after diagnosis and the other 3 year after. The latter patient is the only one documented to have had therapy with vasodilator and antiremodeling drugs, so his case is detailed below. Clinically relevant PAH associated with GSD I tends to progress rapidly, and thus presents after significant rightsided heart failure has already developed. Therefore, physical examination, electrocardiogram, chest X-ray, and echocardiogram should suggest right ventricular hypertrophy. As always, suspected PAH must be confirmed with RHC, which allows for direct measurement of pulmonary artery pressure and accurate calculation of cardiac output and pulmonary vascular resistance.

Fig.  1  (a) Small pulmonary artery, showing medial hypertrophy, intimal fibrosis, and adjacent plexiform lesion. (b) High-power view of plexiform lesion. (From Pizzo [1]. Reproduced with permission from the American Academy of Pediatrics)

As with most of the risk factors identified for pulmonary vascular remodeling, many are necessary yet no single one is sufficient to cause disease. Furthermore, many factors such as BMPR2 mutations and elevated serum serotonin concentrations are systemic, yet vascular disease is confined to the pulmonary circulation. Taken together with the observation that IPAH does not recur after lung transplantation, one must conclude a significant portion of the risk of severe pulmonary vascular remodeling comes from factors intrinsic to the affected individual’s pulmonary vasculature.

2.4  C  linical Presentation and Evaluation and Diagnostic Testing Severe PAH associated with GSD I tends to have a fulminant presentation with rapid deterioration often resulting in death. Of the eight cases reported in the current literature,

2.5  Management and Therapy Although good metabolic disease control is desirable, unlike Gaucher’s disease, a relationship between disease control and severe PAH in GSD I has not been established. There is only a single case report of a patient with GSD I  APAH being treated with vasodilating/antiremodeling agents. Uneo et  al. provided a report of a 17-year-old boy with GSD I and severe PAH (ePAP 92  mmHg) in 2009. Unfortunately, the patient (and family) refused invasive testing or therapy; thus, RHC was not performed and intravenous therapy was not an option. His initial therapy consisted of a combination of beraprost (a stable orally available prostacyclin analogue) in addition to diuretics, anticoagulation, and digoxin. After 6  months, the patient’s ePAP fell and his functional status ­significantly improved from New York Hear Association (NYHA) class IV to III. Sadly, 1 year later (18 months after initiation of therapy) the patient again worsened despite an escalating beraprost dose. He presented with NYHA class IV symptoms and exertional syncope, and was switched from beraprost to sildenafil (an oral phosphodiesterase type 5 inhibitor) therapy. At the time of the publication of their case report, the patient was alive with NYHA class III symptoms and stable hemodynamics after 2 years of sildenafil therapy [6].

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This is an atypical case, and it is always hazardous to generalize one patient’s experience. In IPAH, the response to sildenafil monotherapy may not be maintained over time; therefore, one may speculate that it may have more efficacy in patients with PAH associated with GSD I. Warfarin should be used cautiously in patients with GSD I since they frequently have an elevated bleeding time. Despite this theoretical concern, all seven patients reported in the literature were anticoagulated and none had reported bleeding complications.

2.6  Summary of Key Concepts 1. Severe progressive PAH occurring in association with GSD I is exceedingly rare, with a total of eight cases reported in the literature to date. 2. Mild asymptomatic elevations in pulmonary artery pressure may be seen more frequently in GSD I, but this is of unclear significance. 3. The pathophysiologic processes of severe PAH in GSD I are not well established and it is difficult to implicate glycogen accumulation, or the predictable metabolic derangements that accompany the disease, to pulmonary vascular remodeling. 4. Although elevated circulating levels of serotonin may contribute to the disease, levels appear elevated in many patients with GSD I, complicating the association. 5. Pathologically, all cases demonstrated changes indistinguishable from IPAH, with no unique features attributable to GSD I. 6. Despite a lack of clinical experience, given its underlying similarities to IPAH, it is our opinion that severe progressive pulmonary hypertension associated with GSD I should be treated aggressively in a manner similar to IPAH with vasodilating/antiremodeling agents and close hemodynamic and functional monitoring. 7. The risk-benefit ratio of warfarin in these individuals must be weighed carefully given their increased bleeding risk.

3  PAH in Gaucher’s Disease Type I 3.1  Background and Introduction Gaucher’s disease is the most common lysosomal storage disorder in humans [7]. Gaucher’s disease type I (representing 90% of phenotypes) is an autosomal recessive disorder, most common in the Ashkenazi Jewish population, where it affects approximately one in 800 individuals, with a carrier frequency of approximately 7% [8]. Patients with Gaucher’s disease suffer from a deficiency of acid b-glucosidase, causing

J.W. Landsberg and J. Mandel

glucosylceramide-laden lysosomes to accumulate in macrophages known as Gaucher cells. Gaucher cells cause disease by accumulating in multiple organs (e.g. liver, spleen, and bone marrow). In addition to pathologic organ infiltration, these macrophages are persistently activated, leading to elaboration of mediators capable of causing or propagating disease. The natural history of the condition is extremely variable, ranging from death in infancy to asymptomatic indolent disease incidentally discovered in the elderly. Interestingly, phenotypic expression cannot be predicted by genotype, and severity may vary between identical twins, pointing to the importance of nonheritable factors [9]. The treatment of Gaucher’s disease type I has been revolutionized by the availability of specific enzyme replacement therapy (ERT) in 1991, involving glucocerebrosidase purified from human placentas. In 1994, recombinant human glucocerebrosidase became available, replacing the human derived product [10]. ERT is capable of reversing or preventing the progression of most disease manifestations, although lung disease may require higher dosing because of poor lung penetration [11]. Although over half of patients with Gaucher’s disease type I have abnormalities in pulmonary function [reduced carbon monoxide diffusing capacity (DLCO) in 45%, reduced forced expiratory volume in 1 s in 36%, and reduced total lung capacity in 22%] [12], severe symptomatic pulmonary involvement is uncommon, affecting less than 1% of patients [13]. Pulmonary disease varies depending upon the site of macrophage accumulation, but it most commonly involves the alveolar septum and interstitium in a lymphatic distribution, with subsequent restrictive lung disease. Less common but reported pulmonary manifestations include submucosal infiltration and bronchoconstriction, alveolar filling and chronic pneumonia, and even direct pulmonary alveolar capillary occlusion by Gaucher cells, causing pulmonary hypertension [14].

3.2  T  he Prevalence of Gaucher’s Disease APAH The prevalence of Gaucher’s disease APAH (GAPAH) varies widely depending upon the population of patients studied, the severity of their disease, their experience and compliance with ERT, and whether or not they have undergone splenectomy. Using echocardiography, Mistry et  al. screened 134 consecutive patients evaluated at their referral center with Gaucher’s disease type I for pulmonary hypertension. They stratified their patients by estimated pulmonary artery systolic pressure (ePASP) into mild disease (ePASP greater than 35 mmHg but less than 50 mmHg) or severe disease (ePASP

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greater than 50 mmHg). In patients without a history of ERT, 12 of 40 patients (30%) had mild PAH. Only one of 134 patients (0.76%) had severe PAH on presentation. Pulmonary hypertension was far less common in Gaucher’s disease patients who had undergone ERT; seven of 94 (7.4%) had mild PAH and none had severe PAH. In an attempt to better understand the predisposing factors to severe pulmonary hypertension in Gaucher’s disease type I, Mistry et al. analyzed a tertiary referral cohort of nine patients with Gaucher’s disease and ePASP greater than 50 mmHg. All nine patients had a history of splenectomy for cytopenias or splenic congestion, compared with 31% of patients with mild or no pulmonary hypertension. Five of these patients (55%) were diagnosed with PAH after splenectomy but before ERT. The remaining 45% were found to have PAH while receiving ERT. Importantly, none had been prescreened for the PAH prior to therapy, and all had significant histories of noncompliance. Other risk factors for severe GAPAH included female sex (2:1), a family history of PAH (22%), and specific mutations in the acid ß-glucosidase gene and in angiotensin-converting enzyme I polymorphisms.

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Gaucher’s disease, who at autopsy was found to have his pulmonary alveolar capillary bed nearly completely occluded with Gaucher cells (Fig. 2), in addition to pathologic findings typical of IPAH, including plexigenic arteriopathy (Fig. 3). Similarly, Ross et al. demonstrated the presence of Gaucher cells from pulmonary capillary blood aspirated through a Swan–Ganz catheter in the wedge position during RHC [18]. Mistry et  al. report the presence of intravascular Gaucher cells, as well as Gaucher cells lining pulmonary capillaries, leading to luminal narrowing [19]. Despite this, several subsequent autopsy reports in patients with Gaucher’s disease and severe pulmonary hypertension have shown pathological small vessel remodeling indistinguishable from that in IPAH. These cases are confounded by variable rates of liver disease (raising the possibility of portopulmonary hypertension) and splenectomy (an accepted risk factor for PAH) [15].

3.3  Pathophysiology Gaucher’s disease type I confers a strong, multifactorial predisposition to mild/moderate pulmonary hypertension, by both its ability to cause parenchymal lung and liver disease as well direct effects on the pulmonary vasculature. Patients with cirrhosis (Gaucher’s disease associated or otherwise) may develop intrapulmonary arteriovenous malformations (AVMs) with right-to-left shunting and difficult-to-correct hypoxemia [15]. Less commonly, they may develop portopulmonary hypertension where pathological examination of the small pulmonary arteries and arterioles reveals findings is indistinguishable from those in IPAH [16]. Although mild, asymptomatic pulmonary involvement may be common in Gaucher’s disease (17% with abnormal chest X-rays, 68% with abnormal findings in pulmonary function tests), end-stage parenchymal lung disease capable of causing pulmonary hypertension by destruction and fibrosis of the vascular bed (as well as hypoxemia) occurs in less than 1% of cases [13]. In the absence of liver or parenchymal lung disease, Gaucher’s disease type I is capable of causing pulmonary vascular disease by three distinct mechanisms: (1) pulmonary capillary obstruction with Gaucher cells, (2) pulmonary arteriolar luminal narrowing from Gaucher cell accumulation in the vascular wall, and (3) pathologic changes indistinguishable from those in IPAH (without obvious Gaucher cell vascular involvement). An early report by Roberts and Frederickson [17] detailed the case of 30-year-old attorney with a family history of

Fig.  2  Pulmonary alveolar capillaries occluded by Gaucher cells (arrows). (Reproduced from [17] with permission)

Fig. 3  (a) The muscular artery and its branching arteriole shows extensive intimal proliferation. Numerous large thin-walled dilated vessels surround the obstructed arteriole. (b) Classic plexiform lesion arising from a muscular artery, which has hypertrophied media and intimal fibrous proliferation at the site of origin of the plexiform lesion. (Reproduced from [17] with permission)

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Interestingly, severe PAH has been reported in patients undergoing therapy with human-placenta-derived glucocerebrosidase, raising the concern for a “drug-induced” pulmonary vascular remodeling from contaminants in the preparation [20]. Data from Mistry et al. point to the overwhelmingly beneficial effects of ERT with regard to pulmonary hypertension and overall disease progression, showing that ERT decreased the incidence of mild pulmonary hypertension by 24% [19]. Additionally, recombinant human glucocerebrosidase has replaced placenta-derived enzyme, putting to rest many of the concerns regarding the older preparation. The association between splenectomy and severe pulmonary hypertension in Gaucher’s disease deserves special mention. Mistry et al. and Pastor et al. both report a 100% incidence of splenectomy preceding the diagnosis of severe pulmonary hypertension in their cohorts. In contrast, only 31% of patients with mild PAH had a preceding history of splenectomy. Thus, splenectomy appears necessary but not sufficient to cause severe PAH in this condition. The precise mechanism by which splenectomy predisposes to pulmonary hypertension is not known, and a detailed discussion of splenectomy and PAH can be found elsewhere in this chapter.

3.4  Clinical Presentation and Evaluation Symptomatic pulmonary vascular disease at the time of the initial diagnosis of Gaucher’s disease is rare. In the preechocardiography era, the diagnosis of pulmonary hypertension would often be made pathologically at autopsy. Severe pulmonary hypertension occurs in patients with severe Gaucher’s disease who have required splenectomy, and who have not been treated or are noncompliant with ERT. In such individuals, pulmonary hypertension is a well-recognized cause of premature death [20]. Like for patients with IPAH, sudden life-threatening deterioration can occur. PAH initially manifests itself as exercise intolerance, as the pulmonary vasculature fails to passively distend and recruit to accommodate the increased blood flow with exertion. As PAH progresses, symptoms of right ventricular failure and fluid overload, such as ascites and edema, predominate, progressing to exercise-induced syncope and ultimately death from progressive right ventricular failure.

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magnitude of PAH. Elstein et  al. found that the pulmonary artery pressure measured at RHC tended to be 5–10  mmHg higher than that estimated by echocardiography [21]. Therefore, patients with echocardiographic evidence of pulmonary hypertension should have a confirmatory RHC allowing for measurement of pulmonary artery pressure, pulmonary artery occlusion pressure (PAOP; to exclude occult left ventricular dysfunction), and accurate calculation of cardiac output and pulmonary vascular resistance. Patients in whom PAH is suspected clinically but whose resting echocardiogram is normal are generally evaluated with a stress echocardiogram or a RHC with exercise provocation to rule out significant PAH during exercise. Although this recommendation is true for all suspected pulmonary vascular disease, it may be specifically important in screening patients with Gaucher’s disease. Sirrs et al. reported of two patients with Gaucher’s disease type I in whom the resting echocardiogram was normal yet provocative testing revealed significant PAH. Interestingly, one of these patients had a lung biopsy demonstrating widespread occlusion of the pulmonary arterioles with Gaucher cells [22]. Given the possibility (albeit rare) of Gaucher’s disease causing interstitial lung disease, pulmonary function testing in conjunction with a computed tomography scan of the chest should be considered in all patients with suspected PAH to establish the degree of parenchymal lung disease. Although symptomatic pulmonary disease is rare, Kerem et al. showed that the majority of Gaucher’s disease patients (68%) have pulmonary function abnormalities; most commonly a decreased DLCO in 42%, followed by a decreased total lung capacity in 22% [12]. The degree to which pulmonary hypertension is attributable to parenchymal lung disease may impact therapeutic decisions, as discussed below. Additionally, Gaucher’s disease patients with PAH should be screened for hypoxemia and pulmonary AVMs, since these can commonly occur with liver disease, which may be occult. By comparing biomarkers to echocardiographic findings, Elstein et  al. examined the utility of N-terminal pro-brain natriuretic peptide and C-reactive protein in predicting the presence of pulmonary hypertension in Gaucher’s disease type I. Although their data did not reach statistical significance (both tests lack specificity), C-reactive protein appeared to have better predictive value then brain natriuretic peptide, possibly implicating inflammation in the pathophysiologic process of PAH in these patients [23]. Shitri et al. found no correlation between echocardiographic evidence of PAH and D-dimer levels in patients with Gaucher’s disease [24].

3.5  Diagnostic Testing Specialty centers for Gaucher’s disease recommend screening echocardiography, chest X-ray imaging, and electrocardiography evaluation in all patients with type I disease. As in other situations, echocardiography can underestimate or overestimate the

3.6  Management Specific ERT is the mainstay of Gaucher’s disease management, having significantly reduced both the morbidity

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and the mortality of the disease [11]. Where controversy once existed (and may never be settled with regard to human-placenta-derived products), it is now generally accepted that recombinant ERT prevents and improves pulmonary hypertension, rather than causing it [19]. Because of its ability to prevent splenectomy (the largest modifiable risk factor for severe PAH in Gaucher’s disease) and its ability to reduce pulmonary artery pressure after splenectomy, ERT should be the first modality used in patients with Gaucher’s disease and PAH. Patients who fail to respond to standard dosing may receive a trial of highdose ERT, based on several case reports of success with this strategy. There is at least one case of pulmonary hypertension developing during ERT in a patient with AVMs from hepatopulmonary syndrome. The authors hypothesized that the closure of pulmonary AVMs and the subsequent decrease in right-to-left shunting may have unmasked coexisting small vessel pulmonary vascular disease [15]. For this reason, patients undergoing ERT should be closely monitored. If pulmonary hypertension develops or fails to improve, additional therapy with antiremodeling and vasodilating agents should be implemented.

3.7  Antiremodeling and Vasodilating Agents The literature regarding treatment of GAPAH with antiremodeling and vasodilating agents is largely composed of case reports. Mistry et al. reported the only series available, in which they followed nine patients with severe pulmonary hypertension (ePAP ³ 50  mmHg), on treatment, over time (mean 4.6 years). All patients were receiving ERT: 67% were treated with intravenously administered epoprostenol and 33% were additionally anticoagulated with warfarin. Patients treated with enzyme replacement and intravenously administered epoprostenol (with or without warfarin) demonstrated a significant reduction in ePAP from 92 to 56  mmHg. Importantly, the two patients who were treated with ERT alone also had dramatic reductions in their ePAP over time, from 77 to 15 mmHg and from 70 to 45 mmHg, respectively. The latter patient was on therapy at the time of presentation and responded to higher-dose ERT alone. On treatment, all patients demonstrated an improvement in their NYHA functional class. Two patients were removed from the lung transplant waiting list. Interestingly, the authors noted significant individual variation with regard to response time to therapy. Some patients experienced rapid reductions in ePAP, whereas others required up to 2 years of therapy before significant reduction in ePAP was achieved. This may reflect the heterogeneous pathophysiologic processes underlying pulmonary hypertension in these patients. Perhaps individuals with Gaucher cell pulmonary vascular obstruction improve

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r­ apidly, whereas patients with IPAH-like pulmonary vascular remodeling take longer to improve. Fernandez et al. reported a patient with severe GAPAH treated successfully over a 9-month follow-up period with sildenafil. The patient’s systolic pulmonary artery pressure, measured by RHC, fell from 115 to 78  mmHg, she resolved her oxygen requirement, and had significant improvement in her 6-min walk distance [25]. Similarly, Leuchte et al. reported a patient with GAPAH treated successfully over a 9-month follow-up period with inhaled iloprost [26]. The patient’s systolic pulmonary artery pressure fell from 61 to 42  mmHg, and her 6-min walk distance improved significantly.

3.8  Anticoagulation The role of anticoagulation has not been well studied in patients with GAPAH. However, most GAPAH patients have undergone splenectomy. Splenectomy itself increases the risk of thrombosis and PAH; therefore, splenectomized patients with severe GAPAH may benefit from anticoagulation. Untreated Gaucher’s disease patients with an intact spleen may have a bleeding diathesis from thrombocytopenia and other acquired bleeding risks [27]. Thus, warfarin should be reserved for splenectomized patients with severe disease.

3.9  Summary of Key Concepts 1. Gaucher’s disease type I confers a remarkable multifactorial predisposition to mild-moderate PAH, affecting up to 30% of untreated patients (often without symptoms). 2. Severe progressive PAH, mandating specific therapy and consideration of transplantation, occurs rarely (less than 1%), typically in association with additional PAH risk factors such as splenectomy, female sex, and a family a history of PAH. 3. The pathophysiologic processes of PAH in Gaucher’s disease involve both pulmonary vascular accumulation of Gaucher cells in addition to small vessel remodeling by an unknown mechanism. 4. Gaucher’s disease patients with suspected PAH, in whom the resting echocardiogram is normal, should undergo exercise assessments to rule out dramatic elevations in pulmonary artery pressure that are only evident during exercise. 5. Pulmonary hypertension that appears or worsens during ERT should not be attributed to the enzyme therapy itself.

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The enzyme dose may need to be increased, and vasodilator/antiremodeling agents may need to be added. 6. The primary way to prevent severe pulmonary hypertension in Gaucher’s disease is by avoiding splenectomy, a therapy hopefully relegated to the pre-ERT era. 7. When treated with ERT (with or without vasodilator/antiremodeling agents), PAH occurring with Gaucher’s disease may completely resolve, even when severe.

4  PAH in the Splenectomized 4.1  Background The association between splenectomy and PAH was initially observed in adult patients with hereditary hemolytic disorders (e.g. thalassemia and hereditary stomatocytosis) who underwent splenectomy in childhood to decrease transfusion requirements. Case series looking at such patients who developed right-sided heart failure nearly uniformly report a history of splenectomy [28]. Subsequently, splenectomy has been associated with both chronic thromboembolic pulmonary hypertension (CTEPH) and IPAH. Still more intriguing, pathologic specimens of patients with splenectomy and PAH suggest a unique vasculopathy, combing features of the classic arteriopathy seen in IPAH with abundant small vessel thrombosis, suggesting a possible overlap syndrome.

4.2  T  he Prevalence of Splenectomy-Induced PAH Hoeper et al. found that a history of splenectomy was significantly more common in patients undergoing lung transplantation for IPAH (11%) than other causes of end-stage lung disease (0%). Importantly, 43% of the splenectomies in their series were traumatic (and thus not associated with hemolytic conditions) [29]. More recently, Phrommintikul et  al. performed screening echocardiography on 68 asymptomatic patients with thalassemia and found that just under half had PAH, 75% of whom had a history of splenectomy. In their multivariate analysis, splenectomy was the only factor significantly related to PAH [30]. Interestingly, Bonderman et al. in two large retrospective series, found splenectomy to be a significant independent risk factor for the development of CTEPH [31, 32]. To further explore the association between PAH and splenectomy, Jais et al. examined the prevalence of splenectomy in patients with CTEPH and IPAH versus patients with nonvas-

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cular pulmonary disease. Importantly, they found ­significant differences in the prevalence of prior splenectomy in patients with CTEPH (8.6%) and IPAH (2.5%) compared with patients with nonvascular pulmonary disease (0.6%) [33]. As discussed earlier, the prevalence of splenectomy in patients with GAPAH approaches 100%. It is worth noting that Schwartz et al. found no increase in the incidence of pulmonary hypertension (regardless of cause) in patients who underwent splenectomy for idiopathic thrombocytopenic purpura. However their median length of follow-up was only 7.5 years. Hoeper et al. showed that the median length of time between splenectomy and the diagnosis of PAH was 25 years, and one patient in this series had undergone splenectomy for idiopathic thrombocytopenic purpura [29].

4.3  Pathophysiology Loss of the splenic filter leads to an increased number of circulating platelets as well as damaged red blood cells, which together increase the risk of thrombosis in all vascular beds [34]. Much of these data comes from patients with hereditary hemolytic syndromes, in whom abnormal red cell morphology, hemolysis, and free hemoglobin likely pose an additional vascular risk, by scavenging nitric oxide and releasing arginase, which reduces nitric oxide production [35]. These systemic changes may preferentially affect the pulmonary vascular, which, after splenectomy, is vulnerable to accumulation of damaged red cells and red cell fragments, capable of trapping and activating platelets, promoting in situ thrombosis, and pulmonary vascular remodeling [29]. Overall, most splenectomized patients do not develop pulmonary hypertension, arguing for an intrinsic pulmonary vascular vulnerability in those who do. Unlike most risk factors for severe pulmonary hypertension, splenectomy increases the risk of both CTEPH and IPAH. These classically distinct disease entities are united by the common feature of pulmonary arterial thrombotic obstruction. In CTEPH this occurs in the large vessels (presumably embolic in origin), whereas in IPAH thrombosis occurs in small to medium-sized vessels (presumably occurring in situ). Surgical lung biopsy and autopsy findings in patients with splenectomy-induced PAH (SIPAH) reveal a unique vasculopathy with intimal thickening, medial hypertrophy, and plexiform lesions, typical of IPAH, in the setting of abundant thrombotic lesions affecting small and medium-sized vessels, not typically seen in IPAH [29, 35] (Fig. 4). This microthrombotic vasculopathy is strongly associated with splenectomy. Sumiyoshi et  al. found evidence of abundant small vessel thrombosis in 57% of thalassemia patients with a history of splenectomy compared with 16% in those with an intact spleen [36].

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Fig. 4  (a) Prominent medial hypertrophy with intimal fibrosis resulted in total luminal occlusion of this pulmonary artery. (b) Pulmonary hypertension, grade IV. In addition to medial hypertrophy and intimal fibrosis, numerous endothelial-lined vascular channels with focal fibrinoid necrosis were noted. (c) Severe intimal fibrosis, muscular hyper-

trophy with focal recanalization of the lumen, and fibrin thrombi were noted. (d) Plexiform lesion of this pulmonary artery was characterized by aneurysmal expansion of the vascular wall with fibrin thrombi occluding the lumen. (From [77]. Reprinted courtesy of the International Journal of Hematology)

It is possible that the pathologic setting of SIPAH represents a unique, overlap vasculopathy combining features of IPAHtype vascular remodeling with widespread small vessel thrombosis. This concept is supported by the observation that patients with splenectomy-induced CTEPH may behave differently from typical CTEPH patients. Lang and Kerr noted that patients with CTEPH and a history of splenectomy tended to have distal, surgically inaccessible disease [37]. In addition, the diagnosis of SIPAH typically occurs 15–20 years after the splenectomy.

As in all diseases that increase the risk of PAH, echocardiography remains the cornerstone of screening, whereas RHC remains the gold standard to confirm the diagnosis. Given the association between splenectomy and surgically amenable CTEPH, patients with suspected SIPAH require a radionuclide perfusion scan or angiography studies to rule out chronic (proximal) thrombus.

4.4  C  linical Presentation, Evaluation, and Diagnostic Testing The development of symptomatic pulmonary hypertension after splenectomy takes over a decade, with the median interval between splenectomy and the diagnosis of PAH ranging from 10 to 25 years depending upon the series. The risk is greatest in patients with Gaucher’s disease and hemolytic anemia, and thus higher rates of asymptomatic PAH also may be seen in these patients. Phrommintikul et al. showed that 43% of asymptomatic thalassemia patients had evidence of PAH on screening echocardiography, 75% of whom had a history of splenectomy. Given this, it is important to consider PAH as the cause of dyspnea on exertion or exercise limitation in patients with hemolytic anemia, especially those with a history of splenectomy (rather than simply attributing symptoms to the underlying anemia).

4.5  M  anagement and Therapy with Antiremodeling and Vasodilating Agents There are no data regarding the treatment of SIPAH, in fact most of these patients have probably been diagnosed as having either IPAH or CTEPH and treated according to standard algorithms. If one extrapolates data from IPAH and inoperable CTEPH patients, the 5-year survival of patients with severe SIPAH is likely less than 10%; thus, treatment should be attempted, and lung transplantation considered. Screening SIPAH patients for surgically amenable CTEPH should be the initial priority. If proximal surgical accessible disease is identified (and persists despite systemic anticoagulation), referral for pulmonary thromboendarterectomy (PTE) should be considered. If proximal thrombus is not identified, patients should be treated with antiremodeling/ vasodilator agents. Given the predominance of thrombosis in pathological specimens of patients with SIPAH, systemic anticoagulation seems prudent.

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4.6  Summary of Key Concepts 1. Splenectomy increases the risk of severe pulmonary hypertension by causing a unique small vessel thrombotic vasculopathy, which may manifest itself clinically as either IPAH or CTEPH. 2. This risk is greatest in patients with Gaucher’s disease and hereditary hemolytic disorders. 3. The time interval between splenectomy and the development of PAH is typically on the order of decades. 4. As in most disease states, the development of severe PAH dramatically shortens life such that therapy should be attempted. 5. There are no data regarding recurrence of PAH after lung transplantation in patients with SIPAH, but it is likely that this would appear in the literature as recurrent IPAH after transplantation (which has not yet been reported).

5  P  AH in Pulmonary Langerhans Cell Histiocytosis 5.1  Background Pulmonary Langerhans cell histiocytosis (PLCH) is a smokingrelated disease in which Langerhans cells infiltrate the pulmonary parenchyma, causing both fibrotic and cystic obstructive pathological changes. The disease may have extrapulmonary manifestations (bone, pituitary gland, skin, and lymph nodes) but isolated lung involvement is the rule, representing 85% of cases. The clinical course is variable, ranging from spontaneous remission to progression to endstage lung disease. Langerhans cells are CD1a- and s100expressing antigen-presenting cells, attracted and activated by components of cigarette smoke. PLCH is a disease which classically has been reported in Caucasian male smokers (current or former); however, this gender bias may reflect smoking trends, with recent studies suggesting a more balanced sex distribution. The most consistent epidemiologic risk factor for the disease remains cigarette smoking, with more than 90% of affected patients having a history of tobacco use [38]. Pathologically, Langerhans cells proliferate in the small airways, forming loose granuloma (with occasional eosinophilic involvement), ultimately coalescing into macroscopic stellate nodules that progress to fibrosis. Although Langerhans cells classically have been reported to infiltrate the pulmonary vasculature, causing a granulomatous inflammation and vasculitis, more recent pathology studies have shown significant degrees of IPAH-like pulmonary vascular remodeling without significant degrees of vasculitis or direct vascular

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infiltration of Langerhans cells. Additionally, mounting ­clinical evidence suggests that severe pulmonary hypertension may be both common in patients with PLCH and responsible for most of their dyspnea on exertion and exercise limitation, symptoms previously ascribed to end-stage parenchymal lung disease [39–41]. Interestingly, the vascular lesions seen in PLCH affect both the arterioles and venules, mimicking features of both IPAH and pulmonary venoocclusive disease (PVOD), significantly limiting potential treatment options.

5.2  The Prevalence of PLCH APAH The prevalence of severe pulmonary hypertension associated with PLCH appears exceedingly high, ranging from 88% in patients with varying disease severity [41], to as high as 100% in patients with end-stage PLCH [40]. This exists in stark contrast to the prevalence of severe pulmonary hypertension seen in association with other causes of end-stage lung disease. In the same report, Fartoukh et  al. reported severe pulmonary hypertension present in only approximately 30% of patients with end-stage lung disease secondary to chronic obstructive pulmonary disease or idiopathic pulmonary fibrosis. This dramatic difference in prevalence suggests that PLCH is capable of causing pulmonary vascular injury and remodeling by a direct mechanism, beyond simple parenchymal destruction.

5.3  Pathophysiology Although patients with severe PLCH are prone to hypoxemia and significantly abnormal respiratory mechanics, recent evidence suggests that the severe pulmonary ­hypertension seen nearly uniformly in these patients is not secondary to vascular loss in the setting of widespread parenchymal destruction. Instead, in patients with PLCH the results of pulmonary function and exercise physiology testing tend to be consistent with pulmonary vascular disease rather than ventilatory limitation (or oxygen desaturation). Crausman et al. reported the results of pulmonary function and exercise testing in 23 patients with moderate  PLCH. Despite relatively normal lung volumes and spirometry findings, the average DLCO was significantly reduced to 59% of that predicted. More importantly, exercise testing of the group demonstrated a reduced maximum exercise capacity (to 54% of that predicted) that was not associated with a ventilatory limitation. Instead, the group had abnormal physiologic dead space at rest, which failed to decrease significantly with exercise, implying pulmonary

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vascular disease [39, 42]. Similarly, Chawalit et al. found no correlation between pulmonary artery systolic pressure and the results of pulmonary function testing. Taken together, these data strongly suggest that PCLH causes pulmonary vascular disease independent of parenchymal disease, and that this vascular pathophysiologic condition may be the main cause of exercise limitation and dyspnea on exertion. Supporting this pulmonary function evidence, pathological analysis of patients with severe PLCH requiring lung transplantation shows three distinct pulmonary vascular lesions giving rise to a unique pulmonary vasculopathy that combines features of both IPAH and PVOD. Historically, case reports and small autopsy series have demonstrated foci of Langerhans cells causing areas of lymphocytic vasculitis with luminal obliteration, in addition to differing degrees of small vessel muscularization and remodeling similar to the changes seen in IPAH [43] (Fig.  5). More recently, Fartoukh et  al. presented autopsy data from 12 patients with PLCH and severe pulmonary hypertension referred to their center for lung transplantation over a 7-year period. Not surprisingly, the parenchymal disease revealed differing degrees of fibrosis mixed with Langerhans cell granulomatous changes; more specifically, 42% of patients had predominantly fibrotic lesions, 25% had predominantly Langerhans cell granulomatous changes, whereas the remaining 33% had a combination of both. Interestingly, the vascular disease showed a paucity of Langerhans cell granulomatous inflammation or vascular invasion. Only 25% of patients demonstrated any evidence of direct Langerhans cell granulomatous arteriolar inflammation (predominately mild), with only one patient showing severe granulomatous arteriolar inflammation. Instead, the dominant pathologic features resembled those seen in IPAH, with 66% of patients demonstrating muscularization of small and mediumsized pulmonary arteries, and 83% demonstrating intimal fibrosis, usually moderate to severe changes. Perhaps the most

surprising feature of these autopsy data is the severity and degree to which the small to medium-sized pulmonary veins were also affected in a pattern similar to that typically described in PVOD (Figs. 6, 7). In this series, 66% of patients demonstrated moderate to severe intimal fibrosis, causing complete obliteration of pulmonary veins in over half. Additionally, 33% showed severe medial hypertrophy and muscularization of small to medium-sized pulmonary veins. The authors also noted that nearly half of the time vascular disease occurred in regions of lung with unaffected parenchyma. Additionally, serial biopsies of six patients over time showed that 50% had progressive vasculopathy in the face of stable parenchymal disease. These later observations highlight the ability of PCLH to cause a unique pulmonary vasculopathy (separate from parenchymal destruction), possibly implying a role for cytokines and other locally produced growth factors. The granulomatous lesions seen in PLCH are capable of elaborating interleukins 1 and 6, platelet-derived growth factor (PDGF), and transforming growth factor b (TGF-b), all of which are capable of

Fig. 5  Extreme narrowing of a vascular lumen sustained by a transmural Langerhans cells and lymphocytic vasculitis. (From [43]. Reproduced with permission of the American College of Chest Physicians)

Fig. 7  Venular intimal fibrosis; partial occlusion in a lobular septum (From [40]. Official journal of the American Thoracic Society copyright American Thoracic Society)

Fig. 6  Intimal fibrosis of muscular pulmonary artery within Langerhans granuloma. (From [40]. Official journal of the American Thoracic Society copyright American Thoracic Society)

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promoting pulmonary vascular remodeling [40]. The substantial degree of pulmonary venous remodeling seen on autopsy in patients with PLCH may have a significant clinical implication with regard to presentation and therapy. Hamada et al. reported the case of a 29-year-old female with PLCH who died from ­progressive right-sided heart failure over a 7-year period, despite therapy with corticosteroids. At autopsy she had dramatic pulmonary venous involvement, indistinguishable from PVOD [44].

5.4  Clinical Presentation and Evaluation The two published series of patients with pulmonary hypertension and PLCH encompass a spectrum of disease severity. Fartoukh et al. (n = 21) presented data on a series of end-stage patients referred for lung transplantation, whereas Chaowalit et al. (n = 13) described patients with milder disease. In both series, pulmonary hypertension was found in nearly all patients, as discussed earlier. Interestingly, in Chaowalit et al.’s cohort, 53% were female, with a median age at the time of diagnosis with PLCH APAH of 48 years (approximately 10 years older than the median age of PLCH onset). Not surprisingly, all were smokers. Although the range varied considerably, the median time between the diagnosis of PLCH and the diagnosis of PAH was 4 years (ranging from 0 to 25 years). At the time of the diagnosis of PLCH APAH, the most common symptoms were dyspnea on exertion and cough (92%), followed by lower extremity edema (69%) and chest pain (38%). Disease was fairly advanced at diagnosis, with 46% of patients suffering from NYHA class III symptoms, and the rest split between class IV (23%) and class II (23%). Just over half of the cohort had quit smoking after the diagnosis of PLCH (several years before the onset of PAH). Sixty-two percent had been treated with steroids during the course of their disease. After approximately 4.5 years of follow-up, nearly 40% of the cohort had died. The median survival after the diagnosis of PLCH APAH was only 7.6 months. Thus, the diagnosis of PAH in patients with PLCH dramatically increases mortality [41].

5.5  Diagnostic Testing Given the near uniform involvement of the pulmonary vasculature in advanced PLCH, echocardiographic screening at the time of diagnosis is prudent. In particular, patients with exercise limitation, dyspnea on exertion, or DLCO abnormalities should undergo echocardiographic evaluation. All patients in whom PAH is suspected by echocardiography should proceed to confirmatory RHC. Radionuclide ­ventilation–perfusion imaging is warranted in all cases of significant PAH to rule out

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coincidental thromboembolic pulmonary hypertension. There are few data regarding variation in PAOP obtained at different sites, as can be seen in patients with PVOD. It is important to remember that pulmonary vascular disease can progress independent of parenchymal disease; therefore, clinicians must have a low threshold to employ serial echocardiographic evaluations regardless of imaging and or PFT stability.

5.6  M  anagement; Corticosteroids, Antiremodeling, and Vasodilator Agents The mainstay of management of PLCH is smoking cessation; unfortunately, more than half of patients have ceased smoking (to no avail) at the time of their diagnosis of PLCH APAH. Similarly, corticosteroid therapy (despite clear evidence of a benefit) is often attempted in symptomatic patients in conjunction with or after smoking cessation. Benyounes et  al. reported the encouraging case of a 31-year-old male smoker initially diagnosed with PLCH, who was then lost to follow-up for 10 years before representing himself with PLCH APAH. His pulmonary function testing on reevaluation showed a severe obstructive defect with a severely reduced diffusing capacity. RHC confirmed PAH, demonstrating a pulmonary artery systolic pressure of 55 mmHg in the setting of a normal PAOP. The patient then underwent surgical lung biopsy which showed active Langerhans cell granulomatous inflammation, interstitial fibrosis, and pulmonary arterial vascular remodeling. Importantly, there was no evidence for direct arteriolar obliteration from granulomatous involvement. The patient stopped smoking and was started on prednisone therapy (1 mg/kg/day for 1 month followed by 0.5 mg/kg/day over 6 months). Within 4 weeks the patient had marked subjective improvement. After 9 months, the patient had improved imaging (partial clearing of groundglass opacification on CT) with stable pulmonary function test results. More importantly, repeat RHC showed a dramatic reduction in pulmonary artery pressures to near normal (pulmonary artery systolic pressure 38  mmHg, mean pulmonary artery pressure 26 mmHg) [45]. This case highlights two important points. First, symptoms, functional limitations, and pulmonary hypertension improved with therapy despite stable pulmonary function test results, again demonstrating the disconnect between parenchymal lung disease, PAH, and exercise limitation in patients with PLCH APAH. Second, corticosteroids apparently improved pulmonary vascular disease (despite a lack of direct granulomatous vascular involvement on pathological inspection), implying a beneficial effect of corticosteroids in PLCHassociated pulmonary vascular remodeling. Unfortunately, over 50% of PLCH patients report a history of corticosteroid therapy at the time of their diagnosis of PAH, suggesting this

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approach will have significant limitations. There are no data regarding retreating these patients with corticosteroids at the time of their PAH diagnosis, but this may be an option given the PVOD-like changes observed in these patients and its implications for vasodilator treatment. To move this concern from the theoretical realm to the practical, Fartoukh, Humbert, and Simonneau (unpublished data) reported two patients with PLCH APAH who experienced acute pulmonary edema with the initiation of intravenous epoprostenol therapy in a manner similar to that reported in patients with PVOD. Although lung biopsy with an assessment of venous involvement would identify patients with a predominately PVOD-like disease, the surgical risk in patients with severe pulmonary hypertension may be prohibitive. Given the complicating nature of pulmonary venous involvement, lung transplantation remains an important therapeutic option for patients with severe PLCH APAH (although a 20% rate of recurrence has been reported in patients with extrapulmonary manifestations at the time of their transplant) [46]. If the clinical scenario demands consideration for therapy with vasodilator/antiremodeling agents, close observation on initiation is wise, in addition to applying treatment algorithms used for PVOD instead of IPAH.

5.7  Summary of Key Concepts 1. Significant PAH is a nearly uniform complication of severe PLCH. 2. PLCH APAH is caused by a unique vasculopathy involving prominent arterial and venous vascular changes, which occur independently of parenchymal involvement. 3. Because of this asynchrony, pulmonary vascular disease may worsen independently of parenchymal disease activity; thus, diligent pulmonary vascular screening in required. 4. The development of PLCH APAH worsens prognosis and increases mortality. 5. Given the PVOD-like behavior of patients with PLCH APAH, lung transplantation must be strongly considered, and intravenously administered prostacyclin used with extreme caution.

6  PAH in Sarcoidosis 6.1  Background Sarcoidosis is a multisystem granulomatous disease which affects the lungs in 90% of cases [47]. The severity of pulmonary disease ranges widely from benign, self-limited, asymptomatic hilar adenopathy (stage I), to progressively fatal end-stage fibrotic lung disease with cor pulmonale (stage IV).

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Although sarcoid-associated pulmonary hypertension (SAPH) may be the obvious sequela of end-stage lung disease, secondary to some combination of parenchymal vascular destruction, hypoxia, and vascular remodeling, granulomatous inflammation itself is capable of causing both small and large vessel pulmonary vascular disease. Although less common, this type of pulmonary vascular involvement may occur in the absence of significant pulmonary fibrosis. Given the array of medications now available to treat small-vessel-mediated PAH, there is a new emphasis on characterizing and treating the pulmonary hypertension that may complicate sarcoidosis.

6.2  The Prevalence of SAPH Establishing the prevalence of SAPH is difficult given the heterogeneous nature of the patients studied (i.e. stage IV vs. stage I disease), as well as differing definitions and modalities used to establish the presence of pulmonary hypertension (i.e. resting echocardiography values vs. autopsy results suggesting cor pulmonale). Despite these limitations, when reviewing the available data, several patterns emerge. Looking at all patents with the diagnosis of sarcoidosis (combining historical autopsy data with echocardiographic screening data on Japanese outpatients with mild pulmonary disease), the prevalence of pulmonary hypertension is likely close to 5%. In the Japanese outpatient cohort, pulmonary restriction was the (only) independent risk factor for SAPH [48]. These data exists in stark contrast to RHC data obtained from patients awaiting lung transplantation for end-stage sarcoidosis. In these cohorts the prevalence of pulmonary hypertension may be as high as 80% [49]. Although the severity of SAPH typically mirrors the severity of pulmonary restriction, a subset of patients may have SAPH despite only mild to moderate restrictive disease. In patients with sarcoidosis and dyspnea out of proportion to the results of pulmonary function testing, the prevalence of SAPH may be as high as 50% [50]. Additionally, a significant subset of sarcoidosis patients with mild to moderate interstitial disease have normal resting pulmonary artery pressures, which rise dramatically during exercise [51]. Epidemiologic data on SAPH reflect the overall demographics of sarcoidosis in the population studied. In the USA, SAPH is more common in African-Americans and women, with an average age at diagnosis of 50 years of age.

6.3  Pathophysiology Pulmonary hypertension occurring in end-stage interstitial lung disease (regardless of its cause) is presumed to occur by

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Fig. 8  Compression of central pulmonary vascular structures by bulky lymph nodes. Coronal section of thorax of a postmortem specimen from a patient who died from end-stage sarcoid-associated pulmonary hypertension. There is extensive hilar compression of the main pulmonary arteries. The arrow denotes the left lower lobe pulmonary artery, which is subtotally occluded by extrinsic compression of calcified lymph nodes that are present throughout the mediastinum. (Reprinted from [65] with permission from Elsevier)

fibrotic destruction of the distal pulmonary vasculature. This capillary loss leads to a reduction in the overall cross-sectional area of the pulmonary vascular bed, causing a dramatic rise in vascular resistance. However, up to 30% of patients with SAPH have no radiographic evidence of fibrosis. Additionally, up to 50% of SAPH patients who have evidence of parenchymal involvement have pulmonary hypertension out of proportion to the degree of their parenchymal lung disease. These points highlight that sarcoidosis, distinct from other interstitial lung diseases and because of its predilection for lymphatic and bronchovascular bundle involvement, is uniquely able to cause both large and small vessel pulmonary vascular disease. Although up to 21% of patients with SAPH may have radiographic evidence of main or segmental pulmonary artery compression [52], significant pulmonary hypertension attributable to large pulmonary artery obstruction appears to be much less common. It has been reported to occur both from extrinsic compression by extensive bulky calcified hilar adenopathy as well as from granulomatous inflammation and stenosis of the main pulmonary arteries [53–55] (Fig. 8). In contrast, autopsy series suggest the dominant pathologic condition involves granulomatous and giant cell inflammation, affecting small to medium sized arteries, leading to obliteration of the lumen (Fig. 9). This pattern occurs commonly, between 70% and 100% of the time [56, 57]. Patients with SAPH have also been found to have plexiform lesions and nongranulomatous vascular changes associated with IPAH [52, 58].

Fig. 9  Granulomatous arteritis in sarcoidosis. (a) Granulomas obliterating the internal and external elastic laminae and smooth muscle (arrow) of medium-size artery. There is secondary intimal fibroplasias seen in the center of the artery. (b) In some cases, only giant cells and mononuclear cell infiltrates may be seen in sarcoidosis-associated arteritis. The arrowhead indicates the vascular lumen. Numerous giant cells may be seen in the vessel wall. (Reprinted from Diaz-Guzman et al. [65] with permission from Elsevier)

The incidence of pulmonary arterial vasoreactivity is much higher in SAPH than in IPAH. Although the numbers are small, Preston et al. showed that seven of eight patients (87%) demonstrated a 20% or greater reduction in their pulmonary vascular resistance with inhaled nitric oxide [59]. These were a typical group of SAPH patients, all having had abnormal pulmonary function test findings, with the majority having stage IV sarcoidosis. This percentage of vasoreactivity is far greater than that seen in IPAH or pulmonary hypertension associated with end-stage lung disease. When extensive, this type of small and medium vessel granulomatous angiitis is capable of causing a clinical syndrome of small vessel PAH, which unlike IPAH has prominent features of vasoconstriction and inflammation. These features may have important treatment implications. A significant percentage of patients with SAPH have normal resting pulmonary artery pressures that rise ­dramatically with

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exercise. Many of these patients have only mild restrictive disease. The incidence of exercise-induced SAPH depends upon the population studied, but ranges from 16% in unselected cohorts [60], to as high as 75% in patients with unexplained exercise limitation [61]. It remains unclear whether these patients have a unique pathophysiologic profile, rather than simply representing an earlier presentation. It is also unclear how (if at all) vasoreactivity relates to exercise-induced SAPH. The level of endothelin 1 (ET-1), a potent pulmonary vasoconstrictor and pulmonary artery smooth muscle cell mitogen, is elevated in the plasma, urine, and bronchoalveolar lavage (BAL) fluid of patients with sarcoidosis. A study of BAL fluid analysis in 22 subjects with sarcoidosis demonstrated that nearly 25% had elevated levels of ET-1. However, this finding is not specific, since the same authors found BAL fluid ET-1 levels elevated in patients with other interstitial lung diseases, specifically systemic sclerosis and usual interstitial pneumonia [62]. Immunohistochemical staining suggests that alveolar macrophages may be a source for increased ET-1 production in these patients. Interestingly, there is a small series showing that ET-1 levels fall in patients treated with corticosteroids who experience disease remission [63]. Although causality is difficult to establish, ET-1 may link the inflammatory interstitial lung disease of sarcoidosis with pulmonary hypertension and vascular remodeling, beyond granulomatous angiitis. It should also be noted that patients with sarcoidosis are extremely vulnerable to left ventricular diastolic dysfunction, especially during exercise, given potential cardiac involvement and exercise-induced hypoxemia. Unrecognized rightto-left shunting via a patent foramen ovale, which may only be present transiently with exercise-induced elevations in right-sided pressures, may further complicate the situation. It is imperative that an elevated PAOP and right-to-left shunting be excluded at rest and exercise before diagnosing SAPH.

6.4  Clinical Presentation and Evaluation SAPH should be considered in any patients with pulmonary sarcoidosis and exercise limitation, exercise-induced syncope, right-sided heart failure, or known pulmonary hypertension out of proportion to the severity of their parenchymal lung disease. The most common presenting symptoms of SAPH are nonspecific and include progressive dyspnea on exertion, chest pain, cough, edema, and exertional syncope. Similarly, the most common physical findings are signs of right-sided heart failure which lack sensitivity. In a retrospective analysis of 106 SAPH patients who had echocardiography because of suggestive symptoms, 79% lacked physical findings of right-sided heart failure [64]. Although there are case reports of pulmonary sarcoidosis causing a predominantly pulmonary venous

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vasculopathy mimicking PVOD, this is rarely a relevant clinical entity. The diagnosis of pulmonary venous involvement should be made clinically (i.e. pulmonary edema in response to pulmonary vasodilators) and not based on granuloma distribution on lung biopsy. It should be emphasized that the vast majority of patients who develop pulmonary edema in response to vasodilator therapy with will have left ventricular diastolic dysfunction as the cause and not a PVOD variety of SAPH. These patients experience pulmonary edema  when left ventricular end-diastolic volume increases in response to decreased pulmonary vascular tone, which had been appropriately elevated to prevent overfilling of an already failing left ventricle. Given that there may be an association between venous thromboembolism and sarcoidosis, the clinician must always consider CTEPH when evaluating a patient for SAPH. Although precise prevalence data do not exist, we recommend a radionuclide ventilation–perfusion scan as part of the evaluation. Patients with multiple unmatched segmental defects, and no CT evidence of segmental pulmonary artery compression from lymphadenopathy, should be considered for pulmonary angiography to rule out chronic thromboembolism.

6.5  Diagnostic Testing Many tests such as echocardiography, chest CT scanning, and pulmonary function testing have the ability to suggest the presence of pulmonary hypertension, but none has the requisite sensitivity to exclude the disease. Echocardiography may estimate pulmonary artery pressure if a tricuspid regurgitation jet is visible. Alternatively it may show right ventricular hypertrophy or a decreased right ventricular ejection fraction. Chest CT scanning may reveal minimal parenchymal lung disease, out of proportion to a patient’s exercise limitations or diffusing capacity. Chest CT scanning may also show right ventricular hypertrophy or main pulmonary artery compression by bulky hilar adenopathy. Pulmonary function testing may show a low diffusing capacity, out of proportion to the degree of restriction. RHC is the gold standard for the diagnosis of PAH, since it is the only way to both accurately measure PAH and exclude left atrial hypertension by means of a normal PAOP. The significance of both of these points was highlighted by Baughman et  al. [50] in a series of 53 SAPH patients who underwent both echocardiography and RHC. Echocardiography missed pulmonary hypertension in 30% of these patients, whereas 24% had PAOP in excess of 20 mmHg (i.e. left ventricular diastolic dysfunction). Given the potential prevalence of exercise-induced pulmonary hypertension in patients with SAPH (75%) as discussed earlier, we recommend exercise provocation during the RHC if the resting values are normal and the clinic radiographic scenario is compelling.

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The role of vasodilator testing is not well established. It is the practice of our pulmonary vascular center to routinely perform vasodilator testing in any patient’s initial diagnostic RHC when PAH is diagnosed.

6.6  Management Because of limited data, the optimal management strategy for SAPH is not known. The general strategy employed involves (1) oxygen (nearly uniformly indicated in these patients), (2) treatment of the underlying inflammatory lung disease, and (3) therapy aimed specifically at small vessel disease. Treatment of pulmonary sarcoidosis typically focuses on the interstitial pneumonia component of the disease and thus is restricted to patients with stage II–III disease. The mainstay of therapy is prednisone, usually in the range of 60 mg for 3–6 months tapered over 1 year on the basis of the clinical status, imaging, and pulmonary function test data. Gluskowski et al. [51] showed that of 24 subjects with SAPH (both rest and exercised-induced), nearly all patients had improved pulmonary function test values after 1 year of prednisone therapy, whereas only 50% of them enjoyed a significant reduction in pulmonary artery pressures. Other investigators have found even lower rates of steroid-responsive SAPH [50, 52]. It is intriguing to speculate that SAPH patients with steroid-responsive pulmonary hypertension have a primarily granulomatous vasculopathy, in contrast to nonresponders, who may have a more traditional IPAH-like pulmonary vasculopathy. Much more study is necessary, but this type of distinction may have important treatment implications. Although no data exist, it may make sense to attempt treatment for stage I disease if bulky adenopathy is believed to be causing significant SAPH. The question of whether to attempt steroid treatment in SAPH patients with stage I disease, and no obvious pulmonary artery compression, aimed at a granulomatous angiitis, is supported by theory, but not data. Steroid treatment of SAPH that occurs with stage IV fibrotic disease is supported by neither data nor theory. In patients with presumed small-vessel-mediated SAPH who fail steroid therapy or whose sarcoidosis stage (i.e. I or IV) does not warrant steroid therapy, therapy may be approached as it would be for patients with IPAH, although with some important caveats. Although our practice is to use anticoagulation in most patients with IPAH, anticoagulation must be used with extreme caution in patients with severe parenchymal lung disease given the risk of bronchiectasis and hemoptysis.

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6.7  Antiremodeling and Vasodilating Agents Many patients with SAPH are poor candidates for therapy directly aimed at reversing small vessel vasoconstriction and vascular remodeling, whereas a subset of patients will likely benefit from therapy. Pulmonary vasodilators must be used cautiously in patients with end-stage fibrotic lung disease. These patients have heterogeneous ventilation based on parenchymal destruction, which is generally well matched by heterogeneous perfusion, based upon hypoxic vasoconstriction. Indiscriminate pulmonary vasodilatation may send blood flow away from ventilated units to unventilated units, causing severe hypoxemia from ventilation–perfusion mismatch. Additionally, as described above, patients with end-stage sarcoidosis are uniquely vulnerable to left ventricular diastolic dysfunction (e.g. granulomatous cardiac involvement, exercise-induced hypoxemia). Selective pulmonary vasodilatation in the setting of an elevated left ventricular end-diastolic volume may lead to pulmonary edema. Finally, although the incidence is not clear, there are case reports of pulmonary edema occurring in patients with SAPH treated with vasodilators, presumably from predominant pulmonary venous involvement, causing a pathophysiologic state similar to PVOD. However, available data, although limited by low numbers and heterogeneous patient populations, suggest that a subset of patients may benefit from treatment of their SAPH. Combining data from all studies that followed patients for over 1 year reveals that of 27 patients followed on average for 31 months, 60% died or required lung transplantation. Most of these patients were treated with epoprostenol (12), bosentan (eight), inhaled nitric oxide (five), or calcium channel antagonists (two). Looking at studies that followed patients for 1 year or less shows considerably better survival. Of a total of 21 patients followed for an average of 8.5 months, 95% survived. Most were treated with sildenafil (12), bosentan (six), epoprostenol (two), iloprost (one), or calcium channel antagonists (one). The total in this series was 22, as one patient was on dual therapy. Combining both series reveals only one medicationrelated sudden death (occurring within 4  h of initiation of epoprostenol therapy) and only one reported episode of pulmonary edema [65]. Despite the large percentage of vasoreactive SAPH, there are very few data on the use of calcium channel antagonists  in this disease. In the absence of data it is under­ standable  that  clinicians hesitate to use high-dose calcium channel antagonists in patients with right ventricular failure (given the potential negative inotropic effects). Sildenafil, a phosphodiesterase-5 inhibitor, increases levels of cyclic GMP in pulmonary vascular cells, leading to nitric oxide generation and vasodilatation. Milman et al. reported encouraging data from 12 Danish patients with stage IV sarcoidosis awaiting

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lung transplantation treated with sildenafil for 12 months. In this group, despite severe restriction (mean forced vital capacity 47% of that predicted), no patient developed worsened hypoxemia or pulmonary edema. All patients displayed hemodynamic improvement, although 6-min walk values did not increase significantly [66]. It is worth noting that because of profound hypoxemia, the 6-min walk end point may not be appropriate for patients with SAPH. In general, we approach the treatment of severe, steroidrefractory SAPH in a manner similar to IPAH. Patients with significant right ventricular dysfunction are offered epoprostenol therapy. Patients with milder disease may be treated with either sildenafil or an endothelin receptor antagonist. Patients should be monitored closely (especially on initiation of therapy) for cardiopulmonary intolerance.

6.8  Summary of Key Concepts 1. SAPH should be considered in any patients with known or suspected pulmonary hypertension out of proportion to the severity of their parenchymal lung disease. 2. Patients with sarcoidosis and interstitial lung disease are uniquely vulnerable to left ventricular diastolic dysfunction, which may lead to compensatory pulmonary hypertension secondary to an elevated PAOP. 3. The diagnosis can only be excluded by a RHC with exercise provocation and PAOP measurement. 4. The small vessel vasculopathy associated with SAPH has a unique granulomatous angiitis component, making steroid therapy a necessary consideration for all patients with stage I–III disease. 5. Vasodilator and antiremodeling therapy is indicated in select patients, but must be initiated cautiously, given the risk of ventilation–perfusion mismatch and hypoxemia as well the risk of pulmonary edema from unmasked leftsided heart failure or PVOD phenotype.

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number of uncommon diseases, associations between MPD and pulmonary hypertension began with case reports and progressed to small case series. Marvin and Spellberg reported the case of one patient with ET and long-standing PAH whose pulmonary artery pressure fell with a reduced platelet count on hydroxyurea therapy, suggesting an important role for circulating platelets in the development of PAH in these patients [67]. However, several complicating factors exist when examining the PAH that occurs in the setting of MPD. Patients with MPD have high rates of splenectomy, portal hypertension, and left ventricular dysfunction in addition to being prone to acute thromboembolism, CTEPH, and alveolar hemorrhage. García-Manero et  al. nicely illustrated these potential pitfalls in their series of six patients with MPD and PAH (four with PMF, one with ET, and one with PV). Of the two patients that underwent RHC, one patient had a PAOP of 18 mmHg or greater and the patient had a positive ventilation–perfusion scan. More importantly, all had splenectomies. Autopsy data on the five patients who died during the follow-up period revealed pulmonary vascular pathologic changes typical of SIPAH, specifically pulmonary vascular remodeling with plexiform arteriopathy, and abundant small vessel thrombosis [68]. MPDs can impact the pulmonary vasculature directly, via cellular obstruction of the alveolar capillary bed, or indirectly by pulmonary parenchymal myeloid infiltration and associated thrombosis and vascular remodeling (presumably from cytokine and growth factor release). Although the prognoses for ET and PV are improving, the complication of PAH increases morbidity and mortality. It is worth noting that there may be an association between pulmonary vascular remodeling and marrow fibrosis beyond MPDs. Popat et al. found marrow fibrosis present in 100% of patients with IPAH or collagen vascular disease APAH. This association is controversial and requires additional validation [69].

7.2  The Prevalence of MPD APAH 7  PAH in Myeloproliferative Disorders 7.1  Background and Introduction Myeloproliferative disorders (MPDs) constitute a group of acquired syndromes involving clonal proliferation of hematopoietic stem cells, most of which can be classified as polycythemia vera (PV), essential thrombocytosis (ET), or primary myelofibrosis with myeloid metaplasia (PMF), previously known as agnogenic myeloid metaplasia. As with a

With many caveats, it appears that asymptomatic PAH complicates MPD up to 48% of the time. Severe, progressive symptomatic PAH complicates MPD less commonly, perhaps 13% of the time (depending upon the series, method, and specific patient population studied, i.e. PV vs. ET). Cortelezzzi et al. performed echocardiography on 36 patients with MPD (22 with PMF, seven with ET, and seven with PV), finding PAH in 36% of them (only 23% symptomatic). The development of PAH did not correlate with disease duration or cell counts (or any clinical variable assessed). Importantly the presence of left-sided heart failure and or

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embolic disease was not assessed [70]. Gupta et al. studied 27 asymptomatic MPD patients with echocardiography, finding PAH in 12 of them (48%), excluding two patients (14%) with left ventricular systolic dysfunction. Seven patients had ET and five patients had PV. Although the authors found no evidence for acute pulmonary embolism on reviewing the records of the 12 PAH patients, perfusion defects (i.e. CTEPH) were not investigated [71]. Garypidou et  al. performed a similar echocardiographic study on 22 MPD patients (eight male, 14 female), two with PV, 14 with ET, and six with PMF. Excluding patients with cell-mediated lympholysis or splenectomy, they found PAH in 40% of them (nine patients), four males and five females (six with ET, three with PMF). Importantly, RHC and radionuclide perfusion studies were not performed; thus, CTEPH and left ventricular diastolic dysfunction were not excluded. Fiftyfive percent of their PAH patients were symptomatic (five mild-moderate, two severe). No significant correlations with PAH and clinical variables were found. In the mean 5-month follow-up period, one patient died of progressive right-sided heart failure [72]. Altintas et al. screened 46 asymptomatic patients with ET, finding 49% (22) with PAH. This was compared with patients with reactive thrombocytosis, in whom PAH was not found [73].

7.3  Pathophysiology The spectrum of disease underlying MPD APAH is best illustrated by Guilpain et al. in a retrospective case series of ten patients with severe pulmonary hypertension. All patients underwent RHC and individuals with a PAOP above 15  mmHg were excluded. Additionally, all patients underwent ventilation–perfusion scanning, with all positive studies confirmed by pulmonary angiography. No patient in the series had a history of splenectomy; unfortunately, one patient had portal hypertension. Of the ten individuals studied, eight had PV, two had ET, and none had PMF. All patients were treated with anticoagulation, diuretics, and oxygen. Interestingly, six patients (all with PV) were found to have MPD-associated CTEPH (MPDA-CTEPH), whereas the remaining four had MPD APAH Of the six individuals with MPDA-CTEPH, 50% were female, four had proximal surgically accessible disease (all PV-associated), and the remaining two had distal, inoperable disease (one ET- and the other PV-associated). Of the four individuals with MPD APAH, 100% were female (75% had PV and 25% had ET). All four patients with MPD APAH died at 1 year (two each from hemorrhagic complications and right-sided heart failure). In contrast, only two patients (33%) with MPDA-CTEPH died at 1 year; one individual died immediately after PTE, and the other died after medical therapy alone [74].

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The most significant pathophysiologic distinction between the groups appears to be the time of onset of PAH with regard to the diagnosis of MPD. All patients with MPDA-CTEPH were diagnosed with PAH concomitantly with their PV; in other words, all had untreated PV at the time of their CTEPH diagnosis. Fifty percent of these patients had a documented history of pulmonary embolism. Of the four patients with proximal disease who underwent PTE, one died immediately postoperatively (over twice the typical perioperative mortality rate). This outcome strongly suggests the presence of significant, unappreciated, coexisting small vessel vascular remodeling, possibly occurring at a higher frequency in MPDA-CTEPH than expected in the undifferentiated pool of CTEPH patients. In contrast, among the four individuals with MPD APAH (three with PV and one with ET), the median time between the diagnosis of MPD and the discovery of PAH was 13.5 years, far from the simultaneous presentations seen in the PV-associated CTEPH patients. Interestingly, 75% of the MPD APAH patients presented with concomitant evidence of progression to myeloid metaplasia. Overall, 100% of MPD APAH patients had evidence of myeloid metaplasia, whereas none of the MPDA-CTEPH patients did [74]. Taking together these data suggests that there are three distinct mechanisms by which the pulmonary vasculature may be affected by MPD: (1) lung infiltration by hematopoietic cells (myeloid metaplasia) causing associated vascular remodeling and abundant thrombosis, (2) direct alveolar capillary obstruction by megakaroycytes [75] or hematopoietic stem cells [76], and (3) CTEPH. Unfortunately, autopsy data are limited in these patients, making more specific characterizations difficult. The underlying pathophysiologic process of MPD APAH likely involves the elaboration of cytokines and growth factors from clonally expanded abnormal hematopoietic stem cells. Evidence for this is largely circumstantial, coming from the observation that the development of PAH in patients with MPD often occurs simultaneously with the progression to marrow fibrosis and myeloid metaplasia, ultimately leading to mechanical obstruction of the pulmonary capillary bed with hematopoietic stem cells [76]. In Guilipan et al.’s series, 100% of MPD APAH patients had evidence of myeloid metaplasia, whereas none of the MPDA-CTEPH patients did, and 75% of the MPD APAH patients presented with concomitant evidence of progression to myeloid metaplasia. An autopsy of one of their MPD APAH patients showed infiltration of lung by hematopoietic stem cells. Aside from mechanical obstruction, little is known about how this hematopoietic stem cell infiltration impacts the local vasculature [74]. In many patients with MPD (especially those with ET), thrombocytosis with abnormal platelet activation may lead to mechanical obstruction of the pulmonary alveolar capillary

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bed, as well as promoting small vessel thrombosis and vascular remodeling. Activated platelets are rich sources of potent pulmonary vascular mitogens and vasoconstrictors, such as PDGF, TGF-b, and serotonin, all of which have been implicated in the development of pulmonary vascular remodeling. To highlight the clinical relevance of this, Marvin and Spellberg reported the case of one patient with ET and longstanding PAH as diagnosed by RHC and negative pulmonary angiography, whose ePAP fell, and right-sided heart failure resolved on hydroxyurea therapy, in conjunction with falling platelet counts [67]. Additionally, Altintas et  al.’s series found that patients with ET and PAH had statistically higher platelet counts than patients with ET and no evidence of PAH, again implicating the circulating platelet burden in the development of PAH in these patients. Interestingly, they found no PAH in their control group with reactive thrombocytosis, underscoring the importance of the abnormal platelets and megakaryocytes in the formation of PAH in ET patients [73]. One can speculate that abnormal platelets in ET may have increased activation and or elaboration of mediators and growth factors. Cortelezzi et al. measured circulating levels of vascular endothelial growth factor (VEGF), PDGF, TGF-b, and ET-1 in 13 patients with MPD APAH (most were asymptomatic), compared with 23 patients with MPD and no PAH. Only VEGF levels were significantly elevated in MPD APAH patients compared with those with MPD alone. It is still possible that local elevations of the other mediators may occur in the areas of pulmonary vascular microthrombosis, platelet trapping, and platelet activation, although this remains speculation. Although CTEPH and PAH are distinct entities, significant overlap exists, and in this regard MPDA-CTEPH deserves special mention. Although the sample size was small, Guilpain et al.’s MPDA-CTEPH patients appeared to have higher rates of distal (surgically inaccessible) disease, and possibly higher rates of persistent pulmonary hypertension after PTE (the most common cause of immediate postPTE death). Taken together, these observations suggest a unique disease, possibly microthrombosis or in situ thrombosis, akin to that seen in splenectomy-induced PAH. This in turn may be related to increased numbers of activated, circulating platelets [74].

7.4  C  linical Presentation and Evaluation of MPD APAH Individuals with MPD APAH are typically middle-aged (median age 57) on presentation. Patients with MPDACTEPH are uniformly diagnosed with pulmonary hypertension as a presenting complication of their MPD diagnosis (typically PV). This group shows an even male-to-female

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distribution. In contrast, MPD APAH patients tend to be female (100%), and present much later in the course of their disease, a median of 13.5 years after their MPD diagnosis. In these patients the development of pulmonary hypertension suggests the progression or conversion to myeloid metaplasia (as discussed earlier). Although the sample size is small, the data suggest that both MPDA-CTEPH and MPD APAH patients, despite treatment, may have a worse prognosis than their counterparts with treated CTEPH and IPAH alone. Guilpan et al. reported a 25% 1-year mortality for individuals with MPDA-CTEPH despite thromboendartectomy, and a 100% 1-year mortality for MPD APAH patients on medical therapy with vasodilator/antiremodeling drugs. Importantly, 50% of deaths occurring in the MPD APAH group where as a consequence of fatal hemorrhage, and not progressive right-sided heart failure [74].

7.5  Diagnostic Testing and Management Given the significant rate of asymptotic PAH in individuals with MPD coupled with the poor prognosis of those who develop severe PAH (despite treatment), one can argue for screening echocardiography in all patients with MPD. Given the high rates of left ventricular dysfunction reported among MPD patients with PAH (15–50%), confirmatory RHC with estimation of PAOP is essential. When PAH is confirmed, chronic thromboembolic disease must be excluded with perfusion studies (typically a screening ventilation–perfusion scan with confirmatory pulmonary angiography). This is especially crucial for patients presenting with PV. Individuals with MPD and PAH with a normal PAOP, and negative perfusion findings, should be considered MPD APAH patients. Few or no data exist on the optimal therapeutic approach to these patients. Given the pathophysiologic process of PAH in these patients involves capillary obstruction from abnormal circulating cells, is seems prudent to aggressively mange the cell counts in these individuals, especially those with ET [67]. When cytoreductive therapy is exhausted and PAH persists, it is reasonable to treat these patients with vasodilator/antiremodeling agents, much like IPAH. However, the risk of anticoagulation in these patients may outweigh the benefit, given the significant incidence of fatal hemorrhage in this group (possibly related to underlying platelet dysfunction vs. treatmentrelated cytopenias) [74].

7.6  Summary of Key Concepts 1. Asymptomatic pulmonary hypertension (PAH) is common in patients with MPDs.

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2. Individuals with MPD APAH need to be screened for left-sided heart failure, and chronic thromboembolic disease. 3. Individuals presenting with PV and PAH particularly need to have CTEPH (MPDA-CTEPH) excluded. 4. Individuals with MPD who develop PAH later in their course should be presumed to have had progression of their underlying disease to myeloid metaplasia. 5. Patients with MPD APAH suffer from pulmonary capillary obstruction with hematopoietic stems cells and platelets, in addition to abundant microthrombosis, often associated with pulmonary myeloid metaplasia, highlighting the importance of treatment of the underlying disease.

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J.W. Landsberg and J. Mandel 17. Roberts WC, Fredrickson DS (1967) Gaucher’s disease of the lung causing severe pulmonary hypertension with associated acute recurrent pericarditis. Circulation 35:783–789 18. Ross DJ, Spira S, Buchbinder NA (1997) Gaucher cells in pulmonary-capillary blood in association with pulmonary hypertension. N Engl J Med 336:379–381 19. Mistry PK, Sirrs S, Chan A et al (2002) Pulmonary hypertension in type 1 Gaucher’s disease: genetic and epigenetic determinants of phenotype and response to therapy. Mol Genet Metab 77:91–98 20. Harats D, Pauzner R, Elstein D et al (1997) Pulmonary hypertension in two patients with type I Gaucher disease while on alglucerase therapy. Acta Haematol 98:47–50 21. Elstein D, Klutstein MW, Lahad A, Abrahamov A, Hadas-Halpern I, Zimran A (1998) Echocardiographic assessment of pulmonary hypertension in Gaucher’s disease. Lancet 351:1544–1546 22. Sirrs S, Irving J, McCauley G et al (2002) Failure of resting echocardiography and cardiac catheterization to identify pulmonary hypertension in two patients with type I Gaucher disease. J Inherit Metab Dis 25:131–132 23. Elstein D, Nir A, Klutstein M, Rudensky B, Zimran A (2005) C-reactive protein and NT-proBNP as surrogate markers for pulmonary hypertension in Gaucher disease. Blood Cells Mol Dis 34:201–205 24. Shitrit D, Rudensky B, Zimran A, Elstein D (2003) D-dimer assay in Gaucher disease: correlation with severity of bone and lung involvement. Am J Hematol 73:236–239 25. Fernandes CJC, Jardim C, Carvalho LAS, Farias AQ, Filho MT, Souza R (2005) Clinical response to sildenafil in pulmonary hypertension associated with Gaucher disease. J Inherit Metab Dis 28:603–605 26. Leuchte HH, Baumgartner RA, Behr J (2003) Treatment of severe pulmonary hypertension with inhaled iloprost. Ann Intern Med 139:306 27. Hollak CE, Levi M, Berends F, Aerts JM, van Oers MH (1997) Coagulation abnormalities in type 1 Gaucher disease are due to low-grade activation and can be partly restored by enzyme supplementation therapy. Br J Haematol 96:470–476 28. Aessopos A, Stamatelos G, Skoumas V, Vassilopoulos G, Mantzourani M, Loukopoulos D (1995) Pulmonary hypertension and right heart failure in patients with b-thalassemia intermedia. Chest 107:50–53 29. Hoeper MM, Niedermeyer J, Hoffmeyer F, Flemming P, Fabel H (1999) Pulmonary hypertension after splenectomy? Ann Intern Med 130:506–509 30. Phrommintikul A, Sukonthasarn A, Kanjanavanit R, Nawarawong W (2006) Splenectomy: a strong risk factor for pulmonary hypertension in patients with thalassaemia. Heart 92:1467–1472 31. Bonderman D, Jakowitsch J, Adlbrecht C et al (2005) Medical conditions increasing the risk of chronic thromboembolic pulmonary hypertension. Thromb Haemost 93:512–516 32. Bonderman D, Wilkens H, Wakounig S et  al (2009) Risk factors for  chronic thromboembolic pulmonary hypertension. Eur Respir J 33:325–331 33. Jaïs X, Loos V, Jardim C et  al (2005) Splenectomy and chronic thromboembolic pulmonary hypertension. Thorax 60:1031–1034 34. Schilling RF, Gangnon RE, Traver MI (2008) Delayed adverse ­vascular events after splenectomy in hereditary spherocytosis. J Thromb Haemost 6:1289–1295 35. Smedema JP, Louw VJ (2007) Pulmonary arterial hypertension after splenectomy for hereditary spherocytosis. Cardiovasc J Afr 18:84–89 36. Sumiyoshi A, Thakerngpol K, Sonakul D (1992) Pulmonary microthromboemboli in thalassemic cases. Southeast Asian J Trop Med Public Health 23:29–31 37. Lang I, Kerr K (2006) Risk factors for chronic thromboembolic pulmonary hypertension. Proc Am Thorac Soc 3:568–570

74  Pulmonary Arterial Hypertension Related to Gaucher’s Disease, Sarcoidosis, and Other Disorders 38. Vassallo R, Ryu JH, Colby TV, Hartman T, Limper AH (2000) Pulmonary Langerhans’-cell histiocytosis. N Engl J Med 324:1969–1978 39. Crausman RS, Jennings CA, Tuder RM, Ackerson LM, Irvin CG, King TE Jr (1996) Pulmonary histiocytosis X: pulmonary function and exercise pathophysiology. Am J Respir Crit Care Med 153:426–435 40. Fartoukh M, Humbert M, Capron F et al (2000) Severe pulmonary hypertension in histiocytosis X. Am J Respir Crit Care Med 161:216–223 41. Chaowalit N, Pellikka PA, Decker PA et al (2004) Echocardiographic and clinical characteristics of pulmonary hypertension complicating pulmonary Langerhans cell histiocytosis. Mayo Clin Proc 79: 1269–1275 42. Crausman RS, King TE Jr (1997) Pulmonary vascular involvement in pulmonary histiocytosis X. Chest 112:1714 43. Harari S, Brenot F, Barberis M, Simonneau G (1997) Advanced pulmonary histiocytosis X is associated with severe pulmonary hypertension. Chest 111:1142–1144 44. Hamada K, Teramoto S, Narita N, Yamada E, Teramoto K, Kobzik L (2000) Pulmonary veno-occlusive disease in pulmonary Langerhans’ cell granulomatosis. Eur Respir J 15:421–423 45. Benyounes B, Crestani B, Couvelard A, Vissuzaine C, Aubier M (1996) Steroid-responsive pulmonary hypertension in a patient with Langerhans’ cell granulomatosis (histiocytosis X). Chest 110:284–286 46. Dauriat G, Mal H, Thabut G et al (2006) Lung transplantation for pulmonary langerhans’ cell histiocytosis: a multicenter analysis. Transplantation 81:746–750 47. Thomas PD, Hunninghake GW (1987) Current concepts of the pathogenesis of sarcoidosis. Am Rev Respir Dis 135:747–760 48. Handa T, Nagai S, Miki S et al (2007) Incidence of pulmonary hypertension and its clinical relevance in patients with interstitial pneumonias: comparison between idiopathic and collagen vascular disease associated interstitial pneumonias. Intern Med 46:831–837 49. Shorr AF, Helman DL, Davies DB, Nathan SD (2005) Pulmonary hypertension in advanced sarcoidosis: epidemiology and clinical characteristics. Eur Respir J 25:783–788 50. Baughman RP, Engel PJ, Meyer CA, Barrett AB, Lower EE (2006) Pulmonary hypertension in sarcoidosis. Sarcoidosis Vasc Diffuse Lung Dis 23:108–116 51. Gluskowski J, Hawrylkiewicz I, Zych D, Zieliński J (1990) Effects of corticosteroid treatment on pulmonary haemodynamics in patients with sarcoidosis. Eur Respir J 3:403–407 52. Nunes H, Humbert M, Capron F et al (2006) Pulmonary hypertension associated with sarcoidosis: mechanisms, haemodynamics and prognosis. Thorax 61:68–74 53. Damuth TE, Bower JS, Cho K, Dantzker DR (1980) Major pulmonary artery stenosis causing pulmonary hypertension in sarcoidosis. Chest 78:888–891 54. Westcott JL, DeGraff AC Jr (1973) Sarcoidosis, hilar adenopathy, and pulmonary artery narrowing. Radiology 108:585–586 55. Levine BW, Saldana M, Hutter AM (1971) Pulmonary hypertension in sarcoidosis. A case report of a rare but potentially treatable cause. Am Rev Respir Dis 103:413–417 56. Rosen Y, Moon S, Huang CT, Gourin A, Lyons HA (1977) Granulomatous pulmonary angiitis in sarcoidosis. Arch Pathol Lab Med 101:170–174 57. Takemura T, Matsui Y, Oritsu M et al (1991) Pulmonary vascular involvement in sarcoidosis: granulomatous angiitis and microangiopathy in transbronchial lung biopsies. Virchows Arch A Pathol Anat Histopathol 418:361–368

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58. Tayal S, Voelkel NF, Rai PR, Cool CD (2006) Sarcoidois and pulmonary hypertension – a case report. Eur J Med Res 11:194–197 59. Preston IR, Klinger JR, Landzberg MJ, Houtchens J, Nelson D, Hill NS (2001) Vasoresponsiveness of sarcoidosis-associated pulmonary hypertension. Chest 120:866–872 60. Rizzato G, Pezzano A, Sala G et al (1983) Right heart impairment in sarcoidosis: haemodynamic and echocardiographic study. Eur J Respir Dis 64:121–128 61. Gluskowski J, Hawrylkiewicz I, Zych D, Wojtczak A, Zieliński J (1984) Pulmonary haemodynamics at rest and during exercise in patients with sarcoidosis. Respiration 46:26–32 62. Reichenberger F, Schauer J, Kellner K, Sack U, Stiehl P, Winkler J (2001) Different expression of endothelin in the bronchoalveolar lavage in patients with pulmonary diseases. Lung 179:163–174 63. Letizia C, Danese A, Reale MG et  al (2001) Plasma levels of endothelin-1 increase in patients with sarcoidosis and fall after disease remission. Panminerva Med 43:257–261 64. Sulica R, Teirstein AS, Kakarla S, Nemani N, Behnagar A, Padilla ML (2005) Distinctive clinical, radiographic, and functional characteristics of patients with sarcoidosis-related pulmonary hypertension. Chest 128:1483–1489 65. Diaz-Guzman E, Farver C, Parambil J, Culver DA (2008) Pulmonary hypertension caused by sarcoidosis. Clin Chest Med 29:549–563 66. Milman N, Burton CM, Iversen M, Videbaek R, Jensen CV, Carlsen J (2008) Pulmonary hypertension in end-stage pulmonary sarcoidosis: therapeutic effect of sildenafil? J Heart Lung Transplant 27:329–334 67. Marvin KS, Spellberg RD (1993) Pulmonary hypertension secondary to thrombocytosis in a patient with myeloid metaplasia. Chest 10:642–644 68. García-Manero G, Schuster SJ, Patrick H, Martinez J (1999) Pulmonary hypertension in patients with myelofibrosis secondary to myeloproliferative diseases. Am J Hematol 60:130–135 69. Popat U, Frost A, Liu E et al (2005) New onset of myelofibrosis in association with pulmonary arterial hypertension. Ann Intern Med 143:466–467 70. Cortelezzi A, Gritti G, Del Papa N et al (2008) Pulmonary arterial hypertension in primary myelofibrosis is common and associated with an altered angiogenic status. Leukemia 22:646–649 71. Gupta R, Perumandla S, Patsiornik Y, Niranjan S, Ohri A (2006) Incidence of pulmonary hypertension in patients with chronic myeloproliferative disorders. J Natl Med Assoc 98:1779–1782 72. Garypidou V, Vakalopoulou S, Dimitriadis D, Tziomalos K, Sfikas G, Perifanis V (2004) Incidence of pulmonary hypertension in patients with chronic myeloproliferative disorders. Haematologica 89:245–246 73. Altintas A, Karahan Z, Pasa S et al (2007) Pulmonary hypertension in patients with essential thrombocythemia and reactive thrombocytosis. Leuk Lymphoma 48:1981–1987 74. Guilpain P, Montani D, Damaj G et al (2008) Pulmonary hypertension associated with myeloproliferative disorders: a retrospective study of ten cases. Respiration 76:295–302 75. Halank M, Marx C, Baretton G, Müller KM, Ehninger G, Höffken G (2004) Severe pulmonary hypertension in chronic idiopathic myelofibrosis. Onkologie 27:472–474 76. Dingli D, Utz JP, Krowka MJ, Oberg AL, Tefferi A (2001) Unexplained pulmonary hypertension in chronic myeloproliferative disorders. Chest 120:801–808 77. Atichartakarn V, Likittanasombat K, Chuncharunee S et al (2003) Pulmonary arterial hypertension in previously splenectomized patients with beta-thalassemic disorders. Int J Hematol 78: 139–145

Chapter 75

Pediatric Pulmonary Hypertension: An Integrated View from Pediatric Subspecialists Judy L. Aschner, Candice D. Fike, Eric D. Austin, Frederick E. Barr, and J. Donald Moore

Abstract  Much of current knowledge and momentum in the field of pulmonary hypertension (PH) has not penetrated the increasingly heterogeneous pediatric population managed by pediatric subspecialists worldwide. It is critical to prospectively test specific agents in children. Yet, the broad spectrum of pediatric pulmonary vascular diseases and the lack of clinically meaningful end points appropriate for the pediatric population pose challenges for the conduct of sufficiently powered pediatric studies. It is therefore no surprise that in the midst of significant advances in the field, there are no agents approved for use in children afflicted with PH with the exception of inhaled nitric oxide (iNO) for infants with persistent PH of the newborn (PPHN). The current and widely accepted WHO framework by which pediatric subspecialists are guided is not easily translated into the expanding range of situations encountered in pediatric practice. Like PPHN, it may be necessary to approach these pediatric pulmonary hypertensive conditions from outside the frameworks proposed for typical adult-onset diseases. Exploration of the potential intrinsic differences between children and adults may yield important discoveries regarding the genesis of disease in children and adults alike Fortunately, most children with PH are seen within specialized pediatric centers where there are untapped opportunities for multicenter collaboration. Our aim in this chapter is to approach pediatric PH over the age and development continuum from the vantage point of the pediatric subspecialists who encounter these patients in practice. We will review the abnormalities of pulmonary vascular tone and structure that impact the health and survival of children currently cared for in pediatric PH centers. Many of these disease processes occur commonly from our vantage point, yet are underrepresented in the broader field of PH research or current classification schemes. We will also address the challenges that pediatric subspecialists face in defining PH and assessing its severity in infants or children across the developmental and disease spectrums. J.L. Aschner (*) The Monroe Carell Jr. Children’s Hospital at Vanderbilt, Vanderbilt University School of Medicine, 11111 Doctor’s Office Tower, 2200 Children’s Way, Nashville TN 37232-9544 e-mail: [email protected]

Keywords  Pediatric pulmonary hypertension • Pediatric patient • Specialized pediatric center • Neonatology • Pediatrics • Treatment for children • Persistent pulmonary hypertension in the newborn

1 Introduction The field of pulmonary hypertension (PH) is rapidly advancing with major developments in both the basic and the clinical realms. From development of therapeutics that target key mechanistic pathways to large-scale observational registries [1], there is great anticipation for the future. Unfortunately, much of this knowledge and momentum has not penetrated the increasingly heterogeneous pediatric population managed by pediatric subspecialists worldwide. Large-scale clinical trials typically exclude children with PH; extrapolation of findings from these studies in adults to the pediatric PH population is inherently problematic. It is critical to prospectively test specific agents in children. Yet, the broad spectrum of pediatric pulmonary vascular diseases (Fig.  1) and the lack of clinically meaningful end points appropriate for the pediatric population pose challenges for the conduct of sufficiently powered pediatric studies. It is therefore no surprise that in the midst of significant advances in the field, there are no agents approved for use in children afflicted with PH with the exception of inhaled nitric oxide (iNO) for infants with persistent PH of the newborn (PPHN). In the absence of appropriate clinical trials, it is not surprising that the off-label use of agents in pediatrics has increased over the last decade, with encouraging, albeit anecdotal, results. Assigning benefits of drugs and dosing regimens tested in adults to children assumes that the disease processes and pharmacokinetics are comparable in all age groups, a premise that is undoubtedly untrue. Moreover, opportunities for early intervention and prevention are frequently missed because the current approach ignores critically important windows of transition to irreversible disease (Fig. 2). An expansion in our approach to pediatric PH is now needed.

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_75, © Springer Science+Business Media, LLC 2011

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Fig. 1  Common neonatal presentations and the mechanisms underlying abnormal postnatal cardiopulmonary adaptation leading to the broad spectrum of postnatal conditions associated with pediatric pulmonary hypertension (PH). Meconium Aspiration Syndrome; alternatively spell out meconium aspiration syndrome in the Table

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tunities for multicenter collaboration. Furthermore, the broad field of pediatric PH crosses multiple disciplines and benefits from collaboration among multiple pediatric subspecialties. For example, the traditional focus of the pediatric cardiologist has been the patient with idiopathic PH or with PH associated with congenital heart disease (CHD). This horizon is expanding to include a growing population of neonatal intensive care unit (NICU) graduates with various underlying pulmonary abnormalities and PH who may benefit from predischarge input and longitudinal postdischarge follow-up by pediatric cardiologists and pulmonologists. Likewise, patients with underlying pulmonary, cardiac, and genetic diseases who present with PH later in infancy and childhood may benefit from an integrated team approach to care which includes input from pediatric intensivists, pediatric pulmonologists, and pediatric cardiologists with special interest in the PH disease spectrum. Our aim in this chapter is to approach pediatric PH over the age and development continuum from the vantage point of the pediatric subspecialists who encounter these patients in practice. Rather than using the current WHO classifications, the perspective of the pediatric subspecialist – neonatologist, intensivist, pulmonologist, and cardiologist – will be the starting point, highlighting the growing body of work from within these disciplines. In so doing, we will review the abnormalities of pulmonary vascular tone and structure that impact the health and survival of children currently cared for in pediatric PH centers. Many of these disease processes occur commonly from our vantage point, yet are underrepresented in the broader field of PH research or current classification schemes (Fig. 1). We will also address the challenges that pediatric subspecialists face in defining PH and assessing its severity in infants or children across the developmental and disease spectrums.

Fig. 2  Critical windows of transition across the developmental age and disease spectrum of pediatric PH. Failure to appreciate these transitional zones may lead to missed diagnoses and missed opportunities for early intervention and prevention of severe or irreversible disease

2 Definition of PH in Infants and Children

A clear and consistent classification scheme for pediatric PH disease states is of paramount importance [2]. The current and widely accepted WHO framework by which pediatric subspecialists are guided is not easily translated into the expanding range of situations encountered in pediatric practice [3]. Like PPHN, it may be necessary to approach these pediatric pulmonary hypertensive conditions from outside the frameworks proposed for typical adult-onset diseases. Exploration of the potential intrinsic differences between children and adults may yield important discoveries regarding the genesis of disease in children and adults alike Fortunately, most children with PH are seen within specialized pediatric centers [4] where there are untapped oppor-

The normal developing lung and myocardium undergo dynamic transition from the high-resistance fetal pulmonary circulation to the lower-resistance circuit of infancy and childhood. In the setting of associated congenital lung disease or CHD, this normal postnatal decline in pulmonary vascular resistance (PVR) may be delayed or may not occur at all. Mechanisms that underlie abnormal postnatal cardiopulmonary adjustments include chronic or intermittent hypoxemia, altered oxidant, inflammatory, or vasoactive signaling, increased pulmonary blood flow, and structural abnormalities of the pulmonary vascular bed (Fig. 1). A fall in PVR for most of these infants will not be realized until the underlying cardiopulmonary defect is corrected. Interpretation of normal levels of right ventricular (RV) pressure or pulmonary

75  Pediatric Pulmonary Hypertension: An Integrated View from Pediatric Subspecialists

artery pressure (PAP) must therefore be made in this context; even the definition of PH must be classified relative to age. It is for this reason that a diagnosis of PH is not made with certainty in the first few months of life, with the exception of PPHN, a syndrome of failed circulatory transition at birth, which is the topic of another chapter in this volume. The classic adult PH definition is a PAP greater than 25 mmHg at rest or above 30 mmHg with exercise with normal left-sided heart filling pressures [5]. Implicit in this definition is the presence of an indwelling catheter and the ability to exercise with a pulmonary artery catheter in place. These criteria are clearly not applicable to all pediatric PH diagnoses. For example, a diagnosis of PPHN does not require cardiac catheterization. Young children are not routinely subjected to exercise challenge testing; indeed neonates do not exercise beyond feeding and crying. Yet, pediatric subspecialists must have some defining line that demarcates the limits of normal; for most pediatric patients beyond infancy, the current adult definitions are often used.

3 Assessment of PH in Infants and Children 3.1 Echocardiography Most commonly, as with adult patients, the first study to suggest deviation from normalcy is the echocardiogram. The young patient may have unique sources of shunting that allow for quantification of PAP, such as the velocity across the patent ductus arteriosus or a ventricular septal defect. In their absence, the most widely used echocardiographic measure is the tricuspid regurgitant (TR) jet velocity. A velocity greater than 2.8–3.4  m/s (estimated RV pressure of 36–50 mmHg assuming right atrial pressure to be 5 mmHg) has been described as the line above which RV pressure and thus PAP are deemed elevated in the absence of pulmonary stenosis or other forms of RV outflow obstruction. Although these ranges are the accepted definitions in adults, there is admittedly overlap with the ranges for normal subjects with increasing age and body mass index. Furthermore, pressures above the stated upper limits of normal do not necessarily have predictable adverse outcomes [6, 7]. An exception to this, however, has been recently demonstrated in adult patients with sickle cell disease (SCD) in whom a tricuspid velocity above 2.5 m/s predicted less favorable survival [8]. In most reports, estimated systolic pulmonary pressures of 30–40 mmHg define the limits of normal. Although widely applicable, the above-mentioned criteria were developed and validated for the most part in adults and, as such, can prove problematic in the pediatric age group [9]. This is particularly true of neonates and younger infants, who may not have elevation of absolute PAP yet may still

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have significant elevation relative to their low systolic systemic pressures. Expressing RV pressure as a percentage relative to systemic pressure is often used by pediatric cardiologists, with a peak systolic RV pressure greater than 50–75% of the systemic pressure considered grounds for further investigation or intervention [10, 11]. Most commonly, the TR velocity is either absent or not optimal and other direct or indirect measures are utilized. These include assessment of ventricular septal geometry [12, 13], measurement of the pulmonary insufficiency jet if present [14], myocardial performance index [15–17], and tricuspid annular plane systolic excursion [18, 19] or velocity [20]. MRI and real-time 3D echocardiography may also hold promise for the noninvasive assessment of RV function and PH as in adults [21]. In general, the echocardiographic assessment is the starting point for defining disease, or at least initiating a more in depth evaluation which typically includes cardiac catheterization. Despite its importance as a noninvasive determinant of pulmonary vascular disease, echocardiography cannot accurately determine the presence, absence, or severity of PH in infants and young children with chronic lung diseases. Mourani et al. [9] reported that echocardiography correctly diagnosed PH 79% of the time; correlation with PH severity, as determined by cardiac catheterization, was poor (47%). In children in whom no systolic PAP measurement could be determined, 58% were subsequently diagnosed with PH by cardiac catheterization. Thus, cardiac catheterization with acute vasodilator testing remains the gold standard for disease definition and ongoing follow-up in the majority of children regardless of PH cause [22].

3.2 Cardiac Catheterization Pediatric cardiologists frequently perform right-sided heart catheterizations in structurally normal hearts as well as in hearts with more complex structural defects. Simultaneous arterial and venous access is the norm for most pediatric interventional laboratories. A wide variety of potential access sites is necessary as many children lose venous access options, particularly those patients with associated CHD. A PAP of more than 25  mmHg or more than 30  mmHg with exercise commonly defines disease, although the prognostic value of such absolute numbers is not clear in the child [9, 23]. An increasing number of pediatric catheterizations are being performed with the patient under general anesthesia and it is not common practice to measure PAP directly during exercise in children. Vasoreactivity testing has been a key component of PH assessment in the catheterization laboratory, dating back to the 1950s. Studies in the 1980s defined reactivity as a drop in mean PAP and PVR of more than 20% in response to highdose calcium channel blocking agents. A beneficial response

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to calcium channel blockers was associated with improved 5-year survival, adding clinical relevance to this phenotypic distinction [24]. Early studies in children and young adults with idiopathic PH [currently classified as idiopathic and heritable pulmonary arterial hypertension (PAH)] defined a positive response to prostacyclin infusion as a 20% or greater fall in mean PAP with an increase in cardiac index and no change or a decrease in the PVR to systemic vascular resistance (Rp/Rs) ratio [25]. An increasingly accepted and more rigorous definition of reactivity is a drop in mean PAP greater than 10 mmHg to an absolute level below 40 mmHg with no change or an improvement in cardiac index. This definition predicts which adult patients are more likely to have a sustained benefit from calcium channel blockade [7, 26]. This definition of reactivity and its long-term importance in the pediatric patient remain to be determined. Lesser degrees of reactivity that do not meet strict criteria have unclear prognostic value in the pediatric patient. Inhaled vasodilator testing is routinely employed in pediatric catheterization laboratories using a variety of agents, most commonly oxygen and nitric oxide [27, 28] either separately or in combination [29]. The more recent definition of reactivity is being used in an increasing number of pediatric centers [7, 26]. Unfortunately, there are no consistently applied standards for such testing in pediatric catheterization laboratories and thus a wide range of practice is observed. Standardization of practice with regard to vasoreactivity testing would be helpful for our community and would facilitate interpretation of registry data and design of clinical trials.

4 Progressive PH in the Neonatal Period PH in infants, regardless of the cause or age at onset, presents diagnostic and therapeutic challenges. Infants with PPHN, a syndrome of failed circulatory adaptation at birth, have benefited from advances in basic and clinical research, culminating in late 1999 in the approval of iNO for treatment of PH in newborns of 35 weeks’ gestation and older. This inhalation therapy represented the first evidence-based medical therapy specific for PH in a pediatric population. It is difficult to overestimate the importance of this therapeutic advance to the field of neonatology. Yet, up to 40% of infants with PPHN and most infants with later-onset PH associated with lung and heart disease are refractory to short-term iNO therapy. These infants with persistent and often progressive PH represent the next frontier for PH researchers in neonatology and are a focus of this chapter. For infants with PH that persists beyond the first week of life, a more systematic approach to the surveillance, diagnosis, identification of risk factors, and assessment of disease sever-

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ity and progression are needed for the development and application of new therapies. Advancements in this field will require a better understanding of normal and abnormal vascular development, the processes involved in vascular remodeling, and the molecular mechanisms governing cross talk between the developing lung parenchyma and the pulmonary vasculature. For neonatal patients with PH caused by pulmonary hypoplasia, such as infants with congenital diaphragmatic hernia (CDH) or severe bronchopulmonary dysplasia (BPD), the therapeutic Holy Grail is reprogramming of disturbed pulmonary vascular and alveolar development. Innovative therapies are needed to prevent and reverse progressive pulmonary vascular disease and disturbances in cardiac performance while stimulating new lung growth. With a focus on infants with extreme forms of CDH and BPD as the poster children for persistent and progressive PH in early infancy, we will discuss the (a) pathophysiology, (b) clinical presentation, (c) assessment, and (d) management from the vantage point of the pediatric subspecialists most often involved in their NICU and post-hospital-discharge care: the neonatologist, the pediatric cardiologist, and the pediatric pulmonologist.

5 Congenital Diaphragmatic Hernia 5.1 Pathophysiology of CDH CDH is a life-threatening congenital anomaly resulting from failure of the posterolateral (Bochdalek), anterior/midline (Morgagni), or crural (paraesophageal) diaphragm to fuse with the chest wall. In the case of complete diaphragmatic agenesis, there is total absence of a muscular diaphragm. In 80% of infants with CDH, the defect is on the left side. The incidence of CDH is approximately one in 2,500 live births [30, 31]. Prenatal herniation of bowel loops and/or liver into the thorax compresses the lungs, shifts the mediastinum and leads to hypoplasia of both the ipsilateral and the contralateral lung. The anatomic pulmonary vascular changes in patients with PH associated with CDH include a decreased number of pulmonary arteries per unit lung volume, intra-acinar extension of smooth muscle cells, and increased medial hypertrophy with extensive collagen deposition in the media and adventitia of pulmonary arteries [32, 33]. In utero lung compression acting either directly or indirectly (by decreasing pulmonary blood flow) to inhibit lung growth is thought to be responsible for many of the pulmonary vascular structural changes; however, in utero compression may be too simplistic an explanation. Abnormal lung development and fundamental alterations in the pulmonary circulation may constitute a primary abnormality in CDH that is not entirely

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attributable to lung compression [34]. This is supported by the impaired contralateral lung development which occurs independently of compression by intrathoracic viscera. Indeed, there are reports of altered expression of proangiogenic factors in the lungs of infants with CDH [35–37]. Retinoids, which have been shown to play a role in lung development, have also been implicated in the pathogenesis of CDH [38]. Diaphragmatic hernias occur in 25–49% of the offspring of rats with vitamin A deficient diets [39–41]; this incidence is reduced when vitamin A supplementation is provided during gestation. CDH is also produced in doublenull-mutant mice that lack both retinoic acid receptors, RARa and RARb [42, 43]. Nitrofen (2,4-dichlorophenyl-p-nitrophenyl ether), a teratogen that induces CDH in rodents [44–47], inhibits retinal dehydrogenase [48] and suppresses activation of the retinoid response element; these effects can be antagonized in vitro by supplemental retinoic acid. The coadministration of vitamin A and nitrofen reduces the incidence of CDH by 15–30% and attenuates lung hypoplasia [49, 50]. In a small human study of infants with CDH, retinol and retinolbinding-protein plasma levels were reduced in umbilical cord blood [51]. These findings justify a search for candidate genes related to retinoid signaling as a genetic cause of CDH [38]. Unfortunately, the genetic and molecular mechanisms responsible for the abnormal lung parenchymal and vascular development of infants with CDH remain unknown. The nitrofen-induced CDH model in the rat [52, 53] and the surgically induced CDH model in fetal lambs [54, 55] have been used to evaluate functional abnormalities in the pulmonary circulation of animals with CDH. In both of these animal models, pulmonary vascular responses are abnormal [54, 56]. In the rat model, functional abnormalities in the NO-cyclic GMP (cGMP) pathway have been identified [52, 57, 58]. Some [59] but not all studies in the lamb model substantiate abnormalities in the NO-cGMP signaling pathway [54, 55]. Elevated pulmonary expression of endothelin-1 (ET-1) and endothelin A (ETA) receptors [53] and exaggerated pulmonary arterial responses to ET-1 were found in the rat model [60]. ET-1 receptor activation favoring pulmonary vasoconstriction also contributes to PH in the lamb model [61, 62]. Elevated circulating levels of ET-1 [63], greater lung expression of ETA than ETB receptors [64], decreased lung endothelial NO synthase (eNOS) expression [37], elevated urine levels of the competitive NO synthase inhibitor, asymmetric dimethylarginine [65], and elevated levels of the prostanoid constrictor, thromboxane [66, 67], have been reported in human infants with CDH. These latter reports suggest that an imbalance between vasodilators and vasoconstrictors is functionally relevant to PH in human infants with CDH. Moreover, via influences on smooth muscle cell mitogenesis, alterations in production of vasoactive mediators could contribute to the structural vascular abnormalities found in infants with CDH [68].

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5.2 Clinical Presentation of Infants with CDH Today, most infants with CDH are diagnosed prenatally by ultrasound, allowing for prenatal counseling and delivery of the infant in a center with the multidisciplinary team of neonatologists, geneticists, pediatric cardiologists, pediatric surgeons, and pediatric anesthesiologists required for optimal management of symptomatic newborns. The morbidity and mortality from CDH is related to the pulmonary and vascular hypoplasia and the presence of other major anomalies, including cardiovascular defects, intestinal atresias, and chromosomal anomalies, not to the anatomic diaphragmatic defect per se. Even in the absence of other congenital anomalies, the degree of lung hypoplasia is extremely variable with a resultant mortality rate that ranges from 8 to 80% [69]. The precise incidence of PH in infants with CDH is not known. However, PH complicates the course of CDH in many newborns; the presence and persistence of PH contributes significantly to the high rate of morbidity and mortality in this population [70, 71]. The clinical presentation of infants with CDH is not entirely predictable [70, 72]. Infants with CDH at birth may have acute hypoxemic respiratory failure and PPHN or may be asymptomatic, particularly if herniation of abdominal contents does not occur or occurs late in gestation. Some infants with CDH and severe PPHN experience little to no postnatal decline in PVR. The resultant right-to-left shunting of blood across the patent ductus arteriosus and foramen ovale results in life-threatening limitations in pulmonary blood flow and severe systemic hypoxemia. These infants may have urgent, preoperative need for support with extracorporeal membrane oxygenation (ECMO). Other infants have marked improvements in respiratory function over the first few days to weeks of life with a progressive fall in PVR, undergo later surgical repair, and recover uneventfully [73]. Commonly, however, infants with CDH have unresolved pulmonary vascular abnormalities with persistence of elevated PAP [70, 74]. Not all of these infants do poorly. It has been estimated that of those infants with a persistently elevated PAP, 50% slowly resolve their PH over weeks to months without evidence of longterm pulmonary compromise [70]. For other infants, the course of chronic PH may be accompanied by acute exacerbations that necessitate extreme measures including a second course of ECMO. Moreover, there are reports of infants who are discharged and months later are identified as having severe PH [72]. The variability and breadth of the clinical spectrum of PH and the underlying structural alveolar and pulmonary vascular abnormalities in infants with CDH makes them a challenging group of patients to diagnose, treat, and study.

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5.3 Assessment of PH in Infants with CDH For infants with CDH and a patent ductus arteriosus, the diagnosis of PPHN can be presumptively made on the basis of a preductal versus postductal saturation gradient of 5% or more. However, absence of a preductal/postductal saturation gradient does not preclude PPHN. An atrial level shunt, in the absence of a ductal level shunt, will fail to produce differential preductal and postductal oxygen saturations, but will result in venous admixture and hypoxemia. Most institutions rely on the use of 2-D echocardiography with color flow Doppler imaging demonstrating a right-toleft shunt at the ductal and/or atrial level, bowing of the atrial septum, or tricuspid regurgitation to diagnose PPHN in infants with CDH. As noted previously, lack of standardization of echocardiography or other noninvasive techniques for initial diagnosis and ongoing assessment of PH severity and response to therapy in neonates is a barrier to advances in clinical management and design and interpretation of clinical trials.

5.4 Management of Infants with CDH The optimal strategy for medical and surgical treatment of CDH is controversial and has been insufficiently studied. Assessment of airway patency and the extent of alveolar recruitment and lung hypoplasia is as important as determination of the degree of PH. Likewise, evaluation of myocardial function, including filling volumes, myocardial contractility, and diagnosis of structural cardiac abnormalities, is critical to the optimal management of infants with CDH. Poor cardiac function and ventilation–perfusion mismatch are common reasons for failed medical management. The potential for severe, acute PPHN in the first few hours and days of life and the knowledge that the course of PH may be protracted for days to months drives many of the current clinical strategies used to treat infants with CDH. Surgical repair is generally delayed for 24 h or longer to allow for a postnatal decline in PVR to occur. A common approach is to carefully manage the cardiopulmonary support of these infants, minimizing iatrogenic lung damage while waiting for clinically significant PH to abate over time [75, 76]. In accordance with this approach, gentle ventilation and mild permissive hypercapnia are fundamental parts of the treatment strategy for CDH in many institutions [30, 71]. High-frequency ventilation is sometimes incorporated as part of the gentle ventilation strategy [71, 76, 77]. Successful management of infants with CDH and PH must address both the alveolar and the vascular components of their disease. Failure of any infant with PH, including

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those with CDH, to respond to medical management should trigger careful reassessment of alveolar recruitment and myocardial function and institution of appropriate targeted therapies, which might include surfactant, antibiotics, lung volume recruitment strategies, and judicious use of volume replacement and inotropic support. On the basis of a number of studies that have implicated abnormal or delayed surfactant synthesis in infants with CDH [78–80], surfactant is sometimes used as an adjunct to optimize ventilation. Unfortunately, administration of surfactant has not been shown to improve lung compliance, time to extubation, or survival in infants with CDH [81, 82]. Moreover, PH, whether acute, late, or chronic [72], can be so severe that the infant fails to improve after all conventional therapies, including all modes of ventilation. In these cases, ECMO may be used as an alternative strategy to stabilize the infant and optimize gas exchange while waiting for the PH to resolve. Use of ECMO is done with the knowledge that the time period for resolution of PH is not known and may exceed the current limitation of this invasive, supportive measure. Indeed, although nonrandomized studies support its use, there are no conclusive data to support or refute the use of ECMO in infants with CDH [69]. As an additional supportive measure, pulmonary vasodilator therapy is often used prior to, concomitant with, or following ECMO, even though no such therapy, including iNO therapy, has been proven to be efficacious in this patient population. Initial case reports suggested that iNO therapy could improve oxygenation in infants with CDH. It is disappointing that in large randomized, placebo-controlled trials early administration of iNO failed to reduce death or ECMO utilization in CDH patients [83]. Indeed, some studies suggest increased ECMO utilization in CDH patients treated with iNO [84]. Downstream signaling abnormalities in the NO-cGMP pathway, including decreased soluble guanylate cyclase activity, have been identified in experimental CDH [59]. Limited evidence indicates that iNO may be useful in stabilizing the infant for transport or ECMO [83, 85]. However, careful evaluation of left ventricular performance has been recommended when iNO is used in patients with CDH [72] because in some infants left ventricular output (and left ventricular mass) is diminished, causing a RV-dependent systemic circulation. These patients may benefit from therapy with afterload-reducing agents and/or prostaglandin E1 to open or maintain ductal patency to prevent RV failure [86, 87]. There is little information about the efficacy of other pulmonary dilators in infants with CDH. Prostacyclin was reported to be helpful as a temporary bridge to ECMO [88]. The phosphodiesterase type 5 (PDE5) antagonist, sildenafil, was successfully used to wean an infant from iNO [89]. Whether any pulmonary vasodilator will have long-term benefit in CDH patients with acute, protracted, or chronic PH remains to be shown.

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5.5 Management of the Patient with CDH and PH After NICU Discharge There is a paucity of data concerning the optimal approach to transition of the infant with PH following CDH repair from the NICU to outpatient management. What is clear, however, is that the presence of PH portends a worse outcome [70]. Infants with CDH and persistent PH who survive to NICU discharge typically struggle with respiratory, neurologic, and gastrointestinal comorbidities, and often fail to thrive [87]. They require a cohesive approach to follow-up care by multiple pediatric subspecialists, as these comorbidities can exacerbate the underlying PH. Echocardiographic surveillance and in some cases cardiac catheterization with or without bronchoscopy are required to screen for associated pulmonary arterial or venous stenoses in the affected lung [72]. No studies to date have prospectively evaluated the utility of sildenafil, bosentan, calcium channel blockers, anticoagulants, or other oral pharmacologic agents in this population. iNO delivered by nasal cannula has been offered as longterm treatment to enhance resolution of protracted PH in newborns following CDH repair [84]. Use of the PDE5 inhibitor, sildenafil, has also been reported in this population [89]. However, clear guidelines for the dosage, duration, or weaning strategy of any pulmonary vasodilator for children with CDH have not yet been established. For those in whom vasodilator therapy is instituted, we recommend serial echocardiography after baseline cardiac catheterization and follow-up catheterization performed prior to discontinuation of vasodilator therapy, usually within 6–12 months. Consideration should be given for evaluation of extended use of iNO and other agents for promoting lung growth and resolution of vascular remodeling beyond their potential acute vasodilator properties.

6 Bronchopulmonary Dysplasia 6.1 Pathogenesis of PH Associated with BPD Neonatal chronic lung disease is an important cause of morbidity and mortality in the USA. The vast majority of chronic lung disease in newborns is due to BPD, currently (and inadequately) defined as the need for supplemental oxygen at 36 weeks’ corrected age of gestation. Most infants who develop BPD are born at a gestational age of less than 30 weeks and have a birth weight of less than 1,500 g. It is well known that infants with BPD are at significant risk for a variety of longterm health problems, including lifelong alterations in lung

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function [90]. Infants with BPD are also at risk of developing cardiovascular complications, including chronic PH and cor pulmonale. Despite decades of research, the pathogenesis of the airway and alveolar damage that characterize BPD is incompletely understood [90]. Hence, it is not surprising that it remains unknown both why and when some infants with BPD develop clinically significant PH. All newborns, both preterm and term, are at risk of developing PH when exposed to injurious stimuli because the lung, even in term infants, is not fully developed at birth. A significant amount of alveolar and vascular development occurs between birth and approximately 2–4 years of life [91]. Whether the development of the blood vessels in the lung passively follows that of airways or whether lung blood vessels promote alveolar growth is not yet certain [91–93]. Regardless, when exposed to injurious stimuli, both alveoli and intra-acinar vessels may fail to develop normally [92, 93]. Similar to many types of PH in older children and adults, vascular remodeling and increased vasomotor tone contribute to PH in infants with BPD [92, 94, 95]. Features of vascular remodeling in infants with BPD include distal extension of smooth muscle, thickening of the media and adventitia, and excessive accumulation of matrix protein in the pulmonary vessel walls [92, 95, 96]. These structural changes elevate PVR by narrowing vessel diameter and by decreasing vascular compliance. Although not well studied, the intimal hyperplasia and plexiform lesions seen in adults with PH are not commonly found in the pulmonary vasculature of infants with BPD [97]. The precise stimuli responsible for either pulmonary vascular remodeling or abnormal tone in infants with BPD are not known but include all of the factors being considered for maldevelopment and damage to the airways and alveoli, of which hyperoxia and inflammation are the most often cited. Notably, prolonged or intermittent hypoxia, which is not generally considered to be a major contributor to airway and alveolar disease, is felt to play a prominent role in the pathogenesis of PH in infants with BPD [96, 98]. It is possible that the diminished intra-acinar vessel development that is believed to be present in infants with BPD contributes to PH, at least in part, by impairing gas exchange. However, reports of markedly increased angiogenesis in lungs of ventilated preterm infants make the role of intra-acinar vascular growth arrest in the pathogenesis of PH in infants with BPD uncertain [99]. The molecular and genetic mechanisms responsible for the pulmonary vascular abnormalities that contribute to PH in infants with BPD are not yet known. Newborn rodent and premature lamb and baboon animal models have been developed to study the mechanisms underlying arrested alveolar and intra-acinar vessel growth and to evaluate possible treatment strategies to restore lung growth. Reduced NO production has been implicated in the pathogenesis of the impaired

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alveolar and vascular growth found in these animal models [100–103]. Notably, long-term treatment with iNO improved lung structure in all of these animal models [102–105]. Studies in eNOS knockout mice suggest that a deficiency of eNOS leads to a failure of lung vascular and alveolar growth; prolonged iNO treatment restores lung growth in this animal model as well [106]. Vascular endothelial growth factor (VEGF) is an endothelial-cell-specific survival factor that stimulates angiogenesis and protects endothelial cells against injury. NO is a downstream mediator of VEGF-dependent angiogenesis [107]. VEGF receptor (VEGFR) activation increases the expression of eNOS messenger RNA and protein, and stimulates NO release [108]. Inhibition of the VEGFR reduces alveolarization and vascular growth, resulting in histologic changes in lung structure that mimic BPD [109]. Decreased lung VEGF messenger RNA and protein expression, as well as a reduction of the VEGFR-1, has been described in the lungs of infants with fatal BPD [110]. Decreased lung VEGF and VEGFR expression has also been found in the primate model of BPD [111]. iNO treatment after hyperoxia or exposure to the VEGFR inhibitor, SU-5416, prevents PH and improves distal lung growth in rats. These results shed light on possible mechanisms by which prolonged iNO treatment improves pulmonary outcomes in some preterm infants at risk for BPD [112, 113]. Unfortunately, randomized controlled trials performed in preterm infants have not shown consistent efficacy of iNO in preventing BPD in human infants [112–115]. It seems likely that the effects of iNO in the premature human infant will depend on timing, dose, duration of therapy, and underlying disease. Identifying which infants, if any, may benefit from iNO therapy to prevent or treat evolving BPD remains a challenge in neonatology. Randomized controlled trials of iNO to treat established severe BPD with documented PH have not been conducted. Animal models have also been used to evaluate the mechanisms underlying abnormal pulmonary vasomotor tone and pulmonary vascular remodeling in infants with BPD. Pulmonary vascular remodeling similar to that found in human infants with BPD develops in newborn piglets exposed to chronic hypoxia [116] and in preterm lambs that are chronically ventilated [100]. Pulmonary vascular responses are abnormal in both of these animal models and are due in part to impairments in NO signaling [100, 117]. Altered pulmonary vascular production of other known vasoactive mediators, including prostanoids and ET-1, has also been found [118, 119]. Alterations in any of these vasoactive agents could impact vascular remodeling via an influence on smooth muscle cell mitogenesis. Indeed, the relative contributions of vasomotor tone and vascular remodeling to the pathogenesis of PH in these animal models or in human infants with BPD are not known. Inhibiting ET-1 signaling

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was shown to ameliorate pulmonary vascular remodeling but had minimal effect on the development of PH in chronically hypoxic newborn piglets [119]. This latter study indicates that although remodeling may be important, the contribution from vasomotor tone should not be discounted and likely plays a prominent role in the development of PH in infants with BPD.

6.2 Clinical Presentation of PH in Infants with BPD Clinical signs and symptoms of PH in infants with BPD are nonspecific and difficult to distinguish from the underlying chronic lung disease. An awareness of the risk of PH in these infants is crucial. In particular, infants who have persistent oxygen requirements, recurrent cyanotic episodes, poor growth, or are unable to wean from ventilatory support should be considered at high risk for developing PH. Unfortunately, the incidence of PH in infants with BPD is not known. Nor is there an ability to predict which infants with BPD will develop clinically significant PH. Echocardio­graphic studies during the first few days and weeks of life are not predictive of BPD and/or the postnatal development of PH [120]. Although premature infants with severe respiratory distress often have elevated PVR in the first hours and days of life, resolution of the acute lung disease is generally accompanied by a decline in PVR to normal postnatal levels [121]. However, if the infant develops BPD over the ensuing weeks to months of life and is subsequently found to have an elevated PAP, the outcome can be quite poor. In the 1980s, the presence of persistent echocardiographic evidence of PH beyond the first few months of life was associated with up to 40% mortality in infants with chronic lung disease [122, 123]. Sadly, this high risk of death has not improved over the past few decades. In a 2007 publication, the diagnosis of PH in an infant with chronic lung disease was associated with a survival rate of 64% at 6 months and 53% at 2 years [124]. Yet, not all infants with BPD and an elevated PAP do poorly. Despite a high prevalence of subclinical PH in infants with BPD [125, 126], the increased PVR associated with BPD appears to resolve with time in many infants [127]. At present, there is no reliable way to differentiate between those infants with BPD who will resolve their PH and those who will suffer a progressive course accompanied by right-sided heart failure.

6.3 Assessment of PH in the Infant with BPD Although the prevalence of PH in infants with BPD is unknown, anecdotal data suggest that the condition is

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underappreciated and underdiagnosed. There are no standardized definitions of PH in infants of differing postnatal ages and with differing underlying conditions. Nor are there consensus recommendations for how to screen for this problem or how to prospectively follow infants at risk for or with documented PH. These issues must be addressed before progress can be made on the therapeutic front. Cardiac catheterization, the standard method for assessing pulmonary vascular disease in older children and adults, is considered by some to be too invasive for routine use in the neonatal population. Cardiac catheterization is generally reserved for those BPD patients with severe cardiorespiratory disease and concern for significant PH despite optimal clinical management, or to exclude or document anatomic cardiac lesions. Instead, echocardiography is commonly used to screen for and follow PH in infants with BPD. Unfortunately, there is poor correlation between echocardiographic and cardiac catheterization findings. The absence of TR jet velocity on sonographic evaluation cannot rule out the presence of severe PH in a neonate [9].

6.4 Management of PH in Infants with BPD No randomized clinical trials have evaluated therapies for PH in infants with BPD. For decades, treatment has focused on trying to resolve the underlying respiratory disorder and to judiciously using oxygen as a vasodilator. The amount of oxygen to use and the appropriate oxygen saturation targets are not known. Many institutions target oxygen saturations of approximately 95% [128, 129]. This target is based on one study in infants with BPD and PH that showed that during cardiac catheterization the maximal decrease in PAP was achieved when the oxygen saturation exceeded 95% [130]. However, another study found little improvement in PAP when oxygen saturation increased from 93 to 98% [131]. Data from a cardiac catheterization study in older infants with PH associated with severe BPD support the avoidance of hypoxia, but do not suggest that hyperoxia is likely to cause further improvement in PH as little further decrease in PAP occurs with a pO2 greater than 70  mmHg [132]. The desire to achieve a maximal decline in PAP must be counterbalanced by concern about oxygen toxicity; oxygen therapy may not only worsen parenchymal lung disease, but may contribute to the development of retinopathy of prematurity in at-risk preterm infants. At this time there is no evidence to support a pO2 target that exceeds 70–80  mmHg in infants with BPD; neither the safety nor the efficacy of a higher oxygen target has been demonstrated. Longitudinal studies comparing progression of PH at various targeted oxygen saturations are not available to guide therapy. Avoidance of hypoxemia and its associated acute increase in PAP is,

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however, a commonly accepted goal of oxygen therapy. To achieve this, the knowledge that oxygenation varies with activity, feeding, and during sleep must be considered, particularly for infants who are not improving as they could be suffering from unrecognized episodes of hypoxia. Experience with vasodilators other than oxygen to treat PH in infants with chronic lung disease is limited. As previously mentioned, none have been studied in a randomized controlled trial. The response to iNO in infants with BPD is variable, but some patients appear to clinically improve, at least temporarily [133, 134]. The reasons why some infants fail to respond to iNO are not well understood, but are likely related to (a) dysfunctional signaling downstream of NO release or delivery, (b) abnormalities in other vasoactive pathways, and/or (c) vascular remodeling or maldevelopment. Endothelial dysfunction leading to increased expression of ET-1 and reduced synthesis or activity of the vasodilators NO and prostacyclin are thought to play an important role in vasoconstriction and progressive vascular remodeling. Novel therapies manipulating these three pathways have formed the core of therapeutic advances in the treatment of PH in older children and adults. Success in older patients coupled with a low response rate to iNO in infants with severe BPD and PH has fueled interest in these therapies for this population. The PDE5 inhibitor, sildenafil, appears to be well tolerated and may be an effective therapy in these infants both in the NICU and after discharge [135, 136]. Ease of administration makes it an attractive choice for more chronic forms of postnatal PH associated with pulmonary maldevelopment or vascular remodeling, such as BPD. Long-term efficacy and safety studies of sildenafil in infants and young children should be a high priority. Studies of longer-acting PDE5 inhibitors are also needed. Oral therapy with the nonselective endothelin receptor antagonist, bosentan, may also be beneficial, but concerns about liver toxicity and lack of readily available oral suspensions limit the potential for widespread use of this agent in neonates [135]. There are almost no published data on the use of endothelin receptor antagonists in neonates with PH. Despite this, bosentan is being prescribed for refractory PH associated with CDH, BPD, and CHD. There is an urgent need for data on the efficacy and safety of endothelin antagonists in this population and for randomized controlled trials as monotherapy or as adjunctive therapy for long-term management of neonatal and pediatric PH. No recommendations can be made for any of these therapies as none have been adequately studied. Therapy for these infants is further confounded by the realization that responses to various dilator therapies may vary with the stage of BPD and pulmonary vascular disease as the relative contribution of functional and structural changes to PH in infants with BPD likely changes with evolution of the disease process [137]. In many cases, the infant

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with progressive BPD experiences a normal transition with initial successful adaptation of the pulmonary circulation to postnatal life. With subsequent injury, a vasoconstrictive component of early PH develops and predominates. Over time, structural changes occur so that a fixed, vasodilatorunresponsive component develops [94]. Nonetheless, there is evidence that high PVR continues to play a prominent role in older infants with BPD, on the basis of studies showing their responsiveness to altered oxygen tension and iNO [131, 132]. The awareness of a potentially reversible vasoconstrictor component should be kept in mind, even in infants with advanced stages of BPD and PH. An association between pulmonary vein stenosis and chronic lung disease of prematurity has recently been appreciated (Fig. 3). Drossner et al. reviewed the occurrence of pulmonary vein stenosis over a 10-year period at their institution [138]. They reported 26 such cases with an estimated occurrence of 1.7 per 100,000 children under the age of 2 years. This was felt to be an acquired disorder strongly associated with preterm birth. Surgical and percutaneous treatment outcomes were universally poor, with a 2-year survival rate after diagnosis of 43%. This particular acquired, and often fatal, vascular abnormality must be considered in the setting of PH associated with BPD in premature infants prior to instituting medical therapy. At all stages of disease, avoidance of vasoconstrictive stimuli, such as hypoxia, should be foremost in treating these infants. Vigilance for gastroesophageal reflux, dysphagia, and any potential cause of aspiration as well as prophylaxis for respiratory diseases, such as that from respiratory syncytial virus, are warranted.

Fig.  3  MRI 3D reconstruction from an 8-month-old infant seen at Vanderbilt Children’s Hospital with severe bronchopulmonary dysplasia (BPD) and suprasystemic right ventricular pressures. The posterior view demonstrates diminutive left pulmonary veins (small arrows) and engorged right pulmonary veins (asterisks). The majority of blood flow is directed to the already cystic and severely damaged right lung

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6.5 Management of the Patient with BPD and PH After NICU Discharge Patients with BPD and PH benefit immensely from a wellplanned, well-communicated, multidisciplinary approach to the transition from the NICU to the outpatient setting. Involvement of a pediatric pulmonologist and a pediatric cardiologist prior to NICU discharge greatly facilitates discharge planning and can ease the anxiety of parents who are taking home a child on supplemental oxygen after what is commonly a hospital stay of many months’ duration. At the Vanderbilt Pediatric Pulmonary Hypertension Center, patients with BPD are seen every 4–6 weeks by a pulmonologist, and every few months jointly by a pediatric cardiologist and a pediatric pulmonologist. Although there are currently no published screening or management guidelines for patients with BPD following NICU discharge, Doppler echocardiography is used in the Vanderbilt Pediatric Pulmonary Hypertension Center as the surveillance method of choice. All BPD patients with an ongoing oxygen requirement followed in our outpatient clinic have an echocardiogram performed at the first office visit if not obtained within 1 month of NICU discharge. Other indications for PH screening by echocardiogram include (a) persistent oxygen requirement out of proportion to the severity of lung disease, (b) persistent poor growth despite adequate caloric intake, (c) frequent pulmonary exacerbations, and (d) coexistent CHD. Growth is critical for the postnatal maturation of the lungs, and is particularly important for infants and children with PH secondary to BPD who frequently have postnatal growth failure. However, nutrition is frequently a challenge for these patients. BPD patients frequently suffer from dysphagia and gastroesophageal reflux disease, both of which can impair adequate growth and damage the lungs. The multidisciplinary approach to these children at the Vanderbilt Pediatric PH Center not only includes close collaboration between the pediatric cardiologist and the pediatric pulmonologist, but also the dietary specialists. For some children, parenteral nutrition or continuous gastric feeds at night must be pursued to maximize growth. Rigorous investigations of pharmacologic agents to treat PH in this diverse patient population are needed and hold promise for the future. As many of these children are seen in academic pediatric PH centers, the opportunities for collaborative clinical trials are great. At the moment, however, the therapeutic approach remains center-specific and uncontrolled. At a minimum, these centers are obligated to collect data and share their experiences. Enrollment of these patients into pediatric-specific registries, such as the TOPP Registry (tracking outcomes in pediatric PH) through the Association for Pediatric Pulmonary Hypertension

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(https://www.peph-association.org/association.jsp) will provide insights into current practice and is strongly encouraged. Chronic therapy with PDE5 inhibitors is being increasingly used, with perceived success by many centers, including ours. Mourani et  al. recently reported that sildenafil, when used as part of an aggressive program to treat PH in children under 2 years with chronic lung disorders such as BPD and CDH, was associated with echocardiographic improvement of PH in most cases (88%) without significant rates of adverse events [136]. Confirmation of these findings in a randomized, placebo-controlled trial is needed before use of sildenafil can be endorsed in this population.

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7 Pediatric PH That Presents or Persists Beyond the Neonatal Period The pediatric pulmonologist caring for children outside the neonatal period sees a wide variety of chronic lung disorders, the majority of which do not present with PH. However, owing to the intimate relationship between the pulmonary and cardiovascular systems, most severe lung diseases of children may cause, or coexist with, PH. Table  1 lists the major categories of pulmonary diseases associated with PH, organized according to age at presentation and disease processes. Although the degree to which each disease process

Table 1  Major categories of pulmonary diseases associated with pulmonary hypertension (PH), organized according to age at presentation and disease processes Infancy and early childhood Older childhood and adolescence Disorders of lung development Congenital diaphragmatic hernia Bronchopulmonary dysplasia Pulmonary hypoplasia Acinar dysplasia Congenital alveolar dysplasia Alveolar capillary dysplasia Multisystem chromosomal disorders

Obstructive chronic lung diseases Bronchopulmonary dysplasia Cystic fibrosis Bronchiolitis obliterans Postinfectious Posttransplant (lung, hematopoietic stem cell transplantation) Severe, chronic aspiration Bronchiectasis

Childhood interstitial lung diseases Surfactant metabolism and related interstitial lung disorders SFTPB mutations SFTPC mutations ABCA3 mutations Pulmonary alveolar proteinosis Chronic pneumonitis of infancy Desquamative interstitial pneumonitis Nonspecific interstitial pneumonitis Diffuse interstitial lung disorders of unclear origin Neuroendocrine cell hyperplasia Pulmonary interstitial glycogenolysis

Childhood interstitial lung diseases Surfactant metabolism and related interstitial lung disorders SFTPC mutations ABCA3 mutations Pulmonary alveolar proteinosis Desquamative interstitial pneumonitis Nonspecific interstitial pneumonitis

Severe airway disorders Profound malacia Tracheoesophageal fistula with severe lung disease Severe, chronic aspiration Intrinsic or extrinsic causes of airway obstruction

Disorders of sleep Obstructive sleep apnea Sleep-disordered breathing

Chronic hypoventilation Neuromuscular diseases Chest wall abnormalities Abnormal diaphragm function Congenital central hypoventilation syndrome (Ondine’s curse)

Chronic hypoventilation Neuromuscular diseases Chest wall abnormalities Abnormal diaphragm function

Diffuse interstitial lung disorders of unclear origin

Hemoglobinopathy-associated lung disease Sickle cell lung disease Chronic hypoxia due to low FiO2 Chronic high-altitude exposure Miscellaneous Sarcoidosis Systemic autoimmune diseases Storage diseases

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relates to PH is heterogeneous, there is growing recognition that the coexistence of lung diseases and PH is associated with a high morbidity and mortality [124, 139–142]. Although somewhat contrived, the causes of PH in children can be organized according to guidelines in adults, which include categories such as chronic obstructive pulmonary disease, interstitial lung disease, disorders of hypoventilation, and developmental abnormalities [3]. Despite the category similarities, the specific causes of PH associated with lung disease in children are different from those in adults, and include disorders of lung development with diagnoses such as BPD, CDH, pulmonary hypoplasia, and alveolar capillary dysplasia, childhood interstitial lung disease (ChILD), obstructive sleep apnea (OSA), congenital central hypoventilation syndrome, and SCD (Table 1). It is critical that the pediatric pulmonologist recognize the association of chronic lung disorders and PH, so as to facilitate diagnosis and treatment, reduce the number of aggravators and environmental triggers, and optimize disease-specific care. The prevalence and severity of PH varies according to the underlying chronic lung disease process. This likely reflects a variety of factors, some unique to the pediatric population, such as the developmental timing of lung disease initiation [143]. It has become increasingly clear that disruption of airway development may have devastating consequences for the developing pulmonary vasculature, such as described earlier for infants with BPD and CDH, because the processes of airway and pulmonary vascular development are intimately intertwined [92]. Fortunately, the developing lung appears to have a greater potential for recovery following the removal or dissipation of an insult, which may contribute to the resolution of PH over time seen in some situations, such as mildto-moderate BPD [144]. However, the presence of PH in association with a severe irreversible lung disease, such as cystic fibrosis (CF) or severe BPD, portends a poor prognosis [139].

7.1 Clinical Presentation of PH in Pediatric Patients with Chronic Lung Disorders Similar to patients with BPD, PH in older pediatric patients with chronic pulmonary disease is often clinically undetectable early in its course. However, acute pulmonary exacerbations, exercise, and sleep-disturbed breathing often reveal an underlying tendency toward increased PAP and RV dysfunction. For example, during acute respiratory exacerbations in patients with chronic obstructive pulmonary disease, mean PAP rises by as much as 20 mmHg, but returns to baseline patient values with recovery [145]. This

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variability makes routine assessment by noninvasive measures, which, as noted already, are poor estimates of pulmonary vascular disease, even less sensitive in determining the presence of PH in children with chronic lung disorders. Clinicians caring for patients with chronic lung disorders must have a heightened awareness that PH can develop at any time in a child’s clinical course and proactively pursue its detection. It is likely that earlier diagnosis of PH may facilitate more successful management and outcomes, although there is a paucity of data to definitively support this conclusion in most types of childhood chronic lung disorders.

7.2 Assessment of PH in Pediatric Patients with Chronic Lung Disorders Diagnosis of pulmonary vascular disease requires a high index of suspicion, and recognition that symptoms of PH may be subtle. Because the clinical course in children with chronic respiratory disorders is frequently characterized by exacerbations, symptoms and signs of PH may be missed or inappropriately attributed to the underlying lung disease. This is particularly true for infants and young children, such as those with BPD, CDH, ChILD, and pulmonary hypoplasia [70, 130, 146]. As a result, routine surveillance for PH is a critical component of management. Chest radiographs may suggest an enlarged cardiothymic silhouette, and electrocardiography may suggest RV strain and hypertrophy, but neither of these are sensitive tools for the diagnosis of PH in children [147]. At present, Doppler echocardiography is the preferred method for detecting PH in pediatric patients, given its noninvasive nature. However, for most chronic lung disorders which may cause secondary PH, there are few studies to definitively determine when and how often to perform surveillance PH screening. The value of biomarkers, such as plasma brain natriuretic peptide (BNP), atrial natriuretic peptide (ANP) and troponin, for identifying the onset or changing severity of PH in children is also unclear. However, there is growing interest in the need for such biomarkers as noninvasive measures to supplement echocardiographic assessments. For example, Bernus et al. recently demonstrated the utility of serial BNP levels as a means of monitoring for hemodynamic compromise over time in pediatric patients with PH [148]. One or several biomarkers used in combination as a mechanism for biologic surveillance of the child at risk for or with established PH would be a tremendous advancement. This is particularly the case for children with BPD, ChILD, CF, SCD, and profound disorders of sleep.

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7.2.1 Childhood Interstitial Lung Disease (ChILD)

7.2.3 Sickle Cell Disease

Advances in the study of ChILD have placed this spectrum of disorders at the forefront of pediatric pulmonary research in recent years, and it is apparent that the presence of PH is associated with increased mortality [146]. However, the prevalence of PH among these patients has not been definitively determined. A recent multicenter study of ChILD among children less than 2 years of age found that only 5% of 187 patients with ChILD had symptomatic PH; however, subjects with PH had a 6.8-fold higher odds of death [142]. The findings of an accompanying study of older children have not yet been published. Surveillance echocardiography should be routinely performed in this patient population at the time of diagnosis, as well as on a periodic basis similar to the guidelines proposed later herein for BPD patients. In addition, serial overnight oxygen saturation monitoring may help identify underappreciated hypoxemia, especially during sleep, which may predispose to PH.

PH is a well-known complication of hereditary hemolytic anemia syndromes, including SCD. In contrast to adult studies and pertinent to the pediatric pulmonologist, there is no detectable association between abnormal pulmonary function test results and PH in children [153]. Nearly 30% of adults with SCD have PH, and its presence is associated with a tenfold increased risk of death; in children retrospective studies have estimated a prevalence between 16 and 26% [8, 154–156]. This estimate was recently confirmed in a prospective investigation of children which found evidence of elevated PAPs of 30 mmHg or more at rest among 30% of patients evaluated by echocardiography. Although the mean age at diagnosis was 13.3 years, the youngest patient was 6.8 years and there was no correlation between age and estimated PAP [157]. A subsequent follow-up study of 18 of the 19 patients with PH identified the following risk factors for persistent PH: higher TR jet velocity on initial echocardiogram, lower hemoglobin levels, and lower oxygen saturation [158]. These findings are consistent with adult studies, which have emphasized the severity of hemolytic anemia as a particular risk factor [159]. As with adults, children with SCD should be screened for PH using Doppler echocardiography [8]. However, the optimal timing of screening is unclear. Children with profound ongoing hemolysis, nighttime hypoxemia, sleep-disordered breathing, hepatic dysfunction, and renal failure are at particular risk for PH and should be screened [159]. We also suggest Doppler echocardiography for all older children and adolescents with restrictive lung defects and impaired diffusing capacity. Acutely, children with prolonged and recalcitrant acute chest syndrome should be assessed for PH and right-sided heart dysfunction.

7.2.2 Cystic Fibrosis PH and cor pulmonale are known to complicate severe cases of CF, although early detection remains a challenge. Chronic alveolar hypoxia due to mismatched ventilation and perfusion promotes pulmonary vascular remodeling and hypoxic pulmonary vasoconstriction. Progressive lung damage and destruction of the pulmonary vascular bed due to CF and other destructive lung diseases compounds hypoxia, further promoting increased PVR. The areas of greatest pulmonary vascular destruction in CF occur in the areas with the greatest lung parenchyma damage [149]. Detection of PH in patients with CF portends a poor prognosis, although earlier diagnosis may modulate the disease course. Recognition of early, mild PAP elevation in this patient population is difficult for a variety of reasons, including hyperinflation that dulls S2 on examination and deemphasizes the heart size on chest radiography, and insensitivity of the electrocardiogram [150]. ANP levels in plasma may be elevated in PH, although this may be a late finding. The Doppler echocardiogram remains the most reliable noninvasive screening mechanism [151]. Furthermore, echocardiogram-detected RV dysfunction correlates with the severity of lung disease and markers of inflammation in young adults with CF [152]. CF patients with severe defects on pulmonary function testing and daytime and/or nighttime hypoxemia should be assessed for PH by Doppler echocardiography. Additional reasons to evaluate for PH include failure to thrive despite adequate caloric intake, recurrent “pulmonary” exacerbations, and chronic infection with Burkholderia cepacia.

7.2.4 Obstructive Sleep Apnea Children with profound sleep-disordered breathing, such as OSA, can develop PH. The exact mechanisms are unclear, but certainly hypoxemia is a major factor. No comprehensive study to date has examined the prevalence of PH in OSA or the broad range of causes of sleep-disordered breathing. As a result, guidelines for PH surveillance in this population do not exist, and clinical judgment should guide decisions and the choice of screening modality. For otherwise healthy children with profound nighttime hypoxemia, clinical evidence of right-sided heart dysfunction, or an enlarged cardiothymic silhouette, strong consideration should be made for screening. Fortunately, PH due to OSA typically resolves rapidly upon correction of the airway obstruction. There should be a particularly low threshold for the clinician to obtain a screening

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Doppler echocardiogram for those children with comorbid conditions such as Down syndrome, SCD, chest wall restriction, and neuromuscular disease.

7.3 Management of PH in Pediatric Patients with Chronic Lung Disease For the pulmonologist caring for patients with chronic pulmonary diseases complicated by PH, it is critical to recognize and reduce conditions which might promote further lung damage, as well as to stabilize the primary lung disease. Efforts to effectively manage the primary pulmonary disease process must remain a focus, and vary by disease. Regardless of the underlying disease process, detecting and removing factors which promote intermittent and chronic hypoxemia is critical, and the utility of cardiac catheterization to invasively assess the pulmonary vasculature should be considered. The factors which may exacerbate or cause hypoxemia in this patient population are broad, and vary with the underlying disease and patient age. For example, among children of all ages with chronic hypoventilation, chronic aspiration is a major consideration. The reasons for aspiration in this patient population include dysphagia, poor management of oral secretions, and gastroesophageal reflux. Carefully screening for these aggravators is appropriate. Sleep-disordered breathing, including OSA, can potentiate pulmonary vascular disease of any cause, in any age group. Continuous nighttime oximetry and/or formal sleep studies are an important component of the management plan. Table 2 identifies the Table 2  Diagnostic studies that play an important role in the initial and ongoing management of infants and children with PH associated with chronic lung disorders of childhood Required evaluations

Situation-specific evaluations

Infancy and early childhood All age groups •  Physical exam •  Cardiac catheterization •  PA and lateral CXR •  Formal sleep study •  ABG •  Bronchoscopy ± lavage •  Video fluoroscopic swallow •  Chest CT study •  Video fluoroscopic •  Upper GI tract swallow study •  Doppler echocardiography •  Upper GI tract •  Nighttime oximetry study •  pH probe Older childhood and adolescence •  Gastric emptying scan •  Physical examination •  Salivagram •  PA and lateral CXR •  Genetic testing •  ABG •  Lung biopsy •  Doppler echocardiography •  Immune defect studies •  Nighttime oximetry study •  V/Q scan • Pulmonary function testing including lung volumes and DLCO •  6-min walk test CXR chest radiograph, ABG arterial blood gas, CT computed tomography, GI gastrointestinal, PA pulmonary artery

studies that play an important role in the initial and ongoing management of infants and children with PH secondary to chronic lung disorders. Alveolar hypoxia and hypoxemia are the most prominent contributors to PH associated with chronic lung disorders; therefore, optimizing lung function and gas exchange by treating the underlying condition is paramount. For example, the use of hydroxyurea for primary disease control has been associated with a reduction in mean RV pressure among SCD patients with PH following 6–12 months of treatment [158]. In addition, the utility of chronic oxygen supplementation cannot be overstated. Although oxygen should be used judiciously and hyperoxia and oxidant stress should be avoided, the use of supplemental oxygen is a critical component of therapy. We aim to maintain oxygen saturation values between 95 and 97% for patients awake and asleep. Overnight oxygen saturation monitoring can be used to identify desaturation with sleep, and a formal sleep study may be necessary to explore its cause when this is unclear. In certain situations, invasive airway management and ventilatory support may be required. In such situations, careful consideration of the utility of flexible and/or rigid bronchoscopy should be considered. As is true for patients with BPD, growth is critical for infants and children with PH associated with other chronic lung disorders. Comorbid conditions such as dysphagia and gastroesophageal reflux disease often accompany chronic lung diseases interfering with growth and pulmonary recovery. The multidisciplinary approach to these patients should include a nutritionist and aggressive dietary management. When growth failure is pervasive, continuous gastric feeds at night or parenteral nutrition should be considered to optimize growth and development.

8 Congenital Heart Disease The primary inciting triggers of later pulmonary vascular disease in patients with CHD are excessive flow and shear stress. The associated histopathology and pathophysiology of flow-mediated pulmonary vascular disease has been extensively reviewed [160–162], and is also treated elsewhere in this text. The avoidance of excessive pulmonary blood flow remains a foundational tenant in the early and ongoing management of CHD, as is appreciation of the heightened risk of PH in certain patient subgroups, such as those with Down syndrome. Perioperative management of these patients requires collaboration between the pediatric intensivist, the pediatric cardiovascular surgeon, and the pediatric cardiologist. Optimal pre- and postoperative care must be sensitive to the patient’s developmental age and progression along the pulmonary vascular disease continuum.

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8.1 Presentation of PH in Patients with CHD The presentation of PH in pediatric patients with CHD is dependent on the age and developmental status of the pulmonary vascular bed in combination with the particular hemodynamic alterations produced by coexisting structural or functional heart disease. Among term infants and older children, primary closure of existing shunts or relief of pulmonary venous obstruction surgically or percutaneously usually prevents development of PH in most, but certainly not all, patients. Recognition of CHD followed by early intervention to minimize the occurrence of late pulmonary vascular disease has been the traditional role and focus of the pediatric cardiologist and cardiothoracic surgeon in the larger scope of pediatric pulmonary vascular disease. The development of “Eisenmenger physiology” in the current era represents a treatment failure. However, disturbances in blood flow in utero and severe pulmonary vascular disease occur in several structural cardiac defects [163–167]. It is difficult to surmise that the resulting pulmonary vascular abnormalities these children experience are equivalent to those of their adult counterparts. One could further argue that any child with a congenital cardiac defect does not enter the PH continuum as “normal” but is inherently at risk for development of later disease, more so if repair or palliation is performed at a later age [160, 161]. The study of surviving adult patients with advanced or late disease therefore represents one end of the PH disease spectrum related to CHD. Not included in these adult congenital cohorts are patients who develop disease at an accelerated rate and do not survive childhood. This potential selection bias then might suggest a more optimistic outcome for patients with PH associated with CHD. This concept was raised in a recent retrospective review of the UK Pulmonary Hypertension Service for Children [4]. Those patients referred with CHD and severe postoperative PH fared much worse than those with other forms of PH. The notion that over time pulmonary vascular disease progresses through stages from normal, to reversible, and then to irreversible disease is widely embraced in adult PH circles and has been applied to children as well [168]. However, the fact that children can present with advanced, seemingly irreversible disease at young ages implies a different, perhaps more aggressive process, with or without associated structural heart disease. The oft-quoted dismal statistics for children from within earlier registry data support this notion [169, 170]. On the other hand, it is now known that younger children with advanced disease tend to demonstrate a more robust response to vasoreactivity testing and a favorable response to prostacyclin therapy compared with adults, irrespective of their responsivity to vasoreactivity testing [171]. It is expected that these differences between adults and children will be further clarified through ongoing registries in the coming years. In addition, improved

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subgroup phenotyping of pediatric patients may also influence the diagnostic and therapeutic approaches. A subgroup that deserves particular mention is the preterm infant with CHD who develops BPD prior to cardiac repair. BPD alone may predispose an infant to the development of PH [90, 172, 173]. Pulmonary overcirculation can compound this underlying predisposition for pulmonary vascular disease. Conversely, early exposure of this at-risk group of patients to cardiopulmonary bypass may have equally adverse, if not more devastating, consequences. In a retrospective multicenter study, 52 cardiac procedures in 43 formerly premature infants were reviewed. A heterogeneous group of patients ranging in gestational age from 23 to 35 weeks with birth weights of 431–2,500 g were included. Thirty-day mortality was highest (50%) in patients with univentricular physiology and severe BPD [174]. Even ­transcatheter interventions for congenital heart defects are not without significant complications in this patient population [175].

8.2 Assessment of PH in Pediatric Patients with CHD The challenges inherent in defining PH in pediatric (particularly neonatal) patients and the uses and limitations of echocardiography, cardiac catheterization, and vasodilator testing in infants and young children were addressed at the beginning of this chapter. The approach to assessment and management in pediatric CHD tends to be driven by PH disease prevention, as reflected in the shift in the surgical timing for many congenital cardiac defects to protect from the deleterious effects of ongoing left-to-right shunt. What follows are some unique considerations in the assessment of PH in pediatric patients with CHD that deserve special mention. The management of the infant or older child with CHD begins with removal or limitation of left-to-right shunt by complete repair or pulmonary arterial banding. For most neonates and younger children, this decision is made without need for cardiac catheterization. Although timely surgical intervention will prevent PH in the majority of cases, pulmonary vascular disease may occur in patients with residual or acquired defects or in those with single ventricle physiology and borderline hemodynamics. Among children with hypoplastic left-sided heart syndrome, those with an intact atrial septum at birth are at particular risk owing to pre-existing pulmonary vascular disease, and PH may become apparent with and between each stage of their palliative pathway [164]. Once a diagnosis of PH is suspected, cardiac catheterization is the next step along with surveillance for other noncardiac triggers such as upper airway obstruction, thromboembolic disease, and silent aspiration in the younger patient.

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For patients under consideration of operative repair of a structural defect, catheterization is sometimes required and frequently incorporates vasodilator testing. The generally accepted definition of operability for standard left-to-right shunt lesions includes low PVR, below 6–8 Wood units m2 at the baseline, or a drop to below such ranges with vasodilator testing [176]. More recently, a study of the utility of preoperative testing in high-risk patients using inhaled oxygen alone or in combination with iNO concluded that an Rp/Rs ratio less than 0.33 in response to inhalation of combined oxygen and iNO would identify the optimal number of acceptable risk candidates [177]. However, decision making regarding operability often requires a more comprehensive consideration of factors beyond PVR [178]. In the absence of clear guidelines, it is not surprising that great variability is encountered in practice with regard to acceptable risk for surgical repair. The assessment of the child with advanced or so-called irreversible PH typically follows the guidelines provided by adult algorithms. At this stage, although echocardiography is the usual starting point, it is typically followed by cardiac catheterization to define the degree of PH, the level of reactivity if any, and to exclude residual structural lesions which may be the primary problem. In the setting of established disease following surgical repair of cardiac defects, the frequency of follow-up catheterization ranges from 3- to 12-month intervals in most pediatric centers, more frequently with clinical or therapeutic changes and less frequently with stable or improving disease. Vasodilator testing is performed during follow-up catheterization in many centers. Although lack of reactivity has been associated with shorter survival in children [179], it is unclear if this holds true with currently available therapeutic strategies. The definition of reactivity and its importance in children may yet require pediatric-specific refinement as new insights emerge from pediatric centers and the scope of disease broadens to less typical disorders associated with PH.

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shunt include an unrestrictive ventricular septal defect and an atrioventricular septal defect. Children with cardiac defects that result in pulmonary overcirculation usually do not require immediate surgical attention; delayed repair in the first year of life offers specific advantages, including a larger surgical window and cardiovascular structural anatomy and reduced PVR. Correction of these cardiac defects is usually attempted at 6–12 months of age after postnatal PVR has decreased and hopefully before significant pulmonary endothelial injury and pulmonary vascular remodeling can occur. Despite this timing, some patients with significant pulmonary overcirculation will experience postoperative PH. At particular risk are children with Down syndrome.

9.1 Clinical Presentation of Perioperative PH The main clinical manifestations of perioperative PH are hypoxemia and low cardiac output syndrome. Hypoxemia occurs when a pulmonary hypertensive crisis causes right-toleft atrial shunting through an intracardiac defect, such as a patent foramen ovale. Perioperative PH should be suspected when (a) SaO2 levels are below 92% in infants with correction of dual ventricle lesions, (b) SaO2 levels are below 75% in infants undergoing palliation of single ventricle lesions where there is complete mixing of blood at the atrial or ventricular levels, and (c) low cardiac output syndrome is apparent, especially when the duration of cardiopulmonary bypass or the surgical approach would not be expected to result in left ventricular dysfunction. Systemic or suprasystemic PAP causes RV failure with deviation of the ventricular septum into the left ventricle and subsequent decrease in left ventricular output. For patients without a patent foramen ovale or a residual postoperative septal defect, systemic desaturation may not be apparent and low cardiac output may be the predominant manifestation of perioperative PH.

9 Perioperative PH The pediatric intensivist, in collaboration with pediatric cardiologists and cardiothoracic surgeons, must often diagnose and manage perioperative PH in infants and children undergoing surgical repair of CHD. Children with congenital heart defects are at risk for perioperative PH if they require corrective or palliative surgery in the first weeks of life or if they have defects associated with significant pulmonary overcirculation. Congenital cardiac defects that require either complete correction or palliation in the first week of life include transposition of the great arteries, truncus arteriosus, critical aortic stenosis, coarctation of the aorta, hypoplastic left-sided heart syndrome, pulmonary stenosis or atresia, total anomalous pulmonary venous return, and severe tetrology of Fallot. Cardiac lesions associated with a large left-to-right

9.2 Assessment of Perioperative PH Perioperative PH is most often assessed and monitored by echocardiography and, less commonly, by direct measurement of postoperative PAP utilizing a pulmonary artery catheter. Echocardiographic assessment provides an indirect measurement of PAP and was discussed in detail earlier in this chapter. In infants and children, direct continuous monitoring of PAP pressure is rare. Exceptions are sometimes made when there is a known preoperative diagnosis of PH and postoperative pulmonary hypertensive crises are anticipated or in infants with total anomalous pulmonary venous return and severe preoperative PH. In these rare instances, a

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3-French single-lumen pulmonary artery catheter is inserted directly into the right ventricle at the completion of surgery and is threaded by the surgeon into the main pulmonary artery. The risk–benefit ratio of monitoring must be considered because significant bleeding and cardiac tamponade are potential complications upon removal of the catheter.

9.3 Management of Perioperative PH Supportive care is often sufficient for management of mild to moderate perioperative PH. Supportive care in many institutions includes supplemental oxygen, mechanical ventilation ­including induction of a respiratory alkalosis, and sedation with intravenous narcotics, such as fentanyl. Unfortunately, none of these common therapeutic approaches have been subjected to randomized clinical trials targeting appropriate long-term outcomes. Moderate to severe perioperative PH is often treated with pharmacologic therapy such as iNO, which is discussed briefly next and more extensively in another chapter in this volume. In addition, less well studied perioperative therapies such as arginine, citrulline, sildenafil, and inhaled iloprost are sometimes used, and these are discussed later.

9.3.1 Inhaled Nitric Oxide As with many drugs and therapies utilized in the postoperative care of children undergoing surgical repair of congenital heart defects, iNO is not specifically approved by the US Food and Drug Administration to treat postoperative PH. In fact, iNO is only approved to treat PPHN in term or late preterm neonates older than 34 weeks’ completed gestational age. All other uses are considered off-label. That being said, iNO has been both extensively studied and even more extensively used to treat postoperative PH in children. What follows is a brief summary of the data evaluating the clinical use of iNO in pediatric perioperative PH. A more detailed description of the clinical evidence supporting its use and recommendations for both initiation and weaning of iNO may be found in Chap. 105. Several small, single-center, randomized, placebocontrolled trials of iNO for the treatment or prevention of postoperative PH in children have been performed in the past 10 years [180]. Most, but not all, reported fairly dramatic short-term physiologic responses to iNO including rapid and sustained reductions in PAP compared with baseline values. However, many of these early studies were not blinded and several allowed crossover to the iNO treatment group if conventional management was deemed to fail. None were adequately powered to address safety and efficacy [180]. The largest randomized trial evaluating iNO in the postoperative period was also a single-center study [181].

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Compared with patients receiving a placebo, patients who received 10 ppm iNO had a lower median PVR, fewer pulmonary hypertensive crises, and a shorter median time to extubation readiness. This study of 124 patients was not adequately powered to demonstrate a difference in mortality. Despite its limitations, this is the largest and best executed study to date that supports the use of iNO in selected pediatric patients at high risk of postoperative PH following repair of CHD. Currently, recommendations for patient selection, dose, duration, and weaning from iNO in the postoperative setting are limited by the paucity of data.

9.3.2 Respiratory Alkalosis Standard therapy for postoperative PH has historically included induction of respiratory alkalosis due to known effects of pH on PVR, despite the lack of evidence for safety or efficacy. In an unblinded, randomized crossover study in 12 children who had undergone biventricular repair of congenital heart defects, Morris et al. attempted to assess the effects of hyperventilation and iNO as potential treatments for postoperative PH documented by an indwelling PA catheter [182]. Hyperventilation to achieve a pH of 7.50 reduced PAP and PVR, but at the expense of an undesirable decrease in cardiac output and increase in systemic vascular resistance. iNO was equally effective at reducing PAP and PVR but without the untoward effects on cardiac output and systemic vascular resistance. Hyperventilation as a strategy for treatment of PH requires a critical reassessment.

9.3.3 Intravenously Administered Arginine Arginine is the substrate for NO synthase, making it a biologically plausible treatment for PH. In the postoperative period there is decreased availability of arginine [183]. Hemolysis of red blood cells during cardiopulmonary bypass releases arginase, which further decreases plasma arginine levels. In addition, pulmonary endothelial dysfunction in the postoperative period, conventionally defined as a loss of vasodilator response to acetylcholine, may also result in decreased synthesis of NO despite an adequate amount of substrate. Arginine supplementation was the focus of one clinical study of 20 patients with CHD and known PH [184]. Patients received an infusion of arginine at 15 mg/kg/min after being ventilated with an FiO2 of 0.65 for 15  min. Increasing the FiO2 to 0.65 resulted in a dramatic decrease in PVR index. An additional small decrease in PVR index with arginine infusion was seen in postoperative but not in preoperative patients. Use of arginine as a postoperative pulmonary vasodilator is not well supported in the literature. Clearly further research is needed.

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9.3.4 Citrulline Therapy* A series of studies investigating the role of the urea cycle in postoperative PH have been conducted by investigators at Vanderbilt Children’s Hospital. The urea cycle generates both citrulline and arginine and is therefore critical to NO synthesis (Fig.  4). In observational studies of urea cycle function, Barr et  al. noted that both plasma citrulline and arginine levels were significantly decreased after congenital cardiac surgery [183]. This observation led to a randomized, placebo-controlled, double-blind trial of orally administered citrulline in 40 patients undergoing repair of congenital cardiac defects [185]. Plasma citrulline levels were significantly higher in the citrulline group, demonstrating adequate absorption. No adverse side effects were noted. The study was not adequately powered to detect an effect on the incidence of postoperative PH; however, it was noted that none of the patients with a plasma citrulline level above 37 mmol/L developed PH (Table 3). This study of orally administered citrulline was followed by a pharmacokinetic study of intravenously administered citrulline [186]. Again, no side effects or adverse events related to the intravenous administration of citrulline were

Fig. 4  The hepatic urea cycle, primarily known for processing of nitrogen waste, is also the major source of the amino acids arginine and citrulline, key intermediates in nitric oxide synthesis. (Reprinted from Smith et al. [185] with permission from Elsevier)

J.L. Aschner et al. Table  3  In a study of orally administered citrulline in patients undergoing surgery for congenital heart disease, a plasma citrulline level of more than 37 mmol/L, measured 12 h following surgery, was protective against development of postoperative PH Citrulline level at 12 h postoperatively (mmol/L) No PH With PH 18 patients 9 patients £37 >37 12 patients 0 patients p = 0.036 (Fisher’s exact) From Smith et al. [185] with permission from Elsevier

noted. This pharmacokinetic study laid the groundwork for an ongoing randomized, placebo-controlled, double blind trial of intravenously administered citrulline in children at risk for postoperative PH after congenital cardiac surgery. Further research with both orally administered and intravenously administered citrulline is needed.

9.3.5 Sildenafil Therapy Sildenafil is a PDE5 inhibitor that blocks degradation of cGMP. Use of both orally administered sildenafil (clinically available) and intravenously administered sildenafil (clinically not available) has been reported in the postoperative period in small series of patients. In ten infants with severe postoperative PH also being treated with iNO, orally administered sildenafil lowered PAP without significant effects on systemic pressure or oxygenation [187]. Larger doses of orally administered sildenafil up to 2 mg/kg did not offer any added benefit. However, absorption of an oral medication in the immediate postoperative period may be erratic and limit effectiveness. In a randomized-sequence open-label study in 15 patients at risk for postoperative PH, intravenously administered sildenafil was equally effective as iNO at reducing PAP but was also associated with a drop in systemic arterial pressure [188]. More concerning was an increase in the A–a gradient associated with use of sildenafil, presumably due to reversal of hypoxic pulmonary vasoconstriction and increased intrapulmonary shunting. Intravenously administered sildenafil is presently not commercially available, which limits further investigation. Despite biologic plausibility as a therapy for PH, sildenafil has not been adequately studied in pediatric patients with perioperative PH.

9.3.6 Aerosolized Prostacyclin * Vanderbilt University has filed for patents and licensed citrulline as a therapeutic agent with Asklepion Pharmaceuticals. Frederick Barr has had grant support from Asklepion Pharmaceuticals for the study of intravenously administered citrulline.

Iloprost, a commercially available prostacyclin analog, is of potential interest as a selective pulmonary vasodilator but, like the previously discussed therapeutic agents, has not been

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adequately studied in children with postoperative PH. Two small case series reported functional responses to inhaled iloprost with a drop in mean PAP without systemic side effects. No randomized controlled trials have been reported in this population. Further investigation is warranted.

9.4 Management of PH in the Setting of Repaired CHD There are no evidence-based therapies for children with severe disease following surgical repair of their cardiac defects. Generally, positive anecdotal results have been reported using prostacyclin analogs [171, 189, 190], endothelin receptor blockade [22, 191–193], and phosphodiesterase inhibition [194] in pediatric patient groups, which contrasts with the universally dismal prognosis in the absence of therapy. The premature infant with PH and left-to-right shunt lesions deserves special mention. Beyond standard anticongestive therapies, the benefits of operative or transcatheter repair of structural heart disease must be weighed against the inherent risks. Attention to coexisting comorbidites, such as intraventricular hemorrhage, necrotizing enterocolitis, chronic aspiration, and airway abnormalities must also be considered. Lim and Matherne reported on the closure of an atrial septal defect in such a child with mechanical ventilatory dependence using transcatheter techniques [195]. Although it is not recommended that all patients with atriallevel shunts with BPD undergo closure, as many defects can and will close spontaneously, this therapeutic strategy represents an important shift in perspective toward less invasive early intervention in an at-risk patient population.

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disease and elevations in PAP can progress from mild to severe in a relatively short time frame. Despite its limitations, Doppler echocardiography is the most readily available tool for noninvasive and serial screening. A framework for prospective identification and ongoing surveillance of infants with persistent pulmonary disease has been developed and implemented at Vanderbilt Children’s Hospital (Fig. 5). We propose that infants who remain ventilated or who have a fractional inspired oxygen requirement greater than 0.3 be prospectively screened at 30 days of life and at least monthly thereafter for echocardiographic evidence of PH (Fig. 5, group A). More frequent assessments are recommended for those in whom mild PH is evident (Fig. 5, group B) and for those with moderate to severe PH or for whom therapy has been initiated (Fig. 5, group C). This approach is predicated on the knowledge that disease severity can progress or regress over time and that a previously normal echocardiogram may not remain normal in the face of ongoing lung or heart disease. Therefore, disease assessment must bridge the transition from NICU or pediatric intensive care unit (PICU) to outpatient management. A multidisciplinary clinic format involving pediatric pulmonology­ and pediatric cardiology departments may offer the optimal approach to long-term follow-up and management. There are no well-publicized guidelines for the appropriate medical workup of an infant or child newly diagnosed with later-onset PH. We suggest that this evaluation should include an assessment of gastroesophageal reflux and chronic aspiration, and the adequacy of ventilation and oxygenation. Poor nutrition is frequently coexistent; its correction is critical to lung growth and recovery. Although the ideal oxygen saturation target for this population is unknown, it is clearly important to avoid recurrent hypoxemic events. For infants with BPD and a diagnosis of PH, it may be necessary to adjust upward the oxygen saturation targets currently thought

10 Proposed Guidelines for Surveillance and Management It is unfortunate that the diagnosis of PH in children is often not made until advanced stages of the disease. This is largely because of lack of guidelines for proactive surveillance and limitations of the current techniques for assessment of PH in the pediatric population. An unproven tenet is that identification of infants and children at earlier stages of PH will result in more effective interventions and improved outcomes. For this to occur, guidelines for prospective identification of patients at risk and algorithms for longitudinal surveillance and management must be developed. The dynamic nature of PH in the pediatric patient must be considered when designing such algorithms; PH can develop at nearly any time in the course of underlying lung or heart

Fig.  5  A framework for prospective identification and ongoing surveillance by echocardiogram of infants with BPD at risk for later-onset PH

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Fig. 6  Suggested algorithm for medical evaluation of the infant and young child with PH and chronic lung disease. (Modified with permission from McLaughlin and McGoon [199])

to be beneficial in the first weeks of life to avoid the development of BPD. On an individual basis, consideration should be given to a metabolic workup for inborn errors of metabolism, storage diseases and thyroid dysfunction, hematologic diseases such as SCD, coagulation defects including genetic thrombophilias, such as factor V Leiden mutations, and infectious diseases, including HIV (Fig. 6).

11 Gaps in Knowledge Much work must be done before we can translate pharmacologic interventions into evidenced-based therapies for infants and children with PH and structural lung and cardiac disease. This goal will require not just additional basic laboratory research but also a focused effort from the clinical community. We urge the pediatric cardiology community to define and adopt standardized echocardiographic end points for age-based classifications of mild, moderate, and severe PH. This will facilitate prospective monitoring of disease progression and response to therapy. Also needed are age-appropriate and clinically relevant end points for pediatric clinical trials. The 6-min walk test is not useful for infants and most young children with PH. Attention should be given to developing therapies for RV dysfunction in children. We also applaud efforts to develop new imaging modalities for PH, including MRI assessment of lung volume, lung vascular area, and right-sided heart geometry. The validity and predictive power of biomarkers,

such as plasma BNP and troponin, for infants and children with PH and myocardial dysfunction must be determined. Criteria should be developed for referral of patients with progressive PH to an interventional pediatric cardiologist for cardiac catheterization and vasodilator testing. The optimal multidisciplinary approach involving the pediatric pulmonologist, pediatric cardiologist, dietician, and others in the outpatient management of patients with chronic lung or cardiac disorders who are at risk of or who have documented PH must be studied and implemented across centers. More data are needed on the natural history and outcomes of children, including preterm infants, who develop PH beyond the first weeks of life. A wide range of treatment regimens including combination therapies are being used clinically, despite the lack of appropriately powered and randomized efficacy and safety trials. A mechanism beyond the occasional case series is needed for recording the responses to these therapies and the outcomes of these patients. Enrollment of infants and children with PH that persists or develops beyond the first weeks of life in to a pediatric PH registry, such as the TOPP Registry, could potentially meet this need. Although it is true that such registries are observational and descriptive in nature, the systematic accrual of data could help clarify patient phenotypes, genetic and clinical risk factors, and practice patterns. A registry could provide much needed information about the timing and antecedents of diagnosis, the types of therapies employed and whether early detection and treatment confers a benefit [196]. Designed correctly, a registry would provide an accessible cohort to generate and test hypotheses, as demonstrated

75  Pediatric Pulmonary Hypertension: An Integrated View from Pediatric Subspecialists

by the tremendous strides made in the genetic understanding of heritable PAH with simultaneous discoveries of BMPR2 mutations made possible by the Vanderbilt and Columbia PAH registries [197, 198]. A database registry will ultimately enable the conduct of clinical trials by better defining prevalence and phenotypes and assembling random treatment strategies into a comprehensible pattern. Formation of a pediatric PH clinical trials network would be an important step toward addressing the significant obstacles to the conduct of randomized clinical trials in this patient population. These obstacles include limited patient numbers in any one center, disease heterogeneity, lack of standardized definitions for disease onset, severity and progression, multiple treatment paradigms, limited funding, and ethical considerations. Much work remains to achieve similar success for neonates and children with PH associated with structural changes in the lungs as has been realized for infants with reactive pulmonary vascular disease and PPHN. Effective treatment will most likely require long-term administration of a combination of vasodilators and innovative therapies that block progression of the vascular remodeling process and promote regression of established vascular changes. Potential targets for pharmacologic intervention include extracellular matrix components, vasoregulatory and antiproliferative proteins, angiogenic proteins, and intracellular signal transduction cascades. Laboratory and clinical studies are needed to define the roles of genetic polymorphisms, inflammation, and oxidants, vasodilator/vasoconstrictor imbalance, ion channel dysfunction, angiogenesis and differentiation, thrombosis, and hemodynamics in the pathogenesis of neonatal and pediatric pulmonary hypertensive diseases. The spectrum of pediatric PH is broad, crossing disciplines, age groups, and the boundaries between acute and chronic care. A successful institutional pediatric PH program must acknowledge these realities and be both longitudinal and comprehensive in scope. Collaboration between cardiologists, neonatologists, pulmonologists, and intensivists is needed as the diseases producing PH in children span these subspecialty borders and require multidisciplinary expertise. A comprehensive pediatric PH program would also include a multidisciplinary team of investigators with expertise in basic and clinical trials research, epidemiology, pharmacology, genetics, and informatics. The goals and scope of such a program would include basic scientific discovery, translation of discovery into interventional clinical trials, epidemiologic-outcomes-based research, longitudinal clinical care, and data collection for purposes of quality improvement and dissemination of potentially better practices. Core principles would include disease prevention and stabilization, communication, and continuity of care, with special attention to these tenets during vulnerable windows between NICU or PICU discharge and outpatient management. These

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collaborative efforts will likely stimulate advances in the broader field of PH. As pediatric and adult subspecialists and investigators collectively approach the disease from different directions and their unique perspectives, we may then begin to fill the gaps in our knowledge and in the care we provide our patients. Acknowledgements  Supported by award numbers R01 HL075511 (J.L.A.), R01 HL068572 (C.D.F), and 5RO1HL073317 (F.E.B.) from the National Heart, Lung, and Blood Institute (NHLBI). The content is solely the responsibility of the authors and does not necessarily represent the official views of the NHLBI or the National Institutes of Health. The authors acknowledge Robin Roller for her editorial assistance and to Neeru Kaushik for her assistance with the MRI image.

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1108 ventricular septal defect and elevated pulmonary vascular resistance. Am J Cardiol 63:327–331 177. Balzer DT, Kort HW, Day RW et al (2002) Inhaled nitric oxide as a preoperative test (INOP Test I): the INOP Test Study Group. Circulation 106:I76–I81 178. Viswanathan S, Kumar RK (2008) Assessment of operability of congenital cardiac shunts with increased pulmonary vascular resistance. Catheter Cardiovasc Interv 71:665–670 179. Houde C, Bohn DJ, Freedom RM, Rabinovitch M (1993) Profile of paediatric patients with pulmonary hypertension judged by responsiveness to vasodilators. Br Heart J 70:461–468 180. Day RW, Hawkins JA, McGough EC, Crezee KL, Orsmond GS (2000) Randomized controlled study of inhaled nitric oxide after operation for congenital heart disease. Ann Thorac Surg 69: 1907–1912 181. Miller OI, Tang SF, Keech A, Pigott NB, Beller E, Celermajer DS (2000) Inhaled nitric oxide and prevention of pulmonary hypertension after congenital heart surgery: a randomised double-blind study. Lancet 356:1464–1469 182. Morris K, Beghetti M, Petros A, Adatia I, Bohn D (2000) Comparison of hyperventilation and inhaled nitric oxide for pulmonary hypertension after repair of congenital heart disease. Crit Care Med 28:2974–2978 183. Barr FE, Beverley H, VanHook K et al (2003) Effect of cardiopulmonary bypass on urea cycle intermediates and nitric oxide levels after congenital heart surgery. J Pediatr 142:26–30 184. Schulze-Neick I, Penny DJ, Rigby ML et al (1999) L-arginine and substance P reverse the pulmonary endothelial dysfunction caused by congenital heart surgery. Circulation 100:749–755 185. Smith HA, Canter JA, Christian KG et al (2006) Nitric oxide precursors and congenital heart surgery: a randomized controlled trial of oral citrulline. J Thorac Cardiovasc Surg 132:58–65 186. Barr FE, Tirona RG, Taylor MB et  al (2007) Pharmacokinetics and safety of intravenously administered citrulline in children undergoing congenital heart surgery: potential therapy for postoperative pulmonary hypertension. J Thorac Cardiovasc Surg 134:319–326 187. Raja SG, Danton MD, MacArthur KJ, Pollock JC (2007) Effects of escalating doses of sildenafil on hemodynamics and gas exchange in children with pulmonary hypertension and congenital cardiac defects. J Cardiothorac Vasc Anesth 21:203–207

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Chapter 76

Persistent Pulmonary Hypertension of the Newborn: Mechanisms and Treatment Steven H. Abman, Robin H. Steinhorn, and Judy L. Aschner

Abstract  Some infants fail to achieve or sustain the normal decrease in pulmonary vascular resistance (PVR) at birth, leading to severe respiratory distress and hypoxemia, which is referred to as persistent pulmonary hypertension of the newborn (PPHN). PPHN is a major clinical problem, affecting up to 10% of full-term and preterm infants in neonatal intensive care, and contributing significantly to high morbidity and mortality. Newborns with PPHN are at risk for severe asphyxia and its complications, including death, chronic lung disease, and neurodevelopmental sequelae, and other problems. This chapter will review the pathophysiology of PPHN, and clinical strategies that utilize a physiologic approach to the treatment of newborns with severe PPHN. Keywords  Persistent pulmonary hypertension in the newborn • Preterm infants • Development defect

1 Introduction Some infants fail to achieve or sustain the normal decrease in pulmonary vascular resistance (PVR) at birth (described in Chap. 9), leading to severe respiratory distress and hypoxemia, which is referred to as persistent pulmonary hypertension of the newborn (PPHN). PPHN is a major clinical problem, affecting up to 10% of full-term and preterm infants in neonatal intensive care, and contributing significantly to high morbidity and mortality [1, 2]. Newborns with PPHN are at risk for severe asphyxia and its complications, including death, chronic lung disease, and neurodevelopmental sequelae, and other problems. This chapter will review the pathophysiology of PPHN, and clinical strategies that utilize a physiologic approach to the treatment of newborns with severe PPHN.

S.H. Abman (*) Department of Pediatrics, The Children’s Hospital, 1717 East Arizona Avenue, Denver, CO 80210, USA e-mail: [email protected]

2 Pathophysiology of Experimental PPHN Mechanisms that lead to the failure of PVR to fall at birth have been pursued in various animal models to better understand the pathogenesis and pathophysiology of PPHN. Such models have included exposure to acute or chronic hypoxia after birth, chronic hypoxia in utero, placement of meconium into the airways of neonatal animals, sepsis, and other treatment. Although each model demonstrates interesting physiologic changes that may be especially relevant to particular clinical settings, most studies have examined only brief changes in the pulmonary circulation, and mechanisms underlying altered lung vascular structure and function of PPHN remain poorly understood. Clinical observations that neonates with severe PPHN who die during the first few days after birth already have pathologic signs of chronic pulmonary vascular disease suggest that ­intrauterine events may play an important role in this syndrome [3]. Adverse intrauterine stimuli during late gestation, such as abnormal hemodynamics, changes in substrate or hormone delivery to the lung, hypoxia, and inflammation, may potentially alter lung vascular function and structure, contributing to abnormalities of postnatal adaptation. Several investigators have examined the effects of chronic intrauterine stresses, such as hypoxia and hypertension, in animal models in the attempt to mimic the clinical problem of PPHN. Whether chronic hypoxia alone can cause PPHN is controversial. A past report suggested that maternal hypoxia in rats increases pulmonary vascular smooth muscle thickening in newborns, but this observation has not been reproduced in maternal rats or guinea pigs with more extensive studies [4]. Pulmonary hypertension induced by early closure of the ductus arteriosus in fetal lambs alters lung vascular reactivity and structure, causing the failure of postnatal adaptation at delivery, and providing an experimental model of PPHN [5–7]. Over days, pulmonary artery pressure and PVR progressively increase, but flow remains low and PaO2 is unchanged [7]. Marked right ventricular hypertrophy and structural remodeling of small pulmonary arteries develops after 8 days of hypertension. After delivery, these lambs have persistent elevation of PVR despite mechanical ventilation

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with high oxygen concentrations. Studies with this model have shown that chronic hypertension without high flow can alter fetal lung vascular structure and function. This model is further characterized by endothelial cell dysfunction and altered smooth muscle cell reactivity and growth, including findings of impaired NO production and activity and downregulation of lung endothelial NO synthase messenger RNA and protein expression [8–12]. Fetal pulmonary hypertension is also associated with decreased cyclic GMP (cGMP) concentrations, associated with decreased soluble guanylate cyclase and upregulated cGMP-specific phosphodiesterase type 5 (PDE5) activities, suggesting further impairments in downstream signaling [13–15] (Fig. 1). Thus, multiple alterations in the NO-cGMP cascade appear to play an essential role in the pathogenesis and pathophysiology of experimental PPHN, contributing to altered structure and function of the developing lung circulation, and leading to failure of postnatal cardiorespiratory adaptation. Recent evidence indicates that excessive production of reactive oxygen species such as superoxide in the pulmonary vasculature may further contribute to the disruption in NO-cGMP signaling in this model [12, 16]. Upregulation of endothelin-1 (ET-1) may also contribute to the pathophysiology of PPHN. Circulating levels of ET-1, a potent vasoconstrictor and co-mitogen for vascular smooth muscle cell hyperplasia, are increased in human newborns with severe PPHN [17]. In the experimental model of PPHN due to compression of the ductus arteriosus in fetal sheep, lung ET-1 messenger RNA and protein content is markedly increased, and the balance of endothelin receptors is altered, favoring vasoconstriction [18, 19]. Chronic inhibition of the endothelin A receptor attenuates the severity of pulmonary hypertension, decreases pulmonary artery wall thickening, and improves the fall in PVR at birth in this model [20]. Thus, experimental studies have shown the important role of

Fig. 1  Abnormalities of the NO-cyclic GMP cascade in the pathophysiology of persistent pulmonary hypertension of the newborn (PPHN). (Reprinted with permission from Abman [65])

S.H. Abman et al.

the NO-cGMP cascade and the ET-1 system in the regulation of vascular tone and reactivity of the fetal and transitional pulmonary circulation. There is mounting evidence that oxidant stress plays an important role in the pathogenesis of PPHN. Increases in the levels of reactive oxygen species such as superoxide and hydrogen peroxide (H2O2) in the smooth muscle and adventitia of pulmonary arteries have been demonstrated in the ductal ligation model of persistent pulmonary hypertension [16, 21]. There appear to be multiple sources for elevated production of reactive oxygen species, including mitochondrial dysfunction, increased expression and activity of NADPH oxidase, and uncoupled nitric oxide synthase activity [16, 22–24]. Once present in the lung, elevated concentrations of reactive oxygen species promote vasoconstriction directly, and through multiple mechanisms, including increased endothelin levels [25], and oxidization of free fatty acids to create vasoconstrictor metabolites such as isoprostanes [26]. Superoxide anions rapidly combine with NO, inactivating it and in the process forming peroxynitrite, a potent oxidant with the potential to produce vasoconstriction and cytotoxicity. Increased levels of reactive oxygen species in the pulmonary vasculature of the ductal ligation model promote dysfunction of NO-cGMP signaling at multiple steps in the pathway, including blunted nitric oxide synthase expression, uncoupled endothelial nitric oxide synthase activity (thus further promoting reactive oxygen species production), and increased activity and expression of cGMPspecific phosphodiesterases [12, 27]. Superoxide dismutases (SOD) catalyze the conversion of superoxide anions to H2O2 and O2. Owing to the efficiency of the reaction between NO and superoxide, the local concentration of SOD is a key determinant of the biological half-life of endogenous NO [28]. Decreased SOD activity has been observed in models of PPHN, although characterization of alterations for specific isoforms is ongoing. Further evidence for the critical role of SOD is the recent observation that administration of a single intratracheal dose of recombinant human SOD (at birth or at 4 h of age) in neonatal lambs with PPHN produced a sustained increase in oxygenation over a 24-h period, reduced production of isoprostanes and peroxynitrite, and restored normal endothelial nitric oxide synthase expression and function. Finally, in addition to the vasoactive mediators described above, it has become clear that alterations of growth factors, such as vascular endothelial growth factor and platelet-derived growth factor (PDGF), are likely to play key roles in the modulation of vascular maturation, growth, and structure. For example, inhibition of PDGF-B attenuates smooth muscle hyperplasia in experimental pulmonary hypertension in fetal lambs, suggesting a potential role in the pathogenesis of PPHN [29]. Additional new data suggest that maternal exposure to selective serotonin reuptake inhibitors (SSRIs) during late gestation is associated with a sixfold increase in the prevalence of PPHN [30], although it is not clear how many infants

76  Persistent Pulmonary Hypertension of the Newborn: Mechanisms and Treatment

developed severe disease. Newborn rats exposed in utero to fluoxetine develop pulmonary vascular remodeling, abnormal oxygenation, and higher mortality when compared with vehicle-treated controls [31]. However, these findings showed only mild changes in right ventricular hypertrophy and pulmonary vascular remodeling in the neonatal rat pups, with minimal changes in vasoreactivity after maternal SSRI exposure. Whether these effects were related to a direct impact on the fetal lung circulation or were secondary to altered maternal or umbilical–placental function remains unknown. As SSRIs have been reported to reduce pulmonary vascular remodeling in adult models of pulmonary hypertension, these findings also serve to highlight the unique vulnerability of fetal pulmonary vascular development.

3 Clinical Aspects of PPHN The first reports of PPHN described term newborns with profound hypoxemia who lacked radiographic evidence of parenchymal lung disease and echocardiographic evidence of structural cardiac disease (Fig. 2). In these patients, hypoxemia was caused by marked elevations of PVR leading to right-to-left extrapulmonary shunting of blood across the

Fig. 2  Chest X-rays illustrating common disorders associated with PPHN

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patent ductus arteriosus (PDA) or foramen ovale (PFO) during the early postnatal period. Owing to the persistence of high PVR and blood flow through these “fetal shunts,” the term “persistent fetal circulation” was originally used to describe this group of patients. Consequently, it was recognized that this physiologic pattern can complicate the clinical course of neonates with diverse causes of hypoxemic respiratory failure. As a result, the term “PPHN” has been considered as a syndrome, and is currently applied more broadly to include neonates that have a similar physiology in association with different cardiopulmonary disorders, such as meconium aspiration, sepsis, pneumonia, asphyxia, congenital diaphragmatic hernia (CDH), and respiratory distress syndrome (RDS). Striking differences exist between these conditions, and the mechanisms that contribute to high PVR can vary between these diseases. However, these disorders are included in the syndrome of PPHN owing to common pathophysiologic features, including sustained elevation of PVR leading to hypoxemia due to right-to-left extrapulmonary shunting of blood flow across the ductus arteriosus or PFO. In many clinical settings, hypoxemic respiratory failure in term newborns is often presumed to be associated with PPHN-type physiology; however, hypoxemic term newborns can lack echocardiographic findings of extrapulmonary shunting across the PDA or PFO. Thus, “PPHN” should be reserved to

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describe neonates in whom extrapulmonary shunting contributes to hypoxemia and impaired cardiopulmonary function. Recent estimates suggest an incidence for PPHN of 1.9 per 1,000 live births, or an estimated 7,400 cases per year [32]. Diseases associated with PPHN are often classified within one of three categories: (1) maladaptation – vessels are presumably of normal structural but have abnormal vasoreactivity; (2) excessive muscularization – increased smooth muscle cell thickness and increased distal extension of muscle to vessels which are usually nonmuscular; and (3) underdevelopment – lung hypoplasia associated with decreased pulmonary artery number. This designation is imprecise, however, and high PVR in most patients likely involves overlapping changes among these categories. For example, neonates with CDH are primarily classified as having vascular “underdevelopment” due to lung hypoplasia, yet lung histologic examination of fatal cases typically shows marked muscularization of pulmonary arteries, and clinically, these patients can respond to vasodilator therapy. Similarly, neonates with meconium aspiration often have clinical evidence of altered vasoreactivity, but excessive muscularization is often found at autopsy. As described above, autopsy studies of fatal PPHN demonstrate severe hypertensive structural remodeling even in newborns who die shortly after birth, suggesting that many cases of severe disease are associated with chronic intrauterine stress. However, the exact intrauterine events that alter pulmonary vascular reactivity and structure are poorly understood. Epidemiology studies have demonstrated strong associations between PPHN and maternal smoking and ingestion of cold remedies that include aspirin or other nonsteroidal anti-inflammatory products [33, 34]. Since these agents can induce partial constriction of the ductus arteriosus, it is possible that pulmonary hypertension due to antenatal ductal narrowing contributes to PPHN (see later). Other perinatal stresses, including placenta previa and abruption, and asymmetric growth restriction, are associated with PPHN; however, most neonates who are exposed to these prenatal stresses do not develop PPHN. Circulating levels of l-arginine, the substrate for NO, are decreased in some newborns with PPHN, suggesting that impaired NO production may contribute to the pathophysiology of PPHN. It is possible that genetic factors increase susceptibility for pulmonary hypertension. A recent study reported strong links between PPHN and polymorphisms of the carbamoyl phosphate synthase gene [35]. However, the importance of this finding is uncertain, and further work is needed in this area. Studies of adults with idiopathic primary pulmonary hypertension have identified abnormalities of bone morphogenetic protein receptor genes; whether polymorphisms of genes for the bone morphogenetic protein or transforming growth factor b receptors, other critical growth factors, vasoactive substances, or other products increase the risk for some newborns to develop PPHN is unknown.

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4 Clinical Presentation and Evaluation Clinically, PPHN is most often recognized in term or nearterm neonates, but clearly can occur in premature neonates as well (Table 1). PPHN typically presents as respiratory distress and cyanosis within 6–12 h of birth. Although PPHN is often associated with indicators of perinatal distress, such as asphyxia, low Apgar scores, and meconium staining, idiopathic PPHN can present without signs of acute perinatal distress. Laboratory findings can include low levels of glucose, hypocalcemia, hypothermia, polycythemia, or thrombocytopenia. Radiographic findings are variable, depending upon the primary disease associated with PPHN. Classically, Table 1  Disorders associated with neonatal pulmonary hypertension Pulmonary

Meconium aspiration syndrome Respiratory distress syndromea Lung hypoplasia – primary Congenital diaphragmatic hernia Pneumonia/sepsis Idiopathic Transient tachypnea of the newborn Alveolar-capillary dysplasia Associated abnormalities in lung development: – Congenital lobar emphysema (rare association) – Cystic adenomatoid malformation (rare association) – Idiopathic, with impaired distal alveolarization –  Pulmonary interstitial glycogenosis –  Surfactant B and C deficiency –  ABCA 3 deficiency –  Others

Cardiovascular

Myocardial dysfunction (asphyxia, infection, stress) Structural cardiac disease: –  Mitral stenosis, cor triatriatum –  Endocardial fibroelastosis –  Pompe’s disease – Aortic atresia, coarctation of the aorta, interrupted aortic arch –  Transposition of the great vessels –  Ebsteins’s anomaly, tricuspid atresia Hepatic arteriovenous malformations Cerebral arteriovenous malformations Total anomalous pulmonary venous return Pulmonary vein stenosis (isolated) Pulmonary atresia

Neuromuscular disease Metabolic disease Polycythemia Thrombocytopenia Maternal drug use or smoking (especially selective serotonin reuptake inhibitors) a Term and preterm newborns

Associations with other diseases

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the chest X-ray in idiopathic PPHN shows an oligemic lung, normally or slightly hyperinflated, and lacking parenchymal infiltrates. In general, the degree of hypoxemia is disproportionate to the severity of radiographic evidence of lung disease. Not all term newborns with hypoxemic respiratory failure have PPHN-type physiology [36]. Hypoxemia in the newborn can be due to extrapulmonary shunt, as described earlier, in which high pulmonary artery pressure at systemic levels leads to right-to-left shunting of blood flow across the PDA or PFO. However, in many infants intrapulmonary shunt or ventilation–perfusion mismatch is the predominant abnormality, in which hypoxemia results from the lack of mixing of blood with aerated lung regions owing to parenchymal lung disease, without the shunting of blood flow across the PDA and PFO. In the latter setting, hypoxemia is related to the amount of pulmonary arterial blood that perfuses nonaerated lung regions. Although PVR is often elevated in hypoxemic newborns without PPHN, high PVR does not contribute significantly to hypoxemia in these cases. Several factors can contribute to high pulmonary artery pressure in neonates with PPHN-type physiology. Pulmonary hypertension can be due to vasoconstriction or structural vascular lesions that directly increase PVR. Changes in lung volume in neonates with parenchymal lung disease can also be an important determinant of PVR. PVR increases at low lung volumes owing to dense parenchymal infiltrate and poor lung recruitment, or at high lung volumes owing to hyperinflation associated with overdistension or gas trapping. Cardiac disease is also associated with PPHN. High pulmonary venous pressure due to left ventricular dysfunction (e.g., asphyxia or sepsis) can also elevate pulmonary artery pressure, causing right-to-left shunting, with little vasoconstriction. In this setting, enhancing cardiac performance and systemic hemodynamics may lower pulmonary artery pressure more effectively than promoting pulmonary vasodilation. Thus, understanding the cardiopulmonary interactions is key to improving outcome in PPHN. PPHN is characterized by hypoxemia that is poorly responsive to supplemental oxygen. In the presence of right-to-left shunting across the PDA, “differential cyanosis” is often present, which is difficult to detect by physical examination, and is defined by a difference in PaO2 between right radial artery and descending aorta values of more than 10 Torr, or an O2 saturation gradient of more than 5%. However, postductal desaturation can be found in ductus-dependent cardiac diseases, including hypoplastic left-sided heart syndrome, coarctation of the aorta, and interrupted aortic arch. The response to supplemental oxygen can help to distinguish PPHN from primary lung or cardiac disease. Although supplemental oxygen traditionally increases PaO2 more readily in lung disease than in cyanotic heart disease or PPHN, this may not be obvious with more advanced parenchymal lung disease. Marked

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improvement in SaO2 (increase to 100%) with supplemental oxygen suggests the presence of V/Q mismatch due to lung disease or highly reactive PPHN. Most patients with PPHN have at least a transient improvement in oxygenation in response to interventions such as high levels of inspired oxygen and/or mechanical ventilation. Acute respiratory alkalosis induced by hyperventilation to achieve PaCO2 < 30 Torr and pH >7.50 may increase PaO2 to more than 50 Torr in PPHN, but rarely in cyanotic heart disease. The echocardiogram plays an important diagnostic role and is an essential tool for managing newborns with PPHN. The initial echocardiographic evaluation rules out structural heart disease causing hypoxemia or ductal shunting (e.g., coarctation of the aorta and total anomalous pulmonary venous return). Further, as stated already, not all term newborns with hypoxemia have PPHN physiology. Although high pulmonary artery pressure is commonly found in association with neonatal lung disease, the diagnosis of PPHN is uncertain without evidence of bidirectional or predominantly right-to-left shunting across the PFO or PDA. Echocardiographic signs suggestive of pulmonary hypertension (e.g., increased right ventricular systolic time intervals and septal flattening) are less helpful. In addition to demonstrating the presence of PPHN physiology, the echocardiogram is critical for the evaluation of left ventricular function and diagnosis of anatomic heart disease, including such “PPHN mimics” as coarctation of the aorta, total anomalous pulmonary venous return, and hypoplastic left-sided heart syndrome,. Studies should carefully assess the predominant direction of shunting at the PFO as well as the PDA. Although right-to-left shunting at the PDA and PFO is typical for PPHN, predominant right-to-left shunting at the PDA but left-to-right shunting at the PFO may help to identify the important role of left ventricular dysfunction in the underlying pathophysiology. In the presence of severe left ventricular dysfunction with pulmonary hypertension, pulmonary vasodilation alone may be ineffective in improving oxygenation. In this setting, efforts to reduce PVR should be accompanied by targeted therapies to increase cardiac performance and decrease left ventricular afterload. In the setting of impaired left ventricular performance, cardiotonic therapies that increase systemic vascular resistance may further worsen left ventricular function and increase pulmonary artery pressure. Thus, careful echocardiographic assessment provides invaluable information about the underlying pathophysiology and will help guide the course of treatment.

5 Treatment of PPHN In general, management of the newborn with PPHN includes the treatment and avoidance of hypothermia, hypoglycemia,

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hypocalcemia, anemia, and hypovolemia; correction of metabolic acidosis; diagnostic studies for sepsis; and serial monitoring of arterial blood pressure, pulse oximetry (pre- and postductal), and transcutaneous PCO2. Therapy includes optimization of systemic hemodynamics with volume and cardiotonic therapy (dobutamine, dopamine, and milrinone), to enhance cardiac output and systemic O2 transport. Failure to respond to medical management, as evidenced by failure to sustain improvement in oxygenation with good hemodynamic function, often leads to treatment with extracorporeal membrane oxygenation (ECMO) [37]. Although ECMO can be a life-saving therapy, it is costly, labor-intensive, and can have severe side effects, such as intracranial hemorrhage. Since arteriovenous ECMO usually involves ligation of the carotid artery, the potential for acute and long-term CNS injuries continues to be a major concern. The goal of mechanical ventilation is to improve oxygenation, achieve “optimal” lung volume to minimize the adverse effects of high or low lung volumes on PVR, and minimize the risk for lung injury (“volutrauma”). Mechanical ventilation using inappropriate settings can produce acute lung injury (ventilator-induced lung injury, VILI), causing pulmonary edema, decreased lung compliance, and promoting lung inflammation owing to increased cytokine production and lung neutrophil accumulation. The development of VILI is an important determinant of the clinical course and the eventual outcome of newborns with hypoxemic respiratory failure, and postnatal lung injury worsens the degree of pulmonary hypertension [38]. On the other hand, failure to achieve adequate lung volumes (functional residual capacity) contributes to hypoxemia and high PVR in newborns with PPHN. Some newborns with parenchymal lung disease with PPHN physiology improve oxygenation and decrease right-to-left extrapulmonary shunting with aggressive lung recruitment during high-frequency oscillatory ventilation (HFOV) [39] or with an “open lung approach” of higher positive end-expiratory pressure with low tidal volumes, as more commonly utilized in older patients with acute RDS [40]. Marked controversy and variability exists between centers regarding the use of hyperventilation to achieve alkalosis to improve oxygenation. Past studies have clearly shown that acute hyperventilation can improve PaO2 in neonates with PPHN, providing a diagnostic test and potential therapeutic strategy. However, there are many issues with the use of hypocarbic alkalosis for prolonged therapy. Depending upon the ventilator strategy and underlying lung disease, hyperventilation is likely to increase VILI, and the ability to sustain decreased PVR during prolonged hyperventilation is unproven. Experimental studies suggest that the response to alkalosis is transient, and that alkalosis may paradoxically worsen pulmonary vascular tone, reactivity, and permeability edema [41, 42]. In addition, prolonged hyperventilation

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reduces cerebral blood flow and oxygen delivery to the brain, potentially worsening neurodevelopmental outcome. Additional therapies, including infusions of sodium bicarbonate, surfactant therapy, and the use of intravenous vasodilators, are also highly variable between centers. Although surfactant improved oxygenation and reduced ECMO in some lung diseases, such as meconium aspiration and RDS, a multicenter trial showed benefit in infants with relatively mild disease, and failed to show a reduction in ECMO utilization in the subset of newborns with idiopathic PPHN [43]. The use of intravenous vasodilator drug therapy, with agents such as tolazoline, magnesium sulfate, prostacyclin, and sodium nitroprusside, is also controversial owing to the nonselective effects of these agents on the systemic circulation. Systemic hypotension may worsen right-to-left shunting, impair oxygen delivery, and worsen gas exchange in patients with parenchymal lung disease. In addition, the initial response to such agents as tolazoline is often transient, and severe adverse effects such as gastrointestinal hemorrhage have been reported. Endotracheal administration of vasodilators, including tolazoline, sodium nitroprusside, and prostacyclin, may cause selective pulmonary vasodilation and minimize systemic hypotension. However, these data are largely limited to animal studies, and evidence is needed to confirm the safety and efficacy of this approach in humans. Inhaled nitric oxide (iNO) therapy at low doses (5–20 ppm) improves oxygenation and decreases the need for ECMO therapy in patients with diverse causes of PPHN [44–49] (Fig. 3). Multicenter clinical trials support the use of iNO in near-term (more than 34 weeks’ gestation) and term newborns, although the use of iNO in infants of less than 34 weeks’ gestation remains largely investigational. Studies support the use of iNO in infants who have hypoxemic respiratory failure with evidence of PPHN, who require mechanical ventilation and high inspired oxygen concentrations. The most common criterion employed has been the oxygenation index (OI; mean airway pressure times FiO2 times 100 divided by PaO2). Although clinical trials commonly allowed for

Fig.  3  Summary of multicenter randomized trials of inhaled NO in term newborns with hypoxemia and PPHN, showing reduction of extracorporeal membrane oxygenation utilization

76  Persistent Pulmonary Hypertension of the Newborn: Mechanisms and Treatment

enrollment with an OI of more than 25, the mean OI at entry to the study in multicenter trials was approximately 40. It is unclear whether infants with less severe hypoxemia benefit from iNO therapy [50]. The first studies of iNO treatment in term newborns reported initial doses that ranged from 20 to 80 ppm [46, 51]. In the former report, rapid improvement in PaO2 was achieved at low doses (20  ppm) for 4  h, and this response was sustained with prolonged therapy at 6 ppm. Subsequent multicenter studies confirmed the efficacy of this dosing strategy [44, 48], and showed that increasing the dose beyond 20 ppm in nonresponders did not improve outcomes [48]. The available evidence, therefore, supports the use of doses of iNO beginning at 20 ppm in term newborns with PPHN, since this strategy decreased ECMO utilization without increasing adverse effects. Although brief exposures to higher doses (40–80  ppm) appear to be safe, sustained treatment with 80 ppm NO increases the risk of methemoglobinemia [45]. In our practice, we discontinue use of iNO if the FiO2 is less than 0.60 and the PaO2 is more than 60 without evidence of “rebound” pulmonary hypertension or an increase in FiO2 of more than 15% after iNO withdrawal. Weaning can generally be accomplished in 4–5 days, and prolonged need for iNO therapy without resolution of disease should lead to a more extensive evaluation to determine whether previously unsuspected anatomic lung or cardiovascular disease is present (e.g.,, pulmonary venous stenosis, alveolar capillary dysplasia, and severe lung hypoplasia) [52]. In newborns with severe lung disease, HFOV is frequently used to optimize lung inflation and minimize lung injury. The combination of HFOV with iNO often enhances the improvement in oxygenation in newborns with severe PPHN complicated by diffuse parencyhmal lung disease and underinflation (e.g., RDS and pneumonia). A randomized, multicenter trial of infants with severe PPHN demonstrated that treatment with iNO in combination with HFOV was successful in many patients who failed to respond to HFOV or iNO alone [47]. For patients with PPHN complicated by severe lung disease, response rates for the combination of HFOV and iNO were better than those for HFOV alone or iNO with conventional ventilation. In contrast, for patients without significant parenchymal lung disease, both iNO and HFOV in combination with iNO were more effective than HFOV alone. This response to combined treatment with HFOV and iNO likely reflects both improvement in intrapulmonary shunting in patients with severe lung disease and PPHN (using a strategy designed to recruit and sustain lung volume, rather than to hyperventilate the lung) and augmented NO delivery to its site of action. Although iNO may be an effective treatment for PPHN, it should be considered only as part of an overall clinical strategy that simultaneously addresses the role of parenchymal lung disease, cardiac performance, and systemic hemodynamics.

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Although clinical improvement during iNO therapy occurs with many disorders associated with PPHN, not all neonates with acute hypoxemic respiratory failure and pulmonary hypertension respond to iNO therapy. Several mechanisms may explain the clinical variability in responsiveness to iNO therapy (Table  2). As noted above, an inability to deliver NO to the pulmonary circulation due to poor lung inflation is a major cause of poor responsiveness. In addition, poor NO responsiveness may be related to myocardial dysfunction or systemic hypotension, severe pulmonary vascular structural disease, and unsuspected or missed anatomic cardiovascular lesions (such as total anomalous pulmonary venous return, coarctation of the aorta, alveolar capillary dysplasia, pulmonary interstitial glycogenosis, and surfactant protein deficiency). Since iNO is usually delivered with high concentrations of oxygen, there is also the potential for enhanced production of reactive oxygen and reactive nitrogen metabolites, both of which may contribute to vasoconstriction and/or inadequate responses to iNO. Although hyperoxic ventilation is standard therapy for PPHN, it may be toxic to the developing lung through formation of reactive oxygen species, such as superoxide anions [53]. As noted previously, superoxide rapidly combines with and inactivates NO and in the process forms peroxynitrite; both are potent oxidants with the potential to produce vasoconstriction, cytotoxicity, and vascular smooth muscle cell proliferation. Studies in newborn lambs with pharmacologic pulmonary hypertension have indicated that iNO responsiveness is significantly blunted after even brief (30-min) periods of ventilation with 100% O2, and that oxidant stress alters NO responsiveness in part through increasing expression and activity of cGMPspecific phosphodiesterases [27, 54]. These and other studies indicate that another important mechanism of poor responsiveness to iNO may be altered smooth muscle cell responsiveness, and there are a number of emerging therapies that take advantage of our increased understanding of the cellular effects of iNO. As the response to iNO is mediated primarily by activation of soluble guanylate cyclase and cGMP-dependent protein kinase, it is logical to pursue other mechanisms that might enhance cGMP accumulation, such as inhibition of cGMP-metabolizing Table 2  Mechanisms underlying poor responses to inhaled NO Primary disease and pathophysiology Poor lung inflation Anatomic cardiac disease Left ventricular dysfunction Structural vascular disease (pulmonary venous obstruction, severe remodeling) Biochemical: increased PDE5 activity, increased O2− and ET-1 production, others

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PDE5 activity [55, 56]. Recently approved by the FDA for the treatment of male erectile dysfunction, sildenafil is a potent and highly specific PDE5 inhibitor that has been used in several preclinical studies in animal models. For instance, in lambs with experimental pulmonary hypertension, both enterically administered and aerosolized sildenafil dilate the pulmonary vasculature and augment the pulmonary vascular response to iNO [57, 58]. Intravenously administered sildenafil was found to be a selective pulmonary vasodilator with efficacy equivalent to iNO in a piglet model of meconium aspiration, although hypotension and worsening oxygenation resulted when sildenafil was used in combination with iNO [59, 60]. Data have only recently begun to emerge on the use of sildenafil alone or in combination with iNO in clinical populations, and investigations in newborn infants have been limited because sildenafil is currently available only in enteric administration form. A recent report demonstrated that enterically administered sildenafil improved oxygenation and survival in human infants with PPHN compared with a placebo [61]. Preliminary findings from a pilot study of intravenously administered sildenafil in newborns with pulmonary hypertension indicate that the infusion was generally well tolerated, with improvements in oxygenation noted in the cohorts that received higher infusion doses [62]. Similar to cGMP, cyclic AMP (cAMP) also stimulates vasodilatation, and amplification of the cAMP signaling pathway may enhance pulmonary vasodilation. Prostacyclin is a second central vasodilator that is upregulated after birth in response to ventilation of the lung. Prostacyclin stimulates adenylate cyclase to increase intracellular cAMP levels, which, similar to increased intracellular cGMP levels, lead to vasorelaxation through a decrease in intracellular calcium concentrations. Emerging data indicate that prostacyclin stimulates membrane-bound adenylate cyclase, increases cAMP levels, and inhibits pulmonary artery smooth muscle cell proliferation in  vitro. Rebound pulmonary hypertension following withdrawal of iNO has been mitigated by intravenously administered prostacyclin in children with pulmonary hypertension associated with congenital heart disease. However, as noted above, the use of systemic infusions of prostacyclin may be limited by systemic hypotension. Inhaled prostacyclin has been shown to have vasodilator effects limited to the pulmonary circulation. The actions of inhaled prostacyclin and iNO appear to be additive in humans and even synergistic in some studies. Reports of inhaled prostacyclin use in neonates with PPHN are limited, but case reports indicate it may enhance oxygenation in infants that are poorly responsive to iNO [63]. Another potential approach taking advantage of cAMP signaling is inhibition of phosphodiesterase type 3 (PDE3), which metabolizes cAMP. Milrinone, a PDE3 inhibitor, has been shown to decrease pulmonary artery pressure and resistance, and to act additively with iNO in animal studies. A recent report indi-

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cates that the intravenous administration of milrinone to neonates with severe PPHN and poor iNO responsiveness was associated with improvements in oxygenation without compromising hemodynamic status [64]. Finally, although newer therapies, including HFOV and iNO, have led to a dramatic reduction in the need for ECMO therapy, ECMO remains an effective and potentially lifesaving rescue modality for severe PPHN. Current patterns of ECMO use demonstrate persistent use in neonates with CDH and patients with severe hemodynamic instability, with less need for ECMO in meconium aspiration, RDS, idiopathic PPHN, and other disorders. Ongoing laboratory and clinical studies are currently exploring newer agents that may soon have therapeutic roles in PPHN such as SOD, soluble guanylate cyclase activators and stimulators, Rho kinase inhibitors and others (Table 3).

6 Summary PPHN is a clinical syndrome that is associated with diverse cardiopulmonary diseases, with pathophysiologic mechanisms including pulmonary vascular, cardiac, and lung disease. Experimental work on basic mechanisms of vascular regulation of the developing lung circulation and models of perinatal pulmonary hypertension has improved our therapeutic approaches to neonates with PPHN. iNO has been shown to be an effective pulmonary vasodilator for infants with PPHN, but successful clinical strategies require meticulous care of associated lung and cardiac disease. More work is needed to expand our therapeutic repertoire to further improve the outcome of the sick newborn with severe hypoxemia, especially in patients with lung hypoplasia and advanced structural vascular disease.

Table 3  New mechanisms and emerging therapies for persistent pulmonary hypertension of the newborn Mechanism Specific therapy Increased superoxide generation High PDE5 activity Impaired/oxidized sGC

rhSOD PDE5 inhibitors (sildenafil) sGC activators/stimulators (BAY 58-2667, BAY 41-2272) rhVEGF ET receptor antagonists (bosentan) Prostacyclin analogs

Impaired VEGF signaling Increased ET-1 Altered prostacyclin production High Rho kinase activity Rho kinase inhibitors (fasudil) ET endothelin; rhSOD recombinant human superoxide dismutase; rhVEGF recombinant human vascular endothelial growth factor; sGC soluble gunylate cyclase; VEGF vascular endothelial growth factor Reprinted with permission from Abman [65]

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19. Ivy DD, Ziegler JW, Dubus MF, Fox JJ, Kinsella JP, Abman SH (1996) Chronic intrauterine pulmonary hypertension alters endothelin receptor activity in the ovine fetal lung. Pediatr Res 39:435–442 20. Ivy DD, Parker TA, Ziegler JW, Galan HL, Kinsella JP, Tuder RM, Abman SH (1997) Prolonged endothelin A receptor blockade attenuates pulmonary hypertension in the ovine fetus. J Clin Invest 99:1179–1186 21. Fike CD, Slaughter JC, Kaplowitz MR, Zhang Y, Aschner JL (2008) Reactive oxygen species from NADPH oxidase contribute to altered pulmonary vascular responses in piglets with chronic hypoxiainduced pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 295:L881–L888 22. Konduri GG, Ou J, Shi Y, Pritchard KA Jr (2003) Decreased association of HSP90 impairs endothelial nitric oxide synthase in fetal lambs with persistent pulmonary hypertension. Am J Physiol Heart Circ Physiol 285:H204–H211 23. Wedgwood S, Steinhorn RH, Bunderson M et al (2005) Increased hydrogen peroxide downregulates soluble guanylate cyclase in the lungs of lambs with persistent pulmonary hypertension of the newborn. Am J Physiol Lung Cell Mol Physiol 289:L660–L666 24. Konduri GG, Bakhutashvili I, Eis A, Pritchard KA (2007) Oxidant stress from uncoupled nitric oxide synthase impairs vasodilation in fetal lambs with persistent pulmonary hypertension. Am J Physiol Heart Circ Physiol 292:H1812–H1820 25. Wedgwood S, Black SM (2003) Role of reactive oxygen species in vascular remodeling associated with pulmonary hypertension. Antioxid Redox Signal 5:759–769 26. Lakshminrusimha S, Russell JA, Wedgwood S et  al (2006) Superoxide dismutase improves oxygenation and reduces oxidation in neonatal pulmonary hypertension. Am J Respir Crit Care Med 174:1370–1377 27. Farrow KN, Groh BS, Schumacker PT et  al (2008) Hyperoxia increases phosphodiesterase 5 expression and activity in ovine fetal pulmonary artery smooth muscle cells. Circ Res 102:226–233 28. Faraci F, Didion S (2004) Vascular protection: Superoxide dismutase isoforms in the vessel wall. Arterioscler Thromb Vasc Biol 24:1367–1373 29. Balasubramaniam V, Le Cras TD, Ivy DD, Kinsella J, Grover TR, Abman SH (2003) Role of platelet-derived growth factor in the pathogenesis of perinatal pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 284:L826–L833 30. Chambers CD, Hernandez-Diaz S, Van Marter LJ et  al (2006) Selective serotonin-reuptake inhibitors and risk of persistent pulmonary hypertension of the newborn. N Engl J Med 354:579–587 31. Fornaro E, Li D, Pan J, Belik J (2007) Prenatal exposure to fluoxetine induces fetal pulmonary hypertension in the rat. Am J Respir Crit Care Med 176:1035–1040 32. Walsh-Sukys MC, Tyson JE, Wright LL et al (2000) Persistent pulmonary hypertension of the newborn in the era before nitric oxide: practice variation and outcomes. Pediatrics 105:14–20 33. Van Marter LJ, Leviton A, Allred EN (1996) PPHN and smoking and aspirin and nonsteroidal antiinflammatory drug consumption during pregnancy. Pediatrics 97:658–663 34. Alano MA, Ngougmna E, Ostrea EM Jr, Konduri GG (2001) Analysis of nonsteroidal antiinflammatory drugs in meconium and its relation to persistent pulmonary hypertension of the newborn. Pediatrics 107:519–523 35. Pearson DL, Dawling S, Walsh WF et al (2001) Neonatal pulmonary hypertension–urea-cycle intermediates, nitric oxide production, and carbamoyl-phosphate synthetase function. N Engl J Med 344:1832–1838 36. Abman SH, Kinsella JP (1995) Inhaled nitric oxide for persistent pulmonary hypertension of the newborn: the physiology matters. Pediatrics 96:1153–1155

1118 37. UK Collaborative ECMO Trial Group (1996) UK collaborative randomised trial of neonatal extracorporeal membrane oxygenation. Lancet 348:75–82 38. Patterson K, Kapur SP, Chandra RS (1988) PPHN: pulmonary pathologic effects. In: Rosenberg HS, Berstein J (eds) Cardiovascular diseases, perspectives in pediatric pathology, vol 12. Karger, Basel, pp 139–154 39. Kinsella JP, Abman SH (2000) Clinical approach to inhaled NO therapy in the newborn. J Pediatr 136:717–726 40. Acute Respiratory Distress Syndrome Network (2000) Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the ARDS. N Engl J Med 342:1301–1308 41. Gordon JB, Martinez FR, Keller PA, Tod ML, Madden JA (1993) Differing effects of acute and prolonged alkalosis on hypoxic pulmonary vasoconstriction. Am Rev Resp Dis 148:1651–1656 42. Laffey JG, Engelberts D, Kavanaugh BP (2000) Injurious effects of hypocapnic alkalosis in the isolated lung. Am J Resp Crit Care Med 162:399–405 43. Lotze A, Mitchell BR, Bulas DI, Zola EM, Shalwitz RA, Gunkel JH (1998) Multicenter study of surfactant (beractant) use in the treatment of term infants with severe respiratory failure. Survanta in Term Infants Study Group. J Pediatr 132:40–47 44. Clark RH, Kueser TJ, Walker MW et  al (2000) Low dose nitric oxide therapy for persistent pulmonary hypertension of the newborn. N Engl J Med 342:469–474 45. Davidson D, Barefield ES, Kattwinkel J et al (1998) Inhaled nitric oxide for the early treatment of persistent pulmonary hypertension of the term newborn: a randomized, double-masked, placebo-controlled, dose-response, multicenter study. Pediatrics 101:325–334 46. Kinsella JP, Shaffer E, Neish SR, Abman SH (1992) Low-dose inhalational nitric oxide in persistent pulmonary hypertension of the newborn. Lancet 340:819–820 47. Kinsella JP, Truog WE, Walsh WF et al (1997) Randomized, multicenter trial of inhaled nitric oxide and high-frequency oscillatory ventilation in severe, persistent pulmonary hypertension of the newborn. J Pediatr 131:55–62 48. Neonatal Inhaled Nitric Oxide Study Group (1997) Inhaled nitric oxide in full-term and nearly full-term infants with hypoxic respiratory failure. New Engl J Med 336:597–604 49. Roberts JD, Fineman J, Morin FC III et  al (1997) Inhaled nitric oxide and persistent pulmonary hypertension of the newborn. New Engl J Med 336:605–610 50. Konduri GG, Solimani A, Sokol GM et  al (2004) A randomized trial of early versus standard inhaled nitric oxide therapy in term and near-term newborn infants with hypoxic respiratory failure. Pediatrics 113:559–564 51. Roberts JD, Polaner DM, Lang P, Zapol WM (1992) Inhaled nitric oxide in persistent pulmonary hypertension of the newborn. Lancet 340:818–819

S.H. Abman et al. 52. Goldman AP, Tasker RC, Haworth SG, Sigston PE, Macrae DJ (1996) Four patterns of response to inhaled nitric oxide for persistent pulmonary hypertension of the newborn. Pediatrics 98:706–713 53. Lakshminrusimha S, Russell JA, Steinhorn RH, Ryan RM, Gugino SF, Morin FC III, Swartz DD, Kumar VH (2006) Pulmonary arterial contractility in neonatal lambs increases with 100% oxygen resuscitation. Pediatr Res 59:137–141 54. Lakshminrusimha S, Russell JA, Steinhorn RH et  al (2007) Pulmonary hemodynamics in neonatal lambs resuscitated with 21%, 50%, and 100% oxygen. Pediatr Res 62:313–318 55. Ziegler JW, Ivy DD, Wiggins JW, Kinsella JP, Clarke WR, Abman SH (1998) Effects of dipyridamole and inhaled nitric oxide in pediatric patients with pulmonary hypertension. Am J Resp Crit Care Med 158:1388–1395 56. Atz AM, Wessel DL (1999) Sildenafil ameliorates effects of inhaled nitric oxide withdrawal. Anesthesiology 91:307–310 57. Ichinose F, Erana-Garcia J, Hromi J, Raveh Y, Jones R, Krim L, Clark MWH, Winkler JD, Bloch KD, Zapol WM (2001) Nebulized sildenafil is a selective pulmonary vasodilator in lambs with acute pulmonary hypertension. Crit Care Med 29:1000–1005 58. Weimann J, Ullrich R, Hromi J, Fujino Y, Clark MWH, Bloch KD, Zapol WM (2000) Sildenafil is a pulmonary vasodilator in awake lambs with acute pulmonary hypertension. Anesthesiology 92:1702–1712 59. Shekerdemian LS, Ravn HB, Penny DJ (2002) Intravenous sildenafil lowers pulmonary vascular resistance in a model of neonatal pulmonary hypertension. Am J Resp Crit Care Med 165:1098–1102 60. Shekerdemian LS, Ravn HB, Penny DJ (2004) Interaction between inhaled nitric oxide and intravenous sildenafil in a porcine model of meconium aspiration syndrome. Pediatr Res 55:413–418 61. Baquero H, Soliz A, Neira F, Venegas ME, Sola A (2006) Oral sildenafil in infants with persistent pulmonary hypertension of the newborn: a pilot randomized blinded study. Pediatrics 117: 1077–1083 62. Steinhorn RH, Kinsella JP, Butrous G, Dilleen M, Oakes M, Wessel DL (2007) Open-label, multicentre, pharmacokinetic study of IV sildenafil in the treatment of neonates with persistent pulmonary hypertension of the newborn (PPHN). Circulation 116:II-614 63. Kelly LK, Porta NF, Goodman DM, Carroll CL, Steinhorn RH (2002) Inhaled prostacyclin for term infants with persistent pulmonary hypertension refractory to inhaled nitric oxide. J Pediatr 141: 830–832 64. McNamara PJ, Laique F, Muang-In S, Whyte HE (2006) Milrinone improves oxygenation in neonates with severe persistent pulmonary hypertension of the newborn. J Crit Care 21:217–222 65. Abman SH (2007) Recent advances in the pathogenesis and treatment of persistent pulmonary hypertension of the newborn. Neonatology 91(4):283–290

Chapter 77

The Pulmonary Circulation in Congenital Heart Disease Thomas J. Kulik and Mary P. Mullen

Abstract  The right-to-left shunting in patients with a large ventricular septal defect (VSD) is caused by increased pulmonary vascular resistance (PVR) secondary to pulmonary vascular disease. The pulmonary circulation hugely impacts the clinical phenotype of many congenital heart lesions. For example, PVR is a major determinant of pulmonary blood flow (PBF; hence left ventricular output and therefore the likelihood of congestive heart failure) in infants with a large interventricular or great vessel communication (e.g., VSD; patent ductus arteriosus). In babies with a single cardiac ventricle, the magnitude of PVR often is a major determinant of both ventricular volume load and systemic oxygen delivery. On the other hand, over the longer run, many cardiac malformations can cause PVR to increase, often resulting in serious disability for the patient so afflicted. This is especially true for single-ventricle patients outside the neonatal period, for whom operative palliation with a cavopulmonary circulation (e.g., surgically directing the systemic venous return directly to the lungs, without an inter­ mediary pumping chamber) requires a healthy (i.e., low PVR) pulmonary circulation. But patients with two ventricles can also suffer disability and early demise from increased PVR related to a cardiac defect. So, depending upon the circumstances, with heart malformations the pulmonary vascular bed may prove problematic owing to too little (actually, normal) or too much PVR. The primary focus of this chapter is on the latter, increased PVR secondary to increased pulmonary artery (PA) pressure and/or flow caused by cardiac structural malfor­ mations. We use pulmonary vascular disease (PVD) to denote this increased PVR, which is accounted for by both pathological remodeling of the pulmonary circulation and active vasoconstriction of small PAs, in varying relative degrees. Keywords  Eisenmenger syndrome • Septal defect of the heart • Patent ductus arteriosus • Right-to-left shunt • Pulmonary vasoconstriction • Pulmonary blood flow T.J. Kulik (*) Department of Cardiology, Children’s Hospital Boston, 300 Longwood Avenue, Boston, MA 02115, USA e-mail: [email protected]

1 Introduction In 1958 the great Paul Wood related, in the Croonian Lecture at the Royal College of Physicians, how investigators rather haltingly came to discern the physiology and pathology of Eisenmenger syndrome [1]. It was not until the late 1940s to early 1950s that people tumbled to the now-obvious notion that right-to-left shunting in patients with a large ventricular septal defect (VSD) is caused by increased pulmonary vascular resistance (PVR) secondary to pulmonary vascular disease; previous hypotheses attributed the observed cyanosis to reduced oxygenation within the lungs or decreased cardiac output. Although Wood, and others, got much of the story right even 50 years ago, our understanding of how structural malformations of the heart impact the pulmonary circulation, and vice versa, has advanced enormously since then, and this knowledge critically informs practitioners of cardiology and cardiac surgery, especially in pediatrics. Indeed, the “lesser” circulation hugely impacts the clinical phenotype of many congenital heart lesions. For example, PVR is a major determinant of pulmonary blood flow (PBF; hence left ventricular output and therefore the likelihood of congestive heart failure) in infants with a large interventricular or great vessel communication (e.g., VSD; patent ductus arteriosus). In babies with a single cardiac ventricle, the magnitude of PVR often is a major determinant of both ventricular volume load and systemic oxygen delivery. The fact that PVR is normally much lower than systemic resistance can actually be disadvantageous in this setting, as higher than normal PVR (up to a point) enhances systemic oxygen delivery in unoperated infants with a single ventricle [2]. On the other hand, over the longer run, many cardiac ­malformations can cause PVR to increase, often resulting in serious disability for the patient so afflicted. This is ­especially true for single-ventricle patients outside the neonatal period, for whom operative palliation with a cavopulmonary circulation (e.g., surgically directing the systemic venous return directly to the lungs, without an intermediary pumping chamber) requires a healthy (i.e., low PVR) pulmonary circulation. But patients with two ventricles can also suffer disability and early demise from increased PVR related to a cardiac defect.

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So, depending upon the circumstances, with heart malfor­ mations the pulmonary vascular bed may prove problematic owing to too little (actually, normal) or too much PVR. The primary focus of this chapter is on the latter, increased PVR secondary to increased pulmonary artery (PA) pressure and/ or flow caused by cardiac structural malformations. We use pulmonary vascular disease (PVD) to denote this increased PVR, which is accounted for by both pathological remo­ deling of the pulmonary circulation and active vasoconstriction of small PAs, in varying relative degrees. Our use of this term in this way, which rolls together both pathological remodeling and active vasoconstriction, requires some explanation, as “pulmonary vascular disease” (PVD) usually refers specifically to pathological anatomic changes in the vascular bed. We decline to cleave vasoconstriction from structural vascular changes when conceptualizing the problem of increased PVR for two reasons. Firstly, for any population of patients so afflicted, many or most will have both vasoconstriction and remodeling. Moreover, even if we measure the effect of a pulmonary vasodilator on an indi­ vidual’s pulmonary hemodynamics, we will still have only a partial understanding of how PVR is partitioned into active and anatomic components for that patient: our ability to dilate the lung’s circulation is imperfect, and we can never be certain how much residual smooth muscle ­contractile tone remains with a dilator. To dichotomize active and fixed pathological anatomic changes therefore implies a level of understanding actually lacking, and leeches away an important nuance from thinking about this problem. One could argue that it is useful to (as best as ­possible) separate (irreversible) anatomic changes from (reversible) ­vasoconstriction because fixed changes are not amenable to resolution with known therapy, with the ­important clinical implications that follow. Although there is utility in that, when one considers that chronic vasodilator therapy can substantially reduce PVR which does not respond acutely to dilators, the line between “fixed” and reversible causes of increased PVR blurs, whether because “irreversible” anatomic changes are not so irreversible after all, or because release of vasoconstriction can sometimes take days or weeks rather than seconds or minutes. Secondly, though our understanding of the pathobiology of PVD is pitifully limited, it seems likely that active vasoconstriction both directly reduces vascular luminal area and causes pathological remodeling (and, conversely, that vasoconstriction in the remodeled circulation has a disproportionate effect on PVR). It therefore seems important to consider these two factors as being intimately associated, realizing that they are in some ways distinct. We will also briefly discuss abnormalities of the pulmonary circulation that occur in conjunction with cardiac malformations with right-sided obstruction. These cardiac lesions tend to be associated with decreased PA pressure and flow (but not invariably so) and any PA disease usually resides in the large (main, lobar, or proximal segmental) PAs.

T.J. Kulik and M.P. Mullen

2 The Pathology and Physiology of PVD The term “PVR” is used here as is customary: PVR = mean PA pressure – mean left atrial pressure/PBF. PVR is commonly expressed in dyne . sec/cm5, or Wood units (mmHg/l min; 1 Wood unit = 80 dyne . sec/cm5 usually indexed to body surface area in pediatrics. Normal PVR is 1–3 units . m2 after the first approximately 2 months of life [3]. (NB: Some older papers report total pulmonary resistance, which is PA pressure divided by PBF.). Precisely measuring PVR, and interpreting changes in this variable – e.g., after administration of a pharmacological agent – in patients with cardiac shunting lesions requires care and can be difficult. With unrestrictive interventricular or great vessel communications (i.e., the systolic pressures in the pulmonary and systemic circulations are the same), mean PA pressure may change little with change in PVR, and an alteration in resistance may be largely manifest in a change in PBF [4]. However, alteration in systemic vascular resistance, independent from alteration in PVR, will also change PBF in these circumstances, complicating interpretation of observed hemodynamics. Also, because PVR falls with an increase in flow due to passive mechanical effects [5], it may be difficult to parse any alteration in vascular tone from these passive changes. Finally, accurately measuring PBF with certain lesions (e.g., patent ductus arteriosus) using clinically available methods may be impossible. Increased PVR can result not only from structural changes (decreased luminal area of arteries ± veins, ± a diminished number of vessels) but also from active vaso­ constriction of pulmonary resistance vessels. We know this because if patients with cardiac malformations and increased PVR are given vasodilators, some show an acute decrease in both mean PA pressure and PVR [6–11]. This is particularly true for infants, but even older children and adults with markedly increased PVR are often “reactive” [6, 12–14]. A variety of agents can vasodilate the lung under these circumstances (increased FiO2, inhaled nitric oxide, acetylcholine, tolazoline hydrochloride, isoproterenol), and multiple vasodilators in combination have been shown to be more effective than either alone (e.g., high FiO2plus acetylcholine [6], and high FiO2 plus inhaled nitric oxide) [15, 16]. Since these agents differ in their mechanism of action (opening K+ channels; increasing the level of intracellular cyclic GMP directly or via endothelial release of nitric oxide; a-adrenergic blockade; increasing the level of intracellular cyclic AMP), these observations afford little clue as to the mechanism of the smooth muscle activation with increased pressure and flow. This reactivity, when present, is variable in magnitude, and because it is probably impossible to eliminate all active tone from pulmonary vessels in  vivo, one cannot know precisely how PVR is partitioned between structural vascular changes and vasoconstriction.

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3 The Microarchitecture of PVD

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The progressive changes in pulmonary vascular structure that occur with defects involving an unrestrictive communication between the systemic and pulmonary circulations (e.g., large VSD; patent ductus arteriosus) were described (Fig. 1). Heath–Edwards (H-E) grade 1 changes consist of medial hypertrophy, extension of smooth muscle into nor­ mally nonmuscular arteries, and adventitial thickening and fibrosis. Grade 2 changes include increased medial hypertrophy and cellular intimal proliferation, sometimes sufficient to occlude the vessel, in arteries of less than 300-mm diameter. Grade 3 changes include extension of cellular intimal proliferation into arteries of 300–500-mm diameter, and intimal fibrosis in vessels smaller than 300 mm, resulting in widespread occlusion of these small arteries. Longitudinal fasciculi of smooth muscle develop in the

hypertrophied media of medium- and large-diameter arteries. In late stage 3, generalized dilation lesions are seen. Though not emphasized, splitting “or elastosis” of the internal elastic lamina was also described. The arteries in grade 4 show thinning of the media and generalized dilation, as well as local areas of dilation (“dilation lesions”). Dilation lesions are of three types: “plexiform,” “angiomatoid,” and veinlike branches of hypertrophied (usually occluded) muscular PAs. Thrombi are sometimes seen in these lesions. In grade 4, plexiform lesions are present, but the other types of dilation lesions are found in grade 5, where medial fibrosis is also found. The rarely seen grade 6 indicates the presence of the preceding plus necrotizing arteritis. Little attention was given to the pulmonary veins, except to say that with VSDs the veins are normal, but that intimal fibrosis is seen with atrial septal defects (ASDs). The virtue of this description is that it clearly identifies multiple fundamental morphological characteristics of PVD caused by increased pressure and flow: medial hypertrophy, intimal hyperplasia, intimal and medial fibrosis, dilation of small PAs, and dilation and plexiform lesions. Heath and Edwards also suggested that these abnormalities occur in a stereotyped sequence (although they did not actually show that in their paper). Its most important limitations are that no quantification of the pathological changes is provided, and that other important characteristics of the remodeled ­circulation were not uncovered (the reduction in the number of small PAs) or described in much detail (distal extension of smooth muscle).

Fig. 1  Heath–Edwards classification of pulmonary vascular pathological changes observed in patients with congenital cardiac malformations with intracardiac shunting. (a) Grade 1 – medial hypertrophy (magnification ×150). (b) Grade 2 – cellular intimal proliferation (×250). (c) Grade 3

– intimal fibrosis (×150). (d) Grade 4 – dilation and intimal fibrosis (×150). (e) Grade 5 – plexiform lesion (×95). (f) Grade 6 – necrotizing arteritis (×250). See the text for details. (Reproduced from [158] with permission)

Donald Heath and Jesse Edwards gave the first systematic description of pulmonary vascular remodeling with left-toright shunting lesions in 1958 [17]. Subsequent work by multiple investigators has provided additional important insights, and a better means of quantifying pathological changes in small pulmonary blood vessels.

3.1 Observations of Heath and Edwards

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3.2 Morphometric Insights A second method of assessing the pulmonary vascular bed was subsequently developed which has provided additional information regarding the earliest changes in growth and development of the lung associated with pulmonary arteriopathy. The morphometric approach quantifies the thickness of the arterial muscular coat, the degree of abnormal distal extension of smooth muscle, and the density of small PAs relative to the number of alveoli. Although these studies initially used whole lung injected with a barium–gelatin mixture, modified morphometric analysis has also been applied to uninjected postmortem lungs and biopsy material [18–21]. Morphometric structural findings are graded as follows: grade A – appearance of smooth muscle more peripherally than normal (into normally nonmuscular arteries) with or without modest medial hypertrophy (medial thickness less than or equal to 1.5 times the normal thickness); grade B – distal extension of muscle plus medial thickness 1.5 – 2.0 times normal thickness (mild), or greater than 2  times normal thickness (severe); grade C – grade B plus a decreased number of peripheral arteries relative to alveoli. Grade C “mild” indicates a less than 50% reduction in the number of ­peripheral arteries, and “severe” denotes a reduction greater than 50%. Grade A changes are seen with increased PBF but normal PA pressure (these are normal vessels from the standpoint of H-E criteria). Grade B mild changes usually occur with increased PA pressure (there is always PA hypertension with severe grade B). Any of the three morphometric changes can be observed with any of the H-E 1–3 grades, although grades A and B mild are rarely seen with any H-E abnormality, and H-E 3 is almost always accompanied by grade C findings [20, 22]. Pulmonary venous abnormalities with increased PBF ± pressure are quite variable: often the veins are normal, but increased medial thickness is sometimes found, as well as arterialization (development of an external elastic lamina), and occasionally intimal fibrosis in older patients [20, 21, 23, 24] (Fig. 2). Other observations made using morphometric techniques are relevant to the architecture of pulmonary vascular remodeling. For example, Haworth carefully examined lungs from patients who died with a variety of different types of heart lesions and described the pattern of early intimal pathology: intimal proliferation and fibrosis reduces the cross-sectional area of the vascular bed both by reducing the lumen of very small PAs and by completely occluding small branch vessels, reducing the number of small PAs [24]. By establishing that abnormal extension of smooth ­muscle and a reduction of small artery density are as much a part of PVD as medial hypertrophy and intimal changes, ­morphometric analysis has contributed importantly to our understanding of the architecture of pulmonary vascular remodeling. Also, by quantifying early changes caused by increased flow and pressure (distal extension and mild

T.J. Kulik and M.P. Mullen

hypertrophy) this analysis has enhanced our understanding of the evolution of pathological remodeling. There are some puzzles related to data derived from this approach, however, which require further exploration. For example, some [20] but not all [24] reports indicate that grade C changes are almost always seen in the context of increased PVR. The external diameter of intra-acinar arteries (i.e., arteries accompanying the most distal respiratory unit – respiratory bronchiole, alveolar duct, and alveolus) with increased pressure and flow appears to be quite variable [20, 21, 24]. Not all studies of increased pressure and flow lesions have observed a reduced number of peripheral arteries [25], and there are other differences in morphometric findings between different types of defects with similar hemodynamic profiles that lack clear explanation [24].

3.3 Angiographic Assessment of Pathological Remodeling Investigators have performed angiography of small PAs in patients with congenital heart disease and compared angiographic findings with hemodynamics and lung biopsy. Nihill and McNamara [26] found dilation of small vessels with increased PBF (but normal pressure), but that with increased PVR there is also abrupt tapering of small PAs (about 1–2 mm in diameter), a reduction in the number of supernumerary arteries, and an incomplete, patchy, and reticular (as compared with ground glass) capillary blush. They also found these pathological changes, which they attributed to obstruction of small vessels, to be randomly distributed within the lung. Little correlation was found between H-E biopsy findings (which ranged from normal to grade 5) and PA pressure and PVR, but that with a patchy, incomplete capillary blush and PVR of 6 units . m2 or more, H-E grade 3 or greater changes were almost always present on biopsy. Rabinovitch et al. [27] quantified the rate of taper in small axial PAs (1.5–2.5 mm in diameter), and found that the rate of taper increased as did PVR, which correlated with morphometric findings on biopsy. Capillary blush was reduced in patients with morphometric abnormality on biopsy, especially with the most severe pathological remodeling.

4 When Is PVD Reversible? The issue of “reversibility,” i.e., reduction or normalization of PVR with elimination of the associated cardiac structural lesion, is of great clinical and biological interest. Perhaps the best place to start a discussion of reversibility of PVD is Eisenmenger syndrome, the ultimate incarnation of

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Fig. 2  Comparison of lung biopsy at time of corrective surgery for conditions associated with pulmonary artery (PA) hypertension (VSD, ventricular septal defect; DTGA, d-transposition of the great arteries; CAVC, complete atrioventricular canal. “Other” lesion include: left-sided regurgitation or obstruction; atrial septal defect; truncus arteriosus; patent ductus arteriosus, aortopulmonary window). (a) Mean PA pressure measured 1 year after surgery. (b) Mean pulmonary vascular resistance measured 1 year after surgery. Vertical lines separate normal from abnormally elevated values. Horizontal lines separate biopsy grades. Age at repair is shown. Patients who underwent repair within the first 8 months of life, regardless of biopsy grade, had normal or near­normal PA pressure and normal resistance 1 year after surgery. (Reproduced from [22] with permission from Lippincott Williams and Wilkins)

irreversibility. Paul Wood’s definition of Eisenmenger syndrome is probably as good as any of those definitions which have followed: pulmonary hypertension at the systemic level, due to high PVR (more than 800 dyne · sec/cm5) with reversed (i.e., right-to-left) or bidirectional shunt [1]. These patients have severely remodeled pulmonary vasculature (which includes the entire range of H-E grades) and, except in childhood, seldom respond to vasodilators with a decline in PVR, especially if the shunting is purely from right to left [1, 12– 14]. Surgical therapy for such patients is precluded: closure of ventricular or great vessel shunts attended by pure rightto-left shunting lacks a theoretical rationale (since, lacking the “pop off,” right ventricular pressure will increase to suprasystemic level, which is poorly tolerated). Clinical

experience confirms persistently elevated or increased PVR following repair, and poor outcome [1]. Adults with increased PA pressure and PVR of more than 800  dyne · sec/cm5, but substantial left-to-right shunting, are also at risk for progressive PVD even after surgical correction, although the response of the pulmonary vascular bed to elimination of the shunt can be much more favorable under those circumstances, as is discussed later. For patients with only modestly elevated PVR, especially those under about 2 years of age, elimination of the shunt almost always results in normal PVR. In fact, the possible responses of the pulmonary vascular bed to repair of a shunting lesion exist on a continuum, ranging from complete resolution of the PVD to no change or even an increase in resistance.

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What is the anatomic/physiological basis of alleviation of PVD, and what factors influence the likelihood of resolution of PVD following elimination of the cardiac lesion? As previously noted, there are two components of increased PVR in this setting (active vasoconstriction and structural remodeling of the pulmonary vascular bed) and both, depending upon the circumstances, may be ameliorated after repair of the cardiac defect. In some settings, vasoconstriction probably accounts for much or all of the increase in PVR related to the cardiac defect. As detailed in Sect. 5.1, relief of vasoconstriction is a very large component of the fall in PVR following relief of pulmonary venous hypertension. Dalen et al. [28] studied five adults having had mitral valve replacement and found that their average PVR fell from 1,234 dyne · sec/cm5 (preoperative) to about 675 dyne · sec/cm5 by the second postoperative day, and by the fourth day it was down to about 400 dyne · sec/ cm5 in three patients in whom this measurement was made. Atz et al. [29] investigated the effect of inhaled nitric oxide on 15 children with mitral stenosis, most of whom were studied immediately after surgical or catheter intervention on the valve. They found that inhaled nitric oxide acutely decreased mean PVR from 5.8 ± 0.7 to 2.9 ± 0.4 units · m2 (Fig. 3). Increased PVR in cardiac malformations with increased PA pressure and flow is also often due to vasoconstriction. Neutze et al. [10] studied the effect of isoproterenol infusion on pulmonary hemodynamics in patients with VSD. In 22 patients with moderately increased PVR (5.0–5.9 units · m2), ranging in age from 3 to 84 months (mean 28), with isoproterenol infusion PVR decreased in all patients, falling from 6.2 to 3.2 units · m2; cardiac catheterization at an average of 5.5 years after closure of the defect showed PVR to be about the same as the preoperative isoproterenol value. Thirty-six patients had severe PVD, with PVR of 8 units or more (range, 8–43 units · m2), 2–392 months old, yet nearly half decreased

Fig. 3  Atz et al. gave 15 patients with congenital mitral stenosis and pulmonary hypertension nitric oxide by inhalation for 15 min. Most of the patients were studied immediately after cardiopulmonary bypass or interventional catheterization. Mean PA pressure fell in all patients. (Reprinted from [29] with permission from Elsevier)

T.J. Kulik and M.P. Mullen

their PVR to 7 units · m2 or less with isoproterenol infusion, and in four patients it fell to less than 3 units · m2. Vogel et al. made observations consistent with these [8]. Lock et al. [4] found that 20% of patients with VSD and increased mean PA pressure (44  mmHg or more) and PVR (pulmonary-to-systemic vascular resistance ratio of 0.3 or more ) decreased their pulmonary-to-systemic vascular resistance ratio to the normal level (0.2 or less) with 100% inspired oxygen. There is not much room left for a fixed reduction in the cross-sectional area of the pulmonary vascular bed to account for increased PVR when acute interventions can reduce PVR to the normal level, or nearly so. When one considers that only a single vasodilator was used in the above-mentioned studies, and that at least some residual vascular tone therefore likely remained, it emphasizes the importance of vasoconstriction in increased PVR with congenital defects, even in patients beyond the first year of life. However, as described above, there are patients (who tend to be older and with higher PVR) who are not “responders,” with a severely pathologically remodeled vascular bed, for whom structural changes presumably account for the vast majority of the increased resistance. To what extent the microvasculature undergoes “reverse” architectural remodeling is poorly defined. It has generally been assumed that medial hypertrophy, and perhaps intimal proliferation, can regress after removal of increased pressure and/or flow [30], whereas intimal and medial fibrosis and plexiform lesions cannot. There is actually very little information directly pertaining to remodeling of the pulmonary circulation following repair, and the authors are aware of only two papers that directly address this issue. Investigators compared lung biopsies taken before and 2.5–10 years after placement of a PA band, to reduce PA pressure, in patients with a variety of different types of congenital shunting lesions [31]. A reduction of pressure was correlated with regression of medial hypertrophy, intimal proliferation, and, possibly, mild intimal fibrosis. However, there were a limited number of patients, the histological findings were variable (especially regarding intimal changes), and few hemodynamic data were provided. Damman et  al. examined lung biopsies taken before and several years after PA banding, and also concluded that medial hypertrophy and intimal “thickening” had regressed, but their data are too scanty to shed much light on the question of regression of intimal lesions [32]. Inferential data suggesting that remodeling can occur also come from studies of chronic infusion of prostacyclin, as is discussed in Sect. 7.3.2 . The authors are aware of no data regarding whether the number of small PAs is altered with favorable remodeling. It is unfortunate that we do not have a better understanding of precisely how the microcirculation undergoes reverse remodeling in the human. That said, it seems unlikely that histological observations could supplant assessment of the most physiologically (and clinically) relevant variable, i.e.,

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resistance to blood flow across the lungs. And even if biopsy did provide a perfect measure of postrepair remodeling and hemodynamics, the natural reluctance to subject patients to this procedure would greatly limit its utility. Our under­ standing of reversibility is largely informed by observations of pulmonary hemodynamics before and some time after repair, not by direct inspection of the microarchitecture of the vascular bed. A considerable body of literature has accumulated regarding this question, and although our understanding of reversibility is still imperfect, we have learned that four variables have a major impact on the likelihood that a given patient will have reversal of PVD after repair:

3. The PVR at the time of operation. Normal PVR stays ­normal after operation, whereas – generally speaking – severely increased PVR stays high or increases (beyond the first year of life or so). For patients whose PVR is moderately increased, there is considerable variability in the response to repair. 4. As yet undefined factors inherent in the individual. Reports of large series of patients studied before and following closure of cardiac shunting lesions show that there is considerable individual variability in the pulmonary circulatory response to shunt elimination (Fig.  4). The biological basis for this is unclear.

1. The type of cardiac lesion, mostly because the nature of the lesion determines the PA pressure and flow characteristics. As detailed later, PVD caused by lesions which increase PA pressure but not flow (e.g., mitral stenosis) is much more likely to resolve upon repair than that associated with increased PA pressure and flow. There are a few lesions [e.g., d-transposition of the great arteries (d-TGA)] asso­ ciated with a particularly high risk of or precocious deve­ lopment of PVD, for reasons that are unclear (Table 1). 2. The age of the patient at time of repair. For lesions that cause PVD in the first few years of life (e.g., VSD), earlier repair confers a greater likelihood of resolution of PVD. To what extent this is a simple matter of prevention of pathological remodeling, versus reversal of same, is unknown, although the fact that advanced (and irreversible) H-E changes tend to occur only in older patients ­suggests that prevention rather than resolution predominates.

These considerations are further discussed in the context of specific heart lesions next.

5 Cardiac Structural Lesions Causing PVD Consideration of the general types of structural lesions ­associated with PVD (and their impact on PA pressure and flow) is important partly because of the obvious clinical implications, and because it also affords clues to the biological processes involved in the vascular remodeling. As elaborated upon in the following subsections: (1) increased PA pressure (alone) is quick to cause active vasoconstriction and medial hypertrophy, but slow to provoke irreversible pathological remodeling of the pulmonary circulation; (2) increased PBF (alone) is very slow to cause significant vascular

Table 1  Summary of the pulmonary circulatory and clinical characteristics of some representative congenital cardiac malformations Physiological Pathological Time course Hemodynamics Lesion characteristics characteristics Risk of PVD of PVD Reversibility of PVD Increased PA pressure

MS

Increased PBF

ASD

Increased PA pressure and PBF

VSD

H-E 1–3; Active pulmonary plexiform vasoconstriction lesions rare accounts for a large fraction of the increased PVR Active ­vasoconstriction H-E 1–6 and structural changes Active ­vasoconstriction H-E 1–6, morphometric and structural A–C changes increase PVR; fixed structural changes with very high PVR Active vasoconstriction H-E 1–6, morphometric and structural A–C changes

Unclear

Unclear, but can occur in early childhood

Even very high PVR in adults is usually reversible

~10–15%

Seldom before ~20 years of age

See the text

Infancy on

Moderately increased PVR at least partially reversible, even in adults Almost always reversible for the first 1–2 years of age; likelihood of reversal falls with age and increasing PVR Reversible, but requires correction or palliation at very early age

Unclear, See the text d-TGA/VSD Complex likely high malformations with increased PA pressure and flow ASD atrial defect, d-TGA d-transposition of the great arteries, H-E Heath–Edwards, MS mitral stenosis, PA pulmonary artery, PBF pulmonary blood flow, PVD pulmonary vascular disease, PVR pulmonary vascular resistance, VSD ventricular septal defect

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Fig. 4  Grosse-Brockhoff et al. reported the effect of surgical repair of VSD in 65 patients operated on at various ages. The first postoperative catheterization was 6–8 weeks after the operation. In general, the likelihood of normal mean PA pressure after closure of the defect was greatest for patients operated on at younger age, and for those with the lowest preoperative pressure. (Reproduced from [72] with permission)

remodeling, and does so in only a relatively small minority of people; (3) increased pressure and flow together are considerably more pernicious in causing pathological remodeling, especially high-grade lesions, than either increased pressure or flow alone; and (4) other factors besides PA pressure and flow importantly influence the propensity to develop PVD. Both the clinician and the biologist need to take these observations into account when they set about to do their good work (Table 1).

5.1 Increased PA Pressure Increased pressure in the pulmonary circulation is alone ­sufficient to cause pathological remodeling. Cardiac lesions associated with increased pulmonary venous pressure but normal, or decreased PBF (e.g., mitral stenosis, cor ­triatriatum, or obstructed pulmonary venous connection), cause increased PA pressure out of proportion to pulmonary venous pressure, and pathological pulmonary vascular remodeling, even in early childhood [33]. Mitral stenosis is the most ­common structural lesion causing pulmonary venous ­hypertension, where medial hypertrophy and intimal fibrosis are observed in both arteries and veins, although plexiform lesions only rarely develop [33–38]. With mitral stenosis, PVR is highly variable, ranging from normal (even with left atrial pressure greater than 30  mmHg) to more than 1,000 dyne · sec/cm5 [28, 33, 39, 40]. As noted above, the increased PVR is usually mostly due to active pulmonary vasoconstriction, as revealed by an acute fall in PA pressure and PVR with vasodilators, and the very rapid fall in PA pressure and resistance after surgery to relieve the

stenosis [28, 29]. In fact, adult patients with mitral stenosis, even those with greatly elevated PVR, typically show a large reduction in PVR, often to normal levels, following relief of the left atrial hypertension [28, 40, 41]. This is of some ­interest, since intimal fibrosis, which is commonly thought to infer irreversible vascular disease, is commonly observed with mitral stenosis [37, 38]. Much less information is ­available for pediatric patients, although substantial ­reduction of PVR following reduction of pulmonary venous hypertension also appears to be true for infants and children, at least in the case of isolated elevation in pulmonary venous pressure (i.e., without the presence of other cardiac defects) [33, 42]. Cardiac malformations which increase pulmonary venous pressure in utero cause remodeling of the pulmonary vascular bed in the fetus and newborn. Hypoplastic left-sided heart syndrome with highly restrictive connection between the left and right atria is associated with pulmonary venous hypertension in utero, and the same is likely true for at least some patients with total anomalous pulmonary venous connection with obstruction. Marked medial hypertrophy and “arterialization” of the pulmonary veins, and dilation of pulmonary lymphatics are noted even in neonates. Abnormalities of the small PAs are less notable in the newborn, but develop early in the neonatal period [43–45].

5.2 Increased PBF Small increases in PBF (and without elevation in pressure), as occur with a small ASD, VSD, or patent ductus arteriosus, pose no appreciable risk for the pulmonary circulation [46].

77  The Pulmonary Circulation in Congenital Heart Disease

On the other hand, a large increase in PBF, even when PA pressure is initially normal, will cause PVD in a significant percentage of people. With a sufficiently large ASD – the most common lesion associated with this physiological state – marked elevation of PA pressure and PVR and advanced H-E changes may develop over time [47]. The risk of developing PVD with an ASD is relatively low. Data vary widely in the percentage of adults with ASD and PVD, probably partly because of variability in case ascertainment and partly because “PVD” is variably defined in published studies, but 10–15% seems a reasonable estimate [48–51]. What is quite clear is that the risk of PVD in the first two decades of life with ASD is very low [47, 52], in contradistinction to lesions causing increased pressure and flow (e.g., unrestrictive VSDs), where irreversible PVD in the first several years of life is not uncommon. Having said that, severe PVD in the first few months or years of life with ASD is well described [19, 53], and comes up periodically for practitioners who treat pulmonary hypertension. It is unclear whether this precocious pathological pulmonary vascular remodeling is caused by the increased PBF, or if the presence of the cardiac defect is only coincidental in these unusual cases. There are several reports of pulmonary hemodynamics in adults studied months to years after repair of ASD [54–57]. Preoperatively, these patients had at most moderate PA hypertension (mean PA pressure 60 mmHg or lower). Patients with normal or near-normal mean PA pressure ­preoperatively (lower than about 30 mmHg) had a fall in pressure after ASD closure and PA pressures were within the normal range; those with significant PA hypertension before repair also had a reduction in PA pressure, although PA pressure often remained abnormally high. With both normal and elevated preoperative PA pressure, postoperative PVR fell after repair in two of the studies [55, 56], but was unchanged or increased in two others [54, 57]. Lueker at al. [57] reported total pulmonary resistance rather than PVR. Some postoperative ASD patients, even with normal resting PA pressure, show an increase rather than the normal decline [58] in PVR with exercise [56, 57], suggesting that the pulmonary vascular bed remains abnormal despite closure of the defect.

5.3 Increased PA Pressure and Flow In cardiac lesions with an unrestrictive communication between the systemic and the pulmonary circulations, there is increased PA pressure and flow (the latter as long as PVR is less than systemic vascular resistance). Examples include, but are not limited to, isolated, unrestrictive VSD, large patent ductus arteriosus, double-outlet right ventricle without pulmonary stenosis, single ventricle malformations without significant obstruction of PBF, and excessively large surgi-

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cally placed systemic-to-pulmonary shunts. The risk of development and rate of progression of PVD is highly variable and poorly defined, although it is known that several factors clearly impact the likelihood of and the rate of progression of PVD: the type of malformation, the size of the communication between the systemic and pulmonary circuits, the propensity of the individual to develop PVD, and environmental factors (e.g., alveolar hypoxia). The best characterized lesion of this type is the isolated VSD. The microvascular remodeling associated with this lesion begins in early infancy, and consists of distal extension of smooth muscle, seen with increased PBF but normal pressure [20]. Medial hypertrophy is also a consistent finding in infants with increased PA pressure, and a reduction in the number of small PAs is often observed [20, 22, 23, 59]. Intimal proliferation (H-E grade 2) is very unusual in early infancy (but Newfeld et al. [60] found it common) but begins to appear near the end of the first year of life in patients with increased pressure and PVR [21, 22]. Two important points are worth noting: (1) Progressive PVD occurs over time. Wood [1] and others thought that adult patients with irreversible PVD from unrestrictive communication between the systemic and pulmonary circulations had never had the ­normal postnatal decline in PVR, and thus PVR had been markedly elevated since birth. It is now clear that most patients with an unrestrictive VSD who develop elevated PVR have a substantial fall in PVR in infancy, followed later by an increase in resistance [61, 62]. (2) Remodeling in the first year of life is generally limited to distal extension of smooth muscle, medial hypertrophy, and a reduction of the number of small arteries; medial hypertrophy, at least, is reversible with alleviation of the increased PA pressure. Higher grade H-E changes are usually not observed until the third year of life [21, 63], which is consistent with clinical observations documenting a reliable fall in PVR with VSD closure in the first 2 years of life, but less reliably so at older ages (described below). Precise data regarding the risk of developing PVD with VSD are limited. Babies with large a VSD often develop congestive heart failure and either succumb to or have ­surgical closure of the defect, hence the “natural history” of pulmonary vascular remodeling is often either interrupted by death or modified by therapy. Keith et al. reported experience at the Hospital for Sick Children in Toronto with about 1,500 patients with isolated VSD [62, 64]. Two hundred patients were catheterized in the first year of life, and again “some years later.” No infant with a highly restrictive defect ­(normal PVR and PBF less than twice the systemic value) (n = 43) developed elevated PVR over the course of observation. There were 146 infants with PBF greater than twice the ­systemic value, with or without an increase in PVR; 21 (14%) of them had elevated PVR (more than 20% of the systemic value) at follow-up cardiac catheterization, which was an

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increase in PVR in all but three of these patients. This is likely an underestimate of the risk of developing PVD, since some of the patients had an operation to close the VSD ­during the period of observation. (They also described 11 neonates with low flow/high PVR who at follow-up evaluation had normal PVR; they attributed this to a delay in the normal postnatal fall in PVR.) The Joint Study on the Natural History of Congenital Heart Defects made similar observations. Five hundred and thirty-seven patients with an isolated VSD (21 years old or younger, half of whom were 2–11 years old) were observed over a time period of 4–8 years [46]. None of these patients had surgical closure or palliation of the defect. In patients with normal PVR, PVR rarely increased over the period of observation, but as resistance at initial observation increased, so did the risk of progression (Table 2). Hence, although we do not know the precise relationship between PA pressure, PBF, and PVR as a function of time, it is clear (for isolated VSD; see later for other increased pressure/flow lesions) that for patients with increased PBF but normal PA pressure the risk of developing PVD – at least over a period of several years – is exceedingly small, but that increased PVR tends to increase with time, at least in patients outside the early neonatal period. Reversibility has been extensively investigated following repair of VSD. Closure of a VSD in the first 2 years of life, even when PVR is markedly elevated, reliably (but with a few exceptions) results in normal or near-normal PVR at follow-up, and the likelihood of favorable pulmonary vascular remodeling is probably even better if operation is performed in the first year of life [22, 63, 65–67]. If PVR is increased, the status of the pulmonary circulation is much less favorable at older ages of operation. Although an occasional older child or adult with substantially elevated PVR has a marked fall after repair [65, 67–69], in general patients much beyond the first few years of life or so with increased PVR preoperatively continue to have increased resistance after surgery (although the PA pressure often falls as a result of the reduced volume of PBF), and some show a progressive rise despite closure. Friedli et al. [70] reported their experience with VSD closure in 57 children (average age 4.8 years, range 11 months to 17 years, at operation). PA pressure fell

Table 2  Summary of observations from The Joint Study on the Natural History of Congenital Heart Defects regarding risk of progression of PVD in patients with VSDs PVR-to-SVR ratio at initial evaluation <0.2 0.2–0.49 0.5–0.69 >0.7 485 29 18 32 N (patients) 1.5 38 89 100 Percentage of patients with PVR-to-SVR ratio more than 0.2 at follow-up (4–8 years) Rarely did normal PVR increase. Substantially increased PVR was unusual, but tended to increase with time. See the text for details

T.J. Kulik and M.P. Mullen

in nearly all patients after closure, although it often remained elevated; seven of ten patients with a preoperative pulmonary-to-systemic resistance ratio of more than 1:3 had a progressive increase in PVR following VSD closure, as compared with two of eight with a pulmonary-to-systemic resistance ratio of less than 1:3. Their experience was somewhat less favorable than that of many others, but persistent elevation of PA pressure (although it may fall after closure) and persistent elevation of PVR are the rule rather than the exception when higher-resistance VSDs are closed beyond the first about 3–4 years of life; [54, 65, 67–69, 71, 72] for the exception, see Ikawa and et al. [73] (Fig. 4). Further evidence of residual abnormality of the pulmonary circulation after VSD repair comes from studies showing in many patients PVR increases with exercise [57, 71, 74, 75] and/or they have an exaggerated increase in PVR as a response to subambient oxygen [57]. Maron et  al. found an abnormal increase in PA pressure with exercise even in postoperative patients with normal resting PA pressure, and found that the magnitude of increase in pressure with exercise correlated positively with the age at operation [76]. The clinical consequences of persistent elevation of PVR after VSD closure appear to be related to the magnitude of residual PA pressure elevation, although available data are mostly inferential. Moller et al. [77] observed a large ­reduction in 30–35-year postoperative survival in patients ­operated on with preoperative PVR of more than 7 units, many of whose deaths were related to PVD; the negative effect on survival was considerably less marked for lesser elevation of PVR at operation. The report of Cartmill et al. [68] is consistent with this. It is important to note that although VSD is the best characterized of the increased pressure/flow cardiac lesions relative to the pulmonary circulation, it is not perfectly representative of other simple left-to-right post-atrial-­shunting lesions. For example, large patent ductus arteriosus may be associated with early development of PVD, probably because the pulmonary vascular bed is exposed to increased pressure and flow in both systole and diastole. And, as discussed next, a few more complex pressure/flow lesions are associated with a greater propensity to early development of PVD than isolated VSDs.

5.4 Complex Lesions with Increased PA Pressure and Flow Variables other than PA pressure and flow importantly impact the risk of, and the pace of developing PVD. For example, patients with unrepaired d-TGA have increased PBF but normal PA pressure after the early neonatal period, yet are at risk of developing markedly increased PVR and advanced H-E changes in the first year or two of life, a tempo far more precocious than that observed with ASD. Patients with d-TGA and VSD and/or patent ductus arteriosus are even

77  The Pulmonary Circulation in Congenital Heart Disease

more notorious for developing early PVD, and are at high risk for developing grade 4 H-E lesions in the first year of life [24, 78–82]. The reason for this propensity is unclear, and a number of possible factors have been adduced to explain this unusual tendency for pathological remodeling: polycythemia (common in these patients), increased bronchial flow, observed microthrombi in small PAs, and preferential ejection of blood into the right PA (hence increased flow to the corresponding lung) [30]. It is unclear which, if any, of these factors are involved. Two other observations also suggest that the pulmonary vascular bed in d-TGA is unusually prone to increased PVR. Progressive and severe PVD following repair in patients without PVD, although uncommon, is well described [79,  83–86], whereas it is hardly ever seen with other cardiac lesions repaired in neonates. And acute and labile pulmonary vasoconstriction in newborns with d-TGA (resulting in severe hypoxemia) is not uncommonly seen [87, 88], even though such labile PVR is rarely apparent with other neonatal ­congenital heart defects. (However, it is ­possible that the increased propensity for pulmonary vasoconstriction for d-TGA is more apparent than real, since arterial oxygen saturation in neonates with d-TGA is ­probably more dependent on PBF than for most other cyanotic lesions.) Other malformations, e.g., truncus arteriosus [25], are associated with an increased tendency to develop PVD. The notion is well entrenched that patients with trisomy 21 are prone to early development of fixed pulmonary vascular changes, especially in the context of complete atrioventricular canal defects. Multiple papers have appeared on this subject, yet convincing confirmatory evidence is lacking [89]. Alveolar hypoxia, even at the modestly elevated altitude of Denver, Colorado (5,280 ft), augments PVR with congenital cardiac defects [8, 90, 91].

5.5 Right-Sided Obstructive Lesions This is a complex group of lesions relative to the pulmonary circulation. Some (e.g., severe tetralogy of Fallot) may have decreased PBF in utero and certainly ex utero, until surgical palliation or repair is undertaken, which may be at several months of age. Others (e.g., pulmonary atresia with an intact ventricular septum) may have decreased flow in utero (since the only source of PBF is the ductus arteriosus, which is usually restrictive in this setting), but surgical palliation in the early neonatal period often results in a normal or increased volume of PBF. Patients with pulmonary atresia and VSD (aka tetralogy of Fallot and pulmonary atresia) are complex cases: depending upon the anatomy of the central PAs, and their blood supply from the aorta, the microvascular bed in any given region of the lung may be exposed to low, normal, or increased pressure and flow. Much less

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attention has been directed to the pulmonary microvascular bed with right-sided obstructive lesions than those with leftto-right shunts, probably because increased PVR due to small-vessel abnormalities is much less likely to impact the clinical phenotype of these patients (possibly excepting those with pulmonary atresia and VSD) than those with unrestrictive communication between the systemic and pulmonary circulations. Tetralogy of Fallot is the archetypical cardiac lesion ­causing decreased PBF. One morphometric study investigated injected lungs from six children with this lesion, aged 1.5–4.5 years, two having had previous surgical palliation. The intra-acinar arteries and veins were increased in number, and the PAs were somewhat thicker than normal, with distal extension of muscle into normally nonmuscular arteries. Other studies have not found medial hypertrophy in this ­setting [92]. Any abnormality of resistance PAs in tetralogy of Fallot must seldom be of physiological consequence, however, as increased PVR after repair is rarely seen unless the patient had a previously-placed shunt (and therefore exposure to increased pressure and flow) [93]. Haworth and Reid [94] used morphometric techniques to examine the pulmonary circulation in six neonates with pulmonary atresia with an intact ventricular septum and two patients with pulmonary atresia with VSD. In all cases, the only source of PBF was via a ductus arteriosus, which they found to be restrictive (3 mm or less in diameter) in all but one case. Hence, these patients presumably had decreased PBF in utero relative to the normal fetus. One patient had had an aortopulmonary shunt placed surgically 25 days prior to death. Their findings were variable, but in most cases the pre- and intra-acinar arteries were smaller than normal, had medial muscle, and were fewer in number than normal. With pulmonary atresia and VSD there are two basic anatomic arrangements for blood supply to the lungs (albeit with some overlap): (1) a patent ductus arteriosus supplies central PAs, which distribute to all or nearly all segments of the lungs, and (2) the blood supply to the lungs derives solely from aortopulmonary collateral vessels, arising from the aorta (most often the descending aorta), or occasionally its branches. In some cases, the ductus arteriosus may provide the sole supply to a large percentage of lung, the remainder being supplied by aortopulmonary collateral vessels. The aortopulmonary collateral vessels, most likely remnants of the primitive arterial supply to the lungs which normally regress when the intrapulmonary PAs become connected to the sixth aortic arches, may be one or multiple in number, and are extraordinarily variable in their size and distribution. They may be so large as to transmit high pressure and flow to the distal vascular bed, or small or stenotic in their proximal course, and therefore flow- and pressure-restrictive. In most cases small central PAs are present, and are connected to the aortopulmonary collateral vessels, but their distribution to

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the lung segments is variable and may be quite deficient; parts of the lung may be connected only to aortopulmonary collateral vessels. In addition, multiple stenoses are often present in the central PAs and collateral vessels, which substantially increase resistance to flow through the pulmonary circuit [95, 96] (Fig. 5). Haworth and McCartney [95] reported somewhat variable morphometric findings in seven specimens from patients with pulmonary atresia and VSD. The percentage medial thickness was normal or reduced in small arteries in five cases, without abnormal distal extension of smooth muscle, but in three cases there was medial hypertrophy and distal extension of muscle. In a few segments there was mild eccentric intimal “change” or fibrosis. The diameter of the peripheral arteries was normal or small, and quite variable. The arterial number was normal. The authors interpreted these findings as being compatible with a reduction in pulmonary blood flow. The size of the aortopulmonary collateral vessels supplying the examined segments was not clear from their report, and it seems likely that their observations were of areas supplied by pressure- and flow-restrictive collateral vessels. Although published data are sparse, anecdotal observations suggest that a significant fraction of patients who have “repair” of this lesion (i.e., connecting many or all of the aortopulmonary collateral vessels, and any central PA, to a conduit connected to the right ventricle and closing the VSD) are left with increased PVR. In some cases this seems to be mostly related to increased PVR at the level of the small, resistance PAs; in others, abnormalities in the central PAs (small size, focal stenosis, lack of distribution to all segments of the lung) are operative. Peripheral PA stenosis can occur as an isolated lesion, or in conjunction with other congenital cardiac malformations, especially valvar pulmonary stenosis and tetralogy of

Fig. 5  Descending aortogram of an infant with pulmonary atresia and VSD. Multiple aortopulmonary collateral vessels arising from the descending aorta supply multiple segments of the right and the left lungs, and are connected to a large central left pulmonary artery

T.J. Kulik and M.P. Mullen

Fallot. Isolated PA stenosis can affect the main or proximal branch PAs or distal lobar or segmental PA segments; narrowing at the takeoff of lobar or segmental branches is common. The stenotic areas may be focal and discrete, or long segments of the PA may be diffusely narrow [97, 98]. Peripheral PA stenosis is most commonly diagnosed in infancy and childhood, but is well described in adults [99]. The histological findings are variable, and include fibrous intimal hyperplasia, medial hypertrophy, or hypoplasia, but diffusely hypoplastic PAs may be histologically normal [99, 100]. This condition is strongly associated with a variety of congenital syndromes, most commonly Williams syndrome, Noonan syndrome, Alagille syndrome, and congenital rubella [99]. Right ventricular pressure ranges from normal (with mild or unilateral stenosis) to suprasystemic. Interestingly, at least in patients with Williams syndrome, there is a ­tendency for PA stenoses to resolve with time, sometimes remarkably so [101].

6 The Biology of PVD As noted already, there are multiple distinct, but biologically related components of PVD caused by congenital heart lesions, which are variably present: (1) active constriction of pulmonary resistance arteries, (2) extension of smooth muscle distally, into normally nonmuscular PAs, (3) medial hypertrophy (arteries and veins), (4) a reduction in the number of small PAs, (5) cellular intimal proliferation, (6) intimal and medial fibrosis, (7) plexiform and other complex lesions, and (8) miscellaneous abnormalities, such as intraluminal thrombi, adventitial thickening, and atheromatous lesions in large, elastic PAs. For none of these features do we have a mechanistic understanding of their genesis. Although factors distinct from PA pressure and flow clearly impact the development of PVD secondary to cardiac malformations, the reasonably stereotyped response of the human pulmonary vascular bed to hemodynamic abnormalities suggests that increased wall stress and endothelial shear stress must somehow play a key role. Indeed, it has been long appreciated that hemodynamic forces help shape systemic arterial architecture [102], and experimental studies have suggested that increased wall stress and endothelial shear stress are important modulators of pulmonary vascular remodeling (see later). The following is a very brief outline of a few ways in which mechanical factors may play a role of the pathogenesis of PVD, considering first vasoconstriction, and then pathological remodeling. It is beyond the scope of this review to discuss the massive accumulation of other information related to the general issue of pathological pulmonary vascular remodeling (for recent reviews, see [103–105]), although cellular and molecular mechanisms unrelated to hemodynamic

77  The Pulmonary Circulation in Congenital Heart Disease

forces must also be important components of pulmonary vascular remodeling.

6.1 Mechanical Forces and Vasoconstriction Vasoconstriction is a common feature of PVD with congenital malformations, and likely increases PVR both by decreasing the cross-sectional area of resistance vessels and via stimulating structural changes in the microcirculation. The latter could occur in two ways. First, constriction of small PAs increases intraluminal pressure (and therefore a stimulus for remodeling) in the arteries proximal to them. Second, smooth muscle contractile activation itself shares intracellular biochemical pathways with the control of growth and phenotype [106]. (Indeed, most endogenous vasoconstrictors, e.g., endothelin and epinephrine, also stimulate growth of vascular smooth muscle [107, 108].) At least under certain circumstances, contractile activation may therefore stimulate smooth muscle hypertrophy and/or hyperplasia, and connective tissue synthesis. There are at least three mechanisms by which increased PA pressure could stimulate smooth muscle contraction: 1. Stretch-inducted (myogenic) contraction. There is a large body of work investigating myogenic tone in systemic arteries, and stretch-induced contraction of small PAs has been shown in vitro [109, 110] and in vivo [111]. Some have speculated that stretch-induced contraction serves to normalize wall tension in blood vessels, by reducing the vessel radius in response to increased intraluminal pressure [112]; if so, myogenic tone could be relatively high in the hypertensive pulmonary circulation. However, it is not clear if or how this property of smooth muscle operates to activate contraction in the steady state in  vivo. Both the length of and the load born by smooth muscle cells in the vascular wall are a function of multiple factors and would not necessarily be abnormally high with increased intraluminal pressure. 2. Reflex contraction of resistance PAs caused by distention of the central PAs. Distention of the main- or large-branch PAs causes PVR to increase substantially [113–115]. This effect has been demonstrated in immature and mature animals [116], and human infants [117]. If the distended region of the PA is infiltrated with lidocaine, or is dissected through to the media of the artery to disrupt any nerves traversing the region, the vasoconstrictor reflex is abolished [114]. Although 6-hydroxydopamine (which destroys adrenergic nerve terminals) abolishes the hypertensive response [114], a-adrenergic blocking agents do not affect the response [113, 114], suggesting that nonadrenergic, noncholinergic transmission may be involved. This reflex could serve as a positive-feedback loop for

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increasing PVR since increased intraluminal pressure distends the large PAs. Unfortunately, this “reflex” is incompletely characterized and what if any role it plays in PVD is unknown, although it is tempting to adduce both myogenic tone and this reflex-mediated vasoconstriction – actually, their sudden reduction – to explain the rapid fall in PVR after relief of mitral stenosis. 3. Endothelial dysfunction. Pulmonary endothelial elaboration of vasoconstrictors, or reduction of endothelial production of vasodilators, could increase vascular tone. Abnormalities of pulmonary endothelial cells, including their morphology, and an increased density of microfilament bundles and rough endoplasmic reticulum, have been observed with increased PA pressure and flow in humans [118]. Although these ultrastructural abnormalities tell us little about the behavior of these cells, subsequent observations suggest deficient endothelium-mediated pulmonary vasodilation with increased pressure/flow lesions in humans [119], and in an animal model of increased PA pressure and flow [120].

6.2 Mechanical Forces and Vascular Remodeling In vitro studies of cultured systemic [121] and PA smooth muscle cells, and segments of PAs, have shown that mechanical stretch can serve as a growth stimulus and increase matrix protein synthesis [122–126]. In vivo experiments are also consistent with the notion that increased PA pressure and flow stimulate a variety of cellular responses associated with remodeling. For example, Reddy et al. developed a neonatal lamb model of increased PA pressure and flow (using a surgically placed aortopulmonary shunt) which has a pulmonary vascular phenotype consistent with that seen in the human (medial hypertrophy and distal extension of smooth muscle, although an increased number of small PAs is also observed) [127]. This model has been used to investigate the effect of altered PA hemodynamics on the expression of a number of factors which may play a role in vascular remodeling. The levels of endothelin-1, transforming growth factor b1, vascular endothelial growth factor, and fibroblast growth factor 2 are increased (relative to controls) in PAs and/or lung tissue from shunted lambs, as are alterations in expression of the cognate receptors consistent with growth promotion [128– 131]. Experiments using other in  vivo models have also shown that PA pressure and/or flow critically modulate the microarchitecture of the pulmonary vascular bed [132–134]. Shear stress modulates endothelial cell phenotype, and expression of a variety of factors which affect the biological processes of adjacent cells and are thought to be operative in vascular remodeling. The notion has evolved that temporally

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and spatially uniform shear stress of the appropriate magnitude promotes endothelial expression of substances which negatively modulate growth and matrix protein synthesis (e.g., nitric oxide and prostacyclin); conversely, disturbed flow or low shear stress decreases synthesis of these negative growth regulators, and increases endothelial expression of smooth muscle mitogens, endothelin-1, and other substances operative in pathological remodeling [135–137]. Most of the work in this arena has focused on the systemic vascular bed, but surely many of the same biological processes apply to the pulmonary circulation. There is still much to be learned before a comprehensive and detailed theory of pulmonary vascular modeling related to hemodynamic disturbance can evolve. The search for such a theory is confounded by the complexity of the phenomenon, by our relatively crude understanding of how mechanical forces affect biological processes in vivo, and the lack of specific antagonists for (or genetically engineered models of) stretch- or shear-stress-mediated cellular perturbations. Insights into the pathogenesis of other forms of PVD will doubtlessly apply to uncovering the biological processes of that related to cardiac malformations. In particular, it will be interesting if gene mutations associated with idiopathic ­pulmonary hypertension (e.g., BMPR2 and ALK-1 mutations) are in any way linked with cardiac-malformationrelated PA hypertension; available data at this time suggest no association with BMPR2 mutations [138], or are too limited to be definitive [139].

7 Clinical Management 7.1 Prevention The best cure for PVD related to congenital heart lesions is prevention, by correction of the defect early enough to prevent permanent pulmonary vascular remodeling. As outlined already, the “window” of opportunity for prevention is reasonably wide, and to some extent lesion-specific. Although the time of operation is, to some extent, center-specific, in general ASDs are repaired by age 4–5 years, VSDs within the first year of life, complete atrioventricular canal defects and large patent ductus arteriosi by 6 months of age, and d-TGA/VSD is either repaired or is palliated using a pulmonary arterial band in the first few weeks of life. For neonates with a single cardiac ventricle the aim is to absolutely minimize pathological remodeling, and surgery to secure normal PA pressure (if needed) is usually undertaken in the first few weeks. Patients of any type who show evidence of higher than expected PVR may require operation sooner, and other factors (most often, congestive heart failure) may also dictate earlier surgery.

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7.2 Deciding When Surgical Intervention Is Appropriate For the unusual patient with a severely remodeled pulmonary circulation, things are more complicated and less satisfactory, starting with the decision of whether repair is appropriate. (We refer here to patients with increased PA pressure/flow lesions, not those with left-sided inflow obstruction; as noted previously, for the latter even markedly increased PVR in adults generally responds well to relief of pulmonary venous hypertension.) Few patients less than 1–2 years old have severely elevated PVR (pulmonary-to-­ systemic vascular resistance ratio of more than 0.75) and, even for those who do, given the considerable capacity of the young pulmonary circulation to “reverse” remodel, surgical repair is usually offered unless PVR is suprasystemic. On the other hand, older patients with severely elevated PVR are at risk for having persistently increased PA ­pressure, or even a progressive increase in PVR after repair. Significantly increased PVR also increases the risk of mortality immediately after operation in patients of all ages [10, 16] owing to labile and at times severe acute pulmonary vasoconstriction. This variably increased PVR is apparently caused by one or more endogenous vasoconstrictors, or perhaps lack of an endogenous vasodilator(s), in combination with the pathologically remodeled vascular bed [140]. More than increased smooth muscle cell mass may be involved in this hyperreactivity; e.g., abnormal endothelial function may play a role [9, 119, 141], but see [142]. At increased risk are patients with an unrestrictive ventricular or great vessel communication, truncus arteriosus having increased PVR before operation, and pulmonary venous hypertension (e.g., total anomalous pulmonary connection with obstruction or mitral stenosis) [22, 143, 144]. For patients with high, but not systemic-level PVR, we do not have a sufficiently sensitive and specific method for determining whom will respond favorably to operation (i.e., will have a substantial and lasting fall in PA pressure). Wood [1], 50 years ago, suggested that surgical repair is indicated for those whose PVR does not exceed 10 units, and whose pulmonary-to-systemic flow ratio is at least 2:1. Borderline (for repair) cases have PVR of 10–12 units, and a pulmonary-to-systemic flow ratio of 1.75–2. A more modern approach to determining operative suitability is to consider resting PVR along with the reactivity of the ­pulmonary circulation (enhanced inspired oxygen, tolazoline, isoproterenol, and inhaled nitric oxide have been used). Vogel et al. found that patients with VSD, even those ­having PVR exceeding 1,000 dyne · sec/cm5, who were “reactive” (i.e., responded to a 1 mg/kg bolus of tolazoline with a reduction of PVR to 450 dyne · sec/cm5 or less) had a substantial fall in PVR postoperatively, whereas nonreactors did

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not. But the patients of Vogel et al. were studied in Denver (alveolar hypoxia adds a reactive component to the PVR not present at sea level [91]) and nearly all of the reactive patients were 5 years old or younger. Neutze et  al. [10] studied patients with VSD and found that for those with PVR of less than 8 units (average age, 34 months) reactive patients (i.e., those whose decreased PVR to less than 7 units with isoproterenol infusion) had normal or modestly increased PVR after surgical closure (n = 15). Four of the unreactive patients had surgical repair, at least three of whom died or had evidence of progressive PVD (one was lost to follow-up). Balzer et  al. [16] collected data on patients with a variety of cardiac malformations (median age 28 months), including a small percentage evaluated for heart transplantation, who had vasodilator testing with enhanced FiO2 and inspired nitric oxide (n = 124). They found that if a combination of FiO2 = 1.0 and inhaled nitric oxide were used to evaluate reactivity, PVR of less than 5.3 units . m2 and a pulmonary-to-systemic resistance ratio of less than 0.27 (with vasodilators) was 97% sensitive and 90% specific for identifying patients with a good ­outcome (alive and without right-sided heart failure) after operation. On the other hand, Lock et al. found no relationship between pulmonary vascular response to 100% inspired oxygen and outcome after repair of VSD [4]. Lung biopsy (usually as an adjunct to hemodynamic data) has been advocated as a means of determining suitability for surgical repair. Indeed, it has been shown that preoperative biopsy (analyzed by H-E criteria) may be predictive of persistent PA hypertension in patients older than 2 years of age following repair of interventricular communications [22]. Unfortunately, there are only limited follow-up data ­correlating preoperative biopsy with eventual outcome, and the available information suggests that biopsy findings are neither highly sensitive nor specific for determining the presence of irreversible vascular disease. For example, Braunlin et al. [145] provided long-term follow-up data on 57 patients who had had preoperative lung biopsy with VSD. Although patients with H-E grades 1 and 2 changes generally did well, four of 47 patients died of PVD, and it is unclear how many (if any) of the survivors had increased PVR. There were only five patients with grade 3 changes, with limited follow-up information (though one clearly died of PVD). Of four patients with grade 4 changes, at least three died of PA hypertension, suggesting that advanced lesions reliably portend a poor outcome, but their numbers were few, and exceptions have been reported [146]. Other reports have shown considerable overlap between patients with severe pathological changes who do well following repair and those who do not [21, 147]. Despite multiple papers advocating the use of this modality over the last three decades, the authors are aware of no centers that routinely employ biopsy for decision making in difficult cases.

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7.3 Therapy There are multiple therapeutic modalities available for patients with PVD for whom surgery is not suitable. Unfortunately, none are nearly as effective as one would like. Most of these modalities are supportive rather than designed to decrease PVR (e.g., diuretics, erythrophoresis, and balloon atrial septostomy) and will not be approached here, nor will lung transplantation. Only therapies whose primary intended effect is to decrease PVR are discussed. 7.3.1 PA Banding Experimental work in rats suggests that the pathologically remodeled pulmonary circulation can undergo “reverse” remodeling (regression of hypertrophy and distal ­muscu­larization of distal PAs) if the PA pressure is reduced [133]. This suggests that if PA pressure can be reduced via placing a restrictive band on the main PA, so that pressure distal to the band is decreased, favorable remodeling might follow. Castaneda et al. [67] banded 13 infants (average age 3.5 months) with a large VSD (and, although not stated, probably significantly increased PBF), and found that PVR fell from an average of 1,950 to 700 dyne · sec cm5 when the patients were studied about 3–5 years later. Also, as discussed already, there are two other published reports suggesting that PA banding can reverse some pathological remodeling [32, 35]. However, this approach is rarely feasible for patients without an unrestrictive interventricular communication, since right ventricular pressure would be acutely increased by the band, which is poorly tolerated. And with a large VSD, PA banding will cause unacceptable hypoxemia if the combination of PVR and resistance to flow across the band critically reduces PBF. We do not know which patients are most likely to tolerate and usefully decrease their PVR with PA banding, but certainly only a very small number stand to derive benefit (leaving aside infants with little or no increase in PVR, banded in anticipation of surgery at an older age). 7.3.2 Pharmacological Therapy 1. Enhanced inspired oxygen. Alveolar hypoxia causes acute pulmonary vasoconstriction, and chronic hypoxia causes increased PVR and medial hypertrophy. The effect of hypoxia on the pulmonary circulation with increased pressure/flow is likely even greater than on the otherwise normal lung, given the experience in Denver with ­shunting lesions and mitral stenosis [90, 91]. Increasing the fraction of inspired oxygen might therefore be rational therapy, especially for patients with cardiac malformations with hypoventilation or lung disease. The authors are

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unaware of any published data on the effect of chronic oxygen therapy on PVR in patients with congenital ­shunting lesions, although there is one report describing its feasibility in children [13]. 2. Inhaled nitric oxide. Virtually all known vasoactive agents, notably excepting hypoxia and acidosis, have the same directional (constriction vs. dilation) affect on the pulmonary and systemic circulations. Nitric oxide, which increases the levels of intracellular cyclic GMP, relaxes both pulmonary and systemic vessels, but when given by inhalation selectively dilates the pulmonary circulation since it is mostly inactivated by blood hemoglobin before it can affect systemic vascular smooth muscle. Inhaled nitric oxide has thus proven valuable in treating postoperative pulmonary hypertensive “crises” (the acute and labile increase in PVR after cardiac surgery described earlier) [148], since systemic vasodilation in this setting is usually counterproductive. This drug has largely supplanted most other agents and maneuvers (e.g., tolazoline hydrochloride, hyperventilation) previously used for acute pulmonary vasoconstriction, although it is not invariably effective therapy. 3. Calcium channel blockers. These drugs were the first effective therapy for idiopathic pulmonary vasoconstriction (albeit for only a small minority of adult patients), and thus might prove effective with PVD due to congenital heart malformations. However, these drugs are ineffective in those patients with idiopathic PA hypertension who are not acutely responsive to vasodilators, a quality which describes most patients (beyond childhood) with severely increased PVR due to increased PA pressure and flow. Published experience with long-term use of nifedipine does not permit an accurate assessment of its effect on PVR in patients with congenital cardiac lesions [149]. 4. Prostacyclin. Two studies found chronic (3 months or more) infusion of prostacyclin in children and adults with PVD secondary to congenital cardiac shunting lesions (repaired and unrepaired) to decrease PVR, improve oxygen delivery, and increase exercise capacity [150, 151]. No effect on survival was demonstrated in these studies (which were without placebo controls). The fall in PVR was substantial (about 50%, although PVR remained very elevated), and the fact that it occurred in patients who seldom show an acute response to vasodilators suggests that prostacyclin may have effected partial remodeling of the pulmonary circulation (although other explanations are possible). There is anecdotal experience that chronic infusion of prostacyclin decreased PVR sufficiently in some patients to permit closure of defects considered inoperable before therapy [152, 153]. 5. Bosentan. There are several studies of this endothelin receptor (A and B) blocker in patients with Eisenmenger

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syndrome, two of which include hemodynamic data. One study of adult patients found 16 weeks of therapy with orally administered bosentan very modestly decreased both PA pressure and PVR [154], whereas another found a very small reduction in PVR but no change in PA pressure after 12 months of therapy [155]. Both showed an increase in systemic oxygen delivery, exercise capacity, and functional class. It is not known if bosentan affects survival in this setting. 6. Sildenafil. A report of this phosphodiesterase type V inhibitor in seven adults with Eisenmenger syndrome found that after 6 months mean PA pressure and PVR were substantially decreased and systemic oxygen delivery was increased; New York Heart Association functional class was improved, but exercise capacity was not significantly different from the baseline [156]. Multiple other studies have shown symptomatic improvement and increase in exercise capacity with sildenafil in Eisenmenger syndrome patients [157].

8 Summary Generally speaking, congenital cardiac malformations are associated with an abnormal pulmonary circulation in one of two ways: (1) Defects which increase PA pressure and/or PBF cause PVD to develop over time. The evolution of PVD generally occurs over months and years, although a few defects are associated with abnormal pulmonary hemodynamics in utero and PVD in those cases can be manifest even in the neonate. (2) Congenital abnormalities of the large PAs are a feature of certain congenital heart malformations (e.g., pulmonary atresia and VSD). Regarding malformations which increase PA pressure and flow, although there is considerable variability in the pulmonary vascular phenotype expressed even within a population of apparently similar patients, three observations stand out: (1) both active vasoconstriction and pathological remodeling operate to increase PVR, (2) increased PA pressure and increased PBF, in isolation, are considerably less likely to cause PVD than when both occur together, and (3) excepting a few high-risk malformations (e.g., d-TGA and VSD), permanent or progressive pathological changes seldom occur when malformations are corrected in the first 1–2 years of life. Our mechanistic understanding of the biological processes involved in pulmonary vasoconstriction and vascular remodeling is primitive; although many pieces of the puzzle have been accumulated, nobody knows how many are ­missing, nor how to fit the accumulated pieces together. Because any successful mechanistic explication of PVD in this setting must account for the effect of physical forces on pulmonary

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vascular tone and remodeling, it is heartening that multiple in vitro and in vivo models have been developed which have and will yield yet more information pertinent to the biological effects of increased PA flow and pressure. New genetic discoveries will also doubtlessly play an increasing role in uncovering the mechanisms involved in PVD, as well as a better understanding of vascular biological processes in general. Therapy for PVD related to congenital heart defects is primarily prevention, which has been much enhanced by advances in the diagnosis and repair of congenital cardiac malformations. Early experience indicating that some drugs may ameliorate PVD previously thought to be irreversible gives hope that reverse remodeling – at least to some degree – may be possible even in older patients.

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77  The Pulmonary Circulation in Congenital Heart Disease 78. Clarkson PM, Neutze JM, Wardill JC, Barratt-Boyes BG (1976) The pulmonary vascular bed in patients with complete transposition of the great arteries. Circulation 53:539–543 79. Lakier JB, Stanger P, Heymann MA, Hoffman JI, Rudolph AM (1975) Early onset of pulmonary vascular obstruction in patients with aortopulmonary transposition and intact ventricular septum. Circulation 51:875–880 80. Ferencz C (1966) Transposition of the great vessels: pathophysiologic considerations based upon a study of the lungs. Circulation 33:232–241 81. Keane JF, Ellison RC, Rudd M, Nadas AS (1973) Pulmonary blood flow and left ventricular volumes in transposition of the great arteries and intact ventricular septum. Br Heart J 35:521–526 82. Newfeld EA, Paul MH, Muster AJ, Idriss FS (1979) Pulmonary vascular disease in transposition of the great vessels and intact ventricular septum. Circulation 59:525–530 83. Rivenes SM, Grifka RG, Feltes TF (1998) Development of advanced pulmonary vascular disease in D-transposition of the great arteries after the neonatal arterial switch operation. Tex Heart Inst J 25:201–205 84. Hutter PA, Kreb DL, Mantel SF, Hitchcock JF, Meijboom EJ, Bennink GBWE (2002) Twenty-five years' experience with the arterial switch operation. J Thorac Cardiovasc Surg 124:790–797 85. Mair DD, Danielson GK, Wallace RB, McGoon DC (1974) Longterm follow-up of Mustard operation survivors. Circulation 50:II46–II53 86. Lambert V, Serraf A, Durand P, Losay J (2001) Aerosolized iloprost therapy in an infant with chronic pulmonary hypertension after a neonatal arterial switch operation. Arch Pediatr 8:1218–1221 87. Roofthooft MTR, Bergman KA, Waterbolk TW, Ebels T, Bartelds B, Berger RMF (2007) Persistent pulmonary hypertension of the newborn with transposition of the great arteries. Ann Thorac Surg 83:1446–1450 88. Soongswang J, Adatia I, Newman C, Smallhorn JF, Williams WG, Freedom RM (1998) Mortality in potential arterial switch candidates with transposition of the great arteries. J Am Coll Cardiol 32:753–757 89. Freedom R, Yoo S-J, Collins MH (2004) Atrioventricular septal defect. In: Freedon R, Yoo S-J, Mikailian H, Williams WG (eds) The natural and modified history of congenital heart disease. Futura, Elmsford, pp 46–47 90. Shoultz CA Jr, Kelminson LL, Vogel JH, Pryor R, Blount SG Jr (1971) Hemodynamics in congenital mitral stenosis. Chest 59:47–49 91. Vogel JH, McNamara DG, Blount SG Jr (1967) Role of hypoxia in determining pulmonary vascular resistance in infants with ventricular septal defects. Am J Cardiol 20:346–349 92. Hislop A, Reid L (1973) Structural changes in the pulmonary arteries and veins in tetralogy of Fallot. Br Heart J 35:1178–1183 93. Kinsley RH, McGoon DC, Danielson GK, Wallace RB, Mair DD (1974) Pulmonary arterial hypertension after repair of tetralogy of Fallot. J Thorac Cardiovasc Surg 67:110–120 94. Haworth SG, Reid L (1977) Quantitative structural study of pulmonary circulation in the newborn with pulmonary atresia. Thorax 32:129–133 95. Haworth SG, Macartney FJ (1980) Growth and development of pulmonary circulation in pulmonary atresia with ventricular septal defect and major aortopulmonary collateral arteries. Br Heart J 44:14–24 96. Amark K, Freedom RM, Yoo S-J (2004) Tetralogy of fallot with pulmonary atresia (pulmonary atresia and ventricular septal defect). In: Freedom RM, Yoo S-J, Mikailian H, Williams WG (eds) The natural and modified history of congenital heart disease. Futura/Blackwell, New York, pp 217–218 97. Baum D, Khoury G, Ongley P, Swan H, Kincaid O (1964) Congenital stenosis of the pulmonary artery branches. Circulation 29:680–687

1137 98. Eldredge WJ, Tingelstad JB, Robertson LW, Mauck HP, McCue CM (1972) Observations on the natural history of pulmonary artery coarctations. Circulation 45:404–409 99. Kreutzer J, Landzberg MJ, Preminger TJ et  al (1996) Isolated peripheral pulmonary artery stenoses in the adult. Circulation 93:1417–1423 100. Edwards BS, Lucas RV Jr, Lock JE, Edwards J (1985) Morphologic changes in the pulmonary arteries after percutaneous balloon angioplasty for pulmonary arterial stenosis. Circulation 71:195–201 101. Giddins NG, Finley JP, Nanton MA, Roy DL (1989) The natural course of supravalvar aortic stenosis and peripheral pulmonary artery stenosis in Williams's syndrome. Br Heart J 62:315–319 102. Langille BL (1996) Arterial remodeling: relation to hemodynamics. Can J Physiol Pharmacol 74:834–841 103. Rabinovitch M (2008) Molecular pathogenesis of pulmonary ­arterial hypertension. J Clin Invest 118:2372–2379 104. Chan SY, Loscalzo J (2008) Pathogenic mechanisms of pulmonary arterial hypertension. J Mol Cell Cardiol 44:14–30 105. Yuan JXJ, Rubin LJ (2005) Pathogenesis of pulmonary arterial hypertension: the need for multiple hits. Circulation 111:534–538 106. Wamhoff BR, Bowles DK, Owens GK (2006) Excitationtranscription coupling in arterial smooth muscle. Circ Res 98:868–878 107. Douglas SA, Ohlstein EH (1997) Signal transduction mechanisms mediating the vascular actions of endothelin. J Vasc Res 34:152–164 108. Li Z, Yu C, Han Y, Ren H, Shi W, Fu C et al (2008) Inhibitory effect of D1-like and D3 dopamine receptors on norepinephrineinduced proliferation in vascular smooth muscle cells. Am J Physiol Heart Circ Physiol 294:H2761–H2768 109. Kulik TJ, Evans JN, Gamble WJ (1988) Stretch-induced contraction in pulmonary arteries. Am J Physiol 255:H1391–H1398 110. Belik J, Keeley FW, Baldwin F, Rabinovitch M (1994) Pulmonary hypertension and vascular remodeling in fetal sheep. Am J Physiol 266:H2303–H2309 111. Tourneux P, Chester M, Grover T, Abman SH (2008) Fasudil inhibits the myogenic response in the fetal pulmonary circulation. Am J Physiol Heart Circ Physiol 295:H1505–H1513 112. Johnson PC (1980) The myogenic response. In: Bohr DF, Somlyo AP, Sparks HV Jr, Geiger SR (eds) Handbook of physiology - the cardiovascular system II. American Physiologic Society, Bethesda, p 433 113. Hyman AL (1968) Pulmonary vasoconstriction due to nonocclusive distention of large pulmonary arteries in the dog. Circ Res 23:401–413 114. Juratsch CE, Jengo JA, Castagna J, Laks MM (1980) Experimental pulmonary hypertension produced by surgical and chemical denervation of the pulmonary vasculature. Chest 77:525–530 115. Juratsch CE, Grover RF, Rose CE, Reeves JT, Walby WF, Laks MM (1985) Reversal of reflex pulmonary vasoconstriction induced by main pulmonary arterial distension. J Appl Physiol 58:1107–1114 116. Juratsch CE, Emmanouilides GC, Thibeault DW, Baylen BG, Jengo JA, Laks MM (1980) Pulmonary arterial hypertension induced by distention of the main pulmonary artery in conscious newborn, young, and adult sheep. Pediatr Res 14:1332–1338 117. Baylen BG, Emmanouilides GC, Juratsch CE, Yoshida Y, French WJ, Criley JM (1980) Main pulmonary artery distention: a potential mechanism for acute pulmonary hypertension in the human newborn infant. J Pediatr 96:540–544 118. Rabinovitch M, Bothwell T, Hayakawa BN et al (1986) Pulmonary artery endothelial abnormalities in patients with congenital heart defects and pulmonary hypertension. A correlation of light with scanning electron microscopy and transmission electron microscopy. Lab Invest 55:632–653 119. Celermajer DS, Cullen S, Deanfield JE (1993) Impairment of endothelium-dependent pulmonary artery relaxation in children with congenital heart disease and abnormal pulmonary hemodynamics. Circulation 87:440–446

1138 120. Steinhorn RH, Russell JA, Lakshminrusimha S, Gugino SF, Black SM, Fineman JR (2001) Altered endothelium-dependent relaxations in lambs with high pulmonary blood flow and pulmonary hypertension. Am J Physiol Heart Circ Physiol 280:H311–H317 121. Li C, Xu Q (2007) Mechanical stress-initiated signal transduction in vascular smooth muscle cells in vitro and in vivo. Cell Signal 19:881–891 122. Quinn TP, Schlueter M, Soifer SJ, Gutierrez JA (2002) Cyclic mechanical stretch induces VEGF and FGF-2 expression in pulmonary vascular smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 282:L897–L903 123. Kolpakov V, Gordon D, Kulik TJ (1995) Nitric oxide-generating compounds inhibit total protein and collagen synthesis in cultured vascular smooth muscle cells. Circ Res 76:305–309 124. Tozzi CA, Poiani GJ, Harangozo AM, Boyd CD, Riley DJ (1989) Pressure-induced connective tissue synthesis in pulmonary artery segments is dependent on intact endothelium. J Clin Invest 84: 1005–1012 125. Belik J (1994) Myogenic response in large pulmonary arteries and its ontogenesis. Pediatr Res 36:34–40 126. Weiser MC, Majack RA, Tucker A, Orton EC (1995) Static tension is associated with increased smooth muscle cell DNA synthesis in rat pulmonary arteries. Am J Physiol 268:H1133–H1138 127. Reddy VM, Meyrick B, Wong J et al (1995) In utero placement of aortopulmonary shunts. A model of postnatal pulmonary hypertension with increased pulmonary blood flow in lambs. Circulation 92:606–613 128. Mata-Greenwood E, Meyrick B, Steinhorn RH, Fineman JR, Black SM (2003) Alterations in TGF-b1 expression in lambs with increased pulmonary blood flow and pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 285:L209–L221 129. Mata-Greenwood E, Meyrick B, Soifer SJ, Fineman JR, Black SM (2003) Expression of VEGF and its receptors Flt-1 and Flk-1/ KDR is altered in lambs with increased pulmonary blood flow and pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 285:L222–L231 130. Black SM, Bekker JM, Johengen MJ, Parry AJ, Soifer SJ, Fineman JR (2000) Altered regulation of the ET-1 cascade in lambs with increased pulmonary blood flow and pulmonary hypertension. Pediatr Res 47:97–106 131. Wedgwood S, Devol JM, Grobe A et al (2007) Fibroblast growth factor-2 expression is altered in lambs with increased pulmonary blood flow and pulmonary hypertension. Pediatr Res 61:32–36 132. Botney MD (1999) Role of hemodynamics in pulmonary vascular remodeling: implications for primary pulmonary hypertension. Am J Respir Crit Care Med 159:361–364 133. O'Blenes SB, Fischer S, McIntyre B, Keshavjee S, Rabinovitch M (2001) Hemodynamic unloading leads to regression of pulmonary vascular disease in rats. J Thorac Cardiovasc Surg 121:279–289 134. Rabinovitch M, Konstam MA, Gamble WJ et al (1983) Changes in pulmonary blood flow affect vascular response to chronic hypoxia in rats. Circ Res 52:432–441 135. Chatzizisis YS, Coskun AU, Jonas M, Edelman ER, Feldman CL, Stone PH (2007) Role of endothelial shear stress in the natural history of coronary atherosclerosis and vascular remodeling: molecular, cellular, and vascular behavior. J Am Coll Cardiol 49:2379–2393 136. White CR, Frangos JA (2007) The shear stress of it all: the cell membrane and mechanochemical transduction. Philos Trans R Soc Lond B Biol Sci 362:1459–1467 137. Chien S (2007) Mechanotransduction and endothelial cell homeostasis: the wisdom of the cell. Am J Physiol Heart Circ Physiol 292:H1209–H1224 138. Therrien J, Rambihar S, Newman B et al (2006) Eisenmenger syndrome and atrial septal defect: nature or nurture? Can J Cardiol 22:1133–1136

T.J. Kulik and M.P. Mullen 139. Roberts KE, McElroy JJ, Wong WPK et  al (2004) BMPR2 mutations in pulmonary arterial hypertension with congenital heart disease. Eur Respir J 24:371–374 140. Kulik TJ (2009) The pathophysiology of acutely increased pulmonary vascular resistance. Pediatr Crit Care Med (in press) 141. Lakshminrusimha S, Wiseman D, Black SM et al (2007) The role of nitric oxide synthase-derived reactive oxygen species in the altered relaxation of pulmonary arteries from lambs with increased pulmonary blood flow. Am J Physiol Heart Circ Physiol 293: H1491–H1497 142. Schulze-Neick I, Penny DJ, Rigby ML et al (1999) L-arginine and substance P reverse the pulmonary endothelial dysfunction caused by congenital heart surgery. Circulation 100:749–755 143. Jones OD, Shore DF, Rigby ML et al (1981) The use of tolazoline hydrochloride as a pulmonary vasodilator in potentially fatal episodes of pulmonary vasoconstriction after cardiac surgery in children. Circulation 64:II134–II139 144. Hopkins RA, Bull C, Haworth SG, de Leval MR, Stark J (1991) Pulmonary hypertensive crises following surgery for congenital heart defects in young children. Eur J Cardiothorac Surg 5:628–634 145. Braunlin EA, Moller JH, Patton C, Lucas RV Jr, Lillehei CW, Edwards J (1986) Predictive value of lung biopsy in ventricular septal defect: long-term follow-up. J Am Coll Cardiol 8:1113–1118 146. Epting CL, Wolfe RR, Abman SH, Deutsch GH, Ivy D (2002) Reversal of pulmonary hypertension associated with plexiform lesions in congenital heart disease: a case report. Pediatr Cardiol 23:182–185 147. Geggel RL, Mayer JE Jr, Fried R, Helgason H, Cook EF, Reid LM (1990) Role of lung biopsy in patients undergoing a modified Fontan procedure. J Thorac Cardiovasc Surg 99:451–459 148. Kulik TJ (1996) Inhaled nitric oxide in the management of congenital heart disease. Curr Opin Cardiol 11:75–80 149. Wimmer M, Schlemmer M (1992) Long-term hemodynamic effects of nifedipine on congenital heart disease with Eisenmenger's mechanism in children. Cardiovasc Drugs Ther 6:183–186 150. Rosenzweig EB, Kerstein D, Barst RJ (1999) Long-term prostacyclin for pulmonary hypertension with associated congenital heart defects. Circulation 99:1858–1865 151. Fernandes SM, Newburger JW, Lang P et al (2003) Usefulness of epoprostenol therapy in the severely ill adolescent/adult with Eisenmenger physiology. Am J Cardiol 91:632–635 152. Schwerzmann M, Zafar M, McLaughlin PR, Chamberlain DW, Webb G, Granton J (2006) Atrial septal defect closure in a patient with "irreversible" pulmonary hypertensive arteriopathy. Int J Cardiol 110:104–107 153. Frost AE, Quinones MA, Zoghbi WA, Noon GP (2005) Reversal of pulmonary hypertension and subsequent repair of atrial septal defect after treatment with continuous intravenous epoprostenol. J Heart Lung Transplant 24:501–503 154. Galiè N, Beghetti M, Gatzoulis MA et al (2006) Bosentan therapy in patients with Eisenmenger syndrome: a multicenter, double-blind, randomized, placebo-controlled study. Circulation 114:48–54 155. D'Alto M, Vizza CD, Romeo E et al (2007) Long term effects of bosentan treatment in adult patients with pulmonary arterial hypertension related to congenital heart disease (Eisenmenger physiology): safety, tolerability, clinical, and haemodynamic effect. Heart 93:621–625 156. Chau EMC, Fan KYY, Chow WH (2007) Effects of chronic sildenafil in patients with Eisenmenger syndrome versus idiopathic pulmonary arterial hypertension. Int J Cardiol 120:301–305 157. Wort SJ (2007) Sildenafil in Eisenmenger syndrome: safety first. Int J Cardiol 120:314–316 158. Rabinovitch M (2008) Pathophysiology of pulmonary hypertension. In: Allen HD, Driscoll DJ, Shaddy RE, Feltes TF (eds) Moss and Adam’s heart disease in infants, children, and young adults. Wolters Kluwer, Lippincott Williams & Wilkins, Philadelphia

Chapter 78

Pulmonary Hypertension Secondary to Congenital Systemic-to-Pulmonary (Left-to-Right) Shunts Antonio A. Lopes

Abstract  Pulmonary arterial hypertension associated with congenital left-to-right shunts remains a matter of concern not only in underserved areas, but also in developed countries, in both un-operated on and operated on patients. The risk of developing advanced pulmonary vascular disease generally but not invariably depends on the size and location of the defect. Patients with restrictive ventricular septal defects (VSDs) are unlikely to acquire pulmonary arterial hypertension (the prevalence is only 3%). On the other hand, the likelihood of severe pulmonary hypertension and development of Eisenmenger syndrome is considerable in patients with nonrestrictive defects (greater than 1.5 cm in diameter); in this case, 50% will be affected. This is in contrast with subjects with ASDs, for whom the risk of acquiring pulmonary hypertension is not higher than 10% with a late onset (90% during the adulthood). However, pulmonary artery pressure and vascular resistance tend to be more frequently elevated in patients with sinus venosus defects than in those with secundum defects. Despite the general concept that pulmonary hypertension is not a matter of concern if patients undergo repair of the cardiac shunts during the first year of life, certain anomalies are known to be associated with early elevation of pulmonary vascular resistance. There is general agreement that this is the case for truncus arteriosus, atrioventricular septal defects, and transposition of the great arteries with VSD. For unknown reasons, some patients with simple defects such as VSD or patent ductus arteriosus have no history of pulmonary congestion or failure to thrive during the first months of life, suggesting early-onset pulmonary arterial hypertension. These patients cannot be safely assigned for repair of the defects on the basis of noninvasive evaluation only. Rather, complete evaluation including direct measurement of pulmonary vascular resistance is necessary to make a decision about the therapeutic strategies. This chapter explores how the appropriate recognition of a state of A.A. Lopes (*) Department of Pediatric Cardiology and Adult Congenital Heart Disease, Heart Institute (InCor), University of São Paulo School of Medicine, São Paulo-SP 05403-000, Brazil e-mail: [email protected]

increased pulmonary blood flow or a state of increased pulmonary vascular resistance allows for decisions to be made between the therapeutic options that are applicable to patients with left-to-right shunts associated with pulmonary hypertension (correction of the defect, pulmonary artery banding, medical treatment, or combinations). Although there is not much evidence to support such decisions, expertise that has been accumulated in tertiary reference centers with high standards of medical assistance allows for proper selection of the therapeutic strategies. The issue of adults with ASD is particularly focused on. The pathogenic mechanisms of pulmonary vascular disease in left-to-right shunts and the correlations of pathological features with the outcomes are also addressed. Keywords  Left-to-right shunt • Ventricular septal defect • Atrial septal defect • Eisenmenger syndrome

1 Introduction “In developed countries, pulmonary arterial hypertension is not a matter of concern in patients with congenital cardiac defects associated with left-to-right shunting, since the defects are repaired in early infancy, thus eliminating the hemodynamic stimuli for development of severe pulmonary vasculopathy.” Although this statement emphasizes the rationale for early repair of congenital cardiac shunts, it is not totally correct. Three decades ago, there was a general concept that at least 30% of patients with left-to-right shunts were at risk for development of pulmonary vascular disease [1]. In 1993, the Second Natural History Study of Congenital Heart Defects (NHS-2) showed that more than 50% of patients with a large, nonrestrictive ventricular septal defect (VSD) developed Eisenmenger syndrome [2]. Of note, 13.8% of patients with successfully repaired VSDs (no residual defects) had persistent pulmonary hypertension. More recently, the Euro Heart Survey on Adult Congenital Heart Disease showed that 12% of 377 patients with repaired atrial septal defects (ASDs) and 13% of 275 patients with repaired

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_78, © Springer Science+Business Media, LLC 2011

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VSDs had residual pulmonary hypertension. For subjects with open defects, the prevalence of pulmonary hypertension was 34 and 28% respectively. The overall prevalence of Eisenmenger syndrome in patients followed up for 5 years in this survey was 7% (133 of 1,877 adults) [3]. On the other hand, of 201 patients with Eisenmenger syndrome followed up from 1976 to 1992 in a single tertiary center in India, the prevalence of ASDs, VSDs, and patent ductus arteriosus was 29.8, 33.3, and 14.2%, respectively [4]. Thus, pulmonary arterial hypertension associated with congenital left-to-right shunts remains a matter of concern not only in underserved areas, but also in developed countries, in both unoperated on and operated on patients. The risk of developing advanced pulmonary vascular ­disease generally but not invariably depends on the size and location of the defect. Patients with restrictive VSDs are unlikely to acquire pulmonary arterial hypertension (the prevalence is only 3%). On the other hand, the likelihood of severe pulmonary hypertension and development of Eisenmenger syndrome is considerable in patients with nonrestrictive defects (greater than 1.5 cm in diameter); in this case, 50% will be affected [5]. This is in contrast with ­subjects with ASDs, for whom the risk of acquiring pulmonary hypertension is not higher than 10% with a late onset (90% during the adulthood) [6]. However, pulmonary artery pressure and vascular resistance tend to be more frequently elevated in patients with sinus venosus defects than in those with secundum defects [7]. Despite the general concept that pulmonary hypertension is not a matter of concern if patients undergo repair of the cardiac shunts during the first year of life, certain anomalies are known to be associated with early elevation of pulmonary vascular resistance. There is general agreement that this is the case for truncus arteriosus, atrioventricular septal defects, and transposition of the great arteries with VSD. For unknown reasons, some patients with simple defects such as VSD or patent ductus arteriosus have no history of pulmonary congestion or failure to thrive during the first months of life, suggesting early-onset pulmonary arterial hypertension. These patients cannot be safely assigned for repair of the defects on the basis of noninvasive evaluation only. Rather, complete evaluation including direct measurement of pulmonary vascular resistance is necessary to make a decision about the therapeutic strategies. This chapter explores how the appropriate recognition of a state of increased pulmonary blood flow or a state of increased pulmonary vascular resistance allows for decisions to be made between the therapeutic options that are applicable to patients with left-to-right shunts associated with pulmonary hypertension (correction of the defect, pulmonary artery banding, medical treatment, or combinations). Although there is not much evidence to support such decisions, expertise that has been accumulated in tertiary reference centers with high standards of medical assistance allows

A.A. Lopes

for proper selection of the therapeutic strategies. The issue of adults with ASD is particularly focused on. The pathogenic mechanisms of pulmonary vascular disease in left-to-right shunts and the correlations of pathological features with the outcomes are also addressed.

2 Pathogenic Mechanisms For many years it was reasoned that patients with congenital heart defects and left-to-right shunting had pulmonary vascular disease as a consequence of increased mechanical forces acting on the wall of the pulmonary vessels. Until recently, except for Down syndrome, which seems to be associated with early development of pulmonary vasculopathy, no other pathogenic factors were proposed. In recent years, there has been increasing interest in genetic susceptibility, in view of its importance in the pathogenesis of idiopathic and familial pulmonary arterial hypertension. With or without a genetic factor, it seems reasonable to assume that the shear stress and the circumferential stretch associated with increased pulmonary flow and pressure are important initiating/triggering events. Vascular cells are equipped with receptors that allow them to convert mechanical inputs into biochemical events. Many pathways, including the mitogen-activated protein kinase cascade can be activated by flow or stretch leading to activation of transcription factors with subsequent gene expression [8, 9]. Endothelial cells use multiple sensing mechanisms to detect changes in mechanical forces, and the cytoskeleton plays a pivotal role in mechanotransduction [10]. In pulmonary hypertension associated with congenital heart defects, transmission election microscopy has been used to demonstrate increased density of endothelial microfilament bundles corresponding to the cytoskeleton [11]. Shear- and stretch-induced cytoskeletal reorganization in endothelial cells is followed by formation of the so called focal adhesion complexes where a number of substrates are phosphorylated by several kinases. Downstream of such signaling cascades, multiple transcription factors are activated, including AP-1, NF-kB, Sp-1 and Egr-1. The actions of these factors on their responsive elements result in the transcription of several genes [12]. Of note, nitric oxide production can be induced in pulmonary vascular endothelial cells by circumferential stretch. The phosphatidylinositol 3-hydroxy kinase pathway and endothelial nitric oxide synthase (eNOS) phosphorylation seem to be involved [13]. The results of studies on the expression of nitric oxide synthase genes (eNOS and the inducible isoform, iNOS) in pulmonary hypertension associated with increased pulmonary blood flow/pulmonary artery pressure are not totally uniform. The differences may be partly due to differences in

78  Pulmonary Hypertension Secondary to Congenital Systemic-to-Pulmonary (Left-to-Right) Shunts

the models (humans, animals, isolated-perfused lungs, etc.). Black et al. [14] used aortopulmonary vascular grafts in fetal lambs to demonstrate a 2.4:1 and 2.08:1 increase, respectively, in lung eNOS messenger RNA (mRNA) and protein in comparison with age-matched controls. On the other hand, Berger et al. [15] used immunohistochemistry to analyze both isoforms in lung tissue of patients with congenital heart disease, and showed differential (increased) expression of iNOS (but not eNOS) in patients with increased pulmonary blood flow and pulmonary artery pressure compared with those with increased flow but normal pressure, those with congestive pulmonary vasculopathy and controls. It is not known if overexpression of nitric oxide synthase genes and production of nitric oxide induced by increased shear stress or circumferential stretch are effective in counteracting the actions of the vasoconstrictors present in the scenario. Interestingly, using the same model of in utero aortopulmonary shunts in lams, Steinhorn et  al. [16] were able to demonstrate impaired endothelium-dependent relaxation of pulmonary arteries which was significantly improved by pretreatment with superoxide dismutase and catalase, but not l-arginine. Flow- and pressure-related damage to the pulmonary vascular endothelium has been shown to induce proteolytic activities within the vascular wall leading to cellular proliferation and deposition of extracellular matrix. The loss of endothelial integrity is associated with entry of circulating factors to the subendothelial space. The presence of these factors in the subendothelium likely accounts for stimulation of smooth muscle cells to produce proteases such as endovascular elastase [17]. Pericellular proteolysis leads to release of peptide growth factors from their proteoglycan storage sites within the matrix. In addition, extracellular matrix degradation results in tight interactions between matrix proteins (collagens) and the smooth muscle cell membrane via integrin superfamily receptors, leading to further synthesis of matrix glycoproteins such as fibronectin and tenascin. Subsequent interactions of the cell with the modified matrix (collagen, tenascin) via integrins result in cytoskeleton organization, intracellular signaling with the formation of focal adhesion complexes, and clustering of growth factor receptors (e.g., epidermal growth factor) which become sensitive to agonists [17]. Colocalization of tenascin and epidermal growth factor with smooth muscle cells of hypertensive pulmonary arteries has been demonstrated [18]. The end results of these processes include changes in smooth muscle cell phenotype to a synthetic pattern, cellular proliferation, and migration to the intima which is facilitated by the presence of fibronectin in the matrix [19]. There has been evidence of increased production of endothelin in lungs of patients with congenital heart disease [20]. However, in humans, it is not known whether endothelin and its receptors (ETA and ETB, which mediate vasoconstriction and vasodilatation, respectively) play a substantial

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role in the early stages of pulmonary vascular remodeling associated with congenital cardiac shunts. Anyway, because endothelin (particularly endothelin-1) is a potent vasoconstrictor and proliferation-stimulating agent, there has been general agreement that it contributes to the progression of the disease. Controversy remains about the stimulus that triggers the expression of endothelin and its receptors in lungs of patients with cardiac shunts (increased flow, increased flow and pressure, increased diastolic pressure in the pulmonary circulation). In fetal lambs with aortopulmonary shunts (presumably increased pulmonary flow and pressure), increased levels of endothelin-converting enzyme-1 and ETA receptor mRNA and protein have been observed in lung tissue. However, ETB receptor mRNA and protein have been shown to be decreased [21]. Lutz et  al. demonstrated increased density of ETA receptors in lung arteries and parenchyma of patients with increased pulmonary vascular resistance in comparison with those with increased flow but low resistance, whereas the density of ETB receptors was low and not related to hemodynamics [22]. Vincent et  al. observed increased circulating endothelin levels in children with congenital heart defects associated with a pulmonary to systemic flow ratio higher than 1.5 [23]. On the other hand, Ishikawa et al. demonstrated increased plasma levels of endothelin-1 in patients with VSDs (increased pulmonary flow and pressure) in contrast to those with high flow and low pressure associated with ASDs. Interestingly, in patients with severe pulmonary congestion due to pulmonary venous stenosis, plasma levels of endothelin-1 were even higher compared with those in patients with VSDs [24]. Thus, it can be hypothesized that increased diastolic pressure in the pulmonary circulation is the principal stimulus for induction of endothelin. Further studies are required to refine the mechanisms that trigger the expression of endothelin and its receptors in patients with congenital cardiac shunts, and for a better understanding of their behavior on a long-term basis following repair of the anomalies. Several other mechanisms described for pulmonary arterial hypertension in general likely play a role in the development of pulmonary vasculopathy associated with congenital heart defects. For example, abnormalities in the behavior of ion channels (particularly Kv channels) which have been extensively studied in idiopathic pulmonary arterial hypertension are probably present in congenital heart disease. Limsuwan et al. reported that the activity of K+ channels was reduced in pulmonary vascular smooth muscle cells exposed to serum from patients with pulmonary arterial hypertension associated with congenital cardiac defects [25]. In the specific setting of peptide growth factors, increased expression of vascular endothelial growth factor which has been demonstrated in patients with congenital heart disease [26] associated with defective transforming growth factor b (TGF-b) signaling may account for loss of the equilibrium

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between cellular growth and apoptosis in favor of proliferation. Mutations in the gene that encodes the bone morphogenetic protein receptor type II (BMPR-II), a member of the TGF-b superfamily of receptors, have been identified in approximately 60% of patients with familial pulmonary arterial hypertension and in 25% of individuals with the sporadic form of the disease [27]. The BMPR-II mutations are known to alter the pattern of phosphorylation of cytoplasmic signaling proteins within the smooth muscle cells, leading to signal transduction, gene ­transcription, and cellular proliferation, thus explaining the tendency for cellular growth observed in patients with pulmonary arterial hypertension [28]. Interestingly, BMPR-II mutations have been reported in pulmonary hypertension associated with congenital heart disease [29]. Even more exciting are the observations that members of the bone morphogenetic protein/ TGF-b pathway may be involved in developmental anomalies of the heart that are known to be associated with pulmonary arterial hypertension such as atrioventricular septal and conotruncal defects [30–32]. This leads to the fascinating hypothesis that abnormalities of a single gene family might account for both the congenital cardiac anomaly and the tendency for development of advanced pulmonary vasculopathy, with flow and pressure acting as triggering factors.

3 Pathology The pulmonary vascular lesions that accompany the congenital cardiac anomalies with left-to-right shunting were classified by Donald Heath and Jesse E. Edwards in 1958 [33]. The lesions were classified in six grades according to the severity. This classification remains useful and has been considered as the most important qualitative approach to pulmonary vasculopathy in congenital heart disease. It con-

A.A. Lopes

stitutes the basis for definition of reversibility of vascular lesions following repair of the septal defects. Grade I corresponds to medial hypertrophy of the pulmonary arteries, whereas in grade II lesions intimal proliferation is present, with progressive accumulation of cellular elements and fibroelastic tissue in the vascular lumen leading to complete occlusion (grade III) (Fig. 1). Grades IV–VI correspond to vascular dilatation sometimes associated with angiomatoid structures or plexiform lesions (thin-walled lesions with capillary-like proliferation inside) and, finally, necrotizing arteritis with reactive inflammatory exudate throughout the vascular layers [34]. Subsequently, Rabinovitch et al. proposed a morphometric (quantitative) approach to the pulmonary vascular abnormalities that occur in congenital heart disease, which consisted of measuring the amount of smooth muscle in the pulmonary arteries and determining the number of small intraacinar arteries relative to the number of alveoli [35]. The first observed change is an abnormal extension of smooth muscle into peripheral, normally nonmuscular arteries (grade A). In grade B, medial thickness is present as a result of hypertrophy and hyperplasia of preexisting smooth muscle cells and increase in the amount of extracellular matrix, and is determined relative to the arterial diameter. Grade C corresponds to a decrease in arterial concentration likely due to impaired growth, and is established on the basis of the alveolar to arterial ratio. This ratio is around or above 20:1 in neonates, but normally decreases to less than 10:1 by the age of 5 or 6 years owing to accelerated growth of intraacinar arteries relative to the alveoli. In children with congenital cardiac shunts and pulmonary hypertension, the alveolar to arterial ratio remains elevated. There has been general agreement that Heath–Edwards grade III–VI lesions are associated with irreversible disease and persistent pulmonary hypertension following surgical

Fig. 1  a Muscular pulmonary artery with marked medial hypertrophy and absence of intimal proliferation (Heath–Edwards grade I). b Small pulmonary artery totally occluded by intimal proliferation and fibrosis (Heath–Edwards grade III). (Courtesy of Vera D. Aiello, Heart Institute, São Paulo, Brazil)

78  Pulmonary Hypertension Secondary to Congenital Systemic-to-Pulmonary (Left-to-Right) Shunts

repair of the cardiac defects. This is particularly so when a morphometric grade C is detected. However, Rabinovitch et al. observed that when patients are subjected to surgical treatment early in life (before 9 months of age), pulmonary vascular resistance decreases 1 year postoperatively to normal levels regardless of the severity of pulmonary vascular lesions in biopsy specimens [36]. Furthermore, all of the drugs that are currently available for treatment of pulmonary arterial hypertension (namely, prostanoids, endothelin receptor antagonists, and phosphodiesterase inhibitors) probably have antiproliferative properties as suggested in experimental conditions. Therefore, the criteria for reversibility of pulmonary vascular abnormalities in congenital heart disease will probably change in the years to come with the emerging possibilities of combining surgical and medical (drug) interventions. There has been much discussion about the usefulness of lung biopsies to establish the severity of pulmonary vasculopathy and predict outcomes, in particular in view of the possibility of challenging the whole pulmonary circulation with vasodilators during cardiac catheterization. However, it should be considered that as new therapies become available, more information is needed about their actions on pulmonary vascular remodeling. New biological markers in lung tissue will probably be validated on the basis of the correlations between their expression and long-term outcomes. Thus, in many institutions, intraoperative lung biopsies are still performed in selected patients suspected of having ­moderate to severe disease and likely to require long-term postoperative treatment. Obviously, biopsies should be performed only in tertiary centers with expert pathologists. Moreover, lung biopsy specimens (preferably obtained from the upper lobes) must be adequate for analysis. The results must include not only qualitative information, but also morphometric data, a detailed description of intraacinar arteries (dilated thin-walled vessels may be suggestive of severely occlusive proximal lesions not seen in the material), and reference to the presence or absence of perivascular inflammatory elements.

4 Clinical Assessment of Pulmonary Hemodynamics and Prediction of Outcomes The decision to repair large, nonrestrictive septal defects, ­frequently referred to as “operability,” is based on the likelihood of persistent pulmonary hypertension following the procedure, since this may be associated with right-sided cardiac failure, low cardiac output, and, eventually, fatal outcome. This is particularly important in patients who require openheart operations, since cardiopulmonary bypass is often fol-

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lowed by a rise in pulmonary vascular resistance. Theoretically, moderate to severe pulmonary vascular lesions known to be associated with postoperative pulmonary hypertension, in particular in the presence of vasoconstrictor stimuli, should be detected by direct examination of the pulmonary microvasculature. However, lung biopsies are not routinely performed preoperatively. On the other hand, postoperative pulmonary hypertensive crises may occur even in patients with mildly elevated pulmonary vascular resistance, and are difficult to predict in these instances. Therefore, the propensity to pulmonary vasoconstriction per se does not define a given patient as inoperable. Rather, it reinforces the need for specific perioperative therapeutic interventions. The definition of inoperability is based on the likelihood of moderate to severe pulmonary vasculopathy (obstructive lesions) which is established after complete preoperative noninvasive and invasive evaluation. Direct examination of lung biopsy specimens may be necessary in selected cases. There are no doubts about the importance of differentiating between “predominantly increased pulmonary blood flow” (either spontaneously or during vasodilator administration) and “predominantly increased pulmonary vascular resistance” (fixed) since, as mentioned, patients in the second category are at a higher risk for persistent pulmonary hypertension following repair of the cardiac defects. However, the application of this concept in practice requires caution in view of questions that remain unanswered. First, there is no clear-cut value of pulmonary vascular resistance that defines which levels are “considerably high.” Second, the magnitude of vasodilator-induced drop in pulmonary vascular resistance that defines a “significant response” in terms of predicting favorable outcomes has been a matter of debate. Also, there has been considerable discussion about the intensity of the vasodilator stimulus that should be used in vasoreactivity tests. Last, neither the baseline level of pulmonary vascular resistance nor its change by vasodilators can accurately predict the occurrence of postoperative pulmonary hypertensive crises which may be associated with fatal outcomes in some cases. Despite these difficulties, attempts to correlate preoperative hemodynamics with outcomes deserve consideration. Examples of such attempts are shown in Table 1 [37–41]. In patients with congenital cardiac shunts associated with pulmonary hypertension, pulmonary vasoreactivity has been tested using a number of vasodilator stimuli and protocols. Inhaled oxygen and inhaled nitric oxide, either alone or in combination, have been the most frequently used vasodilators. Nitric oxide has been used in concentrations ranging from 5 to 80 ppm. Maximal pulmonary vasodilatation can be achieved by using 80  ppm nitric oxide in the presence of high oxygen concentrations (90–100%). However, controversy remains about the use of such a strong stimulus to define operability.

11

Turanlahti et al. [40] Not pre-established >10% drop in PVR/SVR in 11 patients >20% drop in PVR/SVR in 9 patients ³20% drop in PVR/SVR or PVR/SVR <0.33 (proposed in retrospect)

Not pre-established ³20% drop in PVR in 14 patients

Not pre-established ³20% drop in PVR in 14 of 15 operated on patients Not pre-established >10% drop in PVR/SVR in 11 operated on patients

Balzer et al. [41]

124

• ~100% O2 • ~100% O2 + 10–80 ppm NO (median 60 ppm NO)

Not specified PVR/SVR < 0.27 (optimal balance in sensitivity and specificity in retrospect) RA room air (21–23% oxygen), NO inhaled nitric oxide, PVR pulmonary vascular resistance, PVR/SVR pulmonary to systemic vascular resistance ratio

• RA + 20, 40, 80 ppm NO • 90–100% O2 + 20, 40, 80 ppm NO

100% O2 RA + 80 ppm NO 91% O2 + 80 ppm NO 100% O2 RA + 5 ppm NO RA + 40 ppm NO

7 (immediate) and 4 (late) of 74 operated on patients

2 of 11 operated on patients

1 of 15 operated on patients 3 of 15 operated on patients (all of them with a ³20% drop in PVR preoperatively) 6 of 11 operated on patients

15

Yasuda et al. [39]

• • • • • •

Not mentioned

19

Atz et al. [38]

Fatal outcome 1 of 7 operated on patients None of 10 operated on patients

Positive response + PVR/ SVR £0.3 Positive response + PVR <6 U·m2 in 9 of 10 operated on patients

All 7 operated on patients

>10% drop in PVR and PVR/SVR ³20% drop in PVR

13

Berner et al. [37]

• RA + 35 ppm NO

Postoperative pulmonary hypertensive crises

Table 1  Correlation between pulmonary vascular reactivity and outcome in patients with left-to-right shunts Patients in the study References with unrepaired shunts Vasodilatation protocols Positive response Criteria for operability

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Some specialists would not consider an open-heart operation for patients who respond to strong stimuli only. On the other hand, the use of oxygen at high concentrations in patients with large septal defects may lead to error in the calculation of pulmonary blood flow. In these instances, even mild variations in oxygen saturation in the pulmonary artery lead to considerable inaccuracy in the estimation of pulmonary flow by the Fick method. Furthermore, it is not possible to measure oxygen consumption in patients receiving about 100% O2. The magnitude of the drop in pulmonary vascular resistance during vasodilator administration has been used to define operability. Ideally, the response should be linked to the intensity of the vasodilator stimulus used in the test. For example, using 80 ppm nitric oxide with about 100% ­oxygen, Balzer et al. [41] reported a 97% sensitivity and 90% accuracy in predicting favorable outcomes when a final pulmonary to systemic vascular resistance ratio of less than 0.33 was used as the criterion for operability. In their study, the specificity was only 8% when a 20% decrease in pulmonary to systemic resistance ratio was used as the criterion. Other authors prefer to adopt a 20% or greater drop in pulmonary vascular resistance as the responsiveness criterion, but to define operability only in the presence of pulmonary vascular resistance levels below 6 units·m2 during vasodilator administration. On the basis of the observations by the authors listed in Table 1 and others, very stringent hemodynamic criteria for operability can be proposed. The majority of specialists agree that patients with cardiac shunts associated with pulmonary hypertension are expected to have a favorable postoperative outcome if they have a baseline pulmonary vascular resistance index of less than 6 units·m2 and a pulmonary to systemic vascular resistance ratio of less than 0.3. For patients above these levels, a 20% or greater drop in both parameters during inhalation of low to intermediate concentrations of nitric oxide (e.g., 20–40 ppm), with final levels of less than 6 units·m2 and less than 0.3, respectively, would be predictive of favorable outcome as well. Pulmonary vasodilator tests are generally performed if baseline pulmonary vascular resistance is in the range of 6.0–9.0 units·m2, which corresponds to a pulmonary to systemic vascular resistance ratio of 0.3–0.5. Although patients above these limits can be considered for surgical treatment if the mentioned criteria are met, the risk of postoperative complications and unfavorable outcome is considerable. These policies are obviously stringent, and would probably exclude patients who might benefit from repair of their defects. However, for adoption of less stringent criteria, randomized controlled studies are required. Finally, it is noticeable that even stringent criteria cannot totally eliminate the risk of postoperative pulmonary hypertensive crises. In this way, some intensivists strongly argue in favor of having a pulmonary arterial line for a better postoperative management of patients who are considered “at risk.”

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Also, many argue in favor of the “prophylactic” use of nitric oxide postoperatively in these patients, although there are no controlled studies to support such a recommendation. Thus, prediction of outcomes in patients with congenital cardiac shunts associated with pulmonary hypertension remains a challenge. For the majority of patients with large defects causing significant left-to-right shunting who are assisted early in life, repair can be indicated on the basis of noninvasive evaluation. However, for patients who do not have a clear history of congestive heart failure, a careful analysis of results of noninvasive and sometimes invasive evaluation becomes mandatory. In these instances, the decision to operate and prediction of outcomes cannot be based purely on hemodynamic data. Rather, the presence of associated syndromes (especially Down syndrome), the patient age, the type of the anomaly, the direction of flow across the defect (left-to-right or bidirectional), and the peripheral oxygen saturation recorded sequentially over several days must be taken into account for decision making.

5 Treatment Modalities Ideally, patients with simple defects such as VSDs should undergo complete repair, particularly if they are seen early in life. However, as discussed, persistent pulmonary hypertension following repair is still a problem in some of them. Patients with persistent pulmonary hypertension are generally treated with the drugs that are available for management of pulmonary arterial hypertension, namely, prostanoids, endothelin receptor antagonists, and phosphodiesterase inhibitors. In fact, in many clinical studies designed to investigate the efficacy of these drugs, patients with residual pulmonary hypertension following repair of congenital cardiac defects have been enrolled [42–44]. Alternatively, patients who are considered “at risk” for persistent pulmonary hypertension can be offered pulmonary artery banding initially. If this is performed in young children, one expects that removal of the hemodynamic stimuli (mainly increased pressure) will be followed by improvement of pulmonary vascular abnormalities [45]. If so, complete repair will become possible months later, with a lower risk of poor outcomes due to heightened pulmonary artery pressure and vascular resistance. Favorable effects of hemodynamic unloading were demonstrated by Wagenvoort et al. [46] with reversal of pulmonary vasculopathy following pulmonary artery banding in patients with congenital cardiac shunts. In experimental pulmonary hypertension, hemodynamic unloading is associated with decreased number of proliferating cells in the wall of pulmonary arteries and decreased medial thickness [47]. However, there are unsolved problems with the use of

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pulmonary artery banding in humans. There have been no controlled studies demonstrating the efficacy of this procedure in specific patient subgroups (different ages and different defects). The magnitude of the reduction in pulmonary artery pressure that would be necessary to induce reversal of vascular remodeling is not known. In some instances, distal pulmonary artery pressure does not decrease substantially, unless a pharmacological intervention is proposed as part of the treatment. Telemetrically adjustable pulmonary artery banding devices are now available [48], and may be advantageous in such instances. Ineffectiveness of banding may be associated with extreme thickness of the media of small pulmonary arteries [49]. As a general rule, pulmonary artery banding may be beneficial in selected cases, if a substantial reduction in distal pulmonary artery pressure can be achieved (the magnitude and appropriate duration have not been defined yet) in the absence of hypoxemia. There are, in fact, several possible therapeutic approaches to congenital cardiac shunts associated with pulmonary hypertension. For the majority of patients seen within the

first months of life with large defects, complete repair is the treatment of choice. At the other side of the range, there is a smaller group of children, even young ones, with no history of pulmonary congestion or failure to thrive who present with peripheral oxygen desaturation and heightened pulmonary vascular resistance unresponsive or poorly responsive to vasodilators. In these instances, medical treatment should be always considered first. In many cases, it is the only treatment acceptable. Between these extremes, there are several situations where patients could possibly benefit from combined medical and surgical approaches. Unfortunately again, there are no controlled studies to support such recommendations. However, expertise coming from reference centers with high standards of assistance can be used a general guide for decision making, at least in the less complex situations. On the basis of the authors listed in Table  1 and others [50–52], Fig. 2 was constructed as an attempt to summarize the strategies that can be used in the management of patients with VSD and moderate to severe pulmonary hypertension. Accordingly, only patients in categories A and B can be

Fig. 2    Possible therapeutic strategies for patients with ventricular septal defects (VSDs) at different levels of pulmonary vascular resistance (PVR), pulmonary to systemic vascular resistance ratio (PVR/SVR), and response to vasodilators. Patients with the profiles in a and b are likely to become free of medication (assuming that in b pulmonary artery banding will be followed by VSD closure). Patients with the profiles in c and d are unlikely to benefit from complete repair, and will probably need medication for

treatment of pulmonary arterial hypertension on a long-term basis. Even defined as operable, some patients “are likely” to require special care postoperatively (e). This is particularly so for subjects with marked pulmonary vasoreactivity shown in preoperative tests. The therapeutic proposals shown are based on the opinion of experts, but are not supported by evidence. ASD atrial septal defect, PA pulmonary artery. (The artwork is courtesy of Valéria de Melo Moreira, Heart Institute, São Paulo, Brazil)

78  Pulmonary Hypertension Secondary to Congenital Systemic-to-Pulmonary (Left-to-Right) Shunts

expected to become free of medication on a long-term basis (assuming that patients in category B will have the VSD corrected sometime after pulmonary artery banding). Patients in categories C and D are likely to need drugs for treatment of pulmonary arterial hypertension for their whole life. Again, the situations depicted in Fig.  2 are illustrative and should not be used as evidence-based guidelines.

6 The Special Issue of Atrial Septal Defects ASDs are prevalent in adults with congenital heart disease, corresponding to more than 40% of all the anomalies detected in adulthood [3]. In normal subjects, the pulmonary artery systolic to diastolic pressure ratio is about 2:1. In patients with uncomplicated ASDs (left-to-right shunting), this ratio increases, with heightened pulmonary artery systolic pressure due to increased right ventricular ejection volume, and normal or even decreased pulmonary artery diastolic pressure due to the septal defect itself. Pulmonary vascular disease is a relatively rare complication in this disease. It occurs in 5–10% of patients with unrepaired defects [6], but a prevalence of 35% has been reported in adults [3]. As pulmonary vasculopathy develops, the pulmonary artery systolic to diastolic pressure ratio decreases owing to increased diastolic pressure. In a recent report, pulmonary arterial hypertension was identified as the best independent predictor of functional limitation in adults with ASDs, with an odds ratio of 25.2 [53]. Importantly, pulmonary arterial hypertension may be detected in about 12% of adults with repaired defects [3]. This leads to a considerable change in the natural history of the disease, with need for long-term vasodilator therapy and eventually atrial septostomy. There has been general agreement that adults with ASDs should be offered corrective procedures, in particular since long-term functional limitations and hemodynamic abnormalities seem to be more prevalent among patients with an open defect compared with those with a closed defect [53]. Unfavorable outcomes in patients above the age of 40 years with unrepaired defects are associated with a mean pulmonary artery pressure of more than 35 mmHg and peripheral oxygen saturation of 80% or less [54]. On the other hand, anatomic closure of ASDs in adulthood is generally performed in patients with pulmonary artery systolic pressure of less than 70 mmHg and a pulmonary to systemic blood flow ratio of more than 1.7 [55]. Even in the era of new drugs for treatment of pulmonary arterial hypertension, there is no reason for closing defects in patients with right-to-left shunting or a pulmonary vascular resistance of more than 14 Wood units·m2 [56]. Temporary balloon occlusion of the ASD ­during cardiac catheterization may be of help to assess the impact of the acute shunt elimination on right atrial and ­pulmonary artery pres-

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sures. The procedure may also be ­helpful in patients with left-sided diseases, to ­analyze left atrial pressure and decide which subjects will tolerate a corrective procedure. There are alternatives for the management of patients with an ASD and elevated pulmonary artery pressure and vascular resistance. Although there are no controlled studies to support recommendations of long-term therapy with vasodilators, there have been some reports suggesting that these drugs may be beneficial [57, 58]. In particular, there have been reports of patients treated with intravenous administration of epoprostenol or oral administration of bosentan with successful closure of the defect subsequently [59, 60]. Alternatively, fenestrated devices can be used for partial closure of the defects in patients with elevated pulmonary artery pressure [61], since in late follow-up, many patients undergoing percutaneous closure with conventional devices do not have complete normalization of pulmonary hemodynamics [62]. All these single or combined procedures require controlled studies for them to be recommended. Only case series have been reported until now. Anyway, in view of the poor outcomes in patients with closed defects and moderate to severe residual pulmonary hypertension, with survival curves similar to those for patients with idiopathic pulmonary arterial hypertension, there has been increasing interest in the use of vasodilators before making a decision about corrective procedures.

7 Drug Therapy There are different potential objectives of drug therapy in congenital cardiac shunts associated with pulmonary hypertension, depending on the clinical and hemodynamic conditions. An obvious objective of the treatment is to improve peripheral oxygen saturation, the physical capacity, and the quality of life in severely hypoxemic patients with advanced Eisenmenger syndrome. In terms of clinical studies, there has been considerable effort to achieve this goal. For example, prostanoids [63] and the endothelin receptor antagonist bosentan [64–66] have proved beneficial up to 1 year of treatment. However, the efficacy of bosentan seems to decline over the second year of therapy [44, 67], particularly in children. A second goal to be achieved is the management of elevated pulmonary artery pressure postoperatively. Pulmonary hypertensive crises may occur even after successful repair of the cardiac anomalies in patients for whom operability is defined on the basis of complete noninvasive and invasive evaluation. Increased pulmonary vasoreactivity following cardiopulmonary bypass is a well-known phenomenon likely related to decreased nitric oxide production and/or its inactivation by free radicals produced during the ischemic process

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[68, 69]. Furthermore, increased plasma levels of ­endothelin-1 have been demonstrated after cardiopulmonary bypass in patients with congenital heart defects and pulmonary hypertension [70]. Substances that increase nitric oxide production such as its precursor l-arginine might theoretically improve the postoperative pulmonary endothelial dysfunction that occurs in these patients [71]. Alternatively, nitric oxide can be administered by inhalation to counteract the effects of endogenous vasoconstrictors [72–74], with daily measurement of the methemoglobin level. One randomized study demonstrated favorable effects of inhaled nitric oxide after congenital heart surgery [75], although its use against a background of hyperoxic alkalosis does not seem to be associated with any benefits [76]. Also, there seems to be no benefit from doses exceeding 10–20 ppm, and effects have been demonstrated with 2 ppm nitric oxide [77]. Preliminary data suggest that prostanoids, endothelin receptor antagonists, and phosphodiesterase inhibitors may be useful to control postoperative changes in pulmonary hemodynamics. Aerosolized iloprost seems to be as effective as nitric oxide in selectively lowering pulmonary vascular resistance [52]. In experimental models of pulmonary hypertension secondary to increased pulmonary blood flow, endothelin receptor antagonists seem to effectively block the increase in pulmonary vascular resistance that occurs after bypass [78, 79]. Intravenously administered sildenafil appears to be a potent pulmonary vasodilator when used in children with congenital heart disease in the catheterization laboratory, as well as postoperatively [80]. In addition, sildenafil has proved useful to facilitate weaning from inhaled nitric oxide and prevent rebound pulmonary hypertension [81, 82]. Rebound pulmonary hypertension is an undesired condition that leads to hemodynamic instability and hypoxemia, and requires reinstitution of inhaled nitric oxide therapy with prolonged ventilation and delayed tracheal extubation. Despite the usefulness of all these pharmacological agents, there have been no controlled studies demonstrating benefits with their prophylactic use in children who are considered “at risk” for postoperative pulmonary hypertensive crises. The third issue that needs to be addressed is the advantage (if any) of giving vasodilators preoperatively (and eventually postoperatively) to patients who are considered operable, but are at risk for persistent pulmonary hypertension after hospital discharge. The typical scenario is that of a 2-year-old child with preoperative pulmonary vascular resistance of 9–10  Wood units·m2 decreasing to less than 5 Wood units·m2 during nitric oxide inhalation, discharged from hospital after successful repair of an atrioventricular septal defect. Survival curves for patients who are seen with residual pulmonary hypertension after repair of congenital cardiac shunts are considerably worse than those for unoperated on subjects [83]. Thus, decreasing pulmonary artery pressure by means of vasodilator therapy initiated

A.A. Lopes

before or immediately after operation could be tremendously beneficial. Unfortunately, there are no controlled studies to support such recommendations, and decisions are based on careful analysis of individual data. However, on the basis of the benefits that have been demonstrated with the use of prostanoids, endothelin receptor antagonists, and phosphodiesterase inhibitors in children with pulmonary hypertension in general [84–87], it seems reasonable to suppose that these drugs may be of help in the management of pulmonary hypertension associated with congenital cardiac shunts before and after operation. The recent advances in the treatment of pulmonary arterial hypertension lead to a new dilemma, which is the last point to be addressed in this session. Can the new drugs reverse advanced pulmonary vascular lesions associated with congenital cardiac shunts so that inoperable patients become operable? Although there are potential merits of the “treatand-repair” approach using the recent therapies, the discussion will probably remain open for a long time [88]. This is not the case for hypoxemic subjects with advanced Eisenmenger syndrome. Thus, if pulmonary vasodilatation actually occurs, surgical palliation will become necessary to protect pulmonary vessels (those not completely damaged) from increased blood flow. Adjustable pulmonary artery banding could be an option in such instances. Alternatively, a fenestrated patch could be used for partial closure of an ASD or a VSD (Fig.  3). To test these hypotheses, specific studies are needed taking into consideration the drugs (single or combined), the patient age, the nature of the cardiac anomaly, and the severity of pulmonary vasculopathy. On the basis of current knowledge and survival curves, there is no rationale for closing septal defects in patients with advanced pulmonary vascular disease.

8 Summary Early recognition of congenital cardiac shunts and advances in postoperative care have allowed for successful correction of the anomalies with decreased risk of residual pulmonary hypertension associated with progressive pulmonary vasculopathy. In the vast majority of cases, assignment for corrective procedures is based on noninvasive evaluation, with no need for cardiac catheterization. In some cases, however, a noninvasive diagnostic approach does not suffice. These include patients who seek medical care later in life (e.g., by the age of 2 years or above) and those, even younger, with specific defects such as truncus arteriosus, atrioventricular septal defects, transposition of the great arteries, and large, unrestrictive VSDs. In these instances, despite recent advances with imaging, cardiac catheterization is needed. Direct measurement of pulmonary vascular resistance and

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these disorders toward much poorer outcomes. With the availability of new drugs for treatment of pulmonary ­arterial hypertension, the criteria for operability tend to change toward a “treat-and-repair” paradigm. But controlled studies are required to determine which therapies will prevent immediate postoperative pulmonary hypertensive crises often associated with fatal outcomes, and long-term residual pulmonary hypertension. Until then, patients should be carefully evaluated, and decisions should be made on an individual basis using multiple data. A full picture is better!

References

Fig.  3  Definition of operability in patients with congenital cardiac shunts associated with pulmonary hypertension. In patient 1, the decision to catheterize was based on the absence of a clear history of failure to thrive, the presence of bidirectional flow across the VSD, and periods of peripheral oxygen desaturation. Operability was defined on the basis of the patient’s age, the presence of pulmonary congestion on the chest X-ray, and favorable hemodynamic findings. A pulmonary vasoreactivity test was considered unnecessary. In patient 2, cardiac catheterization was indicated on the basis of age, the absence of pulmonary congestion, bidirectional shunting, and periods of peripheral oxygen desaturation. The more than 20% drop in PVR index (PVRi) and PVR/SVR during nitric oxide (NO) inhalation indicated that vasoconstriction was present. However, since the final values of PVRi and PVR/SVR were above 6  units m2 and 0.3, respectively, complete VSD closure was not indicated. A fenestrated patch was used for partial closure of the defect after 45 days of vasodilator therapy with sildenafil. The patient had an uneventful postoperative course. MR mitral regurgitation, PAP pulmonary artery pressure, Qp/Qs pulmonary to systemic blood flow ratio, SatO2 peripheral oxygen saturation

determination of pulmonary vasoreactivity using appropriate vasodilatation protocols are crucial for making decisions about the therapeutic strategies. The survival curves for unoperated on patients with congenital cardiac shunts and pulmonary hypertension are considerably better than those for patients with idiopathic pulmonary arterial hypertension, suggesting that inappropriate closure of septal defects may shift the natural history of

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Chapter 79

Surgical Evaluation of Congenital-Heart-Disease-Associated Pulmonary Hypertension Clive J. Lewis and Andrew A. Klein

Abstract  Congenital heart disease (CHD) is the most common birth defect, with an incidence of approximately 0.8% of live births. Improvements in surgery have altered the impact of CHD enormously, not only improving mortality in many conditions but also allowing some infants to survive into adulthood when previously they would have died within the first few days after birth. This increased survival has led to a huge increase in the number of survivors of surgery for CHD in childhood now reaching adulthood. This group of “grown-up CHD” (GUCH) patients have distinct needs outside both pediatric and adult cardiology environments and are best served by specialists in this field who are trained to understand the long-term complications these patients face. Pulmonary arterial hypertension (PAH) associated with CHD (CHD-PAH) is relatively common, occurring in 5–10% of patients. It may complicate both simple and more complex lesions and may occur early or late in the natural or operative history of the condition. It results in significant morbidity and early mortality of patients compared with those without evidence of PAH. This chapter discusses the evaluation of CHD patients with dynamic PAH where surgical correction of lesions may be safely undertaken. Surgical or interventional correction is desirable, particularly early in the natural history of CHD before advanced changes in the pulmonary vasculature are established, provided there is evidence to support reversibility of PAH afterward, although this may be difficult to predict in some difficult or marginal patients. Special consideration is given to established, moderate to severe PAH, in particular patients with Eisenmenger syndrome. Discussion of operability most often arises in older children and adults with large shunts and therefore this chapter concentrates on this group of patients. Consideration is also given to advanced therapies, including transplantation, and the importance of anesthetic and intensive care management in patients with CHD-PAH.

C.J. Lewis (*) The Transplant Unit, Papworth Hospital NHS Foundation Trust, Papworth Everard, CB3 8RE, Cambridge, UK e-mail: [email protected]

Keywords  Surgical treatment • Operability • Inoperable patients • Interventional correction

1 Introduction Congenital heart disease (CHD) is the most common birth defect, with an incidence of approximately 0.8% of live births [1]. Advances over the last five decades have led to increased recognition of congenital defects both in utero and in early life, particularly through improved imaging but also through earlier screening and improved recognition. This has led to improved outcomes through better medical and surgical treatment but also a better understanding about the timing of intervention to reduce longer-term complications. Improvements in surgery have altered the impact of CHD enormously, not only improving mortality in many conditions but also allowing some infants to survive into adulthood when previously they would have died within the first few days after birth. This increased survival has led to a huge increase in the number of survivors of surgery for CHD in childhood now reaching adulthood. This group of “grown-up CHD” (GUCH) patients have distinct needs outside both pediatric and adult cardiology environments and are best served by specialists in this field who are trained to understand the long-term complications these patients face. Pulmonary arterial hypertension (PAH) associated with CHD (CHD-PAH) is relatively common, occurring in 5–10% of patients [2]. It may complicate both simple and more complex lesions and may occur early or late in the natural or operative history of the condition. It results in significant morbidity and early mortality of patients compared with those without evidence of PAH [3]. The majority of patients with CHD-PAH are those who have large intracardiac or extracardiac shunt lesions which have not been repaired or where they have been repaired late. In CHD-PAH, pulmonary vascular resistance (PVR) starts to increase at a variable time point following birth and there are many factors which influence the development of PAH, including the anatomical lesion, length of life, degree of

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_79, © Springer Science+Business Media, LLC 2011

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shunting, previous intervention, and genetics. Early in the natural history of CHD, changes in the pulmonary vasculature giving rise to increased PVR may be reversible and therefore lesion correction would be the optimal management. Once changes in the vascular bed are established, correction of the shunt lesion is high risk and may worsen the natural history. The earlier detection and treatment of CHD and a better understanding about the timing of intervention will hopefully translate into a significant reduction of the incidence of CHD-PAH. Landzberg described CHD-PAH in five different scenarios: [4] (1) dynamic (relating to high shunt flow and responding to closure of shunt); (2) late postoperative PAH (therapeutic options include pulmonary vasodilator therapy); (3) immediate postoperative (reactive); (4) unusual forms of CHD where hemodynamics depend on low PVR such as the Glenn and Fontan shunts in tricuspid atresia or single ventricle circulations; (5) The physiologic characteristics of Eisenmenger syndrome includes left-to-right shunting, which results in significant elevation of pulmonary artery pressure (PAP) and PVR to systemic or near-systemic levels, resulting in bidirectional or reversed right-to-left shunting. Both simple and more complex lesions such as atrioventricular septal defects, truncus arteriosus, and single ventricle circulations can give rise to Eisenmenger physiology. Symptoms may not appear until the second decade of life, especially if the presence of CHD has not been recognized early in life. The outlook is poor, with progressive right ventricular (RV) failure and premature death due to sudden arrhythmia, heart failure, and hemoptysis. Thus, early recognition of CHD, especially in patients with large, unrestrictive shunts, is imperative to prevent the sequelae of progressive PAH. Earlier diagnosis, recognition, and treatment of CHD has already resulted in a significant decrease in patients presenting later in life with Eisenmenger physiology and it is hoped that this trend will continue [5]. However, even in developed countries, late presentation of missed shunts means that a number of patients continue to present with previously undiagnosed CHD and established changes in the pulmonary vascular bed. In developing countries, owing to a lack of resources and health-care infrastructures, early diagnosis and treatment of even simple CHD is not available, which still leads to large numbers of children and adults with untreated CHD, many of whom will have significant PAH. This chapter discusses the evaluation of CHD patients with dynamic PAH where surgical correction of lesions may be safely undertaken. Surgical or interventional correction is desirable, particularly early in the natural history of CHD before advanced changes in the pulmonary vasculature are established, provided there is evidence to support reversibility of PAH afterward, although this may be difficult to predict in some difficult or marginal patients in whom it is not clear that here will be reversal of PAH. Special consideration is given to established, moderate to severe PAH, in particular patients

C.J. Lewis and A.A. Klein

with Eisenmenger physiology previously thought to be very high risk or inoperable, where recent advances in the management of PAH, particularly with pulmonary vasodilators, result in reduction of PAP and/or PVR to levels which may allow safe correction of the congenital lesion. Discussion of operability most often arises in older children and adults with large shunts and therefore this chapter concentrates on this group of patients. Consideration is also given to advanced therapies, including transplantation, and the importance of anesthetic and intensive care management in patients with CHD-PAH.

2 Investigations To Evaluate Operability of CHD-PAH Appropriate selection of patients is essential for good outcomes after surgery or intervention, and full clinical assessment and invasive and noninvasive investigations are necessary to aid selection. Of particular importance are anatomical definition, shunt definition, and exclusion of confounding precipitants of PAH. Evaluation of patients with CHD-PAH should therefore be carried out in centers with particular expertise in CHD to safely characterize patients who may be suitable for corrective procedures after extensive assessment. Generally, CHD-PAH is reversible provided that correction occurs prior to changes in the pulmonary vascular bed becoming established and “fixed.” Improved understanding of the natural history of lesions and when PAH tends to become irreversible has therefore changed clinical practice, with increased vigilance and surveillance for progressive increase in PAH, and this has led to earlier intervention. However, it is difficult to determine exactly when PAH has become irreversible and there are no clearly established parameters. Indeed, recent experience with the use of novel oral pulmonary vasodilators in CHD-PAH has shown that even patients assumed to have established “fixed” PAH can have dramatic falls in PAP, which has the potential to allow late, high-risk surgical correction of the underlying lesion. Several factors help determine whether PAH is established, including pulmonary blood flow (Qp), PVR, and PAH reversibility by pulmonary vasodilatation. Poor postoperative outcome is likely when there is high pulmonary blood flow and PVR, little evidence of reversibility, and poor RV function, in terms of both immediate postoperative problems with right-sided heart failure and pulmonary hypertensive crises but also longer-term residual significant PAH.

2.1 Clinical Assessment Full comprehensive clinical examination and noninvasive investigation (oxygen saturations, electrocardiogram, chest X-ray) provide important information. Favorable findings

79  Surgical Evaluation of Congenital-Heart-Disease-Associated Pulmonary Hypertension

suggesting operability include those indicating ongoing leftto-right shunting: shunt murmur with absence of loud single S2 and pulmonary regurgitation on clinical examination; normal saturations exceeding 95%; no desaturation on the 6-min walk test; absence of right-sided heart strain and RV hypertrophy on the electrocardiogram and prominent lung vascularity on the chest X-ray [6] (Table 1). Consideration should also be given to exclude conditions with can increase PVR, such as upper airway obstruction (tracheomalacia, enlarged adenoids), hypoventilation (sleep apnea, neuromuscular disease), restrictive lung disease (chest wall abnormalities, scoliosis, interstitial lung disease), small airway obstruction (asthma, bronchiolitis), and parenchymal lung disease (consolidation/collapse, pulmonary edema).

2.2 Open-Lung Biopsy Open-lung biopsy has been used to gauge the potential for PAH reversibility using histopathological grading. Generally, PVR reactivity to vasodilatation provides a more physiologic method of assessing reversibility, so open-lung biopsy should only be considered in selected cases where the PVR reversibility studies are not conclusive. Open-lung biopsy is high risk in CHD-PAH and should only be considered in appropriate centers. The extent of structural change in the pulmonary vascular bed correlates with pulmonary hemodynamics including postoperative residual pulmonary hypertension and can therefore be useful to select patients for surgical correction but by itself is not reliable in predicting operability [7, 8]. Three progressively severe stages are recognized and incorporate the Heath and Edwards classification with pulmonary hemodynamics. Grade A describes smooth muscle cell migration to the subendothelium of arterioles. It is associated

with increased pulmonary blood flow and raised pulse pressure, but with normal mean PAP. Grade B describes proximal artery medial hypertrophy with increased intercellular connective tissue and is associated with an increase in PAP. In grade C there are fewer distal pulmonary arterioles, with a resultant increase in PVR. Vascular remodeling of the pulmonary vascular bed may be seen on lung biopsy following pulmonary vasodilator therapy in patients with very high PAP.

2.3 Cardiac Imaging Imaging has evolved greatly in the last few decades; 2D and 3D echocardiography, cardiac computed tomography (CT), and magnetic resonance imaging (MRI) with 3D reconstruction have revolutionized the way CHD is assessed. In many cases, imaging methods have replaced conventional cardiac catheterization and angiography. Echocardiography and MRI can be used not only for anatomical definition but can also allow calculation of the ratio of pulmonary to systemic blood flow (Qp/Qs), an important concept to help understand whether congenital lesions can be corrected in the presence of PAH. Echocardiography is very frequently used in the assessment of CHD-PAH and gives a number of important pieces of information to aid clinical evaluation. 2D echocardiography allows clear definition of cardiac anatomy, including understanding the nature of a shunt. The size of any communication may be ascertained as well as the pressure gradient across a shunt, which will determine whether a lesion is restrictive or unrestrictive; the latter is more likely to be associated with PAH. PAP can be estimated from assessment of tricuspid valve regurgitation velocity, which gives RV systolic pressure, calculated from systolic PAP = 4V2 + RAP, where RAP is right atrial pressure and V is the maximum tricuspid

Table 1  Clinical assessment of hemodynamics in left-to-right shunts Modality Clues suggesting operability History

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Clues suggesting inoperability

Feeding difficulty, failure to thrive, tachypnea, and frequent respiratory infections in infancy

Absence of symptoms or “improvement” in status as suggested by improved feeding, weight gain, and resolution of tachypnea Visible cyanosis, quiet precordium, loud single S2, Physical examination Increased precordial activity, labored respiratory efforts absence of flow murmurs, early diastolic murmur of (subcostal and intercostal recession), split second heart pulmonary regurgitation sound (S2), mid-diastolic “flow” murmurs Oxygen saturation a Normal (>95%) Reduced (<90–95%) Normal heart size, normal or reduced vascularity, Chest X-ray Cardiac enlargement, increased vascularity suggested by evidence of pruning with dilated hilar pulmonary prominent end-on vessels, visible vascularity in the artery and rapid decline in vessel size peripheral lung fields ECG Prominent left ventricular forces, q waves in lateral Right ventricular dominance, absence of q waves in precordial leads lateral precordial leads a  Oxygen saturations cutoffs may not be absolute. For example, patients with ventricular septal defects with significant aortic override may have some resting desaturation at rest. However, saturations below 90% are seldom compatible with operation in simple shunt lesions. For patent ducts, oxygen saturation should be estimated in the lower limbs. From Viswanathan and Kumar [6]. Reprinted with permission of John Wiley & Sons, Inc.

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valve regurgitation jet velocity on continuous-wave Doppler echocardiography. Mean PAP can also be calculated from the pulmonary valve regurgitant velocity or pulmonary valve acceleration time. It is important to establish that there is no RV outflow tract (RVOT), PA, or branch PA obstruction as this would give raised RV pressures and mimic the effects of PAH, but the distal PAP and pulmonary vascular bed would be normal (Fig. 1). Echocardiography can also be used to detect a significant left-to-right shunt by calculating Qp/Qs using RVOT velocity integral × RVOT area/LVOT velocity integral × LVOT area (where the area is D2p5 and LVOT is the left ventricular outflow tract). Echocardiography can exclude other causes of right-sided heart failure or PAH, including pulmonary stenosis, Ebstein’s anomaly, RV cardiomyopathy, and most importantly left-sided heart disease causing PAH. Long-standing left ventricle systolic or diastolic dysfunction, mitral valve disease, and restrictive cardiomyopathy can all give rise to PAH which may be amenable to therapy by addressing the underlying cause. The

following echocardiography characteristics are associated with more significant PAP elevation: RV hypertrophy and dilatation (tricuspid annulus greater than 40 mm), D-shaped right ventricle (septal flattening) with paradoxical septal motion, enlarged pulmonary arteries (PAs), Qp/Qs > 2, small gradient across lesion (less than 25 mmHg), bidirectional color flow across a lesion, and more severe tricuspid regurgitation. Cardiac CT and MRI are especially useful for assessment of patients who have poor echocardiographic windows. Both techniques are able to define cardiac anatomy, including shunt flow, sizes, and location. CT is especially useful to look for complications of PAH which may have a bearing on suitability for surgery, including pulmonary embolism/infarction and thrombosis in situ. MRI, through cine imaging, allows myocardial motion, valves, and blood flow to be imaged. Both cine imaging and 3D magnetic resonance angiography allow collateral arteries to be visualized, which is essential when planning intervention. Phase velocity mapping allows functional questions to be answered such as the severity of a

Fig. 1   Typical transthoracic echocardiographic findings in pulmonary arterial hypertension associated with congenital heart disease (CHD-PAH). a A 2D apical long-axis image showing grossly dilated right atrium and ventricle with flattening of the interventricular septum. Failure of tricuspid valve apposition results in severe

regurgitation as shown in color Doppler echocardiography (b) and in continuous-wave Doppler echocardiography (c). (d) Tricuspid annular plane systolic excursion is used to assess right ventricular function. (Echocardiographic images courtesy of Dr. Rosemary Rusk, Papworth hospital, Cambridge, UK)

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stenotic/regurgitant lesion and measurement of shunt or collateral flow. MRI is especially useful for assessment of RV and left ventricular size, function, and mass measurement. In many cases, the images and measurements obtained complement those obtained by other noninvasive investigations and avoid the need for invasive cardiac catheterization. However, cardiac MRI in CHD patients is complex and requires expertise, planning, and clinical supervision from appropriately specialized practitioners owing to the variability of anatomy and clinical conditions. Special consideration should be given to assessment of the right ventricle as preserved postoperative RV function is critical for the success of correcting CHD lesions. Patients with idiopathic PAH (iPAH) have a much worse prognosis than patients with CHD-PAH (discussed in Sect. 9.1), which in part may be due to the length of time the right ventricle has been exposed to progressive but slow increases in PAP, leading to adaptive RV hypertrophy and preservation of RV function. Assessment of RV function is difficult; however, recent improvements in our understanding of RV function have improved imaging diagnostic ability. Cardiac MRI is the “gold standard” test for RV function, allowing measurement of RV size and ejection fraction. However, better echocardiography including 3D techniques to measure RV volumes and ejection fraction and tissue Doppler echocardiography assessment has also improved quantification of RV function.

2.4 Invasive Hemodynamic Assessment Cardiac catheterization provides a direct invasive measurement of pulmonary hemodynamics, the ability to determine the potential reversibility of PAP and PVR using pulmonary vasodilators (such as high-flow oxygen, nitric oxide, calcium antagonists, prostacyclin analogues, and phosphodiesterase inhibitors) and the ability to test therapeutic potentials (temporary balloon occlusion of shunt). Since the severity and the significance of a shunt are used to make decisions on surgical or interventional closure of lesions, it is important to document accurate values; thus, catheter-based studies of PAP and PVR are the accepted “gold standard.” Important confounding factors in cardiac catheterization include accuracy of data during acquisition, distal or branch PA stenosis, pulmonary vein stenosis, and multiple sources of pulmonary blood flow. In addition, there are a number of different calculations used to assess hemodynamics, including the Fick principle and thermodilution, which each have limitations and assumptions about oxygen consumption and mixing of blood in shunts. Using the Fick principle, one may estimate the blood flow to shunt relationship as Qp/Qs = (aortic saturation–mixed venous saturation)/(pulmonary vein saturation–PA saturation). Sources of error have to be con-

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sidered when considering the validity of the Fick result, including blood gas analysis in anemia, cyanotic conditions, hepatic derangement, and sampling and measurement errors. In addition, PAP and PVR are often labile and change in response to stress stimuli or exercise, and cardiac catheterization provides hemodynamic assessment only at one time point. Exercise performed during invasive monitoring can therefore be useful and may be performed by a patient using a bicycle pedal ergometer on the catheterization table. Further consideration should also to be given to the degree of shunting. Shunts with high flow and therefore greatly increased volume of blood passing through the lung may recruit upper and middle lobe vascular beds, which could result in an underestimate of PVR and PAP. Following shunt closure, subsequent to decreased flow, the previously recruited vascular beds no longer contribute to the PVR, resulting in a significantly lower than expected fall in PAP postoperatively. Pulmonary blood flow in CHD may be made up from multiple sources, including the native RVOT, surgically created shunts (e.g., Blalock–Taussig shunt), and aortopulmonary (AP) or other venous/systemic collaterals, and each may have different flow and oxygen saturation characteristics. In these cases, it is important to isolate each source of blood flow and to measure pulmonary blood flow and PVR in isolated lung segments to assess reversibility. This can be achieved using a balloon to temporarily occlude shunts/collaterals and by using a balloon PA catheter manipulated into different lung segments to obtain the pulmonary capillary wedge pressure (PCWP) and hence calculate PVR (mean PAP–mean PCWP/ cardiac output), which can then be used with the calculated PVR in each segment to accurately determine overall PVR. The following have been used as thresholds beyond which surgical or interventional outcomes are significantly worse: Qp/Qs > 1.5–2, PVR > 15 Wood units (WU), PVR indexed 6–8 WU/m2, and pulmonary to systemic resistance ratio (Rp/ Rs) £ 1/3 [9]. Other factors to be considered include the type of congenital lesion, reversibility of PAH/PVR on vasodilator testing, and temporary shunt occlusion; this is discussed further in Sect. 3.

2.5 PVR Reversibility Studies PVR studies for potential reversibility depend on pulmonary bed vasodilatation using a variety of agents [2]. Calcium antagonists such as nifedipine have been used in the past, although ideally a selective pulmonary vasodilator should be used since systemic vasodilatation and consequent fall in blood pressure will also result in a fall in PAP. Assessment of pulmonary vasoreactivity can be performed with short-acting pulmonary vasodilators, including 100% inspired oxygen, nitric oxide, nebulized or intravenously

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administered prostacyclin analogues, adenosine, and phosphodiesterase inhibitors such as sildenafil. There is no consensus as to which agent or specific protocol provides the optimal information. A good vasoreactive response could be considered as a fall in PAP of more than 10  mmHg or to a level below 40 mmHg, without a significant fall in systemic blood pressure or cardiac output [10]. However, there is limited guidance on vasoreactivity criteria in the literature when considering surgical correction of CHD-PAH. One study demonstrated that with the use of a combination of oxygen and inhaled nitric oxide, a greater number of appropriate candidates for corrective cardiac surgery or transplantation could be identified during preoperative vasoreactivity testing when the ratio of pulmonary and systemic vascular resistance (Rp/Rs) was less than 0.33 [11]. Data relating vasoreactivity to long-term outcome after surgery are lacking. However, Post et al. reported correlation between the baseline response to nitric oxide and the mid-term outcome to 5 years in adult patients with CHD-PAH [12].

2.6 Temporary Shunt Occlusion Balloon test occlusion of shunts may provide additional information to aid clinical decision making. If the shunt is temporarily occluded, this allows assessment of PAP and PVR under test conditions representing the immediate postintervention state, allowing better prediction of postintervention results. A rise in right-sided heart filling pressures, PCWP, PAP, RV systolic pressure or a fall in cardiac index would suggest an adverse outcome for proceeding with shunt correction. Temporary shunt occlusion is easily facilitated using a balloon for a patent ductus arteriosus (PDA) or an atrial septal defect (ASD) but is difficult in ventricular septal defects (VSDs). Several case series have reported the utility of this test to aid decisions regarding the potential for permanent shunt closure. In one series, responders to balloon test occlusion were defined as having a fall of at least 25% in PAP or at least a 50% fall in the PAP to systemic arterial pressure ratio [6]. Fourteen of 18 responders were shown to have sustained reductions in PAP (even in the presence of high PVR and low Qp/Qs), with a progressive rise in nonresponders. The utility of balloon test occlusion to determine suitability for percutaneous transcatheter PDA device closure in patients with severe PAH has been demonstrated by Yan et al. [13]. Twenty of 29 patients who underwent successful PDA closure demonstrated a mean fall in PAP of 37 mmHg with saturations maintained above 95% following temporary PDA occlusion. The criteria used to determine that the remaining patients were unsuitable for PDA closure included worsening of symptoms, increase in PAP (mean 10 mmHg), and saturations below 86% or below 95% with supplemental oxygen.

C.J. Lewis and A.A. Klein

3 Consideration When Undertaking Repair of CHD Lesions Associated with Left-to-Right Shunting All lesions associated with significant left-to-to right shunting lead to increased pulmonary blood flow and may lead to pathological and physiological changes in the pulmonary vascular bed, which produce progressive pulmonary hypertension and increasing PVR. Large defects, unrestrictive flow dynamics, and unrepaired defects are most likely to lead to the development of PAH, but the response to increased pulmonary blood flow is not predictable, resulting in significant difficulty in deciding whether to close a lesion. Patients with more complex lesions such as truncus arteriosus and atrioventricular septal defects are at high risk for developing PAH early. VSDs are common and approximately 50% of large, unrestrictive VSDs will develop Eisenmenger physiology if they are not closed within the first 18 months of life. Overall, for all types of VSDs there is a moderate risk (approximately 20%) for a patient developing PAH, with a similar risk for a large PDA. Simple shunts such as ASDs are often associated with mild to moderate elevation of PAP, in part due to increased pulmonary blood flow, and these may be safely closed. However, assessment of PAP and PVR is important prior to closure and during long-term follow-up since PAP may remain elevated or even rise progressively. In some forms of CHD, primary repair is best carried out when an infant has grown and so initial banding of the PA is performed, for example, in a large VSD. This allows protection of the pulmonary vascular bed and prevents the increase in PVR which would otherwise ensue, so that correction of the defect and debanding can be delayed. Intraoperative assessment of the PA band is important so that a sufficient gradient is generated to reduce pulmonary blood flow and prevent the development of PAH while maintaining adequate oxygenation. Frequent postoperative evaluation is also important to ensure that there is sufficient blood flow as the infant grows and that PAH does not develop. Cardiac catheterization is required to assess PAP and PVR if there is clinical suspicion that PAH may have developed (low gradient on echocardiography across the PA band). PA banding in severe PAH with Eisenmenger physiology has been shown in a small number of patients to allow regression of the histological findings of PAH and reduction in PAP, allowing surgical correction [14]. Two of four patients with severe PAH who were considered high risk for conventional surgery benefited from PA banding, with subsequent reduction in PVR allowing later surgical correction and a good medium-term outcome; the remaining patients failed to respond. Other evidence suggests that PVR only improves to a small extent and only in very young patients after PA banding. There are no selection criteria to determine which patients will respond to banding and therefore this strategy

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cannot be recommended to improve the chances of operability in established CHD-PAH. Patients with variants of tetralogy of Fallot may need additional pulmonary blood supply owing to the RVOT obstruction and subsequent cyanosis and “spelling.” Various systemic–pulmonary shunts have been developed to increase pulmonary blood flow: the Blalock–Taussig shunt (modified – subclavian to PA); Waterston and Pott’s shunts (AP connections, now rarely encountered). The AP shunts are particularly associated with the development of PAH due to overshunting at systemic pressure. In the modern era, overshunting is unusual owing to appropriate selection of shunt size, but may still result in pulmonary vascular disease or distortion of the branch PA and it is important to exclude these prior to undertaking surgical repair. Pulmonary atresia is often associated with single or multifocal AP collateral arteries, which must be carefully assessed when considering operative repair. Collaterals may subject the pulmonary vasculature to systemic-level pressure and increasing PVR but may also contain stenoses protecting the lung. Different lung segments may have differing arterial supply, including dual supply (from collaterals and native pulmonary blood flow), which makes assessing PVR in these patients more complex. Strategies for early or late surgical repair of this condition may allow prevention of PAH developing, e.g., unifocalization of collaterals, formation of a central PA, or interventional coil embolization of collateral arteries. There is a lack of evidence and guidance when deciding on the operability of patients where there is late presentation, moderate to severe PAH, and the suspicion of “established” or “fixed” changes in the pulmonary vascular bed. Factors which have been shown to affect outcome after surgical correction of CHD are explored in Table 2 and include age at repair, preoperative PAP to systemic pressure ratio, and Rp/Rs ratio, although there is no predictable relationship or cutoff value [15, 16]. However, when considering surgical correction, if there is significant systemic cyanosis (i.e., representing

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right-to-left shunting at atrial level), the severity of PAH and its irreversibility is likely to prevent successful surgery. Established CHD-PAH has previously been viewed as irreversible and therefore these patients have been considered unsuitable for surgical or interventional treatment owing to the high perioperative risk and poor short- and medium-term outcome, including residual PAH and RV failure. Recent advances in the management of CHD-PAH include pulmonary vasodilator therapy (discussed in Sect. 5), intervention to reduce pulmonary blood flow, and atrial septostomy. In severe PAH, shunting at atrial level can partially relieve high right atrial pressure and improve symptoms at the expense of systemic cyanosis due to right-to-left shunting. Indeed, the clinical benefit of creating a right-to-left shunt to “off-load” the right side of the heart by performing atrial septostomy is recognized in selected severe PAH patients who are very symptomatic [17]. Atrial septostomy is usually performed by interventional catheterization using a blade but has significant morbidity associated with it. Up to nearly a third of Eisenmenger patients have evidence of pulmonary vasoreactivity in response to inhaled nitric oxide, meriting the suggestion that responders could be targeted with specific treatments to reduce PAP [18]. The use of pulmonary vasodilators, in particular, has shown promise in reducing PVR and PAH in congenital patients with the additional potential benefit of the proposed vascular remodeling effects of endothelin antagonists [19]. With the expected increase in the number of CHD patients on these therapies, we are likely to see a number of patients, who with appropriate follow-up and reinvestigation or rechallenging, may meet conventional criteria which would allow consideration for surgical correction in the future in patients previously thought inoperable. This may allow not only improvement in the significant morbidity associated with CHD-PAH, but may also potentially improve mortality. Very symptomatic patients who are deemed too high risk for corrective intervention and who fail a trial of pulmonary vasodilator therapy should be referred for consideration of lung or combined heart and lung transplantation.

Table 2  Criteria suggesting adverse outcome following shunt closure Hemodynamics Vasodilator testing Temporary shunt occlusion Rise in filling pressures SPAP >70 mmHg Decrease in SPAP (PCWP, PAP, RVSP) <10 mmHg Mean PAP Decreased SPAP <25% >45 mmHg SPAP remains >40 mmHg Decrease in Pp/Ps <50% Pp/Ps >2/3 Fall in systemic Fall in cardiac index Qp/Qs >1.5–2 blood pressure or Saturations <85% or <95% PVR >15 WU cardiac output with supplemental PVRI 6–8 WU/m2 oxygen Rp/Rs £ 1/3 SPAP systolic pulmonary artery pressure, PAP pulmonary artery pressure, Pp/Ps pulmonary to systemic pressure ratio, Qp/Qs pulmonary to systemic blood flow, PVR pulmonary vascular resistance, PVRI pulmonary vascular resistance (indexed), WU wood units, Rp/Rs pulmonary to systemic resistance ratio, PCWP pulmonary capillary wedge pressure, RVSP right ventricular systolic pressure

4 Other Forms of CHD Associated with PAH Consideration and assessment of PAH is important in some other forms of CHD which do not involve cardiac or extracardiac shunts prior to cardiac surgery. Some forms of CHD affecting the left side of the heart may be associated with PAH by causing pulmonary venous hypertension. If this is recognized early, surgical correction of the underlying lesion will result in normalization of the PAP, so early recognition and treatment remain imperative. Mitral valve disease results in raised left atrial and pulmonary venous pressures and

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particular attention should be given to regular assessment of mitral stenosis or cor triatriatum. Inherited cardiomyopathies including dilated, restrictive, and hypertrophic forms, especially those with restrictive characteristics, also commonly give rise to PAH. Strategies to manage the underlying disease may slow progression of PAH, but patients with long-standing left-sided heart disease may have PVR which is relatively fixed and thus corrective surgery carries significant risk and later cardiac transplantation may not be an option. Some patients with unusual forms of CHD, e.g., univentricular circulations (tricuspid atresia, hypoplastic left-sided heart syndrome) may be palliated with direct systemic venous to PA shunts. These unique pulmonary circulations bypass the right side of the heart and thus depend on right-sided filling pressure and nonpulsatile flow. The Glenn shunt (superior vena cava to PA) and Fontan operation (total cavopulmonary connection) both critically depend on low PVR for hemodynamic effectiveness. Thus, careful assessment for even mild elevation in PVR is extremely important before undertaking surgery to form Glenn or Fontan connections. The following have been associated as risk factors for successful completion of the Fontan procedure: mean PAP ³20 mmHg, PVR ³5 WU, small PA (Nakata index less than 200 mm2/m2), moderate to severe atrioventricular valve incompetence, distortion of the PA, and poor right and left ventricular function. Owing to the poor outlook for patients who develop raised PVR in these conditions, long-term use of pulmonary vasodilator therapy may be required to maintain or reduce PVR. There is, however, little evidence to support its use for this indication. One case report supports the use of sildenafil to resolve proteinlosing enteropathy complicating a post-Fontan patient with presumed high PVR [20]. A further study used the orally administered prostacyclin analogue beraprost to demonstrate a reduction in PVR in potential candidates for the Fontan procedure with mild PAH [21].

5 New Therapies in CHD-PAH Vasodilators which have effects on both pulmonary and systemic circulations have been used as therapy in iPAH for many years. Prostacyclin analogues administered intravenously and subcutaneously have been shown to improve hemodynamics in iPAH [22]. More recent advances with oral treatments including endothelin antagonists, such as ambrisentan, bosentan and sitaxsentan, and phosphodiesterase inhibitors, such as sildenafil, have revolutionized the treatment and outlook for patients with iPAH. Evidence for their use includes randomized, controlled trials showing they are well tolerated, with improvement in survival, functional class, exercise tolerance, hemodynamics, echocardiographic parameters, and quality of life measures and relatively few side effects [2, 23].

C.J. Lewis and A.A. Klein

These therapies are being used in patients with Eisenmenger physiology with an evolving evidence base. Trials using these agents to date have only included small numbers of Eisenmenger physiology patients. There have been few convincing data for the use of these agents in CHDPAH until more recently and concerns have been raised about the potential for increased right-to-left shunting with agents that promote both systemic as well as pulmonary vasodilatation. Inhaled iloprost, orally administered endothelin antagonists, and phosphodiesterase inhibitors, however, are thought to have a more selective pulmonary vasodilator response and, although limited, there is increasing support in the literature for their use in CHD-PAH [5]. Prostacyclin analogues have been used in selected severely impaired and symptomatic patients with Eisenmenger physiology for some time [24]. Intravenously administered prostacyclin has been shown to significantly improve functional capacity, oxygen saturation, and cardiopulmonary hemodynamics. However, major limitations with this treatment include the need for continuous intravenous administration of prostacyclin analogues, with inherent infective risk. Since mortality in CHD-PAH is significantly lower than that observed in iPAH, the potential long duration of therapy limits the use of intravenously administered prostacyclin. Inhaled and subcutaneously administered prostacyclin analogues have also been used but with less supportive evidence. Similarly, inhaled nitric oxide reduces PVR and improves outcome in CHD-PAH but with inherent difficulties for long-term administration [12]. Endothelin receptor antagonists have been used to successfully treat iPAH, resulting in reduction in PAP and PVR and evidence of pulmonary vascular remodeling with an acceptable side-effect profile [23, 25]. Bosentan, a dual ETA and ETB receptor antagonist, is now recommended therapy in iPAH for patients with World Health Organization (WHO) functional classes III and IV. The EARLY study has furthered the evidence base to include a benefit in mildly symptomatic class II PAH patients, although the number of patients included with CHD was small [26]. Similar benefits have been demonstrated in CHD-PAH, including improvement in pulmonary hemodynamics and markers of disease and 6-min walk distance [19, 27]. The BREATHE-5 study was the first randomized, controlled trial of an endothelin antagonist in CHD-PAH and showed that during short-term follow-up (16 weeks), WHO class III Eisenmenger patients taking bosentan had an improvement in functional class, 6-min walk distance, and pulmonary hemodynamics without serious side effects or fall in systemic saturations. In addition to the beneficial effects of pulmonary vasodilatation, the BREATHE-5 investigators suggest that bosentan has properties which promote vascular remodeling and antiproliferative effects such that patients with Eisenmenger–PAH may benefit from this therapy regardless of PVR or vasoreactivity.

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The favorable hemodynamic response has led to the widespread use of bosentan in WHO class III and class IV CHD-PAH patients [2]. The proven benefits of pulmonary vasodilators therefore provide the main rationale why previously inoperable CHD-PAH patients could be reassessed for corrective procedures according to the response to therapy. Furthermore, consideration should be given to other agents which have a favorable influence on perioperative mortality due to postoperative pulmonary hypertension and pulmonary crises. The phosphodiesterase type-5 inhibitor sildenafil has an established use in iPAH and improves exercise capacity, WHO functional class, and hemodynamics in patients with symptomatic PAH [28]. However, there is a very limited amount of data on patients with CHD-PAH treated with sildenafil in the literature, although sildenafil has found use in patients with postoperative PAH. PAH and increased PVR are often observed postoperatively following cardiac surgery, and may be exacerbated by ventilation/perfusion mismatch and is an important cause of morbidity and mortality in this period. The resultant effects of sudden rises in PAP lead to increased right-sided filling pressures, RV enlargement, tricuspid regurgitation, and right-sided heart failure with resultant decreased cardiac output, acidosis, and ischemia. The likely pathogenic cause of postoperative pulmonary hypertensive crises relates to endothelial dysfunction, impaired nitric oxide synthesis, and increased levels of endothelin, a potent vasoconstrictor. This is the rationale behind the therapeutic use of nitric oxide, inhaled iloprost, phosphodiesterase inhibitors, and endothelin antagonists to treat postoperative PAH. The use of inhaled nitric oxide and iloprost is of particular interest, since they may be more pulmonary selective and would produce fewer systemic effects compared with intravenously administered prostacyclin. Short-term postoperative inhaled nitric oxide has been used to stabilize pulmonary hypertensive crises in a patient undergoing ASD closure with severe PAH, with a good outcome at 2 years [29]. Sildenafil has been shown to reduce PAP and PVR, prevent episodes of arterial desaturation, treat pulmonary hypertensive crises, and allow weaning from nitric oxide after corrective CHD surgery [30, 31]. Thus, a strategy to administer pulmonary vasodilators earlier to prevent postoperative PAH may be useful. To support this approach, there is a dose-dependent decrease in PAP and PVR following treatment with the ETA receptor antagonist sitaxsentan when given to patients coming off cardiac bypass [32]. Patients with previously inoperable CHD-PAH undergoing corrective surgery, following a period of treatment with pulmonary vasodilators to reduce PAP and PVR, would appear to warrant continued use of these agents in the immediate postoperative period. This could prevent postoperative PAH and pulmonary hypertensive crises and, if continued in the long-term, potentially allow further pulmonary vascular remodeling with an improvement in outcome. Thus, a

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strategy of using endothelin antagonists combined with phosphodiesterase inhibitors could lower perioperative risk and increase the potential number of patients who could undergo corrective procedures, but this approach remains to be proven in appropriate trials.

6 Why Consider Correction of Severe CHD-PAH Patients? A strategy to correct lesions in severe CHD-PAH has several attractions. Patients with Eisenmenger physiology are often very symptomatic and have a number of important complications relating to PAH, right-sided heart function, and to the shunt itself, with significant associated morbidity affecting quality of life (Table  3). The particular complications associated with reversed right-to-left shunting in Eisenmenger physiology are to a great extent due to the sequelae of cyanosis, which is also a major contributor to the decreased exercise capacity seen in this condition. Cyanosis may also lead to ventricular and other organ dysfunction (renal, hepatic, etc.), erythrocytosis and hyperviscosity, platelet dysfunction, and clotting abnormalities. Erythrocytosis contributes to the procoagulant state in some Eisenmenger patients which may exacerbate PAH by causing multiple small pulmonary emboli or thrombosis in situ and cause paradoxical embolism, resulting in stroke and cerebral abscess. Treatment with pulmonary vasodilators alone may improve pulmonary hemodynamics and symptoms, but correcting the congenital defect may have additional benefit by preventing the sequelae of worsening PAH related to the shunt. Significant morbidity in PAH relates to dilatation and dysfunction of the right ventricle, resulting in arrhythmia, exercise limitation, and symptoms of right-sided heart failure. It is interesting to note that the significantly more aggressive disease and high mortality seen in iPAH compared with Table 3  Evaluating operability of cardiac defects in adults with congenital heart disease and pulmonary arterial hypertension For Against Potential conversion of Eisenmenger Abort right-to-left shunting physiology to idiopathic Decreased cerebrovascular pulmonary arterial hypertension events (stroke/abscess) (and thus worse long-term Prevent cyanosis outcome)   Increased exercise capacity High perioperative risk   Decreased erythrocytosis Very limited experience and no   Decreased hemostatic long-term data available problems   Decreased systemic organ failure Protect pulmonary circulation From Dimopoulos et  al. [36]. Copyright (2008), with permission of Elsevier

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CHD-PAH may relate to preservation of RV function by offloading of the right ventricle through a shunt [33]. Currently, there is no evidence to support the use of pulmonary vasodilators to improve RV dysfunction in Eisenmenger patients. However, there is evidence of RV remodeling, reduction in RV size, and improved function following intervention for other conditions which have increased RV pressure and RV dysfunction. In chronic obstructive PAH, pulmonary thromboendarterectomy results in improved pulmonary hemodynamics and RV function [34]. Similarly, relief of pulmonary stenosis/regurgitation by pulmonary valve replacement late after tetralogy of Fallot repair allows recovery of RV size and function [35]. Therefore surgical correction of patients with CHD-PAH may still be considered in the presence of impaired RV function, with careful postoperative management. A strategy to close defects in CHD-PAH, however, has many potential dangers. Surgery in patients with even moderate PAH is high risk, with significant perioperative mortality. Surgery must take place in an institution with particular expertise in the anesthesia and intensive care management of PAH in addition to having PAH and CHD cardiology specialists. Pulmonary hypertensive crises are difficult to manage and there are no criteria to predict the incidence of crises or significant residual PAH in the postoperative period. Local protocols for management may be useful with routine initial postoperative supportive measures including inhaled nitric oxide, intravenously administered prostacyclin analogues, or nebulized iloprost. Intravenous inodilators (enoximone or milrinone) may also be helpful to manage postoperative raised PVR in association with ventricular impairment. The addition of epinephrine may also be required to support right-sided heart function. The use of sildenafil in the postoperative period was discussed in Sect. 5, but clearly continued use of the preoperative pulmonary vasodilator therapy should be instigated. Although there is evidence to support short-term use of pulmonary vasodilator therapy, there are no data on longerterm use and outcome. It has been suggested that the initial reduction in PAP and PVR with pulmonary vasodilators in VSD associated PAH may later, paradoxically, result in increased PVR [36]. The initial reduction in PVR allows an increase in left-to-right shunting, increased pulmonary blood flow at systemic pressure, and thus worsening the changes in the pulmonary vascular bed [37]. This would be seen as failure of therapy with return to the baseline following the initial response. Furthermore, there have been no studies of the effect of correcting the underlying lesion and the subsequent natural history of CHD-PAH. Indeed, Dimopoulos et al. suggest that following closure of a shunt, the modified natural history may now follow the course of iPAH, which has a much more rapid progression and significantly higher mortality [36].

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7 Evidence Supporting Shunt Closure in CHD-PAH There are no randomized, controlled data to support the safety or outcome of the approach to improving pulmonary hemodynamics using new therapies and then correcting underlying congenital lesions. However, there are a number of case reports in the literature, mostly relating to ASDs and PDAs, which report successful closure of lesions either with no additional therapy or with pulmonary vasodilator therapy given before or after closure with resultant reduction in PAH and good short-term outcome but no evidence regarding longer-term outcome. There will, of course, be a positive reporting bias in these reports with failure to report poor outcome in other cases. The use of prostacyclin analogues immediately after shunt closure is described in a number of case reports. Yamauchi et  al. described four patients with ASD and severe PAH (mean PAP 84 mmHg) with advanced changes in the pulmonary vascular bed on lung biopsy, including more than 70% pulmonary arteriolar occlusion [38]. Immediately after surgery, long-term use of orally administered prostacyclin (mean 3.4-year follow-up) was commenced, with a reduction in mean PAP from 74 to 57  mmHg and New York Heart Association (NYHA) class from II–III to I. Frost et  al. reported a patient with an ASD, systemic-level PAP (systolic 105 mmHg), NYHA class III, and a small right-to-left shunt [39]. Following 4 years of intravenous administration of prostacyclin, systolic PAP was 45–50 mmHg and Qp/Qs was 3 so surgical ASD closure was undertaken. The PAP was 60–70 mmHg after closure; the use of prostacyclin was continued for a further 6 months, with further reduction in PAP, followed by treatment with a calcium antagonist. Long-term follow-up to 8 years showed systolic PAP of 45 mmHg and NYHA class II. Schwerzmann et al. described a patient with a large ASD, mean PAP of 53  mmHg, PVR index of 8.8, NYHA class III, and advanced changes on lung biopsy [40]. Long-term use of intravenously administered prostacyclin was commenced and reassessment after 1 year demonstrated mean PAP of 32 mmHg, PVR index of 2.8 (improving further on test occlusion), and NYHA class I, so percutaneous ASD device closure was undertaken. The use of prostacyclin was continued for 1 year and then orally administered bosentan was used, with evidence of sustained improvement with NYHA class I, increased 6-min walk distance, and normalized RV size and function. The use of bosentan has also been described in a number of case reports. Nemoto et al. reported the successful use of bosentan, following the unsuccessful use of orally administered sildenafil alone, to reduce early postoperative PAH in two infants following surgical correction of CHD [41]. Eicken et al. reported the use of bosentan in an adult started 5 months following percutaneous PDA device closure for residual PAH

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with an improvement in symptoms [42]. Ussia et al. reported the first successful use of bosentan before shunt occlusion in a 63-year-old patient with a PDA, NYHA class III, and severe PAH with mean PAP of 65 mmHg [43]. Bosentan was given for 3 months before percutaneous PDA device closure (mean PAP 55 mmHg), with sustained improvement in pulmonary hemodynamics and functional class at 8-month follow-up (mean PAP 35 mmHg) and normalization of RV function.

8 Paradigm for Shunt Closure in CHD-PAH? A suggested paradigm for managing patients with CHDPAH is given in Fig. 2. In all patients considered for shunt closure in CHD-PAH, there is a need to balance the benefit of correcting the lesion against the risk of closing the shunt and causing right-sided heart failure. In cases of mild PAH it would be reasonable to correct the congenital lesion as soon as possible to prevent progression to severer disease. In moderate PAH, if the criteria in Table 2 (discussed in Sect. 3) are met, then surgical or interventional correction may be warranted to prevent disease progression. Some patients will remain with mild or moderate residual PAH, both in the immediate postoperative period and in the long term. Regular ongoing clinical and echocardiographic surveillance for PAH is therefore mandatory. There is recent evidence of the benefit of bosentan in WHO symptom class II patients with PAH of any cause [26]. Whether these patients would benefit from ongoing therapy with bosentan or sildenafil after surgery remains to be determined, but initial case reports suggest this would be a reasonable strategy.

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Patients with severe CHD-PAH, especially those with Eisenmenger physiology, have previously been considered excessively high risk for correction. Each patient with severe CHD-PAH should be assessed against the conventional criteria for successful closure of a shunt. With the accumulating evidence for the use of pulmonary vasodilator treatment, in particular bosentan in class II to class IV patients with CHDPAH, a strategy should be adopted to target therapy in those patients with evidence of favorable characteristics, including reversibility and improvement in hemodynamics with temporary shunt occlusion. Careful follow-up may allow later consideration of shunt closure. A sufficient length of treatment would be required to ensure sustained response to treatment. If echocardiographic follow-up, perhaps every 3 months, were to demonstrate sustained reduction in PAH, further right-sided heart catheterization assessment should be undertaken to determine hemodynamics and PVR. There is no evidence to suggest how much PAP or PVR needs to fall before shunt correction could be contemplated, but reasonable criteria would be a fall to levels when conventional shunt closure would be indicated, i.e., mild to moderate level of PAH with evidence of reversibility and no adverse change in hemodynamics including cardiac output with temporary shunt occlusion. If surgical or interventional shunt closure were undertaken, preclosure pulmonary vasodilator therapy should be continued and ongoing close follow-up would be needed to monitor PAP and ensure an ongoing favorable outcome. At completion of cavopulmonary connection via a lateral tunnel in the Fontan operation, many centers fenestrate the lateral tunnel to the common atrium. If PVR and filling pressures rise, the fenestration shunts from right to left, allowing off-loading of the Fontan circulation. A similar approach could be considered in the CHD-PAH patient undergoing late surgical repair with fenestration of an ASD or a VSD patch or interventionally using a percutaneous inserted fenestrated ASD device. Novick et al. reported the use of a fenestrated patch closure of VSDs with elevated PVR [44].

9 Alternative Strategies for CHD-PAH 9.1 Transplantation in CHD-PAH

Fig. 2  Treatment algorithm for evaluating patients with CHD-PAH for lesion correction

For selected highly symptomatic patients with NYHA classes III and IV and CHD-PAH, transplantation offers the chance of increased quality of life with the potential for improved mortality. Only a small number of transplants are currently performed for CHD: during the period 1995–2007, 0.2% of single-lung and 1.3% of double-lung transplants; during 1982–2007, 34.4% of a total of only 2,639 combined heart and lung transplants reported to the International Society for Heart and Lung Transplantation Registry [45]. However,

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there is currently an unmet need: increasing numbers of patients with CHD are surviving into adulthood, therefore increasing demand for transplantation at a time when many countries are seeing a decrease in the number of suitable donors; many patients are not referred to transplant centers at an appropriate time and others die on waiting lists for transplant. Current indications for combined heart and lung transplant include Eisenmenger syndrome with an uncorrected intracardiac defect and uncorrectable CHD with atresia or severely hypoplastic PAs (which would make connection to the donor PA difficult) and progressive heart failure despite maximum medical therapy. Clearly there are specific anatomical and physiologic considerations and comorbid conditions which are important when assessing CHD-PAH patients for transplant surgery. Controversially, assessment for transplantation in this difficult patient group would be better maintained in a few larger-volume transplant centers with cardiological expertise in CHD and surgeons and intensivists with significant experience in CHD surgery. One of the challenges when assessing CHD-PAH patients for transplantation is the understanding of the natural history of Eisenmenger physiology and therefore characterizing the most appropriate time for a patient to be listed for transplantation. The survival for patients with CHD-PAH is considerably better than that for patients with iPAH and they are less likely to require transplantation [3]. Hopkins et  al. demonstrated that the actuarial survival for patients who did not receive a transplant during follow-up to 3 years was 35% for iPAH and 77% for CHD-PAH, although other reports suggest survival rates of 75% to 30 years of age for Eisenmenger physiology patients. The following risk factors for rapid deterioration may help in recognizing when CHD-PAH patients should be considered for transplant listing: worsening functional capacity, cyanosis and RV function resistant to medical therapy, arrhythmia, RV hypertrophy, renal dysfunction, and raised levels of uric acid [3, 46]. A reasonable approach would be to consider transplantation in patients in whom the expected survival would be less than 2 years, although this is not easy to predict and needs the involvement of CHD/GUCH physicians in patient selection. There is limited evidence of the impact of pulmonary vasodilator therapy on the need for transplantation in Eisenmenger syndrome. Adriaenssens et al. retrospectively analyzed data on 43 patients with unstable Eisenmenger syndrome [47]. They showed no difference in survival with or without pulmonary vasodilator therapy, but did show a significant longer time before death or listing for transplantation (7.8 ± 1.0 vs. 3.4 ± 0.9 years).

9.2 Lung Transplantation and Repair of CHD The reported survival for double-lung transplantation is superior to that for combined heart and lung transplantation

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[45]. For double-lung and combined heart and lung transplantation, respectively, the 1-year survival is 78 and 64%, the half-life is 5.2 and 3.5 years, but the 1-year conditional survival is 7.3 and 9.1 years. In recent years there has been a significant fall in both the number of patients undergoing combined heart and lung transplants and the availability of organs, whereas there has been an improvement in the 1-year mortality in the most recent era (2000–2006) to 71%. However, patients are likely to wait for a considerable length of time before a donor graft becomes available and periodic reassessment for suitability is necessary to ensure that they do not become excessively high risk for transplant. Nonetheless, a strategy to improve the outcome for transplantation in simple CHD-PAH would suggest from these data that double-lung transplantation with intracardiac repair of the congenital defect would result in better outcome to 5 years after the transplant. There may be initial problems postoperatively requiring careful management of right-sided heart function, but as discussed elsewhere, significant strides have been made in the understanding and management of RV function after surgery. Following transplantation, the size and function of the right ventricle are known to recover over the subsequent 3–12 months. There also reports of intracardiac repair and single-lung transplantation, although the intermediate-term results appear to be inferior to tose for combined heart and lung transplantation and so intracardiac repair and single-lung transplantation would not be the strategy of choice [48].

9.3 Evaluation for Combined Heart and Lung or Lung Transplantation Selection for lung or combined heart and lung transplantation generally follows guidelines to those for patients with noncongenital causes of advanced heart and lung failure, although there are a number of particular considerations in the CHD patient. Complete anatomical definition is essential when considering thoracic transplantation, including systemic and pulmonary venous return, PA and branch anatomy, extracardiac and collateral shunts, presence of pulmonary arteriovenous malformations which may be occur in cyanotic patients, and the relationship of cardiac structures and vessels immediately posterior to the sternum. Previous thoracic surgery, pleural disease (effusions, thickening, empyema) and previous pleurodesis may all impact on the feasibility of lung transplantation. Physiologic concerns include previous phrenic nerve damage, diaphragmatic palsy, and cardiac function. Longstanding right-sided heart failure can give rise to both hepatic and renal dysfunction with glomerular filtration rate less than 40–50 mL/min/1.73 m2 and cardiac cirrhosis precluding transplantation. Cyanosis may lead to severe thrombocytopenia

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and high perioperative bleeding risk. Patients who have undergone pregnancy or previous surgery may have received blood transfusions (additional risk of hepatitis) and/or homograft material, both of which may give rise to HLA antibodies, which may restrict the number of compatible donors or make transplantation unfeasible if titers are at very high levels. Chronic debility may lead to malnutrition, which impacts on the risk of transplantation and is seen particularly in Fontan patients with high PVR and protein-losing enteropathy, which may lead to acquired immunodeficiency and high risk of perioperative infection.

9.4 Interventional Lung Assist To Reduce PAH The Novalung is a nonpulsatile interventional lung assist membrane ventilator which has been developed for lung support in lung failure. The Novalung is connected to the patient with an arteriovenous shunt and has a low-resistance diffusion membrane which removes carbon dioxide and contributes to oxygenation [49]. This device can divert up to 30% of lung blood flow and has been used for up to 90 days. The Novalung could produce a sustained reduction in PA blood flow, which may allow a fall in PVR followed by shunt closure. This approach has not been reported in the literature and would require testing, perhaps in extremely symptomatic patients with a presumed high mortality in which combined heart and lung transplantation is not an option.

10 Anesthetic Considerations for CHD-PAH Patients Undergoing Cardiac and Noncardiac Surgery 10.1 Preoperative Assessment As part of the careful evaluation by the multidisciplinary team, the anesthetist should assess all patients before surgery. Good exercise tolerance and lack of oxygen desaturation at rest and with exercise are predictive of good outcome. Most medication should be continued until the time of surgery, particularly pulmonary vasodilators. Consideration should be given, however, to stopping antiplatelet drugs and anticoagulants at least 5 days before surgery; heparin administration may be required if there is a high risk of thrombosis. Patients presenting with at least one of the following major risk factors and requiring surgery are at increased risk of morbidity/mortality, and specialized input is essential: cyanosis, resternotomy, arrhythmia, or ventricular dysfunction (right and/or left).

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10.2 Noncardiac Surgery and General Considerations There is controversy as to the ideal location for noncardiac surgery in patients with CHD. However, when PAH is also present or one of the aforementioned risk factors for increased mortality is identified, a specialist cardiac center is probably preferable. Certainly, senior anesthetic input is required, and ideally this should include cardiothoracic CHD experience. General anesthesia is generally regarded as safer than regional blockade with or without sedation, with the exception of limb blockade. This is because of the marked systemic vasodilatation associated with intrathecal and even epidural anesthesia. This is due to arterial vasodilatation and reduction of venous return, and may cause or exacerbate pulmonary hypertensive crises and cardiovascular collapse [50]. General anesthesia with controlled ventilation affords the advantage of hyperventilating the patient and reducing PAH; this outweighs the risk of increasing the RV afterload because the PVR is already so elevated that the increase in intrathoracic pressure has only a minimal effect on the impedance to ejection of the hypertrophied right ventricle. All patients are at increased risk of endocarditis and prophylactic antibiotics should be administered. Transesophageal echocardiography may be useful for hemodynamic monitoring in all but the most routine surgery, in order to assess volume status and ventricular performance. In addition, invasive hemodynamic monitoring should be considered in the majority of procedures. The chronic hypoxia associated with PAH leads to a decrease in von Willebrand factor and poor platelet function, as well as increased fibrinolysis [51]. Thus, intraoperative bleeding is more common; desmopressin may be indicated to improve platelet function, and the antifibrinolytic agent tranexamic acid should be considered (aprotinin has now been withdrawn from clinical use in most countries). Monitoring of coagulation is often necessary, and this may most effectively be performed by use of the thromboelastograph.

10.3 Cardiac Surgery Induction of anesthesia is certainly hazardous in such patients, and can lead to a spiral of worsening PAH, systemic hypotension, decreased cardiac output, and cardiac arrest. This type of deterioration may be very difficult to manage successfully, and cardiac arrest in such a situation is very often irreversible. Therefore, great care must be taken to avoid physiologic derangement and worsening of hemodynamic indices during anesthesia. Premedication is very rarely administered to these patients, and in fact may be contraindicated because the drowsiness

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produced by sedative drugs most commonly used may be accompanied by oxygen desaturation in an unmonitored ward bed. In the operating theater, full monitoring should be instituted while the patient is awake; this should include as a minimum invasive arterial pressure, but consideration should also be given to placement of the central venous catheter under local anesthesia. Intravenous access should also include a wide-bore cannula. Head-down tilt should be avoided at all times, including during central venous cannulation, as this may precipitate RV overload and consequent failure. Ultrasound guidance should facilitate successful cannulation while the patient is laid flat. Intravenous fluid administration should also be limited throughout surgery, because the great majority of these patients are volume-overloaded. The majority of anesthetic induction agents cause systemic vasodilatation; the resulting drop in systemic vascular resistance may lead to myocardial ischemia as well as multiorgan hypoperfusion. Therefore, commencement of an infusion of an inotropic agent should be considered before administration of anesthetic agents. Dopamine may be ideally suited; at 2.5–5  mg/kg/min, vasoconstriction and increased cardiac output are unlikely to be accompanied by tachyarrhythmia; in addition it can be safely administered via a peripheral intravenous cannula for a short period of time. There is no ideal single anesthetic agent or combination of anesthetic agents. However, the majority of experienced practitioners rely on benzodiazepines and opioids in this clinical situation. Midazolam (0.1 mg/kg) and fentanyl (10 mg/kg) intravenously followed by pancuronium (0.15 mg/kg) for muscle relaxation is a combination in frequent clinical use. Etomidate is frequently suggested as the induction agent of choice in these patients, but has been associated with adrenocortical inhibition, even after one-off administration, and should probably be avoided [52]. Tracheal intubation may itself cause pulmonary vasoconstriction, and should not be attempted until adequate anesthesia and muscle relaxation has been administered and given time to be effective. The large dose of opioid mentioned above is likely to blunt the hemodynamic response to intubation, and lead to stability during the intervention. Hypoxia and hypercarbia should, of course, be avoided at all times, as they may also worsen PAH. Intermittent positive ventilation per se may also lead to hemodynamic compromise, because of the increase in mean intrathoracic pressure [53]. At all times during the induction process, hypotension should be avoided; any decrease in arterial pressure of more than 10% from preinduction values (the mean arterial pressure is most useful) should be immediately treated by small incremental doses of vasoconstrictor; phenylepherine or meteraminol is most useful in this regard. Central venous access should also include PA catheterization, as monitoring of PAP during surgery is essential. Transesophageal echocardiography is an invaluable tool

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during surgery for assessing right-sided heart function as well as the lesion itself and the degree of shunting may also be estimated. In addition, unexpected lesions may be identified and the surgical procedure altered if indicated. Anesthesia, as well as causing systemic vasodilatation, will also cause pulmonary vasodilatation, and a significant improvement in PAH and PVR is likely, providing systemic perfusion and pressure are maintained. This should be borne in mind if the severity of disease or the echocardiographic findings are assessed under anesthesia. Use of nitrous oxide should be avoided throughout the procedure, as cardiac output may fall and there may be an increase in pulmonary vasoconstriction. The halogenated ethers isoflurane and sevoflurane and propofol by infusion are safe drugs for maintenance of anesthesia. Desflurane, however, should be avoided as rapid increases in concentration are associated with increased PAP [54]. If PAP does increase during surgery, there are a number of therapeutic options to help manage this situation; these are outlined in Table 4.

11 Conclusion Improvement in the early recognition, investigation, and treatment of infants with CHD in the developed world has already started to decrease the incidence of the later development of PAH, although this remains a significant problem in lessdeveloped countries. Comprehensive and careful clinical, noninvasive, and invasive investigation allows identification of patients with PAH and at low or moderate risk of surgical or interventional correction of the underlying congenital defect with good long-term outcome and reduction or normalization of PAP. Limited evidence supports the use of pulmonary vasodilator therapy to improve symptoms in patients with NYHA class II–IV exercise limitation and possibly survival. A subgroup of patients using these therapies, who normally would not be considered suitable for correction, will have significant

Table 4  Therapeutic options for rising PAP during anesthesia Deepening of anesthesia Hyperventilation, PaCO2 25–30 mmHg, pH > 7.45 Hyperoxia, increase FiO2 to 1.0 Reduce mean intrathoracic pressure to 6–10 mmHg by altering ventilator parameters Nebulized prostacyclin analogue (iloprost 20 mg over 15 min) Phosphodiesterase inhibitor loading dose/infusion (enoximone, milrinone) Intra-aortic counterpulsation device (intra-aortic balloon pump) Intravenous administration of sildenafil Intravenous infusion of dipyridamole (0.5 mg/kg/h) Open pericardium/sternum Nitric oxide 10–50 ppm in inspiratory limb of ventilator circuit Cardiopulmonary bypass

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reduction in pulmonary hemodynamics which may allow shunt closure, although this remains an unproven strategy. Carefully selected CHD-PAH patients with severe symptoms may benefit from combined heart and lung transplantation or lung transplantation with correction of CHD.

References 1. Perloff JK, Warnes C (2001) Congenital heart diseases in adults: a new cardiovascular specialty. Circulation 84:1881–1890 2. Galie N, Torbicki A, Barst R et al (2004) Guidelines on diagnosis and treatment of pulmonary artery hypertension. The Task Force on Diagnosis and Treatment of Pulmonary Arterial Hypertension of the European Society of Cardiology. Eur Heart J 25:2243–2278 3. Hopkins WE, Ochoa LL, Richardson FW et al (1996) Comparison of the haemodynamics and survival of adults with severe primary pulmonary hypertension or Eisenmenger syndrome. J Heart Lung Transplant 15:100–105 4. Landzberg MJ (2007) Congenital heart disease associated pulmonary arterial hypertension. Clin Chest Med 28:243–253 5. Diller GP, Gatzoulis MA (2007) Pulmonary vascular disease in adults with congenital heart disease. Circulation 115:1039–1050 6. Viswanathan S, Kumar RK (2008) Assessment of operability of congenital cardiac shunts with increased pulmonary vascular resistance. Catheter Cardiovasc Interv 71:665–670 7. Rabinovitch M, Haworth SG, Castandea AR et  al (1978) Lung biopsy in congenital heart disease: a morphometric approach to pulmonary vascular disease. Circulation 58:1107–1122 8. Frescura C, Thiene G, Giulia Gagliardi M et al (1991) Is lung biopsy useful for surgical decision making in congenital heart disease? Eur J Cardiothorac Surg 5:118–122 9. Steele PM, Fuster V, Cohen M et  al (1987) Isolated atrial septal defect with pulmonary vascular obstructive disease: long term follow-up and prediction of outcome after surgical correction. Circulation 76:1037–1042 10. Moss AJ, Adams FH, Emmanouilides GC et al (1989) Moss’ heart disease in infants, children and adolescents. Williams and Wilkins, Baltimore 11. Balzer DT, Kort HW, Day RW et al (2002) Inhaled nitric oxide as a preoperative test (INOP Test I): the INOP Test Study Group. Circulation 106:I76–I81 12. Post MC, Janssens S, Van de Werf F et al (2004) Responsiveness to inhaled nitric oxide is a predictor for mid-term survival in adult patients with congenital heart defects and pulmonary arterial hypertension. Eur Heart J 25:1651–1656 13. Yan C, Zhao S, Jiang S et al (2007) Transcatheter closure of patent ductus arteriosus with severe pulmonary arterial hypertension in adults. Heart 93:514–518 14. Khan SA, Gelb BD, Nguyen KH (2006) Evaluation of pulmonary artery banding in the setting of ventricular septal defects and severely elevated pulmonary vascular resistance. Congenit Heart Dis 5:244–250 15. Bando K, Turrentine MW, Sharp TG et  al (1996) Pulmonary hypertension after operations for congenital heart disease: analysis of risk factors and management. J Thorac Cardiovasc Surg 112:1600–1607 16. Fried R, Falkovsky G, Newburger J et al (1986) Pulmonary arterial changes in patients with ventricular septal defects and severe pulmonary hypertension. Pediatr Cardiol 7:147–154 17. Sandoval J, Rothman A, Pulido T (2001) Atrial septostomy for pulmonary hypertension. Clin Chest Med 22:547–560

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18. Budts W, Van Pelt N, Gillyns H et al (2001) Residual pulmonary vasoreactivity to inhaled nitric oxide in patients with severe obstructive pulmonary hypertension and Eisenmenger syndrome. Heart 86:553–558 19. Galiè N, Beghetti M, Gatzoulis MA et  al (2006) Bosentan Randomized Trial of Endothelin Antagonist Therapy-5 (BREATHE-5) Investigators. Bosentan therapy in patients with Eisenmenger syndrome: a multicenter, double-blind, randomized, placebo-controlled study. Circulation 114:48–54 20. Uzun O, Wong JK, Bhole V et al (2006) Resolution of protein-losing enteropathy and normalization of mesenteric Doppler flow with sildenafil after Fontan. Ann Thorac Surg 82:e39–e40 21. Takahashi K, Mori Y, Yamamura H et al (2003) Effect of beraprost sodium on pulmonary vascular resistance in candidates for a Fontan procedure: a preliminary study. Pediatr Int 45:671–675 22. Jones DK, Higenbottam TW, Wallwork J (1987) Treatment of primary pulmonary hypertension intravenous epoprostenol (prostacyclin). Br Heart J 57:270–278 23. Steiropoulos P, Trakada G, Bouros D (2008) Current pharmacological treatment of pulmonary arterial hypertension. Curr Clin Pharmacol 3:11–19 24. Fernandes SM, Newburger JW, Lang P et al (2003) Usefulness of epoprostenol therapy in the severely ill adolescent/adult with Eisenmenger physiology. Am J Cardiol 91:632–635 25. Channick RN, Simonneau G, Sitbon O et al (2001) Effects of the dual endothelin-receptor antagonist bosentan in patients with pulmonary hypertension: a randomised placebo-controlled study. Lancet 358:1119–1123 26. Galiè N, Rubin LJ, Hoeper M et  al (2008) Treatment of patients with mildly symptomatic pulmonary arterial hypertension with bosentan (EARLY study): a double-blind, randomized controlled trial. Lancet 371:2061–2062 27. Schulze-Neick I, Gilbert N, Ewert R et al (2005) Adult patients with congenital heart disease and pulmonary arterial hypertension: first open prospective multicenter study of bosentan therapy. Am Heart J 150:716 28. Galiè N, Ghofrani HA, Torbicki A et  al (2005) Sildenafil Use in Pulmonary Arterial Hypertension (SUPER) Study Group. Sildenafil citrate therapy for pulmonary arterial hypertension. N Engl J Med 353:2148–2157 29. Imanaka K, Kotsuka Y, Takamoto S et al (1998) Atrial septal defect and severe pulmonary hypertension in an adult who needed nitric oxide inhalation after repair. Kyobu Geka 51:403–405 30. Lim ZS, Salmon AP, Vettukattil JJ et al (2007) Sildenafil therapy for pulmonary arterial hypertension associated with atrial septal defects. Int J Cardiol 118:178–182 31. Kovacikova L, Zahorec M, Nosal M (2007) Sildenafil as a pulmonary vasodilator after repair of congenital heart disease. Bratisl Lek Listy 108:453–454 32. Ikonomidis JS, Hilton EJ, Payne K et al (2007) Selective endothelinA receptor inhibition after cardiac surgery: a safety and feasibility study. Ann Thorac Surg 83:2153–2160 33. Hopkins WE (2005) The remarkable right ventricle of patients with Eisenmenger syndrome. Coron Artery Dis 16:19–25 34. Thistlethwaite PA, Kaneko K, Madani MM et al (2008) Technique and outcomes of pulmonary endarterectomy surgery. Ann Thorac Cardiovasc Surg 14:274–282 35. Frigiola A, Tsang V, Nordmeyer J et al (2008) Current approaches to pulmonary regurgitation. Eur J Cardiothorac Surg 34: 576–580 36. Dimopoulos K, Peset A, Garzoulis MA (2008) Evaluating operability in adults with congenital heart disease and the role of pretreatment with targeted pulmonary arterial hypertension therapy. Int J Cardiol 129:163–171 37. Beghetti M (2006) Pulmonary arterial hypertension related to congenital heart disease. Elsevier, Munich

1168 38. Yamauchi H, Yamani S, Fujii M et  al (2004) Atrial septal defect with borderline pulmonary vascular disease: surgery and long-term oral prostacyclin therapy for recalcitrant pulmonary hypertension. Jpn J Thorac Cardiovasc Surg 52:213–216 39. Frost AE, Quiñones MA, Zoghbi WA et al (2005) Reversal of pulmonary hypertension and subsequent repair of atrial septal defect after treatment with continuous intravenous epoprostenol. J Heart Lung Transplant 24:501–503 40. Schwerzmann M, Zafar M, McLaughlin PR et al (2006) Atrial septal defect closure in a patient with “irreversible” pulmonary hypertensive arteriopathy. Int J Cardiol 110:104–107 41. Nemoto S, Yoshimura S, Matsumura M et al (2007) An initial experience of endotherine-1 dual-receptor blockade in the treatment of sustained pulmonary hypertension early after congenital cardiac surgery in infancy. Kyobu Geka 60:453–456 42. Eicken A, Balling G, Gildein HP et al (2007) Transcatheter closure of a non-restrictive patent ductus arteriosus with an Amplatzer muscular ventricular septal defect occluder. Int J Cardiol 117:e40–e42 43. Ussia GP, Mulè M, Caruso E et  al (2007) Combined endothelin receptor antagonist and transcatheter interventional therapy of patent ductus arteriosus with severe pulmonary artery hypertension. Int J Cardiol 116:427–429 44. Novick WM, Sandoval N, Lazorhysynets VV et  al (2005) Flap valve double patch closure of ventricular septal defects in children with increased pulmonary vascular resistance. Ann Thorac Surg 79:21–28

C.J. Lewis and A.A. Klein 45. Christie J, Edwards L, Aurora P, Hertz MI et al (2008) Registry of the International Society for Heart and Lung Transplantation: twentyfifth official adult lung and heart/lung transplantation report – 2008. J Heart Lung Transplant 27:957–969 46. Daliento L, Somerville J, Presbitero P et  al (1998) Eisenmenger syndrome. Factors relating to deterioration and death. Eur Heart J 19:1845–1855 47. Adriaenssens T, Delcroix M, Van Deyk K et al (2006) Advanced therapy may delay the need for transplantation in patients with the Eisenmenger syndrome. Eur Heart J 27:1472–1477 48. Lupinetti FM, Bolling SF, Bove EL et  al (1994) Selective lung or heart-lung transplantation for pulmonary hypertension asso­ciated with congenital cardiac anomalies. Ann Thorac Surg 57:1545–1548 49. Walles T (2007) Clinical experience with the iLA membrane ventilator pumpless extracorporeal lung-assist device. Expert Rev Med Devices 4:297–305 50. Chassot PG, Bettex DA (2006) Anesthesia and adult congenital heart disease. J Cardiothorac Vasc Anesth 20:414–437 51. Tempe K, Virmani S (2002) Coagulation abnormalities in patients with cyanotic congenital heart disease. J Cardiothorac Vasc Anesth 16:752–765 52. Lundy JB, Slane ML, Frizzi JD (2007) Acute adrenal insufficiency after a single dose of etomidate. J Intensive Care Med 22:111–117 53. Lovell AT (2004) Anaesthetic implications of grown-up congenital heart disease. Br J Anaesth 93:129–139 54. Ciofolo M, Reiz S (1999) Circulatory effects of volatile anesthetic agents. Minerva Anestesiol 65:232–238

Chapter 80

Pulmonary Veno-occlusive Disease Peter F. Clardy and Jess Mandel

Abstract  Pulmonary veno-occlusive disease (PVOD) is a rare and highly lethal disorder of the pulmonary vasculature. In contrast to the insights into pathophysiology and management that have characterized the diagnosis and treatment of idiopathic pulmonary arterial hypertension (IPAH; formerly known as primary pulmonary hypertension) over the past decade, the pathophysiologic mechanisms underlying PVOD are incompletely understood, the clinical diagnosis is notoriously difficult, and therapy is largely unsatisfactory. Historically, what is now called PVOD has been variably termed “isolated pulmonary venous sclerosis,” “obstructive disease of the pulmonary veins,” or “the venous form of primary pulmonary hypertension.” Over the past decade, “pulmonary obstructive venopathy” has been proposed as a more accurate alternative description, but this phrase has not been adopted into wide clinical use. It is important to note that PVOD is completely distinct from stenosis of one or more of the four main pulmonary veins. Stenosis of these large pulmonary veins is primarily a result of congenital malformation, but may also develop as a complication following cardiothoracic surgery. In addition, large-vessel pulmonary venous stenosis has been increasingly reported as a complication following radio-frequency ablation for atrial fibrillation or other dysrhythmias. Keywords  Stenosis • Pulmonary vein • Venous obstruction • Venous sclerosis

1 Introduction Pulmonary veno-occlusive disease (PVOD) is a rare and highly lethal disorder of the pulmonary vasculature [1, 2]. In contrast to the insights into pathophysiology and management that have characterized the diagnosis and treatment of

P.F. Clardy (*) Department of Medicine, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, KsB-23, Boston, MA 02215, USA e-mail: [email protected]

idiopathic pulmonary arterial hypertension (IPAH; formerly known as primary pulmonary hypertension) over the past decade, the pathophysiologic mechanisms underlying PVOD are incompletely understood, the clinical diagnosis is notoriously difficult, and therapy is largely unsatisfactory. The initial clinicopathologic description of PVOD was documented by Julius Höra at the University of Munich in 1934 [3]. The initial patient presented with progressive dyspnea, edema, and cyanosis, resulting in death over the course of approximately 1 year. The presumptive clinical diagnosis was mitral stenosis, but at autopsy, diffuse obstruction of the pulmonary venules with loose fibrous tissue was noted, in the absence of mitral valve disease or other causes of left atrial hypertension. Höra postulated that an infection, perhaps streptococcal, was responsible for the pathologic findings, but no organisms could be demonstrated on stains or cultures. Historically, what is now called PVOD has been variably termed “isolated pulmonary venous sclerosis,” “obstructive disease of the pulmonary veins,” or “the venous form of primary pulmonary hypertension.” The term “pulmonary venoocclusive disease” (PVOD) was popularized in the 1960s by Heath, Brown, and others [4–7]. Over the past decade, “pulmonary obstructive venopathy” has been proposed as a more accurate alternative description, but this phrase has not been adopted into wide clinical use [8]. It is important to note that PVOD is completely distinct from stenosis of one or more of the four main pulmonary veins. Stenosis of these large pulmonary veins is primarily a result of congenital malformation, but may also develop as a complication following cardiothoracic surgery [9, 10]. In addition, large-vessel pulmonary venous stenosis has been increasingly reported as a complication following radio-frequency ablation for atrial fibrillation or other dysrhythmias [11–13].

2 Epidemiology Although PVOD is clearly a rare disease, the true incidence and prevalence of PVOD are challenging to precisely measure for two reasons: the fact that mild cases may not come

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to medical attention, and the suspicion that many cases of PVOD are misclassified as IPAH given the difficulty in distinguishing the two syndromes without tissue biopsy [14]. Despite these challenges, an estimate of the annual incidence of PVOD can be extrapolated from analysis of several series of patients with IPAH from which the fractions of patients fulfilling criteria for PVOD were reported. Pooling of seven such series published between 1970 and 1991 (comprising a total of 465 patients) suggests that the incidence of PVOD is approximately 10% of that of true IPAH [15–22]. Using an annual incidence of IPAH of one to two cases per million persons in the general population, this predicts an annual incidence of PVOD of around 0.1–0.2 cases per million persons in the general population [23, 24]. However, this may represent an underestimate of the true burden of disease, since a number of patients with PVOD are probably misclassified as having either interstitial lung disease or heart failure because of similarities in the radiographic appearance of all of these disorders. No well-designed studies have examined the magnitude of this likely misclassification phenomenon. Unlike IPAH, which is more common in women, there does not appear to be a clear gender imbalance among patients with PVOD [2, 25, 26]. The age at diagnosis has ranged from within 9 days of birth to the seventh decade of life.

3 Pathology The pathologic characterization of pulmonary vasculopathy remains challenging, even for experienced pathologists. To minimize errors in interpretation, lung specimens should be fixed in a distended state, and at least five blocks from each lobe should be sectioned and examined. In addition to hematoxylin and eosin staining, examination using Movat, Masson, Verhoeff–van Gieson, and Perls iron stains is recommended. Immunohistochemical markers for smooth muscle and endothelial antigens (e.g., factor VIII, CD31, CD34) may also be useful [8].

Fig. 1  Pulmonary veno-occlusive disease. Elastic stain distinguishes a small artery (a) from an involved small vein (b) (×20). (Reprinted with permission from Mandel and Taichman [154]. Copyright Elsevier 2006)

P.F. Clardy and J. Mandel

The dominant pathologic abnormality observed in PVOD is the extensive and diffuse obliteration of small pulmonary veins or venules by fibrous tissue, which may be either loose and edematous, or dense, sclerotic, and acellular [2, 8, 15, 27, 28]. Of note, lesions within the venous system tend to be eccentric or trabeculated, similar to the changes that are observed in arterial structures that have undergone recanalization following thrombotic occlusion [29]. The media of the venules may undergo arterialization with an increase in the number of elastic fibers, and these fibers become calcified in some cases (Fig. 1). In addition, a foreign body giant cell response to these calcified fibers has been reported (Fig. 1) [8]. Pulmonary arteriolar changes frequently accompany venous changes, although it is not clear if they occur concomitantly or develop later as a consequence of venous obstruction. These arteriolar changes contribute to the misclassification of PVOD as IPAH. In approximately 50% of patients with PVOD, pulmonary arterioles demonstrate marked medial hypertrophy, which is easily confused with IPAH unless the venous structures are carefully surveyed. Plexiform lesions are less commonly encountered than is the case in IPAH, and arteritis or venulitis is distinctly uncommon [14, 30, 31]. Pleural and parenchymal lymphatic channels can become markedly dilated, presumably because of the increased volume of fluid moving from pulmonary capillaries to the interstitial space as a result of pulmonary venous hypertension [32]. In PVOD, capillaries become engorged and tortuous, occasionally leading to a misdiagnosis of pulmonary capillary hemangiomatosis (PCH; also called pulmonary microvasculopathy) [33]. Both of these disorders are also associated with prominent lymphadenopathy, and a study comparing lymph node disease in patients with PVOD and PCH showed similar nonspecific changes in lymph node architecture, including vascular transformation of the sinuses, intrasinusal hemorrhage with erythrophagocytosis, and lymphoid follicular hyperplasia [34]. These changes are very rare in

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patients with IPAH and suggest, along with other lines of evidence, that PVOD and PCH may exist along a continuum of disorders [35]. Areas of microscopic pulmonary hemorrhage and hemosiderosis are rare in IPAH but are frequently observed in PVOD, and presumably develop as a consequence of capillary endothelial damage and extravasation of erythrocytes due to pulmonary capillary hypertension [14]. Determining whether observed areas of pulmonary hemorrhage are due to PVOD or reflect tissue disruption by biopsy procedures can be challenging. Patients with PVOD commonly demonstrate hemosiderin within alveolar macrophages or the interstitium, and in some cases this feature is sufficiently prominent that the diagnosis of idiopathic pulmonary hemosiderosis or a vasculitis associated with alveolar hemorrhage (e.g., Wegener’s granulomatosis) is considered (Fig. 2) [2, 36]. In contrast to many other causes of pulmonary hypertension, pulmonary parenchymal abnormalities are commonly observed in PVOD [14]. Interstitial edema is commonly seen, and is particularly prominent within lobular septa. Collagen deposition in these regions, and occasionally also involving alveolar walls, may appear similar to changes observed in long-standing mitral stenosis [37]. Lymphocytes and monocytes are commonly seen within the interstitium, and may be sufficiently numerous to erroneously suggest usual interstitial pneumonitis as the primary diagnosis [28]. Because of a number of shared features, it has been proposed that IPAH, PVOD, and PCH may represent a continual spectrum of manifestations of a single disease process termed “pulmonary vascular occlusive disease” [8, 35, 38]. Advocates of this paradigm emphasize that obliterative pulmonary

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vascular lesions frequently are not limited to only the arterial, capillary, or venous structures, but can involve a number of different vascular areas within the same patient. Further evidence that these disorders may represent a single disease process includes the presence of bone morphogenic protein receptor type 2 (BMPR2) abnormalities in both IPAH and PVOD (see later). Although such a conceptualization has not yet found general acceptance, the third and fourth World Health Organization Conferences on Pulmonary Hypertension (Venice 2003, Dana Point 2008) classified PVOD and PCH together in the subcategory of “pulmonary arterial hypertension associated with significant venous or capillary involvement” [9].

4 Etiology The cause of PVOD remains unknown, and no well-designed and adequately powered case-control or cohort studies have been performed specifically to explore the potential causes of this condition. The situation is further complicated by the hypothesis that PVOD represents a final common clinicopathologic pathway triggered in a susceptible host by a number of discrete causes. Despite these uncertainties, a variety of risk factors have been proposed, based upon case reports and small case series.

5 Etiology 5.1 Genetic Susceptibility

Fig.  2  Pulmonary veno-occlusive disease. Hematoxylin and eosin staining shows obstructive intimal fibrosis of small vein and extensive hemosiderin-laden intra-alveolar macrophages (×20). (Reprinted with permission from Mandel and Taichman [154]. Copyright Elsevier 2006)

Reports of PVOD developing in siblings have led for many years to suspicions that a genetic predisposition may underlie development of the disease in some instances. In most cases involving siblings, PVOD developed before the third decade of life, and nonaffected siblings have also been detailed in a number of these families [7, 21, 28, 39, 40]. No definite genetic abnormality has been demonstrated in these sibling pairs, and the development of disease in these cases could also be the result of common environmental exposures to inciting agents. Since the identification of mutations of the BMPR2 gene as a major cause of familial IPAH in 2000, there has been speculation that abnormalities at this site may also be involved in the pathogenesis of PVOD [14, 41–43]. A total of six distinct BMPR2 mutations have been described in patients with PVOD [14]. In one compelling case report, a BMPR2 mutation resulting in a truncated and nonfunctional protein was described in a patient who developed PVOD at age 36 [44]. The patient’s mother had died of pulmonary

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hypertension several decades earlier, but lung tissue had not been examined to determine if she had suffered from PVOD or IPAH. The authors reported that at the time of the publication, the index patient had been stable for 5 years while receiving continuous intravenous administration of epoprostenol, a course that is somewhat atypical in PVOD. The description of BMPR2 mutations in patients with PVOD suggests that IPAH and PVOD may share a common genetic basis, and the pathologic phenotype that develops may be due to modifier genes or differing environmental exposures. It has been suggested that loss of a functional BMPR2 allele might contribute to the development of PVOD by diminishing the normal antiproliferative effects of bone morphogenic protein on pulmonary vascular endothelial and smooth muscle cells, resulting in unchecked proliferation in response to other ligands of the transforming growth factor b family [45]. A common BMPR2 defect might also help explain the overlap in pathologic features found in IPAH and PVOD, including the development of scattered venous lesions found in some IPAH patients with predominantly precapillary disease, and the presence of precapillary lesions and abnormal precapillary resistance in many patients with PVOD [46, 47]. Expanded genotyping of patients with PVOD should help clarify the genetic overlap of these two diseases, but at present this is informed only by occasional reports in isolated patients.

5.2 Infectious Agents Since Höra’s initial description of PVOD, infection has been postulated to play an etiologic role in the initiation or progression of disease [3, 48]. Höra believed that streptococcal infection played a role in his patient’s illness; since that time, nonbacterial pathogens have been more commonly hypothesized to play a role in the development of PVOD. In some cases, an antecedent influenza-like illness or serologic evidence of recent infection with agents such as measles and Toxoplasma gondii has been noted to coincide with the diagnosis of PVOD, and other patients have had manifestations of Epstein–Barr or cytomegalovirus infection, such as lymphadenopathy, fever, and erythrophagocytosis [4, 5, 49–51]. Several cases of PVOD have been reported among patients infected with the human immunodeficiency virus (HIV) [52–54]. However, despite these reports, it is difficult to reconcile the time courses of acute infectious illnesses and PVOD, given that it requires months to years to develop right ventricular hypertrophy and the symptom complex associated with PVOD. It is more likely that the significance of an acute illness near the time of PVOD diagnosis is that it may bring an undiagnosed patient to medical attention, and stimulate a diagnostic evaluation that ultimately demonstrates the presence of PVOD.

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In contrast to acute infection, HIV and other lentiviruses could pursue a chronologic course of infection that is more congruent with the time course required for the development of observed cardiovascular adaptations to PVOD. Despite this, viral inclusions or DNA has not been definitively documented in the pulmonary vascular lesions of affected patients.

5.3 Toxic Exposures An association between exposure to certain drugs or chemicals and the development of PVOD has been suggested by several case reports. As an example, the disease developed in a 14-yearold boy with a 2-year history of ingesting and sniffing a powdered cleaning product containing silica, soda ash, dodecyl benzyl sulfonate, and trichloro-s-triazinetriome [55]. Exposure to drugs delivered with therapeutic intent is also linked to the development of PVOD. As awareness developed that patients could manifest hepatic veno-occlusive disease following antineoplastic chemotherapy, reports began to emerge linking PVOD to these agents as well. Many of the patients with PVOD diagnosed after the treatment of malignancy received radiation and a variety of different antineoplastic drugs over several years, making identification of a specific culprit agent problematic; nonetheless, the most commonly implicated compounds are bleomycin, mitomycin, carmustine, and gemcitabine [56–62]. Anecdotal reports suggest that PVOD may be more common following either allogeneic or autologous bone marrow or stem cell transplantation than following conventional cytoreductive chemotherapy; however, there are insufficient data to definitively test this assertion [63–66]. The mechanism by which certain drugs might cause pulmonary venous remodeling is unknown. It is possible that these chemotherapeutic agents are metabolized to toxic intermediates in pulmonary capillaries and then cause damage to pulmonary venous structures. Cocaine, amphetamines, and anorectic agents are associated with pulmonary arterial hypertension, but have not been definitively linked to PVOD [23, 24]. Similarly, “bush teas” containing pyrrolizidine alkaloids have been responsible for case clusters of hepatic veno-occlusive disease but have not been associated with the development of PVOD [67, 68].

5.4 Thrombophilia It has long been hypothesized that thrombophilia may play a role in the pathogenesis of PVOD, both because venules and small veins in PVOD have an appearance similar to recanalized, thrombotically occluded arteries, and because

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lung specimens occasionally have evidence of fresh thrombi in affected vessels. Although coagulation and rheologic parameters have not been assessed in PVOD patients by state-of-the-art techniques, several reports from the 1960s described increased platelet adhesiveness in patients with the disorder, in one case to a degree four standard deviations above the mean for normals [4, 69]. In addition, several cases of PVOD have occurred in patients with a risk factor for hypercoagulability, such as pregnancy or oral contraceptive use [70, 71]. Although cases of PVOD have indeed occurred in the setting of documented thrombophilia, such comorbidities are unusual [72]. The thrombophilia hypothesis also fails to explain the fact that extrapulmonary venous or arterial thrombi, which would be expected to develop commonly in hypercoagulable states, are rare in patients with PVOD. Finally, careful examination of lung specimens fails to demonstrate acute pulmonary venous thrombi in the majority of patients with PVOD [32].

5.5 Autoimmune Disorders Venulitis, either primary or occurring secondary to an infectious vasculitis, followed by thrombosis, remodeling, or both, could presumably explain the pathologic changes seen in PVOD, but inflammation is not a typical feature of this disorder. Granulomatous venulitis rarely has been described to accompany sarcoidosis, and a similar vascular lesion has been described in a 21-year-old man with an extensive history of cannabis smoking and the clinical syndrome of PVOD [31, 73]. Although most patients with PVOD do not display manifestations of autoimmunity, a number of individuals have developed PVOD in the context of positive antinuclear antibodies, alopecia, myopathy, rheumatoid arthritis, Felty’s syndrome, systemic lupus erythematosus, mixed connective tissue disease, or the scleroderma spectrum of conditions (including features such as calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly, and telangiectasias) [74–86]. However, the fact that only a minority of cases display associated autoimmune findings argues against autoimmunity playing a fundamental role in the development of PVOD.

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many patients are initially diagnosed with an acute respiratory infection. In general, PVOD is subsequently diagnosed only after patients fail to improve significantly after treatment with antimicrobial agents [5, 51, 87]. Progressive pulmonary hypertension may result in right upper quadrant pain (secondary to hepatic congestion), pedal edema, and/or exertional syncope. Orthopnea is more common and severer in patients with PVOD than in those with IPAH or other causes of pulmonary arterial hypertension [36]. Unusual clinical presentations of PVOD include symptomatic diffuse alveolar hemorrhage and sudden cardiac death [88–90]. Chronic subacute alveolar hemorrhage appears common on the basis of lung histopathologic findings and bronchoalveolar lavage (BAL) results, but massive and life-threatening hemoptysis rarely develops [88, 91]. Physical examination tends to reveal nonspecific findings of pulmonary hypertension, with or without overt right ventricular failure. Classic findings include increased intensity of the pulmonic component of the second heart sound, a right ventricular heave or lift, a right-sided third or fourth heart sound, elevated jugular venous pressure, hepatomegaly, and/ or peripheral edema. Murmurs of tricuspid regurgitation and, less commonly, pulmonic insufficiency may be appreciated in some patients. Murmurs or gallops present in this setting generally are augmented with inspiration, as increased venous return to the thorax increases turbulent flow through right-sided valvular defects [92]. Digital clubbing is sometimes present, and basilar rales, which are unusual in IPAH, are appreciated in some patients (Fig. 3) [51]. Pleural effusions are relatively common in patients with PVOD, in contrast to their infrequent association with purely precapillary causes of pulmonary hypertension [93, 94]. The likely pathophysiologic explanation for this observation is that PVOD and other postcapillary causes of pulmonary

6 Clinical Manifestations The majority of patients with PVOD present with nonspecific symptoms of pulmonary hypertension, including dyspnea on exertion and fatigue [14, 26]. Patients may develop a chronic cough (either productive or nonproductive), and

Fig. 3  Prominent clubbing of the digits in a patient with pulmonary veno-occlusive disease. (Reprinted with permission from Mandel and Taichman [154]. Copyright Elsevier 2006)

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hypertension produce elevated pulmonary capillary and visceral pleural hydrostatic pressures, leading to transudation of fluid into the pleural space. In contrast, the predominant high-resistance areas of the pulmonary circulation are precapillary in IPAH, and thus pleural effusions do not generally accumulate until elevated hydrostatic pressure develops in the central systemic veins, leading to elevations in hydrostatic pressure within the parietal pleural capillaries.

6.1 Radiographic Features Chronic pulmonary capillary hypertension can produce transudation of fluid into the pulmonary interstitium, with consequent engorgement of pulmonary lymphatics. This results in the radiographic appearance of Kerley B lines on plain chest radiographs. Other possible findings on chest radiographs include enlargement of central pulmonary arteries, peribronchial cuffing, and scattered parenchymal opacities (Fig. 4) [4, 95]. However, these findings are not pathognomonic of PVOD, and the absence of any or all of these findings does not eliminate the possibility that PVOD is present [26, 80, 96, 97]. Computed tomography images may display smooth thickening of the septa, diffuse or mosaic ground-glass opacities, multiple well-defined or poorly defined small noncalcified nodules, pleural effusions, or areas of alveolar consolidation which may be gravitationally dependent or centrilobular in their distribution (Fig. 5) [93, 98–106]. The pathologic correlate of ground-glass attenuation seen in these patients is not

Fig. 4  A posteroanterior chest radiograph from a 28-year-old woman with pulmonary veno-occlusive disease demonstrated at autopsy shows increased interstitial markings diffusely and Kerley B lines that are most prominent in her lower lateral right chest. (Reprinted with permission from Mandel and Taichman [154]. Copyright Elsevier 2006)

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entirely clear, although alveolar hemorrhage, focal areas of hydrostatic edema corresponding to regions with markedly obstructed venous outflow, nonspecific interstitial inflammation, and alveolar septal thickening with associated epithelial hyperplasia have each been proposed to explain the finding. Importantly, the central pulmonary veins and the left atrium are not enlarged, in contrast to patients with mitral stenosis, cor triatriatum, left atrial myxoma, or large pulmonary vein stenosis secondary to electrophysiologic ablation, prior cardiothoracic surgery, or congenital malformation. Prominent mediastinal lymphadenopathy has been reported in a number of cases [1, 9, 14, 34, 101, 102, 107, 108]. Radionuclide ventilation–perfusion images display normal ventilation but commonly show multiple focal areas of hypoperfusion, sometimes approximating a segmental pattern [109]. This finding may be misinterpreted as supportive of the diagnosis of chronic thromboembolic pulmonary hypertension [26, 36, 87, 88, 97, 98, 110–113].

6.2 Cardiac Catheterization It is notoriously difficult to obtain a satisfactory pulmonary artery occlusion (wedge) pressure in patients with PVOD. Flushing of the catheter when the distal port of the catheter is in the wedged position and the balloon is inflated results in a disproportionate rise in recorded pressure, which then falls extremely slowly to the baseline. These phenomena are the result of impaired runoff of infused saline due to downstream

Fig.  5  Computed tomographic image through the lung bases shows numerous thickened septal lines (double arrow) and patchy foci of ground-glass attenuation (thin arrow). The arteries are enlarged relative to bronchi (thick arrow), whereas pulmonary veins appear of normal caliber (curved arrow). (Used with permission from Mandel et al. [2]. Copyright American Thoracic Society)

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obstruction and diminished cross-sectional area of the pulmonary venules and small veins [4, 88, 114]. To document successful catheter wedging in this situation, it may be helpful to demonstrate that blood gas analysis of a sample slowly drawn from the distal catheter port while the balloon is inflated results in values similar to those for a specimen drawn simultaneously from an arterial source. If a pulmonary artery occlusion pressure can be successfully measured, one should record values in several different locations to ensure that spurious values have not been obtained as a result of local phenomena. In patients with PVOD, the pulmonary artery occlusion pressure is generally normal or decreased, despite the fact that postcapillary obstruction does in fact produce pulmonary capillary hypertension [14, 51, 115]. The cause of this seeming paradox is that the pulmonary artery occlusion pressure is determined by left atrial pressures, not by hydrostatic pressures within the pulmonary capillaries, as the term “pulmonary capillary wedge pressure” is sometimes taken to imply. Rather, with the balloon inflated and the catheter in the wedged position, a static column of blood is created that extends from the catheter tip, through the pulmonary capillaries, venules, and veins to the left atrium, the latter of which determines the recorded pressure, and is generally normal in PVOD. Extensive stenosis of the small pulmonary veins in PVOD tends to dampen this pressure tracing to some degree, but normal left atrial pressures remain the fundamental determinant of the pulmonary artery occlusion pressure [116, 117]. Short-acting pulmonary arterial vasodilators are routinely administered to patients with pulmonary arterial hypertension at the time of cardiac catheterization to determine if pulmonary vasoreactivity is present [24]. However, in patients with PVOD, such medications may lead to the development of acute life-threatening pulmonary edema [14, 51, 108, 118]. Presumably, pulmonary edema develops in this setting because of pulmonary arterial vasodilation without concomitant pulmonary venodilation, producing a rapid increase in transcapillary hydrostatic forces and transudation of fluid into the pulmonary interstitium and alveoli.

6.3 Other Studies Patients with PVOD generally display normal spirometry. The single-breath diffusing capacity for carbon monoxide is usually reduced, and a mild to moderate restrictive ventilatory defect is observed in some cases [51, 97, 98]. Laboratory parameters are usually unremarkable, although isolated cases have displayed otherwise unexplained features such as microangiopathic hemolytic anemia [60], heavy proteinuria [5], or elevated serum IgG or IgM concentrations [76]. Bronchoscopy may reveal intense hyperemia and longitudinal vascular engorgement of the lobar and segmental

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bronchi in a series of patients with PVOD [111]. This finding is thought to result from engorgement of the bronchial arterial system, which in segmental and more distal bronchi normally drains into the pulmonary circulation, but which has poor distal runoff in PVOD. In this series, hyperemia was not seen in the trachea or main bronchi, where systemic bronchial veins are well developed.

7 Diagnosis The classic triad of severe pulmonary hypertension, radiographic evidence of pulmonary edema, and a normal pulmonary artery occlusion pressure is frequently sufficient to warrant a clinical diagnosis of PVOD [117]. However, many patients with PVOD do not have all three components of this triad, and thus its absence cannot reliably exclude the diagnosis. Delays in diagnosis are almost universally encountered by patients with PVOD, with many patients believed to suffer from heart failure because of radiographic evidence of pulmonary edema and pleural effusions, or chronic thromboembolic pulmonary hypertension because of nonresolving radionuclide perfusion defects. In cases of PVOD where interstitial changes are radiographically or histologically prominent, alternative diagnoses of diffuse parenchymal lung diseases such as sarcoidosis, pneumoconioses, cystic fibrosis, and idiopathic pulmonary fibrosis may be considered [119]. Evidence of pulmonary hemorrhage may be noted in BAL fluid in patients with PVOD, but this finding is not specific and may also be noted in patients with IPAH or other forms of pulmonary vascular disease. However, the demonstration of occult pulmonary hemorrhage by BAL in the proper clinical setting may support the diagnosis if lung tissue has not been obtained [91]. Transbronchial biopsy almost never yields enough tissue for a firm pathologic diagnosis, and carries significant bleeding risk when performed in the setting of pulmonary hypertension. Definitive diagnosis requires surgical lung biopsy, and this procedure should be contemplated to confirm the clinical suspicion of PVOD. Although the need for lung biopsy has been questioned because therapy for PVOD is so unsatisfactory, establishing the diagnosis has significant implications for prognosis, medical therapy, and timing of lung transplantation, and therefore biopsy should be considered in patients in whom the diagnosis is suspected and surgical risk is not prohibitive.

8 Prognosis and Treatment The prognosis of PVOD is poor, with most patients dying within 2 years of diagnosis. However, cases have been reported in which individuals have survived for 5 years or

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more, generally when therapy with orally administered calcium channel antagonists or intravenous epoprostenol therapy has been well tolerated [44, 120, 121]. Because PVOD is a rare disease, large treatment trials cannot be easily performed, and the degree to which any of the current therapies influence survival and quality of life is speculative [122]. With the possible exceptions of lung transplantation and intravenous administration of epoprostenol (if tolerated), the positive impact of current therapies does not seem profound.

8.1 Conventional Supportive Therapy Patients with PVOD should receive conventional supportive therapy similar to that prescribed for patients with IPAH, unless contraindications are present [123]. Of note, none of these therapies have been documented in highquality randomized trials to be efficacious in patients with pulmonary hypertension of any cause; they are employed because of theoretical consideration and/or case reports, case series, or retrospective analyses that have suggested possible benefit. Warfarin generally is titrated to an International Normalized Ratio of approximately 2.0, based upon the suggestion of several nonrandomized studies in IPAH that survival may be modestly improved [124, 125]. Episodic small-volume hemoptysis generally does not require discontinuation of anticoagulation, but the medication is usually stopped if more than 50  mL of blood is expectorated over a 24-h period, if significant extrapulmonary hemorrhage occurs, or if syncope or other risk factors for head trauma develop. Long-term oxygen therapy should be initiated if an oxygen saturation of 89% or less, or an arterial oxygen partial pressure of 59 mmHg or less, is documented. These recommendations are based upon clinical trials of oxygen therapy in patients with chronic obstructive pulmonary disease rather than primary pulmonary vascular disease, and not all patients with pulmonary hypertension show improvement in pulmonary hemodynamics after oxygen therapy is begun [126–128]. Transtracheal oxygen therapy permits delivery of oxygen at higher flow rates than via nasal cannulae and may be considered when epistaxis is problematic, although local bleeding complications can also occur [129]. Diuretics should be used as necessary to maintain euvolemia. Both dehydration and hypervolemia should be avoided, and patients should monitor their weight daily. On the basis of the improvement in cardiac output described in patients with IPAH treated with digoxin, the drug is routinely prescribed to patients with PVOD unless contraindications are present [130].

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8.2 Calcium Channel Antagonists and Epoprostenol A minority of patients with IPAH will display acute pulmonary vasoreactivity in response to vasodilator medications such as adenosine, epoprostenol, inhaled nitric oxide, and calcium channel antagonists [123, 124]. Pulmonary vasoreactivity was defined in older literature as a 20–25% decrease in mean pulmonary artery pressure and pulmonary vascular resistance, or more recently as a reduction in mean pulmonary artery pressure of more than 10  mmHg to a value of 40  mmHg or below, with a normal or high cardiac output [131]. Vasoreactive patients tend to have an excellent prognosis when treated with calcium channel antagonists. In IPAH patients without an acute hemodynamic response and who thus cannot be treated with calcium channel antagonists, therapy with continuous intravenous administration of epoprostenol has been associated with improved survival [132]. Therapy with other prostanoids (e.g., treprostinil, iloprost), endothelin-1 receptor antagonists (e.g., bosentan, ambrisentan), or phosphodiesterase-5 inhibitors (e.g., sildenafil) likewise is associated with improved outcomes [133, 134]. The generalizability of such data to PVOD is unclear. Theoretically, a reduction in pulmonary arterial resistance without a concomitant reduction in pulmonary venous resistance could cause pulmonary edema, whereas a parallel decrease in both arterial and venous resistance should be well tolerated and advantageous. The clinical experience with vasodilator/remodeling medications in patients with PVOD has been mixed. Modest improvements in hemodynamics and exercise tolerance have been reported in a small number of patients with nifedipine, hydralazine, and prazosin, but in general these benefits have not been well maintained over time [121, 135]. Epoprostenol has been reported to have salutary effects on pulmonary hemodynamics and to decrease vasomotor tone in pulmonary venules in some patients, but has produced fulminant pulmonary edema and death in others [44, 51, 108, 120, 136, 137]. Unfortunately, pulmonary edema may result from chronic vasodilator therapy, even if the initial diagnostic challenge was associated with beneficial changes in the patient’s hemodynamic profile [14]. There is limited experience with inhaled nitric oxide or iloprost in this condition, although initial descriptions of the use of these agents in this context suggested that improvement in cardiac output resulted without the development of pulmonary edema [138]. Because the therapeutic options in PVOD are so limited, a cautious trial of epoprostenol is generally indicated. If the use of epoprostenol is tolerated, epoprostenol is generally initially given to patients at a dosage of 2–4  ng/kg/min, then this is uptitrated by 1–2  ng/kg/min every 4 weeks as permitted by side effects. Whether endothelin-1 antagonists exert a beneficial or detrimental effect has not been firmly established.

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8.3 Lung Transplantation Either single-lung and double-lung transplantation can be performed to treat PVOD [139]. Heart–lung transplantation is rarely required, as impaired right ventricular function usually improves following the transplantation of normal lungs [139, 140]. Rare recurrence of PVOD after transplantation has been reported, although the risk of recurrence is difficult to estimate because the number of transplants performed for this diagnosis has been relatively small [99, 141, 142].

8.4 Immunosuppressive Agents Immunosuppressive medications such as glucocorticoids and azathioprine have occasionally have been employed in the treatment of PVOD, although protocols have been neither standardized nor randomized, making it difficult to form unequivocal conclusions about their effectiveness. Nonetheless, only rare responses to therapy have been reported, and only a minority of these cases have shown sustained improvements [76, 84, 143, 144]. Most patients do not have prominent autoimmune features or biochemical indices suggestive of an inflammatory process, and thus would not be expected to respond to such interventions [2, 119]. In general, immunosuppressive agents do not have a role in the treatment of PVOD, although a 4-week trial of 0.75– 1.0 mg/kg of prednisone may be considered in patients with associated nonspecific interstitial pulmonary inflammation, or an autoimmune feature such as arthritis, alopecia, or an elevated erythrocyte sedimentation rate. If improvement is seen in symptoms, radiographs, diffusing capacity, and/or alveolar–arterial oxygen gradient, the dosage is then slowly tapered to 20–40 mg/day.

8.5 Experimental Therapies Although endothelin-1 receptor antagonists such as bosentan and phosphodiesterase-5 inhibitors such as sildenafil are finding roles in the treatment of pulmonary arterial hypertension, there is little published experience with these medications in the treatment of PVOD [134, 145–147]. Both classes of medications could prove either beneficial or deleterious in PVOD for similar reasons as vasodilators, and at present no recommendation regarding their use can be made. A number of experimental therapies have been investigated for hepatic veno-occlusive disease, such as defibrotide, recombinant tissue plasminogen activator, and antithrombin-III concentrate [148, 149]. No data are available regarding the potential utility of these treatments in

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PVOD, and given the likely differences in pathogenesis of the two conditions, none of these agents are recommended to treat patients with PVOD pending specific data supporting their efficacy. The beneficial therapeutic effect of the platelet-derived growth factor receptor inhibitor, imatinib (Gleevec), in patients with IPAH has been described in several case studies [150–152]. Use of this agent was reported in a single patient with presumed PVOD, with rapid improvement in clinical status and radiographic abnormalities [153]. The value of this approach in other patients remains speculative, and the absence of both tissue biopsy prior to therapy and hemodynamic evaluation following the initiation of therapy are limitations of this report. However, given the otherwise poor prognosis and lack of response to treatment seen in most patients with PVOD, these findings are suggestive that platelet derived growth factor receptor inhibition may play a role in the future management of PVOD.

9 Conclusion Despite more than seven decades since its initial description, PVOD remains a highly lethal and poorly understood syndrome, and fundamental questions regarding the cause and optimal treatment remain unanswered. The diagnosis of PVOD requires a high degree of clinical suspicion and in most cases necessitates surgical lung biopsy to definitively establish the presence of this disorder. Because the prognosis and management of PVOD are sufficiently different from those of precapillary causes of pulmonary hypertension, the risk associated with surgical biopsy is generally justified. In particular, the benefits and risks associated with medical therapy using prostanoids or endothelin-1 receptor antagonists are different for patients with PVOD than for those with IPAH. Although these treatments may be of benefit to selected patients with PVOD, significant harm or death may occur if PVOD goes unrecognized or is misdiagnosed as IPAH. Additional research is required to more precisely delineate the epidemiologic factors, cause, risk factors and optimal therapy for this syndrome.

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Chapter 81

Left Ventricular Diastolic Heart Function and Pulmonary Hypertension Stuart Rich and Mardi Gomberg-Maitland

Abstract  Pulmonary venous hypertension is now one of the most common causes of pulmonary hypertension (PH) diagnosed by both pulmonologists and cardiologists. Patients with pulmonary venous hypertension will typically have elevated pulmonary venous pressure (as reflected in the pulmonary capillary wedge pressure), most frequently as a reflection of increased left ventricular end-diastolic pressure. Although mitral stenosis was the most common cause of this entity decades ago, left ventricular diastolic dysfunction is the most common cause of pulmonary venous hypertension seen in the Western world today. The mechanisms for the hypertension in mitral stenosis and in left ventricular diastolic abnormalities are thought to be similar. A chronic elevation in the diastolic filling pressure of the left side of the heart causes a backward transmission of the pressure to the pulmonary venous system which appears to trigger vasoconstriction in the pulmonary arterial bed. The current accepted designation is PH secondary to heart failure with a preserved ejection fraction. Keywords  Pulmonary venous hypertension • Pulmonary venous pressure • Mitral stenosis • Diastolic function

1 Introduction Pulmonary venous hypertension is now one of the most common causes of pulmonary hypertension (PH) diagnosed by both pulmonologists and cardiologists. Patients with pulmonary venous hypertension will typically have elevated pulmonary venous pressure (as reflected in the pulmonary capillary wedge pressure), most frequently as a reflection of increased left ventricular end-diastolic pressure [1]. Although mitral stenosis was the most common cause of this entity

S. Rich (*) Section of Cardiology, University of Chicago Medical Center, 5841 South Maryland Avenue, Chicago, IL 60637, USA e-mail: [email protected]

decades ago, left ventricular diastolic dysfunction is the most common cause of pulmonary venous hypertension seen in the Western world today. The mechanisms for the hypertension in mitral stenosis and in left ventricular diastolic abnormalities are thought to be similar. A chronic elevation in the diastolic filling pressure of the left side of the heart causes a backward transmission of the pressure to the pulmonary venous system which appears to trigger vasoconstriction in the pulmonary arterial bed [2]. The current accepted designation is PH secondary to heart failure with a preserved ejection fraction (HFpEF) [3].

2 Pathology Histologically, abnormal thickening of the veins and formation of a neointima is seen [4]. The latter can be quite extensive. As a secondary feature there is medial hypertrophy and with time thickening of the neointima on the arterial side of the pulmonary circulation [5]. Reversibility of these changes with treatment of the underlying cause of the venous hypertension may be possible. The variability in the response of the pulmonary arterial circulation to the elevated venous pressure indicates that there may be genetic factors that control individual response and potential reversibility of the disease. For example, insulin resistance, frequently observed in association with impaired left ventricular diastolic function, is independently linked to the development of pulmonary arterial hypertension (PAH) [6].

3 Mechanism of PH in HFpEF The development of PH in HFpEF has largely focused on the role of left ventricular diastolic dysfunction with a passive effect leading to pulmonary venous hypertension [7]. As with mitral stenosis, in many patients the passive contribution of pulmonary venous hypertension may not by itself account for the increased pulmonary artery systolic pressure.

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The greater severity of PH in HFpEF may be caused by an additional precapillary component of PAH [8]. In patients with long-standing pulmonary congestion, precapillary PH may be mediated by a reactive increase in pulmonary arterial tone or development of a congestive arteriopathy characterized by pulmonary arteriolar remodeling, medial hyperplasia, and intimal fibrosis [9]. The same abnormality is seen in patients with mitral stenosis and systolic heart failure.

4 Left Ventricular Compression Versus Left Ventricular Underfilling The classic echocardiographic appearance of PH with HFpEF is one of a markedly dilated right ventricle and a small left ventricle. It has been debated whether the mechanism of the small left ventricle is compression which is contributing to the increased left ventricular end-diastolic pressure, or underfilling as a consequence of the failure of the right ventricle. Bernheim described this interdependence between the right and left ventricles, hypothesizing that the geometric changes from progressive left ventricular enlargement and hypertrophy could directly compress the right ventricle and thereby reduce right ventricular filling. This “Bernheim effect” could lead to elevated central venous pressure and peripheral edema in cases of right ventricular failure [10]. The “reverse” Bernheim phenomenon (i.e., enlargement of the right ventricle leading to compression and underfilling of the left ventricle) has also been described in both animals and humans with PH and right ventricular failure [11]. The mechanisms of interdependence are better understood by use of new imaging techniques. On the one hand, direct ventricular interaction could limit left ventricular diastolic filling. Several echocardiographic studies have shown a high incidence of decreased early left ventricular filling in PH and right ventricular enlargement [12, 13]. The left ventricle appears distorted and compressed in the setting of severe right ventricular pressure overload, and the interventricular septum functions more as part of the right ventricle than as part of the left ventricle. Typically there is reversal of the E/A transmitral pattern, a lower stroke volume index, and a lower cardiac index in PAH versus control patients. Using magnetic resonance imaging, patients with idiopathic PAH had a reduction in left ventricular peak filling rate and left ventricular stroke volume as compared with control patients [14]. Investigators also found that leftward curvature of the interventricular septum correlated with left ventricular filling rate and concluded that direct ventricular interaction impairs ventricular filling in PH. On the other hand, underfilling of the left side of the heart is also a rational explanation for the observed physiologic characteristics [15]. The high pulmonary vascular resistance and low cardiac output observed in severe PH may lead to

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underfilling of the left side of the heart. A study in patients with chronic thromboembolic PH (CTEPH) pre- and postoperatively observed this phenomenon [15]. Preoperatively, the interventricular septum was not particularly hypertrophied, and its thickness matched that of the posterior wall of the left ventricle. Also, although the left ventricle in CTEPH appeared compressed within the pericardium by the right ventricle, pericardiectomy did not lead to changes in left ventricular shape or diastolic pressure/volume relationships. After successful thromboendarterectomy, transmitral Doppler E velocity increased significantly and the E/A ratio normalized. The normal preoperative thickness of the interventricular septum and posterior wall of the left ventricle also did not change after surgery. The finding of an abnormal transmitral filling pattern (E/A) together with a normal mitral Em velocity before thromboendarterectomy supports the concept that the abnormal relaxation pattern of transmitral left ventricular filling in CTEPH appears to be due to a low left ventricular preload and underfilling rather than right ventricular hypertrophy, enlargement, and left ventricular compression. Postoperatively, the dramatic increases in transmitral E and pulmonary venous flow velocities after thromboendarterectomy are consistent with a marked improvement in left ventricular preload. The rapid normalization of the E/A ratio after thromboendarterectomy and the presence of normal preoperative interventricular septal thickness by echocardiography would also argue against any significant lingering effects from septal hypertrophy. Finally, the coexistence of an E/A transmitral filling pattern and an essentially normal mitral annular Em velocity before thromboendarterectomy is perhaps the most persuasive evidence of left ventricular underfilling with relatively preserved diastolic function.

5 Prevalence of PH in HFpEF The prevalence of PH in HFpEF has not been evaluated in prospective studies and is unknown. Severe PH from HFpEF was described in early isolated case reports of elderly hypertensive patients with HFpEF [16]. In two recent series of patients with HFpEF, PH was diagnosed by echocardiography in 44% in the clinic [17] and in 25% of patients hospitalized for symptomatic heart failure [18]. Of note, the latter study also identified elevated pulmonary artery systolic pressure as an independent predictor of mortality in HFpEF (Fig. 1).

6 Evaluation The clinical profile of left ventricular diastolic dysfunction is characteristically observed in an older patient with hypertension, diabetes, coronary artery disease, and/or obesity (Fig.  2).

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Table 1  The diagnosis of pulmonary hypertension from heart failure with preserved ejection fraction Clinical assessment Features Symptoms

Fig. 1  Kaplan–Meier survival curves in patients with heart failure with a preserved ejection fraction with pulmonary artery systolic pressure above and below the median. Patients were stratified on the basis of the pulmonary artery systolic pressure determined by Doppler echocardiography and their being followed for 3 years. Those with severer pulmonary hypertension had worse survival. (Reprinted from Lam et al. [3], with permission from Elsevier)

Fig. 2  The differential diagnoses to the syndrome of heart failure with normal ejection fraction. Conditions that can cause or contribute to dyspnea in patients with normal left ventricular systolic function are illustrated. They can be characterized as cardiac and noncardiac in origin. Many patients will have more than one cause. (Reprinted from Maeder and Kaye [38], with permission from Elsevier)

The clinical signs and symptoms that are characteristic of pulmonary venous hypertension include dyspnea with effort, and eventually right ventricular failure with edema, similar to category 1 PAH [19]. However, important and distinctive symptoms are orthopnea and paroxysmal nocturnal dyspnea, which is not a feature of PAH. Atrial arrhythmias also seem to be more common in patients with pulmonary venous hypertension than has been noted in PAH.

Orthopnea Paroxysmal nocturnal dyspnea Signs Elevated jugular venous pressure Edema Electrocardiogram RVH may be absent LVH may be present Chest X-ray Pulmonary vascular congestion Pulmonary edema Pleural effusions Chest CT scan Mosaic perfusion pattern Ground-glass opacities Echocardiogram RV enlargement Elevated PA pressure by Doppler echocardiography Normal LV ejection fraction Cardiac Normal LV systolic function catheterization Elevated PCWP Normal PCWP and normal cardiac output (consider challenge with exercise or short acting pulmonary vasodilator) Normal PCWP and reduced cardiac output (consider challenge with exercise or inotropic agents) RVH right ventricular hypertrophy, LVH right ventricular hypertrophy, RV right ventricular, LV left ventricular, PA pulmonary arterial, PCWP pulmonary capillary wedge pressure

The clinical evaluation of these patients will often reveal findings that support pulmonary venous hypertension [20] (Table  1). The electrocardiogram may show left ventricular hypertrophy rather than right ventricular hypertrophy. The chest X-ray will often show pulmonary vascular congestion, pleural effusions, and on occasion pulmonary edema. A highresolution chest CT scan can be particularly helpful because it will often reveal ground-glass opacities consistent with chronic pulmonary edema, and a mosaic perfusion pattern. These constellations of findings should alert the clinician that the patient likely has pulmonary venous hypertension and not PAH. The use of Doppler echocardiography has become a popular screening tool for the diagnosis of PH and in some clinical trials has been the only measure of the severity of the PH in response to treatment. However, in spite of the common perception that Doppler echocardiography can accurately measure pulmonary pressures, the data suggest that it is very imprecise [21]. In addition, because Doppler echocardiography cannot determine pulmonary capillary wedge pressure and cardiac output, two critical measurements in making an accurate diagnosis, the use of Doppler echocardiography alone to diagnose and initiate treatment of patients should be discouraged. Cardiac catheterization is essential as demonstration of an elevated pulmonary capillary wedge pressure secures the diagnosis. Clinicians need to be reminded that an accurate assessment of left ventricular end-diastolic pressure or

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pulmonary capillary wedge pressure can only be made at end expiration; using a digitally derived mean pulmonary capillary wedge pressure will generally yield erroneous information and usually a misclassification of the patient as having normal filling pressures [22]. Establishing the pulmonary capillary wedge pressure in a patient with PH is paramount, as it has critical implications. As most of the treatments for PAH increase cardiac output, when given to patients with left ventricular diastolic dysfunction they run the risk of causing acute or chronic pulmonary edema and worsening hypoxemia. The potential for clinical worsening and pulmonary edema from the PAH therapies is so great that, in our opinion, a direct measurement of the left ventricular end-diastolic pressure should be performed in any patient in whom an adequate pulmonary capillary wedge pressure cannot be obtained, or is obtained but its validity is in doubt. Two hemodynamic profiles have been described that are common in these patients [19]. Some patients will have an elevation in pulmonary arterial pressure with only a minimal increase in the transpulmonary gradient (mean PA pressure minus pulmonary capillary wedge pressure), as a reflection of the passive increase in pulmonary arterial pressure necessary to overcome the increase downstream resistance. Indeed, a preserved right ventricle must generate high systolic pressures to ensure adequate forward blood flow in these patients, and thus moderate degrees of PH are not only characteristic, but also favorable. However, a subset of these patients will have reactive pulmonary vasoconstriction resulting in marked elevations in pulmonary arterial pressure beyond that which is necessary to maintain cardiac output. These patients are frequently distinguished by a marked elevation in pulmonary arterial diastolic pressure. It is believed that these patients have a permissive genotype such that, when exposed to high pulmonary venous resistance, they develop reactive PH with severer arterial changes, including neointimal formation, than the other subgroup [23]. There are also patients with left ventricular diastolic dysfunction who have normal left ventricular end-diastolic pressure at rest [24] (Fig. 3). When this occurs in the setting of reactive PH, it is very difficult to know if these patients actually have PAH or pulmonary venous hypertension. Using an inotropic challenge at the time of diagnostic cardiac catheterization may be helpful. If the response shows a significant increase in cardiac output without an accompanying increase in pulmonary capillary wedge pressure, the patient likely has PAH. On the other hand, if the increase in cardiac output is accompanied by an increase in pulmonary capillary wedge pressure, the patient likely has pulmonary venous hypertension. Many physicians will use the information from acute vasodilator testing to provide insight into the effects of therapies other than calcium channel blockers. Because of the many different effects these medications can have, it is important to note their impact on the interaction of pre- and

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Fig. 3  End-diastolic pressure–volume relationships from patients with left ventricular diastolic dysfunction and from normal patients. The steep curve in those with diastolic dysfunction illustrates how it is possible to have normal left ventricular end-diastolic pressure at rest. It also shows how increasing left ventricular filling, either by exercise (as done here) or with inotropic drugs, raises the left ventricular end-diastolic pressure to abnormal levels. (Adapted with permission [24])

postcapillary pressures, transpulmonary blood flow, and gas exchange in every patient who undergoes vasoreactivity testing. Determining the effects of vasodilators on pulmonary capillary wedge pressure is essential. Since it can be difficult to distinguish patients with idiopathic PAH from those with left ventricular diastolic dysfunction, one needs to be particularly careful when evaluating pulmonary venous and/or left ventricular end-diastolic pressures during drug testing.

7 Treatment The current dilemma relates to how best to treat these patients. Again, lessons learned from treating patients with mitral stenosis can be instructive [25]. Patients who present with pulmonary edema should satisfactorily respond to the use of diuretics as a temporizing measure. However, the only definitive treatment of patients with mitral stenosis and PH is mitral valve repair or replacement [26, 27]. Several clinical studies have shown that removing the mitral valve gradient, either surgically or percutaneously, will result in an immediate fall in the pulmonary arterial pressure. The magnitude of the fall, however, can be quite variable, with some patients achieving normal hemodynamics within 24 h, and others taking many months to improve [28]. The magnitude and rate of improvement may be related to the severity of the vascular disease dictated both by the duration of the PH and genetic factors that either induce severer vascular disease or that impede regeneration and remodeling following removal of the mitral valve obstruction [29]. It is likely that these same issues are relevant to PH in patients with left ventricular diastolic dysfunction.

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To date, there are no proven therapies in HFpEF [30]. Treatment recommendations as outlined in heart failure guidelines are empiric and have not changed over time. Vasodilator therapy aimed at PH in HFpEF is therefore an appealing consideration but has been tempered by concern that increases in right-sided heart output with pulmonary vasodilators may result in further increases in left atrial pressure in patients with left-sided heart disease and hear failure. Since the major hemodynamic effect of these therapies is to raise cardiac output, it will predictably cause a worsening of pulmonary edema if the pulmonary venous obstruction is not being relieved [31, 32]. In fact these therapies are contraindicated in patients with pulmonary venous hypertension, and there are multiple reports of rapid deterioration and death when pulmonary vasodilators were used in the presence of pulmonary venous hypertension [33]. Indeed, the use of epoprostenol was associated with increased mortality in systolic heart failure, although the mechanism for increased mortality was unclear [34]. Similarly, trials of endothelin receptor antagonists in systolic heart failure failed to show clinical benefit [35]. Recent small trials using phosphodiesterase-5 inhibitors in systolic heart failure have shown beneficial effects, including improvement in exercise capacity and quality of life [36]. In fact, evidence exists that phosphodiesterase-5 inhibition may not only improve pulmonary tone and right-sided heart function but that it also exerts pleiotropic effects on left ventricular structure, and ventricular function [37]. Our approach to treating patients with PH in HFpEF has been to use medical measures to lower left ventricular filling pressures (such as nitrates, diuretics, and aggressive treatment of systemic hypertension). When successful, we have found that the pulmonary arterial pressure will also fall, and the cardiac output will increase.

8 Outcome A recent series on PH from HFpEF listed independent predictors of death, including age, stroke, chronic obstructive pulmonary disease, cancer, diabetes, low glomerular filtration rate, and hyponatremia, findings that mirror those of other large epidemiology studies of patients with HFpEF [3]. Taken together, these studies emphasize that cardiac and noncardiac comorbidities, along with hear failure, appear to play an important role in the increased morbidity and mortality in patients with HFpEF. Because patients with HFpEF often have important comorbid conditions, and because these comorbidities strongly influence outcomes, clinicians should aggressively identify and treat conditions such as hypertension, coronary artery disease, atrial fibrillation, diabetes, chronic kidney disease, and cerebrovascular disease in these patients rather than waiting for new PH- and HFpEF-specific treatments to emerge.

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As the frequency of diagnosis of PH secondary to HFpEF is increasing, we propose the following guidelines: • One should always attempt to treat the underlying disease first. • The use of conventional therapies (diuretics, oxygen) should be tried initially to correct related clinical problems. Exercise testing may be helpful to uncover exerciseinduced hypoxemia which may benefit from treatment. • The acute testing of vasodilators with hemodynamic guidance is recommended prior to initiating chronic use to evaluate the potential for beneficial or adverse effects. • Patients who respond favorably to pulmonary vasodilators should manifest clear improvement in their symptoms related to the PH and this should be corroborated with clinical testing (echocardiography, exercise testing, and catheterization). • Patients who fail to improve, or who demonstrate worsening clinical findings (tachycardia, hypoxemia, hypotension, or worsening edema), should have the vasodilator therapy promptly discontinued. Beneficial effects should be objectively apparent within 4–6 weeks.

References 1. Redfield MM (2004) Understanding "diastolic" heart failure. N Engl J Med 350:1930–1931 2. Wood P (1958) Pulmonary hypertension with special reference to the vasoconstrictive factor. Br Heart J 20:557 3. Lam CSP, Roger VL, Rodeheffer RJ, Borlaug BA, Enders FT, Redfield MR (2009) Pulmonary hypertension in heart failure with preserved ejection fraction. J Am Coll Cardiol 53:1119–1126 4. Wagenvoort C, Wagenvoort N (1977) Pathology of pulmonary hypertension, 2nd edn. Wiley, New York 5. Pietra GG Capron F, Stewart S, Leone O et  al (2004) Pathologic assessment of vasculopathies in pulmonary hypertension. J Am Coll Cardiol 43:25S–32S 6. Hansmann G, Wagner RA, Schellong S et  al (2007) Pulmonary arterial hypertension is linked to insulin resistance and reversed by peroxisome proliferator-activated receptor-g activation. Circulation 115:1275–1284 7. Kessler KM, Willens HJ, Mallon SM (1993) Diastolic left ventricular dysfunction leading to severe reversible pulmonary hypertension. Am Heart J 126:234–235 8. Willens HJ, Kessler KM (1993) Severe pulmonary hypertension associated with diastolic left ventricular dysfunction. Chest 103:1877–1883 9. LaBourene JI CJ, Johnson DJ, Mehra A, Keeley FW, Rabinovitch M (1990) Alterations in elastin and collagen related to the mechanism of progressive pulmonary venous obstruction in a piglet model. A hemodynamic, ultrastructural, and biochemical study. Circ Res 66:438–456 10. Santamore WP, Dell’Italia LJ (1998) Ventricular interdependence: significant left ventricular contributions to right ventricular systolic function. Prog Cardiovasc Dis 40:289–308 11. Little WC, Badke FR, O’Rourke RA (1984) Effect of right ventricular pressure on the end-diastolic left ventricular pressure volume relationship before and after chronic right ventricular pressure overload in dogs without pericardia. Circ Res 54:719–730

1188 12. Boussuges B, Pinet C, Molenat F et  al (2000) Left atrial and ventricular filling in chronic obstructive pulmonary disease. An echocardiographic and Doppler study. Am J Respir Crit Care Med 162:670–675 13. Schena M, Clini E, Errera D, Quadri A (1996) Echo-Doppler evaluation of left ventricular impairment in chronic cor pulmonale. Chest 109:1446–1451 14. Benza R, Biederman R, Murali S, Gupta H (2008) Role of cardiac magnetic resonance imaging in the management of patients with pulmonary arterial hypertension. J Am Coll Cardiol 52:1683–1692 15. Gurudevan SV, Malouf PJ, Auger WR, Waltman TJ, Madani M, Raisinghani AB, DeMaria AN, Blanchard DG (2007) Abnormal left ventricular diastolic filling in chronic thromboembolic pulmonary hypertension: true diastolic dysfunction or left ventricular underfilling? J Am Coll Cardiol 49:1334–1339 16. Shapiro BP, McGoon MD, Redfield MM (2007) Unexplained pulmonary hypertension in elderly patients. Chest 131:94–100 17. Klapholz M, Maurer M, Lowe AM et al (2004) Hospitalization for heart failure in the presence of a normal left ventricular ejection fraction: Results of the New York Heart Failure Registry. J Am Coll Cardiol 43:1432–1438 18. Kjaergaard J, Akkan D, Iversen KK et al (2007) Prognostic importance of pulmonary hypertension in patients with heart failure. Am J Cardiol 99:1146–1150 19. Rich S, Rabinovitch M (2008) The diagnosis and treatment of secondary (non-category 1) pulmonary hypertension. Circulation 118:2190–2199 20. Angeja BG, Grossman W (2003) Evaluation and management of diastolic heart failure. Circulation 107:659–663 21. Fisher MR, Forfia PR, Chamera E et al (2009) Accuracy of Doppler echocardiography in the hemodynamic assessment of pulmonary hypertension. Am J Respir Crit Care Med 179:615–621 22. Halpern SD, Taichman DB (2009) Hemodynamic assessment of pulmonary hypertension. Am J Respir Crit Care Med 179:615–621 23. Du L, Sullivan C, Chu D et al (2003) Signaling molecules in nonfamilial pulmonary hypertension. New Engl J Med 348:500–509 24. Westermann D, Kasner M, Steendijk P et  al (2008) Role of left ventricular stiffness in heart failure with normal ejection fraction. Circulation 117:2051–2060 25. Mahoney P, Loh E, Blitz L, Herrmann H (2001) Hemodynamic effects of inhaled nitric oxide in women with mitral stenosis and pulmonary hypertension. Am J Cardiol 87:188–192 26. Braunwald E, Braunwald N, Ross J et al (1965) Effects of mitral valve replacement on pulmonary vascular dynamics of patients with pulmonary hypertension. N Engl J Med 273:509–514

S. Rich and M. Gomberg-Maitland 27. Zener JC, Hancock EW, Shumway NE, Harrison DC (1972) Regression of extreme pulmonary hypertension after mitral valve surgery. Am J Cardiol 30:820–826 28. Levine M, Weinstein J, Diver D, Berman AD, Wyman RM (1989) Progressive improvement in pulmonary vascular resistance following percutaneous mitral valvuloplasty. Circulation 79:1061–1067 29. Fawzy ME, Hassan W, Stefadouros M, Moursi M (2004) El Shaer F, Chaudhary MA. Prevalence and fate of severe pulmonary hypertension in 559 consecutive patients with severe rheumatic mitral stenosis undergoing mitral balloon valvotomy. J Heart Valve Dis 13:942–947 30. Senni M, Redfield MM (2001) Heart failure with preserved systolic function. A different natural history? J Am Coll Cardiol 38: 1277–1282 31. Palmer S, Robinson L, Wang A, Gossage J, Bashore T, Tapson V (1998) Massive pulmonary edema and death after prostacyclin infusion in a patient with pulmonary veno-occlusive disease. Chest 113:237–240 32. Humbert M, Maître S, Capron F, Rain B, Musset D, Simonneau G (1998) Pulmonary edema complicating continuous intravenous prostacyclin in pulmonary capillary hemangiomatosis. Am J Respir Crit Care Med 157:1681–1685 33. Preston I, Klinger J, Houtchen J, Nelson D, Mehta S, Hill N (2002) Pulmonary edema caused by inhaled nitric oxide therapy in two patients with pulmonary hypertension associated with CREST syndrome. Chest 121:656–659 34. Califf RM, Adams KF, McKenna WJ et  al (1997) A randomized controlled trial of epoprostenol therapy for severe congestive heart failure: The Flolan International Randomized Survival Trial (FIRST). Am Heart J 134:44–54 35. Anand I, McMurray J, Cohn JN et al, for the EARTH Investigators (2004) Long-term effects of darusentan on left-ventricular remodelling and clinical outcomes in the Endothelin A Receptor Antagonist Trial in Heart Failure (EARTH): randomised, double-blind, placebo controlled trial. Lancet 364:347–354 36. Guazzi M, Tumminello G, Di Marco F, Fiorentini C, Guazzi MD (2004) The effects of phosphodiesterase-5 inhibition with sildenafil on pulmonary hemodynamics and diffusion capacity, exercise ventilatory efficiency, and oxygen uptake kinetics in chronic heart failure. J Am Coll Cardiol 44:2339–2348 37. Takimoto E, Champion HC, Li M et al (2005) Chronic inhibition of cyclic GMP phosphodiesterase-5A prevents and reverses cardiac hypertrophy. Nat Med 11:214–222 38. Maeder MT, Kaye DM (2009) Heart failure with normal ejection fraction. J Am Coll Cardiol 53:905–918

Chapter 82

Pulmonary Hypertension Associated with Chronic Obstructive Pulmonary Diseases Norbert F. Voelkel, Catherine Grossman, and Herman J. Bogaard

Abstract  Chronic obstructive lung diseases and so-called cor pulmonale have traditionally been connected by the link of pulmonary hypertension (PH). The concept that right ventricular hypertrophy is attributed to the afterload of the right ventricle, i.e., attributable to PH, has not been challenged and clinicians continue to attempt to explain cor pulmonale as a consequence of chronic PH. Both pulmonary parenchyma disease and primary vascular disease can lead to cor pulmonale. Among the obstructive lung diseases, chronic obstructive pulmonary disease (COPD)/ emphysema has been recognized as the most common cause of PH in countries where smoking, but not schistosomiasis, is endemic. Until recently, clinical investigators conceptually associated PH in the setting of COPD/emphysema with chronic hypoxia. Pulmonary angiograms documented the extent of vascular involvement and vascular loss in patients with emphysema. Chronic oxygen supplemen­tation therapy was introduced into clinical management of COPD patients on the basis of the finding that oxygen treatment increased survival and that improved survival was associated with a small reduction in mean pulmonary artery pressure at rest. This chapter discusses the pathogenic mechanisms, diagnostic and therapeutic approaches for pulmonary hypertension associated with chronic obstructive pulmonary diseases. Keywords  Chronic hypoxia and hypoxemia • Emphysema • Cor pulmonale • Pulmonary parenchyma disease

N.F. Voelkel (*) Division Pulmonary and Critical Care Medicine, Department of Internal Medicine, Virginia Commonwealth University, 1101 E. Marshall Street, Richmond, VA, USA e-mail: [email protected]

1 Chronic Obstructive Pulmonary Disease, Pulmonary Hypertension, and Cor Pulmonale: The History Chronic obstructive lung diseases and so-called cor pulmonale have traditionally been connected by the link of pulmonary hypertension (PH). The first clinical description of cor pulmonale goes back to White in 1931 [1]. The concept that right ventricular hypertrophy is attributed to the afterload of the right ventricle, i.e., attributable to PH, has not been challenged and clinicians continue to attempt to explain cor pulmonale as a consequence of chronic PH. An expert committee of the WHO defined “cor pulmonale” in 1963 [2] as stress or strain of the right ventricle as a consequence of diseases that affect the structure or function of the lung and thus effect a pressure increase in the lung circulation. According to this definition, both pulmonary parenchyma disease and primary vascular disease can lead to cor pulmonale. Among the obstructive lung diseases, chronic obstructive pulmonary disease (COPD)/emphysema has been recognized as the most common cause of PH in countries where smoking, but not schistosomiasis, is endemic. Until recently, clinical investigators conceptually associated PH in the setting of COPD/emphysema with chronic hypoxia [3] although substantial pathological vascular abnormalities, difficult to ascribe to hypoxia, had already been described by Liebow in 1958 (see later) [4]. At the same time, pulmonary angiograms documented the extent of vascular involvement and vascular loss in patients with emphysema [5]. Chronic oxygen supplementation therapy was introduced into clinical management of COPD patients on the basis of the finding that oxygen treatment increased survival and that improved survival was associated with a small reduction in mean pulmonary artery pressure at rest [6]. Although it is unclear if improved survival of these patients was attributable to a reduction of the pulmonary artery pressure, the pharmaceutical industry continues to be interested in the development of treatments for PH in COPD patients. MacNee [7, 8] reviewed PH, COPD, and cor pulmonale and there are also more recent reviews of this topic [9–11].

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The German literature distinguishes between “manifest PH” (at rest), which is most likely also “fixed,” because of remodeling of the vasculature or vessel loss, and “latent PH,” which emerges with exercise. Both Burrows et al. [12] and Weitzenblum et al. [13] have emphasized the importance of exercise testing in the evaluation of PH in COPD patients. Figure 1 depicts the pulmonary vessel loss which was assessed by a postmortem angiogram of a patient with emphysema. A lung function parameter which relates to lung vessel loss in COPD is the diffusing capacity of carbon monoxide (DLCO). Early on, investigators of PH in COPD distinguished between a “hypoxic form of cor pulmonale” and a “vascular form of cor pulmonale.” Alveolar hypoventilation leading to hypoxia, hypercapnia, and pulmonary arteriolar vasoconstriction explained the hypoxic form, whereas vascular occlusions explained the vascular form [14]. The “vascular occlusions” concept is apparently undergoing a renaissance as pulmonary embolic events are now more frequently being appreciated as a cause of COPD exacerbations (see later). Although the term “cor pulmonale” was coined to distinguish right ventricular impairment caused by lung disease, new data may point to an involvement of the left ventricle as well in patients with COPD (see later). This begins to put into question the strict use of the term “cor pulmonale” in the setting of COPD. In fact, in some COPD patients there may be a combination of precapillary and postcapillary PH, and

Fig. 1  Pulmonary arteriogram of a segment of artery of a normal lung and segmental arteriogram of a lung from a patient with severe emphysema. A artery, V vein. (Reproduced with permission [5])

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this aspect may change our treatment and practice guidelines. A “classic” paper which examines the basic pulmonary hemodynamics is entitled “Alveolar pressure, pulmonary venous pressure, and the vascular waterfall,” by Permut et al. [15]; these key words all need to be considered when investigating and treating PH in COPD.

2 The Clinical Presentation of PH in COPD PH in COPD patients may not only characterize a particular COPD patient phenotype, but importantly PH in COPD patients is a survival-determining factor [16]. Figure 2 shows that mean pulmonary artery pressure and age predict survival of COPD patients. Patients with COPD/emphysema are short of breath, initially with strenuous work or exercise and, as the disease progresses, at rest. Understandably the clinician focuses his/her attention on the airflow limitation, bronchoconstriction, and symptoms associated with bronchitis. Since exercise limitation and dyspnea are also cardinal symptoms of severe PH, the clinical investigation of PH in COPD patients is usually delayed until signs of heart failure appear. Nevertheless, many attempts have been made over the years to define the lung function, and blood gas variables, which characterize a COPD patient who is likely to develop PH. The approach has generally been to collect data from a reasonably large cohort of COPD patients and to correlate the forced expiratory volume in 1 s (FEV1), SO2, PaO2, PCO2, or DLCO data with the pulmonary artery pressure. Figures 3 and 4 provide examples of such plots. These correlations (plots) are based on the assumption that the pulmonary artery pressure

Fig.  2  Survival of chronic obstructive pulmonary disease (COPD) patients with pulmonary hypertension related to the mean pulmonary artery pressure and related to the age of the patient. (Reproduced with permission of the American College of Chest Physicians [16])

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Fig.  3  Relationship between the mean pulmonary artery pressure and the PaO2 in patients with severe pulmonary hypertension and COPD. (a) Patients have no other causes of pulmonary hypertension, reflecting a rare subgroup of COPD patients with severe

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pulmonary hypertension. These patients were hypoxemic and had a median survival of 26 months. (b) Patients show a relationship between pulmonary artery pressure and PaO2. (Reproduced with permission [23])

Fig. 4  Relationship between mean pulmonary artery pressure, diffusing capacity of carbon monoxide (a) PaO2 (b) and oxygen saturation (c and d) (Reproduced with permission [50])

at rest should relate to known pathobiological concepts; that is pulmonary artery pressure relates to hypoxia, loss of lung vessels, and so on. The histology and morphology of the lung tissue and of lung vessels is not considered because lung tissue is usually not available for analysis. On the other hand, the degree of hyperinflation or inspiratory capacity, which affect the venous return and the nocturnal oxygen desaturation, are also part of the equation [17, 18]. Astutely,

the early clinical COPD investigators noticed and distinguished the edematous, blue and bloated and the thin (often cachectic) pink and puffing phenotype, and autopsy studies demonstrated a greater degree of right ventricular hypertrophy in the blue and bloated patients [18]. An untested hypothesis is that the so-called blue bloater phenotype of COPD disappeared after the introduction of oxygen therapy for COPD. Another untested hypothesis is that PH is severer or

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occurs more frequently in COPD patients living at altitude. Kessler et al. investigated the natural history of PH in a series of 131 COPD patients and found that the pulmonary artery pressure at rest and that during exercise are independent predictors of the subsequent development of PH and that the progression of PH in patients with mild or moderate hypoxemia is slow [19].

3 Diagnosis of PH in COPD The diagnosis of PH in patients with COPD/emphysema is established like in all other forms of PH using noninvasive and invasive methods. The diagnosis of PH should be considered in patients with obstructive airway diseases (including cystic fibrosis, bronchiectasis, and obliterating bronchiolitis) as well as in patients with alveolar hypoventilation and sleep apnea syndromes. Both electrocardiogram and chest radiographic findings are unreliable when it comes to establishing a diagnosis of PH in COPD patients. Unfortunately, echocardiographic examination is also fraught with problems and the chance of obtaining tricuspid valve regurgitation signals of sufficient quality is slim [20–22]. The frustration of the experienced echocardiographer to interpret signals and patterns in patients with hyperinflated lungs together with the widespread therapeutic nihilism are often factors which prevent the vigorous pursuit of the diagnosis of PH in clinical practice, and the gold-standard method of hemodynamic assessment is not contemplated. Weitzenblum’s group in Strassbourg, France, has the greatest experience in the hemodynamic study of COPD patients and recently reported right-sided heart catheter data of 998 COPD patients [23]. These data are extremely helpful although they may not reflect the entire spectrum of PH in COPD patients since only patients living at or near sea level were included. Nevertheless, these data show that severe PH (defined as a resting mean pulmonary artery pressure of more than 40  mmHg) is rare in COPD patients. Likely severe PH defines a subgroup of COPD patients as also recently described by Thabut et al. [24]. This group described a “cluster of patients” characterized by moderate airway obstruction but profound hypoxemia without hypercapnea. In fact, these patients were hypocapnic and thus resembled patients with idiopathic PAH. If the diagnosis of PH matters in the overall treatment plan of a patient, then a right-sided heart catheterization with exercise should be performed. Figure  5 illustrates the variable pulmonary artery pressure response of COPD patients during exercise and the fact that exercise-induced dyspnea in some COPD patients can indeed be attributed (also) to their PH. The factors that account for the variability of exercise-induced PH and the degree of the pulmonary artery pressure response in an individual patient remain unclear. This variability is

Fig. 5  The most important components of the cardiovascular system response and how they interact when a COPD patient who has a hyperinflated lung exercises

likely due to a number of components, including (but not limited to) hyperinflation (auto-positive end-expiratory pressure), loss of lung vessels, and even an element of left ventricular dysfunction. Although technically demanding, measurement of the pulmonary capillary wedge pressure during exercise may unmask a left-sided heart failure component.

4 Pathophysiology of PH in COPD Hypoxic pulmonary arteriolar vasoconstriction, chronic pulmonary vascular remodeling, and in situ thrombosis are determinants of the pulmonary artery pressure. Polycythemia can be another factor, yet COPD patients are more likely to be anemic. A still unexplained aspect of the pathophysiological characteristics of the PH is the observation that oxygen supplementation blunts the exercise-induced rise in the pulmonary artery pressure as originally reported by Burrows et al. [12]. Oxygen could blunt the dyspnea of the patient, affecting a lower respiratory rate, which in turn may cause less air trapping; however, oxygen can also reduce the cardiac output and flow-dependent PH as well as hypoxic vasoconstriction. Respi­ratory and metabolic acidosis can worsen hypoxic vasoconstriction, which may be prevented by oxygen treatment (Fig. 5).

5 Pathobiology of PH Both pulmonary vascular remodeling and lung vessel loss are cellular processes controlled by proteins and genes. As already mentioned, the three principal histological manifestations of the lung vessel abnormalities in COPD/emphysema are muscularization and fibrosis of arterioles, capillary loss, and intravascular abnormalities related to thrombosis [11]. Until recently, hypoxic vasoconstriction had been consi­ dered the major driver of pulmonary vascular remodeling in

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COPD/emphysema although convincing cellular concepts were lacking. “Vasoconstriction causes pulmonary vascular smooth muscle hypertrophy” has been a popular creed [3, 16]. However, it is now clear that pulmonary muscularization of arterioles can develop as a consequence of inflammation and independently of hypoxia [25], and indeed Santos et al. [26] have described pulmonary vascular remodeling in the lungs from patients with mild COPD without hypoxia. In addition, exposure of experimental animals to cigarette smoke causes pulmonary vascular remodeling [27]. Capillary loss can now be explained by endothelial cell apoptosis as a consequence of chronic oxidant stress or secondary to the destruction of the matrix on which the endothelial cells sit (anoikis). The third manifestation – in situ thrombosis – is the least understood aspect of pulmonary vascular disease in COPD/ emphysema, although a hypercoagulable state is perhaps best explained with the concept of endothelial cell dysfunction [28, 29]. We believe that an entirely rational and productive approach to the pathobiology of lung vessels in COPD/ emphysema is the investigation of endothelial cell dysfunction. Endothelial cell dysfunction and endothelial cell “disease” could be important organizing principles of the COPD/ emphysema vascular disease aspect. There is both functional as well as histological evidence for endothelial cell dysfunction in COPD. It has been shown that NO-dependent vasodilation of small arteries is impaired [29] and histologically that the expression of the prostacyclin synthase [30] and endothelial nitric oxide synthase [31] proteins is lost in small pulmonary arteries in the lungs from patients with COPD/ emphysema (Fig.  6). The levels of vascular endothelial growth factor and vascular endothelial growth factor receptor 2 proteins are reduced in the tissue from end-stage COPD patients [32]. Taken together, these findings mean that the case for endothelial cell disease in the COPD lung is quite strong. It has been shown that pulmonary microvascular endothelial cell apoptosis stimulates vascular smooth muscle growth via transforming growth factor b [33]. Thus, during the last few years, the concept of “vasoconstriction” has been complemented by the concepts of “inflammation” and “endothelial cell dysfunction.” It is apparent that these new disease models call for new treatment principles rather than for pulmonary vasodilators. To end on a historical note regarding loss of capillaries (in emphysema): “Whether the process is initiated by air trapping or the result of atrophy otherwise induced, the effect is that the rich vascular beds vanish with the tissues they once supplied. The loss of vascularity and alveoli in emphysema noted so many years ago is still not certainly explained.” And: “In advanced emphysema, whether or not there is congestive heart failure, the bronchial veins become markedly expanded and can be traced along the bronchi…” and “it may be said that such changes as appear to be induced by very high levels of pressure in the pulmonary arteries occur very uncommonly in pulmonary emphysema” [4].

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Fig.  6  Mean pulmonary artery pressure measurements in COPD patients at rest and during exercise demonstrating the variable pulmonary artery pressure response to exercise. (Reproduced with permission from Macmillan Publishing Group [3])

How can we fit these histopathological data with recent polymorphism studies which conclude that a serotonin transporter (5-HTT) polymorphism and an interleukin-6 gene polymorphism are associated with the development of PH in COPD [34, 35]? How can we fit these morphological derangements of the lung with data that indicate that lungs from COPD patients have acquired a decrease in the expression of histone deacetylase 2 expression [36]? Could this be the explanation for the altered emphysema lung gene expression which is characterized by a “no-growth” signature [37]? Are acroleinated proteins (Fig. 6) decorating endothelial cells and vascular smooth muscle cells of the emphysema lung signals of permanent molecular and protein “scars,” which, together with suppressed growth signals, preclude vascular repair of the lung?

6 Cor Pulmonale: COPD, Associated PH, and “COPD-Associated Heart Disease” As readers may have noticed by now, it is our intention to steer away from PH as an outcome-defining aspect of the COPD/emphysema syndrome and the question rather becomes: What are the underlying causes of the high cardiovascular mortality in patients with COPD/emphysema? We offer in the form of postulates three interrelated causes. We postulate that COPD is an endothelial disease, that in

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COPD/emphysema, as a consequence of the endothelial cell disease, there is a significant in situ thrombosis component, and lastly, that “cor pulmonale” is not strictly a right ventricular (by increased afterload determined) problem, but is also a problem of the left ventricle. This topic was recently reviewed by Lee-Chiong and Matthay [38]. The question of a potential left-sided heart involvement was addressed first 40 years ago and has still not been entirely resolved. Some studies have shown that left ventricular function remains normal in COPD patients and others suggest left ventricular dysfunction [39, 40]. Even studies of the contractility of the right ventricle, using end-systolic pressure–volume analysis, indicated that the right ventricular contractility was preserved [41, 42]. Rutten et al. [43] examined patients with mild/moderate degrees of COPD and confirmed the diagnosis of chronic heart failure using cardiac MRI in 20% of these COPD patients. COPD patients with heart failure have high levels of brain natriuretic peptide, a greater BMI, and a slightly lower cardiac index when compared with patients without heart failure. Paudel et al. [44] performed echocardiography in 60 patients with COPD and found evidence for left ventricular systolic dysfunction in 26% of these patients. A comprehensive literature search [45] of cross-sectional or prospective studies which had used CT scanning or pulmonary angiography (550 patients) found an overall prevalence of pulmonary embolism of about 20%. Thus, taken together, and entirely from a clinical view point, both pulmonary embolism and left-sided heart involvement are significant

Fig. 7  Immunohistology of lungs from patients with COPD/ emphysema. (a) Tissue stained with an antibody directed against prostacyclin synthase; loss of prostacyclin synthase expression in small-vessel endothelial cells (left) when compared with control lungs. (b) Loss of endothelial nitric oxide synthase expression in a COPD/ emphysema lung (left) when compared with a normal lung. (c) Vessels and a long-term smoking history. The antibody is directed against acroleinated proteins. Note that both vascular endothelial cells and smooth muscle cells test positive for acroleinated proteins

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problems in patients with COPD; unfortunately the prevalence of severe PH in patients with COPD is hard to come by and the “heart and lungs” in COPD – close friends in real life – are still separated in clinical practice [46] (Fig. 7). We propose that a common denominator of COPD, pulmonary embolism, and chronic heart failure is the vasculature, and more precisely the endothelium. The Emphysema and Cancer Action Project study report demonstrated an impairment of flow-mediated vasodilation in COPD patients which remarkably was related to the CT-scan-determined degree of emphysema but not to the forced expiratory volume [45]! Lusuardi et al. reached a similar conclusion of impaired endothelial cell (EC) function in a smaller cohort of 44 COPD patients: here, however, flow-mediated vasodilation was inversely related to the FEV1 to forced vital capacity ratio [46]. Lack of vasodilator production by the endothelium, a prothrombotic state, and capillary loss are likely important components of the COPD syndrome (Fig. 8).

7 Therapy of PH in COPD On the basis of the concepts and postulates we have outlined, the treatment plan for patients with COPD and PH may not fundamentally differ from the treatment plan we may now contemplate for all patients with severely disabling COPD. Chronic oxygen treatment is likely indicated for all patients with resting hypoxia. Should all patients with

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endothelial disease may be central in particular when COPD/ PH is associated with other morbidities such as diabetes, hyperlipidemia, obesity, and high blood pressure. Perhaps we can postulate that it will be productive – reducing both morbidity and mortality – to consider that a microangiopathy, that involves several organs, not only the lung, and chronic systemic inflammation become more defined treatment targets for investigating the effects of statins in COPD patients with PH as a start [53].

References Fig. 8  The relationship between COPD, pulmonary hypertension, and chronic heart disease

exercise-induced hypoxemia be treated with supplemental oxygen? The pulmonary artery pressure per se may no longer become a treatment target in contrast to the endothelial cell dysfunction that we need to understand better and need to treat. At this juncture we cannot provide treatment recommendations, we can only ask questions. Should COPD patients who do not have contraindications be anticoagulated? Should COPD patients be treated with “statins” if for no other reason than that a retrospective data base analysis found that statins, angiotensin-converting-enzyme inhibitors, or angiotensin receptor blockers appear to reduce the mortality of patients with COPD [47]. What about the heretical thought to treat these patients with cardioselective b-adrenergic receptor blockers? A reasonably large cohort of COPD patients was followed in the Netherlands for more than 15 years and the analysis of the data shows a reduction of mortality in COPD patients undergoing vascular surgery [48]. This finding is also of interest in the context of another recent report which suggests that the treatment of COPD patients with inhaled b-adrenergic agonists was associated with increased cardiovascular risk [49]. Holverda et al. compared the exercise characteristics of COPD patients with and without PH and demonstrated a significantly reduced peak workload in COPD patients with PH [50]. Sildenafil acutely decreased the mean pulmonary artery pressure during submaximal exercise [51], but after 12 weeks of therapy did not affect the cardiac stroke volume or exercise capacity [52], illustrating again that the cardiac output rather than the pulmonary artery pressure determines exercise limitation.

8 Summary and Perspective We have provided a contextual view of the problem of PH associated with COPD/emphysema. The “big picture” of the patient with COPD plus PH is, in our opinion, that the

1. White PD (1931) Heart disease, 1st edn. Macmillan, New York 2. Chronic cor pulmonale: report of an expert committee. Circulation 1963;27:594–615 3. Weitzenblum E, Chaouat A (2004) Pulmonary hypertension due to chronic hypoxia lung disease. In: Peacock A, Rubin LJ (eds) Pulmonary circulation, 2nd edn. Arnold, London, pp 374–386 4. Liebow AA (1958) Pulmonary emphysema with special reference to vascular changes. Am Rev Resp Dis 80:67–92 5. Junghanss W (1959) Das Lungenemphysem im postmortalen Angiogramm. Virchows Arch Pathol Anat 332:538 6. Anon (1981) Long term domiciliary oxygen therapy in chronic hypoxic cor pulmonale complicating chronic bronchitis and emphysema. Report of the Medical Research Council Working Party. Lancet 1:681–686 7. MacNee W (1994) Pathophysiology of cor pulmonale in chronic obstructive pulmonary disease. Part one. Am J Respir Crit Care Med 150:833–852 8. MacNee W (1994) Pathophysiology of cor pulmonale in chronic obstructive pulmonary disease. Part two. Am J Respir Crit Care Med 150:1158–1168 9. Peinado VI, Pizarro S, Barbera JA (2008) Pulmonary vascular involvement in COPD. Chest 134:808–814 10. Chaouat A, Naeije R, Weitzenblum E (2008) Pulmonary hypertension in COPD. Eur Respir J 32:1371–1385 11. Voelkel NF, Cool CD (2003) Pulmonary vascular involvement in chronic obstructive pulmonary disease. Eur Respir J 22:28s–32s 12. Burrows B, Kettel LJ, Niden AH, Rabinowitz M, Diener CF (1972) Patterns of cardiovascular dysfunction in chronic obstructive lung disease. N Engl J Med 286:912–918 13. Weitzenblum E, Chaouat A, Canuet M (2008) Pulmonary hypertension in chronic obstructive disease. In: Voelkel NF, MacNee W (eds) Chronic obstructive lung diseases, 2nd edn. Decker, London, pp 299–316 14. Szam I (1975) Cor pulmonale chronicum., F.K. Schattauer Verlag. Stuttgart, New York 15. Permutt S, Bromberger-Barnea B, Bane HN (1962) Alveolar pressure, pulmonary venous pressure, and the vascular waterfall. Med Thorac 19:239–260 16. Oswald-Mammosser M, Weitzenblum E, Quoix E et  al (1995) Prognostic factors in COPD patients receiving long-term oxygen therapy. Importance of pulmonary artery pressure. Chest 107:1193–1198 17. Chaouat A, Weitzenblum E, Kessler R et al (1997) Sleep-related O2 desaturation and daytime pulmonary haemodynamics in COPD patients with mild hypoxaemia. Eur Respir J 10:1730–1735 18. Mitchell RS, Stanford R, Johnson JM, Silvers GW, Darl G, George M (1975) The morphology of chronic airways obstruction. In: Schüren KP, Hüttemann U, Schröder R (eds) Chronisch obstructive Lurgenerkrankungen und Cor Pulmonale. Schattauer, New York, pp 31–44

1196 19. Kessler R, Faller M, Weitzenblum E et  al (2001) “Natural history” of pulmonary hypertension in a series of 131 patients with chronic obstructive lung disease. Am J Respir Crit Care Med 164:219–224 20. Torbicki A, Skwarski K, Hawrylkiewicz I, Pasierski T, Miskiewicz Z, Zielinski J (1989) Attempts at measuring pulmonary arterial pressure by means of Doppler echocardiography in patients with chronic lung disease. Eur Respir J 2:856–860 21. Laaban JP, Diebold B, Zelinski R, Lafay M, Raffoul H, Rochemaure J (1989) Noninvasive estimation of systolic pulmonary artery pressure using Doppler echocardiography in patients with chronic obstructive pulmonary disease. Chest 96:1258–1262 22. Tramarin R, Torbicki A, Marchandise B, Laaban JP, Morpurgo M (1991) Doppler echocardiographic evaluation of pulmonary artery pressure in chronic obstructive pulmonary disease. A European multicentre study. Working Group on Noninvasive Evaluation of Pulmonary Artery Pressure. European Office of the World Health Organization, Copenhagen. Eur Heart J 12:103–111 23. Chaouat A, Bugnet AS, Kadaoui N et al (2005) Severe pulmonary hypertension and chronic obstructive pulmonary disease. Am J Respir Crit Care Med 172:189–194 24. Thabut G, Dauriat G, Stern JB et al (2005) Pulmonary hemodynamics in advanced COPD candidates for lung volume reduction surgery or lung transplantation. Chest 127:1531–1536 25. Daley E, Emson C, Guignabert C et al (2008) Pulmonary arterial remodeling induced by a Th2 immune response. J Exp Med 205:361–372 26. Santos S, Peinado VI, Ramirez J et al (2002) Characterization of pulmonary vascular remodelling in smokers and patients with mild COPD. Eur Respir J 19:632–638 27. Wright JL, Churg A (1991) Effect of long-term cigarette smoke exposure on pulmonary vascular structure and function in the guinea pig. Exp Lung Res 17:997–1009 28. Peinado VI, Barbera JA, Ramirez J et al (1998) Endothelial dysfunction in pulmonary arteries of patients with mild COPD. Am J Physiol 274:L908–L913 29. Dinh-Xuan AT, Higenbottam TW, Clelland CA et  al (1991) Impairment of endothelium-dependent pulmonary-artery relaxation in chronic obstructive lung disease. N Engl J Med 324:1539–1547 30. Nana-Sinkam SP, Lee JD, Sotto-Santiago S et al (2007) Prostacyclin prevents pulmonary endothelial cell apoptosis induced by cigarette smoke. Am J Respir Crit Care Med 175:676–685 31. Barbera JA, Peinado VI, Santos S, Ramirez J, Roca J, RodriguezRoisin R (2001) Reduced expression of endothelial nitric oxide synthase in pulmonary arteries of smokers. Am J Respir Crit Care Med 164:709–713 32. Kasahara Y, Tuder RM, Cool CD, Lynch DA, Flores SC, Voelkel NF (2001) Endothelial cell death and decreased expression of vascular endothelial growth factor and vascular endothelial growth factor receptor 2 in emphysema. Am J Respir Crit Care Med 163:737–744 33. Sakao S, Taraseviciene-Stewart L, Wood K, Cool CD, Voelkel NF (2006) Apoptosis of pulmonary microvascular endothelial cells stimulates vascular smooth muscle cell growth. Am J Physiol Lung Cell Mol Physiol 291:L362–L368 34. Eddahibi S, Chaouat A, Morrell N et al (2003) Polymorphism of the serotonin transporter gene and pulmonary hypertension in chronic obstructive pulmonary disease. Circulation 108:1839–1844 35. Eddahibi S, Chaouat A, Tu L et al (2006) Interleukin-6 gene polymorphism confers susceptibility to pulmonary hypertension in chronic obstructive pulmonary disease. Proc Am Thorac Soc 3:475–476

N.F. Voelkel et al. 36. Barnes PJ (2009) Role of HDAC2 in the pathophysiology of COPD. Annu Rev Physiol 71:451–464 37. Golpon HA, Coldren CD, Zamora MR et  al (2004) Emphysema lung tissue gene expression profiling. Am J Respir Cell Mol Biol 31:595–600 38. Lee-Chiong T, Matthay RA (2003) Pulmonary hypertension and cor pulmonale in COPD. Semin Respir Crit Care Med 24:263–272 39. Murphy ML, Adamson J, Hutcheson F (1974) Left ventricular hypertrophy in patients with chronic bronchitis and emphysema. Ann Intern Med 81:307–313 40. Fluck DC, Chandrasekar RG, Gardner FV (1966) Left ventricular hypertrophy in chronic bronchitis. Br Heart J 28:92–97 41. Burghuber OC, Salzer-Muhar U, Gotz M (1988) Right ventricular contractility is preserved in patients with cystic fibrosis and pulmonary artery hypertension. Scand J Gastroenterol Suppl 143:93–98 42. MacNee W, Wathen CG, Hannan WJ, Flenley DC, Muir AL (1983) Effects of pirbuterol and sodium nitroprusside on pulmonary haemodynamics in hypoxic cor pulmonale. Br Med J (Clin Res Ed) 287:1169–1172 43. Rutten FH, Vonken EJ, Cramer MJ et al (2008) Cardiovascular magnetic resonance imaging to identify left-sided chronic heart failure in stable patients with chronic obstructive pulmonary disease. Am Heart J 156:506–512 44. Paudel B, Dhungel S, Paudel K, Pandru K, Paudel R (2008) When left ventricular failure complicates chronic obstructive pulmonary disease: hypoxia plays the major role. Kathmandu Univ Med J 6:37–40 45. Rizkallah J, Man SF, Sin DD (2009) Prevalence of pulmonary embolism in acute exacerbations of chronic obstructive pulmonary disease: a systematic review and meta-analysis. Chest 135(3):786–793 46. Lusuardi M, Garuti G, Massobrio M, Spagnolatti L, Bendinelli S (2008) Heart and lungs in COPD. Close friends in real life– separate in daily medical practice? Monaldi Arch Chest Dis 69:11–17 47. Mancini GB, Etminan M, Zhang B, Levesque LE, FitzGerald JM, Brophy JM (2006) Reduction of morbidity and mortality by statins, angiotensin-converting enzyme inhibitors, and angiotensin receptor blockers in patients with chronic obstructive pulmonary disease. J Am Coll Cardiol 47:2554–2560 48. van Gestel YRBM, Hoeks SE, Sin DD et al (2008) Impact of cardioselective beta-blockers on mortality in patients with chronic obstructive pulmonary disease and atherosclerosis. Am J Respir Crit Care Med 178:695–700 49. Macie C, Wooldrage K, Manfreda J, Anthonisen N (2008) Cardiovascular morbidity and the use of inhaled bronchodilators. Int J Chron Obstruct Pulm Dis 3:163–169 50. Holverda S, Bogaard HJ, Groepenhoff H, Postmus PE, Boonstra A, Vonk-Noordegraaf A (2008) Cardiopulmonary exercise test characteristics in patients with chronic obstructive pulmonary disease and associated pulmonary hypertension. Respiration 76:160–167 51. Holverda S, Rietema H, Bogaard HJ et al (2008) Acute effects of sildenafil on exercise pulmonary hemodynamics and capacity in patients with COPD. Pulm Pharmacol Ther 21:558–564 52. Rietema H, Holverda S, Bogaard HJ et al (2008) Sildenafil treatment in COPD does not affect stroke volume or exercise capacity. Eur Respir J 31:759–764 53. Lee TM, Chen CC, Shen HN, Chang NC (2009) Effects of pravastatin on functional capacity in patients with chronic obstructive pulmonary disease and pulmonary hypertension. Clin Sci (Lond) 116:497–505

Chapter 83

Pulmonary Hypertension Associated with Interstitial Lung Disease Mary E. Strek and Julian Solway

Abstract  Interstitial lung diseases (ILDs) are a distinct type of chronic respiratory disorder that can result in pulmonary hypertension. There are numerous causes of ILD but all are characterized by dyspnea and abnormal lung function, with arterial oxygen desaturation occurring as the disease advances. Patients with idiopathic pulmonary fibrosis (IPF), a relentlessly progressive form of ILD, are particularly likely to develop pulmonary hypertension. Both chronic hypoxemia with subsequent pulmonary vasoconstriction and obliteration of the pulmonary vascular bed as a result of interstitial fibrosis have traditionally been considered the pathways by which pulmonary hypertension develops in ILD. Not all patients with pulmonary hypertension from ILD have a severe restrictive ventilatory deficit or pulmonary fibrosis, however, suggesting that other pathologic mechanisms may contribute to the development of pulmonary vascular disease and cor pulmonale in patients with ILD. Sarcoidosis, pulmonary Langerhans cell histiocytosis (PLCH), and lymphangioleiomyomatosis (LAM) are types of ILD that may directly involve the pulmonary vasculature, in addition to causing interstitial fibrosis. Collagen vascular diseases such as scleroderma may cause pulmonary hypertension as a result of progressive ILD or by direct pulmonary vascular involvement. In patients with chronic lung disease, worsening dyspnea and hypoxemia are often mistaken for progressive ILD rather than recognized as heralding the onset of pulmonary hypertension. Although physical findings, echocardiography results, and serum brain natriuretic peptide (BNP) levels can suggest the presence of pulmonary hypertension, right-sided heart catheterization remains the diagnostic gold standard. In this chapter, we review the current classification schemes of both pulmonary hypertension and ILD. The epidemiology of pulmonary hypertension in specific types of ILD is discussed. New information about the origin, pathogenesis, and pathologic findings in ILD-related pulmonary hypertension is described. Clinical manifestations and means of diagnosis M.E. Strek (*) Department of Medicine, University of Chicago, Chicago, IL 60637, USA e-mail: [email protected]

are reviewed. We end the chapter with a focus on current treatment options and prognosis. Keywords  Interstitial lung disease • Sarcoidosis • Langerhans cell histiocytosis • Lymphangioleiomyomatosis • Idiopathic pulmonary fibrosis

1 Introduction Chronic lung disease is a major cause of pulmonary hypertension and subsequent right-sided heart failure or cor pulmonale. Interstitial lung diseases (ILDs) are a distinct type of chronic respiratory disorder that can result in pulmonary hypertension. There are numerous causes of ILD but all are characterized by dyspnea and abnormal lung function, with arterial oxygen desaturation occurring as the disease advances. Patients with idiopathic pulmonary fibrosis (IPF), a relentlessly progressive form of ILD, are particularly likely to develop pulmonary hypertension. Both chronic hypoxemia with subsequent pulmonary artery vasoconstriction and obliteration of the pulmonary vascular bed as a result of interstitial fibrosis have traditionally been considered the pathways by which pulmonary hypertension develops in ILD. Not all patients with pulmonary hypertension from ILD have a severe restrictive ventilatory deficit or pulmonary fibrosis, however, suggesting that other pathologic mechanisms may contribute to the development of pulmonary vascular disease and cor pulmonale in patients with ILD. Sarcoidosis, pulmonary Langerhans cell histiocytosis (PLCH), and lymphangioleiomyomatosis (LAM) are types of ILD that may directly involve the pulmonary vasculature, in addition to causing interstitial fibrosis. Collagen vascular diseases such as scleroderma may cause pulmonary hypertension as a result of progressive ILD or by direct pulmonary vascular involvement (see Chap. 70). In patients with chronic lung disease, worsening dyspnea and hypoxemia are often mistaken for progressive ILD rather than recognized as heralding the onset of pulmonary hypertension. Although physical findings, echocardiography

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results, and serum brain natriuretic peptide (BNP) levels can suggest the presence of pulmonary hypertension, right-sided heart catheterization remains the diagnostic gold standard. Supplemental oxygen for arterial oxygen desaturation and treatment of the underlying ILD have been the primary modes of therapy, but improved understanding of the pathogenic mechanisms of pulmonary hypertension in patients with ILD suggests there is a role for pulmonary artery vasodilators, as well as therapies that prevent inflammation and remodeling of the pulmonary vasculature. Pulmonary hypertension as a result of ILD, especially of IPF, portends a poor prognosis. In this chapter, we review the current classification schemes of both pulmonary hypertension and ILD. The epidemiology of pulmonary hypertension in specific types of ILD is discussed. New information about the origin, pathogenesis, and pathologic findings in ILD-related pulmonary hypertension is described. Clinical manifestations and means of diagnosis are reviewed. We end the chapter with a focus on current treatment options and prognosis.

2 Background Our understanding of pulmonary hypertension and its causes has improved dramatically in recent years leading to the development of effective therapies [1, 2]. Pulmonary hypertension is defined as a sustained mean pulmonary artery pressure (mPAP) greater than 25 mmHg at rest or greater than 30 mmHg with exercise [2]. The World Health Organization has classified pulmonary hypertension into five different categories with diseases grouped together on the basis of similar pathophysiologic changes, clinical presentation, and potential therapy [1]. Pulmonary arterial hypertension may be idiopathic, which was previously referred to as “primary” pulmonary hypertension. Since idiopathic pulmonary arterial hypertension shares similar features with pulmonary hypertension associated with collagen vascular disease, portal hypertension, congenital shunts, and drugs and toxins they are grouped together in the Dana Point 2008 “Updated Clinical Classification of Pulmonary Hypertension” (Table 1). Other major causes include left-sided heart disease, chronic lung disease, chronic thromboembolic disease, and a group of miscellaneous causes. Pulmonary hypertension associated with lung diseases and/or hypoxemia can result from a variety of chronic pulmonary disorders including chronic obstructive pulmonary disease, obstructive sleep apnea, and ILD [3]. The ILDs are a heterogeneous group of diseases that primarily affect the pulmonary interstitium [4]. These diseases result in varying degrees of inflammation and fibrosis of the lung parenchyma. They can be caused by occupational exposure to asbestos or silica dusts, a hypersensitivity response to environmental exposures, or collagen vascular diseases [5–7]. In many

M.E. Strek and J. Solway Table  1  Updated clinical classification of pulmonary hypertension (Dana Point, 2008) Pulmonary arterial hypertension Idiopathic, heritable, collagen vascular disease, congenital heart diseases, portal hypertension, drugs and toxins Pulmonary hypertension with left-sided heart disease Pulmonary hypertension associated with lung diseases and/or hypoxia Pulmonary hypertension due to chronic thrombotic and/or embolic disease Multifactorial Mechanisms Sarcoidosis, pulmonary Langerhans cell histiocytosis, lymphangioleiomyomatosis

cases, despite a thorough evaluation including surgical lung biopsy, the type or cause of the ILD remains unidentified. Recent advances in our understanding of the idiopathic ILDs has led to an international consensus statement on idiopathic interstitial pneumonias that suggests these be grouped according to clinical, radiographic, and pathologic patterns with a multidisciplinary approach to diagnosis [8]. The most common idiopathic ILD is IPF, which is characterized pathologically by usual interstitial pneumonia (UIP) with architectural destruction, fibroblastic foci, and honeycomb fibrosis. In addition to this specific pathologic criteria, characteristic high-resolution chest computed tomography (CT) scan findings in UIP obviate surgical lung biopsy in many cases [4, 9]. Nonspecific interstitial pneumonia (NSIP) is a pathologic pattern that is most often seen with collagen vascular disease or a hypersensitivity response to an environmental antigen, but it too can be idiopathic [10]. Patients with NSIP tend to be younger and have a better prognosis than those with IPF. There are numerous poorly understood inflammatory causes of ILD including sarcoidosis, which is a multisystem granulomatous disorder that most commonly involves the lungs [11]. PLCH results from proliferation of a specific population of dendritic cells in the small airways and lung parenchyma and is predominantly seen in cigarette smokers [12]. LAM is caused by mutations in tuberous sclerosis genes with smooth muscle proliferation in many compartments of the lung and a remarkable restriction to the female gender [13]. Sarcoidosis, PLCH, and LAM may directly involve the pulmonary vasculature, in addition to causing interstitial fibrosis and are grouped separately under the “miscellaneous” category in the classification scheme noted above (Table 1). Collagen vascular diseases, especially scleroderma, may directly involve the pulmonary arterial bed, resulting in a clinical picture very similar to that of idiopathic pulmonary arterial hypertension or result in pulmonary hypertension from ILD and hypoxemia [7, 14]. Some of these patients have ILD but disproportionately severe pulmonary hypertension, suggesting the connective tissue disease affects both the pulmonary interstitial and the vascular compartments [15, 16]. This is discussed in more detail in Chap. 70.

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The best studied populations with pulmonary hypertension complicating ILD are patients with IPF, scleroderma, and sarcoidosis [17, 18].

3 Epidemiology of Pulmonary Hypertension in ILD Pulmonary hypertension is a common complication of ILDs (Table  2). The apparent incidence and prevalence vary depending on the type and severity of ILD as well as the method used for diagnosis of the pulmonary hypertension. A retrospective analysis examined the incidence of pulmonary hypertension in 163 consecutively enrolled patients with either idiopathic or collagen vascular disease associated interstitial pneumonias [19]. Among the 78 cases of idiopathic interstitial pneumonia, 43 patients had IPF. Pulmonary hypertension defined by systolic pulmonary artery pressure (sPAP) of 40 mmHg or more on echocardiography was seen in 28% of the patients with idiopathic interstitial pneumonia and in 21% of the patients with collagen vascular disease associated ILD. In scleroderma ILD, pulmonary hypertension was noted in approximately 20% of the patients who underwent screening echocardiography in two separate studies [15, 16]. In IPF, the most serious form of ILD, sPAP greater than 35 mmHg was noted in 74 of 88 patients at presentation to the Mayo Clinic, after excluding 48 patients with IPF who also had left ventricular or valvular heart disease and those in whom pulmonary pressures could not be estimated [20]. A much lower number was noted in 78 unselected patients with IPF who were prospectively followed for up to 14 years [21]. In this study, right-sided heart catheterization data in 61 patients revealed 8.1% with mPAP greater than 25  mmHg. Among patients with advanced IPF, 32% of patients u­ ndergoing lung Table  2  Common causes of interstitial lung disease (ILD) and percentage with pulmonary hypertension Percentage with pulmonary hypertensiona Causes of ILD Screeningb Before transplant Idiopathic pulmonary fibrosis 8–84 32–46 Collagen vascular disease 20 Unknown related ILD Hypersensitivity Rare Unknown pneumonitis Asbestosis 6 Unknown Sarcoidosis 6 74 Pulmonary Langerhans 12 92 cell histiocytosis Lymphangioleiomyomatosis Rare Uncommon a Most of the data are from retrospective case series of selected populations. b These figures are mostly based on echocardiographic rather than rightsided heart catheterization determination of pulmonary artery pressures. IPF, idiopathic pulmonary fibrosis.

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transplant evaluation had elevated mPAP greater than 25 mmHg measured by right-sided heart catheterization [22]. In a larger series of patients with IPF undergoing right-sided heart catheterization for lung transplantation evaluation, 46.1% had mPAP of 25  mmHg or greater, with 9% having values of more than 40 mmHg [23]. In a small study of 16 men with asbestosis, 15 of whom were cigarette smokers, right-sided heart catheterization showed that only one patient had pulmonary hypertension using the currently accepted definition [24]. In a study of 48 ex-miners with suspected coal workers’ pneumoconiosis, 63% of whom were cigarette smokers, 21 patients had mPAP of 20 mmHg or more [25]. The presence of emphysema or progressive massive fibrosis was not higher in this group compared with those with mPAP lower than 20 mmHg. The prevalence of pulmonary hypertension diagnosed by echocardiography in patients with both diffuse parenchymal lung disease with fibrosis (ILD) and cigarette-smoking-related emphysema was 47% at diagnosis and 55% during follow-up [26]. Compared to patients with IPF, patients with combined pulmonary fibrosis and emphysema have an increased incidence of pulmonary hypertension, normal lung volumes and higher mortality. The incidence of pulmonary hypertension defined as sPAP of 40 mmHg or greater in a Japanese population of patients with sarcoidosis was 6% in over 200 patients screened by echocardiography [27]. A retrospective study of 363 patients with advanced sarcoidosis listed for lung transplantation from 1995 to 2002 showed that 73.8% of the patients had mPAP of 25 mmHg or greater measured by right-sided heart catheterization [28]. In a study comparing pulmonary hemodynamics measured by right-sided heart catheterization in patients with severe chronic respiratory disease listed for lung transplantation, patients with PLCH had more severe pulmonary hypertension compared with patients with IPF or COPD [29]. At a center with 123 patients with PLCH, 17 underwent echocardiography for suspected pulmonary hypertension or symptoms [30]. Fifteen patients had sPAP greater than 35 mmHg, with nine having sPAP greater than 50 mmHg. Of 39 patients who underwent lung transplantation for PLCH, pulmonary hypertension was observed in 92% of cases, with moderate to severe elevations (mPAP og 35 mmHg or greater) in 72.5% of cases [31]. LAM is characterized by proliferation of smooth muscle cells around bronchovascular structures; however, clinically significant pulmonary hypertension is not well described [3, 13].

4 Etiology and Pathogenic Mechanisms The mechanism by which the various types of ILD result in inflammation and fibrosis of the lung parenchyma is poorly understood [4, 8]. In patients with ILD, ventilation–perfusion heterogeneity from disordered lung architecture results in

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hypoxemia. That this and not diffusion limitation is the main contributor to the gas-exchange abnormalities has been confirmed in studies of patients with ILD using multiple inert gas techniques [32, 33]. During exercise, however, structural vascular changes may cause diffusion limitation and prevent redistribution of blood away from poorly ventilated lung units, causing worsening ventilation–perfusion inequality and lower PaO2. It is likely that in some patients with ILD, decreased respiratory muscle tone with sleep may result in nocturnal hypoxemia [34]. By whatever mechanism, chronic or sustained hypoxemia results in pulmonary artery vasoconstriction, which may persist in adults as a physiologic mechanism to preserve ventilation–perfusion homogeneity in the lung [35]. Chronic pulmonary artery vasoconstriction results in pulmonary hypertension. Acidosis from CO2 retention, which may occur in patients with severe pulmonary fibrosis, has an additive effect on increasing pulmonary pressures [35]. In animal models, chronic hypoxia results in “muscularization” of small pulmonary arteries that usually do not have a smooth muscle layer [36]. This creates pulmonary vascular resistance in vascular segments that were previously lowresistance units, thereby increasing pulmonary artery pressure. Compromise of the pulmonary vascular bed from inflammation and fibrosis of the lung parenchyma also contributes to increased pulmonary artery pressures and in some patients there is a correlation between the severity of the ILD and the presence of pulmonary hypertension [9, 21, 22]. Hypoxic pulmonary vasoconstriction and architectural destruction of the pulmonary capillary bed, however, are likely not the whole story. In some patients, pulmonary hypertension is more severe than the ILD and cannot be explained by the extent of pulmonary fibrosis or the degree of hypoxemia [37, 38]. In a study of patients with advanced IPF undergoing lung transplant evaluation, neither total lung capacity nor forced vital capacity (FVC) differed between those with pulmonary hypertension and those without pulmonary hypertension [22]. In addition, supplemental oxygen does not typically reverse the pulmonary hypertension once it is present. These observations have prompted numerous studies, some of which are discussed below, that suggest the following may contribute to pulmonary vascular disease in ILD independent of chronic hypoxemia: (1) inflammation and fibrosis of the pulmonary vascular bed with altered growth factor and cytokine expression, and (2) activation of the coagulation cascade which may contribute to a thrombotic angiopathy. In animal models, chronic hypoxia has been shown to produce an inflammatory response within the pulmonary vasculature with recruitment of inflammatory cells and mediators [36]. Gene expression patterns in patients with IPF who had estimated right ventricular systolic pressure greater than 30 mmHg on echocardiography revealed 19.8% of the genes known to be associated with the vascular endothelium were underexpressed, including vascular endothelial growth ­factor

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(VEGF) and platelet endothelial cell adhesion molecule, whereas genes that regulate inflammation such as phospholipase A2 were overexpressed [39]. Analysis of messenger RNA expression in lung tissue from patients with UIP or NSIP showed a decrease in the level of VEGF in the alveolar wall compared with normal lung tissue [40]. Vascular density was evaluated in open lung biopsies in patients with IPF, scleroderma-related ILD, and normal lung tissue. Reduced vessel density was noted in both scleroderma-related ILD (3.9%) and IPF (4.5%) compared with controls (20.4%) [41]. Expression of the angiostatic protein pigment epitheliumderived factor was increased in the fibroblastic foci from lungs of patients with IPF, whereas the VEGF level was decreased [42]. Ebina et al. confirmed that vascular density was reduced in areas of dense fibrosis in lung tissue from patients with UIP compared with normal controls [43]. This study suggested that vascular remodeling was heterogeneous since vascular density was significantly higher in areas of low-grade fibrosis than in control lungs. Plasma concentrations of the angiogenic cytokine interleukin-8 (IL-8) and endothelin-1 levels were increased in patients with idiopathic ILD compared with healthy volunteers [44]. Increased VEGF levels correlated with increasing chest CT scan fibrosis score and decreased FVC. Lung tissue from patients with IPF demonstrate higher levels of IL-8 than controls [45]. In lung tissue from patients with IPF, immunostains for endothelin-1 were prominent compared with minimal staining in control lungs [46]. In patients who also had pulmonary hypertension, staining was present in pulmonary vascular endothelial cells. Animal models of bleomycin lung injury, a form of ILD, suggest that thrombin expression may be increased, with thrombin inhibition shown to decrease lung collagen deposition [47]. Increased tissue factor expression and fibrin deposition were seen in lung tissue from patients with IPF and scleroderma-related ILD compared with control lung tissue [48]. Immunofluorescence staining for thrombomodulin, which exerts a protective effect against blood coagulation, was not seen in capillary endothelial cells from lung tissue in patients with UIP or fibrotic NSIP [40]. In a study of 19 patients thought to have IPF, immune-based microvascular injury was noted with patchy areas of capillary injury and elevated factor VIII levels in most patients, with antiphospholipid antibodies noted in 18 patients [49]. Although none of the patients were diagnosed with a collagen vascular disorder, many had serologic findings suggestive of collagen vascular disease and nine had NSIP on lung biopsy, a pathologic finding often seen in association with connective tissue disease, suggesting the antiphospholipid antibodies and pulmonary vascular disease were related to an autoimmune process in these patients. Sarcoidosis and PLCH result in pulmonary hypertension by a number of mechanisms, hence their separate ­classification from other chronic respiratory disorders that are associated with pulmonary hypertension. Severe, chronic

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ILD from sarcoidosis and PCLH can result in pulmonary fibrosis and hypoxemia. Additionally, there is evidence that sarcoidosis and PLCH can directly and primarily involve the pulmonary vasculature. In 40 autopsy cases of sarcoidosis, pulmonary vascular involvement was noted in all patients, even those who were clinically thought to lack lung involvement, and was noted at all levels from large elastic pulmonary arteries to venules [50]. In addition, compression of large blood vessels by fibrogranulomatous involvement of proximal lymph nodes was seen. In this study, the degree of pulmonary vascular involvement on pathological changes correlated with the degree of pulmonary parenchymal involvement. In a study of 22 patients with pulmonary hypertension from sarcoidosis, 15 patients had pulmonary fibrosis, whereas two had no radiographic evidence of pulmonary sarcoidosis [51]. Granulomatous vascular involvement with occlusive venopathy was noted in the five patients undergoing lung transplantation [51]. Short-term vasoresponsiveness to pulmonary vasodilators was noted in the majority of patients in a small study of moderate to severe pulmonary hypertension from sarcoidosis [52]. Both diastolic and systolic cardiac dysfunction can cause pulmonary hypertension from elevated left-sided heart pressures in patients with cardiac sarcoidosis [53]. Direct pulmonary vascular involvement of the muscular arteries and veins has been noted in PLCH [29, 31]. Left-sided heart disease may contribute to pulmonary hypertension in some patients, especially those with sarcoidosis. Elevated pulmonary capillary wedge pressures were seen in 16.1% of 48 patients with both IPF and pulmonary hypertension [54]. In 22 patients with IPF and mild to moderate pulmonary hypertension, enhanced images obtained by tissue Doppler echocardiography showed impaired right ventricular function, reversal of left ventricular diastolic filling to late diastole, and lower peak myocardial velocities in early systole compared with healthy controls, suggestive of impaired left ventricular diastolic function [55]. In many patients, pulmonary hypertension worsens over time and right-sided heart failure develops. Rapid clinical deterioration from right-sided heart failure has been noted in some patients once pulmonary hypertension is present. Right ventricular pressure overload can cause decreased left ventricular function through ventricular interdependence, decreasing cardiac output, and mixed venous oxygen saturation, resulting in severe hypoxemia and worsened pulmonary hypertension [56]. A fall in cardiac output in a patient with ILD has greater physiologic consequences than for a patient without chronic lung disease since the associated decrease in mixed venous oxygen saturation may cause a dramatic increase in arterial oxygen desaturation owing to ventilation–perfusion mismatch in the affected lung. In summary, hypoxic pulmonary vasoconstriction, destruction of the vascular bed from pulmonary fibrosis, vascular inflam-

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mation and remodeling, alteration of the coagulation cascade, and left-sided heart disease all may play a role in the development of pulmonary hypertension in patients with ILD.

5 Pathology Pulmonary vascular remodeling, with dilation of elastic pulmonary arteries, medial hypertrophy of elastic and muscular arteries, and right ventricular hypertrophy, is described in all forms of pulmonary hypertension [57]. Lung biopsies from patients with ILD frequently reveal pulmonary artery hypertrophy (Fig. 1). Pulmonary artery

Fig. 1  Lung biopsy in a patient with idiopathic pulmonary fibrosis and pulmonary hypertension. a Background of usual interstitial pneumonia with moderate interstitial fibrosis and a fibroblastic focus (thick arrow). Smaller branch of the pulmonary artery with mild to moderate medial hypertrophy (thin arrow) (hematoxylin–eosin stain, original ×100). b Background of microscopic honeycombing. Pulmonary artery (arrow) with marked medial hypertrophy and intimal fibrosis which significantly narrows the lumen (hematoxylin–eosin stain, original ×100). Photographs courtesy of Dr. Aliya Husain.

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medial and intimal thickening can be a nonspecific finding in ILD in the absence of clinically evident pulmonary hypertension [57]. In IPF, increased numbers of fibroblasts and myofibroblasts and increased extracellular matrix deposition result in advential thickening around pulmonary vessels [17]. Lung explants from 26 patients with IPF undergoing lung transplantation were examined for vascular abnormalities both in areas of dense fibrosis and in relatively preserved lung [58]. In the dense fibrotic areas, more than two thirds of the muscular pulmonary arteries had moderate or greater intimal fibrosis and medial hypertrophy. A similar number of pulmonary arterioles had moderate muscularization. The majority had moderate luminal narrowing by intimal fibrosis of the small pulmonary veins and venules. In architecturally preserved lung zones, occlusion of the pulmonary venules occurred in lung tissue from 17 of 26 patients, with associated findings suggestive of an occlusive venopathy. Plexiform lesions were not seen. In situ thrombosis has been described in small muscular pulmonary arteries consistent with the prothrombotic environment described above [17]. Sarcoidosis and PLCH both may directly involve pulmonary vessels. Granulomas were seen throughout the pulmonary vasculature from large pulmonary arteries to venules, with venous involvement more prominent than arterial involvement in one autopsy study of patients with sarcoidosis [50]. Granulomas were seen more prominently in the veins, with occlusive intimal fibrosis and recanalization resulting in an occlusive venopathy in a study of explanted lungs from patients undergoing lung transplantation for sarcoidosis [51]. No plexiform or thrombotic lesions were noted. In a study of 12 patients with severe pulmonary hypertension from PLCH undergoing lung transplantation, a proliferative arteriopathy with intimal fibrosis and medial hypertrophy with arterial obliteration was noted in 60% of the cases and intimal fibrosis and medial hypertrophy and obliteration of septal pulmonary veins were noted in 75% of patients [29]. In addition, venular obliteration, hemosiderosis, and capillary dilatation similar to veno-occlusive disease were present in one third of the patients. Thus, in striking contrast to the pathologic findings in pulmonary arterial hypertension that is idiopathic, when it is caused by ILD, plexiform lesions are not seen, whereas perivascular fibrosis and an occlusive venopathy may be noted.

6 Clinical Manifestation The development of pulmonary hypertension in patients with ILD is typically not recognized in the early stages, with ­clinical manifestations of pulmonary hypertension mistakenly attributed

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to the underlying ILD. Dyspnea, especially with exertion, is the first and most common symptom of both ILD and pulmonary hypertension. Patients with pulmonary hypertension may also report fatigue and exercise limitation as well as chest pain and palpitations. Complaints of weight gain, increased abdominal girth, or pedal edema suggest volume retention from right-sided heart dysfunction. Findings on physical examination include an elevated jugular venous pulsation from increased right atrial pressure, a right ventricular heave at the left lower sternal border, fixed splitting or a loud pulmonic component of the second heart sound, a right ventricular fourth heart sound, a holosystolic tricuspid regurgitation or early diastolic pulmonic regurgitation murmur, ascites, and lower-extremity edema (Table  3). Many of these findings do not occur until later in the course of the disease. Syncope is concerning for the development of severe pulmonary hypertension. New or worsening arterial oxygen desaturation, especially with exercise, should increase the suspicion of pulmonary hypertension in patients with ILD. The arterial oxygen saturation may be disproportionately reduced compared with the severity of the reduction in lung function. In patients with IPF undergoing lung transplant evaluation, those who had mPAP greater than 25 mmHg had a lower mean arterial oxygen saturation and decreased exercise capacity as measured on a standardized 6-min walk test, compared with those who did not have pulmonary hypertension, with similar total lung capacity and FVC in both groups [22]. In a retrospective study of patients with IPF and pulmonary hypertension from a registry of patients listed for lung transplant, need for supplemental oxygen was as an independent variable associated with pulmonary hypertension, whereas both increased age and carbon dioxide tension were risk factors for severe pulmonary hypertension [23]. Formal evaluation of exercise ability with the 6-min walk test may reveal a decrease in the previously measured walk distance, increased symptoms at the end of the walk, or new or worsened oxygen desaturation in patients who have developed pulmonary hypertension [59].

Table 3  Clinical manifestations of pulmonary hypertension Symptoms   Dyspnea, especially with exertion   Exercise limitation   Syncope Physical examination   Decreased arterial oxygen saturation   Increased jugular venous pulsation   Loud pulmonic component of the second heart sound   Pedal edema Laboratory findings   Increased serum brain natriuretic peptide level   Enlarged pulmonary artery on chest radiograph   Decreased diffusing capacity

83  Pulmonary Hypertension Associated with Interstitial Lung Disease

These findings were also noted in patients with pulmonary hypertension from sarcoidosis [28, 60]. Chest radiography can suggest the diagnosis of pulmonary hypertension but is not particularly sensitive. A chest radiograph finding of the right interlobar pulmonary artery diameter greater than 15  mm on the frontal view suggests pulmonary artery enlargement [37]. Attenuation of peripheral pulmonary vasculature and right ventricular enlargement may also be seen. Electrocardiography may show right-axis deviation and right ventricular hypertrophy. Pulmonary function tests may reveal a disproportionate reduction in the diffusing capacity in patients with ILD or sarcoidosis who develop pulmonary hypertension [54, 61]. Nathan et al. noted a modest association between mean diffusing capacity less than 30% of predicted and a twofold higher prevalence of pulmonary hypertension [54]. There was a trend toward higher prevalence and greater severity of pulmonary hypertension in those with mean FVC more than 70% of that predicted compared with those with FVC less than 40% of predicted again, suggesting that reduction in lung volumes may not correlate with the presence of pulmonary hypertension. In the study by Shorr et al., a low forced expiratory volume in 1 s was a risk factor for severe pulmonary hypertension, perhaps owing to concomitant airflow obstruction [23].

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IPF [62]. Zisman et al. then validated this screening method in a follow-up study of 60 patients with IPF [63]. They found that the estimated mPAP using this equation was within 5 mmHg of the mPAP measured by right-sided heart catheterization 72% of the time [63]. An estimated mPAP greater than 21 mmHg was associated with a sensitivity of 95% and a negative predictive value of 96% for mPAP greater than 25  mmHg measured by right-sided heart catheterization. Percent predicted diffusing capacity, but not FVC, correlated with the presence of pulmonary hypertension [62]. Chest CT scans findings that suggest the presence of pulmonary hypertension include increased size of the main pulmonary artery trunk compared with the aorta, increased pulmonary artery size compared with the accompanying airway, right ventricular enlargement, and leftward shift of the ventricular septum (Fig. 2). In a study of patients with advanced lung disease of many types, the sensitivity of the diameter of the main pulmonary artery 29 mm or greater for

7 Diagnosis Pulmonary hypertension in patients with ILD requires a high index of suspicion and is suggested by increasing dyspnea, worsening hypoxemia, and a disproportionate reduction in the diffusing capacity. Other causes such as left-sided heart disease, valvular heart disease, obstructive sleep apnea, pulmonary embolism, and portal hypertension must be excluded [1]. Pulmonary capillary hemangiomatosis and pulmonary venoocclusive disease can be mistaken for ILD-associated pulmonary hypertension. Nocturnal oximetry should be performed in those without evidence for hypoxemia at rest or with exercise. Chest radiographs, chest CT scans, echocardiograms and serum BNP levels all can suggest the diagnosis but the gold standard remains the measurement of pulmonary artery pressures by right-sided heart catheterization. None of these other diagnostic tests have demonstrated sufficient accuracy to preclude invasive and direct measurement of pulmonary pressures in the majority of patients in whom pulmonary hypertension is suspected to document the presence, assess the severity, and demonstrate improvement with treatment. Although individual measures of lung volumes cannot be used to screen for pulmonary hypertension, Zisman et  al. used the ratio of percent predicted FVC to percent predicted diffusing capacity and room air resting pulse oximetry to develop an equation that predicted mPAP in patients with

Fig.  2  Chest computed tomography (CT) scan findings in a patient with IPF and pulmonary hypertension. a The chest CT scan shows a typical UIP pattern with peripheral reticular interstitial changes, traction bronchiectasis, and honeycombing (arrow). The main pulmonary artery trunk is larger than the aorta. b The same patient with a section through the lung bases. Peripheral pulmonary artery branches are larger than the accompanying airway (circle)

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pulmonary hypertension was 65.9% and increased to 85.7% when combined with the ratio of the diameter of the lobar artery to the diameter of the lobar bronchiole greater than 1 [64]. A recent study of 65 patients with advanced IPF, refuted the claim that pulmonary artery size on chest CT scan could predict the presence of pulmonary hypertension [65]. No significant correlation between main pulmonary artery diameter or the ratio of the main pulmonary artery diameter to aorta diameter and mPAP as measured by right-sided heart catheterization was found. In addition, the degree of pulmonary fibrosis on chest CT scan also did not correlate with pulmonary hypertension in this study. Transthoracic Doppler echocardiography is a noninvasive method to screen patients for pulmonary hypertension. Right ventricular size, wall thickness, and function can also be assessed [66]. If pulmonary outflow tract obstruction is not present, estimated right ventricular systolic pressure measured by echocardiography has been shown to correlate with sPAP measured invasively [67]. Transthoracic Doppler echocardiography detects regurgitation of blood backward across the tricuspid valve during right ventricular systole. The tricuspid regurgitant jet velocity is used to estimate the right ventricular pressure after the addition of an estimate of central venous pressure. Using a modification of the Bernoulli equation, the change in pressure equals 4 times the tricuspid regurgitant jet velocity squared [68]. A value of more than 3.0 m/s correlates reasonably well with elevated right-sided heart pressures measured by catheterization. Significant discordance is not uncommon and the absence of a tricuspid regurgitant jet in 20–30% of cases does not rule out pulmonary hypertension. In a cohort study of 374 lung transplant candidates with advanced lung disease, with a prevalence of pulmonary hypertension of 25%, estimation of the sPAP was possible in 166 patients (44%) [69]. The correlation between sPAP measured by cardiac catheterization and that estimated by echocardiography was good (r = 0.69, P < 0.0001). In 52% of the cases, the pressure estimates were more than 10 mmHg different from the measured pressure and 48% of the patients were misclassified as having pulmonary hypertension by echocardiography. Estimated sPAP was 85% sensitive, 55% specific, and had a positive predictive value of 52% and a negative predicted value of 87% for the diagnosis of pulmonary hypertension. The accuracy was lower in patients with ILD than in patients with obstructive lung disease (56 vs. 37%). In patients with IPF, right-sided heart catheterization was performed in all 40 patients with right ventricular systolic pressure greater than 40  mmHg on echocardiography [70]. Pulmonary hypertension was confirmed in 75% of patients, whereas normal hemodynamics were seen in 12% of patients. The remaining patients had elevated pulmonary venous pressures without pulmonary hypertension. These data suggests that echocardiography cannot replace direct measurement of pulmonary pressures by right-sided heart catheterization.

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There is great interest in a noninvasive biomarker of pulmonary hypertension. It is possible to measure plasma levels of the N-terminal fragment of BNP released from cardiac myocytes during pressure or volume stretch. In pulmonary hypertension, the level of BNP correlates with the degree of pulmonary vascular resistance and risk of death. Two studies have looked at BNP in lung disease. Circulating BNP levels were measured in 176 consecutive patients with a variety of lung diseases [71]. Pulmonary hypertension, defined as mPAP greater than 35 mmHg, was diagnosed in 25% of the patients by right-sided heart catheterization. Elevated BNP concentrations were 85% sensitive and 88% specific for pulmonary hypertension. In another study, BNP concentration predicted moderate to severe pulmonary hypertension with 100% sensitivity and 89% specificity in patients with pulmonary fibrosis, the majority of whom had IPF [59]. Right-sided heart catheterization measures pulmonary artery pressures directly and can detect left-sided heart disease as well. It is the most accurate method of diagnosing pulmonary hypertension and is superior to chest radiography, echocardiography, and serum BNP levels. It has the additional benefit of allowing assessment of the short-term response to therapy as administration of oxygen, nitric oxide, and short-acting vasodilators in the laboratory during catheterization can demonstrate patients who might benefit from therapy and those whose oxygenation may worsen from increased ventilation–perfusion inhomogeneity. In addition, those with diastolic dysfunction may experience pulmonary edema from increased forward flow and thus may not be candidates for chronic vasodilator therapy. It is invasive, however, with risk of infection and bleeding. It is indicated in those with physical examination findings of pulmonary hypertension, elevated sPAP or right-sided heart dysfunction on echocardiography, or an elevated BNP level. A positive response to therapy as measured by right-sided heart catheterization has been defined as a fall in mPAP of 10 mmHg or more to a mPAP of 40 mmHg or less with an unchanged or increased cardiac output [72]. Lung biopsy in patients with ILD may reveal the pathologic findings noted earlier (Fig. 1). This is often performed for the diagnosis of ILD but not for pulmonary hypertension since it is nonspecific except for patients with pulmonary capillary hemangiomatosis and pulmonary veno-occlusive disease. In patients with pulmonary hypertension, there is an increased risk of bleeding with transbronchial biopsy via bronchoscopy and increased morbidity and mortality with surgical lung biopsy.

8 Treatment Once the diagnosis of pulmonary hypertension has been confirmed, a broad and thorough assessment of potential

83  Pulmonary Hypertension Associated with Interstitial Lung Disease

therapy should be begun. Current cigarette smokers need assistance with smoking cessation since this may improve the underlying ILD, especially in patients with PLCH, respiratory bronchiolitis, and desquamative interstitial pneumonia. Accurate diagnosis of the cause of the ILD is essential since treatment depends on the type of ILD that is present. Therapy may include corticosteroids and immune modulators such as azathioprine, mycophenolate mofetil, and cyclophosphamide. In patients with IPF, no therapy has been proven effective, so patients should be referred to a center of excellence for consideration of enrollment in a clinical trial (http://www.clinicaltrials.gov). In addition to treating the parenchymal lung disease, corticosteroids may improve pulmonary hypertension related to sarcoidosis in patients with pulmonary involvement that has not progressed to fibrosis [51, 73]. Methetrexate was used in addition to prednisone in one of these cases [51]. Improvement in symptoms, the chest CT scan appearance of ILD, and pulmonary hemodynamics, with unchanged pulmonary function test findings, was noted in a case of PLCH in response to corticosteroids and smoking cessation [74]. There are case reports of collagen vascular disease related pulmonary hypertension that improved with administration of corticosteroids and intravenous administration of cyclophosphamide [75]. These patients did not have significant parenchymal lung disease, with FVC greater than 60% of predicted and chest CT scans that were normal or showed at most mild ILD. This is consistent with the view that an inflammatory vasculopathy may contribute to the pulmonary hypertension in these disease states. In all patients with pulmonary hypertension from ILD, assessment of oxygen saturation at rest, with exercise, and at night should be performed with supplemental oxygen given to keep saturation above 89% at all times. Oxygen is considered a mainstay of therapy, in keeping with the view that hypoxic vasoconstriction contributes to the pulmonary hypertension, although unlike COPD, studies have not been done to confirm the clinical benefit. It is important to treat both left-sided and right-sided heart failure if they are present. Diuretics are given for volume overload and may help decrease right ventricular dilation; however, care must be taken to avoid decreasing right ventricular preload such that cardiac output declines and results in hypotension. There is no agreement on the use of digitalis for right ventricular failure. In the last decade, there has been an explosion of vasomodulator therapies for patients with isolated pulmonary arterial hypertension (see Chap. 68). These include the prostacyclin analogues epoprostenol, treprostinil, and iloprost, the nonselective endothelin A/B receptor antagonist bosentan, the selective endothelin A receptor antagonists ambrisentan and sitaxsentan, and the phosphodiesterase-5 inhibitor sildenafil. There are theoretical issues with their use in patients with chronic lung disease [76, 77]. These agents

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increase forward flow, which may worsen diastolic dysfunction. This is of particular concern in patients with pulmonary venous disease such as pulmonary veno-occlusive disease, which is sometimes mistaken for an ILD, especially in the early stages. Pulmonary vasodilators also may result in flow through lung units with abnormal ventilation–perfusion ratios, thereby worsening hypoxemia. Intravenously administered prostacyclin has been shown to both decrease arterial oxygen tension and increase pulmonary right-to-left shunt flow [76, 77]. In deciding whether to use these agents in a given patient, it is important to assess the severity of the symptoms, exercise limitation, and degree of pulmonary hypertension. Hemodynamic assessment of a positive response to therapy is encouraging but not as routinely evaluated in patients with ILD as in patients with pulmonary arterial hypertension. There have been no randomized, placebo-controlled trials of vasomodulator therapy in patients with pulmonary hypertension from ILD. As in most studies of therapy in patients with pulmonary arterial hypertension, benefit has been defined by short-term end points that may not be markers for improved mortality [78, 79]. Clinical trials of vasodilators in pulmonary hypertension have shown an increased 6-min walk distance but little is known about how these drugs affect the underlying disease [79]. Case series and small clinical trials evaluating vasomodulator therapy in patients with pulmonary hypertension from ILD have shown clinical improvement with modest decreases in pulmonary pressures (Table 4). These medications improve exercise capacity as measured by 6-min walk distances but their effect on survival is not known. In addition to their vasoactive properties, these medications may also act through antiproliferative effects on the vasculature and interstitium. Intravenously administered epoprostenol (prostacyclin) has been shown to improve survival in patients with idiopathic pulmonary arterial hypertension compared with historical control subjects in two large long-term observational series [72]. In a study of eight patients with pulmonary hypertension from pulmonary fibrosis from a variety of causes, including six with ILD, the hemodynamic effects of intravenously administered prostacyclin, inhaled nitric oxide, and aerosolized prostacyclin were compared with the effects of oxygen and the calcium antagonist nifedipine [77]. Aerosolized prostaglandin caused a decrease in mPAP without affecting blood pressure, arterial oxygen saturation, or pulmonary shunt fraction. Both intravenously administered prostacyclin and nifedipine resulted in a fall in systemic blood pressure with a significant increase in shunt flow from the prostacyclin infusion. One patient received inhaled longacting prostacyclin (iloprost) with a dramatic improvement in clinical status. A retrospective chart review of all cases of pulmonary hypertension from sarcoidosis at one institution revealed eight patients who had received epoprostenol intravenously, four of whom had stage IV pulmonary sarcoidosis

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M.E. Strek and J. Solway Table 4  Summary of treatment of pulmonary hypertension in ILD Authors Year Disease Medication

Outcomea

Olschewski et al. [77] 1999 ILD Inhaled prostacyclin ↓ mPAP, CI Fisher et al. [80] 2006 Sarcoid Epoprostenol ↓ PVR, CI King et al. [82] 2008 IPF Bosentan No benefit Madden et al. [88] 2006 Chronic lung disease Sildenafil ↓ PVR, ↑ 6-min walk Ghofrani et al. [89] 2002 ILD Sildenafil ↓ PVR, ↑ PaO2 Collard et al. [90] 2007 IPF Sildenafil ↑ 6-min walk Milman et al. [90] 2008 Sarcoid Sildenafil ↓ mPAP, no change 6-min walk mPAP mean pulmonary arterial pressure, CI clinical improvement, PVR pulmonary vascular resistance a Most of these studies involve unblinded data from small case series.

with fibrosis [80]. Six of the seven patients who underwent a vasodilator trial had a more than 25% reduction in pulmonary vascular resistance, with five of the six patients continuing on therapy with significant clinical improvement. Bosentan has been shown to be beneficial in patients with pulmonary arterial hypertension [81]. A randomized controlled trial of bosentan in patients with IPF, the BUILD-1 study, showed no benefit in the primary end point of 6-min walk distance [82]. The trial excluded patients with severe pulmonary hypertension defined as sPAP of 50  mmHg or greater on echocardiography. In a post hoc analysis, bosentan delayed the time to death or disease progression in a subgroup of patients diagnosed with IPF by pathologic findings on surgical lung biopsy. Improvement in quality-of-life scores generally favored those treated with bosentan. Elevations in aminotransferase levels were noted in 20% of the bosentan group, so regular monitoring of liver function tests in patients taking these medications is advised. The BUILD-3 study, a larger investigation of the effect of bosentan on time to occurrence of disease worsening or death in patients with IPF did not show an improvement in their primary end points in the treated group (http://www.clinicaltrials.gov). A case report of the use of bosentan in a patient with sarcoidosis with moderate parenchymal lung disease (FVC 64% of the predicted value) but severe pulmonary hypertension (mPAP 55 mmHg) noted marked improvement in symptoms, functional class, and hemodynamics that was sustained over 2 years time [83]. Sitaxsentan has recently been studied for use in patients with pulmonary arterial hypertension [84]. It has a theoretical advantage over bosentan since it is a selective antagonist of endothelin A receptor. In patients with pulmonary arterial hypertension that was idiopathic, related to a collagen vascular disease without ILD or from congenital heart disease, an increased 6-min walk distance as well as improved functional class and pulmonary vascular resistance were noted. Elevated aminotransaminase levels more than 3 times the normal level were noted in 10% of the group receiving 300 mg, in 0% of the group receiving 100 mg, and in 3% of the patients receiving a placebo [84]. Sildenafil improves exercise capacity, functional class, and hemodynamics in patients with symptomatic pulmonary arterial hypertension [85]. It inhibits hypoxia-induced

p­ ulmonary hypertension in humans, mice, and rats [86, 87]. A study of sildenafil in seven patients with pulmonary hypertension from chronic lung disease, three of whom had IPF, demonstrated a decrease in pulmonary vascular resistance and an increase in 6-min walk distance after treatment [88]. In 16 patients with lung fibrosis, sildenafil caused preferential pulmonary vasodilation and improved gas exchange, whereas intravenously administered epoprostenol increased ventilation–perfusion mismatch and decreased arterial oxygenation [89]. Sildenafil improved the 6-min walk distance in patients with documented pulmonary hypertension from IPF [90]. Recent study in patients with severe IPF who have a DLCO of less than 35% of the predicted value did not show a difference in the primary outcome which was the proportion of patients with an increase in the 6-min walk distance of 20% or more in the group taking sildenafil compared with placebo. There was a difference in many secondary outcomes, including arterial oxygenation, DLCO, degree of dyspnea and quality of life in the group taking sildenafil versus placebo. (http://www.clinicaltrials.gov). Sildenafil was given to a group of patients with pulmonary hypertension from sarcoidosis who were referred for lung transplantation [91]. Hemodynamic improvement as assessed by right-sided heart catheterization was noted without a change in 6-min walk distance. A case of pulmonary hypertension from PLCH responding to vasodilator therapy is described, but the cases series does not specify which vasodilator this patient was given [92]. Warfarin is recommended in patients with idiopathic pulmonary arterial hypertension but it is not routinely given to patients with pulmonary hypertension from ILD [72]. In a widely cited study of patients hospitalized in Japan for IPF, improved survival was noted in those who received prednisolone and either warfarin or low molecular weight heparin compared with those who received prednisolone alone [93]. This study was unblinded and routine assessment of the presence of pulmonary hypertension was not performed, thus limiting the applicability of this study. A recent study of vascular disease in patients with IPF which showed an increased risk of acute coronary artery syndrome and deep vein thrombosis provides insight into why anticoagulation might improve survival in IPF [94].

83  Pulmonary Hypertension Associated with Interstitial Lung Disease

Lung transplantation is considered for patients with advanced ILD who are otherwise healthy. It reduced the risk of death from IPF by 75% in a French study of 46 patients with end-stage disease [95]. In patients with IPF who underwent a single lung transplant, there was a linear increase in risk of 90-day mortality with increasing mPAP up to 35  mmHg, above which there were too few patients to ­estimate the relative risk [96]. Five patients with moderate pulmonary hypertension from end-stage fibrotic lung disease, four of whom had IPF, had improvement in mPAP from 33 to 18  mmHg and right ventricular function following single lung transplantation [97]. Magnetic resonance imaging showed a significant reduction in right ventricular enddiastolic and end-systolic volume indices as well as an expanded left ventricular end-diastolic volume index following single or double lung transplantation in patients with chronic lung disease [98]. Lung transplantation has been successful in patients with pulmonary sarcoidosis, with survival rates generally comparable to those for other indications for lung transplantation [99]. In a study of patients who underwent lung transplantation for PLCH, 92% of whom had pulmonary hypertension, survival was similar to that for other chronic lung diseases [31].

9 Complications and Prognosis The development of pulmonary hypertension in patients with ILD portends a worse prognosis. Numerous studies have demonstrated that patients with pulmonary hypertension in the setting of ILD have increased symptoms, decreased exercise ability, decreased oxygen saturation, reduced quality of life, and decreased survival compared with those without pulmonary hypertension. A subset of patients have a very rapid decline once clinical signs of pulmonary hypertension become apparent, such that patients who are candidates for lung transplantation should be referred for evaluation at this point regardless of the severity of the ILD. In a study of patients with advanced IPF undergoing lung transplant evaluation, an elevated mPAP of more than 25 mmHg was associated with a 1-year mortality rate of 28% compared with 5.5% in patients without pulmonary hypertension [22]. In a study of the effect of pulmonary hypertension on survival in patients with IPF, patients were more likely to die with a hazard ratio of 1.34 for each 10-mmHg increase in sPAP [100]. A median survival of 0.7 years was seen in the patients with a right ventricular systolic pressure on echocardiography of more than 50 mmHg compared with 4.1 years in those with lower pressures. The 5-year survival of patients with IPF was 62.2% in the group with mPAP below 17 mmHg but 16.7% in the group with mPAP above 17 mmHg [21]. An elevated serum BNP level identified the

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presence of pulmonary hypertension and predicted mortality in patients with chronic lung disease [71]. In patients with sarcoidosis awaiting lung transplantation, mPAP was a predictor of survival [101]. Complications of severe pulmonary hypertension include right-to-left shunt in patients with a patent foramen ovale and refractory hypoxemia despite receiving high-flow ­supplemental oxygen. Vasodilator therapy may decrease cardiac output as well as pulmonary artery pressures and cause increased perfusion of fibrotic lung that may worsen gas exchange. It is important to consider appropriate palliative care for those with ILD and severe pulmonary hypertension not responsive to medical therapy or who are not candidates for lung transplantation [102].

10 Summary Pulmonary hypertension is increasingly recognized as a common and serious complication of ILD. It is especially prevalent in the later stages of IPF, sarcoidosis, and PLCH, although precise determination of the prevalence is difficult as most studies are retrospective and involved highly selected populations. In addition, diagnosis of pulmonary hypertension is often made by echocardiograpy except in the patients with advanced ILD undergoing lung transplant evaluation, where pulmonary artery pressures were directly measured during right-sided heart catheterization. ILD leads to pulmonary hypertension by causing alveolar hypoxia which results in hypoxic vasoconstriction and destruction of the pulmonary capillary bed increases pulmonary vascular resistance. There is emerging evidence for vascular remodeling from chronic hypoxia, as well as inflammation and fibrosis. The effects on the pulmonary vasculature may be a by-product of the parenchymal lung disease but may also reflect primary vascular involvement which is seen in collagen vascular diseases, sarcoidosis, and PLCH. Histology studies have demonstrated pulmonary venous involvement in sarcoidosis and PLCH, and perhaps IPF, as well as muscularization of the pulmonary arterial bed and capillary destruction. In situ thrombosis seen in some case series of pathologic findings in IPF suggests that a microscopic angiopathy may contribute. Diagnosis relies on an understanding of both the epidemiology and the pathophysiology of pulmonary vascular disease in patients with ILD and a strong clinical suspicion. The principal symptoms of dyspnea and exercise limitation are also seen in ILD; therefore recognition that pulmonary hypertension has developed requires a careful physical examination and further laboratory testing. An elevated right ventricular systolic pressure associated with right

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ventricular dysfunction on echocardiography or an elevated serum BNP level suggests the diagnosis. In most cases, direct measurement of pulmonary pressures by right-sided heart catheterization is required to confirm the diagnosis, exclude left ventricular or valvular heart disease, and assess the acute response to therapy. Studies of the positive and negative ­predictive values of an abnormal echocardiogram combined with an abnormal BNP level might result in a noninvasive method of diagnosis of pulmonary hypertension in patients with ILD in the future. Treatment is directed at the underlying parenchymal lung disease. Oxygen is given to correct resting, exercise, and nocturnal hypoxemia, although it has not been proven to reverse pulmonary hypertension in this setting or improve outcome. The numerous vasomodulators approved for use in pulmonary arterial hypertension have been studied in small case series and a few clinical trials in patients with ILD. Some show improvement by decreasing mPAP and increasing the 6-min walk distance, but no effect on disease progression or mortality has been noted. Future studies in selected populations and with combination therapy may yet demonstrate benefit. Treatment to prevent the development of pulmonary hypertension in ILD and decrease its severity is likely to have a significant positive effect on mortality in patients with ILD.

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1210 76. Ryu JH, Krowka MJ, Pellikka PA et  al (2007) Pulmonary hypertension in patients with interstitial lung diseases. Mayo Clin Proc 82:343–350 77. Olschewski H, Ghofrani HA, Walmrath D et  al (1999) Inhaled prostacyclin and iloprost in severe pulmonary hypertension secondary to lung fibrosis. Am J Respir Crit Care Med 160:600–607 78. Ghofrani HA, Wilkins MW, Rich S (2008) Uncertainties in the diagnosis and treatment of pulmonary arterial hypertension. Circulation 118:1195–1201 79. Rich S (2006) The current treatment of pulmonary arterial hypertension. Chest 130:1198–1202 80. Fisher KA, Serlin DM, Wilson KC et  al (2006) Sarcoidosisassociated pulmonary hypertension. Chest 130:1481–1488 81. Rubin LJ, Badesch DV, Barst RJ (2002) Bosentan therapy for pulmonary arterial hypertension. N Engl J Med 342:892–903 82. King T, Behr J, Brown KK (2008) BUILD-1: a randomized placebocontrolled trial of bosentan in idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 177:75–81 83. Foley RJ, Metersky ML (2008) Successful treatment of sarcoidosis-associated pulmonary hypertension with bosentan. Respiration 75:211–221 84. Barst RJ, Llangleben D, Frost A et al (2004) Sitaxsentan therapy for pulmonary arterial hypertension. Am J Respir Crit Care Med 169:441–447 85. Galie N, Ghofrani HA, Torbicki A et al (2005) Sildenafil citrate therapy for pulmonary arterial hypertension. N Engl J Med 353: 2148–2157 86. Zhao L, Mason NA, Morrell NW et  al (2001) Sildenafil inhibits hypoxia-induced pulmonary hypertension. Circulation 104:424–428 87. Sebkhi A, Strange JW, Phillips SC et al (2003) Phosphodiesterase type 5 as a target for the treatment of hypoxia-induced pulmonary hypertension. Circulation 107:3230–3235 88. Madden BP, Allenby M, Loke T-K et al (2006) A potential role for sildenafil in the management of pulmonary hypertension in patients with parenchymal lung disease. Vasc Pharmacol 44:372–376 89. Ghofrani HA, Wiedemann R, Rose F et  al (2002) Sildenafil for treatment of lung fibrosis and pulmonary hypertension: a randomized controlled trial. Lancet 360:895–900

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Chapter 84

High-Altitude Pulmonary Hypertension Fabiola León-Velarde and Francisco C. Villafuerte

Abstract  Life at high altitude challenges the organism to cope with the environmental and physiological stress of hypoxia, the low ambient O2 partial pressure that results from the drop in barometric pressure as altitude increases. The physiology of high altitude residents responds to this situation in an attempt to maintain a normal cellular oxygenation through numerous mechanisms. Among these, the increase in hematocrit and hemoglobin concentration, and the elevation of pulmonary artery pressure (PAP) represent hallmark mechanisms in the response to high-altitude chronic hypoxia directed to increase blood O2 carrying capacity and redistribute blood to optimally ventilated lung areas, respectively. A large or excessive increase in hemoglobin concentration and/or in PAP, however, is associated with potentially fatal illnesses such as chronic mountain sickness (CMS), or Monge’s disease, and high-altitude pulmonary hypertension (HAPH). Rotta et al. were the first to describe hemodynamics in one CMS patient in the Peruvian Andes at 4,540 m. This patient showed a mean PAP of 35 mmHg, arterial blood oxygen saturation (SaO2) of 75%, and a hemoglobin concentration of 26 g/dL. Some years later, Peñaloza et al. reported their hemodynamic observations in ten cases of CMS at 4,300 m. In these studies they found mean values of PAP of 47 ± 17.7 mmHg, SaO2 of 69.6 ± 4.9%, and hemoglobin concentration of 24.7 ± 2.4 g/dL. At that time, the authors described these patients as having a type of hypoxic cor pulmonale, in contrast to the hypertensive cor pulmonale observed in primary pulmonary hypertension or idiopathic pulmonary arterial hypertension. Historical terms used in different mountainous regions for this high-altitude disease, which has excessive elevation of PAP as a common characteristic, are CMS of vascular type, high-altitude heart disease, hypoxic cor pulmonale, infant subacute mountain sickness, pediatric high-altitude heart disease, and adult subacute mountain sickness. F. León-Velarde (*) Laboratorio de Fisiologia Comparada-LID, Department of Biological Sciences and Physiology, Universidad Peruana Cayetano Heredia, Avenue Honorio Delgado 430, 13 Lima, Peru e-mail: [email protected]

Keywords  Mountain sickness • Monge’s disease • Chronic hipoxia • Pediatric high-altitude heart disease • Hypoxic pulmonary vasoconstriction

1 Introduction Life at high altitude challenges the organism to cope with the environmental stress of hypoxia. Several physiological responses are triggered in an attempt to maintain a normal cellular oxygenation through numerous mechanisms. Among these, the increase in hematocrit and hemoglobin concentration, and the elevation of pulmonary artery pressure (PAP) represent hallmark mechanisms in the response to high-altitude chronic hypoxia directed to increase blood O2 carrying capacity and redistribute blood to optimally ventilated lung areas, respectively [1–4]. A large or excessive increase in hemoglobin concentration and/or in PAP, however, is associated with potentially fatal illnesses such as chronic mountain sickness (CMS), or Monge’s disease, and high-altitude pulmonary hypertension (HAPH) [5–10]. Rotta et al. [11] were the first to describe hemodynamics in one CMS patient in the Peruvian Andes at 4,540 m. This patient showed a mean PAP of 35 mmHg, arterial blood oxygen saturation (SaO2) of 75%, and a hemoglobin concentration of 26  g/dL. Some years later, Peñaloza et  al. reported their hemodynamic observations in ten cases of CMS at 4,300 m [7, 8]. In these studies they found mean values of PAP of 47 ± 17.7 mmHg, SaO2 of 69.6 ± 4.9%, and hemoglobin concentration of 24.7 ± 2.4 g/dL. At that time, the authors described these patients as having a type of hypoxic cor pulmonale, in contrast to the hypertensive cor pulmonale observed in primary pulmonary hypertension or idiopathic pulmonary arterial hypertension. Historical terms used in different mountainous regions for this high-altitude disease, which has excessive elevation of PAP as a common characteristic, are CMS of vascular type, high-altitude heart disease, hypoxic cor pulmonale, infant subacute mountain sickness, pediatric high-altitude heart disease, and adult subacute mountain sickness.

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_84, © Springer Science+Business Media, LLC 2011

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2 Etiology The cause of HAPH is the development of vascular resistance; the underlying mechanism involves sustained and exaggerated pulmonary vasoconstriction and remodeling of the pulmonary vascular wall in response to chronic hypoxia due to proliferation and decreased apoptosis of pulmonary artery smooth muscle cells (PASMCs) in the tunica media. The effects of chronic hypoxia on PAP are reflected in the negative correlation that exists between PAP and SaO2 at high altitude [12, 13]. Arterial hypoxemia, in addition to hypoxic vasoconstriction, leads to several structural changes in the pulmonary vessels, including an increase in the number of intimal smooth muscle cells, medial thickening in the small muscular arteries, and fibrinoid necrosis of the vessel walls [13, 14]. These structural changes in the pulmonary vessels, rather than hypoxic vasoconstriction alone, are believed to be responsible for the sustained rise in PAP in chronic hypoxia. These changes can have different intensities and different onset times depending on the length of hypoxic exposure, comprising the full range from acute to chronic. Ca2+ channels have a central role in the development of increased pulmonary vascular resistance during chronic hypoxia [15–18]. These channels mediate Ca2+ influx into PASMCs, increasing cytosolic Ca2+ concentration and contributing to sustained vasoconstriction and vascular remodeling. Studies have shown that prolonged exposure to hypoxia upregulates the expression of two types of voltage-independent Ca2+ channels in PASMCs: store-operated calcium channels (SOCs) and receptor-operated calcium channels [15]. The enhanced expression of these channels during chronic hypoxia contributes to elevate cytosolic Ca2+ concentrations, which in turn stimulates PASMC hypertrophy and proliferation, leading to the enhanced vascular tone observed in hypoxic pulmonary hypertension. Upregulation of SOC expression in pulmonary artery endothelial cells (PAECs) may also play an important role in the development of chronic pulmonary hypertension during life at high altitude. Although PAECs do not contribute directly to vasoconstriction or medial hypertrophy, they play a key role in the modulation of vascular tone due to the production of several vasoactive substances such as endothelin-1 (ET-1), nitric oxide (NO), thromboxane A2, platelet-derived growth factor (PDGF), and vascular endothelial growth factor (VEGF), derived from the intimae of the pulmonary vessels [19]. It has been shown that chronic hypoxia enhances the expression of TRPC4 (a SOC) and increases cytosolic Ca2+ concentration. TRPC4 activation in PAECs stimulates proliferation and the increased cytosolic Ca2+ concentration promotes synthesis of growth and vasoactive factors [20]. Additionally, prolonged exposure to hypoxia and increased cytosolic Ca2+ concentration enhances the expression and DNA-binding activity of the transcription-factor-activating

F. León-Velarde and F.C. Villafuerte

proteins (APs)-1 in PAECs. It is known that many genes modulated by APs-1 encode for endothelium-derived mitogens such as ET-1, PDGF, and VEGF. The increased synthesis and release of these compounds can result in their vaconstrictor and mitogenic effect on PASMCs via a paracrine mechanism, therefore contributing to pulmonary vasoconstriction and hypertrophy of the medial layer. Prolonged exposure to a hypoxic environment also modulates the expression of voltage-dependent K+ channels in PASMCs [21–23]. Downregulation of voltage-dependent K+ channels results in K+ current (IKv) reduction and membrane depolarization [24, 25]. The change toward a more positive membrane potential plays a key role in modulating pulmonary medial hypertrophy by promoting proliferation and by attenuating apoptosis of PASMCs. This is mainly achieved by two pathways. Depolarization due to decreased IKv causes Ca2+ influx through voltage-dependent Ca2+ channels and Ca2+ release from intracellular stores [26]. The resulting increase in cytosolic Ca2+ concentration modulates vascular tone and constitutes a triggering signal for cell proliferation [27]. In addition, since K+ efflux is a prerequisite for apoptotic cell shrinkage, reduction of IKv attenuates apoptosis of PASMCs by slowing down this process, and also by inhibiting cytoplasmic caspases (Fig. 1) [28]. Membrane depolarization can also modulate cytosolic Ca2+ concentration by altering the activity of the Na+/Ca2+ exchanger [29–33]. Under resting conditions, this transporter operates in forward mode, extruding Ca2+ from the cell in exchange for Na+, contributing to the maintenance of low basal cytosolic Ca2+ concentration. Because of its electrogenic nature (three Na+/Ca2+), under conditions where the cells are depolarized, or when the cytosolic Ca2+ concentration and/or Na+ concentration are altered, the activity of the exchanger slows down, resulting in less extrusion of Ca2+. Under certain conditions it can also operate in reverse mode, resulting in Ca2+ influx. Indeed, reverse-mode Na+/Ca2+ exchange has been described in PASMCs following activation of SOCs [33], whereas acute hypoxia was found to decrease forward-mode operation, resulting in diminished Ca2+ efflux [32]. Inhibiting the reverse mode of the Na+/Ca2+ exchanger in chronically hypoxic PASMCs has been shown to decrease cytosolic Ca2+ concentration [34]. Increase in cytosolic Ca2+ concentration during chronic hypoxia could also be the result of changes in the rate of Ca2+ uptake and release from sarcoplasmic reticulum. Ca2+ release is mediated by ryanodine receptors and inositol trisphosphate receptors, and several subtypes have been identified in PASMCs [35–38]. It has been demonstrated that acute hypoxia results in Ca2+ release in both PASMCs and PAECs [37–42], and that this release in PASMCs appears to occur in part via activation of ryanodine receptors [38, 39]. Thus, it is possible that these mechanisms also play an important role during chronic hypoxia.

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globin concentration and PAP (r = 0.64; p < 0.001) observed in healthy high-altitude subjects is lost, suggesting that erythrocytosis-associated increased blood viscosity is not the predominant cause of HAPH in high-altitude residents with CMS [50]. In addition, when the age-related increase of the logarithms of PAP, hematocrit, and viscosity are calculated, PAP has a steeper slope than hematocrit and viscosity, which indicates that viscosity is a minor contributor to HAPH, when compared with the important role of the medial hypertrophy of small pulmonary arteries and muscularization of the pulmonary arterioles [51].

3 Risk Factors and Genetics

Fig. 1  Role of Ca2+ and K+ channels in chronic hypoxia-induced pulmonary vascular remodeling. Chronic hypoxia downregulates Kv channel expression and upregulates TRPC expression in pulmonary artery smooth muscle cells (PASMCs). The resultant decrease in Kv currents causes membrane potential depolarization, opens voltage-dependent Ca2+ channels, and increases cytosolic Ca2+ concentration, whereas the increase in store-operated and receptor-operated calcium channel activity enhances voltage-independent capacitative calcium entry and raises cytosolic Ca2+ concentration, causing sustained pulmonary vasoconstriction and stimulating PASMC proliferation. The decrease in the expression of sarcolemmal K+ channels decreases transmembrane K+ efflux, which then increases cytosolic K+ concentration and thus reduces apoptosis. The increased PASMC proliferation and decreased apoptosis results in pulmonary vascular remodeling and consequently in sustained pulmonary hypertension. ET-1 endothelin-1; HIF-1 hypoxia-inducible factor-1; PDGF platelet-derived growth factor. (Reprinted with permission from Weir and Olschewski [106])

The development of pulmonary vascular resistance due to vasoconstriction, and the subsequent vascular proliferation and remodeling, has also been attributed to increased blood viscosity. Erythrocytosis, due to exposure to chronic hypoxia, increases blood viscosity, which has been suggested to bring about a rise in PAP in cor pulmonale in the case of chronic obstructive pulmonary disease [43]. In many CMS patients, however, either elevated hemoglobin concentration and HAPH are present concurrently [7, 8] or there is little or no elevation of PAP beyond the normal increase due to high-altitude hypoxic exposure [44, 45]. Alternatively, severe HAPH may occur with little or no increase in hemoglobin concentration as has been described frequently in children and young adults (subacute mountain sickness) [46–48], and also in older adults [49]. This actually means that at high altitude in a given patient, excessive erythrocytosis and excessive PAP may or may not coexist. In cases where elevated hemoglobin concentration and HAPH coexist, it is reasonable to assume that both excessive erythrocytosis and low blood O2 saturation would contribute to HAPH. In CMS, however, the correlation between hemo-

A number of factors are known to influence the development of HAPH. These include altitude, age, sleep, and genetic susceptibility. The prevalence of PAP increases with altitude [12, 13] (Fig. 2), as well as with age [51] (Fig. 3). Both the decrease in SaO2 with altitude and the decrease of pulmonary function with age, which in turn also decreases SaO2, seem to explain the changes observed in PAP in altitude-sensitive populations. Sleep disorders at high altitude also contribute to increase PAP (more than 10  mmHg) in patients with CMS, and are also related to the decrease of SaO2 during the night [52, 53]. HAPH leads to right ventricular hypertrophy, which may be a cause of right ventricular failure [54]. The degree of rightsided heart hypertrophy is a direct function of altitude and age [55]. However, right ventricular hypertrophy is greater in high-altitude children than in adults [56, 57], and this is due to the persistent high PAP that exists for some time after birth, and in some cases is due to the persistent elevated PAP during infancy (see Sect.7 ). HAPH occurs mostly in males. Women have a lower prevalence than men and develop CMS mainly after the menopause [58, 59]. Several factors may be responsible for this gender difference, but it has been demonstrated that the main cause is the stimulating effect of female sex hormones on ventilation [60]. As women are less hypoxemic than men, they tend to have a lower vasoconstrictor and vascular remodeling response of small muscularized pulmonary vessels. The magnitude of hypoxic pulmonary vasoconstriction, either in sea-level or in high-altitude natives, is highly variable, probably owing to differences in the acclimatization process, risk factors, and/or genetics [61]. At high altitude, the variation in PAP could be as great as 12–60 mmHg, but normally, in approximately 80% of the cases, PAP is between 20 and 40 mmHg. In around 10% of the cases the values are higher that 40 mmHg. This fact has led some authors to suggest the existence of two different populations regarding their PAP response to hypoxia: the hyporesponders and the

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Fig. 2  Variation of mean pulmonary artery pressure (mPAP; mmHg) as a function of altitude and arterial oxygen saturation (SaO2; %). (Reprinted from Wu [13], with permission)

Fig. 3  Variation of mPAP (mmHg) as a function of age. (Reprinted with permission from Monge-C et al. [51])

h­ yperresponders, the hyperresponders being more prone to develop HAPH [4, 62, 63] (Fig. 4). In the Andean countries of South America, approximately 35 million people live above 2,500 m, approximately 15 million in North America, Mexico, and the western USA, and in Asia, approximately 80 million people live above this altitude [64]. It is estimated that between 5 and 35% of highaltitude inhabitants may develop CMS or HAPH [13, 65]. Ethnic differences and familial clustering are suggestive of a genetic involvement in HAPH. In addition, as observed in different populations, large interindividual differences exist in the PAP response to hypoxia, with some subjects demonstrating a low or exaggerated response. These features could imply

Fig.  4  Individual variation of mPAP (mmHg) in normoresponders (52.5%), hyperresponders (24.5%), and patients with high-altitude pulmonary hypertension (HAPH; 47.5%) in Kyrgyz highlanders. *p < 0.05 compared with the baseline. (Reprinted from Aldashev et al. [62], with permission)

a genetic susceptibility to HAPH. In fact, it has been shown that the level of exhaled NO is twofold higher in Tibetans than in lowlanders, which may explain the low mean PAP values in Tibetans [66], which correlates in turn with the lack of smooth muscle in small pulmonary arteries of Tibetans [67]. Research on the genetic susceptibility of HAPH has included studies on the hypoxic regulation of the activity and expression of endothelial nitric oxide synthase (eNOS), angiotensin-converting enzyme (ACE), and b-2 adrenergic receptors (BAR2). Also, the association of polymorphisms in these genes with HAPH has been studied. These genes

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code for proteins that modulate vascular mechanical force and vascular remodeling, and might be involved in the pathogenesis of HAPH [62, 63, 68]. In fact, Aldashev et al. [62] have shown that in 125 Kyrgyz highlanders that underwent right-sided heart catheterization, HAPH is associated with a higher frequency of Gln27Gln genotype of the BAR2 gene (0.56 vs. 0.41, p < 0.04) and of I/I genotype of the ACE gene (0.52 vs. 0.26, p < 0.003), but is not associated with changes in the frequency of GG polymorphism of the eNOS gene. Despite these interesting results, published data to date are insufficient for a rigorous test of any hypothesis regarding the implication of gene polymorphisms in HAPH.

4 Pathophysiology The physiological response of the pulmonary circulation to hypoxia is to enhance pulmonary arteriolar resistance which follows an arterial vasoconstriction. When excessive, however, the elevated PAP observed during exposure to hypoxia has been reported to cause congestive right-sided heart failure within months or years in newcomers, and also in high-altitude residents [7, 46]. In unacclimatized and susceptible climbers, increased PAP can cause high-altitude pulmonary edema [69]. The location of the hypoxic pulmonary vasoconstriction is small pulmonary arterioles of a diameter less than 900 mm; however, the veins also account for approximately 20% of the total increase in pulmonary vascular resistance [70–72]. The remodeling of small muscularized pulmonary vessels begins

Fig. 5  Proposed scheme for the pathophysiology of HAPH. PAECs pulmonary artery endothelial cells, NO nitric oxide, VEGF vascular endothelial growth factor, ET-1 endothelin-1, TXA2 thromboxane A2

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within hours of hypoxic exposure, and further remo­deling of the precapillary pulmonary vessels is probably crucial during prolonged hypoxia of months or years to achieve a homogenous vasoconstrictor response, and to protect pulmonary capillaries from overperfusion [73, 74]. On the other hand, however, thickening of the pulmonary arteries increases pulmonary vascular resistance, which in turn causes PAP to increase and, eventually, the right ventricle to fail (Fig. 5). In children and animal models with severe hypoxic pulmonary hypertension, the presence of collagen deposits has been described, and also elastin in the extracellular matrix, including the adventitia. This reduces the diameter of the vascular lumen, and increases the stiffness of the pulmonary tree [48, 75]. This explains the elevated pulmonary vascular resistance and vascular tone, as well as the gradual reversion of the increased PAP and vascular hypertrophy once the hypoxic stimulus is over [76, 77]. The increased thickness of medial pulmonary arteries seems to predict the development of pulmonary artery hypertension, and may underlie the concept of pulmonary artery disease as the fundamental pathogenic factor [78]. Remodeling and thickening of the pulmonary vascular wall seems to play a more prominent role than pulmonary artery vasoconstriction in the development of chronic and subacute increased PAP, in opposition to acute hypoxic pulmonary hypertension [79]. Indeed, pulmonary vasoconstriction is an immediate response to hypoxia but with sustained hypoxic exposure, hypoxia-sensitive species develop increased thickness of medial pulmonary arteries, whereas hypoxia-tolerant ones do not [78].

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5 Clinical Manifestations and Complications The first symptoms associated with HAPH are fatigue, insomnia, dizziness and cognitive dysfunction, slowed mental function, confusion, and impaired memory. Clinical examination reveals tachypnea, tachycardia, stasis of the jugular veins, cyanosis of the face and fingers, and clubbing of the fingers; hepatomegaly and ascites are present in the late stage of the disease. Arterial blood gas analysis should be performed to confirm hypoxemia. Chest radiography shows enlargement of the heart, dilatation of the pulmonary trunk and general dilatation of the small lung vessels, prominent vascular pedicles, but no pulmonary infiltrates. The electrocardiogram evidences right-axis deviation of QRS, clockwise rotation of the ventricles, marked right ventricular hypertrophy, and normal dimensions and ejection fraction of the left ventricle [7, 8, 10, 50, 59, 63, 80, 81, 107]. Echocardiography is the most useful imaging modality for detecting pulmonary hypertension and excluding underlying cardiac disease. Confirmation of pulmonary hypertension is based on identification of tricuspid regurgitation. The addition of mean right atrial pressure to the peak tricuspid jet velocity gives an accurate noninvasive estimate of peak pulmonary pressure. Echocardiography also reveals signs of right ventricular hypertrophy. Episodes suggestive of right-sided heart failure with dyspnea, cough, turgid jugular veins and peripheral edema could be present in association with excessively elevated PAP. These symptoms and signs suggest a loss of acclimatization in individuals born or living at high altitude for years.

6 Diagnosis Healthy Amerindian natives of South America have an average PAP of 20 mmHg at altitudes between 3,800 and 4,200 m and approximately 28 mmHg at 4,500 m [4]. At 3,100 m in Leadville, Colorado, the mean PAP in healthy men is approximately 18 mmHg [82]. In contrast, at altitudes between 3,600 and 4,000m, natives from Ladakh and Tibetans have a mean PAP of 14 mmHg [67, 83], a value that is close to that observed in healthy sea-level natives. As mentioned already, in these cases the small pulmonary arteries have a thin layer of muscularization and the arterioles do not have muscularization. Given that this value is considered low for the altitude of residence, some authors have postulated that it could reflect a complete acclimatization to high altitude that has been attained because of the considerable amount of times that has past since their arrival in the mountainous regions (Fig. 6) [84]. In view of the consistent PAP values reported in the literature, HAPH has been defined as a clinical syndrome occurring in children and adults travelling to or resident at altitudes above 2,500 m. HAPH can be diagnosed when the mean PAP is above 30  mmHg or the systolic PAP is above 50  mmHg

Fig. 6  Individual variation of mPAP values of sea-level residents and high-altitude residents. (a) The mPAP in healthy lowlanders at sea level, and at altitudes between 3,650 and 4,500 m in residents of high altitude living in Tibet and in the central Andes of Peru. (b) The mPAP values at altitudes between 3,000 and 4,600 m in subjects with HPAH from the Qinghai-Tibetan Plateau (Han) and the Andes (Quechua). The horizontal lines indicate the median mean PAP value for each group of subjects. (Reprinted from Maggiorini and Leon-Velarde [50], with permission from European Respiratory Society Journals Ltd)

measured at the altitude of residence. Right ventricular hypertrophy, heart failure, and moderate hypoxemia are associated with the elevated PAP [50, 65]. Typical values for CMS patients in the Andean population are shown in Table 1. In infants living at or traveling to high altitude, the mean PAP could be considered high when it is above 50  mmHg and the systolic PAP could be considered high when it is above 65  mmHg, measured at the altitude of residence in infants up to 6 months of age. Maignan et  al. [85] have shown that chronic hypoxia with appropriate acclimatization, as seen in healthy highaltitude residents, induces an increase in pulmonary vascular resistance without change in pulmonary artery systolic pressure, the dimensions of the cavities of the right side of the heart, and right systolic ventricular function. However, an increase in right ventricular total ejection isovolume index, a global myocardial performance indicator (calculated as the ratio of the systolic time minus the ejection time divided by the ejection time), could reflect an early right ventricular impairment. When hypoxemia becomes severe, as observed in some patients with excessive PAP, a further impairment could be found, with a significant increase in systolic PAP and enlargement of the cavities of the right side of the heart. This confirms the variability of the severity of HAPH, which is highly dependent on risk factors and

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Table 1  Clinical, hematological, and echocardiographic characteristics of chronic mountain sickness patients (CMS), highaltitude normal subjects (HA), and sea-level normal subjects (SL) SL (n = 15) HA (n = 15) CMS (n = 47) p Age (years) 42 ± 7 44 ± 9 45 ± 10 0.46 Body-mass index (kg/m2) 29.3 ± 3.9 24.7 ± 2.5 25.1 ± 2.9 0.49 Heart rate (beats/min) 68 ± 7.9 64 ± 10 71 ± 12 0.06 Hemoglobin (g/L) 131 ± 9 165 ± 9 231 ± 16 <0.001 SaO2 (%) 97 ± 1 89 ± 2 81 ± 4 <0.001 PaO2 (mmHg) 93 ± 4 49 ± 2 42 ± 4 <0.001 PaCO2 (mmHg) 38 ± 4 24 ± 1 29 ± 3 <0.001 CaO2 (mL O2/L) 176 ± 12 215 ± 12 256 ± 18 <0.001 pHa 7.45 ± 0.02 7.50 ± 0.03 7.47 ± 0.02 <0.001 Bicarbonates (mmol/L) 25.7 ± 2.5 18.4 ± 1.1 20.3 ± 1.9 <0.001 Cardiac output (L/min) 4.4 ± 0.8 4.2 ± 0.9 4.0 ± 0.9 0.36 Right ventricular dilation (cm2) 12 ± 2 13 ± 2 17 ± 2 <0.001 Right ventricular dilation/left ventricular diameter 0.95 ± 0.11 0.95 ± 0.11 1.12 ± 0.11 <0.001 Right auricular dilation (cm2) 14 ± 2 13 ± 2 17 ± 3 <0.001 Left auricular dilation (cm2) 15 ± 2.0 10 ± 2.2 12 ± 2.9 <0.001 Cardiac index (L/min/m2) 2.5 ± 0.5 2.5 ± 0.5 2.3 ± 0.5 0.51 Tricuspid pressure gradient (mmHg) 19 ± 3 25 ± 4 34 ± 10 0.002 Pulmonary acceleration time (ms) 132 ± 18 127 ± 21 96 ± 17 <0.001 Pulmonary acceleration time/ejection time 0.40 ± 0.06 0.41 ± 0.06 0.32 ± 0.05 <0.001 Right ventricular TEI index 0.21 ± 0.12 0.52 ± 0.12 0.56 ± 0.15 0.64 Pulmonary vascular resistance (WU) 124 ± 014 163 ± 024 175 ± 032 0.31 From Richalet et al. (2008) and Maignan et al. (2009). The values are the mean ± the standard deviation. p values are for HA controls versus CMS. PaCO2 carbon dioxide arterial pressure, PaO2 oxygen arterial pressure, pHa arterial pH, SaO2 oxygen arterial saturation, CaO2 arterial oxygen content, TEI total ejection isovolume, WU Wood units.

susceptibility. To corroborate these preliminary results, large epidemiology studies should be conducted to determine the prevalence and features of heart failure in CMS. HAPH should not be mistaken with other types of pulmonary hypertension, including persistent pulmonary hypertension of the newborn; chronic obstructive pulmonary diseases such as chronic bronchitis, chronic obstructive emphysema, and chronic cor pulmonale, interstitial lung disease, including pneumoconiosis, and other cardiovascular diseases complicated with pulmonary hypertension, such as coronary heart disease, valvular heart disease, dilative and hypertensive cardiomyopathy, and congenital heart diseases. It must be made clear that the cause of HAPH is the development of vascular resistance due to an exaggerated response to hypoxia. Thus, hypoxemia must be a sign that has to be considered when obtaining the anamnesis, and the diagnostic approaches should be performed at the altitude of residence of the patient, or a few days after the patient has returned to sea level.

7 HAPH in Newborns and Children In newborns at high altitude, transition from the fetal to the mature pattern of pulmonary circulation is slower compared with that for infants at sea level. These findings explain the higher frequency of patent ductus arteriosus at altitude [49,

86, 87], as well as its increased prevalence with altitude [88, 89]. It has been reported that infants born at high altitudes show lower SaO2 values [90], persistent high PAP values for some time after birth [91], and in some cases an elevated mean PAP (approximately 40 mmHg) which persists during infancy [92]. Thus, at high altitude, the adult PAP level is attained gradually during childhood and adolescence, in parallel with the maturation of the structure of the small arteries and pulmonary arterioles, which takes place slowly [14, 93, 94]. The same occurs with the anatomic and electric predominance of the right ventricle that decreases gradually, but persists in adulthood at high altitudes [57, 89, 95–97]. In contrast, Tibetan children at high altitude appear to be more adapted to life at high altitude [84]. In this case, small pulmonary arteries attain maturity between 4 and 6 months of age, and the right-to-left ventricle predominance shift takes place in the same time period. This suggests that the evolution of the PAP in Tibetan children is similar to that at sea level [48]. Because of the characteristics of their pulmonary tree, children are hyperresponders compared with adults. At 4,500 m, a 6-year-old child has a higher PAP than an adult, because its small pulmonary arteries are more muscularized and have a higher muscular tone [14, 92]. Infants with this characteristic, whether residents or sea-level natives, could develop HAPH and right-sided heart deficiency when transported to high altitude [48, 49].

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Nevertheless, active monitoring of the health status of a pediatric population at high altitude has proved valuable in the timely detection of cardiac abnormalities. Huicho and Niermeyer [98] have shown that no symptomatic HAPH was identified in high-altitude children (4,000 m) in the absence of underlying structural cardiac anomalies. Yet, it is worth noting that their findings are applicable to children with some degree of high-altitude genetic background and patterns of high mobility to lower altitudes and living in relatively good nutritional and socioeconomic conditions.

8 Treatment The ideal treatment is migration to low altitude. Once at sea level, a gradual reversion of the increased PAP and vascular hypertrophy will take place once the hypoxic stimulus is over [76, 77]. Alternative treatments however, should be considered for the reduction of excessive pulmonary hypertension. Calcium-channel blockers such as nifedipin [44, 99] (20–30 mg/12 h), NO inhalation (40 pp for 15 min), 15 ppm NO plus 50% O2 [100], as well as prostaglandin [101] and phosphodiesterase type 5 (PDE-5) inhibitors (25–100  mg/8  h) [100, 102, 103] have been demonstrated to decrease hypoxemia, pulmonary hypertension, pulmonary vascular resistance, and the alveolar–arterial PO2 gradient. However, it should be noted that no long-term controlled trials have been carried out. Recently, the effects of acetazolamide have been studied in randomized, placebo-controlled trials in the Peruvian Andes. The effects of this carbonic anhydrase inhibitor on CMS include an improvement of ventilation (through metabolic acidosis), less hypoxemia, and thus a decrease of erythropoietin concentration and excessive erythrocytosis. In addition, a decrease in pulmonary vascular resistance and an increase in pulmonary acceleration time have been shown [104]. Despite the encouraging results, extensive research and clinical trials are required before recommendations can be made regarding drug treatment of HAPH [65]. A recent study has shown that, apart from pharmacological treatment, some other factors such as iron can modulate PAP. Smith et  al. [105] have recently shown that the effect of hypoxia on PAP depends in part on iron status. To determine whether increasing or decreasing iron availability modifies altitude-induced hypoxic PAP, sea-level residents were transported to an altitude of 4,340  m for 1 week, where they received intravenous infusions of iron-sucrose (200 mg) or a placebo on the third day after ascent. In addition, high-altitude residents diagnosed with CMS were studied over 1 month at the same altitude. These patients undertook bloodletting of 2 L of blood. Two weeks later, patients received intravenous infusions of iron sucrose (400 mg) or a placebo. PAP was assessed by Doppler echocardiography.

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The results showed that in sea-level residents at high altitude, 42% of the pulmonary hypertensive response to hypoxia was reversed by infusion of iron. In CMS patients, progressive iron deficiency induced by bloodletting was associated with a 25% increase in pulmonary artery systolic pressure. A reduction in pulmonary artery systolic pressure was observed in patients who received iron supplementation. Thus, this study shows that HAPH is attenuated by iron supplementation and is exacerbated by iron depletion. These findings may have implications in the clinical management of CMS, where the necessity to limit red cell mass should be balanced with the potential for worsening pulmonary hypertension. These findings also suggest that adjustment of iron balance may have a role in the management of hypoxic pulmonary hypertension.

9 Prognosis Generally, the prognosis of pulmonary hypertension varies depending on the underlying condition that is causing it. Thus, for HAPH, the overall prognosis depends on the vasoconstrictor pulmonary response, the intensity of vascular resistance, the type of onset (acute or chronic), and the altitude. A patient with primary pulmonary hypertension can expect a survival time of about 3 years without any therapy. For patients with HAPH, once the chronic hypoxic stimulus has ceased, the prognosis is most favorable. In fact, Sime et  al. [77] have shown that the mean PAP returns to sea-level values in highaltitude natives (at 4,445 m) after 2 years at sea level. For those who do not relocate to sea level, the impact of the treatment with selective inhibitors of PDE-5 on survival is not known at present. Taking into account however, the significant decrease in the mean PAP and in pulmonary vascular resistance in patients with HAPH treated with this drug [102], it would be reasonable to expect a favorable prognosis.

10 Summary HAPH and CMS, present as different entities, or together in the same individual, have been well established as high-altitude diseases in most mountainous regions of the world. This chapter presented a general overview of HAPH and briefly summarized the existing information related to the clinical features and pathophysiological characteristics of the illness, with emphasis on the findings from studies in the Andes. Pulmonary hypertension, remodeling of pulmonary arterioles, and right ventricular enlargement are hallmarks of HAPH. Risk factors such as altitude, age, sleep disorders, and genetic susceptibility increase the prevalence of this illness. The cause of HAPH is the development of vascular resistance; the underlying

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mechanism involves sustained and exaggerated pulmonary vasoconstriction and pulmonary vascular remodeling in response to chronic hypoxia, due to proliferation and decreased apoptosis of PASMCs in the tunica media. HAPH is aggravated by right ventricular hypertrophy, which may be a cause of right ventricular failure. A diagnosis of HAPH can be made when the mean PAP is more than 30 mmHg or the systolic PAP is more than 50 mmHg measured at the altitude of residence. The prognosis of HAPH varies depending on the underlying conditions; thus, it depends on the vasoconstrictor pulmonary response, the intensity of vascular resistance, the type of onset (acute or chronic), and the altitude. In patients with HAPH who are no longer exposed to chronic hypoxia, the prognosis is most favorable because the mean PAP return to sea-level values. Although no clinical trials have been performed, selective inhibitors of PDE-5 have proved to be effective in decreasing the mean PAP and pulmonary vascular resistance in patients with HAPH.

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84  High-Altitude Pulmonary Hypertension 71. Hakim TS, Michel RP, Minami H, Chang HK (1983) Site of pulmonary hypoxic vasoconstriction studied with arterial and venous occlusion. J Appl Physiol 54(5):1298–1302 72. Zhao Y, Packer CS, Rhoades RA (1993) Pulmonary vein contracts in response to hypoxia. Am J Physiol 265(1 Pt 1):L87–L92 73. Rabinovitch M, Konstam MA, Gamble WJ et al (1983) Changes in pulmonary blood flow affect vascular response to chronic hypoxia in rats. Circ Res 52(4):432–441 74. Sobin SS, Tremer HM, Hardy JD, Chiodi HP (1983) Changes in arteriole in acute and chronic hypoxic pulmonary hypertension and recovery in rat. J Appl Physiol 55(5):1445–1455 75. Stenmark KR, Fasules J, Hyde DM et al (1987) Severe pulmonary hypertension and arterial adventitial changes in newborn calves at 4,300 m. J Appl Physiol 62(2):821–830 76. Fried R, Reid LM (1984) Early recovery from hypoxic pulmonary hypertension: a structural and functional study. J Appl Physiol 57(4):1247–1253 77. Sime F, Penaloza D, Ruiz L (1971) Bradycardia, increased cardiac output, and reversal of pulmonary hypertension in altitude natives living at sea level. Br Heart J 33(5):647–657 78. Tucker A, Rhodes J (2001) Role of vascular smooth muscle in the development of high altitude pulmonary hypertension: an interspecies evaluation. High Alt Med Biol 2(2):173–189 79. Reid L (1994) Structural remodeling of the pulmonary vasculature by environmental change and disease. In: Wagner W, Weir E (eds) The pulmonary circulation and gas exchange. Futura, New York, pp 77–110 80. Ge RL, Helun G (2001) Current concept of chronic mountain sickness: pulmonary hypertension-related high altitude heart disease. Wilderness Environ Med 12(3):190–194 81. Sarybaev AS, Mirrakhimov MM (1998) Prevalence and natural course of high altitude pulmonary hypertension and high altitude cor pulmonale. In: Ohno H, Kobayashi T, Masuyama S, Nakashima M (eds) Progress in mountain medicine and high altitude physiology. Press Committee of the 3rd World Congress on Mountain Medicine and High Altitude Physiology, Matsumoto, pp 126–131 82. Hartley LH, Alexander JK, Modelski M, Grover RF (1967) Subnormal cardiac output at rest and during exercise in residents at 3,100 m altitude. J Appl Physiol 23(6):839–848 83. Gupta ML, Rao KS, Anand IS, Banerjee AK, Boparai MS (1992) Lack of smooth muscle in the small pulmonary arteries of the native Ladakhi. Is the Himalayan highlander adapted? Am Rev Respir Dis 145(5):1201–1204 84. Moore L, Sun S (1990) Physiologic adaptation to hypoxia in Tibetan and acclimatized Han residents of Lhasa. In: Sutton J, Coates G, Remmers J (eds) Hypoxia: the adaptations. Decker, Toronto, pp 66–71 85. Maignan M, Rivera-Ch M, Privat C, Leon-Velarde F, Richalet JP, Pham I (2009) Pulmonary pressure and cardiac function in chronic mountain sickness patients. Chest 135(2):499–504 86. Gamboa R, Marticorena E (1972) The ductus arteriosus in the newborn infant at high altitude. Vasa 1(3):192–195 87. Miao CY, Zuberbuhler JS, Zuberbuhler JR (1988) Prevalence of congenital cardiac anomalies at high altitude. J Am Coll Cardiol 12(1):224–228 88. Marticorena E, Peñaloza D, Severino J, Hellriegel K (1963) Frequency of patent ductus arteriosus at high altitude. Paper presented at the proceedings of the IV world congress of cardiology, Mexico City

1221 89. Peñaloza D, Arias-Stella J, Sime F, Recavarren S, Marticorena E (1964) The heart and pulmonary circulation in children at high altitudes. Physiological, anatomical and clinical observations. Pediatrics 34:568–581 90. Niermeyer S, Shaffer EM, Thilo E, Corbin C, Moore LG (1993) Arterial oxygenation and pulmonary arterial pressure in healthy neonates and infants at high altitude. J Pediatr 123(5):767–772 91. Gamboa R, Marticorena E (1971) Pulmonary arterial pressure in newborn infants in high altitude. Arch Inst Biol Andina 4(2):55–66 92. Sime F, Banchero N, Penaloza D, Gamboa R, Cruz J, Marticorena E (1963) Pulmonary hypertension in children born and living at high altitudes. Am J Cardiol 11:143–149 93. Heath D, Smith P, Rios Dalenz J, Williams D, Harris P (1981) Small pulmonary arteries in some natives of La Paz, Bolivia. Thorax 36(8):599–604 94. Heath D, Williams D, Rios-Dalenz J, Calderon M, Gosney J (1990) Small pulmonary arterial vessels of Aymara Indians from the Bolivian Andes. Histopathology 16(6):565–571 95. Arias-Stella J, Recavarren S (1962) Right ventricular hypertrophy in native children living at high altitude. Am J Pathol 41:55–64 96. Penaloza D, Gamboa R, Dyer J, Echevarria M, Marticorena E (1960) The influence of high altitudes on the electrical activity of the heart. I. Electrocardiographic and vectorcardiographic observations in the newborn, infants, and children. Am Heart J 59:111–128 97. Penaloza D, Gamboa R, Marticorena E, Echevarria M, Dyer J, Gutierrez E (1961) The influence of high altitudes on the electrical activity of the heart. Electrocardiographic and vactorcardiographic observations in adolescence and adulthood. Am Heart J 61:101–115 98. Huicho L, Niermeyer S (2006) Cardiopulmonary pathology among children resident at high altitude in Tintaya. Peru: a cross-sectional study. High Alt Med Biol 7(2):168–179 99. Zhao L, Mason NA, Morrell NW et al (2001) Sildenafil inhibits hypoxia-induced pulmonary hypertension. Circulation 104(4): 424–428 100. Wilkins MR, Aldashev A, Morrell NW (2002) Nitric oxide, phosphodiesterase inhibition, and adaptation to hypoxic conditions. Lancet 359(9317):1539–1540 101. Das UN (1980) Possible role of prostaglandins in the pathogenesis of pulmonary hypertension. Prostaglandins Med 4(3):163–170 102. Aldashev AA, Kojonazarov BK, Amatov TA et  al (2005) Phosphodiesterase type 5 and high altitude pulmonary hypertension. Thorax 60(8):683–687 103. Richalet JP, Gratadour P, Robach P et al (2005) Sildenafil inhibits altitude-induced hypoxemia and pulmonary hypertension. Am J Respir Crit Care Med 171(3):275–281 104. Richalet J-P, Rivera M, Bouchet P, Chirinos E, Onnen I, Petitjean O, Bienvenu A, Lasne F, Moutereau S, Leon-Velarde F (2005) Acetazolamide: a treatment for chronic mountain sickness. Am J Respir Crit Care Med 172(11):1427–1433 105. Smith T, Talbot N, Privat C, Rivera-Ch M, Nickol A, Ratcliffe P, Dorrington K, León-Velarde F, Robbins P (2009) Effects of iron supplementation and depletion on hypoxic pulmonary hypertension: two randomized controlled trials. JAMA 302(13):1444–1450 106. Weir EK, Olschewski A (2006) Role of ion channels in acute and chronic responses of the pulmonary vasculature to hypoxia. Cardiovasc Res 71:630–641 107. Penaloza D, Arias-Stella J (2007) The heart and pulmonary circulation at high altitudes: healthy highlanders and chronic mountain sickness. Circulation 115(9):1132–1146

Chapter 85

Pulmonary Hypertension and Congenital Heart Defects at High Altitude Alexandra Heath de Freudenthal, Franz-Peter Freudenthal Tichauer, Carmen R. Condori Taboada, and Janne C. Lopes Mendes

Abstract  Around 140 million people in the world, including 4.9 million Bolivians, live 2,500 m above sea level. The barometric pressure and the partial pressure of oxygen in the blood fall in direct proportion to the altitude. The time necessary for a person living at sea level to adapt to the altitude depends, in direct proportion, on the altitude at which that person arrives. Living, working, and playing sports at high altitudes are considered by many a great feat. In fact, there is controversy about the feasibility of playing competitive sports in cities located at very high altitudes. However, native inhabitants may play soccer at altitudes even higher than the summit of Mt Sajama, located 6,000 m above sea level. Traveling to high altitudes as a lowland immigrant is considered a risk. In one renowned article, discussing altitude, it was stated that the altitude causes more than mere muscular fatigue; it affects memory, calculating capacity, decisionmaking ability, and judging capacity. What are the adaptive mechanisms that help people live and work with relative normality in these high regions? Many working groups have worked to understand the physiological mechanisms of adaptation to the altitude for newcomers and the connotations of living in mountains for many generations. These studies have dealt not only with hypobaric hypoxia, but also with lower temperatures, low humidity levels, and increased solar radiation. Temperature and the amount of UV radiation also vary with the altitude: the temperature decreases by 6.5°C per 1,000 m and the amount of radiation increases at a rate of 4% per 1,000 m. However, the inhabitant at high altitudes has found a balance based on adaptive processes. With regard to congenital heart defects, the factors mentioned previously, combined with the lower socioeconomic level, have contributed to a higher incidence of these types of disorders at altitude.

A. Heath de Freudenthal (*) Kardiozentrum Medical Specialty Center, Obrajes Calle 14 # 665, La Paz, Bolivia e-mail: [email protected]

Keywords  Chronic hypoxia • Congenital heart disease • Ventricular and atrial septal defects • Mountain sickness

1 Introduction Around 140 million people in the world, including 4.9 million Bolivians, live 2,500 m above sea level [1, 2]. The barometric pressure and the partial pressure of oxygen in the blood fall in direct proportion to the altitude. The time necessary for a person living at sea level to adapt to the altitude depends, in direct proportion, on the altitude at which that person arrives [3]. Living, working, and playing sports at high altitudes are considered by many a great feat. In fact, there is controversy about the feasibility of playing competitive sports in cities located at very high altitudes. However, native inhabitants may play soccer at altitudes even higher than the summit of Mt Sajama, located 6,000 m above sea level [4]. Traveling to high altitudes as a lowland immigrant is considered a risk. In one renowned article, discussing altitude, it was stated that the altitude causes more than mere muscular fatigue; it affects memory, calculating capacity, decision-making ability, and judging capacity [5]. What are the adaptive mechanisms that help people live and work with relative normality in these high regions? Many working groups have worked to understand the physiological mechanisms of adaptation to the altitude for newcomers and the connotations of living in mountains for many generations. These studies have dealt not only with hypobaric hypoxia, but also with lower temperatures, low humidity levels, and increased solar radiation. Temperature and the amount of UV radiation also vary with the altitude: the temperature decreases by 6.5°C per 1,000 m and the amount of radiation increases at a rate of 4% per 1,000 m [5]. However, the inhabitant at high altitudes has found a balance based on adaptive processes. With regard to congenital heart defects, the factors mentioned previously, combined with the lower socioeconomic level, have contributed to a higher incidence of these types of disorders at altitude.

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_85, © Springer Science+Business Media, LLC 2011

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2 History

3 Adaptation

Archeologists have found the remains of villages in regions up to 5,000 m above sea level, as well as Andean sanctuaries at even higher altitudes [6]. The highest place where ancient constructions have been found is at 6,700 m above sea level, in Llullayllaco (Puna de Atacama). It has been calculated that, on the basis of the type of instruments found in these regions, the first Andean inhabitants arrived in these regions 22,000 years ago [7]. The first records on the possible health problems associated with altitude were made by the Jesuit priest Josde Acosta during the colonial period. He reported on many of the symptoms that affected those who traveled to high altitudes without having been previously adapted: “I was surprised by a strange and mortal piercing pain, and I thought I would fall off the cliff because the air was so scarce and thin that I could barely breath” [8]. Nevertheless, when reading about the colonial period, the most surprising fact is the extreme neonatal mortality rate in the Spanish population that migrated to Mt Potosì (3,976  m). The survival of children born to Spaniards was so rare that the first surviving child was named “the miracle of Saint Nicholas.” History recalls that this child was the first to survive as a product of Spanish parents, from a couple who had previously lost six children in the neonatal period [9]. The situation was so extreme that during the first years of occupancy, Spaniards only gave birth to their children in the city of Sucre (2,790 m). With the passing of time, Spaniards started adapting to the higher altitudes. The mixing of races also contributed to the higher survival rates of infants born at altitude, and this has allowed cities at high elevations to remain populated until the present. The following description on the prevalence of congenital heart defects in Bolivia is based on the studies of Alfonso Gamarra, a physician of the national mining center. Since 1965, Gamarra has published interesting conclusions based on the 126,000 medical appointments with inhabitants of populations situated between 3,734 and 4,138  m above sea level. Gamarra informs on a prevalence of congenital heart defects of 2.8:1,000 among these inhabitants, of which those with patent ductus arteriosus (PDA) constitute 37.7%, those with a ventricular septal defect (VSD) constitute 27.8%, and those with an atrial septal defect (ASD) constitute 11.4%, leaving the other 23.1% for other congenital heart defects [10]. In the same way, Gamarra affirms that the incidence of congenital heart defects increases in proportion to the increase in altitude, and that the most frequent heart defect is PDA, which predominates in girls [11]. Peruvian authors, such as Vela, also informed on PDA being the most common congenital heart defect at altitude [12]. In 1960, Alzamora-Castro et al. stated that the frequency of PDA is 30 times higher at altitude than at sea level [13]. It is important to mention that these observations were made on the basis of clinical examinations at a time when echocardiography was not a standard procedure.

Ascension to high altitudes results in a gradual but steady decline of arterial oxygen pressure (PaO2). The concentration of oxygen in the environment is 20.9% at any altitude. But the PaO2, which is 100 mmHg at sea level, declines to 60 mmHg at an altitude of 3,600 m. Hypoxia is detected by the chemoreceptors located in the carotid tissues, which send a signal to the respiratory center causing an increase in ventilation, respiratory rate, as well as the depth of inspiration; this is called hypoxic ventilatory response (HVR). As the PaO2 increases,the process also results in a greater PCO2 depuration, which lowers the concentration of CO2 in the arterial blood. Renal counterregulation achieves the normalization of the respiratory rate in a couple of days through acid–base balance conservation mechanisms. This phenomenon is called renal ventilatory adaptation [5]. The lowest oxygenation point is reached within the first few hours after arrival, and oxygen saturation is correlated with the severity of the symptoms (dizziness, agitation, headache, confusion, somnolence, and loss of coordination). It is considered that hypocapnia produces cerebral vasoconstriction, which could be responsible for provoking the symptoms [3, 5]. A decrease in the quality of sleep is a universal characteristic of newcomers to altitude. While sleeping, ventilation and response capacity to low oxygen levels decrease, causing sleep apneas which lower the oxygen pressure even more. The stimuli generated by carotid bodies wake up the individual, who makes several inspirations to normalize oxygen levels. This process makes the individual wake up several times during the night. If the process is successful, the newcomer to altitude attains enough initial oxygenation and continues through the following steps in the adaptation process. After living at high altitude for a few years, new changes take place. Ventilation undergoes changes destined to conserve PO2 at acceptable levels. These changes can be divided into acute, subacute, and chronic changes. The acute changes, described earlier, correspond to HVR. As the days go by, new changes occur. Hypocapnia causes alkalosis and free protons (H+) to appear in the blood; this constitutes an inhibitory element for the respiratory center, producing hypoventilation [3, 5]. A series of regulatory effects occur in response to the blood alkalosis: above all, the renal excretion of bicarbonate. Natives in these regions have a lower HVR in comparison with acclimated people. This characteristic could be related to carotid body hypertrophy. This histological change is observed with greater frequency at altitude [14, 15]. The development of other methods of adaptation for the conservation of tissue oxygenation was well described by Penaloza and Arias Stella [16], such as an increase in capillary density, increased capacity for the interchange of gases in the tissues, greater number and density of mitochondria, or an increment of the vital capacity; these continue during the process of adaptation.

85  Pulmonary Hypertension and Congenital Heart Defects at High Altitude

Other mechanisms include the increase in cardiac capacity and variations in the hematopoietic system (increase in the concentrations of erythropoietin and hemoglobin) [17]. The first achieves a greater liberation of O2 to the tissues through an increase in the cardiac output. The second increases the number of red blood cells and optimizes the transportation and delivery of O2 to the tissues. Zubieta-Calleja et  al. explained a deviation to the left in the hemoglobin dissociation curve with regard to the Chipaya population, bordering Lake Titicaca; a group of people who have lived at an altitude of 4,100  m for around 4,500 years [17]. They proposed the application of a formula to calculate the time required for a person to adapt to altitude from a hematopoietic point of view: the number of days required for adaptation = 11.4 × altitude (km). The time required for complete adaptation at an altitude of 3,500  m would be around 40 days after arrival, at which point the hematocrit reaches normal levels. The descent to sea level would induce a reverse adaptation that would only require 23 days [17]. Hypoxia means a decrease in oxygen delivery to the organs, tissues, and cells. In relation to altitude, hypoxia is associated with altitude sickness, tissue damage, and various degrees of injuries and tissue ischemia. However, chronic hypoxia is also reported as a type of training for the cells and many authors associate intermittent hypoxia with a protective factor, which increases the mitochondrial count, stimulates cellular proliferation, and increases a person’s capacity to be involved in sports and work [18, 19]. The increase in the mitochondrial count and the capillary density results in optimization of tissue O2 exploitation. Lastly, an increase in myoglobin concentration has been observed in athletes. Myoglobin is an intracellular protein with high affinity for oxygen, which achieves diffusion and cellular delivery despite low tissue O2 pressures [5]. Altitude sicknesses are like acute pulmonary and cerebral edema, which occur in the first few days after arrival. Chronic altitude sickness of highlanders involving pulmonary hypertension and a very high erythrocyte count, which many authors call the sickness of the Monge, is beyond the scope of this chapter, so we refer the reader to other texts and previous chapters [20].

4 Pulmonary Hypertension Hypoxia produces vasoconstriction to the smooth muscle of the pulmonary vessels, causing an increase in pulmonary resistance and pulmonary pressure [16]. Relatively high pulmonary pressures are frequent in inhabitants at high elevations. Pulmonary pressures considered normal in La Paz, at an altitude of 3,600  m, are as follows: 32.64  mmHg for median pulmonary pressure [21]; systolic pulmonary pressure reaches up to 45 mmHg in normal conditions [21, 22];

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the pulmonary pressure may vary with age – Penaloza and Arias Stella described a mean pulmonary pressure of 45.16 mmHg for children younger than 5 years at an altitude of 4,500 m, but 28 mmHg for older ones [16]. These criteria are not applicable to breast-feeding children, in whom pulmonary pressure is measured as a percentage of systemic pressure: mild – less than 45% of the systemic systolic pressure, moderate – 50–75% of the systemic pressure, and severe – more than 75% of the systemic pressure [23]. These ranges are considered normal when patients are asymptomatic, show no progression in measurements, or in those who show no secondary signs of pulmonary hypertension. These signs include O2 saturation less than 90%, New York Heart Association (NYHA) physical well-being score greater than 1, as well as clear echocardiography signs that include a mild to severe tricuspid regurgitation with a constricted vein greater than 2 mm in diameter, hypertrophy and dilatation of the right ventricle in correlation to the septum movement making it hypokinetic, and dilatation of the right atrium. In our patient cohort in La Paz, at 3,600 m, we have found the following data pertaining to our group of patients: of 1,658 patients younger than 25 years of age, 33.1% did not have congenital heart defects (innocent murmur). Ninety-six patients had tricuspidal insufficiency. These patients had a systolic pulmonary arterial pressure (SPAP) measured by an average gradient through tricuspidal insufficiency of 32.9  mmHg (25–37.9); 9.17% (n = 152) of the patients had pulmonary hypertension greater than 45 mmHg. Asymptomatic children with a SPAP between 45 and 60  mmHg remained in La Paz city, and presented themselves for follow-up checks every 6 months. Most of these patients remained within these levels of pulmonary arterial pressure, with no antihypertensive treatment up to 2-year follow-up after the initial diagnosis. The children with SPAP greater than 60 mmHg were sent to the coast, where the majority had normal pulmonary pressures after the adaptation period. Only one girl, age 12, did not; she died after 3 years with the diagnosis of idiopathic pulmonary arterial hypertension. The group of newborn babies with pulmonary hypertension, greater than 60  mmHg and dependent on O2, was treated with sildenafil, and the following results were found: with a dosage of 1–2 mg/kg three times per day decreased SPAP in the children, and gradually decreased their need for oxygen.

5 Congenital Heart Defects Congenital heart defects, which at sea level have a prevalence rate of 0.8–1.2% [24–31], have a prevalence rate of 2.5–2.8% at altitude [32–34]. According to many authors, the persistence of PDA rivals that of VSD as the most frequent congenital heat defect, which is reported in the world

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literature [35] and the reported frequency of PDA is 10–15 times greater than at sea level [34, 35]. Our work group has found that the number of patients with PDA is more than 3.5 times greater at high altitudes than at sea level. Patients with ASD are 2.3 times more frequent at altitude than at sea level. In a prospective standard protocol study screening children who were suspected of having congenital heart defects, and who were referred to the study by other pediatricians or cardiologists, we found 33% were normal. Of the group of patients with congenital heart diseases, the most common heart defect at altitude was an isolated VSD, in its many manifestations, constituting 22.8% of congenital heart defects. The second most common was PDA, with 22.6%. In third place was ASD, with 11.6%. Among the cyanotic heart defects, as occur at sea level, tetralogy of Fallot is the most common, at 3.61%. Malformations, such as Ebstein anomaly, at 1.62%, and tricuspid atresia, at 1.26%, are 3  times more frequent at high altitudes than at sea level. Aparicio and Garabito reported an incidence rate of 9% for Ebstein malformations among adults with congenital heart defects [35]. The general distribution of the congenital heart defects at altitude was compared with that reported at the Boston Children Hospital [36], from a group of children who underwent cardiopediatric and echocardiographic testing after showing symptoms and signs, cardiac murmur, cyanosis, failure to thrive or cardiac insufficiency. Both statistics take into account all of the patients, including the normal ones (Table 1). Pulmonary hypertension in patients with congenital heart defects, and specifically a left-to-right shunt, born at sea level are characterized by a constant alteration in the pulmonary vessels, which leads to an increase in pulmonary resistance and irreversible pulmonary hypertension in the scope of the reversal of flow as can be seen in Eisenmenger syndrome. If the patient is not operated on during the breast-feeding stage, or in early infancy, pulmonary hypertension can become irreversible. The ideal time to operate is during the first year of life [37]. Nevertheless, not all children in the world have the opportunity to receive treatment owing to socioeconomic factors, such as reported by Saxena in India [38]. The situation is similar in Bolivia, where only 20% of the population has health insurance or has enough money to pay for cardiac surgery if the congenital heart defect is detected in time. A large percentage of the diagnoses are delayed, and the surgeries are carried out on patients who, in some cases, have reached their second decade of life with considerable pulmonaryto- systemic shunts. Not all patients with large pulmonary-tosystemic shunts develop early and irreversible changes in their pulmonary vasculature. The predisposition of developing irreversible pulmonary hypertension after undergoing considerable histological changes seems to depend, to some degree, on a person’s genetic inheritance, and not only on the degree of pulmonary hyperflow [39].

A.H. de Freudenthal et al. Table 1  Distribution of congenital heart defects at high altitude relative to that found at sea level KZ (absolute Boston Cases no.) KZ (%) (%) Normal 549 33.1 34.1 Ventricular septal defect 253 15.3 4.1 Patent ductus arteriosus 248 15 3.7 Atrial septal defect 129 7.8 3.8 Pulmonary hypertension 152 9.2 0.6 Tetralogy of Fallot 40 2.4 5.9 Aortic valve 17 1.0 4.5 Pulmonary valve 20 1.2 6.4 Coarctation of the aorta 25 1.5 3.5 Tricuspid atresia 14 0.8 0.7 Atrioventricular canal 16 1 3.0 Ebstein malformation 18 1.1 0.3 Rheumatic cardiopathy 18 1.1 1.7 Transposition of the great arteries 5 0.3 3.2 Truncus arteriosus 5 0.3 0.5 Shone syndrome and hypoplastic 15 0.9 1.2 left heart syndrome Double-outlet right ventricle 9 0.5 1 Pulmonary atresia 5 0.3 0.4 Others 120 7.2 11.8 Total 1,658 100 100 Data from the Kardiozentrum Medical Specialty Center (KZ) in Bolivia are compared with data from the Boston Children’s Hospital (Boston). Information for normal cases is also included.

The group of patients we have operated on for VSD includes 21 children and adolescents with systemic-to-pulmonary shunts and hyperdynamic pulmonary hypertension. The size of the interventricular communications was 17.2 ± 10.0  mm [mean ± standard deviation (SD); range from 4 to 35  mm] (Table 2). The SPAP of these patients was 64.1 ± 18.5 mmHg (mean ± SD; range from 22 to 94 mmHg) before surgery and 36.2 ± 11.0 mmHg (range from 20 to 61) after surgical closure (Fig.  1). None of the patients had Eisenmenger syndrome despite the fact that 62% were above 2 years of age and three were above the age of 20. The presurgery NYHA physical well-being score was 2.1. One year after the corrective surgery, all patients had a NYHA physical well-being score of 1.2. It is worth noting that despite the degree of pulmonary hypertension, before the surgery the patients seemed to be in good condition, with NYHA physical well-being scores between 1 and 2. The oxygen saturation was 92.8 ± 5.0% (range from 82 to 99%) (Table 2). At altitude, PDA manifests special characteristics. We found 59% of ductus arteriosi greater than 4  mm in their minimal diameter (angiographic measurement), against 3.8 and 17% at sea level [40]. The ductus arteriosi have a larger minimum diameter and are longer, as reported by other authors as well [41]. The early development of pulmonary hypertension was reported on previously by Coudert in 1970 [21]. In 1986, Lavadenz conducted an important study on the behavior of pulmonary arterial pressure in patients with

85  Pulmonary Hypertension and Congenital Heart Defects at High Altitude

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Table 2  Relevant data of the group of patients who had surgery for a ventricular septal defect (VSD) at high altitude Preoperative NYHA physical well-being Preoperative Postoperative VSD size O2 saturation score (%) SPAP (mmHg) SPAP (mmHg) Patient Sex Age (mm) 1 F 5 35 88 37 32 2 M 5 8 96 40 30 3 F 4 25 85 71 48 4 M 1 15 93 94 38 5 F 11 25 94 90 48 6 F 1 15 95 68 40 7 M 4 30 86 91 61 8 M 1 4.1 94 70 41 9 F 10 9 98 22 20 10 F 1 10 99 60 42 11 M 4 9 96 60 28 12 M 4 15 88 75 35 13 M 23 30 90 70 40 14 F 9 30 82 55 22 15 F 1 7 97 61 57 16 F 1 10 90 48 32 17 F 22 35 92 65 25 18 M 1 12 97 83 34 19 M 2 10 99 45 35 20 M 1 10 99 70 20 21 M 20 18 90 72 32 SPAP systolic pulmonary arterial pressure, NYHA New York Heart Association, M male, F female

2 2 2 2 2 2 2 2 3 2 3 3 2 2 2 2 2 2 2 2 2

Postoperative NYHA physical well-being score 1 1 2 1 1 1 2 1 1 1 1 2 1 1 1 1 2 2 1 1 1

Fig. 1  Systolic pulmonary arterial pressure (SPAP) in patients before and after operation for ventricular septal defect. The average values of pre- and postoperative SPAP are 64.1 ± 18.5 and 36.2 ± 11.0 mmHg, respectively (P < 0.001, 21 patients)

PDA and found that 63.7% developed pulmonary hyper­ tension early on [42]. Above all, even in patients with severe pulmonary hypertension that responded to the administration of oxygen, no necrotizing arteritis or alterations in the intima were found [42]. Among our 60 patients with PDA who were examined by cardiac catheterism for percutaneous closure, we found that, on average, the patients had a minimal ductal diameter of 4.7 mm (from 4 to 8.3 mm), with a preintervention SPAP of 56.6 ± 15.7 mmHg (range from 32 to 94 mmHg) and a postintervention SPAP of 42.3 ± 13.0  mmHg (range from 26 to 61 mmHg) (Fig. 2). All ductus arteriosi were closed interventionally with a Nit-Occlud PDA-R device (Fig. 3). It is important to emphasize that we maintained the ductus arteriosus

closed for 20  min, observing the change of the pulmonary arterial pressure before introducing the device. This way of measuring the reversibility of pulmonary hypertension in older patients is usual in catheterization laboratories, using balloons [43]. It is also important to carry out a hyperoxia test to determine the reactivity of the pulmonary vessels. Patients with trisomy 21 syndrome (T21) often have congenital heart defects. It is calculated that the prevalence of congenital heart defects in these patients is around 42–58% [44–46]. The most common heart defect in children born and who live at sea level is complete atrioventricular (AV) canal, with an incidence rate of 24–38% [45, 46]. In our group of T21 patients born between 3,600 and 4,100 m (mostly Aymara people), we have found some significant differences in

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A.H. de Freudenthal et al.

Fig. 2  SPAP in patients before and after percutaneous intervention for patent ductus arteriosus (PDA) closure. The average values of pre- and postoperative SPAP are 56.6 ± 15.7 and 42.3 ± 13.0 mmHg, respectively (P < 0.01, 23 patients)

Fig. 3  Postinterventional angiography showing percutaneous PDA closure with the Nit-Occlud PDA-R® device in two patients

relation to those reported in the literature. The group included 172 children with T21 from 1 day to 21 years of age (average 2.9 years), with 19.7% being newborn babies (n = 34). Children without congenital heart defects made up 78.4% (n = 135) of the total. The most common heart defect was PDA (37%), followed by VSD (32%) and ostium secundum ASD (29%), whereas ostium primum ASD occurred in only 2% of the patients (Fig.  4). The three abnormalities frequently occurred together. The complete AV canal was only present in two patients out of 172, representing only 1.1% of the total. In Mexico City, which is also at a high altitude, a complete AV canal was present in 9% of the children [44]. All the children developed pulmonary hypertension early on, with an average pulmonary arterial pressure of 57.8 mmHg (24–110  mmHg) among patients with left-to-right shunt. Children who had been operated on showed a significant decrease in pulmonary arterial pressure. Regarding the correction of a single ventricle (Glenn and Fontan procedures), the evolution of patients with cavopulmonary palliation of heart diseases with a single ventricle could be affected by living at high altitude, owing to the fact that the passive pulmonary circulation depends on the resistance of the

Fig. 4  Distribution of different congenital heart defects in patients with trisomy 21 syndrome. ASD atrial septal defect, VSD ventricular septal defect

vascular bed. It is known that the pulmonary vascular resistance and, in consequence, the pulmonary arterial pressure rise with altitude, as another mechanism of adaptation. Even though we do not have the proper experience with the Fontan operation and its variants, in the literature, there are some documented studies regarding this. Malhotra et al. reported that

85  Pulmonary Hypertension and Congenital Heart Defects at High Altitude

the clinical result of Glenn and Fontan procedures was favorable in the majority of the patients who live at 305–2,570 m above sea level. Nevertheless, these authors identified a mean pulmonary arterial pressure above 15 mmHg and a pulmonary vascular resistance index around 2.5 Wood units/m2 [47] as risk factors. On the basis of a follow-up study over more than 20 years of patients who live at moderate altitude and who had undergone Glenn and Fontan procedures, Day stated that the risk of brain strokes, protein-losing enteropathy, and arrhythmias is notably higher in patients with Fontan circulation (and variants) than in those living with a Glenn derivation [48]. The same author identified children with Fontan circulation as having huge difficulties in gaining weight: the higher the altitude at which they live, the less the gain. The difficulty in gaining weight is associated with a worse prognosis [49]. Besides, the author investigated the capacity and tolerance of physical exercise in children with Fontan circulation who live at 910– 2,130 m above sea level, and concluded that the tolerance of physical exercise diminishes in this group of patients, but that early and intermediate results do not diverge from those reported at sea level [50]. McMahon et al. described a case of precipitation and exacerbation of protein-losing enteropathy in a patient who was relocated to an altitude of 3,695 m above sea level [51]. Personally, we think that children whose passive pulmonary circulation depends on pulmonary resistance and pressure should not live at high altitudes. Their early move to sea level is important if the surgery has been realized at high altitude.

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(Fig. 4). It is interesting to discuss the reason why patients at altitude tolerate a large left-to-right shunt and significant pulmonary hypertension without developing alterations in the pulmonary vasculature. Kolar reported a myocardic protection factor in patients with chronic hypoxia [18]. We believe the inhabitants at altitude, who have been acclimated for many generations, maintain a stable muscular layer in the pulmonary vasculature. This provides protection from complex histological changes. In the presence of pulmonary hyperflow, the pulmonary arterial pressure increases owing to a hyperkinetic mechanism and vasoconstriction reflex. In conclusion, we can observe that at altitude patients develop pulmonary hypertension early on without developing all the clinical manifestations of severe pulmonary hypertension. This gives us the chance to operate on them even if they are more advanced in age than the age recommended in the literature, even when they have high pulmonary arterial pressures. We are not certain whether this interesting characteristic is due to living at altitude and having undergone pulmonary vasculature changes for generations, or if it is due to the fact that the race in our context is predominantly Aymara. Patients are born with a thickening of the pulmonary medium muscle, and maintain this pattern, which may confer them with relative stability against more advanced histological changes. We have not found any case of Eisenmenger syndrome in the group of patients under 18 years of age in our medical center, although one case was reported in the Lavadenz series of 1986 [42]. Patients’ pulmonary arterial pressure reaches stability in the course of time, and there is normalization of the right side of the heart.

6 Conclusions The adaptive physiological processes to altitude are fascinating. The adaptive capacity of the living being is amazing, not only during its lifetime, but also the silent genotypic adaptation that transcends generations until the human being is completely adapted to the new environment. In La Paz, located at 3,600 m above sea level, we have observed considerable differences, not only in the distribution of congenital heart defects, but also in patients’ natural evolution and in their behavior after the congenital heart defects have been corrected. The lessons learned regarding chronic hypoxia and pulmonary hypertension reach a level of relativization, even in talking about the factors that protect the highland dweller. Clear examples of this are those demonstrated with regard to protection at the moment of a myocardial ischemic injury in the presence of intermittent hypoxia, the protection conferred by the muscular media when faced with hyperkinetic pulmonary hypertension, and the distribution of congenital heart defects in patients with T21. Although the most frequent form of congenital heart defects at sea level is complete AV canal, at altitude, we find more benign forms of cardiac compromise

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Chapter 86

Pulmonary Embolism and Deep Vein Thrombosis Paul F. Currier and Charles A. Hales

Abstract  This chapter provides an overview of pulmonary embolism (PE) and deep vein thrombosis (DVT) – the history of their clinical recognition and treatment, pathogenesis, epidemiology, risk factors, prognosis, and current therapy. Many of these topics will be addressed in detail in other chapters. PE and DVT will be referred to together as venous thromboembolism (VTE). Keywords  Venous thromboembolism • Chronic thromboembolic pulmonary hypertension • Thrombus • Embolus

1  History of the Disease Although the description of pathologic blood coagulation occurred in China as early as 2650 BC, the first note of venous thrombosis is thought to have occurred around 1271 AD in Italy [1, 2]. Rudolf Virchow is credited with describing the triad of factors (venous stasis, endothelial injury, and alterations in coagulation) which still today are recognized as significant factors in the development of VTE. It is unclear if Virchow actually ever described these as a triad. In fact, by the time that Virchow was investigating pulmonary and venous thrombosis in the 1800s, these pathologic factors had already been described in the literature [1, 3, 4]. Wiseman had suggested the impact of coagulation factors and venous stasis in the development of VTE in as early 1686 [1, 5]. Virchow certainly did make great contributions to the understanding of VTE. As part of his work in the context of autopsies at the Charité Hospital in Berlin, Virchow

P.F. Currier (*) Massachusetts General Hospital, Harvard Medical School, Department of Medicine, 55 Fruit Street, Bulfinch 148, Boston, MA 02114, USA e-mail: [email protected]

c­ oncluded that clots found in the pulmonary vasculature had traveled from the deep veins and coined the term ­“embolism.” This contradicted a widely held belief at the time that most clots in the pulmonary vasculature developed in situ. Further animal experiments helped to confirm these findings. Virchow wrote extensively about the likely pathologic ­factors contributing to the development of VTE, and his prominence certainly helped to lead to this triad of factors bearing his name [1]. In Virchow’s era, therapies for venous thrombosis included warm compresses, leeches, and in what now seems paradoxical, bed rest [1]. Proximal ligation of veins to treat DVT was first attempted as early as the 1680s [1, 6]. It was performed on Richard M. Nixon in 1974 with ligation of the left ­external iliac vein. It was done in the context of an extensive clot in the left lower extremity and difficulties with prior compliance with anticoagulants by the president. He lived for another 19 years on warfarin therapy and died of unrelated causes [7]. Surgical embolectomy was the first described treatment for PE in 1908. Trendelenburg performed the procedure in three patients, all of whom died [8]. The development of cardiopulmonary bypass helped lead embolectomy to become a safer procedure. The first known successful embolectomy using cardiopulmonary bypass was performed in 1961 [8, 9]. Greenfield described the first successful use of catheterbased removal of a thrombus by suction in 1971. Further experience with this technique revealed the importance of proximally blocking the embolization of a disrupted clot to the pulmonary arteries. This resulted in the use of inferior vena cava (IVC) filters (first introduced in 1969 by MobinUddin et al. and then in 1973 by Greenfield et al.) both in and out of the context of this procedure to prevent a clot in the deep venous system from propagating to the pulmonary vasculature [8, 10, 11]. Heparin was isolated from canine liver in 1916 and 1918 by different groups. It was named from the Greek word for “liver,” hepar [12]. It was first used in the treatment of VTE in 1938. Dramatic positive treatment results were reported by multiple

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_86, © Springer Science+Business Media, LLC 2011

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groups [8]. The findings of the first and only randomized controlled trial of heparin versus no treatment for a clinical diagnosis of VTE were published in 1960. The trial was stopped after only 35 patients had been randomized because of the significant decrease in clinical diagnosis of VTE and in death that occurred in the group treated with heparin [13]. Oral anticoagulants were first developed after the veterinary pathologists Schofield and Roderick discovered that spoiled sweet clover was causing a hemorrhagic disease of cattle in the 1920s [14]. This serendipitous discovery was a positive development that came out of the financial challenges of the Great Depression. The moldy hay that contained the sweet clover and which was being fed to the cows would normally have been discarded, had it not been for the farmers trying to save money during these dark financial times [12]. These observations ultimately led to the isolation and synthesis of dicumarol from the clover in 1940 in the laboratory of Karl Link at the University of Wisconsin [12]. Subsequently, the synthesis of the more potent, faster-acting related drug warfarin occurred. Warfarin was first used in 1955 [8, 12, 14, 15]. The first objective evidence of the use of thrombolytic therapy for VTE in dogs and humans was described with the use of streptokinase in 1964 [16]. Streptokinase had been isolated after William Smith Tillett serendipitously noted that clotted human plasma would liquefy in the presence of streptococcal cultures [17]. Because it is isolated from streptococcal bacteria, streptokinase has the propensity to cause immune reactions and has the potential to be inactivated in people with pre-existing antibodies [8]. Therefore, there has been significant work to develop newer types of thrombolytic therapies, which will be detailed in Chap. 108. Much work is currently being done in attempting to develop newer diagnostic and therapeutic options for treatment of VTE. This will be detailed in other chapters in this book.

P.F. Currier and C.A. Hales

flow, such as at the venous valves. The additional proximal DVTs develop de  novo in these veins without prior calf involvement. Iliofemoral thromboses appear to be the source of most clinically apparent pulmonary emboli as they are large enough to allow for formation of large clots [18–20]. Most PE is bilateral. Lower lobes are more often involved than upper lobes, and emboli are usually multiple. Only about 10% of emboli cause infarction, usually in patients with pre-existing cardiopulmonary disease [21]. Infarction, which is defined pathologically by tissue necrosis, can be recognized as a peripherally based, often wedge-shaped, persistent scar on the chest radiograph or CT scan. Once in the pulmonary circulation, large clots, such as may come from the iliofemoral veins, may lodge at the bifurcation of the pulmonary and lobar arteries, causing hemodynamic compromise. This may be transient as the clot breaks up and moves distally, obstructing less of the vascular bed. Smaller clots continue distally to arterioles or capillaries. Physiologic effects are not simply due to physical obstruction by a clot, but are also due to neurohumoral effects. Hypoxemia is due to ventilation–perfusion mismatching and shunt in the context of hypoxemic vasoconstriction, alterations in surfactant, and atelectasis [22, 23]. Pulmonary artery pressures increase in part due to hypoxemic vasoconstriction. In severe cases, this can cause both right ventricular dysfunction and loss of cardiac output leading to hypotension and cardiovascular collapse [22]. Dead space increases owing to the obstruction of the pulmonary arteries by thrombus. With areas of lung that are ventilated but not perfused, elimination of carbon dioxide is impaired. However, chemoreceptors in the medulla will normally cause an increase in minute ventilation after sensing increased levels of carbon dioxide. Irritant receptors in the lung may also be activated by neurohumoral pathways. Therefore, despite the increase in dead space, in most patients with pulmonary embolus, the arterial carbon dioxide levels will be either normal or even below normal [22].

2  Pathogenesis Pulmonary emboli usually develop after clots first form in the deep venous system and then break off and travel to the pulmonary arteries. DVT usually begins in the lower extremities, although occasional thrombi form in pelvic veins, renal veins, upper-extremity veins, and the right side of the heart [18–20]. The majority of calf thrombi seem to resolve spontaneously and clinically apparent embolization to the lung is uncommon. Still, about 20–30% of untreated calf clots will propagate to the proximal popliteal, femoral, or iliac veins. Of clots noted in these popliteal, femoral, or iliac veins, 50–80% of them are thought to originate in the calf veins. Most of these thrombi are thought to originate at sites of low

3  Incidence and Epidemiology The incidence of PE is estimated to be greater than one in 1,000, with a mortality at 3 months of around 15% in those with PE [24]. The incidence of DVT ranges from 10–40% among hospitalized medical and general surgical patients without prophylaxis. Rates of DVT in orthopedic patients in the absence of prophylaxis are among the highest, in the range of 40–60% [25]. Factors that lead to a higher risk of VTE within these groups are discussed in the following section. Of these DVTs, approximately one quarter to one third involve the deep venous system and are therefore more likely to lead to PE. In

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one study of patients discharged from the hospital, VTE was the second most common complication and cause of excess length of stay and the third most common cause of excess mortality [25, 26]. With prophylaxis, rates of DVT are reduced significantly and it is important that all hospitalized patients receive appropriate prophylaxis [25]. In a large review of data from the USA from the National Center for Health Statistics from 1979 to 1998, “pulmonary thromboembolism” (PTE) was listed on the death certificates of 1.3% of the approximately 43 million cases reviewed. Of these cases, 33.9% had PTE listed as the underlying cause of death. Over the years from 1979 to 1998, the age-adjusted death rate from PTE decreased by 56% for men and 46% for women [27]. Age-adjusted mortality rates from VTE were 50% higher for blacks than for whites and 50% higher for whites than for those of other races. Mortality rates were also 20–30% higher in men than in women when adjusted for other factors [27]. Whether these differences are attributable to underlying genetic differences or to differences in delivery of health care is unknown.

4  Risk Factors Risk factors for VTE are both genetic and acquired. Some acquired factors can be controlled, some cannot. Older age is associated with a higher risk of VTE [28]. Cardiovascular risk factors associated with VTE include hypertension, diabetes mellitus, smoking, atherosclerosis, and hypercholesterolemia [29–31]. Chronic illnesses such as congestive heart failure, inflammatory bowel disease, rheumatologic disease, and nephrotic syndrome are all associated with an increased risk of VTE [32]. Obesity has recently been recognized as a risk factor for both primary and recurrent VTE [29, 33]. Cancer is significantly associated with VTE [28, 32]. Hospitalized patients for both surgical and medical illnesses are at increased risk of VTE [34]. Placement of a foreign device such as a central venous catheter or a pacemaker is also associated with VTE [34]. Medicines which have been associated with an increased risk of VTE include oral contraceptives, tamoxifen, and corticosteroids [35, 36]. A more complete list of acquired risk factors is shown in Table 1. Genetic risk factors for VTE have been divided into those which lead to reduced levels of inhibitors of the coagulation cascade (e.g., protein C, protein S, and antithrombin deficiency) and those which increase the level or function of coagulation factors (commonly factor V Leiden gene mutation, and prothrombin gene mutation, and rarely the dysfibrogenemias) [37]. The genetic factors which lead to reduced levels of coagulation inhibitors are as a group less common and are associated with a

1233 Table 1  Acquired factors associated with an increased risk for venous thromboembolism (VTE) Increasing age Neurologic disease with extremity paresis Hypertension Immobility Diabetes mellitus Superficial vein thrombosis/varicose veins Smoking Inflammatory bowel disease Hypercholesterolemia Acute infection Atherosclerosis Pregnancy Obesity Long air travel Hospitalization Rheumatologic disease Recent surgery Nephrotic syndrome Malignancy Corticosteroid use Trauma Fractures Congestive heart failure Oral contraceptives Central venous catheter Tamoxifen Pacemaker Elevated triglyceride levels (postmenopausal women)

Table 2  Genetic risk factors in VTE Activated protein C resistance Factor V Leiden mutation Prothrombin gene mutation Antithrombin deficiency Protein C deficiency Protein S deficiency Dysfibrogenemias

greater risk of venous thrombosis than those which lead to increased function of the ­coagulation cascade [37] (Table 2). Factor V Leiden and prothrombin gene mutation are the most common genetic thrombophilias, found in 10% and 5–10%, respectively, of patients presenting with venous thrombosis [37]. The presence of genetic risk factors varies by ethnicity. For example, factor V Leiden mutation is common in patients of European heritage, but is rare in patients of Asian or African origin [37, 38]. The complexity of genetic risk factors is only now starting to be understood. As an example, factor V Leiden mutation is associated with a significantly increased risk of DVT, but only a mildly increased risk of PE. This is sometimes referred to as the factor V Leiden paradox in that the mechanism for this difference is currently unclear [39]. A greater understanding of the genetic risk factors for VTE and further knowledge of individual genetotypes will likely allow for a better ability to screen for VTE and stratify for intensity of prophylaxis in addition to helping to make decisions on long-term anticoagulation.

5  Current Treatment This section is designed to provide a brief overview of current treatment. Therapy of VTE will be covered in detail in other chapters.

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Therapy can be divided into medical and surgical therapies. Medical therapies consist of acute treatment, including thrombolysis and anticoagulation, and chronic treatments. Surgical therapies consist of intravascular and open procedures.

5.1  Medical Management When a physician is confronted with treatment for VTE, medical management has traditionally been preferred owing to relatively good outcomes in patients in whom thrombolysis or anticoagulation is not contraindicated. As various mechanical, particularly noninvasive surgical methods are further improved, this general therapeutic preference may change, particularly when large emboli are present in the context of hemodynamic instability.

5.1.1  Thrombolytic Therapy A primary decision point when one is confronted with the treatment for VTE is whether to employ fibrinolytic therapy. Thrombolytic agents act to lyse clots by breaking up fibrin and fibrinogen. There are three thrombolytic agents presently approved for treatment of PE: streptokinase, urokinase, and tissue plasminogen activator. The diagnosis of massive PE (as defined by hemodynamic instability, i.e., systolic blood pressure below 90 mmHg or a drop of 40 mmHg from baseline systolic blood pressure, resulting from PE) is now generally regarded as an indication for the use of fibrinolytic therapy in the absence of contraindication to this systemic therapy [40]. Thrombolytic therapy in submassive PE (any PE without hemodynamic instability) is generally not employed because of concerns over hemorrhagic risks outweighing potential benefits. A recent review of available literature showed no significant difference in mortality or major hemorrhagic events (such as intracranial or retroperitoneal bleeds or decreases in hemoglobin by more than 2 g/dL) in studies examining the use of thrombolytic therapy in submassive PE. There was a nonsignificant increase in the number of minor hemorrhagic events [41]. Another metaanalysis showed no significant benefit to thrombolytic therapy in unselected patients with PE, although there was a decrease in either recurrent PE or death noted in five trials that enrolled patients with hemodynamically significant PE [42]. One trial showed that treating submassive PE along with pulmonary hypertension or right ventricular dysfunction with heparin alone did lead to a need for escalation of treatment. In this study there was a significant

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improvement in the primary end point of either death or escalation of treatment when these patients were treated with thrombolytic therapy [43]. Although right ventricular dysfunction has been suggested as a useful tool for determining the need for lytic therapy [44], it is unclear if right ventricular dysfunction in submassive PE is a predictor of a significant outcome [45]. Moreover, although data suggest that thrombolytic therapy may improve right ventricular function acutely in submassive PE, data also suggests that outside the acute period there is no long-term difference in right ventricular function in patients with submassive PE treated with thrombolytics or unfractionated heparin (UFH) [46, 47]. Various biomarkers are also being used to help stratify risk with PE and to potentially help make decisions on the use of thrombolysis. Brain natriuretic peptide (BNP) or N-terminal pro-brain natriuretic peptide (NT-proBNP) is released in the context of myocardial stretch. In a meta-analysis of 16 studies of PE, mortality was increased by sixfold [95% confidence interval (CI) 1.31–27.42, p = 0.0021] for BNP above 100 pg/mL and 16.12-fold (95% CI 3.1–83.68, p = 0.001) for NT-proBNP above 600  ng/L [48]. In a meta-analysis of 13 studies of patients with PE, patients with an elevated BNP or NT-proBNP level were found to have a higher 30-day mortality [odds ratio (OR) 7.6; 95% CI 3.4–17] and a 10% risk of dying [49]. Elevated levels of troponins may also help to predict death. In a meta-analysis of 20 studies, elevated troponin levels were significantly associated with increased short-term mortality (OR 5.24; 95% CI 3.28–8.38) and death from PE (OR 9.4; 95% CI 4.14–21) [49, 50]. Combining the use of NT-proBNP and troponin may help to further predict mortality. In a study of 100 normotensive patients with acute PE, 40-day mortality in patients with NT-proBNP of 600  ng/L and above and troponin T of 0.07 mg/L and above reached 33%. There were no deaths in the group with NT-proBNP below 600 ng/L. For NT-proBNP of 600 ng/L and above and troponin T below0.07 mg/L, mortality was 3.7%. Echocardiographic data did not improve stratification [51]. New biomarkers promise to help further stratify the outcome for PE. Growth differentiation factor  15 is a cytokine induced by cardiac ischemia or heart failure. It has been shown to be an independent predictor for a complicated course due to PE and in multivariate logistic regression has been shown to enhance the ability of tronponin T, NT-proBNP, and right ventricular dysfunction on the echocardiogram to predict complications due to PE at 30 days [52]. However, care is taken to note that such biomarkers alone should not be used to justify more invasive treatment strategies such as thrombolysis [49].

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5.1.2  Anticoagulation UFH has been a mainstay of medical therapy for submassive PE since it was shown to decrease mortality compared with a placebo in 1960 [13]. UFH binds to antithrombin to inactivate thrombin and factor Xa. By inactivating thrombin, heparin prevents fibrin formation and inhibits activation of platelets and factors V and VIII [53]. It is normally given intravenously and is titrated by monitoring the activated thromboplastin time. However, low molecular weight heparins (LMWHs) now seem preferable to UFH for treatment except in select groups of patients. LMWHs are derived from UFH and are approximately one third of its size. Because of their smaller size, they are unable to bind antithrombin and thrombin simultaneously as does heparin. LMWHs therefore have less ability to inactivate thrombin. However, they maintain their ability to inactivate factor Xa – a primary means via which they serve as anticoagulants [53]. Inactivation of factor Xa blocks the generation of thrombin. LMWHs also seem to exert their effects via tissue factor pathway inhibitor, an important mediator of anticoagulant and antithrombotic effects [54]. Overall, when compared with UFH, LWMHs seems to lead to decreased mortality, a decreased risk of bleeding, decreased recurrence of PE, and a decreased rate of heparin-induced thrombocytopenia [55, 56]. Because of its short half-life of 1–2  h compared with the longer half-life of LMWHs, UFH is still the first choice for acute therapy in patients in whom anticoagulation may need to be quickly stopped, e.g., patients in whom thrombolytic therapy may be employed or patients undergoing procedures or at high risk of bleeding. It is also preferred over LMWHs in patients with renal failure (because of dosing difficulties imposed by renal clearance of LMWHs) or with morbid obesity (which can unpredictably affect the elimination and distribution of LMWHs because of delayed uptake from subcutaneous administration sites) and because of its ability to be titrated rapidly on the basis of the activated partial thromboplastin time. Fondaparinux is a synthetic anticoagulant that binds to antithrombin III and potentiates its inhibition of factor Xa. Because it does not bind platelet factor 4, it is unlikely to cause heparin-induced thrombocytopenia [57]. It has been shown to be not inferior to UFH or LMWHs in the treatment of PE [58, 59]. Warfarin remains the only currently approved oral anticoagulant. It acts by inhibiting the vitamin K dependent clotting factors II, VII, IX, and X and by impairing the function of the anticoagulants activated proteins C and S [53]. Use of warfarin can be started concurrently with or after the initiation of intravenous or subcutaneous therapy for treatment of PE. Its use should not be started as the only initial therapy for

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acute PE. This is because warfarin alone can induce a hypercoagulable state. It does so because it decreases the anticoagulant factors activated proteins C and S faster than it inhibits the procoagulant factors, leaving the patient hypercoagulable [60]. Recommendations for the duration of treatment with warfarin for VTE generally vary between 3 months and the lifetime of the patient on the basis of the underlying risk factors for recurrence of thromboembolism [61]. Work is currently being done to stratify the risk of recurrence of VTE following cessation of treatment with warfarin to help guide the duration of therapy. One study stratified patients on the basis of abnormal or normal D-dimer level 1 month ­following cessation of anticoagulation following first unprovoked DVT or PE. The adjusted hazard ratio for recurrent VTE for those who stopped anticoagulation with an abnormal D-dimer level compared with a normal D-dimer level was 2.27 [1.15, 4.46], p = 0.02, at mean follow-up of 1.4 years [62]. Another study combined D-dimer with factor VIII levels approximately 1 month following cessation of anticoagulation following the first episode of idiopathic proximal DVT of the lower extremities. At 2 years, recurrent VTE was found in 6% of patients with a negative D-dimer level and a factor VIII level below 75%, and in 20% of those with a negative D-dimer level and a factor VIII levels above 75%. Recurrent VTE was found in 29% of patients with a D-dimer level above 500 ng/mL, and in over 50% of patients with a D-dimer level above 500 ng/mL and a factor VIII level above 75% [63]. Therapies for VTE that are currently being investigated include direct thrombin inhibitors and new inhibitors of factor Xa. Thrombin inhibitors block activity of existing thrombin. Several of these agents are currently in clinical trials [64].

5.2  Embolectomy Surgical and catheter-based embolectomy for the treatment of PE is typically reserved for patients in whom there is a contraindication to thrombolysis or insufficient time for them to receive thrombolytics and in whom there is hemodynamic instability (systolic blood pressure below 90 mmHg or a drop in systolic blood pressure of 40 mmHg or more) with a subtotal or total filling defect in the right, left, or bilateral pulmonary arteries [61, 65]. The use of these criteria depends upon the often high reported rate of complications and mortality associated with surgical embolectomy. Catheter-based embolectomy is still a relatively new technology and the understanding of its role is still developing. Still, this technology can be life-saving in properly selected patients and if it is used at centers where there is sufficient expertise.

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5.2.1  Surgical Embolectomy Surgical embolectomy involves median sternotomy with ­cardiac bypass followed by pulmonary arteriotomy and embolectomy. It often includes clipping of the IVC as well [66]. Most deaths with massive PE occur in the first 1–2 h after the event. Thus, to be effective, embolectomy must be done in the first hour, which only hospitals with a standing bypass team can accommodate. In a review of 46 case series of pulmonary embolectomy from 1961 to 2006, average mortality was 30%. In those patients undergoing the procedure before 1995, mortality was 32%. In patients undergoing embolectomy from 1985 to 2005, mortality was 20%. Mortality was significantly higher in patients experiencing cardiac arrest prior to embolectomy (59 vs. 29% without a history of arrest) [67]. In what is likely the only trial of surgical embolectomy versus thrombolysis, 37 patients were followed long term. In the nonrandomized trial, patients treated with surgical embolectomy had a 5-year survival rate of 77% compared with 67% for those patients treated with thrombolytic therapy [66]. However, the fact that this trial included only a small number of patients and that it was not randomized and therefore allowed for selection bias makes conclusions from the data difficult to draw. Although it is difficult to draw a definitive conclusion on the general utility of surgical embolectomy, the fact that a meta-analysis of thrombolytic therapy shows rates of combined recurrence of PE or death of only around 9% in patients with massive PE [42] along with the invasive nature of the procedure and specialized resources necessary to perform it makes it unlikely that it will be used except in select patients.

5.2.2  Catheter-Based Embolectomy Percutaneous intervention offers a less invasive means than surgical embolectomy in the treatment of massive PE. Techniques include (1) balloon angioplasty in which the thrombus is compressed against the vessel wall, (2) various types of rotational devices which break up the clot and then aspirate the clot to minimize further embolization, and (3) hydrodynamic or rheolytic catheters in which high-velocity injection via one catheter lumen serves to break up the clot. This injection of fluid via the small lumen of the catheter causes a low-pressure gradient in the larger lumen of the catheter via the Bernoulli effect. This low-pressure vortex facilitates aspiration of the clot via the larger catheter lumen [65]. There is at this time limited data on the efficacy of catheter-based embolectomy in massive PE. A recent review cited only slightly more than 300 cases for which there have been data published. There have been no randomized controlled trials. Overall, the clinical success rate was around 80%, with mortality rates in the case series of 0–25% [65]. Most of

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these interventions were performed in conjunction with thrombolytic therapy, making it difficult to judge the efficacy of the physical techniques themselves. The most significant complications of therapy include perforation of the pulmonary artery or right ventricle. Because risk of pulmonary artery perforation increases as vessel size decreases, it is recommended that percutaneous devices only be used in main and lobar pulmonary arteries [65]. Other complications include bleeding, arrhythmia, induction of hemolysis, and other complications that accompany any catheter-based intervention.

5.2.3  Inferior Vena Cava Filters IVC filters serve to block the embolization of a clot to the pulmonary vasculature. IVC filters are usually inserted percutaneously using fluoroscopy to position the filter. IVC filters can be either permanent or retrievable. Concerns exist about the development of a clot due to disrupted flow caused by the filter on a long-term basis. Therefore, retrievable filters are often preferred in a patient with transient risk factors for VTE or in whom anticoagulation can be used at a later date. The ability to successfully remove retrievable filters declines with time owing to the filter becoming embedded in tissue. In one case series, removal of the retrievable filter was successful in 91% of cases [68]. At our institution, efforts are made to remove retrievable IVC filters within 2 months of placement to facilitate successful removal. There has been one randomized controlled trial of IVC filters. This showed that IVC filters were associated with a significantly lower rate of PE acutely (at 8–12 days), but that this difference was no longer significant at 2 years. IVC filters were associated with significantly more recurrent DVT at 2 years than anticoagulation alone, with no significant difference in PE or mortality [69]. IVC filters are an important part of the management of some patients with VTE disease, such as those in whom anticoagulation is contraindicated or those who are hemodynamically compromised from PE and who still have a clot in the leg veins which if embolized could cause death. However, there is a lack of randomized trials in examining IVC filters versus other treatments and in comparing different kinds of filters. Therefore, the role of IVC filters in all patients with DVT and the relative merits of different filters are unclear.

6  Conclusion Much has been learned since the first recognition of venous thrombosis thousands of years ago. Our understanding of the pathophysiologic processes, risks, and epidemiologic factors

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of the disease has progressed significantly. Developments in the near future will likely focus on a better understanding of the genetic risks factors for VTE and on pharmacogenetics to allow for more individualized treatment of patients. Clarifying which patients benefit from certain treatments will allow for more efficacious use of both pharmacologic and mechanical treatments.

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1237 24. Goldhaber SZ, Visani L, De Rosa M (1999) Acute pulmonary embolism: clinical outcomes in the International Cooperative Pulmonary Embolism Registry (ICOPER). Lancet 353: 1386–1389 25. Geerts WH, Bergqvist D, Pineo GF et  al (2008) Prevention of venous thromboembolism. Chest 133:381S–453S 26. Zhan C, Miller MR (2003) Excess length of stay, charges, and mortality attributable to medical injuries during hospitalization. J Am Med Assoc 290:1868–1874 27. Horlander KT, Mannino DM, Leeper KV (2003) Pulmonary embolism mortality in the United States, 1979-1998. Arch Intern Med 163:1711–1717 28. Alikhan R, Cohen AT, Combe S (2004) Risk factors for venous thromboembolism in hospitalized patients with acute medical illness. Arch Intern Med 164:963–968 29. Ageno W, Becattini C, Brighton T, Selby R, Kamphuisen PW (2008) Cardiovascular risk factors and venous thromboembolism – a meta-analysis. Circulation 117:93–102 30. Prandoni P, Bilora F, Marchiori A et  al (2003) An association between atherosclerosis and venous thrombosis. N Engl J Med 348:1435–1441 31. Doggen CJM, Smith NL, Lemaitre RN (2004) Serum lipid levels and the risk of venous thrombosis. Arterioscler Thromb Vasc Biol 24:1970–1975 32. Prandoni P (2005) Acquired risk factors for venous thromboembolism in medical patients. ASH Education Book 2005:458–461 33. Eichinger S, Hron G, Bialonczyk C et al (2008) Overweight, obesity, and the risk of recurrent venous thromboembolism. Arch Intern Med 168:1678–1683 34. Heit JA, O’Fallon WM, Petterson TM et al (2002) Relative impact of risk factors for deep vein thrombosis and pulmonary embolism. Arch Intern Med 162:1245–1248 35. Huerta C, Johansson S, Wallander MA, García Rodríguez LA (2007) Risk factors and short-term mortality of venous thromboembolism diagnosed in the primary care setting in the United Kingdom. Arch Intern Med 167:935–943 36. Decensi A, Maisonneuve P, Rotmensz N et  al (2005) Effect of tamoxifen on venous thromboembolic events in a breast cancer prevention trial. Circulation 111:650–656 37. Crowther MA, Kelton JG (2003) Congenital thrombophilic states associated with venous thrombosis: a qualitative overview and proposed classification system. Ann Intern Med 138:128–134 38. Ridker PM, Miletich JP, Hennekens CH, Buring JE (1997) Ethnic distribution of factor V Leiden in 4047 men and women. Implications for venous thromboembolism screening. J Am Med Assoc 277:1305–1307 39. van Stralen KJ, Doggen CJM, Bezemer ID, Pomp ER, Lisman T, Rosendaal FR (2008) Mechanisms of the factor V Leiden paradox. Arterioscler Thromb Vasc Biol 28:1872–1877 40. Hirsh J, Guyatt G, Albers GW et  al (2008) Antithrombotic and thrombolytic therapy 8th ed: ACCP guidelines. Chest 133:71S–105S 41. Dong B, Jirong Y, Liu G, Wang Q, Wu T (2006) Thrombolytic therapy for pulmonary embolism. Cochrane Database Syst Rev 2:CD004437 42. Wan S, Quinlan DJ, Agnelli G, Eikelboom JW (2004) Thrombolysis compared with heparin for the initial treatment of pulmonary embolism: a meta-analysis of the randomized controlled trials. Circulation 110:744–749 43. Konstantinides S, Geibel A, Heusel G et  al (2002) Heparin plus alteplase compared with heparin alone in patients with submassive pulmonary embolism. N Engl J Med 347:1143–1150 44. Piazza G, Goldhaber SZ (2006) Acute pulmonary embolism part II: treatment and prophylaxis. Circulation 114:e42–e47 45. Ten Wolde M, Söhne M, Quak E, MacGillavry MR, Büller HR (2004) Prognostic value of echocardiographically assessed right ventricular dysfunction in patients with pulmonary embolism. Arch Intern Med 164:1685–1689

1238 46. Goldhaber SZ, Haire WD, Feddstein ML (1993) Alteplase versus heparin in acute pulmonary embolism: randomized trial assessing right-ventricular function and pulmonary perfusion. Lancet 341:507–510 47. Konstantinides S, Tiede N, Geibel A, Olschewski M, Just H, Kasper W (1998) A Comparison of alteplase versus heparin for resolution of major pulmonary embolism. Am J Cardiol 82:966–970 48. Cavallazzi R, Nair A, Vasu T, Marik PE (2008) Natriuretic peptides in acute pulmonary embolism: a systematic review. Intensive Care Med 34:2147–2156 49. Klok FA, Mos ICM, Huisman MV (2008) Brain-type natriuretic peptide levels in the prediction of adverse outcome in patients with pulmonary embolism. Am J Respir Crit Care Med 178:425–430 50. Becattini C, Vedovati MC, Agnelli G (2007) Prognostic value of troponins in acute pulmonary embolism, a meta-analysis. Circulation 116:427–433 51. Kostrubiec M, Pruszczyk P, Bochowicz A et al (2005) Biomarkerbased risk assessment model in acute pulmonary embolism. Eur Heart J 26:2166–2172 52. Lankeit M, Kempf T, Dellas C et al (2008) Growth differentiation factor-15 for prognostic assessment of patients with acute pulmonary embolism. Am J Respir Crit Care Med 177:1018–1025 53. Hirsh J, Anand SS, Halperin JL, Fuster V (2001) Mechanism of action and pharmacology of unfractionated heparin. Arterioscler Thromb Vasc Biol 21:1094–1096 54. Hoppensteadt DA, Walenga JM, Fasanella A, Jeske W, Fareed J (1995) TFPI antigen levels in normal human volunteers after intravenous and subcutaneous administration of unfractionated heparin and a low molecular weight heparin. Thromb Res 77:175–185 55. Martel N, Lee J, Wells PS (2005) Risk for heparin-induced thrombocytopenia with unfractionated and low molecular-weight heparin thromboprophylaxis: a meta-analysis. Blood 106:2710–2715 56. Van Dongen CJJ, van den Belt AG, Prins MH, Lensing AWA (2004) Fixed dose subcutaneous low molecular weight heparins versus adjusted dose unfractionated heparin for venous thromboembolism. Cochrane Database Syst Rev 4:CD001100 57. Dager WE, Andersen J, Nutescu E (2004) Special considerations with fondaparinux therapy: heparin induced thrombocytopenia and wound healing. Pharmacotherapy 24:88S–94S

P.F. Currier and C.A. Hales 58. Büller HR, Davidson BL, Decousus H et al (2003) Subcutaneous fondaparinux versus intravenous unfractionated heparin in the ­initial treatment of pulmonary embolism. N Eng J Med 349: 1695–1702 59. Büller HR, Davidson BL, Decousus H et al (2004) Fondaparinux or enoxaparin for the initial treatment of symptomatic deep venous thrombosis. Ann Intern Med 40:867–873 60. Brandjes DP, Heijboer H, Büller HR, de Rijk M, Jagt H, ten Cate JW (1992) Acenocoumarol and heparin compared with acenocoumarol alone in the initial treatment of proximal-vein thrombosis. N Engl J Med 327:1485–1489 61. Kearon C, Kahn SR, Agnelli G et al (2008) Antithrombotic therapy for venous thromboembolic disease: American College of Chest Physicians evidence-based clinical practice guidelines (8th edition). Chest 133:454S–545S 62. Palareti G, Cosmi B, Legnani C (2006) D-dimer testing to determine the duration of anticoagualation therapy. N Engl J Med 355:1780–1789 63. Cosmi B, Legnani C, Cini M, Favaretto E, Palareti G (2008) D-dimer and factor VIII are independent risk factors for recurrence after anticoagulation withdrawal for a first idiopathic deep vein thrombosis. Thromb Res 122:610–617 64. Gross PL, Weitz JI (2008) New anticoagulants for the treatment of  venous thromboembolism. Arteroscler Thromb Vasc Biol 28: 380–386 65. Kucher N (2007) Catheter embolectomy for acute pulmonary embolism. Chest 132:657–663 66. Gulba DC, Schmid C, Borst HG, Lichtlen P, Dietz R, Luft FC (1994) Medical compared with surgical treatment for massive pulmonary embolism. Lancet 343:576–577 67. Stein PD, Alnas M, Beemath A, Patel NR (2007) Outcome of pulmonary embolectomy. Am J Cardiol 99:421–423 68. Stein PD, Alnas M, Skaf E et al (2004) Outcome and complications of retrievable inferior vena cava filters. Am J Cardiol 94:1090–1093 69. Decousus H, Leizorovicz A, Parent F et al (1998) A clinical trial of vena caval filters in the prevention of pulmonary embolism in patients with proximal deep-vein thrombosis. N Eng J Med 338: 409–415

Chapter 87

Pulmonary Hypertension Due to Pulmonary Embolism and Thromboembolic Obstruction of Proximal and Distal Pulmonary Arteries Peter F. Fedullo, Kim M. Kerr, and William R. Auger

Abstract  Considerable advances have been made in the diagnostic approach and surgical management of patients afflicted with chronic thromboembolic pulmonary hypertension (CTEPH). CTEPH occurs in a minority of patients following a single or recurrent episode of pulmonary embolism. Although incidence figures 3.1% following a single episode of pulmonary embolism and 13.4% following a recurrent venous thromboembolism have been reported, these figures would appear to substantially overstate the true incidence. Recent series suggest an incidence of 0.8–1.0%, a rate somewhat more consistent with the annual number of patients experiencing pulmonary embolism and the number of pulmonary thromboendarterectomy (PTE) procedures performed in the USA. Given what appears to be a large number of patients with persistent postembolic pulmonary vascular obstruction, it also remains uncertain why only a relatively small number develop pulmonary hypertension and what factors determine the initiation and progression of their disease. Although the overall extent of pulmonary vascular obstruction appears to play a central role in the initiation, there is a growing body of evidence to suggest that the increa­sing pulmonary vascular resistance (PVR) in the majority of patients arises from events in the distal pulmonary vascular bed rather than from central obstruction. That the progression of the pulmonary hypertension is a consequence of this distal arteriopathy is supported by several lines of evidence. What is not subject to debate is that survival without intervention is poor and, as in other forms of pulmonary hypertension, is proportional to the degree of pulmonary hypertension and right ventricular dysfunction at the time of diagnosis. Keywords  Chronic thromboembolic pulmonary hypertension • Pulmonary embolism • Pulmonary endarterectomy • Postoperative outcome

P.F. Fedullo (*) Division of Pulmonary Critical Care Medicine, University of California, San Diego, 200 West Arbor Drive, MC 8372, San Diego, CA 92103-8372, USA e-mail: [email protected]

1 Natural History Considerable advances have been made in the diagnostic approach and surgical management of patients afflicted with chronic thromboembolic pulmonary hypertension (CTEPH). Despite these advances, a great deal of uncertainty remains regarding the epidemiology and natural history of the disease. The natural history of acute pulmonary embolism under most circumstances includes sufficient thromboembolic resolution to restore normal pulmonary hemodynamics, gas exchange, and exercise tolerance. However, complete anatomic recovery after acute pulmonary embolism may not occur. A recent meta-analysis of imaging studies evaluating the resolution rate of acute pulmonary embolism revealed that 52% of patients had evidence of residual emboli 11 months after the acute event [1]. The extent of residual disease may be extensive. In a study by Wartski and Collignon, 8.3% of patients had residual vascular obstruction exceeding 50% of the pulmonary vascular bed 3 months after the acute event as determined by perfusion lung scintigraphy [2]. CTEPH occurs in a minority of patients following a single or recurrent episode of pulmonary embolism. Although it was initially estimated to occur in 0.1–0.5% of embolic survivors, recent data suggest that the disease is somewhat more common. Although incidence figures as high as 3.1% following a single episode of pulmonary embolism and 13.4% following a recurrent venous thromboembolism have been reported, these figures would appear to substantially overstate the true incidence [3]. Recent series suggest an incidence of 0.8–1.0%, a rate somewhat more consistent with the annual number of patients experiencing pulmonary embolism and the number of pulmonary thromboendarterectomy (PTE) procedures performed in the USA [4, 5]. If it is assumed that 300,000 patients experience pulmonary embolism annually in the USA and that 210,000 have long-term survival potential, approximately 1,700–2,100 patients would be expected to develop CTEPH [5, 6]. This figure, however, considerably exceeds the number of PTE procedures performed annually in the USA, estimated to be in the range of 300. Either an estimated incidence of 1% is an overstatement and the true incidence is closer to the

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0.1–0.5% initially estimated or the disease is being substantially overlooked or misdiagnosed. The risk factors for incomplete thromboembolic resolution remain largely unknown. The presence of coexisting cardiopulmonary disease, the initial embolic burden, and the age of the thrombus at the time of embolization may all contribute. Although a defect in fibrinolytic activity has not been identified in patients with CTEPH, recent data suggest that certain patients may have fibrinogen variants that render them resistant to lysis [7, 8]. Identifiable thrombophilic tendencies in patients referred for PTE have included elevated factor VIII levels and the presence of antiphospholipid antibodies and/or a lupus anticoagulant [9, 10]. Given what appears to be a large number of patients with persistent postembolic pulmonary vascular obstruction, it also remains uncertain why only a relatively small number develop pulmonary hypertension and what factors determine the initiation and progression of their disease. Although the overall extent of pulmonary vascular obstruction appears to play a central role in the initiation, the effects of circulating vasoconstrictors, immune-related events, or an individual genetic predisposition resulting in the development of a hypertensive pulmonary arteriopathy may contribute to this outcome. The pathophysiologic events responsible for progression of the pulmonary hypertension also remain uncertain. In certain patients the hemodynamic and symptomatic decline may be related to recurrent embolic events or in situ pulmonary artery thrombosis. However, there is a growing body of evidence to suggest that the increasing pulmonary vascular resistance (PVR) in the majority of patients arises from events in the distal pulmonary vascular bed rather than from central obstruction. That the progression of the pulmonary hypertension is a consequence of this distal arteriopathy is supported by several lines of evidence. First, lung biopsy findings obtained at the time of PTE demonstrate histopathologic changes in the microvasculature similar to those seen in other forms of smallvessel pulmonary hypertension, distal to both obstructed and nonobstructed central arteries [11]. Second, there appears to be a poor correlation between the scintigraphic and angiographic extent of central thromboembolic obstruction and the severity of hemodynamic compromise [12, 13]. Third, in patients with sequential perfusion scans available for review, pulmonary hypertension has progressed in the absence of perfusion scan change or clinical evidence of embolic recurrence. Fourth, approximately 10–15% of patients will experience persistent postoperative pulmonary hypertension despite a satisfactory central endarterectomy. Finally, recent studies have demonstrated that patients with inoperable chronic thromboembolic disease and those with persistent postoperative pulmonary hypertension can derive benefit from vasodilating and/or antiproliferative medications [14, 15]. What is not subject to debate is that survival without intervention is poor and, as in other forms of pulmonary hyperten-

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sion, is proportional to the degree of pulmonary hypertension and right ventricular dysfunction at the time of diagnosis. In one study, the 5-year survival rate in patients with CTEPH was 30% when the mean pulmonary artery pressure was more than 40 mmHg and 10% when it was more than 50 mmHg [16]. In another study, a mean pulmonary artery pressure of more than 30 mmHg appeared to serve as a threshold value portending a poor prognosis [17].

2 Clinical Presentation The diagnosis of CTEPH is usually not made until the degree of pulmonary hypertension is advanced, with most patients manifesting a PVR at the time of diagnosis of more than 600 dyn s cm−5. To achieve this level of pulmonary hypertension, months to years may pass between the initial thromboembolic event and the onset of clinical decline and subsequent diagnosis. Diagnostic delay following symptom onset, often of a prolonged duration, is also common with patients being labeled as physically deconditioned or suffering from asthma, chronic obstructive pulmonary disease, cardiomyopathy, or psychogenic dyspnea. Diagnostic delay appears to be related to various causes: (1) the often nonspecific clinical presentation of the disease and, early in its natural history, its subtle physical examination findings; (2) failure to consider disorders of the pulmonary vascular bed in patients with unexplained dyspnea; (3) a tendency to discount the possibility of chronic thromboembolic disease in the absence of a documented history of acute thromboembolism; and (4) a lack of awareness of the disease entity by many physicians. As in other forms of pulmonary arterial hypertension, the complaint common to patients with CTEPH is exertional dyspnea, which is related to limitation in cardiac output and to increased dead-space ventilation. Patients accustomed to higher levels of activity on a daily basis recognize the decline in exercise capacity at an earlier point in time than those who lead a sedentary lifestyle. In certain patients, tolerable dyspnea at sea level is amplified by ascent to altitude. As the disease progresses, symptoms of light-headedness or presyncope may occur with exertion. Late in the course of the disease, as right ventricular function and coronary perfusion become incapable of responding to increased metabolic demands, syncopal events and exertional chest pain may develop. Physical examination findings early in the course of the disease may be subtle and limited to an accentuation of the pulmonic component of the second heart sound. As the disease progresses, findings consistent with pulmonary hypertension develop: a right ventricular lift, a closely split second heart sound with further accentuation of its pulmonic component, a right ventricular S4 gallop, and varying degrees of

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tricuspid regurgitation. Even at this stage of the disease, however, physical findings can be deceptive, particularly for those unfamiliar with the physical diagnostic manifestations of pulmonary hypertension and in patients who are obese or have coexisting cardiopulmonary disease. As right ventricular failure ensues, elevated jugular venous pressure with a prominent v wave, a right-sided S3, lower-extremity edema, hepatomegaly, ascites, and cyanosis develop. The intensity of the tricuspid regurgitant murmur may diminish as the tricuspid annulus dilates and the transvalvular pressure gradient decreases. The presence of pulmonary flow bruits is an important physical diagnostic finding in approximately 30% of patients with chronic thromboembolic disease [18]. The bruits appear to result from turbulent flow across partially obstructed central pulmonary vascular segments. These bruits are not unique to chronic thromboembolic disease, having been described in other disease states associated with focal narrowing of large pulmonary arteries, such as congenital branch stenosis and large-vessel pulmonary arteritis. They have not, however, been described in primary pulmonary hypertension, the most common competing diagnostic possibility.

3 Diagnosis Once the diagnosis of pulmonary hypertension has been considered, the diagnostic pathway is straightforward. The central goal of the pathway is to determine whether pulmonary hypertension is present and whether it results from central pulmonary arterial obstruction or one of a number of potential disorders of the peripheral pulmonary vascular bed. Once it has been confirmed to be on a large-vessel basis, confirmation of its thromboembolic nature and the associated extent of hemodynamic compromise should follow. Transthoracic echocardiography commonly provides the initial objective evidence that pulmonary hypertension is present and that it is not the result of primary left ventricular systolic or diastolic dysfunction or valvular disease. The use of contrast can help exclude the possibility of an intracardiac shunt. Findings in chronic thromboembolic and other forms of pulmonary arterial hypertension include enlargement of the right cardiac chambers, an increased velocity of the tricuspid regurgitant envelope from which the pulmonary artery systolic pressure can be estimated, flattening or paradoxical motion of the interventricular septum, encroachment of an enlarged right ventricle on the left ventricular cavity, and impaired left ventricular diastolic filling [19]. Once the diagnosis of pulmonary hypertension has been confirmed, distinguishing between the multiple etiologic possibilities represents the next essential step. Depending on the clinical circumstances, pulmonary function testing, chest computed tomography (CT), sleep testing, or serology studies

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may be required. However, once the differential possibility has been narrowed to a primary problem of the pulmonary vasculature, ventilation–perfusion (V/Q) lung scanning represents a simple, noninvasive means of differentiating disorders of the peripheral pulmonary vascular bed from those of the central pulmonary vascular bed (Fig.  1). Despite being supplanted by CT angiography in the diagnostic pathway for acute pulmonary embolism, ventilation–perfusion scintigraphy continues to play a central role in the evaluation of patients with pulmonary hypertension. In chronic thromboembolic disease, at least one segmental or larger mismatched perfusion defect is present (more commonly, several are noted) [20]. In disorders of the distal pulmonary vascular bed, perfusion scans are either normal or exhibit a “mottled” appearance characterized by nonsegmental defects. The one exception to this rule is pulmonary veno-occlusive disease, in which segmental defects have been reported [21]. The magnitude of the perfusion defects in chronic thromboembolic disease often understates to a considerable extent the actual degree of pulmonary vascular obstruction determined angiographically or at surgery [12, 13]. Therefore, the presence of even a single, mismatched, segmental ventilation– perfusion scan defect in a patient with pulmonary hypertension should raise concerns regarding a potential thromboembolic basis. In the initial evaluation of a patient with pulmonary hypertension, CT scanning is valuable in detecting disorders of the pulmonary parenchyma, chest wall, and mediastinum. However, it has a limited role in differentiating CTEPH from other variants of pulmonary hypertension. A variety of CT abnormalities have been described in patients with chronic thromboembolic disease. However, the absence of these findings does not preclude the possibility of surgically accessible chronic thromboembolic disease. In a recent series, V/Q scintigraphy was demonstrated to be more sensitive (96 vs. 51%) in detecting chronic thromboembolic disease than multidetector CT pulmonary angiography [22]. Alternatively, central thrombi have been described in processes other than CTEPH, such as idiopathic pulmonary hypertension, Eisenmenger syndrome, and chronic obstructive pulmonary disease [23–25]. In terms of ancillary data obtained during the evaluation period, the alveolar–arterial oxygen gradient is typically widened and the majority of patients have a decrease in the arterial Po2 with exercise [26]. Profound hypoxemia, however, is not a usual component of the disease. Worsening hypoxemia which is often ascribed to a recurrent embolic event can occur when a large right-to-left shunt develops through a patent foramen ovale. The findings of tests of pulmonary function are usually within normal limits but may demonstrate a mild to moderate restrictive impairment, caused to a large extent by parenchymal scarring related to prior infarcts [27]. Although a mild to

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Fig. 1  Representative ventilation–perfusion scan in a patient with chronic thromboembolic pulmonary hypertension (CTEPH). The perfusion scan demonstrates multiple mismatched segmental and larger defects

moderate reduction in single-breath diffusing capacity for carbon monoxide can be observed, a normal value does not exclude the diagnosis. Chest radiography, although the findings are often normal, may demonstrate one or more findings that suggest the diagnosis of pulmonary hypertension, such as right ventricular prominence, asymmetry in the size of the central pulmonary arteries, or enlargement of both pulmonary arteries. Areas of oligemia or hyperemia also may be present (Fig. 2). Results of routine hematologic and blood chemistry tests are usually unremarkable. Unexplained thrombocytopenia or a prolonged activated partial thromboplastin time in the absence of heparin therapy may suggest the presence of a lupus anticoagulant or antiphospholipid antibody. Once the diagnosis of CTEPH has been suggested by preliminary studies, both anatomic and hemodynamic confirmation should follow. Right-sided heart catheterization with pulmonary angiography has represented the gold standard in terms of both diagnosis and surgical referral. Symptom-limited

exercise hemodynamic measurements may be obtained when the level of pulmonary hypertension at rest is only modest. However, once the normal compensatory mechanisms of recruitment and dilation have been overcome, an almost linear elevation in pulmonary artery pressure will occur with exercise as cardiac output increases. Modest reductions in PVR and mean pulmonary artery pressure may occur following the inhalation of nitric oxide in patients with distal CTEPH [28]. There appears to be little clinical rationale for routine vasoreactivity testing as part of the diagnostic evaluation until trials demonstrate that patients who demonstrate vasoreactivity have a different natural history or therapeutic response when managed either surgically or medically. The angiographic appearance of chronic thromboembolic disease bears little resemblance to that of acute pulmonary embolism. Well-defined, intraluminal filling defects found in acute disease are not present. Instead, the angiographic patterns encountered in chronic thromboembolic disease reflect the complex patterns of organization and recanalization that

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Fig. 2  Chest radiograph in a patient with CTEPH. Asymmetry of the central pulmonary arteries, enlargement of the right pulmonary artery, and absence of the left descending pulmonary artery are seen along with a left-sided opacity most likely from a prior pulmonary infarct

occur following an acute thromboembolic event. Five angiographic patterns have been described in chronic thromboembolic disease that correlate with findings at the time of surgery [29]. These include (1) pouch defects, (2) pulmonary artery webs or bands, (3) intimal irregularities, (4) abrupt, often angular narrowing of the major pulmonary arteries, and (5) complete obstruction of main, lobar, or segmental vessels at their point of origin (Fig. 3). In most patients with extensive chronic thromboembolic disease, two or more of these angiographic findings are present and the findings are present bilaterally. Concerns have been raised regarding the safety of pulmonary angiography in the setting of severe pulmonary hypertension. Much of this concern arose from complications associated with angiography in the setting of acute massive pulmonary embolism associated with acute right ventricular decompensation. Even in the presence of severe pulmonary hypertension, pulmonary angiography can be performed safely in patients with CTEPH if appropriate precautions are taken [30]. In terms of technique, multiple selective injections are not required. A single injection of nonionic contrast into both proximal pulmonary arteries, with the volume and injection rate adjusted on the basis of cardiac output, appears to be suf-

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Fig.  3  Representative right-sided pulmonary angiogram in a patient with CTEPH. Note irregularity of the right upper-lobe artery, a prominent “pouch” defect in the interlobar artery (indicated by the arrow), and absent flow to right middle and lower lobes

ficient. As little as 15–20 ml of contrast may be required for each pulmonary artery injection. Biplane acquisition provides optimal anatomic detail, the lateral projection providing more detailed definition of lobar and segmental anatomy than can be achieved with an anterior–posterior view alone (Fig. 4). Under essentially all circumstances, a properly performed biplane angiogram will provide sufficient information on which to base a decision regarding surgical accessibility. At the University of California, San Diego (UCSD) fiber-optic pulmonary angioscopy was once utilized in approximately 30% of patients to confirm the presence of chronic thromboembolic obstruction and to determine whether it was amenable to surgical intervention [31, 32]. However, advances in imaging technology have substantially reduced the need for this technique. Magnetic resonance (MR) imaging and MR angiography have been used successfully to visualize the pulmonary vascular system in patients with CTEPH [33]. MR imaging can also provide information about right ventricular size and function. The number of studies that have directly compared MR techniques with conventional contrast angiography is

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Fig. 4  Pulmonary angiogram, anterior and lateral view. (a) Right-sided pulmonary angiogram (anterior view) demonstrating abrupt narrowing and irregularity of right upperand lower-lobe arteries. (b) Lateral projection demonstrating the nearly complete lack of patent vessels to the lower lobe (indicated by the arrow) with the exception of the superior segment and the widely dilated middle-lobe artery

small. In one such study involving 29 patients, the combined interpretation of MR perfusion imaging and MR angiography led to a correct diagnosis of idiopathic pulmonary arterial hypertension or CTEPH in 26 (90%) of 29 patients when compared with the reference diagnosis based on V/Q scintigraphy, digital subtraction angiography, or CT angiography. Kappa values for interobserver agreement were 0.73, representing fair to good agreement. The interpretation of MR angiography alone had a sensitivity of 71% for wall-adherent thrombi and 50% for webs and bands when compared with digital subtraction angiography or CT angiography [34]. The role of CT scanning in the evaluation of patients with chronic thromboembolic disease also remains incompletely defined [35]. CT scan findings described in patients with CTEPH include enlargement of the right atrium and right ventricle, dilation of the main pulmonary artery, mosaic perfusion, dilated bronchial arteries, peripheral subpleural densities, and thrombus either within or lining the walls of the pulmonary arteries. Certain CT findings such as the presence of central disease and dilated bronchial arteries have been demonstrated to be possibly associated with an improved postoperative hemodynamic outcome [35]. The number of studies which have directly compared CT with angiography is small. In one such study, CT and digital angiography were nearly equivalent in terms of identifying complete vessel occlusion at the segmental level [36]. However, for nonocclusive changes, CT was significantly inferior to angiography. The accuracy of CT scanning has improved with technological

advances and the introduction of 64-detector-row scanners. In a preliminary study involving 27 patients with suspected CTEPH, the sensitivity of 64-detector-row CT was 98.3% at the main and lobar level and 94% at the segmental level when compared with digital subtraction angiography [37]. There is no question that MR and CT imaging can be utilized as primary imaging techniques in selected patients prior to PTE. However, the wholesale substitution of MR or CT imaging for conventional angiography would appear to be both premature and imprudent. The consequences of denying an effective, life-saving procedure to patients potentially eligible for that procedure on the basis of incompletely documented technology are substantial in terms of mortality, quality of life, and subsequent medical costs. Differential diagnostic possibilities must be considered prior to surgical referral. Other processes associated with pulmonary hypertension and segmental or larger defects on V/Q scanning include pulmonary veno-occlusive disease, pulmonary artery sarcoma, large-vessel pulmonary vasculitides, extrinsic vascular compression by mediastinal carcinoma, lymphadenopathy or fibrosis, and pulmonary vein thrombosis [21, 38–41]. If the patient is considered to be an operative candidate, several other interventions should be considered before surgery. Given the risk of embolic recurrence, both over the long term and especially during the high-risk perioperative period when bleeding complications may contraindicate the administration of even prophylactic doses of anticoagulation,

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an inferior vena cava filter is placed. For those at risk of coronary artery disease, coronary angiography is performed before surgery, usually at the time of the right-sided heart catheterization and pulmonary angiogram. Coronary artery bypass grafting, if necessary, can be performed at the time of the PTE.

4 Surgical Selection The intent of the extensive evaluation process is to establish the need for surgical intervention, determine the surgical accessibility of the chronic thromboemboli, and estimate the risk of PTE as well as the anticipated hemodynamic outcome in the individual patient. The differentiation of operable from nonoperable CTEPH remains one of the most complex, problematic, and strongly contested issues surrounding this disease process. The basis for this controversy is that no precise anatomic–hemodynamic definition of “operable” (and therefore “nonoperable”) disease has been established. In anatomic terms, there is general consensus that disease originating distal to the segmental pulmonary arteries is not amenable to surgical intervention. In hemodynamic terms, however, no specific target has been defined either in anticipated percent or absolute reduction in PVR or absolute PVR goal. For example, there would be little disagreement in performing a PTE in a patient with a preoperative PVR of 900 dyn s cm−5 if the anticipated postoperative PVR (based on the preoperative evaluation) was expected to be 250 dyn s cm−5. Alternatively, considerable disagreement would accompany a decision to perform the procedure in a patient with the same preoperative PVR if the anticipated postoperative PVR was expected to be 500 dyn s cm−5. In the same vein, certain centers will proceed with a PTE in a patient with a preoperative PVR of 600 dyn s cm−5 even in the setting of predominantly “distal” disease; others would not and would instead proceed to primary medical management. Contributing to the uncertainty of preoperative assessment is the awareness that the increased PVR associated with chronic thromboembolic disease arises not only from the central, surgically accessible chronic thromboembolic obstruction but also from the resistance arising from a secondary, small-vessel arteriopathy. PTE will relieve only that portion of the pulmonary hypertension that arises from the accessible component of the chronic thromboembolic disease. A major focus of the preoperative evaluation, therefore, is attempting to partition the proximal component of the elevated vascular resistance from the distal and, by extension, estimating the anticipated hemodynamic outcome. With experience in hemodynamic–anatomic correlation, this determination can be made with reasonable accuracy. This determination is an essential one. Failure to lower PVR,

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especially in patients with severe pulmonary hypertension and right ventricular dysfunction, may be associated with severe hemodynamic instability and death in the early postoperative period. The findings of studies differ as to whether there is a strong correlation between the level of preoperative PVR and postoperative mortality [42–44]. No such difference of opinion exists regarding the correlation between the level of postoperative PVR and mortality. In a series by Jamieson et al. involving 500 consecutive operated on patients in whom the overall mortality rate was 4.4%, 77% of deaths were related to residual high pulmonary artery pressures. Patients with a postoperative PVR of more than 500 dyn s cm−5 had a mortality rate of 30.6% compared with 0.9% in patients with a postoperative PVR of less than 500 dyn s cm−5 [44]. Objective methods to assist in partitioning the different elements of the vascular resistance have been investigated. Kim et  al. utilizing pulmonary artery occlusion waveform analysis demonstrated excellent inverse correlation between the percent upstream resistance and postoperative mean pulmonary artery pressure and PVR [45]. All four deaths in this series occurred in patients in whom the upstream resistance was less than 60%. Unfortunately, this technique is invasive, requires specialized equipment, and obtaining adequate occlusion waveforms has proven to be problematic in patients with chronic thromboembolic disease. Reesink et al. evaluated the correlation between endothelin-1 (ET-1) levels and the hemodynamic severity in patients with thromboembolic pulmonary hypertension [46]. As is the case with nonthromboembolic variants of pulmonary hypertension, ET-1 levels correlated strongly with preoperative hemodynamic values. Preoperative ET-1 levels also proved to be predictive of an adverse postoperative outcome defined as either death or the presence of residual pulmonary hypertension. An ET-1 level exceeding 1.77 pg ml−1 had a sensitivity of 79% and a specificity of 85% for these outcomes. The majority of patients who undergo PTE exhibit a PVR of more than 300 dyn s cm−5. At centers reporting their experience with PTE surgery, mean preoperative PVR is typically in the range of 700–1,100 dyn s cm−5 [44, 47–59]. Certain patients may present with significant dyspnea and normal or minimally abnormal resting pulmonary hemodynamics. Such patients include those with involvement of one main pulmonary artery, those with unusually vigorous lifestyle expectations, and those who live at altitude. PTE is effective in these patients by alleviating the exercise impairment associated with their high dead-space and minute ventilatory demands. Surgery is also offered to patients with normal pulmonary hemodynamics or only mild levels of pulmonary hypertension at rest who develop significant levels of pulmonary hypertension with exercise. There is a natural tendency to avoid this intervention in patients with only modest levels of pulmonary hypertension. However, given what is currently known about the natural history of the disease, the

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potential for progressive pulmonary hypertension due to the development of a small-vessel arteriopathy, and the low risk nature of PTE in this population, early surgical intervention is indicated. At centers experienced with this procedure, the perioperative mortality rate for this group of patients has fallen to the range of 1–2%. If the decision is made to defer surgery, then careful follow-up is required to ensure that progressive pulmonary hypertension does not occur. It is possible, but not yet proven, that long-term medical therapy in these patients will result in stability of or improvement in pulmonary hemodynamics. As noted previously, an absolute criterion for surgery is the presence of accessible chronic thrombi. Current surgical techniques allow removal of organized thrombi whose proximal extent is in the main and lobar arteries and, depending on surgical skill and experience, those involving the segmental arteries. The presence of comorbid conditions that may adversely affect perioperative mortality or morbidity as well as long-term survival must also be considered before surgical referral. Advanced age and the presence of collateral disease do not represent absolute contraindications to PTE, although they do influence risk assessment. The one exception to this guideline is the presence of severe underlying obstructive or restrictive parenchymal lung disease. In this circumstance, PTE may result in hemodynamic improvement but often does not ameliorate the gas-exchange consequences of the underlying parenchymal lung disease.

5 Surgery The surgical procedure, details of which are outlined in Chap. 112, has undergone substantial evolution over the past 25 years. However, several features of the procedure should be emphasized. The procedure is a true endarterectomy and not an embolectomy (Fig. 5). The neointima must be meticulously dissected away from the native intima, and considerable surgical experience with this procedure is required to identify the correct operative plane. The removal of nonadherent, partially organized thrombus within the lumen of the central pulmonary arteries is ineffective in reducing right ventricular afterload, whereas creation of too deep a plane poses the risk of pulmonary artery perforation and massive pulmonary hemorrhage at the time of discontinuation of cardiopulmonary bypass. The current approach involves median sternotomy, cardiopulmonary bypass, and periods of hypothermic circulatory arrest [60, 61]. Deep hypothermia (18–20°C) and periods of circulatory arrest were initiated to avoid back-bleeding from retrograde bronchial artery to pulmonary artery anastomoses, thereby providing the bloodless field necessary to perform the meticulous dissection required for an adequate proce-

P.F. Fedullo et al.

Fig. 5  Right and left pulmonary thromboendarterectomy specimens. Note the fibrotic nature of the dissected tissue

dure. Modifications of this approach intended to avoid deep hypothermia and/or circulatory arrest have been described and include the use of moderate hypothermia (28–32°C), aortic bronchial artery occlusion with a balloon catheter, antegrade cerebral artery perfusion with and without total circulatory arrest, and application of negative pressure in the left ventricle [62–64]. Implementation of these modifications was primarily designed to avoid deep hypothermic circulatory arrest and the potential for neurologic complications that may occur when the period of circulatory arrest exceeds 20–25 min. It has not yet been demonstrated that any of these modifications provide any benefit compared with the traditional technique. With increased surgical experience, most PTE procedures can be performed with a single period of circulatory arrest for the right and the left pulmonary artery. In a series of 500 consecutive patients described by Jamieson et  al. in 2003, the mean circulatory arrest time was 35.7 ± 11.9  min (19.8 ± 8.0  min for the right side and 15.8 ± 6.3  min for the left side) [44]. Additional measures designed to minimize neurologic complications during deep hypothermic arrest include hemodilution to a hematocrit in the range of 18–25% to decrease blood viscosity during hypothermia and to optimize capillary blood flow. Additional cerebral protection is provided by surrounding the head with ice and a cooling blanket. Phenytoin is administered intravenously during the cooling period to reduce the risk of perioperative seizure activity. Although the procedure was initially attempted using a unilateral or bilateral thoracotomy approach, this was abandoned in favor of sternotomy. A sternotomy approach provides access to the central pulmonary vessels of both lungs and avoids the potential for disruption of the extensive bronchial collateral circulation and pulmonary adhesions that may develop

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following pulmonary artery obstruction [65–67]. A sternotomy approach also provides adequate exposure for additional procedures that need to be performed. In a review of 1,190 patients undergoing PTE at UCSD Medical Center, 90 patients (7.6%) required such a combined procedure exclusive of solitary closure of a patent foramen ovale (which is performed in approximately 30% of PTE procedures). Of these 90 patients, 83 underwent coronary bypass graft surgery, three underwent tricuspid valve repair, two underwent mitral valve repair, and two underwent aortic valve replacement [68].

6 Outcome In the majority of patients undergoing PTE, both the short-term and the long-term hemodynamic outcomes are favorable. Dramatic and immediate post-PTE reduction, and at times normalization, of the pulmonary artery pressure and PVR can be achieved. In published series, the mean reduction in PVR has approximated 70% and a PVR in the range of 200– 350 dyn s cm−5 can be achieved [44, 47–59]. A corresponding improvement in right ventricular function determined by echocardiography, gas exchange, exercise capacity, and quality of life has also been reported. As longer-term data have accrued, it appears this improvement is sustained [58, 59, 69]. Most patients initially in New York Heart Association (NYHA) functional class III or IV preoperatively return postoperatively to NYHA class I or II and are able to resume normal activities [70, 71]. In series of patients undergoing PTE since 1996, in-hospital mortality rates as high as 24% have been reported [44, 47– 59]. In experienced, larger-volume programs, however, mortality rates of 3–7% are typical. A number of factors appear to have contributed to this improved outcome, including improved diagnostic techniques, a better understanding of the natural history of the disease, more selective surgical referral, and advances in operative and postoperative care. Attributable causes of death following PTE surgery are variable and similar to those associated with other open-heart procedures. Cardiac arrest, multiorgan failure, uncontrollable mediastinal bleeding, sepsis syndrome, and massive pulmonary hemorrhage are among the causes of death cited. In recent series, the major causes of postoperative morality are residual pulmonary hypertension and acute lung injury [44, 47–59]. Approximately 10% of postoperative patients are left with a residual PVR of more than 500 dyn s cm−5. In the majority of these patients, the postoperative hemodynamic improvement is significant. However, approximately 3–5% of operated on patients experience minimal to no hemodynamic benefit from surgery owing to the severity of their distal vasculopathy, which is unaffected by removal of the central portion of their disease [72].

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Biochemically and clinically, the acute lung injury that may occur following PTE appears to represent a localized form of high-permeability lung injury. Although often referred to as “reperfusion pulmonary edema,” it has not yet been defined whether the phenomenon is related to ischemia– reperfusion, the effects of cardiopulmonary bypass or hypothermic circulatory arrest, details of the surgical procedure itself, or some other combination of causes. Whatever the cause, acute lung injury following PTE may appear immediately after termination of cardiopulmonary bypass to as long as 72 h after surgery. It is highly variable in severity, ranging from a mild form resulting in postoperative hypoxemia to an acute, hemorrhagic and fatal form of lung injury. The unique aspect of this form of lung injury is that it is limited to those areas of lung from which proximal thromboembolic obstructions have been removed. The development of reperfusion injury can represent a significant postoperative challenge in terms of ventilator management. Management of reperfusion edema, as with other forms of acute lung injury, is supportive until resolution occurs. High-dose corticosteroid therapy has been used to modulate the inflammatory component of the process, although its effectiveness is unpredictable and, frequently, minimal. The postoperative avoidance of inotropic and vasodilator support, along with a strategy of low-volume (less than 8 ml kg−1) ventilation, resulted in a lower incidence of reperfusion pulmonary edema in 47 patients undergoing PTE [73]. As in other forms of acute lung injury, a low-volume ventilatory strategy to minimize the risk of ongoing alveolar damage, and incremental levels of positive end expiratory pressure have proved useful in improving ventilation– perfusion relationships and gas exchange. Inhaled nitric oxide has been reported to improve gas exchange, although in our experience this effect is transient and does not affect the progression of the disease [74–76]. In extreme situations, extracorporeal support has been used successfully in patients when aggressive conventional measures have been inadequate to maintain oxygenation [77]. Lifelong anticoagulant therapy is strongly recommended after PTE. A number of patients in whom anticoagulation was discontinued or maintained at a subtherapeutic level experienced recurrent thromboembolism, and several of them required a second PTE [78].

7 Alternative Therapy Lung transplantation remains a therapeutic alternative for patients not deemed candidates for PTE and for those who have undergone PTE with an inadequate hemodynamic outcome. Therapies developed for primary use in idiopathic pulmonary arterial hypertension, including prostacyclin analogs, endothelin receptor antagonists, and phosphodiesterase-5

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inhibitors, have been studied in patients with CTEPH [79]. Prior to any discussion of this topic, however, it is worthwhile reiterating that PTE remains the definitive intervention for CTEPH. The hemodynamic and symptomatic benefits to be accrued from medical therapy, although often positive, are quite modest in comparison with those that can be achieved with PTE. That such is the case is to be expected given that medical therapy would only be expected to affect that portion of the increased PVR conferred by the secondary, smallvessel arteriopathic changes and not the fixed resistance resulting from the central thromboembolic obstruction. The decision to forego PTE and to utilize medical therapy should be made only after a comprehensive evaluation has been performed, only for discrete indications, and only after consultation with a center experienced in the management of this disease process to confirm that the disease is truly inoperable. In a retrospective review of patients undergoing PTE at UCSD in 2007, 37% of patients had been prescribed pulmonary-hypertension-specific medical therapy at the time of referral [80]. Analysis demonstrated that there were no significant differences in hemodynamic parameters between the two groups either at the time of the referral visit or after they had undergone PTE surgery. This intervention also resulted in a considerable delay to referral and unnecessary expense. Of greater concern are those patients who have been prescribed specific medical therapy and have not been referred for evaluation and possible surgery. Potential indications for medical rather than definitive surgical therapy include (1) patients with surgically accessible CTEPH who elect not to undergo surgery owing to personal choice or where comorbidities are such that the risk of PTE is prohibitive, (2) patients with distal chronic thromboembolic disease or limited central disease that is disproportionate to the severity of the pulmonary hypertension and in whom the postendarterectomy PVR is not anticipated to fall below 500 dyn s cm−5, (3) as a preoperative therapeutic bridge to surgery in patients with severe right ventricular dysfunction, and (4) management of residual pulmonary hypertension following PTE. The majority of data concerning pulmonary-hypertension-specific medical therapy for CTEPH come from small uncontrolled studies, retrospective evaluations, trials involving patients with pulmonary arterial hypertension but not dedicated to those with CTEPH, and trials in which patients with inoperable and residual postendarterectomy CTEPH are mixed. Neither can existing data define whether one class of drugs is superior to another or whether combination therapy is preferred. Despite these limitations, the data that are emerging are encouraging. Although not providing the hemodynamic or functional improvement achievable with PTE, pulmonaryhypertension-specific medical therapy appears useful in those patients either ineligible for the procedure or demonstrating residual pulmonary hypertension following it.

P.F. Fedullo et al.

For patients with inoperable CTEPH, an open-label study of sildenafil in 104 patients resulted in a decrease in PVR from 863 ± 38 to 759 ± 62 dyn s cm−5 at 3 months [81]. After 12 months, the 6-min walk distance increased from 310 ± 11 to 366 ± 18 m. In a double-blind, placebo-controlled pilot study comparing sildenafil with a placebo for 12 weeks in 19 patients, no significant difference was seen between groups in terms of exercise capacity [14]. However, there was a significant difference in World Health Organization class and in PVR (a decrease of 179 ± 245 compared with an increase of 18 ± 76 dyn s cm−5). Control patients were then transferred to treatment with openlabel sildenafil and follow-up was obtained at 12 months. At that time, significant differences were noted in the 6-min walk distance, symptom score, and PVR (from 722 ± 383 to 573 ± 330 dyn s cm−5). In an open-label, national study, 15 patients with CTEPH not eligible for or awaiting surgery were treated with bosentan [15]. At 6 months, the PVR decreased from 852 ± 319 to 657 ± 249  dyn s cm−5. The 6-min walk distance increased from 389 ± 78 to 443 ± 79 m. A retrospective analysis of 27 patients with inoperable CTEPH treated with epoprostenol for 3 months resulted in a decrease in mean pulmonary artery pressure (from 56 ± 9 to 51 ± 5 mmHg), total pulmonary resistance (from 29.3 ± 7 to 23.0 ± 5 U m−2), and an increase in the 6-min walk distance of 66 m [82]. In a single-center uncontrolled observational study, 28 patients were treated with subcutaneously administered treprostinil [83]. Follow-up catheterization was performed in 19 patients after 19 ± 6.3 months. Treprostinil therapy was associated with a significant improvement in PVR (from 924 ± 347 to 808 ± 372  dyn s cm−5). The 5-year survival rate was 53% compared with 16% in an untreated historical control group. Only limited data are available regarding the use of medical therapy as a therapeutic bridge prior to PTE [84, 85]. Using intravenously administered prostacyclin for a period of 46 ± 12 days before surgery, Nagaya et al. showed a preoperative decrease in PVR from 1,510 ± 53 to 1,088 ± 58 dyn s cm−5 [84]. Postoperative mortality was 8.3%, compared with no deaths in the 21 CTEPH patients who had a preoperative PVR of 1,200  dyn s cm−5 or less. Postoperative hemodynamic improvement was comparable between the groups. Although this study was able to demonstrate a hemodynamic benefit of prostacyclin in a small number of severely pulmonary hypertensive CTEPH patients, the effect of pulmonaryhypertension-specific medical pretreatment on postoperative morbidity and mortality remains unknown. Until this issue is resolved, the use of pulmonary-hypertension-specific medical therapy in CTEPH patients who are likely to have operable chronic thromboembolic disease should not inappropriately delay surgical intervention. At UCSD, patients with severe pulmonary hypertension, evidence of right ventricular failure,

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and gross volume overload are often hemodynamically monitored for several days prior to surgery and treated with aggressive diuresis and, if necessary, inotropic support. The use of medical therapy in patients with post-PTE residual pulmonary hypertension has been studied as part of mixed series including patients with inoperable CTEPH. In an open-label study involving 47 patients evaluating the long-term efficacy and safety of bosentan in CTEPH at three European centers, 39 patients had inoperable CTEPH and eight had persistent pulmonary hypertension after PTE. A significant improvement was observed in the 6-min walk distance (52 ± 10 m) at 1-year follow-up. Twenty-eight subjects had repeated right-sided heart catheterization at 1 year, and there was significant improvement in the cardiac index (0.2 ± 0.007  L min−1 m−2) and a decrease in total pulmonary resistance (138 ± 42 dyn s cm−5) [86]. Hoeper et al. performed a prospective, multicenter, openlabel trial evaluating 3 months of bosentan therapy in a mixed population of 19 patients with CTEPH. After 3 months of therapy, PVR significantly decreased from 914 ± 329 to 611 ± 220 dyn s cm−5, and the 6-min walk distance increased from 340 ± 102 to 413 ± 130  m. Significant improvements were also seen in serum pro-brain natriuretic peptide levels, mean pulmonary artery pressure, and cardiac output [87]. In terms of this indication, the level of post-PTE pulmonary hypertension that requires therapy has not yet been defined. Neither is it known whether a predisposed population exists who, in the absence of medical therapy, will develop progressive pulmonary hypertension.

8 Conclusion A review of the world literature by Chitwood et al. in 1985 disclosed published reports on 85 patients undergoing PTE with an overall mortality of 22% [88]. Since that time, and in large part owing to the vision of Kenneth M. Moser and the pioneering and educational efforts of the team at the UCSD, approximately 5,000 procedures have been performed worldwide, 2,500 at UCSD alone. CTEPH has come to be recognized as the only truly curable form of pulmonary hypertension, with mortality rates associated with PTE falling to the range of 5%. In lower-risk populations, mortality rates in the range of 1–3% have been reported. Despite these considerable diagnostic and therapeutic advances, a number of uncertainties associated with this disease process remain unanswered. Among others, these include (1) the basis for progression of pulmonary hypertension in certain patients with residual vascular obstruction following pulmonary embolism and whether it is possible to identify these patients prior to the development of pulmonary hypertension, (2) a more precise definition of “operable” versus

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“inoperable” chronic thromboembolic disease based on anatomic and hemodynamic variables, (3) improved diagnostic techniques capable of defining the extent of small-vessel arteriopathy, thereby providing a means of better predicting postoperative hemodynamic outcome, and (4) the risks, benefits, and outcome of primary medical therapy versus surgical therapy versus combined therapy in patients with distal CTEPH or CTEPH associated with pulmonary hypertension disproportionate to the degree of central vascular obstruction. A worldwide community is now actively involved in the care of these patients. It is hoped that the combined efforts of the clinicians and scientists involved in this area will continue to contribute to the substantial advances that have occurred over the past 25 years in better understanding the natural history and demographics of this disease and in optimizing the diagnostic approach and surgical and medical management of it.

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P.F. Fedullo et al. 34. Nikolaon K, Schoenberg SO, Attenberger U et al (2005) Pulmonary arterial hypertension: diagnosis with fast perfusion imaging and high-spatial-resolution MR angiography - preliminary experience. Radiology 236:694–703 35. Heinrich M, Uder M, Tscholl D et al (2005) CT scan findings in chronic thromboembolic pulmonary hypertension. Predictors of hemodynamic improvement after pulmonary thromboendarterectomy. Chest 127:1606–1613 36. Pitton MB, Kemmerich G, Herber S et  al (2002) Chronic thromboembolic pulmonary hypertension: diagnostic impact of multislice CT and selective pulmonary DSA. Roto 174:474–479 37. Reichelt A, Hoeper MM, Galanski M, Keberle M (2008) Chronic thromboembolic pulmonary hypertension: evaluation with 64-detector row versus digital subtraction angiography. Eur J Radiol (in press) 38. Kerr KM (2005) Pulmonary artery sarcoma masquerading as chronic thromboembolic pulmonary hypertension. Nat Clin Pract Cardiovasc Med 2:108–112 39. Castellani M, Vanoli M, Cali G et al (2001) Ventilation-perfusion lung scan for the detection of pulmonary involvement in Takayasu's arteritis. Eur J Nucl Med 28:1801–1805 40. Packer DL, Keelan P, Munger TM et al (2005) Clinical presentation, investigation, and management of pulmonary vein stenosis complicating ablation for atrial fibrillation. Circulation 111:546–554 41. Park HM, Jay SJ, Brandt MJ et al (1981) Pulmonary scintigraphy in fibrosing mediastinits. J Nucl Med 22:349–351 42. Dartevelle P, Fadel E, Mussot S et al (2004) Chronic thromboembolic pulmonary hypertension. Eur Resp J 23:637–648 43. Thistlethwaite P, Kemp A, Lingling D et  al (2006) Outcomes of pulmonary thromboendarterectomy for treatment of extreme thromboembolic pulmonary hypertension. J Thorac Cardiovasc Surgery 131:307–313 44. Jamieson SW, Kapelanski DW, Sakakibara N et al (2003) Pulmonary endarterectomy: experience and lessons learned in 1, 500 cases. Ann Thoracic Surg 76:1457–1462 45. Kim HS, Fesler P, Channick RN et al (2004) Preoperative partitioning of pulmonary vascular resistance correlates with early outcome after thromboendarterectomy for chronic thromboembolic pulmonary hypertension. Circulation 109:18–22 46. Reesink HJ, Meijer RC, Lutter R et al (2006) Hemodynamic and clinical correlates of endothelin-1 in chronic thromboembolic pulmonary hypertension. Circ J 70:1058–1063 47. Nakajima N, Masuda M, Mogi K (1997) The surgical treatment for chronic pulmonary thromboembolism: our experience and current review of the literature. Ann Thorac Cardiovasc Surg 3:15–21 48. Mayer E, Kramm T, Dahm M et al (1997) Early results of pulmonary thromboendarterectomy in chronic thromboembolic pulmonary hypertension. Z Kardiol 86:920–927 49. Gilbert TB, Gaine SP, Rubin LJ et  al (1998) Short-term outcome and predictors of adverse events following pulmonary thromboendarterectomy. World J Surg 22:1029–1032 50. Miller WT, Osiason AW, Langlotz CP et  al (1998) Reperfusion edema after thromboendarterectomy: radiographic patterns of disease. J Thorac Imaging 13:178–183 51. Dartevelle P, Fadel E, Chapelier A et al (1999) Angioscopic videoassisted pulmonary endarterectomy for post-embolic pulmonary hypertension. Eur J Cardiothorac Surg 16:38–43 52. Ando M, Okita Y, Tagusari O et  al (1999) Surgical treatment for chronic thromboembolic pulmonary hypertension under profound hypothermia and circulatory arrest in 24 patients. J Card Surg 14:377–385 53. Rubens FD, Bourke M, Hynes M et al (2007) Surgery for chronic thromboembolic pulmonary hypertension - Inclusive experience from a national referral center. Ann Thorac Surg 83:1075–1081 54. Mares P, Gilbert TB, Tschernko EM et al (2000) Pulmonary artery thromboendarterectomy: a comparison of two different postoperative treatment strategies. Anesth Analg 90:267–273

87  Pulmonary Hypertension Due to Pulmonary Embolism and Thromboembolic Obstruction of Proximal 55. D’Armini AM, Cattadori B, Monterosso C et al (2000) Pulmonary thromboendarterectomy in patients with chronic thromboembolic pulmonary hypertension: hemodynamic characteristics and changes. Eur J Cardiothorac Surg 18:696–702 56. Tscholl D, Langer F, Wendler O et al (2001) Pulmonary thromboendarterectomy: risk factors for early survival and hemodynamic improvement. Eur J Cardiothorac Surg 19:771–776 57. Masuda M, Nakajima N (2001) Our experience of surgical treatment for chronic pulmonary thromboembolism. Ann Thorac Cardiovasc Surg 7:261–265 58. Piovella F, D'Armini AM, Barone M, Tapson VF (2006) Chronic thromboembolic pulmonary hypertension. Semin Thromb Hemost 32:848–855 59. Mellemkjaer S, Ilkjaer LB, Klaaborg KE et  al (2006) Pulmonary endarterectomy for chronic thromboembolic pulmonary hypertension. Ten years experience in Denmark. Scand Cardiovasc J 40:49–53 60. Mayer E, Klepetko W (2006) Techniques and outcome of pulmonary endarterectomy for chronic thromboembolic pulmonary hypertension. Proc Am Thorac Soc 3:589–593 61. Madani M, Jamieson SW (2006) Technical advances of pulmonary endarterectomy for chronic thromboembolic pulmonary hypertension. SeminThorac Cardiovasc Med 18:243–250 62. Hagl C, Khaladj N, Peters T et  al (2003) Technical advances of pulmonary thromboendarterectomy for chronic thromboembolic pulmonary hypertension. Eur J Cardiothorac Surg 23:776–781 63. Thomson B, Tsui SSL, Dunning J et al (2008) Pulmonary endarterectomy is possible and effective without the use of complete circulatory arrest - the UK experience in over 150 patients. Eur J Cardiothorac Surg 33:157–163 64. Mikus PM, Mikus E, Martin-Suarez S et  al (2008) Pulmonary endarterectomy:an alternative to circulatory arrest and deep hypothermia: mid-term results. Eur J Cardiothorac Surg 34:159–163 65. Sabiston DC, Wolfe WG, Oldham NH et al (1977) Surgical management of chronic pulmonary embolism. Ann Surg 185:699–712 66. Cabrol C, Cabrol A, Acar J et  al (1978) Surgical correction of chronic postembolic obstruction of the pulmonary arteries. J Thorac Cardiovasc Surg 76:620–628 67. Daily PO, Johnston GG, Simmons CJ et al (1980) Surgical management of chronic pulmonary embolism. Surgical treatment and late results. J Thorac Cardiovasc Surg 79:620–628 68. Thistlethwaite PA, Auger WR, Madani MM et al (2001) Pulmonary thromboendarterectomy combined with other cardiac operations: indications, surgical approach, and outcome. Ann Thorac Surg 72:13–19 69. Corsico AG, D'Armini AM, Cerveri I et al (2008) Long term outcome after pulmonary thromboendarterectomy. Am J Resp Crit Care Med 178:419–424 70. Archibald CJ, Auger WR, Fedullo PF, Channick RN, Kerr KM, Jamieson SW, Kapelanski DP, Watt CN, Moser KM (1999) Longterm outcome after pulmonary thromboendarterectomy. Am J Respir Crit Care Med 160:523–528 71. Zoia MC, D’Armini AM, Beccaria M, Corsico A, Fulgoni P, Klersy C, Piovella F, Vigano M, Cerveri I, Pavia Thromboendarterectomy Group (2002) Mid term effects of pulmonary thromboendarterec-

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tomy on clinical and cardiopulmonary function status. Thorax 57:608–612 72. Auger WR, Kim NH, Kerr KM et al (2007) Chronic thromboembolic pulmonary hypertension. Clin Chest Med 28:255–269 73. Mares P, Gilbert TB, Tschernko EM et al (2000) Pulmonary artery thromboendarterectomy: a comparison of two different postoperative treatment strategies. Anest Analg 90:267–273 74. Pinelli G, Mertes PM, Carteaux JP, Hubert T, Dopff C, Burtin P, Villemot JP (2008) Inhaled nitric oxide as an adjunct to pulmonary thromboendarterectomy. Ann Thorac Surg 61:227–229 75. Gardeback M, Larsen FF, Radegran K (1995) Nitric oxide improves hypoxaemia following reperfusion oedema after pulmonary thromboendarterectomy. Br J Anaesth 75:798–800 76. Imanaka H, Miyano H, Takeuchi M et  al (2000) Effects of nitric oxide inhalation after pulmonary thromboendarterectomy for chronic pulmonary thromboembolism. Chest 118:39–46 77. Berman M, Tsui S, Vuylsteke A et al (2008) Successful extracorporeal membrane oxygenation support after pulmonary thromboendarterectomy. Ann Thorac Surg 86:1261–1267 78. Mo M, Kapelanski DP, Mitruka SN et al (1999) Reoperative pulmonary thromboendarterectomy. Ann Thorac Surg 68:1770–1776 79. Bresser P, Pepke-Zaba J, Jais X et al (2006) Medical therapies for chronic thromboembolic pulmonary hypertension. Proc Am Thorac Soc 3:594–600 80. Jensen KW, Kerr KM, Fedullo PF, Kim NH, Test VJ, Agure WR (2008) Prevalence of medical therapy for pulmonary arterial hypertension (PAH) in chronic thromboembolic pulmonary hypertension (CTEPH) prior to referral for pulmonary thromboendarterectomy (PTE). Am J Resp Crit Care Med; A181 81. Reichenberger F, Voswinckel R, Enke B et  al (2007) Long-term treatment with sildenafil in chronic thromboembolic pulmonary hypertension. Eur Resp J 30:922–927 82. Cabrol S, Souza R, Jais X et al (2007) Intravenous epoprostenol in inoperable thromboembolic pulmonary hypertension. J Heart Lung Transplant 26:357–362 83. Skoro-Sajer N, Bonderman D, Wiesbauer F et al (2007) Treprostinil for severe inoperable chronic thromboembolic pulmonary hypertension. J Thromb Haemost 5:483–489 84. Nagaya N, Sasaki N, Ando M, Ogino H, Sakamaki F, Kyotani S et al (2003) Prostacyclin therapy before pulmonary thromboendarterectomy in patients with chronic thromboembolic pulmonary hypertension. Chest 123:338–343 85. Bresser P, Fedullo PF, Auger WR et al (2004) Continuous intravenous prostacyclin for chronic thromboembolic pulmonary hypertension. Eur Resp J 23:595–600 86. Hughes RJ, Jais X, Bonderman D et al (2006) Bosentan in inoperable chronic thromboembolic pulmonary hypertension: efficacy at 1 year. Eur Resp J 28:138–143 87. Hoeper MM, Kramm T, Wilkens H et al (2005) Bosentan therapy for inoperable chronic thromboembolic disease. Chest 128: 2362–2367 88. Chitwood WR Jr, Sabiston DC, Wechsler AS (1984) Surgical treatment of chronic unresolved pulmonary embolism. Clin Chest Med 5:507–536

Chapter 88

Risk Factors for Chronic Thromboembolic Pulmonary Hypertension Diana Bonderman and Irene M. Lang

Abstract  Traditional concepts of the pathogenesis of chronic thromboembolic pulmonary hypertension (CTEPH) invoke a thromboembolic origin. However, neither classic plasmatic risk factors for venous thromboembolism nor defects in fibrinolysis are associated with CTEPH. These observations have lent support to the concept of alternative nonthromboembolic pathogenetic grounds for CTEPH. Compared with the general population, no increased prevalence of the factor V Leiden mutation was found. Other hereditary thrombotic risk factors, such as deficiencies in antithrombin, protein C, protein S, and the prothrombin gene mutation, lack an association with CTEPH. Efforts to identify functional defects or genetic polymorphisms in the pulmonary vascular fibrinolytic system of affected individuals have failed. Alterations in plasma levels of neither type 1 plasminogen activator inhibitor nor tissue-type plasminogen activator could be linked with CTEPH development. However, conditions that account for both venous and arterial thrombosis, such as antiphospholipid antibodies, malignancies, infection, elevated plasma lipoprotein levels, and hormone treatment providing a pathophysiological link between arterial and venous thrombosis, have recently appeared as risk factors for CTEPH. Growing knowledge of these conditions has a significant impact on clinical routine. The identification of conditions predisposing to CTEPH development may provide important clues to disease pathology. In this chapter, risk factors are categorized by odds ratios (ORs) as they appear in case-control or cohort studies, and are discussed in the order of the strength of the statistical relationship to a control cohort. According to our own data the ORs did not change significantly irrespective of whether patients with pulmonary hypertension, normal age-matched controls, or patients with pulmonary emboli who did not develop CTEPH were considered as the controls.

I.M. Lang (*) Department of Cardiology, Medical University of Vienna, Vienna, Austria

Keywords  Pulmonary thromboembolic disease • Pulmonary embolism • Thrombosis • Fibrinolysis • Theomboembolism

1 Introduction Traditional concepts of the pathogenesis of chronic thromboembolic pulmonary hypertension (CTEPH) invoke a thromboembolic origin [1]. However, neither classic plasmatic risk factors for venous thromboembolism nor defects in fibrinolysis are associated with CTEPH. These observations have lent support to the concept of alternative nonthromboembolic pathogenetic grounds for CTEPH [2]. Compared with the general population, no increased prevalence of the factor V Leiden mutation was found [3, 4]. Other hereditary thrombotic risk factors, such as deficiencies in antithrombin, protein C, protein S, and the prothrombin gene mutation [4–6], lack an association with CTEPH. Efforts to identify functional defects [7, 8] or genetic polymorphisms [6] in the pulmonary vascular fibrinolytic system of affected individuals have failed. Alterations in plasma levels of neither type 1 plasminogen activator inhibitor nor tissue-type plasminogen activator could be linked with CTEPH development [9]. However, conditions such as antiphospholipid antibodies, malignancies, infection, elevated plasma lipoprotein levels, and hormone treatment providing a pathophysiological link between arterial and venous thrombosis [10], have recently appeared as risk factors for CTEPH [11, 12]. Growing knowledge of these conditions has a significant impact on clinical routine. The identification of conditions predisposing to CTEPH development may provide important clues to disease pathology. In this chapter, risk factors are categorized by odds ratios (ORs) as they appear in case-control or cohort studies (Table 1), and are discussed in the order of the strength of the statistical relationship to a control cohort. According to our own data [13] the ORs did not change significantly irrespective of whether patients with pulmonary hypertension, normal age-matched controls, or patients with pulmonary emboli who did not develop CTEPH were considered as the controls.

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_88, © Springer Science+Business Media, LLC 2011

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Table  1  Risk factors for chronic thromboembolic pulmonary hypertension (CTEPH) listed in the order of the magnitude of published odds ratios Risk factor

Odds ratioa

Infected VA shunt/pacemaker [12, 21]

13.00 (2.5–129) and 76.40 (7.67–10,350.62) Splenectomy [12, 21] 13.00 (2.7–127) and 17.87 (1.56–2,438) Recurrent VTE [12] 14.49 (5.40–43.08) Thyroid disease [12] 6.10 (2.73–15.05) Previous VTE [12] 4.52 (2.35–9.12) Antiphospholipid antibodies [12] 4.20 (1.56–12.21) Survived cancer [12] 3.76 (1.47–10.43) Inflammatory bowel disease [12] 3.19 (0.74–16.03) Non-0 blood groups [12] 2.09 (1.12–3.94) Fibrinogen Aa Thr312Ala polymorphism [6] 1.68 (1.13–2.49) HLA-B*5201 (Japan) [63] 2.14 (1.29–3.55) HLA-DPB1*0202 (Japan) [63] 3.41 (1.71–6.74) VA ventriculoatrial, VTE venous thromboembolism

2 Infected Ventriculoatrial Shunts and Infected Pacemaker Leads A few case reports suggested a link between ventriculoatrial (VA) shunts for the treatment of hydrocephalus and CTEPH [14–20]. In a case-control study comparing 109 CTEPH patients with 187 patients who had resolved pulmonary thrombi after an episode of acute pulmonary embolism, 6.0% of patients but only 0.5% of controls had VA shunts [21]. The presence or a history of a VA shunt was an independent predictor of CTEPH [OR 13.0 (2.5–129)]. When data from the Viennese center were combined with those from Bratislava, Prague, and Homburg, VA shunt/pacemaker leads could be confirmed as a CTEPH risk factor [OR 76.40 (7.67– 10,351)]. The prevalence of VA shunts/pacemaker leads was 3.7% among patients, whereas none of the controls with nonthromboembolic pulmonary hypertension had intravenous lines [21]. Shunt infection is common in patients with VA shunts, and Staphylococcus aureus or S. epidermidis is responsible for up to half of these infections [22], leading to thrombosis and device failure [23]. We have observed that the majority of our CTEPH patients having either a VA shunt or a pacemaker had a history of device infection. Indeed, staphylococcal DNA was present in six out of seven pulmonary endarterectomy specimens from patients with VA shunts/pacemakers. Molecular analyses of human surgical specimens and S. aureus-infected murine thrombi suggest that thrombus infection – as is common in patients with VA shunts– impedes thrombolysis [24]. Taken together, patients who have VA shunts or pacemaker leads and have had a history of device infection are at increased risk for CTEPH.

3 Splenectomy Because several case studies have suggested a link between CTEPH and prior splenectomy [25–28], a first systematic analysis was conducted at the Medical University of Vienna [21]. One hundred and nine CTEPH patients were compared with 187 control subjects who did not develop CTEPH after a pulmonary thromboembolic event. Nine percent of the patients but only 0.5% of controls had a history of splenectomy. With an OR of 13.0 (2.7–127), splenectomy conferred an increased risk for the development of CTEPH. This finding was confirmed by Jais et  al. [29], who compared 257 CTEPH cases with controls consisting of 276 patients with idiopathic pulmonary hypertension or 180 patients with other chronic pulmonary conditions. Of the patients with CTEPH, 8.6% had a history of splenectomy, whereas only 2.5 or 0.6% of the controls, respectively, were splenectomized. The 8.6% frequency of splenectomy was estimated to be 20 times greater than expected in the general population. In the combined database of CTEPH patients from Vienna, Bratislava, Prague, and Homburg (n = 433), the prevalence of splenectomy was 5.5% as opposed to 0.0% in the control group [12]. Previous splenectomy, irrespective of the underlying cause, was confirmed as an independent predictor of CTEPH [OR 17.9 (1.6–2,438)]. In the UK national registry overlooking 469 CTEPH patients, a prevalence of splenectomy of 6.7% was found [30]. The biological mechanisms underlying CTEPH development in asplenic individuals remain enigmatic. Although transient thrombocytosis is expected immediately after splenectomy, it is not usually associated with thrombotic events [31, 32]. Moreover, in centers across Europe, CTEPH developed years or even decades after splenectomy when thrombocytosis was no longer present. However, there is some speculation as to the possible role of splenectomy in promoting venous thromboembolic disease. Coltheart et al. [33] reported a 10.7% frequency of pulmonary thromboembolic disease in a retrospective study of 150 consecutive splenectomies performed over a 5-year period. Likewise, a review of 37,012 necropsies performed over 20 years analyzed 202 deceased adults who had had a history of splenectomy. The number of thromboembolic complications related to death in these patients was compared with that of a matched deceased population who had not undergone splenectomy (n = 403). This study showed that pulmonary embolism was the major cause or a contributory cause of death more often in the splenectomy group than in the control group (35.6 vs. 9.7%, p < 0.001) [34]. A role for components of the erythrocyte membrane has been reported in venous thrombembolism occurring after splenectomy [35, 36]. The loss of the filtering function of the spleen may allow abnormal red cells to remain in the peripheral circulation, resulting in the activation of the coagulation cascade.

88  Risk Factors for Chronic Thromboembolic Pulmonary Hypertension

A medical history of splenectomy in patients with CTEPH may impact clinical management. Condliffe et  al. [30] reported that splenectomized patients are less likely to undergo surgical treatment of CTEPH owing to a more distal location of pulmonary thrombi. Accordingly, in the Viennese cohort of 181 patients, splenectomy was identified as a predictor of adverse outcome in CTEPH [37]. In the context of the pulmonary hypertension screening program at the Medical University of Vienna, we investigated the prevalence of CTEPH among individuals at least 12 months after splenectomy. Of a total of 101 individuals screened, two were diagnosed with CTEPH (Bonderman et al. Abstract, ATS 2008, A 185, unpublished).

4 Previous Venous Thromboembolism The scientific debate over the thromboembolic nature of CTEPH has been partially resolved after documentation of previous venous thrombotic events in the majority of affected patients. Similar to what was reported by Moser et  al. [1] Wolf et al. found 60% of 116 French CTEPH patients with a previous history of venous thromboembolism [4]. In accord with these observations, 58% of the 469 CTEPH patients in the UK national registry [30] reported previous thromboembolic events. An interesting observation of the study authors was that those patients with a history of symptomatic venous thromboembolism were more often surgical candidates than patients without previous symptomatic venous thromboembolism. In the database combining information on patients from Vienna, Bratislava, Prague, and Homburg (n = 433), previous venous thromboembolism was reported in 69.0% of CTEPH patients but only in 10.6% of controls with nonthromboembolic pulmonary vascular disease. A history of recurrent thromboembolism was found in 52.2% of patients but only in 3.2% of controls. Also, a history of arm vein thrombosis was more common in CTEPH patients (3.0%) than in controls (0.4%). Although previous venous thromboembolism conferred a 4.5-fold increased risk for the development of CTEPH, a history of recurrent thromboembolic events increased the risk 14.5-fold [12]. The incidence of CTEPH in patients experiencing a nonfatal symptomatic venous thromboembolic event has been investigated in three prospective studies. Ribeiro et  al. [38] followed 78 patients after acute pulmonary embolism for 5 years. Three patients from the cohort were diagnosed with CTEPH and underwent pulmonary endarterectomy at 12, 24, and 44 months after inclusion. Patients at risk were those with systolic pulmonary arterial pressures of more than 50 mmHg at presentation [OR 3.3 (1.2–9.4)] or more than 70 years of age [OR 4.1 (1.4–12.9)] (Table  2). Researchers at the University Hospital in Padua, Italy [39], followed 223 patients referred for symptomatic pulmonary embolism without prior

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Table 2  Risk factors for CTEPH after acute pulmonary embolism Risk factor

Odds ratioa

sPAP >50 mmHg by echocardiography [38] Age >70 years [38] Previous pulmonary embolism [39] Idiopathic pulmonary embolism [39] Large perfusion defect [39] Younger age [39] sPAP systolic pulmonary artery pressure

3.3 (1.2–9.4) 4.1 (1.4–12.9) 19.0 (4.5–79.8) 5.7 (1.4–22.9) 2.2 (1.5–3.3) 1.8 (1.2–2.6)

episodes of pulmonary embolism or venous thrombosis. The cumulative incidence of symptomatic CTEPH was 1.0% at 6 months, 3.1% at 1 year, and 3.8% at 2 years. Patients at risk had a previous pulmonary embolic event [OR 19.0 (4.5– 79.8)], idiopathic pulmonary embolism [OR 5.7 (1.4–22.9)], larger perfusion defects [OR 2.2 (1.5–3.3)], and were younger [OR 1.8 (1.2–2.6)] (Table 2). A major limitation of the study was that at the time of inclusion, preexisting CTEPH was not convincingly excluded. In a more recent analysis performed in 12 Italian medical centers [40], patients after a first episode of pulmonary embolism were followed for 3  years. Of the 259 patients, only two were finally diagnosed with CTEPH, yielding an incidence of 0.8%.

5 Thyroid Disease Only one study investigated the role of thyroid disease/thyroid hormone substitution in CTEPH development [12]. In the database with contributions from four European CTEPH centers, thyroid hormone replacement therapy which was ongoing at the time of clinical assessment for CTEPH was identified as an independent risk factor [OR 6.10 (2.73–15.05), p < 0.001]. The prevalence of subclinical hypothyroidism is 4–8% in the general population, and up to 15–18% in women who are over 60 years of age [41]. For comparison, the prevalence of recognized thyroid disease (either self-reported history of thyroid disease or current levothyroxine treatment) in a population aged over 49 years was reported to be 10% [42]. Therefore, the observation of 19.9% of patients with CTEPH under thyroid hormone replacement is not only statistically different from the nonthromboembolic precapillary pulmonary hypertension comparator (3.5%), but appears biologically meaningful. It has been shown that patients suffering from moderate hypothyroidism are at an increased risk of thrombosis [43]. In addition, treatment with levothyroxine increases von Willebrand factor (vWF) levels and decreases the in  vitro platelet plug formation measured as collagen– epinephrine-induced closure time [44]. However, on the basis of the available data, it cannot be said whether the risk of developing CTEPH is attributed to hypothyroidism or thyroid hormone replacement therapy, or both.

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6 Antiphospholipid Antibodies Antiphospholipid antibodies are directed against plasma proteins with affinity for anionic phospholipids. They alter the regulation of hemostasis and induce activation of complement. Clinically, they represent a risk factor for recurrent arterial and venous thrombosis [45]. In a cohort of 216 CTEPH patients from the University of California, San Diego, 10.6% carried antiphospholipid antibodies [5]. Another study compared the frequencies of antiphospholipid antibodies in 116 CTEPH patients with those in 83 patients with nonthromboembolic pulmonary hypertension. Although in the nonthromboembolic group only 9.5% of the patients were antiphospholipid antibody carriers, 21.5% of the patients tested positive in the CTEPH group. Moreover, half of the patients with CTEPH had high antibody titers, whereas the titers were low in control patients [4]. The significantly higher levels of antiphospholipid antibodies in CTEPH suggest their involvement in the pathophysiologic mechanisms of the disease. In a more recent analysis of a combined database from four European CTEPH centers (Vienna, Austria; Bratislava, Slovakia; Homburg, Germany; Prague, Czech Republic) with information on a total of 433 CTEPH patients, the frequency of antiphospholipid antibodies was as high as 10.0%. In the control group, which consisted of 254 patients with nonthromboembolic pulmonary hypertension, only 4.0% were antiphospholipid-antibodypositive. Antiphospholipid antibodies conferred a 4.2-fold increased risk for the development of CTEPH [12]. In the context of the pulmonary hypertension screening program at the Medical University of Vienna, 71 asymptomatic antiphospholipid antibody carriers were subjected to transthoracic Doppler echocardiography. A final diagnosis of CTEPH was made in two cases (2.8%) by invasive hemodynamic measurement and pulmonary angiography (Lang et al. Abstract, ATS 2008, A 23, unpublished).

7 Cancer Survivors In the database combining patients from four European CTEPH centers, a history of malignancy was reported as a risk factor for CTEPH [OR 3.76 (1.47–10.43), p < 0.005] [12]: 12.2% of the CTEPH patients but only 4.3% of the controls with nonthromboembolic pulmonary hypertension reported previous cancer. Leading diagnoses were breast cancer and gastrointestinal tumors. Epidemiology studies have identified malignancy as an independent thrombosis risk factor and have shown that cancer patients are at increased risk of both initial and recurrent venous thromboembolic events. The effects of

D. Bonderman and I.M. Lang

chemotherapy and other treatments compound the risk due to cancer. The interaction of tissue factor, thrombin, and other coagulation factors with protease activated receptor proteins expressed by tumor cells and host vascular cells leads to the induction of genes related to angiogenesis, cell survival, and cell adhesion and migration [46]. Whether treatment-related factors, for example, adjuvant hormone replacement therapies, are also contributory could not be clarified. Yet, on the basis of the available data, pulmonary hypertension in a tumor survivor should be a signal to search for CTEPH, rather than for another cause of pulmonary hypertension.

8 Elevated Coagulation Factor VIII/von Willebrand Factor Plasma Levels and Non-0 Blood Groups Coagulation factor VIII (FVIII) and vWF are known for their crucial roles in hemostasis. Both proteins circulate in plasma as a tight noncovalently linked complex with vWF serving as a FVIII stabilizer and activator [47]. Epidemiology studies have revealed that elevated levels of FVIII and/or vWF predispose to venous thrombosis [48] and its recurrence [49]. Interestingly, vWF levels are strongly related to the nature of the AB0 determinant. Blood group oligosaccharide structures on the vWF molecule account for the clearance of the FVIII/vWF complex [50]. In persons with blood group O, average vWF concentrations are approximately 25% lower than in non-0 blood group individuals [51]. Elevated plasma FVIII and vWF levels were found in CTEPH patients as compared with controls with nonthromboembolic pulmonary hypertension or healthy individuals. No change in plasma FVIII or vWF antigen was encountered after successful pulmonary endarterectomy, suggesting that the measured elevations were not a consequence of the high shear stress in the hypertensive pulmonary vasculature. In the same study, 41% of CTEPH patients had plasma FVIII levels of more than 230 IU/dl [13]. It had previously been shown that patients after a venous thromboembolic event had a 6.6-fold increased risk for recurrence if the level of FVIII antigen was above this cutoff value [49]. In the context of FVIII/vWF measurements, the blood group distribution among CTEPH patients was analyzed. Non-0 blood groups were more prevalent in CTEPH patients (77%) than in controls (58%, p = 0.003) [13]. In the combined four-center database, the binary variable non-0 blood group was a predictor for the diagnosis of CTEPH [OR 2.09 (1.12–3.94), p = 0.019] after adjustment for all other variables [12].

88  Risk Factors for Chronic Thromboembolic Pulmonary Hypertension

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9 Fibrinogen Aa Thr312Ala Polymorphism

11 Female Gender and HLA Polymorphisms

Suntharalingam et al. [6] investigated the role of eight functionally relevant hemostatic polymorphisms in CTEPH. Genomic DNA was isolated from 214 CTEPH patients and 200 healthy controls, and analyzed for factor V Leiden, prothrombin G to A substitution at nucleotide 20210 (20210G>A), plasminogen activator inhibitor-1 4G/5G, tissue plasminogen activator 7351C>T, factor XIIIG>T, fibrinogen Aa substitution of the threonine with alanine at position 312 (Thr312Ala), fibrinogen Bb substitution of arginine with lysine at position 448 (Arg448Lys), and fibrinogen Bb 455G>A polymorphisms. The main finding of the study was that the presence of the fibrinogen Aa chain Ala312 allele was associated with increased odds of being diagnosed with CTEPH. Although the functional relevance of this polymorphism remains unclear, clinically, homozygosity for the alanine allele has been associated with acute pulmonary embolism but not deep vein thrombosis [52]. The presence of the alanine allele has also been associated with higher poststroke mortality in subjects with atrial fibrillation [53]. It has, therefore, been suggested that the alanine substitution may predispose to embolic phenomena, through the formation of a brittler clot [54].

In contrast to an equal gender distribution in the Western world, Japanese researchers reported a 2.1 female-to-male ratio in CTEPH patients followed in a multicenter national registry. Moreover, a higher ratio of CTEPH to acute pulmonary thromboembolism was observed [62]. Because such ethnic differences may be controlled by genetic factors, Tanabe et  al. [63] performed HLA typing by serological and/or DNA typing methods in 80 patients with CTEPH diagnosed at Chiba University Hospital, Japan, and 678 controls. The authors found a strong association between HLA-B*5201 (40 vs. 24%) as well as DPB1*0202 (19 vs. 6%) and CTEPH. HLA-B*5201-positive patients showed a significant female predominance. More recently, Shigeta et al. [64] demonstrated that HLA-B*5201-positive female patients had better cardiac function with lower operative mortality than HLA-B*5201-negative female patients, who had more peripheral thrombi, resulting in less improvement after surgery. However, reported data have to be interpreted with caution, as overlaps with Takayasu arteritis, a chronic vasculitis mainly involving large vessels, cannot be excluded. Takayasu arteritis is more prevalent in Asia and South America than elsewhere [65, 66] and is known for its female predominance and the association with HLA-B*5201 [67].

10 Inflammatory Bowel Disease The role of inflammatory bowel disease was investigated in the Viennese cohort consisting of 109 CTEPH patients [21]. Because of its inflammatory nature, the condition was grouped together with present or previous osteomyelitis. The prevalence of either of the two conditions was higher in CTEPH patients (10.0%) than in controls (0.0%). The presence of inflammatory bowel disease or osteomyelitis was associated with an increased risk for CTEPH [OR 67 (7.9–8,832)]. Because of the larger patient number in the database from four European CTEPH centers, inflammatory bowel disease was studied separately [12]. The multivariable analysis failed to demonstrate an association with CTEPH [OR 3.19 (0.74– 16.03), p = 0.121]. However, clearly more cases of inflammatory bowel disease were recorded in CTEPH patients (2.8%) than in controls (1.2%). A high incidence of acute venous thromboembolic events has been reported in patients with inflammatory bowel disease [55–59]. Importantly, approximately half of the patients with inflammatory bowel disease who develop a thromboembolic event have no identifiable risk factor [60]. Control of the inflammatory process is thought to be the key factor in risk reduction for thrombotic events [61]. Whether the ongoing inflammatory process predisposes not only to thrombus formation but also to thrombus persistence and CTEPH remains under debate.

12 Congenital Vascular Malformations Congenital combined vascular malformations, such as Klippel–Trenaunay syndrome, have been reported to be associated with venous thromboembolism, occurring in 8–22% of affected patients [68, 69]. Klippel–Trenaunay syndrome is a rare congenital low-flow vascular malformation of the capillary, venous, and lymphatic type, combined with disturbed growth of the bone and soft tissue. Recently, Oduber et  al. [70] reported a series of four patients with low-flow vascular malformations who developed CTEPH. Given previous case reports in the literature [71–73], the risk for CTEPH in Klippel–Trenaunay patients was investigated in the database contributed by four European CTEPH centers. Of 433 CTEPH patients, two (0.5%) had Klippel–Trenaunay syndrome, but none of the controls had it [12]. Given a low prevalence of Klippel–Trenaunay syndrome in the general population, statistically significant numbers may be difficult to achieve in systematic analyses; however, clinicians should be aware of a potential association.

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13 Conclusion Unfortunately, despite a large increase in the understanding of CTEPH in recent years, current knowledge and evidence primarily stems from experimental settings or from observational case-control analyses of CTEPH patient cohorts. Few data exist on the natural history of CTEPH, and in particular, no long-term longitudinal prospective analyses are available on at risk populations, e.g., patients after splenectomy or patients with inflammatory bowel disease. Few prospective studies exist on the development of CTEPH after idiopathic pulmonary embolism, albeit with contradictory results [39, 40]. The difficulty is the decade-long evolvement of the disease, and the relatively uncommon occurrence. A major confirmative cross-sectional and longitudinal prospective study focusing on CTEPH is currently being undertaken by the Association for Research in CTEPH. Some preliminary results from this first international prospective incident Webbased registry have been announced (Abstract, ERS 2008, e-communication: P1045, unpublished).

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88  Risk Factors for Chronic Thromboembolic Pulmonary Hypertension 34. Pimpl W, Dapunt O, Kaindl H, Thalhamer J (1989) Incidence of septic and thromboembolic-related deaths after splenectomy in adults. Br J Surg 76:517–521 35. Kuypers FA, Yuan J, Lewis RA et al (1998) Membrane phospholipid asymmetry in human thalassemia. Blood 91:3044–3051 36. Atichartakarn V, Angchaisuksiri P, Aryurachai K et  al (2002) Relationship between hypercoagulable state and erythrocyte phosphatidylserine exposure in splenectomized haemoglobin E/betathalassaemic patients. Br J Haematol 118:893–898 37. Bonderman D, Skoro-Sajer N, Jakowitsch J et al (2007) Predictors of outcome in chronic thromboembolic pulmonary hypertension. Circulation 115:2153–2158 38. Ribeiro A, Lindmarker P, Johnsson H, Juhlin-Dannfelt A, Jorfeldt L (1999) Pulmonary embolism: one-year follow-up with echocardiography Doppler and five-year survival analysis. Circulation 99:1325–1330 39. Pengo V, Lensing AW, Prins MH et al (2004) Incidence of chronic thromboembolic pulmonary hypertension after pulmonary embolism. N Engl J Med 350:2257–2264 40. Becattini C, Agnelli G, Pesavento R et  al (2006) Incidence of chronic thromboembolic pulmonary hypertension after a first episode of pulmonary embolism. Chest 130:172–175 41. Villar HC, Saconato H, Valente O, Atallah AN (2009) Thyroid hormone replacement for subclinical hypothyroidism. Cochrane Database Syst Rev 18:CD003419 42. Empson M, Flood V, Ma G, Eastman CJ, Mitchell P (2007) Prevalence of thyroid disease in an older Australian population. Intern Med J 37:448–455 43. Franchini M (2006) Hemostatic changes in thyroid diseases: haemostasis and thrombosis. Hematology 11:203–208 44. Homoncik M, Gessl A, Ferlitsch A, Jilma B, Vierhapper H (2007) Altered platelet plug formation in hyperthyroidism and hypothyroidism. J Clin Endocrinol Metab 92:3006–3012 45. Salmon JE, de Groot PG (2008) Pathogenic role of antiphospholipid antibodies. Lupus 17:405–411 46. Bick RL (2003) Cancer-associated thrombosis. N Engl J Med 349:109–111 47. Lenting PJ, Vans CJ, Denis CV (2007) Clearance mechanisms of von Willebrand factor and factor VIII. J Thromb Haemost 5: 1353–1360 48. Martinelli I (2005) von Willebrand factor and factor VIII as risk factors for arterial and venous thrombosis. Semin Hematol 42:49–55 49. Kyrle PA, Minar E, Hirschl M et al (2000) High plasma levels of factor VIII and the risk of recurrent venous thromboembolism. N Engl J Med 343:457–462 50. Kamphuisen PW, Eikenboom JC, Bertina RM (2001) Elevated factor VIII levels and the risk of thrombosis. Arterioscler Thromb Vasc Biol 21:731–738 51. Jenkins PV, O'Donnell JS (2006) ABO blood group determines plasma von Willebrand factor levels: a biologic function after all? Transfusion 46:1836–1844 52. Carter AM, Catto AJ, Kohler HP, Ariens RA, Stickland MH, Grant PJ (2000) Alpha-fibrinogen Thr312Ala polymorphism and venous thromboembolism. Blood 96:1177–1179 53. Carter AM, Catto AJ, Grant PJ (1999) Association of the alphafibrinogen Thr312Ala polymorphism with poststroke mortality in subjects with atrial fibrillation. Circulation 99:2423–2426 54. Standeven KF, Grant PJ, Carter AM, Scheiner T, Weisel JW, Ariens RA (2003) Functional analysis of the fibrinogen Aa Thr312Ala polymorphism: effects on fibrin structure and function. Circulation 107:2326–2330

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55. Talbot RW, Heppell J, Dozois RR, Beart RW Jr (1986) Vascular complications of inflammatory bowel disease. Mayo Clin Proc 61:140–145 56. Solem CA, Loftus EV, Tremaine WJ, Sandborn WJ (2004) Venous thromboembolism in inflammatory bowel disease. Am J Gastroenterol 99:97–101 57. Bernstein CN, Blanchard JF, Houston DS, Wajda A (2001) The incidence of deep venous thrombosis and pulmonary embolism among patients with inflammatory bowel disease: a populationbased cohort study. Thromb Haemost 85:430–434 58. Miehsler W, Reinisch W, Valic E et al (2004) Is inflammatory bowel disease an independent and disease specific risk factor for thromboembolism? Gut 53:542–548 59. Bernstein CN, Nabalamba A (2007) Hospitalization-based major comorbidity of inflammatory bowel disease in Canada. Can J Gastroenterol 21:507–511 60. Jackson LM, O’Gorman PJ, O’Connell J, Cronin CC, Cotter KP, Shanahan F (1997) Thrombosis in inflammatory bowel disease: clinical setting, procoagulant profile and factor V Leiden. QJM 90:183–188 61. Freeman HJ (2008) Venous thromboembolism with inflammatory bowel disease. World J Gastroenterol 14:991–993 62. Nakamura M, Okada O, Sakuma M et al (2002) Incidence and clinical characteristics of chronic pulmonary thromboembolism in Japan compared with acute pulmonary thromboembolism: results of a multicenter registry of the Japanese Society of Pulmonary Embolism Research. Circ J 66:257–260 63. Tanabe N, Kimura A, Amano S et al (2005) Association of clinical features with HLA in chronic pulmonary thromboembolism. Eur Respir J 25:131–138 64. Shigeta A, Tanabe N, Shimizu H et al (2008) Gender differences in chronic thromboembolic pulmonary hypertension in Japan. Circ J 72:2069–2074 65. Ishikawa K (1978) Natural history and classification of occlusive thromboaortopathy (Takayasu’s disease). Circulation 57:27–35 66. Numano F (1992) Hereditary factors of Takayasu arteritis. Heart Vessels Suppl 7:68–72 67. Kimura A, Ota M, Katsuyama Y et al (2000) Mapping of the HLAlinked genes controlling the susceptibility to Takayasu’s arteritis. Int J Cardiol 75:S105–S110 68. Baskerville PA, Ackroyd JS, Lea Thomas M, Browse NL (1985) The Klippel–Trenaunay syndrome: clinical, radiological and haemodynamic features and management. Br J Surg 72:232–236 69. Jacob AG, Driscoll DJ, Shaughnessy WJ, Stanson AW, Clay RP, Gloviczki P (1998) Klippel-Trenaunay syndrome: spectrum and management. Mayo Clin Proc 73:28–36 70. Oduber CE, Gerdes VE, van der Horst CM, Bresser P (2009) Vascular malformations as underlying cause of chronic thromboembolism and pulmonary hypertension. J Plast Reconstr Aesthet Surg 62(5):684–689 71. Mikula N Jr, Gupta SM, Miller M, Felder S (1991) KlippelTrenaunay-Weber syndrome with recurrent pulmonary embolism. Clin Nucl Med 16:253–255 72. Muluk SC, Ginns LC, Semigran MJ, Kaufman JA, Gertler JP (1995) Klippel-Trenaunay syndrome with multiple pulmonary emboli – an unusual cause of progressive pulmonary dysfunction. J Vasc Surg 21:686–690 73. Walder B, Kapelanski DP, Auger WR, Fedullo PF (2000) Successful pulmonary thromboendarterectomy in a patient with Klippel– Trenaunay syndrome. Chest 117:1520–1522

Chapter 89

Evaluation of Small-Vessel Arteriopathy in Chronic Thromboembolic Pulmonary Hypertension Nick H. Kim

Abstract  The principal treatment for chronic thrombo­ embolic pulmonary hypertension (CTEPH) remains surgery. Pulmonary thromboendarterectomy (PTE), also referred to as pulmonary endarterectomy, offers the possibility of cure for many patients with symptomatic CTEPH who would otherwise succumb to progressive right-sided heart failure. Two subgroups of patients with CTEPH, however, will not fully benefit from PTE: those not offered surgery owing to concern of the disease being too distal, and those with persistent symptomatic pulmonary hypertension identified only after PTE – the group also at the highest risk for postoperative death. This latter group represents patients with significant small-vessel disease. For the former group, determination of inoperability remains often subjective and open to interpretation. To use an analogy from surgical oncology, the field of CTEPH is in need of a better preoperative “clinical staging.” This chapter will discuss the importance of small-vessel disease in CTEPH and the current limitations and opportunities in improving the preoperative evaluations. Keywords  Small arteriopathy • Distal pulmonary artery • Pulmonary embolism • Thromboembolism • Thrombosis • Fibrinolysis

1 Introduction The principal treatment for chronic thromboembolic pulmonary hypertension (CTEPH) remains surgery [1, 2]. Pulmonary thromboendarterectomy (PTE), also referred to as pulmonary endarterectomy, offers the possibility of cure for many patients with symptomatic CTEPH who would otherwise succumb to progressive right-sided heart failure [3–5]. Two subgroups of patients with CTEPH, however, will not fully benefit from PTE: those not offered surgery owing to concern N.H. Kim (*) Division of Pulmonary and Critical Care Medicine, University of California, San Diego, 9300 Campus Point Drive, MC 7381, 92037, La Jolla, CA, USA e-mail: [email protected]

of the disease being too distal, and those with persistent symptomatic pulmonary hypertension identified only after PTE – the group also at the highest risk for postoperative death. This latter group represents patients with significant small-vessel disease [6]. For the former group, determination of inoperability remains often subjective and open to interpretation. To use an analogy from surgical oncology, the field of CTEPH is in need of a better preoperative “clinical staging.” This chapter will discuss the importance of small-vessel disease in CTEPH and the current limitations and opportunities in improving the preoperative evaluations.

2 Persistent Pulmonary Hypertension After PTE The details of PTE are discussed by Madani and Jamieson [7]. This highly specialized and complex surgery can be performed safely in experienced centers with mortality rates below 5% [8]. In a consecutive cohort of 500 PTE patients reported from University of California, San Diego (UCSD), the overall mortality rate was 4.4% (n = 22). The critical determinant of morbidity and mortality after PTE is the degree of postoperative pulmonary hypertension. Over three quarters of the reported deaths were as a result of persistent pulmonary hypertension. A staggering statistic from this report was the in-hospital mortality rate depending on the early postoperative pulmonary vascular resistance (PVR) result. PTE patients from this cohort with postoperative PVR under 500 dyn s cm−5 had a mortality rate of under 1%. In contrast, patients with postoperative PVR above 500 dyn s cm−5 had greater than 30% in-hospital mortality. Persistent pulmonary hypertension is also a key determinant for long-term post-PTE outcome. A long-term post-PTE survey from UCSD with a mean follow-up period of 3.3 years reported that nearly half of all long-term deaths were attributed to persistent pulmonary hypertension [9]. Table  1 lists the possible causes of residual pulmonary hypertension after PTE. In experienced centers, the chance of either inadequate endarterectomy or reocclusion is low. The experienced PTE surgeon is capable of removing the

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_89, © Springer Science+Business Media, LLC 2011

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chronic thromboembolic material within reach with dramatic improvements in hemodynamics [10]. The type of operative specimen, however, is important. Patients with type IV disease – representing intimal removal rather than a true endarterectomy of chronic thromboembolic occlusive material – are at the highest risk of postoperative pulmonary hypertension and mortality. It is important to note that the classification used for the PTE specimen cannot be reliably applied during the preoperative period; it is a surgical classification. Furthermore, finding proximal disease on just one side, and in some instances even bilaterally (see case example 2), of the pulmonary circulation does not necessarily ensure overall hemodynamic and outcome benefits.

3 Small-Vessel Arteriopathy The pathogenesis of CTEPH remains poorly understood. The prevailing hypothesis starts with a pulmonary thromboembolic event followed by incomplete or nonresolution of acute thrombus [11]. This then leads to the development of a chronic obstructive defect and varying degrees of distal small-vessel arteriopathy, contributing to both increased afterload and pulmonary hypertension. There are ongoing investigations attempting to elucidate the various mechanisms likely to be involved in the development of CTEPH. In terms of the small-vessel arteriopathy, the endothelin pathway has been implicated from both a canine CTEPH model using ceramic bead embolization and a cohort of PTE patients noted to have alterations in their endothelin signaling and clearance [12, 13]. CTEPH patients also appear to have upregulation of angiopoietin-1, a protein involved in vascular remodeling [14]. More recently, the discovery of endothelial progenitor cells in chronic thromboembolic endarterectomized tissues has raised the possibility of their involvement in distal remodeling [15]. Such discoveries and other ongoing research dedicated specifically to CTEPH will hopefully lead to a better understanding of the pathogenesis. For now, our understanding of CTEPH pathogenesis remains rudimentary. However, with future breakthroughs in CTEPH pathogenesis, it may be plausible to develop complementary preoperative tests or screening tools, which may help with risk stratification and facilitate the making of treatment decisions.

N.H. Kim Table  2  Possible mechanisms contributing to distal inoperable microvascular disease in chronic thromboembolic pulmonary hypertension Vascular pathology Features Occlusions of small arteries with stenoses, webs, and bands; Similarity/overlap with IPAH Intimal proliferation and/or Classical pulmonary arteriopathy increased media thickeness, of small muscular arteries and plexiform lesions; arterioles distal to nonobstructed elastic pulmonary Endothelial dysfunction possibly arteries related to increased pressure and flow Endothelial dysfunction Pulmonary arteriopathy of small possibly related to poor muscular arteries and arterioles perfusion and/or bronchialdistal to partially or totally to-pulmonary vascular obstructed elastic pulmonary anastomoses arteries Modified from [6] with permission from the American Thoracic Society. Copyright American Thoracic Society

Predominant obstructions of “small” subsegmental elastic pulmonary arteries

Currently, given the success of PTE in experienced centers, the majority of patients with evidence of segmental or more proximal disease are offered PTE. The assumption is that the experienced surgeon will remove whatever mechanical component of CTEPH is within reach. The problem of persistent pulmonary hypertension therefore following PTE by an experienced surgeon is most likely attributed to the presence of significant concomitant small-vessel disease. The histopathologic characteristics of small-vessel involvement in CTEPH have been described from both antemortem lung biopsies and postmortem examinations [16, 17]. Although there are subtle differences in the distribution and degree of small-vessel changes, the small-vessel lesions in CTEPH appear to overlap with those found in pulmonary arterial hypertension (PAH). Previously thought to be only associated with PAH, characteristic plexiform lesions were also seen in CTEPH. Furthermore, the small-vessel changes were not isolated to the open, unobstructed beds. Even small vessels distal to occluded pulmonary arteries could be seen affected with remodeling. Table 2 summarizes the three main types of small-vessel disease in CTEPH and their possible pathogenic mechanisms. These observations highlight the two-compartment problem in CTEPH – a disease of both proximal mechanical obstruction and varying degree of distal arteriopathy, both contributing to pulmonary hypertension. It is important to emphasize, however, that the mere presence of small-vessel arteriopathy in CTEPH does not necessarily portend a poor response to PTE. The more important and challenging preoperative question to answer is whether the degree of concomitant small-vessel disease will lead to unfavorable clinical outcome owing to significant persistent pulmonary hypertension after PTE. The following three case examples highlight the importance of the small-vessel contribution in CTEPH.

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3.1 Case 1: High-Risk Patient, Good Results Figure  1: This was a 68-year-old woman, who was moderately obese and of functional class IV with preoperative hemodynamics as follows: right atrial pressure (Pra) 21 mmHg, pulmonary artery pressure (PAP) 98/38 (mean 59), pulmonary artery occlusion pressure (Paop) 10, CO 2.6 l/m, cardiac index (CI) 1.3, PVR 1,508 dyn s cm−5. PTE was offered with a much higher estimate of perioperative mortality taking into account the severity of pulmonary hypertension, rightsided heart failure, and concerns for some distal disease – especially on the left. She underwent bilateral PTE and had the following hemodynamics in the ICU: Pra 7 mmHg, PAP 36/16 (mean 23), CO 4.4 l/min. The postoperative VQ scan revealed a marked increase in perfusion to the right lung. She was discharged in good condition on hospital day 12.

3.2 Case 2: Large Specimen, Poor Outcome Figure 2: This was a 47-year-old man of functional class IV, with preoperative hemodynamics as follows: Pra 11 mmHg, PAP 90/40 (mean 60), Paop 5, CO 2.7 l/min, CI 1.2, PVR 1,630 dyn s cm−5. PTE was offered with a higher estimate of perioperative mortality owing to the degree of pulmonary hypertension and right-sided heart failure. He underwent bilateral PTE with removal of a large type I specimen from both sides. Although his postoperative hemodynamics improved, with a mean PAP of 41 mmHg with an initial CI of 2.7 on pressors, he had a complicated postoperative course including severe reperfusion pulmonary edema, persistent pulmonary hypertension, with a prolonged pressors requirement for refractory right-sided heart failure, culminating in multiple organ failure and death.

Fig. 1  Case 1: higher preoperative risk. (a) Right and left lateral pulmonary arteriogram demonstrating the presence of chronic thromboembolic disease but also multiple intact segmental vessels, especially on the left. (b) Pulmonary thromboendarterectomy (PTE) specimen – type I right, type II left

3.3 Case 3: Small Specimen, Good Results Figure 3: This was a 30-year-old man with a history of cardiac ablation for supraventricular tachyarrhythmia, of functional class III, with the following hemodynamics: Pra 5 mmHg, PAP 105/38 (mean 61), Paop 9, CO 3.1 l/min, PVR 1,342 dyn s cm−5. The left pulmonary artery was occluded proximally; the left lung was reduced in size. PTE was offered with preoperative concern that the left pulmonary artery may not be reperfused. He underwent PTE with removal of only type III disease from the right. The left pulmonary artery was found intraoperatively to be atretic. Despite the limited endarterectomy, his hemodynamics were significantly improved, with Pra 5 mmHg, PAP 48/15 (mean 25), and CO 5.4 l/min, while off all pressors. His postoperative VQ scan revealed

Fig. 2  Case 2: large PTE specimen – type I bilaterally

increased perfusion to the right lower lobe. He was discharged in good condition by hospital day 17.

3.4 Case Discussions These three cases were selected to address the importance of the small-vessel component in CTEPH. The patient in case 1

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role in that development by damaging the pulmonary veins. Although the PTE in this case may not serve as a long-term cure, his hemodynamic and symptomatic improvements were impressive in spite of the operative specimen.

4 Improving the Preoperative Evaluations

Fig. 3  Case 3: dramatic hemodynamic improvement despite a small PTE specimen. (a) Pulmonary arteriogram demonstrating right lower lobe disease and complete proximal obstruction of the left main pulmonary artery. (b) PTE specimen – type III right, nothing on the left

had multiple poor-outcome risk factors described in the PTE literature [18, 19]. Although she clearly has radiographic evidence of disease amenable to PTE, she represents the type of patient that a less experienced PTE center may even turn down owing to perceived excess risk. Case 2 represents an unfortunate example of a good but incomplete hemodynamic response of a patient to PTE. Although a postmortem examination was not performed, the probable cause of persistent pulmonary hypertension and right-sided heart failure was related to concomitant small-vessel disease distal to the endarterectomized pulmonary arteries. Lastly, case 3 is an exceptional example of how the size of the PTE specimen, especially when compared alongside case 2, does not directly correlate with hemodynamic response. The only way the pulmonary hemodynamics of this patient improved to this degree from his preoperative values is because the right lower lobe distal circulation was relatively free of small-vessel arteriopathy. It is unclear why his left pulmonary artery was atretic and whether the history of cardiac ablation had a

Before some of the limitations are discussed, it is imperative to emphasize that the current preoperative evaluations are adequate in facilitating the experienced center to successfully treat the majority of CTEPH patients with PTE. However, for the approximately 10–15% of PTE patients with significant residual pulmonary hypertension after surgery and, depending on the center, the more than 50% of evaluated patients not offered surgery owing to perceived risks including concerns for distal or small-vessel disease, the current preoperative system has limitations [20]. Table 3 outlines the role and limitations of the current preoperative investigative tools. The conventional tools generally focus on the presence of proximal disease, but have limited objective utility in assessing the degree of concomitant small-vessel disease. The three cases presented were selected to illustrate the limitations of our current evaluation system. We are in need of a better, more objective preoperative measure of significant concomitant small-vessel disease beyond subjective comparison of the degree of radiographic obstruction to the preoperative hemodynamics. The pulmonary artery occlusion waveform analysis may be a tool to more objectively assess the degree of small-vessel Table 3  Preoperative tests and their role in the assessment of small-vessel disease Technique

Utility

VQ scan

Recommended screening test to differentiate CTEPH and PAH; may underestimate the extent of pulmonary vascular obstruction Remains the gold standard for detecting proximal disease; can identify disease down to the subsegmental level Can assess wall thickness, bronchial vessels; can identify disease down to the subsegmental level Powerful prognostic indicator; used with imaging modalities to help assess operability Experience-based; current means of prognosticating and determining operability Experimental; partition PVR into upstream and downstream components

Traditional pulmonary angiography Multidetector spiral CT angiogram PVR by right-sided heart catheterization Hemodynamics and imaging Pulmonary artery occlusion waveform analysis Lung biopsy

Cannot sufficiently differentiate CTEPH and PAH; not indicated Modified [6] with permission from the American Thoracic Society. Copyright American Thoracic Society

89  Evaluation of Small-Vessel Arteriopathy in Chronic Thromboembolic Pulmonary Hypertension

disease [21]. However, this tool has not been validated and remains experimental – only applicable in patients when the signal can be obtained and analyzed. Cases 1 and 2 were from the original cohort studied with the occlusion technique. Figure 4 shows where they fit on the key data comparing preoperative upstream resistance (Rup) and postoperative hemodynamic outcome (total peripheral resistance index). Despite the concerns prior to PTE in case 1, this patient had a high Rup, suggesting that much of her PVR was from large-vessel disease. In contrast, despite the bulky proximal disease in case 2, this patient had one of the lower Rup values measured preoperatively, suggesting the presence of concomitant and significant small-vessel disease. Whether the occlusion technique will have a place in the preoperative evaluations remains uncertain, but represents an intriguing step toward investigating the status of the small-vessel circulation in CTEPH prior to PTE. On the basis of the preliminary data with the occlusion technique, and in recognition of the absence of a preoperative classification in CTEPH, a prototype classifications system was presented (Table  4). CTEPH patients with high preoperative PVR represent a consistently higher risk group for PTE regardless of the center [18, 19, 22]. A tool capable of partitioning the PVR into an operable and an inoperable component would be helpful in separating this high-risk

Fig. 4  Case 1 and case 2 on the preoperative upstream resistance versus postoperative total peripheral resistance index relationship graph. (Modified from [21])

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group into two groups – one likely to benefit from PTE and the other who may not. The latter group may be more suitable candidates for trial of medical therapy targeting the small-vessel arteriopathy before or in lieu of PTE.

5 Role of PAH Medical Therapy There are four subgroups of CTEPH patients for which the role of medical therapy may be of interest: (1) CTEPH patients receiving “bridging” or adjuvant therapy prior to PTE, (2) patients with accessible disease turned down owing to significant comorbidities and/or perceived high risk, (3) patients turned down owing to distal disease deemed inaccessible, and (4) persistent pulmonary hypertension following PTE. The first and fourth groups represent CTEPH patients who will undergo or have undergone PTE surgery. The bridging therapy concept is an attempt to medically pretreat otherwise high-risk cases before PTE [23, 24]. The second and third groups represent patients not offered surgery. However, the distinction needs to be made that patients turned down with otherwise operable disease may respond differently from patients with anatomically distal or smallvessel disease. In other words, an agent designed to target the microscopic arteriopathy component of CTEPH should not be necessarily expected to improve the pulmonary hypertension from predominantly proximal vessel mechanical obstruction from chronic thromboembolic disease. To date, we have no compelling data to support the routine use of currently available PAH-targeted therapies (prostanoids, endothelin receptor antagonists, phosphodiesterase-5 inhibitors) in any of these four instances. The group best studied to date is group 3 – patients turned down owing to distal disease. The results of three short-term randomized placebo-controlled medical treatment studies have been disappointing in terms of efficacy and final analysis [25–27]. Though speculative, the designs of the clinical trials may be partially responsible for the negative results to date from randomized controlled trials investigating medical therapy in CTEPH. First, borrowing PAH clinical trial end points and

Table 4  Proposed preoperative classifications Class Factor A

B

C

Pulmonary artery angiography Right-sided heart catheterization Miscellaneous

+a PVR £ 1,100 dyn s cm−5

+a PVR > 1,100 dyn s cm−5 Rup > 60%

+a PVR > 1,100 dyn s cm−5 Rup < 60%

Treatment

PTE

PTE

PAH

(−) +PH (−) CT angiography or pulmonary angioscopy PTE (greater risk) or trial Medical therapy of medical therapy

Ventilation–perfusion scan supportive of chronic thromboembolic disease PH pulmonary hypertension, Rup upstream resistance Modified from [20]. Reprinted with permission from the American Thoracic Society. Copyright American Thoracic Society a

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study duration may not be valid in CTEPH. Also, given the relative paucity in our knowledge of the pathogenesis of CTEPH, it is possible that these medical agents effective in PAH may not work the same for CTEPH. Lastly, owing to the subjective and variable definitions of inoperability in CTEPH, poor patient selection may have contributed to the relative lack of benefit of these medical therapies. It begs the question: Were those patients truly inoperable? Could they have not responded to medical treatment because they had predominantly obstructive, mechanical disease which requires endarterectomy? Therefore, for this subgroup of CTEPH patients, representing patients with a potentially prohibitive degree of concomitant small-vessel disease not amenable to surgery, the advancement must first come in improving the preoperative evaluations and classifications. Together with better knowledge of CTEPH pathogenesis, we may then be able to more effectively develop and investigate medical therapies in CTEPH.

6 Conclusion Although PTE is the treatment of choice in the majority of patients with symptomatic CTEPH, not all patients will benefit from endarterectomy. Persistent pulmonary hypertension due to distal inaccessible disease remains the major concern and the leading cause of morbidity and mortality with PTE. Although the current preoperative evaluation is typically adequate in identifying patients with accessible disease, it has limitations when trying to screen for significant smallvessel disease. Along with improving our general knowledge of the pathogenesis of CTEPH, we also need improvements in the preoperative screening process to better identify those subgroup of CTEPH patients who may not benefit from PTE. Such patients will be more suitable and better candidates for trials in evaluating the role of medical therapy.

References 1. Klepetko W, Mayer E, Sandoval J, Trulock EP, Vachiery JL, Dartevelle P, Pepke-Zaba J, Jamieson SW, Lang I, Corris P (2004) Interventional and surgical modalities of treatment for pulmonary arterial hypertension. J Am Coll Cardiol 43(12 Suppl S):73S–80S 2. McGoon M, Gutterman D, Steen V, Barst R, McCrory DC, Fortin TA, American LJE, College of Chest Physicians (2004) Screening, early detection, and diagnosis of pulmonary arterial hypertension: ACCP evidence-based clinical practice guidelines. Chest 126(Suppl 1): 14S–34S 3. Archibald CJ, Auger WR, Fedullo PF, Channick RN, Kerr KM, Jamieson SW, Kapelanski DP, Watt CN, Moser KM (1999) Longterm outcome after pulmonary thromboendarterectomy. Am J Respir Crit Care Med 160:523–528

N.H. Kim 4. Riedel M, Stanek V, Widimsky J, Prerovsky I (1982) Longterm follow-up of patients with pulmonary thromboembolism: late prognosis and evolution of hemodynamic and respiratory data. Chest 81:151–158 5. Lewczuk J, Piszko P, Jagas J, Porada A, Wojciak S, Sobkowicz B, Wrabec K (2001) Prognostic factors in medically treated patients with chronic pulmonary embolism. Chest 119:818–823 6. Galie N, Kim NH (2006) Pulmonary microvascular disease in chronic thromboembolic pulmonary hypertension. Proc Am Thorac Soc 3:571–576 7. Madani MM, Jamieson SW (2006) Technical advances of pulmonary endarterectomy for chronic thromboembolic pulmonary hypertension. Semin Thorac Cardiovasc Surg 18(3):243–249 8. Jamieson SW, Kapelanski DP, Sakakibara N, Manecke GR, Thistlethwaite PA, Kerr KM, Channick RN, Fedullo PF, Auger WR (2003) Pulmonary endarterectomy: experience and lessons learned in 1,500 cases. Ann Thorac Surg 76:1457–1462 9. Archibald CJ, Auger WR, Fedullo PF, Channick RN, Kerr KM, Jamieson SW, Kapelanski DP, Watt CN, Moser KM (1999) Longterm outcome after pulmonary thromboendarterectomy. Am J Respir Crit Care Med 160:523–528 10. Thistlethwaite PA, Mo M, Madani MM, Deutsch R, Blanchard D, Kapelanski DP, Jamieson SW (2002) Operative classification of thromboembolic disease determines outcome after pulmonary endarterectomy. J Thorac Cadiovasc Surg 124:1203–1211 11. Lang IM, Kerr K (2006) Risk factors for CTEPH. Proc Am Thorac Soc 3:568–570 12. Kim H, Yung GL, Marsh JJ, Konopka RG, Pedersen CA, Chiles PG, Morris TA, Channick RN (2000) Endothelin mediates pulmonary vascular remodelling in a canine model of chronic embolic pulmonary hypertension. Eur Respir J 15:640–648 13. Bauer M, Wilkens H, Langer F, Schneider SO, Lausberg H, Schafers HJ (2002) Selective upregulation of endothelin B receptor gene expression in severe pulmonary hypertension. Circulation 105: 1034–1036 14. Du L, Sullivan CC, Chu D et al (2003) Signaling molecules in nonfamilial pulmonary hypertension. N Engl J Med 348:500–509 15. Yao W, Firth AL, Sacks RS et  al (2009) Identification of putative endothelial progenitor cells (CD34+CD133+Flk-1+) in endarterectomized tissue of patients with chronic thromboembolic pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 296:L870–L878 16. Moser KM, Bloor CM (1993) Pulmonary vascular lesions occurring in patients with chronic major vessel thromboembolic pulmonary hypertension. Chest 103:685–692 17. Yi ES, Kim H, Ahn H, Strother J, Morris T, Masliah E, Hansen LA, Park K, Friedman PJ (2000) Distribution of obstructive intimal lesions and their cellular phenotypes in chronic pulmonary hypertension. A morphometric and immunohistochemical study. Am J Respir Crit Care Med 162:1577–1586 18. Tscholl D, Langer F, Wendler O, Wilkens H, Georg T, Schafers H-J (2001) Pulmonary thromboendarterectomy – risk factors for early survival and hemodynamic improvement. Eur J Cardiothorac Surg 19:771–776 19. Hartz RS, Byme JG, Levitsky S, Park J, Rich S (1996) Predictors of mortality in pulmonary thromboendarterectomy. Ann Thorac Surg 62:1255–1259 20. Kim NH (2006) Assessment of operability in chronic thromboembolic pulmonary hypertension. Proc Am Thorac Soc 3:584–588 21. Kim NH, Fesler P, Channick RN, Knowlton KU, Ben-Yehuda O, Lee SH, Naeije R, Rubin LJ (2004) Preoperative partitioning of pulmonary vascular resistance correlates with early outcome after thromboendarterectomy for chronic thromboembolic pulmonary hypertension. Circulation 109:18–22 22. Dartevelle P, Fadel E, Mussot S, Chapelier A, Herve P, de Perrot M, Cerrina J, Ladurie FL, Lehouerou D, Humbert M, Sitbon O, Simonneau G (2004) Chronic thromboembolic pulmonary hypertension. Eur Respir J 23:637–648

89  Evaluation of Small-Vessel Arteriopathy in Chronic Thromboembolic Pulmonary Hypertension 23. Nagaya N, Sasaki N, Ando M, Ogino H, Sakamaki F, Kyotani S, Nakanishi N (2003) Prostacyclin therapy before pulmonary thromboendarterectomy in patients with chronic thromboembolic pulmonary hypertension. Chest 123:338–343 24. Bresser P, Fedullo PF, Auger WR, Channick RN, Robbins IM, Kerr KM, Jamieson SW, Rubin LJ (2004) Continuous intravenous epoprostenol for chronic thromboembolic pulmonary hypertension. Eur Respir J 23:595–600

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25. Olschewski H, Simonneau G, Galie N et al (2002) Inhaled iloprost for severe pulmonary hypertension. N Engl J Med 347:322–329 26. Suntharalingam J, Treacy CM, Doughty NJ et al (2008) Long-term use of sildenafil in inoperable chronic thromboembolic pulmonary hypertension. Chest 134:229–236 27. Jais X, D’Armini AM, Jansa P et al (2008) Bosentan for the treatment of inoperable chronic thromboembolic pulmonary hypertension. J Am Coll Cardiol 52:2127–2134

Chapter 90

Hemolytic-Anemia-Associated Pulmonary Hypertension: Sickle-Cell-Disease- and Thalassemia-Associated Pulmonary Hypertension Elizabeth S. Klings and Mark T. Gladwin

Abstract  Pulmonary hypertension (PH) is now recognized as a complication of both chronic and acquired hemolytic anemias. The process of hemolysis appears to be central to disease pathogenesis. Sickle cell disease (SCD), a congenital hemoglobinopathy affecting as many as 30 million individuals worldwide, is the best characterized hemolytic anemia associated with PH. Multiple clinical studies have demonstrated a 10–30% prevalence of PH in the hemoglobin SS (HbSS) population, with cases reported in children as young as 7 years of age. The presence of even mild pulmonary artery (PA) systolic hypertension [PA systolic pressures (PASPs) of 35 mmHg or more] in SCD is an independent risk factor for increased mortality in these patients, with 2-year mortality rates approaching 50%. Right-sided heart catheterization, echocardiographic studies, and autopsy findings reveal that patients with SCD develop an overlap of pulmonary arterial hypertension (PAH) with plexogenic vasculopathy and pulmonary venous hypertension secondary to diastolic dysfunction. The resting hemodynamic profile of the severely anemic SCD patient demonstrates a high cardiac output state with mean pulmonary pressures of 36 ± 2 mmHg, and only modest elevations in pulmonary vascular resistance (259 ± 31 dyn·s/cm5). Pulmonary pressures rise acutely both with exercise and during intercurrent complications, such as vaso-occlusive pain crisis and the acute chest syndrome (ACS). ACS, in particular, is associated with a high rate of right-sided heart failure during hospitalization (13% of subjects develop acute cor pulmonale during the ACS). This chapter will overview the epidemiology, pathogenesis, diagnosis, and treatment of hemolysis-associated PH with a focus on SCD. Keywords  Hemolysis • Nitric oxide • Hemoglobin • Congenital hemoglubinopathy

1 Introduction Pulmonary hypertension (PH) is now recognized as a complication of both chronic and acquired hemolytic anemias (Table 1). The process of hemolysis appears to be central to disease pathogenesis. Sickle cell disease (SCD), a congenital hemoglobinopathy affecting as many as 30 million individuals worldwide [1], is the best characterized hemolytic anemia associated with PH. Multiple clinical studies have demonstrated a 10–30% prevalence of PH in the hemoglobin SS (HbSS) population, with cases reported in children as young as 7 years of age [2–5]. The presence of even mild pulmonary artery (PA) systolic hypertension [PA systolic pressures (PASPs) of 35 mmHg or more] in SCD is an independent risk factor for increased mortality in these patients, with 2-year mortality rates approaching 50% [6]. Right-sided heart catheterization, echocardiographic studies, and autopsy findings reveal that patients with SCD develop an overlap of pulmonary arterial hypertension (PAH) with plexogenic vasculopathy and pulmonary venous hypertension secondary to diastolic dysfunction. The resting hemodynamic profile of the severely anemic SCD patient demonstrates a high cardiac output state with mean pulmonary pressures of 36 ± 2  mmHg, and only modest elevations in pulmonary vascular resistance (259 ± 31  dyn.s/cm5). Pulmonary pressures rise acutely both with exercise and during intercurrent complications, such as vaso-occlusive pain crisis and the acute chest syndrome (ACS). ACS, in particular, is associated with a high rate of right-sided heart failure during hospitalization (13% of subjects develop acute cor pulmonale during the ACS) [7, 8]. This chapter will overview the epidemiology, pathogenesis, diagnosis, and treatment of hemolysis-associated PH with a focus on SCD.

2 The Epidemiologic and Causal Link Between Hemolysis and Pulmonary Hypertension E.S. Klings (*) Pulmonary Center, Boston University, 72 East Concord St., R 304, Boston, MA 02118, USA e-mail: [email protected]

PH has been increasingly recognized as a complication of hemolytic anemia. Although this has been most widely studied in SCD and thalassemia, cases of PH have been observed in

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1270 Table 1  Hemolytic disorders associated with pulmonary hypertension Sickle cell disease Thalassemia intermedia and major Hereditary spherocytosis and stomatocytosis (and other red cell membrane disorders) Paroxysmal nocturnal hemoglobinuria Hemoglobin Mainz hemolytic anemia (and other unstable hemoglobin variants) Alloimmune hemolytic anemia Pyruvate kinase deficiency Glucose 6-phosphate dehydrogenase deficiency Microangiopathic hemolytic anemia (hemolytic uremic syndrome, thrombotic thrombocytopenic purpura) Malaria Hemolysis from mechanical heart valves Left ventricular assist devices and cardiopulmonary bypass procedure

association with hereditary spherocytosis and stomatocytosis, pyruvate kinase deficiency, paroxysmal nocturnal hemoglobinuria, and other conditions associated with hemolysis [9–17] (Table 1). A growing body of mechanistic studies support the observation that intravascular hemolysis and subsequent vascular remodeling play a role in pathogenesis as well [11, 14, 16]. As will be discussed in more detail later in this chapter, the process of intravascular hemolysis impairs endothelial function via a number of mechanisms, a major one being the reaction of hemoglobin released from the red blood cell with endothelial nitric oxide (NO) [9, 18, 19]. Red blood cell arginase 1 is also released into plasma during hemolysis and catabolizes arginine to ornithine, thus reducing its availability for de novo NO synthesis from endothelial NO synthase [19–21]. Many of these diseases are also associated with functional (infarction-related) or surgical splenectomy. Splenectomy is an etiologic factor associated with PH in each of these conditions; moreover, it is an independent risk factor for the development of PH [22, 23]. The role that splenectomy has in this process is unclear but is now being addressed by a number of studies. Westerman et al. have demonstrated that splenectomy results in an increase in the intravascular levels of senescent red blood cells expressing phosphatidylserine, and an increase in the plasma levels of cell free hemoglobin and red blood cell microparticles [24]. The cell free hemoglobin is known to react with and scavenge NO and the microparticles can both scavenge NO as well as activate the coagulation system. The presence of pulmonary vascular thrombi in surgical specimens obtained from splenectomy patients supports the notion that PH in this population is, at least in part, related to thromboses [10, 25].

3 Thalassemia and Pulmonary Hypertension PH occurs commonly in thalassemia. “Thalassemia” is a term delineating a spectrum of diseases characterized by reduced or absent production of one or more a-globin or b-globin chains. b-Thalassemia, due to impaired b-globin chain pro-

E.S. Klings and M.T. Gladwin

duction, results in a relative excess of unstable a-globin chains unable to form the tetramers necessary to create a hemoglobin molecule. These a-globin chains precipitate within the cell, leading to ineffective erythropoiesis and an increased propensity for hemolysis [26]. Thalassemia major or homozygous b-thalassemia is a severe disorder due to the inheritance of two b-thalassemia alleles characterized by transfusion-dependent anemia, hepatosplenomegaly, and skeletal deformities related to bone marrow expansion. b-Thalassemia minor (b-thalassemia trait) occurs in heterozygotes and is usually asymptomatic and characterized by mild anemia. b-Thalassemia intermedia occurs in patients with disease of intermediate severity, such as those who are compound heterozygotes of two thalassemic variants. Such patients may have the skeletal abnormalities and hepatosplenomegaly observed in thalassemia major; however, their hemoglobin concentrations are usually between 5 and 10 g/dL and they only require transfusions when they have a clinical event, such as an infection, which impairs erythropoiesis [26]. Echocardiographic screening studies suggest that approximately 40–50% of patients with thalassemia intermedia [27] and 60–75% of patients with thalassemia major have evidence of PH [27–29]. In spite of its potential complications, chronic transfusion therapy and iron chelation in severe thalassemia has changed the clinical course of the disease and is likely to have a favorable impact in individuals with PH. In support of this theory was a report of complete reversal of PH in well-transfused, iron-chelated patients with thalassemia major [30]. A favorable response to the known PAH treatment sildenafil has been reported in patients with thalassemia major and thalassemia intermedia [31]. However, there are no larger studies evaluating the specific hemodynamic abnormalities observed in association with PH nor its impact on survival in this patient population.

4 Pulmonary Hypertension of Sickle Cell Disease 4.1 Epidemiology Although in the NIH cohort, the presence of PH was an independent risk factor for mortality [6], it remains to be determined if elevations in pulmonary pressures directly contribute to death related to progressive or acute right-sided heart failure or if they represent a marker of systemic vasculopathy characterized by increased risk for generalized cardiovascular events (stroke, vaso-occlusion, etc.). It is increasingly clear that pulmonary pressures rise acutely during a vaso-occlusive crisis and even more so during the ACS [32]. A recent study examined 84 consecutive hospitalized patients with the ACS and found that 13% had clinical manifestations of right-sided heart failure, placing

90  Hemolytic-Anemia-Associated Pulmonary Hypertension

them at the highest risk for mechanical ventilation and death [8]. These data suggest that acute PH and right-sided heart dysfunction represent major comorbidities during the ACS. Several other studies have confirmed the presence of at least mild PA systolic hypertension in approximately 30% of the HbSS population [2, 5] and have elicited some other interesting findings. Approximately 13% of HbSS patients with a normal echocardiogram followed longitudinally will develop PH over 3 years [2], suggesting that this may be an age-related complication of SCD and that routine screening echocardiograms need to be employed on an annual or semiannual basis in this population. Although currently classified by the World Health Organization with other forms of PAH [33], PH of SCD is a distinct clinical entity. The presence of left-sided heart disease either echocardiographically or by right-sided heart catheterization in up to 40% of these patients suggests an overlap, in some, of PAH and pulmonary venous hypertension [3, 5, 34]. Left-sided heart disease in SCD is primarily due to diastolic dysfunction (present in approximately 13% of patients), although cases of systolic dysfunction and mitral or aortic valvular disease can occur as well (present in approximately 2% of patients) [35]. The presence of diastolic dysfunction alone in SCD patients is an independent risk factor for mortality, suggesting that it may be reflective of a more systemic vasculopathy in these patients [36]. Importantly, patients with both pulmonary vascular disease and echocardiographic evidence of diastolic dysfunction are at a particularly high risk of death (odds ratio for death of 12.0; 95% confidence interval, 3.8–38.1; p < 0.001) [35].

4.1.1 Findings on Right-Sided Heart Catheterization Right-sided heart catheterization studies of patients with SCD and PH have revealed a hyperdynamic state similar to the hemodynamic profile of porto-PH [6, 34]. The mean PA pressure (mPAP) in patients with SCD and PAH catheterized at the NIH is approximately 40  mmHg, with a pulmonary vascular resistance of approximately 250 dyn.s/cm5 (Table 2). The relatively low pulmonary vascular resistance observed in these patients is reflective of the high cardiac output of anemia. Of catheterized patients, approximately 60% with a tricuspid regurgitant jet velocity (TRV) greater than 3.0 m/s meet the definition of PAH, indicating that the vasculopathy primarily involves the pulmonary arterial system. In the remaining 40%, the left ventricular end-diastolic pressures are greater than 15  mmHg, indicating a component of left ventricular diastolic dysfunction [34]. Figure 1 shows a flow diagram of prevalence estimates of PAH and pulmonary venous hypertension diagnosed by right-sided heart catheterization based on screening data from the NIH. Studies of SCD patients with different hemoglobin genotypes have yielded some interesting results, although these patients are not as well characterized as the HbSS popula-

1271 Table 2  Cardiovascular hemodynamics of sickle cell disease patients with pulmonary arterial hypertension (PAH), diastolic dysfunction, and mixed vasculopathy Diastolic Mixed PAH dysfunction vasculopathy RAP (mmHg) 8 ± 0.6 11 ± 1.0 18 ± 5 PASP (mmHg) 58 ± 3 51 ± 3 60 ± 7 PADP (mmHg) 25 ± 1 23 ± 1 37 ± 0.9 mPAP (mmHg) 36 ± 2 33 ± 2 42 ± 5 CO (L/min) 7.6 ± 0.6 9.7 ± 0.6 9.9 ± 1.3 PCWP (mmHg) 13 ± 0.7 20 ± 1 20 ± 2 PVR (dyn/s/cm5) 259 ± 31 122 ± 20 187 ± 59 RAP right atrial pressure, PASP pulmonary artery systolic pressure, PADP pulmonary artery diastolic pressure, mPAP mean pulmonary artery pressure, CO cardiac output, PCWP pulmonary capillary wedge pressure, PVR pulmonary vascular resistance

Fig. 1  Overview of the epidemiology of pulmonary hypertension (PH) in the hemoglobin SS (HbSS) adult population. These data are derived from the NIH population. Approximately 30% of HbSS adults will have a tricuspid regurgitant jet velocity (TRV) of 2.5 m/s or more, which corresponds to a pulmonary artery (PA) systolic pressure of 35 mmHg or more. Of those subjects, two thirds (20% of the total number of patients) will have PA systolic pressures between 35 and 45  mmHg (TRV between 2.5 and 2.9 m/s). The remaining 10% will have an estimated PA systolic pressure of 45 mmHg or more (TRV ³ 3.0 m/s) and should undergo right-sided heart catheterization. At right-sided heart catheterization, 4% of the original population will have pulmonary arterial hypertension (PAH), whereas 4% will have pulmonary venous hypertension. The remaining 2% will have normal right-sided heart catheterizations, reflective of a false-positive echocardiogram

tion. Genotypes with a lower hemolytic rate [such as hemoglobin SC (HbSC) and hemoglobin S (HbS)/b-thalassemia] do have some degree of protection from PH [37]. Approximately 10–15% of HbSC adults have elevated pulmonary systolic pressures estimated by echocardiogram, but when these are present, the mortality risk is similar to and possibly worse than that observed with the HbSS population [5, 37]. The presence of coexistent a-thalassemia appears to confer protection against PH in the HbSS population [37]. In patients with nonhemolytic HbSC or HbS a-thalassemia

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disease who present with PH, it is very important to evaluate carefully for other risk factors for PH (not simple intravascular hemolysis), such as chronic thromboembolic disease, sleep apnea, sarcoidosis, and HIV infection. 4.1.2 Epidemiology in Pediatric Sickle Cell Disease Patients In the pediatric population, over 11 small screening studies comprising over 600 patients have been performed [4]. Combining the data from these studies has found the prevalence of mild PH (TRV 2.5–2.9 m/s) in 30% and moderate-tosevere PH (TRV ³ 3.0 m/s) in 8%, similar to what was observed in adults [4]. Whether or not these findings will be confirmed in rigorous prospective studies remains to be determined. Data presented from the Pulmonary Hypertension and the Hypoxic Response in Sickle Cell Disease (PUSH) cohort suggest a much lower prevalence of PH in the pediatric population of 11% [38]. Even if the prevalence rates of an elevated PASP are similar to those observed in adults, many questions about the extrapolation of data obtained from adults to children and adolescents remain. One reason for this is that an elevated TRV in this population may not necessarily be reflective of PH but may relate hemodynamically to the characteristic anemiarelated elevated cardiac output observed in these patients [4]. Additionally, treatment of associated conditions such as obstructive sleep apnea, nocturnal hypoxemia, and asthma in a small series of pediatric patients produced improvement in PA pressures, suggesting that this is a potentially modifiable condition [39]. The effect of pulmonary pressure elevation on long-term prognosis and survival is also unknown in the pediatric population and represents an area of intense interest and clinical importance in the SCD community.

4.2 Pathogenesis Although all SCD patients share the same genetic hemoglobin defect, the clinical heterogeneity observed in this population supports the notion that factors unrelated to the hemoglobin mutation play a role in the development of clinical phenotypes. It is likely that SCD represents a condition of chronic systemic vasculopathy. Studies of the medium and large-sized vessels of the circle of Willis reveal intimal hyperplasia, proliferative changes, and disruption of the internal elastic lamina [40–42]. Importantly, autopsy studies of SCD patients reveal similar pathologic changes in the lung vasculature [43] (Fig.  2), and suggest common pathogenic mechanisms across vascular beds. The mechanisms involved in the development of PH in SCD remain complex at this time and are not completely understood. It is likely that SCD vasculopathy results in hemolysis, oxidative stress and dys-

Fig.  2  Histopathology of PH in sickle cell disease (SCD). Photomicrograph (×40) of a histologic section of lungs of a patient with PAH and SCD demonstrating medial hypertrophy (arrow) and a classic plexiform lesion (A)

regulated NO metabolism, genetic predisposition, chronic inflammation, and altered coagulation. This section will review the role of each of these in PH of SCD.

4.2.1 Hemolysis and Nitric Oxide Bioavailability An interesting paradigm that has been developing over the past decade concerns the pathogenesis of clinical phenotypes in SCD. Epidemiology and mechanistic studies suggest that factors related to intravascular hemolysis are paramount to the development of PH and other vascular complications such as priapism, leg ulcers, and certain types of stroke [9, 44] (Fig.  3). The association between hemolysis and the development of PH in SCD is supported by multiple clinical studies suggesting a pathogenic link [2–4, 9, 18]. One of the mechanisms by which hemolysis is thought to affect pulmonary vascular tone and remodeling is via its effects on NO metabolism. NO, a regulator of basal vasodilator tone and an inhibitor of platelet migration and aggregation, is synthesized from l-arginine via an oxidation reaction catalyzed by a family of enzymes termed the nitric oxide synthases [45]. Within the vasculature, NO is produced primarily by the vascular endothelium by endothelial nitric oxide synthases (eNOS; NOSIII) and exerts its effects on vascular smooth muscle via cyclic GMP activation [45]. Upon lysis, sickle erythrocytes release hemoglobin into the plasma, where it avidly reacts with NO to form methemoglobin and nitrate [19, 44]. This creates a state of decreased NO bioavailability, NO resistance, and endothelial dysfunction. Other mechanisms implicated in the effects of hemolysis upon NO bioavailability include arginine dysregulation and alterations in redox biology. Hemolysis results in the release of arginase I, which converts arginine to ornithine in the plasma, thereby

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Fig. 3  Overview of the mechanisms by which hemolysis contributes to the clinical phenotypes of SCD. A schematic representation of the interplay between hemolysis and decreased nitric oxide bioavailability in SCD and the contributions this mechanism plays in the

development of different clinical phenotypes. PH, priapism, leg ulcers, and strokes appear to be linked to hemolysis, whereas pain crises, acute chest syndrome, and osteonecrosis are primarily vasoocclusive

limiting the substrate for NO synthesis [20]. In a study of 228 SCD patients, arginase I activity was increased as compared with its activity in controls and the arginine to ornithine ratio, reflective of arginine bioavailability, correlated inversely with the degree of PH and survival [20]. Additional support for the hypothesis that hemolysis produces impairments in NO signaling comes from transgenic mouse models of SCD and spherocytosis as well as from mouse models of alloimmune hemolysis and malaria [11, 46, 47]. These models exhibit impaired vasodilation to NO donors and endothelial-dependent vasodilators. Interestingly, they also develop spontaneous PH and right-sided heart failure [11, 46].

4.2.2  Altered Redox Biology Altered redox biology and increased oxidant stress have long been implicated in the pathogenesis of SCD-related vascular disease [40, 48, 49]. Potential sources of oxidative metabolites include the sickle erythrocyte, leukocyte, and vascular endothelium [40, 50–55]. Studies of the microcirculation in transgenic mouse models of SCD reveal that hypoxia or inflammatory triggers, such as tumor necrosis factor a and lipopolysaccharide, increase endothelium–leukocyte–erythrocyte adhesive interactions in the postcapillary venules, to initiate vascular occlusion [49, 56, 57]. This modeling indicates that cycles of ischemia and reperfusion, in

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addition to intravascular hemolysis, produce oxidant stress, characterized by activation of vascular oxidases [46, 58], and inflammatory stress, characterized by the expression of endothelial adhesion molecules, generation of inflammatory cytokines, and systemic leukocytosis [54, 55, 59–61]. The sickle erythrocyte, at the baseline, has both an increased concentration of oxygen-related radicals and a relative deficiency in intracellular antioxidant defense mechanisms [40, 50]. Altered glutathione metabolism and glutamine bioavailability (necessary for NADPH oxidase synthesis) occurs within the erythrocyte, particularly in patients with PH [53]. Although it is likely that reactive oxygen species play a role in altering gene expression, another potential target is the posttranslational modifications of plasma proteins. Oxidative posttranslational modifications of plasma albumin have been identified in a small series of patients with PH of SCD [62]. Moreover, a lower abundance of the oxidant-scavenging protein apoliprotein A1 was identified in PH of SCD patients, supporting the theory of similarities in pathogenesis to atherosclerosis [32]. Note that the superoxide formed from enzymatic oxidases such as NADPH oxidase [63], xanthine oxidase [64], and uncoupled eNOS [46, 63] will also react with and scavenge NO, contributing to the state of NO resistance in SCD. The role of eNOS uncoupling, a state where eNOS generates superoxide and not NO, is of particular interest as new therapeutic regimens using tetrahydrobioterin therapy may present a novel mechanism for therapeutic eNOS “recoupling.”

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(ADRB1) were associated with PH [72]. These data need to be confirmed in larger-scale studies but suggest interesting similarities between the pathogenesis of SCD vasculopathy and familial non-sickle-cell-related PAH.

4.2.4 The Role of Inflammation in Vasculopathy Multiple studies have implicated inflammation as a mediator of sickle cell vasculopathy [40, 49, 58]. Abnormal interactions between monocytes and the vascular endothelium have been observed in both in vitro and mouse models of SCD [49, 58, 73, 74]. Additionally, microarray studies of peripheral blood mononuclear cells from SCD patients have demonstrated an antioxidant, proinflammatory phenotype, suggesting that these are mechanisms important to vascular disease [52]. The role that inflammation plays in specific SCD complications such as PH is less clear. Several groups have identified elevated levels of soluble adhesion molecules, particularly VCAM-1, P-selectin, and E-selectin in the plasma of PH in SCD patients [5, 75]. VCAM-1 levels correlated both with serum lactate dehydrogenase (LDH) levels and TRV, suggesting a relationship with both hemolysis and decreased NO bioavailability. These adhesion molecules are not only indicative of endothelial dysfunction but also play a role in mediating interactions between sickle erythrocytes, leukocytes, and platelets with the endothelium. As NO is an important second messenger regulating their expression, it can be inferred that this is one of the downstream effects of reduced NO bioavailability.

4.2.3 Genetic Epidemiology of Pulmonary Hypertension in Sickle Cell Disease

4.2.5 Alterations in Coagulation

HbS results from the primary genetic abnormality of SCD, a valine for glutamic acid substitution at position 6 of the b-globin chain of the hemoglobin molecule. Sickle cell anemia is an autosomal recessive disorder in which patients carry two copies of this mutant b-globin gene. Approximately 8% of black Americans are heterozygous for this gene and have sickle cell trait. The incidence of sickle cell anemia (HbSS) is approximately one in 600 in the USA, whereas in sub-Saharan Africa, it is estimated that 1–4% of the population is born with this disease [44]. The clinical heterogeneity of SCD suggests the possibility that other genetic modifiers of the disease exist [65, 66]. Genetic linkage studies have identified genetic polymorphisms in the transforming growth factor b (TGF-b), Klotho, and vascular cell adhesion molecule  1 (VCAM-1) pathways associated with complications of SCD such as stroke, priapism, leg ulcers, avascular necrosis, and bacteremia [67–71]. The findings of a large-scale study of genetic predictors for PH of SCD has yet to be published. However, a small study of 49 genes in 111 HbSS patients (44 of whom had PH) revealed polymorphisms in the TGF-b superfamily (ACVRL1, BMP6, BMPR2) and the b-1 adrenergic receptor

SCD is a hypercoagulable state; SCD patients have both alterations in the clotting cascade and an increased risk of thromboembolic disease [76, 77]. The association between hypercoagulability and PH of SCD is less clear at this point. Although anecdotally, most practitioners who treat a large number of SCD patients with PH can identify some with coexistent thromboembolic disease, the published literature to date does not support these findings. Villagra et al. recently reported that basal platelet activation increases linearly with the severity of PH in SCD [78]. Additionally, they found that platelet activation correlated with the intensity of intravascular hemolysis and that plasma hemoglobin directly activated platelets, suggesting a primary role for hemolysis in hypercoagulability. Similar correlations between indices of hemolysis and activation of the coagulation cascade have recently been published by three other independent groups [24, 76, 79]. Despite the severity of hypercoagulability related to PH, Ataga et al. did not find any difference in markers of coagulation between SCD patients with and without PH [76]. Moreover, van Beers et al. did a cross-sectional study of SCD patients using echocardiograms and ventilation–perfusion

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(V/Q) scanning and demonstrated no association between PH and an abnormal V/Q scan [79]. Because of the small size of the Ataga et al. and van Beers et al. patient cohorts, further studies are necessary to more clearly define the role of hypercoagulability in the pathogenesis of PH in SCD.

4.3 Diagnostic Evaluation 4.3.1 History and Physical Examination PH in SCD presents a diagnostic dilemma in many patients for several reasons. In most, the elevation of the pulmonary pressures is modest and may not adversely affect the cardiac output (which at the baseline is increased owing to the patients’ anemia) [3, 34]. Additionally, dyspnea, the most common presenting symptom of PAH [80], is difficult to interpret in this population. Dyspnea is extremely common in SCD, occurring in approximately 50% of HbSS adults and 40% of HbSC adults [5]. However, dyspnea is multifactorial in SCD patients, with asthma, restrictive lung disease, chest wall abnormalities, and anemia also contributing to the symptoms. Other symptoms suggestive of PH, particularly chest pain and exertional syncope, may be difficult to interpret as well. The chest wall is a frequent site of vaso-occlusive pain in SCD patients and often it is difficult to distinguish between different types of pain in these patients. Dehydration and anemia can both lead to symptoms of light-headedness and even rarely syncope. As a result, many patients with mild-to-moderate PH actually have a paucity of symptoms and those with normal hemodynamics can have symptoms suggestive of PH [5], supporting the notion that all SCD patients need to be screened regularly for this complication. The physical findings in PH of SCD are initially subtle. The first signs of disease may be a loud pulmonic component of the second heart sound, a left parasternal systolic murmur of tricuspid regurgitation, jugular venous distension, and a rightsided fourth heart sound. Eventually, a right-sided third heart sound may be audible and a right ventricular heave or pulsatile liver may be present. The findings of ascites and peripheral edema indicate overt right-sided heart failure and are less common in SCD patients. Physical examination must include evaluation for signs associated with other diseases associated with PAH, particularly in the SCD population: HIV disease, thromboembolic disease, cirrhosis, and collagen vascular disease.

4.3.2 Six-Minute Walk Test The 6-min walk test is a well-established objective measure of exercise capacity in the evaluation of PAH patients and is an independent predictor of survival [81]. Although SCD patients can have other factors such as prior strokes and avascular necro-

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sis of the hip which may limit mobility, the 6-min walk test is useful in this population as well. In PH of SCD patients, the distance walked correlates with PH severity and improves with a therapeutic response to treatment, making it a useful biomarker in clinical medication trials [82, 83]. When compared with age, gender, and hemoglobin-matched patients with SCD without PH, individuals with PH and mPAP of more than 36  mmHg exhibit shorter 6-min walk distances (435 ± 31 vs. 320 ± 20 m; p = 0.002) and lower peak oxygen consumption on cardiopulmonary exercise testing (50 ± 3 vs. 41 ± 2% of predicted; p = 0.02) [34]. In addition, in SCD patients, PA vascular resistance sharply rises with exercise, suggesting that pulmonary vascular disease contributes to functional limitation in these patients [7]. Taken together, these data suggest that in patients with chronic anemia, mild-to-moderate PH has a severe adverse impact on functional and aerobic exercise capacity.

4.4 Laboratory Analysis 4.4.1 Markers of Hemolysis SCD patients with PH are more anemic than those without PH [3, 5]. Supportive of the notion that hemolysis is the primary mechanism for anemia in these patients, LDH, a marker of intravascular hemolysis, has been found to be an important biomarker. Although LDH can be produced by multiple organs, including the lungs, heart, and liver, in SCD patients the primary isozymes are LD1 and LD2, suggesting that the erythrocyte is the primary source. Supporting this theory, in SCD, these levels correlated with both the degree of hemolysis and NO resistance. LDH levels were increased in patients with hemolytic complications of SCD including PH, leg ulcers, and priapism and there appeared to be an association between the degree of LDH elevation and the likelihood of having PH [18].

4.4.2 Markers of Systemic Vasculopathy Although most patients with PH of SCD have only modestly elevated pulmonary pressures, their mortality is similar to that observed in patients with the higher-pressure idiopathic PAH, suggesting that in this population, an elevated TRV may be a marker for more diffuse systemic vasculopathy. In support of this notion is the association of PH with relative systemic hypertension and proteinuria, reflective of sickle-cell-related renal dysfunction [84, 85]. SCD patients have lower blood pressures than controls, most likely owing to a variety of mechanisms involving arterial stiffness and ischemic events to the renal medulla. Relative systemic hypertension, as defined by either systolic blood pressures (SBP) between 120 and 139 mmHg or diastolic blood pressures (DBP) between 70 and 89  mmHg, was observed in 37% of patients with a TRV of 2.5  m/s or

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greater. Meanwhile, 93% of those with true systemic hypertension (SBP ³ 140  mmHg or DBP ³ 90  mmHg) had coexistent PH, suggesting that there is a relationship between the degree of systemic hypertension and the occurrence of PH [86].

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The most common PFT patterns were restrictive function (observed in 74%), and an isolated low DLCO (observed in 13%) [89]. PFTs on a small number of SCD patients with and without PH demonstrated that those with PH were more likely to have both restrictive physiology and a decreased DLCO [34].

4.4.3 Markers of Pulmonary Hypertension 4.5.2 Echocardiography Additional laboratory testing is helpful for evaluation of the coexistence of other conditions associated with PAH as well as to provide prognostic information. As is the case with other forms of PH, antinuclear antibody testing is recommended as a screening evaluation for coexistent collagen vascular disease. Additionally, HIV antibody testing is recommended for all subjects [87]. Measurement of N-terminal pro-brain natriuretic peptide (NT-proBNP) levels can be used both to screen SCD patients for PH and for risk stratification [88]. NT-proBNP levels were higher in patients with PH of SCD, and directly correlated with PAH severity and the degree of functional impairment. An NT-proBNP level of 160 pg/mL or higher had a 78% positive predictive value for the diagnosis of PH and was an independent predictor of mortality (risk ratio, 5.1; 95% confidence interval, 2.1–12.5; p < 0.001). In a subset of individuals participating in the Multi-center Study of Hydroxyurea in Sickle Cell Anemia patient follow-up study from 1996 to 2001, 30% of patients had an NT-proBNP level of 160 pg/mL or greater [82]. An NT-proBNP level of 160  pg/mL or greater in the Multicenter Study of Hydroxyarca in Sickle Cell Anemia (MSH) cohort was independently associated with mortality by Cox proportional hazards regression analysis (2.87; 95% confidence interval, 1.2–6.6; p = 0.02) [88]. Although echocardiograms were not available as part of this study, these data suggest that NT-proBNP levels may be used independently as a screening surrogate in the SCD population.

Echocardiogram is currently the primary screening test for PH in SCD. Its primary benefits are that it allows for noninvasive assessment of left- and right-sided cardiac structure and function, provides information about valvular function, and allows for indirect measurement of the PASP. Using echocardiography, one calculates the PASP using the Bernoulli equation (4×TRV2 + right atrial pressure) (Figs. 4, 5). On the basis of the prospective study of 175 HbSS adults published in 2004 by investigators at the NIH, PH of SCD is defined by the presence of a TRV of 2.5 m/s or more, which corresponds to a PASP of approximately 35 mmHg; this occurs in approximately 30–43% of HbSS adults [2, 3, 5]. Although these pressures are lower than those observed in other forms of PAH, the increased mortality observed in this population suggests that this value is indicative of pulmonary vasculopathy [3]. Severe PH is much less common in the SCD population, is defined by a TRV of 3.0 m/s or more (PASP approximately 45 mmHg), and occurs in approximately 8–10% of the SCD population [3, 5]. PH is observed in approximately 10–15% of HbSC adults [5, 37] and can be observed in up to 30% of pediatric HbSS patients, particularly those over the age of 12 [4]. Echocardiography technique was first used in the study to describe the mixed pulmonary vasculopathy observed in SCD: at least 10–15% of

4.5 Pulmonary Hypertension Workup As with other forms of PAH, a complete workup for PH of SCD includes pulmonary function tests (PFTs), an evaluation for thromboembolic disease, echocardiography, antinuclear antibody and HIV testing, and a right-sided heart catheterization [87]. 4.5.1 Pulmonary Function Tests The findings of PFTs are abnormal in most SCD patients. The largest study of HbSS adults to date evaluated 310 patients recruited as part of the Cooperative Study of SCD and found that only 31 of 310 (10%) patients had normal PFT findings. Overall, HbSS adults had decreased total lung capacities (70.2 ± 14.7% predicted) and DLCO (64.5 ± 19.9% predicted).

Fig. 4  Echocardiogram of a patient with PH related to SCD. As demonstrated in this echocardiogram of a patient with an estimated PA systolic pressure of 44 mmHg, in patients with PH of SCD, there is dilation of the right ventricular chamber so that it is larger than the left ventricle (LV), with shift of the interventricular septum (labeled septum) into the LV cavity

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a cohort of 141 SCD patients, Sachdev et al. found that 47% of patients had PH, diastolic dysfunction, or both (29% had PH alone, 11% had diastolic dysfunction and PH, and 7% had diastolic dysfunction alone). PH and diastolic dysfunction were associated with a relative risk of death of 5.1 (95% confidence interval, 2.0–13.3) and 4.8 (95% confidence interval, 1.9–12.1), respectively, whereas the relative risk of death when both were present was 12.0 (95% confidence interval, 3.8–38.1) [35]. These data suggest that both PH and diastolic dysfunction independently carry additive mortality risk.

Fig.  5  Doppler echocardiogram demonstrating determination of the TRV. The Doppler flow of tricuspid regurgitation (shown in blue) from the right ventricle into the right atrium is demonstrated. The velocity of this flow is measured and allows for calculation of an estimated PA systolic pressure

HbSS and HbSC adults have evidence of left-sided heart disease contributing to their elevated PA pressures [35]. Left-sided heart disease in SCD is primarily due to diastolic dysfunction (although systolic dysfunction and valvular disease can occur as well). Diastolic dysfunction, with or without concomitant PH, is an independent risk factor for mortality [35].

4.5.3 Right-Sided Heart Catheterization Right-sided heart or PA catheterization remains the diagnostic gold standard for diagnosing PH [87]. Its advantages over echocardiography include the ability to directly measure PA pressures (to remove the interobserver variability that can occur with assessment of the TRV), cardiac output, and left-sided filling pressures. This allows for a more accurate assessment of left ventricular diastolic function than what is currently available by echocardiogram. Unfortunately, at this point, much of the published literature focuses on echocardiograms in SCD patients although there is a growing body of hemodynamic data. In contrast to patients with traditional forms of PAH (e.g. idiopathic, scleroderma-associated), who are symptomatic with mPAPs in the range of 50–60 mmHg, patients with SCD exhibit mild-to-moderate elevations in mean pulmonary pressures, in the range of 30–40 mmHg, with mild elevations in pulmonary vascular resistance. Many of these patients also have a coexistent mild elevation in pulmonary capillary wedge pressure (PCWP), suggestive of left-sided heart disease (Table 2, Fig. 1). Right-sided heart catheterization data demonstrate a diverse nature to the hemodynamics of the PH in these patients; PAH (defined by mPAP ³ 25  mmHg and PCWP £ 15  mmHg) is present in 54% of catheterized patients at the NIH, whereas pulmonary venous hypertension secondary to left-sided heart disease (defined by mPAP ³ 25 mmHg and PCWP > 15 mmHg) is present in 46% [90]. Further, in echocardiographic studies in

4.5.4 Diagnostic Screening Recommendations Currently, no formal diagnostic guidelines for PH of SCD exist and we base these recommendations on the existing literature and our own clinical practices. Because of the paucity of symptoms and clinical findings associated with PH in SCD, particularly early in the disease, we recommend routine echocardiographic screening of all SCD patients. The prevalence of the disease and the long-term risk of developing PH in those with an initially normal echocardiogram have led us to recommend echocardiography on an annual basis, particularly for HbSS adults. Screening practices for the pediatric and HbSC populations are not as clear at this point, but we think that practitioners can consider echocardiograms on an annual or semiannual basis in these patients as well. Six-minute walk tests and NT-proBNP measurements can be used to provide further prognostic information but should not, at this point, be used in isolation. At this point, we would reserve right-sided heart catheterization for those patients with a TRV of 3.0 m/s or more or those with symptoms related to PH (New York Heart Association class II or higher). To get a better idea of the actual prevalence of PH in SCD from a large-scale screening study, let us say one screens 100 HbSS adults (Fig. 1). On the basis of the epidemiology studies done to date, one would expect that 30–40 of these patients would have a TRV of 2.5 m/s or more. It is likely that at least 15 of these patients will have no cardiopulmonary symptoms. Of the 30–40 patients, only about 10–12 will have a TRV of 3.0 m/s or more, warranting further evaluation. If one performs a right-sided heart catheterization on all of these patients, one will most likely obtain the following results. On the basis of the known inaccuracies of echocardiography in measuring TRV and consequently PASP, if one catheterizes 12 patients, at least two of them will have entirely normal cardiopulmonary hemodynamics. The remaining ten will have PH; it is likely that at least four will have pulmonary venous hypertension. Thus, if one starts with 100 HbSS adults, one will likely find approximately four to six patients with hemodynamically significant PAH (based on conventional definitions). But, one will also find approximately 30 additional patients with pulmonary arterial systolic hypertension who have an increased mortality risk,

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many of whom are without symptoms. This emphasizes the importance of a comprehensive screening program for all HbSS patients, regardless of symptoms and risk factors.

4.6 Treatment 4.6.1 General There are limited data on the specific management of patients with PH of SCD. Most of the recommendations are extrapolated from data derived from other forms of PH or are based on expert opinions from the pulmonary and cardiology communities. The general approach should include maximal treatment of the primary hemoglobinopathy, treatment of associated cardiopulmonary conditions (such as iron overload, chronic liver disease, HIV infection, obstructive sleep apnea/nocturnal hypoxemia, and thromboembolic disorders), and targeted therapy aimed at the pulmonary vasculature (Fig.  6). The overall effect of these therapies on survival is completely unknown and clearly warrants further investigation. 4.6.2 Sickle-Cell-Disease-Specific Therapies Since one of the central pathogenic mechanisms in PH of SCD is chronic intravascular hemolysis, aggressive treatment of the underlying hemoglobinopathy holds promise as a SCD-specific therapy for these patients. Hydroxyurea has been shown to decrease the incidence of pain episodes and ACS, to reduce the need for transfusions, and to lower overall mortality [82, 91]. It is unclear if the survival effects of hydroxyurea relate to effects on PH. One of the primary mechanisms of hydroxyurea, augmentation of hemoglobin F concentrations, has not been proven beneficial in the prevention of PH [92]. Moreover, cross-sec-

Fig. 6  Treatment algorithm for patients with PH of SCD. An overview of current treatment recommendations for patients with PH of SCD based upon echocardiographic findings. HTN hypertension

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tional epidemiology studies of PH in SCD do not demonstrate a treatment benefit in disease prevention [2, 3, 5]. Of interest, however, is a small body of literature supporting the notion that hydroxyurea has a role in potentiating the action of NO, clearly a mediator of PH in SCD [93]. It is possible, via this mechanism or others yet to be described, that some of the decreases in pulmonary and cardiovascular deaths observed in hydroxyureatreated patients could be related to an improvement in PH. The dosage of hydroxyurea is dependent upon renal function. For patients with serum creatinine levels below 1 g/dL, hydroxyurea is started at a dosage of 15 mg/kg/day and is uptitrated to 35 mg/kg/day. Recently, detailed treatment guidelines have been published [94]. Patients with renal dysfunction tend not to tolerate the myelosuppressant effects of hydroxyurea and, as such, we recommend the addition of erythropoietin to the regimen. Chronic or long-term transfusion therapy decreases the risk of most complications of SCD, including ACS and central nervous system vasculopathy [95–98]. A recently published small cross-sectional study suggested that left ventricular mass (a possible predisposing factor for diastolic dysfunction) was decreased in pediatric SCD patients (n = 12) undergoing chronic transfusion therapy compared with those not transfused (n = 95), supporting the notion of a beneficial effect [90]. These data also help to dispel the notion that diastolic dysfunction is a consequence of myocardial iron deposition occurring in the setting of multiple transfusions. It is postulated that transfusion therapy may improve cardiopulmonary function and prevent the progression of PH, but the findings of no large-scale long-term studies have been published in this regard. We advocate a more comprehensive evaluation of transfusion therapy in the PH/SCD population to allow for more universal recommendations to be made. The use of anticoagulation in patients with PH of SCD is highly controversial at present. SCD patients are at increased risk for venous thromboembolic disease and have increased levels of circulating markers of coagulation compared with African American controls [76, 77]. Although chronic thromboembolic disease is clearly a risk factor for the development of PH in non-SCD populations [87], in SCD, the literature to date is confounding. SCD patients with PH have trends toward increased levels of circulating markers of coagulation [76] but this was not significant. In a cross-sectional study of 78 patients who underwent both transthoracic echocardiography and ventilation–perfusion scanning, a high-probability scan was only observed in one PH/SCD patient, suggesting no clear link between PH and thromboembolic disease. However, as this was a cross-sectional study, the long-term risk of thromboembolism is completely unknown in this population. In idiopathic PAH patients, the risk of small-vessel thrombosis is thought to be increased owing to the presence of increased stasis within the pulmonary vasculature [80]. Survival is increased in this population with the use of anticoagulation [99, 100] and because of this, it is empirically recommended for other forms of PAH. Clinically, there is clearly, in some, an overlap of PH related to

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SCD, and the occurrence of thromboembolic disease, usually as a secondary phenomenon. It is clear that, in the setting of documented thrombosis, these patients require lifelong anticoagulation. In terms of prophylactic use of anticoagulation for the PH of SCD patients without documented thrombosis, largescale, longitudinal follow-up studies of this population are needed to determine their actual risk of venous thromboembolism. Until this is done, the potential benefits of anticoagulation will need to be balanced on an individual basis with the risks of hemorrhage (particularly intracerebral) in the SCD population. 4.6.3 Pulmonary Vasodilators As of December 2010, six pulmonary vasodilators have been approved by the Federal Drug Administration of the USA for treatment of PAH. These include the prostaglandins (epoprostenol, treprostinil, and iloprost), the endothelin receptor antagonists (bosentan and ambrisentan), and the phosphodiesterase 5 inhibitors (sildenafil, tadalafil, and inhaled form of treprostinil). Two randomized clinical trials evaluating the use of bosentan and sildenafil, respectively, have been undertaken in the PH of SCD population. However, both of these trials were stopped prematurely. Because of this, much of what is being done clinically with PH of SCD patients is based upon the PAH literature, case reports, and small open-label studies [101]. Prostaglandins Epoprostenol has revolutionized the treatment of PAH over the past 15–20 years and in idiopathic PAH improves symptoms, hemodynamics, and long-term survival [102]. It is a medication given via a continuous intravenous infusion with an extremely short half-life. In the SCD population, epoprostenol has been used successfully in small open-label case series to acutely treat PH, with approximately 75% of patients demonstrating a hemodynamic response [3, 6, 103]. Concerns regarding the use of intravenous prostanoids in SCD surround the increased risk of infection and thrombosis from the presence of an indwelling catheter and the possible exacerbation of a hyperdynamic cardiac state [104]. The longeracting treprostinil and the inhaled iloprost have not been utilized to date in published series of SCD patients. Endothelin Receptor Antagonists Endothelin-1 (ET-1), the most potent endogenous vasoconstrictor, is produced by the vascular endothelium and exerts its action upon smooth muscle cells. Two ET-1 receptors exist, ET-A, which is primarily responsible for vasoconstriction, and ET-B, which modulates vasodilation through NO-related feedback mechanisms [105]. Two endothelin receptor antagonists are currently available in the USA, bosentan, which blocks ET-A and

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ET-B, and ambrisentan, which only blocks ET-A. One of the major shortcomings in the use of these medications in SCD patients relates to their toxicities. Approximately 8% of patients treated with bosentan and 3% of patients treated with ambrisentan develop hepatotoxicity, which may complicate iron overload in SCD [105]. Additionally, use of bosentan in some patients is associated with a decrease in the serum hemoglobin concentration of approximately 1  g/dL. However, the observation that ET-1 may be an important mediator of vaso-occlusion in SCD [106–109] led to the industry-sponsored placebo-controlled ASSET trials of bosentan in treatment of PH in SCD. Unfortunately, this study was discontinued prior to the recruitment goal and consequently, it was underpowered to study medication efficacy [101]. Anecdotally, bosentan has been clinically useful in the treatment of PH in some SCD patients and is generally well tolerated in those without concomitant iron overload as it was in the ASSET studies. Ambrisentan, the newer endothelin receptor antagonist available, has never been studied in SCD.

Agents That Increase Nitric Oxide Bioavailability Inhaled NO, l-arginine, and sildenafil (through phosphodiesterase 5 inhibition and consequent cGMP potentiation) act via an increase in NO bioavailability, thereby correcting one of the primary mechanisms implicated in the pathogenesis of PH in SCD. Inhaled NO has been used successfully to acutely lower PA pressures in case reports of patients with PH of SCD [110]. Its cumbersome delivery system makes its long-term clinical use unpractical at the present time. l-Arginine has been studied via an open-label design in ten patients over 5 days with an average reduction in PASP (by echocardiogram) of 15.2% [111]. However, many difficulties with long-term arginine administration arise. Usually large doses of arginine are necessary to produce a therapeutic effect (6–30 g/day in three divided doses). The large number of capsules necessary to achieve this can result in medication noncompliance as well as the associated abdominal discomfort, headache, and diarrhea observed [104]. Sildenafil, the phosphodiesterase 5 inhibitor, potentiates the action of NO via inhibition of cGMP breakdown. This agent has demonstrated acute and long-term symptomatic and hemodynamic benefits in the treatment of PAH [112] and has tremendous promise for treatment of PH related to SCD. One potential concern about sildenafil use in the SCD population relates to a potential risk for priapism, but this has yet to be borne out by clinical experience. A preliminary open-label study of sildenafil in 12 patients with an estimated pulmonary artery systolic pressure ≥35 mmHg (nine with confirmatory right heart catheterization data at baseline demonstrating a mean PAP ≥25 mm Hg) followed for six months was promising with both acute and chronic effects on pulmonary hemodynamics as well as long-term effects on 6 minute walk distance and NT-proBNP levels [111]. Based upon these results, the multi-center WalkPHaSST (Pulmonary Hypertension and Sickle Cell Disease

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with Sildenafil Therapy), was designed to compare the safety and efficacy of oral sildenafil to placebo for the treatment of Doppler defined PH (tricuspid regurgitant jet velocity ≥2.7 m/s) in adults and children (aged >12 years) with SCD. After 74 (out of a targeted 132) subjects were enrolled, the study was prematurely discontinued due to an increase in significant adverse events, primarily vasoocclusive pain crises in the sildenafil group compared with the placebo arm. One of the difficult issues was whether the adverse effects could have been mitigated if the trial had required intensification of SCDspecific therapy (e.g., hydroxyurea or chronic transfusions) prior to the initiation of sildenafil treatment. The clinical results from this trial, which will likely be limited by the under-powered sample size, have yet to be published.

5 Conclusions At this point, we have only just begun to gain an understanding of the pathogenesis, diagnosis, and treatment of PH related to hemolytic anemias. The extensive work that has been done in the SCD field has greatly enhanced this knowledge, but clearly more work needs to be done as this complication is common and clearly has impacts on the morbidity and mortality of these patients.

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E.S. Klings and M.T. Gladwin 91. Charache S, Terrin ML, Moore RD, Dover GJ, Barton FB, Eckert SV et al (1995) Effect of hydroxyurea on the frequency of painful crises in sickle cell anemia. Investigators of the Multicenter Study of Hydroxyurea in Sickle Cell Anemia. N Engl J Med 332:1317–1322 92. Fujii K, Nakano T, Kawamura T, Usui F, Bando Y, Wang R et al (2004) Multidimensional protein profiling technology and its application to human plasma proteome. J Proteome Res 3:712–718 93. Cokic VP, Andric SA, Stojilkovic SS, Noguchi CT, Schechter AN (2008) Hydroxyurea nitrosylates and activates soluble guanylyl cyclase in human erythroid cells. Blood 111:1117–1123 94. Lanzkron S, Strouse JJ, Wilson R, Beach MC, Haywood C, Park H et al (2008) Systematic review: hydroxyurea for the treatment of adults with sickle cell disease. Ann Intern Med 148:939–955 95. Pegelow CH, Adams RJ, McKie V, Abboud M, Berman B, Miller ST et al (1995) Risk of recurrent stroke in patients with sickle cell disease treated with erythrocyte transfusions. J Pediatr 126:896–899 96. Mehta SH, Adams RJ (2006) Treatment and prevention of stroke in children with sickle cell disease. Curr Treat Options Neurol 8:503–512 97. Lee MT, Piomelli S, Granger S, Miller ST, Harkness S, Brambilla DJ et  al (2006) Stroke Prevention Trial in Sickle Cell Anemia (STOP): extended follow-up and final results. Blood 108:847–852 98. Miller ST, Wright E, Abboud M, Berman B, Files B, Scher CD et al (2001) Impact of chronic transfusion on incidence of pain and acute chest syndrome during the Stroke Prevention Trial (STOP) in sickle-cell anemia. J Pediatr 139:785–789 99. Rubin LJ (1985) Calcium channel blockers in primary pulmonary hypertension. Chest 88:257S–260S 100. Rich S, Kaufmann E, Levy PS (1992) The effect of high doses of calcium-channel blockers on survival in primary pulmonary hypertension. N Engl J Med 327:76–81 101. Barst RJ, Mubarek KK, Machado RF et al (2010) Exercise capacity and haemodynamics in patients with sickle cell disease with pulmonary hypertension treated with bosentan: results of the ASSET studies. Br J Hematol 149:426–435. 102. Badesch DB, McLaughlin VV, Delcroix M, Vizza CD, Olschewski H, Sitbon O et al (2004) Prostanoid therapy for pulmonary arterial hypertension. J Am Coll Cardiol 43:56S–61S 103. Kaur K, Brown B, Lombardo F (2000) Prostacyclin for secondary pulmonary hypertension. Ann Intern Med 132:165 104. Benza RL (2008) Pulmonary hypertension associated with sickle cell disease: pathophysiology and rationale for treatment. Lung 186:247–254 105. Barst RJ (2007) A review of pulmonary arterial hypertension: role of ambrisentan. Vasc Health Risk Manag 3:11–22 106. Sabaa N, de Franceschi L, Bonnin P, Castier Y, Malpeli G, Debbabi H et al (2008) Endothelin receptor antagonism prevents hypoxiainduced mortality and morbidity in a mouse model of sickle-cell disease. J Clin Invest 118:1924–1933 107. Ergul S, Brunson CY, Hutchinson J, Tawfik A, Kutlar A, Webb RC et al (2004) Vasoactive factors in sickle cell disease: in vitro evidence for endothelin-1-mediated vasoconstriction. Am J Hematol 76:245–251 108. Rybicki AC, Benjamin LJ (1998) Increased levels of endothelin-1 in plasma of sickle cell anemia patients. Blood 92:2594–2596 109. Werdehoff SG, Moore RB, Hoff CJ, Fillingim E, Hackman AM (1998) Elevated plasma endothelin-1 levels in sickle cell anemia: relationships to oxygen saturation and left ventricular hypertrophy. Am J Hematol 58:195–199 110. Kato GJ, Gladwin MT (2008) Evolution of novel small-molecule therapeutics targeting sickle cell vasculopathy. JAMA 300:2638–2646 111. Morris CR (2006) New strategies for the treatment of pulmonary hypertension in sickle cell disease: the rationale for arginine therapy. Treat Respir Med 5:31–45 112. Galiè N, Ghofrani HA, Torbicki A, Barst RJ, Rubin LJ, Badesch D et al (2005) Sildenafil citrate therapy for pulmonary arterial hypertension. N Engl J Med 353:2148–2157

Chapter 91

Pulmonary Hypertension Due to Schistosomiasis Ghazwan Butrous and Angela P. Bandiera

Abstract  Schistosomiasis (also known as bilharzia or snail fever) is a parasitic disease caused by several species of the genus. Schistosoma, which are flatworm flukes of the Trematoda class. Schistosomiasis, one of the most important yet neglected tropical diseases, is an ancient and chronic disease of humans, nonhuman primates, other mammals, and birds. It is the second most common parasitic disease after malaria, affecting about 200–300 million people in 74 countries, 85% of whom live in sub-Saharan Africa. Almost 500–800 million people are at risk of acquiring the infection, which causes more than 250,000 deaths per year. It is a major cause of morbidity, having a very significant effect on the economy and welfare in tropical and subtropical countries. It  causes up to 4.5 million disability adjusted life year losses annually with contributory factors such as anemia, pain, diarrhea, exercise intolerance, and undernutrition resulting from chronic infection. The infection is therefore a real health problem in the endemic areas of developing countries mainly because of the constant reinfection of individuals together with poor sanitary conditions, new economic development such as the construction of new dams, or natural disasters such as flooding. All of these factors make the total control of this disease a very difficult task as well as making drug treatment alone inefficient. The current treatment is praziquantel, which is a single-dose monotherapy used worldwide in communitybased programs to control ­schistosomiasis. Schistosomiasis can cause many further complications, including bladder and urethral outflow obstruction, carcinoma of the bladder, prostatitis, portal hypertension, and oesophageal varicose veins. The subject of this chapter coincides with the increasing interest in pulmonary vascular complications of schistosomiasis and this chapter summarizes current knowledge and highlights the need for further investigations.

G. Butrous Division of Cardiopulmonary Sciences, University of Kent, Canterbury, UK e-mail: [email protected]

Keywords  Bilharzia • Snail fever • Parasitic disease • Praziquantel • Infection

1  Introduction Schistosomiasis (also known as bilharzia or snail fever) is a parasitic disease caused by several species of the genus Schistosoma, which are flatworm flukes of the Trematoda class. Schistosomiasis, one of the most important yet neglected tropical diseases, is an ancient and chronic disease of humans, nonhuman primates, other mammals, and birds. It is the second most common parasitic disease after malaria, affecting about 200–300 million people in 74 countries, 85% of whom live in sub-Saharan Africa (Fig. 1). Almost 500–800 million people are at risk of acquiring the infection, which causes more than 250,000 deaths per year. It is a major cause of morbidity, having a very significant effect on the economy and welfare in tropical and subtropical countries. It causes up to 4.5 million disability adjusted life year losses annually with contributory factors such as anemia, pain, diarrhea, exercise intolerance, and undernutrition resulting from chronic infection [1–4]. The infection is therefore a real health problem in the endemic areas of developing countries mainly because of the constant reinfection of individuals together with poor sanitary conditions, new economic development such as the construction of new dams, or natural disasters such as flooding. All of these factors make the total control of this disease a very difficult task as well as making drug treatment alone inefficient [3, 5–7]. The current treatment is praziquantel, which is a singledose monotherapy used worldwide in community-based programs to control schistosomiasis [8–12]. Schistosomiasis can cause many further complications, including bladder and urethral outflow obstruction, carcinoma of the bladder, prostatitis, portal hypertension, and oesophageal varicose veins. The subject of this chapter coincides with the increasing interest in pulmonary vascular complications of schistosomiasis and this chapter summarizes current knowledge and highlights the need for further investigations.

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_91, © Springer Science+Business Media, LLC 2011

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Fig. 1  Geographic distribution of the endemic areas with schistosomiasis (shaded)

2  The Parasite

• S. malayensis, S. mekongi, S. sinensium, S. incognitum, and S. indicum, which are similar to S. japonicum

There are several species of the Schistosoma parasite which infect humans using different species of snails (usually of the Biomphalaria, Bulinus, and Oncomelania genera) as an intermediate host [13–15]. Schistosoma mansoni (also known as Manson’s blood fluke or swamp fever) infects over 120 million people in Africa, Brazil, Suriname, and the Caribbean. The final target of this species is the intestinal and hepatic circulatory system. The major worm more frequently resides in the superior mesenteric veins. The intermediate host is of the Biomphalaria genus. S. japonicum, which is endemic in China and Southeast Asia, affects the intestinal and hepatic circulatory system. The major worm is more frequently in the superior mesenteric veins. The intermediate host is of the Oncomelania genus. S. mekongi, found in Cambodia and Laos, affects both superior and inferior mesenteric veins. It is related to S. japonicum but differs as it has smaller eggs, a different intermediate host, and a longer prepatent period in the mammalian host. S. haematobium (also known as bladder fluke) is endemic in Africa, the Middle East, India, and East Asia. The adult worm resides in the venous plexus of the bladder, but it can also be found in the rectal venules. The intermediate host is of the Bulinus genus. There are, however, many other species of Schistosoma normally infecting cattle, sheep, goats, or rodents in Africa, parts of southern Europe and the Middle East. For example:

Both male and female worms are separate organisms (dioecious), unlike other trematodes. They have two suckers, an anterior oral sucker surrounding the mouth, and a ventral sucker (acetabulum) for attachment and stabilization. The male is 6–20 mm in length, and has a long groove along one side of the body at full length called the gynecophoral canal, in which the female, which is thinner and longer (10–30  mm), resides. Females reach sexual maturity after they have been united with a male. After mating, the two remain locked together for the rest of their lives. They can live for several years and produce many thousands of eggs. The female separates from her mate to migrate to the venules of the organ specific to the species to deposit her eggs, i.e., lumen of the intestine (S. mansoni and S. japonicum) or the bladder and ureters (S. haematobium) [16]. The eggs are eliminated with feces or urine, respectively. Adult worms have a 2–4-mm-thick natural outer covering called the tegument, which covers the entire surface of the worm. It is believed that this structure enables the worm to evade the host immune system [17], which partly explains the longevity of the worm in the host bloodstream (the worm can live for up to 30 years).

• S. edwardiense, S. hippotami, and S. rodhaini, which are similar to S. mansoni • S. bovis, S. curassoni, S. intercalatum, S. leiperi, S. margrebowiei and S. matthei, which are similar to S. haematobium

3  The Life Cycle of Schistosoma The most common way of contracting schistosomiasis in developing countries is by walking, swimming, or even just with feet immersed in shallow water in lakes, ponds, and other bodies of water which are infested with the snails that are the intermediate host of the Schistosoma pathogen (for general reviews, see [18–21]).

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Fig. 2  The life cycle of Schistosoma and its development inside the human host. 1, 2 eggs find their way into the urine or feces of the host, and when they are voided into the water, they hatch into a developmental stage called miracidium. 3 this form infects certain species of freshwater snails. The miracidium inside the snails undergoes various transformational changes and produces a new parasitic form known as cercaria. 4, 5 the cercariae then leave the snail during the warm hours of the day, when most of the potential human hosts are in contact with the water and penetrate the skin of people working or bathing in the infected water. 6 the cercariae undergo transformation, enter postcapillary venules, and travel in the bloodstream to the lungs. 7, 8 they then travel to the mesenteric circulation and liver (9) in the case of Schistosoma mansoni and S. japonicum, or the venous plexus of the bladder in the case of S. hematobium. 9 the adult parasite starts to lay eggs and may leave the body via feces or urine, and the cycle continues

The Schistosoma parasite is digenetic, i.e., a trematode worm in which sexual reproduction as an internal parasite of a vertebrate alternates with asexual reproduction in a mollusk. The life cycle of this parasite (Fig.  2) involves many steps [18, 22]. The eggs are eliminated in the urine (in the case of S. haematobium) and feces (in cases of S. mansoni and S. japonicum) into water. Under optimal conditions, the eggs are capable of hatching within 30 min. Once hatched, they release a ciliated larva called miracidium, which swims and penetrates specific snail intermediate hosts within 1–24 h. The miracidia inside the snails undergo various transformational changes and asexual multiplication several hundred times into primary and secondary saclike structures called sporocytes. The sporocytes migrate to the snail hepatopancreas, where they start to divide again, producing large numbers of new parasitic forms known as cercariae which are 1  mm long and have a characteristic forked tail. They emerge from an infected snail over several weeks; during this time, thousands of cercariae may be released. The cercariae usually leave the snail daily in a circadian rhythm and generally appear when the snails are in sunlight, particularly around noon and in the early afternoon, a period which also coincides with increasing human water-contact activity. In S. haematobium, 4–8 weeks elapse from the penetration of

the miracidium to the liberation of cercariae; in S. mansoni, under optimum conditions, only 4 weeks are necessary. The cercariae remain swimming in freshwater using a whiplike tail (for a maximum of 48 h) at about 4 m/h but can penetrate the skin of people working or bathing in the infected water in 3–5 min using proteolytic enzymes. After penetration, the cercaria may remain in the skin for 1–2 days. The cercaria enters the bloodstream in the postcapillary venules and undergoes transformation, but no multiplication takes place in the human host. It loses its tail and becomes a wormlike schistosomulum (the penultimate stage before becoming a full adult worm). Migration through the venous system continues until the lungs are reached. There, the schistosomulum undergoes further developmental change to enable its subsequent migration to the systemic circulation within 1 week to the targeted organ [23]. The parasite begins to feed on red blood cells and matures in 6–8 weeks and then must find a mate, with the female worm residing in the gynecophoral canal of the male [24]. They begin to produce eggs (about 300 eggs per day for S. mansoni and may produce up to 3,000 eggs per day for S. japonicum). Mature eggs are capable of passing though tissues (possibly through the release of proteolytic enzymes), and may thus find themselves in the urine or in stools. Some eggs become trapped in the small venules. The eggs are antigenic and may vary their antigenicity during their

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maturation. Their antigens can elicit a vigorous immune response which enhances the cellular pathological changes classically associated with schistosomiasis.

4  Immunological Changes The major pathological change of schistosomiasis is the inflammatory and immunological response resulting from a granulomatous reaction. This is due to the various antigenic loads from the Schistosoma eggs rather than the whole worm, which is almost protected by the tegument (see earlier) [25]. The reaction, like in other parasitic helminths, induces a marked CD4+ T helper 2 cell (Th-2) response which contributes to the host protection but also leads to fibrosis and organomegaly [26–28]. However, the immunological response to schistosomiasis is far from fully understood and varies according to the strain, species, age, gender, duration or intensity of infection, genetic predisposition, coinfection, reinfection, the chronological changes of the maturing egg, and other environmental factors [29–34]. The immune response progresses at various stages of the infection. At the early stage, when the host is exposed to migrating parasites in the circulatory system, the immunological reaction shows all the characteristics of T helper 1 cell (Th-1) response. Proinflammatory cytokines such as IL-1, IL-12, interferon-g (IFN-g), and TNF-a can be measured in the plasma and infected tissue and in the peripheral-blood mononuclear cells [35, 36]. They may modulate the release of chemokines such as CXCL2, CXCL5, CXCL9, CXCL10, CXCL11, CXCL22, CCL3, CCL7, and lymphotactin (XCL1) [37]. Later the production of eggs, with their strong antigenicity, induces marked Th-2 responses and the production of a battery of other cytokines, such as IL-4, IL-6, IL-10 IL-13, IL-21, IL-17, IL-31 AMCase, Ym1, and RELMa [31, 38]. The Th-2 response first downregulates the Th-1 response as a protective mechanism. An inability to develop a Th-2 response to regulate the initial proinflammatory response is lethal [29, 39, 40]. Different cytokines have various roles which are far from fully understood and subject to strong Th-1/Th-2 cross-regulation [41]. For example, some of the cytokines can enhance fibrosis but others can promote it. IFN-g plays an important part in the development of the granulomas and has antifibrotic activity. The Th-2 cytokine IL-17 has been shown to significantly contribute to the development of a severe granulomatous reaction in Schistosoma infection [42]. Therefore, it is obvious that balancing the T helper responses is vital for protection against the Schistosoma infection. However, many factors may interfere with this balance, which may lead to a deleterious effect by reducing the granulomatous reaction or by excessive fibrotic effects. The infection is long-lived and the worms continue to produce eggs; thus, Th-2 responses are modulated and the

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granulomas that form around newly deposited eggs are smaller than those deposited earlier in the infection. The disease subsequently enters the chronic phase and the intensity of inflammation in response to newly deposited eggs gradually decreases [43]. Failure to appropriately modulate the immune responses or defective modulation of immunopathological changes associated with granuloma formation results in the increased fibrosis, portal hypertension, and spleen enlargement observed in humans with severe hepatosplenic schistosomiasis [44, 45].

5  Pathological Changes 5.1  The Granuloma The granuloma is the aggregation of mononuclear inflammatory cells, macrophages resembling epithelial cells, lymphocytes, multinucleated giant cell eosinophils, and plasma cells around the egg. These lesions prevent diffusion of the toxic egg substances and eventually destroy them. They also lead to tissue destruction and may cause fibrosis and obstruction. The cellular components participating in egg-granuloma formation differ greatly according to the tissues involved, with the liver being more sensitive than the lung in its response [46, 47]. Furthermore, granuloma formation around S. mansoni eggs is much more intense than that around S. japonicum eggs [47].

5.2  The Liver Pathology The liver changes are the most extensively studied and two forms of pathological changes can be recognized. 1 . Scattered periovular granulomas in the liver parenchyma. 2. Periovular granulomas in periportal spaces, resulting in periportal fibrosis [48], associated with vascular changes which precede development of periportal fibrosis –Symmers pipestem fibrosis [49]. 3. Many other pathological changes have been noticed, including hepatovascular endothelium proliferation, smooth muscle cell dedifferentiation, circulation proliferation of vascular connective tissue cell lines, hyperplasia of elastic tissue, the presence of several collagen isotypes in a richly vascularized connective tissue in addition to the appearance of perisinusoidal fibrosis. Distortion of the small and medium-sized capillaries and the appearance of small vessels sprouting from the largest portal veins but with preservation of arterial and ductal structures were also observed. There are losses of the well-defined limits between periportal connective tissue, and the venous vascular wall and the entire portal vein wall organization is

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destroyed, transforming an open circulation to a partly closed one, which may decrease the blood flow [50, 51].

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Conditions such as portal hypertension and collateral circulation develop and may facilitate the diversion of the egg to the lungs [59]. Eggs taped in the lungs can induce isolated granulo-

mas within the alveolar area. Andrade and Andrade [60] studied the lungs of 78 people with hepatosplenic schistosomiasis and found that 72% had periovular granulomas, of which a third had numerous periovular granulomas involving the alveolar tissue and the pulmonary arterioles, but only 9% had severe chronic obliterative arteritis. Some patients had some sort of chronic cor pulmonale and they suggested that cor pulmonale in schistosomiasis resulted from a massive infection rapidly progressing to portal hypertension with collateral circulation. Endothelial cell hyperplasia and fibrin depositions are the early changes evoked by schistosome eggs within small arteries and arterioles. These are followed by complex fibrin thrombus and revascularization, followed by congestion and dilatation of focal blood vessels, which results in plexiform lesions [61, 62]. These findings were replicated in many experimental studies in mice after cercarial exposure and partial portal vein ligation. Pulmonary periovular granulomas in the alveolar were present. When the granulomas involved arterial structures, proliferation of endothelial and smooth muscle cells occurred and fibrosis associated with angiogenesis became more evident [63]. Severe intimal, medial, and adventitial hypertrophy and proliferation of a plethora of inflammatory cells occur in the pulmonary vasculature [62, 64] (Fig. 3).

Fig. 3  Histopathological sections of pulmonary lesions with schistosomiasis. a, b From an experimental mouse model infected with S. mansoni showing a granuloma around an egg (a, arrow) or a remodeled pulmonary artery with thickened muscularized media surrounded by a granuloma (b). c, d From patients with severe pulmonary hypertension secondary to schistosomiasis, an occluded artery (c) accompanying an

alveolar duct, and diffuse inflammatory infiltrate in the wall of a muscular artery but no granulomatous reaction or parasite remnants (d). In particular, eosinophils are abundant (arrows in the insert) (hematoxylin–eosin). (a, b Courtesy of Rubin Tuder, Department of Medicine University of Colorado at Denver, USA. c, d Courtesy of Vera Demarchi Aiello and Mauro Canzian, of the University of São Paulo Medical School, Brazil)

The pathogenesis of schistosomal periportal fibrosis is, however, only seen in 5–10% of infected subjects. It seems that there are many factors contributing to the development of this severe form. Egg burden is not the only factor, and chronicity, reinfection and coinfection, nutrition, immunological status, environmental factors and probable genetic factors predisposing certain individuals need to be considered [52–56]. These pathological changes usually do not interfere with liver function until the passage of many years of heavy infection. Clinically these lesions will develop hepatosplenic schistosomiasis, which leads to portal hypertension in 21% of cases of S. mansoni esophageal varices, ascites, hepatomegaly, and splenomegaly [57, 58].

5.3  The Lung Pathology

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There was also evidence that eggs are destroyed more rapidly and more completely in the lungs than in the liver [65]. Curative treatment can promote reversion of the periovular granulomatous lesions formed in the alveolar tissue, although the changes in the arterial and arteriolar lesions were defectively repaired, with segmental vascular fibrosis, narrowing, and angiomatoid changes remaining for up to 120 days after treatment. Recent findings in both human and experimental animals suggested that extensive remodeling of the pulmonary vessels may be responsible for most of the pathological changes. Pozzan et al. [66] reported that histopathological changes of ten patients with pulmonary hypertension, attributable only to chronic Schistosoma infection, showed changes including foci of vasculitis in the vessels near the granulomatous lesions, thrombi, intimal fibrosis, and plexiform lesions. Most of these features are similar to the features reported in idiopathic pulmonary hypertension.

6  The Etiology of Pulmonary Hypertension Schistosomiasis pulmonary involvement is not rare. Schistosomal pulmonary vasculopathy is frequently seen with Symmers fibrosis [57, 67]. The cause of pulmonary vascular disease in the schistosomiasis infection is not clear. Four hypothesis were suggested in the past [68]: 1. Mechanical obstruction by the ova or worm of the pulmonary arteries leads to a focal arteritis [69]. 2. Allergic reaction [70, 71]. 3. Magalhaes Filho [72] suggested that pulmonary vascular disease is due to an intense allergic reaction caused by the first passage of the schistosomula through the lungs. 4. A possible secretion of a “vasospastic factor” as suggested by Doss in 1956 [73]. However, the exact pathogenesis is still unknown, so we will discus the potential mechanisms on the basis of our current understanding of the pathological changes in pulmonary vascular diseases that need further work and research. The main hypotheses include the role of inflammatory and immunological reactions (Fig.  4) and that portal hypertension may contribute to the pathological changes.

6.1  Immunological and Inflammatory Role T cells, B cells, mast cells, macrophages, and dendritic cells, as well as inflammatory cytokines and chemokines, are located in the lungs of patients with pulmonary hypertension, in various parts of the remodeled small pulmonary

Fig. 4  The possible mechanisms of pulmonary vasculopathy secondary to schistosomiasis. Top: The immunological modulation. Bottom: The potential role of various cytokines in the pathogenesis of pulmonary vasculopathy. The diagram is based on speculation of the potential role of the inflammatory signals to stimulate further discussion and research. IFN interferon-g, TNF tumor necrosis factor, IL interleukins, sFAS serum FAS, sICAM serum soluble intercellular adhesion molecule-1, RELMa resistin-like molecule

arteries, and in plexiform lesions. Inflammation plays an important role in the pathogenesis of pulmonary hypertension. Elevated serum levels of cytokines IL-1 and IL-6, chemokines (CX3C, fractalkine, RANTES), and various types of autoantibodies (antinuclear, antiendothelial, antifibroblast, and antifibrillin-1) have been detected in patients with severe forms of pulmonary hypertension and in particular in patients with connective tissue disease and HIV [74, 75]. They are associated with heightened smooth muscle cell proliferation and may be associated with certain genetic markers [76–80]. Many investigators have presented experimental proof that the immune response can induce pulmonary arterial remodeling, particularly the role played by the Th-2 immune response. The depletion of T-cell subsets actually worsens the disease [81–84]. Recently, it was proposed that many mechanistic regulators of pulmonary hypertension may involve a common denominator, i.e., a “generalized” activation of the nuclear factor of activated T cells, which increases the transcription of multiple inflammatory mediators and activates T and B cells [85]. These observations suggest the importance of the immunological and inflammatory role in the remodeling of the pulmonary vasculature. The intense immunological and inflammatory reaction seen in the Schistosoma eggs may therefore contribute to the pathological changes in pulmonary hypertension.

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Consequently, one of the possible mechanisms of pulmonary vasculopathy secondary to schistosomiasis may be due to the modulation of various cytokines and inflammatory mediators on the pulmonary vasculature (Fig.  4). The immune response when perturbed and prolonged leads to the later development of a serious chronic condition such as pulmonary hypertension [86, 87].

6.1.1  Th-1 Proinflammatory Mediators The increase in cytokines such as IL-1 and TNF-a may contribute to the vasoconstriction tone and remodeling of the pulmonary vessels in certain animal models. This may be due to various mechanisms, among which is the upregulation of Rho kinase [83]. IL-1b, in addition to transforming growth factor (TGF)-b1 or bradykinin, would impair the cyclic AMP generation pathway in response to prostacyclin analogues in pulmonary artery smooth muscles cells, resulting in an imbalance of the vasoactive components that may lead to pulmonary hypertension [88]. TNF-a is thought to be important in the development of pulmonary fibrosis and also in experimental models of chronic inflammation. Repeated injections of TNF-a resulted in the development of pulmonary vascular changes [89] in transgenic mice with targeted TNF-a increased expression in the lungs, where evidence of pulmonary hypertension and right ventricular hypertrophy was noticed [90, 91]. Some of these cytokines also alter the amounts of various surface components of the blood vessel wall and significantly decrease the level of functional cell surface thrombin receptor (thrombomodulin), thus promoting coagulation in the pulmonary vessels [92].

6.1.2  Th-2 Proinflammatory Mediators IL-4 is produced mainly by mast cells which stimulate activated B-cell and T-cell proliferation, inducing differentiation of naive helper T cells to Th-2, which subsequently produce additional IL-4. This cytokine plays a protective role during schistosomiasis by controlling the production of other cytokines, for example, decreasing the levels of IL-5, IL-10, and IL-13 and elevating the levels of IL-2 and IFN-g. In anti-IL-4-treated infected mice there was not a not consistent reduction in the size of hepatic granulomas around S. mansoni eggs, but there was marked inhibition of granuloma formation in the lungs of the same animals [93]. The role of IL-4 in the pathogenesis of pulmonary vascular disease is not clear; it potentiates pathogenic autoreactive B cells which are present in the plexiform lesions in some models and may produce antiendothelial antibodies [94, 95].

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Angeli et  al. [96] noticed that S. mansoni selectively induces the synthesis of IL-6 in pulmonary microvascular endothelial cells and may reduce the inflammatory reaction that develops in the lungs of infected hosts. IL-6 also increases the number of platelets, and the number of platelets correlated with the extent of pulmonary hypertension [97]. It may enhance muscularization of the proximal and distal arterial, occlusive neointimal angioproliferative lesions that worsened with hypoxia, and may enhance many of the antiapoptotic signals. Thus, IL-6 promotes the development and progression of pulmonary vascular remodeling through pro-proliferative antiapoptotic mechanisms [98]. IL-10 is pleiotropic anti-inflammatory cytokine that can inhibit the expression of other Th-1 cytokines (such as IL-1 and TNF-a) by suppressing their excessive response. It is produced by a variety of cells with a vasculoprotective property. It significantly reduced mean pulmonary artery pressure and reduced the remodeling effect of monocrotaline-induced pulmonary hypertension probably by an increase in the lung level of heme oxygenase-1 [99]. IL-13 is a pleiotropic cytokine produced in large quantities by activated CD4+ Th-2 lymphocytes. It is known to induce airway hyperresponsiveness and endothelial vascular cell adhesion molecule-1 expression, thus playing a role in asthma, acute lung injury, and fibrosis [100]. IL-13dependent fibrosis develops either directly or through mechanisms dependent on TGF-b and probably matrix metalloproteinase-9, both playing important roles in the pulmonary vascular remodeling process. Daley et  al. [81] showed that IL-13 facilitates vascular remodeling by regulating Th-2 responses directly or indirectly. Others reported that IL-13 is a potent antiproliferative factor in pulmonary artery smooth muscle cells, probably via upregulation of the decoy receptor IL-13RA2 on the pulmonary artery smooth muscle cells [101]. IL-21 is produced mostly by activated CD4+ T cells and controls the differentiation and functional activity of the T helper cells. IL-21 has pleiotropic effects on the proliferation, differentiation, and effector functions of B, T, natural killer, and dendritic cells. IL-17 is a CD4+ T-cell-derived cytokine that promotes inflammatory responses in cell lines and its level is elevated in rheumatoid arthritis, asthma, multiple sclerosis, psoriasis, and transplant rejection. RELMa (resistin-like molecule, also referred to as FIZZ1) is a mediator which is highly upregulated by Th-2and IL-13-mediated inflammation in the lungs. Daley et al. [81] found that significantly increased numbers of RELMa positive cells bordered the remodeled pulmonary arteries in immunized and antigen-challenged mice compared with unimmunized controls. Hypoxia-induced mitogenic factor is another Th-2 cytokine contributing to the development of pulmonary

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hypertension secondary to hypoxia by enhancing pulmonary inflammation remodeling and angiogenic and vasoconstrictive properties [102–104]. It also promotes cell proliferation and migration, the production of vascular endothelial growth factor in pulmonary endothelial cells, and the production of reactive oxygen species, which are important components in the remodeling process [104, 105]. Other inflammatory markers such as serum soluble intercellular adhesion molecule-1 (sICAM) play a role in the adhesion of monocytes, lymphocytes, and neutrophils to endothelial cells. Their levels increase in patients with schistosomiasis compared with control subjects [106]. Thus, they may contribute to the endothelial dysregulation of cell–cell adhesion and transendothelial migration, which contribute to the pulmonary vasculopathy. Furthermore, Schistosoma eggs and Th-2 cytokines can induce arginase activity, which is suppressed by interferon. This disequilibrium favors the development of the formation of granulomas and may lead to further damage to the pulmonary vessels [107]. FAS, a membrane-anchored protein related to the TNF receptor family which induces apoptosis, can be antagonized by soluble Fas and thus inhibits apoptosis. In patients with schistosomal pulmonary hypertension, serum levels of soluble Fas are significantly higher compared with those in patients with cor pulmonale resulting from chronic obstructive pulmonary disease [108]. This factor may contribute to the remodeling process of schistosomal pulmonary vascular disease. Figure  4 summarizes the hypothetical scenario whereby various Th-1 and Th-2 contribute to the pathological process of pulmonary vasculopathy.

6.2  The Role of Portohypertension Pulmonary hypertension has been seen in 2–12% of patients with portal hypertension in general [109, 110], but has been reported in 20.6% of the cases with portal hypertension due to hepatosplenic mansonic infection [111]. Aggravation of pulmonary hypertension has also been described after the surgical treatment of schistosomiasis-associated portal hypertension with a shunt procedure [58, 111]. It is thought that the development of portal-systemic collateral circulation leads to a hyperdynamic circulatory state, contributing to an increased flow and sheer stress to the pulmonary circulation. It also facilitates the shunting of eggs from the liver to the lungs, thus increasing the immunological burden on the lungs and pulmonary circulation [67]. This will increase the antigen burden on the lung and thus aggravates pulmonary hypertension [53].

Ghazwan Butrous and Angela P. Bandiera

6.3  The Role of Coinfection Coinfection is common in many regions of the world where both schistosomiasis and HIV/AIDS are endemic. This results in patients harboring coinfections. Both have played a role in the development of pulmonary vascular diseases mediated via their inflammatory responses. The interaction creates complex inflammatory responses that may alter the natural history of the pulmonary vascular sequelae caused by infectious agents alone. This is observed to be of particular interest when immune responses may alter after treatment of either infection or after chemotherapy [112–115].

7  T  he Prevalence of Pulmonary Hypertension Secondary to Schistosomiasis The real prevalence of pulmonary vascular diseases caused by schistosomal infection is not known. Belleli from Alexandria, Egypt [116], in 1885 reported the presence of Schistosoma eggs in the lungs of African natives. After that, there were several reports from Egypt in the first half of the twentieth century [117–124] describing pathological and clinical manifestations of pulmonary vascular diseases secondary to infection by both S. mansoni and S. haematobium. The clinical symptoms and physical signs, the radiological appearances of the lungs and cardiovascular system, and the course and progress have been pieced together and the name “Egyptian Ayerza’s disease” [69, 125] was given for the advanced cases; Abel Ayerza (1861–1918), an Argentinian physician, was one of the first physicians who described the pathological changes in pulmonary vascular disease in around 1901. In the second half of the twentieth century, reports of many small series appeared in the literature, this time mainly from Brazil and occasionally a few cases from Africa [53, 60, 61, 68, 126– 134] and more recently from China [135]. The majority of reported cases are due to S. mansoni, with a few cases due to S. haematobium [64] or S. japonicum [135–137]. Most of the reported clinical prevalence of pulmonary hypertension is from small series, which were uncontrolled, used different methods of data collection or an inappropriate epidemiological method, and were mostly confined to specific groups or patients from a certain area. In these studies the prevalence ranged from 7.7 to 33% (Table 1). Preliminary data from specialized reference pulmonary hypertension centers in Brazil suggest that schistosomiasis is the cause of severe pulmonary hypertension in around 30–47% of referred patients [138] compared with other Western centers [139, 140] (Fig. 5).

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Table 1  Studies suggesting the prevalence of pulmonary hypertension (PH) secondary to schistosomiasis Number of patients studied

Prevalence of PH (%)

282 patients died owing to schistosomiasis 33 37 patients with confirmed schistosomiasis 21.6

Country

Notes

141 patients with hepatosplenic schistosomiasis 246 patients with suspected or confirmed Schistosoma mansoni infection Schistosomal portal hypertension 65 patients with hepatosplenic schistosomiasis

13

Egypt Autopsy study showed pulmonary involvement Côte d’Ivoire Catheterization of the right side of the heart and pulmonary artery Brazil Mean pulmonary pressure 20–40 mmHg

16

Brazil

11.7 7.7

Brazil Brazil

84 patients with schistosomal liver fibrosis

10.7

Brazil

Fig. 5  The distribution of pulmonary hypertension causes in a referral center in an endemic area in Brazil (Oswaldo Cruz University Hospital, Memorial São José Hospital, Real Português Hospital Recife-PE-Brazil). IPAH idiopathic pulmonary arterial hypertension, CTD connective tissue disease, Cong HD secondary to congenital heart diseases, Schis secondary to schistsomiasis. The hatched bar represents the percentage from the French registry published in 2006 in Humbert et al. [139]

Recently, more carefully controlled methodological studies were conducted in patients with hepatosplenic schistosomiasis and liver fibrosis. Ferreira et  al. [141] studied 84 patients from the northeast region of Brazil with schistosomal periportal fibrosis using transthoracic Doppler echocardiography. They found that 14 patients had pulmonary artery systolic pressure above 35  mmHg. However, five patients were excluded (four due to diastolic dysfunction of the left ventricle and one due to interatrial communication). Thus, nine patients (10.7%) were diagnosed with pulmonary hypertension. Sixty-two percent were classified to have functional classes III and IV. There was no  occurrence of pulmonary venous thromboembolism. Hepatosplenomegaly secondary to schistosomiasis was

Using Doppler echocardiography, pulmonary pressure defined as systolic pressure >20 mmHg) A field in the state of Minas Gerais, Brazil Systolic pulmonary artery pressure >40 mmHg on echocardiography; the results were confirmed with invasive haemodynamics measurements Systolic pulmonary artery pressure ranging from 40 to 126 mmHg with Doppler echocardiography

References [69] [151]

[150, 152]

[134]

[153] [142]

[141]

present in just five of the nine patients. These patients underwent contrast-enhanced multidetector-row computed tomography and an increased diameter of the pulmonary artery trunk, tapering of peripheral pulmonary arteries, and cardiac enlargement were observed (Figs. 6, 7). Lapa et al. [142] studied 65 patients followed up in the gastroenterology department for hepatosplenic manifestation of S. mansoni with transthoracic Doppler echocardiography. They found 12 patients with pulmonary artery systolic pressure above 40 mmHg. Thirteen of these patients underwent right-sided heart catheterization, which confirmed significant pulmonary hypertension in five patients (7.7%) (three had been classified as having precapillary pulmonary hypertension and two had postcapillary pulmonary hypertension). On the other hand, in a prospective study of sixty-five patients hospitalized with S. japonicum infection with hepatosplenic disease, only one patient had pulmonary hypertension [136]. The relatively uncommon appearance of severe S. japonicum infections may reflect the difference in the antigenicity between species. It is therefore difficult to estimate the real prevalence of pulmonary vascular diseases, particularly when we have to consider many factors, such as the range of clinical presentation, pathological involvement, difference in the antigenicity, and progress of the pathological changes, as well as the role of coinfection, current treatment, and control and multiple exposures. All these factors can contribute to the real prevalence. It is suggested from our unpublished current experimental observation that the changes in the pulmonary vasculature after Schistosoma infection are far more common but may not always be associated with significant increases in the total vascular resistance and may appear as benign pulmonary hypertension and clinical pulmonary hypertension. This, however, needs further investigation.

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8  The Clinical Presentations The early symptoms of acute schistosomiasis are usually nonspecific symptoms such as nocturnal fever, cough, dyspnea, myalgia, headache, and abdominal tenderness with eosinophilia, and lymphadenopathy. In some patients, chest radiography usually shows patchy or diffuse pulmonary infiltrates. These signs and symptoms appear 3–6 weeks after infection and they are referred to as Katayama syndrome [35, 143]. This most commonly affects older people who are heavily exposed to schistosomiasis for the first time. The condition is usually thought to be due to proinflammatory cytokines which may be related to Th-1 responses [35]. Diagnosis requires a high index of suspicion and, therefore, cannot be made by screening urine or feces for eggs. Serological testing may be required [144]. Chronic pulmonary disease is more common in endemic areas but there are no typical clinical distinguishing features and it may manifest itself as asthma, dyspnea on exertion, anemia, fatigue, weakness, cough, giddiness and fainting, palpitation, thoracic pain, pericardial pain, and hemoptysis. The electrocardiogram may show right ventricular hypertrophy and strain. Finally, the main branches of the pulmonary artery in some patients may become dilated and may develop large aneurysms (Figs. 6, 7). Eventually the right side of the heart hypertrophies and fails (cor pulmonale) [25, 145–148]. Chest radiography may show an enlarged pulmonary arterial trunk, right ventricular hypertrophy, and evidence of pleural effusion (Fig. 8). These clinical and radiological findings are

Fig. 7  The tomographic scan of a 51-year-old female with a pulmonary artery systolic pressure of 71 mmHg showing enlargement of the main pulmonary arteries

Fig. 8  Chest radiograph in a patient with World Health Organization class IV pulmonary hypertension secondary to schistosomiasis from Brazil showing severe right ventricular dilatation and enlargement of the pulmonary trunk with evidence of heart failure

Fig.  6  A 58-year-old woman who had hepatosplenic schistosomal disease and pulmonary artery systolic pressure of 68 mmHg. A multidetector-row tomographic scan of the thorax showing dilated pulmonary arteries and showing abrupt reduction in the diameter of the peripheral pulmonary vasculature

similar to those associated with other causes of pulmonary hypertension [149]. In most reported studies, the majority of the published data indicate that the majority of patients with mild pulmonary hypertension do not develop severe right-sided heart failure [53, 150]. One series from two endemic area in Brazil showed that in 141 patients with hepatosplenic schistosomiasis, pulmonary hypertension occurred in 13% and cor pulmonale in 2.1% [53]. In our experience, particularly in the patients

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referred to specialized pulmonary hypertension centers in endemic areas, 62% of these patients are in New York Heart Association functional class III or IV with an average right ventricular systolic pressure of 83.48 ± 24.61 mmHg.

9  M  anagement of Pulmonary Hypertension Secondary to Schistosomiasis There has been no controlled clinical trial on the use of pulmonary-hypertension-specific drugs in patients with schistosomiasis-associated pulmonary hypertension. In our experience a phosphodiesterase-5 inhibitor (sildenafil) was used in an open-label study in 85 patients (unpublished data), resulting in improvement in exercise capacity as assessed with the 6-min walk test. There were a few cases with successful treatment with the endothelin receptor antagonist bosentan. Affordable safe and efficacious medications are needed to help this group of patients, and thus double-blind clinical trials are needed. Antischistosomal therapy usually has no effect, but it is given to prevent further progression of disease. However, Bouree et al. reported a remission after treatment with praziquantel in patients with S. haematobium pulmonary hypertension [64]. Pulmonary embolisms of dead worms may follow treatment for schistosomiasis, with an abrupt increase in pulmonary pressure and development of acute cor pulmonale [53].

10  The Issues and Problems We can only speculate on the number of patients affected by schistosomiasis-associated pulmonary vascular disease as the real prevalence is not yet known because more than 200 million people are infected worldwide. We can nevertheless consider that schistosomiasis is the major cause of pulmonary hypertension worldwide, and it may even be the leading cause of pulmonary hypertension in endemic areas. A concerted effort is therefore needed in various parts of the word owing to the diversity of the parasite itself and the many factors that are associated with its pathophysiological effects as described in this chapter. The apparent role of inflammation, as discussed in this chapter, can be useful as a good model to assess and understand more thoroughly the role of inflammation; not only in schistosomiasis-associated pulmonary vascular disease but also in general. The clinical profile, pattern, and natural history of the development of pulmonary hypertension need further evaluation. Recent advances in the treatment of pulmonary hypertension have not been implemented in the management of patients with pulmonary hypertension secondary to schistosomiasis. Therefore, the need to develop an inexpensive and affordable treatment warrants further attention.

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Chapter 92

Pulmonary Hypertension Due to Capillary Hemangiomatosis Mourad Toporsian, David H. Roberts, and S. Ananth Karumanchi

Abstract  The pulmonary circulation is a high-compliance, low-resistance vascular bed with enormous volume reserve capacity. These characteristics allow it to accommodate the entire cardiac output while being subjected to a low mean pulmonary artery pressure (mPAP) of 25 mmHg. Elevations in mPAP beyond 25 mmHg at rest or 30 mmHg during exercise are signs of pulmonary hypertension (PH), a devastating disorder that can affect individuals of all ages worldwide. Given the diverse pathophysiological manifestations, causes, and clinical management strategies of PH, it has been divided into five categories on the basis of the modified World Health Organization (WHO) Evian classification. Pulmonary arterial hypertension (PAH), or WHO group 1, consists of all forms of PH which are of unknown cause and involve extensive arteriopathy or loss/remodeling of the pulmonary arterial vasculature. This chapter will focus on an extremely rare form of PAH referred to as pulmonary capillary hemangiomatosis (PCH). Unlike other forms of PAH where changes in the pulmonary vasculature are limited solely to the arteries, PCH is a very intriguing disease of uncontrolled endothelial cell growth that although specific to the lung involves proliferation of capillary-sized blood vessels. Keywords  Pulmonary arteriopathy • Rare disease • Endo­ thelial cell growth • Precapillary arterioles • Capillary-sized blood vessel

1 Introduction The pulmonary circulation is a high-compliance, lowresistance vascular bed with enormous volume reserve capacity. These characteristics allow it to accommodate the entire cardiac output while being subjected to a low mean pulmonary artery pressure (mPAP) of 25 mmHg. Elevations M. Toporsian (*) Beth Israel Deaconess Medical Center, 330 Brookline Avenue, 02215 Boston, MA, USA e-mail: [email protected]

in mPAP beyond 25  mmHg at rest or 30  mmHg during exercise are signs of pulmonary hypertension (PH), a devastating disorder that can affect individuals of all ages worldwide. Given the diverse pathophysiological manifestations, causes, and clinical management strategies of PH, it has been divided into five categories on the basis of the modified World Health Organization (WHO) Evian classification (reviewed in detail in Chap. 65). Pulmonary arterial hypertension (PAH), or WHO group 1, consists of all forms of PH which are of unknown cause and involve extensive arteriopathy or loss/remodeling of the pulmonary arterial vasculature. This chapter will focus on an extremely rare form of PAH referred to as pulmonary capillary hemangiomatosis (PCH). Unlike other forms of PAH where changes in the pulmonary vasculature are limited solely to the arteries, PCH is a very intriguing disease of uncontrolled endothelial cell growth that although specific to the lung involves proliferation of capillary-sized blood vessels.

2  Historical Perspective Three decades ago, PCH was first described by Wagenvoort et al. in a 71-year-old woman with progressive dyspnea, hemoptysis, and hemorrhagic pleural effusions [1]. They described this new clinical entity as an angiomatous growth with ­distinctive atypical proliferation of capillary-like channels in the lung tissue. PCH is now characterized as a diffuse proliferation of capillaries that can form glomeruloid nodules and project into pulmonary veins, arteries, interstitium, and, albeit less commonly, the airways [2, 3]. Similar to what occurs in other types of PAH, intimal thickening and medial hypertrophy of small pulmonary arteries are present, contributing to elevated pulmonary vascular resistance and mPAP. Since its initial description, PCH has been sporadically described in the literature, with fewer than 50 cases reported to date. Given the lack of awareness and difficulty in diagnosis, most cases have been discovered after death, suggesting that the prevalence of PCH may be underestimated and the mechanisms underlying its pathogenesis understudied.

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_92, © Springer Science+Business Media, LLC 2011

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3  Clinical Features and Pathology PCH typically affects young adults (age range 6–71 years), has a very poor prognosis, and often leads to death. Patients usually present with dyspnea, cough, fatigue, weight loss, and abnormal pulmonary function tests, and have a median survival of 3 years from initial clinical discovery. Although the cause of this disease remains unknown, a hereditary form was reported by Langleben et  al. [4], suggesting a genetic susceptibility. However, PCH has not been recognized as a disease of congenital onset, and it is unclear if this hereditary form of the disease is the same as the one seen in adults [5]. PCH often manifests itself as PH and hemoptysis leading to right ventricular hypertrophy and failure [6, 7]. To date, only one case without PH has been reported [8]. In fact, PCH is classified as PAH associated with significant venous or capillary involvement [9]. Consistent with the prevalence of PAH being more common in females than in males, oral contraceptive use has been proposed as an important risk factor for this condition [10]. It is believed that the mechanical obstruction of blood flow in small venules and veins by invading capillaries leads to increased upstream pulmonary artery pressure and contributes to the observed PH. Patients exhibit normal or low pulmonary capillary wedge pressures (PCWP) [6], as this parameter assesses the pressure in the largest pulmonary veins and the left atrium [11], structures that are not affected in PCH. This criterion is helpful in the clinical differential diagnosis as a normal PCWP eliminates the possibility of other abnormalities such as obstructed or stenotic larger pulmonary veins, left atrial myxoma, mitral stenosis, or left ventricular failure. PCH has been described as a slowly progressive neoplasm or angioproliferative defect of the lung. Transbronchial biopsy of the lung may often reveal nonspecific hemosiderosis. Lung biopsies using video-assisted thoracic surgery are needed to make the diagnosis. The most distinctive histologic feature of PCH is proliferation of capillary channels

Fig. 1  (a) Lung biopsy specimen (hematoxylin and eosin stain) from a 44-year-old patient with pulmonary capillary hemangiomatosis (PCH) showing capillary proliferation within the alveolar septae and interstitium. (b) CD31 staining of the lung tissue obtained (from the

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within the alveolar walls. Immunohistochemical studies showing CD31 (platelet/endothelial cell adhesion molecule 1) and factor VIII related antigen staining of PCH/PM = pulmonary microvasculopathy lungs have confirmed that endothelial cells are the cells of origin that invade the pulmonary vasculature, interstitium, and airways (Fig. 1a, b). Highresolution CT scans typically show a diffuse ground-glass pattern and septal thickening (Fig. 1c). The proliferation of at least two layers of thin-walled alveolar capillary blood vessels with infiltration of these structures is diagnostic [2]. Invasion of the alveolar septa and bronchioles by proliferating endothelial cells disrupts gas exchange by reducing the surface area and the diffusion capacity of the lung. Muscularization and concomitant narrowing of pulmonary arteries coupled with the obstruction of veins compromise arterial supply and venous drainage, respectively. These changes result in PH and are accompanied by recurrent pulmonary hemorrhage, scar formation, and thrombosis leading to vascular obliteration. Moreover, there is compensatory redistribution of ventilation/perfusion from abnormal to less affected areas of the lung. Restrictive remodeling as a consequence of endothelial cell invasion of the pulmonary vasculature and interstitium stiffens the lung, compromising inspiratory capacity and increasing lung collapse during expiration. Although PCH is a non-hypoxia-related form of PH, chronic hypoxia and metabolic acidosis, and their associated sequelae, are inevitably a consequence.

3.1  Pulmonary Veno-occlusive Disease The other form of PAH that is associated with significant venous involvement is pulmonary veno-occlusive disease (PVOD). The hemodynamics of PVOD and PCH are the consequence of a widespread vascular obstructive process. However, unlike in PCH, this obstruction originates in the

same subject described in a) confirms capillary proliferation. (c)  Representative high-resolution CT scan image (from the subject with PCH described in a) showing ground-glass opacities and thickening of interlobular septa

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pulmonary venules and small veins in PVOD. The consequential increase in capillary hydrostatic pressure may exceed the osmotic pressure of blood, leading to pulmonary edema as well as engorgement and dilatation of the subpleural and interlobular septal lymphatic channels. Histologically, PVOD is characterized by intimal fibrosis that occludes the pulmonary veins. These venous lesions are accompanied by looplike dilatations of the capillary bed, leading to edema and dilated lymphatic spaces in the interlobular septa [12]. Secondary remodeling of the lung parenchyma and of the pulmonary arteries are caused by increased intravascular pressure due to obstructed venous flow. In contrast, these secondary changes are due to well-circumscribed proliferative capillary lesions in PCH. PVOD and PCH have with very similar signs and symptoms, but the presence of radiologic septal lines primarily in venous structures is indicative of PVOD [13].

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5.2  Obesity A link between obesity and PCH has been previously proposed, yet a direct causal relationship remains controversial. Recently, an autopsy study of 76 patients revealed histologic features of PH, particularly venous hypertension, and PCH in 72% of obese subjects. PCH was observed in 50% of morbidly obese subjects [19]. The authors posit that the pulmonary capillary proliferation in the obese is most likely related to chronic hypoxia and left ventricular dysfunction. Interestingly, however, obesity is also associated with increased levels of inflammatory cytokines and immune activation, suggesting that persistent and chronic inflammation may indeed play a role in PCH.

6  Management 4  Differential Diagnosis Although PCH specifically involves the invasion of the pulmonary vasculature and parenchyma by proliferating capillaries, this disorder is usually misdiagnosed as the more common microvasculopathies such as PVOD (see Chap. 80) or idiopathic PAH. PCH is also to be differentiated from cavernous hemangiomatosis or pulmonary angiomatosis, which typically involve vessels of larger caliber [14]. Other disorders considered as part of the differential diagnosis include usual interstitial pneumonitis, recurrent pulmonary thromboembolism, and idiopathic pulmonary hemosiderosis [15]. Definitive diagnosis of PCH usually requires histological examination of the lung tissue (often at autopsy), and unsuspected incidental findings of PCH have been found after death in patients without clinical disease.

5  Risk Factors 5.1 Infection and Immunity An immune-mediated cause has been suggested owing to the sporadic occurrence of PCH. Moreover, although most cases are idiopathic, PCH has also been described in patients with autoimmune diseases such as systemic lupus erythematosus [16], scleroderma, Takayasu arteritis, and Kartagener syndrome [15, 17]. Recently, a case report showing the occurrence of de  novo PCH in a 45-year-old woman 3 months after bilateral lung transplantation suggests that persistent infection or inflammation may be an inciting factor in the dysregulated angiogenesis [18].

Given the paucity of clinical cases, difficult diagnosis, and unknown cause of PCH, treatments are on an experimental basis and available as case reports. By far, the best outcomes are currently afforded by pneumonectomy [6], heart-lung transplantation [20], or bilateral lung transplantation [21], which may be curative. To this end, pharmacological interventions are considered supportive until surgery. In some cases, immunosuppression with a-interferon [17, 22] has shown some promise whereas, in others, antiangiogenic therapy such as doxycycline has been attempted [23]. Although corticosteroids have been used in this disorder, they have not been found to be of significant benefit. Interestingly, although it is widely accepted that vasodilator therapy with prostacyclin or calcium channel blockers can improve pulmonary hypertensive arteriopathy, selective pulmonary vasodilators such as these medications can cause life-threatening pulmonary edema in patients with pulmonary postcapillary vasculopathies such as PCH and PVOD, and are therefore contraindicated [17, 24, 25]. There are no data available regarding a potential use of several recently approved antiangiogenic agents, such as bevacizumab [anti-vascular endothelial growth factor (VEGF) antibody] and rapamycin (mammalian target of rapamycin inhibitor), in PCH therapy.

7  Mechanisms of Disease 7.1  Hypoxia Adaptation to hypoxia by capillary proliferation has been proposed as a possible mechanism in the development of PCH [3]. This concept is supported by our understanding of

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the mechanisms involved in other diseases characterized by the proliferation of normal capillaries, including childhood hemangiomas, angiofibromas and psoriasis. Potent hypoxiainducible factors, such as VEGF, basic fibroblast growth factor (bFGF), and platelet-derived growth factor (PDGF), have been shown to play prominent roles in the onset and progression of these diseases [26]. Although these factors have been implicated in the pathogenesis of PCH to differing degrees, it remains to be clarified if concomitant aberrant changes in endothelial oxygen sensing and responses contribute to the onset of PCH. Other proposed mechanisms for the pathogenesis of PCH speculate that the lesion is a hamartoma [27] or a low-grade vascular neoplasm [28]. However, the typical histological findings of diffuse vascular proliferation throughout the lung do not lend support to these hypotheses.

7.2  A  ngiogenic Factors (VEGF, bFGF, and PDGF) VEGF is an endothelial-cell-specific mitogen. It promotes angiogenesis via its interactions with two high-affinity receptor tyrosine kinases, VEGF receptors 1 (fms-like tyrosine kinase 1, Flt-1) and 2 (KDR in humans, Flk-1 in mice) [29]. Recent studies have demonstrated alternatively spliced variants of Flt-1, resulting in the synthesis and secretion of an endogenous soluble form, termed sFlt-1, that can bind VEGF and inhibit its angiogenic properties. sFlt-1 can also bind placental growth factor, another member of the VEGF family [30]. Angiogenesis is necessary for normal alveolarization during postnatal lung growth and development [31]. Treatment of newborn rats with a single dose of the VEGF receptor antagonist SU5416 has been shown to reduce lung vascular density, alveolarization, weight, and cause PAH [32]. These effects persist well into adulthood and suggest that the inhibition of lung vascular development during a critical period of postnatal lung growth impairs alveolarization. These findings suggest an intimate cross talk between pulmonary epithelial and vascular endothelial cells, which is at least partly mediated by VEGF. The juxtaposition of the capillary endothelium with alveolar epithelial cells suggests the possibility that PCH may be an epithelial defect related to excessive VEGF secretion. This would be consistent with the lung-specific manifestations of PCH. bFGF belongs to a family of heparin-binding polypeptides and shows multiple functions, including cell proliferation, differentiation, survival, and motility. In particular, it is one of the most potent angiogenic factors, providing a basis for its potential role in PCH. In a special case report by Ginns et al. [23], an elevated urinary bFGF level was observed in a 20-year-old man with PCH. This patient also displayed atypical endotheliomatosis, suggesting the potential efficacy of a matrix metalloprotease (MMP) inhibitor such as doxycycline.

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Interestingly, the use of doxycycline led to a complete remission and normalization of abnormally high bFGF levels. Whether the elevated bFGF levels were specific to the observed atypical endotheliomatosis remains unknown. PDGF is a heterodimeric molecule that is composed of two similar polypeptides (A and B). It plays a central role in angiogenesis and acts as a potent mitogen for pulmonary artery smooth muscle cells via its tyrosine kinase receptor (PDGFR). PDGF and its downstream signaling has been shown to be upregulated in hypoxia- and monocrotalineinduced PH, as well as in lung homogenates of patients with PAH [33–35]. The PDGFR-b antagonist imatinib mesylate, currently used to treat chronic myelogenous leukemia, reverses PAH in these animal models of disease and has also recently shown promise as a new treatment in humans with PAH. Recent tissue microarray analyses have demonstrated increased levels of PDGF-B and PDGFR-b in lungs of patients with PCH [12]. This suggests a common link between the vasculopathy seen in PCH patients and that seen in patients with other forms of PAH. PDGF also plays an important role in the recruitment of pericytes into newly formed blood vessels, regulatory cells involved in the promotion of angiogenesis. The recent finding of prominent pericytic components in the nodules of PCH patients suggests a potential causal role for PDGF-induced excessive angiogenesis in PCH as well as its potential value as a cellular target to induce the regression of capillary ingrowths.

7.3  Matrix Proteases The extracellular matrix (ECM) is a reservoir of growth factors. These factors are released by matrix protease-mediated ECM degradation, which, in turn, promotes cellular invasiveness through the ability of these factors to enhance the expression and activity of matrix-degrading MMP-2 and MMP-9 [36]. Both these MMPs play a pivotal role in endothelial cell invasion and angiogenesis. As mentioned previously, the MMP inhibitor doxycycline has been used successfully in one case of PCH, suggesting that a better understanding of specific MMPs involved in this disease may pave the way to effective therapy [23]. Recent findings in lungs of patients with PCH suggest more biochemical similarities between this disease and other forms of PH. For instance, increased numbers of mast cells, as measured by increased levels of CD117 staining [12], have been documented in lungs of PCH patients. Tryptase is a proangiogenic component of mast cell granules that can induce endothelial cells to form tubelike structures, degrade the ECM, and permits capillary invasion into the pulmonary interstitium. Expression of this factor is increased in tumors and it is involved in tumor progression via its effects on angiogenesis. Its potential role in PCH remains to be elucidated.

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7.4  Endothelial Nitric Oxide Synthase Reduced nitric oxide (NO) bioavailability in the pulmonary vasculature is generally observed in animal models of PAH and in patients with this disease. It is believed that reduced endothelial NO synthase (eNOS) expression and/or its abnormal activation are contributing factors. A report by Kradin et al. [37] demonstrated that patients with PCH have reduced eNOS levels in abnormal capillary vessels. This observation was exclusive to PCH patients displaying pulmonary arterial hypertensive arteriopathy, suggesting that decreased eNOS expression may be a causal factor. Although NO is a very potent vasodilator and likely involved in the regulation of pulmonary vascular tone, its specific role in the prevention of PAH remains to be elucidated. eNOS null mice do not spontaneously develop PH, although their exposure to hypoxia results in an accelerated and severe form of the disease [38]. It is likely that additional factors acting in concert are necessary in PAH. Increased oxidative stress has been shown in numerous animal models of PH and in humans with this disease. Interestingly, the failure of eNOS to couple oxygen to l-arginine metabolism results in an increased amount of eNOS-derived O2•−, and reduced NO release [39]. The enzyme is thus rendered uncoupled and this condition has quickly emerged as an important mechanism in numerous vasculopathies, including PAH [40–43]. Further studies are needed to evaluate a role for eNOS uncoupling in the pathogenesis of PH patients due to a variety of causes, including PCH [44].

7.5  Osler–Rendu–Weber Syndrome Osler–Rendu–Weber syndrome, or hereditary hemorrhagic telangiectasia (HHT), is an autosomal dominant vascular dysplasia characterized by dilated blood vessels, telangiectases, and arteriovenous malformations [45]. Haploinsufficiency in endoglin (ENG) and activin-like kinase 1 receptor (ACVLR1) genes is the underlying cause of HHT type 1 [46] and type 2 [47], respectively. Recently, mutations in the ACVRL1 gene have been reported in patients presenting with PAH [48–50]. The ACVLR1 gene encodes the activin-like kinase 1 (ALK1) receptor, a type I receptor of the transforming growth factor b (TGF-b) superfamily that is mainly expressed in the vascular endothelium and particularly abundant in pulmonary blood vessels. Functionally, ALK1 acts via Smad1/Smad5/Smad8 [51] and regulates endothelial cell proliferation and migration in vitro [52], and angiogenesis in vivo [53, 54], in response to TGF-b1. More recently, bone morphogenetic protein (BMP) 9 and 10 have been identified as high-affinity ligands for ALK1 which in conjunction with BMP receptor 2 (BMPR2) can regulate BMP signaling in endothelial cells [55]. These findings underscore

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the importance of ALK1 in BMP-mediated endothelial cell function and further accentuate its potential involvement in PAH associated with abnormal BMPR2 signaling. However, it is still not well understood how loss of BMPR2 signaling leads to vascular remodeling exclusively in the lung. BMPR2 mutation has also been described for a patient suffering from PVOD, suggesting a close pathogenetic link between idiopathic PAH and PVOD [56]. A recent case report showed PCH arising in a patient with HHT [57]. Immunohistochemical staining for eNOS was also lower in the affected capillary endothelial cells of PCH compared with normal capillary endothelium. We have demonstrated that human and mouse endoglin-deficient (HHT mouse) endothelial cells and tissues have reduced eNOS levels [58]. Endoglin is an endothelial cell-surface glycoprotein and coreceptor for various ligands of the TGF-b superfamily. It resides in caveolae, where it associates with eNOS and promotes its normal activation. Endoglin-deficient endothelial cells display uncoupled eNOS activity [58]. Endoglin is known to regulate angiogenesis [59–61], local vascular tone, and TGF-b and BMP receptor signaling via ALK1, suggesting a critical role for endoglin in endothelial cell function [55] that is potentially impaired not only in HHT but also in PAH and PCH.

8  Conclusion As in other forms of hemangiomatosis, dysregulated angiogenesis significantly contributes to PCH pathogenesis. Aberrant adaptation to hypoxia and chronic infection/inflammation leading to overexpression of angiogenic factors such as VEGF, TGF-b, and bFGF signaling are likely key mechanisms in PCH pathogenesis. A common signaling defect may be an underlying cause of PAH and HHT, providing valuable clues to our understanding of PCH, a rare but devastating disorder. Future work on identifying new genes responsible for this disorder in the hereditary form may shed clues to the pathogenesis of this disease. It is imperative to create new animal models that mimic the human condition to develop new treatments for this devastating disorder.

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Chapter 93

Molecular Basis of Right Ventricular Hypertrophy and Failure in Pulmonary Vascular Disease Yuichiro J. Suzuki

Abstract  Right ventricular hypertrophy and right heart failure occur in patients with pulmonary hypertension. The mechanisms of left ventricular hypertrophy are well understood, however, information on right ventricular hypertrophy is limited. Studies of embryonic and adult hearts provided evidence that the molecular signaling mechanisms are different for right and left ventricles. Thus, the development of therapeutic strategies specific for the right heart is needed. This chapter summarizes current understanding of cell growth signaling mechanisms in the right ventricle. Keywords  Pulmonary hypertension • Right heart failure • Right ventricle • Right ventricular hypertrophy • Signal transduction

1 Introduction to Right Ventricular Hypertrophy and Failure in Pulmonary Vascular Disease In response to pulmonary vascular diseases in which the pulmonary vascular resistance increases, the right ventricle is subjected to increased load or volume, which ultimately has lethal consequences. The term “cor pulmonale,” which was originally introduced by Paul D. White in 1931 [1], has been used to describe these conditions. “Cor pulmonale” is now defined as an alteration in the structure and function of the right ventricle, resulting from diseases affecting the function and/or structure of the lung, except when these pulmonary alterations are the result of diseases that primarily affect the left side of the heart [2]. Acute cor pulmonale, which can occur in response to pulmonary embolism, results in dilation of the right ventricle, whereas chronic cor pulmonale, which is associated with chronic obstructive pulmonary disease and

Y.J. Suzuki (*) Department of Pharmacology, Georgetown University Medical Center, 3900 Reservoir Road NW, 20057 Washington, DC,USA e-mail: [email protected]

idiopathic pulmonary arterial hypertension, exhibits right ventricular hypertrophy. As right ventricular failure is the major cause of death among patients with pulmonary hypertension, understanding the molecular basis of how right ventricular failure develops is highly important. Knowledge of the right ventricle, however, is limited. Many of the molecular mechanisms of cardiac hypertrophy and failure have been studied in the left ventricle, and it is not clear how many of the mechanisms of left and right ventricular hypertrophy and failure are common. Presently, no strategies specific for the right side of the heart are available to treat right-sided heart failure, and it has become apparent that agents to treat left-sided heart failure are not necessarily effective in right-sided heart failure. Although the development of therapeutic strategies to treat right-sided heart failure is needed, information on the ­mechanism of right cardiac hypertrophy and failure is surprisingly lacking. In 2005, National Heart, Lung, and Blood Institute convened a meeting of the Working Group on Cellular and Molecular Mechanisms of Right Heart Failure to foster understanding of the mechanism and the development of new treatments for right-sided heart failure (http://www.nhlbi.nih.gov/meetings/ workshops/right-heart.htm; see also [3]). There is a strong need for the medical community to learn about known mechanisms of right ventricular signaling to foster basic and clinical research on right ventricle to identify molecular targets to develop agents for treating right-sided heart failure. Table 1 lists the learning objectives of this chapter. By definition, left and right ventricles are quite different, as the left ventricle serves the systemic circulation, whereas the right ventricle serves the pulmonary circulation, with markedly lower blood pressure (Table  2). Further, as described in the latter part of this chapter, developmentally, the right and left ventricles are derived from different precursors. This chapter first covers known molecular mechanisms of cardiac hypertrophy and failure, in general, and then provides information on the differences in right and left ventricles. This chapter will focus on the molecular and cellular basis of the right side of the heart. For information on right ventricular anatomy and physiology, the reader should refer to some recent review articles [3–8].

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_93, © Springer Science+Business Media, LLC 2011

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Table 1  Learning objectives 1. 2. 3. 4. 5. 6.

Know the definition of “ventricular hypertrophy and failure” Know the importance of “ventricular hypertrophy and failure” in pulmonary vascular disease Appreciate that the biology of right side of the adult heart is not well understood Know the molecular mechanisms of right ventricular development Know the structural and functional differences between adult right and left ventricles Know the molecular mechanisms of right ventricular hypertrophy and failure

Table 2  Right ventricle versus left ventricle 1. 2. 3. 4. 5.

The right ventricle pumps blood into the low-pressure pulmonary vasculature The mechanical afterload in the right ventricle is significantly smaller The free wall of the right ventricle is much thinner The action potential duration is shorter in the right ventricle There is a higher density of transient outward potassium current in the right ventricle

2 Molecular Basis of Cardiac Hypertrophy and Failure In response to increased vascular resistance, the heart ventricle (right or left) initially adapts by changing its structure. Increased systemic vascular resistance influences the left ventricle, and increased pulmonary vascular resistance influences the right ventricle. Chronic pressure overload by such diseases such as pulmonary and systemic hypertension often initially results in adaptation via cardiac hypertrophy. The term “hypertrophy” is defined by an enlarged organ or part of the body or overgrowth of an organ or part of the body owing to the increased size of the constituent cells. Exercise ­training can cause physiological hypertrophy, which is a temporary increase in the size of an organ or part to provide for a natural increase of function. Pressure overload initially leads to concentric cardiac hypertrophy by increasing the ventricular wall thickness. This type of hypertrophy occurs owing to increased width of cardiac muscle by increasing sarcomere units in parallel, perhaps designed to increase the force of contraction to overcome increased vascular resistance. There has been debate as to whether this type of hypertrophy is a truly adaptive event or a pathologic event. Clinically observed concentric hypertrophy is somewhat different from exercisetraining-induced physiologic hypertrophy. In concentrically hypertrophied cardiac muscle cells, only the width is increased, whereas physiologic hypertrophy is associated with increases in both the width and the length of myocytes. Moreover, physiologic hypertrophy is reversible according to the need for the muscle contraction force. For example,

Fig. 1  Cardiac hypertrophy and dilation

stopping exercise training results in a return of the ventricular thickness to that before the onset of training. In contrast, clinical concentric hypertrophy in response to pressure ­overload is often not readily reversible and can result in the development of a dilated heart (Fig. 1). This process is often referred to as “transition from cardiac hypertrophy to heart failure.” Although the general mechanisms of the development of concentric cardiac hypertrophy have been well ­studied, less is known about how hypertrophied hearts transition to failed hearts. At the cell level, cardiac myocytes can undergo (1) ­physiologic hypertrophy, (2) concentric hypertrophy, and (3) eccentric hypertrophy (Fig. 2). Concentric hypertrophy occurs by the parallel addition of sarcomeres; thus, it requires synthesis of contractile proteins such as myosin, actin, tropomyosin, and troponin. Among a number of transcription factors that have been shown to regulate synthesis of these proteins as well as others, GATA-4 transcription factor regulates gene transcription of important hypertrophic genes such as myosin heavy chains (MHCs), myosin light chains, troponin C, and angiotensin type 1a receptor as well as fetal genes that are reexpressed during adult clinical hypertrophy such as atrial natriuretic factor (ANF) [9].

3 Development of the Right Side of the Heart Although the mechanisms of left ventricular hypertrophy are well understood, it is not clear if the information obtained from studies of the left ventricle directly applies to the right ventricle [3]. In the adult heart, many observations both in humans and in experimental models suggest that the properties of ventricular hypertrophy in the left and right ventricles in response to pressure overload are remarkably similar; however, whether some differences in genetic programming

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Fig. 2  Morphology of ventricular muscle cells in cardiac hypertrophy and failure. Phenotypically distinct changes in the morphology of ­myocytes occur. The expression of embryonic genes is increased in eccentric and

concentric hypertrophy, but not in physiologic ­hypertrophy, in response to exercise training. (Reprinted with permission from Hunter and Chien [72]. Copyright Massachusetts Medical Society. All rights reserved)

and/or signal transduction events occur in the left and right ventricle is not clear. Since no specific treatment strategies for the management of right ventricular hypertrophy and failure have been established, understanding the differences between molecular mechanisms for left and right ventricular hypertrophy and failure may provide clues for developing treatment specific for the right side of the heart. As described below, the molecular mechanisms of the development of left and right ventricles are quite different; thus, it is conceivable that adult left and right ventricles may exhibit differential molecular signaling events during the course of clinical cardiac hypertrophy and failure. In this section, known differences in molecular mechanisms of left and right ventricular formations are described. Considering similarities in molecular responses in adult left and right ventricles, it is remarkable to learn that cells from left and right ventricles originate from differential precursors. Cells in the first heart field (also called the primary heart field) contribute to the formation of left ventricular myocardium, whereas the second heart field (also referred to as the anterior heart field) contributes to the right ventricular myocardium [10, 11]. The first heart field derives from cells in the anterior lateral plate mesoderm that forms a crescent shape at approximately embryonic day 7.5 in the mouse embryo (corresponding to week 2 of human gestation) [12]. Second heart field cells, which lie dorsal to the straight heart tube, migrate into the anterior and posterior ends of the tube to form the right ventricle, conotruncus, and part of the atria (Fig.  3). Precursors of the left ventricle are sparsely populated by the second heart field and are largely derived from the first heart field [12]. The existence of differential molecular mechanisms for the formation of left and right ventricles is well demonstrated ­particularly through studies of basic helix–loop–helix (bHLH) domain containing transcription factors eHand (Hand1) and dHand (Hand2). “dHand” stands for “deciduum membrane,

heart, autonomic nervous system, and other neural-crestderived tissues”; and “eHand” stands for “extraembryonic membrane, heart, autonomic nervous system, and other ­neural-crest-derived tissues.” During mouse cardiogenesis, dHand is expressed specifically in the right ventricle, whereas eHand is expressed in the left ventricle [13]. Gene deletion of dHand in mouse embryos resulted in the inhibition of right ventricular formation without affecting the left ventricle [13]. More recent work on Gata4 knockout mice further delineated the molecular pathways governing chamber-specific regulation by showing that these mice also develop right ventricular hypoplasia [14]. GATA-4 has been shown to regulate dHand gene transcription [15]. These results demonstrate the molecular mechanism for the chamber-specific formation of right and left ventricles. In the adult heart, however, the chamber-specific expression of dHand and eHand is lost and both transcription factors can be detected in the myocardium of both chambers [16]. The free wall thickness and force development of right and left ventricles are similar in the fetus. After birth and in infancy, however, right ventricular hypertrophy regresses and the typical postnatal heart with a crescent-shaped right ventricle and an elliptic left ventricle develops. The crescent shape allows the right ventricle to have a lower ­volume-to-surface area ratio and thus a highly complaint chamber.

4 Electrophysiological and Contractile Properties of the Right Side of the Adult Heart At the cellular level, both adult right and left ventricles develop to have similar excitation–contraction mechanisms for muscle contraction, although slight differences have been

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Fig.  3  Mammalian heart development. Oblique views of whole embryos and frontal views of cardiac precursors during human cardiac development are shown. At fay 15, first heart field (FHF) cells form a crescent shape in the anterior embryo with second heart field (SHF) cells medial and anterior to the FHF. At day 21, SHF cells lie dorsal to the straight heart tube and begin to migrate (arrows) into the anterior and posterior ends of the tube to form the right ventricle (RV), conotruncus (CT), and part of the atria (A). At day 28, following rightward looping of the heart tube, cardiac neural crest (CNC)

cells also migrate (arrow) into the outflow tract from the neural folds to septate the outflow tract and pattern the bilaterally symmetric aortic arch arteries (III, IV, and VI). At day 50, septation of the ventricles, atria, and atrioventricular valves (AVV) results in the fur-chambered heart. V ventricle, LV left ventricle, LA left atrium, RA right atrium, AS aortic sac, Ao aorta, PA pulmonary artery, RSCA right subclavian artery, LSCA left subclavian artery, RCA right carotid artery, LCA left carotid artery, DA ductus arteriosus. (Reproduced from [12] with permission from Elsevier)

observed, as described in this section. Cardiac muscle contraction is initiated by depolarization of the sarcolemmal L-type Ca2+ channel, which in turn transports Ca2+ from extracellular to intracellular space. This transported Ca2+ serves as a second messenger, which in turn targets another Ca2+ channel located on the membrane of an intracellular organelle called the sarcoplasmic reticulum. This ­Ca2+-release channel is called a ryanodine receptor, which opens in response to binding of Ca2+ on the cytosolic side, ­triggering a mechanism called Ca2+-induced Ca2+ release, which amplifies the amount of Ca2+ in the cytosolic space. Increased cytosolic Ca2+ concentration triggers binding of Ca2+ to troponin C and results in actin–myosin interactions and muscle contraction. During the relaxation phase, Ca2+ is extruded out of the cytosolic space to the sarcoplasmic reticulum via Ca2+ATPase (SERCA2 or also called ATP-dependent Ca2+ pump) and to the extracellular space via the Na+–Ca2+ exchanger. In adult rodents, the action potential duration at a fixed heart rate has been reported to be shorter in the right ventricle than in the left ventricle [17–19]. Whole-cell voltage-clamp recordings revealed that the transient outward K+ current, Ito, density was significantly higher in the mouse right ventricle than in the left ventricle [20]. Similarly, Ito density was found to be higher in the canine right ventricle than in the left ventricle [21, 22]. In rats, right ventricular myocytes contracted more rapidly than those from the left ventricle [23].

Contractile activity differences between right and left ventricles were studied by Kondo et al. [24]. They monitored intracellular Ca2+ transients and contractile function in freshly isolated myocytes from mouse right and left ventricles. They found that the Ca2+-induced Ca2+-release amplitude is smaller in the right ventricle than in the left ventricle. Radiolabeled [45] Ca2+ uptake activities in isolated sarcoplasmic reticulum vesicles from right and left ventricles showed no differences. The messenger RNA levels of Ca2+-handling proteins such as the sarcoplasmic reticulum Ca2+-ATPase, phospholamban, ryanodine receptor, and Na+–Ca2+ exchanger did not show any differences between right and left ventricles in mice. In contrast, Sathish et  al. [25] found, in rats, that ATPdependent Ca2+ uptake of the sarcoplasmic reticulum is nearly fourfold lower in the right ventricle than in the left ventricle. Similarly, depolarization-induced Ca2+ transients in the right ventricle decayed more slowly than those in the left ventricle. This may be attributed to the lesser interactions of the Ca2+-ATPase with phospholamban. Lower Ca2+ uptake by the sarcoplasmic reticulum and a lower Ca2+-ATPase phosphoenzyme formation were noted in the right ventricle compared with the left ventricle. Less than 50% of the sarcoplasmic reticulum Ca2+ pump in the right ventricle is catalytically active, despite the expression level of this protein being only about 15% lower in the right ventricle than in the left

93  Molecular Basis of Right Ventricular Hypertrophy and Failure in Pulmonary Vascular Disease

ventricle. Coimmunoprecipitation experiments revealed that the level of phospholamban-bound Ca2+-ATPase molecules in the sarcoplasmic reticulum of the right ventricle is greater than that in the sarcoplasmic reticulum of the left ventricle. In isolated rat cardiac myocytes, the Ca2+-induced Ca2+release kinetics is significantly prolonged in right ventricular myocytes compared with myocytes from the left ventricle. The slow decay of cytosolic Ca2+ concentration in the right ventricle and the consequent decrease in the rate of right ventricular relaxation may promote synchrony of right and left ventricles, and the reduced sarcoplasmic reticulum Ca2+ pump activity in the right ventricle may reflect a higher diastolic reserve which may maintain right ventricular function in the condition of increased pulmonary vascular resistance [25].

5  M  echanisms of Adult Right Ventricular Hypertrophy and Failure in Pulmonary Vascular Disease Despite the right and left ventricles being developed from different precursors, the protein expression patterns of adult right and left ventricles are very similar. Our laboratory performed two-dimensional gel electrophoresis studies to compare the protein expression patterns between right and left ventricles from adult rats. We found that the protein expression patterns in right and left ventricles are remarkably similar and our Coomassie-stained gels did not exhibit any detectable differences (unpublished results). Although a large body of results on the mechanisms of left ventricular hypertrophy exists in the literature, results on right ventricular hypertrophy are substantially less developed. Lack of studies of right cardiac hypertrophy and failure is apparent by examining the number of published studies. For example, as of December 2010, a literature search on PubMed using the keyword “ventricular hypertrophy” resulted in 28,989 hits and “left ventricular hypertrophy” gave 21,132 hits, indicating that approximately half of the studies of ventricular hypertrophy might have been on the left side. However, in contrast, the keyword “right ventricular hypertrophy” resulted in only 4,786 hits, suggesting that studies on cardiac hypertrophy have focused on the left side of the heart and/or the experiments did not consider differences between right and left sidedness. These searches as well as literature searches with similar keywords clearly indicate the lack of published studies concerning the right side of the heart, especially those pertaining to the mechanisms of cardiac hypertrophy and heart failure. The available results from studies of the mechanisms of right ventricular hypertrophy largely resemble what is known for left ventricular hypertrophy. Studies of rodent models of pulmonary hypertension have shown changes in gene expression in the right side of

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the heart. Many studies have demonstrated that ANF is upregulated in the hypertrophied right ventricle [26, 27]. ANF is a well-known marker for ventricular hypertrophy and has been shown to be upregulated in left ventricular hypertrophy. Therefore, these findings in the right ventricle are consistent with what has been shown in the left side of the heart. Some studies have also demonstrated the altered expression of MHC in the hypertrophied right ventricle [26, 28, 29]. Since GATA-4 transcription factor plays important roles in the regulation of ANF and MHC gene expression as well as other hypertrophic markers [9], our laboratory examined if GATA-4 is activated in the hypertrophied right ventricle in response to pulmonary hypertension. Rats were subjected to chronic hypoxia at 10% O2, which caused pulmonary hypertension and right ventricular hypertrophy [30]. In these rats, GATA-4 DNA binding activity was found to be upregulated in the right ventricle, but not in the left [31]. Although the activation of GATA-4 in the right ventricle in response to pulmonary hypertension was expected and is consistent with other forms of cardiac hypertrophy [32], the mechanism of the activation appears to be unique in the right ventricular hypertrophy induced by pulmonary hypertension. Although much of GATA-4 activation has been attributed to the involvement of posttranslational modifications such as nuclear factor of activated T cells/GATA-4 interactions [33] and GATA-4 phosphorylation [34, 35], GATA-4 activation in the right ventricle in response to pulmonary hypertension is due to increased gene transcription of Gata4 [31]. Our laboratory cloned the Gata4 promoter and identified an important regulatory element in the proximal region, namely, the CCAAT box, which can bind to transcription factors such as CBF/NF-Y [36]. The activation of Gata4 gene transcription in the right ventricle in response to pulmonary hypertension involves the activation of CCAATbinding factor (CBF)/nuclear factor-Y (NF-Y) binding to the CCAAT box of the Gata4 promoter through a novel signaling mechanism involving protein carbonylation of annexin A1 [37]. Figure 4 depicts a putative mechanism for the GATA-4 activation via the promotion of annexin A1 carbonylation and degradation, which in turn liberates CBF/ NF-Y for binding to the Gata4 promoter and gene activation in the right ventricle. As discussed in various chapters in this book, pulmonary hypertension and pulmonary vascular remodeling are mediated by various factors. Notably, endothelin-1 and serotonin play important roles in both vasoconstriction as well as pulmonary vascular thickening. Since these molecules also mediate cardiac hypertrophy and failure, the roles of mediators of pulmonary hypertension such as endothelin-1 and serotonin in the development of cardiac hypertrophy and failure should be considered. Endothelin-1, a peptide composed of 21 amino acids, is a potent vasoconstrictor originally identified in vascular

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Fig. 4  Proposed mechanism for GATA-4 activation in the right ventricle in response to pulmonary hypertension. Pulmonary hypertension exerts pressure overload on the right ventricle and also produces growth factors such as endothelin-1 and serotonin. These stimuli generate reactive oxygen species in the right ventricle, which in turn carbonylate annexin A1, which is bound to CBF/NF-Y transcription factor. Carbonylated annexin A1 is degraded by the proteasomes, resulting in liberated CBF/NF-Y that can bind to the CCAAT box within the Gata4 promoter. CBF/NF-Y binding enhances gene transcription of Gata4 and increased levels of GATA-4 transcription factor, which in turn promote gene expression of various hypertrophic regulators

endothelial cells [38]. The plasma endothelin-1 level was found to be elevated in patients with idiopathic pulmonary arterial hypertension [39, 40]. Histology studies of lung tissues from patients with pulmonary hypertension demonstrated the production of excess endothelin-1 and increased expression of preproendothelin-1, the precursor for endothelin-1 [40]. Similarly, increased endothelin-1 expression was observed in lungs of fawn-hooded rats with increased susceptibility to developing pulmonary hypertension [41]. In secondary pulmonary hypertension, plasma levels of endothelin-1 correlated with the severity of the disease [42, 43]. In various animal models of secondary pulmonary hypertension, endothelin receptor antagonists have been shown to block the progression of the disease [44–51]. Human studies showed that the endothelin receptor antagonist bosentan increased exercise capacity and improved hemodynamics in patients with pulmonary hypertension [52, 53]. Bosentan (Tracleer®) has been approved by the FDA for the treatment of pulmonary arterial hypertension. Soon after the discovery of endothelin-1, this vasoactive peptide was found to be a potent promoter of cardiac muscle cell hypertrophy [54–57]. Several years later, endothelin-1 was also found to play a role in the functional deterioration of left ventricles during the transition from compensatory hypertrophy to congestive heart failure in salt-sensitive hypertensive rats [58]. Thus, endothelin-1, the level of which is elevated in pulmonary hypertension patients, plays a role

Y.J. Suzuki

in the development of right ventricular hypertrophy and/or in the transition from compensatory right ventricular hypertrophy to right-sided heart failure. However, studies of the roles of endothelin-1 in right ventricular hypertrophy and failure have not yet been reported. Serotonin (5-hydroxytryptamine) is a potent vasoconstrictor and a mitogen of pulmonary artery smooth muscle cells. Evidence for the role of serotonin in the development of pulmonary hypertension was first recognized in fawnhooded rats, in which a genetic deficit in serotonin platelet storage and high plasma levels of serotonin are associated with the susceptibility to developing pulmonary hypertension [59]. Further studies showed that a continuous intravenous infusion of serotonin during the exposure of rats to hypoxia potentiated the development of pulmonary hypertension [60]. The role of serotonin in pulmonary hypertension is further supported by results showing that serotonin-transporter-deficient mice develop less hypoxic pulmonary hypertension [61]. In mice, overexpression of serotonin transporter enhanced the hypoxia-induced increase in pulmonary vascular remodeling, right ventricular pressure, and right ventricular hypertrophy [62]. In patients with idiopathic pulmonary arterial hypertension, high levels of plasma serotonin were observed [63]. Eddahibi et  al. [64] reported that pulmonary artery smooth muscle cells from patients with pulmonary hypertension grow faster than cells from control subjects in response to serotonin. On the basis of these results, possible therapeutic strategies for pulmonary hypertension by targeting serotonin pathways have been proposed [65, 66]. Although the existence of serotonin metabolic enzymes and serotonin receptors in the heart has been known for some time, the roles of serotonin in cardiac disease only recently emerged. Nebigil et al. [67] found that serotonin 2B receptor mutant mice have dilated cardiomyopathy without a ­compensatory hypertrophic response. In contrast, overexpression of this receptor results in cardiac hypertrophy [68], ­suggesting that serotonin signaling through serotonin 2B receptor may play a role in the development of concentric cardiac hypertrophy. Bianchi et  al. [69] also showed that serotonin can promote cardiac myocyte hypertrophy through serotonin transporter and monoamine oxidase A. Brattelid et al. reported that serotonin 2A receptor may be involved in cardiac hypertrophy, whereas serotonin 4A receptor may contribute to the transition to heart failure [70]. Although serotonin levels have been shown to be increased in patients with pulmonary hypertension [63], the roles of serotonin in right ventricular hypertrophy and failure have not been investigated. Recently, our laboratory found that serotonin promotes protein oxidation specifically in the right ventricle, which might elicit right ventricular hypertrophy and/or rightsided heart failure [71].

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6  Summary Pulmonary hypertension results in right ventricular hypertrophy and right-sided heart failure. Although the mechanisms of left ventricular hypertrophy have been well studied, information on right ventricular hypertrophy is limited. Recent results from embryonic and adult hearts provided evidence that the molecular signaling mechanisms are different for the right and left ventricles, indicating the importance of developing therapeutic agents specific for the right side of the heart. This chapter summarized current understanding of growth ­signaling mechanisms in the right ventricle.

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1312 35. Kitta K, Clément SA, Remeika J, Blumberg JB, Suzuki YJ (2001) Endothelin-1 induces phosphorylation of GATA-4 transcription ­factor in the HL-1 atrial-muscle cell line. Biochem J 359:375–380 36. Park AM, Nagase H, Kumar SV, Suzuki YJ (2007) Effects of intermittent hypoxia on the heart. Antioxid Redox Signal 9:723–729 37. Wong CM, Cheema AK, Zhang L, Suzuki YJ (2008) Protein carbonylation as a novel mechanism in redox signaling. Circ Res 102:310–318 38. Yanagisawa M, Kurihara H, Kimura S et al (1988) A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 332:411–415 39. Stewart DJ, Levy RD, Cernacek P, Langleben D (1991) Increased plasma endothelin-1 in pulmonary hypertension: marker for ­mediator of disease? Ann Intern Med 114:464–469 40. Giaid A, Yanagisawa M, Langleben D et al (1993) Expression of endothelin-1 in the lungs of patients with pulmonary hypertension. N Engl J Med 328:1732–1739 41. Zamora MR, Stelzner TJ, Webb S, Panos RJ, Ruff LJ, Dempsey EC (1996) Overexpression of endothelin-1 and enhanced growth of pulmonary artery smooth muscle cells from fawn-hooded rats. Am J Physiol 270:L101–L109 42. Yoshibayashi M, Nishioka K, Nakao K et al (1991) Plasma endothelin concentrations in patients with pulmonary hypertension associated with congenital heart defects. Evidence for increased production of endothelin in pulmonary circulation. Circulation 84:2280–2285 43. Cody RJ, Haas GJ, Binkley PF, Capers Q, Kelley R (1992) Plasma endothelin correlates with the extent of pulmonary hypertension in patients with chronic congestive heart failure. Circulation 85:504–509 44. Miyauchi T, Yorikane R, Sakai S et  al (1993) Contribution of endogenous endothelin-1 to the progression of cardiopulmonary alterations in rats with monocrotaline-induced pulmonary hypertension. Circ Res 73:887–897 45. Bonvallet ST, Zamora MR, Hasunuma K et  al (1994) BQ123, an ETA-receptor antagonist, attenuates hypoxic pulmonary hypertension in rats. Am J Physiol 266:H1327–H1331 46. Okada M, Yamashita C, Okada M, Okada K (1995) Endothelin receptor antagonists in a beagle model of pulmonary hypertension: contribution to possible potential therapy? J Am Coll Cardiol 25:1213–1217 47. Eddahibi S, Raffestin B, Clozel M, Levame M, Adnot S (1995) Protection from pulmonary hypertension with an orally active endothelin receptor antagonist in hypoxic rats. Am J Physiol 268:H828–H835 48. Chen SJ, Chen YF, Meng QC, Durand J, Dicarlo VS, Oparil S (1995) Endothelin-receptor antagonist bosentan prevents and reverses hypoxic pulmonary hypertension in rats. J Appl Physiol 79:2122–2131 49. Sakai S, Miyauchi T, Sakurai T et al (1996) Pulmonary hypertension caused by congestive heart failure is ameliorated by long-term application of an endothelin receptor antagonist. Increased expression of endothelin-1 messenger ribonucleic acid and endothelin-1like immunoreactivity in the lung in congestive heart failure in rats. J Am Coll Cardiol 28:1580–1588 50. Hill NS, Warburton RR, Pietras L, Klinger JR (1997) Nonspecific endothelin-receptor antagonist blunts monocrotaline-induced pulmonary hypertension in rats. J Appl Physiol 83:1209–1215 51. Ueno M, Miyauchi T, Sakai S, Goto K, Yamaguchi I (2000) EndothelinA-receptor antagonist and oral prostacyclin analog are comparably effective in ameliorating pulmonary hypertension and right ventricular hypertrophy in rats. J Cardiovasc Pharmacol 36:S305–S310 52. Channick RN, Simonneau G, Sitbon O et al (2001) Effects of the dual endothelin-receptor antagonist bosentan in patients with pulmonary hypertension: a randomised placebo-controlled study. Lancet 358:1119–1123

Y.J. Suzuki 53. Rubin LJ, Badesch DB, Barst RJ et al (2002) Bosentan therapy for pulmonary arterial hypertension. N Engl J Med 346:896–903 54. Suzuki T, Hoshi H, Mitsui Y (1990) Endothelin stimulates hypertrophy and contractility of neonatal rat cardiac myocytes in a serumfree medium. FEBS Lett 268:149–151 55. Ito H, Hirata Y, Hiroe M et al (1991) Endothelin-1 induces hypertrophy with enhanced expression of muscle-specific genes in cultured neonatal rat cardiomyocytes. Circ Res 69:209–215 56. Sugden PH, Fuller SJ, Mynett JR, Hatchett RJ 4th, Bogoyevitch MA, Sugden MC (1993) Stimulation of adult rat ventricular myocyte protein synthesis and phosphoinositide hydrolysis by the endothelins. Biochim Biophys Acta 1175:327–332 57. Yamazaki T, Komuro I, Kudoh S et  al (1996) Endothelin-1 is involved in mechanical stress-induced cardiomyocyte hypertrophy. J Biol Chem 271:3221–3228 58. Iwanaga Y, Kihara Y, Hasegawa K et al (1998) Cardiac ­endothelin-1 plays a critical role in the functional deterioration of left ventricles during the transition from compensatory hypertrophy to congestive heart failure in salt-sensitive hypertensive rats. Circulation 98:2065–2073 59. Sato K, Webb S, Tucker A et al (1992) Factors influencing the idiopathic development of pulmonary hypertension in the fawn-hooded rat. Am Rev Respir Dis 145:793–797 60. Eddahibi S, Raffestin B, Pham I et al (1997) Treatment with 5-HT potentiates development of pulmonary hypertension in chronically hypoxic rats. Am J Physiol 272:H1173–H1181 61. Eddahibi S, Hanoun N, Lanfumey L et al (2000) Attenuated hypoxic pulmonary hypertension in mice lacking the 5-hydroxytryptamine transporter gene. J Clin Invest 105:1555–1562 62. MacLean MR, Deuchar GA, Hicks MN et al (2004) Overexpression of the 5-hydroxytryptamine transporter gene: effect on pulmonary hemodynamics and hypoxia-induced pulmonary hypertension. Circulation 109:2150–2155 63. Hervé P, Launay JM, Scrobohaci ML et al (1995) Increased plasma serotonin in primary pulmonary hypertension. Am J Med 99:249–254 64. Eddahibi S, Humbert M, Fadel E et al (2001) Serotonin transporter overexpression is responsible for pulmonary artery smooth muscle hyperplasia in primary pulmonary hypertension. J Clin Invest 198:1141–1150 65. Eddahibi S, Adnot S (2006) The serotonin pathway in pulmonary hypertension. Arch Mal Coeur Vaiss 99:621–625 66. MacLean MR (2007) Pulmonary hypertension and the serotonin hypothesis: where are we now? Int J Clin Pract 61:27–31 67. Nebigil CG, Hickel P, Messaddeq N et al (2001) Ablation of serotonin 5-HT(2B) receptors in mice leads to abnormal cardiac structure and function. Circulation 103:2973–2979 68. Nebigil CG, Jaffré F, Messaddeq N et al (2003) Overexpression of the serotonin 5-HT2B receptor in heart leads to abnormal mito­ chondrial function and cardiac hypertrophy. Circulation 107: 3223–3229 69. Bianchi P, Pimentel DR, Murphy MP, Colucci WS, Parini A (2005) A new hypertrophic mechanism of serotonin in cardiac myocytes: receptor-independent ROS generation. FASEB J 19:641–643 70. Brattelid T, Qvigstad E, Birkeland JA et al (2007) Serotonin responsiveness through 5-HT2A and 5-HT4 receptors is differentially regulated in hypertrophic and failing rat cardiac ventricle. J Mol Cell Cardiol 43:767–779 71. Liu L, Marcocci L, Wong CM, Park AM, Suzuki YJ (2008) Serotonin-mediated protein carbonylation in the right heart. Free Radic Biol Med 45:847–854 72. Hunter JJ, Chien KR (1999) Signaling pathways for cardiac hypertrophy and failure. N Engl J Med 341:1276–1283

Chapter 94

Right Ventricular Dysfunction in Pulmonary Hypertension Francois Haddad, Mehdi Skhiri, and Evangelos Michelakis

Abstract  We now know that there are many key differences between the right ventricle (RV) and the left ventricle (LV), ranging from embryological origin, structure, function (metabolism and perfusion), neurohormonal activation and response to increased afterload. Although the role of the RV in the low-pressure normal pulmonary circulation may not be critical, its critical role may become apparent in the diseased pulmonary circulation (e.g. pulmonary hypertension, PHT) or in high-altitude environments. Similarly, although in utero the RV is hypertrophied, within a few weeks after birth, its metabolism and structure is switching to the adult phenotype. Thus, the RV may be designed to be more “plastic.” However, its dynamic responses to physiologic or nonphysiologic triggers increase the risk of maladaptation. We now know that a maladaptive, failed RV is the most important factor in the morbidity and mortality in PHT, regardless of its specific cause. Right ventricular dysfunction is also a very strong predictor of outcome in patients with heart failure due to left ventricular dysfunction. Yet, at this time, few studies have focused on the RV, without extrapolating concepts form the LV. Thus, the concept of RV-specific therapies remains embryonic. In 2006, the NIH formed a task force focusing on increasing awareness and promoting RV studies specifically. Here we review the basic principles of right ventricular dysfunction, clinical diagnosis, as well as therapy, introducing the concept of RV-specific therapies. However, the biggest question in this field, i.e. what is the difference between the adapted RV (for example, the RV in a patient with severe PHT due to congenital heart disease that offers the patient decades-long survival) and the maladaptive RV [for example, the RV in a patient with idiopathic pulmonary arterial hypertension (PAH) of similar magnitude, which fails relatively quickly and allows only a few years of survival], remains unknown. It is hoped that the next edition of this textbook will provide some answers to this critical question.

F. Haddad (*) Division of Cardiovascular Medicine, Stanford School of Medicine, 770 Welch Road, Suite 400, Palo Alto, CA 94304-5715, USA e-mail: [email protected]

Keywords  Right ventricular hypertrophy • Afterload • Maladaptation • Right heart dysfunction and failure

1 Introduction “…And I ask, as the lungs are so close at hand, and in continual motion, and the vessel that supplies them is of such dimensions, what is the use or meaning of this pulse of the right ventricle? And why was nature reduced to the necessity of adding another ventricle for the sole purpose of nourishing the lungs?” William Harvey Exercitatio Anatomica de Motu Cordis et Sanguinis in Animalibus, 1628 The answers to Harvey’s questions remain unanswered. Since the pulmonary circulation has a very low resistance compared with the systemic circulation, it was fair to question why another heart chamber was dedicated to “pushing blood through,” even in the form of a rhetorical question. This is particularly intriguing if one realizes that the right ventricle (RV)-ejected blood is not “nourishing the lung” as Harvey implied. The lungs consume a minimal amount of oxygen and they are “nourished” by the bronchial circulation. As opposed to every other organ in the body, the lungs are oxygen suppliers, not consumers. This is a fundamental difference between the left and the right circulation. Thus, a critical concept of left ventricular failure, i.e. the neurormonal hypothesis, which explains the cardiovascular remodeling in response to neurohormones secreted by hypoperfused target organs, may not apply fully to right ventricular failure (RVF). We now know that there are many key differences between the RV and the left ventricle (LV), ranging from embryological origin, structure, function (metabolism and perfusion), neurohormonal activation and response to increased afterload [1–3]. Going back to Harvey’s rhetorical question, one can speculate that although the role of the RV in the low-pressure normal pulmonary circulation may not be critical, its critical role may become apparent in the diseased pulmonary circulation (e.g. pulmonary hypertension, PHT)

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_94, © Springer Science+Business Media, LLC 2011

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or in high-altitude environments. Similarly, although in utero the RV is hypertrophied, within a few weeks after birth, its metabolism and structure is switching to the adult phenotype. Thus, the RV may be designed to be more “plastic.” However, its dynamic responses to physiologic or ­nonphysiologic triggers increase the risk of maladaptation. We now know that a maladaptive, failed RV is the most important ­factor in the morbidity and mortality in PHT, regardless of its specific cause [4–9]. Right ventricular dysfunction is also a very strong predictor of outcome in patients with heart failure due to left ventricular dysfunction [5, 6, 9–14]. Yet, at this time, few studies have focused on the RV, without extrapolating concepts form the LV. Thus, the concept of RV-specific therapies remains embryonic. In 2006, the NIH formed a task force focusing on increasing awareness and promoting RV studies specifically [15]. Here we review the basic principles of right ventricular dysfunction, clinical diagnosis, as well as therapy, introducing the concept of RV-specific therapies. However, the biggest question in this field, i.e. what is the difference between the adapted RV (for example, the RV in a patient with severe PHT due to congenital heart disease that offers the patient decades-long survival) and the maladaptive RV [for example, the RV in a patient with idiopathic pulmonary arterial hypertension (PAH) of similar magnitude, which fails relatively quickly and allows only a few years of survival], remains unknown. It is hoped that the next edition of this textbook will provide some answers to this critical question.

2 Definition of Right Ventricular Failure RVF is defined as a complex clinical syndrome that can result from any structural or functional cardiac disorder that impairs the ability of the right side of the heart to fill or eject appropriately [16, 17]. The cardinal clinical manifestations of RVF are (a) fluid retention, manifested as peripheral edema or ascites, (b) decreased systolic reserve or low cardiac output syndrome, which may present as exercise intolerance, fatigue or altered mentation, and (c) atrial or ventricular arrhythmias [16–18]. Functionally, the affected RV may be in the subpulmonary (usual) or systemic position (in congenitally corrected levotranspositions of great vessels or ­dextrotranspositions of great vessels following an atrial-switch repair). Patients may present with a clinical picture of biventricular failure or predominantly right-sided heart failure. The causes of predominant right-sided heart failure include pulmonary embolism, right ventricular myocardial infarction, PAH, chronic pulmonary disease, postcardiotomy RVF, tricuspid valve disease, and selected forms of congenital heart disease, i.e. tetralogy of Fallot, pulmonary stenosis, and systemic RV (Table 1). When RVF is considered as a progressive disorder, patients with asymptomatic ventricular dysfunction are considered to

F. Haddad et al. Table 1  Causes and mechanisms of right-sided heart failure (RHF) Mechanism of right ventricular dysfunction Specific cause Pressure overload

LHF(most common cause) Acute pulmonary embolism (common) PH RVOT obstruction Double chambered Systemic right ventricle (TGA) Volume overload Tricuspid regurgitation Pulmonary regurgitation Atrial septal defect Total or partial anomalous pulmonary return, Carcinoid syndrome (stenotic component possible) Ischemia and infarction Right ventricular myocardial ischemia or infarction Intrinsic myocardial Cardiomyopathy or infiltrative process process Arrhythmogenic right ventricular dysplasia Inflow limitation Tricuspid stenosis Superior vena cava stenosis Complex congenital Ebstein's anomaly malformation Tetralogy of Fallot Double-outlet right ventricle with mitral atresia Hypoplasic right ventricle Pericardial disease Constrictive pericarditis Source: Adapted from Haddad et al. [20] with permission

Fig.  1  The theoretical progression of right ventricular failure (RVF) from normal physiological function to compensated right ventricular remodeling and to overt RVF. As the right ventricle fails, it dilates and the septum shifts to the left because of ventricular interdependence. This stage is often associated with decreased reserve and exercise tolerance of the right ventricle. (Adapted with permission from Champion et al. [23])

be in the early stages of RVF (Fig. 1) [16]. Analogous for the staging proposed for left-sided heart failure, patients may progress from being at risk of RVF (stage A), to asymptomatic right ventricular dysfunction (stage B), to RVF (stage C), and finally refractory RVF (stage D) [16]. Patients with more advanced stages of RVF often have a greater degree of dilatation to hypertrophy (greater volume to mass ratio). Finally, it is also practical to divide RVF as to whether it is acute or chronic.

3 Pathophysiology of Right Ventricular Dysfunction and Failure Following initial myocardial stress or injury, several factors may contribute to progression to RVF, including the timing of myocardial stress, the type of stressor, myocardial ­ischemia,

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as well as possible neurohormonal and immunologic activation [14, 15, 19, 20]. In general, the RV adapts better to volume rather than pressure overload and to chronic rather than acute stressors. Pediatric patients also appear to have a more favorable course or adaptation than adult patients for similar levels of elevation in pulmonary pressure. Although it was previously thought that right ventricular volume overload is very well tolerated by the RV, recent data from MessikaZeitoun et al. have demonstrated that the presence of a flail tricuspid valve is associated with decreased survival and a higher incidence of symptomatic heart failure and atrial fibrillation [21]. This points toward a more aggressive strategy for surgical repair of flail tricuspid valves, a trend similar to that currently followed for mitral regurgitation. With the exception of Eisenmenger’s syndrome, the RV is unable to sustain long-term severe pressure overload. Early on, right ventricular hypertrophy is mainly an adaptive response (compensated state). As the disease progresses, the RV dilates and RVF eventually occurs (maladaptative right ventricular remodeling; Fig.  1) [22]. The compensatory phase is significantly shorter in the RV than in the LV and explains largely the increased mortality observed in patients with PAH compared with patients with systemic hypertension. Similarly, cardiovascular collapse is not uncommon in patients with massive pulmonary embolism but is rare in patients with a hypertensive urgency. Although this increased vulnerability may be in part due to the lower mass of the RV, several other mechanisms may be involved. An intriguing hypothesis involves the loss of the “protective” molecular, metabolic, and structural phenotype of the fetal RV. When the transition to the adult phenotype occurs, the RV becomes more vulnerable to failure in the presence of increased afterload. This is in contrast to what occurs in patients who develop Eisenmenger’s syndrome in the first year of life who may maintain the fetal phenotype and better adaptation to pressure overload. Several mechanisms are now recognized to contribute to maladaptative right ventricular remodeling, including (1) a switch in contractile protein isoforms from a-myosin heavy chain (a-MHC) to b-myosin heavy chain (b-MHC), (2) mitochondrial and metabolic remodeling, (3) electrical remodeling, including alterations in enzymes and ion channels involved in myocyte excitation– contraction coupling, (4) matrix remodeling with increased fibrosis, and (5) neurohormonal and cytokine activation (Fig. 2) [22–24]. As in left-sided heart disease, a decrease in the amount of a-MHC and an increase in the amount of b-MHC, a feature of the “stressed myocardium,” are also observed in the failing adult RV [25]. In the normal adult human RV, the a-MHC isoform makes up approximately 23–34% of total myosin heavy chain, and b-MHC makes up the remainder [22]. In the pressure-overloaded RV, the change in contractile protein isoforms leads to a decrease in cardiomycyte function. In the LV, microRNA-208 has been identified as one of the key

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Fig. 2  Postulated molecular mechanisms of RVF

regulators of this contractile protein switch, but whether the same or a similar molecular trigger also occurs in the RV remains unknown [26]. Attention has also been drawn to the role of mitochondrial and metabolic remodeling in PAH and right ventricular hypertrophy [24, 27–30]. In the hypertrophied RV, there is a switch to glycolysis from fatty acid oxidation, i.e. the primary fuel source becomes glucose (glycolysis taking place in the cytoplasm) as opposed to fatty acid oxidation (taking place in mitochondria). At first, this appears paradoxical since oxidative phosphorylation in the mitochondria produces much more ATP than does glycolysis. However, we now know from lessons in cancer that there are “secondary gains” from such a switch [31]. For example, this metabolic switch is associated with mitochondrial hyperpolarization, a state in which mitochondria-dependent apoptosis is suppressed. This metabolic switch explains in part the resistance to apoptosis in cancer. It is possible that in the presence of increased afterload, the myocardium suppresses mitochondria-dependent apoptosis, at the expense of suboptimal contractility, at least acutely. As in cancer, long-term response leads to increased uptake of glucose in the cytoplasm and upregulation of the glycolysis, catching up to the energetic efficiency of the mitochondrial oxidative phosphorylation. Thus, as right ventricular hypertrophy increases, the right ventricular mitochondria become more hyperpolarized [28] and 18F-fluorodeoxyglucose (FDG) uptake (measured by PET in  vivo) increases [32] in an impressive similarity to what occurs in cancer. Since this metabolic remodeling can be detected and quantified in  vivo by FDG-PET and is restricted to the RV in PAH, metabolic targeting therapies might be RV-specific and feasible. Among the neurohormones involved in RVF, evidence is stronger for angiotensin II, catecholamines, and natriuretic

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peptides [33–35]. However, the role of these neurohormones has not been as extensively studied in RVF and direct extrapolation from the left ventricular literature may not be wise. Cytokine activation may also play an important role in patients with RVF. In patients with selected forms of congenital heart disease and right ventricular dysfunction, elevated levels of tumor necrosis factor and endotoxin were associated with more symptomatic disease (lower functional class or more edema) [36, 37]. A recent study demonstrated that genes may be differentially regulated in the pressure-overloaded RV as compared with the pressure-overloaded LV, again supporting the view that the two ventricles and their response to disease might have significant differences. The differentially expressed genes were involved in the Wnt signaling pathway, apoptosis, migration of actin polymerization, and processing of the ubiquitin system [38]. As discussed earlier, the differential expression of genes in the right side of the heart and the left side of the heart is not surprising in view of the different embryological origin of the RV and the LV and their different physiological environments [2, 3]. A more detailed review of the molecular mechanisms of RVF can be found in this textbook and in a recent review by Bogaard et al. [22]. Ventricular interdependence also plays an essential part in the pathophysiology of RVF. Although always present, ventricular interdependence is most apparent with changes in loading conditions such as those seen with volume loading, respiration, or sudden postural changes [39]. Ventricular interdependence helps maintain hemodynamics in early stages of RVF. Experimental studies have shown that in the absence of a dilated RV, left ventricular systolic contraction contributes 20–40% of right ventricular systolic pressure generation [39, 40]. Diastolic ventricular interdependence contributes to the development of left ventricular systolic dysfunction in patients with RVF. Right ventricular enlargement or increased afterload may shift the interventricular septum and increase pericardial constraint on the LV; both of these changes can alter left ventricular geometry and decrease left ventricular preload and contractility (Fig. 1) [39, 41]. Compression of the left main coronary artery by a dilated main pulmonary artery, which is occasionally observed in PAH, may contribute to left ventricular dysfunction [42]. Tricuspid regurgitation and ongoing ischemia may also contribute to the progression of RVF. Although the RV often fails in the presence of pressure overload, it often, but not always, retains an amazing ability for reverse remodeling once the stressor has been removed. Right ventricular volumes and function can return to normal following lung transplantation for severe PAH or after pulmonary artery thrombarterectomy for chronic ­thromboembolic disease or pulmonary valve replacement for tetralogy of Fallot [43]. Identifying which

F. Haddad et al.

RVs will be able to recover function after the afterload decreases will be an important priority of noninvasive imaging in the near future.

4 Evaluation of Patients with Right Ventricular Failure A complete history, physical examination, and selective clinical studies help establish the cause and severity of right ventricular dysfunction as well as the extent of end-organ damage (renal or liver dysfunction) or the presence of associated conditions. Signs of right ventricular hypertrophy or right ventricular dilatation may occasionally be detected in routine electrocardiography (ECG) or chest X-ray studies. Although echocardiography plays a key role in the diagnosis of right-sided heart disease, magnetic resonance imaging (MRI) is emerging as the gold standard for evaluating right ventricular size and function. It is also becoming clearer that in order to better understand right ventricular physiology, one has to adequately characterize right ventricular contractility and afterload and preload of the RV. In this section, we will first discuss key physiological concepts in the evaluation of the RV as well as specific modalities in the assessment of the right-sided heart disease.

5 Key Physiological Concepts An ideal index of contractility should be independent of afterload and preload, sensitive to change in inotropy, independent of heart size and mass, easy and safe to apply, and feasible in the clinical setting [4, 44]. Ventricular elastance is considered by many investigators as the most reliable index of contractility (Table  2, Fig.  3). Ventricular elastance is measured using pressure–volume loop tracing at different loading conditions; it is the slope of the end-systolic pressure–volume relationship (Fig. 3) [23, 45, 46]. Because conductance catheterization is invasive and time-consuming, it is predominantly used as a research tool for the assessment of ventricular function [47]. More recently, a simplified method of deriving ventricular elastance has been derived using single-beat elastance measures. This method uses the maximal pressure of isovolumic beat (as the second point in the curve) and may be derived using MRI and single pressure measurements [48, 49]. This method could potentially simplify the measure of ventricular elastance and facilitate its clinical use [48]. Recent work has shown that single-beat elastance methods may be useful in assessing ventricular adaptation to PHT [2, 50].

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Table 2  Selected parameters in the assessment of right ventricular function Functional parameters Normal value Load dependence RVEF (%)

61 ± 7% (47–76%) [77] >45% >32% [75]

Clinical utility

+++

Clinical validation, wide acceptance Prognostic value in cardiopulmonary disorders [41] RVFAC (%) +++ Good correlation with RVEF Prognostic value in coronary artery disease [75] TAPSE (mm) > 18 [75] +++ Simple measure not limited by endocardial border recognition: good correlation with RVEF RVMPI 0.28 ± 0.04 [69] ++ Global nongeometric index of systolic and diastolic function, prognostic value PH, CHD [70–72] dP/dt max (mmHg/s) 100–250 [41] ++ Not a reliable index of contractility [76] More useful in assessing directional change when preload accounted for IVA (m/s2) 1.4 ± 0.5 [74] + Promising new noninvasive index of contractility, studies in CHD [47, 74] Ees (mmHg/ml) 1.30 ± 0.84 [46] + Most reliable index of contractility [41] Ees/Ea (measure of ventriculoat- ~1.5 + Useful to study the mechanisms of disease. Value in erial coupling) right-sided heart disease currently under investigation Pulmonary arterial input Ratio of pulsatile Not applicableValue in right-sided heart disease currently under impedance pulmonary pressure measure of arterial investigation to pulsatile flow load RVEF right ventricular ejection fraction, RVFAC, right ventricular fractional area change, TAPSE, tricuspid annular plane systolic excursion, Sm tissue Doppler echocardiography maximal systolic velocity at the tricuspid annulus, RVMPI right ventricular myocardial performance index, dP/ dt max maximal positive first derivative of pressure, IVA, isovolumic acceleration, Ees maximal right ventricular elastance, Ea arterial elastance, CHD, congenital heart disease Fig. 3  Right ventricular pressure–volume (PV) relationship in patients with pulmonary vascular disease. (a) The placement of the conductance catheter in the right ventricle to obtain PV data. (b) The end-systolic PV loop relationship and the end-diastolic PV relationship. Ea refers to arterial elastance and is a measure of arterial afterload; Ea is calculated as the slope of the line that links end-systolic pressure and end-diastolic volume. (c) Representative tracing of PV loops in borderline and late pulmonary arterial hypertension. (d) The key features of summary impedance spectra. PVR pulmonary vascular resistance, PWV pulse wave velocity. (Adapted with permission from Champion et al. [23])

It is now also becoming clear that better understand right ventricular physiology, it is important to consider the RV and the pulmonary artery as a unit [23]. Pulmonary vascular impedance is a way of expressing opposition to flow from the RV to the pulmonary artery (Fig. 3) [51]. The factors that determine the flow from the RV include the resistance of the pulmonary bed (pulmonary vascular resistance), the ability of the arterial vessels to accommodate the ejected blood

bolus with a small rise in pressure at the ventricular outlet (i.e. the pulmonary arterial compliance), the inertance of the blood that has to be accelerated during ejection, pulse wave reflections, and pulmonary vascular recruitment [51]. Considering these properties of the pulmonary circulation may help improve prediction of cardiovascular outcome in cardiovascular disease. A simple calculation of pulmonary compliance as the ratio of stroke volume to pulmonary pulse

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pressure has been suggested to have independent and strong prognostic significance in PAH [52, 53]. Future studies will help establish the clinical value of more complex modeling of pulmonary flow and RV-pulmonary artery coupling on the basis of invasively derived pulmonary impedance.

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6.1 Electrocardiography

Although the symptoms of RVF are similar regardless of the cause, e.g. fluid retention or decreased exercise tolerance, careful history-taking and physical examination can usually provide important hints to the cause of RVF. For example, a classic mitral valve murmur (whether systolic or diastolic) will suggest mitral valve disease as a cause of secondary PHT; or a murmur suggesting an atrial septal defect may suggest congenital heart disease. Signs that are important to keep in mind that suggest RVF regardless of the cause include jugular venous distension, right-sided S3 gallop, the presence of right ventricular heave, and the classic murmur of tricuspid regurgitation (holosystolic systolic murmur that increases with inspiration).

ECG is a specific but less sensitive means of diagnosing right venricular hypertrophy and for this reason, although simple, it is not a good screening test (Fig. 4a). Nonetheless, if right venricular hypertrophy is detected, PHT is likely present [54]. Lehtonen et al. compared four sets of ECG criteria for diagnosing right ventricular hypertrophy in patients whose right ventricular thickness was measured at autopsy. Hearts were considered normal if the total weight was less than 250 g, the RV weighed less than 65 g, and the combined LV plus septum weight was less than 190 g (ratio of weight of LV + weight of septum/weight of RV 2.3–3.3). By combining aspects of the four sets of ECG criteria, they achieved a diagnostic sensitivity of 63% and a specificity of 96%. These combined criteria included right-axis deviation greater than 110°, R-wave equal to or greater than S-wave (in V1 or V2), R-wave equal to or less than S-wave in lead V6, a calculated value AR/PL of 0.7 (where A is the maximum R-wave amplitude in lead V1 or lead V2, R is the maximal S-wave amplitude in lead I or lead V6, and PL is the minimal S-wave amplitude in lead I or lead V6). This set of criteria was quite specific, despite the

Fig. 4  Use of echocardiography in assessing right ventricular hypertrophy. A chest X-ray and right ventricular hypertrophy in a patient with pulmonary arterial hypertension and right ventricular hypertrophy. Note the signs of right ventricular hypertrophy on the electrocardiogram and the signs of right ventricular enlargement and pulmonary arterial dilatation on the chest X-ray, as discussed in the text. Representative echocardiographic tracings of tricuspid annular systolic excursion (a, b) and Doppler signals

of the right ventricular outflow and tricuspid regurgitation (c, d). Decreased acceleration time and the presence of pulmonary notch are associated with severer pulmonary vascular disease. Right ventricular myocardial performance index is calculated as the ratio of the isovolumic acceleration and the relaxation time divided by the ejection time. ET ejection time, AT acceleration time, Vmax maximal tricuspid pressure gradient at peak tricuspid regurgitation signal (modal frequency), RV right ventricle

6 History and Physical Examination

94  Right Ventricular Dysfunction in Pulmonary Hypertension

presence of left ventricular hypertrophy and myocardial infarction in the study population. A limitation of this study is that the autopsy was restricted to patients dying from respiratory failure and perhaps these criteria might not be applicable in other causes of PHT. For example, the chest size is enlarged in patients with chronic airway disease and influences the voltage and the axis in ECG. The ECG criteria for right ventricular hypertrophy become less specific in the presence of posterior myocardial infarction, left posterior hemiblock, and Wolff–Parkinson–White syndrome. These conditions, all rare in PHT patients, may cause right-axis deviation and/or a predominant RV in lead V1, similar to the findings in right ventricular hypertrophy. Lastly, patients with right ventricular hypertrophy typically have right ventricular enlargement (known as P-pulmonale), i.e. asymmetrical peaked P-waves greater than 0.25 mV in lead 2.

6.2 Chest X-ray Enlargement of the RV is an important clue to the presence of severe PHT and is suggested by the filling in of the retrosternal space on the lateral view of the chest X-ray (Fig. 4a). Additional signs may suggest a specific cause. For example, “pruning” of the peripheral pulmonary arteries is a classic sign, which reflects vascular obliteration of the small arteries in the periphery of the lungs in PAH. Important signs of secondary PHT can be also documented with a chest X-ray; for example, the presence of septal thickening or pleural effusions will suggest venous hypertension to left ventricular dysfunction, venoocclusive disease, or pulmonary capillary angiomatosis.

6.3 Laboratory Evaluation Obtaining baseline renal and liver function tests and albumin and uric acid levels as well as B-type natriuretic peptide levels may be of particular interest in determining the prognosis of right-sided heart disease [55–61]. Data are also emerging on the prognostic value of renal dysfunction and hyponatremia in patients with PAH [60, 62]. In the last 5 years, studies have also identified interleukin-1 receptor like protein soluble isoform (sST2) as a novel biomarker in heart failure; elevation in the sST2 level predicted mortality in heart failure [63–65]. In mouse models, IL-33/ST2L signaling antagonizes cardiomyocyte hypertrophy induced by transverse aortic constriction and agonists (angiotensin II and phenylepinephrine), while improving systolic function [22, 64]. The value of sST2 is currently being investigated in patients with right-sided heart failure. Other laboratory studies should be individualized depending of the suspected cause of RVF.

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For example, serological tests for HIV and connective tissue disease (e.g. antinuclear antibody) are often routinely performed in patients with PAH. Finally, in patients with edema and severe hypoalbuminemia, protein-losing enteropathy should be excluded with the assay of stool a1-antitrypsin.

6.4 Echocardiography Echocardiography plays a key role in the diagnosis of rightsided heart disease. Signs of right-sided heart disease on an echocardiogram can include right ventricular enlargement, right ventricular systolic dysfunction, tricuspid regurgitation, and PHT, congenital heart defects, valvular heart disease, or left-sided heart disease. Common indices of right ventricular function include right ventricular fractional area change (RVFAC), tricuspid annular plane systolic excursion (TAPSE), and the right ventricular myocardial performance index (RVMPI). RVFAC represents the ratio of systolic area change to diastolic right ventricular area. It is measured in the four-chamber view and can be systematically incorporated in the basic echocardiographic study. In end-stage pulmonary disease, a reasonable correlation has been reported between RVFAC and right ventricular ejection fraction [66]. TAPSE is another useful quantitative measurement of right ventricular systolic performance. TAPSE reflects the longitudinal systolic excursion of the lateral tricuspid valve annulus toward the apex (Fig. 4b). It is usually measured using M-mode imaging in the four-chamber view [67]. Studies showed moderate correlation between TAPSE and right ventricular ejection fraction measured by radionuclide angiography [68]. Finally, RVMPI, which is the ratio of isovolumic time intervals to ventricular ejection time, has been described as a nongeometric index of global ventricular function (Fig. 4) [69]. RVMPI appears to be relatively independent of preload, afterload, and heart rate and has been useful in assessing patients with congenital heart disease and PHT [70–72]. In recent years, a novel noninvasive index of contractility based on the myocardial isovolumic acceleration assessed by tissue Doppler echocardiography has been described (Fig.  5) [47, 73, 74]. In close-chested animal models, isovolumic acceleration was found to reflect right ventricular myocardial contractile function and was less affected by preload and afterload within a physiologic range when compared with either dP/dt max or elastance. Its value was validated clinically in congenital heart disease, i.e. after repair of tetralogy of Fallot and in transposition of great arteries [47, 74]. Further validation of this new index is being actively pursued in PHT and heart failure. Table 2 summarizes common indices used in the study of right ventricular function [41, 69–72, 75–77].

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c­ ardiac output, shunt fraction, and pulmonary vasoreactivity [79]. Left-sided heart catheterization is often performed to precisely measure left ventricular end-diastolic pressure.

6.7 Exercise Testing

Fig. 5  Tissue Doppler echocardiography spectral curve at the level of the proximal right ventricular myocardial proximal wall. The isovolumic myocardial acceleration is calculated as the difference between the baseline and the peak velocity (stars in the right panel) during isovolumic contraction divided by the time interval. (Reproduced from Vogel et al. [74] with permission from Elsevier)

6.5 Magnetic Resonance Imaging MRI is becoming the goal standard for evaluating right-sided heart structure and function. Except when contraindicated, MRI is now the modality of choice for determining right ventricular ejection fraction, a position previously held by radionuclide angiography. Pulmonary angiography may also be performed using MRI. Performing MRI is particularly useful in patients with complex congenital heart defects (e.g. Ebstein’s anomaly, hypoplasic RV), in patients in whom precise quantification of valvular regurgitation is important, in assessment of the pulmonary vasculature or arteriovenous malformation, for planning of complex surgery or procedures, or for research purposes [20]. Recent studies using MRI have also demonstrated the prognostic value of right ventricular end-diastolic volumes and pulmonary compliance assessed by MRI in PAH [52, 78]. Although currently the utilization of MRI is limited by availability and cost, it is possible that the ability of the MRI to offer a “single stop shop” comprehensive assessment of the right ventricular cardiopulmonary unit might in the future prove to be an essential part of the evaluation of RVF (see Chaps. 35, 100).

6.6 Right-Sided Heart Catheterization Right-sided heart catheterization is an important part of the evaluation of right-sided heart disease. Indications for rightsided heart catheterization include assessment of pulmonary vascular resistance or impedance, pulmonary pressures,

Exercise testing is also very useful in objectively assessing clinical deterioration in patients with PAH or congenital heart disease. Caution is, however, advised in performing maximal exercise testing in patients with severe pulmonary vascular disease and RVF (contraindicated in the recent American Heart Association consensus on congenital heart disease) [80]. Other studies should be individualized depending of the suspected cause of RVF. In patients with PAH, a ventilation– perfusion scan, pulmonary function tests, and overnight oximetry are often performed. Lung or heart biopsy is rarely indicated in patients with isolated right-sided heart disease. Genetic counseling should be pursued in patients with congenital heart disease or arrhythmogenic right-sided heart dysplasia. Depending on the cause and severity of RVF, patients are usually followed at differing intervals (usually 3 months to 1 year). The recent guidelines for congenital heart disease and PAH offer individualized timing for follow-up depending on the conditions [80].

7 Managing Right Ventricular Failure Management of RVF should always take into account the cause and setting in which RVF occurs. Figures 6 and 7 summarize the management of acute and chronic RVF. In contrast to patients with chronic ischemic or nonischemic cardiomyopathy, patients with RVF often have significantly abnormal afterload (e.g. PHT) or valvular heart disease (acquired or congenital pulmonary or tricuspid disease). It is therefore not surprising that therapy for RVF should primarily target its cause. It is useful to divide the RVF syndrome into four clinical categories, i.e. biventricular failure, systemic RVF, predominant subpulmonary RVF, and hypoplasic RV syndrome [80]. The management of patients with a hypoplasic RV is beyond the scope of this chapter and the reader is referred to the recent consensus statement of Warnes et al. [80]. As in left-sided heart failure, specific treatment goals include optimization of preload, afterload, and contractility and maintenance of sinus rhythm and atrioventricular synchrony, to increase cardiac output and decrease venous pressure (i.e. right atrial pressure). The management of patients with RVF remains more empiric than the management of patients with left-sided heart failure [16, 17]. Clinical trials of patients with

94  Right Ventricular Dysfunction in Pulmonary Hypertension

Fig.  6  Management of chronic RVF. ACC, American College of Cardiology, AHA American Heart Association, ACEI angiotensin converting enzyme inhibitor, ARB angiotensin receptor blocker, CTEPH

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chronic thromboembolic pulmonary hypertension, ERB, endothelin receptor blockers, PDE5, phosphodiesterase 5, TOF, tetralogy of Fallot, TV, tricuspid valve

Fig. 7  Management of acute RVF. PE pulmonary embolism, PH pulmonary hypertension, PR, pulmonary regurgitation, TR, tricuspid regurgitation, endocarditis, RVAD, right ventricular assist device, ECMO extracorporal membrane oxygenation, TV, tricuspid valve, A-V, atrioventricular

RVF have also not been powered for mortality end points. Among patients with RVF, the evidence is best established for patients with PAH [16, 80, 81]. In PAH it is, however, more difficult to distinguish whether the beneficial effects of therapy are due to changes in pulmonary vasculature or RV-specific effects; we therefore often consider the effects PAH therapy

in the context of the RV-pulmonary artery unit. In patients with congenital heart disease, the effects of therapy have not been consistently studied across functional class severity (New York Heart Association functional class). Because the prevalence of right-sided heart failure is relatively small compared with the prevalence of left-sided heart failure, finding

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appropriate surrogate end points has been an important focus of research [79]. Surrogate end points being considered include exercise capacity, clinical worsening, ventricular remodeling, and measures of vascular impedance in PHT.

8 Chronic Right Ventricular Failure In patients with biventricular failure, the management is based on the recommendations of the American College of Cardiology [16]. In addition to the standard therapies, new therapies are emerging that may be particularly important for patients with biventricular failure. Recent studies have shown that the use of sildenafil may improve pulmonary hemodynamics and exercise capacity in patients with chronic systolic heart failure [82, 83]. Whether sildenafil will improve hemodynamics and outcome of patients with heart failure and PHT or RVF is the subject of ongoing investigation. Table 3 summarizes the results of selected clinical studies on sildenafil in patients with PAH, left-sided heart failure, and chronic thromboembolic disease [84–87]. In patients with predominately RVF, the management is closely related to the cause of RVF (Fig. 6). Patients with PAH benefit from prostanoid therapy, endothelin receptor blockers,

sildenafil, and anticoagulation. Although both endothelin receptor blockers and sildenafil have proven to improve the 6-min walk distance, recent studies suggest that sildenafil may also have positive inotropic effects on the RV [88]. Whether these differences will translate into beneficial long-term effects still remains to be proven. Digoxin has not been shown to acutely improve hemodynamics in patients with right-sided heart failure and PAH; chronic benefits have not been clearly demonstrated (Table 4) [89, 93]. Inhibition of the angiotensin system or beta blockade may be considered in selected patients with systemic RVs although their benefit remains uncertain and controversial [80, 94]. Tables 5 and 6 summarize the clinical evidence on the use of angiotensin system inhibition or beta blockade in patients with right-sided heart failure [95–100]. Resynchronization therapy or right-sided heart pacing may become useful in selected patients with RVF, if further validated. It has currently been tested mainly in patients with congenital heart disease (Table 7) [101–104]. A multicenter trial is currently planned to assess the benefits of resynchronization therapy in patients with PAH. Timely repair of congenital lesions is also essential to avoid progressive right ventricular dysfunction in congenital heart disease. Tricuspid valvuloplasty, either surgical or percutaneous, or cardiac support devices may also have a role in managing chronic RVF in the future.

Table 3  Selected studies on phosphodiesterase type 5 inhibition in patients with right ventricular failure (RVF) or PH Study Study population Characteristics n Design Main findingsa Galiè et al. [84]

PAH

NYHA class II/III MPAP 49–56 mmHg CI 2.2–2.5 L/min/m2 6MWT 339–347 m

278

RCT Orally administered sildenafil vs. placebo 12 weeks

Beneficial effect: increase in 6MWT by 14%. Dose-dependent increase in CI, and decrease in MPAP, PVR, and RAP

Lewis et al. [85]

LHF with PH

NYHA class II/III MPAP 33 mmHg PCWP 19 mmHg Stroke volume 44 mL

34

RCTOrally administered sildenafil vs. placebo 12 weeks

Beneficial effect: increase in peak VO2 by 14%, decrease in exercise stroke volume by 60%, decrease in PVR by 18% at rest and 28% with exercise

Lepore et al. [86] LHF with PH

NYHA class III or IV MPAP 37 mmHg PCWP 22 mmHg CI 2.1 L/min/m2

11

RCT Orally administered sildenafil vs. iNO vs. combination

Additive beneficial effect: decrease in PVR by 50%, decrease in SVR by 24%, increase in CI by 30%, decrease in MPAP 14% (NS)

Ghofrani et al. [87]

CTEPH

NYHA class NA MPAP 52 mmHg CI 2.0 L/min/m2 6MWT 312 m

12

Beneficial effect: increase in 6MWT Prospective study by 17%, increase in CI by 20%, Orally administered decrease in PVR by 30% decrease sildenafil for 6 months in MPAP by 15%

Stocker et al. [123]

CHD

Infants at risk of PH Detrimental effect: decrease in PaO2 16 RT after cardiac surgery by 29.9 mmHg when intravenously iNO and intravenously administered sildenafil given first, Ventilated infants administered sildenafil leading to an early termination MPAP 68 mmHg of the study CI 3.9 L/min/m2 a Unless otherwise specified, the results refer to statistically significant findings (p < 0.05). The changes reported refer to relative changes in the mean effect size or when indicated by changes in the absolute effect size RCT randomized placebo-controlled trial, RT randomized trial, NS not significant; NYHA New York Heart Association (the most common NYHA class reported), MPAP mean pulmonary arterial pressure, PCWP pulmonary capillary wedge pressure, RAP, right atrial pressure, CI, cardiac index, CI cardiac index, CTEPH chronic thromboembolic pulmonary hypertension, VO2 maximal oxygen consumption, iv, intravenous, PAH pulmonary arterial hypertension, 6MWT 6-min walk test, PVR pulmonary vascular resistance, iNO inhaled nitric oxide, SVR systemic vascular resistance Source: Adapted from Skhiri et al. [18] with permission from Elsevier

94  Right Ventricular Dysfunction in Pulmonary Hypertension Table 4  Digoxin therapy in RVF Study Study population

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Characteristics

n

Design Single arm prospective study Digoxin 1 mg intravenously

Rich et al. [89]

Idiopathic PAH

MPAP 60 mmHg PCWP 12 mmHg CO 3.49 L/min

17

Rich et al. [90]

Idiopathic PAH

64

Kawut et al. [91]

Idiopathic PAH

MPAP 57 mmHg CI 2.6 L/min/m2 PVRI 27 units/m2 MPAP 55 mmHg Peak VO2 11.4 mL/kg/min CI 1.8 L/min/m2 Evidence of right ventricular dysfunction but no history of heart failure

Main findingsa

Beneficial acute effect: increased CO by 9%, decreased circulating norepinephrine level by 15% Substudy of a prospective CCB No difference in survival at 5 trial years

84

Retrospective study. Follow-up No difference in survival 764 days

15

Cross-over RCT 8 weeks. Digoxin vs. placebo

No improvement in RVEF in the absence of left ventricular systolic dysfunction Brown et al. [93] COPD Decreased RVEF and no 12 Crossover RCT 2 weeks No significant difference in evidence of CHF digoxin vs. placebo RVEF or exercise duration a Unless otherwise specified, the results refer to statistically significant findings (p < 0.05). The changes reported refer to relative changes in the mean effect size or when indicated by changes in the absolute effect size. CCB calcium channel blockers, COPD, chronic pulmonary obstructive disease, CO cardiac output, PVRI pulmonary vascular resistance, CHF congenital heart failure Source: Adapted from Skhiri et al. [18] with permission from Elsevier Mathur et al. [92]

COPD

Table 5  Angiotensin converting enzyme inhibitors or angiotensin receptor blockers in RHF Study Study population Characteristics n Design Multicenter RCT crossover design Losartan vs. placebo for 15 weeks RCT, crossover design. Losartan vs. placebo for 8 weeks

Main findingsa No difference in peak VO2, exercise duration or NT-proBNP levels

Dore et al. [95]

CHD

Systemic right ventricle (TGA) Majority NYHA class I RVEF 42%

29

Lester et al.[96]

CHD

D-TGA (atrial baffle) Age > 13 years NYHA class < IV RVEF 48%

7

Robinson et al. [97]

CHD

8

Single arm prospective study Enalapril for 1 year

Therrien et al. [98]

CHD

17

Multicenter RCT No difference in RVEF, right ventricular volume, or Ramipril vs. placebo for 1 year peak VO2

Hechter et al. [99]

CHD

D-TGA (atrial baffle) NYHA class I Age 7–21 years CI 2.2 L/min/m2 D-TGA (atrial baffle) Mainly NYHA class I Age > 18 years RVEF 44% D-TGA (atrial baffle) Age > 26 years RVEF 47% Left ventricular fractional shortening of 33% TTPG 43 mmHg

26

Retrospective study ACE-I from 6 to 126 months

Beneficial effect: increase in exercise time by 18%, decrease in regurgitant volume by 63.5%, increase in RVEF by 6% ‡ No difference in exercise duration, peak VO2, or cardiac index

No difference in peak VO2 or exercise duration

No difference in TTPG or exercise duration but subgroup of patients who were better responders a Unless otherwise specified, the results refer to statistically significant findings (p < 0.05). The changes reported refer to relative changes in the mean effect size or when indicated by the ‡ changes in the absolute effect size. D-TGA dextrotransposition of great arteries; TTPG transtricuspid pressure gradient, NT-proBNP N-terminal pro Brain Natriuretic Peptide Source: Adapted from Skhiri et al. [18] with permission from Elsevier Morrell et al. [100]

COPD with PH

In patients with advanced refractory RVF, transplantation can be considered after exclusion of all reversible causes and careful consideration of contraindications. In appropriate patients with severe pulmonary vascular disease, heartlung transplantation or double lung transplantation is

40

RCT Losartan vs. placebo for 48 weeks

considered [105]. Because of the scarcity of organs, ­heart-lung transplantation is usually considered only in patients with congenital heart defects and in patients in whom the physician considers recovery of right-sided heart function unlikely. Predictors of persistent RVF after double

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Table 6  Bata blockade in patients with RVF Study Study population Characteristics Beck-da-Silva et al. [124] Quaife et al. [125] Provencher et al. [126]

Shaddy et al. [127]

Doughan et al. [128]

NYHA class II–III RVEF 31% LVEF 22% NYHA class II–III Biventricular heart RVEF 27% failure LVEF 23% Portopulmonary NYHA class III hypertension MPAP 52 mmHg CO 5.24 L/min 6WMT 338 m CHD or LHF Dilated CM and systemic right ventricle Age < 13 years LVEF 27% NYHA class II/III CHD D-TGA (atrial baffle) Adults RVEF 35% NYHA class I/III

Biventricular heart failure

n

Design

Main findingsa

30

Prospective study Bisoprolol for 4 months

Beneficial effect: increase in RVEF by 7.1%‡ and decrease in LVEF by 7.9%‡

22

RCT. Carvedilol vs. placebo for 4 months

Beneficial effect: increase in RVEF by 11%‡. Prevents further chamber dilatation

10

Prospective study Propranolol or atenolol

Detrimental effect: increase in CO by 28%, increase in 6MWT by 79 m‡ at 1 month once beta-blockers had bean withdrawn

161

No difference. In patients with Multicenter RCT systemic left ventricle, a trend Carvedilol vs. placebo for toward improvement was noted 8 months

60

Retrospective study. Carvedilol or metoprolol for 4 months

Beneficial effect: decrease in NYHA class especially if there was a pacemaker or initially NYHA class III Prevents further chamber dilatation Beneficial effect: decreased NYHA class in 5 patients

8 Retrospective study. D-TGA (atrial baffle) Median follow-up of NYHA mainly class II, Adults 3 years. Carvedilol Right ventricular diameter metoprolol, sotalol 41.5 mm Beneficial effect: decrease in RVED Giardini CHD D-TGA (atrial baffle), L-TGA 8 Single arm prospective volume by 6%, increase in RVEF et al. [130] study Age > 18 years by 6% ‡. No change in peak VO2 Carvedilol for 12 months NYHA class II and III RVEF 34% No difference in peak VO2, RVEF, Norozi CHD Tetralogy of Fallot 33 RCT et al. [131] ventricular volumes, NYHA class NYHA class I and II Bisoprolol vs. placebo for 6 months Adults LVEF 57% CI 3.8 L/min/m2 a Unless otherwise specified, the results refer to statistically significant findings (p < 0.05). The changes reported refer to relative changes in the mean effect size or when indicated by a double dagger changes in the absolute effect size. CI cardic index, RVED, right ventricular end-diastolic, L-TGA levotranspoition of great arteries, LVEF left ventricular ejection fraction Source: Adapted from Skhiri et al. [18] with permission from Elsevier Josephson et al. [129]

CHD

Table 7  Resynchronization therapy in patients with RHF [101–104] Study Study population Characteristics Janousek et al. [104]

CHD

Dubin et al. [103]

CHD

Janousek et al. [102] Dubin et al. [101]

CHD

Children with acute postoperative heart failure with conduction delay Chronic right ventricular dysfunction RBBB Tetralogy of Fallot, aortic stenosis after Ross procedure D-TGA (atrial baffle) L-TGA CHD and dilated CMP and congenital atrioventricular. block

n

Design

Main findingsa

20

Prospective study

7

Prospective study Multipacing sites, conductance catheter

Beneficial effects. Improvement in hemodynamics (systolic blood pressure) Beneficial effect. Atrioventricular block augments right ventricular and systemic performance (increase in CI by 17 ± 8%,increase in right ventricular dP/dt)

8

Prospective

Beneficial effect. increase in RVEF by 9.6%, decrease in RVMPI by 7.7% 103 Multicenter study Beneficial effects in all 3 groups. Average CHD and (22 centers) increase in election fraction of pediatric 11.9–16.1%. Responders had a lower baseline ejection fraction a Unless otherwise specified, the results refer to statistically significant findings (p < 0.05). The changes reported refer to relative changes in the mean effect size. RBBB, CMP cardiomyopathy dapted from Skhiri et al. [18] with permission from Elsevier

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lung transplantation have, however, are not well established at this time. The observation of improved survival of patients with PAH and patent foramen ovale has led to the hypothesis that atrial septostomy, which “decompresses” the RV and increases right-to-left shunting, could be helpful in severe RVF. The response to atrial septostomy in PAH is variable. At this time, atrial septostomy should be considered palliative [106].

or worsening ventilation–perfusion mismatches. In patients with acute right-sided hear failure refractory to medical treatment, mechanical support of the RV is sometimes used as a bridge to transplantation or a bridge to recovery. The most common indications for right ventricular assist device use are severe RVF after left ventricular assist-device use, RVF after heart transplantation, and RVF after massive pulmonary embolism [117].

9 Acute Right Ventricular Failure

10 Therapies Targeting the Right Ventricle

Acute RVF may occur in patients with established chronic right ventricular dysfunction or as a result of an acute event such as pulmonary embolism, after cardiotomy, or postsurgical compression of the pulmonary artery. In the acute setting, every effort should be made to minimize hypotension. Systemic hypotension may lead to a vicious circle of ischemia and circulatory failure. Patients with acute RVF are also very vulnerable to loss of sinus rhythm or atrioventricular synchrony. Excessive volume loading should be avoided as it may increase pericardial constraint and decrease left ventricular preload and cardiac output through the mechanisms of ventricular interdependence. Inotropic therapy is indicated in patients with acute rightsided heart failure and signs of low cardiac output. Among the inotropic or vasopressor agents, dobutamine has been the most extensively studied in RVF [107, 108]. In right ventricular myocardial infarction, dobutamine increased cardiac index and stroke volume while maintaining preload [107]. In PAH, dobutamine at dosages of 2–5 mg/kg/min increases cardiac output while decreasing pulmonary vascular resistance [108]. The combination of dobutamine and inhaled nitric oxide in PHT has also been shown to increase cardiac index, decreased pulmonary vascular resistance indices, and significantly increased the PaO2/FiO2 ratio [108]. Dopamine use is often reserved for hypotensive patients, whereas milrinone is preferred in the presence of tachyarrythmias. Although epinephrine infusion is commonly used in the intensive care unit, its specific effects in PHT have not been well studied. In patients with pure RVF, the effects of several of these inotropic agents on the normal LV (for example, tachycardia and decrease in the left ventricular filling time, particularly if the left ventricular volume is already restricted by fight ventricular dilatation) might have adverse effects and actually decrease cardiac output. Continuous hemodynamic monitoring is therefore strongly advised. In the absence of RV-specific inotropic therapies, the management of such patients will remain quite challenging. Nitric oxide may also be beneficial in patients with acute RVF [109–116]. Inhaled nitric oxide could provide selective pulmonary vasodilatation without causing systemic hypotension

Currently, specific RV-targeting therapies are being actively investigated. Such therapies are desirable in specific clinical settings of predominant or primary RVF. For example, in the failing RV after lung transplantation, large pulmonary embolism, or PAH, attempts to increase right ventricular contractility using inotropes are limited by their adverse effects on the LV and systemic circulation, as discussed already. Targeting mechanisms that are abnormal only in the hypertrophied RV and pulmonary circulation and that do not affect or minimally affect the LV and systemic circulation are thus ideal therapeutic strategies [118]. There are currently two such therapies that appear to fulfill these criteria: (1) metabolic modulators and (2) phosphodiesterase type 5 (PDE5) inhibitors (Fig. 8). The small molecule dichloroacetate (DCA) is a metabolic modulator that reverses the mitochondrial hyperpolarization and the glycolytic phenotype in cancer [119], the remodeled pulmonary arteries in PAH [120], and the hypertrophied RV [28]. As a consequence, DCA decreases cancer growth, decreases pulmonary artery remodeling, and increases right ventricular inotropy. Importantly, DCA has no significant effects in noncancer tissues, the nonhypertrophied myocardium, and the systemic nondiseased blood vessels, since the metabolism of these tissues is not altered. It is thus a potentially ideal therapy for PAH or possibly acute RVF. Clinical trials of DCA in cancer and PAH are currently ongoing. The ability to monitor the effectiveness of a metabolic intervention using in  vivo imaging (PET; Fig.  8) enhances the feasibility of such a therapy. Other clinically used metabolic modulators that have the same metabolic effects as DCA, such as ranolazine and trimetazidine, might have similar beneficial effects on the contractility of the diseased RV. Phosphodiesterase inhibitors have also been shown to specifically target the RV. Although expression of PDE5 is minimal in the normal RV (where it is only expressed in the smooth muscle cells of the coronary arteries), its expression is markedly induced in the hypertrophied RV in rat and humans (as well as in the neonatal RV). It is possible that its upregulation is a part of the stressed myocardium and it

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Fig. 8  Targeted right ventricular therapy. Summary of the changes in the expression of phosphodiesterase inhibitors and metabolism observed in the hypertrophied right ventricle and comparison of the effects on targeted right ventricular therapy. The lower-right panel shows immunohistochemistry of right ventricle biopsies showing increased phosphodiesterase type 5 expression (green) in a hypertrophied right ventricle compared with a normal right ventricle; RV right ventricle, MHC myosin heavy chain, DAPI 4¢,6-diamidino-2-phenylindole. (Adapted with permission from Archer and Michelakis [118] copyright Massachusetts Medical Society. All rights reserved)

p­ arallels the induction of brain natriuretic peptide (BNP) expression in the hypeptrophied myocardium [88]. PDE5 is also induced in the remodeled pulmonary arteries in PAH. PDE5 inhibitors have beneficial effects in the right ventricular afterload by dilating and reversing the remodeled pulmonary arteries, with minimal effects on the systemic arteries (other than the penile circulation). Recently, they were also found to increase contractility in the hypertrophied RV in rat hearts [88]. Subsequently, confirmation in patients where right ventricular contractility was measured invasively was reported [121]. The previously unrecognized effects of PDE5 inhibitors on the RV remind us that the effects of other therapies used in PAH also have to be studied on the RV. Lack of effects on the normal RV or the LV does not exclude effects (beneficial or adverse) on the RV, where upregulation or downregulation of drug targets may also take place, For example, the effects of endothelin receptor antagonists on the RV need to be studied given the fact that these drugs are used widely in PAH patients but have actually worsened morbidity and mortality in left ventricular failure [122]. Other investigational drugs currently in clinical trials for PAH (e.g. imatinib) have also been reported to have toxic effects on the LV and thus their potential effects on the diseased RV cannot be ignored.

Future therapies in RVF may target either a differentially expressed pathway of the LV or common ventricular pathways. Other attractive potential targets for therapy may include the mitochondria, the extracellular matrix, microRNA regulation, and neurohormonal or immune emodulation. The possibility of inducing a more compensated (with some similarities to the fetal phenotype) metabolic and molecular phenotype in the adult diseased RV will remain an intriguing goal of research.

11 Pearls and Pitfalls Management of RVF may be at the same time simple and complex. To optimize patient care, several pitfalls should be avoided. Among them, the most common ones include (1) ordering maximal exercise testing in patients with severe pulmonary vascular disease and RVF, (2) failure to exclude chronic thromboembolic disease as a cause of PHT, (3) delaying referral to a specialized center for appropriate surgical, interventional, or medical therapy, (4) excessively volume loading a patient with acute RVF, and (5) closing an atrial septal defect in a patient with severe pulmonary vascular

94  Right Ventricular Dysfunction in Pulmonary Hypertension

d­ isease. Caution should also be advised when using inhaled nitric oxide or sildenafil in patients with severely elevated left ventricular filling pressures as increased cardiac output can precipitate pulmonary edema.

12 Conclusion Right ventricular dysfunction is an important predictor of functional capacity and survival in patients with cardiovascular disease. RVF is now a priority for preclinical and translational research in congenital heart disease and PAH. A  better understanding of the mechanisms underlying the transition from compensated right ventricular hypertrophy to maladaptive remodeling and dilatation could lead to the development of RV-specific therapies. A comprehensive approach to the RV-pulmonary circulation unit may facilitate the translation of experimental therapies for PAH to humans and will likely change our current diagnostic and imaging approach to RVF [23].

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F. Haddad et al. 101. Dubin AM, Janousek J, Rhee E, Strieper MJ, Cecchin F, Law IH, Shannon KM, Temple J, Rosenthal E, Zimmerman FJ, Davis A, Karpawich PP, Al AA, Vetter VL, Kertesz NJ, Shah M, Snyder C, Stephenson E, Emmel M, Sanatani S, Kanter R, Batra A, Collins KK (2005) Resynchronization therapy in pediatric and congenital heart disease patients: an international multicenter study. J Am Coll Cardiol 46(12):2277–2283 102. Janousek J, Tomek V, Chaloupecky VA, Reich O, Gebauer RA, Kautzner J, Hucin B (2004) Cardiac resynchronization therapy: a novel adjunct to the treatment and prevention of systemic right ventricular failure. J Am Coll Cardiol 44(9):1927–1931 103. Dubin AM, Feinstein JA, Reddy VM, Hanley FL, Van Hare GF, Rosenthal DN (2003) Electrical resynchronization: a novel therapy for the failing right ventricle. Circulation 107(18):2287–2289 104. Janousek J, Vojtovic P, Hucin B, Tlaskal T, Gebauer RA, Gebauer R, Matejka T, Marek J, Reich O (2001) Resynchronization pacing is a useful adjunct to the management of acute heart failure after surgery for congenital heart defects. Am J Cardiol 88(2):145–152 105. McLaughlin VV, Rich S (2004) Pulmonary hypertension. Curr Probl Cardiol 29(10):575–634 106. Sandoval J, Gaspar J, Pulido T, Bautista E, Martinez-Guerra ML, Zeballos M, Palomar A, Gomez A (1998) Graded balloon dilation atrial septostomy in severe primary pulmonary hypertension. A therapeutic alternative for patients nonresponsive to vasodilator treatment. J Am Coll Cardiol 32(2):297–304 107. Dell’Italia LJ, Starling MR, Blumhardt R, Lasher JC, O’Rourke RA (1985) Comparative effects of volume loading, dobutamine, and nitroprusside in patients with predominant right ventricular infarction. Circulation 72(6):1327–1335 108. Vizza CD, Rocca GD, Roma AD, Iacoboni C, Pierconti F, Venuta F, Rendina E, Schmid G, Pietropaoli P, Fedele F (2001) Acute hemodynamic effects of inhaled nitric oxide, dobutamine and a combination of the two in patients with mild to moderate secondary pulmonary hypertension. Crit Care 5(6):355–361 109. Inglessis I, Shin JT, Lepore JJ, Palacios IF, Zapol WM, Bloch KD, Semigran MJ (2004) Hemodynamic effects of inhaled nitric oxide in right ventricular myocardial infarction and cardiogenic shock. J Am Coll Cardiol 44(4):793–798 110. Morales-Blanhir J, Santos S, de JL, Sala E, Pare C, Roca J, Rodriguez-Roisin R, Barbera JA (2004) Clinical value of vasodilator test with inhaled nitric oxide for predicting long-term response to oral vasodilators in pulmonary hypertension. Respir Med 98(3):225–234 111. Bocchi EA, Bacal F, Auler Junior JO, Carmone MJ, Bellotti G, Pileggi F (1994) Inhaled nitric oxide leading to pulmonary edema in stable severe heart failure. Am J Cardiol 74(1):70–72 112. Rea RS, Ansani NT, Seybert AL (2005) Role of inhaled nitric oxide in adult heart or lung transplant recipients. Ann Pharmacother 39(5):913–917 113. Khan TA, Schnickel G, Ross D, Bastani S, Laks H, Esmailian F, Marelli D, Beygui R, Shemin R, Watson L, Vartapetian I, Ardehali A (2009) A prospective, randomized, crossover pilot study of inhaled nitric oxide versus inhaled prostacyclin in heart transplant and lung transplant recipients. J Thorac Cardiovasc Surg 138(6):1417–1424 114. Wagner F, Dandel M, Gunther G, Loebe M, Schulze-Neick I, Laucke U, Kuhly R, Weng Y, Hetzer R (1997) Nitric oxide inhalation in the treatment of right ventricular dysfunction following left ventricular assist device implantation. Circulation 96(Suppl 9):II-6 115. Argenziano M, Choudhri AF, Moazami N, Rose EA, Smith CR, Levin HR, Smerling AJ, Oz MC (1998) Randomized, double-blind trial of inhaled nitric oxide in LVAD recipients with pulmonary hypertension. Ann Thorac Surg 65(2):340–345 116. Fattouch K, Sbraga F, Bianco G, Speziale G, Gucciardo M, Sampognaro R, Ruvolo G (2005) Inhaled prostacyclin, nitric

94  Right Ventricular Dysfunction in Pulmonary Hypertension oxide, and nitroprusside in pulmonary hypertension after mitral valve replacement. J Card Surg 20(2):171–176 117. Kaul TK, Fields BL (2000) Postoperative acute refractory right ventricular failure: incidence, pathogenesis, management and prognosis. Cardiovasc Surg 8(1):1–9 118. Archer SL, Michelakis ED (2009) Phosphodiesterase type 5 inhibitors for pulmonary arterial hypertension. N Engl J Med 361(19): 1864–1871 119. Bonnet S, Archer SL, Allalunis-Turner J, Haromy A, Beaulieu C, Thompson R, Lee CT, Lopaschuk GD, Puttagunta L, Bonnet S, Harry G, Hashimoto K, Porter CJ, Andrade MA, Thebaud B, Michelakis ED (2007) A mitochondria-K + channel axis is suppressed in cancer and its normalization promotes apoptosis and inhibits cancer growth. Cancer Cell 11(1):37–51 120. McMurtry MS, Bonnet S, Wu X, Dyck JR, Haromy A, Hashimoto K, Michelakis ED (2004) Dichloroacetate prevents and reverses pulmonary hypertension by inducing pulmonary artery smooth muscle cell apoptosis. Circ Res 95(8):830–840 121. Tedford RJ, Hemnes AR, Russell SD, Wittstein IS, Mahmud M, Zaiman AL, Mathai SC, Thiemann DR, Hassoun PM, Girgis RE, Orens JB, Shah AS, Yuh D, Conte JV, Champion HC (2008) PDE5A inhibitor treatment of persistent pulmonary hypertension after mechanical circulatory support. Circ Heart Fail 1(4):213–219 122. Rich S, McLaughlin VV (2003) Endothelin receptor blockers in cardiovascular disease. Circulation 108(18):2184–2190 123. Stocker C, Penny DJ, Brizard CP, Cochrane AD, Soto R, Shekerdemian LS (2003) Intravenous sildenafil and inhaled nitric oxide: a randomised trial in infants after cardiac surgery. Intensive Care Med 29(11):1996–2003 124. Beck-da-Silva L, de BA, Davies R, Chow B, Ruddy T, Fraser M, Struthers C, Haddad H (2004) Effect of bisoprolol on right ven-

1331 tricular function and brain natriuretic peptide in patients with heart failure. Congest Heart Fail 10(3):127–132 125. Quaife RA, Christian PE, Gilbert EM, Datz FL, Volkman K, Bristow MR (1998) Effects of carvedilol on right ventricular function in chronic heart failure. Am J Cardiol 81(2):247–250 126. Provencher S, Herve P, Jais X, Lebrec D, Humbert M, Simonneau G, Sitbon O (2006) Deleterious effects of beta-blockers on exercise capacity and hemodynamics in patients with portopulmonary hypertension. Gastroenterology 130(1):120–126 127. Shaddy RE, Boucek MM, Hsu DT, Boucek RJ, Canter CE, Mahony L, Ross RD, Pahl E, Blume ED, Dodd DA, Rosenthal DN, Burr J, LaSalle B, Holubkov R, Lukas MA, Tani LY (2007) Carvedilol for children and adolescents with heart failure: a randomized controlled trial. JAMA 298(10):1171–1179 128. Doughan AR, McConnell ME, Book WM (2007) Effect of beta blockers (carvedilol or metoprolol XL) in patients with transposition of great arteries and dysfunction of the systemic right ventricle. Am J Cardiol 99(5):704–706 129. Josephson CB, Howlett JG, Jackson SD, Finley J, Kells CM (2006) A case series of systemic right ventricular dysfunction post atrial switch for simple D-transposition of the great arteries: the impact of beta-blockade. Can J Cardiol 22(9):769–772 130. Giardini A, Lovato L, Donti A, Formigari R, Gargiulo G, Picchio FM, Fattori R (2006) A pilot study on the effects of carvedilol on right ventricular remodelling and exercise tolerance in patients with systemic right ventricle. Int J Cardiol 131. Norozi K, Bahlmann J, Raab B, Alpers V, Arnhold JO, Kuehne T, Klimes K, Zoege M, Geyer S, Wessel A, Buchhorn R (2007) A prospective, randomized, double-blind, placebo controlled trial of beta-blockade in patients who have undergone surgical correction of tetralogy of Fallot. Cardiol Young 17(4):372–379

Chapter 95

Large Vessel Pulmonary Arteritis Kim M. Kerr

Abstract  Takayasu’s arteritis (TA) and giant cell arteritis (GCA) are the most well known large vessel vasculitides. Although similar in histologic appearance, they have been divided into two distinct clinical entities on the basis of their age of onset, clinical presentation, and distribution of vascular involvement. Although both may involve large and mediumsized arteries, including the aorta and its branches, the vast majority of large vessel pulmonary arteritis cases reported in the literature are attributed to TA. TA will therefore be the primary focus of this chapter. TA is named after Mikito Takayasu, a Japanese ophthalmologist who first reported a case of a 21-year-old woman with an interesting fundoscopic finding of coronary anastomosis of the retinal central vessels. Takayasu did not report on any other physical findings in the patient during his presentation at the Ophthalmology Society in Japan in 1905. However, at this same meeting two other ophthalmologists, Onishi and Kagoshima, presented two additional cases with the same retinal abnormalities associated with absent radial pulses. TA is now recognized to be a vasculitis whose characteristic ophthalmic findings are a result of retinal artery ischemia. TA is an acute and chronic form of vasculitis of the elastic arteries resulting in stenosis, occlusion, and occasionally aneurysmal changes of the aorta, its main branches, pulmonary arteries, and coronary arteries. It is a disease that predominantly affects young women, with symptoms determined by the distribution of the vessels involved, the type of vascular lesion, and the presence of active inflammation. Treatment strategies are focused on suppressing inflammation with corticosteroids or other immunosuppressive agents, but revascularization procedures may be required in some patients. Keywords  Large vessel vasculitis • Pulmonary artery • Stenosis • Occlusion • Pulmonary vascular lesion • Inflammation

1  Introduction Takayasu’s arteritis (TA) and giant cell arteritis (GCA) are the most well known large vessel vasculitides. Although similar in histologic appearance, they have been divided into two distinct clinical entities on the basis of their age of onset, clinical presentation, and distribution of vascular involvement [1]. Although both may involve large and mediumsized arteries, including the aorta and its branches, the vast majority of large vessel pulmonary arteritis cases reported in the literature are attributed to TA. TA will therefore be the primary focus of this chapter. TA is named after Mikito Takayasu, a Japanese ophthalmologist who first reported a case of a 21-year-old woman with an interesting fundoscopic finding of coronary anastomosis of the retinal central vessels. Takayasu did not report on any other physical findings in the patient during his presentation at the Ophthalmology Society in Japan in 1905. However, at this same meeting two other ophthalmologists, Onishi and Kagoshima, presented two additional cases with the same retinal abnormalities associated with absent radial pulses. TA is now recognized to be a vasculitis whose characteristic ophthalmic findings are a result of retinal artery ischemia [2]. TA is an acute and chronic form of vasculitis of the elastic arteries resulting in stenosis, occlusion, and occasionally aneurysmal changes of the aorta, its main branches, pulmonary arteries, and coronary arteries. It is a disease that predominantly affects young women, with symptoms determined by the distribution of the vessels involved, the type of vascular lesion, and the presence of active inflammation. Treatment strategies are focused on suppressing inflammation with corticosteroids or other immunosuppressive agents, but revascularization procedures may be required in some patients [3, 4].

2  Etiology and Pathogenic Mechanisms K.M. Kerr (*) Division of Pulmonory Critical Care Medicine, University of California, San Diego, La Jolla, CA 92037-7381, USA e-mail: [email protected]

Despite a number of advances in the understanding of TA, the etiologic mechanisms are unclear. TA is an autoimmune disease in which the etiology appears to be multifactorial and involve

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_95, © Springer Science+Business Media, LLC 2011

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primarily cellular immunity [3]. Inflammatory infiltrates with occasional giant cell formation involving the media and adventitia of large and medium-sized arteries are the histologic features of TA [5]. Infectious agents such as bacteria, mycobacteria, treponemes, and viruses have all been impugned as causative agents of TA and other primary vasculitides, but no specific microorganism has been confirmed to induce TA [4, 6]. The role of humoral immunity in TA remains unclear. Autoantibodies against aortic endothelial cells have long been reported in TA; however, the precise antigen targeted remains to be identified [3]. Chauhan et al. [7] demonstrated that most TA patients have antiaortic endothelial cell antibodies (AAECAs) directed against the 60–65-kDa heatshock proteins. Exposing aortic endothelial cells to the sera from patients with AAECAs results in a significant increase in expression of E-selectin and vascular cell adhesion ­molecule-1, and increased production of interleukin (IL)-4, IL-6, and IL-8, as well as induction of apoptosis of aortic endothelial cells [7]. Presently, there is no evidence that AAECAs cause TA, but they may be a response to the enhanced expression of heat-shock proteins in the aorta which may be induced by other mechanisms [4]. Examination of the cellular composition of the arterial wall in TA reveals mainly gd T cells, natural killer (NK) cells, cytotoxic T cells, T-helper cells, macrophages, and dendritic cells. T cells, NK cells, and cytotoxic T cells express and release large amounts of perforin (a membrane-disrupting protein) onto the surface of arterial vascular cells. Major histocompatibility class I chain-related A (MICA), a ligand for receptors expressed on NK and gd T cells, is induced by cellular stress and appears to be induced in the aortic tissue of patients with TA. Engagement of MICA by the cell receptors triggers cytotoxic responses of NK and gd T cells [8]. Patients with TA have an increased serum concent­ ration of IL-6, RANTES (regulated on activation, normal ­T-cell-expressed and secreted), a chemokine that attracts mononuclear cells [9], and IL-8 [10] which correlates with disease activity. RANTES and IL-6 can induce production of matrix metalloproteinases (MMP), proteolytic enzymes than can degrade the arterial wall components elastin and collagen. TA patients exhibit higher levels of MMP-3 and MMP-9 compared with controls. IL-6, RANTES, and IL-8 are thought to contribute to TA vessel wall inflammation and destruction by the recruitment and activation of mononuclear cells and by the induction of MMP in the vascular wall [3]. Tripathy et  al. [11] demonstrated that peripheral blood monocytes from patients with TA show higher messenger RNA gene expression of TNF-a, IFN-d, IL-2, IL-3, IL-4, and IL-12 after lipopolysaccharide stimulation than controls. Verma et al. reported a significant elevation in plasma IL-12 levels in TA patients, with levels correlated to disease activity [12]. Park et al. reported that IL-6 and IL-18 levels were elevated in the serum of TA patients and that IL-18, but not IL-12, levels correlated with disease activity [13]. The contradictory findings in

K.M. Kerr

these two studies may be due to ethnic variation in TA or to the fact that the increased levels of these cytokines is simply a reflection of inflammation and is not specific to TA [4]. What triggers the development of TA remains unknown. It appears to be a complex autoimmune disease involving cellular immunity. An improved understanding of the pathophysiology of TA will lead to more specific and effective therapies for the treatment of TA.

3  Epidemiology The incidence and prevalence of TA is unclear owing to lack of reliable data. The lack of rigid diagnostic criteria and perhaps genetic differences may explain differences in reported prevalence. A population study of Olmsted County, Minnesota, found three cases over a 13-year period, yielding an incidence rate of 2.6 cases per million individuals per year [14]. A similar epidemiology study in Sweden identified a yearly prevalence of 6.4 cases per million population [15]. Series from Japan, China, and India have reported patient numbers much greater than those from Europe and North America, suggesting that the disease is more common in Asia [16]. Regional differences in phenotypic patterns have also been identified. Patients in India and China have a higher incidence of abdominal aortic involvement, whereas Japanese patients have a significantly higher frequency of aortic regurgitation due to dilation of the ascending aorta [17]. TA is typically a disease of young women, but can occur in childhood or in adults of more advanced age. A report of 60 cases from the National Institutes of Health (NIH) found that 97% of the patients were female, with onset of the first symptoms at a median age 25 years (range 7–64 years). Nineteen patients (32%) were juveniles at disease onset and 13% of patients had onset of their disease after the age of 40 years [18]. The Italian Takayasu’s Arteritis (ITAKA) study group reviewed 104 Italian patients with TA. They found over 87% of the patients to be female, with a mean age of disease onset of 29 years, although 17% of the cohort experienced initial symptom onset at more than 40 years of age and 15% were under 15 years of age [19]. A study of the clinical characteristics of 108 patients with TA in Korea excluded patients with disease onset after the age of 40 years. Women constituted 84% of the study population and the mean age at the onset of disease was 30 years [20].

4  Clinical Manifestations TA is a chronic, idiopathic, systemic inflammatory disease with a predilection for arteries with elastic tissue in their walls such as the aorta, its main branches, and pulmonary arteries.

95  Large Vessel Pulmonary Arteritis

It has classically been described as a disease that progresses through three stages: (1) an early systemic illness with constitutional symptoms such as fever, weight loss, night sweats, and arthralgias; (2) a vascular inflammatory phase; and (3) an inactive or “burned out” phase. However, many patients may present for medical care with symptoms of vascular inflammation without a prodrome of constitutional symptoms. NIH investigators found that only 33% of their cohort of 60 patients with TA recalled having constitutional symptoms at the time of presentation and systemic symptoms occurred at different times (early to late) in their disease course. Furthermore, 18% never progressed to a “burned out” phase [18, 21]. Most symptoms are related to arterial insufficiency and the clinical presentation reflects the location and extent of arterial lesions and the presence of collateral circulation. Park et al. found in 108 Korean patients with TA that of the main branches of the aorta the subclavian artery was most commonly involved, followed by the renal artery and the common carotid artery [20]. Patients may present with arm claudication or numbness with involvement of the upper extremities. Subclavian artery involvement should be suspected when bruits are auscultated over the subclavian artery or blood pressure discrepancy is noted between arms. Neurologic complaints are common and include headache, visual disturbances, light-headedness, seizures, transient ischemic attacks, and stroke [18]. These symptoms are related to carotid and vertebral artery disease and hypertension. Involvement of the ascending aorta results in aortic insufficiency in up to one quarter of the patients and congestive heart failure in up to half of patients with TA. Heart failure may be caused by aortic regurgitation, hypertension, and coronary artery disease. Lower-extremity claudication with diminished or absent pulses reflecting disease of the distal aorta and its branches may be distinguished from atherosclerotic disease by its development in younger patients lacking risk factors for atherosclerosis. Narrowing of the abdominal aorta or disease involvement of the renal arteries commonly results in hypertension [14, 18, 22, 23]. Pulmonary artery involvement is common in TA, ranging from 1 to 86% incidence, depending upon diagnostic criteria and study design [20, 24, 25]. Although pulmonary artery manifestations of TA are frequently overshadowed by the involvement of systemic large arteries, pulmonary artery involvement may be the initial manifestation of TA and occur in the absence of systemic artery involvement. Making the correct diagnosis in these patients is particularly challenging and many are initially misdiagnosed with a more common alternative disease such as acute or chronic thromboembolic disease [26–28]. Patients may present with dyspnea, chest pain, and hemoptysis of acute onset or duration of months to years. Bruits over the lung fields due to turbulent flow through the involved pulmonary arteries may be noted. Such bruits can also be heard in other diseases involving the pulmonary arteries such as

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chronic thromboembolic disease, fibrosing mediastinitis, and pulmonary artery sarcoma. However, the finding of bruits over systemic arteries or absent pulses is helpful in distinguishing TA from competing diseases that also cause pulmonary bruits. Pulmonary examination may also reveal findings consistent with pulmonary hypertension or pleural effusion [26]. Clinically silent pulmonary artery involvement is common. Tyagi performed right-sided heart catheterization and pulmonary angiography on 38 patients with known TA. Ten (23.7%) of the patients had pulmonary artery involvement, yet only one of the patients had a clinical sign of pulmonary artery involvement (a pulmonary bruit) prior to evaluation. Pulmonary artery occlusion was the most common angiographic finding, followed by stenotic lesions. Although nine of the patients had pulmonary hypertension defined as a mean pulmonary artery pressure greater than 20  mmHg, only three had precapillary pulmonary hypertension, and all cases were described as mild. Four of the ten patients had a demonstrable gradient of more than 15 mmHg in the pulmonary arteries [29]. Small series of patients with TA and significant precapillary pulmonary hypertension have been published [26, 30].

5  Diagnosis 5.1  Clinical Criteria The diagnosis of TA is challenging owing to the variable symptoms and absence of pathognomonic features. It is based upon clinical symptoms, physical findings, and ­evidence of large vessel involvement on radiologic imaging. Several criteria have been published to improve the diagnosis­of TA and are summarized in Table 1. Ishikawa published criteria in 1988 based upon 108 Japanese patients [31]; 96 with TA and 12 with other diseases of the aorta. The criteria consist of one obligatory criterion (age less than 40 years), two major criteria, and nine minor criteria. The American College of Rheumatology (ACR) published its own criteria for North American patients in 1990 [32]. Sharma et al. modified the criteria proposed by Ishikawa to include patients of age greater than 40 years to prevent the possibility of underdiagnosis of TA [33]. Using the criteria of Ishikawa or Sharma et  al. one can diagnose TA when a patient fulfills two major criteria or one major criterion and two minor criteria or four minor criteria. The sensitivity of Ishikawa’s criteria ranges from 60 to 96%, whereas it is 92–96% utilizing the criteria of Sharma et al. depending upon the ethnicity of the patients studied [34]. The presence of three or more of the six criteria proposed by the ACR demonstrated a sensitivity of 90.5% [32].

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Table 1  Diagnostic criteria for Takayasu’s arteritis Ishikawa’s criteria ACRa criteria Sharma et al.’s criteria Age <40 years Major criteria 1. Left mid subclavian artery lesion 2. Right mid subclavian artery lesion Minor criteria 1. Elevated ESRc 2. Carotid tenderness 3. Hypertension 4. Aortic regurgitation/annular disease 5. Pulmonary artery lesion 6. Left common carotid lesion 7. Distal brachiocephalic trunk lesion 8. Descending thoracic aortic lesion 9. Abdominal aortic lesion

Age <40 years Claudication of extremities Decreased brachial pulse BPb difference >10 between arms Bruit over subclavian artery or aorta Arteriogram abnormalities

Major criteria   1. Left mid subclavian artery lesion   2. Right mid subclavian artery lesion   3. Signs and symptoms >1 month Minor criteria   1. Elevated ESR   2. Carotid tenderness   3. Hypertension   4. Aortic regurgitation/annular disease   5. Pulmonary artery lesion   6. Left mid common carotid disease   7. Distal brachiocephalic trunk lesion   8. Descending thoracic aortic disease   9. Abdominal aortic lesion 10. Coronary artery lesion

American College of Rheumatology Blood pressure (in mmHg) c  Erythrocyte sedimentation rate a  b

5.2  Laboratory Findings Laboratory findings may include an elevation of acute-phase reactants and anemia. The Cleveland Clinic reported on 75 patients with TA evaluated at its center; the mean Westergren erythrocyte sedimentation rate (ESR) was 67  mm/h (range 5–135  mm/h) with 8% of patients having a normal ESR (20 mm/h or less) at the time of diagnosis [35]. During longitudinal follow-up, increases of the ESR or C-reactive protein correlated with active disease in 70% of patients, but levels were found to be normal in 23% of patients with active disease. Seventy-two percent of patients with active disease in the NIH cohort had an elevated ESR. On the other hand, the ESR was normal in only 56% of patients with disease remission [18]. Thus, it appears that acute-phase reactants cannot be used to diagnose nor exclude active vasculitis. A review of 126 patients with TA treated at Tokyo Medical and Dental University found on HLA typing a higher frequency of Bw52 (46%) in TA patients compared with

the healthy Japanese population (13%) and the Bw52 was associated with a higher incidence of aortic regurgitation and increased mortality [36]. Testing for the Bw52 antigen in 12 patients in the NIH cohort was negative [18].

5.3  Imaging Although clinical manifestations may suggest the diagnosis of TA, accurate diagnosis depends upon imaging studies. The ACR proposed angiography as the gold standard of diagnosis in its 1990 criteria [32]. However, newer techniques such as ultrasound, computed tomography (CT), magnetic resonance angiography (MRA), and positron emission tomography (PET) have become useful noninvasive imaging techniques for diagnosing and monitoring disease activity and extent. Conventional angiography has revealed stenosis, occlusion, dilation, and aneurysm either alone or in combination in the aorta, its major branches, pulmonary arteries [37], and coronary arteries. Systemic-to-pulmonary shunts such as ­coronary–pulmonary and bronchopulmonary shunts have also been described in TA patients [38]. Sixty-one patients from the ITAKA group underwent complete angiography, which revealed stenosis as the most common angiographic abnormality (93% patients), followed by occlusion (57%), dilation (16%), and aneurysm. The left subclavian artery was the vessel most commonly involved and was abnormal in 65% of the patients. Four of 25 patients who underwent investigation for symptoms of ischemic heart disease were found to have abnormalities (three stenosis, one occlusion) [19]. The most characteristic findings in the pulmonary arteries­in TA are stenosis (Fig. 1) and occlusion (Fig. 2) and, less commonly, aneurysms (Fig. 3), dilations, and peripheral pruning. There appears to be greater involvement of the right lung and upper lobes compared with the left lung and lower lobes [28]. A personal observation of this author is the lack of enlargement of the main pulmonary arteries in the presence of severe pulmonary hypertension, which may help in distinguishing pulmonary hypertension due to other causes from TA (Fig. 1). Lack of proximal pulmonary artery distension appears to be due to decreased compliance of the vessel wall thickened by the inflammatory and fibrotic changes of TA. Although conventional angiography depicts vessel luminal changes well and is useful in guiding interventional procedures, angiography is an invasive procedure that carries risks of complications such as hematoma, arteriovenous fistula, pseudoaneurysm, and vessel thrombosis [39, 40]. Color duplex ultrasound is noninvasive and can assess both vessel anatomy and the status of the lumen as well as early vessel wall alterations. The advantages of ultrasound include its relatively low cost, absence of radiation, and absence of

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Fig. 1  Pulmonary angiogram of the left lung demonstrating multiple stenoses of the lingula and left lower lobe vessels with diminished flow to the left upper lobe. Note the lack of enlargement of the left main pulmonary artery despite severe pulmonary hypertension

Fig.  3  Two saccular dilations of the right pulmonary artery with obstruction of the right middle and lower lobe vessels. (Reprinted with permission from Kerr et  al. [26]. Official journal of the American Thoracic Society, copyright American Thoracic Society)

Fig.  2  Digital subtraction angiogram of the left lung revealing total occlusion of the left descending pulmonary artery after the left upper lobar artery

contrast risks. However, ultrasound is operator-dependent and cannot image all arteries such as the thoracic aorta, the pulmonary arteries, and the proximal left subclavian artery [40]. CT can be used to assess changes of the aorta and of large, deep vessels. CT angiography can evaluate both the vessel wall and the lumen and may show arterial wall changes such as mural wall thickening and mural enhancement that would not be appreciated on conventional angiography if the lumen is unaffected. CT does not visualize small vessels well and requires the administration of iodinated contrast as well as exposure to a relatively large dose of radiation [40]. Like CT scanning, magnetic resonance imaging (MRI) is useful for early diagnosis because of its ability to evaluate vascular wall changes and not just luminal narrowing. Vessels can be viewed in any desired plane and MRA provides details about the location, extent, and degree of vascular involvement as well as the presence of collateral vessels. Vessel wall edema on T2 sequences and mural contrast enhancement on T1 sequences represent early signs of inflammation or hypervascularity. It should be noted that vessel wall thickness on CT or MRI has not consistently correlated with the development of

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new vascular lesions [39, 40]. Yamada et al. [37] prospectively studied 20 patients with TA with conventional angiography and MRA. MRA accurately depicted 323 of 330 arteries (98%), but seven stenotic arteries (2%) were overestimated as occluded. Ten of the 20 patients had pulmonary artery involvement confirmed by conventional angiography and MRA. MRA has been used in serial evaluations of patients to assess the effectiveness of immunosuppressive therapy. MRI/MRA has the advantage of multiplanar imaging without the use of ionizing radiation, but it is a costly procedure that also does not visualize smaller vessels well [39, 40]. The newest imaging technique being explored in TA is [18F]fluorodeoxyglucose ([18F]FDG) PET. Fluorine-labeled glucose analogs are administered to patients and whole-body imaging with PET is used to identify increased uptake by active cells to detect malignancies and infections [40]. The current data on [18F]FDG-PET in large vessel vasculitis are sparse, and even more limited in TA owing to the rarity of the disease in comparison with GCA. Small studies have suggested that metabolic imaging may identify more affected vascular regions than morphologic imaging with MRI in TA. Although perhaps more sensitive in detecting early inflammation, [18F]FDG-PET does not provide any information about changes in wall structure or vascular lumen. [18F]FDG-PET CT scanning now being used in oncology may combine techniques to allow for the integration of morphologic and metabolic data in the evaluation of involved vessels in TA [41]. Kobayashi et al. found that the combined imaging modality of [18F]FDG-PET CT was particularly helpful in localizing mediastinal uptake to the pulmonary arteries [42]. The value of conventional chest radiography in ­identifying pulmonary arterial involvement is debated. Kozuka et  al. found 100% correlation between chest radiographs and ­pulmonary angiograms [43]. Sharma et al. found abnormal chest radiographs (areas of oligemia) in 33% of their patients with pulmonary arteritis [25]. Moriwaki et  al. reported on 126 TA patients treated in their clinics between 1971 and 1990. Pulmonary perfusion scanning was performed on 81 patients and was there were abnormal findings in 62% of the patients [36].

K.M. Kerr

case reports and small series in the absence of clinical data. The histopathologic changes of TA are distinctly different at various stages of the disease process. Biopsies done during the early, active stage will show granulomatous arteritis with transmural inflammation and patchy destruction of the medial musculoelastic lamellae. Lymphoplasmacytic infiltrates of the media with giant cells (both Langhans and foreign-body type) can be seen (Fig.  4). Biopsies at this phase may be diagnostic in the appropriate clinical setting. Later in the disease course, with healing or end-stage arteritis, progressive intimal and adventitial fibrosis occurs with extensive media scarring and minimal lymphocytic infiltrate. Biopsies at this stage of the disease are not diagnostic of TA [44] and one must interpret these findings in conjunction with the clinical features of the disease. Matsubara et al. looked specifically at the pathologic features of the pulmonary arteries at autopsy in six patients with TA [45]. The elastic arteries showed histologic changes similar to those in the aorta with intimal fibrosis, thinning of the media with elastic fiber disruption, and fibrosis of the adventitia.

5.4  Pathology Kunio Oata is attributed with clarifying that the disease ­initially reported by Takayasu was caused by a vasculitis involving mainly the aorta and its main branches and was the first to report involvement of the pulmonary arteries in this disease [2]. There are few histologic descriptions of documented TA cases diagnosed by rigorous clinical criteria, especially from Western countries. Many pathology reports are limited to

Fig.  4  Pulmonary artery biopsy shows marked infiltration of ­mononuclear cells mainly in the media and adventitia. Granulomas replete with Langhans giant cells are seen (arrows). Disruption of ­elastic fibers is also noted (elastic staining; original magnification ×40). (Reprinted with permission from Kerr et al. [26]. Official journal of the American Thoracic Society, copyright American Thoracic Society)

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Furthermore, three findings unique to the pulmonary elastic arteries were noted: (1) marked intimal fibrosis with evidence of new vessel formation within the vessel, (2) eccentric intimal fibrous thickening, and (3) loose fibrous ­tissue in the lumen resulting in complete vessel occlusion. The investigators reported that the newly formed vessels within the obstructed pulmonary artery lumen (stenosis–­recanalization or vessel-in-vessel lesions) arose from the vaso vasorum and were branches of the bronchial arteries, distinctly different from what is seen with recanalized thromboembolism [45]. Yamada et al. [46] and Rose et al. [47] have also described this unique vessel-in-vessel lesion in the pulmonary arteries of patients with TA.

6  Treatment 6.1  Medical Therapy Corticosteroids are the cornerstone of therapy for TA. No rigorous clinical trials have been performed to define the optimal dose and duration of therapy, but steroids have been shown to improve systemic symptoms and return of pulses and blood flow in some patients [5, 18, 48]. In the NIH study of 60 patients, remission was achieved at least once in 60% of patients treated with corticosteroids, with a median time to remission of 22 months. Forty-five percent of those who achieved remission suffered a relapse [18]. The Cleveland Clinic reported a series of 75 patients diagnosed with TA, 30 of whom received longitudinal care at its facility. Twenty-eight of the 30 achieved disease remission, but most of the patients (73%) required other immunosuppressive agents in addition to corticosteroids to achieve or maintain remission [35]. Small studies and case reports have demonstrated variable success with the use of methotrexate, cyclophosphamide, azothioprine, cyclosporine, and mycophenolate mofetil in either new cases of TA or in patients who failed or who could not be weaned from corticosteroid therapy [5, 49–52]. Successful use of anti-TNF therapy in the treatment of TA has been reported by multiple investigators [35, 53, 54]. The largest series examined 25 patients with active, relapsing TA treated with infliximab (n = 21) or etanercept (n = 9). Four of the nine patients treated with etanercept underwent complete remission, three of them experiencing relapses. Two patients experienced partial remission and three patients did not respond to etanercept and were switched to inflixmab, resulting in complete remission. Of the 21 patients treated with infliximab, 12 achieved complete remission and six partial remission. However, relapse was also common, requiring treatment with increased doses at shorter intervals [55].

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6.2  Vascular Interventions Indications for revascularization include severe hypertension due to renal artery stenosis refractory to medical therapy, ischemia of extremities affecting daily activities, ischemia of cerebral vasculature, cardiac ischemia due to coronary artery involvement of the disease, coarctation of the aorta, dissecting aneurysm, progressive aneurysmal dilation, and congestive heart failure. Revascularization techniques employed include surgical bypass grafting, patch angioplasty, endarterectomy, percutaneous transluminal angioplasty, and vascular stenting [5, 18, 56, 57]. Outcomes of arterial bypass and reconstruction procedures have shown better sustained patency rates than those of other forms of revascularization in patients with TA, but longterm studies have revealed the limitations of these procedures as well when compared bypass procedures for atherosclerotic disease. Autologous grafts appear to be more successful than synthetic grafts. Complications of bypass grafting include a 20–36% chance of reocclusion or restenosis as well as other complications, including anastomototic aneurysms, thrombosis, hemorrhage, and infection [5, 18, 35, 56]. Outcomes appear to be significantly better when surgery is performed during disease quiescence (absence of signs and symptoms of active disease, no maintenance glucocorticoid requirement) versus procedures performed during active disease [58]. There are few cases of revascularization of pulmonary artery obstruction in TA reported in the literature. Patients may be taken to the operating room for the presumed diagnosis of chronic thromboembolic disease, with the correct diagnosis being made at the time of surgery [26]. Isolated case reports of successful patching or bypassing of partially or totally occluded pulmonary arteries have been published [27]. Surgical therapy may be indicated in patients with focal pulmonary artery obstruction and significant cardiopulmonary compromise who do not respond to medical therapy.

7  Prognosis Prior to the availability of noninvasive vascular imaging, TA was perceived as a chronic but self-limited disease in many patients [35, 59, 60]. The Cleveland Clinic longitudinal cohort demonstrated discordance between acute-phase reactants and findings with routine surveillance imaging [35]. The NIH study reached similar conclusions that clinical parameters and nonspecific acute-phase reactants correlate poorly with disease activity. TA is now appreciated to be a chronic, relapsing disease associated with significant morbidity. Twenty-eight of 30 patients followed at the Cleveland Clinic achieved remission, with 73% of those patients requiring additional immunosuppressive agents to achieve and

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maintain disease remission. Relapse was documented in 27 of the 28 patients who had attained remission, with most of the relapses occurring while patients were on immunosuppressive therapy. Twenty-one of the 30 patients (70%) required a total of 64 revascularization procedures, most commonly involving the coronary arteries [35]. The findings from the NIH cohort and a study of 104 Italian patients are similar to those from the Cleveland Clinic cohort, demonstrating that most patients require both corticosteroid and additional immunosuppression to achieve or maintain remission, and revascularization procedures were also frequently required [18, 19]. Morbidity is substantial. Of 34 patients interviewed from the NIH cohort [18], 74% had some degree of morbidity and 26% were not functionally affected. Approximately half of the NIH patients had permanent disability and 26% had interruptions in their daily activities during periods of active disease. Two patients died during follow-up, one likely from disease-related complications and one committed suicide. Of the 30 patients followed for 3 years in the Cleveland Clinic cohort [35], 60% had significant vascular claudication and 17% experienced cerebral ischemic disease. Twenty-three percent were unable to work. Two disease-related deaths were noted during the 3-year follow-up. Subramanyan et al. published a long-term follow-up study of 88 TA patients in 1989 [22]. The 5-year survival was 91% and the 10-year survival was 80%. Severe hypertension, severe functional disability, and cardiac involvement were predictors of morbidity and mortality.

8  Other Large Vessel Arteritis GCA is the most common vasculitis of large and mediumsized vessels. In contrast to TA, it preferentially affects the elderly (typical age more than 55 years), women more than men. GCA classically involves the arteries originating from the aortic arch, most commonly the temporal artery. Patients present with fever, anemia, elevated ESR, headache, ocular symptoms, and a palpably tender temporal artery with or without symptoms of polymyalgia rheumatica [4, 61]. Large artery complications such as aortic aneurysms and large vessel stenoses occur in up to 27% of patients with GCA. Pulmonary artery involvement is rare and has been reported in patients with [62] and without [63] concomitant involvement of other large arteries and may precede other symptoms of GCA [64]. It has been confused with pulmonary embolism [62, 63], pneumonia, and lung cancer [64]. As in TA, treatment is immunosuppression. Behçet’s disease is a chronic inflammatory disorder of unknown cause. It is seen predominantly in Asian and

K.M. Kerr

Mediterranean populations, typically presenting in the ­second to third decade of life. Initially described by a Turkish dermatologist, Hulusi Behçet, as a symptom complex of oral and genital ulcers with uveitis, it is now recognized as widespread vasculitis that affects vessels of all sizes. The pulmonary arteries are the second most common site of involvement, with aneurysms being the most frequent abnormality. Pulmonary aneurysms affect mainly young men, with hemoptysis a predominant symptom [65]. Deep venous thrombosis may also be present as a consequence of the disease, leading to the misdiagnosis of pulmonary thromboembolism. Helical CT scanning is commonly used for pulmonary vascular imaging. The aneurysms are seen most commonly in the right lower lobar artery followed by the right and left main pulmonary arteries and may be associated with thrombosis [65, 66]. Associated symptoms classic for Behçet’s disease and a positive pathergy test aid in distinguishing it from TA. Treatment is immunosuppression, most commonly a combination of cyclophosphamide and methyprednisolone. Anticoagulation is associated with significant hemorrhagic risk in these patients and it is recommended anticoagulants only be used following treatment with immunosuppression [65]. Hughes–Stovin syndrome, first described in 1959, is a syndrome of multiple pulmonary artery aneurysms and venous thrombosis [66]. It is exceptionally rare and the cause is unknown. Presenting symptoms include hemoptysis, chest pain, fever, and cough. The natural course of the syndrome is usually fatal owing to aneurysm rupture [67]. Significant similarities exist between Hughes–Stovin syndrome and Behçet’s disease and some have suggested that ­Hughes–Stovin syndrome is actually a variant of Behçet’s disease rather than a discrete disease process [66, 68]. Immunosuppression has also been shown to be successful in the treatment of this ­syndrome [66, 67]. Granulomatous vascular involvement is a common ­feature in the lung in patients with sarcoidosis. Transbronchial and open lung biopsies have predominantly demonstrated venous involvement with granulomas. However, Takemura et al. [69] demonstrated in an autopsy series of 40 sarcoidosis­ patients that all patients had pulmonary vascular involvement and the extent of granulomatous vascular involvement was related to that of the parenchyma. The venules and arterioles were most commonly involved, but 30% of the patients had granulomatous involvement of the elastic pulmonary arteries, located primarily in the adventitia and outer layer of the media with intimal fibrous thickening [69]. A more ­common finding of the larger pulmonary arteries was extrinsic compression of proximal arteries by enlarged lymph nodes due to sarcoidosis. Focal proximal compression of large ­pulmonary arteries has been successfully treated with angioplasty and stenting [70].

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9  Summary Large vessel arteritis is an extremely rare disease that is most commonly due to TA, a systemic disease seen predominantly in young women. The presentation may vary from asymptomatic to acute onset of dyspnea, chest pain, and hemoptysis mimicking acute thromboembolism or may have a more insidious onset of symptoms over months to years. Pulmonary artery involvement most commonly occurs in the setting of concomitant involvement of large systemic arteries, but pulmonary artery involvement may occur in the absence of systemic artery disease. A high index of suspicion for pulmonary arteritis must be maintained in patients, particularly young women, who present with symptoms of pulmonary embolism in the absence of risk factors for thromboembolism. Constitutional symptoms such as fever, weight loss, arthralgias, or more specific symptoms related to systemic artery involvement such as limb claudication and neurologic complaints are clues that a diagnosis of arteritis should be entertained. Chest examination may reveal evidence of pulmonary hypertension, aortic insufficiency, and bruits over the pulmonary arteries. Systemic hypertension in a young patient, blood pressure discrepancy between arms, bruits over systemic vessels, and diminished pulses are very suggestive of large vessel arteritis. The diagnosis of TA is confirmed with characteristic findings of stenosis, occlusions, dilation, and aneurysmal changes seen on angiographic imaging of the pulmonary and systemic arteries. Corticosteroids are the most frequently used drugs in the treatment of TA; however, most patients require additional immunosuppressive medications to achieve or sustain remission. Relapses are common and may occur in the absence of symptoms or elevated levels of acute-phase reactants. Rare patients may benefit from pulmonary artery reconstruction or bypass in the setting of focal pulmonary artery stenosis with a patent distal vascular bed.

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1341 7. Chauhan SK, Tripathy NK, Nityanand S (2006) Antigenic targets and pathogenicity of antiaortic endothelial cell antibodies in Takayasu’s arteritis. Arthritis Rheum 54:2326–2333 8. Seko Y, Sugishita K, Sato O et al (2004) Expression of costimulatory molecules (4-IBBL and Fas) and major histocompatibility class I chain-related A (MICA) in aortic tissue with Takayasu’s arteritis. J Vasc Res 41:84–90 9. Noris M, Daina E, Gamba S, Bonazzola S, Remuzzi G (1999) Interleukin-6 and RANTES in Takayasu’s arteritis; a guide for therapeutic decisions? Circulation 100:55–60 10. Tripathy NK, Sinha N, Nityanand S (2004) Interleukin-8 in Takayasu’s arteritis: plasma levels and relationship with disease activity. Clin Exp Rheumatol 22:S27–S30 11. Tripathy NK, Chauhan SK, Nityanand S (2004) Cytokine mRNA repertoire of peripheral blood mononuclear cells in Takayasu’s arteritis. Clin Exp Immunol 138:369–374 12. Verma DK, Tripathy NK, Verma NS, Tiwari S (2005) Interleukin 12 in Takayasu’s arteritis: plasma concentrations and relationship with disease activity. J Rheumatol 32:2361–2363 13. Park MC, Lee SW, Park YB, Lee SK (2006) Serum cytokine profiles and their correlations with disease activity in Takayasu’s arteritis. Rheumatology 45:545–548 14. Hall S, Barr W, Lie JT, Stanson AW, Kazmier FJ, Hunder GG (1985) Takayasu’s arteritis. A study of 32 North American patients. Medicine 64:89–99 15. Waern AU, Andersson P, Hemmingsson A (1983) Takayasu’s arteritis: a hospital-region based study on occurrence, treatment and prognosis. Angiology 34:311–320 16. Hall S, Buchbinder R (1990) Takayasu’s arteritis. Rheum Dis Clin 16:411–422 17. Numano F (1997) Differences in clinical presentation and outcome in different countries for Takayasu’s arteritis. Curr Opin Rheumatol 9:12–15 18. Kerr GS, Hallahan CW, Giordano J et al (1994) Takayasu arteritis. Ann Intern Med 120:919–929 19. Vanoli M, Daina E, Salvarani C et al (2005) Takayasu’s arteritis; a study of 104 Italian patients. Arthritis Rheum 53:100–107 20. Park M-C, Lee S-W, Park Y-B, Chung NS, Lee S-K (2005) Clinical characteristics and outcomes of Takayasu’s arteritis: analysis of 108 patients using standardized criteria for diagnosis, activity assessment, and angiographic classification. Scand J Rheumatol 34:284–292 21. Hoffman GS (1996) Takayasu’s arteritis: lessons from the American National Institutes of Health experience. Int J Cardiology 54:S83–S86 22. Subramanyan R, Joy J, Balakrishnan KG (1989) Natural history of aortoarteritis (Takayasu’s disease). Circulation 80:429–437 23. Lupi-Herrera E, Sánchez-Torres G, Marcushamer J, Mispireta J, Horwitz S, Vela JE (1977) Takayasu’s arteritis. Clinical study of 107 cases. Am Heart J 93:94–103 24. Yamato M, Lecky JW, Hiramatsu K, Kohda E (1986) Takayasu arteritis: radiographic and angiographic findings in 59 patients. Radiology 161:329–334 25. Sharma S, Kamalakar T, Rajani M, Talwar KK, Shrivastava S (1990) The incidence and patterns of pulmonary artery involvement in Takayasu’s arteritis. Clin Radiology 42:177–181 26. Kerr KM, Auger WR, Fedullo PF, Channick RH, Yi ES, Moser KM (1995) Large vessel pulmonary arteritis mimicking chronic thromboembolic disease. Am J Respir Crit Care Med 152:367–373 27. Nakajima N, Masuda M, Imamaki M, Ishida A, Tanabe N, Kuriyama T (2007) A case of pulmonary artery bypass surgery for a patient with isolated Takayasu pulmonary arteritis and a review of the literature. Ann Thorac Cardiovasc Surg 13:267–271 28. Singh J, Brasington RD (2001) Pulmonary Takayasu’s arteritis masquerading as acute pulmonary embolism. J Clin Rheumatol 7:388–394 29. Tyagi S, Kaul UA, Gambhir DS, Arora R, Khallilulah M (1987) Pulmonary artery involvement in aorto-arteritis. Ind Heart J 39:46–50

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K.M. Kerr 50. Shelhamer JH, Volkman DJ, Parrillo JE, Lawley TJ, Johnston MR, Fauci AS (1985) Takayasu’s arteritis and its therapy. Ann Intern Med 103:102–106 51. Valsakumar AK, Valappil UC, Jorapur V, Garg N, Nityanand S, Sinha N (2003) Role of immunosuppressive therapy on clinical, immunological, and angiographic outcome in active Takayasu’s arteritis. J Rheumatol 30:1793–1798 52. Daina E, Schieppati A, Remuzzi G (1999) Mycophenalate mofetil for the treatment of Takayasu’s arteritis; report of three cases. Ann Intern Med 130:422–426 53. Tanaka F, Kawakami A, Iwanaga N et al (2006) Infliximab is effective for Takayasu arteritis refractory to glucocorticoid and methotrexate. Intern Med 45:313–316 54. Jolly M, Curran JJ (2005) Infliximab-responsive uveitis and vasculitis in a patient with Takayasu’s arteritis. J Clin Rheumatol 11:213–215 55. Molloy ES, Langford CA, Clark TM, Gota CE, Hoffman GS (2008) Antitumor necrosis factor therapy in patients with refractory Takayasu’s arteritis; long term follow-up. Ann Rheum Dis 67:1567–1569 56. Liang P, Tan-Ong M, Hoffman GS (2004) Takayasu’s arteritis; vascular interventions and outcomes. J Rheumatol 31:102–106 57. De Franciscis S, Serra R, Luongo A, Sabino G, Puzziello A (2007) The management of Takayasu’s arteritis: personal experience. Ann Vasc Surg 21:754–760 58. Fields CE, Bower TC, Cooper LT et al (2006) Takayasu’s arteritis: operative results and influence of disease activity. J Vasc Surg 43:64–71 59. Jain S, Kumari S, Ganguly NK, Sharma BK (1996) Current status of Takayasu’s arteritis in India. Int J Cardiol 54:S111–S116 60. Dabague J, Reyes P (1996) Takayasu’s arteritis in Mexico: a 38-year clinical perspective through literature review. Int J Cardiol 54:S87–S93 61. Bongartz T, Matteson EL (2006) Large-vessel involvement in giant cell arteritis. Curr Opin Rheumatol 18:10–17 62. Glover MU, Muñiz J, Bessone L, Carta M, Casellas J, Maniscalco BS (1987) Pulmonary artery obstruction due to giant cell arteritis. Chest 91:924–925 63. Wagenaar S, van den Bosch JMM, Westermann CJJ, Bosman HG, Lie JT (1986) Isolated granulomatous giant cell vasculitis of the pulmonary elastic arteries. Arch Pathol Lab Med 110:962–964 64. Doyle L, McWilliam L, Hasleton PS (1988) Giant cell arteritis with pulmonary involvement. Br J Dis Chest 82:88–92 65. Erkan F, Gul A, Tasali E (2001) Pulmonary manifestations of Behçet’s disease. Thorax 56:572–578 66. Emad Y, Ragab Y, Shawki Ael H, Gheita T, El-Marakbi A, Salama MH (2007) Hughes-Stovin syndrome; is it incomplete Behçet’s? Report of two cases and review of the literature. Clin Rheumatol 26:1993–1996 67. Lee J, Noh JW, Hwang JW et al (2008) Successful cyclophosphamide therapy with complete resolution of pulmonary artery aneurysm in Hughes-Stovin syndrome patient. Clin Rheumatol 27:1455–1458 68. Erkan D, Yazici Y, Sanders A, Trost D, Yazici H (2004) Is HughesStovin syndrome Behçet’s disease? Clin Exp Rheumatol 22:S64–S68 69. Takemura T, Matsui Y, Saiki S, Mikami R (1992) Pulmonary vascular involvement in sarcoidosis. Hum Pathol 23:1216–1223 70. Hamilton-Craig CR, Slaughter R, McNeil K, Kermeen K, Walters DL (2009) Improvement after angioplasty and stenting of pulmonary arteries due to sarcoid mediastinal fibrosis – case report. Heart Lung Circ 18:222–225

Chapter 96

Tumors of the Pulmonary Vascular Bed Eunhee S. Yi

Abstract  Primary or secondary tumors of the lung can affect all levels of the pulmonary vascular bed, including the pulmonary arteries, veins, and capillaries. Most primary tumors of the pulmonary vasculature are poorly differentiated, highly fatal sarcomas of the large main pulmonary arteries and veins. Pulmonary arterial sarcoma (PAS) is far more prevalent than its venous counterpart, although both arterial and venous sarcomas are quite rare. Secondary involvement of the pulmonary vasculature by other primary lung cancers or metastatic malignant neoplasm is not as rare, however. Direct extension of pelvic sarcomas to pulmonary arteries via the central venous system and the right side of the heart has been documented as well. Pulmonary capillary hemangiomatosis (PCH) is an elusive entity and has been considered either as a low-grade neoplasm or a as tumorlike lesion of pulmonary capillaries. PCH typically causes severe pulmonary hypertension that is often accompanied by fatal right-sided heart failure. Most aortic sarcomas and PASs are intimal sarcomas and that most sarcomas of the pulmonary veins and inferior vena cava are mural leiomyosarcomas. Mural sarcomas of the inferior vena cava constitute the largest subgroup of the primary tumors arising in the great blood vessels. The incidence of pulmonary vein sarcomas (PVSs) is far lower than that of PASs. Most intimal sarcomas of the aorta and the pulmonary arteries in that study were poorly differentiated or undifferentiated sarcomas with a prominent intraluminal growth pattern, immunohistochemical evidence of myofibroblastic differentiation, and a poorer prognosis than the mural leiomyosarcomas in the inferior vena cava and the pulmonary veins. This chapter focuses on the current clinicopathological concepts of the primary sarcomas of pulmonary arteries and veins. In light of the unclear biologic nature of PCH, nonneoplastic as well as neoplastic aspects of PCH are discussed although the main focus of this chapter is on the neoplastic diseases of the pulmonary vasculature. Manifestations of secondary tumors mimicking a primary E.S. Yi (*) Department of Anatomic Pathology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905, USA e-mail: [email protected]

pulmonary vascular tumor or idiopathic pulmonary hypertension are also covered briefly. Keywords  Pulmonary arterial and venous sarcoma • Pulmonary capillary hemangiomatosis • Neoplasm • Leiomyosarcoma • Myofibroblastic proliferation and differentiation

1  Introduction Primary or secondary tumors of the lung can affect all levels of the pulmonary vascular bed, including the pulmonary arteries, veins, and capillaries. Most primary tumors of the pulmonary vasculature are poorly differentiated, highly fatal sarcomas of the large main pulmonary arteries and veins. Pulmonary arterial sarcoma (PAS) is far more prevalent than its venous counterpart, although both arterial and venous ­sarcomas are quite rare [1]. Secondary involvement of the pulmonary vasculature by other primary lung cancers or metastatic malignant neoplasm is not as rare, however [2]. Direct extension of pelvic sarcomas to pulmonary arteries via the central venous system and the right side of the heart has been documented as well [3]. Pulmonary capillary hemangiomatosis (PCH) is an elusive entity and has been considered either as a low-grade neoplasm or a as tumorlike lesion of pulmonary capillaries. It is still controversial whether PCH is a neoplastic or a nonneoplastic process; it is also possible that PCH might represent more than one entity. Regardless of its true biologic nature, PCH typically causes severe pulmonary hypertension that is often accompanied by fatal right-sided heart failure [4]. In a clinicopathology study on the sarcomas of the great vessels, Burke and Virmani put the sarcomas of the aorta, pulmonary arteries and veins, and inferior vena cava into two major categories: intimal sarcoma and mural sarcoma [1]. They concluded that most aortic sarcomas and PASs are intimal sarcomas and that most sarcomas of the pulmonary veins and inferior vena cava are mural leiomyosarcomas. Mural sarcomas of the inferior vena cava constitute the largest subgroup of the primary tumors arising in the great blood vessels [1].

J.X.-J. Yaun et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_96, © Springer Science+Business Media, LLC 2011

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A worldwide registry of 218 patients demonstrating ­sarcomas of the inferior vena cava indicated that the incidence of ­sarcomas in the inferior vena cava is greater than that of ­sarcomas in great arteries [5]. The incidence of ­pulmonary vein sarcomas (PVSs) is far lower than that of PASs [1, 6]. Most intimal sarcomas of the aorta and the pulmonary arteries in that study were poorly differentiated or undifferentiated sarcomas with a prominent intraluminal growth pattern, immunohistochemical evidence of myofibroblastic differentiation, and a poorer prognosis than the mural leiomyosarcomas in the inferior vena cava and the pulmonary veins [1]. This chapter will focus on the current clinicopathological concepts of the primary sarcomas of pulmonary arteries and veins. In light of the unclear biologic nature of PCH, nonneoplastic as well as neoplastic aspects of PCH are discussed although the main focus of this chapter is on the neoplastic diseases of the pulmonary vasculature. Manifestations of ­secondary tumors mimicking a primary pulmonary vascular tumor or idiopathic pulmonary hypertension are also covered briefly.

E.S. Yi

probably underestimated, since many PAS cases are still misdiagnosed as pulmonary embolism on preoperative workups and some may remain unrecognized without pathological examination. In a series of 200 consecutive pulmonary thromboendarterectomy (PTE) operations performed at the University of California, San Diego (UCSD) Medical Center, two patients (1%) were found to have PAS instead of chronic thromboembolic pulmonary hypertension (CTEPH) [8]. Another earlier study from UCSD reported that approximately 3–4% of patients with the diagnosis of CTEPH turned out to have PAS at the time of PTE [9]. A review of 138 primary sarcomas of the pulmonary artery reported that the average age at diagnosis was 49.3 years (range 13–81 years), with a roughly equal sex distribution [10], whereas other previous studies have shown a slight female or male predominance [11–13]. A recent series of 43 PAS cases demonstrated no sex predilection as well [7].

2.3  Clinical Manifestations 2  Primary Sarcomas of Pulmonary Arteries 2.1 G  eneral Characteristics  

PAS encompasses intimal sarcoma and mural sarcoma ­arising in the intima and wall of the large pulmonary artery, respectively. Primary sarcomas are rare, with only a few series correlating histologic features with follow-up data [7]. Intimal sarcomas show the characteristic intraluminal polypoid growth pattern and histological evidence of fibroblastic or myofibroblastic differentiation in most cases. “Intimal sarcoma of the pulmonary artery” has been used interchangeably with “PAS” in many previous studies, since intimal ­sarcomas constitute the vast majority of PASs, with exceedingly rare cases of mural sarcomas; only two cases have been reported in the literature specifically as mural sarcomas [1]. Accordingly, the clinical and pathologic features of PAS in the literature mostly represent those of intimal sarcomas. Mural sarcomas are considered distinct from intimal ­sarcomas clinically and histogenetically; thus they are classified­ ­separately according to the histologic subtype as used in the classification of general soft tissue sarcomas. It may be difficult to distinguish mural sarcomas of pulmonary arteries from the sarcomas originating in the pulmonary parenchyma [1].

2.2  Epidemiology PAS is a rare tumor, with only a few hundred cases reported in the literature. The true incidence of PAS is unknown and

The most common presenting symptom is dyspnea (72%), followed by chest/back pain (45%), cough (42%), hemoptysis (24%), weight loss (21%), malaise (10%), syncope (9%), fever (8%), and sudden death in rare cases [10]. These clinical findings are often indistinguishable from those of CTEPH, but progressive weight loss, anemia, and fever are unusual for benign pulmonary vascular diseases and should raise a suspicion for malignancy [14]. Common physical signs include systolic ejection murmur, cyanosis, extremity edema, jugular venous distension, hepatomegaly, and clubbing [14]. PAS primarily metastasizes to the lung and mediastinum (50%) [10]. Distant extrathoracic metastases were reported in 16% of PAS cases, which is lower than in its counterpart in the aorta [10].

2.4  Imaging Studies Radiologic findings of PAS also overlap with those of CTEPH, but the rate of preoperative diagnosis has increased remarkably in the last decade with advances in imaging ­technology [10, 15, 16]. Although most chest radiographic findings of PAS are nonspecific, solid sarcomatous expansions of the proximal pulmonary artery branches are highly suggestive of PAS, especially in the presence of associated pulmonary nodules, cardiac enlargement, and decreased vascularity [1]. The features in computed tomography (CT) and magnetic resonance imaging that favor the diagnosis of PAS over CTEPH include heterogeneous soft tissue density (from areas of

96  Tumors of the Pulmonary Vascular Bed

necrosis, hemorrhage, and ossification), enhancement after gadopentetate dimeglumine, vascular distension, and smooth vascular tapering without abrupt narrowings and cutoffs [15]. Unilateral central pulmonary embolus is uncommon and suggests PAS [10, 15]. High-resolution CT may show a mosaic pattern of lung attenuation as seen in the cases of CTEPH [17]. Neovascularization in PAS may be seen with bronchial arteriography [10]. Pulmonary angiography shows intraluminal masses and lung perfusion abnormalities with a smooth tapering of pulmonary arteries and “to-and-fro” motion of pedunculated or lobulated lesions [10]. Recently, intravascular ultrasound study and 18F-2-fluoro-2-deoxy-d-glucose–positron emission tomographic tumor imaging have been reported as helpful ancillary techniques to achieve the preoperative diagnosis of PAS [18, 19].

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Fig. 1  Gross photograph of pulmonary arterial sarcoma (PAS) resected by the pulmonary thromboendarterectomy procedure

2.5  Pathologic Findings Intimal sarcomas of the pulmonary artery grossly resemble mucoid or gelatinous clots filling the lumens (Fig. 1). The main tumor masses extend distally along the branches of the pulmonary artery with a smooth tapering pattern (Fig.  1). The cut ­surface of a tumor may show firm fibrotic areas as well as bony/ gritty and chondromyxoid areas depending on the histological composition of the sarcoma. Hemorrhage and necrosis are common in high-grade PAS. The tumors are usually confined to the intima and lumen, but sometimes infiltrate into the adventitia or pulmonary parenchyma in advanced cases. PAS excised by PTE often has the shape of a thromboembolus, with a complete or partial obliteration of the lumens. Most PAS cases have bilateral pulmonary artery involvement, although one side is usually dominant. The reported sites of tumor include ­pulmonary trunk (85%), right pulmonary artery (71%), left pulmonary artery (65%), pulmonary valve (32%), and right ventricular outflow tract (10%) [10]. Histologically, intimal sarcomas typically show patchy areas of spindle cell proliferation in a myxoid background, alternating with hypocellular collagenous stroma. The tumor may be intimately incorporated with recanalizing thrombi, especially toward distal branches of the pulmonary artery. Most intimal sarcomas are poorly differentiated or ­undifferentiated (Fig. 2) and generally, but not always, show immunohistochemical or ultrastructural evidence of ­myofibroblastic differentiation [1, 12, 13, 20–23]. The luminal­ surface of PAS often shows a layer of fibrinous materials (Fig. 3). Histopathologic classification of PAS has been variable in that some authors have emphasized the presumed intimal origin [1], whereas others have used generic classifications of soft tissue tumors. Foci of osteosarcoma, chondrosarcoma, or rhabdomyosarcoma are reported to be more common in PAS

Fig. 2  PAS showing high-grade undifferentiated malignant cells (right) that merge with spindle cells in a myxoid stroma (hematoxylin and eosin stain, ×400 original magnification)

Fig. 3  Abundant fibrin deposit at the edge of high-grade PAS (hematoxylin and eosin stain, ×200 original magnification)

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than in its aortic counterpart. Intimal sarcomas with a specific component can be designated with an appropriate modifier, for example, intimal sarcoma with focal osteosarcoma, chondrosarcoma, or rhabdomyosarcoma. It is of note that there are relatively few reports of primary angiosarcomas of the pulmonary artery, in contrast to more frequent occurrence in the setting of primary aortic sarcomas [7, 24, 25]. Although uncommon, low-grade sarcomas do occur in the pulmonary arteries and have been associated with long-term survival [7, 26]. Mattoo et al. reported a low-grade PAS in a 32-year-old woman who was initially treated with a local resection of the tumor followed by a pneumonectomy 2 years later for the management of invasive aspergillosis associated with a cavitary lesion [26]. This patient was alive and well without any evidence of disease at 5 years after the initial surgery. Pathological examination disclosed an intraluminal tumor within the left main pulmonary artery and the tumor was composed of low-grade spindle cells showing myofibroblastic phenotype and profuse plasmacytic infiltrates (Fig. 4). These features are compatible with inflammatory fibrosarcoma, which is regarded as a synonym for “inflammatory myofibroblastic tumor” according to the current WHO classification of soft tissue tumors [27]. Tavora et  al. reported three of their 43 cases had the histopathologic features of ­low-grade myofibroblastic sarcomas [7]. Histologically, mitotic rate was significantly lower in this low-grade myofibroblastic sarcoma than in other histologic subtypes and there was inflammatory background and myofibroblastic phenotype. Follow-up revealed that two of three low-grade ­myofibroblastic sarcomas were cured, with no evidence of ­disease at 10 years [7]. Immunohistochemically, the undifferentiated tumor cells of intimal sarcomas are usually positive for vimentin and osteopontin, an extracellular matrix protein binding to av

Fig. 4  A low-grade PAS with features of inflammatory myofibroblastic sarcoma. Numerous plasma cells are present in this case that are not shown in this field (hematoxylin and eosin stain, ×200 original magnification)

E.S. Yi

integrins [28]. Variable positivity for smooth muscle actin and desmin has been observed [1, 12, 13, 20–23]. In typical intimal sarcomas, endothelial markers (CD31, CD34, and factor VIII) test negative [1, 12, 13, 20–23]. The two cases of mural sarcoma reported in the literature were described as leiomyosarcoma and pleomorphic sarcoma [1]. One case of mural sarcoma encountered at UCSD Medical Center showed features of pleomorphic sarcoma. There is no specific histological grading system for PAS; the two widely used generic grading systems for soft tissue sarcomas (NCI and FNCLCC systems) can be applied with tumor differentiation, mitotic count, and tumor necrosis as the parameters for scoring.

2.6  Differential Diagnosis Pathologic diagnosis of PAS is fairly straightforward in most cases, although some resected pulmonary thrombi have highly cellular foci of remodeling that can mimic a lowgrade intimal sarcoma. The possibility of metastasis from other sites should always be excluded. Mural sarcomas of the pulmonary artery are very difficult to distinguish from ­sarcomas of the lung parenchyma. Clinical and radiological differential diagnoses include CTEPH, primary lung or mediastinal tumors, congenital pulmonary stenosis, ­fibrosing ­mediastinal tumors, primary pulmonary hypertension, and pulmonary infections (usually tuberculosis).

2.7  Treatment and Prognostic Factors Currently, surgical resection is the single most effective modality for short-term palliation [10, 29]. The types of surgery for PAS recorded in the literature include pneumonectomy, local excision of the mass from the vascular bed with plastic reconstruction, and endarterectomy [10, 29]. The role of adjuvant therapy is yet to be determined, but according to a review of 136 reported PAS cases, adjuvant radiotherapy and/or chemotherapy after surgery improved the 1-year ­survival rate (58% with adjuvant therapy from 31% with ­surgery alone; p = 0.11) and 2-year survival rate (39% from 24%; p = 0.4), but had no effect on the 5-year survival rate (0% with adjuvant therapy vs. 6% with surgery alone) [10]. The small number of patients treated with radiotherapy and chemotherapy alone allowed no conclusion to be drawn as to the therapeutic efficacy [6]. Improved survival can probably be achieved by early diagnosis and radical surgical resection possibly with adjuvant chemotherapy and radiation. Anecdotal reports on successful treatment of PAS include a two-drug combination chemotherapy consisting of ifosfamide and epirubicin [30].

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Patients with PAS rarely survive beyond 3 years. The overall prognosis of PAS is very poor regardless of therapy at this time, with the mean survival ranging from 14 to 18 months [1, 12, 29]. Long-term survival at 5 years or more after surgery has been reported in two of two mural sarcomas, suggesting their favorable clinical course as compared with intimal sarcomas [1]. In the report by Huo et al., one of their 12 patients was alive at 68 months with widespread metastasis, and another patient was alive at 40 months with lung metastases, highlighting that occasional patients with PAS may have a prolonged survival before eventually succumbing to metastatic disease [31]. These findings support the aggressive treatment of patients with PAS [32]. Most patients who were alive without disease beyond 3 years had tumors showing features of low-grade sarcoma with the myofibroblastic phenotype such as inflammatory fibrosarcoma or inflammatory myofibroblastic tumor [7, 26, 33]. Although long-term survival is possible in patients with high-grade PAS with aggressive treatment, a clinical cure can be achieved in patients with low-grade myofibroblastic sarcomas showing low mitotic rate, inflammatory background, and myofibroblastic phenotype, as for the case illustrated in Fig. 4.

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expression of the apoptosis-related proteins p53, Bax, Bcl-2, Fas (CD95), Fas-ligand, and perforin in seven PAS cases and concluded that the apoptosis occurs in PAS via an induction of Bax, mostly independent of p53 [35]. Host defense against the sarcoma could be mediated by perforin-expressing lymphocytes, but not through the Fas/Fas-ligand pathway [35]. A recent study explored the adenomatous polyposis coli (APC)/b-catenin (WNT) pathway in 18 cases of PAS and attempted to subclassify PAS according to the genetic abnormalities as well as to correlate the classification with the clinical outcome [36]. They did not detect any mutation in either the APC or the b-catenin pathway and concluded that alterations of the APC/b-catenin (WNT) pathway do not appear to be causally involved in the pathogenesis of PAS. Target genes for b-catenin such as cyclin D1 were overexpressed particularly in the myxofibrosarcoma type, possibly via transcriptional activation due to factors other than b-catenin according to this study [36].

3  Primary Sarcomas of Pulmonary Veins 3.1  General Characteristics

2.8  P  ostulated Cell of Origin and Molecular Pathogenesis Intimal sarcomas presumably arise from pluripotential mesenchymal cells of the intima [1, 12]. The origin of endoluminal pleomorphic sarcomas, including those with apparent stromal differentiation such as osteoid and chondroid, is considered likely be the intimal myofibroblasts, in light of the in situ atypia in intima adjacent to endoluminal cardiac sarcomas [1], and also because grossly excised PASs often shell out like thrombi without significant gross or histological evidence of spread beyond the media. Primitive cells of the bulbus cordi in the trunk of the pulmonary artery have been also proposed as the origin [20]. The cell of origin for mural sarcomas is not clear, but mural sarcomas showing a leiomyosarcoma component probably derive from medial smooth muscle cells [1]. A comparative genomic hybridization (CGH) study demonstrated that six of eight intimal sarcomas had gains or amplifications of the chromosomal region 12q13-q15 by a conventional technique [34]. In the same study, a microarraybased CGH technique was also applied to investigate further the amplification of putative oncogenes located in the chromosomal regions of 12q13-q15 (SAS/CDK4, MDM2, and GLI) and 4q12 (platelet-derived growth factor receptor a, PDGFR-alpha). The data suggested that the amplifications of SAS/CDK4, MDM2, GLI, and PDGFR-alpha were strongly associated with the tumorigenesis of pulmonary artery ­intimal sarcomas [34]. An immunohistochemical study examined the

Primary sarcomas of the pulmonary veins are mostly leiomyosarcomas that arise from the medial smooth muscle cells [6, 37]. Intimal sarcomas derived from the pluripotential intimal cells may occur in the pulmonary veins as in the pulmonary arteries, but are exceedingly rare [1]. In most reported PVS cases, PVS extended to the left atrium [6, 37, 38]. The venous sarcomas are typically larger than sarcomas of the arteries and may remain asymptomatic for a long period when they lack luminal growth [1].

3.2  Epidemiology PVSs are extremely rare and are outnumbered in the literature by the PASs by approximately 20:1 [6]. There have been only 17 reported PVS cases in the literature do date [37]. However, the incidence of sarcomas in the inferior vena cava is known to be somewhat greater than that of sarcomas in great arteries [1]. In fact, the true incidence may be even higher, considering the potential origin of some retroperitoneal leiomyosarcomas from the inferior vena that was obscured by the large size. Similarly, the pulmonary venous origin would be difficult to establish in advanced cases of PVS, which might have attributed to the low number of documented cases. According to the largest compiled series of PVS including 17 cases, the mean age was 50.0 ± 15.6 years (median 48.0 years; range 23–74 years) at the time of

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p­ athologic diagnoses [37]. Six patients were men and 11 were women [37]. Compared with the marked female predominance in leiomyosarcomas of the inferior vena cava (1:4.8) and a slight female dominance or equivocal gender difference in PAS patients, the female dominance in PVS patients appeared moderate (1:1.8) [37].

3.3  Clinical Manifestations The presenting symptoms of those 17 patients were as follows: dyspnea in eight patients, hemoptysis or bloody sputa in five patients, cough in four patients, chest pain and weigh loss each in three patients, ipsilateral pleural effusion and palpitation each in two patients, and mental disturbance, dizziness, weakness, paroxysmal tachycardia, orthopnea, and sputa each in one patient [37]. In three cases, hemoptysis was the major symptom and necessitated blood transfusion or emergency surgery [37]. Primary sites of leiomyosarcomas of the pulmonary vein were reported in the right side in nine cases, the left side in six cases, and were unknown in two cases [37]. Right superior and left inferior pulmonary veins (six and four, respectively) were reported as the frequent origins for leiomyosarcomas [37]. The left PVSs tended to enter the left atrium, which was recognized in nine of 17 cases [37]. Five of 17 patients developed local or mediastinal recurrence [37]. Three patients showed metastases to the liver, scalp, or axillary lymph node [37].

3.4  Imaging Studies There has been no comprehensive report regarding the radiologic findings for PVS and only sporadic descriptions are available in the literature. One patient presented with an asymptomatic right hilar mass on a routine chest radiograph and another patient had pleural effusion detected by chest CT [5, 37]. Right upper lobe density on chest radiograph and left atrial tumor on CT scan have each been reported in one case [6, 38].

3.5  Pathologic Findings Grossly, the tumors typically present as fleshy white masses that range from 2 to 8.3 cm in the greatest dimension [6, 37]. Some tumors partially or completely occlude the lumen of the involved vessels in addition to the mural involvement, whereas others only have mural components [6, 37]. Extension of the tumor into the left atrial lumen is commonly seen, but invasion of the left atrial wall was seen in only one case [5, 25, 26]. Extension of the tumor into the pulmonary parenchyma was seen in some cases [6, 37].

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Microscopically, most tumors are classifiable as intermediate- to high-grade leiomyosarcomas [5, 25]. Tumors show either predominantly spindle cells with cigar-shaped nuclei or epithelioid cells. Multifocal calcifications are also observed in some tumors. Mitotic activity ranges from less than one to 25 mitotic figures per ten high-power fields. Necrosis was extensive in most reported tumors. The metastases, when present, had the same morphology as the primary lesions. Immunohistochemically, the tumor cells were reported to test positive for smooth muscle actin, muscle specific actin (HHF35), desmin, and vimentin, but to test negative for epithelial membrane antigen, S-100 protein, and factor VIII-related antigen. Focal positivity to keratin and dotlike cytoplasmic MIC-2 reactivity have been reported in some cases [6].

3.6  Treatment and Prognosis Little is known as to the optimal therapy for PVS because of the rarity of documented cases with adequate treatment history and follow-up. Surgical resection with wide margins probably offers the only chance of cure as in other leiomyosarcomas [6, 37]. Chemotherapy and radiation therapy have not been shown to be effective [6, 37]. In two cases in the literature estrogen receptor protein and/or progesterone receptor protein were expressed on immunohistochemical staining, suggesting a potential role for hormone ablation therapy [6]. According to a review of 17 PVS cases, local or mediastinal recurrence occurred in five cases and metastasis to the liver, scalp, or axillary lymph node was recorded in three cases [37]. Among 16 reported cases with available prognostic data, six patients had already died from disease at the time of the report [37]. The length of follow-up ranged from 2 months to 21 years after the surgery in the remaining ten patients who were alive [37]. The postoperative survival rates at 6 months and 1, 2, 3, and 5 years were 75, 73, 50, 33, and 20%, respectively [37]. The age at the time of operation, the gender of the patients, or the presence of subjective symptoms at the time of the surgery did not seem to have any significant influence on the postoperative survival up to 2  years [37]. The size of the tumor, the presence of tumor growth in the left atrium, and mitotic count did not appear to be significant prognostic factors in PVSs [37].

4  Pulmonary Capillary Hemangiomatosis 4.1  General Characteristics PCH was first described in 1978 by Wagenvoort et al. in a 71-year-old woman with progressive dyspnea, hemoptysis,

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and hemorrhagic pleural effusions [39]. PCH is known as an extremely rare cause of pulmonary hypertension and fewer than 50 cases have been reported since the first case was reported by Wagenvoort et al. This rarity makes it difficult to understand the true nature of this disease. It is not clear whether PCH represents a reactive, neoplastic, or congenital hamartomatous process [39]. PCH usually presents as a bilateral pulmonary disease of unknown cause and shows a proliferation of capillaries within the alveolar septa and into other structures, including bronchial and venous walls, pleura, and even regional lymph nodes, rendering the histopathologic features of a low-grade capillary neoplasm [39]. Extrathoracic spread or atypical nuclear features have not been described in PCH cases previously [4]. Some authors purported that PCH represents a secondary angioproliferative process caused by postcapillary obstruction rather than a separate disease [40]. PCH-like changes are not uncommon in the setting of acute lung injury, especially diffuse alveolar damage. Increased alveolar septal capillaries mimicking PCH can be seen in idiopathic pulmonary fibrosis as well and the clinical significance of this finding merits further study. An autopsy study of 76 obese subjects reported that 72% of these obese individuals had histopathologic features of pulmonary hypertension, particularly venous hypertension and PCH as compared with 6% in the age-matched, nonobese controls in the same time period. It was postulated that the pulmonary venous hypertensive changes and pulmonary capillary proliferation in the obese are most likely related to chronic hypoxia and left ventricular dysfunction. The capillary endothelial cells were cytologically bland and did not exhibit excessive mitosis. This histologic finding was also observed in another autopsy series of obese patients and it was ascribed to chronic hypoxia-associated pulmonary hypertension [41]. The pathogenesis of PCH remains controversial, as some investigators consider it to be neoplastic, whereas others surmise that it is a response to an unknown angiogenic stimulus, such as hypoxia [42, 43]. A recent molecular study using high-throughput gene expression ­profiling reported increased expression of PDGFB and PDGFR-beta­ in PCH, suggesting their role in the pathogenesis and in therapeutic potential [44].

4.2  Clinical Features PCH has been reported to occur in patients of 2–71 years of age, with a peak in the third and fourth decades [4, 40, 45–47]. Both males and females appear to be equally affected. A hereditary form of PCH with a probable autosomal–­ recessive inheritance pattern has been reported by Langleben et  al. [48]. Cases associated with hereditary hemorrhagic telangiectasia and in premature infants have been reported as well [49]. Clinical manifestations include progressive

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s­ hortness of breath, pleuritic chest pain, and frequent ­hemoptysis, which often lead to clinical evaluation of chronic thromboembolic diseases. Digital clubbing is infrequently reported in PCH [4, 45]. Patients with PCH later develop clinical signs of cor pulmonale [3, 27, 29, 30]. Right-sided heart catheterization invariably reveals highly elevated pulmonary arterial pressures but normal pulmonary capillary wedge pressures [4]. Recognition of early signs and symptoms of PCH is important to initiate the consideration for bilateral lung transplantation that may prevent a fatal outcome. The time from symptom onset to death ranges from 2 to 12 years [4].

4.3  Imaging Studies Chest radiographs typically show a diffuse, bilateral, reticulonodular pattern with enlarged central pulmonary arteries and increased number of septal lines [4, 39, 45–47]. Similar radiographic findings are observed in many other pulmonary diseases, including pulmonary venoocclusive disease (PVOD) and idiopathic interstitial fibrosis, microthromboembolic pulmonary vascular disease, and pulmonary lymphangimatosis. Although subtle and somewhat nonspecific, the chest radiograph findings may correlate with the characteristic CT features. CT shows enlarged pulmonary arteries and multiple, bilateral, small, poorly defined nodular opacities [4]. High-resolution CT shows a mosaic pattern of attenuation of pulmonary parenchyma, which likely represents variable pulmonary perfusion [4]. The septa may be nodular or smooth depending on the predominance of focal capillary invasion and/or pulmonary edema. As the disease progresses, the classic CT features of secondary pulmonary hypertension will be manifest, including the enlargement of the main pulmonary artery and the right chamber of the heart. Ventilation–perfusion scans may show match defects [4]. Radionuclide lung scan shows the combination of enhanced perfusion in the hemangiomatous tissue and decreased ­perfusion in the regions with occluded vessels. Inhomogeneous perfusion in radionuclide lung scan may be helpful to distinguish PCH from other types of pulmonary hypertension [4]. Angiographic study may also be helpful in distinguishing PCH from other forms of pulmonary hypertension.

4.4  P  athologic Findings and Histopathologic Differential Diagnosis Definite diagnosis requires a histological examination [4, 39, 45–48]. Histologically, the lung shows a multifocal, widespread, dense proliferation of capillary-like endothelial-lined vessels within the alveolar walls, interlobular septa,

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p­ eribronchial and perivascular connective tissue, and pleura (Fig. 5). The alveolar septal capillaries appear engorged and may extend around and into both arteries and veins. Pulmonary veins and venules may show narrowing or obliteration of the lumen that are reminiscent of PVOD. Hemosiderosis and interstitial fibrosis may be seen. Muscular pulmonary arteries usually show mild to moderate medial hypertrophy and intimal thickening, but plexiform lesion is not seen in PCH [4, 39, 45–48]. Histopathologic differential diagnosis of PCH includes PVOD, acute congestion, and atelectasis artifact. In some cases of PVOD, one may see foci of increase capillary number in the alveolar septa that are indistinguishable from PCH. However, the infiltrative growth into bronchial and venous walls in PCH is not prominent in PVOD. The luminal occlusion of pulmonary veins in PCH is not as widespread as in PVOD and is typically caused by proliferative capillaries. In contrast, the venous lumens in PVOD are mostly occluded by recanalizing or fibrotic thrombi. However, there is still a significant overlap in the clinicopathologic features of PCH and PVOD, and the distinction between PVOD and PCH may be arbitrary. Passive congestion causing alveolar capillary engorgement and atelectasis may give the superficial impression of PCH. However, there is no evidence of capillary proliferation; thus, there is no increased density of capillaries. Marked alveolar hemosiderosis seen in some PCH cases may mimic Goodpasture’s disease or idiopathic pulmonary hemosiderosis in a limited tissue sample. In fact, the case of PCH first described by Wagenvoort et al. was initially diagnosed as primary pulmonary hemosiderosis on open lung biopsy, but autopsy revealed features typical of PCH [39]. Diffuse pulmonary hemangiomatosis occurring in pediatric patients is a form of vascular malformation with

Fig.  5  Pulmonary capillary hemangiomatosis characterized by an ill-defined area showing septal thickening due to increased alveolar capillary number. Thickened pulmonary arterioles are also apparent (hematoxylin and eosin stain, ×100 original magnification)

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e­ xtrapulmonary as well as pulmonary involvement and ­consists of numerous large and dilated vascular channels resembling a cavernous hemangioma, which should be ­easily distinguishable from small capillaries in PCH [39].

4.5  Treatment and Prognosis Only bilateral lung or heart–lung transplantation currently offers long-term survival, although no optimal therapy for PCH has been established owing to its rarity. Prostacyclin therapy is generally contraindicated owing to the possible complication of pulmonary edema as reported previously [50]. Other potential therapies include inhaled NO and angiogenesis inhibitors such as interferon [51]. A case report ­documented remission achieved by doxycycline therapy in a case of PCH with atypical endotheliomatosis [51].

5  S  econdary Tumor Involvement of the Pulmonary Vasculature Tumor embolization to the pulmonary vasculature is common and has been reported in 26% of autopsies on patients who had extrathoracic malignancy [2]. Direct extension of tumor thrombus into the proximal pulmonary artery may occur in renal cell carcinomas as a continuous column of tumor thrombus filling the renal vein, inferior vena cava, and right atrium [52]. Pelvic sarcomas such as endometrial stromal sarcoma and uterine leiomyosarcomas may form tumor emboli to involve the inferior vena cava and right side of the heart which may eventually extend to the pulmonary artery [53]. Polypoid growth within the main pulmonary arterial lumen has been reported as a rare growth pattern in primary lung carcinomas [54]. However, microscopic foci of pulmonary arterial invasion are not uncommon in resected lung specimens from primary lung carcinoma cases [55]. Surgical excision of the centrally located tumor embolus or thrombus could offer a markedly improved prognosis; however, the preoperative diagnosis is difficult [52–55]. The most common primary carcinomas giving rise to tumor emboli in the large pulmonary arteries include choriocarcinomas and carcinomas of the stomach, breast, prostate, liver, and kidney [56]. Two distinct patterns of pulmonary arterial metastasis have been described: thrombotic microangiopathy and vascular intimal carcinomatosis [56–58]. Thrombotic microangiopathy, also known as carcinomatous arteriopathy, carcinomatous endarteritis, or embolic carcinomatosis, has been reported in 3.3% of cancer patients in autopsy series

96  Tumors of the Pulmonary Vascular Bed

[56, 57]. It is most frequently associated with adenocarcinomas from various primary sites, including the stomach, breast, colon, pancreas, and liver [56]. On histological ­examination, pulmonary arteries and arterioles are occluded by fibrocellular intimal proliferation and fibrin thrombosis that contain only minor component of malignant tumor cells. However, these malignant cells are postulated to initiate the thrombotic process via local activation of coagulation [57]. Clinical manifestations include progressive dyspnea, cough, hypoxia, and pulmonary hypertension [56, 57]. Pulmonary hypertension associated with thrombotic microangiopathy should be considered in the differential diagnosis of primary pulmonary hypertension, particularly in patients who develop subacute cor pulmonale and have a known history of malignancy [56, 57]. Chest radiography findings may be unremarkable despite there being severe clinical symptoms and signs [57]. A so-called tree-in-bud pattern, a finding more typically seen in cases of infectious bronchiolitis, has been reported as a helpful finding in chest CT [58]. Pulmonary vascular intimal carcinomatosis is histologically characterized by a diffuse replacement of the arterial intima by the tumor cells that do not invade the medial wall or occlude the arterial lumen. This pattern was described by Kobayashi et al. in a case of transitional cell carcinoma of the renal pelvis that had an isolated metastasis to the muscular pulmonary arteries greater than 300  mm in diameter [59]. Rarely, nonepithelial tumors can involve the pulmonary arteries, causing pulmonary hypertension; a case of intravascular bronchioloalveolar tumor of the lung, also known as epithelioid hemangioendothelioma, reported an unusual presentation as thromboembolic disease and rapidly progressive pulmonary hypertension [60]. Metastatic or primary tumor may also involve the large pulmonary veins with or without extension to the left atrium of the heart. Pulmonary infarction due to pulmonary venous tumor thrombus from metastatic osteogenic sarcoma has been reported [61]. Metastatic chondrosarcoma to the lung has been reported to present as an embolic cerebral infarction that was caused by invasion of the pulmonary veins and subsequent extension into the left atrium [62]. Segmental PVOD secondary to lung cancer has been documented in two patients who had squamous cell carcinoma and leiomyosarcoma, respectively [63].

6  Summary The rare primary tumors arising from the pulmonary vasculature are mostly sarcomas of the main pulmonary arteries and veins. Sarcomas arising in the pulmonary arterial intima are far more prevalent than those arising in the venous intima or those arising in the muscular media of pulmonary arteries

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and veins. Clinically, intimal sarcomas of pulmonary arteries are often indistinguishable from pulmonary chronic ­thromboembolic diseases, which causes delayed diagnosis and a fatal outcome. Recently, long-term survival has been documented especially in those who have low-grade PAS showing myofibroblastic phenotype. An aggressive therapeutic approach has been associated with improved survival in these patients who would otherwise follow a dismal clinical course. Most sarcomas of the pulmonary veins are mural sarcomas, which tend to follow a somewhat less dismal clinical course than intimal sarcomas of the pulmonary artery. PCH is a rare disease of unknown cause and unclear pathogenesis. Regardless of its biologic nature, most PCH patients will develop severe pulmonary hypertension often followed by a fatal right-sided heart failure unless they receive lung transplantations with a timely diagnosis. However, some PCH patients may not have a significant clinical manifestation and are only found to have histologic changes of PCH at the time of postmortem examination as incidental findings. Secondary tumors in the pulmonary vasculature may masquerade as primary vascular tumors or as primary/idiopathic pulmonary hypertension. Thrombotic microangiopathy and intimal carcinomatosis have been described as the distinct patterns seen in the secondary arterial involvement. Pulmonary venous tumor thrombosis may result in pulmonary infarctions or even systemic infarctions via left atrial involvement with subsequent systemic tumor embolism.

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1353 58. Franquet F, Giménez A, Prats R, Rodríguez-Arias JM, Rodríguez C (2002) Thrombotic microangiopathy of pulmonary tumors: a vascular cause of tree-in-bud pattern on CT. Am J Radiol 179:897–899 59. Kobayashi H, Tamashima S, Shigeyama J, Shimizu S, Suchi T (1997) Vascular intimal carcinomatosis: an autopsy case of unusual form of pulmonary metastasis of transitional cell carcinoma. Pathol Int 47:655–657 60. Yi ES, Auger WR, Friedman PJ, Morris TA, Shin SS (1995) Intravascular bronchioloalveolar tumor of the lung presenting as pulmonary thromboembolic disease and pulmonary hypertension. Arch Pathol Lab Med 119:255–260 61. Nelson E, Klein JS (2000) Pulmonary infarction resulting from metastatic osteogenic sarcoma with pulmonary venous tumor thrombus. Am J Radiol 174:531–533 62. Woodring JH, Bognar B, van Wyk CS (2002) Metastatic chondrosarcoma to the lung with extension into the left atrium via invasion of the pulmonary veins presentation as embolic cerebral infarction. J Clin Imag 26:338–341 63. Ibrahim NBN, Burnley H, Gaber KA et al (2005) Segmental pulmonary veno-occlusive disease secondary to lung cancer. J Clin Pathol 58:434–436

Chapter 97

Cor Pulmonale Mardi Gomberg-Maitland, Thenappan Thenappan, John J. Ryan, Ankush Goel, Nicole Cipriani, Aliya N. Husain, Amit Patel, Savitri E. Fedson, and Stephen L. Archer

Abstract  The World Health Organization (WHO) defined cor pulmonale in 1963 as, “hypertrophy of the right ventricle (RV) resulting from diseases affecting the function and/or the structure of the lungs except when these alterations are the results of diseases which primarily affect the left side of the heart or congenital heart disease”. At that time this definition had somewhat less clinical value than currently, since RV hypertrophy (RVH) was largely a pathological diagnosis based on EKG (which is insensitive) or autopsy. In 1970, Behnke et al. replaced the term “hypertrophy” in the definition by substituting it “with alteration in the structure and function of the RV”. However, this definition too was considered inaccurate as it included a spectrum of RV abnormalities from mild to severe right-sided heart failure. Pulmonary hypertension (PH) is an inciting stimulus for the RVH and RV failure in patients with cor pulmonale. PH is defined as a mean pulmonary artery pressure (PAP) greater than 25 mmHg at rest or greater than 30 mmHg during exercise, in the presence of a pulmonary capillary wedge pressure (PCWP) less than 15 mmHg. Chronic respiratory diseases and/or hypoxia cause pulmonary arterial vasoconstriction and remodeling, resulting in elevated PAP and pulmonary vascular resistance (PVR). The elevated PAP positively reinforces the vascular remodeling and sustains RVH. In cor pulmonale, the PAP should be greater than 25 mmHg and the PVR greater than 3 Wood units. The most recent WHO classification of PH has classified PH associated with chronic respiratory diseases and/or hypoxia as a separate category. Thus, cor pulmonale, as currently defined requires demonstration of both RVH and/or RV dilatation and PH in association with a chronic respiratory disease and/or hypoxia. Ultimately cor pulmonale identifies a subset of patients with lung disease who are at increased risk of RV dysfunction and death. We favor the following definition of cor pulmonale: the combination of right ventricular hypertrophy and/or failure with pulmonary hypertension that results from lung disease, impaired ventilation, or environmental hypoxia. M. Gomberg-Maitland (*) University of Chicago Medical Center, MC 5403, 5841 South Maryland Ave, 60637 Chicago, IL, USA e-mail: [email protected]

Keywords  Pulmonary heart disease • Right ventricular hypertrophy • Right heart failure • Pulmonary vascular resistance

1  Classification 1.1  Definition The World Health Organization (WHO) defined cor pulmonale in 1963 as, “hypertrophy of the right ventricle (RV) resulting from diseases affecting the function and/or the structure of the lungs except when these alterations are the results of diseases which primarily affect the left side of the heart or congenital heart disease” [1]. At that time this definition had somewhat less clinical value than currently, since RV hypertrophy (RVH) was largely a pathological diagnosis based on EKG (which is insensitive) or autopsy [2]. In 1970, Behnke et al. replaced the term “hypertrophy” in the definition by substituting it “with alteration in the structure and function of the RV” [3]. However, this definition too was considered inaccurate as it included a spectrum of RV abnormalities from mild to severe right-sided heart failure. Pulmonary hypertension (PH) is an inciting stimulus for the RVH and RV failure in patients with cor pulmonale. PH is defined as a mean pulmonary artery pressure (PAP) greater than 25 mmHg at rest or greater than 30 mmHg during exercise, in the presence of a pulmonary capillary wedge pressure (PCWP) less than 15  mmHg [4]. Chronic respiratory diseases and/or hypoxia cause pulmonary arterial vasoconstriction and remodeling, resulting in elevated PAP and pulmonary vascular resistance (PVR) [5]. The elevated PAP positively reinforces the vascular remodeling and sustains RVH. In cor pulmonale, the PAP should be greater than 25 mmHg and the PVR greater than 3 Wood units. The most recent WHO classification of PH (Table 1) has classified PH associated with chronic respiratory diseases and/or hypoxia as a separate category (group 3) [6]. Thus, cor pulmonale, as currently defined requires demonstration of both RVH and/or RV dilatation and PH in association with a chronic respiratory disease and/or hypoxia [7].

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_97, © Springer Science+Business Media, LLC 2011

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1356 Table 1  Clinical classification of pulmonary hypertension Group 1: Pulmonary arterial hypertension Idiopathic • Familial • Associated with collagen vascular disease, congenital systemic-topulmonic shunt, portal hypertension, human immunodeficiency virus infection, and drugs and toxins • Other: thyroid disorders, glycogen storage disease, Gaucher’s disease, hereditary hemorrhagic telangiectasia, hemoglobinopathies (sickle cell disease and hemolytic anemias), myeloproliferative disorders, splenectomy • Pulmonary veno-occlusive disease, pulmonary capillary hemangiomatosis, and persistent pulmonary hypertension of the newborn Group 2: Pulmonary hypertension with left-sided heart disease • Left-sided ventricular or atrial disease • Left-sided valvular disease Group 3: Pulmonary hypertension associated with lung disease and/or hypoxemia • Chronic obstructive lung disease • Interstitial lung disease • Sleep-disordered breathing • Alveolar hypoventilation disorders • Chronic exposure to high altitude • Developmental abnormalities Group 4: Pulmonary hypertension due to chronic thrombotic and/or embolic disease • Thromboembolic obstruction of proximal pulmonary arteries • Thromboembolic obstruction of distal pulmonary arteries Group 5: Miscellaneous • Sarcoidosis, histiocytosis X, lymphangiomyomatosis, compression of pulmonary vessels (adenopathy, tumor, fibrosing mediastinitis), and schistosomiasis Adapted from Simonneau et al. [6] with permission from Elsevier

M. Gomberg-Maitland et al. Table 2  Diseases of the respiratory system associated with pulmonary hypertension Obstructive lung diseases • COPDa (chronic obstructive bronchitis, emphysema, and their association) • Asthma (with irreversible airway obstruction) • Cystic fibrosisb • Bronchiectasis • Bronchiolitis obliterans Restrictive lung diseases • Neuromuscular diseases: amyotrophic lateral sclerosis, myopathy, bilateral diaphragmatic paralysis, etc. • Kyphoscoliosisb • Thoracoplasty • Sequelae of pulmonary tuberculosis • Sarcoidosis • Pneumoconiosisb • Drug-related lung diseases • Extrinsic allergic alveolitis • Connective tissue diseases • Idiopathic interstitial pulmonary fibrosisb • Interstitial pulmonary fibrosis of known origin Respiratory insufficiency of “central” origin • Central alveolar hypoventilation • Obesity–hypoventilation syndromeb (formerly “Pickwickian syndrome”) • Sleep apnea syndrome a Very frequent cause of pulmonary hypertension b Relatively frequent cause of pulmonary hypertension Adapted from Weitzenblum [7] with permission from BJM Publishing Group Ltd

Ultimately cor pulmonale identifies a subset of patients with lung disease who are at increased risk of RV dysfunction and death. We favor the following definition of cor pulmonale: the combination of right ventricular hypertrophy and/or failure with pulmonary hypertension that results from lung disease, impaired ventilation, or environmental hypoxia.

1.2  Etiology Chronic respiratory diseases associated with cor pulmonale can be classified under three major categories: obstructive lung diseases, restrictive lung diseases, and respiratory insufficiency of central origin [7]. Table 2 lists the various disorders under each category. Chronic obstructive pulmonary disease (COPD), which includes emphysema (Fig.  1) and chronic bronchitis, is the most common cause of cor pulmonale, accounting for 84% of patients in one study [8]. The most common restrictive lung diseases associated with cor pulmonale include idiopathic pulmonary fibrosis (IPF) (Fig.  2), ­sarcoidosis, and connective-tissue-related interstitial lung

Fig. 1  Lung from a patient with severe emphysema showing apical bleb

disease [9]. Patients with scleroderma also frequently develop cor pulmonale related to pulmonary fibrosis. Interestingly, the scleroderma patients are those with antitopoisomerase antibodies and diffuse scleroderma [10], not the subset with CREST (telangiectasia and anticentromere antibodies) who develop a more classic pulmonary arterial hypertension (PAH) syndrome. Compared with scleroderma without pulmonary disease, cor pulmonale increases the mortality risk

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ratio. In a study of 619 patients with echocardiographic measurement of PH and lung function testing to detect IPF, relative mortality for scleroderma patients with isolated PH, combined IPF and PH, and isolated IPF (compared with that for those with scleroderma but no IPF and no PH) was 2.9, 2.4, and 1.61, respectively [10]. The other lung diseases frequently associated with cor pulmonale are obesity hypoventilation syndrome and obstructive sleep apnea. Finally, cor pulmonale can also occur in the absence of lung disease in individuals living at high altitude. In general, the severity of PH in cor pulmonale is mild compared with that in idiopathic PAH. Moreover, it is unclear whether the PH, which undoubtedly portends a poor prognosis, is a cause of excess mortality or a marker of the severity of the lung disease, or both.

1.3  Epidemiology Cor pulmonale accounts for 10–30% of all heart failure admissions in the USA [11]. Accurate diagnosis of cor pulmonale requires right-sided heart catheterization (RHC)

Fig. 2  Lung from a patient with idiopathic pulmonary fibrosis

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because Doppler-based estimation of PAP can be inaccurate, particularly in patients with chronic lung disease [12]. Moreover, estimation of PCWP cannot be reliably performed by echocardiography. In addition to hemodynamics, diagnosis also requires documentation that the RV is enlarged, hypertrophied, or hypokinetic. Currently no large populationbased study exists in which RHC has been performed on patients with chronic lung diseases; thus, the available epidemiological data on cor pulmonale are based on hospitalized patient cohorts. The estimated prevalence of cor pulmonale in patients with chronic respiratory disease varies widely across different studies depending on the definition of PH, the diagnostic modality used to categorize PH, and the severity of the underlying lung disease. COPD is the most common respiratory disorder leading to cor pulmonale. The prevalence of cor pulmonale in patients with COPD ranges from 2.7 to 90.8% depending on the level of mean PAP used to define PH and the severity of COPD (Table 3). PH in patients with COPD is typically mild to moderate [13]. In a minority of patients, however, it can be severe and out of proportion to the lung disease. In a recent retrospective series of 998 patients with COPD who underwent RHC as a part of the workup for chronic respiratory failure approximately 3% (27 of 998 patients) had severe PH defined as a mean PAP greater than 40 mmHg [14]. Of the 27 patients, 11 patients had COPD as the only cause for PH. These 11 patients had an unusual pattern of cardiopulmonary abnormalities, with mild to moderate airway obstruction, severe hypoxemia, hypocapnia, and a very low diffusing capacity for carbon monoxide compared with a control group of COPD patients who had no PH. Survival was shorter in those with cor pulmonale than in the control subjects (P = 0.0026) [14]. PH tends to progress slowly in patients with COPD. When Kessler et al. prospectively followed patients with COPD with mild to moderate hypoxemia and no resting PH, only 25% developed a mean PAP greater than 20 mmHg over a 6-year period [15]. The group as a whole had an average change in the mean PAP of 0.4 mmHg per year. In this study, baseline PAP (resting and exercise) predicted the future development of PH. There are only limited data on the prevalence of cor ­pulmonale in patients with interstitial lung diseases. In a

Table 3  Prevalence of pulmonary hypertension in patients with COPD Study Study cohort Severity of COPD

Definition of PH

Prevalence of PH

Weitzenblum et al. [122] Patients with moderate to severe COPD FEV1/VC = 40.2% MPAP > 20 mmHg 35% FEV1 = 24.3% MPAP > 25 mmHg 50% Thabut et al. [157] Severe COPD patients undergoing evaluation for lung transplant or lung MPAP > 35 mmHg 13.5% volume reduction surgery Scharf et al. [158] Patients with severe emphysema FEV1 = 27% MPAP > 20 mmHg 90.8% Chaouat et al. [14] COPD patients with chronic respiratory FEV1 = 33% MPAP > 40 mmHg 2.7% failure All studies are based on right-sided heart catheterization PH pulmonary hypertension, FEV1, forced expiratory volume in 1 s, VC, vital capacity, MPAP mean pulmonary artery pressure

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retrospective study of 88 patients with IPF referred to the Mayo Clinic, 84% had a Doppler-estimated PAP greater than 35 mmHg [16]. However, pretransplant RHC data in patients with severe IPF suggest that fewer patients (30–46%) have PH, defined as a mean PAP greater than 25 mmHg [17, 18]. A  retrospective cross-sectional study of 619 patients with scleroderma found PH and interstitial lung disease in 18% of patients, based on echocardiography and pulmonary function test performed within 6 months of one another [10]. The prevalence of cor pulmonale in patients with sarcoidosis varies from estimates as high as 73.8%, based on RHC in patients listed for lung transplant, to as low as 5.7%, in a cross-sectional study based on Doppler echocardiography [19, 20].

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The histological manifestations of cor pulmonale vary depending on the underlying lung disease, and a thorough histological examination of both the pulmonary vasculature

and the parenchyma at the time of biopsy or autopsy is recommended. The vascular disease is primarily manifest in small arteries and arterioles. The microscopic findings involve both the tunica media and the intima of small muscular arteries and arterioles, which are located adjacent to small bronchioles and within alveolar septa, respectively. One characteristic feature of cor pulmonale is the muscularization of small arterioles. Normally, arterioles within alveolar septa are nonmuscular. This feature (muscularization of small pulmonary arteries) is recapitulated in animal models of cor pulmonale created by exposing animals to chronic hypoxia [23]. The finding of smooth muscle fibers surrounding arterioles within alveolar septa indicates the presence of distal extension of muscularization (Fig.  4a). This tissue changes are prominent in emphysema, and are often the only vascular abnormality seen [24, 25]. On the other hand, IPF manifests both distal extension of arteriolar muscularization and medial hypertrophy of small arteries [26, 27]. Medial hypertrophy consists of increased numbers of smooth muscle fibers and nuclei in the tunica media, resulting in an increased thickness of the arterial wall (Fig. 4b, c). Sarcoidosis with PH manifests itself as direct infiltration of nonnecrotizing granulomas in the pulmonary vasculature [28, 29]. The walls of small to medium-sized arteries are invaded by collections of histiocytes, often with prominent foreign-body giant cells (Fig. 4d). Asteroid bodies, cytoplasmic inclusions within the granulomas in sarcoidosis, are characteristic but not diagnostic [30]. Finally, the histologic finding that characterizes the lung disease associated with connective tissue disease is intimal fibrosis of pulmonary artery branches [28, 31]. In this case, the tunica intima becomes concentrically thickened by collagen and proliferating endothelial cells (Fig. 4e). In summary, emphysema is characterized by arteriolar muscularization, IPF by medial hypertrophy, sarcoidosis by direct invasion of vessels by granulomas, and connective tissue disease by intimal fibrosis. Findings that differentiate PAH from cor pulmonale include the involvement of large vessels in PAH, which often have intimal plaques and the

Fig. 3  The heart in cor pulmonale. (a) Cross-section of the heart from a patient with cor pulmonale showing right ventricular hypertrophy and dilation and a D-shaped left ventricle (chamber on the right of the pho-

tograph). (b) Histology of right ventricular myocardium in cor pulmonale showing myofiber hypertrophy with enlarged “boxcar” nuclei (inset), hematoxylin and eosin (H&E) stain, high power with magnified insert

1.4  Histopathology 1.4.1  The RV The pulmonary vascular remodeling in cor pulmonale increases RV afterload, leading to RVH and RV dilation [21, 22]. The RV wall thickness increases and may even double in cor pulmonale. The trabeculae become more prominent, and may bridge the ventricular lumen (Fig. 3a). Histologically, myocardial hypertrophy is evidenced by an increase in nuclear size and change in shape to rectangular, or “boxcar” nuclei (Fig. 3b). With increasing RV pressure and blood flow, the interventricular septum may bow into the left ventricle, with the compression creating a D-shaped left ventricle. This finding reflects the severity of the PH and is not specific for cor pulmonale.

1.4.2  The Lung

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Fig. 4  Lung histology in cor pulmonale. (a) Arteriolar muscularization in emphysema, H&E stain, high power. (b) Medial hypertrophy, H&E stain, high power. (c) Medial hypertrophy, trichrome stain, high power. (d) Vessel involved by granulomas in sarcoidosis, H&E

stain, low power. (e) Intimal fibrosis in connective tissue disease associated lung disease, H&E stain, high power. (f) Plexiform lesion in idiopathic pulmonary arterial hypertension, H&E stain, high power

presence of plexiform lesions in PAH [32, 33]. Plexiform lesions consist of capillary-like, angioproliferative vascular channels that form within the lumen of small muscular arteries, and often appear at branch points (Fig. 4f). The luminal diameter of these channels may vary, giving them a disordered appearance. Fibrin thrombi are often present within the lumina of plexiform lesions. These lesions are not seen in cor pulmonale and are mentioned here only to contrast them with the histologic findings of cor pulmonale.

of catecholamines may contribute to systemic hypertension and perhaps to cor pulmonale. Alveolar hypoxia is the major mechanism involved in the pathogenesis of PH in cor pulmonale [11]. Acutely, hypoxia causes pulmonary vasoconstriction (hypoxic pulmonary vasoconstriction, HPV) [35], whereas chronic hypoxia leads to pulmonary vascular remodeling by promoting cell proliferation [36, 37] in addition to activating constrictor pathways, such as endothelin synthesis and activation of Rho kinase [38]. Although endothelin-1 is detectable in both the peripheral blood and the lavage of COPD patients, the levels are only modestly elevated compared with those of controls and are not statistically greater in patients with cor pulmonale than in COPD controls [39]. In contrast to its vasodilatory effect on the systemic circulation, acute hypoxia causes vasoconstriction of precapillary pulmonary resistance vessels (HPV) through contraction of pulmonary smooth muscles. HPV is a homeostatic mechanism to optimize ventilation– perfusion matching by shunting the blood flow away from poorly ventilated lungs [35]. HPV is initiated by inhibition of voltage-gated potassium (Kv) channels, notably Kv1.5, in pulmonary artery smooth muscle cells (PASMCs) [40]. In the PASMC, hypoxia inhibits outward potassium currents, thereby causing membrane depolarization and entry of calcium through the L-type voltage-gated calcium channels. In addition, hypoxia causes calcium sensitization, which sustains HPV [41], and also promotes release of calcium stores

1.5  Pathophysiology Cor pulmonale results from a variety of active and passive mechanisms that alter pulmonary hemodynamics and ultimately RV function (Fig. 5).

1.5.1  Role of Catecholamines, Hypoxia, Hypercarbia, and Acidosis In normal individuals, altitude increases systemic blood pressure and plasma arterial noradrenaline and adrenaline concentrations (3.7-fold and 2.4-fold, respectively) [34]. Thus, chronic hypoxia activates the sympathetic nervous system in healthy humans; this elevation in the concentrations

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Fig. 5  Confirmed and suspected mechanisms leading to pulmonary hypertension in chronic obstructive pulmonary diseases

from the sarcoplasmic reticulum via store-operated channels [42]. Hypoxia also stimulates Rho kinase, which in turn inhibits myosin light chain phosphatase, thereby increasing phosphorylation of the light chain and augmenting contraction [35, 40]. HPV is transient and resolves rapidly. Although HPV persists in COPD patients [43], there is a general tendency for HPV to decrease in patients (and animals) exposed to chronic hypoxia. Indeed, HPV is largely absent in species that are native to highlands, such as the yak. Thus, HPV likely plays only a minor role in the pathogenesis of chronic cor pulmonale and is generally beneficial in these patients (as manifest by a worsening shunt and hypoxemia when HPV is suppressed in COPD using a calcium channel blocker [44]). However, excessive HPV may contribute to the acute worsening of PH in patients with COPD during acute exacerbations, exercise, and sleep [11].

1.5.2  The Basic Science of Cor Pulmonale Chronic hypoxia results in pulmonary vascular remodeling characterized by muscular medial hypertrophy of the small pulmonary arteries, neomuscularization of the pulmonary arterioles, and a variable degree of intimal fibrosis [45]. The mechanism underlying chronic-hypoxia-induced pulmonary vascular remodeling is unclear. Stenmark et al. have documented excessive proliferation in PASMCs of the calf in response to hypoxia, but remind us that the affects of hypoxia touch each layer of the pulmonary artery [46, 47]. They noted that there is smooth muscle heterogeneity within the pulmonary artery wall and that only certain populations of PASMCs proliferate in response to hypoxia (PASMCs that

stain negative for the cytoskeletal protein metavinculin) [47]. We have found a similar diversity in the expression of O2-sensitive Kv channels in the pulmonary artery, noting that Kv1.5 channel expression is enriched in resistance PASMCs, the site of maximal HPV [48, 49]. In vitro studies on human pulmonary artery cells and in vivo rat models, as well as human studies, have suggested that chronic hypoxia produces an imbalance of endotheliumderived vasoactive and vasoproliferative mediators that favors constriction and proliferation. Hypoxia increases the synthesis of vasoconstrictive endothelin and angiotensin II, whereas it decreases the production of the potent vasodilators nitric oxide and prostacyclin [50–54]. In addition, hypoxia induces the synthesis of vascular endothelial growth factor (VEGF) and platelet-derived growth factor [51]. Several other vasoactive and proliferative mediators, such as serotonin and serine elastases, have also been observed to be released by hypoxia [55, 56]. These vasoactive and vasoproliferative mediators interact in a highly complex fashion, leading to increased pulmonary vascular tone and proliferation of pulmonary vascular cells [46].

1.5.3  T  he Mitochondria: Hypoxia-Inducible Factor 1a: Kv Pathway in PH Abnormalities in a mitochondria–hypoxia-inducible factor 1a (HIF-1a)–Kv pathway, recently discovered in an experimental model of PAH, may also contribute to excess smooth muscle cell proliferation and hypertrophy in cor pulmonale. From animal models we know that the PASMCs in cor pulmonale are calcium-overloaded and prone to constriction

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and proliferation. The fawn-hooded rat is a mutant strain which offers useful information about the basic mechanism of cor pulmonale [57]. These rats have mild PH and polycythemia and are hypoxia-sensitive (i.e. they develop PH in response to modest hypoxia or even spontaneously in normoxia as they age) [36, 57–61]. In the fawn-hooded rat, the ionic remodeling appears to relate to normoxic activation of HIF-1a, which promotes a metabolic shift and causes downregulation of Kv1.5 (one of the Kv channels involved in HPV). The fawn-hooded rats are reminiscent of patients from the Chuvash region of Russia, in whom homozygous germline mutations (598 C->T) of the von Hippel–Lindau gene lead to HIF-1a upregulation during normoxia, resulting in high erythropoietin levels, polycythemia, and PH [62]. In chronic hypoxia (in rats) there is also evidence that hypoxia activates the master transcription factor HIF-1a and that this (in part) explains the loss of Kv1.5 and development of PH. The decrease in expression of specific Kv channels (notably Kv1.5) in chronic hypoxia has been confirmed by several groups [61, 63], causes PASMC depolarization [64], and favors PASMC proliferation and vascoconstriction [57]. Further supporting the pathological role of HIF-1a in cor pulmonale, mice that are haploinsufficient for HIF-1a are resistant to the development of hypoxic PH and do not downregulate their PASMC Kv current in response to hypoxia [65, 66]. Thus, there is some basic science to suggest that abnormalities in mitochondrial metabolism and redox signaling in PASMCs whether inherited, as in the fawn-hooded rat, or acquired, as occurs with exposure to chronic hypoxia, leads to normoxic activation of HIF-1a and ultimately Kv channel downregulation, which contributes to PH [57]. This mitochondria–HIF-1a–Kv pathway may promote proliferating apoptosis-resistant PASMCs that contribute to cor pulmonale. This pathway offers several appealing therapeutic targets to treat cor pulmonale. Although most of the evidence for this mechanism comes from rodent studies,

similar mitochondrial abnormalities, resulting in impaired respiration and enhanced glycolysis, occur in PASMCs [57] and endothelial cells [67] of humans with PAH. There are no human cor pulmonale data assessing this pathway. In rodents, experimental cor pulmonale can be regressed by restoring oxidative mitochondrial metabolism (using the pyruvate dehydrogenase kinase inhibitor dichloroacetate [37, 57]) or even by Kv1.5 gene therapy [36]. Dichloroacetate appears to be beneficial by reducing proliferation and enhancing apoptosis in PASMCs. Hypercarbia and acidosis also play a role in the development of cor pulmonale, although the impact of these factors is less than that of hypoxia. Hypercarbia induces hyperventilation, which in turn produces changes in lung mechanics, leading to increased PAP. It has also been suggested that hypercarbia augments HPV [68]. Likewise, acidemia acts synergistically with hypoxia to enhance pulmonary vasoconstriction [69].

Fig. 6  Airway pressure to pulmonary blood flow relationships relevant to cor pulmonale. The relationship of flow and pressure in the pulmonary circulation obtained after increasing the flow through the left pulmonary artery by balloon occlusion of the right pulmonary artery. (a) The influence of alveolar pressure on pulmonary blood flow and pressure in cor pulmonale is assessed by increasing mouth pressure.

(b) The relationship between flow and pressure of the pulmonary circulation in normal subjects with increased mouth pressure is similar to the relation in patients with chronic obstructive pulmonary disease. (Adapted with permission from MacNee [11]. The official journal of the American Thoracic Society, copyright American Thoracic Society)

1.5.4  Abnormal Lung Mechanics In addition to the structural disease in the pulmonary vasculature, elevated alveolar pressure in patients with COPD may contribute to the increased PVR [70]. A linear relationship exists between arterial pressure and flow in the pulmonary circulation, when the intraalveolar pressure is normal. However, in patients with obstructive lung disease, when the alveolar pressure increases owing to increased airway resistance, the PAP increases out of proportion to flow, owing to compression of the pulmonary vasculature by the overdistended alveoli. Heath and coworkers [71] elegantly demonstrated this by increasing the alveolar pressure by increasing the gas pressure in the mouth (Fig. 6). As alveolar pressures are increased, the pressure in the pulmonary circulation initially increases steeply and then changes in a curvilinear

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pattern as opposed to a normal linear pattern. Airway resistance affects pulmonary hemodynamics, particularly when there is increased ventilatory pressure, as occurs during COPD exacerbations [11].

1.5.5 Inflammation The role of inflammation as a mediator of cor pulmonale in patients with chronic lung disease is controversial [72]. The number of inflammatory cells, especially CD8 lymphocytes, in the pulmonary vascular wall has been shown to correlate with the enlargement of the intimal layer [73]. Systemic levels of inflammatory cytokines, such as C-reactive protein and interleukin 6, correlate with mean PAP in patients with COPD [74, 75]. Recent studies suggest that oxidants in cigarette smoke cause inflammation, releasing endothelium-derived vasoconstrictive and vasoproliferative mediators such as endothelin and VEGF [76]. Oxidants in cigarette smoke also interfere with nitric oxide mediated vasodilatation by decreasing nitric oxide bioactivity. Pulmonary vascular remodeling has been reported in smokers even when their lung function is normal [77].

1.5.6  Miscellaneous Additional mechanisms that contribute to cor pulmonale include increased blood viscosity from polycythemia, emphysematous reduction of the pulmonary capillary bed, and the anatomical distortion of the pulmonary vasculature by parenchymal changes seen in interstitial lung diseases. Polycythemia, secondary to hypoxia, increases blood viscosity, which in turn augments PVR on the basis of Poiseuille’s equation. Reducing blood volume and thereby blood viscosity decreases PAP and PVR in COPD patients with polycythemia [78]. Theoretically, destruction of the pulmonary vascular bed in emphysema can result in increased PVR. However, the report of Hicken et al. contradicts this thinking, noting a poor correlation between the presence of RVH, a

Fig. 7  The response of the right and left ventricles to an acute increase in afterload. (Adapted with permission from MacNee [11]. The official journal of the American Thoracic Society, copyright American Thoracic Society)

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sign of significant cor pulmonale, and the total alveolar surface area, a reflection of the size of the pulmonary vascular bed [79]. Thus, the role of emphysematous reduction of the pulmonary capillary bed in the pathogenesis of cor pulmonale is unclear. Nonetheless, loss of the capillary vascular bed may contribute to the worsening of PH in these patients during exercise, which may expose a reduced reserve capacity to accommodate increased pulmonary blood flow [5].

1.6  RV Dysfunction The RV is a thin-walled, crescentic, compliant chamber formed by a concave free wall opposite to a convex interventricular septum. The RV both contracts against the septum in systole and also shortens in the long axis. Although the RV pumps the same stroke volume as the left ventricle, its stroke work is significantly less than that of the left ventricle since the PVR is significantly lower than the systemic vascular resistance. In view of its geometric configuration, the RV has a lower volume to surface area ratio than the left ventricle; thus, it acts as a volume pump rather than a pressure pump [80]. When the RV afterload increases acutely, as in acute pulmonary embolism, the RV cannot generate a mean PAP greater than 40 mmHg, and in such circumstances, the RV stroke volume decreases linearly with the increase in the afterload [11] (Fig. 7). In addition, the shortening of the RV in the long axis is diminished in PH, as reflected by a diminished tricuspid annular plane systolic excursion (TAPSE) on M-mode echocardiography. On the other hand, sustained pressure overload, secondary to chronic PH, leads to RVH and RV enlargement. In the presence of RVH, the perfusion pressure gradient between the right coronary artery, which supplies the RV both during systole and diastole, and the RV cavity decreases. The increased demand due to the hypertrophy coupled with the decreased supply results in a relative RV ischemia, leading to RV diastolic and systolic dysfunction, and eventually right-sided heart failure [81].

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PH associated with chronic lung disease is generally mild to moderate, not the systemic levels of PAP seen in patients with PAH [13]. In addition, PH in cor pulmonale tends to progress slowly over years, allowing the RV to adapt [15]. Thus, it is unclear whether the concept of sustained pressure overload should be used at all to describe the pathophysiological features of cor pulmonale [5]. Patients with moderate to severe COPD, but normal arterial PO2, usually have normal RV function during periods when their lung disease is stable [82]. Likewise, RV contractility, assessed by the end systolic pressure–volume relation, is generally preserved in patients with cor pulmonale when their underlying lung disease is stable [83, 84]. In patients with chronic lung disease, RV dysfunction and right-sided heart failure primarily occur during acute exacerbations of the lung disease, with sleep, during exercise, and when there is an acute increase in PAP (due to HPV, acidosis, or altered lung mechanics) [85–87]. Severe PH in a patient with chronic lung disease should elicit a search for other causes of PH, making cor pulmonale a diagnosis of exclusion.

1.7  Clinical Presentation Dyspnea is the most common symptom of cor pulmonale, but clinical recognition is difficult as most patients with chronic lung disease often have dyspnea. The presence of other symptoms such as chest pain, light-headedness, syncope, and worsening dyspnea may indicate the presence of cor pulmonale [88]. Typical physical examination findings suggestive of PH and RVH such as an elevated jugular venous distension, a palpable RV heave, a loud pulmonic component of the second heart sound, a holosystolic murmur secondary to tricuspid regurgitation, and an early diastolic murmur, due to pulmonary regurgitation, may be masked by the hyperinflation of the chest [7]. However, cor pulmonale patients tend to have elevated jugular venous pressure, ascites, and edema when they develop decompensated right-sided heart failure. It is vital to recognize that edema in patients with cor pulmonale is not always synonymous with right-sided heart failure. Some patients may develop edema even in the absence of right-sided heart failure. Although the exact mechanism for this is unclear, hypercapnia and hypoxia have been suggested to cause renal vasoconstriction, leading to salt and water retention [89].

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2.1  Electrocardiography The most common electrocardiographic signs of cor pulmonale include an enlarged P wave in lead II (P-pulmonale), rightward P-wave axis deviation, and S1Q3T3 pattern (measuring a dominant S wave in lead I, a Q wave in III, and an inverted T wave in lead III) (Fig.  8). Other electrocardiographic signs include evidence of RVH, rightward QRS axis deviation, and right bundle branch block [90]. There are several criteria for RVH, but generally tall R waves in the anteriorly and rightward directed leads (aVR, V1, V2) and deep S waves with small R waves in the leftward-directed leads (I, aVL, and lateral precordial leads) suggest RVH. Patients with cor pulmonale associated with COPD have been reported to have low QRS voltage because of hyperinflated lungs that can decrease the amplitude of EKG deflections and often have delayed R-wave progression, rather than R-wave predominance in the precordial leads. The amplitude of the R wave in V1 and the R/S ratio in V2 correlate with PAP in patients with cor pulmonale [91]. Unfortunately, EKG findings are not very sensitive for diagnosing cor pulmonale. In a study of 68 patients with COPD, electrocardiography had a specificity of 86% but a sensitivity of only 51% for diagnosing cor pulmonale compared with RHC. The sensitivity was particularly low (38%) in patients with mild PH [92]. Thus, EKG changes suggestive of right-sided heart involvement in patients with chronic lung disease if present can identify cor pulmonale; however, a normal EKG does not exclude it.

2.2  Chest Radiography Chest radiography is inferior to electrocardiography for the diagnosis of cor pulmonale. The classic radiographic signs of cor pulmonale on chest X-ray (Fig. 9) include enlargement of the main pulmonary artery and its branches, with marked tapering of the vessels in the lung periphery, known as “pruning.” In more advanced stages, when the RV is enlarged, one can see filling in of the retrosternal space on the lateral view. In patients with COPD, the presence of cor pulmonale has been related to a right descending pulmonary artery diameter greater than18 mm [93]. Unfortunately a normal chest X-ray does not rule out the presence of mild cor pulmonale. The value of the chest radiograph is that it is relatively inexpensive, easily performed, and can help to exclude other underlying lung disease and pulmonary venous hypertension.

2  Diagnostic Evaluation Because the clinical signs of cor pulmonale are relatively insensitive, noninvasive testing is a useful adjunct to enable early diagnosis and to quantify PH, RVH, and RV volume/ function.

2.3  Echocardiography Transthoracic echocardiography (TTE) is the most commonly used noninvasive screening test for quantifying cor pulmonale.

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Fig. 8  The electrocardiogram in cor pulmonale. A typical electrocardiogram from a patient with cor pulmonale demonstrating right-axis deviation, right ventricular strain pattern, low voltage in the limb leads, and delayed R-wave transition

Fig. 9  The chest X-ray in cor pulmonale. Typical chest X-ray seen from a patient with sarcoidosis-associated cor pulmonale. Note the prominent pulmonary arteries, pruning of vessels at the periphery, and right ventricular enlargement

TTE can detect elevated PAP, evaluate RV function, and rule out other causes of PH such as left-sided heart disease, shunting, and congenital heart disease (Fig.  10). Pulmonary artery systolic pressure (PASP) is equivalent to the RV systolic pressure (RVSP) in the absence of pulmonary outflow

obstruction. RVSP can be estimated by the measurement of the peak velocity of the tricuspid regurgitant jet, on continuous-wave Doppler echocardiography, using the simplified Bernoulli equation. The RVSP is calculated as 4V2, where V is the peak velocity of the tricuspid regurgitant flow with contin-

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Fig. 10  The echocardiogram in cor pulmonale. Echocardiogram seen in the apical four chambers and short-axis views from a patient with severe pulmonary hypertension. Left: Apical four-chamber view. Right: Parasternal short axis view

Fig.  11  Assessment of pulmonary artery pressure in cor pulmonale using continuous-wave Doppler echocardiography of tricuspid regurgitation

uous-wave Doppler echocardiography. The formula requires estimation of the mean right atrial pressure (RAP). PASP = RVSP = 4V2 + RAP [94] (Figs. 11, 12). The RAP can be estimated by observing the dimension and collapse of the inferior vena cava with a maneuver such as inspiration [95] (Table 4). If the inferior vena cava collapses more than 50% with inspiration, the RAP is usually less than 10 mmHg [96]. Doppler-estimated PASP correlates well with cathetermeasured PASP in patients with cardiac disease [97, 98]; however, this method has two major limitations in patients with chronic lung disease. First, it can be difficult to visualize tricuspid regurgitant jet in patients with chronic lung disease owing to hyperinflation of the lungs and a poor acoustic window, with the rate of an adequate examination ranging from 24 to 76%. This is particularly true in patients with obstructive lung disease and in those with a residual volume greater than 150% of that predicted. Second, Doppler-estimated PASP is frequently inaccurate in patients with chronic lung disease. In a cohort study of 374 lung transplant candidates, only 48% of patients had less than 10  mmHg discrepancy

between Doppler-estimated and catheter-measured PASP (Fig.  13). In the same study, the accuracy increased only modestly when the estimated RAP was replaced with cathetermeasured RAP. Fifty-two percent of pressure estimations were found to be inaccurate (more than 10 mmHg difference compared with the measured pressure), and 48% of patients were misclassified as having PH by echocardiography [12]. The sensitivity, specificity, and positive and negative predictive values of systolic PAP estimation for diagnosis of PH were 85, 55, 52, and 87%, respectively [12]. Morphologic and functional evaluation of the RV is important in the TTE screening of patients suspected to have cor pulmonale. When evaluating the morphology of the chambers of the right side of the heart, it is important to detect RVH, RV and right atrium volume, and the presence of a pericardial effusion. RVH is determined by measuring the thickness of the RV free wall at end-diastole. A value greater than 5 mm is abnormal and is considered to be a sign of increased afterload [99]; this, however, is not true for acute RV pressure overload, where these morphologic changes to the RV wall may not have had time to develop. RV volume is difficult to determine quantitatively by echocardiography, as the RV is a complex cavity that changes in orientation in the presence of different loading conditions and its relationship to the left ventricle. There have been several attempts at volume estimations by single plane dimensions (Fig. 14) and areas of geometric figures have shown poor correlation with volumes determined by angiography [100]. The use of real-time, threedimensional echo has improved the noninvasive echocardiographic assessment of RV volume and function [101]. RV function can be noninvasively evaluated in patients with cor pulmonale using several Doppler echocardiographic measurements. These measurements either assess the motion of the tricuspid annulus or measure time intervals in regard to contraction and relaxation times of the RV [102, 103]. The RV Tei index, defined as the ratio of the sum of the isovolumetric contraction time and the isovolumetric relaxation time to the ejection time, is a measure of global RV function. In general, Tei index increases with PH; a cutoff value of 0.47

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Fig. 12  Estimation of pulmonary artery pressure in cor pulmonale using Bernoulli’s equation. Estimation of right ventricular pressure by using the velocity of the tricuspid regurgitant jet. The velocity of the jet is 4.26 m/s. Using Bernoulli’s equation, the estimated right ventricular systolic pressure is 4 × (4.04)2 = 72 mmHg + the right atrial pressure (estimated from physical examination or assessment of inferior vena cava size and motion)

Table 4  Determination of the right atrial pressure using the inferior vena cava (IVC) size and collapsibility Estimation of mean right atrial pressure Right atrial pressure IVC diameter IVC collapsibility with (mmHg) (mm) respiration (%) 5 10 15 20

<20 <20 ³20 ³20

>50 <50 >50 <50

Fig. 13  Inaccuracy of Doppler echocardiography in predicting pulmonary artery systolic pressure. (Adapted with permission from Arcasoy et al. [12]. The official journal of the American Thoracic Society, copyright American Thoracic Society)

has been proposed in heart failure [104]. Tei index is reproducible and is independent of heart rate and the severity of tricuspid regurgitation. It correlates with clinical symptoms and prognosis in patients with idiopathic PAH. However, in a prospective cohort study of 87 patients with chronic pulmonary disease, Tei index predicted survival only in the univari-

ate analysis [103]. When other Doppler echocardiographic measurements were included in the multivariate analysis, Tei index did not predict survival independently. TAPSE is an M-mode measurement of the displacement of the lateral tricuspid annulus [102]. A TAPSE of less than 18 mm correlated with abnormal RV function and predicted survival in patients with idiopathic PAH, but its clinical utility in patients with cor pulmonale has not been studied [102]. To obtain TAPSE, the apical four-chamber view is used, and an M-mode cursor is placed through the lateral tricuspid annulus in real time. TAPSE is measured as the total displacement of the tricuspid annulus (in centimeters) from end-diastole to end-systole. Overall, echocardiography, although it has some shortcomings, is a fast noninvasive screening tool that helps to not only provide an approximate measurement of PAP, but also gives information regarding RV morphology and function. Cor pulmonale can be reliably excluded in the absence of a Dopplerestimated PASP greater than 45 mmHg in patients with COPD, but it is less discriminative in patients with advanced interstitial lung disease [12]. The presence of a Doppler-estimated PASP greater than 45 mmHg requires further verification by RHC.

2.4  Cardiovascular Magnetic Resonance Imaging Cardiovascular magnetic resonance imaging (CMR) is being used increasingly for the evaluation of patients with cor pulmonale (Figs. 15, 16). The advantages of CMR imaging over echocardiography are the fact that it is intrinsically a threedimensional technique, can image any plane desired, has a

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Fig. 14  Quantification of right ventricular size by echocardiography. RVD1 mid tricuspid annulus, RVD2 apical endocardium. RVD3 right ventricular length

Fig. 15  Cardiac magnetic resonance imaging a patient with cor pulmonale secondary to sarcoidosis illustrating right ventricular enlargement and intraventricular interdependence. Top: Four-chamber view in diastole and systole in a patient with right ventricular dilation and elevated right-sided pressures. Note flattening of the interventricular septum with a “D” shaped left ventricle. Bottom: Same patient, images in short axis. Left: Diastole. Right: Systole

large field of view, and is not limited by poor acoustic windows. CMR imaging can be used to quantify volumetric, morphologic, and functional changes of the RV, and measure flow in the heart and great vessels. The disadvantages of CMR imaging include the need to perform multiple breath holds, which can be difficult for patients with hypoxemia, scan times which are approximately 45  min, poor image quality in patients with arrhythmia or poor EKG signal, inability to image people with significant claustrophobia, and incompatibility with certain metallic hardware (i.e. prostacyclin pumps, pacemakers, defibrillators, and aneurysm clips). CMR imaging allows measurement of end-systolic and end-diastolic volumes of any of the cardiac chambers using EKG-gated cine acquisitions. The ventricular volumes are computed using Simpson’s method of disks [82], which cal-

culates the sum of ventricular cavity areas multiplied by slice thickness through the entire chamber. Vonk-Noordegraaf et  al. imaged 25 clinically stable COPD patients [mean forced expiratory volume in 1 s (FEV1) 1.23, PO2 82 mmHg] and 26 age-matched control subjects [82]. RV ejection fraction in these stable patients was the same (53%) in COPD and control subjects; however, concentric RVH was evident in COPD, suggesting it is the earliest sign of RV pressure overload [82]. CMR may thus allow for early diagnosis of cor pulmonale. This confirms prior studies indicating that RV mass is significantly greater in COPD patients compared with healthy subjects, despite having a normal RV ejection fraction [105, 106]. Moreover, increased RV mass on CMR imaging correlates with increased mean PAP and PVR [107].

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Fig. 16  Cardiac magnetic resonance imaging a patient with cor pulmonale secondary to sarcoidosis illustrating right ventricular enlargement and intraventricular interdependence. Top: Four-chamber view in diastole and systole in a patient with right venricular dilation and elevated right-sided pressures. Note flattening of the interventricular septum with a “D” shaped left ventricle. Bottom: Same patient, images in short axis. Left: Diastole. Right: Systole

Fig.  17  Velocity flow curve in the main pulmonary artery derived using velocity-encoded cardiac magnetic resonance imaging. Note patients with chronic obstructive pulmonary disease have decreased

acceleration time and smaller ratio of acceleration time to ejection time compared with that in controls. (Adapted with permission [109] http:// www.interscience.wiley.com)

Similarly, RV wall thickness is higher in COPD patients than in controls and correlates with mean PAP and PVR [108]. CMR imaging permits noninvasive assessment of RV function by measuring stroke volume, cardiac output, and ejection fraction. These variables may be calculated from RV volumes or by the measurement of flow through the main pulmonary artery using velocity-encoded techniques. The volumetric flow through the pulmonary artery is obtained in each time frame by multiplying the spatial mean velocity (cm/s) of blood flow with the cross-sectional area of the vessel (cm2). Integrating the volumetric flow curve over systole gives the RV stroke volume. In patients with significant tricuspid regurgitation, the RV stroke volume measured using flow measurements in the pulmonary artery is considered to be more reliable than the stroke volume calculated using Simpson’s method of disks, which overestimates the RV stroke volume, as it cannot differentiate between volume that moves backward through the tricuspid valve and volume that moves forward through the pulmo-

nary valve. In one study, RV stroke volume was significantly decreased in COPD patients compared with controls (57 ± 13 vs. 71 ± 13 ml); however, the RV ejection fraction was well preserved at rest [109]. Pulmonary flow rate measurements obtained using velocity encoding also aid in the diagnosis of cor pulmonale. Pulmonary artery acceleration time (AT), defined as the time from the onset of flow to the peak velocity of the flow near the origin of the main pulmonary artery, and the ratio of AT to ejection time were significantly decreased in COPD patients with cor pulmonale compared with controls [109] (Fig.  17). Pulmonary artery AT can be measured using pulsed-wave Doppler echocardiography or using MRI, and it is 120  ms or greater in normal subjects. Pulmonary artery AT less than 120  ms indicates PH. The mean PAP can be calculated from the AT using Mahan’s regression equation [mean PAP = 79 – (0.45 ×AT)], but this is not always an accurate measure of PAP compared with that of RHC.

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Late gadolinium enhancement (LGE) is CMR imaging technique predominantly used to noninvasively image myocardial fibrosis [110]. The presence of LGE has not been investigated specifically in patients with cor pulmonale; however, LGE has been demonstrated at the RV insertion points and the interventricular septum in patients with idiopathic PAH and chronic thromboembolic PH. In these patients, the extent of LGE correlated directly with RV mass, mean PAP, and PVR [111].

2.5  Radionuclide Ventriculography Radionuclide ventriculography is a method used to assess RV ejection fraction. In this method, a computerized scintillation camera acquires a time-activity curve following intravenous administration of technetium-99m labeled red blood cells or albumin [112]. The camera measures the radioactive counts as the tracer passes from peripheral venous circulation into the RV and then into the pulmonary circulation during either one (first-pass technique) or more (gated-equilibrium technique) cardiac cycles. The RV ejection fraction is then estimated by measuring the difference in counts between end-diastole and end-systole. This technique for measuring RV ejection fraction is accurate, but involves significant exposure to ionizing radiation. This method does not allow for the diagnosis for elevated PAPs, owing to the overlap of this measurement in COPD patients in a stable disease state compared with normal subjects [84]. This technique has largely been supplanted by CMR imaging. Thus, by whatever technique measured, RV ejection fraction is not a good index of RV contractility and is an insensitive diagnostic criteria for cor pulmonale. RV ejection fraction gives only an estimate of the systolic function and is afterload-dependent, decreasing when PAP and PVR increase. Accordingly, the decreased RV ejection fraction observed in many COPD patients is caused primarily by increased afterload and is not an indicator of “true” RV function (i.e. it does not reflect contractility) [113].

2.6  Biomarkers There are few data on biomarkers in patients with cor pulmonale. The one class that has been studied in cor pulmonale patients with COPD is the natriuretic peptides: atrial natriuretic peptide (ANP), brain (B-type) natriuretic peptide (BNP), and C-type natriuretic peptide (CNP). ANP and BNP are hormones that are released from cardiac myocytes both in the atria and in the ventricles in response to elevated pressure and volume [114]. ANP and BNP act on the kidneys to

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enhance diuresis and natriuresis, and relax vascular smooth muscle, causing arterial/venous dilation leading to decreased preload and blood pressure [114]. Serum levels of these peptides have been shown to be elevated in congestive heart failure and cor pulmonale [115]. An elevated serum BNP level may indicate the presence of cor pulmonale in patients with chronic lung disease who lack other causes of increased BNP level (left-sided heart failure, myocardial ischemia, and pulmonary embolism). Patients with cor pulmonale secondary to COPD have a higher BNP level compared with the patients without cor pulmonale and normal control subjects. In a small study of 38 patients with COPD, BNP level correlated with PAP (r = 0.68), partial pressure of oxygen (r = −0.70), FEV1 (r = −0.65), and forced vital capacity (r = −0.52) [116]. Leutche et al. found that an elevated BNP level had a sensitivity of 85% and a specificity of 88% for diagnosing cor pulmonale in patients with chronic lung disease and noted that an increased BNP level predicted mortality [117]. The same group reported that the patients with lung fibrosis and elevated BNP levels had severe PH, and in such patients, an elevated BNP level had a sensitivity of 100% and a specificity of 88% for diagnosing moderate to severe PH [118]. Finally, chronic respiratory failure patients with cor pulmonale had higher levels of BNP as well as higher ANP levels (81.5 ± 13.1 and 80.8 ± 12.1 pg/ml) compared with patients without cor pulmonale (13.3 ± 2.7 and 26.1 ± 4.4 pg/ml). To date, all these studies with ANP and BNP in cor pulmonale have yielded concordant results and suggest BNP is a useful biomarker of cor pulmonale; but confirmation in larger trials is indicated. It is evident that BNP and ANP levels can help in confirming the diagnosis of cor pulmonale in patients with respiratory failure, but they cannot be used to discriminate cor pulmonale patients from those with left-sided heart failure. CNP, on the other hand, is released predominantly from the vascular endothelium [119]. CNP causes reduced cardiac output and natriuresis and its level is not elevated in conventional congestive heart failure [120]. CNP levels were elevated threefold in 16 patients with hypoxemic cor pulmonale in comparison with 12 elderly normal controls and 12 patients with New York Heart Association grade III–IV congestive heart failure [119]. It is yet unclear what role CNP plays in the pathophysiological processes in cor pulmonale and larger studies will assess its use as a biomarker.

2.7 Right-Sided Heart Catheterization RHC is the gold standard for diagnosing cor pulmonale (in patients with lung disease in whom RVH or RV dilatation is

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documented). It allows for accurate measurement of pressures in the right atrium, the RV, the pulmonary artery, and the wedge position to assess the left-sided filling pressure [88]. All pressure measurements are typically performed using a Swan–Ganz catheter at the end of expiration. In addition to diagnosing cor pulmonale, RHC can be instrumental in assessing the severity of underlying PH and RV function, monitoring therapeutic response, and prognostication. Patients with cor pulmonale can often have concomitant leftsided heart disease. Measurement of PCWP and left ventricular end-diastolic pressure during catheterization is needed to exclude PH secondary to left ventricular dysfunction (WHO category II), which is prevalent and often difficult to diagnose with Doppler echocardiography [32]. Unlike PAH, acute vasodilatory testing with inhaled nitric oxide, intravenously administered prostacyclins, or intravenously administered adenosine is not a routine part of the RHC in patients with cor pulmonale, and indeed, it may worsen hypoxia owing to increased ventilation–perfusion mismatch. The drawbacks of catheterization are that it is an invasive procedure and there has been no evidence-based study demonstrating its clinical value in the management of severe COPD.

3  Prognosis Traditionally, the development of cor pulmonale in patients with COPD and other lung diseases is viewed as a marker of poor prognosis. The mortality from cor pulmonale is high despite the fact that the patients with chronic lung disease have only mild to moderate elevation in PAP. It is unclear, however, whether the PH is the cause of the increased mortality or simply a marker of underlying disease severity.

3.1  Chronic Obstructive Pulmonary Disease Physical examination signs and diagnostic testing demonstrating right-sided heart failure in COPD patients portend a poor prognosis. Patients with peripheral edema secondary to COPD have a 5-year survival of 30% [5, 11]. Oxygen therapy improves right-sided heart function, allowing for longer survival after the initial episode of peripheral edema [7]. EKG changes reflective of cor pulmonale such as rightward QRS axis and increased P-wave amplitude are associated with 4-year survival of approximately 40%, whereas for patients without these findings the survival was 75% [121]. Mean PAP is an important prognostic factor in patients with COPD. Despite similar airflow limitations, patients with higher PAP have increased mortality compared with patients

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with normal PAP [122]. PH is also a negative prognostic factor in patients receiving long-term oxygen therapy (LTOT); patients with PAP greater than 25  mmHg had a lower 5-year survival rate than patients with PAP less than 25 mmHg (33% vs. 66%) [123]. A progressive increase in PAP and PVR is also associated with decreased survival [124]. Finally, COPD patients with even a modest increase in mean PAP (greater than 18 mmHg) have an increased risk of presenting with frequent and severe exacerbation of underlying COPD [125]. However, it is unclear that there is any benefit in treating the mild PH in cor pulmonale patients.

3.2  Interstitial Lung Disease Cor pulmonale is also a predictor of mortality in patients with interstitial lung diseases secondary to IPF, sarcoidosis, and systemic sclerosis. In these patient populations, the severity of PH is related to the impairment of diffusion capacity and the need for oxygen therapy, but not to the reduction in the lung volumes. Mild elevations of PAP predict survival in patients with IPF. In a study by Hamada et al. IPF patients with mean PAP greater than 17 mmHg had a twofold higher relative risk of mortality at 5-years compared with patients with mean PAP less than 17  mmHg (62.2% vs. 16.7%) [126]. In the same study, six patients with mean PAP greater 25  mmHg died within 5 years after RHC. Several other retrospective studies have validated these findings [16, 17]. The presence of cor pulmonale is an immediate indication to list these patients for lung transplantation [127]. Unfortunately, the presence of preoperative cor pulmonale is associated with higher mortality after transplantation [128]. In a retrospective analysis of sarcoidosis patients awaiting lung transplantation by Shorr et al. mean PAP in nonsurvivors was 41.4  mmHg compared with 31.7 mmHg in survivors. PAP was associated with increased mortality, independent of the extent of lung disease [129]. Patients with sarcoidosis followed in clinical practice appear to have a survival benefit from a pulmonary vasodilator such as epoprostenol and improve pulmonary hemodynamics and functional class [130]. Trad et  al. prospectively followed patients with interstitial lung disease associated with systemic sclerosis, and demonstrated that the presence of cor pulmonale independently predicted death (hazard ratio 5.07, 95% confidence interval 1.09–23.8, P = 0.038) [131].

4 Treatment Management of patients with cor pulmonale aims to treat the underlying disease, decrease resistance to pulmonary vascular flow, and improve circulatory response to RV volume overload.

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The goals of therapy are to decrease symptoms, improve hemodynamics, reduce hospitalizations, and ultimately prolong survival.

4.1  Oxygen Alveolar hypoxia is one of the key determinants of elevated PAP and PVR in patients with COPD. LTOT decreases chronic alveolar hypoxia, thereby decreasing pulmonary vasoconstriction. This in turn leads to lower PVR and improved RV stroke volume and cardiac output [132]. Oxygen therapy also relieves sympathetic vasoconstriction, thus improving renal perfusion, which assists in symptomatic volume management of these patients by increased excretion of urinary sodium [133]. LTOT stabilizes the progression of cor pulmonale in patients with COPD. The Nocturnal Oxygen Therapy Trial (NOTT) [134] and the Medical Research Council (MRC) [135] studies assessed pulmonary hemodynamics by RHC in COPD patients receiving LTOT. In the MRC study, 42 patients had follow-up RHC after more than 500 days. Of these 42 patients, PAP was stable in 21 patients who received LTOT in contrast to an increase of 2.8 mmHg in control patients. LTOT decreased resting as well as exercise mean PAP (−3 and −6  mmHg, respectively) in 177 patients who had 6-month follow-up RHC in the NOTT trial. A similar reversal of progression of PH has been demonstrated by Weitzenblum et al. and more recently by Zielinski et al [136, 137]. In all these studies, the effect of LTOT on pulmonary hemodynamics was directly related to the duration of LTOT. Patients treated with only nocturnal oxygen or those not treated with oxygen demonstrated no change in pulmonary pressures. It is unclear whether oxygen therapy affects the progression of cor pulmonale due to other pulmonary disorders. Oxygen does improve functional status and provides symptomatic relief in patients who are hypoxic from a lung disease such as interstitial lung disease [9]. In these patients, with or without cor pulmonale, nasal oxygen therapy should be used to maintain oxygen saturation greater than 90% both at rest as well as during exercise [9].

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Calcium Channel Blockers These drugs have been studied, may be beneficial in some patients, but are not widely used because they can lower PO2 by increasing shunting and there is no evidence that they improve prognosis. In one small study, in eight patients with stable COPD and cor pulmonale, nitrendipine (given for 6 weeks) significantly decreased both mean PAP (40.4 ± 10.3 to 31.2 ± 6.6 mmHg) and PVR (6.8 ± 3.9 to 3.0 ± 1.1 Wood units) [138]. Although PO2 fell (53.1 ± 18.7 to 45.5 ± 11.3 mmHg), systemic oxygen transport increased by over 30%. Systemic arterial pressure, PCWP, and heart rate were unchanged. However, despite these hemodynamic changes, three patients died from their underlying disease while on therapy after 8, 9, and 10 months. This reflects the challenge for all putative cor pulmonale therapies: Do they change the symptom? Do they improve prognosis?

Prostacyclins In a study of 16 COPD patients with acute respiratory failure, we found that PH was mild and intravenously administered prostacyclin (Flolan) was not beneficial in accelerating extubation or other measures of disease in these patients [139]. The frequency with which the pulmonary vasodilators may worsen hypoxemia by increasing ventilation–perfusion mismatch is unknown but was experienced with administration of Flolan to decompensated patients with COPD [139].

Sildenafil and Bosentan Sildenafil and bosentan do not improve outcomes in cor pulmonale. In a recent uncontrolled 12-week follow-up study of 15 patients with COPD, sildenafil did not improve either exercise capacity or stroke volume [140]. Alps et al. investigated the effect of sildenafil, a single intravenous dose of 50 mg followed by 50 mg twice daily orally for 3 months, in six patients with COPD. The intravenous dose decreased mean PAP, and the mean PAP decreased from 30.2 to 24.6  mmHg in five patients who completed the 3-month study. In a randomized control study of patients with COPD, bosentan failed to improve exercise capacity, and indeed, worsened hypoxemia and functional status [141].

4.2.1  Studies in COPD Inhaled Nitric Oxide Although several pulmonary vasodilators such as synthetic prostacyclins, prostacyclin analogues, endothelin receptor antagonists, and phosphodiesterase inhibitors have been approved for PAH, they are not recommended in patients with cor pulmonale secondary to COPD.

Inhaled nitric oxide is a selective pulmonary vasodilator that has some potential for short-term treatment of right-sided heart failure in COPD patients [142]. Nitric oxide added to oxygen therapy as pulsed inhalation over a 3-month period

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decreased mean PAP from 27.6 to 20.6  mmHg and PVR index from 7.1 to 4 Woods units. Despite concerns that nitric oxide would worsen ventilation–perfusion mismatch and accentuate hypoxia, this did not occur [142]. However, the logistical challenges of extended treatment with inhaled nitric oxide and the cost are substantial.

4.2.2 Studies in Pulmonary Fibrosis Inhaled iloprost, inhaled nitric oxide, and orally administered sildenafil have been shown to decrease PVR without worsening hypoxemia. In a small open-label study, 3 months of sildenafil therapy improved the mean 6-min walk distance by 49  m in patients with IPF and cor pulmonale [143]. Long-term aerosolized therapy with inhaled iloprost improved hemodynamics and functional capacity in a patient with cor pulmonale due to interstitial lung disease without worsening hypoxemia [144]. Ghofrani et  al. compared the acute effects of orally administered sildenafil and intravenously administered epoprostenol in patients with PH secondary to lung fibrosis and found that sildenafil preferentially causes pulmonary vasodilatation without affecting gas exchange. Both sildenafil and intravenously administered epoprostenol decreased PVR; however, sildenafil improved oxygenation, whereas epoprostenol decreased it. Nevertheless, these studies were small, uncontrolled, and nonrandomized, and no long-term outcomes were measured. Thus, pulmonary vasodilators are presently not recommended for treating cor pulmonale secondary to interstitial lung disease except in clinical trials.

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outcomes with double-lung transplant [148]; however, older patients may benefit more with a single-lung transplant [149]. Patients with cor pulmonale associated with interstitial lung disease have a poor prognosis after lung transplant [127].

4.4  S  ymptomatic Therapies Lacking Trial-Based Evidence 4.4.1  Diuretic Therapy When patients with cor pulmonale develop RV volume overload, the dilated RV impairs the left ventricular diastolic filling. In such patients, diuretic therapy has the potential to improve cardiovascular performance by decreasing the intravascular volume and RV preload [150].

4.4.2  Digoxin Therapy In patients with cor pulmonale, digitalis has only been shown to be beneficial in patients with concomitant left ventricular failure. When 15 patients with cor pulmonale were randomized to digoxin or a placebo for 8 weeks, the only objective benefit observed was an improvement in RV function. No demonstrable effect was observed in pulmonary function, exercise capacity, or symptom relief [151].

4.4.3  Lung Volume Reduction Surgery

4.3  Lung Transplant Although controversy may exist regarding the effects of lung volume reduction surgery (LVRS) on pulmonary hemodynamics, there is remarkable consensus regarding the benefits of lung transplantation in patients with cor pulmonale. In fact, the first successful lung transplant performed was a combined heart–lung transplant on a patient with cor pulmonale. Transplant surgery for PAH and cor pulmonale is either single-lung or double-lung transplant [145]. Despite initial concern that the dilated RV in patients with cor pulmonale would not regain contractility, in fact there is normalization of pulmonary hemodynamics, reduction of RV size, and improvement of RV contractility almost immediately [146]. Pulmonary hemodynamics normalized in patients with COPD after single-lung transplant, with persistent benefit 2 years after transplant [147]. It is unclear if these patients benefit more from a single-lung or a doublelung transplant. There are data supporting better long-term

Successful LVRS improves symptoms and quality of life in patients with emphysema; however, its role in attenuating cor pulmonale in such patients is unclear [152]. Theoretically, LVRS could worsen pulmonary hemodynamics by reducing the size of the pulmonary vascular bed. On the other hand, it could improve pulmonary hemodynamics by lowering hyperinflation, increasing elastic recoiling of the lungs, increasing capillary recruitment, and decreasing functional compression of the pulmonary vessels. Contemporary studies evaluating pulmonary hemodynamics before and after LVRS have reported mixed results. Two groups observed no change in resting or exercise pulmonary hemodynamics after LVRS [153, 154]. Similarly, in the recent, National Emphysema Treatment Trial (NETT), LVRS was not associated with an increase in PAP compared with medical treatment alone [155]. In contrast, Weg et al. reported that PAP increased 3 months following LVRS [156]. Currently, LVRS is not recommended for COPD patients with mean PAP of 35 mmHg or more, and these patients should be referred for lung transplantation.

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4.5  Summary of COPD Treatment In summary, oxygen should be used for the treatment of cor pulmonale. Evidence regarding the role of other measures such as diuretics, prostacyclins, digoxin, and calcium channel blockers in treating these patients is not consistent and therefore is not recommended on an individual patient basis or at all. The only definitive therapy recognized consistently is lung transplantation in patients with cor pulmonale if they are less than 65 years of age without comorbidities.

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Chapter 98

Pregnancy and Contraception in Patients with Pulmonary Arterial Hypertension Barbara A. Cockrill and Charles A. Hales

Abstract  The risk for complications and death associated with pregnancy in patients with pulmonary hypertension remains prohibitive. This chapter will review the physiological changes associated with pregnancy, past and current reported experience in pregnancy associated with pulmonary arterial hypertension (PAH), the management of patients with PAH during pregnancy, and finally, the approach to contraception in patients with PAH. In general, the hemodynamic issues and outcomes in pregnancy associated with PAH are similar despite the primary cause of PAH. Thus, pregnancy in idiopathic PAH (iPAH), PAH related to congenital heart disease (PAH–CHD), and connective tissues diseases are considered together unless noted. Keywords  Birth control • Pregnant • Contraceptive drug • Congenital heart disease

1  Introduction The risk for complications and death associated with pregnancy in patients with pulmonary hypertension remains prohibitive. This chapter will review the physiological changes associated with pregnancy, past and current reported experience in pregnancy associated with pulmonary arterial hypertension (PAH), the management of patients with PAH during pregnancy, and finally, the approach to contraception in patients with PAH. In general, the hemodynamic issues and outcomes in pregnancy associated with PAH are similar despite the primary cause of PAH. Thus, pregnancy in idiopathic PAH (iPAH), PAH related to congenital heart disease (PAH–CHD), and connective tissues diseases are considered together unless noted.

B.A. Cockrill (*) Pulmonary Vascular Disease Center, Brigham and Womens Hospital, 75 Francis Street, Boston, MA 02115, USA e-mail: [email protected]

2  Physiological Changes Associated with Normal Pregnancy Pregnancy is a hyperdynamic state that is akin hemodynamically to exercising continuously at a slow to moderate jog. To adequately perfuse the placenta, and accomplish labor and delivery, the mother undergoes marked physiological changes in blood volume and cardiac function and vascular tone. Most of these changes are particularly poorly tolerated by the patient with pulmonary hypertension. The first change is an increase in circulating blood volume. By week 6 after conception, plasma volume has begun to increase primarily through the actions of aldosterone causing salt and water retention. The increase in blood volume reaches a peak of 40–50% over the baseline during the second trimester [1]. Red cell mass also increases, but the magnitude is smaller than the plasma volume increase. Therefore, the effect is a physiologic anemia of pregnancy, with a typical hemoglobin level being 11 mg/dl during pregnancy [2]. The relative anemia is necessary to decrease the viscosity of blood, allowing better perfusion of the placenta. The lack of the development of a slight anemia is associated with intrauterine growth retardation and placental insufficiency. Cardiac output has increased by week 10, and progresses during gestation to reach a peak of 30–50% of that of the baseline resting state. Initially, the increase is accomplished with an increase in stroke volume. Later in gestation, the heart rate also increases by 10% above the baseline [3]. Both pulmonary vascular resistance (PVR) and systemic vascular resistance (SVR) fall dramatically during normal pregnancy. The increases in blood volume and cardiac output are, therefore, not normally associated with increases in either pulmonary artery pressure or systemic arterial pressure. In a prospective study, ten healthy pregnant women underwent pulmonary artery catheterization between 36 and 38 weeks of gestation and again between 11 and 13 weeks postpartum [4]. This study showed a 34% lower PVR and a 21% lower SVR during pregnancy as compared with the postpartum state. The pulmonary artery pressure was not changed. Typically, a pregnant patient will have no change in

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_98, © Springer Science+Business Media, LLC 2011

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either systemic arterial pressure or pulmonary artery pressure during the course of gestation. This study did not reassess hemodynamics until 3 months after delivery, so the exact timing of the increase in PVR compared with baseline cannot be ascertained. However, others have found a return to baseline hemodynamics occurs within 2 weeks [5] (Table 1). The mechanisms of the hemodynamic changes in pregnancy are being elucidated. Enhanced endothelial synthesis of nitric oxide (NO) has been implicated [6–8]. Parturients with polymorphisms in the endothelial NO synthase gene have abnormalities in flow-mediated vasodilation in early pregnancy [9, 10]. The increase in NO synthesis is at least in part regulated by the hormone relaxin [11–13]. Relaxin is produced by the corpus luteum and is present at increased concentrations during pregnancy [14] and appears to have a role in eclampsia as well as other vascular diseases [11]. Serial measurements of central hemodynamics during pregnancy in a single patient with moderate pulmonary hypertension have been reported [15]. This patient was intensively monitored and had a successful outcome with delivery of a healthy infant at term by cesarean section (c-section) at 38 weeks. The pulmonary artery pressure and PVR were elevated at the baseline in this patient, but both decreased significantly during pregnancy (Table  2). Importantly, this report documents an abrupt increase in PVR and pulmonary artery pressure in the immediate postpartum period. As noted in the study of healthy patients, the increase in PVR that occurs after delivery is normal [4]. This change would be especially poorly tolerated in the patient with PAH, and likely accounts for the repeatedly

described increased mortality seen in the postpartum period in patients with PAH [16, 17]. In the normal pregnant state, the left ventricle undergoes eccentric remodeling allowing a larger end-diastolic volume and larger stroke volume without an increase in pressures [4, 5]. These changes return to the baseline within about 12 weeks [5]. It is not known if similar changes occur in patients with PAH. Changes in the respiratory system during normal pregnancy will also make the PAH patient more dyspneic. Maternal minute ventilation increases by about 50% during pregnancy and this increase starts in the first trimester. This is accomplished by an increase in tidal volume, rather than an increase in respiratory rate and is caused by the effects of rising progesterone levels in the central nervous system. A normal PaCO2 in pregnancy is 28–32 mmHg [3]. Up to 75% of normal patients become dyspneic during pregnancy primarily owing to these changes [18]. All of the above-mentioned changes are poorly tolerated in the patient with pulmonary hypertension. For the patient with an elevated and fixed PVR, the increased blood volume and cardiac output will cause right ventricular strain, and often failure. The normal increases in PVR and SVR that occur in the postpartum period make this the most dangerous time for the mother with PAH.

Table 1  Summary of hemodynamic changes during normal pregnancy Plasma volume Increases by 30–50% Cardiac output Increases by 30–50% Stroke volume Increases by 20–50% Systemic vascular resistance Decreases by 20–30% Pulmonary vascular resistance Decreases by 30% Central venous pressure Unchanged Pulmonary artery occlusion pressure Unchanged Adapted with permission [4]

Despite advances in therapy and monitoring, the maternal death rate and fetal complication rate remain unacceptably high. In an early case series before any treatment was available, a maternal mortality rate of 50% was reported [19]. A large review of published reports of 125 pregnancies spanning 1978–1996 documented a maternal mortality of 30% in iPAH, 36% in patients with Eisenmenger syndrome, and 52% in patients with PAH related to collagen vascular disease or other diseases, with an overall maternal mortality of 38% [16]. Most patients died from progressive, irreversible right ventricular dysfunction or suddenly from a presumed pulmonary embolism or cardiac arrhythmia. Late diagnosis of pulmonary hypertension and delayed hospital admission were independent risk factors for maternal mortality. Infant survival ranged from 87 to 89%, and was similar for all groups. Importantly, only three of 48 maternal deaths occurred prior to labor and delivery: 45 maternal deaths occurred within 35 days after delivery [16]. Unfortunately, a recent retrospective review of cases reported between 1997 and 2007 still validates a high maternal mortality rate [17]. This review found mortality rates of 17% in patients with iPAH, 28% in patients with PAH–CHD,

Table 2  Serial hemodynamics from a single pregnancy in a patient with pulmonary arterial hypertension (PAH) Immediately 38 Baseline weeks’ after cesarean 17 h after (prior to delivery pregnancy) gestation delivery Systemic blood 104/64 pressure (mmHg) Heart rate (beats/ 57 min) Right atrial pressure 2 (mmHg) Pulmonary artery 61/22 pressure (mmHg) Adapted with permission [15]

90/58

106/66

97/60

61

78

77

4

9

5

50/25

48/26

80/32

3  Experience in Pregnancy in Patients with Pulmonary Hypertension

98  Pregnancy and Contraception in Patients with Pulmonary Arterial Hypertension

and 53% in patients with other causes of PAH. Many patients were receiving advanced therapy for PAH, including inhaled NO, prostacyclin analogs, sildenafil, and bosentan (iPAH 72%, PAH–CHD 52%, and other 47%). Notably, the use of advanced therapies was not associated with improved survival. Similar to previous studies, the infant survival rate was close to 90%, although more than 85% of infants were delivered prematurely. Of 18 maternal deaths, only two occurred during pregnancy – the other 16 occurred within 1–15 days postpartum [17]. Reviews of published case reports may overestimate maternal survival owing a publication bias favoring reports of good maternal and fetal outcomes. Bonnin et al. reported a case series of 15 pregnancies in 14 patients from a single center with special expertise in the treatment of PAH [20]. This series was published after the availability of advanced therapy, and all of the patients had severe pulmonary hypertension. The patients had the following types of PAH: iPAH (n = 4), PAH–CHD (n = 6), fenfluramine-associated PAH (n = 1), mixed connective tissue disease (n = 1), HIVassociated PAH (n = 1), and chronic thromboembolic disease (n = 2). Even under the best circumstances, a maternal mortality rate of 36% was found [20].

3.1  Treatment of PAH During Pregnancy There have been no controlled studies or even prospective large case series comparing treatment and outcome in pregnancy and PAH. Data are therefore limited to small series and case reports, and likely are influenced by reporting bias such that the actual complication rate is higher than can be gleaned from published reports. The largest retrospective series of Bedard et al. discussed earlier did not find a survival benefit in patients treated with advanced therapy [17]. A retrospective review of four patients that attempted to compare treatment with either nifedipine or intravenously administered prostacyclin suggested that early recognition and treatment of PAH may improve pregnancy outcome [21]. Four women carried a total of five pregnancies, and all had severe pulmonary hypertension at the time of diagnosis, with mean pulmonary artery pressures greater than 70 mmHg. Two were diagnosed with PAH and began treatment during pregnancy. Of the two diagnosed during pregnancy, one patient died. She was newly diagnosed at 28 weeks of gestation, and presented with severe right ventricular failure and shock. Despite treatment with intravenously administered prostacyclin and dobutamine, neither the mother nor the fetus survived. The other patient who was diagnosed during pregnancy survived, but had a very difficult course after delivery. The other two patients had been treated for PAH for 12 and 15 months prior to conception

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and became pregnant despite being counseled to avoid ­pregnancy. One of these patients had been treated with nifedipine and one with intravenously administered prostacyclin and both of these patients had relatively uneventful pregnancies and deliveries. Importantly, both of the patients with successful pregnancies had responded to treatment, with a marked improvement in pulmonary artery pressures and cardiac output, and normalized right ventricular function prior to becoming pregnant [21]. A number of case reports also document successful pregnancies in patients with PAH who were treated with epoprostenol. Most of these patients had mild to moderate pulmonary hypertension, and had a favorable response to treatment [22– 26]. However, there is still a high risk of maternal mortality even with prostanoid therapy. In the large retrospective review from London, 30 of 73 patients (41%) received prostacyclin analogs at some point during pregnancy. However, six of the 30 patients (20%) died [17]. Another report describes a patient with severe PAH with a systolic pulmonary artery pressure greater than 100 mmHg at the time of diagnosis. This patient was managed with intravenously administered epoprostenol and delivered a healthy baby at 32 weeks of gestation. However, she died 14 days after delivery of intractable right-sided heart failure [27]. Two cases of PAH–CHD resulted in maternal death despite prostacyclin treatment [28, 29]. There is very limited published experience with inhaled prostanoids [30, 31]. One report describes successful outcomes in three patients treated with inhaled iloprost during pregnancy [32]. Two of the three patients were New York Heart Association functional class II–III while pregnant but had relatively uneventful pregnancies and deliveries. The third patient’s course was complicated by a cardiac arrest at 25 weeks from which she was successfully resuscitated. The baby was delivered by c-section at 25 weeks, but survived. One report describes the use of sildenafil and l-arginine in a patient with severe PAH due to an atrial septal defect and Eisenmenger syndrome [33]. Treatment was started at 7 weeks of gestation with sildenafil and l-arginine, and inhaled NO was used after delivery to treat an abrupt increase in pulmonary artery pressure. Both mother and infant survived. In another report, addition of sildenafil to bosentan at 28 weeks of gestation in a woman with Eisenmenger syndrome was reported. She required an urgent c-section at 30 weeks, and survived. The infant survived initially, but succumbed at 26 weeks of age to respiratory syncytial virus infection [34]. Sildenafil may have other beneficial effects in pregnancy: it has been shown to cause uterine artery vasodilation and improve uterine muscle wall thickness during in vitro fertilization treatments [35]. Inhaled NO is a treatment which is generally not available for outpatient use, but is frequently administered during labor and delivery [36–38]. Its use during a successful

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pregnancy has been reported in a parturient who was ­admitted to hospital for bed rest at 28 weeks of gestation. She was treated continuously with inhaled NO and oxygen via nasal cannulae, and this treatment was continued for 2 weeks postpartum [39]. As summarized above, there are a number of case reports and small series describing a successful pregnancy and survival by the patient with PAH and her infant. However, a careful reading of the details of these reports often reveals a difficult course during which the outcome could have gone either way. In addition, most of the cases in which the pregnancy and delivery progressed without major complications were in women who had relatively mild PAH or in women who had been very successfully treated prior to becoming pregnant. Thus, although advanced therapies are now widely available, the risk of pregnancy is still prohibitive, and it is not clear what effect, if any, these treatments have in changing this risk.

[43]. Despite the small size of the molecules, LMWH do not cross the placenta [44]. LMWH can be dosed by weight alone without monitoring in the nonpregnant patient; however, during pregnancy weight, volume of distribution, and glomerular filtration rate all change. Thus, dosing in high-risk PAH patients should be adjusted according to manufacturer-­ recommended anti-factor Xa levels [45]. Although heparininduced thrombocytopenia (HIT) is a rare complication in pregnant women, our practice is to monitor platelet counts monthly to assess for the development of HIT. LMWH treatment should be suspended at least 24 h prior to delivery. For the rare patient who is intolerant of LMWH treatment owing to either HIT or local reactions to subcutaneous injections of LMWH, fondaparinux may be considered. Fondaparinux is a synthetic pentasaccharide which binds to antithrombin and inhibits factor Xa without inhibiting thrombin. This drug has been used in pregnancy and appears to be safe [46, 47], although there have been no large studies.

3.2 Anticoagulation

3.3 Diagnosis and Monitoring During Pregnancy

Many patients with PAH are chronically anticoagulated, and the agent of choice is warfarin. Unfortunately, warfarin is contraindicated in all patients during pregnancy. Warfarin is a small molecule that easily passes through the placenta and enters the fetal circulation. Its use during gestation is associated with a characteristic embryopathy (fetal warfarin syndrome), central nervous abnormalities, and fetal bleeding [40]. The fetal warfarin syndrome is due to warfarin effects on collagen synthesis and consists of nasal hypoplasia, and stippled epiphysis. The risk is highest during weeks 6–9 of gestation, and warfarin treatment may be safe prior to 6 weeks. The central nervous system abnormalities include optic atrophy due to ventral midline dysplasia, and may occur with exposure at anytime during gestation [41]. Fetal bleeding associated with warfarin exposure can also occur at anytime during pregnancy. Intracranial hemorrhage, especially during labor and delivery, is a major concern. Heparins do not cross the placenta and are considered safe for the fetus during pregnancy. An earlier retrospective review of 100 pregnancies during which unfractionated heparins were used for the prevention and treatment of clotting due to venous thromboembolism and mechanical heart valves found no increase in the number of congenital defects or other adverse events. There were two episodes of maternal bleeding; however, neither episode was associated with fetal harm or the need for transfusion [42]. Low molecular weight heparins (LMWH) are now standard of care for patients with normal renal function who need anticoagulation during pregnancy. LMWH have a more predictable response, a longer half-life, and fewer side effects

Cardiac echo is an excellent screening test for elevated pulmonary artery pressures in nonpregnant patients. However, cardiac echo may be especially unreliable in the hyperdynamic pregnant state. In a retrospective chart review of 27 pregnant women with suspected PAH, the findings on cardiac echo were compared with the results of pulmonary artery catheterization [48]. There was a significant overestimation of pulmonary artery systolic pressure by echo as compared with catheterization (59.6 mmHg vs. 54.8 mmHg, p < 0.04). The most important finding in this study, however, was that 32% (eight of 25) of patients were classified as having PAH by echo, but subsequently had normal hemodynamics on catheterization. In addition, two patients had a marked underestimation of pulmonary artery pressure by echo: one did not appear to have elevated pressures by echo, but was found to have moderate PAH, and the other patient had a moderate elevation in pulmonary artery pressure by echo (52 mmHg) and was found to have severe pulmonary hypertension with catheterization (134 mmHg) [48]. As is the case in all patients with suspected PAH, the cardiac echo cannot be relied upon for important clinical decisions – especially termination of pregnancy. The diagnosis of PAH cannot be made without invasive hemodynamic assessment. The safe use of a pulmonary artery catheter in parturients with PAH has been described in numerous reports [15, 17, 25]. A diagnostic catheterization does not appear to be ­riskier in pregnancy than it is in other patients with PAH. There is one report of a critical arrhythmia associated with an ­indwelling pulmonary artery catheterization used during labor and

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d­ elivery. The authors noted that the hyperdynamic state of pregnancy and the need to move the patient for placement of an epidural catheter contributed to displacement of the catheter into the right ventricle, and caused the ­arrhythmia. Once the pulmonary artery catheter had been removed, normal sinus rhythm was restored [49].

3.4 Labor and Delivery Labor, delivery, and the postpartum period are the most dangerous periods and present a special management challenge in the patient with PAH. The physiological changes associated with labor and delivery are difficult to summarize owing to the complex and changing interplay between pain, different types of anesthesia and analgesia, the cyclic effects of uterine contraction, and potential blood loss [3]. The normal increases in PVR and SVR which occur in the postpartum period are likely the primary contributor to the hemodynamic instability. In the few case reports in which invasive hemodynamic values were reported, a decrease in PVR from a pre-pregnancy baseline followed by an increased PVR within hours after delivery were found in parturients with PAH [15, 39]. Because there have been no randomized trials, the best mode of delivery in patients with PAH is not known. Earlier experience suggested an increased mortality associated with c-sections [16] but this may have been related to general anesthesia rather than the surgery itself. An increased risk with c-section was not confirmed in later studies [17]. One series of 15 pregnancies found the best outcome to be associated with a scheduled cesarean delivery during combined spinal and epidural anesthesia as compared with vaginal delivery or c-section with general anesthesia. The authors noted, however, that no conclusions could be made, since this was a small, retrospective review [20]. It does appear that general anesthesia is associated with an increased mortality [17]. Even in healthy patients, general anesthesia may be associated with a drop in right ventricular preload and increase in PVR due to positive pressure ventilation and a depression of myocardial contractility. These hemodynamic effects are especially poorly tolerated in the patient with PAH. If possible, general anesthesia should be avoided during delivery in patients with PAH. The commonly used drug oxytocin is especially problematic in patients with PAH. Oxytocin is routinely used during labor to enhance the contraction of uterine muscle to facilitate delivery. This drug also stimulates smooth muscle in the pulmonary arteries, and is associated with an increase in pulmonary artery pressure and PVR [50]. Great care should be taken when this drug must be used in parturients with PAH as complications associated with the use of oxytocin have been reported by a number of authors [16, 20, 51].

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Vigilant assessment and monitoring of systemic blood pressure, arterial oxygen saturation, and cardiac rhythm is recommended, and most authors recommend a central venous and peripheral arterial catheter. However, the value of a pulmonary artery catheter to monitor central hemodynamics during labor and delivery is uncertain. In high-risk surgical patients with other medical problems, invasive monitoring does not appear to confer a benefit compared with usual management [52]. Patients with PAH are at increased risk of complications related to pulmonary artery catheters, including pulmonary artery rupture [53] and hemodynamically significant arrhythmias [54]. Right atrial enlargement frequently seen in patients with PAH may predispose to arrhythmias and catheter displacement [48]. A detailed discussion of the critical care management of patients with PAH is beyond the scope of this chapter and is covered elsewhere. However, the use of vasopressin for systemic blood pressure support merits mention here. Unlike other vasopressors, vasopressin does not appear to cause an increase in PVR. In a report of nine cardiac surgery patients with postcardiotomy syndrome with hypotension in the setting of refractory low SVR and high PVR, vasopressin was effective in raising SVR and systemic blood pressure without having a deleterious effect on PVR [55]. Another report documents of the use of vasopressin specifically in two patients with iPAH after c-sections. Both patients developed severe right ventricular failure after delivery. It was thought that placental autotransfusion and oxytocin contributed to the deterioration. Both patients were successfully managed, and the use of vasopressin was associated with improvement in SVR without negative effects on PVR or right ventricular function [56].

4 Adverse Effects of Advanced Therapies on the Developing Fetus Terotogenicity is a class effect of endothelin receptor antagonists and these drugs are clearly contraindicated in pregnancy. The patient should be immediately transitioned to another therapy unless a therapeutic abortion is planned. In more than one species, animal studies have documented congenital abnormalities of the heart and great vessels as well as the palate and jaw [57]. In one case report, a patient had been treated with bosentan prior to becoming pregnant. Pregnancy was diagnosed at 28 weeks of gestation, and the bosentan treatment was continued until the mother’s deteriorating condition mandated a c-section at 30 weeks. No fetal abnormalities were reported [34]. Endothelin receptor antagonists s are considered pregnancy category X by the US Federal Drug Administration (FDA; Tables 3, 4).

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Table 3  Federal Drug Administration (FDA) drug ratings during pregnancy Category Interpretation A B

C

D

X

Adequate, well-controlled studies in pregnant women have not shown an increased risk of fetal abnormalities to the fetus in any trimester of pregnancy Animal studies have revealed no evidence of harm to the fetus; however, there have been no adequate and well-controlled studies in pregnant women or Animal studies have shown an adverse effect, but adequate and well-controlled studies in pregnant women have failed to demonstrate a risk to the fetus in any trimestera Animal studies have shown an adverse effect and there have been no adequate and well-controlled studies in pregnant women or No animal studies have been conducted and there have been no adequate and well-controlled studies in pregnant women Adequate well-controlled or observational studies in pregnant women have demonstrated a risk to the fetus; however, the benefits of therapy may outweigh the potential risk. For example, the drug may be acceptable if needed in a life-threatening situation or serious disease for which safer drugs cannot be used or are ineffective Adequate well-controlled or observational studies in animals or pregnant women have demonstrated positive evidence of fetal abnormalities or risks. The use of the product is contraindicated in women who are or may become pregnant

Table 4  FDA pregnancy drug ratings of advanced PAH drugs Drug FDA rating Endothelin antagonist Prostacyclin analogs Sildenafil

X B B

Prostacyclin analogs do not have documented adverse effects on the developing fetus. Animal studies in rats and rabbits found no evidence of fetal harm with 2.5–4.8 times the usual human dose of intravenously administered epoprostenol [58]. In human case reports of patients in which epoprostenol treatment was initiated either prior to pregnancy or in early gestation, there is no evidence of congenital injury to the infant [21, 23, 26]. Prostacyclins are considered FDA pregnancy category B. Sildenafil is considered FDA pregnancy category B. In rats and rabbits there was no evidence of fetal harm in doses considered to be more than 30 times a typical human dose. There have been no adequate studies of sildenafil in pregnant women [59]. The use of assisted reproductive technology to harvest eggs from a patient with PAH and a successful pregnancy in a surrogate has been described. The 28-year-old woman with severe iPAH underwent ovarian stimulation and subsequent harvesting under general anesthesia. The first cycle failed to result in pregnancy in the surrogate, but a second cycle was successful [60].

5 Contraception Because the risk of maternal death remains prohibitive in patients with PAH, it is essential that the clinician have an ongoing discussion regarding contraception with patients who are women of childbearing age. This may be a very

d­ ifficult conversation as the emotional impact of being told they cannot have children may be devastating for some patients. Once again, there are special considerations in these patients but there are a few options available which are effective and appear to be safe. The combination of estrogen and progesterone pills, the most commonly used form of contraception, is not recommended in patients with pulmonary hypertension owing to the increased risk of both arterial and venous thrombosis events conferred by the estrogen component [61]. Importantly, anticoagulation with warfarin does not eliminate this risk. In patients with known patent foramen ovale, and right-to-left shunting who are at risk for paradoxical embolism, the use of estrogen-containing contraceptives is contraindicated. In contrast, when used for contraception, progesteroneonly preparations containing levonorgestrel are not associated with an increased risk of thrombosis. In a review of over 70,000 women who took progesterone preparations, there was no increased risk at doses required for contraception [62]. Additionally, one-time use of higher doses of levonorgestrel, as is used in postcoital contraception, is not associated with a higher incidence of deep venous thrombosis. However, not all progesterones may be safe: a newer medication, desogestrel, has been associated with a higher risk of venous thromboembolism when compared with contraceptives containing levonorgestrel alone [63]. Unfortunately, pills containing levonorgestrel alone are less effective contraceptives than estrogen–progesterone preparations. In patients with pulmonary hypertension, other forms of levonorgestrel are recommended. Depo-Provera® is a highly effective injectable contraceptive method that contains levonorgestrel. It must be injected into patients every 12 weeks to guarantee efficacy. Patients who are anticoagulated are at risk for troublesome hematomas at the injection site. In our experience, however, this has not been a limiting factor.

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Another reasonable option is a levonogestrel-releasing intrauterine device (Mirena®). This device provides effective contraception for 3 years. Caution must be used when placing the device, as vasovagal reactions are common during insertion in all patients. In patients with PAH, a vasovagal response can be catastrophic. A team approach to patient care with input from an anesthetist, a pulmonary hypertension specialist, and the gynecologist, is key. Permanent sterilization may be considered in young women with PAH. However, the most commonly performed procedure is a laparoscopic tubal ligation. This procedure is fraught with risk in PAH patient as it requires general anesthesia. As discussed above, general anesthesia is associated with a higher risk of maternal death in patients with PAH. It requires endotracheal intubation and positive-pressure ventilation. In addition, for the surgeon to visualize the fallopian tubes, the patient is placed in the Trendelenberg position and ­carbon dioxide is insufflated into the pelvic cavity. All of these maneuvers put the patient with PAH at increased risk for serious complications. Recently, the successful use of hysteroscopic sterilization has been introduced. In this procedure, a fiber-optic scope is vaginally inserted and tiny devices can be placed in the fallopian tubes under fiber-optic visualization [64]. In a small series of 18 women with severe cardiac disease, including PAH, this procedure was safe, and resulted in 100% tubal occlusion. No pregnancies occurred during the follow-up period of 8–33 months (mean 20 months) [65].

6 Conclusion and Recommendations The normal hemodynamic changes that occur during gestation are particularly poorly tolerated by patients with PAH. Pregnancy for these patients still carries a prohibitive risk of maternal death. All patients should be counseled to avoid pregnancy and treated with appropriate contraception if necessary. This chapter described potential treatments if pregnancy does occur. However, none of the treatments have been shown to improve outcome. The most dangerous time for the parturient is in the postpartum period.

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1384 28. Goodwin TM, Gherman RB, Hameed A, Elkayam U (1999) Favorable response of Eisenmenger syndrome to inhaled nitric oxide during pregnancy. Am J Obstet Gynecol 180:64–67 29. Lust KM, Boots RJ, Dooris M, Wilson J (1999) Management of labor in Eisenmenger syndrome with inhaled nitric oxide. Am J Obstet Gynecol 181:419–423 30. Cotrim C, Simões O, Loureiro MJ et al (2006) Acute resynchronization with inhaled iloprost in a pregnant woman with idiopathic pulmonary artery hypertension. Rev Port Cardiol 25:529–533 31. Bildirici I, Shumway JB (2004) Intravenous and inhaled epoprostenol for primary pulmonary hypertension during pregnancy and delivery. Obstet Gynecol 103:1102–1105 32. Elliot CA, Stewart P, Webster VJ et al (2005) The use of iloprost in early pregnancy in patients with pulmonary arterial hypertension. Eur Respir J 26:168–173 33. Lacassie HJ, Germain AM, Valdés G, Fernández MS, Allamand F, López H (2004) Management of Eisenmenger syndrome in pregnancy with sildenafil and L-arginine. Obstet Gynecol 103:1118–1120 34. Molelekwa V, Akhter P, Kenna P et  al (2005) Eisnmenger’s synfrome in a 27 weeks pregnancy – management with bosentan and sildenafil. Ir Med J 98:87–88 35. Lynch TD, Laffey JG (2006) Sildenafil for pulmonary hypertension in pregnancy? Anesthesiology 104:382 36. Huang S, DeSantis ER (2007) Treatment of pulmonary arterial hypertension in pregnancy. Am J Health Syst Pharm 64:1922–1926 37. Lam GK, Stafford RE, Thorp J, Moise KJ Jr, Cairns BA (2001) Inhaled nitric oxide for primary pulmonary hypertension in pregnancy. Obstet Gynecol 98:895–898 38. Duggan AB, Katz SG (2003) Combined spinal and epidural anaesthesia for caesarean section in a parturient with severe primary pulmonary hypertension. Anaesth Intensive Care 31:565–569 39. Robinson JN, Banerjee R, Landzberg MJ, Thiet MP (1999) Inhaled nitric oxide therapy in pregnancy complicated by pulmonary hypertension. Am J Obstet Gynecol 180:1045–1046 40. Stevenson RE, Burton M, Gerlauto GJ, Taylor HA (1980) Hazards of oral anticoagulants during pregnancy. J Am Med Assoc 243:1549 41. Hall JG, Pauli RM, Wilson KM (1980) Maternal and fetal sequelae of anticoagulation during pregnancy. Am J Med 68:122–140 42. Ginsberg JS, Kowalchuk G, Hirsh J, Brill-Edwards P, Burrows R (1989) Heparin therapy during pregnancy. Arch Intern Med 149:2233–2236 43. Gris JC, Lissalde-Lavigne G, Quere I, Mares P (2006) Monitoring the side effects of anticoagulation during pregnancy. Obstet Gynecol Clin North Am 33:397–411 44. Omri A, Delaloye JF, Andersen H, Bachmann F (1989) Low molecular weight heparin Novo (LHN-1) does not cross the placenta during the second trimester of pregnancy. Thromb Haemost 61:55–56 45. Greer IA, Neslon-Piercy C (2005) Low-molecular-weight heparins for thromboprophylaxis and treatment of venous thromboembolism in pregnancy: a systematic review of safety and efficacy. Blood 106:401–407 46. Harenberg J (2007) Treatment of a woman with lupus and thromboembolism and cutaneous intolerance to heparins using fondaparinux during pregnancy. Thromb Res 119:385–388 47. Mazzolai L, Hohlfeld P, Spertini F, Hayoz D, Schapira M, Duchosal MA (2006) Fondaparinux is a safe alternative in case of heparin intolerance during pregnancy. Blood 108:1569–1570

B.A. Cockrill and C.A. Hales 48. Penning S, Robinson KD, Major CA, Garite T (2001) A comparison of echocardiography and pulmonary artery catheterization for evaluation of pulmonary artery pressures in pregnant patients with suspected pulmonary hypertension. Am J Obstet Gynecol 184:1568–1570 49. George RB, Olufolabi AJ, Muir HA (2007) Critical arrhythmia associated with pulmonary artery catheterization in a parturient with severe pulmonary hypertension. Can J Anaesth 54:486–487 50. Roberts NV, Keast PJ, Brodeky V, Oates A, Ritchie BC (1992) The effects of oxytocin on the pulmonary and systemic circulation in pregnant ewes. Anaesth Intensive Care 20:199–202 51. O’Hare R, McLoughlin C, Milligan K, McNamee D, Sidhu H (1998) Anaesthesia for caesarean section in the presence of severe primary pulmonary hypertension. Br J Anaesth 81:790–792 52. Sandham JD, Hull RD, Brant RF et al (2003) A randomized, controlled trial of the use of pulmonary-artery catheters in high-risk surgical patients. N Engl J Med 348:5–14 53. Barash PG, Nardi D, Hammond G et  al (1981) Catheter-induced pulmonary artery perforation. Mechanisms, management, and modifications. J Thorac Cardiovasc Surg 82:5–12 54. Tongers J, Schwerdtfeger B, Klein G et  al (2007) Incidence and clinical relevance of supraventricular tachyarrhythmias in pulmonary hypertension. Am Heart J 153:127–132 55. Tayama E, Ueda T, Shojima T et al (2007) Arginine vasopressin is an ideal drug after cardiac surgery for the management of low systemic vascular resistant hypotension concomitant with pulmonary hypertension. Interact Cardiovasc Thorac Surg 6:715–719 56. Price LC, Forrest P, Sodhi V et al (2007) Use of vasopressin after caesarean section in idiopathic pulmonary arterial hypertension. Br J Anaesth 99:552–555 57. Gilead Corporation (2008) Letairis (ambrisentan) package insert. Gilead, Foster City 58. Briggs GG, Freeman RK, Yaffe SJ (2002) Drugs in pregnancy and lactation: a reference guide to fetal and neonatal risk, 6th edn. Lippincott Williams & Wilkins, New York 59. Pfizer Labs (2007) Revatio prescribing information. Pfizer Labs, New York 60. Metzler E, Ginsburg E, Tsen LC (2004) Use of assisted reproductive technologies and anesthesia in a patient with primary pulmonary hypertension. Fertil Steril 81:1684–1687 61. Thorne S, Nelson-Piercy C, MacGregor A et al (2006) Pregnancy and contraception in heart disease and pulmonary arterial hypertension. J Fam Plann Reprod Health Care 32:75–81 62. Vasilakis C, Jick H, del Mar Melero-Montes M (1999) Risk of idiopathic venous thomboembolism in users of progestagens alone. Lancet 354:1610–1611 63. Jick SS, Kaye JA, Russmann S, Jick H (2006) Risk of nonfatal venous thromboembolism with oral contraceptives containing norgestimate or desogestrel compared with oral contraceptives containing levonorgestrel. Contraception 73:566–570 64. Cooper JM, Carignan DS, Cher D, Kerin JF (2003) Selective tubal occlusion procedure 2000 investigatiors group. Microinsert nonincisional hysteroscopic sterilization. Obstet Gynecol 102:59–67 65. Famuyide AO, Hopkins MR, El-Nashar SA et  al (2008) Hypertoscopic sterilization in women with severe cardiac disease: experience at a tertiary center. Mayo Clin Proc 83:431–438

Part V

Diagnosis and Treatment

Chapter 99

Cardiac Catheterization in the Patient with Pulmonary Hypertension Christopher Barnett and Ori Ben-Yehuda

Abstract  Invasive diagnostic tests, including right-sided heart catheterization (RHC), vasoreactivity testing, and, if indicated, pulmonary angiography, are mandatory in the diagnosis and ongoing management of patients with pulmonary hypertension (PH). We review the techniques used to perform RHC, selective pulmonary angiography, and pulmonary angioscopy in the evaluation of the patient with PH and with suspected chronic thromboembolic PH (CTEPH). This chapter describes detailed procedure for diagnosis of pulmonary hypertension using right heart catheterization. Keywords  Right heart catheterization • Diagnosis of pulmonary hypertension • Pulmonary hemodynamics • Right heart function

1  Introduction Invasive diagnostic tests, including right-sided heart ­cathete­rization (RHC), vasoreactivity testing, and, if ­indicated, pulmonary angiography, are mandatory in the diagnosis and ongoing management of patients with pulmonary hypertension (PH). We review the techniques used to perform RHC, selective pulmonary angiography, and pulmonary angioscopy in the evaluation of the patient with PH and with suspected chronic thromboembolic PH (CTEPH).

2  RHC and the Role of Noninvasive Imaging RHC should be performed in any patient in whom a diagnosis of PH is being considered to help confirm the suspected diagnosis, provide prognostic information, and aid in

C. Barnett (*) Division of Cardiology, University of California, San Diego, 200 W Arbor Dr, MPF 360, San Diego, CA 92103, USA e-mail: [email protected]; [email protected]

p­ lanning therapy [1, 2]. Similarly, pulmonary angiography is ­mandatory in the setting of possible CTEPH to assess disease burden and surgical accessibility. Other diagnostic tests such as echocardiography, radionuclide ventilation–perfusion (V/Q) scanning and computed tomography (CT) pulmonary angiography play an important role and provide useful information, but are, at this time, not considered a substitute for RHC and pulmonary angiography. They do, however, play a role in screening patients prior to invasive studies such as RHC. Echocardiography is often the first study ordered in cases of suspected PH from a variety of causes. Echocardiography provides an indirect estimate of pulmonary artery systolic pressure (PASP) and a limited assessment of right ventricular size and function. Although some centers have demonstrated good correlation between echocardiographic estimates of PASP and PASP measured by RHC [3], echocardiography is limited by technical expertise in measurement of the ­tricuspid regurgitant jet, patient factors such as body habitus, it perhaps not being sensitive enough to detect mild PH [4] and it perhaps overestimating the presence of PH among some patients, such as those with scleroderma [5]. An accurate assessment of left ventricular end-diastolic pressure is critical especially when there is concern for a venous cause of PH, such as diastolic left ventricular dysfunction. Additionally, if therapy with pulmonary vasodilators is being considered, vasoreactivity to an agent such as inhaled nitric oxide should be assessed. Although echocardiography is a useful initial screening test, expert consensus supports at least one RHC prior to the initiation of PH-specific therapy [1, 2]. V/Q scanning is considered a required test in any patient with PH for whom a diagnosis of CTEPH is being considered. The V/Q scan in patients with CTEPH demonstrates one or more mismatched, segmental or larger defects, whereas patients with other small vessel forms of PH will have normal perfusion or subsegmental defects. A mottled appearance is often seen in pulmonary arterial hypertension (PAH). The magnitude of the perfusion defects in patients with chronic thromboembolic disease, however, often

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understates to a considerable extent the actual degree of pulmonary vascular obstruction, determined angiographically or at surgery. Therefore, the presence of even a single mismatched, segmental perfusion defect in a patient with PH should raise the question of a CTEPH and patients should proceed to angiography. Catheterization is not necessary in patients with residual, postembolic perfusion defects in the absence of symptomatic limitation or echocardiographic abnormalities, unless the perfusion defect is substantial, accounting for 40% or more of total pulmonary perfusion [6]. CT scanning may play an increasingly important role in the diagnosis and management of patients with PH and has several potential advantages over traditional angiography. High-resolution CT scanning without contrast is useful in the evaluation of the pulmonary parenchyma and in the exclusion of primary lung diseases such as COPD and pulmonary fibrosis. Unilateral perfusion defects on V/Q scanning are especially suggestive of a variety of disorders such as pulmonary artery sarcoma, other cancers, vasculitis, and mediastinal fibrosis that can be diagnosed from standard ­thoracic CT scans with contrast. The role of CT pulmonary angiography is well established in the setting of acute pulmonary embolism and has demonstrated high sensitivity and specificity [7]. Studies of CT pulmonary angiography in chronic pulmonary thromboembolic disease have shown a variety of abnormalities: pulmonary thromboemboli located within the central pulmonary arteries, right ventricular enlargement, dilated central pulmonary arteries, bronchial artery collateral flow, parenchymal abnormalities consistent with prior infarcts, and mosaic attenuation of the pulmonary parenchyma [8]. However, the specificity may be inadequate as some findings, such as central thrombus, have been reported in patients with idiopathic PH and other chronic pulmonary disorders [9, 10]. The diagnostic utility of newly emerging CT technology may prove to be more useful in patients with suspected CTEPH. In a recent study, Reichelt et al. compared 64-slice CT angiography and invasive digital subtraction angiography in 27 patients undergoing evaluation for likely CTEPH [8]. With invasive angiography as the gold standard test, the sensitivity and specificity for 64-slice CT scanning were 96.3 and 94.8% for lobar findings and 94.1 and 92.9% for segmental findings consistent with CTEPH. The study did not evaluate the utility of CT angiography for subsegmental disease. Although CT evaluation offers the advantage of noninvasive imaging and provides detailed anatomic information, the use of this modality in assessing suitability for pulmonary thromboendarterectomy has yet to be determined and presently it cannot be considered a substitute for traditional angiography. Additionally, it cannot provide direct hemodynamic assessment, which is essential in the diagnosis of CTEPH, and may assist in the preoperative classification of patients undergoing surgery [11].

C. Barnett and O. Ben-Yehuda

3  Safety Consideration Shortly after the development of techniques to perform RHC and pulmonary angiography, reports of deaths associated with the procedures appeared in the literature [12–16]. Patients with severe PH were thought to be particularly susceptible to ventricular arrhythmias and acute right ventricular failure associated with mechanical complications of the procedure or radiographic contrast administration. Until recently, data regarding the safety of these procedures came from only a small number of retrospective single-center studies with relatively few patients. Five fatal complications occurred in a series by Fuster et al. [17] that included 120 patients undergoing RHC for evaluation of PH. The National Institutes of Health (NIH) registry included 187 patients with PH [18] who underwent invasive evaluation. In this series, ten adverse events but no deaths were reported. Pulmonary angiography was associated with three deaths of 1,350 patients with ­suspected CTEPH in a report from 1980 by Mills et al. [19]. Nicod et al. found that there were no deaths in the original series of 67 patients undergoing CTEPH evaluation at the University of California, San Diego (UCSD) [20]. Since that time, more than 3,000 pulmonary angiograms have been performed at UCSD in patients with severe PH without any ­procedure-related mortalities. The low incidence of adverse effects in the UCSD experience has been attributed to the use  of nonionic contrast as well as careful attention and adherence to a variety of safety precautions which are described below [21, 22]. Several other recent series of pulmonary angiography performed in patients with PH have resulted in conflicting safety data. One study of 402 patients reported no procedure-related mortality [23], whereas angiography in another series of 202 patients resulted in three fatal complications [24]. The largest study to date is a multicenter collaboration reporting on 7,218 RHCs and 1,214 pulmonary angiographies in patients with PH that has established the safety of these procedures when performed by expert operators in experienced centers [25]. The authors reported combined retrospective and prospective data collected on all patients with PH undergoing procedures from 15 centers in the USA and Europe between 2000 and 2005. A total of four fatalities (0.055%, 95% confidence interval 0.01–0.099%) related to the 7,218 RHCs were noted. Two of the fatalities, one from pulmonary artery rupture and the other from unexplained intraparenchymal pulmonary hemorrhage, were directly related to the procedure. Fifty-one total adverse events were related to venous access and RHC, including ten puncture-site hematomas, eight vagal reactions, and seven episodes of supraventricular tachycardia. In addition to complications related specifically to the ­performance of RHC and pulmonary angiography, administration of intravenous contrast agents carries the risk of contrast-­associated allergic reactions and acute kidney injury, which have recently been reviewed [26]. The reported

99  Cardiac Catheterization in the Patient with Pulmonary Hypertension

i­ncidence of contrast-associated acute kidney injury varies greatly ­depending on the patient population, baseline risk factors, and the definitions used. Appropriate standard precautions for patients with intravenous contrast allergies and renal dysfunction should be followed in patients undergoing pulmonary angiography. On the basis of our own experience and the available data, we believe that RHC and pulmonary angiography performed by clinicians in an experienced center is safe even in patients with severe PH.

4 Right-Sided Heart Catheterization Evaluation of hemodynamics by RHC plays an integral part of the evaluation of patients with PH. The goals of RHC are (1) to measure pulmonary artery pressure (PAP) directly and estimate pulmonary vascular resistance (PVR), (2) to make an evaluation for left-to-right shunts, (3) to test the response to therapeutic agents, and (4) to determine the site of increased vascular resistance (upstream vs. small vessel). Essential to the appropriate use of a pulmonary artery catheter is a complete understanding of all aspects of catheter use, including transducer calibration, leveling, zeroing, and troubleshooting as well as proper interpretation of pressure waveforms [27]. RHC is performed in PH using standard techniques. Although it has been shown to be safe in this population, several important modifications and special precautions are routinely practiced in our laboratory when PH and CTEPH are suspected. (1) All procedures are performed in the cardiac catheterization laboratory with the assistance of trained, experienced staff, easy access to emergency resuscitation equipment, and fluoroscopy. (2) A preprocedure echocardiogram is performed in all patients to look for possible right atrial or ventricular clots and to alert the operator for the presence of an intracardiac shunt. (3) Vital signs, including PAPs, are monitored continuously. Supplemental oxygen is administered to maintain a SaO2 of 92% or greater. (4) Procedures are performed preferentially via the right internal jugular vein to avoid potential dislodgement of unrecognized venous thrombi from the femoral vein, iliac vein, or inferior vena cava and to facilitate angioscopy. Patients with prior internal jugular catheterization or the presence of upper body clot may have an occluded internal jugular vein, often unilaterally. Examination of the jugular venous pulsation may be absent in this situation and should guide the operator to access the neck on the side with an intact jugular venous pulsation. When jugular venous pulsations are present ­bilaterally, we prefer to use the right internal jugular vein as it allows for a straight-line access to the superior vena cava and right atrium. The use of portable ultrasound is helpful in obese

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patients with poor landmarks, but is superfluous in the patient with marked jugular venous pulsations, a common finding in the patient with PH [28]. Jugular venous access also facilitates the performance of exercise on a supine bicycle during the RHC if clinically indicated. (5) Hemodynamics are assessed using a balloon flotation catheter (Swan–Ganz catheter). The use of a 0.025-cm wire helps stiffen the catheter and guide the placement of the catheter in the right side of the heart when balloon flotation is difficult secondary to an enlarged right ventricle, significant tricuspid regurgitation, severely elevated PAPs, and low cardiac output. The use of a wire is often essential when access is obtained from the leg and when attempting to advance the catheter to the left pulmonary artery. Carbon dioxide is used for balloon inflation to minimize the possibility of paradoxical embolism in case of balloon rupture. During RHC by balloon flotation, rapid determination and continuous monitoring of pressures in the right ventricle, right atrium, and pulmonary artery and of postcapillary wedge pressure (PCWP) is performed. In patients with idiopathic PAH (IPAH), particular attention should be paid to the right atrial pressure and right ventricular end-diastolic pressure. In the National Heart, Lung, and Blood Institute registry, mean right atrial pressure and decreased cardiac index were the most important predictive variables for survival in patients with PAH [18]. The patient with PAH commonly has elevated right atrial pressure, elevated mean PAP (mPAP), reduced cardiac index, and low or normal PCWP. PCWP is obtained with an end-hole catheter positioned in a side branch of the pulmonary artery facing a pulmonary capillary bed. One pitfall to avoid is wedging the balloon too proximally, creating a hybrid tracing of pressures between the actual PAP and PCWP, resulting in an overestimation of the PCWP (Fig. 1). Deflating the balloon to decrease its size will allow the catheter to be wedged in a smaller, more distal branch of the pulmonary artery. Caution should be taken to avoid overwedging and possible pulmonary artery rupture, a potentially lethal event [29]. Blood gas analysis of a sample obtained from the distal port of the RHC with the balloon inflated may be useful when the PCWP tracing is of poor quality. In the wedged position, aspirated blood comes from the capillary bed and the saturation should be equivalent to that of arterial blood. The authors have found that typically the narrow, tapering anatomy of the left pulmonary artery allows more reliable wedge determinations. The use of a J-tipped 0.25-mm guide wire to guide the Swan–Ganz catheter to the left pulmonary artery is essential. Determination of an accurate PCWP is critical to establish the cause of PH and determine appropriate therapy [30]. In the presence of substantially elevated PCWP, left-sided heart ­catheterization may be performed to exclude mitral stenosis or left ventricular dysfunction. Obtaining an accurate PCWP in patients with CTEPH can be particularly challenging,

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C. Barnett and O. Ben-Yehuda

Fig. 1  (a) Hybrid tracing of pulmonary artery wedge pressure and wedge tracing overestimating wedge pressure. (b) Actual pulmonary capillary wedge pressure tracing once the balloon has been

deflated and allowed to wedge in a smaller, more distal pulmonary artery. (Reprinted from Guillinta et  al. [22] with permission of Elsevier)

which may reflect the irregular proximal vasculature [11]. Elevated wedge pressures must be interpreted with caution in some patients with severe right ventricular enlargement and dysfunction. Elevated left ventricular pressures secondary to severe right ventricular enlargement and pressure overload have been described and may not reflect true left ventricular dysfunction [31]. At UCSD, cardiac output is generally determined by both the Fick method and the thermodilution method. Cardiac output is best determined by the Fick method in the setting of low cardiac output states or when there is significant ­tricuspid regurgitation. However, measurement of a patient’s oxygen consumption is frequently not possible, and estimation of oxygen consumption may not apply to patients with PH, introducing error in the calculations.

Chronic left-to-right shunting can result in PH secondary to right-sided heart pressure and volume overload. Shunting through a patent foramen ovale may also occur as a result of elevated right-sided heart pressures. In patients with echocardiographic evidence of shunting or clinical evidence of shunting, such as hypoxemia that is not ­corrected with oxygen supplementation, a shunt study should be performed in addition to routine hemodynamic measurements. To diagnose, localize, and quantify shunt flow, blood samples are obtained from the superior vena cava, multiple positions in the right atrium, the right ­ventricle, and the pulmonary ­arteries. An increase in ­oxygen saturation between the ­superior vena cava and the ­pulmonary artery suggests a left-to-right shunt and allows its quantification [32].

99  Cardiac Catheterization in the Patient with Pulmonary Hypertension

In some cases, pulmonary hemodynamic abnormalities may appear underwhelming, but in fact may reflect the ­presence of severe disease which must be accounted for in decisions regarding periprocedural management. Patients from the NIH registry [18] of PH had an average mPAP of 60 mmHg and a cardiac index of 2.27 l/min/m2. In contrast, patients with PAH secondary to chronic anemia have a high cardiac output and relatively low mPAP. In one study of patients with sickle cell disease [33], the mPAP was 40 mmHg and the cardiac output was 7.6 l/min. Although the increase in mPAP is modest and the cardiac output remains above normal, this degree of PH in sickle cell anemia is associated with marked increases in PAP and PVR with exercise or hemolytic crisis [34, 35] and with a increased mortality [35]. PH associated with chronic liver disease produces a similar hemodynamic profile of relatively low mPAP and PVR associated with a high cardiac output and high mortality [36].

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pulmonary artery to estimate the pulmonary occlusion or ­pulmonary capillary pressure (Poccl). Such techniques could be useful in locating the predominant site of increased resistance (large pulmonary arteries, pulmonary artery capillaries, and pulmonary veins), which may help clarify the diagnosis of PH. Rup is calculated using the following formula:

Rup % = 100 ´ (mPpa - Poccl) / (mPpa - Ppao),

Analysis of PAP decay curves after pulmonary arterial ­occlusion with the balloon of the Swan–Ganz catheter can be used to partition the PVR into an arterial or upstream (Rup) component and a capillary–venous or downstream component (Rdo) (Fig. 2). The calculation requires backward extrapolation to the pressure at the point of balloon occlusion of the

where mPpa is the mean pulmonary pressure, Poccl is the pressure at the point of balloon occlusion, and Ppao is the pulmonary artery occluded pressure, or pulmonary capillary wedge pressure (Fig. 2). Fesler et  al. [38] measured routine hemodynamics and capillary pressure in 42 patients with a diagnosis of PH confirmed by invasive testing. The cause was IPAH in 19 patients, PAH associated with anorexigen use in 11 patients, portal–pulmonary hypertension in three patients, scleroderma in three patients, pulmonary veno-occlusive disease (PVOD) in two patients, and CTEPH in four patients. Capillary pressure was determined by analysis of the decay of a PAP curve after occlusion of the pulmonary artery. The authors showed that the mean value of Rup was lowest in PVOD and highest in CTEPH, correlating with the presumed anatomic location of increased resistance (small veins in PVOD, arterioles in PAH, and proximal large arteries in CTEPH). However, isolated measures of Rup were not adequate to make a diagnosis in an individual patient. Using the same technique, Kim et al. [11, 37] demonstrated in 26 patients undergoing ­thromboendarterectomy that

Fig. 2  Sample pulmonary artery pressure occlusion waveforms from two patients with (a) primarily upstream resistance and (b) significant downstream resistance [longer time needed for the pressure to reach a

stable pulmonary artery occluded pressure (Ppao)]. Poccl occlusion pressure, Ppa pulmonary artery pressure. (Reprinted with permission from Kim et al. [37]. Copyright Wolters Kluwer Health)

5  Partitioning of PVR

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C. Barnett and O. Ben-Yehuda Table  1  Proposed preoperative classification of chronic thrombo­ embolic pulmonary hypertension (CTEPH) Class A B C Pulmonary vascular resistance (dyn·s/cm5) Pulmonary vascular resistance partitioning Treatment

£1,100

PEA

>1,100

>1,100

Rup > 60%

Rup < 60%

PEA

PEA or trial of medical therapy

All patients have a ventilation–perfusion scan and pulmonary angiography consistent with CTEPH. Class A has a low pulmonary vascular resistance associated with low postoperative mortality. Classes B and C have increased pulmonary vascular resistance; partitioning of pulmonary vascular resistance identifies patients with higher upstream or lower upstream resistance that correlates to lower and higher risk of postpulmonary endarterectomy pulmonary hypertension and death Adapted with permission from Kim [11]. Copyright American Thoracic Society PEA pulmonary endarterectomy, Rup upstream resistance

Fig.  3  Correlations between preoperative upstream resistance (Rup, %) and (a) postoperative total pulmonary resistance index (TPRI) and (b) mean pulmonary artery pressure (mPpa). Closed triangles pulmonary endarterectomy (PEA) survivors (n = 22); open squares PEA nonsurvivors (n = 4). (Reprinted with permission from Kim et al. [37]. Copyright Wolters Kluwer Health)

those with a higher downstream resistance (indicating ­significant concomitant inoperable small vessel disease) were at high risk for postoperative death and persistent PH (Fig. 3). PVR partitioning, in combination with the overall PVR and angiographic appearance, has been proposed as a method to predict which patients will respond favorably to pulmonary thromboendarterectomy [11] (Table 1).

5.1 Exercise During RHC PH has been defined as a mPAP of 25 mmHg or greater at rest or 30  mmHg or greater with exercise [1]. Hemodynamic assessment during exercise, however, has been described infrequently. Echocardiographic assessment of PAP during exercise is complicated by many of the same factors that limit

its use at rest, including difficulty in evaluating the ­tricuspid regurgitant jet and inability to measure pulmonary capillary wedge pressure [39]. However, the presence of exercise-associated hemodynamic abnormalities may be clinically relevant and may identify populations of individuals with asymptomatic, preclinical disease, or those with exercise intolerance, but normal resting hemodynamics. Although the natural history and role of treatment in these patients remains to be defined, recent studies invasively confirmed the existence of exerciseassociated PH [39] and the benefit of using pulmonary vasodilator therapy in patients with less severe disease [40]. Published guidelines do not address how hemodynamic assessment during exercise should be performed. Until recently, only a few small studies had invasively assessed patients with possible pulmonary vascular disease during exercise using a variety of methods to exercise patients and evaluate the response. Machado et  al. [34] evaluated the response to exercise in 21 patients with sickle cell disease. After a catheter had been placed in the right side of the heart, patients performed upper-extremity exercise with 2-lb weights for 5  min or until exhaustion and complete hemodynamic data were collected at rest and at peak exercise. Raeside et al. used a 7-Fr multiuse micromanometer-tipped high-fidelity catheter for ambulatory PAP monitoring [41] and supervised exercise testing [42]. The first report was of six patients studied [41] with ambulatory monitoring for 24 h in addition to supine exercise bicycle testing at 30% VO2 max (as determined during cardiopulmonary exercise test (CPET) prior to RHC) with measurement of mPAP at the baseline at 4 min of exercise. Another ten patients [42] were studied during RHC with 3 min of straight leg raising exercise and hemodynamic assessment at the baseline and at maximum exercise. The largest study of exercise hemodynamics in patients with PH was published recently by Tolle et al. [39]. Four ­hundred

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and six patients with dyspnea on exertion or fatigue underwent full cardiopulmonary exercise testing using a supine bicycle with a catheter in the right side of the heart and an arterial line in place. Workload on the bicycle was increased by between 6.25 and 25 W every minute until exhaustion. Mean systemic pressure and end-expiratory right atrial pressure and mPAP were measured continuously. PCWP was measured every minute and blood was collected from the peripheral and pulmonary arteries once each minute. A validated method for the performance of exercise evaluation of PH is needed. Until the time when it becomes available, we suggest that all testing for exercise-induced PH be performed during continuous telemetry and hemodynamic monitoring with a catheter in the right side of the heart. Complete hemodynamic measurements should be obtained at minimum during rest and at peak exercise, and if possible, at each stage of exercise. The use of hand weight and the use of bicycle exercise have been described by several authors and appear to be equally effective. It is unclear what the goal of exercise should be. For many patients with significant PH and other comorbidities, exhaustion may occur after only a brief period of low-intensity exercise and patients may demonstrate a pulmonary hypertensive response to exercise early. Exercise should start at a low level and be appropriately titrated up at fixed intervals as the patient tolerates the exercise. Exercise should then continue until exhaustion or hemodynamics are conclusive for a diagnosis of exercise-induced PH.

5.2  Vasodilator Challenge Pulmonary vasoconstriction may play a significant role in the pathophysiology of many patients with PH, and vasodilator therapy with calcium channel blockers may be useful in a small number of patients. Unfortunately, there are no clinical or hemodynamic factors that can predict which patients will have a response to calcium channel blockers. Therefore, current guidelines support the performance of a vasodilator study whenever PAH is confirmed on RHC in a patient for whom treatment is planned. The criteria for a positive vasodilator study have recently changed. In their landmark trial which established the efficacy of calcium channel blockers in idiopathic PH, Rich et al. found that the 17 of 64 patients who had a decrease in both the mPAP and PVR by more than 20% during an acute vasodilator challenge also had a marked reduction in mortality during chronic therapy [43]. A more recent study by Sitbon et al. led to the revision of the criteria [44]. In a retrospective single+center study of 557 patients with IPAH who underwent acute vasodilator testing and chronic therapy with calcium channel blockers, only 38 patients (6.8%) had a long-term favorable response. The authors found that a more sensitive criterion to identify

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long-term responders was a decrease in mPAP by more than 10 mmHg to a mPAP of less than 40 mmHg with a stable or increasing cardiac output. These parameters were subsequently adopted by the most recent guidelines [1]. Pulmonary vasoreactivity testing during RHC may be a useful tool in the assessment of PH from CTEPH. SkoroSajer et  al. performed RHC with vasodilator testing using inhaled nitric oxide in 62 patients who underwent pulmonary thromboendarterectomy [45]. Repeat invasive hemodynamic assessment was performed immediately postoperatively, at 1 year, and at 3 years to evaluate for the recurrence of PH. A decrease in mPAP by more than 10.4% during preprocedure vasodilator testing was associated with better long-term survival free from lung transplant. This finding may reflect increased vascular tone and small vessel arteriopathy in some patients with CTEPH and could be used as a tool to select those likely to benefit from surgery. PH is a known risk factor for early graft failure in orthotopic heart transplant. Reversibility of PH determined by vasoreactivity testing may identify a population of patients who can safely undergo heart transplantation. Klotz et  al. examined 217 patients undergoing orthotopic heart transplantation [46]. Those with PH defined as PVR greater than 2.5 Woods units and/or transpulmonary gradient greater than 12 mmHg underwent vasodilator testing with intravenously administered prostacyclin. Reversibility was present if PVR was less than 2.5 Woods units and the transpulmonary gradient was less than 12 mmHg with the infusion. There was no difference in mortality between patients without PH and those with reversible PH. Original vasodilator studies were performed with escalating oral doses of calcium channel blockers during RHC until PAP and PVR decreased. Owing to the relatively long duration of action and the potential for hemodynamic instability and even death, this approach has largely been replaced by the use of potent, rapid-acting, titratable intravenous or inhaled vasodilators with a short half-life such as intravenously administered epoprostenol and inhaled nitric oxide [47]. In their recent study of RHC in patients with PH, Hoeper et al. reported on the agents used to assess pulmonary vasoreactivity in 15 specialized PH centers in the USA and Europe [25] (Table  2). Nitric oxide was the most Table 2  Agents used for vasoreactivity testing Agent Number of procedures Inhaled nitric oxide Inhaled iloprost Intravenously administered epoprostenol Intravenously administered iloprost Calcium channel blockers Sildenafil Oxygen nitrates, inhaled treprostinil Data from Hofmann et al. [24]

2,357 (44.8%) 1,476 (28%) 99 (1.9%) 50 (1.0%) 79 (1.5%) 270 (5.1%) 851 (16.2%)

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c­ ommonly used agent, being used in 42.8% of procedures. There were no significant adverse reactions with the use of inhaled nitric oxide in this study. Inhaled iloprost was the second most commonly used agent because it is used in German centers which were overrepresented in this sample. There are, however, inadequate data on the safety and predictive value of calcium channel blocker response to recommend this agent in published guidelines. Intravenously administered epoprostenol was used in 97 patients (2.4%), also with no adverse effects. None of the centers in this series used intravenously administered nitroprusside, intravenously administered adenosine, or orally administered sildenafil for acute vasoreactivity testing. Owing to the risk of hemodynamic instability, acute ­vasodilator testing should be performed only with a pulmonary artery catheter in place in a closely monitored setting. Complete hemodynamic data including right atrial pressures, right ventricular pressures, cardiac output, and pulmonary capillary wedge pressure should be recorded at the baseline and then again after administration of the chosen vasodilator. Table  3 gives recommendations for dosing of intravenously administered epoprostenol, inhaled nitric oxide, and intravenously administered adenosine [2]. Inhaled nitric oxide at a dose of 20 parts per million for 10 min is the most commonly used regimen during vasodilator testing at our institution. Acute vasodilator testing in patients with a pulmonary venous cause of PH, such as PVOD or left ventricular diastolic dysfunction, can be complicated by pulmonary edema and hemodynamic compromise. In one small study, pulmonary vasodilator testing with 5–10 min of inhaled nitric oxide use was safely performed in 24 patients with PVOD, although seven of them developed pulmonary edema shortly after the initiation of continuous vasodilator therapy. Thus, even though acute vasodilator studies might be performed safely in patients with PVOD, they do not appear to predict a positive response to vasodilator therapy. The development of pulmonary edema during acute vasodilator testing should raise suspicion for the presence of PVOD or pulmonary capillary hemangiomatosis and is a contraindication to long-term vasodilator therapy. Vasodilator testing is still recommended in all cases of PAH to identify patients who might be candidates for longterm oral calcium channel blocker therapy; however, this is a relatively small number of patients [44]. However, the predictive value of acute vasodilator testing is unclear with the use of newer agents such as epoprostenol and sildenafil which

combine vasodilator and antiproliferative effects. Furthermore, the likelihood of a response during acute vasodilator testing in patients with PAH secondary to a variety of conditions such as HIV and connective tissue disease is small.

6  Pulmonary Angiography Pulmonary angiography is a critically important test if chronic thromboembolic disease is suspected to both ­confirm the diagnosis and assess the surgical accessibility of the ­disease. A variety of other causes of PH have distinct angiographic­ ­features which can be useful in identifying the underlying cause. Biplane imaging provides optimal anatomical detail. When dilated and overlapping vessels are present, the lateral view provides more detailed images of lobar and segmental anatomy than those obtained with an anteroposterior view alone.

6.1  Anatomy Pulmonary vascular anatomy parallels bronchial anatomy [48] (Fig.  4). The main pulmonary artery arises from the right ventricle, anterior and to the left of the aorta. It takes a posteromedial direction until its bifurcation into the right and left pulmonary arteries. The right pulmonary artery courses anterior to the right mainstem bronchus. It gives rise to the right upper lobe branch within the mediastinum. The left pulmonary artery passes under the aortic arch over the left mainstem bronchus and descends posterior to the bronchus before the origin of the left upper lobe branch. At our institution, pulmonary angiography (Fig. 5) generally follows hemodynamic assessment when a diagnosis of CTEPH is suspected. A stiff 8-Fr Berman catheter with a balloon tip and distal side holes is guided into the right pulmonary artery under fluoroscopic guidance and positioned so that the side holes are about 1 cm distal to the right upper lobe pulmonary artery, which will fill retrograde during contrast injection. The injection through the side holes allows good opacification with reduced injection velocity and pressure and helps minimize catheter retraction and whip. Nonionic contrast is used because of the lower risk of adverse reactions and hemodynamic instability. A 10-ml test

Table 3  Dosing regimen for nitric oxide, intravenously administered epoprostenol, and intravenously administered adenosine during pulmonary vasodilator testing Drug Route Half-life Dosage range Titration increments Duration per increment Epoprostenol Adenosine Nitric oxide

Intravenous Intravenous Inhaled

3 min 5–10 s 15–30 s

2–12 ng/kg/min 50–350 mg/kg/min 10–20 ppm

2 ng/kg/min 50 mg/kg/min No titration

Adapted from [2] with permission of the European Society of Cardiology

10 min 2 min 5 min

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Fig. 4  (a) Anteroposterior view of the anatomy of the right pulmonary artery. (b) Lateral view. (c) Anteroposterior view of the anatomy of the right pulmonary artery. (d) Lateral view. (Reprinted from Peterson and Nicod [48] with permission from Elsevier)

particularly helpful in separating the overlapping tributaries of the right middle and right lower lobes and the left lingual and left lower lobes. It also allows a clear separation between the superior segmental artery and the artery to the middle lobe (Fig. 6).

6.2 Patterns of Angiographic Findings

Fig.  5  Normal pulmonary angiogram. All branches are present, smooth, and extend to the periphery without pruning

injection of 50% contrast and 50% saline is then performed by hand to check the catheter position and to estimate the volume of contrast needed for angiography. A standard dose of contrast during angiography is 40 ml of contrast injected over 2  s, which is adjusted up or down depending on how rapidly the test injection clears. Biplane angiography is then performed. The Berman catheter is then moved into the left pulmonary artery with the assistance of a 0.25-mm-diameter wire. Angiographic recordings are taken using the anteroposterior and lateral projections. The lateral projection is

In series of 250 patients with CTEPH, Auger et al. described five angiographic patterns seen in patients with chronic thromboembolic disease. These patterns included (1) ­pouching abnormalities, (2) vascular webs or band-like constrictions, (3) intimal irregularities or lining clot, (4) abrupt narrowing of major pulmonary vessels, and (5) obstruction of major pulmonary vessels [49]. A pouch appearance describes occluding or partially occluding thrombi that have organized in a concave configuration (Fig.  7). The pouch occlusion may initially appear complete, but as contrast injection continues, more distal vessels may become apparent. A pouch in the right or left main pulmonary artery may mimic the appearance of pulmonary artery agenesis. Pulmonary artery webs appear as lines or bands in the lobar or segmental vessels that fail to opacify completely and remain stable in appearance throughout contrast injection (Fig.  8). Webs traverse the entire vessel and may be associated with vessel narrowing and distal poststenotic dilatation. Common in CTEPH, webs may also be seen in congenital stenotic lesions of the pulmonary vessels and with vasculitides such as Takayasu’s arteritis. Patients with congenital stenotic lesions of the pulmonary vasculature present at an early age and typically have coexisting cardiac abnormalities. Narrowing of the large pulmonary arteries may be a prominent finding. This narrowing can have the appearance of scalloping of the wall of the pulmonary artery, a finding that has been associated with a chronic, irregularly organized

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Fig. 6  Importance of the lateral projection. (a) In the posteroanterior projection, the lower lobe vessels are overlapped and appear normal.

(b) In the lateral projection, complete occlusion of the left lower lobe posterolateral branch of the pulmonary artery is readily apparent

Fig. 7  Pulmonary angiogram in a patient with “pouch” occlusion of the right pulmonary artery distal to the upper lobe branch in the posteroanterior projection (a) and the lateral projection (b). Notable are faintly filling distal

branches of the lower lobe vessel, reflecting minimally recanalized clot. Pulmonary angiogram demonstrating complete pouch occlusion of the left pulmonary artery distal to the left upper lobe artery takeoff (c)

thrombus found at the time of surgery (Fig. 9). Alternatively, there may be replacement of the normally gently tapering vessel by abrupt, dramatic narrowing representing a large recanalized thrombus, a concentric thrombus lining the arterial wall, or contraction of the artery by an organized thrombus (Fig. 9). A final finding is the absence of lobar vessels that would normally be expected to arise from the main pulmonary arteries. This finding may be associated with abnormally straight borders or luminal irregularities of the main pulmonary arteries (Fig. 9). A variety of other abnormalities may be apparent on pulmonary angiography that can suggest an alternative diagnosis­. The classic angiographic findings in patients with IPAH include ­normal or dilated central arteries with pruning of the small, more distal, nonelastic arteries. Pruning of the pulmonary vessels may also be seen with chronic thromboembolic disease, but in this scenario pruning is regional and more

proximal (Fig. 10). Large vessel vasculitis, such as Takayasu’s arteritis, may present with evidence of lobar or segmental pulmonary artery occlusions, stenotic lesions, pulmonary artery aneurysms, and luminal irregularities (Fig.  11). Vasculitis affecting the pulmonary vasculature can often be distinguished on the basis of the systemic symptoms which are generally present. Total obstruction or abrupt narrowing of the pulmonary vessel, typically seen in chronic thromboemboli, can also be seen in extrinsic compression from extensive mediastinal or hilar lymphadenopathy, fibrosing mediastinitis, and pulmonary vascular or primary lung malignancies. These findings are demonstrated in the pulmonary angiogram of a patient who was diagnosed with a pulmonary artery sarcoma at the time of surgery (Fig. 12). At our institution, coronary angiography is generally ­performed to rule out obstructive coronary artery disease in any male patient over the age of 45, female patients over the

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Fig. 8  Pulmonary angiogram demonstrating linear opacities in the pulmonary artery termed “webs” (a, b). Prominent web in the posteroanterior projection (c) and the lateral projection (d)

Fig. 9  Pulmonary angiogram demonstrating narrowing of the main pulmonary arteries and tapering segmental vessels consistent with thrombus

lining the vessel wall. Also noted is complete obstruction of lobar vessels. Views in the posteroanterior projection (a) and the lateral projection (b)

age of 40, or any other patient with possible coronary ­ischemia who is to undergo thromboendarterectomy. An interesting ­finding that has been noted in occasional patients studied at UCSD is the presence of coronary artery to pulmonary artery collaterals (Fig.  13). Also, in the case of marked enlargement of the pulmonary arteries, the left main

coronary artery may be compressed, giving the appearance of ostial left main coronary artery stenosis (Fig.  14). This ­finding is generally best viewed in the left anterior oblique cranial view. The diagnosis is reserved for those patients who do not otherwise have evidence of significant coronary artery disease and is generally not an indication for

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Fig. 10  Pulmonary angiogram in a patient with idiopathic pulmonary hypertension which demonstrates loss of small-caliber arteries in the lung periphery termed pruning. Views in the posteroanterior (a) and lateral (b) projections

(70%), no major vessel thrombus is appreciated. Only thickened intima can be seen, occasionally with webs and the endarterectomy plane is raised in the main, lobar, or segmental vessels. Type III disease (20% of cases) is very distal and is confined to the segmental and subsegmental branches. Type IV disease is not primary pulmonary thromboembolic and is inoperable. Although pulmonary angiography does not correlate fully with this intraoperative classification, it can predict surgical accessibility particularly when coupled with V/Q scan results.

6.4 Pulmonary Angioscopy

Fig. 11  Pulmonary angiogram in a patient with vasculitis demonstrating the classic “beads on a string” appearance of the affected branches

bypass  surgery, especially if the patient’s PAP decreases after surgery.

6.3  Significance of Angiography in Patients with CTEPH The presence of bilateral disease and proximal involvement (main pulmonary trunks, lobar arteries, and segmental arteries) predicts surgical accessibility. Correlation with findings on the perfusion scan is essential because the presence of segmental defects is predictive of surgical success. In the operating room, CTEPH has been classified by Jamieson [11]. In type 1 disease, approximately 10% of cases of thromboembolic PH, major vessel clot is present and readily visible on opening of the pulmonary arteries. In type II disease

The goals of pulmonary angiography are to determine, primarily, if thromboembolic material is located proximally and to judge surgical accessibility and, secondarily, the likely response to surgery as well as surgical risk [11]. Most often angiography is adequate to make this determination; however, in about 20% of cases, the pulmonary angiogram is not definitive. Recanalization of chronic clot may allow contrast to permeate through the lesions, and the pulmonary angiogram can underestimate the extent of disease just as the perfusion scan can. In addition, the presence of defects at the transition between segmental and subsegmental vessels may raise doubt regarding the surgical accessibility. Fiber-optic angioscopy was developed to allow direct visualization of the interior of the central pulmonary vessels up to the segmental and subsegmental levels and is used to define surgical accessibility better in borderline cases. The angioscope used is a 120-cm fiber-optic device, 3 mm in diameter (Fig.  15) which is introduced through an 11-Fr venous sheath. A disposable balloon is tied securely to the spool-like stainless steel tip, which can be inflated through a tube with carbon dioxide and used to assist in floating the angioscope downstream as well as occluding the blood flow

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Fig. 12  Pulmonary angiogram in a patient who was found to have a pulmonary sarcoma on surgery. Notable is marked but relatively smooth narrowing of the pulmonary artery. Views in the posteroanterior (a) and lateral (b) projections

Fig.  13  Coronary angiogram demonstrating collaterals between the right coronary artery and the right pulmonary artery

Fig. 15  The pulmonary angioscope (a) and the angioscope with the balloon inflated (b). (a Courtesy of Olympus Corporation, Lake Success, NY, USA)

Fig. 14  Coronary angiogram demonstrating compression and “pseudostenosis” of the ostial left main coronary artery by a severely enlarged pulmonary artery in a patient with chronic thromboembolic pulmonary hypertension

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Fig.  16  Images obtained during angioscopy in chronic thromboembolic pulmonary hypertension (a–c) and normal bifurcation of pulmonary artery (d). Note fibrotic white material with membranes,

webs, and pitting, with marked irregularities, in contrast with the smooth symmetric and unobstructed bifurcation of the normal artery (d)

completely for better visualization of the vessel of interest. Although angioscopy can be accomplished from the femoral approach, it is much easier to navigate the instrument through the right ventricle using the internal jugular vein approach, particularly from the right. The angioscope can be flexed 90° at the tip, allowing navigation through the ­pulmonary tree. Two operators are required to guide the angioscope, with one advancing and applying torque to the instrument and the other flexing and extending it. Manipulating the angioscope is complicated by the need to maneuver through the frequently hypertrophied right ventricle, the wide variation in pulmonary vessel size with varying anatomic branching, the large number of branches that require visualization, and the robust collateral circulation from the bronchial circulation. During the procedure, the fluoroscopic images are recorded to localize the lesions seen on angioscopy and are correlated with the angiogram. Normal pulmonary findings are of smooth, pale white, glistening intima. Bifurcations are ­typically round and regular in appearance. In patients with PH, either IPAH or secondary to CTEPH, the arterial walls may demonstrate small, yellowish atherosclerotic-like plaques as well as more diffuse yellowish colorations (Fig. 16).

Patients with chronic thrombi have irregular pulmonary artery walls with transluminal and obstructive lesions and irregular vessel ostia. Thin membranes can be visualized. At  times, reddish-purplish subacute thrombi can be seen which are distinct from the white fibrotic lesions of chronic organized and recanalized clot. Angioscopy is most helpful in defining the starting point of chronic thrombi and establishing whether or not they are in surgical reach. A detailed map of the pulmonary vessels can be performed safely in approximately 20  min without significant morbidity in this patient population. The main complications encountered have been transient ventricular ectopy during transit in the ventricle and minor local bleeding from the 11-Fr sheath site in the neck.

7 Conclusion RHC and pulmonary angiography have been established as safe procedures, even in those patients with severe PH. Invasive pulmonary vascular assessment is mandatory to

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confirm the diagnosis of PAH and hemodynamic ­evaluation is mandatory prior to the initiation of vasodilator therapy and is useful to follow the response to therapy. Additionally, although technologies such as CT angiography show some promise, traditional pulmonary angiography remains the gold standard test in the diagnosis and assessment of surgical accessibility in patients with CTEPH.

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19. Mills SR, Jackson DC, Older RA, Heaston DK, Moore AV (1980) The incidence, etiologies, and avoidance of complications of ­pulmonary angiography in a large series. Radiology 136:295–299 20. Nicod P, Peterson K, Levine M et al (1987) Pulmonary angiography in severe chronic pulmonary hypertension. Ann Intern Med 107:565–568 21. Moser KM, Auger WR, Fedullo PF (1990) Chronic major-vessel thromboembolic pulmonary hypertension. Circulation 81:1735–1743 22. Guillinta P, Peterson KL, Ben-Yehuda O (2004) Cardiac catheterization techniques in pulmonary hypertension. Cardiol Clin 22:401–415 23. Hudson ER, Smith TP, McDermott VG et  al (1996) Pulmonary angiography performed with iopamidol: complications in 1,434 patients. Radiology 198:61–65 24. Hofmann LV, Lee DS, Gupta A et al (2004) Safety and hemodynamic effects of pulmonary angiography in patients with pulmonary hypertension: 10-year single-center experience. Am J Roentgenol 183:779–786 25. Hoeper MM, Lee SH, Voswinckel R et al (2006) Complications of right heart catheterization procedures in patients with pulmonary hypertension in experienced centers. J Am Coll Cardiol 48:2546–2552 26. McCullough PA (2008) Contrast-induced acute kidney injury. J Am Coll Cardiol 51:1419–1428 27. Kern MJ (1999) Hemodynamic rounds, 2nd edn. Wiley-Liss, New York 28. McGee DC, Gould MK (2003) Preventing complications of central venous catheterization. N Engl J Med 348:1123–1133 29. Bossert T, Gummert JF, Bittner HB et al (2006) Swan-Ganz catheter-induced severe complications in cardiac surgery: right ventricular perforation, knotting, and rupture of a pulmonary artery. J Card Surg 21:292–295 30. Simonneau G, Robbins IM, Beghetti M et al (2009) Updated clinical classification of pulmonary hypertension. J Am Coll Cardiol 54:43–54 31. Alpert JS (2001) The effect of right ventricular dysfunction on left ventricular form and function. Chest 119:1632–1633 32. Pirwitz MJ, Willard JE, Landau C, Hillis LD, Lange RA (1997) A critical reappraisal of the oximetric assessment of intracardiac leftto-right shunting in adults. Am Heart J 133:413–417 33. Anthi A, Machado RF, Jison ML et al (2007) Hemodynamic and functional assessment of patients with sickle cell disease and pulmonary hypertension. Am J Respir Crit Care Med 175:1272–1279 34. Machado RF, Mack AK, Martyr S et al (2007) Severity of pulmonary hypertension during vaso-occlusive pain crisis and exercise in patients with sickle cell disease. Br J Haematol 136:319–325 35. Gladwin MT, Vichinsky E (2008) Pulmonary complications of sickle cell disease. N Engl J Med 359:2254–2265 36. Golbin JM, Krowka MJ (2007) Portopulmonary hypertension. Clin Chest Med 28:203–218 37. Kim NH, Fesler P, Channick RN et al (2004) Preoperative partitioning of pulmonary vascular resistance correlates with early outcome after thromboendarterectomy for chronic thromboembolic pulmonary hypertension. Circulation 109:18–22 38. Fesler P, Pagnamenta A, Vachiéry JL et  al (2003) Single arterial occlusion to locate resistance in patients with pulmonary hypertension. Eur Respir J 21:31–36 39. Tolle JJ, Waxman AB, Van Horn TL, Pappagianopoulos PP, Systrom DM (2008) Exercise-induced pulmonary arterial hypertension. Circulation 118:2183–2189 40. Galiè N, Rubin L, Hoeper M et al (2008) Treatment of patients with mildly symptomatic pulmonary arterial hypertension with bosentan (EARLY study): a double-blind, randomised controlled trial. Lancet 371:2093–2100 41. Raeside DA, Chalmers G, Clelland J, Madhok R, Peacock AJ (1998) Pulmonary artery pressure variation in patients with connective tissue disease: 24 hour ambulatory pulmonary artery pressure monitoring. Thorax 53:857–862

1402 42. Raeside DA, Smith A, Brown A et  al (2000) Pulmonary artery ­pressure measurement during exercise testing in patients with suspected pulmonary hypertension. Eur Respir J 16:282–287 43. Rich S, Kaufmann E, Levy PS (1992) The effect of high doses of calcium-channel blockers on survival in primary pulmonary hypertension. N Engl J Med 327:76–81 44. Sitbon O, Humbert M, Jais X et al (2005) Long-term response to calcium channel blockers in idiopathic pulmonary arterial hypertension. Circulation 111:3105–3111 45. Skoro-Sajer N, Hack N, Sadushi-Kolici R et al (2009) Pulmonary vascular reactivity and prognosis in patients with chronic ­thromboembolic pulmonary hypertension: a pilot study. Circulation 119:298–305

C. Barnett and O. Ben-Yehuda 46. Klotz S, Wenzelburger F, Stypmann J et  al (2006) Reversible ­pulmonary hypertension in heart transplant candidates: to transplant or not to transplant. Ann Thorac Surg 82:1770–1773 47. Sitbon O, Humbert M, Jagot JL et al (1998) Inhaled nitric oxide as a screening agent for safely identifying responders to oral calciumchannel blockers in primary pulmonary hypertension. Eur Respir J 12:265–270 48. Peterson KL, Nicod P (1997) Cardiac catheterization: methods, diagnosis, and therapy. Saunders, Philadelphia 49. Auger WR, Fedullo PF, Moser KM, Buchbinder M, Peterson KL (1992) Chronic major-vessel thromboembolic pulmonary artery obstruction: appearance at angiography. Radiology 182: 393–398

Chapter 100

Imaging of Pulmonary Vascular Diseases Michael A. Bettmann

Abstract  Imaging is a crucial element in the diagnosis of pulmonary vascular disease, and it is used most often for two purposes: to diagnose or exclude pulmonary embolism (PE), and to make an evaluation for the presence and severity of cardiac dysfunction. For the latter, the main tool is the chest radiograph and the focus is the pulmonary arterial vascular pattern. For the former, there has been a dramatic progression over the last decade from the use of ventilation–perfusion lung scintigraphy combined with leg ultrasound and/or catheter pulmonary angiography, to the use of multislice CT pulmonary angiography (CTPA). The role of imaging for other pulmonary vascular abnormalities is limited. The aim of this chapter is to review the relevant imaging modalities and their utility in specific clinical conditions. Keywords  Diagnostic approach • Pulmonary embolism • Cardiac dysfunction • Chest radiograph • Ventilationperfusion lung scintigraphy • Pulmonary angiography

1 Introduction Imaging is a crucial element in the diagnosis of pulmonary vascular disease, and it is used most often for two purposes: to diagnose or exclude pulmonary embolism (PE), and to make an evaluation for the presence and severity of cardiac dysfunction. For the latter, the main tool is the chest radiograph and the focus is the pulmonary arterial vascular pattern. For the former, there has been a dramatic progression over the last decade from the use of ventilation–perfusion lung scintigraphy combined with leg ultrasound and/or catheter pulmonary angiography, to the use of multislice CT pulmonary angiography (CTPA). The role of imaging for other pulmonary vascular abnormalities is limited. The aim of this M.A. Bettmann (*) Department of Radiology, Medical Center Boulevard, Wake Forest University-NC Baptist Hospital, Winston-Salem, NC 27157, USA e-mail: [email protected]

chapter is to review the relevant imaging modalities and their utility in specific clinical conditions.

2 The Chest X-ray The standard posterior–anterior and lateral chest radiographs are widely used, generally cost effective, and safe, with a low radiation dose to the patient. They are readily available, and their interpretation, although extremely complex, is reasonably well codified. If the chest radiograph is of good quality, a large amount of information is reliably extractable. In contrast, portable chest X-rays are very widely used, but the amount of information they provide is very limited. The pulmonary vascular pattern and heart size cannot be reliably evaluated (short of pulmonary edema), and infiltrates, effusions, or pneumothoraces may not be visible. Portable studies should be reserved for evaluation of certain gross findings, such as the position of lines and tubes and the presence of large masses or infiltrates. One reason is the position of the patient (generally semierect or supine, so air will rise and fluid will be positioned posteriorly, and neither is likely to be seen), with the X-ray source in front and the recording device (either film or digital plate) behind. Also, the distance from the X-ray tube to the recording plate is far less than with the standard 6-ft posterior–anterior and lateral chest study, leading to worsened X-ray geometry and loss of spatial resolution, and therefore loss of accurate definition of, for example, heart size. Also, the power of the X-rays used with a portable machine is less, so the exposure is longer, leading to not only greater patient exposure but also to worsened temporal resolution. This leads to loss of definition of the margins of the pulmonary vessels. For vascular evaluation, then, portable chest radiographs have little value. Posterior–anterior and lateral chest radiographs are useful, when interpreted by trained observers, for evaluating the pulmonary arterial vascularity, as a reflection of both cardiac and pulmonary pathology and pathophysiology. In standing 6-ft posterior–anterior chest radiography, the pulmonary

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_100, © Springer Science+Business Media, LLC 2011

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arteries can be evaluated in three zones, from the hila to the pleura: a central perihilar zone including the main pulmonary arteries, a middle zone, and a peripheral zone. In the middle zone, the vessels (pulmonary arteries) are distinct with a clearly definable margin, and the lower (inferior) vessels are larger than upper lung ones. As left atrial pressure increases, the upper and lower lung vessels equalize in size and the vascular margins begin to lose clear margins. With further pressure elevation, vessels extend out closer to the pleura, the upper lung vessels in the middle zone enlarge still more relative to the vessels in the lower lung, and the margins blur further. Evidence of interstitial fluid may emerge, with small effusions, Kerley B lines, or evident thickening of the fissures. These findings are most predictable in patients with acute increases in left atrial pressure (as occurs with acute myocardial ischemia). In patients with increased pulmonary flow, such as those with a high-output state (pregnancy, anemia) or a left-to-right shunt, the flow pattern is different. Vessels tend to enlarge uniformly, with preserved margins, and are seen extending closer to the pleura. The main pulmonary arteries may also dilate. In primary pulmonary vascular disease, the findings are both less predictable and more limited. The classic findings of pulmonary emboli are focal hypoperfusion and a wedge-shaped pleural-based density (Hampton’s hump) (Fig. 1) caused by pulmonary infarct with accompanying consolidation. These findings are rare on chest radiographs, but not on CT. The major role of the chest radiograph in the setting of suspected pulmonary vascular disease, then, is to assess the vascular pattern as a reflection of cardiac (primarily left atrial and left ventricular) hemodynamics, and to rule out other disease. For example, a patient with dyspnea and chest pain may have pneumonia, a tumor, a hiatal hernia, or heart failure rather than pulmonary emboli. Also, if the patient is going to have lung scintigraphy, a chest radiograph is necessary for two reasons: first, as above, to exclude other cases that might eliminate the need for a lung scan, such as large, non-pleural-based infiltrates or a large effusion, and secondly, to determine if there is disease that would explain abnormal perfusion. That is, a chest X-ray is necessary for accurate interpretation of a lung scan.

3 Radionuclide Lung Scans Radionuclide studies played a central role in the diagnosis of PE from the 1960s through the 1990s. They have since been largely but not completely replaced by multislice CTPA. They still have a limited role, and debate about what this role precisely is continues. Radionuclide studies can still be used to rule out arteriovenous malformations and, rarely, to help localize perfusion abnormalities. Although they play a major

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Fig. 1  Pulmonary embolism (PE) plus additional parenchyma findings. Images at the same level, with different windows and leveling that demonstrates pleural-based infiltrate on the left (possible infarct, equivalent of Hampton’s hump as seen on a chest X-ray). Note additional small infiltrate in the right lower lobe, and different appearance of pulmonary markings in (a). (b) Small emboli can be seen peripherally

role, for example, in defining where and when a gastrointestinal bleed is occurring, they are less helpful in suspected hemoptysis, since the bleeding is usually lower in volume and intermittent. They are used mainly in particular settings for the diagnosis of PE. Pulmonary scintigraphy initially consisted of the intravenous injection of a radionuclide. The radionuclide (now 99m Tc, a g emitter with a relatively short half-life) is bound to a particle large enough to lodge in the pulmonary capillaries. Currently, the most widely used particle is macroaggregated albumin (MAA). When injected intravenously, the 99m Tc-MAA particles lodge in the pulmonary capillaries on first pass. The number of particles injected is small enough

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to avoid any deleterious effects from the long-term occlusion of a small percentage but large number of pulmonary capillaries, but is sufficiently large to allow imaging. With use of an externally placed recording device (a g camera), the emerging g particles can be reconstructed in multiple views to give an image of areas of the lung that are, or are not, perfused. The utility and accuracy of the method depend on a number of factors. First, any alterations in the lung that alter perfusion will be reflected in the images. Possible reasons include pulmonary emboli that block flow (i.e. occlusive thrombi), but also large bullae, tumors (since almost all have limited macrovascularity), and infiltrates. The perfusion pattern will also be altered by congestive heart failure, and even by positioning. That is, if the labeled MAA is injected intravenously into a patient who is sitting, flow will be slightly greater to the lower lungs than to the upper lungs, so the g emissions will be slightly greater inferiorly in the lungs. If the patient is supine during injection, there will be more counts posteriorly than anteriorly. In the presence of chronic heart failure, perfusion may be decreased in peripheral zones, and may be somewhat patchy. Since ventilation is usually not altered focally after a pulmonary embolus, ventilation scans have been used as an adjunct to perfusion lung scintigraphy and have been shown to increase the accuracy of the diagnosis of PE. A matched ventilation–perfusion defect suggests an explanation other than PE, whereas normal ventilation with no perfusion, particularly to an area that corresponds to one or more lung segments, reliably establishes the diagnosis of PE. There is an extensive literature concerning improved accuracy and utility of lung scintigraphy. Studies have evaluated the use of single photon emission CT as opposed to planar imaging and the use of various alternatives for ventilation scanning, including using alternative isotopes and means of administration, e.g. an injectable rather than an inhaled radiopharmaceutical agent for ventilation evaluation [1–4]. Many studies have also been performed to try to improve the accuracy of interpretation of lung scans. To this end, quantified interpretation systems have been developed and have been paired with various clinical risk factor rating criteria [5, 6]. The Wells criteria appear to be most widely used. With the combination of the Wells criteria, serum D-dimer assay, and chest X-ray, the accuracy of lung scintigraphy is widely regarded as sufficient to exclude or confirm the presence of PE [6–8]. There are very limited uses of lung scintigraphy currently other than for acute PE, and there are also significant limitations. First, PE can be established with a lung scan that meets certain criteria (generally, two or more segmental or lobar perfusion defects, with no matching ventilation defects and no focal abnormalities on the chest radiograph) and excluded if the scan is entirely normal or near normal (using PIOPED

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or other validated criteria) [9, 10]. Many scans, however, fall into the indeterminate category and are therefore ­nondiagnostic, even when put into the context of the validated Wells criteria. As a corollary, in the presence of significant abnormalities on the chest X-ray, a lung scan is very likely to be indeterminate (e.g. with matched ventilation– perfusion defects, a single subsegmental perfusion defect, or multiple subsegmental defects with and without ventilation defects). Second, other imaging modalities, most notably CT, are available and generally do not share these specific limitations. Third, compared with CT, lung scans require more time to perform, generally require greater expertise for high-quality interpretation, and depend more on the clinical setting for accuracy. Both CT and lung scintigraphy cause radiation exposure to the patient; the level depends on the specific protocols used for each. Finally, CT has the potential to define additional abnormalities and thus alternative explanations for the clinical concerns that lead to the use of an imaging study (Fig.  2) [11]. In fact, a “triple rule out,” to make an evaluation for PE, aortic dissection, and coronary artery disease, has been shown to be feasible, if not often necessary [11, 12].

4 CT Pulmonary Angiograms Pulmonary arterial CT studies have been available since the introduction of CT as an imaging modality in the 1970s. Accuracy was severely limited by the slow acquisition of images. This changed with the availability in the late 1990s of helical CT, and subsequently of multislice CT, both of which led to much faster imaging. This increased speed of imaging allows coverage of a larger area in less time (the entire thorax in a single breath hold, even in tachypneic patients), eliminating motion artifact and improving image resolution, and therefore accuracy. With careful timing of the contrast bolus, an automated function, CTPA is highly accurate (Figs. 1, 3) [13, 14]. As noted, CT also defines not only the pulmonary arteries from the main posterior–anterior to subsegmental vessels; it also allows evaluation of underlying lung disease. It is also possible to make an assessment for signs of right-sided heart strain (with septal bowing and increased right ventricle to left ventricle diameter) (Fig. 4). This provides the ability to further define PE by evaluating the hemodynamic effect [15]. The accuracy of conventional CT scanners that acquired images relatively slowly from single segments was limited. With conventional scanners, a single-array X-ray source rotated around the patient in a single plane, with the X-rays received at 180°, and then the X-ray source and the image plane moved and the process was repeated. With helical scanners, the gantry containing the source and the

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Fig.  2  Multiple large and small PE by CT pulmonary angiography (CTPA). Fifty-six-year-old Caucasian male with metastatic lung

cancer, bilateral pleural effusions, atelectasis, and bilateral pulmonary emboli (arrows). Note the large saddle embolus on the right

Fig.  3  Multiple, diffuse large and small PE by CTPA. Twenty-oneyear-old with hemodynamic instability with large bilateral pulmonary emboli (c). Emboli are fairly central, involve upper and lower lobe

v­ essels, and extend into small peripheral branches (a, d). Note the ­partially occlusive saddle embolus on the left in b

receiving device rotates around the patient continuously, so imaging is faster. With multichannel or multislice CT scanners, a longitudinal array of X-ray sources and receivers rotates around the patient, allowing a larger area of head-to-toe coverage with each rotation, combined with faster rotation. Thus, with the newer, larger field scanners (e.g. 64- or 128-slice scanners) a large area can be scanned (i.e. the entire chest in less than 5–8 s). Sixty-four slice (or channel) scanners are currently widely used, and ones with up to 320 channels are undergoing clinical trials. These multislice scanners cover the area of interest so quickly

and in such thin sections that there is no artifact from breathing, and even vessel wall and cardiac motion is minimized, thereby allowing superb temporal and spatial resolution. Resolution can be further improved by using software that allows cardiac and respiratory gating, either prospectively or retrospectively. Comparison studies of helical and four-slice CT scanners with catheter pulmonary angiography and even nuclear scintigraphy suggested that the accuracy of diagnosis of PE, particularly to segmental or smaller arteries, was limited [16]. Accuracy with 64-slice scanners, and even with 16-slice

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Fig. 4  Acute right-sided heart strain by CTPA. Two patients with PE by CT. (a) Fifty-three-year-old male with large left-sided PE, hemodynamically stable. (b) Twenty-one-year-old female, hemodynamically

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unstable s/p large bilateral PE. Note the dilated right ventricle (without septal bulging) in b

Fig. 5  Fifty-three-year-old male with large but not central left lung pulmonary emboli. Note that PE is occlusive of some branches but not others

scanners, however, has been shown to be very high. These new scanners have been shown to demonstrate small, peripheral pulmonary emboli more clearly and more frequently than earlier scanners (Fig.  5) [16, 17]. Studies have also shown that a negative CTPA study correlates with a very low incidence of thromboembolic events in subsequent months, confirming both a high negative predictive value and a high degree of accuracy, as well as very high interobserver variability agreement [18–20]. There are, however, several concerns with CTPA. First, there is significant radiation exposure, on the order of 10 mSv or more, depending on numerous factors, including the specific scanner, the protocol, and patient body habitus. By way of comparison, a chest radiograph usually results in a dose of 1 mSv or less and a ventilation–perfusion lung scan usually has a dose of 5–10 mSv. For any individual, a dose of 10 mSv is almost certainly not significant in terms of carcinogenesis. Over the last two decades, however, there has been a ­dramatic increase in the use of imaging using ionizing radiation (CT, coronary angiography, radionuclide studies), so the cumulative dose to patients from many examinations does start to create concerns. For example, patients commonly undergo serial chest CTs, often with studies of the abdomen and pelvis, as well as PET scans for evaluation and follow-up of

malignancy. Since carcinogenic effects of radiation (if they exist as is likely but not certain for diagnostic radiation) are thought to have a latency of several decades, the risk in elderly patients is almost certainly insignificant [21]. As longevity and utilization increase, younger patients may be at risk, and the possible effects become more of a concern. Another concern is that CT scanners are now so widely available, rapid, and reliable that they are being used increasingly, even in patients with low pretest probability [22–24]. In addition to increased risks of the possible sequelae of radiation, for the patient and the population, this increases costs to the system. Additionally, owing to the high sensitivity of multidetector CT (MDCT), it is likely that very small and peripheral emboli are being defined, ones that almost certainly could not be diagnosed in the past. Natural history studies in the pre-MDCT era suggested that the risk of venous thromboembolic disease was very low after negative pulmonary angiography. With the visualization of emboli that would not have been seen, it is likely that patients who do not actually require extended anticoagulation are being treated. This is appropriate, in the sense that the best available evidence suggests that the risk of not treating PE is high, but the evidence for treating very small PE is very limited. It is hard to develop a study that would ethically allow randomization

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to treatment with anticoagulation or with a placebo in patients with even very small PE. It is likely, then, that some patients who would not have been treated are now at greater risk of the significant complications of anticoagulation than of the sequelae of small pulmonary emboli. Further, there may be adverse effects related to the use of iodinated contrast for CTPA. The risk of significant reactions is real, although very small (fatalities occur in fewer than one in 100,000 patients) [25]. There is also the risk of renal damage, in the form of contrast-induced nephrotoxicity in patients who have underlying renal dysfunction, and there is concern for volume in patients with limited cardiac reserve. Although current iodinated contrast agents are among the safest of all pharmaceuticals, they should still only be used when the possible benefits to patients, through making or excluding specific diagnoses, clearly outweigh the risks.

5 Magnetic Resonance Angiography Magnetic resonance imaging, specifically magnetic resonance angiography (MRA), has been investigated for the diagnosis of PE and other pulmonary vascular abnormalities. It has the clear advantage of avoiding the use of ionizing radiation, but there are also significant limitations and concerns. Contrast agents (in small volumes of less than 20 ml) are usually used with MRA. Currently, all those in use for pulmonary MRA rely on gadolinium, a heavy metal that is tightly bound. Although there are no concerns about volume overload and the incidence of significant adverse events has been thought to be very low, there are other complications that are worrisome. It has been found that in patients with stage 3–5 chronic kidney disease; there is an association with nephrogenic systemic fibrosis, a scleroderma-like illness that can be fatal [26]. The association has been found with particular agents, and the incidence appears to increase with worsening renal function, so most reported cases are in patients on chronic renal replacement therapy. The risk seems to be markedly lessened, perhaps eliminated, if certain agents are avoided in patients with creatinine clearance less than 25 ml/min. Since magnetic resonance imaging uses a very powerful magnetic field, there are also real concerns with its use in patients with certain ferrous-metal-containing implants, such as pacemakers, some heart valves, and some older aneurysm clips, and with the proximity to the magnetic field of metal equipment, such as oxygen tanks or other intravenous poles. There are also concerns about the direct effect of the rapidly oscillating magnetic field and the radio-frequency pulses used, but in general it is thought that these factors do not impose a real risk for patients. These concerns may become more relevant if scanners using higher ­magnetic

M.A. Bettmann

fields, such as 7 T, rather than the usual 1.5 T, are more widely used clinically. The other major concern with MRA is that the imaging is relatively slow, leading to motion artifacts from breathing that may reduce the image resolution. This has been addressed with postprocessing of images to achieve retrospective ­gating, as well as with the use of more powerful (and thus potentially faster) magnets and with the use of improved pulse sequences. MRA is clearly able to define PE in some patients, particularly larger emboli in patients who are relatively stable with a slow respiratory rate [27–30]. Overall, however, studies suggest that MRA has a limited role, ­primarily in patients who are not good candidates for CT, either because of concerns about radiation (i.e. children) or because of concerns with iodinated contrast agents.

6 Pulmonary Angiography Pulmonary angiography has long been considered the gold standard for any evaluation of pulmonary vascularity, for PE or other abnormalities (Fig.  6). As all other imaging ­modalities, it has advantages and drawbacks. First, it is not 100% accurate [31, 32]. Second, it requires the use of iodinated contrast agents and radiation. Third, it is an invasive

Fig. 6  Very large, central PE by pulmonary angiography. The pulmonary angiogram demonstrates a large saddle embolus to the right ­middle and lower lobes, with additional embolic occlusion of most of the upper lobe

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procedure, and thus carries some risk. The risks in the hands of experienced operators are perhaps less than is generally perceived. Major morbidity is confined mainly to patients with hemodynamic compromise and mortality is rare [33], but clearly the risks are greater than with noninvasive modalities. These risks relate both to catheter manipulation and to the hemodynamic effects of contrast injection. On the other hand, there are clear advantages to pulmonary angiography: it has a very high degree of accuracy for the diagnosis of PE, it is generally safe, it can demonstrate abnormalities other than acute pulmonary emboli (Fig. 7), and it also can be used as a tool for treatment (Fig. 8). It is likely that CTPA is more accurate than conventional catheter pulmonary angiography, since cross-sectional imaging allows both visualization of more limited areas and reconstruction of images in various plains, thus eliminating confusion or misdiagnosis caused by overlapping vessels. Also, the temporal resolution of CTPA is superior. It is empirically clear that small pulmonary emboli that would not be likely to be seen with pulmonary angiography are frequently seen with CTPA (Figs. 1b, 3d). Accuracy is not complete with either modality, as various patient, technique, and interpreter considerations play a role, but recent studies suggest that CTPA is more accurate in general [18, 20]. CTPA also has the advantages of being less invasive, less operator-dependent, and more likely to delineate alternative explanations to pulmonary emboli. Pulmonary angiography at this time has three indications. First, it has a role in the rare cases in which the diagnosis of PE cannot be made by other imaging modalities. Second, it plays a major role in the treatment of massive or submassive,

symptomatic pulmonary emboli. In such cases, the options are surgical thrombectomy, systemic therapy with a ­thrombolytic agent, or catheter-directed therapy. Because of

Fig. 8  Extensive pulmonary emboli, with response to catheter-directed thrombolysis. Early (a) and late (b) arterial phases of the pulmonary arteriogram in an elderly male with multiple right middle and lower

lobe PE. (c) Repeat right pulmonary angiogram after 2 h of catheterdirected thrombolysis

Fig. 7  Abnormal pulmonary angiogram, secondary to invasive carcinoma. The pulmonary angiogram confirms invasive hilar carcinoma, with postobstructive atelectasis and partial occlusion of the central right upper lobe pulmonary artery

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the associated risks, surgery should be reserved for patients with free-floating thrombi in the right atrium or ventricle, or those in whom thrombolytic therapy is either contraindicated or ineffective. Systemic thrombolytic therapy has been shown to be effective, probably as effective as catheterdirected thrombolysis [34]. Catheter-directed therapy, however, has the advantage of allowing the use of lower doses of the lytic agent, and combination therapy with mechanical devices to remove clot. Third, pulmonary angiography has a role in defining (in association with MDCT) and treating pulmonary arteriovenous malformations. These are often associated with heritable syndromes, most notably hereditary hemorrhagic telangectasia, and can be associated with exercise intolerance or exertional dyspnea [35–37]. These lesions, when clinically suspected, are often but not invariably well defined with CTPA. Pulmonary angiography is also occasionally used to define chronic pulmonary emboli (Fig.  9), particularly prior to surgical thrombectomy. With the improvements in CT, however, definition of the pulmonary vascularity is often both more accurate and more useful for surgical panning with CT than with catheter angiography. Pulmonary angiography, as lung scans, remains a well-established and reliable technique with clear limitations and certain specific roles.

7 Summary and Conclusion

Fig. 9  Pulmonary angiography of chronic pulmonary emboli. Elderly, dyspneic male. Early (a) and late (b) phase of the left main pulmonary arteriogram (in different obliquities). No acute PE is seen. Note pruning

of vessels and very limited peripheral (lung parenchymal) flow. No ­evidence of acute pulmonary emboli

The pulmonary vasculature is imaged for the evaluation of cardiac function, for evaluation and treatment of chronic, symptomatic abnormalities, primarily arteriovenous malformations and chronic pulmonary emboli, and to rule out or confirm acute pulmonary emboli. To evaluate ­hemodynamics, the chest X-ray is the first tool, and a very valuable one. Although portable chest X-rays have very limited utility beyond define lines, tubes, and very gross abnormalities, standard posterior–anterior and lateral chest films provide a large amount of information, not only concerning acute cardiac and pulmonary abnormalities, but also for tracking changes (e.g. in left ventricular end diastolic pressure and in heart size) over time. For evaluation of chronic vascular abnormalities, both CTPA and catheter pulmonary angiography are useful and reliable. For the diagnosis of PE, radionuclide scans were the central tool until the development over the last decade of multislice CT. Radionuclide scans still have a role when CT is unavailable, contraindicated, or rarely, not definitive. CTPA, however, allows very rapid, accurate, and reproducible evaluation of not only the pulmonary arterial tree but also of the heart, aorta, and lungs; while excluding or confirming PE, it may additionally define other causes for the presenting symptoms or signs. MRA has the

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advantage of not requiring ionizing radiation, but it is currently limited by both required expertise and relatively slow image acquisition. It may play a larger role in the future, particularly if imaging becomes faster and evaluation of tissue perfusion becomes clinically important. Pulmonary angiography, formerly recognized as the gold standard for the diagnosis of PE, is now used almost exclusively for treatment. It is used to allow access for treatment of symptomatic arteriovenous fistulae and for treatment of symptomatic massive or submassive pulmonary emboli. All four modalities have advantages and disadvantages, so they must be used carefully, in association with appropriate clinical questions. CT has become accepted as the gold standard in the diagnosis of pulmonary emboli. For this reason, as well as its wide availability and relative ease of interpretation, its use has grown dramatically over the last decade, with a commensurate increase in the diagnosis of pulmonary emboli. It is likely that very small emboli that were not usually diagnosed previously are now being seen, and treated. It is important to carefully consider the risk–benefit ratio in the use of any imaging modality, but particularly CT. The benefits of accurate, rapid diagnosis and possible definition of additional abnormalities must be weighed against the cost of the CT scan, radiation exposure, and the risks of treatment.

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1411 8. Hagen PJ, Hartmann IJ, Hoekstra OS, Stokkel MP, Postmus PE, Prins MH, ANTELOPE Study Group (2003) Comparison of observer variability and accuracy of different criteria for lung scan interpretation. J Nucl Med 44(5):739–744 9. Sostman HD, Stein PD, Gottschalk A, Matta F, Hull R, Goodman L (2008) Acute pulmonary embolism: sensitivity and specificity of ventilation-perfusion scintigraphy in PIOPED II study. Radiology 246(3):941–946 10. Pontana F, Faivre JB, Remy-Jardin M, Flohr T, Schmidt B, Tacelli N, Pansini V, Remy J (2008) Lung perfusion with dual-energy multidetector-row CT (MDCT): feasibility for the evaluation of acute pulmonary embolism in 117 consecutive patients. Acad Radiol 15(12):1494–1504 11. Schertler T, Frauenfelder T, Stolzmann P, Scheffel H, Desbiolles L, Marincek B, Kaplan V, Kucher N, Alkadhi H (2009) Triple rule-out CT in patients with suspicion of acute pulmonary embolism: findings and accuracy. Acad Radiol 16(6):708–717 12. Halpern EJ (2009) Triple-rule-out CT angiography for evaluation of acute chest pain and possible acute coronary syndrome. Radiology 252(2):332–345 13. Perrier A, Roy PM, Sanchez O, Le Gal G, Meyer G, Gourdier AL, Furber A, Revel MP, Howarth N, Davido A, Bounameaux H (2005) Multidetector-row computed tomography in suspected pulmonary embolism. N Engl J Med 352(17):1760–1768 14. Stein PD, Fowler SE, Goodman LR, Gottschalk A, Hales CA, Hull RD, Leeper KV Jr, Popovich J Jr, Quinn DA, Sos TA, Sostman HD, Tapson VF, Wakefield TW, Weg JG, Woodard PK, PIOPED II Investigators (2006) Multidetector computed tomography for acute pulmonary embolism. N Engl J Med 354(22):2317–2327 15. Nural MS, Elmali M, Findik S, Yapici O, Uzun O, Sunter AT, Erkan L (2009) Computed tomographic pulmonary angiography in the assessment of severity of acute pulmonary embolism and right ventricular dysfunction. Acta Radiol 50(6):629–637 16. Van Strijen MJ, De Monye W, Kieft GJ, Pattynama PM, Prins MH, Huisman MV (2005) Accuracy of single-detector spiral CT in the diagnosis of pulmonary embolism: a prospective multicenter cohort study of consecutive patients with abnormal perfusion scintigraphy. J Thromb Haemost 3(1):17–25 17. Stein PD, Kayali F, Hull RD (2007) Spiral computed tomography for the diagnosis of acute pulmonary embolism. Thromb Haemost 98(4):713–720 18. Subramaniam RM, Chou T, Swarbrick M, Karalus N (2006) Pulmonary embolism: accuracy and safety of a negative CT pulmonary angiogram and value of a negative D-dimer assay to exclude CT pulmonary angiogram-detectable pulmonary embolism. Australas Radiol 50(5):424–428 19. Subramaniam RM, Blair D, Gilbert K, Coltman G, Sleigh J, Karalus N (2007) Withholding anticoagulation after a negative computed tomography pulmonary angiogram as a stand-alone imaging investigation: a prospective management study. Intern Med J 37(9):624–630 20. Mos IC, Klok FA, Kroft LJ, DE Roos A, Dekkers OM, Huisman MV (2009) Safety of ruling out acute pulmonary embolism by normal computed tomography pulmonary angiography in patients with an indication for computed tomography: systematic review and meta-analysis. J Thromb Haemost 7(9):1491–1498 21. Fazel R, Krumholz H, Wang Y, Ross JS, Chen J, Ting HH, Shah ND, Nasir K, Einstein AJ, Nallamothu BK (2009) Exposure to lowdose ionizing radiation from medical imaging procedures. N Engl Med J 361:849–857 22. DeMonaco NA, Dang Q, Kapoor WN, Ragni MV (2008) Pulmonary embolism incidence is increasing with use of spiral computed tomography. Am J Med 121(7):611–617 23. Donohoo JH, Mayo-Smith WW, Pezzullo JA, Egglin TK (2008) Utilization patterns and diagnostic yield of 3421 consecutive multidetector row computed tomography pulmonary angiograms in a busy emergency department. J Comput Assist Tomogr 32(3):421–425

1412 24. Siegel A, Holtzman SR, Bettmann MA, Black WC (2004) Clinicians’ perceptions of the value of ventilation-perfusion scans. Clin Nucl Med 29(7):419–425 25. Bettmann MA (2004) Frequently asked questions: iodinated contrast agents. RadioGraphics 24(Suppl 1):S3–S10 26. Sadowski EA, Bennett LK, Chan MR, Wentland AL, Garrett AL, Garrett RW, Djamali A (2007) Nephrogenic systemic fibrosis: risk factors and incidence estimation. Radiology 243(1): 148–157 27. Ley S (2008) Kauczor HU MR imaging/magnetic resonance angiography of the pulmonary arteries and pulmonary thromboembolic disease. Magn Reson Imaging Clin N Am 16(2): 263–273 28. Blum A, Bellou A, Guillemin F, Douek P, Laprévote-Heully MC, Wahl D, GENEPI Study Group (2005) Performance of magnetic resonance angiography in suspected acute pulmonary embolism. Thromb Haemost 93(3):503–511 29. Altes TA, Mai VM, Munger TM, Brookeman JR, Hagspiel KD (2005) Pulmonary embolism: comprehensive evaluation with MR ventilation and perfusion scanning with hyperpolarized helium-3, arterial spin tagging, and contrast-enhanced MRA. J Vasc Interv Radiol 16(7):999–1005 30. Stein PD, Woodard PK, Hull RD, Kayali F, Weg JG, Olson RE, Fowler SE (2003) Gadolinium-enhanced magnetic resonance angiography for detection of acute pulmonary embolism: an indepth review. Chest 124(6):2324–2328

M.A. Bettmann 31. Wittram C, Waltman AC, Shepard JA, Halpern E, Goodman LR (2007) Discordance between CT and angiography in the PIOPED II study. Radiology 244(3):883–889 32. Henry JW, Relyea B, Stein PD (1995) Continuing risk of thromboemboli among patients with normal pulmonary angiograms. Chest 107(5):1375–1378 33. Stein PD, Athanasoulis C, Alavi A, Greenspan RH, Hales CA, Saltzman HA, Vreim CE, Terrin ML, Weg JG (1992) Complications and validity of pulmonary angiography in acute pulmonary embolism. Circulation 85(2):462–468 34. Konstantinides S (2008) Acute Pumonary Embolism. N Engl J Med 359:2804–2813 35. Coulier B (2003) Detection of pulmonary arteriovenous malformations by multislice spiral CT angiography. JBR-BTR 86(1):28 36. Lacombe P, Lagrange C, Beauchet A, El Hajjam M, Chinet T, Pelage JP (2009) Diffuse pulmonary arteriovenous malformations in hereditary hemorrhagic telangiectasia: long-term results of embolization according to the extent of lung involvement. Chest 135(4):1031–1037 37. Faughnan ME, Palda VA, Garcia-Tsao G, Geisthoff UW, McDonald J, Proctor DD, Spears J, Brown DH, Buscarini E, Chesnutt MS, Cottin V, Ganguly A, Gossage JR, Guttmacher AE, Hyland RH, Kennedy SJ, Korzenik J, Mager JJ, Ozanne AP, Piccirillo JF, Picus D, Plauchu H, Porteous ME, Pyeritz RE, Ross DA, Sabba C, Swanson K, Terry P, Wallace MC, Westermann CJ, White RI, Young LH, Zarrabeitia R (2009) International guidelines for the diagnosis and management of hereditary hemorrhagic telangiectasia. J Med Genet

Chapter 101

Histological and Pathological Diagnosis of Pulmonary Hypertension: Pathological Classification of Pulmonary Vascular Lesions Brian B. Graham, Li Zhang, and Rubin M. Tuder

Abstract  Pulmonary pathology plays a central role in the diagnosis and management of patients with lung diseases, including pulmonary vascular diseases. Methods of obtaining tissue include transbronchial, computed-tomography-guided transthoracic, and surgical (either by open or by video-assisted thoracoscopic techniques) biopsies. Accurate histopathological interpretation of lung tissue requires understanding the foundation of fundamental cellular processes, including inflammation and repair, neoplasia, and vascular alterations. The underlying complexity of normal gross and microscopic anatomy of the lung imposes significant challenges for the analysis of histopathological alterations due to disease processes. The normal lung contains at least 30 different cells, and lung tissue can appear significantly different depending on the degree of alveolar inflation. Tissue obtained by transbronchial and transthoracic biopsy is subject to crush artifact and limited tissue sampling. Such challenges can be overcome only with experience and insight into the specific clinical data pertinent to a given case. Keywords  Histology • Pathology • Pulmonary vascular remodeling • Vascular lesion • Inflammation • Structural alteration

1 Introduction Pulmonary pathology plays a central role in the diagnosis and management of patients with lung diseases, including pulmonary vascular diseases. Methods of obtaining tissue include transbronchial, computed-tomography-guided transthoracic, and surgical (either by open or by video-assisted thoracoscopic techniques) biopsies. Accurate histopathological interpretation of lung tissue requires understanding the foundation of B.B. Graham (*) Division of Pulmonary Sciences and Critical Care Medicine, Department of Medicine, Program in Translational Lung Research, University of Colorado Denver, School of Medicine, 12700 E 19th Ave, Campus Box C-272, Denver, CO 80045, USA e-mail: [email protected]

fundamental cellular processes, including inflammation and repair, neoplasia, and vascular alterations. The underlying complexity of normal gross and microscopic anatomy of the lung imposes significant challenges for the analysis of histopathological alterations due to disease processes. The normal lung contains at least 30 different cells, and lung tissue can appear significantly different depending on the degree of alveolar inflation. Tissue obtained by transbronchial and transthoracic biopsy is subject to crush artifact and limited tissue sampling. Such challenges can be overcome only with experience and insight into the specific clinical data pertinent to a given case.

2 Preparation of Specimens Lung tissue requires some degree of inflation, ideally reproducing the alveolar structure at tidal volumes. Inflation can be at least partially achieved by instilling formalin or another preservative solution. Whole lungs should be inflated with a 10% buffered formalin solution under 25-cmH2O pressure, with each lung requiring approximately 2–4  L to achieve inflation (total lung capacity). Lung biopsies can be partially inflated using 10% formalin injected with a syringe and a 15-gauge needle, or by gently tapping the tissue with a stick when it is submerged in formalin to expel air and allow ­formalin absorption. Transbronchial or transthoracic biopsies can be partially inflated by tapping each fragment against the wall of a vial containing formalin, which will expand the periphery of the fragment. Formalin is a disinfectant and causes immediate fixation by cross-linking protein amino groups, preserving the molecular and morphological details. Most immunohistochemical stains work well with formalin preservation. Formalin penetrates tissue at a rate of about 1 mm per hour, up to a maximum thickness of about 4 mm, so sections thicker than 4 mm will not be properly fixed. The volume of formalin used should be about 10 times that of the tissue volume. To perform frozen sectioning, alternative materials are used, generally with a 50% solution of optimal cutting temperature or a buffered solution containing 0.5% agarose instilled at approximately 40–60°C.

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_101, © Springer Science+Business Media, LLC 2011

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After being fixed, the fragments are processed through gradients of alcohol and then sequential immersions in xylene, leading to dehydration of the tissue and removal of hydrophobic molecules such as fat and fatty acids. The fragments are then embedded into molds of molten paraffin, allowing the samples to be sectioned on a microtome, placed onto histological slides, and stained. Most histopathological interpretations are carried out using hematoxylin (a basic stain that identifies acidic substances, principally nucleic acids) and eosin (an acidic stain that identifies basic structures, such as cytoplasmic proteins). The use of special stains such as Masson’s trichrome stain to identify connective tissue is discussed in the specific context herein.

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features found in pulmonary disease. Representative images of normal pulmonary vascular histologic findings are shown in Fig. 1.

3.1 Pulmonary Arteries and Precapillary Vessels

The components of the normal pulmonary vascular system have been described extensively elsewhere [1], but it is important to briefly review them before discussing the pathologic

The diameter of the pulmonary trunk is slightly less than that of the aorta, but the pulmonary trunk wall is only about half as thick as the aorta wall, reflective of the lower pulmonary artery pressures. The large pulmonary arteries have extensive elastic membranes, which on histology appear irregular and interrupted, as opposed to the aorta, which has contiguous elastic laminae. As the pulmonary arterial vasculature and airways divide, the pulmonary arteries follow the bronchioles, and continue to have elastic laminae in the media until reaching a diameter of approximately 1 mm. From 1,000 to 500 mm, the pulmonary arteries transition to muscular pulmonary arteries. Prior to connecting into a rich network of precapillary vessels and capillaries, pulmonary arteries have progressive

Fig. 1  Normal pulmonary vascular histologic findings. Structures are indicated with arrows. (a) Elastic artery. (b) Muscular artery. (c) Muscular artery (arrow) transitioning to a precapillary vessel (arrowhead). (d) Capillaries

(arrows) and precapillary vessel (star). The wall of the precapillary vessel contains endothelial cells and connective tissue, but no smooth muscle cells. (e) A thin-walled pulmonary vein within the interlobular septae

3 Components of the Normal Pulmonary Vascular System

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disappearance of the medial elastic laminae and muscle cells, with the persistence of only thin internal and external elastic laminae. In medium-sized arteries the media is replaced by smooth muscle cells (SMCs) oriented circumferentially. These pulmonary arteries continue to branch ­continuously down to a ­diameter of about 70 mm. Below 70 mm they lose the smooth muscle component of the media entirely, are surrounded by pericytes, and transition into capillaries. The normal pulmonary arterial endothelial lining is only one cell thick in all vessel segments. The media-to-diameter ratio remains constant at about 0.10–0.15, independent of the absolute arterial diameter, with occasional wall thickness up to 0.20 or 0.25 [2]. The pulmonary adventitia is primarily composed of fibroblasts, and the normal adventitia-to-diameter ratio is approximately 0.15 in pulmonary arteries and precapillaries.

3.2 Pulmonary Capillaries Pulmonary capillaries are about 8 mm in diameter, comparable to the diameter of an erythrocyte but marginally smaller than a leukocyte (10-mm diameter), which needs to deform as it migrates. Capillaries are 500–1,000-mm long and serve ­multiple alveoli on their passage from a precapillary to a venule. The  thickness of the capillary and alveolar wall membrane together is about 1.7 mm, with five layers of structure: a layer of endothelial cells (ECs) with an intracellular junction, the endothelial basement membrane, a thin interstitial space with reticulin and occasional collagen fibers, the epithelial basement membrane, and a layer of alveolar epithelial cells [1].

3.3 Pulmonary Venules and Veins The pulmonary capillaries gather in collecting venules, which then merge to form pulmonary venules. The wall of a pulmonary venule is a single layer of endothelium overlying a basement membrane, with occasional SMCs outside the basement membrane, and overall is similar in appearance to precapillary vessels. As the venules coalesce, at 60–100-mm diameter they enter the fibrous interlobular septa, and can be distinguished from pulmonary precapillaries by their location. As the pulmonary venules enlarge, they appear different from precapillary vessels as the venous walls are composed of thin elastic lamina, with scattered smooth muscle fibers and collagen, and no clear distinction between the media and adventitia. The pulmonary vein endothelium remains a single layer of cells overlying the internal elastic lamina. Pulmonary veins do not have valves. Identification of the pulmonary vein structure requires three-dimensional reconstruction and determination of connectivity with interseptal veins.

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3.4 Correlation Between Pathology and Physiology The pulmonary arterial circulatory system is predicted to have approximately 100 million segments distributed along 17 orders of branching prior to transitioning into the alveolar capillaries. Elastic arteries are distributed along orders 1–8 and consist of approximately 3,000 segments. Muscular arteries represent orders 9–13 and include about 800,000 segments. Precapillary vessels are the 14th to 17th orders of branching and account for 70 million vascular segments [3, 4]. In healthy individuals, cardiac output can increase fourfold to fivefold before pulmonary artery pressure rises, and thus it is anticipated for pulmonary hypertension (PH) to develop, the resistance to flow needs to increase at least fourfold to fivefold, or equivalently at least 80% of the pulmonary circulation has to be compromised. However, there remains limited understanding of the distribution and extent of involvement of focal pulmonary vascular lesions throughout the lung in PH. Pulmonary arterial hypertension (PAH), for example, may represent a disease involving multiple ­levels of the pulmonary arterial circulation, which in summation­ ultimately accounts for the necessary decrease in cross-sectional area and increase in resistance. However, PAH may also be principally due to lesions at a single level, such as the muscular arterial or precapillary level. The effect on resistance to flow (which goes as the inverse to the fourth power of the diameter) resulting from a small change in vessel diameter is much more pronounced in smaller vessels, but many more individual lesions would be required to affect the necessary 80% of vessels at that level. At the muscular arterial level alone it would require about 640,000 distinct lesions for PH to develop, and they would occur predominantly at the periphery of the lung. This prediction is consistent with observations from angiography and vascular casting showing decreased perfusion at the lung periphery in patients with idiopathic PHA (IPAH), but the same distribution may not hold for other types of PAH. Furthermore, small studies suggest most lesions occur at branch points [5, 6].

4 Pathological Classification System for Pulmonary Vascular Disease The pathologic interpretation of pulmonary vascular remodeling morphologies in the context of the natural history and pathophysiology of different types of PH continues to pose significant challenges, and consequently at this time lung biopsy provides limited usefulness in the workup and evaluation of the disease. However, identifiable pulmonary disease is present in all forms of PH, including arterial, venous, and other forms of PH.

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PAH specifically is clinically defined by hemodynamic criteria (i.e. a mean pulmonary artery pressure greater than 25 mmHg), and is classified by the World Health Organization (WHO) criteria using specific clinical characteristics or underlying medical conditions associated with the disease, such as collagen vascular disease, HIV infection, anorexigen toxicity, and schistosomiasis. The underlying rationale for this classification relies on the assumption that the multiple forms of PAH share clinical and/or pathological presentations, and thus justifies a common clinical approach for diagnosis and/or treatment. Pathologic interpretation at this time is limited to identifying the underlying or associated lung diseases that give rise to PAH rather than identifying specific disease characteristics, which, for example, independently predict prognosis or response to specific treatments. Although pulmonary disease may be present in the context of pulmonary venous hypertension, usually such analysis plays little role in making the diagnosis. However, pulmonary pathology can be critical in making the diagnosis of rarer causes of PH, such as pulmonary capillary hemangiomatosis (PCH). This section reviews the range of pathologic features that can be seen in each compartment of the pulmonary system in the context of PH, to create a framework for the identification­ of pathologic features specific for different types of PH. The following section will discuss the pathologic appearance of specific types of PH.

4.1 Pulmonary Arterial and Precapillary Vessel Pathology All three luminal components of pulmonary arteries (intima, media, and adventitia) can develop lesions which are likely significant in the pathogenesis of PAH, and are also frequently seen in other causes of PH, including chronic hypoxia and chronic thromboembolic disease. Intimal lesions account for most of the reduction in the luminal area of the small pulmonary arteries and most likely contribute to most of the increases in pulmonary vascular resistance. However, medial and adventitial lesions may also play significant roles in the PAH disease process. Examples of pathologic arterial and precapillary lesions are given in Fig. 2. 4.1.1 Arterial and Precapillary Intimal Lesions Intimal lesions can be histologically classified into four categories: eccentric intima thickening and fibrosis, concentric intima thickening, plexiform lesions, and dilated or angiomatoid lesions [7]. Focal eccentric lesions can be detected in normal lungs, but in PAH the lesions are more widespread and impact to a larger extent on the vascular luminal area. Some of these

Fig. 2  Pulmonary arterial disease histology. Structures are indicated with arrows. (a) Eccentric intimal thickening. (b) Concentric intimal thickening, with “onion-skin” appearance. (c) Significant decrease in luminal area due to intimal thickening with a concentric arrangement. (d) Plexiform lesion. (e) Dilated lesions (arrows), adjacent to a plexiform lesion (star) and concentric intimal thickening (arrowhead). (f) Precapillary vessel muscularization. (g) Medial hyperplasia with intimal thickening. (h) Adventitial thickening (arrowheads) overlying medial and intimal thickening (arrow)

lesions may result from organization after localized thrombi (i.e. lysis of fibrin, recanalization by newly formed blood vessels, and proliferation of myofibroblasts, discussed further later), which form the nidus for a secondary localized growth of SMCs. More advanced lesions acquire a fibrotic

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pattern, with myofibroblasts and accumulation of mucopolysaccharides, and such fibrotic-like lesions are widely present in explanted lungs of patients with severe PAH [8]. The organization of myofibroblasts or ECs in “onionskin” layers characterizes concentric lesions [7]. These lesions are usually seen in severe PAH, and result in a significant reduction of the luminal vascular area. Concentric lesions typically are seen in vascular segments proximal to plexiform lesions, suggesting concentric lesions arise from remodeled plexiform lesions [5]. Severe concentric lesions may have near complete occlusion of small arteries by the accumulation of an acellular matrix, suggesting a terminal scarring process, resulting in fibrosis of the lesions [7]. Collagen can be easily visualized by Masson’s trichrome stain or picrosirius red stain. Plexiform lesions typically occur at branch points of ­muscular arteries [5], and are a network of vascular channels lined by ECs [9], around a core of myofibroblastic or less well-differentiated cells [10]. Immunohistochemistry for both EC markers (such as CD31) and cell proliferation (such as proliferating cell nuclear antigen) readily reveals the lesions, as the cells are of EC lineage but multiplying much faster than normal ECs [11]. Dilated or angiomatoid lesions are characterized by the formation of a rosary of dilated channels, often distal to a plexiform lesion. These lesions also occur in patients with severe PAH [12]. Dilated lesions contain ECs and abundant collagen type 4 expression, more commonly a component of basement membranes. 4.1.2 Arterial and Precapillary Medial Lesions Medial SMC hypertrophy is a characteristic pathologic feature of many forms of PH that involves muscularized arteries (ranging between 70 and 500 mm in diameter) and precapillary vessels (less than 70 mm in diameter). The medial SMC layer represents approximately 10–15% of the normal outside diameter of muscularized pulmonary arteries, whereas it approaches 30–60% of the diameter in vessels of IPAH lungs [13–15]. More mild medial thickening can occur in the lungs of smokers who do not have PH [16]. Smooth muscle in precapillary media also becomes prominent, and these usually have only thin medial elastic laminae; this finding is termed “muscularization of precapillary vessels.” 4.1.3 Arterial and Precapillary Adventitial Lesions The adventitia is primarily composed of fibroblasts. Growing evidence shows that, rather than being just a structural support to pulmonary vessels, the adventitia may also play a role in regulating pulmonary vascular function [17]. The normal adventitia represents approximately 15% of the external diameter of pulmonary arteries larger than 50 mm in diameter,

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but in IPAH arteries the adventitial thickness increases to 25–30% of arterial diameter, predominantly due to collagen deposition [13]. It is unclear if the increased thickness is due to adventitial cell remodeling or the deposition of stromal cells. In PH, inflammatory cells are often seen around the adventitia, and likely play a role in the vasoproliferative and vasoconstrictive response.

4.2 Pulmonary Capillary Pathology Pulmonary capillaries are generally spared in PH, but the one disease associated with significant capillary disease is PCH (WHO classification 5.2). PCH is described further later, but the primary pathologic finding is a heterogeneous distribution of numerous thin-walled capillary-sized vascular structures occupying a thickened interstitium [18].

4.3 Pulmonary Venous Pathology The pulmonary veins generally do not develop lesions in PAH, but venous disease does occur in the setting of pulmonary venous hypertension. The disease seen in pulmonary venous hypertension is venous medial hypertrophy, due to both muscularization and fibrosis, and is called “arteriolization­ of the veins and venules” [1]. Two layers of elastic laminae appear, similar to the internal and external laminae of muscular pulmonary arteries. Intimal fibrosis of the veins and venules may develop, similar to eccentric or concentric arterial intimal lesions, and collections of tortuous pulmonary veins, which may parallel the intimal plexiform lesions of PAH, have also been described [19]. Examples of pulmonary venous lesions are given in Fig. 3a and b. Pulmonary veno-occlusive disease (VOD) has a specific pathologic appearance: narrowing or complete occlusion of the lumen of smaller veins and venules from thickening of the venous intima with a fibrous and acellular tissue, but containing little collagen [1].

4.4 Inflammation Leukocytes and macrophages are important effector cells in lung antimicrobial defense, but are commonly seen in the context of PH of multiple causes, and likely play an important but still unclear role in lung vascular inflammatory and remodeling processes [20]. Pulmonary arteries of patients with severe PAH have B cells, T cells, and macrophages infiltrating the vessel wall, particularly within intimal lesions [9]. Many of the T cells express CD45RO, suggesting cell activation [7]. Some of these inflammatory cells may be

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Fig. 3  Pulmonary venous, vascular inflammatory, and thrombotic diseases. (a, b) Pulmonary venous thickening. (c) Perivascular inflammation. (d) Recent thrombus. (e) Chronic and recanalized arterial thrombus. (f) Chronic and recanalized venous thrombus

derived from circulating blood progenitor cells. In chronic obstructive pulmonary disease (COPD), vascular progenitor cells identified by CD133 and CD45 markers have been found to infiltrate the remodeled intima [21] and then transdifferentiate into ECs or SMCs [22]. Other infiltrating cells can include fibrocytes, which express the mononuclear cell markers CD45, CD11b, and CD14, as well as a1-procollagen [23]. Fibrocytes can locally proliferate as well as express smooth muscle actin, suggesting transdifferentiation to SMCs. The role of inflammation in the pathogenesis of PH is further demonstrated by the depletion of monocyte precursors preventing pulmonary vascular remodeling in rats exposed to chronic hypoxia [23]. A histologic example of pulmonary vascular inflammation is given in Fig. 3c.

4.5 Thromboembolic Disease Thromboembolic disease is the primary cause of PH in the diseases of WHO category IV, but thrombosis is often seen in IPAH and other forms of PH, and hence the rationale for the prophylactic anticoagulation of patients with IPAH [24].

The pathologic distinction must first be made between thrombi which are present acutely, chronically, or after the pathologic specimen was acquired (after death, in the case of autopsy examination). Thrombus, which forms after the specimen has been acquired, also called clot, can ­generally be distinguished on gross and microscopic ­examination by layering of the blood components into two zones by the effect of gravity: a lower, dense region with many red blood cells, and an overlying, coagulated clear plasma. Within the red blood cell coagulum, the clot has a uniform appearance lacking the characteristic lines of Zahn, which occur in thrombus that has formed more slowly in ­circulating blood. Acute thromboembolic disease (such as occurring in massive pulmonary embolism) is pathologically characte­ rized by a previously formed thrombus which appears lodged inside the blood vessels, rather than integrated into the blood vessel wall, and could easily be manually extracted from the gross specimen. These thrombi often fill the large arteries in addition to fragmenting into more ­distant small arteries and precapillary vessels. Depending on the ­duration of time between the embolic event and the pathologic examination, the pulmonary parenchyma ­supplied by the obstructed vasculature may become ­hemorrhagic or necrotic.

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Chronic thromboembolic disease affects principally smaller elastic and muscular arteries, and to a lesser extent precapillaries. A single specimen from a patient with chronic thromboembolic disease may have thrombi that appear to have been present for a range of durations, from acute to very old, as the disease often involves recurrent events over a long period of time. As time progresses after an acute embolus, the thrombotic lesions transition to become an eccentric fibrotic mass that narrows or even obstructs the vessel lumen [1]. The early host response appears as a polymorphonuclear cell infiltration into the thrombus, followed by fibroblasts, with concurrent endothelialization of the exposed surface of the thrombus. The fibroblasts subsequently deposit collagen and elastic fibers, resulting in a relatively homogenous eccentric ­intimal fibrosis. Such fibrosis can be clearly seen with trichrome or picrosirius red staining. Complete occlusions may subsequently recanalize, often with multiple ­endothelialized lumens within the original structure of a single vessel. The overlying media usually remains intact, but thinning and destruction of the internal elastic laminae may occur. Veins may also show evidence of recanalization with multiple lumens, suggestive of prior and chronic pulmonary venous thrombosis. Infected thrombi that embolize cause a localized pulmonary arteritis around the thrombus, with polymorphonuclear cell invasion of the thrombus and all layers of the surrounding­ pulmonary artery. Nonthrombotic emboli also occur, and can also cause PH, although such material usually embolizes in a single acute event rather than in chronic recurrent events. Other material that may embolize includes endogenous biologic material such as adipose tissue, amniotic fluid, and clusters of tumor cells. Foreign matter can embolize acutely or chronically depending on the pattern of the disease course, such as recurrent talcum powder embolization from intravenous drug abuse, and parasitic infections with chronic helminth and/or ova embolization. The pulmonary pathology from these cases can often determine the specific biologic or foreign material that is present. Examination with polarized light will reveal foreign material that causes rotation of the light waves, such as talcum powder. Histologic examples of pulmonary arterial and venous thromboembolic lesions are shown in Fig. 3d and f.

4.6 Pulmonary Parenchyma Pathology Careful examination of the surrounding pulmonary parenchyma can provide clues to the underlying cause or disease associations of PH. Interstitial lung disease (ILD) patterns, such as nonspecific interstitial pneumonitis or usual interstitial pneumonitis, may be present with an associated

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connective tissue disease. Pulmonary venous hypertension with significant pulmonary edema may appear as swelling of the interstitium.

5 Characteristic Pathologic Appearances of Different Types of PH In the context of the classification system for pulmonary vascular disease discussed already, the specific pathologic appearances of different types of PAH are presented, using the WHO system for organization [25].

5.1 Class 1.1 and 1.2: IPAH and Familial PAH The pathologic appearances of IPAH and familial PAH (FPAH) are indistinguishable. As in all forms of severe PAH, the diameter of the pulmonary trunk is enlarged owing to the effect of chronic PH, and significant atherosclerosis of the large vessels may be present [1]. In IPAH the muscular ­pulmonary arteries demonstrate medial hypertrophy, and the precapillary ­vessels  become muscularized. The intima of small muscular arteries and precapillaries typically has ­proliferative lesions, the spectrum of which can include ­plexiform; ­concentric, which is often widespread and can be associated with fibrosis; eccentric, which appears in patchy areas of intimal fibrosis owing to resolved thrombus; and  dilated. In severe disease there may be necrotizing ­arteritis, with replacement of the arterial media by fibrinoid necrosis [1]. The pulmonary capillaries and veins are generally ­uninvolved in IPAH and FPAH, as is the pulmonary parenchyma.

5.2 Class 1.3 and 1.4: PAH Induced by Drugs or Toxins and Associated with Other Diseases Patients with PAH related to other diseases have in general the same pathologic findings of IPAH or FPAH described already, including plexiform lesions, concentric intimal fibrosis, medial hypertrophy, and arteriolar muscularization. However, clinically these patients have diseases known or suspected to be associated with the development of PAH. It is striking, however, that from this extremely diverse group of PAH-related diseases, including scleroderma, HIV infection, portal hypertension, and anorectic agent ingestion, the same pathologic findings are seen.

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Careful histologic examination may reveal clues to identify underlying PAH-related diseases which might otherwise be diagnosed as IPAH. Distinguishing IPAH from other PH-related diseases is important for prognostic and treatment information. For example, patients with IPAH in general have a worse prognosis, whereas patients with PAH related to scleroderma as a whole have worse outcomes with lung transplantation. Patients with scleroderma, a connective tissue disease classically primarily associated with dermal thickening, may also develop a fibrotic lung disease that appears histo­pathologically as a nonspecific interstitial pneumonia. However, scleroderma patients with PAH may or may not have ILD, and patients with scleroderma-ILD may or may not have PAH. PH that has been present since birth, as occurs with congenital right-to-left shunt, may result in more profound pulmonary arterial dilation, potentially including aneurysms, as well as severe atheroma and atherosclerotic lesions, even at a young age. Such massive dilation of the pulmonary arteries can cause compression of the adjacent bronchi, particularly in the right lung, resulting in an inability to clear mucous [1]. The muscular artery medial hypertrophy and arteriolar muscularization seen in all forms of PAH develops initially in patients with congenital disease, whereas the intimal lesions (including plexiform, concentric, and dilated) develop after several years of age [26]. Patients with portal hypertension may also develop ­significant pulmonary arteriovenous fistulae, which can be seen on pathologic examination and may or may not be ­concurrent with PAH. Pathologic examination of the lungs of patients with schistosomiasis-associated PH reveals two categories of lesions. The first category is pulmonary arterial lesions similar to those seen in IPAH and other forms of PAH with intimal lesions (plexiform, concentric thickening and fibrosis, and dilated), and medial hypertrophy (SMC hypertrophy in muscular arteries and muscularization of precapillaries). The second type of lesions are unique: discrete foci of granulomatous inflammation, occurring around schistosome ova which have embolized from the portal system by portocaval shunting, and become trapped in pulmonary precapillaries. The pathogenic mechanism by which schistosomiasis infection in general and these granulomatous lesions in particular lead to the diffuse pulmonary arterial disease remains unclear, although such PH is almost certainly not exclusively owing to chronic embolism of the ova alone.

5.3 Class 1′: PCH and PVOD PCH is exceedingly rare and unfortunately usually diagnosed after death. The characteristic pathologic finding is a heterogeneous distribution of numerous thin-walled

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capillary-sized vascular structures occupying a thickened interstitium [18]. The widespread vascular lesions effectively represent a diffuse neoplastic process [33]. Pulmonary VOD, a specific and rare form of pulmonary venous hypertension, has the characteristic pathologic appearance of narrowed or completely occluded lumens of smaller veins and venules, due to intimal thickening by a fibrous and acellular tissue [1]. The venous vascular segments located in intralobular septae become muscularized. The veins in VOD may also show evidence of eccentric intimal thickening and recanalization with multiple lumens, suggestive of prior and chronic venous thrombosis. Presumably owing to poor outflow there may be variable regions of capillary engorgement, particularly adjacent to septal lesions.

5.4 Class 2: Pulmonary Venous Hypertension Due to Left-Sided Heart Disease Pulmonary venous hypertension most commonly occurs as a result of left-sided heart disease, including mitral valvular disease. Rarer causes include congenital malformations such as cor triatriatum, in which the pulmonary vein makes an incomplete communication with the left atrium, and mediastinal diseases such as fibrosing mediastinitis and massive adenopathy. The pulmonary vascular disease seen in pulmonary venous hypertension is largely independent of the underlying cause. As described already, the primary pathologic abnormality is arterialization of the veins and venules, appearing as medial hypertrophy, development of double elastic laminae, and intimal thickening and fibrosis. Septal thickening by reticular collagen, thought to be the result of chronic pulmonary edema or superimposed infection, may be seen in the pulmonary parenchyma [27]. Pulmonary venous hypertension can be more rarely caused by resistance to flow due to extrinsic compression from structures in the mediastinum. The tissues that can impinge on the veins to cause such severe compression include lymph nodes, tumors, and fibrous tissue. Fibrosing mediastinitis can be associated with numerous inciting causes, most commonly histoplasmosis, or may be idiopathic [28]. Such massive focal fibrosis is thought to arise from rupture of infectious granulomas, allowing the antigen into the tissue planes of the mediastinum and causing an inflammatory and fibrotic reaction. Fibrosing mediastinitis is typically diagnosed by radiologic imaging, but ­pathology of mediastinal tissue reveals granulomas surrounded by a large amount of collagen. The histologic examination of such tissue should include silver staining (for histo­plasmosis) and acid fast staining (for tuberculosis)

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if the origin of the ­fibrosis is unclear, although these tests have a poor ­sensitivity [29].

5.5 Class 3: PH Associated with Disorders of the Respiratory System and/or Hypoxia Chronic lung diseases, high altitude, and hypoxia due to sleep apnea can cause PH with a resulting pathologic appearance similar to that of IPAH [30]. However, hypoxia appears to have a more profound effect on smaller vessels, as small muscular pulmonary arteries develop significant medial hypertrophy, whereas larger muscular arteries may be dilated but otherwise normal in appearance. Intimal lesions and fibrosis in general are not features of hypoxic pulmonary vascular disease. However, patients with chronic hypoxia do develop a characteristic and widespread lesion with longitudinal SMCs that develop in the intima of small muscular arteries [1]. Fibrinoid necrosis of arterial media is rarer than in other forms of PAH. Venous medial thickening and intimal fibrosis may be seen, although to a lesser extent than in pulmonary venous hypertension. Distinguishing the specific cause of respiratory disease or chronic hypoxemia may be difficult on the basis of pathology alone, but patients with COPD or ILD will have the pulmonary parenchymal pathologic feature specific for their disease. Patients with underlying conditions such as chronic high-altitude exposure or sleep-disordered breathing will likely have no clues to the specific cause of PH from the pulmonary pathology alone, but these diseases are diagnosed on the basis of clinical history. Congenital alveolar capillary dysplasia is a rare disease with the characteristic pathologic appearance of numerous anomalous veins appearing in the bronchovascular bundles, with concurrent and extensive intra-alveolar septal vessel muscularization, and a reduction in the number of alveolar capillaries [31]. Examination of the pulmonary parenchyma reveals a decreased number of alveoli, with the remaining alveoli having thickened walls.

5.6 Class 4: PH Due to Chronic Thrombotic and/or Embolic Disease Many forms of PH have chronic thromboembolism, a feature of the clinical disease and tissue dissue, but PH due to chronic thromboembolic disease will result in the ­pathologic lesions characteristic of acute and resolving thrombus, without the other findings such as plexiform or concentric lesions seen in PH of other causes. As discussed already, chronic

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thromboembolic disease affects principally smaller elastic and muscular arteries, and to a lesser extent precapillary vessels. The thrombotic lesions first appear as acute clots, which then evolve to become an eccentric fibrotic mass that narrows or even obstructs the vessel lumen. Such lesions can resolve by recanalization, often with multiple newly endothelialized lumens within the original vessel space. Such vessels should be carefully inspected for foreign or otherwise abnormal intravascular material, including using polarized light, to diagnose nonthrombotic causes of embolic disease.

5.7 Class 5.2: PH Due to Sarcoidosis Sarcoidosis is a disease of unknown origin that pathologically appears as noncaseating granulomas, which may also become fibrotic. The granulomas and fibrosis typically occur in a bronchovascular distribution, usually without hemodynamic­ consequences. However, particularly severe cases may cause compressive or constrictive narrowing of the pulmonary vasculature, with nonspecific intimal fibrosis, and eccentric granulomas may also develop in the arterial intima, further contributing to the vascular narrowing [32]. If any evidence of necrosis in the granuloma is present, infection must be ruled out by methods such as acid fast staining for tuberculosis.

6 Conclusion with Systematic Diagnostic Approach The range of pathologic features that can be seen in patients with PH is extensive, but it is remarkable how many dissimilar diseases can result in a similar spectrum of pathologic findings. The cause of PH can often be obtained from the history and physical examination alone, and the addition of other commonly used tools such as computed tomography of the chest, echocardiogram, and right ventricular catheterization significantly decreases the number of cases where the diagnosis remains in doubt. The resulting diagnostic approach has evolved such that when such a workup is otherwise negative for an underlying cause, it is often concluded by declaring the diagnosis of IPAH, rather than including lung biopsy and histologic examination of the pulmonary vasculature and parenchyma. As a result, some patients with IPAH may actually have PAH related to other diseases. However, the clinical impact of including tissue examination in the diagnosis of PAH has not been studied. Furthermore, in many patients the histopathologic appearance of different forms of PH, particularly the types of PAH-related diseases, share several of the diagnostic vascular lesions. However, there may be specific and subtle

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Fig. 4  A systematic approach to determining the cause of pulmonary hypertension on the basis of pathologic examination

clues found by pathologic examination that can be helpful in making a precise diagnosis. A systematic approach to determining the cause of PH on the basis of pathologic examination is outlined in Fig. 4.

References 1. Wagenvoort CA, Wagenvoort N (1977) Pathology of pulmonary hypertension. Wiley, New York 2. Elliott FM, Reid L (1965) Some new facts about the pulmonary artery and its branching pattern. Clin Radiol 16:193–198 3. Horsfield K (1978) Morphometry of the small pulmonary arteries in man. Circ Res 42:593–597 4. Singhal S, Henderson R, Horsfield K, Harding K, Cumming G (1973) Morphometry of the human pulmonary arterial tree. Circ Res 33:190–197 5. Cool CD, Stewart JS, Werahera P et al (1999) Three-dimensional reconstruction of pulmonary arteries in plexiform pulmonary hypertension using cell-specific markers. Evidence for a dynamic and heterogeneous process of pulmonary endothelial cell growth. Am J Pathol 155:411–419 6. Yaginuma G, Mohri H, Takahashi T (1990) Distribution of arterial lesions and collateral pathways in the pulmonary hypertension of congenital heart disease: a computer aided reconstruction study. Thorax 45:586–590

7. Tuder RM, Marecki JC, Richter A, Fijalkowska I, Flores S (2007) Pathology of pulmonary hypertension. Clin Chest Med 28:23–42 8. Cool CD, Kennedy D, Voelkel NF, Tuder RM (1997) Pathogenesis and evolution of plexiform lesions in pulmonary hypertension associated with scleroderma and human immunodeficiency virus infection. Hum Pathol 28:434–442 9. Tuder RM, Groves B, Badesch DB, Voelkel NF (1994) Exuberant endothelial cell growth and elements of inflammation are present in plexiform lesions of pulmonary hypertension. Am J Pathol 144:275–285 10. Heath D, Smith P, Gosney J (1988) Ultrastructure of early plexogenic pulmonary arteriopathy. Histopathology 12:41–52 11. Mitani Y, Ueda M, Komatsu R et al (2001) Vascular smooth muscle cell phenotypes in primary pulmonary hypertension. Eur Respir J 17:316–320 12. Hutchins GM, Ostrow PT (1976) The pathogenesis of the two forms of hypertensive pulmonary vascular disease. Am Heart J 92:797–803 13. Chazova I, Loyd JE, Zhdanov VS, Newman JH, Belenkov Y, Meyrick B (1995) Pulmonary artery adventitial changes and venous involvement in primary pulmonary hypertension. Am J Pathol 146:389–397 14. Yamaki S, Wagenvoort CA (1985) Comparison of primary plexogenic arteriopathy in adults and children. A morphometric study in 40 patients. Br Heart J 54:428–434 15. Yi ES, Kim H, Ahn H et al (2000) Distribution of obstructive intimal lesions and their cellular phenotypes in chronic pulmonary hypertension. A morphometric and immunohistochemical study. Am J Respir Crit Care Med 162:1577–1586 16. Santos S, Peinado VI, Ramirez J et al (2002) Characterization of pulmonary vascular remodelling in smokers and patients with mild COPD. Eur Respir J 19:632–638

101  Histological and Pathological Diagnosis of Pulmonary Hypertension 17. Stenmark KR, Davie N, Frid M, Gerasimovskaya E, Das M (2006) Role of the adventitia in pulmonary vascular remodeling. Physiology (Bethesda) 21:134–145 18. Ito K, Ichiki T, Ohi K et al (2003) Pulmonary capillary hemangiomatosis with severe pulmonary hypertension. Circ J 67:793–795 19. Kelvin FM, Boone JA, Peretz D (1972) Pulmonary varix. J Can Assoc Radiol 23:227–229 20. Zhang P, Summer WR, Bagby GJ, Nelson S (2000) Innate immunity and pulmonary host defense. Immunol Rev 173:39–51 21. Peinado VI, Ramírez J, Roca J, Rodriguez-Roisin R, Barberà JA (2006) Identification of vascular progenitor cells in pulmonary arteries of patients with chronic obstructive pulmonary disease. Am J Respir Cell Mol Biol 34:257–263 22. Diez M, Barbera JA, Ferrer E et al (2007) Plasticity of CD133(+) cells: role in pulmonary vascular remodeling. Cardiovasc Res 76(3):517–527 23. Frid MG, Brunetti JA, Burke DL et  al (2006) Hypoxia-induced pulmonary vascular remodeling requires recruitment of circulating mesenchymal precursors of a monocyte/macrophage lineage. Am J Pathol 168:659–669 24. Johnson SR, Mehta S, Granton JT (2006) Anticoagulation in pulmonary arterial hypertension: a qualitative systematic review. Eur Respir J 28:999–1004

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25. Simonneau G, Robbins IM, Beghetti M et al (2009) Updated clinical classification of pulmonary hypertension. J Am Coll Cardiol 54(Suppl. 1):S43–S54 26. Rabinovitch M (2008) Molecular pathogenesis of pulmonary ­arterial hypertension. J Clin Invest 118:2372–2379 27. Heard BE, Steiner RE, Herdan A, Gleason D (1968) Oedema and fibrosis of the lungs in left ventricular failure. Br J Radiol 41:161–171 28. Berry DF, Buccigrossi D, Peabody J, Peterson KL, Moser KM (1986) Pulmonary vascular occlusion and fibrosing mediastinitis. Chest 89:296–301 29. Dines DE, Payne WS, Bernatz PE, Pairolero PC (1979) Mediastinal granuloma and fibrosing mediastinitis. Chest 75:320–324 30. Xu X-Q, Jing Z-C (2009) High-altitude pulmonary hypertension. Eur Respir Rev 18:13–17 31. Antao B, Samuel M, Kiely E, Spitz L, Malone M (2006) Congenital alveolar capillary dysplasia and associated gastrointestinal anomalies. Fetal Pediatr Pathol 25:137–145 32. Valeyre D, Uzunhan Y, Bouvry D, Naccache JM, Nunes H (2008) Up-to-date in pulmonary and extrapulmonary sarcoidosis. Acta Clin Belg 63:408–413 33. Eltorky MA, Headley AS, Winer-Muram H, Garrett HE Jr, Griffin JP (1994) Pulmonary capillary hemangiomatosis: a clinicopathologic review. Ann Thorac Surg 57:772–776

Chapter 102

Echocardiography in Pulmonary Vascular Disease Paul R. Forfia

Abstract  This chapter will focus on the principles and uses of the Doppler echocardiography (DE) examination in patients with pulmonary vascular disease (PVD). That is, patients who have pulmonary hypertension that is related to a marked increase in the pulmonary vascular resistance (PVR) and loss of large pulmonary artery (PA) compliance. We will also address the role of DE in the initial assessment of patients with pulmonary hypertension (PH) of all causes, as the diagnosis of PH typically precedes confirmation or exclusion of PVD as the cause of PH. We will emphasize noninvasive clues that provide insight into the pathophysiology of an individual patient’s PH prior to invasive hemodynamic assessment. To accomplish this, a basic understanding of the right side of the heart and the pulmonary circulation is required, then how various forms of PH, including PVD, can disrupt the right ventricle (RV)–PA interaction. Using a more integrated approach helps the clinician avoid some of the pitfalls of the DE examination, especially the overreliance on the Doppler pressure estimation in the initial diagnosis and serial assessment of patients with PH. This approach also improves the diagnostic sensitivity of the DE examination for PVD and improves patient selection for further invasive hemodynamic testing. Keywords  Doppler echocardiography • Pulmonary vascular resistance • Cardiac output • Hemodynamic assessment

1 Introduction This chapter will focus on the principles and uses of the Doppler echocardiography (DE) examination in patients with pulmonary vascular disease (PVD). That is, patients who have pulmonary hypertension that is related to a marked increase in the pulmonary vascular resistance (PVR) and loss of large P.R. Forfia (*) Cardiovascular Division, University of Pennsylvania School of Medicine, 3400 Spruce Street, Perelman Ctr., 2nd Flr., East Pavillion, 19104 Philadelphia, PA, USA e-mail: [email protected]

p­ ulmonary artery (PA) compliance. We will also address the role of DE in the initial assessment of patients with pulmonary hypertension (PH) of all causes, as the diagnosis of PH typically precedes confirmation or exclusion of PVD as the cause of PH. We will emphasize noninvasive clues that provide insight into the pathophysiologic origin of an individual patient’s PH prior to invasive hemodynamic assessment. To accomplish this, a basic understanding of the right side of the heart and the pulmonary circulation is required, then how various forms of PH, including PVD, can disrupt the right ventricle (RV)–PA interaction. Using a more integrated approach helps the clinician avoid some of the pitfalls of the DE examination, especially the overreliance on the Doppler pressure estimation in the initial diagnosis and serial assessment of patients with PH. This approach also improves the diagnostic sensitivity of the DE examination for PVD and improves patient selection for further invasive hemodynamic testing.

2 Anatomy and Physiology of the Pulmonary Circulation and the Right Side of the Heart The right ventricle comprises inflow (sinus) and outflow (conus) regions, separated by a muscular ridge, the crista supraventricularis. The moderator band is part of the crista supraventricularis, extending from the interventricular ­septum to the anterior wall of the RV and anterior papillary muscle. This anatomic connection serves as one of the anatomic links (along with interlacing epicardial and endocardial muscle fibers) between the contracting left ventricle (LV) and RV, and in part explains how circulatory homeostasis can be maintained in experimental models of RV destruction when the damaged RV remains coupled to a normal pulmonary circuit [1, 2]. The inflow region includes the tricuspid valve, the chordae/papillary muscles, as well as the body of the RV [3, 4]. The anterior papillary muscle is the largest and most prominent of the three papillary muscles, whereas the posterior and septal papillary muscles are

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_102, © Springer Science+Business Media, LLC 2011

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far smaller and more numerous. The boundaries of the body of the RV are formed by the RV free wall, extending with a radius of curvature approximating that of a large sphere, from the anterior and posterior aspects of the interventricular septum. The normal septal curvature is convexed toward the RV cavity, imparting a crescentic shape to the RV in cross section. The interior surface of the RV is heavily trabeculated; this feature along with the moderator band and more apical insertion of the tricuspid valve annulus impart key morphologic differences that distinguish the RV from the LV by echocardiography. In contrast, the infundibulum is a smooth, funnel-shaped outflow portion of the RV that ends at the pulmonic valve. Thus, the RV has a complex geometry, with the RV inflow geometry approximating a pyramid and the outflow portion a tapered cylindrical spout. This complex shape does not permit typical geometric assumptions needed to calculate RV volumes (and thus RV ejection fraction) by 2D echo; however, it does not preclude alternative, nonvolumetric methods of RV function assessment (vide infra). Normal RV free wall thickness is 0.3–0.5  cm, versus 0.7–1.1 cm in the LV and RV mass is one sixth the mass of the LV. These differences in wall thickness in part lead to the far greater distensability and larger cavity volumes in the RV than in the LV, despite lower end-diastolic filling pressures. This translates to a RV ejection fraction that is typically 35–45% (versus 55–65% in the LV) yet generates the identical stroke volume as the LV. RV mass is typically onesixth that of the LV. Systolic function of the RV, like the LV, is influenced by changes in preload, afterload, and the intrinsic contractility of the ventricle. RV contraction occurs in a peristalsis-like fashion, with the outflow region contracting about 30–40 ms after the outflow region, accounting for only about 15% of stroke volume generation; most of the blood is ejected by the force generated by the body of the RV [5]. Differences in RV muscle fiber orientation dictate that the body of the RV shortens symmetrically in the longitudinal and radial planes; thus, longitudinal shortening accounts for a much larger proportion of ejection in the RV than in the LV [6]. This relatively conspicuous RV shortening along the longitudinal axis can be exploited to measure RV systolic function using relatively simple techniques that do not require geometric assumptions or meticulous endocardial definition, both of which are known limitations to the noninvasive assessment of RV systolic function [6, 7]. The principal function of the pulmonary circulation is to deliver blood flow to the alveolar–capillary interface for gas exchange. Thus, under normal circumstances, the entire cardiac output must travel through the lung. The pulmonary circulation is by design a far more compliant and lower-resistance vascular circuit than the systemic circulation, typically with half the large artery compliance, 5–15% the vascular

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­resistance, and one third the degree of arterial wave ­reflection of the systemic vasculature. Therefore, the resulting interaction between blood flow and vascular impedance (an aggregate measure of afterload that encompasses small vessel resistance, large artery stiffness, and wave reflection) results in a pulse pressure that is half and a mean PA pressure that is normally one sixth that of the systemic circulation. The RV and the pulmonary circulation function as a coupled unit. In health, a normal pulmonary vasculature couples to an appropriately thin-walled, distensible RV that is designed to generate large amounts of blood flow without a resulting high pressure (“flow generator”). As long as this normal interaction holds, so will this physiologic paradigm. As PVD develops, the PVR increases and the large arteries stiffen. As a result, the (formerly appropriate) nonmuscular RV is typically incapable of completely matching or coupling its contractile performance to its new afterload [8, 9]. This relative RV–PA uncoupling leads to a stereotypical triad of changes that occur at the level of the RV, including RV systolic dysfunction, increased size and altered shape of the RV, as well as differing degrees of systolic and diastolic bowing of the interventricular septum. This triad of changes forms the basis of the echocardiographic diagnosis of PVD. The degree to which these changes occur is contingent upon the degree of RV–PA uncoupling. Thus, at a PVR of 10 WU, a patient with depressed RV contractility is far more uncoupled to his or her vascular load than a patient with an ­intrinsically normal RV coupled to the same afterload. As such, the RV dysfunction triad helps to adjudicate and assess the physiologic and clinical significance of any given degree of PVD. The greater the RV impairment, the greater the overall significance of the PVD, no matter what the noninvasive pressure estimate is reported to be. Hypertrophy typically denotes chronicity; thus, in more chronic forms of PH, RV hypertrophy predominates over RV dilatation, whereas in more acute forms such as pulmonary embolism, or even pulmonary arterial hypertension (PAH), dilatation of the RV occurs disproportionate to hypertrophy. In general, the net effect is that chronicity affects the degree of adaptation that can occur at the level of the right side of the heart. For example, the RV in Eisenmenger’s syndrome is often massively hypertrophied, not dilated, and maintains relatively normal function despite decades of systemic level afterload; this is the most likely explanation for the more favorable long-term prognosis of these patients as compared with patients with other forms of PAH [8, 9]. Doppler data provides further physiologic and hemodynamic characterization of the varying causes of PH. Importantly, differing degrees of PH often result from an abnormal RV–PA interaction. However, the increased PA pressure is the result, not the cause, of the RV–PA mismatch. Thus, a simple, but critically important distinction is that PA pressure is a poor measure of RV afterload, which is why PA

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pressure is also a poor predictor of clinical RV failure and prognosis in PAH [10]. Understanding this physiologic paradigm puts the clinician at a distinct advantage when assessing a patient with known or suspected PH, as the presence or absence (whether a true negative or false negative) of elevated PA pressure will not necessarily dissuade the clinician from suspecting PVD if there is evidence of RV dysfunction, especially when occurring in the form of the above-noted triad.

3 Pressure Assessment by Doppler Examination Despite its shortcomings as a measure of RV afterload, PA pressure assessment is a central component of the evaluation of patients with known or suspected PVD. This relates to the fact that differing degrees of PH are nearly always present in patients with PVD, and the workup of these patients typically emanates from the initial noninvasive pressure assessment. A PA systolic pressure (PASP) greater than 40 mmHg is generally accepted as the upper limit of normal in most subjects; however, the cutoff may be higher in elderly subjects [11, 12]. The most common method used in Doppler pressure assessment utilizes continuous-wave DE to determine the peak tricuspid regurgitant jet velocity, which estimates the pressure difference between the RV and the right atrium using the modified Bernoulli equation (4v2; v is the peak velocity of the tricuspid regurgitant jet) [13, 14] (Fig. 1). The

Fig.  1  Continuous-wave Doppler interrogation across the tricuspid valve, obtained from the apical four-chamber view. Note the full Doppler envelope with a clearly defined peak velocity. The peak velocity of the jet is 3.3 m/s, which estimates a pressure gradient between the right ventricle (RV) and the right atrium (RA) of 44 mmHg. The systolic pulmonary artery pressure is 44 mmHg plus the RA pressure estimate. In this example, the well-developed Doppler envelope and clearly defined peak velocity lead to an accurate Doppler pressure estimate

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tricuspid valve must be interrogated from multiple different views (i.e. RV inflow, short axis, apical four-chamber, subcostal views) to align the ultrasound beam parallel to the regurgitant signal to obtain the most complete Doppler envelope, and thus peak transtricuspid velocity. In general, the highest velocity obtained should be used to calculate the peak RVSP. One exception is the inclusion of a post-­ extrasystolic beat, which will often have a substantially higher peak velocity than steady-state beats, relating to the larger stroke volume generated during the compensatory pause. In this scenario, the inclusion of the single highest velocity to the exclusion of the others can lead to marked PA pressure overestimation. If pulmonic stenosis is present, the gradient across the pulmonic valve must be subtracted from the peak RVSP to obtain the peak PA pressure, otherwise a distinction between “RV hypertension” and PH can be missed. For example, a gradient from the RV to the right atrium of 70 mmHg and a peak gradient across the pulmonic valve of 50 mmHg estimates a PASP of only 20 mmHg. In this case, intervention for severe pulmonic stenosis is warranted, whereas pulmonary vasodilators are not. In a technically limited study, agitated saline can be injected intravenously to enhance the tricuspid regurgitant jet signal and improve the measurement of the maximum tricuspid regurgitant jet velocity [15]. The absence of tricuspid ­regurgitation precludes pressure assessment via this method. An estimated right atrial pressure (RAP) is then added to the RV pressure–RAP gradient to approximate PASP. Thus, PASP » 4v2 + estimated RAP. When all “conditions” required for this method are met, namely, sufficient tricuspid regurgitation for interrogation, proper Doppler alignment, optimal visualization of the peak of the tricuspid regurgitant jet, and accurate RAP estimation, this method indeed correlates strongly with invasive PASP assessment [16, 17]. However, as one would expect, one or more limitations in the “Doppler–patient interface” frequently occur in clinical practice, leading to differing degrees of discrepancy between Doppler and invasive pressure. Clinicians experienced in assessing patients with PH will attest to the limitations of the Doppler pressure estimate, and recognize that the Doppler PASP estimates should not be viewed as synonymous with invasive pressure recordings. This was demonstrated by Arcasoy et al. in a sizeable cohort of patients with advanced lung disease whom underwent DE within 72  h of right heart catheterization [18]. Perhaps the most striking finding from their study was that DE assessment of PASP was possible in only 44% of these patients, with obstructive lung disease posing the biggest challenge to optimal imaging. Most patients had relatively mild PH by right-sided heart catheterization. In the patients where DE was feasible, DE pressure was 10 mmHg or more different from invasive pressure in nearly half the cases. More recently, Fisher et al. examined the correlation between

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Fig. 2  Example of pulmonary artery systolic pressure overestimation by the continuous-wave Doppler method. Note that the peak velocity has been overestimated. The reported peak velocity is 3.4 m/s, estimating a gradient between the RV and the RA of 46 mmHg. However, the true peak velocity is only 2.5 m/s, estimating an RV to RA gradient of only 18 mmHg. This figure highlights the importance of measuring the peak velocity carefully, as the peak velocity is squared, thus amplifying the error

Doppler-estimated and invasively measured PASP in a cohort of 65 patients with severer PH (62% with PAH) [19]. Although the correlation between Doppler and invasive PASP measurements was reasonable (r = 0.66, P < 0.001), Bland–Altman analysis revealed that 48% of patients had a Doppler-estimated PASP that was at least 10 mmHg different from the catheterization value (16 underestimates, 15 overestimates). Pressure overestimations arose from either overestimation of the peak velocity signal (Fig.  2) or an overestimated RAP arising from inferior vena cava (IVC) diameter and collapsibility assessment (vide infra). The magnitude of underestimation (−30  mmHg) was significantly greater than the degree of overestimation (+19 mmHg), with the underestimates leading to more frequent and marked misclassification of the degree of PH in the individual patient. Forty-seven percent of subjects whose pressure was underestimated by DE were misclassified by two or more diagnostic categories (i.e. mild PH by DE and severe PH by catheterization). In contrast, thirteen percent of subjects with DE pressure overestimation had gross misclassification of their PH severity by DE examination. Logically, subjects with Doppler pressure underestimation had comparatively less tricuspid regurgitation, and even more importantly, had either fair or poor quality continuous-wave Doppler envelopes across the tricuspid valve due to signal dropout; thus, the peak velocity was not fully appreciated, leading to underestimation (Fig. 3). Of note, of the six subjects in the study with no tricuspid regurgitation, four of these patients had PH by ­catheterization. Thus, the absence of tricuspid regurgitation is not sufficient to exclude

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Fig. 3  Example of pulmonary artery systolic pressure underestimation by the continuous-wave Doppler method. Note the poor quality of the Doppler envelope, with signal dropout and a poorly defined peak tricuspid regurgitation velocity. This scenario leads to pressure underestimation as the peak velocity may be much higher than what is visualized by the echocardiographer. The peak velocity is estimated at 3.3 m/s, estimating an RV to RA gradient of 43 mmHg and pulmonary artery systolic pressure of 53 mmHg. A right-sided heart catheterization within 24 h of the Doppler echocardiography examination revealed a pulmonary artery systolic pressure of 95 mmHg. Note the degree of RV dilation on the “scout” image; this degree of RV dilatation should also raise suspicion for an underestimated pulmonary artery systolic pressure

s­ ignificant PH, even though it typically denotes a more ­compensated RV. Twelve of the 16 patients in whom PA pressure was underestimated by DE had evidence of RV enlargement and/or dysfunction on their DE examination. A variety of techniques have been used to estimate RAP, most often using the IVC dimensions and/or the degree of IVC collapsibility with inspiration or “sniff” [20, 21]. None of these techniques have proved particularly accurate, with RAP overestimation being the most frequently observed limitation [18, 19]. In fact, an overestimated RAP was the primary source of error in nearly 50% of the subjects with an overestimated PASP [19]. The relative inaccuracy of the IVC-estimated RAP relates to a number of factors, including the fact that IVC dilatation and collapsibility are dictated not only by the intravascular distending pressure, but also by the relative compliance of the IVC, the degree of chronic remodeling of the IVC, and also the relative degree and transmission of the fall in pleural pressure to the IVC, all of which can vary significantly and cannot be accounted for by simple visualization of the IVC. Therefore, it is our preference to add the clinically estimated RAP from the jugular venous pressure examination to the Doppler-derived transtricuspid gradient to obtain the most accurate PASP estimate. These data underscore the point that Doppler pressure estimation should not be viewed as a “standalone” test in the assessment of a patient with known or suspected PVD.

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Rather, the Doppler pressure estimate should be integrated into the context of the remainder of the examination; if the Doppler examination estimates mild PH in the context of moderate RV enlargement and systolic dysfunction, it is more likely that the pressure has been underestimated and a right-sided heart catheterization should be performed. In contrast, mild PH (i.e. Doppler PASP 45 mmHg) in the context of normal RV size, shape, and systolic function is far less likely to be indicative of clinically significant PH. PA diastolic pressure (PADP) can be derived from the end-diastolic point of the pulmonary regurgitation signal, added to an estimate of RAP [22]. Alternatively, PADP can be estimated from the velocity obtained from the tricuspid regurgitant jet at the time of pulmonic valve opening [23, 24]. In either case, the accuracy of the method is contingent on the presence of pulmonary regurgitation or tricuspid regurgitation and the quality of the respective Doppler envelopes. The mean PA pressure can also be estimated by DE. The most common method takes advantage of the inverse correlation between the time to peak velocity (acceleration time, AcT) of the pulsed-wave Doppler profile obtained in the RV outflow tract (RVOT) and the mean PA pressure (Fig.  4). Kitabatake et al. showed that patients with a mean PA pressure of 19 mmHg or lower had an average AcT of 137 ± 24 ms, whereas an AcT of 97 ± 20 and 65 ± 14  ms was seen in patients with a mean PA pressure of 20–39 and 40 mmHg or greater, respectively [25]. This is relatively easy to perform and unlike pressure estimates based on tricuspid regurgitation velocity, Doppler recordings from the RVOT are possible in virtually all patients. Mean PA pressure can also be estimated by applying the standard formula for mean PA pressure ([1/3(systolic PA

Fig.  4  Pulsed-wave Doppler interrogation in the RV outflow tract. Acceleration time represents the time to the peak velocity of the Doppler envelope. In this example, the acceleration time is 120  ms, which is consistent with a normal mean pulmonary artery pressure

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pressure − diastolic PA pressure) + diastolic PA pressure]) to Doppler estimates of PASP and PADP. This approach is more labor-intensive as it requires separate PASP and PADP estimates, is subject to the same inherent limitations of each method alone, and can lead to compounding error if both methods are inaccurate. Alternatively, the mean PA pressure can be estimated from the Doppler PASP estimate [mean PA pressure = 0.61(systolic PAP) + 2 mmHg], which takes advantage of the predictable hemodynamic relationship between mean and systolic PA pressure in most patients [26]. Naturally, the accuracy of the mean PA pressure estimate via this approach is contingent on the accuracy of the PASP estimate.

4 RV Function Assessment 4.1 Direct Measures of RV Systolic Function Assessment of RV function is the single most important aspect of the DE examination in patients with known or suspected PVD, given the morbidity and mortality associated with this condition is heavily dependent on the degree of adaptation of the RV to its excessive pulmonary vascular load. Virtually all echocardiographic measures shown to have prognostic significance in PAH are either direct or ­indirect measures of RV systolic performance [10]. Historically, noninvasive measures of ventricular function have focused on assessment of chamber volumes to derive ventricular ejection fraction. This is especially true for the LV, where chamber volume can be derived from modeling of the ventricle as a prolate ellipsoid. However, as mentioned already, RV geometry does not lend itself to simple modeling, and thus formulae to estimate RV volumes are not nearly as reliable. As a result, accurate RV ejection fraction assessments have proved elusive. With growing use of 3D echocardiography, RV ejection fraction assessment will likely become more accurate and applicable in clinical practice. The most common and arguably least ideal method of RV function assessment by echocardiography is visual estimation. The echocardiographer visually integrates the relative change in RV cavity area to estimate global RV systolic function, typically reporting RV function as normal, mild, moderate, or severely reduced. However, the interplay between the inherent limitations of RV imaging and reader subjectivity leads to degrees of interobserver variability that limit reliable application of this approach in clinical practice. Thus, one must be very cautious in how to interpret RV function by this method, as one reader’s “severe” may be another reader’s­ “mild” RV dysfunction.

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A more quantitative approach is to measure the total s­ ystolic area change of the RV, referred to as the RV fractional area change (RVFAC). This measure is derived from the planimetered areas of the RV at end-diastole and endsystole [(RVFAC = RV areaED − RV areaES/RV areaED) × 100] from the apical four-chamber view. The RVFAC does not require geometric assumptions and correlates with the RV ejection fraction [27]. However, incomplete visualization of the RV cavity (more common in the setting of RV enlargement) as well as suboptimal endocardial definition lead to relatively high inter- and intraobserver variablility [28, 29]. As mentioned already, differences in muscle fiber orientation of the RV dictate that there is proportionally greater RV longitudinal shortening in the RV than in the LV [6]. This

unique contraction pattern of the RV can be exploited to assess RV systolic function. Prior studies have shown that the total longitudinal systolic displacement of the RV base toward the RV apex (referred to as tricuspid annular plane systolic excursion, or TAPSE) correlates strongly (r = 0.79– 0.92) with RV ejection fraction derived from radionuclide angiography [7, 30]. TAPSE can be derived from 2D echocardography or M-mode (Fig. 5), is simple to ­perform, and has been shown to be highly reproducible, owing in part to the lack of reliance on RV endocardial definition or geometric assumptions [7, 29]. Evidence from prior studies indicates that a normal TAPSE is 2.4–2.7 cm, with lesser values indicating mild (2.0–2.3 cm), moderate (1.5–1.9 cm), and severe (less than 1.5 cm) RV dysfunction [31, 32]. A TAPSE less

Fig.  5  Tricuspid annular plane systolic excursion is measured in the ­apical four-chamber view, and can be derived from either the 2D images (a, b) or M-mode (c). Using the 2D method, tricuspid annular plane ­systolic excursion (TAPSE) represents the difference between the enddiastolic and end-systolic length of the RV from the tricuspid annulus to

the RV apex (TAPSE 2.6 cm). In c, an M-mode cursor is placed through the point where the tricuspid valve inserts into the tricuspid annulus. The distance between end-diastolic (trough) and end-systolic (peak) points of the M-mode image represents the TAPSE. The M-mode method is most commonly used and is easier to perform

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than 1.8  cm predicts a stroke volume index greater than 29 ml/m2 with 87% accuracy, and is associated with increased hospitalization rates for right-sided heart failure and decreased survival in patients with PAH [29, 30]. Tissue Doppler imaging (TDI) can also be used to measure the velocity of RV contraction in the longitudinal axis (denoted S¢), correlates with TAPSE (r = 0.90), and also is a simple and reproducible method of RV function assessment [33] (Fig. 6). S¢ less than 10 cm/s predicts a cardiac index less than 2.0 l/min/m2 with 89% sensitivity and 87% specificity [34]. In addition, the RV TDI signal can be integrated to measure the longitudinal tissue displacement [35]. Using receiver operating characteristic curve analysis, an RV ­tissue displacement of greater than 1.5  cm predicted an RV stroke volume index of 30 ml/m2 or greater (area under the curve 0.79); changes in RV tissue displacement related strongly to changes in RV stroke volume index in response to intravenous epoprostenol infusion. The myocardial performance index (MPI) uses a different approach to RV function assessment, integrating systolic and diastolic function parameters in a single measure, using the formula IVRT + IVCT/RVET, where IVRT is the RV isovolumic relaxation time, IVCT is the isovolumic contraction time, and RVET is the RV ejection time. The time intervals are typically derived from tissue Doppler signals (Fig.  6). Increasing values represent worsening function, with an increased RV MPI associated with decreased survival in PAH [36]. In certain centers, RV MPI is a standard noninvasive measure of global RV function, whereas other centers have found the method somewhat cumbersome and thus it is used less often in clinical practice.

Fig. 6  RV tissue Doppler imaging (TDI) along the long axis of the RV to obtain the peak velocity of shortening of the RV in the longitudinal plane (S¢). S¢ = 14  cm/s, which is consistent with normal RV systolic function. The RV myocardial performance index can also be obtained from RV TDI, derived from the RV isovolumic contraction time and isovolumic relaxation time and RV ejection time

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All of the clinically used noninvasive methods of RV (and LV) function are load-dependent, and thus will change in response to different loading conditions, most notably RV afterload. The afterload sensitivity of these RV function measures may be viewed as a strength or weakness: a strength in that the vast majority of RV dysfunction in the setting of PVD is indeed afterload-dependent and thus it is desirable that these measures will track with changes in RV afterload; a weakness in that a fall in TAPSE, for example, does not indicate the intrinsic, load-independent performance of the RV (RV contractility). This distinction becomes clinically relevant when a patient with PAH is referred for lung rather than heart–lung transplantation, and the question is raised as to whether normalizing RV afterload with lung transplant will be sufficient to allow for RV function recovery. Isovolumic myocardial acceleration, derived from TDI, has been shown in experimental animal models to remain constant across wide ranges of preload and afterload, yet correlated well with invasive measures of RV contractility such as RV end-systolic elastance [37]. Future work will help determine how isovolumic myocardial acceleration and other measures of RV contractility can be used in similar contexts. Moreover, these methods may be combined with noninvasive measures of RV afterload to obtain noninvasive indices of RV–PA coupling.

4.2 Indirect Measures of RV Systolic Function The fundamental response of the relatively nonmuscular RV to progressive increases in RV afterload is a fall in RV systolic function. As mentioned already, the tempo with which this occurs is a key important determinant as to the relative degree of RV adaptation to the increased load. In most forms of PVD (except chronic systemic-to-pulmonary shunts), the rise in RV afterload occurs relatively quickly, which in turn impedes RV ejection and increases RV wall tension. Owing to the relative compliance of the RV and right atrium, this leads to increased chamber size, which in part helps restore the RV stroke volume closer to the baseline level (heterometric autoregulation). As a result, increases in size of the rightsided heart chambers can be thought of as an important indirect measure of RV systolic dysfunction. The normal RV measures approximately 3.5 cm at end-diastole, with a planimetered area of 15–18 cm2 [38]. Typically, the RV dimension and area are two thirds those of the LV. Expressing RV size as a ratio of RV-to-LV dimension is especially practical and is easily derived from the apical four-chamber view, where the RV and LV are viewed side by side. The normal RV-to-LV ratio is approximately 0.6–0.8, with increasing RV-to-LV ratios in patients with mild (0.8–1.0), moderate (1.1–1.4), and severe (1.5 or greater) RV dilatation. A useful

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rule of thumb is that the RV-to-LV ratio should be less than 1.0, and any value greater than 1.0 is strongly suggestive of RV dilatation which often coincides with RV dysfunction. Similarly, the angle and position of the RV apex change in response to increased RV afterload, thus leading to an overall change in RV shape. Normally, the RV apex forms a relatively acute angle and does not form or occupy the apex of the heart. Thus, in the apical four-chamber view, the normal RV has a triangular or sickle shape. In the setting of PVD, the RV apical angle often “opens” and the RV and LV will often “share” the apex of the heart, taking on a more oblong shape (Fig. 7). López-Candales et al. showed that a relatively large or “open” RV apical angle was a common finding in the setting of chronic PH, relating inversely to decreases in TAPSE and RVFAC [39]. The right atrium to left atrium ratio should

also not exceed 1.0. Moreover, the direction of interatrial septal bowing can provide important information as to the relative pressure differences in the right and the left atrium. Unlike mitral regurgitation, which occurs due to either intrinsic mitral valve disease (i.e. myxomatous mitral valve disease, rheumatic heart disease) or secondary regurgitation from LV dysfunction, the vast majority of cases of tricuspid regurgitation occur secondary to RV dysfunction and tricuspid annular dilatation. As a result, tricuspid regurgitation is a common accompanying feature of RV dysfunction, with greater degrees of tricuspid regurgitation typically signifying greater degrees of RV decompensation. Moreover, the rate of rise in tricuspid regurgitation velocity provides qualitative insight into RV systolic function, and forms the basis for estimating RV dP/dt by Doppler [40, 41] (Fig. 8).

Fig. 7  Apical four-chamber view. (a) Normal RV size [RV to left ventricle (LV) dimension ratio approximately 0.6] and shape (note the relatively acute RV apical angle). Also note that the RV apex does not form or “share” the

apex of the heart with the LV. (b) A patient with moderate RV enlargement, with an RV-to-LV ratio of 1.3. Note the relatively open RV apical angle and apex-sharing RV. This patient has pulmonary arterial hypertension

Fig.  8  Continuous-wave Doppler interrogation across the tricuspid valve. Note the difference in the rate of rise of the Doppler velocity

between the two tracings. Panel B demonstrates a markedly slowed rise in velocity, consistent with severe RV systolic dysfunction

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As the right side of the heart progressively dilates, the n­ ondistensible pericardium becomes an increasingly important determinant of ventricular diastolic function and diastolic ventricular interaction (pericardial constraint). Once the pericardial space has been fully occupied by a dilated right side of the heart, further increases in RV size lead to diastolic septal bowing from right to left, with reciprocal reductions in LV (and left atrium) chamber size [42]. Systolic flattening of the septum is also common in PVD, and likely occurs owing to a delay in the time to peak RV contraction, creating mechanical RV/LV asynchrony [43] (Fig. 9). Magnetic resonance imaging studies have shown relatively strong correlations between the degree of RV systolic septal bowing and PVR [44]. Thus, its presence typically signifies RV dysfunction on the basis of markedly increased RV afterload. Importantly, even mild systolic septal flattening is a distinctly abnormal finding, and occurring in relative isolation is often one of the earliest signs of underling PVD and PH. Eventually, septal bowing leads to impaired early diastolic filling of the left side of the heart. Thus, a common Doppler feature in PVD with RV dysfunction is Doppler evidence of “diastolic dysfunction” (i.e. “E to A reversal” of the transmitral Doppler pattern) [45]. However, Doppler evidence of diastolic dysfunction should not be considered synonymous with left atrial hypertension. Thus, it is important to develop a sense, noninvasively, for whether a patient’s Doppler ­evidence of diastolic dysfunction is the cause of the PH or instead is a marker of impaired LV filling related to RV dysfunction and septal bowing. This will be discussed in further detail below. In the setting of PAH, mild to moderate circumferential pericardial effusions are seen in up to half of patients, and are particularly common at the time of initial clinical presentation [46] (Fig. 9). In general, a pericardial effusion typically indicates right-sided heart decompensation, and is likely

conferred on the basis of long-standing right atrial ­hypertension and impaired myocardial lymphatic drainage [47]. The presence of a pericardial effusion has been one of the most consistent echocardiographic findings indicative of a poor prognosis in PAH [10, 46]. Importantly, the temptation to pursue percutaneous or surgical pericardial drainage should be avoided unless there is especially compelling evidence of tamponade, as the effusion typically is the result (not the cause) of RV failure and right atrial hypertension. Hemnes et al. reported that attempted drainage of pericardial effusions in patients with PAH has been associated with very poor outcomes, with most deaths occurring within 12–24 h of the procedure [48]. In our experience, pericardial effusions in PAH progressively decrease in size or resolve over weeks or months as the RV failure responds to diuresis and pulmonary vasodilator therapy. Thus, there are numerous ways to quantify RV function, either directly or indirectly. Often, the limitations of the DE examination are so frequently cited that echocardiography laboratories, including those at major academic medical centers, do not have a single quantitative measurement of RV function in their clinical echocardiography protocol. This is unfortunate, as the inability to obtain an RV ejection fraction, for instance, should not be interpreted to mean that RV function cannot be assessed. It is important that clinical echocardiography protocols include basic measurements of RV size, the relative proportion of the RV to the LV, and a direct ­measurement of RV systolic function. Whether the direct RV function assessment be RVFAC, TAPSE, RV TDI, or another measurement is perhaps not as important as the laboratory making that particular measurement consistently, and properly, such that the sonographers, echocardiographers, and clinicians can develop an understanding of “their” measurement so that it may be optimally integrated into clinical practice.

Fig. 9  Short-axis view at the midventricular level. Note the flattening of the septum from right to left in diastole (a) and systole (b). Also note

the moderate, circumferential pericardial effusion (PE), a common accompanying feature of the decompensated right heart

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5 Doppler Hemodynamic Assessment 5.1 Pulmonary Vascular Resistance An elevated PVR is the sine qua non of PVD. Thus, a reliable, simple, and noninvasive method of PVR assessment would be of great value. Abbas et al. showed that the ratio of transtricuspid flow velocity (surrogate of pressure) to the velocity–time integral (VTI) obtained from the RVOT (surrogate of flow) closely correlates to PVR in a population of patients with an average PVR of 2 mmHg/l/min [49]. A more recent study showed that this same method reliably estimated the PVR in patients with a PVR of less than 8 mmHg/l/min; however, the estimated PVR correlated poorly with invasive PVR measurements in those with a PVR greater than 8  mmHg/l/min [50]. Another important drawback of this method is that this ratio, by definition, is an estimate of total pulmonary resistance (not PVR) as there is no accounting for left atrial pressure. Thus, in a patient or population of patients with significant left atrial hypertension, this formula would consistently lead to significant overestimations of the PVR and wrongly implicate PVD as the cause of the PH in a patient with pulmonary venous hypertension.

5.2 Cardiac Output Cardiac output can be estimated by Doppler examination, employing the formula Q = AV (Q is flow, A is cross-sectional area, V is flow velocity). Typically, the LV outflow tract (LVOT) is used as the site for Doppler interrogation, as the dimensions and flow velocity in the LVOT are more easily obtained than in the RVOT. The flow velocity envelope (FVE) is integrated to obtain the VTI. Heart rate is recorded. The cardiac output is then estimated using the following formula: CODoppler = [VTILVOT × (diameter of LVOT)2 × (0.785)] × heart rate. Several studies have shown reasonable correlations between Doppler and invasively derived cardiac output [51]. In a cohort with moderate or severe PH, Doppler-estimated cardiac output correlated reasonably well with invasively derived cardiac output (r = 0.74); however, there were substantial discrepancies between the values on an individual patient basis. Using a simpler approach, an RVOT VTI less than 12 cm predicted a cardiac index less than 2.2 with a sensitivity and specificity of 86 and 71%, respectively [29]. Likewise, a TAPSE less than 1.8 cm or an S¢ less than 10 cm/s is a strong predictor of a low cardiac index [29, 34]. Thus, even if precise cardiac output estimation is not feasible, easily obtained Doppler and 2D echocardiographic measures can still provide useful information for identifying patients with a low cardiac index.

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5.3 Integrated Assessment of RV–PA Interaction Doppler interrogation in the RVOT can provide important information in the patient with known or suspected PVD, as this is a key anatomic and physiologic interface between the RV and the pulmonary vasculature. Pulsed-wave Doppler interrogation in the RVOT is performed just proximal to the pulmonic valve, with the goal being to obtain an FVE without pulmonic valve artifact. The AcT (measured as the time from baseline to peak velocity) is measured from this envelope, and has been shown to strongly correlate with mean PA pressure, as mentioned already [25]. Aside from the AcT however, the shape of the FVE can be used to qualitatively differentiate differing levels of PH. A rounded FVE has been associated with normal PA pressures, whereas patients with mild-moderate PH more often have a triangular, or dagger-shaped FVE (often with late-systolic notching), and those with severe PH typically manifest prominent midsystolic notching of the FVE (Fig. 10). However, these same authors pointed out that only 53% of patients with PH evidenced a notched FVE, suggesting that pressure is an associate, but not the actual physiologic cause, of this notching pattern. Later work in animal models showed that midsystolic flow deceleration in the RVOT temporally coincides with the (early) arrival of reflected arterial waves from the pulmonary vasculature, leading to “real-time” impedance to flow [52]. It is generally believed that wave reflection, and thus midsystolic notching, is more prominent in the setting of acute and chronic pulmonary emboli, relating to the introduction of a more proximal site of wave reflection owing to proximal clot burden [53, 54]. However, from a physiologic standpoint, early arrival of reflected waves from the pulmonary vasculature may occur without proximal obstruction as long as the two physiologic requisites for early arrival of reflected arterial waves are present: high PVR and decreased large artery compliance, which impact the proportion­of the incident wave that is reflected backwards and the reflected wave speed, respectively. In support of this, Torbicki et al. showed that the time to midsystolic flow deceleration of the FVE did not differ between patients with chronic thromboembolic PH (CTEPH) and patients with idiopathic PAH (IPAH) [53]. Thus, exaggerated wave reflection is likely present in other forms of PH (i.e. PAH) aside from thromboembolic disease, and that analyses of RVOT Doppler FVE may be applied more broadly to detect PVD in a referral population of PH patients. As such, this information may help quickly determine not only if a patient has PH, but also whether the PH is occurring owing to PVD. Recent work reported on the hemodynamic differences seen among a cohort of 79 patients (patients with acute and chronic pulmonary emboli excluded) with undifferentiated

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p­ ulmonary pressure, and helps explain why notching is present in some, but not all, patients with PH. Furthermore, when we examined hemodynamic differences based on the position or timing of the notch in systole, the PVR was far higher in those with early/midsystolic (Fig.  10c) notching (PVR 12.4 WU) than i those with late systolic (Fig. 10b) notching (PVR 6.4 WU). Those with early/midsystolic notching also had a lower stroke volume index (27 vs. 39  ml/m2) and TAPSE (1.65 vs. 1.80 cm), consistent with more advanced RV dysfunction. Taken together, these data suggest that this simple measurement may possess a large amount of physiologic information; the presence of a notched FVE strongly suggested PH from a pulmonary vascular cause, with earlier arrival of the notch typically coinciding with the patient phenotype with the most advanced PVD and right-sided heart dysfunction. As such, notching may be a simple noninvasive indicator of RV–PA uncoupling. Potential future applications of FVE analysis include its use as a screening tool to predict present or future development of PAH in high-risk conditions such as scleroderma, and whether serial changes in notching coincide with clinical and hemodynamic changes in PVD patients on ­medical therapy.

6 DE Findings in PVD 6.1 Pulmonary Arterial Hypertension

Fig. 10  Pulsed-wave Doppler tracings obtained from the RV outflow tract. The flow velocity envelope can be rounded, which is typically seen in patients with either normal pulmonary artery pressure or mild pulmonary hypertension (a), triangular or dagger-shaped (often with an end-systolic notch), associated with moderate pulmonary hypertension (b), or may have a more pronounced midsystolic notching pattern, associated with severer pulmonary hypertension (c)

PH [55]. In contrast to early reports, patients without notching of the FVE may still have PH, albeit more mild than in patients with a notched FVE (mean PA pressure 33 vs. 46 mmHg). More striking, however, were the differences in PA pressure composition; patients without notching (Fig. 10a) had an average PVR of 3.8 WU, with a PA wedge pressure of 20 mmHg, whereas those with a notched FVE (Fig. 10b, c) averaged a PVR of 10.1 WU with a wedge pressure of 15 mmHg. Similarly, the large artery compliance of subjects with a notched FVE was approximately half that of subjects without notching. The sensitivity and specificity of notching for a PVR greater than 6 WU was 98 and 88%, respectively. These findings suggest that notching of the FVE is more indicative of PVD (high PVR, low compliance) than

The relatively nonmuscular, compliant RV is put at a distinct physiologic disadvantage in PAH and other PVD states, yet these same characteristics allow the RV to act as a relatively reliable “transducer” of its increased vascular load. Therefore, the triad of declining RV systolic function, size and shape changes, and septal bowing forms the fundamental basis for the echocardiographic recognition of PAH, with or without demonstration of an increase in Doppler-estimated PASP. In 1973, Goodman et al. described the echocardiographic features of IPAH [56]. They noted that all patients had RV dilatation, and half had an RV dimension greater than their LV dimension. In addition, patients commonly demonstrated abnormal, or paradoxical, septal motion, where the interventricular septum moved away from the center of the LV cavity in systole. This occurs when the curvature of the septum is convexed toward the LV at end-diastole, such that LV systole serves to “push” the septum toward the RV. Aside from being a common echocardiographic feature in PAH, the relative loss of inward septal motion can account for a mild loss of LV systolic function in PAH and thus a low-normal or mildly reduced LV ejection fraction in PAH [57]. Bossone et  al. reported in a larger series of IPAH patients that 96% of patients had a Doppler-estimated pressure greater than

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60  mmHg at diagnosis; however, the overall correlation between Doppler and invasive pressure estimates was relatively low (r = 0.31). The time to peak velocity (AcT) in the RVOT was less than 100 ms in 94% of subjects. Ninety-eight percent of subjects had RV enlargement, whereas three quarters of the patients had qualitatively reduced RV systolic function. Systolic flattening of the interventricular septum occurred in 90% of subjects. Moderate or severe tricuspid regurgitation occurred in 80% of subjects, whereas mild to moderate mitral regurgitation was present in only one patient. A small or moderately sized pericardial effusion was noted in about 16% of subjects. The average ratio of transmitral Doppler E wave velocity to A wave velocity (E/A ratio) was 0.93, with 70% of subjects demonstrating “E to A reversal,” typically thought to denote LV diastolic dysfunction. Importantly, all subjects in this study were shown to have normal left atrial pressure by right-sided heart catheterization, underscoring the fact that Doppler evidence of “diastolic dysfunction” is not synonymous with left atrial hypertension. In fact, the pattern of E to A reversal, also referred to as “grade I diastolic dysfunction,” typically equates to normal left atrial pressure, reflecting the necessary redistribution of LV filling into late diastole that must occur when LV relaxation is impaired in the context of a normal LV filling pressure [58]. In clinical practice, the presence of any or all of the triad of findings of RV dysfunction, accompanied by grade I diastolic dysfunction, is a strong indicator of severe PH in the context of a normal left atrial pressure and high PVR. Subsequent studies have reported on the clinical and prognostic significance of many of the typical DE findings in PAH. Earlier studies focused on more indirect measures of RV dysfunction, such as right-sided heart dilatation, septal bowing, pericardial effusion, and tricuspid regurgitation severity. Eysmann et al. showed that the presence and severity of a pericardial effusion is a strong independent predictor of mortality in PAH. Similarly, a mitral E/A ratio less than 1.0 was a strong predictor of adverse outcome [59]. Raymond et al. also showed that the presence of a pericardial effusion and with an increased right atrial area index were predictive of mortality [60]. Eysmann et  al. showed that a peak pulmonic flow velocity greater than 60 cm/s was predictive of a drop in PVR of more than 30% on acute vasodilator testing; none of the patients with a peak velocity of less than 60 cm/s were vasoreactive [61]. Likewise, decreased LV and left atrial area, increasing RV-to-LV area ratio, and the degree of leftward septal bowing are also associated with an increased risk of systemic hypotension in response to calcium channel blocker therapy, likely reflecting the inability to sufficiently recruit RV stroke volume to overcome the drop in systemic vascular resistance [62]. Subsequent studies focused on more direct assessment of RV systolic function. In a retrospective study by Tei et al. the mean RV MPI of patients with IPAH (0.83) was higher than

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in healthy controls (0.28), and patients with an RV MPI of 0.83 or greater had less than 10% event-free survival at 5 years, versus more than 70% in those with an RV MPI less than 0.83 [36, 63]. A prospective study in patients with IPAH and PAH associated with connective tissue ­disease showed that a TAPSE less than 1.8  cm was associated with lower invasively derived stroke volume index, higher right atrial pressure, greater RV dilatation, right heart to left heart disproportion, septal bowing, and a higher incidence of pericardial effusion. Two-year ­survival estimates were 88 and 50% for patients with a TAPSE of 1.8 cm or more and 1.8 cm or less, respectively [29]. The prognostic value of TAPSE persisted after adjusting for several echocardiographic and invasive hemodynamic variables of known prognostic significance. The typical changes in RV size, function, and septal position have been shown to improve in response to various therapies. Following 4 months of therapy with bosentan in patients with PAH (84% IPAH, remainder PAH associated with connective tissue disease), serial DE examination showed an improved RV MPI, reduced RV end-systolic area and RV-to-LV diastolic area ratio, improved early diastolic LV filling, and decreased pericardial effusion scores [64]. Similarly, following 3 months of intravenous epoprostenol, RV size decreased as did peak tricuspid regurgitation ­velocity and the noninvasive PVR estimate; the RV MPI did not change in response to epoprostenol infusion [65]. Interestingly, none of these echocardiographic parameters correlated with changes in New York Heart Association (NYHA) functional class in this relatively small cohort. In contrast, preliminary data showed that 21 of 27 subjects with PAH increased their TAPSE by more than 0.2  cm (mean +0.5  cm) after 6 months of PH-specific therapy, and that these subjects exhibited improvements in 6-min walk distance (median +115  m), B-type natriuretic peptide level (−210 pg/ml), and NYHA class (−1.0 functional class). There were no deaths or right-sided heart failure admissions in those with a TAPSE of 2.0 cm or more at 6-month follow-up, but there were two deaths and four right-sided heart failure hospitalizations in those with a TAPSE less than 2.0 cm on follow-up [66]. Given the limited clinical and prognostic value of serial pressure assessment in PAH, along with concerns with the accuracy of Doppler-estimated PASP, most centers do not routinely follow or titrate therapy on the basis of changes in DE-estimated PA pressure. Further study is needed to more clearly determine whether serial changes in various DE parameters will provide important functional and prognostic information in patients with PAH. Studies need to address whether changes in various DE parameters have similar clinical and prognostic value as invasive hemodynamic assessment, and how such information can be used to determine who is responding favourably and who requires escalation of medical therapy or referral for lung transplant evaluation. It will also be important to

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determine whether certain threshold values exist for various DE parameters, such that we can try and add these values to the goal-directed therapeutic approach in PAH.

6.2 Chronic Pulmonary Embolism CTEPH is a relatively rare complication of prior acute ­pulmonary emboli, occurring in some 0.5% of cases [67, 68]. The major site of vascular impedance is often the proximal pulmonary vasculature, owing to the persistence of clot and vessel remodeling in the affected area. However, small vessel pulmonary arteriopathy is also known to occur in CTEPH, such that the degree of PVR elevation may be equal to or greater than that seen in IPAH [69]. The end result is often that the overall burden of PVD is comparable to that in PAH, and in some cases is worse owing to the added hemodynamic load from the proximal obstruction. Thus, the typical triad of RV dysfunction, RV dilatation, and septal bowing is present in CTEPH and is largely indistinguishable from the various forms of PAH. Nevertheless, there are ­hemodynamic ­differences between CTEPH and PAH that can aid in differentiating the two conditions. Namely, in CTEPH, the presence of a discrete proximal obstruction leads to more exaggerated, earlier arrival of arterial reflected waves and thus a more pulsatile pulmonary vascular bed [54]. This increased pulsatility can be expressed as the fractional pulse pressure (FPP = PA pulse pressure/mean PA pressure); the typical FPP is 0.8 in PAH and 1.4 or more in CTEPH [70, 71]. The FPP can also be estimated by Doppler, with a noninvasive FPP of 1.35 predicting CTEPH with a sensitivity of 95% and specificity of 100% over IPAH [72]. Serial DE examination following pulmonary endarterectomy (PEA) has shown increases in RVFAC as well as reductions in RV size, decreased septal bowing, and improved early diastolic LV filling [73, 74]. An interesting study by Hardziyenka et al. showed that patients with early arrival of arterial reflected waves, as measured by a shorter time to notching of the pulmonary FVE, were the only CTEPH patients to benefit from PEA [75]. These findings prove that simple FVE analysis was able to predict clinical response to PEA and that a requisite degree of pulmonary vascular impedance and arterial wave reflection is needed for sufficient RV–PA recoupling to occur following surgical intervention.

6.3 Acute Pulmonary Embolism Acute pulmonary embolism, and especially large, proximal emboli, leads to a sudden increase in the pulmonary vascular impedance without a marked rise in the PA pressure. This

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relates to the fact that the normal RV is typically incapable of generating a sufficient stroke volume to maintain a PASP greater than 60 mmHg (mean pressure of 40 mmHg) when suddenly uncoupled from a normal pulmonary vascular bed [76, 77]. As a result, RV failure and cardiogenic shock may ensue despite a mean PA pressure between 20 and 40 mmHg [78]. Thus, it follows that the degree of RV dysfunction predicts the extent of perfusion defects by ventilation–perfusion scan [79]. In contrast, small subsegmental pulmonary emboli that are not associated with a marked rise in PA impedance will not lead to abnormal DE findings, highlighting the fact that the DE examination is insufficient to rule out an acute pulmonary embolism [80]. The relative degree of PH helps establish the chronicity of the embolic event, as the average transtricuspid flow velocity in acute proximal pulmonary emboli (3.0 m/s) is significantly lower than in patients with subacute pulmonary emboli (4.2  m/s) [77]. In contrast, patients with acute pulmonary emboli with a PASP greater than 50 mmHg at the time of diagnosis are at increased risk of persistent PH at 1-year follow-up [81]. From a physiologic viewpoint, acute pulmonary embolism is an important and often dramatic clinical example underscoring the concept that PA pressure is a poor measure of RV afterload. In fact, the relative disconnect between vascular impedance and PA pressure forms the basis for several relative “signature” DE findings in the setting of an acute pulmonary embolism. RV dilatation is almost universally present in the setting of a large pulmonary embolism, as the normally compliant RV will rapidly distend in response to the increased vascular load. However, unlike in PAH or other forms of chronic PVD, RV hypertrophy will be conspicuously absent, and thus a simple qualitative clue for acute pulmonary embolism is RV dilatation and dysfunction in the relative absence of RV hypertrophy. Another common DE finding in acute pulmonary embolism is “McConnell’s sign,” which is visually appreciated as RV dysfunction along the RV base and mid segments, with a hinge point or “buckling” of the RV free wall near the RV apex [82]. This relative “sparing” of RV apical function most likely relates to tethering of the RV apex by a normal or hyperdynamic LV, via forces transmitted across the interventricular septum. Others have shown that the RV ejection fraction, RV TDI (S¢), and TAPSE are reduced in the setting of acute pulmonary embolism [83]. A characteristic Doppler finding in acute pulmonary embolism is the “60/60 sign,” which refers to a markedly shortened AcT (less than 60  ms) in the FVE owing to the increased PA impedance, combined with a PASP below 60 mmHg [84]. Another important finding to look for on the initial DE examination is right-sided heart thrombi or thrombi in transit, which complicate approximately 4% of patients with acute pulmonary emboli [85]. Thrombi may be visualized within the right atrium, RV, and occasionally are seen straddling

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the interatrial septum. Thrombi in the right side of the heart are a very high risk feature, typically associated with prior massive pulmonary embolism, severe RV dysfunction, and high in-hospital mortality [85]. In such cases, thrombolysis, catheter-based intervention or surgical embolectomy is typically warranted [85, 86].

6.4 Systemic-to-Pulmonary Venous Shunts A detailed overview of the various DE perturbations associated with the numerous forms of congenital systemic-to-pulmonary shunts (SPS) is beyond the scope of this chapter. Suffice to say that a variety of 2D and Doppler findings are associated with shunting at the venous, atrial, and ventricular levels. We will focus on the general principles of RV structure and function and basic shunt assessment in this context. In the case of smaller shunts, or larger shunts at an earlier stage in the disease process, the RV is primarily subjected to an increased volume load. Owing to the relative compliance of the RV, volume loading is generally well tolerated, which is likely why these lesions can go unnoticed for decades. In this circumstance, the primary DE finding is RV dilatation, typically with normal or hyperdynamic function. The increased RV function is due to increased RV performance occurring on the basis of increased preload; so-called Starling effect. Thus, a dilated and hyperdynamic RV is an important initial clue to the presence of a left-to-right shunt. On the short axis, there is often bowing of the interventricular septum from right to left that is isolated to diastole. These patients have differing degrees of PH, typically occurring on the basis of marked increases in transpulmonary blood flow. Herein lies another example of the disconnect between PA pressure and RV afterload, as the increased pressure is due to, but not opposing, pulmonary blood flow. With time, excess pulmonary blood flow can lead to marked remodeling of the pulmonary vasculature, with the PVR reaching levels equal to or greater than that seen in other forms of PAH. Under these conditions, the RV must accommodate the persistent volume load as well as a marked increase in RV afterload. However, unlike other forms of PAH, in the setting of a SPS, the rise in RV afterload occurs relatively slowly, often over decades. As a result, the structure and function of the RV is often markedly different in patients with chronic SPS. Typically, the RV cavity is normal in size, maintains a relatively normal proportion relative to the LV, and exhibits marked concentric hypertrophy [87]. Marked RV hypertrophy can often be appreciated by both thickening and excess RV trabeculations, along with a very conspicuous moderator band; these findings alone can often help distinguish PAH related to a SPS from other forms of PAH.

P.R. Forfia

An initial shunt assessment is often performed using rapid injection of agitated saline (“bubble study”), which opacifies the right chambers of the heart and allows for detection of both right-to left-shunts (early passage of bubbles from right to left) and left-to-right shunts (via a negative contrast jet created from the passage of blood into the opacified right atrium or right ventricle). Shunt quantification by Doppler is expressed in the same way as measured by cardiac catheterization, by the ratio of pulmonary to systemic blood flow. Doppler flow in the RVOT (Qp) and that in the LVOT (Qs) are assessed to derive the Doppler Qp/Qs ratio. The RVOT diameter is typically about 0.5–1.0 cm greater than the LVOT diameter; given that flow is equal to the product of crosssectional area and velocity, it follows that the integrated flow velocity signal (VTI) in the LVOT is typically less than the VTI in the RVOT. Thus, an initial clue to a sizeable left-toright shunt is when the RVOT VTI is equal to or greater than the LVOT VTI. Shunt localization by DE is performed by first assessing for 2D evidence of interatrial or interventricular septal defects. Further characterizations, including blood flow direction, blood flow velocity, and estimated pressure gradients across shunts can be performed by color, pulsedwave, and continuous-wave Doppler examinations. Proper DE examination in the case of known or suspected congenital heart disease is best performed by a sonographer/echocardiographer who has been trained in the assessment of patients with congenital heart disease. Determination of the full extent and the characterization of shunts requires subsequent cardiac catheterization.

7 DE Findings in Pulmonary Venous Hypertension By far the most common cause of PH relates to passive elevation of the PA pressure owing to an increase in left atrial pressure. A variable degree of “reactive” pulmonary vascular remodeling may occur, typically relating to the severity and chronicity of the left atrial hypertension [88, 89]. As a result, patients with pulmonary venous hypertension compose a large percentage of patient referrals for PH evaluation. Thus, an understanding of the noninvasive manifestations of pulmonary venous hypertension is critically important in the initial approach to patients with undifferentiated PH. It follows that the noninvasive predictors of pulmonary venous hypertension include 2D and Doppler findings that either reflect or predispose to left atrial hypertension, as well as more direct Doppler estimates of increased left atrial pressure [90, 91]. Importantly, the LV ejection fraction is a very poor predictor of left atrial hypertension, and thus a normal ejection fraction in no way provides reassurance that the left atrial pressure is normal.

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The 2D echocardiographic examination can provide c­ ompelling initial evidence that either supports or refutes the presence of left atrial hypertension. One of the first clues to past or present left atrial hypertension is left atrial enlargement. This should certainly not be considered a physiologic measurement of left atrial pressure; however, increasing left atrial size typically denotes chronically increased left atrial impedance and either the presence of or predisposition for increased left atrial pressure. Left atrial enlargement is best appreciated in the parasternal long-axis view, with a left atrial dimension at end-systole of 4.0 cm or greater generally being the accepted cutoff. As an initial visual clue to left atrial enlargement, one can simply look at the size of the left atrium relative to the aortic root, which is seen just anterior to the left atrium in this view; when the left atrial size exceeds the aortic root size, this typically indicates left atrial enlargement. Another important finding is the presence of LV hypertrophy; even when mild, it denotes chronic remodeling of the LV, typically the result of long-standing systemic hypertension. This is a significant form of structural left-sided heart disease, and is typically associated with increased LV chamber­ stiffness. Melenovsky et al. showed that left atrial size (expressed as left atrial volume), LV mass index, and the product of these two variables were the strongest discriminants of nonsystolic (diastolic) heart failure from hypertensive heart disease without a history of heart failure [92]. In the context of pulmonary venous hypertension, the interventricular septum will typically maintain a normal configuration on short-axis imaging, convexed toward the RV. Thus, the LV cavity will retain its round shape in both systole and diastole, with no evidence of septal flattening. In the apical four-chamber view, the ratio of RV-to-LV dimension (or area) will typically remain less than 1.0, with the RV apex maintaining its sharp apical angle, with the LV forming the apex of the heart alone. The Doppler examination also provides important physiologic information with respect to valvular function, diastolic function, and left atrial pressure assessment. As a general statement, moderate or greater dysfunction of either the aortic valve or the mitral valve is relatively uncommon when PVD is the primary source of the increased PA pressure. This is particularly true of the mitral valve. Of course, moderate or greater mitral stenosis is well known to cause severe PH, largely on the basis of passive pulmonary venous congestion. However, moderate or greater mitral regurgitation is also distinctly uncommon in the setting of PAH, and provides fairly compelling evidence for a pulmonary venous source of PH [58]. Importantly, mitral regurgitation severity can be underestimated by transthoracic Doppler imaging, especially when the mitral regurgitation jet is eccentric and off axis. Thus, complete Doppler interrogation using multipledifferent imaging planes is often required to appreciate the full extent of the mitral regurgitation. If there is clinical

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suspicion that the degree of mitral regurgitation is greater than reported by transthoracic DE examination, transesophageal echocardiography should be performed to obtain a more comprehensive structural and physiologic assessment of the mitral valve [93, 94]. Pulsed-wave Doppler evidence of systolic pulmonary vein flow reversal also indicates severe mitral regurgitation, representing the Doppler associate to large V waves in the left atrial pressure tracing [94]. Lastly, Doppler interrogation of the mitral valve inflow provides a key component to the patient with known or suspected PH. In normal sinus rhythm, LV filling can be divided into an early, passive phase and a late, active phase. Pulsedwave Doppler interrogation across the mitral valve allows for quantification of these events, with the E wave and A wave Doppler envelopes corresponding to the early and late diastolic events, respectively. In normal individuals, in the absence of structural left-sided heart disease, the E/A ratio is greater than 1.0 with a transmitral E wave deceleration time (DT) between 160 and 240 ms, indicating that the bulk of LV filling occurs passively in early diastole as left atrial pressure exceeds diastolic ventricular pressure; atrial contraction serves to complete LV filling at end-diastole. The transmitral pattern can be altered in one of three ways, often expressed as grade I, II, and III diastolic dysfunction [59]. In grade I diastolic dysfunction, the E/A ratio is less than 1.0 with a DT greater than 240 ms, denoting a redistribution of LV filling into late diastole due to impaired LV relaxation and typically a normal left atrial pressure. Grade I diastolic dysfunction is a common feature seen in healthy persons over the age of 60; however, it may occur at any age in persons with structural left-sided heart disease (i.e. LV hypertophy/hypertensive heart disease, impaired LV systolic function). As mentioned previously, an E/A ratio less than 1.0 is also a common finding in PAH, and is typically caused by RV enlargement, septal bowing, and an abnormal diastolic ventricular interaction that impedes LV relaxation. Thus, the presence of grade I diastolic dysfunction should not dissuade one from suspecting PAH or another PVD-related cause of PH, particularly when accompanied by features of the RV dysfunction triad. In fact, this constellation of findings suggests the PH is occurring in the context of a normal left atrial pressure. Grade II diastolic dysfunction, referred to as the pseudonormal transmitral pattern, occurs when there is structural left-sided heart disease and/or age exceeding 60 years with an E/A ratio between 1 and 1.5 and a DT between 160 and 240 ms. This represents a more advanced stage of diastolic dysfunction, as left atrial hypertension is typically manifest. In grade III diastolic dysfunction, or restrictive transmitral filling, the E/A ratio typically exceeds 2.0 with a DT below 160  ms; a restrictive transmitral Doppler filling pattern is strongly suggestive of an increased left atrial pressure (Fig. 11). Thus, grade II and grade III diastolic dysfunction are strongly suggestive of an increased left atrial pressure at

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P.R. Forfia Table 1  2D and Doppler features of pulmonary arterial and pulmonary venous causes of pulmonary hypertension (PH) Pulmonary arterial cause 2D echocardiography Normal LA size Normal or small LV cavity size No LVH Normal or increased LVEF RV-to-LV dimension ³1.0 RV apex forming Rounded RV apical angle D-shaped LV cavity (septal bowing; systole and/or diastole) Pericardial effusion

Pulmonary venous cause Dilated LA Normal or dilated LV cavity size LVH Variable LVEF RV-to-LV dimension <1.0 RV not apex forming Sharp RV apical angle Round LV cavity (no septal bowing) No pericardial effusion

Doppler Variable PASP MR grade £1 + E < A Notching of FVE in RVOT

Fig. 11  Pulsed-wave Doppler recordings across the mitral valve. (a) The mitral inflow pattern representing grade I diastolic dysfunction, often referred to as “E to A reversal.” This is a common finding in healthy persons over the age of 65, as well as in patients with structural left-sided heart disease at any age with impaired LV relaxation. In addition, this pattern is commonly seen in pulmonary arterial hypertension, highlighting how decreased RV function and increased RV size increase ventricular interdependence and impair early LV diastolic filling. Importantly, grade I diastolic dysfunction typically denotes normal resting left atrial pressure. The scout image shows marked RV enlargement; this patient has pulmonary arterial hypertension. (b) Grade II diastolic dysfunction, often referred to as a “pseudonormalized” filling pattern. This typically occurs when an individual with impaired LV relaxation, typically on the basis of structural left-sided heart disease, develops an increased left atrial pressure; commonly, these subjects will revert from grade II to grade I diastolic dysfunction after diuresis and return of the left atrial pressure to normal. This patient had mild-moderate pulmonary hypertension with a pulmonary artery wedge pressure of 22 mmHg and a pulmonary vascular resistance of 1.2 WU. (c) Restrictive transmitral filling, highly suggestive of a markedly increased left atrial pressure. In this case, the patient had moderate to severe pulmonary hypertension that was almost entirely explained on the basis of a markedly increased pulmonary artery wedge pressure (33 mmHg). The transmitral filling patterns shown in b and c are uncommon in patients with pulmonary arterial hypertension

the time of DE examination is performed and are thus uncommon findings in PAH. A PASP between 40 and 70  mmHg (mean PA pressure of approximately 25–45  mmHg) in the presence of pseudonormal or restrictive transmitral flow is strongly suggestive of pulmonary venous hypertension as the dominant source of PH. If this is coupled with normal septal

Variable PASP MR grade ³2 + E > A with pseudonormal or restrictive transmitral flow No notching of FVE in RVOT Pulmonary arterial causes include pulmonary arterial hypertension (WHO group I PH), chronic thromboembolic PH and WHO group III PH with a normal left atrial pressure and pulmonary vascular resistance greater than 3 WU LA left atrium, LV left ventricle, LVH left ventricular hypertrophy, LVEF left ventricular ejection fraction, RV right ventricle, PASP, Dopplerestimated pulmonary artery systolic pressure, MR mitral regurgitation, E early mitral diastolic inflow velocity, A atrial, or late, mitral diastolic inflow velocity, FVE flow velocity envelope, RVOT right ventricular outflow tract

position as well as normal RV size and shape, the diagnosis of pulmonary venous hypertension becomes almost certain.

8 Integration of the DE Examination into the Initial Diagnostic Assessment of Undifferentiated PH From a clinician’s perspective, a useful initial diagnostic framework considers whether a patient’s PH is from a ­pulmonary venous (“postcapillary”) or a pulmonary arterial (“precapillary”) source. This approach is both rational and practical, given the prevalence of pulmonary venous ­hypertension, as well as the fact that the diagnostic and ­therapeutic considerations for these patients differ considerably­. Table 1 highlights some of the salient 2D and Doppler features that help distinguish pulmonary venous sources from pulmonary arterial sources of PH. In the context of known or suspected PH, the accumulation­ of typical 2D and Doppler features suggestive of a pulmonary venous source of PH makes the presence of PAH or another pulmonary vascular source (i.e. CTEPH) of PH progressively less likely. Conversely, the relative absence of stigmata of

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left-sided heart disease and/or left atrial hypertension along with features of the RV dysfunction triad dramatically increases the likelihood of underlying PVD. We recently integrated selected 2D and Doppler findings from the routine clinical DE examinations of 76 consecutive patients with undifferentiated PH to determine if a simple echocardiographic scoring system could help determine the pathophysiology of the PH in these patients [95]. All patients underwent diagnostic right-sided heart catheterization following their initial assessment. The DE was scored on the basis of 2D/Doppler evidence suggesting pulmonary venous hypertension (LV hypertrophy; left atrial enlargement; pseudonormal/ restrictve transmitral Doppler; yes = 0, no = 1) or suggesting PVD/RV dysfunction (septal bowing; RVto-LV ratio 1 or greater; TAPSE less than 2.0; yes = 1, no = 0) and Doppler “notching” of the FVE in the RVOT (yes = 2, no = 0). The scoring was designed such that a low score (0–2) would indicate pulmonary venous hypertension, whereas a high score (6–8) would strongly suggest PVD; an intermediate score (3–5) would suggest a combination of pulmonary venous hypertension and PVD. Patients with an echocardiographic score from 0 to 2 were older (70 ± 10 years), had mild-moderate PH (mean PA pressure 33 ± 8 mmHg), an increased PA wedge pressure (18 ± 7 mmHg), with an average PVR of only 2.8 WU. In contrast, patients with an echocardiographic score of 6–8 were younger (57 ± 15 years), had severe PH (50 ± 7 mmHg), a normal wedge pressure (12 ± 5 mmHg), with an average PVR of 10 ± 4 WU. The patients with a score of 3–5 also had severe PH, yet associated with higher wedge pressure and less marked elevations in the PVR. All subjects with a score of 6 or greater had a PVR of 4.5 WU or greater; one of 24 subjects with score of less than 3 had PAH; no subjects with a score of 6 of greater had pulmonary venous hypertension. Thus, these preliminary data suggest that adding a relatively simple scoring algorithm to a standard DE examination may assist in the rapid identification and triage of patients with PH related to PVD for further invasive diagnostic testing.

9 Exercise Echocardiography Exercise DE is being used with increasing frequency in the assessment of patients with known or suspected PVD as well in the context of dyspnea of unclear cause. In the former case, the presence or absence of exercise-induced PH is thought to increase the diagnostic sensitivity of the DE examination for the early stages of PVD; in the latter case, a rise in PA pressure is often thought to provide “proof” that PH as an important source of a patient’s dyspnea. Although conceptually attractive, exercise DE assessment and interpretation has multiple important limitations that need be addressed to best apply this modality in clinical practice.

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The first issue to address is whether there should be a rise in PA pressure with exercise. The time-honored teaching on this subject is that the PA pressure should not increase with exercise. However, studies do not support this and indicate that exercise-induced PH is more common than was once appreciated. In 1966, Damato et al. reported on the hemodynamic responses to 24 normal healthy male volunteers who underwent upright treadmill exercise with a PA catheter in place. The average peak oxygen consumption (VO2) was 30  ml O2/kg/min (approximately 8–9 metabolic equivalents), indicating normal, but not elite exercise performance. At peak exercise, ten of the 24 normal subjects had a mean PA pressure greater than 30 mmHg, thus meeting the definition of exercise-induced PH; the increases in PA pressure correlated with increasing cardiac output and the pressureflow relationship and total pulmonary resistance remained normal in all subjects [96]. In another cohort of more fit subjects, bicycle exercise up to 240  W led to an average increase in the mean PA pressure of 37  mmHg [97]. Similarly, Bossone et  al. showed that highly conditioned college athletes routinely increased their Doppler-estimated PASP to more than 50  mmHg at peak exercise, whereas healthy nonathletes typically did not exceed a PASP of 30  mmHg [98]. The degree of PASP elevation correlated best with changes in stroke volume, again indicating a normal pressure-flow relationship. The second important limitation of exercise echocardiographic assessment of PA pressure is that changes in left atrial pressure are not accounted for during exercise DE. This is critical, given that exercise-induced PH (in health and in disease) most commonly arises from the interaction between an increased cardiac output and a rise in the left atrial pressure [97, 99]. To the contrary, Laskey et al. showed that the PVR of patients with IPAH remained elevated during exercise, but was unchanged from resting values [100]. Thus, exercise-induced pulmonary vasoconstriction is not typical, even in patients with established, severe PVD. The third limitation of exercise DE is the accuracy of Doppler PA pressure assessment. Also, age likely exerts an important influence on the relative change in PA pressure with exercise, owing to age-related increases in pulmonary vascular stiffness; thus, exercise-induced PH, even at relatively low workloads, may be a part of the normal aging process, similar to the exaggerated increase in systemic arterial pressure with aging [101]. Lastly, it seems that the systemic blood pressure response to exercise should be factored into the interpretation of an exercise-induced rise in PA pressure. For example, a rise in PASP from 35 to 70  mmHg in the context of a rise in systemic systolic pressure from 120 to 210 mmHg indicates that the relative proportion of pulmonary to systemic blood pressure has remained quite similar from rest to exercise, thus questioning the clinical significance of the rise in PA pressure in this context.

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Investigators have also used exercise echocardiography to help identify latent PVD in high-risk groups such as those with scleroderma. Steen et al. showed that 24 of 54 scleroderma patients at high risk for PAH had an increase of 20 mmHg or more with exercise DE examination; 21 of the 24 underwent right-sided heart catheterization. Of these subjects, four had resting PH and 17 showed mild exerciseinduced PH by catheterization. The average PVR at the end of exercise was less than 3 WU [102]. Others have defined exercise-induced PAH to be present if the mean PA pressure increased to more than 30  mmHg with a wedge pressure below 20 mmHg and a PVR that need only be more than 1 WU [103]. Though defined differently, both studies confirmed some degree of exercise-induced PH in these subjects; however, at such low PVR values, the physiologic and clinical significance of these findings is less clear. Interestingly, Reichenberger et  al. observed Doppler-estimated increases in PASP with exercise in 16 of 33 patients with scleroderma (mean PASP 47 mmHg at a peak VO2 of 64% predicted), yet only one of the 16 subjects with exercise-induced PH developed PAH over a 3-year follow-up [104]. Taken together, one must exercise caution in the interpretation of a rise in Doppler-estimated PA pressure with exercise, as even when accurate, a rise in PA pressure with exercise does not necessarily implicate the pulmonary vasculature as the source of PH, that the PH is appropriate for PH-specific therapies, nor whether the PH is the source of dyspnea in an individual patient. Regarding this last point, patients with chronic obstructive pulmonary disease and PH demonstrated the same aerobic exercise capacity as chronic obstructive pulmonary disease patients without PH; in both groups, exercise capacity was limited by exhaustion of the ventilatory reserve and did not correlate with PA pressure [105]. Thus, the demonstration of a rise in PA pressure with exercise in many cases amounts to an association, but not a diagnosis, as to the source of dyspnea. For many of these reasons, the recently published expert consensus document from the American College of Cardiology/ American Heart Association recommends that at this time no treatment decisions be made on the basis of exercise echocardiography [13]. An alternative approach to exercise DE may be to focus on the RV functional response to exercise, especially considering that impaired RV functional reserve is the mechanism of exercise limitation in PAH and other forms of PVD [106]. This approach may provide a more direct and clinically relevant answer as to whether a patient has clinically significant latent PVD or PH as their cause of dyspnea. If the RV function augments normally with exercise, the rise in PA pressure would likely be flow-mediated and of far less clinical significance. In contrast, a dilating and non-augmenting RV would seem to indicate an impedance-mediated process of greater clinical significance.

P.R. Forfia

10 Summary The DE is a central part of the initial diagnosis and assessment of patients with PVD and other forms of PH. Often, the results of the initial DE examination are pivotal in the decision-making process for which patients are referred for invasive hemodynamic assessment of their PH. As a result, it is important for the clinician to fully appreciate the echocardiographic features associated with underlying PVD, as a missed or delayed diagnosis can have devastating consequences. Overreliance on any single DE metric, and especially PA pressure estimation, detracts from the overall diagnostic potential of the DE examination. The RV often bears witness to perturbations in the RV–PA interaction, with stereotypical changes in the size, shape, and function of the RV often leading to the diagnosis of PVD even without a reliable noninvasive pressure assessment. Thus, clinical DE protocols should include basic measurements of RV size, the relative proportion of the RV to the LV, and a direct measurement of RV systolic function. This is especially important when considering that the ­morbidity and mortality in PVD rests almost entirely on the ability­of the right side of the heart to adapt to its afterload. Attention should be directed toward the shape or pattern of the FVE leaving the RVOT, as this likely provides important physiologic information about the pulmonary vasculature and its interaction with the ejecting RV. Doppler evidence of diastolic dysfunction should be considered along a continuum that runs in parallel with rising left atrial pressure, and that right-sided heart failure itself is an important cause of impaired LV relaxation. Integrating the relative “balance” of findings for the right side and the left side of the heart along with proper Doppler interpretation often provides a wealth of clinical and pathophysiologic insight into the individual patient prior to invasive hemodynamic assessment. The end results are heightened awareness and improved identification of which patients should be referred for further invasive testing, as well the use of the DE information to complement the findings from invasive testing.

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1443 artery pressure from Doppler analysis of tricuspid regurgitant velocity spectrum in patients with chronic heart failure. Am Heart J 144:1087–1094 24. Stephen B, Dalal P, Berger M, Schweitzer P, Hecht S (1999) Noninvasive estimation of pulmonary artery diastolic pressure in patients with tricuspid regurgitation by Doppler echocardiography. Chest 116:73–77 25. Kitabatake A, Inoue M, Asao M et al (1983) Noninvasive evaluation of pulmonary hypertension by a pulsed Doppler technique. Circulation 68:302–309 26. Chemla D, Castelain V, Humbert M et al (2004) New formula for predicting mean pulmonary artery pressure using systolic pulmonary artery pressure. Chest 126:1313–1317 27. Schenk P, Globits S, Koller J et al (2000) Accuracy of echocardiographic right ventricular parameters in patients with different endstage lung diseases prior to lung transplantation. J Heart Lung Transplant 19:145–154 28. Ghio S, Recusani F, Klersy C et al (2000) Prognostic usefulness of the tricuspid annular plane systolic excursion in patients with congestive heart failure secondary to idiopathic or ischemic dilated cardiomyopathy. Am J Cardiol 85:837–842 29. Forfia PR, Fisher MR, Mathai SC et al (2006) Tricuspid annular displacement predicts survival in pulmonary hypertension. Am J Respir Crit Care Med 174:1034–1041 30. Lee CY, Chang SM, Hsiao SH, Tseng JC, Lin SK, Liu CP (2007) Right heart function and scleroderma: insights from tricuspid annular plane systolic excursion. Echocardiography 24:118–125 31. Hammarström E, Wranne B, Pinto FJ, Puryear Y, Popp RL (1991) Tricuspid annular motion. J Am Soc Echocardiogr 4:131–139 32. López-Candales A, Dohi K, Rajagopalan N, Edelman K, Gulyasy B, Bazaz R (2008) Defining normal variables of right ventricular size and function in pulmonary hypertension: an echocardiographic study. Postgrad Med J 84:40–45 33. Saxena N, Rajagopalan N, Edelman K, López-Candales A (2006) Tricuspid annular systolic velocity: a useful measurement in determining right ventricular systolic function regardless of pulmonary artery pressures. Echocardiography 23:750–755 34. Rajagopalan N, Saxena N, Simon MA, Edelman K, Mathier MA, López-Candales A (2007) Correlation of tricuspid annular velocities with invasive hemodynamics in pulmonary hypertension. Congest Heart Fail 13:200–204 35. Urheim S, Cauduro S, Frantz R et al (2005) Relation of tissue displacement and strain to invasively determined right ventricular stroke volume. Am J Cardiol 96:1173–1178 36. Yeo TC, Dujardin KS, Tei C, Mahoney DW, McGoon MD, Seward JB (1998) Value of a Doppler-derived index combining systolic and diastolic time intervals in predicting outcome in primary pulmonary hypertension. Am J Cardiol 81:1157–1161 37. Vogel M, Schmidt MR, Kristiansen SB (2002) Validation of myocardial acceleration during isovolumic contraction as a novel noninvasive index of right ventricular contractility: comparison with ventricular pressure-volume relations in an animal model. Circulation 105:1693–1699 38. Bommer W, Weinert L, Neumann A, Neef J, Mason DT, DeMaria A (1979) Determination of right atrial and right ventricular size by two-dimensional echocardiography. Circulation 60:91–100 39. López-Candales A, Dohi K, Iliescu A, Peterson RC, Edelman K, Bazaz R (2006) An abnormal right ventricular apical angle is indicative of global right ventricular impairment. Echocardiography 23:361–368 40. Imanishi T, Nakatani S, Yamada S et al (1994) Validation of continuous wave Doppler-determined right ventricular peak positive and negative dP/dt: effect of right atrial pressure on measurement. J Am Coll Cardiol 23:1638–1643 41. López-Candales A, Rajagopalan N, Gulyasy B, Edelman K, Bazaz R (2008) A delayed time of the peak tricuspid regurgitation

1444 signal: marker of right ventricular dysfunction. Am J Med Sci 336:224–229 42. Bove AA, Santamore WP (1981) Ventricular interdependence. Prog Cardiovasc Dis 23:365–388 43. Marcus JT, Gan CT, Zwanenburg JJ et al (2008) Interventricular mechanical asynchrony in pulmonary arterial hypertension: leftto-right delay in peak shortening is related to right ventricular overload and left ventricular underfilling. J Am Coll Cardiol 51:750–757 44. Benza R, Biederman R, Murali S, Gupta H (2008) Role of cardiac magnetic resonance imaging in the management of patients with pulmonary arterial hypertension. J Am Coll Cardiol 52:1683–1692 45. Gan CT, Lankhaar JW, Marcus JT et al (2006) Impaired left ventricular filling due to right-to-left ventricular interaction in patients with pulmonary arterial hypertension. Am J Physiol Heart Circ Physiol 290:H1528–H1533 46. Hinderliter AL, Willis PW 4th, Long W et  al (1999) Frequency and prognostic significance of pericardial effusion in primary pulmonary hypertension. PPH Study Group. Primary pulmonary hypertension. Am J Cardiol 84:481–484 47. Miller AJ (1970) Some observations concerning pericardial effusions and their relationship to the venous and lymphatic circulation of the heart. Lymphology 3:76–78 48. Hemnes AR, Gaine SP, Wiener CM (2008) Poor outcomes associated with drainage of pericardial effusions in patients with pulmonary arterial hypertension. South Med J 101:490–494 49. Abbas AE, Fortuin FD, Schiller NB, Appleton CP, Moreno CA, Lester SJ (2003) A simple method for noninvasive estimation of pulmonary vascular resistance. J Am Coll Cardiol 41:1021–1027 50. Rajagopalan N, Simon MA, Suffoletto MS et al (2009) Noninvasive estimation of pulmonary vascular resistance in pulmonary hypertension. Echocardiography 26:489–494 51. Ihlen H, Amlie JP, Dale J et  al (1984) Determination of cardiac output by Doppler echocardiography. Br Heart J 51:54–60 52. Furuno Y, Nagamoto Y, Fujita M, Kaku T, Sakurai S, Kuroiwa A (1991) Reflection as a cause of mid-systolic deceleration of pulmonary flow wave in dogs with acute pulmonary hypertension: comparison of pulmonary artery constriction with pulmonary embolisation. Cardiovasc Res 25:118–124 53. Torbicki A, Kurzyna M, Ciurzynski M et al (1999) Proximal pulmonary emboli modify right ventricular ejection pattern. Eur Respir J 13:616–621 54. Castelain V, Hervé P, Lecarpentier Y, Duroux P, Simonneau G, Chemla D (2001) Pulmonary artery pulse pressure and wave reflection in chronic pulmonary thromboembolism and primary pulmonary hypertension. J Am Coll Cardiol 37:1085–1092 55. Opotowsky AR, Arkles J, Liu T et al (2009) Midsystolic “notching” of the pulmonary artery Doppler flow profile identifies pulmonary vascular disease and RV dysfunction in a mixed PH cohort. Am J Respir Crit Care Med 179:A3399 56. Goodman DJ, Harrison DC, Popp RL (1974) Echocardiographic features of primary pulmonary hypertension. Am J Cardiol 33:438–443 57. Bossone E, Duong-Wagner TH, Paciocco G et  al (1999) Echocardiographic features of primary pulmonary hypertension. J Am Soc Echocardiogr 12:655–662 58. Nishimura RA, Tajik AJ (1997) Evaluation of diastolic filling of left ventricle in health and disease: Doppler echocardiography is the clinician’s Rosetta Stone. J Am Coll Cardiol 30:8–18 59. Eysmann SB, Palevsky HI, Reichek N, Hackney K, Douglas PS (1989) Two-dimensional and Doppler-echocardiographic and cardiac catheterization correlates of survival in primary pulmonary hypertension. Circulation 80:353–360 60. Raymond RJ, Hinderliter AL, Willis PW et  al (2002) Echocardiographic predictors of adverse outcomes in primary pulmonary hypertension. J Am Coll Cardiol 39:1214–1219

P.R. Forfia 61. Eysmann SB, Palevsky HI, Reichek N, Hackney K, Douglas PS (1991) Echo/Doppler and hemodynamic correlates of vasodilator responsiveness in primary pulmonary hypertension. Chest 99:1066–1071 62. Ricciardi MJ, Bossone E, Bach DS, Armstrong WF, Rubenfire M (1999) Echocardiographic predictors of an adverse response to a nifedipine trial in primary pulmonary hypertension: diminished left ventricular size and leftward ventricular septal bowing. Chest 116:1218–1223 63. Tei C, Dujardin KS, Hodge DO et al (1996) Doppler echocardiographic index for assessment of global right ventricular function. J Am Soc Echocardiogr 9:838–847 64. Galiè N, Hinderliter AL, Torbicki A et al (2003) Effects of the oral endothelin-receptor antagonist bosentan on echocardiographic and Doppler measures in patients with pulmonary arterial hypertension. J Am Coll Cardiol 41:1380–1386 65. Hinderliter AL, Willis PW 4th, Barst RJ et  al (1997) Effects of long-term infusion of prostacyclin (epoprostenol) on echocardiographic measures of right ventricular structure and function in primary pulmonary hypertension. Primary Pulmonary Hypertension Study Group. Circulation 95:1479–1486 66. Basil A, Liu T, Arkles J et al (2009) Serial changes in TAPSE correlate with functional assessment and clinical events in patients with PAH. Am J Respir Crit Care Med 179:A4882 67. Dartevelle P, Fadel E, Mussot S et al (2004) Chronic thromboembolic pulmonary hypertension. Eur Respir J 23:637–648 68. Fedullo PF, Auger WR, Kerr KM, Rubin LJ (2001) Chronic thromboembolic pulmonary hypertension. New Engl J Med 345: 1465–1472 69. Bresser P, Pepke-Zaba J, Jais X, Humbert M, Hoeper MM (2006) Medical therapies for chronic thromboembolic pulmonary hypertension: an evolving treatment paradigm. Proc Am Thorac Soc 3:594–600 70. Nakayama Y, Nakanishi N, Sugimachi M et al (1997) Characteristics of pulmonary artery pressure waveform for differential diagnosis of chronic pulmonary thromboembolism and primary pulmonary hypertension. J Am Coll Cardiol 29:1311–1316 71. Tanabe N, Okada O, Abe Y, Masuda M, Nakajima N, Kuriyama T (2001) The influence of fractional pulse pressure on the outcome of pulmonary thromboendarterectomy. Eur Respir J 17:653–659 72. Nakayama Y, Sugimachi M, Nakanishi N et al (1998) Noninvasive differential diagnosis between chronic pulmonary thromboembolism and primary pulmonary hypertension by means of Doppler ultrasound measurement. J Am Coll Cardiol 31:1367–1371 73. Casaclang-Verzosa G, McCully RB, Oh JK, Miller FA Jr, McGregor CG (2006) Effects of pulmonary thromboendarterectomy on right-sided echocardiographic parameters in patients with chronic thromboembolic pulmonary hypertension. Mayo Clin Proc 81:777–782 74. Menzel T, Kramm T, Wagner S, Mohr-Kahaly S, Mayer E, Meyer J (2002) Improvement of tricuspid regurgitation after pulmonary thromboendarterectomy. Ann Thorac Surg 73:756–761 75. Hardziyenka M, Reesink HJ, Bouma BJ et  al (2007) A novel echocardiographic predictor of in-hospital mortality and mid-term haemodynamic improvement after pulmonary endarterectomy for chronic thrombo-embolic pulmonary hypertension. Eur Heart J 28:842–849 76. Guyton AC, Lindsey AW, Gilluly JJ (1954) The limits of right ventricular compensation following acute increase in pulmonary circulatory resistance. Circ Res 2:326–332 77. Kasper W, Geibel A, Tiede N et al (1993) Distinguishing between acute and subacute massive pulmonary embolism by conventional and Doppler echocardiography. Br Heart J 70:352–356 78. Jardin F, Dubourg O, Guéret P, Delorme G, Bourdarias JP (1987) Quantitative two-dimensional echocardiography in massive pulmonary embolism: emphasis on ventricular interdependence and leftward septal displacement. J Am Coll Cardiol 10:1201–1206

102  Echocardiography in Pulmonary Vascular Disease 79. Ribeiro A, Juhlin-Dannfelt A, Brodin LA, Holmgren A, Jorfeldt L (1998) Pulmonary embolism: relation between the degree of right ventricle overload and the extent of perfusion defects. Am Heart J 135:868–874 80. Miniati M, Monti S, Pratali L et al (2001) Value of transthoracic echocardiography in the diagnosis of pulmonary embolism: results of a prospective study in unselected patients. Am J Med 110:528–535 81. Ribeiro A, Lindmarker P, Johnsson H, Juhlin-Dannfelt A, Jorfeldt L (1999) Pulmonary embolism: one-year follow-up with echocardiography Doppler and five-year survival analysis. Circulation 99:1325–1330 82. McConnell MV, Solomon SD, Rayan ME, Come PC, Goldhaber SZ, Lee RT (1996) Regional right ventricular dysfunction detected by echocardiography in acute pulmonary embolism. Am J Cardiol 78:469–473 83. Hsiao SH, Lee CY, Chang SM, Yang SH, Lin SK, Huang WC (2006) Pulmonary embolism and right heart function: insights from myocardial Doppler tissue imaging. J Am Soc Echocardiogr 19:822–828 84. Kurzyna M, Torbicki A, Pruszczyk P et al (2002) Disturbed right ventricular ejection pattern as a new Doppler echocardiographic sign of acute pulmonary embolism. Am J Cardiol 90:507–511 85. Torbicki A, Galiè N, Covezzoli A et al (2003) Right heart thrombi in pulmonary embolism: results from the International Cooperative Pulmonary Embolism Registry. J Am Coll Cardiol 41:2245–2251 86. Pierre-Justin G, Pierard LA (2005) Management of mobile right heart thrombi: a prospective series. Int J Cardiol 99:381–388 87. Hopkins WE (2005) The remarkable right ventricle of patients with Eisenmenger syndrome. Coron Artery Dis 16:19–25 88. Delgado JF, Conde E, Sánchez V et al (2005) Pulmonary vascular remodeling in pulmonary hypertension due to chronic heart failure. Eur J Heart Fail 7:1011–1016 89. Moraes DL, Colucci WS, Givertz MM (2000) Secondary pulmonary hypertension in chronic heart failure: the role of the endothelium in pathophysiology and management. Circulation 102:1718–1723 90. Tribouilloy CM, Enriquez-Sarano M, Rossi A, Tajik AJ, Seward JB (1997) Determinants of the pulmonary artery pressure rise in left ventricular dysfunction. Cardiologia 42:1051–1058 91. Forfia PR (2007) Approach to patients with heart failure and pulmonary hypertension. Curr Treat Options Cardiovasc Med 9:302–309 92. Melenovsky V, Borlaug BA, Rosen B et al (2007) Cardiovascular features of heart failure with preserved ejection fraction versus nonfailing hypertensive left ventricular hypertrophy in the urban Baltimore community: the role of atrial remodeling/dysfunction. J Am Coll Cardiol 49:198–207

1445 93. Grayburn PA (2008) How to measure severity of mitral regurgitation. Postgrad Med J 84:395–402 94. Smith MD, Cassidy JM, Gurley JC, Smith AC, Booth DC (1995) Echo Doppler evaluation of patients with acute mitral regurgitation: superiority of transesophageal echocardiography with color flow imaging. Am Heart J 129:967–974 95. Prasanna V, Opotowsky A, Ojeda J, Arkles J, Forfia P (2009) Echo-Doppler scoring system provides simple noninvasive assessment of the pathophysiology of pulmonary hypertension in a mixed pulmonary hypertension cohort. Chest Meeting Abstracts 2009 136:32S-h 96. Damato AN, Galante JG, Smith WM (1966) Hemodynamic response to treadmill exercise in normal subjects. J Appl Physiol 21:959–966 97. Wagner PD, Gale GE, Moon RE, Torre-Bueno JR, Stolp BW, Saltzman HA (1986) Pulmonary gas exchange in humans exercising at sea level and simulated altitude. J Appl Physiol 61:260–270 98. Bossone E, Rubenfire M, Bach DS, Ricciardi M, Armstrong WF (1999) Range of tricuspid regurgitation velocity at rest and during exercise in normal adult men: implications for the diagnosis of pulmonary hypertension. J Am Coll Cardiol 33:1662–1666 99. West JB (1998) Left ventricular filling pressures during exercise: a cardiological blind spot? Chest 113:1695–1697 100. Laskey WK, Ferrari VA, Palevsky HI, Kussmaul WG (1993) Pulmonary artery hemodynamics in primary pulmonary hypertension. J Am Coll Cardiol 21:406–412 101. Kovacs G, Berghold A, Scheidl S, Olschewski H (2009) Pulmonary arterial pressure during rest and exercise in healthy subjects. A systematic review. Eur Respir J 34:888–894 102. Steen V, Chou M, Shanmugam V, Mathias M, Kuru T, Morrissey R (2008) Exercise-induced pulmonary arterial hypertension in patients with systemic sclerosis. Chest 134:146–151 103. Tolle JJ, Waxman AB, Van Horn TL, Pappagianopoulos PP, Systrom DM (2008) Exercise-induced pulmonary arterial hypertension. Circulation 118:2183–2189 104. Reichenberger F, Voswinckel R, Schulz R, Mensch O, Ghofrani HA, Olschewski H, Seeger W (2009) Noninvasive detection of early pulmonary vascular dysfunction in scleroderma. Respir Med 103(11):1713–1718 105. Pynnaert C, Lamotte M, Naeije R Aerobic exercise capacity in COPD patients with and without pulmonary hypertension. Respir Med 2010 Jan;104(1):121–126 106. Sun XG, Hansen JE, Oudiz RJ, Wasserman K (2001) Exercise pathophysiology in patients with primary pulmonary hypertension. Circulation 104:429–435

Chapter 103

Calcium Channel Blockers in the Treatment of Pulmonary Arterial Hypertension Stuart Rich and Mardi Gomberg-Maitland

Abstract  Calcium channel blockers (CCBs) are considered a standard treatment of pulmonary arterial hypertension (PAH) in a specifically defined subset of patients, and the only treatment that has resulted in a chronic reduction in pulmonary artery pressure (PAP) associated with dramatically improved survival. Keywords  Antihypertensive drug • Calcium homeostasis • Responders • Vasodilation • Calcium antagonist

1 Introduction Calcium channel blockers (CCBs) are considered a standard treatment of pulmonary arterial hypertension (PAH) in a specifically defined subset of patients, and the only treatment that has resulted in a chronic reduction in pulmonary artery pressure (PAP) associated with dramatically improved survival.

SMC relaxation, which leads to vasodilation and a decrease in vascular resistance [2]. Cardiomyocytes, which depend on intracellular calcium for excitation–contraction coupling, are also affected by CCBs, which reduce oxygen demand in a dose-dependent manner by decreasing contractility. The nondihydropyridine CCBs reduce automaticity of the sinus node and slow conduction through the atrioventricular node resulting in a reduction of heart rate. CCBs improve myocardial perfusion through their effects on myocardial microcirculation and metabolism [3]. CCBs enhance the use of free fatty acids in reversibly ischemic myocardium and attenuate myocardial stunning independent of hemodynamic changes. In addition, CCBs inhibit platelet aggregation and thrombus formation, which may indirectly improve myocardial perfusion and be beneficial in patients with pulmonary vascular disease.

3 Initial Clinical Studies with CCBs 2 Cellular Mechanisms Calcium channels are consistently open after depolarization, resulting in an increase in intracellular calcium concentration [1] (Fig. 1). Calcium forms a complex with calmodulin, thereby activating myosin light chain kinase. Activated myosin light chain kinase phosphorylates myosin light chains, resulting in myosin–actin interaction and smooth muscle cell (SMC) contraction. CCBs bind to the calcium channel, causing these channels to open infrequently dramatically, reducing the influx of intracellular calcium. This results in sustained

S. Rich (*) Section of Cardiology, University of Chicago Medical Center, 5841 South Maryland Avenue, MC 5403 L08, Chicago, IL 60637, USA e-mail: [email protected]

The initial development and use of CCBs as a treatment for chronic essential hypertension proved highly effective [4]. Because of an unmet medical need of the time, they were also utilized in hypertensive emergencies, where the dose of drug used was titrated against the clinical response [5]. Numerous studies have shown that the use of nifedipine in severe hypertension and in hypertensive crises produces a prompt, consistent, and predictable decrease in blood pressure. In general, the higher the initial blood pressure, the greater the response. The decrease in blood pressure is due to a marked decrease in systemic vascular resistance (SVR) that is inversely proportional to the initial SVR and blood pressure. This potent arterial vasodilation results in a mild reflex increase in heart rate in most patients, and the decreased SVR and decreased impedance to left ventricular work results in an increased stroke volume and cardiac index. Myocardial oxygen consumption, right arterial pressure (RAP), PAP, pulmonary vascular resistance (PVR), and pulmonary capillary wedge

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_103, © Springer Science+Business Media, LLC 2011

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and constriction of the SMCs in the pulmonary vascular bed. The wide spectrum of responsiveness is interpreted as a reflection of the chronicity and the underlying severity of the disease. Although it has not been possible to relate the presence of vasoreactivity specifically to the vascular changes noted on histology, one study reported a qualitative relationship in patients with more advanced lesions with a reduced likelihood to respond to acute vasodilator testing [10]. This study did not clarify whether the presence of vasoreactivity represents a different stage of the disease, or a different disease altogether. Some speculate that patients who present earlier appear to be more likely to respond to an acute vasodilator challenge, and those that are at a more advanced stage are less likely to be responsive. An important concept in the development of PAH is that the disease develops in patients with an underlying genetic predisposition following exposure to specific stimuli, which serve as triggers [11]. Presumably, a genetic basis underlies these differences in pulmonary vascular reactivity, similar to the genetic basis for the increased reactivity of the systemic vascular bed.

Fig. 1  Various pathways controlling pulmonary vascular smooth muscle contraction via intracellular calcium. Blockade of the calcium channel is only one mechanism by which calcium-channel-mediated contraction and proliferation of the smooth muscle cell is achieved

pressure (PCWP) all decrease and coronary artery blood flow increases as coronary artery resistance decreases. The dose and method of administration have a minor effect on the antihypertensive response, time of response onset, and peak action in a hypertensive crisis [6]. Several factors that may contribute to the rapid hypotensive effect of nifedipine include hypovolemia and concomitant treatment with other vasodilators. Hypotension is uncommon, ­however, if nifedipine is used alone [7]. The experience with CCBs in severe hypertension provided the background rationale for their use in PAH.

4 Chronic Vasoconstriction in Primary Pulmonary Hypertension (Now Called Idiopathic and Familial PAH) In nearly all reported pathologic case series of primary pulmonary hypertension (PPH), differing degrees of medial hypertrophy exist in the pulmonary vasculature, which is interpreted as an expression of underlying vasoconstriction [8]. Early physiologic studies in patients with PPH demonstrated that the administration of a vasodilator, such as acetylcholine, can cause immediate and pronounced vasodilatation [9]. Since the vessels are characterized by marked thickening of the media, it was originally postulated that this represented uncontrolled growth

5 CCBs for PAH This first class of drugs for a subset of PPH patients, CCBs, changed the treatment paradigm of PPH. The initial experience demonstrated that nifedipine or diltiazem, given hourly to patients with PPH and monitored hemodynamically with a pulmonary artery catheter, could lower pressure and resistance in the pulmonary vasculature [12]. The effect would not occur until the patient received a threshold dose, which caused the PAP and PVR to fall dramatically. With a fall in the PAP, cardiac output increased, with no change in the systemic blood pressure. Patients who did not respond after six consecutive hourly doses were not likely to respond to further dosing, and were classified as “nonresponders.” Responders received high doses of CCBs in divided doses throughout the day. Although the optimal dose remains uncertain, the typical dosage used was amlodipine 20–30 mg/day, nifedipine 180–240  mg/day, or diltiazem 720–960  mg/day. CCBs at these high dosages are remarkably well ­tolerated, with the only major side effect being ankle edema, a wellknown side effect of CCBs, which is easily treated with diuretics. The patients had dramatic clinical improvements, as reflected by fewer symptoms of dyspnea, better exercise tolerance, and regression of right ventricular ­hypertrophy on EKG and echocardiography. Most importantly, however, is the dramatic and sustained fall in PAP (more than 30%). No therapy in any other subset has improved pressure and resistance to this degree. The initial experience by Rich et  al. revealed near perfect survival out to 5 years [13] (Fig. 2). The practice of hourly administration of CCBs in intensive care units with hemodynamic monitoring in the hospital is no

103  Calcium Channel Blockers in the Treatment of Pulmonary Arterial Hypertension

Fig.  2  The long-term survival of patients with primary pulmonary hypertension (PPH) receiving high doses of calcium channel blockers based on acute vasoreactivity testing compared with historical controls from the NIH registry, and from the nonresponders to acute vasoreactivity testing followed prospectively. The improvement in survival is dramatic. (From Rich et al. [13]. Copyright 1992 Massachusetts Medical Society. All rights reserved)

Fig. 3  The long-term survival of patients receiving high doses of calcium channel blockers based on acute vasoreactivity testing compared with nonresponders followed over a similar time frame. Like the American experience, the French experience showing improvement in survival is also dramatic [18]

longer necessary. Patients most likely to respond to high doses of CCBs can be identified by hemodynamic testing with short-acting vasodilators at the time of initial evaluation [14]. The responses to inhaled nitric oxide [15], intravenously administered epoprostenol [16], or intravenously administered adenosine [17] all identify patients with the ability to acutely lower their PAP in response to ­vasodilators. It has been reported that 10–20% of patients with ­idiopathic PAH (IPAH) who are identified as vasoreactive and treated with very high doses of CCBs may manifest a ­dramatic reduction in PAP and PVR. This response, demonstrated­ by cardiac catheterization, can be maintained in some patients for more than 15 years (Fig. 3). High doses of CCBs need to be used to realize the full hemodynamic benefit. With a favorable response, the patient’s quality of life is restored with improved

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functional class and survival (94% rate at 5 years) compared with “nonresponders” and historical control patients (36% rate). Patients who respond will show marked clinical improvement within the first few months of treatment. It is unknown whether the response to CCBs identifies two subsets of patients with IPAH, different stages of IPAH, or a combination of both. However, it is essential to point out that patients who do not exhibit a dramatic acute hemodynamic response to CCBs do not appear to derive similar benefit from their long-term use in the anticipation that it may take time for the therapy to have an effect. Sitbon et al. reported an almost identical experience from their national referral center in France [18]. Using acute testing with intravenously administered epoprostenol or inhaled nitric oxide, patients with an acute response had a significant decrease in the mean PAP and PVR (33 ± 11% and 45 ± 15%, respectively). In all these patients, mean PAP decreased by 10 mmHg or more from the baseline. The mean change in mean PAP was similar with both testing agents in responders, with a decrease of 33 ± 12% from the baseline with inhaled nitric oxide and a decrease of 32 ± 11% with intravenously administered epoprostenol. PVR decreased more with intravenously administered epoprostenol (54 ± 11%) than with inhaled nitric oxide (44 ± 15%) owing to the direct effect of epoprostenol on increasing cardiac output. The responders received long-term treatment with oral CCBs (diltiazem, nifedipine, or amlodipine). After 1 year of treatment with CCBs, the investigators reclassified patients on the basis of their clinical response. Long-term CCB responders represented 54% of acute responders. These patients appeared to have less advanced disease as evidenced by a larger proportion of New York Heart Association (NYHA) functional class II patients, and less severe hemodynamic parameters. Long-term CCB responders had a larger decrease in mean PAP and PVR than patients who failed therapy (mean PAP drop of 39% and a 50% drop in PVR) Their maximum follow-up was 18 years (mean 7.0 ± 4.1 years) after initiation of CCB therapy, and there was only one death. At the time of the last ­evaluation, all patients were NYHA functional class I or class II, had a mean 6-min walk distance of 467 ± 101 m, and displayed a sustained improvement in hemodynamics, with a mean RAP of 5 ± 3 mmHg, a mean PAP of 35 ± 7 mmHg, a mean PCWP of 8 ± 3 mmHg, and a mean cardiac output of 6.6 ± 1.8 L/min. After review, no clinical characteristics or baseline hemodynamic features enabled the investigators to predict those patients with or without a sustained response. CCBs provide benefit primarily through vasodilatation, as represented by the fall in mean PAP as they do not have positive inotropic effects. The increase in cardiac output that results is due to pressure unloading of the right ventricle. It is possible that these patients represent a subset of patients whose pulmonary hypertension has been triggered by increased Ca2+ concentration in pulmonary artery SMCs due

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to abnormalities in the voltage-gated potassium channel [19]. The high doses of CCBs necessary to achieve the maximum beneficial effect in responders may be related to impaired absorption, and in part to an increased requirement of Ca2+ in the pulmonary vascular bed. The duration of the benefit in these patients appears to be indefinite.

6 CCB Use in Patients Without Acute Vasoreactivity The use of CCBs in patients with PPH can be harmful, ­particularly when CCBs are administered in high doses [20]. Systemic hypotension producing reflex tachycardia, sympathetic stimulation, and right ventricular ischemia are effects of CCBs that may cause clinical worsening. Reports of serious adverse events when CCBs are used inappropriately underscore the fact that CCBs need to be used with caution, and only following an acute trial of a short-acting vasodilator to confirm the presence of reversible vasoconstriction. Their indiscriminate prescription without close patient follow-up with documentation of the beneficial hemodynamic effects is dangerous and not recommended. Increasing CCB doses in nonresponders increases morbidity and may be fatal. The use of CCBs at conventional doses in nonvasoreactive patients has been reported in a meta-analysis [21]. It is interesting to note that many patients report clinical benefit from the CCBs in the absence of a marked reduction in PAP. Many patients had subjective improvement in dyspnea and exercise tolerance, and a stabilization of the clinical status for up to 30 months. This experience raises the possibility that a vasodilator may have the ability to produce clinical improvements in patients with PAH without having direct effects on the pulmonary circulation.

7 Conclusion CCBs remain an important treatment of PAH. The clinical benefit in the subset of patients who respond acutely is unmatched by any other treatment, and appears to be lifelong. The genotype of these patients remains unknown, but its identification could enlighten researchers with a better understanding of the cause of pulmonary hypertension and its successful treatment [22].

References 1. Dolphin AC (2006) A short history of voltage-gated calcium channels. Br J Pharmacol 147:S56–S62

S. Rich and M. Gomberg-Maitland 2. Webb RC (2003) Smooth muscle contraction and relaxation. Adv Physiol Educ 27:201–206 3. Yamakage M, Namiki A (2002) Calcium channels–basic aspects of their structure, function and gene encoding; anesthetic action on the channels-a review. Can J Anaesth 49:151–164 4. Abernethy DR, Schwartz JB (1999) Calcium-antagonist drugs. N Engl J Med 341:1447–1457 5. Cherney D, Straus S (2002) Management of patients with hypertensive urgencies and emergencies: a systematic review of the literature. J Gen Intern Med 17:937–945 6. Elliott WJ (2001) Hypertensive emergencies. Crit Care Clin 17:435–451 7. Pedrinelli R, Dell’Omo G, Mariani M (2001) Calcium channel blockers, postural vasoconstriction and dependent oedema in essential hypertension. J Hum Hypertens 15:455–461 8. Pietra GG, Edwards WD, Kay JM et al (1989) Histopathology of primary pulmonary hypertension. A qualitative and quantitative study of pulmonary blood vessels from 58 patients in the National Heart, Lung, and Blood Institute, Primary Pulmonary Hypertension Registry. Circulation 80:1198–1206 9. Dresdale DT, Schultz M, Michtom RJ (1951) Primary pulmonary hypertension. I. Clinical and hemodynamic study. Am J Med 11:686–705 10. Palevsky HI, Schloo BL, Pietra GG et al (1989) Primary pulmonary hypertension: vascular structure, morphometry, and responsiveness to vasodilator agents. Circulation 80:1207–1221 11. Tuder RM, Lee SD, Cool CC (1998) Histopathology of pulmonary hypertension. Chest 114:1S–6S 12. Rich S, Brundage BH (1987) High-dose calcium channel-blocking therapy for primary pulmonary hypertension: evidence for longterm reduction in pulmonary arterial pressure and regression of right ventricular hypertrophy. Circulation 76:135–141 13. Rich S, Kaufmann E, Levy PS (1992) The effect of high doses of calcium-channel blockers on survival in primary pulmonary hypertension. N Engl J Med 327:76–81 14. Rich S, Kaufmann E (1991) High dose titration of calcium channel blocking agents for primary pulmonary hypertension: guidelines for short-term drug testing. J Am Coll Cardiol 18: 1323–1327 15. Atz AM, Adatia I, Lock JE, Wessel DJ (1999) Combined effects of nitric oxide and oxygen during acute pulmonary vasodilator testing. J Am Coll Cardiol 33:813–819 16. Rubin LJ, Groves BM, Reeves JT, Frosolono M, Handel F, Cato AE (1982) Prostacyclin-induced acute pulmonary vasodilation in primary pulmonary hypertension. Circulation 66:334–338 17. Nootens M, Schrader B, Kaufman E, Vestal R, Long W, Rich S (1995) Comparative acute effects of adenosine and prostacyclin in primary pulmonary hypertension. Chest 107:54–57 18. Sitbon O, Humbert M, Jaïs X et al (2005) Long-term response to calcium channel blockers in idiopathic pulmonary arterial hypertension. Circulation 111:3105–3111 19. Yuan JX-J, Aldinger AM, Juhaszova M et al (1998) Dysfunctional voltage-gated K+ channels in pulmonary artery smooth muscle cells of patients with primary pulmonary hypertension. Circulation 98:1400–1406 20. Packer M, Medina N, Yushak M (1984) Adverse hemodynamic and clinical effects of calcium channel blockade in pulmonary hypertension secondary to obliterative pulmonary vascular disease. J Am Coll Cardiol 4:890–901 21. Malik AS, Warshafsky S, Lehrman S (1997) Meta-analysis of the long-term effect of nifedipine for pulmonary hypertension. Arch Intern Med 157:621–625 22. Stenmark K, Frid M (1998) Smooth muscle cell heterogeneity. Role of specific smooth muscle cell subpopulations in pulmonary vascular disease. Chest 114:82S–90S

Chapter 104

Prostacyclin and Prostaglandins Horst Olschewski

Abstract  Prostacyclin was the first effective therapy for patients with pulmonary arterial hypertension. The first experiences were reported in 1980 and the first patient with severe idiopathic pulmonary arterial hypertension [IPAH; formerly primary pulmonary hypertension (PPH)] to receive long-term therapy was reported in 1984. After 25 years, prostanoids remain a mainstay in the treatment of these patients. Prostacyclin and its analogs (prostanoids) are potent vasodilators and possess antithrombotic, antiproliferative, and anti-inflammatory properties. Pulmonary hypertension is associated with vasoconstriction, thrombosis, and proliferation, and this may be partly due to a lack of endogenous prostacyclin secondary to prostacyclin synthase downregulation. This supports a strong rationale for prostanoid use as therapy for this disease. Keywords  Prostanoids • Cyclic AMP • Vasodilation • Anti-proliferative • Anti-inflammatory

1 Introduction Prostacyclin was the first effective therapy for patients with pulmonary arterial hypertension. The first experiences were reported in 1980 [1] and the first patient with severe idiopathic pulmonary arterial hypertension [IPAH; formerly primary pulmonary hypertension (PPH)] to receive long-term therapy was reported in 1984 [2]. After 25 years, prostanoids remain a mainstay in the treatment of these patients. Prostacyclin and its analogs (prostanoids) are potent vasodilators and possess antithrombotic, antiproliferative, and anti-inflammatory properties. Pulmonary hypertension is associated with vasoconstriction, thrombosis, and proliferation, and this may be partly due to a lack of endogenous prostacyclin [3] secondary to prostacyclin synthase ­downregulation

H. Olschewski (*) Department of Pulmonology, Medical University Graz, Auenbruggerplatz 20, Graz 8010, Austria e-mail: [email protected]

[4]. This supports a strong rationale for prostanoid use as therapy for this disease.

2 Properties of Prostanoids The term “prostaglandin” was introduced by Ulf S. von Euler in 1935 [5], and described a short-lived vasodilative and antiaggregative substance from the human prostate gland. The discovery of the chemical structure of the main component, prostacyclin, was made in 1976 by Moncada et  al. [6]. According to the systematic chemical nomenclature, the substance corresponds to PGI2. Beyond its action on vascular smooth muscle cells (SMC) and platelets, later investigations demonstrated its antiproliferative actions and the reduction of matrix secretion in SMCs, endothelial cells, and fibroblasts as well as an anti-inflammatory profile in leukocytes (Fig.  1). Because the endothelial cells are the major source of endogenous prostacyclin and owing to the short half-life, the action of this mediator is directed on both the local vascular wall and blood cells, particularly those that adhere to the endothelium. In 1976, the same year when the chemical structure of prostacyclin was discovered, a chemical analog of prostacyclin, epoprostenol, was synthesized and tested for its biologic actions [8]. Because epoprostenol is chemically identical to prostacyclin, its pharmacologic half-life is just 2–5  min. Several chemically stable analogs were synthesized in the following years. Clinical studies for patients with pulmonary arterial hypertension (PAH) were performed with epoprostenol, iloprost, beraprost, and treprostinil, whereas cicaprost, a specific IP receptor agonist, was not clinically developed. The main target of prostanoids is the human IP receptor, which is abundantly expressed in blood vessels, leukocytes, and thrombocytes and is rapidly activated by the compounds. The IP receptor is coupled with Gs proteins and activates adenylate cyclase, leading to increased cyclic AMP levels in the target cells, which explains most of the biologic effects. Principally, it also couples with Gq proteins and might activate vasoconstrictive pathways under certain instances [9, 10]. However, prostacyclin is not highly specific to the IP

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_104, © Springer Science+Business Media, LLC 2011

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2.1 Differences Between Prostanoids

Fig.  1  Effects of prostanoids on the vessel wall and adherent blood cells. SMC smooth muscle cells, EC endothelial cells, mono mononuclear cells, MF macrophages, PMN polymorphonuclear neutrophils, T-cells T lymphocytes, TNF transforming nuclear factor a, IL interleukin, MAPK mitogen-activated protein kinase, burst generation of reactive oxygen species, elastase elastase secretion, IFN interferon g. (Reprinted from [7] with permission from Elsevier)

Fig. 2  Intracellular targets of prostacyclin. Extracellular prostacyclin mainly, but not specifically, acts on the IP receptor and the EP1 receptor, while prostaglandin E1 is more specific to EP receptors. Gs, Gq, and Gi G proteins, PLC phospholipase C, PKC protein kinase C, AC adenylyl cyclase, PKA protein kinase A, AA2 arachidonic acid, COX cyclooxygenases 1 and 2, PGH2 prostaglandin H2, PGIS prostacyclin synthase, PPAR peroxisome-proliferator-activated receptors, PGD prostaglandin D, PGJ2 prostaglandin J2, PDE phosphodiesterases. (Reproduced with permission from European Respiratory Society Journals Ltd [16])

receptor. To a lesser extent it also activates EP receptors [11] which are located on the cell surface as well as in the nucleus [12, 13] and peroxisome-proliferator-activated receptor (PPAR) d in the nucleus [14]. Both PPARa and PPARd may also be activated via IP-receptor-dependent protein kinase A activation, but the intracellular prostacyclin from the endogenous prostacyclin synthase seems to specifically activate the apoptosis pathway by activation of PPARd [15] (Fig. 2).

Epoprostenol is provided as a dry powder with a highly basic glycine buffer as a solvent drug. After the drug powder has been mixed with the solvent, the solution has to be used within 12–24  h owing to spontaneous degradation of the compound. When it is given via peripheral veins, there will be painful vein irritation after a short time. Therefore, epoprostenol therapy can only be used as continuous intravenous infusion through a central venous catheter with freshly dissolved drug filled into the pump system. All other prostanoids are chemically stable in solution and their plasma half-life is much longer. It is about 30 min for iloprost and beraprost and up to 4.5 h for treprostinil. Vein irritation is common to all prostanoids. All stable prostanoids have been provided as oral preparations; however, only beraprost has received approval for PAH in Japan and some Eastern countries (see later) and orally administered treprostinil is currently being investigated. Iloprost has been approved as inhalational therapy and treprostinil as subcutaneous infusion (see later). Apart from differences in pharmacokinetics and pharmacodynamics, there may be other specific differences between prostanoids. The antiproliferative effects of treprostinil appeared more impressive than those of the other prostanoids but owing to differences in chemical stability such results must be interpreted with caution [17]. Non-IPreceptor effects of prostanoids have attracted some interest in tumor biology [18] and might be interesting for pulmonary hypertension as well. Iloprost and cicaprost were found to have different effects in the murine corneal model of angiogenesis. Whereas iloprost caused significant angiogenesis, comparable to that caused by vascular endothelial growth factor (VEGF), cicaprost had no such effects [19]. The explanation may be that iloprost and carbacyclin but not cicaprost activate PPARs [20], which results in VEGF secretion [21, 22]. Increased levels of VEGF may antagonize endothelial dysfunction and this represents a potential beneficial effect [23, 24]. There are some biologic effects that have been described only in one of the compounds but that probably can be applied to several if not all prostanoids. Treprostinil, for example, was found to augment the positive inotropic effects of catecholamines in isolated ventricular myocytes [25], although it had no positive inotropic effects of its own. This effect may have clinical relevance because during right-sided heart failure there is an increased catecholamine drive, and from the clinical perspective it has been speculated for long that prostanoids might have positive inotropic effects that explain their instantaneous beneficial clinical effects in rightsided heart failure patients. These effects may add on indirect effects that originate from systemic vasodilation and subsequent baroreflex activation (Fig. 3) and improved ventriculoarterial coupling [26].

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Fig.  3  Hemodynamic effects of systemically applied prostanoids. Within minutes, prostanoids cause a strong vasodilatation, which in most patients is more prominent on reducing the systemic vascular resistance (SVR) compared with the pulmonary vascular resistance (PVR). The decrease in systemic arterial pressure (SAP) activates the arterial baroreflex, which increases SVR and has a positive inotropic effect. The reduction in PVR would decrease pulmonary arterial pressure (PAP); however, in the acute phase, the increase in cardiac output

Iloprost has been shown to suppress neutrophil adhesion, respiratory burst, and elastase secretion [27]. This may be important because inflammation appears to play a role among the pathologic mechanisms of PAH [28]. Recently, in SMCs, a number of beneficial changes in gene expression (hyaluronic acid, cyclooxygenase 2, and VEGF upregulation; monocyte chemotactic protein and plasminogen activator inhibitor-1 downregulation) were seen after incubation with iloprost [29, 30]. If we consider that endothelial dysfunction or damage may play a major role in the pathologic development of pulmonary artery remodeling, this translates into a number of beneficial effects of iloprost. It remains speculative if this translates to all prostanoids (Fig. 4).

2.2 Hemodynamic Effects The hemodynamic effects including the side effects of the different prostanoids are similar. However, the mode of application, the applied doses, as well as long-term adaptive changes of the patients may have an impact on pulmonary effects and tolerability (Fig. 3). If the dose of a continuous intravenous infusion is gradually increased over some weeks, in the vast majority of patients there will be beneficial hemodynamic effects with a significant decrease in pulmonary vascular resistance and a minor decrease in systemic pressure. However, if the dose is rapidly increased, systemic

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is normally more prominent, resulting in an unchanged or even increased PAP. Within weeks, the PVR decrease becomes more prominent than the SVR decrease, which may result in a significant PAP decrease. For ventriculoarterial coupling and inotropic effects of prostacyclin, see the text. Within weeks, the neurohormonal activation increases systemic tone, whereas the slow vasorelaxing effects in the pulmonary arteries cause an additional decrease in PVR. Indirect positive inotropic effects may ameliorate systemic hypotension (see the text)

Fig. 4  Hypothesis for neointimal formation and possible role of prostanoids. After endothelial damage, monocytes, fibroblasts, and the coagulation system will form a neointima which leads to intima fibrosis. Iloprost has been shown to inhibit the messenger RNA expression of important mediators of these processes. VEGF vascular endothelial growth factor, COX2 cyclooxygenase 2, PAI-1 plasminogen activator inhibitor, Cyr 61 cysteine-rich angiogenic protein, MCP-1, monocyte chemotactic protein 1. (Reproduced with permission from European Respiratory Society Journals Ltd [16])

vasodilatation and its side effects as well as other side effects may cause intolerable symptoms and systemic pressure drop. If a prostanoid is infused at a constant dose, there may be IP receptor desensitization with a complete loss of the vasodilatory effect. This has recently been shown using iloprost [31]. This desensitization is dependent on protein

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kinase C. It is possible that other effects of the compounds (e.g. on the EP1 receptor) become more and more important during constant infusion as suggested by the authors [31]. In clinical practice, there is no loss of pharmacologic effect during constant infusion, but the dosage has to be gradually increased (from 4 ng/kg/min as an average tolerated dosage of epoprostenol to about 20–60 ng/kg/min after 1 year and from about 0.5 ng/kg/min of iloprost to about 2.5 ng/kg/min after 1 year) to keep the same level of pulmonary vasodilatation and systemic side effects. If the IP receptor downregulation is also present in humans, this would suggest that most if not all prostanoid effects are mediated by their effects on EP receptors and PPARs. If the prostanoid drug is applied by inhalation every 3  h, there is no loss of effect [32–34] and only in a few patients is there a need for dose increase (about 16% per year, unpublished data), suggesting that this mode of application may not cause receptor desensitization.

3 Clinical Application 3.1 Epoprostenol: Intravenous Continuous intravenous infusion of prostacyclin (epoprostenol) was the first therapy for PPH, a disease that was later renamed idiopathic and heritable PAH. Case reports and small case series [2, 35, 36] demonstrated improvement in symptoms and hemodynamics and were followed by a prospective, randomized open-label controlled trial where 81 subjects with PPH in New York Heart Association (NYHA) functional class III or IV were randomized to epoprostenol in addition to conventional therapy or conventional therapy alone (warfarin, digoxin, oxygen, and oral vasodilators) [37] for 12 weeks. The 6-min walk distance (6MWD) improved by 32  m in the epoprostenol group compared with a decrease of 15  m in the control group. Pulmonary arterial pressure decreased by 8% in the epoprostenol group in contrast to an increase of 3% in the control group. The mean pulmonary vascular resistance decreased by 21% in the epoprostenol group in contrast to an increase of 9% in the conventional group. Although this study was not powered for this purpose, it suggested a survival benefit as eight patients died during the 12-week trial, all in the conventional therapy group. Long-term effects of epoprostenol have never been ­compared with those of a placebo because administration of a placebo is considered unethical owing to the substantial treatment benefit after 3 months of therapy. Observational cohort analyses comparing the survival of epoprostenoltreated IPAH and hereditary PAH (HPAH) patients with historical controls suggested long-term benefits. In the USA,

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the survival in epoprostenol-treated patients after 1, 2, and 3 years was 88, 76, and 63% compared with the expected survival of 59, 46, and 35% [38]. Prognostic factors included functional class and exercise tolerance at the baseline, and after 1 year of therapy cardiac index and mean pulmonary arterial pressure. In France, patients were followed for up to 10 years after the start of intravenously administered epoprostenol [39]. Compared with a historical cohort with the same severity (survival rates at 1, 2, 3, and 5 years of 58, 43, 33, and 28%, respectively), survival rates improved at 1, 2, 3, and 5 years to 85, 70, 63, and 55%. Right-sided heart failure, NYHA functional class III or IV a 6MWD of 250 m or less, and right atrial pressure of 12 mmHg or greater at the baseline predicted poor outcome. After 3 months on epoprostenol, persistence of NYHA functional class III or IV, a 6MWD of less than 380 m, and the absence of a fall in total pulmonary resistance of more than 30%, relative to the baseline, were associated with poor survival. Subjects with scleroderma treated with epoprostenol appear to have a worse prognosis than subjects with PPH in uncontrolled analyses [40, 41]. In scleroderma-associated PAH, intravenously administered epoprostenol improved exercise capacity [42]. Similar improvements in exercise functional capacity and functional class have been seen in subjects with congenital left-to-right cardiac shunts [43] and infection with the human immunodeficiency virus (HIV) [44, 45], whereas the results are ambiguous in portopulmonary hypertension [46] and show unfavorable effects in pulmonary hypertension secondary to left-sided heart failure [47]. Over the past decade, increasing use of epoprostenol has delayed the use of lung transplantation in subjects with severe disease at presentation. A US survey indicated that treatment with epoprostenol allowed two thirds of subjects to be removed from the transplant list [48]. It is unclear how long the disease will remain stable and therefore frequent clinical follow-up with examination, exercise, and hemodynamics is recommended [49, 50].

3.1.1 Combination with Other PAH Medication In the BREATHE 2 study, subjects with PAH were started on epoprostenol with uptitration for 16 weeks and randomized in a 2:1 ratio to bosentan (62.5 mg twice daily for 4 weeks and then 125 mg twice daily) or a placebo [51]. Addition of bosentan caused a small, but not significant, beneficial effect in clinical or hemodynamic measurements. Open uncontrolled studies of addition of sildenafil to ­epoprostenol improved hemodynamics [52]. Simonneau et al. reported the results of a 16-week multinational, double-blind, placebo-controlled trial assessing the safety and efficacy of sildenafil in addition to epoprostenol (PACES) [53]. Patients

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had improvements in exercise capacity (adjusted increase 26  m, p < 0.001), hemodynamics, and time to ­clinical ­worsening in favor of sildenafil. The long-term open-label extension at 1 year maintained this benefit.

3.1.2 Limitations of Intravenously Administered Epoprostenol The limitations are mainly based on the pharmacologic properties of epoprostenol, with its chemical instability and extremely short half-life after infusion. Long-term administration of epoprostenol requires a permanent central venous catheter and a portable infusion pump. Medication needs to be prepared daily and to be kept cold, requiring ice packs to be worn with medication in 24-h cassettes. Patients need to be educated on sterile technique, operation of the pump, and care of the catheter, and a strong support system is necessary to help ensure a safe environment prior to initiation. A “caregiver,” a family member or friend who lives near the patient, is strongly recommended to obviate problems if the subject is too ill or unable to prepare medication on a given day. Serious complications include infection and thrombosis around the catheter and temporary interruption of the infusion because of inadvertent disconnection or pump malfunction [41]. The incidence of catheter-related sepsis ranges from 0.1 to 0.6 cases per patient-year in experienced centers [39,  41], but may be much higher in less experienced centers. Side effects are usually well tolerated, provided the dose is uptitrated at an adequate rate. The most common side effects include flushing, headache, nausea, loose stool, jaw pain, and foot pain [37–39]. Although jaw pain is mostly transient and well tolerated, foot pain may deteriorate physical activity and quality of life.

3.2 Iloprost: Intravenous Intravenously administered iloprost has been approved for pulmonary hypertension in New Zealand, but it has also been used in other countries, such as Germany, Switzerland, England, Australia, Thailand, Israel, Argentina, and Brazil. Intravenously administered iloprost has acute hemodynamic effects similar to those of epoprostenol [54]. It has practical advantages as it does not need cooling and owing to its longer half-life is less risky in the case of accidental therapy disruption. In Europe, the typical starting dosage is 0.5 ng/kg/min and the long-term dosage of infused iloprost is up to 3 ng/kg/ min [55]. It has not been formally tested if infused iloprost has the same clinical efficacy as infused epoprostenol or treprostinil.

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3.3 Treprostinil: Intravenous Most recently, the FDA approved the use of intravenously administered treprostinil on the basis of bioequivalence to subcutaneous treprostinil therapy. The advantage over epoprostenol is the chemical stability and the longer half-life (up to 4.5 h) that may also decrease the risk of rebound pulmonary hypertension in the case of inadvertent cessation of the infusion. Hemodynamics also improved on therapy [56]. Patients can successfully transition in the hospital from intravenously administered epoprostenol to intravenously administered treprostinil [57]. Of 27 functional class II and functional class III patients, four transitioned back to epoprostenol, three due to leg pain and one with worsening PAH symptoms in the setting of pneumonia. Transition maintained exercise capacity despite a reduction in cardiac index (−0.4 ± 0.1 L/min/m [2]; p = 0.01). Patients finished on more than twice the dose of treprostinil compared with epoprostenol. In a French study, a similar transition from epoprostenol to treprostinil resulted in 1.5–3-fold higher doses of treprostinil as compared with epoprostenol [58]. It is unclear why a higher treprostinil dose is required compared with epoprostenol as, theoretically, its longer half-life would suggest the opposite. When septic events in some experienced centers in the USA were analyzed, there were more Gram-negative sepsis episodes in patients on intravenously administered treprostinil as compared with epoprostenol therapy [59]. This might be associated with the pump system, the slower infusion rate, or pharmacologic effects of the drug. There was no indication that the drug itself was contaminated.

3.4 Treprostinil: Subcutaneous Treprostinil may also be delivered by subcutaneous infusion [60]. Subcutaneous therapy avoids a permanent central venous catheter. Medication is infused via a small self-inserted subcutaneous needle with continuous injection by syringe through an ambulatory pump. The infusion site should to be changed every 3–4 days according to the package insert. In clinical practice, patients often leave the needle in for 2–4 weeks [61]. The drug comes as a ready-to-use solution; there is no need for cooling or intravenous catheter care. The most common side effect occurring in up to 85% of the patients is local infusion site pain [62]. Patients are treated with variable response with a combination of local anesthetic solutions, nonsteroidal anti-inflammatory agents, gabapentin, pregabalin, or low-dose narcotics. The pain does not appear to be dose-related and cannot be predicted before therapy starts. A 12-week multicenter, randomized, double-blind trial demonstrated modest but significant improvement in 6MWD

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with subcutaneously administered treprostinil as compared with a placebo. Treprostinil also improved dyspnea, fatigue, signs, symptoms of pulmonary hypertension, and quality of life. Class IV patients and patients receiving higher doses of treprostinil had a more significant improvement. There was also an improvement in hemodynamic parameters, including right atrial pressure, mean pulmonary arterial pressure, pulmonary vascular resistance, and cardiac output. In subset analyses, patients who had PAH related to connective tissue disease had improvement in exercise capacity, symptoms of PAH, and hemodynamics [63]. The drug was FDA-approved for the treatment of NYHA class II–IV PAH patients in 2002 and was EMEA-approved for NYHA class III PAH in 2005. Recent retrospective and observational studies of subcutaneously administered treprostinil suggest long-term clinical improvement and survival benefits. Lang et  al. evaluated clinical outcomes in 99 PAH patients and 23 patients with inoperable chronic thromboembolic pulmonary hypertension (CTEPH) for a mean follow-up of 26 ± 17 months in the open-label phase of a randomized clinical trial [61]. The mean dosage was 40 ± 3 ng/kg/min (range 16–84 ng/kg/min). Patients maintained their improved exercise tolerance (6MWD) and functional class at 3 years. Event-free survival (no hospitalization for clinical worsening, transition to intravenously administered epoprostenol, or need for combination therapy or atrial septostomy) was 83% at 1 year and 69% at 3 years and the overall survival was 89% at 1 year and 71% at 3 years; these rates are similar to those observed with intravenously administered epoprostenol. Evaluation of all patients who had been enrolled in randomized controlled trials with open-label extension and available long-term observations revealed 860 patients who were treated for up to 4 years [64]. The diseases of the patients enrolled were IPAH (48%), associated with congenital heart disease (21%), associated with connective tissue disease (19%), CTEPH (6%), and portal pulmonary hypertension (5%). Survival in patients on monotherapy [censoring patients with addition of targeted PAH therapy (130 patients) or premature discontinuation owing to adverse events (199 patients)] was 88, 79, 73, and 70% at 1, 2, 3, and 4 years, respectively; however, only a few patients remained on therapy for more than 2 years. The most frequently reported side effect was site pain. Addition of sildenafil to subcutaneously administered treprostinil improved exercise capacity (6MWD) and functional class [65] and the same was found for the addition of sildenafil to inhaled treprostinil [66].

3.5 Beraprost: Oral Beraprost is absorbed rapidly after oral administration and has an elimination half-life of 20–40  min. In Europe, 130

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functional class II and functional class III subjects were enrolled in a 12-week randomized double-blind placebocontrolled trial of beraprost [67]. Subjects had PAH caused by IPAH, connective tissue diseases, congenital left-to-right shunts, portal hypertension, and HIV. At a median dose of 80 mg, four times daily, the 6MWD improved by 25 m, compared with a placebo (p = 0.04). Subjects with IPAH had a mean increase of 46 m, whereas PAH from other causes had no significant improvement. In the USA, a similar trial was designed but with inclusion of functional class II and functional class III patients and a 12-month study duration. This study demonstrated improved 6MWD at 3 and 6 months compared with a placebo that was not sustained at 9 and 12 months [68]. Beraprost is not approved in the USA and Europe but is an approved therapy in Japan and several other Far East countries.

3.6 Iloprost: Inhaled The lung anatomy allows direct access to the precapillary vessels of the lung via drug deposition in the alveolar space or the surface of the bronchiolus terminalis (Fig. 5). Inhalation of prostacyclin as compared with infusion was shown to provide intrapulmonary selectivity, and pulmonary selectivity with less severe systemic side effects [32, 34, 69, 70]. The inhalational approach was used with epoprostenol from the early 1990s, with iloprost from 1994, and with treprostinil from 2003. Iloprost is a chemically stable prostacyclin analog that can be delivered by suitable nebulizers [34]. To ensure alveolar deposition, the delivery system produces small aerosolized particles (median diameter about 3.0 mm) [71]. Iloprost must be inhaled six to nine times a day to achieve good clinical results. The doses for significant clinical effects were much smaller with inhaled iloprost (about 0.31  ng/kg/min) [72] than with the intravenous infusion of iloprost for PAH. Inhaled iloprost was approved for PAH NYHA functional classes III and IV, in the USA, for PAH, NYHA functional classes III and IV, as well as inoperable CTEPH in Australia, and for IPAH, NYHA functional class III, in Europe. Inhaled iloprost was investigated in a double-blind randomized controlled study in Europe in therapy-naïve patients and in patients treated with bosentan in the USA. In the European study, patients with IPAH, HPAH, PAH associated with connective tissue diseases, or inoperable CTEPH in functional class III or IV were enrolled in a 12-week multicenter placebo-controlled trial [72]. The primary end point was a combined clinical end point where four criteria had to be met to be for a patient to be counted as a responder: a 10% increase in 6MWD, an improvement in NYHA functional class, no deterioration, no death. Seventeen percent of patients on iloprost

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Fig.  5  Considerations before and during prostanoid therapy. RHC right-sided heart catheter investigation, CTPA multidetector lungscan-based pulmonary angiography, PFT pulmonary function test, DLCO CO diffusion capacity, 6MWD 6-min walk distance, NYHA FC ­modified New York Heart Association functional class assessment, LV ­dysfunction left ventricular systolic or diastolic dysfunction,

PVOD pulmonary veno-occlusive disease, CTEPH chronic thromboembolic pulmonary hypertension, ICM intensive care medicine including catecholamines, dialysis, ventilatory support, as needed, AST atrial septostomy, LTX lung transplantation, hemodynamics, most importantly cardiac index pulmonary arterial oxygen saturation, and right atrial pressure, QOL quality of life

reached this end point in contast to 4% in the placebo group (p = 0.007). The mean increase in 6MWD was 37 m (p = 0.004) and 59  m among subjects with PPH. Subjects’ well-being improved as evidenced by quality-of-life scores and the Mahler dyspnea index. Hemodynamic measures at 12 weeks improved slightly but significantly in the treatment group compared with the placebo group (p < 0.001) when preinhalation values are considered. There was a major improvement when postinhalation values are considered. Side effects consisted of symptoms related to systemic vasodilation. More ­syncopal episodes (eight vs. five, not significant) occurred in the iloprost group although they were not associated with clinical deterioration. Iloprost also improved hemodynamics and physical capacity in a small series of lung fibrosis patients [73], decompensated right-sided heart failure patients [33], and HIV patients [74]. A long-term observational study from Germany described patients who were started on treatment with inhaled iloprost between 1996 and 2002 as compassionate treatment, a time when no approved therapies were available in that country [75]. After 1, 2, and 3 years, survival was 79, 70, and 59%. Event-free survival, i.e. survival without transplantation on iloprost monotherapy, was only 53, 29, and 20%. When these results are discussed, it should be considered that during the observation period alternative options became available (in particular orally administered beraprost and bosentan), a dose increase was not allowed, and financial restrictions may have biased the result.

In a long-term observational study from Germany sponsored by the pharmaceutical industry, the survival after 1 and 2 years was 92 and 87% and event-free survival was 84 and 74% in IPAH patients (Olschewski et al. unpublished data). These results are comparable with recent long-term data obtained with endothelin receptor antagonists.

3.7 Combination with Inhaled Iloprost Hoeper et  al. studied 20 patients with IPAH that were on either inhaled iloprost or orally administered beraprost. Three months’ therapy with bosentan added to the prostacyclin resulted in improvement in exercise capacity (6MWD) and maximal oxygen consumption [76]. The reverse order, addition of inhaled prostacylin to an orally administered endothelin antagonist was used in the STEP trial (“Iloprost inhalation solution safety and pilot efficacy trial in combination with bosentan for evaluation in pulmonary arterial hypertension”), a double-blind placebo-controlled trial of 67 functional class III and functional class IV patients on a stable dose of bosentan for 3 months [77]. Subjects were randomized to either iloprost 5  mg six to nine times daily or placebo with the 6MWD distance after inhalation as the primary end point. The mean improvement of 26 m versus the distance achieved by the placebo group did not meet statistical significance (p = 0.051),

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but improvements in secondary end points including ­functional class and time to clinical worsening favored inhaled iloprost. In their recent 12-week COMBI study (The Combination Therapy of Bosentan and Aerosolized Iloprost in Idiopathic Pulmonary Arterial Hypertension), Hoeper et al. randomized stable functional class III IPAH patients, currently on bosentan (125 mg twice daily) to either inhaled iloprost 5 mg six times daily or no additional therapy [78]. The investigators powered this study with an assumption that addition of iloprost to bosentan would yield the same improvement as iloprost monotherapy compared with a placebo, an increase in the 6MWD of 45 ± 75 m. Because of slow recruitment and the results of the interim analysis (36 completed and 40 enrolled patients) this trial was terminated early. Sildenafil may act synergistically with prostanoids owing to its interaction with cyclic AMP breakdown via inhibition of phosphodiesterase 4 via cyclic GMP [79]. Ghofrani et al. investigated the effect of adjunct therapy with sildenafil in patients on inhaled iloprost in 14 out of 73 PAH patients who were on monotherapy with inhaled iloprost. Over 9–12 months, follow-up exercise capacity and hemodynamics improved [80].

3.8 Treprostinil: Inhaled Inhaled treprostinil has similar effects to inhaled iloprost; however, there are significant differences in the pharmacodynamics. This allows for a shorter inhalation time and fewer inhalations per day [81]. For inhaled treprostinil the maximal acute hemodynamic effects occurred later than for iloprost and the effects lasted longer. A transient drop in systemic arterial pressure occurred after iloprost inhalation but not with treprostinil. The maximally tolerated dose with a near maximal acute decrease in pulmonary vascular resistance occurred with the 30-mg dose. After inhalation with a pulsed ultrasonic nebulizer with 18 pulses, nine pulses, three pulses, two pulses, or one pulse (i.e. one breath), each mode achieved comparable, sustained vasodilation without significant side effects. These pharmacodynamic properties allowed for the application of treprostinil via a metered dose inhaler [82]. In a small cohort, the addition of inhaled iloprost to oral PAH therapy produced significant improvements in 6MWD, with nearly all patients improving physical capacity and functional class [83]. The results of a randomized placebocontrolled study with inhaled treprostinil (TRIUMPH-1) have not been published but the major results were demonstrated at clinical conferences. The study drug or the placebo was inhaled with a special ultrasonic nebulizer four times daily. Patients were on a stable treatment with bosentan or sildenafil before enrollment and this therapy was not changed

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during the study period. Patients on inhaled treprostinil improved significantly as compared with those on a placebo (p < 0.001). Side effects were mostly mild and did not involve toxic effects. Combination of sildenafil with inhaled treprostinil had synergistic hemodynamic effects [60].

4 Management 4.1 Decision for a Prostanoid Therapy The decision for a prostanoid therapy is – in most cases – equivalent to a lifelong commitment for this kind of drug. Because all approved prostanoids employ demanding application modes and are prone to side effects, this decision must be based on solid ground.

4.2 Potential Treatment Hazards Left ventricular dysfunction, either systolic or diastolic, represents a contraindication for prostanoids owing to the results of the FIRST study [47]. In this study, 471 patients with left ventricular failure and mild pulmonary hypertension were randomized to continuous intravenously administered epoprostenol or a placebo. After 6 months, there was a significantly higher mortality in the epoprostenol group compared with the control group. There have been no studies in patients with mild left ventricular failure and severe pulmonary hypertension and there is no consensus for how to treat “out of proportion” pulmonary hypertension in left-sided heart failure. However, there is consensus, although there has been no formal study, not to employ prostanoids in patients with a pulmonary artery wedge pressure (PAWP) greater than 15 mmHg. As a consequence, all candidates for targeted therapy should undergo right-sided heart catheterization and a number of further diagnostic tests before prostanoid therapy is initiated. Prostanoids are very potent pharmacologic agents and may cause worsening of gas exchange, which may become life-threatening.

4.2.1 Pulmonary Veno-Occlusive Disease Pulmonary veno-occlusive disease in adults is 10 times less common than IPAH. Although PAWP is normal, these patients have a significantly increased pulmonary capillary pressure and they are prone to lung edema, disturbed gas exchange, pleural effusion, and even pericardial ­effusion. Thin-slice CT of the lungs normally reveals ­typical signs such as thickened interlobular septae and centrilobular

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opacities and the bronchoalveolar lavage reveals excess ironladen macrophages [84]. Although prostanoids may worsen all of these disturbances, they have been used with success, mostly in combination w+ith high-dose diuretics [85].

4.2.2 Chronic Thromboembolic Pulmonary Hypertension CTEPH patients by definition have a heterogeneous lung perfusion with low- or no-perfusion areas and hyperperfused areas at the same time. Any vasodilators will primarily increase the blood flow of the hyperperfused areas, resulting in worsening of hypoxia. In many cases this is partly compensated by an increased cardiac index and the secondary increase in SvO2. Currently the most sensitive test for CTEPH is perfusion scintigraphy. In the AIR study, about 30% of the patients suffered from nonsurgical CTEPH. This subgroup had the same benefit from inhaled iloprost therapy as the PAH patients with respect to the primary end point. However, there was no significant increase in 6MWD of this subgroup compared with the placebo group [72]. A similar result was recently found for bosentan in CTEPH patients [86]. Currently, there is no approval for any targeted therapy for CTEPH, except for inhaled iloprost in Australia.

4.2.3 Lung Diseases Patients with obstructive lung disease or lung fibrosis have both heterogeneous ventilation and heterogeneous perfusion, and are prone to ventilation–perfusion mismatch and shunt blood flow. In obstructive lung diseases any vasodilator, even inhaled NO, will cause worsening of gas exchange; in lung fibrosis this depends on the individual pattern of the patient. Inhaled prostanoids have been used as compassionate treatment in lung fibrosis with some success [73], but there have been no formal studies and there is no approval in this indication worldwide.

4.2.4 Liver Diseases Liver diseases may cause severe hypoxemia owing to inhomogeneous dilatation of the pulmonary vessels (hepatopulmonary syndrome) but they may also cause diffuse narrowing of the pulmonary vessels with decreased cardiac index ­(portopulmonary hypertension). The latter has been considered an indication for epoprostenol therapy, albeit with moderate success [46]. Such patients have also been treated with inhaled iloprost which was combined with sildenafil when this became available [87]. This treatment led to a major improvement in some and clinical stabilization in most

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patients and was associated with moderate side effects. However, prostanoids have not been approved in this indication worldwide.

4.2.5 Hereditary Telangiectasia (Osler’s Disease) Osler’s disease may be associated with pulmonary hypertension, either due to an ALK-1 mutation or due to chronic thromboembolism or hepatic involvement. If bleeding is not a clinical issue, patients may be treated like PAH patients. However, prostanoids deteriorate thrombocyte function and may cause worsening of bleeding. Although inhaled prostanoids would appear advantageous, there has been no formal study or there is no approval for this orphan indication.

4.3 Start of Prostanoid Treatment Most patients who start a prostanoid treatment have been treated with non-prostanoid medication such as endothelin receptor antagonists or phosphodiesterase 5 inhibitors. Then they are switched to a prostanoid or they receive combination therapy (Fig. 6). Patients who are primarily started on a prostanoid are mostly in NYHA functional class III or IV showing hemodynamic signs of right-sided heart decompensation according to their increased right atrial pressure in combination with a reduced cardiac index and central venous oxygen saturation. Sometimes these patients primarily need intensive care medicine. The typical procedure is to treat water retention with intravenous diuretics and start with a low-dose prostanoid infusion or a prostanoid inhalation therapy. If the systemic pressure is not sufficient, patients receive low-dose catecholamines in parallel. There is positive experience with dobutamine, dopamine, and adrenalin in such a setting. Noradrenalin should be used with great caution because it may further constrict the ­pulmonary vessels and promote right-sided heart decompensation and death. The prostanoid dose is then uptitrated and the resting medication is downtitrated as soon as possible. It may be adequate to start early oral PAH medication in combination with the prostanoid, although there are no published data to support this.

4.4 Transition Between Targeted PAH Therapies If a patient has been on a prostanoid treatment, it may be possible to switch to a less demanding therapy (Fig.  6). This option will apply if a patient had in right-sided heart decompensation at diagnosis and had a major improvement on

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Fig. 6  Black arrows mark areas where locally deposited drug penetrates the airway wall and diffuses into the pulmonary artery wall. Note that the terminal arterioles are completely surrounded by alveolar surfaces. (Reproduced with permission from European Respiratory Society Journals Ltd [16])

prostanoid treatment. Non-prostanoid therapy may be an endothelin receptor antagonist, a phosphodiesterase inhibitor, or a calcium channel blocker. Calcium channel blocker responders are characterized by a major acute vasodilator response to NO or prostanoids. However, this response becomes weaker or is completely lost with disease duration and severity and may be restored with prostanoid therapy. With ongoing intravenous medication, it is difficult to assess vasoreactivity, because the vessels are maximally dilated by the drug. If there was a substantial improvement compared to the baseline with a short-lived substance such as epoprostenol, it would be possible to assess the patient’s vasoreactivity by halting the infusion for a short period of time after the catheter has been placed in the right side of the heart. For safety reasons, this test has rarely been performed. If the hemodynamic response to the prostanoid was less favorable or there is no indication of major vasoreactivity despite an established prostanoid therapy, an add-on nonprostanoid may be suitable. The most convincing data exist for the combination of epoprostenol with sildenafil as discussed earlier (PACES [53]), but there has been no formal study that has compared the combination of a prostanoid with a phosphodiesterase 5 inhibitor versus endothelin receptor antagonists.

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that transition from epoprostenol to treprostinil is safe. However, the dose of treprostinil is about double the ­epoprostenol dose and there is an increased risk of Gramnegative septicemia, as discussed previously. Another alternative is iloprost infusion in countries where this is available. The practical aspects are the same as for treprostinil, the dose requirements are much lower than those for epoprostenol, but there have been no transition studies and intravenously administered iloprost is only approved for PAH in New Zealand. Transition to subcutaneously administered treprostinil is an option for patients who have experienced life-threatening complications from the intravenous infusion. The major drawback is infusion site pain, as discussed earlier. Transition to inhaled iloprost is another option. The major drawback is the need to inhale six to nine times a day. The advantage is there are fewer systemic side effects. There are only a few published data on this transition. In a small series of children it worked in most of the cases [88]. If patients have been on higher doses of intravenously administered epoprostenol (less than 15–30 ng/kg/min), it may not be possible to completely taper off the infusion.

4.4.2 Switch from Inhaled Iloprost to Another Prostanoid There are two major reasons to consider this switch: (1) the frequent inhalations are too time-consuming and demanding and (2) the effect is not sufficient. The first reason normally becomes evident within the first few days or weeks of therapy; the second may become evident after months or years. Although the dose of inhaled iloprost is quite stable over the first 2 years, there are patients who need more drug over time. If the upper dose limit is reached (nine times 5 mg) and the effect is not sufficient, a switch to a prostanoid infusion or an infusion as an add-on to the inhalation are the options.

4.4.3 Switch from Subcutaneously Administered Treprostinil If subcutaneously administered treprostinil is not well tolerated (site pain), the infusion can be switched to intravenous, if a central venous access is in place. The dose requirement is the same. If alternative prostanoids are considered, there is uncertainty about the equivalent dose.

4.4.1 Switch from Intravenously Administered Epoprostenol to Another Prostanoid If a patient is on intravenously administered epoprostenol, switching to another prostanoid therapy may be considered. Treprostinil infusion offers practical advantages (no cooling, easy handling of the infusion) and two studies demonstrated

4.5 Lung Transplantation Patients who need a prostanoid may be considered potential candidates for lung transplantation and atrial septostomy.

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On the other hand, patients who are considered candidates for lung transplantation should be treated with a prostanoid. If they do not improve sufficiently, they qualify for listing for transplantation. Otherwise, their prognosis may be better with medical therapy.

in the pulmonary vessels. Prostanoid therapy has been shown to be efficacious, particularly in advanced disease. Combination of other targeted therapies with prostanoids appears to be effective and safe. Despite demanding application methods, prostanoids remain a mainstay in the treatment of PAH.

4.6 Daily Management Issues

References

As prostanoid patients represent a small population with a life-threatening disease and a risky medication, a strong partnership between the patient and the health care professionals is very important. In most centers, there is a long-lasting personal relationship between individual patients and nurses and/or physicians. Most patients experience some degree of side effects. They must learn to accept this for the sake of their physical capacity and survival. They often need to talk with an experienced professional about these issues. Both water retention and overnutrition can cause significant clinical deterioration. Patients must learn to control their water, salt, and calorie intake and to adapt the diuretic dose to their needs. Ascites may be caused by treatment with prostanoid infusions but may also be due to right-sided heart decompensation. The management of ascites may be very difficult. Infection management is also very important, particularly in patients on intravenous or subcutaneous medication. Patients profit from a written plan for the correct response to a given situation including antibiotic treatment and eventually removal of the intravenous line or change of the subcutaneous line. Daily physical activity prevents deconditioning and decreasing physical capacity. However, physical overload can promote right-sided heart decompensation both acutely (syncope) and chronically and must be avoided. Reimbursement issues can interfere with therapeutic decisions. In some countries a necessary combination therapy will not be reimbursed; in other countries there is no approved therapy at all or none of the approved therapies are reimbursed. This is a very demanding issue and the medical community as well as pharmaceutical companies should increase their efforts to make effective therapies available to all patients.

5 Conclusion In pulmonary hypertension, a lack of prostacyclin is an important component of the pathologic changes. Therapy with prostanoids restores physiological pathways involving the pulmonary arterial vessel wall, the platelets, and inflammatory cells and exerts beneficial clinical effects antagonizing vasoconstriction, inflammation, and aggregation of circulating cells

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104  Prostacyclin and Prostaglandins 57. Gomberg-Maitland M, Tapson VF, Benza RL et al (2005) Transition from intravenous epoprostenol to intravenous treprostinil in pulmonary hypertension. Am J Respir Crit Care Med 172:1586–1589 58. Sitbon O, Manes A, Jais X et al (2007) Rapid switch from intravenous epoprostenol to intravenous treprostinil in patients with pulmonary arterial hypertension. J Cardiovasc Pharmacol 49:1–5 59. Centers for Disease Control (CDC) (2007) Bloodstream infections among patients treated with intravenous epoprostenol or intravenous treprostinil for pulmonary arterial hypertension–seven sites, United States, 2003–2006. MMWR Morb Mortal Wkly Rep 56:170–172 60. Laliberte K, Arneson C, Jeffs R, Hunt T, Wade M (2004) Pharmacokinetics and steady-state bioequivalence of treprostinil sodium (Remodulin) administered by the intravenous and subcutaneous route to normal volunteers. J Cardiovasc Pharmacol 44:209–214 61. Lang I, Gomez-Sanchez M, Kneussl M et  al (2006) Efficacy of long-term subcutaneous treprostinil sodium therapy in pulmonary hypertension. Chest 129:1636–1643 62. Simonneau G, Barst RJ, Galiè N et al (2002) Continuous subcutaneous infusion of treprostinil, a prostacyclin analogue, in patients with pulmonary arterial hypertension. A double-blind, randomized, placebo-controlled trial. Am J Respir Crit Care Med 165:800–804 63. Oudiz RJ, Schilz RJ, Barst RJ et al (2004) Treprostinil, a prostacyclin analogue, in pulmonary arterial hypertension associated with connective tissue disease. Chest 126:420–427 64. Barst RJ, Galiè N, Naeije R et  al (2006) Long-term outcome in pulmonary arterial hypertension patients treated with treprostinil. Eur Respir J 28:1195–1203 65. Gomberg-Maitland M, McLaughlin V, Gulati M, Rich S (2005) Efficacy and safety of sildenafil added to treprostinil in pulmonary hypertension. Am J Cardiol 96:1334–1336 66. Voswinckel R, Reichenberger F, Enke B et al (2008) Acute effects of the combination of sildenafil and inhaled treprostinil on haemodynamics and gas exchange in pulmonary hypertension. Pulm Pharmacol Ther 25:824–832 67. Galiè N, Humbert M, Vachiery JL et al (2002) Effects of beraprost sodium, an oral prostacyclin analogue, in patients with pulmonary arterial hypertension: a randomized, double-blind, placebo-controlled trial. J Am Coll Cardiol 39:1496–1502 68. Barst RJ, McGoon M, McLaughlin V et al (2003) Beraprost therapy for pulmonary arterial hypertension. J Am Coll Cardiol 41:2119–2125 69. Walmrath D, Schneider T, Pilch J, Grimminger F, Seeger W (1993) Aerosolised prostacyclin in adult respiratory distress syndrome. Lancet 342:961–962 70. Walmrath D, Schneider T, Schermuly R, Olschewski H, Grimminger F, Seeger W (1996) Direct comparison of inhaled nitric oxide and aerosolized prostacyclin in acute respiratory distress syndrome. Am J Respir Crit Care Med 153:991–996 71. Gessler T, Schmehl T, Hoeper MM et al (2001) Ultrasonic versus jet nebulization of iloprost in severe pulmonary hypertension. Eur Respir J 17:14–19 72. Olschewski H, Simonneau G, Galiè N et al (2002) Inhaled iloprost for severe pulmonary hypertension. N Engl J Med 347:322–329

1463 73. Olschewski H, Ghofrani HA, Walmrath D et al (1999) Inhaled prostacyclin and iloprost in severe pulmonary hypertension secondary to lung fibrosis. Am J Respir Crit Care Med 160:600–607 74. Ghofrani HA, Friese G, Discher T et al (2004) Inhaled iloprost is a potent acute pulmonary vasodilator in HIV-related severe pulmonary hypertension. Eur Respir J 23:321–326 75. Opitz CF, Wensel R, Winkler J et al (2005) Clinical efficacy and survival with first-line inhaled iloprost therapy in patients with idiopathic pulmonary arterial hypertension. Eur Heart J 26:1895–1902 76. Hoeper MM, Taha N, Bekjarova A, Gatzke R, Spiekerkoetter E (2003) Bosentan treatment in patients with primary pulmonary hypertension receiving nonparenteral prostanoids. Eur Respir J 22:330–334 77. McLaughlin VV, Oudiz RJ, Frost A et al (2006) Randomized study of adding inhaled iloprost to existing bosentan in pulmonary arterial hypertension. Am J Respir Crit Care Med 174:1257–1263 78. Hoeper MM, Leuchte H, Halank M et al (2006) Combining inhaled iloprost with bosentan in patients with idiopathic pulmonary arterial hypertension. Eur Respir J 28:691–694 79. Ghofrani HA, Wiedemann R, Rose F et  al (2002) Combination therapy with oral sildenafil and inhaled iloprost for severe pulmonary hypertension. Ann Intern Med 136:515–522 80. Ghofrani HA, Rose F, Schermuly RT et al (2003) Oral sildenafil as long-term adjunct therapy to inhaled iloprost in severe pulmonary arterial hypertension. J Am Coll Cardiol 42:158–164 81. Voswinckel R, Enke B, Reichenberger F et  al (2006) Favourable effects of inhaled treprostinil in severe pulmonary hypertension. J Am Coll Cardiol 48:1672–1681 82. Voswinckel R, Reichenberger F, Gall H et al (2009) Metered dose inhaler delivery of treprostinil for the treatment of pulmonary hypertension. Pulm Pharmacol Ther 22:50–56 83. Channick RN, Olschewski H, Seeger W, Staub T, Voswinckel R, Rubin LJ (2006) Safety and efficacy of inhaled treprostinil as addon therapy to bosentan in pulmonary arterial hypertension. J Am Coll Cardiol 48:1433–1437 84. Rabiller A, Jais X, Hamid A et al (2006) Occult alveolar haemorrhage in pulmonary veno-occlusive disease. Eur Respir J 27:108–113 85. Humbert M, Maître S, Capron F, Rain B, Musset D, Simonneau G (1998) Pulmonary edema complicating continuous intravenous prostacyclin in pulmonary capillary hemangiomatosis. Am J Respir Crit Care Med 157:1681–1685 86. Jais X, D’Armini AM, Jansa P et al (2008) Bosentan for treatment of inoperable chronic thromboembolic pulmonary hypertension: BENEFiT (Bosentan Effects in iNopErable Forms of chronIc Thromboembolic pulmonary hypertension), a randomized, placebocontrolled trial. J Am Coll Cardiol 52:2127–2134 87. Reichenberger F, Voswinckel R, Steveling E et al (2006) Sildenafil treatment for portopulmonary hypertension. Eur Respir J 28:563–567 88. Ivy DD, Doran AK, Smith KJ et  al (2008) Short- and long-term effects of inhaled iloprost therapy in children with pulmonary arterial hypertension. J Am Coll Cardiol 51:161–169

Chapter 105

Endothelin Receptor Antagonists for the Treatment of Pulmonary Arterial Hypertension Victor F. Tapson and Robyn J. Barst

Abstract  There are distinct endothelin (ET) isoforms, ET-1, ET-2 and ET-3, of which ET-1 is considered the most active. Local ET-1 synthesis in vascular endothelial cells appears to play an important role in the pathobiological and pathophysiological processes of pulmonary arterial hypertension (PAH), promoting vasoconstriction, smooth muscle cell proliferation, as well as inflammation and fibrosis. ET acts via two different receptors: ETA receptors on vascular smooth muscle cells and ETB receptors on vascular smooth muscle cells and endothelial cells. Both ETA and ETB receptors mediate vascular smooth muscle cell proliferation. ETA receptors mediate vasoconstriction. ETB receptors may have a role in either vasoconstriction (via actions on smooth muscle cell ETB receptors) or vasodilation, owing to release of nitric oxide and prostacyclin via actions on endothelial cell ETB receptors. Clearance of ET-1 appears to be due to the ETB receptors on endothelial cells. Plasma levels of ET-1 correlate with the severity of PAH and prognosis and clearance of ET-1 in the pulmonary vasculature in this disease is reduced. Although the clinically available drugs that work via ET receptor antagonism bear substantial similarities, and although it would appear that a number of class effects exist, they are distinct entities, and will be discussed separately in this chapter. Adverse effects such as elevated liver function test levels, peripheral edema, and anemia appear to be ET receptor antagonist (ERA) class effects. Although most of the available data on teratogenicity are with bosentan, this is quite likely to be a class effect. Studies in animals have shown dose-dependent teratogenic effects, including malformation of the head, mouth, face, and large blood vessels, occurring during the first trimester of pregnancy. Thus, education and careful monitoring of treatment are crucial. Keywords  Endothelial cell • Vasoconstriction • Cell proliferation • Vasodilation • Antiproliferative • Teratogenicity • Bosentan • Ambrisentan R.J. Barst (*) Columbia University, New York, NY 10032, USA e-mail: [email protected]

1 Background and Pharmacology Endothelins (ETs) are a family of 21-amino acid peptides, primarily secreted by endothelial cells. These peptides and their receptors are present in a variety of tissues, where they play important physiological and pathophysiological roles, principally involving the cardiovascular system [1–3]. Preproendothelins are hydrolyzed by endopeptidases into proendothelins (39 amino acids), which are converted to the ETs by ET-converting enzyme. There are distinct ET isoforms, ET-1, ET-2, and ET-3, of which ET-1 is considered the most active. Local ET-1 synthesis in vascular endothelial cells appears to play an important role in the pathobiological and pathophysiological processes of pulmonary arterial hypertension (PAH), promoting vasoconstriction, smooth muscle cell proliferation, as well as inflammation and fibrosis [4–6]. A schematic of the ET system is shown in Fig. 1. ET acts via two different receptors: ETA receptors on vascular smooth muscle cells and ETB receptors on vascular smooth muscle cells and pulmonary vascular endothelial cells. Both ETA and ETB receptors mediate vascular smooth muscle cell proliferation [6, 8]. ETA receptors mediate vasoconstriction. ETB receptors may have a role in either vasoconstriction (via actions on smooth muscle cell ETB receptors) or vasodilation, owing to release of nitric oxide and prostacyclin via actions on endothelial cell ETB receptors. Clearance of ET-1 appears to be due to the ETB receptors on endothelial cells. Presently, there is no clear clinical advantage of either selective (ETA) or nonselective (ETA and ETB) receptor blockade. Plasma levels of ET-1 correlate with the severity of PAH and prognosis [5, 9] and clearance of ET-1 in the pulmonary vasculature in this disease is reduced [4, 5]. Continued basic research has improved the understanding of the complex interactions between the ET axis and other biologic systems involving human pathobiological and pathophysiological processes. Although the clinically available drugs that work via ET receptor antagonism bear substantial similarities, and although it would appear that a number of class effects exist, they are distinct entities, and will be discussed separately.

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_105, © Springer Science+Business Media, LLC 2011

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Adverse effects such as elevated liver function test levels, peripheral edema, and anemia appear to be ET receptor antagonist (ERA) class effects. Although most of the available data

Endothelin System in Vascular Tissue ET-1

Lumen ECE

Vascular endothelium

Big-ET-1

ET-1

Smooth muscle cell

ETB

2 Nonselective (Dual) Endothelin Receptor Blockade

NO PGI2 ETA

ETB

Vasoconstriction proliferation

on teratogenicity are with bosentan, this is quite likely to be a class effect. Studies in animals have shown dose-dependent teratogenic effects, including malformation of the head, mouth, face, and large blood vessels, occurring during the first trimester of pregnancy. Thus, education and careful monitoring of treatment are crucial. An overview of the differences between the three available ERAs is presented in Table 1.

Vasodilation antiproliferation

ET-1=endothelin-1; Big-ET-1=proendothelin-1; ECE=endothelin-converting enzyme; NO=nitric oxide; PGI2=prostacyclin Dupuis. Lancet 2001

Fig.  1  The endothelin system in vascular tissue. ET-1 endothelin-1, BIG-ET-1 proendothelin-1, ECE endothelin-converting enzyme, NO nitric oxide, PGI2 prostacyclin. (Reprinted from [7] with permission from Elsevier)

2.1 Bosentan Bosentan is an antagonist of both ETA and ETB receptors, with approximately 50 times higher affinity for ETA receptors than for ETB receptors (i.e. compared with other ERAs studied in PAH, bosentan is much less selective for ETA). Bioavailability is approximately 50%, with the elimination process of bosentan in humans being hepatic and entirely

Table 1  Endothelin receptor antagonists: a comparison Bosentan

Sitaxsentanf

Ambrisentan

ETA selectivity

No (ETA > ETB, 50 times)

Yes (ETA > ETB, 6,500 times)

Approved in USA Approved in the European Union Dose Elimination half-life Protein binding Absolute bioavailability Metabolism

Yes Yesa

No Yes

Yes (ETA > ETB, 260–4,000 times) Yes Yes

62.5 mg bid × 1 month, then 125 mg bid 5.4 h Very high (>98%) 50% CYP2C9/CYP3A4 Biliary excretion Yes

100 mg qd 10 h Very high (99%) 70–100% CYP2C9/CYP3A4 Renal elimination Yes

5 or 10 mg qd 15 hb Very high (99%) Unknown Nonrenalc

Effect on/of CYP2C9 and Possible CYP3A4d Significant warfarin No Yes No interaction Interaction with sildenafil Yes Minimal None Teratogenic Yes Yes Yes Causes anemia Yes Yes Yes Causes peripheral edema Yes Yes Yes Causes elevated LFT levels Yes Yes Minimal Yes Yes Yes Liver function monitoring requirede ETA endothelin A receptor, ETB endothelin B receptor, bid twice daily, qd once daily, CYP cytochrome, LFT liver function test a Also approved in Switzerland, Canada, Australia, and Japan b  Although ambrisentan has a 15-h terminal half-life, the trough concentration at the steady state is about 15% of the mean peak concentration and the accumulation factor is about 1.2 after long-term daily dosing; thus, the effective half-life of ambrisentan is about 9 h c  The elimination of ambrisentan is predominantly by nonrenal pathways, but the relative contributions of metabolism and biliary elimination have not been well characterized. It appears to be independent of cytochrome P450 pathways and to occur predominantly by glucuronidation and transport via the bile into the feces d  Bosentan is an inducer of CYP2C9 and CYP3A4 and possibly also of CYP2C19. Sitaxsentan is a strong competitive inhibitor of CYP2C9 and has been reported to also moderately inhibit CYP2C19 and CYP3A4. For ambrisentan, interactions with strong inhibitors of CYP3A4 and CYP2C19 are possible. The drug interaction potential of ambrisentan is not well characterized as fewer data are available e  See the text for rates of transaminase level elevation and monitoring with these drugs f  Sitaxsentan was withdrawn from the market and future clinical drug development stopped in 2010

105  Endothelin Receptor Antagonists for the Treatment of Pulmonary Arterial Hypertension

dependent on metabolism mediated by two cytochrome P450 enzymes (i.e. CYP3A4 and CYP2C9). The elimination halflife is approximately 5.4 h. There is biliary excretion and less than 3% of an administered oral dose is recovered in urine. Most interactions with concomitantly administered drugs are due to inhibition of these cytochrome P450 enzymes. Coadministration of cyclosporine A and bosentan resulted in markedly increased plasma concentrations of bosentan (i.e. concomitant use of bosentan and cyclosporine A is contraindicated). An increased risk of liver enzyme elevations was observed in patients receiving glyburide concomitantly with bosentan, i.e. glyburide is thus also contraindicated with bosentan. Bosentan has now been administered to more than 55,000 patients worldwide. It has been approved by the Food and Drug Administration in the USA for treatment of functional class II, III and IV PAH. It is also approved in Canada, the European Union, Switzerland, Australia, and Japan. In 2007, it was approved in the European Union for reduction of new digital ulcers in patients with systemic sclerosis and ongoing digital ulcer disease.

2.1.1 Clinical Trials in Pulmonary Arterial Hypertension with Bosentan Two prospective, randomized, double-blind, placebo-controlled trials demonstrated the safety and efficacy with bosentan for the treatment of PAH (shown in Tables 2, 3) [10, 11]. In 2001, Channick et al. [10] reported the findings of the first

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randomized, double-blind, placebo-controlled, multicenter study evaluating bosentan. In the 33 patients randomized 2:1 to bosentan (62.5 mg twice daily for the first 4 weeks with uptitration to 125 mg twice daily thereafter) versus a placebo, there was a 70-m improvement in the 6-min walk distance (6MWD) from 360 ± 19 m at the baseline to 430 ± 14 m at week 12. With the placebo the baseline d­istance was 355 ± 25 m and 349 ± 44 m at week 12 for a ­placebo-corrected improvement of 76 m (p = 0.02). There were also significant improvements in Borg dyspnea score, New York Heart Association functional class, and hemodynamic parameters, including mean pulmonary artery pressure (mPAP) (p = 0.013), cardiac index (p = 0.0001), and pulmonary vascular resistance (PVR) (p = 0.0002). Asymptomatic increases in liver function test levels were observed in two patients receiving bosentan. The Bosentan Randomized Trial of Endothelin Antagonist Therapy for Pulmonary Hypertension-1 Study (BREATHE-1) was the second prospective, double-blind, placebo-controlled bosentan trial [11]. In BREATHE-1, bosentan (125 or 250 mg twice daily) therapy was evaluated in 213 patients with functional class III and IV symptoms with either idiopathicPAH (IPAH) or PAH associated with connective tissue disease (CTD), for a minimum of 16 weeks. Bosentan improved the 6MWD by 36 m, whereas deterioration (−8 m) was seen with a placebo, for a placebo-corrected improvement of 44  m (95% confidence interval, 21–67  m; p = 0.0002). The composite end point of time to clinical worsening, defined as death, initiation of intravenous epoprostenol therapy, hospitalization for worsening PAH, lung transplantation, or atrial

Table 2  Pivotal randomized endothelin receptor antagonist trials in pulmonary arterial hypertension (PAH): study design Drug/triala N Population Dosage WHO classb Primary end point Bosentan Channick et al. [10] Rubin et al. [11] (BREATHE-1)

  32c 213

IPAH, PAH-CTD IPAH, PAH-CTD

125 mg bidd 125 mg bidd 250 mg bid

III III–IV

6MWD 6MWD

Sitaxsentan Barst et al. [12] (STRIDE-1)

178

Peak

247

100 mg qd 300 mg qd 50 mg qd 100 mg qd Bosentan bidd,e

III–IV

Barst et al. [13] (STRIDE-2)

IPAH, PAH-CTD, CHD IPAH, PAH-CTD, CHD

II–IV

6MWD

·

V O2

Ambrisentan Galiè et al. [14] (ARIES-2) 192 IPAH, PAH-CTD 2.5 mg, 5 mg I–IV 6MWD Galiè et al. [14] (ARIES-1) 202 IPAH, PAH-CTD 5 mg, 10 mg I–IV 6MWD N number enrolled, WHO World Health Organization, IPAH idiopathic pulmonary arterial hypertension, PAH-CTD pulmonary arterial hypertension related to underlying connective tissue disease, CHD congenital heart disease, 6MWD 6-min walk distance, · V O2 oxygen consumption, qd once daily, bid twice daily a Study durations: all 12 weeks except Rubin et al. (16 weeks), and Barst et al. (STRIDE-1) (18 weeks) b Percentage of class IV patients included in these studies: Channick et al., 0%; BREATHE-1, 8.5%; STRIDE-1, 1%; STRIDE-2, 7%; ARIES-2, less than 10%; ARIES-1, less than 10% c 2:1 bosentan to placebo d In the bosentan trials, 62.5 mg twice daily was administered for 1 month; at that time, the dosage was increased to 125 mg twice daily e The bosentan arm was open-label

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Table 3  Pivotal randomized endothelin receptor antagonist trials in PAH: results Drug/triala Primary end point Secondary end points Bosentan Channick et al. [10]

Rubin et al. [11] (BREATHE-1)

Sitaxsentan Barst et al. [12] (STRIDE-1)

Barst et al. [13] (STRIDE-2)

Ambrisentan Olschewski et al. [15] (ARIES-2)

PC 6MWD increase of 76 m ( p = 0.021; 95% CI, 12–139)b

• • • • • • •

PAP (mean) improved (0.013) Cardiac index improved (p < 0.001) PVR improved (p < 0.001) WHO class improved (p < 0.019) TCW improved (p = 0.033) Hemodynamics not studied Borg index: mean effect –0.6 favoring bosentan (95% CI, –1.2 to –0.1)c • TCW improved (p = 0.01 for each dose) • WHO class: 42% of bosentan and 30% of placebo patients with improved functional class at week 16 [mean treatment effect of 12% favoring bosentan (95% CI, −3 to 25)]

PC 6MWD increase of 44 m ( p < 0.001; 95% CI, 21–67)a

·

Increased peak V O2 ( p < 0.01 for 300-mg dose only)

PC 6MWD increased:50 mg: 24.2 m ( p = 0.07) (NS) 100 mg: 31.4 m ( p = 0.03; 95% CI, 5.37–57.44) Bosentan 29.5 m ( p = 0.05) (NS) PC 6MWD increased:2.5 mg: 32.3 m ( p = 0.022) 5 mg: 59.4 m ( p = 0.0002)

Oudiz et al. [43] (ARIES-1)

100 mg: PC 6MWD increase of 35 m p < 0.01 300 mg: PC 6MWD increase of 33 m p < 0.01 PAP (mean) improved with 300-mg dose (p < 0.001) RAP (mean) improved (300 mg, p < 0.001; 100 mg, p < 0.005) Cardiac index improved (300 mg, p < 0.001; 100 mg, p < 0.02) PVR improved (300 mg, p < 0.001; 100 mg, p < 0.001) WHO class: p < 0.02 for both doses TCW NS (only four total events) Hemodynamics not studied WHO class: 100 mg improved (p < 0.014); 50 mg NS; bosentan NS TCW NS for all arms Borg index NS for all arms WHO class NS Borg index improved (2.5 mg, p = 0.036; 5 mg, 0.038) TCW improved (2.5 mg, p = 0.005; 5 mg, 0.008) SF-36 (QOL) improved (2.5 mg, p = 0.005; 5 mg, 0.04) WHO class NS dBorg index NSd TCW NS

PC 6MWD increased:5 mg: 30.6 m ( p = 0.008; 95% CI, 2.9–58.3) 10 mg: 51.4 m ( p = 0.0001; 95% CI, 26.6–76.2) PC 6MWD placebo-corrected 6-min walk distance, CI confidence interval, PAP pulmonary artery pressure, PVR pulmonary vascular resistance, WHO World Health Organization, TCW time to clinical worsening, RAP right artrial pressure, NS not significant, SF-36 36-item Short-Form · Health Survey, QOL (quality of life) V O2 oxygen consumption a Although both bosentan doses induced a significant treatment effect, the placebo-corrected improvement was more pronounced (not significant) for the 250-mg dose twice daily than for 125 mg twice daily regimen (54 and 35 m, respectively) b Both ambrisentan studies have been published in abstract form; at the time of this publication, neither has been published c Placebo-corrected improvement greater for 250 mg (–0.9, p = 0.012) than for 125 mg (–0.4, p = 0.42) d These secondary end points had clinically relevant improvements that achieved p < 0.05 but were not considered statistically significant owing to the prespecified approach for multiple comparisons

septostomy, was reduced by bosentan compared with a placebo (p = 0.0015, log-rank test). Abnormal liver function test levels, syncope, and flushing occurred more frequently in the bosentan group. The development of abnormal liver test levels was dose-dependent, occurring more commonly in patients treated with the 250 mg twice-daily dose, resulting in only the 125  mg twice-daily dose being recommended. Although patients with class IV symptoms were included in BREATHE-1, they were few, and eligible patients in class IV were “required to have a sufficiently stable clinical status to participate in a placebo-controlled trial.” [11]

2.1.2 Survival with Bosentan To determine the effect of first-line bosentan therapy on survival, 169 patients with IPAH from the two placebo-controlled trials described earlier were followed [17]. Observed survival up to 36  months was reported as Kaplan–Meier estimates and compared with predicted survival as determined for each patient by the NIH Primary Pulmonary Hypertension (previous term for IPAH) Registry equation. On the basis of these estimates, survival was 96% at 12  months and 89% at 24 months. In contrast, predicted survival was 69% and 57%,

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for 12 and 24 months, respectively. Furthermore, at the end of 12 and 24 months, 85% and 70% of patients, respectively, remained alive on bosentan monotherapy. Prognostic parameters included World Health Organization (WHO) functional class IV and baseline 6MWD below the median (358 m). Sitbon et al. [18] also evaluated survival with bosentan in functional class III IPAH patients. However, this was done on the basis of a comparison with historical data from similar patients treated with continuous intravenous administration of epoprostenol. Baseline data from the 139 patients treated with bosentan and the 346 patients treated with epoprostenol suggested that the epoprostenol patients had severer disease. Kaplan–Meier survival estimates after 12 and 24  months were 97% and 91%, respectively, in the bosentan cohort, and 91% and 84% in the epoprostenol cohort. Cox regression analyses adjusting for differences in baseline factors showed a greater probability of death in the epoprostenol cohort. When matched cohorts of 83 patients each were selected, survival estimates were similar. In the bosentan cohort, 87% and 75% of patients were followed up for 1 and 2 years, respectively, on bosentan monotherapy. Although these data are encouraging, they do not represent head-to-head prospective data and comparisons with epoprostenol therapy cannot accurately be made without randomized trial data. The results from a retrospective, “real-life” IPAH population offer additional support to the prospective clinical trial data. Provencher et al. [19] evaluated (1) overall survival and (2) event-free survival (i.e. survival without lung ­transplantation, acute right-sided heart failure with hospitalization and ­intravenous diuretic therapy, dobutamine infusion, and/or initiation of intravenous administration of epoprostenol). The 6MWD increased from 322 ± 15  m at the baseline to 364 ± 109  m at 4  months (p < 0.001), concomitant with improved hemodynamic parameters that were maintained at 1  year. The overall survival estimate at 2  years was 89% compared with 45% predicted by the equation derived from the NIH registry. Although 44% of patients survived “eventfree” at 2 years, more than half were in need of additional intervention, including epoprostenol therapy or lung transplantation. Prognostic outcome parameters included 6MWD, right atrial pressure at the baseline, and change in total pulmonary resistance after 4 months of bosentan therapy.

2.1.3 Functional Class II Patients Although the first two trials focused on functional class III patients, with a small number of class IV patients, data were lacking on patients less ill (i.e. functional class II patients). In the EARLY study, bosentan was compared with a placebo in class II patients with PAH, in a randomized, double-blind trial [20]. The effects of bosentan versus the placebo on the time to clinical worsening and functional class worsening were significant, with a clinical worsening risk reduction of 77%

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(p = 0.01), and less functional class worsening (3.4% vs. 13.2%, p < 0.03). Quality-of-life indices also improved. Finally, bosentan reduced PVR with a treatment effect of −22.6% (p < 0.0001); the change in 6MWD was not significant. These data provide evidence consistent with efficacy in treating mild pulmonary hypertension (class II) patients.

2.1.4 Connective Tissue Disease Bosentan has proven effective in PAH associated with CTDs [21]. In a long-term, open-label follow-up of the CTD subgroup from the two initial randomized PAH trials [10, 11], Kaplan–Meier estimates for observed long-term outcome suggested a benefit from bosentan [22, 23]. In addition, a benefit with bosentan was suggested in a 6-year, longitudinal, observational study comparing the long-term outcome in a cohort of scleroderma patients with PAH on first-line bosentan therapy with a matched historical control group treated with conventional therapy (including prostanoid therapy if needed) [24]. The effects of bosentan on quality of life and functional class were evaluated in class III PAH patients with CTD in an open-label study over 48  weeks (Tracleer use in PAH associated with scleroderma and CTD). After 48  weeks of bosentan therapy, a significant proportion of patients had improved functional class, whereas 57% remained stable and 16% worsened [25]. Quality of life, assessed by the 36-item Short-Form Health Survey, improved significantly at week 16 and was maintained at 48 weeks. The Kaplan–Meier estimate for survival was 92% at week 48 (95% confidence interval, 85–100%). These data support the use of bosentan in this patient population.

2.1.5 Pediatric Patients and Patients with Congenital Heart Disease BREATHE-3 was an open-label evaluation of bosentan in 19 pediatric patients with IPAH or PAH associated with congenital heart disease (CHD) [26]. The results showed that the single-dose and the multiple-dose pharmacokinetics of bosentan were similar to those observed in healthy adults. Although this study was primarily designed to assess pharmacokinetics and safety, improvements in mPAP and PVR were evident after 12 weeks of bosentan therapy. A number of uncontrolled, prospective, and retrospective studies have subsequently suggested improvement in functional parameters, hemodynamics, or both in children with PAH (IPAH and PAH associated with CHD) as well as in adult patients with CHD [27–31]. On the basis of the above data suggesting a potential benefit in PAH due to underlying CHD, the BREATHE-5 trial was initiated [32]. This was a multicenter, double-blind,

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randomized, placebo-controlled study of bosentan in patients with functional class III Eisenmenger syndrome. Fifty-four patients were randomized 2:1 to bosentan (n = 37) or a placebo (n = 17) for 16 weeks. The first objective was safety, that is, to prove that bosentan did not aggravate shunting (i.e. worsen oxygen saturation) and it did not. Compared with the placebo, bosentan reduced PVR (–472.0  dyn · s/cm5; p = 0.0383) and mPAP (–5.5  mmHg; p = 0.0363). The 6MWD increased by 53.1  m (p = 0.0079). Treatment was discontinued in two patients (5%) in the bosentan group and in two patients (12%) in the placebo group owing to adverse events. An open-label extension of BREATHE-5 revealed that exercise capacity was maintained for at least 6 months [33]. In another small study, improvement in clinical status, exercise tolerance, and hemodynamics was demonstrated in 22 adult patients with PAH associated with CHD after 1  year of bosentan therapy [34]. However, in yet another small study, after 2 years, the 6MWD returned to the baseline in most of the patients with PAH associated with CHD patients on bosentan therapy [35].

2.1.6 HIV and Pulmonary Arterial Hypertension In the multicenter, open-label, noncomparative, phase IIIb BREATHE-4 trial, the safety and efficacy of bosentan were evaluated in 16 patients with PAH related to HIV infection [36]. At week 16, cardiac index and PVR improved versus the baseline (p < 0.001). These hemodynamic changes were associated with improvement in right ventricular geometry and function as assessed by echocardiography.

2.1.7 Other Studies Ongoing investigational trials with bosentan include BENEFIT (bosentan effects in inoperable forms of chronic thromboembolic pulmonary hypertension), a double-blind, placebo-controlled trial designed to evaluate the safety and efficacy of bosentan treatment in patients with inoperable chronic thromboembolic pulmonary hypertension [37]. The treatment effect (co-primary end points of 6MWD and PVR) of bosentan was assessed in 113 patients who had inoperable chronic thromboembolic pulmonary hypertension and 44 who had previously undergone pulmonary endarterectomy. Patients were randomized to bosentan or a placebo for 16  weeks. The co-primary end point, PVR, decreased by 146  dyn · s/cm5 in the bosentan group and increased by 30 dyn · s/cm5 in the placebo group (p < 0.0001 for comparison of mean change between groups). The 6MWD increases were 2.9 m in the bosentan group and 0.8 m in the placebo group (p = 0.54 for comparison of mean change between groups). For secondary end points, functional class improvement was 14.5% versus 11.3% (p = 0.63), and the proportion

V.F. Tapson and R.J. Barst

of patients that clinically worsened was 2.6% versus 8.8% (p = 0.17), respectively, for bosentan versus placebo. On the basis of data reporting a significant impact of pulmonary hypertension on the course of sickle cell disease [38], the ASSET (bosentan to treat pulmonary hypertension in sickle cell disease) trial (ASSET-1 and ASSET-2) was undertaken to evaluate the use of bosentan in pulmonary hypertension associated with sickle cell disease [39]. However, owing to slow enrollment, the study was stopped prematurely.

2.1.8 Adverse Effects, Safety, and Drug Interactions A pharmacovigilance system has been set up in 18 countries to monitor the safety of bosentan; data are currently available on approximately 5,000 patients [12]. Over 2.5  years, an elevation of the levels of liver transaminases to more than 3  times the upper limit of normal values was observed in 7.6% of patients, corresponding to an annualized rate of 10.1%. These results were consistent across subgroups and causes. The liver transaminase level elevations were consistent with the levels reported in the randomized trials [10, 11]. Liver function test results should be routinely monitored on a monthly basis. Other side effects of bosentan include headache, flushing, lower-extremity edema, and anemia. The ­anemia is thought to be due to hemodilution. The hematocrit should be checked on a quarterly basis, and a pregnancy test in women of childbearing potential should be performed on a monthly basis. Bosentan is teratogenic. It also reduces the efficacy of oral contraceptives, so a barrier method should be added to hormonal contraception. Lower-extremity edema may develop, usually within the first several weeks after initiation. The edema can be usually controlled with diuretics. A potential concern regarding the combination of bosentan and sildenafil is a pharmacokinetic interaction between the two substances, resulting in increased plasma concentrations of bosentan with concomitant decreased plasma concentrations of sildenafil.

3 ETA-Selective Endothelin Receptor Blockade 3.1 Sitaxsentan Sitaxsentan, a selective ETA antagonist, was approved in the European Union, Canada, and Australia in 2007. It is approximately 6,000-fold more specific for the ETA receptor than the ETB receptor, with a longer half-life than bosentan, thereby permitting once-daily oral administration.

105  Endothelin Receptor Antagonists for the Treatment of Pulmonary Arterial Hypertension

3.1.1 Metabolism Metabolism of sitaxsentan is primarily via the cytochrome P450 CYP2C9 and CYP3A4 isoforms; its metabolites are less than one tenth as potent as the parent compound. Elimination of 50–60% of a sitaxsentan dose occurs renally (less than 1% as unchanged drug). Sitaxsentan is a strong competitive inhibitor of CYP2C9 and has been reported to also moderately inhibit CYP2C19 and CYP3A4.

3.1.2 Clinical Trials in Pulmonary Arterial Hypertension with Sitaxsentan Two double-blind, placebo-controlled randomized clinical trials with sitaxsentan included more than 400 patients (as shown in Tables  2, 3). STRIDE-1 (sitaxsentan to relieve impaired exercise) enrolled 178 patients with functional class II, III, or IV PAH, including IPAH or PAH related to CTD or CHD [40]. The primary end point was maximum oxygen consumption as measured by cardiopulmonary exercise testing; it improved by 3.1% in the sitaxsentan group receiving a 300-mg dose (p < 0.01) but was unchanged in the sitaxsentan group receiving a 100-mg dose. There were ­significant improvements in the secondary end point of 6MWD, with improvement in the sitaxsentan groups of 35 m (p < 0.01) for the 100-mg dose and 33  m (p < 0.01) for the 300-mg dose. Functional class and hemodynamics also improved. Both doses of sitaxsentan improved hemodynamics. Sitaxsentan interacts with warfarin, and the incidence of international normalized ratio elevation was greater in the sitaxsentan groups than in the placebo group. This effect is related to the inhibition by sitaxsentan of the CYP2C9 enzyme, the primary hepatic enzyme involved in the metabolism of warfarin. During this placebo-controlled trial and its extension (median exposure 26  weeks), 5% of patients on 100 mg and 21% of patients on 300 mg developed increased hepatic transaminase levels to more than 3 times the upper limit of normal. As with the higher dose (250 mg) of bosentan, the unacceptably high incidence of hepatic enzyme elevations with the 300-mg dose of sitaxsentan led to its discontinuation from clinical development. As with bosentan, a liver function test and pregnancy (when indicated) monitoring are performed monthly, and hematocrit testing performed quarterly. STRIDE-2 was the second double-blind, placebo-controlled trial with sitaxsentan in PAH; 247 PAH patients were randomized (245 treated), including patients with IPAH and PAH associated with CTD or CHD [41]. The arms included a placebo, sitaxsentan 50 or 100  mg (all double-blind), and open-label (third-party-blind) bosentan. The primary end point was the change in 6MWD from the baseline to week 18; patients treated with sitaxsentan, 100 mg, had an increased

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6MWD compared with those receiving the placebo (31.4 m, p = 0.03), and an improved functional class (p = 0.04). The placebo-corrected treatment effect for sitaxsentan, 50  mg, was 24.2 m and was not significant (p = 0.07), and for openlabel bosentan was 29.5 m (p = 0.05). The incidence of elevated levels of hepatic transaminases to more than 3  times the upper limit of normal was 6% for the placebo; 5% for sitaxsentan, 50 mg; 3% for sitaxsentan, 100 mg; and 11% for bosentan.

3.1.3 Connective Tissue Disease A post hoc analysis from the STRIDE-1 study was performed on 42 patients with PAH associated with CTD. Data from 33 patients assigned to sitaxsentan 100 mg or 300 mg daily were pooled and compared with data from nine placebo-treated patients. There were 41 patients entered into the blinded extension study, in which all patients received either 100 or 300  mg sitaxsentan once daily. The patients treated with sitaxsentan had a mean increase in 6MWD of 20 m from the baseline to week 12 (p = 0.037), whereas the placebo group had a decrease of 38  m, i.e. a placebo-corrected treatment effect of 58  m (p = 0.027). Improvements in quality of life and hemodynamics were also observed. No patients stopped using the drug during the 12-week trial. At the end of the blinded extension period (median treatment duration 26 weeks), more patients were in functional class I–II than in functional class III–IV (p < 0.001) compared with at the initiation of therapy. An increase in hepatic transaminase levels occurred in two patients. These results, although post hoc, suggest that sitaxsentan is effective in patients with PAH due to CTD.

3.1.4 Adverse Effects, Safety, and Drug Interactions In the STRIDE-1 trial, the most common adverse effects that occurred more frequently with sitaxsentan than with the placebo were headache, peripheral edema, nausea, nasal congestion, and dizziness [40]. Edema, fatigue, nasal congestion, and insomnia occurred more frequently with both doses of sitaxsentan than with the placebo in STRIDE-2; only the occurrence of insomnia reached statistical significance (p = 0.049) [41]. Effects on liver function tests and the warfarin interaction were described earlier; if warfarin is prescribed, the initial dose should be 80% lower than the regular dose prior to initiation of sitaxsentan, with adjustment thereafter as appropriate. A crossover pharmacokinetic analysis of 24 healthy subjects who received concomitant sitaxsentan and sildenafil did not show a significant decrease in blood pressure compared with sitaxsentan alone; elevations in the plasma concentrations of sildenafil were modest when

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sildenafil was given with sitaxsentan. In 2010, sitaxsentan was withdrawn from the market due to hepatotoxicity.

3.2 Ambrisentan Ambrisentan is a nonsulfonamide, propanoic acid based, ETA-selective ERA with a bioavailability and half-life that allow once-daily dosing [42]. This drug is currently approved for use in the USA and the European Union.

3.2.1 Metabolism The elimination of ambrisentan is predominantly by nonrenal pathways, although the relative contributions of metabolism and biliary elimination have not been well characterized. It appears to be independent of cytochrome P450 pathways and to occur predominantly by glucuronidation and transport via the bile into the feces. In vitro and in vivo animal model investigations with ambrisentan have reported a minimal effect on hepatic enzymes such as CYP2C9, CYP3A4, and CYP1A2 [43]. In vitro data indicate that metabolism is predominantly hepatic via CYP3A4, CYP2C19, and uridine 5¢-diphosphate glucuronosyltransferases 1A9S, 2B7S, and 1A3S. Additionally, ambrisentan is a substrate for the organic anion transport protein and P-glycoprotein. Although ambrisentan has a 15-h terminal half-life, the trough concentration at the steady state is about 15% of the mean peak concentration and the accumulation factor is about 1.2 after long-term daily dosing, indicating an effective half-life of ambrisentan of about 9 h.

3.2.2 Clinical Trials in Pulmonary Arterial Hypertension with Ambrisentan In a double-blind, dose-ranging study, 64 patients with IPAH or PAH associated with CTD, anorexigen use, or HIV infection were randomized to receive 1, 2.5, 5, or 10  mg of ambrisentan once daily for 12 weeks followed by 12 weeks of open-label ambrisentan [44]. The primary end point was a change from the baseline in 6MWD. At 12 weeks, ambrisentan increased the walk distance (+36.1 m, p < 0.0001) with all doses. Improvements were also noted in Borg dyspnea score, WHO functional class, subject global assessment, mPAP (–5.2 mmHg, p < 0.0001), and cardiac index (+0.33 l/min/m [2], p < 0.0008). Adverse events were mild, and the incidence and severity of liver enzyme abnormalities were low. Two pivotal randomized, double-blind, placebo-controlled phase III trials (ARIES-1 and ARIES-2) evaluated the safety and efficacy of ambrisentan in PAH patients (as shown in

V.F. Tapson and R.J. Barst

Tables 2, 3) [14]. The ARIES trials were of identical design except for the doses of ambrisentan and the geographic locations: ARIES-1 enrolled patients in North America and selected international sites, and ARIES-2 was conducted in Europe, Israel, and South America. ARIES-1 evaluated oncedaily doses of 5 and 10  mg of ambrisentan and ARIES-2 evaluated once-daily doses of 2.5 and 5  mg. The primary efficacy end point was the change from the baseline to 12  weeks in 6MWD compared to the placebo. Secondary end points included the time to clinical worsening, WHO functional class, and the Borg dyspnea index. In ARIES-1 (n = 202), patients were randomized to one of three treatment groups for 12 weeks [14]. Sixty-three percent of patients had IPAH and 37% had PAH associated with CTD, anorexigen use, or HIV infection. The majority of patients (90%) were functional class II or III, with a mean baseline 6MWD of 341 ± 76 m. For the 10-mg dose, the placebo-adjusted change from the baseline in 6WMD was +51.4 m (95% confidence interval, 26.6–76.2 m; p = 0.0001), whereas for the 5-mg dose it was +30.6 m (95% confidence interval, 2.9–58.3  m; p = 0.0084). Improvements in WHO functional class and Borg dyspnea index were also demonstrated. Six subjects in the placebo group developed clinical worsening, compared with three subjects in each of the ambrisentan groups (p > 0.05). No patients receiving ambrisentan ­developed hepatic transaminase levels more than 3 times the upper limit of normal, whereas two patients in the placebo group did. In ARIES-2, 2.5 and 5  mg of ambrisentan once daily improved the placebo-corrected 6MWD by 32.3  m (p = 0.0219) and 59.4 m (p = 0.0002), respectively [14]. For the placebo group, the 6MWD at week 12 decreased from the baseline by 10.1 m. Improvements in the time to clinical worsening compared with the placebo were also observed for both the 5-mg-dose group (p = 0.0076) and the 2.5-mgdose group (p = 0.0048). The most frequent adverse event in ARIES-2 was headache, occurring in 12.7% of patients in the 5-mg-dose group and 7.8% of patients in the 2.5-mgdose group, compared with 6.2% of patients in the placebo group. There were no aminotransferase concentration elevations to greater than 3  times the upper limit of the normal range in the ambrisentan patients, but there was one elevation in the placebo group. Thus, in these two studies, ambrisentan doses were well tolerated, with most adverse events mild to moderate in severity. The increased frequency of nasal congestion, peripheral edema, and headache observed with ambrisentan is likely due to systemic vasodilatation. In the ARIES studies, a twofold increase in ambrisentan dose was associated with a notable (20–27  m) increase in 6MWD, consistent with a dose response [14]. The change in 6MWD appeared different for the 5  mg ambrisentan dose group in ARIES-1 (31 m) and in ARIES-2 (59 m). However,

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the substantial overlap of the 95% confidence intervals for these data suggests that these results were not discordant. The two studies had very similar patient populations; therefore, this difference may be due to random variability. Similar variability between studies for 6MWDs has also been observed for bosentan at the same dose (70 vs. 35 m) [10, 11]. Nevertheless, examination of the dose response for ambrisentan is best limited to comparisons within the individual studies. In both ARIES studies, improvements in 6MWD were observed in functional class II and III patients. This result differs from the results of previous studies in which smaller improvements were observed in less dyspneic patients, i.e. in class II patients [11, 45]. The effect of ambrisentan on exercise capacity appeared to be maintained after 48 weeks of treatment in the patients continuing with ambrisentan monotherapy, further supporting the clinical significance of the exercise improvements observed in the 12-week studies [14]. The rates of clinical worsening ranged from 4% to 5% in all ambrisentan dose groups and were reduced compared with those of the placebo groups. In ARIES-2, a significant improvement in the time to clinical worsening was observed for patients receiving ambrisentan compared with the placebo (p < 0.001); similar results were observed for the 2.5 and 5 mg ambrisentan groups (p = 0.005 and p = 0.008, respectively) [14]. No significant improvement in the time to clinical worsening was also observed in ARIES-1 for ambrisentan-treated patients compared with patients receiving the placebo [14]. A clear difference was observed in the overall rates of clinical worsening between the placebo groups in ARIES-1 (9%) and ARIES-2 (22%); this may explain why statistical significance was observed only in the latter study. The difference in clinical worsening among the placebo groups of ARIES-1 and ARIES-2 was due mainly to a different rate of hospitalization (3% and 14%, respectively) [14]. ARIES-1 and ARIES-2 patients were enrolled predominantly in North America and Europe, respectively; therefore, differences in the criteria for hospital admissions in these two geographic areas may be responsible in part for this discrepancy. The symptomatic improvement with ambrisentan in these two trials was supported by the favorable effects on WHO functional class, Borg dyspnea score, and the 36-item Short-Form Health Survey [44]. Plasma B-type natriuretic peptide concentrations were significantly reduced at week 12 with each ambrisentan dose compared with the placebo. Limitations of these studies include the exclusion of certain patient populations such as patients with PAH associated with portal hypertension and congenital systemic-to-pulmonary shunts. In addition, although the long-term effect has been shown in a relatively high proportion of patients remaining on ambrisentan monotherapy for 48 weeks, these data may represent a selection of patients who respond to the drug [14].

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3.2.3 Adverse Effects, Safety, and Drug Interactions A 1-year follow-up of the use of ambrisentan as rescue therapy for patients who previously failed bosentan and/or sitaxsentan therapy for liver function abnormalities was conducted [46]. The primary end point was discontinuation of ambrisentan for hepatic transaminase level increases to more than 3 times the upper limit of normal. No patient met this end point. A secondary end point was a transaminase level elevation of more than 3 times the upper limit of normal throughout the course of the study, and only one patient met this end point. That patient experienced an increase to 3.2 times the upper limit of normal at week 12, resulting in an ambrisentan dosage reduction to 2.5 mg/day. The ambrisentan dose was subsequently increased to 5 mg and then 10 mg without further transaminase level elevations. Safety data from the combined analysis of the ARIES-1 and ARIES-2 trials were stratified by their initial WHO classification [14]. For both functional class II and III groups, peripheral edema was the most frequently seen adverse effect, occurring in 6.4% and 12.8% of functional class II and class III placebo patients, respectively, and in 10.6% and 21.7% of class II and class III PAH ambrisentan-treated patients. Headache, nasal congestion, palpitations, and flushing were also seen in more than 5% of patients treated with ambrisentan. Except for nasal congestion, no apparent dose response was noted for adverse events in the ARIES studies [14]. From these data, it appears that both the 5 and the 10 mg ambrisentan doses have appropriate ratios of efficacy to safety. In the clinical setting, an increase to the 10-mg dose may be considered if the drug is well tolerated at the initial 5-mg dose. Caution should be used when ambrisentan is coadministered with cyclosporine, strong CYP3A4 inhibitors (ketoconazole), and CYP2C19 inhibitors (omeprazole) because of the risk of ambrisentan accumulation [43]. The clinical significance of this remains unknown owing to a lack of data. Unlike the other ERAs, ambrisentan does not appear to interact with warfarin and sildenafil, which are other commonly used PAH treatments [43, 47, 48].

4 New Approaches to Endothelin Receptor Antagonism ACT-064992 (Actelion-1) is a tissue-targeting ERA that works via complete blockade of tissue ET. In vivo, ACT064992 is metabolized into a major and pharmacologically active metabolite, ACT-132577. In rats with pulmonary hypertension, ACT-064992 prevented both increase of pulmonary pressure and right ventricular hypertrophy, and markedly improved survival [49]. A phase III randomized, PAH study is in progress [50].

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5 Endothelin Receptor Antagonists in Clinical Practice The ERAs are widely used in clinical practice for PAH. Currently, when oral therapy is initiated, it is with an ERA or with a phosphodiesterate type 5 inhibitor, either sildenafil or tadalafil [45–51]. No clear guidelines specify that one class or the other is more or less advantageous in this setting; the American College of Chest Physicians’ most recent recommendations have suggested the use of bosentan as a grade A recommendation for functional class III patients and as a grade B recommendation for class IV patients. However, few class IV patients have been studied, and initiation of an oral agent as the sole therapy in this population should not be considered except when circumstances are unusual or options are limited. Ambrisentan has a class A recommendation for class II and III patients. The EARLY study resulted in the approval of bosentan for class II patients [20]. ERA therapy has been studied extensively and first-line therapy for functional class II or III patients with bosentan or ambrisentan can be recommended. Use of ERA therapy in combination with other PAH therapy is discussed elsewhere in this textbook.

6  Conclusions ET appears to play an important role in the pathobiological and pathophysiological process of PAH. Three ERAs have been studied in randomized controlled trials. Although the most extensive clinical experience to date is with the nonselective ET antagonist bosentan, all three drugs have been shown to be effective, and this drug class has found an important niche in the therapy of PAH.

References 1. Kedzierski RM, Yanagisawa M (2001) Endothelin system: the double-edged sword in health and disease. Annu Rev Pharmacol 41:851–876 2. Rossi GP, Seccia TM, Nussdorfer GG (2001) Reciprocal regulation of endothelin-1 and nitric oxide: relevance in the physiology and pathology of the cardiovascular system. Int Rev Cytol 209:241–272 3. Ribatti D, Conconi MT, Nussdorfe GG (2007) Nonclassic endogenous novel regulators of angiogenesis. Pharmacol Rev 59:185–205 4. Giaid A, Yanagisawa M, Langleben D et al (1993) Expression of endothelin-1 in the lungs of patients with pulmonary hypertension. N Engl J Med 328:1732–1739 5. Stewart DJ, Levy RD, Cernacek P, Langleben D (1991) Increased plasma endothelin-1 in pulmonary hypertension: marker or mediator of disease? Ann Intern Med 114:464–469 6. Rubanyi GM, Polokoff MA (1994) Endothelins: molecular biology biochemistry, pharmacology, physiology, and pathophysiology. Pharmacol Rev 46:325–415

V.F. Tapson and R.J. Barst 7. Dupuis J (2001) Endothelin-receptor antagonists in pulmonary hypertension. Lancet 358:1113–1114 8. Davie N, Haleen SJ, Upton PD et al (2002) ETA and ETB receptors modulate the proliferation of human pulmonary artery smooth muscle cells. Am J Respir Crit Care Med 165:398–405 9. Rubens C, Ewert R, Halank M et al (2001) Big endothelin-1 and endothelin-1 plasma levels are correlated with the severity of primary pulmonary hypertension. Chest 120:1562–1569 10. Channick RN, Simonneau G, Sitbon O et al (2001) Effects of the dual endothelin-receptor antagonist bosentan in patients with pulmonary hypertension: a randomised placebo-controlled study. Lancet 358:1119–1123 11. Rubin LJ, Badesch DB, Barst RJ et al (2002) Bosentan therapy for pulmonary arterial hypertension. N Engl J Med 346:896–903 12. Humbert M, Segal ES, Kiely DG et al (2007) Results of European post-marketing surveillance of bosentan in pulmonary hypertension. Eur Respir J 30:338–344 13. Barst RJ, Langleben D, Frost A et al (2004) Sitaxsentan therapy for pulmonary arterial hypertension. Am J Respir Crit Care Med 169:441–447 14. Galiè N, Olschewski H, Oudiz RJ et  al (2008) Results of the ambrisentan in pulmonary arterial hypertension, randomized, double-blind, placebo-controlled, multicenter, efficacy (ARIES) study 1 and 2. Circulation 117:3010–3019 15. Billman GE (2002) Ambrisentan (myogen). Curr Opin Investig Drugs 3:1483–1486 16. Gilead Sciences (2008) Package insert. Letairis (ambrisentan). Gilead Sciences, Inc., Foster City 17. McLaughlin VV, Sitbon O, Badesch DB et al (2005) Survival with first-line bosentan in patients with primary pulmonary hypertension. Eur Respir J 25:244–249 18. Sitbon O, McLaughlin VV, Badesch DB et  al (2005) Survival in patients with class III idiopathic pulmonary arterial hypertension treated with first line oral bosentan compared with an historical cohort of patients started on intravenous epoprostenol. Thorax 60:1025–1030 19. Provencher S, Sitbon O, Humbert M, Cabrol S, Jais X, Simonneau G (2006) Long-term outcome with first-line bosentan therapy in idiopathic pulmonary arterial hypertension. Eur Heart J 27:589–595 20. Galiè N, Rubin LJ, Hoeper MM et al (2008) Treatment of patients with mildly symptomatic pulmonary arterial hypertension with bosentan (EARLY study): a double-blind, randomised controlled trial. Lancet 371:2093–2100 21. Denton CP, Humbert M, Rubin L, Black CM (2006) Bosentan treatment for pulmonary arterial hypertension related to connective tissue disease: a subgroup analysis of the pivotal clinical trials and their open-label extensions. Ann Rheum Dis 65:1336–1340 22. Koh ET, Lee P, Gladman DD, Abu-Shakra M (1996) Pulmonary hypertension in systemic sclerosis: an analysis of 17 patients. Br J Rheumatol 35:989–993 23. Kawut SM, Taichman DB, Archer-Chicko CL et  al (2003) Hemodynamics and survival in patients with pulmonary arterial hypertension related to systemic sclerosis. Chest 123:344–350 24. Williams MH, Das C, Handler CE et al (2006) Systemic sclerosis associated pulmonary hypertension: improved survival in the current era. Heart 92:926–932 25. Guillevin L, Gabrielli A, Peter H et al (2006) Long-term effects of bosentan on quality of life, survival, safety and tolerability in pulmonary arterial hypertension associated with connective tissue disease. Ann Rheum Dis 65:392 26. Barst RJ, Ivy D, Dingemanse J et  al (2003) Pharmacokinetics, safety, and efficacy of bosentan in pediatric patients with pulmonary arterial hypertension. Clin Pharmacol Ther 73:372–382 27. Schulze-Neick I, Gilbert N, Ewert R et al (2005) Adult patients with congenital heart disease and pulmonary arterial hypertension: first open prospective multicenter study of bosentan therapy. Am Heart J 150:716

105  Endothelin Receptor Antagonists for the Treatment of Pulmonary Arterial Hypertension 28. Sitbon O, Beghetti M, Petit J et al (2006) Bosentan for the treatment of pulmonary arterial hypertension associated with congenital heart defects. Eur J Clin Invest 36:25–31 29. Maiya S, Hislop AA, Flynn Y, Haworth SG (2006) Response to bosentan in children with pulmonary hypertension. Heart 92:664–670 30. Gilbert N, Luther YC et al (2005) Initial experience with bosentan (Tracleer) as treatment for pulmonary arterial hypertension (PAH) due to congenital heart disease in infants and young children. Z Kardiol 94:570–574 31. Rosenzweig EB, Ivy DD, Widlitz A et  al (2005) Effects of longterm bosentan in children with pulmonary arterial hypertension. J Am Coll Cardiol 46:697–704 32. Galiè N, Beghetti M, Gatzoulis MA et al (2006) Bosentan therapy in patients with Eisenmenger syndrome: a multicenter, double-blind, randomized, placebo-controlled study. Circulation 114:48–54 33. Gatzoulis MA, Beghetti M, Galiè N et  al (2008) Longer-term bosentan therapy improves functional capacity in Eisenmenger ­syndrome: results of the BREATHE-5 open-label extension study. Int J Cardiol 127:27–32 34. D’Alto M, Vizza CD, Romeo E et al (2006) Long term effects of bosentan treatment in adult patients with pulmonary arterial hypertension related to congenital heart disease (Eisenmenger physiology): safety, tolerability, clinical, and haemodynamic effect. Heart 93:621–625 35. Apostolopoulou SC, Manginas A, Cokkinos DV, Rammos S (2007) Long-term oral bosentan treatment in patients with pulmonary arterial hypertension related to congenital heart disease: a 2-year study. Heart 93:350–354 36. Sitbon O, Gressin V, Speich S et al (2004) Bosentan for the treatment of human immunodeficiency virus-associated pulmonary arterial hypertension. Am J Respir Crit Care Med 170:1212–1217 37. Jais X, Ghofrani A, Hoeper MM et al (2007) Bosentan for inoperable chronic thromboembolic pulmonary hypertension (CTEPH): a randomized, placebo-controlled trial – BENEFIT. Am J Respir Crit Care Med 175:A896 38. Gladwin MT, Sachdev V, Jison ML et al (2004) Pulmonary hypertension as a risk factor for death in patients with sickle cell disease. N Engl J Med 350:886–895

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39. Long-term, open-label, multicenter, extension study of bosentan in patients with pulmonary hypertension associated with sickle cell disease completing a double-blind ASSET study (AC 052-368 or AC 052-369). NCT00360087. Available via http://clinicaltrials.gov 40. Barst RJ, Langleben D, Frost A et al (2004) Sitaxsentan therapy for pulmonary arterial hypertension ASRCCM 169:44–447 41. Barst RJ, Langleben D, Badesch D et al (2006) Treatment of pulmonary arterial hypertension with the selective endothelin receptor antagonist sitaxsentan. J Am Coll Cardiol 47:2049–2056 42. Galiè N, Badesch D, Oudiz R et al (2005) Ambrisentan therapy for pulmonary arterial hypertension. J Am Coll Cardiol 46: 529–535 43. Oudiz RJ, Torres F, Frost AE et  al (2006) ARIES-1: a placebo-­ controlled, efficacy and safety study of ambrisentan in patients with pulmonary arterial hypertension. Chest 130:121S 44. Olschewski H ARIES-2 Am J Respir Crit Care Med Proc Am Thorac Soc 2006;3:A728 45. Galiè N, Ghofrani HA, Torbicki A, et al, for the Sildenafil Use in Pulmonary Arterial Hypertension (SUPER) Study Group (2005) Sildenafil citrate therapy for pulmonary arterial hypertension. N Engl J Med 353:2148–2157 46. McGoon MD, Frost AE, Oudiz RJ et al (2009) Ambrisentan therapy in patients with pulmonary arterial hypertension who discontinued bosentan or sitaxsentan due to liver function test abnormalities. Chest 135:122–129 47. Dufton C, Gerber M, Yin O, Brandquist C, Ghofrani HA (2006) No clinically relevant pharmacokinetic interaction between ambrisentan and sildenafil. Chest 130:254S 48. Gerber M, Dufton C, Pentikis H, Zhu L, Ghofrani HA (2006) Ambrisentan has no clinically relevant effects on the pharmacokinetics or pharmacodynamics of warfarin. Chest 130:256S 49. Iglarz M, Binkert C, Morrison K et  al (2008) Pharmacology of macitentan, an orally active, tissue-targeting dual endothelin receptor antagonist. J Pharmacol Exp Ther 327:736–745 50. Actelion (2006) Actelion-1 – first tissue-targeting endothelin receptor antagonist – in phase III for pulmonary hypertension (PH). http://www.actelion.com 51. Jalie N, Brundage BH, Ghofrani HA et al (2009) Tadalafil therapy for pulmonary arterial hypertension circulation. 119(22):2894–903

Chapter 106

Phosphodiesterase Inhibitors in the Treatment of Pulmonary Hypertension Lan Zhao, Zhenguo Zhai, John Wharton, and Martin R. Wilkins

Abstract  As mediators of relaxation of vascular smooth muscle, cGMP and cAMP signaling pathways have become an important focus of drug development for pulmonary hypertension. Phosphodiesterases (PDEs), the enzymes that catalyze hydrolytic cleavage of the 3’-phosphodiester bond of the cyclic nucleotides, controlling intracellular levels, are obvious drug targets. PDEs have a wide distribution in normal tissues and are subdivided into 11 distinctive families on the basis of substrate affinity and selectivity, sequence homology, and regulatory mechanisms. Of these enzymes, PDE5, PDE6, and PDE9 are highly selective for cGMP, PDE1, PDE2, and PDE11 bind cGMP and cAMP with equal affinity, and PDE3 and PDE10 are cGMP-sensitive but cAMP-selective. These differences among the PDE families have been exploited to target the pulmonary vascular bed. PDE5 and PDE1 have attracted the most interest for the treatment of pulmonary hypertension by virtue of their tissue distribution. Experience with PDE5 inhibition has provided the paradigm for other similar treatments for pulmonary arterial hypertension (PAH). One PDE5 inhibitor, sildenafil, originally developed to augment the nitric oxide (NO)/cGMP pathway for the treatment of angina and then approved for erectile dysfunction treatment in 1989, has been shown to improve pulmonary hemodynamics and exercise capacity in patients with PAH and is now approved for clinical use in PAH. The other two PDE5 inhibitors, tadalafil and vardenafil, have now also been studied. An interesting although still provisional observation is the potential of sildenafil to reduce pulmonary vascular resistance without adversely affecting ventilation/perfusion matching. Another is the expression of PDE5 in the hypertrophied right ventricle. These data suggest that PDE5 inhibitors may have effects that distinguish them from other treatments for pulmonary hypertension and merit further study.

M.R. Wilkins  (*) Department of Experimental Medicine, Imperial College London, Hammersmith Campus, London, W12 0NN, UK

Keywords  Cyclic GMP • Cyclic AMP • Second messenger • Sildenafil • Tadalafil • Nitric oxide • Pulmonary vasodilation

1 Introduction Discovered almost 50 years ago, the nucleotide second messengers cyclic guanosine monophosphate (cGMP) and cyclic adenosine monophosphate (cAMP) play a central role in signal transduction. As mediators of relaxation of vascular smooth muscle, cGMP and cAMP signaling pathways have become an important focus of drug development for pulmonary hypertension. Phosphodiesterases (PDEs), the enzymes that catalyze hydrolytic cleavage of the 3¢-phosphodiester bond of the cyclic nucleotides, regulating intracellular levels, are obvious drug targets. PDEs have a wide distribution in normal tissues and are subdivided into 11 distinctive families on the basis of substrate affinity and selectivity, sequence homology, and regulatory mechanisms [1–3]. Of these enzymes, PDE5, PDE6, and PDE9 are highly selective for cGMP, PDE1, PDE2, and PDE11 bind cGMP and cAMP with equal affinity, and PDE3 and PDE10 are cGMP-sensitive but cAMP-selective. These differences among the PDE families have been exploited to target the pulmonary vascular bed. PDE5 and PDE1 have attracted the most interest for the treatment of pulmonary hypertension by virtue of their tissue distribution. Experience with sildenafil has provided the paradigm for other similar treatments for pulmonary arterial hypertension (PAH). Originally developed to augment the nitric oxide (NO)/cGMP pathway for the treatment of angina and then approved for erectile dysfunction treatment in 1989, sildenafil has been shown to improve pulmonary hemodynamics and exercise capacity in patients with PAH and is now approved for clinical use in PAH. Two other PDE5 inhibitors, tadalafil and vardenafil, have now also been studied. An interesting although still provisional observation is the potential of PDE5 inhibition to reduce pulmonary vascular resistance without adversely affecting ventilation/ perfusion matching. Another is the expression of PDE5 in the

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hypertrophied right ventricle. These data suggest that PDE5 inhibitors may have effects that distinguish them from other treatments for pulmonary hypertension and merit further study.

L. Zhao et al.

The major enzyme sources of cGMP in tissues are soluble guanylate cyclase and membrane-bound, receptor-mediated (particulate) guanylate cyclase (Fig. 1). NO stimulates soluble guanylate cyclase, whereas the natriuretic peptides (atrial, brain or B-type, and C-type) stimulate specific natriuretic peptide receptors (NPRA and NPRB) to catalyze conversion of guanosine 5¢-triphosphate to cGMP. In turn, cGMP stimulates cGMP-dependent protein kinases (or protein kinase G, PKG) and cyclic-nucleotide-gated ion channels. cGMP is metabolized by PDEs, particularly PDE5. cGMP binding affinity and hydrolysis are increased by PKG-induced phosphorylation of PDE5.

shown that PDE5 levels in lung are 15 times higher than in heart extract, close to levels in penile corpus cavernosum [4]. PDE5 is the predominant cGMP PDE in the lung and the most abundant in pulmonary smooth muscle cells [4]. Three splice variants have been identified (PDE5A1, PDE5A2, and PDE5A3) similar that encode proteins with cGMP catalytic activities and sensitivity to sildenafil, but distinct N-terminal sequences [5]. The expression of the PDE5A1 and PDE5A2 variants occur in most tissues [5]. All three molecular forms of PDE5 immunoreactivity have been demonstrated in isolated pulmonary vascular smooth muscle cells [6]. In lung tissue from PAH patients, the expression of PDE5, specifically of the predominant 98-kDa (PDE5A1) and 77-kDa PDE5 isoforms, is significantly increased compared with healthy lung [6]. PDE5 immunoreactivity colocalizes with a smooth muscle actin to cells in intimal lesions and neomuscularized distal vessels, as well as smooth muscle cells in the medial layer of the diseased pulmonary vasculature. Thus, PDE5 is expressed in the diseased vascular lesion and may represent a marker of vascular remodeling as well as a potential therapeutic target.

3 Phosphodiesterases in the Lung

3.2 Phosphodiesterase Type 1

3.1 Phosphodiesterase Type 5

The PDE1 family is activated by calcium/calmodulin and termed “calcium/calmodulin-dependent PDEs.” Three different PDE1 isoforms, PDE1A, PDE1B, and PDE1C have been identified [2]. PDE1A and PDE1B have higher affinity for cGMP than cAMP, whereas PDE1C hydrolyzes cAMP and cGMP with similar efficiency. There are five different PDE1C splice variants, PDE1C1, PDE1C2, PDE1C3, PDE1C4, and PDE1C5, in human and mouse species. PDE1C is highly expressed in proliferating human arterial smooth muscle cells and regulates both vascular tone and proliferation. PDE1 is expressed at low levels in pulmonary tissue under normal conditions, but significant upregulation of PDE1 has been reported in proliferating pulmonary vascular smooth muscle cells [7–9]. Specifically, significant vascular upregulation of PDE1C and PDE1A has been demonstrated by immunohistochemistry as well as expression analysis in lung explants and isolated pulmonary smooth muscle cells from patients with idiopathic PAH [7]. The PDE1-selective inhibitor PI79 inhibits DNA synthesis in proliferating human pulmonary arterial smooth muscle cells in culture. Inhibition of PDE1 with 8-methoxymethyl 3-isobutyl-1-methylxanthine in animal models of pulmonary hypertension improved hemodynamics, right-sided heart hypertrophy, and vascular remodeling. These findings suggest upregulation of PDE1C participates in the structural remodeling process. Sildenafil inhibits PDE1, albeit at a higher IC50 than that required to inhibit PDE5. It is interesting to speculate that inhibition of PDE1 contributes to the therapeutic effects of this drug in the treatment of PAH [10].

2 The cGMP Pathway

PDE5 is expressed in a wide variety of tissues. The original discovery, cloning, and characterization of PDE5 used lung tissue as the source of the enzyme. Using radioligand binding assays with [3H]vardenafil or [3H]tadalafil, Corbin et al. have

Fig. 1  Nitric oxide interacts with soluble guanylyl cyclase and natriuretic peptides interact with particulate guanylate cyclase to convert GTP to cyclic GMP (cGMP), which is hydrolyzed and inactivated by phosphodiesterase type 5 (PDE5). cGMP activates cGMP-dependent protein kinase and this results in vasorelaxation and inhibits proliferation of vascular smooth muscle cells. The effects are enhanced by inhibition of cGMP breakdown via PDE5. Prostacyclin from endothelial cells also promotes relaxation and inhibits cell proliferation, via cyclic AMP (cAMP). Adrenomedullin and vasoactive intestinal polypeptide promote vasorelaxation via cAMP

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3.3 cGMP Pathway and PDE5 In addition to spatial regulation by virtue of intracellular localization of its synthetic enzymes, soluble and particulate guanylyl cyclases, compartmentalization of intracellular cGMP signaling in cardiovascular tissues is dependent on its binding to at least three groups of proteins: cGMP-dependent protein kinases (PKG), cGMP-regulated PDEs, and cyclicnucleotide-gated ion channels [11]. PKG is the foremost of these intracellular mediators, whereas the cGMP-regulated PDE5 is mainly responsible for modulating intracellular cGMP levels and PKG-dependent signaling produced by NO and natriuretic peptides. In addition to being hydrolyzed by PDE5, cGMP and its downstream mediator PKG also modulate the activity of this enzyme. Thus, cGMP binding affinity and hydrolysis are increased by PKG-induced phosphorylation of PDE5 [12]. The cGMP binding to the so-called GAF A domain also promotes phosphorylation of PDE5 and increases catalytic activity [13, 14]. In addition to these feedback mechanisms, there is the potential for gene regulation as the promoter region of human PDE5 contains sites responsive to cyclic nucleotides [15, 16].

4 PDE5 Inhibitors 4.1 Pharmacology Three PDE5 inhibitors are currently of interest to PAH – sildenafil, tadalafil, and vardenafil. All three inhibit PDE5 in the low nanomolar range with IC50 values of 1–9 nM for sildenafil [17–21], 1–7 nM for tadalafil [20–22], and 0.09– 0.8  nM for vardenafil [20–23]. Sildenafil shows 80-fold selectivity for PDE5 over PDE1 and inhibits retinal PDE6 at about tenfold higher concentrations. Vardenafil also inhibits PDE6 at a 4–25-fold higher IC50 compared with PDE5 but has no significant effect on other PDEs. Tadalafil has little effect on PDE6 but inhibits PDE11 with fivefold difference in IC50. Tadalafil, in contrast to sildenafil, has no effect on PDE1C.

4.2 Pharmacokinetics and Pharmacodynamics 4.2.1 Absorption Sildenafil has a chemical structure similar to that of cGMP and inhibits PDE5 by binding to the cGMP catalytic sites, thereby allowing the accumulation of cGMP in the tissue. Vardenafil is very similar to sildenafil in terms of chemical

Fig. 2  Sildenafil has a structure similar to that of cGMP. Vardenafil is very similar to sildenafil in terms of chemical structure, whereas tadalafil is markedly different

structure, whereas tadalafil is markedly different. This is reflected in their clinical pharmacokinetics (Fig. 2). All three PDE5 inhibitors are rapidly absorbed after oral administration. Ninety-five percent of sildenafil is absorbed following a single oral dose, although absolute bioavailability is reduced to about 40% because of first-pass metabolism [24]. The time to maximum plasma concentration (Tmax) is about 1 h (range 0.5–2 h). The plasma half-life is around 4 h but if the IC50 for inhibition of PDE5 in vitro and the high protein binding of the parent drug are taken into account, the plasma concentration of sildenafil is maintained above the IC50 for between 12 and 18 h following a 100-mg dose. With orally administered tadalafil, Tmax in plasma is around 2  h, bioavailability is 36%, protein binding (principally a1-acid glycoprotein and albumin) is 94%, and the plasma half-life is 17.5  h [25]. Systemic exposure increases linearly over the dosage range 2.5–20  mg daily and at steady state (around 5 days), plasma levels are 1.6-fold higher than at the start of treatment. Vardenafil Tmax is reached slightly earlier (about 40 min) than that of sildenafil and tadalafil and the plasma half-life is approximately 4  h [26]. The absolute bioavailability of vardenafil is 14.5%, lower than for both sildenafil and tadalafil, and this seems to be the major factor responsible for the greater inter- and intrasubject variability in oral

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c­ learance and systemic exposure (linear over the dose range 5–20 mg) of vardenafil. High-fat meals do not influence the absorption of tadalafil, but reduce sildenafil Cmax by 29% and vardenafil Cmax by 18%, and prolong sildenafil and vardenafil Tmax by approximately 1 h. 4.2.2 Metabolism All PDE5 inhibitors are metabolized by CYP3A4. Approximately 75% of sildenafil metabolism is by CYP3A4 and the remainder is by CYP2C9 [27–29]. The major metabolite of sildenafil is N-desmethylsildenafil, which exhibits the same PDE specificity as sildenafil but with half the potency. Plasma levels reach around 40–50% of the plasma levels of the parent drug and account for approximately 20% of the pharmacological effects of sildenafil [26, 27]. Tadalafil is metabolized to a catechol, which further undergoes methylation and glucuronidation. The methyl catechol glucuronide is 13,000-fold less potent for PDE5 than the parent molecule. The major metabolite of vardenafil, an N-desethyl derivative of vardenafil, is fourfold less potent an inhibitor of PDE5 than its parent compound, contributing approximately 7% to the overall effect of vardenafil.

4.2.3 Drug Interactions and Elimination Coadministration of potent CYP3A4 inhibitors such as erythromycin, ketoconazole, and saquinavir results in significant increases in the peak plasma concentration and the area under curve of sildenafil, with no effect on plasma half-life; this is consistent with inhibition of presystemic metabolism without an effect on elimination. Cotreatment with bosentan, a CYP3A4 inducer, significantly reduces plasma sildenafil levels by about 50% at therapeutic bosentan doses [30]. By contrast, sitaxsentan, a weak in vitro CYP3A4 inhibitor, produces a small (18%) but clinically insignificant rise in sildenafil levels. Sildenafil is a weak inhibitor of several major drugmetabolizing enzymes, but the plasma concentrations reached even following maximum recommended dosing (100 mg) are unlikely to be clinically important, with one important exception. Competition with bosentan through CYP3A4 metabolism when coadministered leads to a significant increase in bosentan levels [31]. Modestly increased plasma sildenafil levels are seen in elderly (older than 65 years) patients and patients with hepatic impairment or severe renal impairment as a result of reduced drug clearance. CYP3A4 inhibitors reduce tadalafil clearance; for example, therapeutic doses of ketoconazole increase tadalafil area under the curve 4.1-fold and Cmax 1.2-fold. Conversely, cotreatment with bosentan reduces tadalafil levels by approximately 40%, with little reciprocal effect on bosentan levels.

L. Zhao et al.

In vitro studies predict that tadalafil would not reversibly inhibit the metabolism of other drugs metabolized by the major human cytochrome P450 enzymes but has the potential to be a weak mechanism-based inhibitor and an inducer of CYP3A4. However, in clinical studies tadalafil in doses of 10 and 20  mg had little effect on the clearance of other CYP3A4 substrates, midazolam and lovostatin. The metabolites of vardenafil are removed principally by biliary excretion. Coadministration of CYP3A4 inhibitors such as ritonavir can affect hepatic metabolism. With respect to pharmacodynamic interactions, PDE5 inhibitors is well tolerated in combination with a wide range of antihypertensive drugs except NO donors, particularly nitrates, but also alpha blockers. The combination augments the fall in blood pressure [32–34].

4.3 Clinical Pharmacology 4.3.1 Blood Pressure Sildenafil administration in single doses of 100–200  mg increases plasma cGMP levels about 50% above the baseline. A modest fall in both systolic and diastolic blood pressure of 10/7 mmHg at 3 h is seen in healthy men, returning to the baseline by 6 h, with no accompanying changes in heart rate or cardiac index [35]. Small falls in systemic blood pressure have also been reported with vardenafil in men with erectile dysfunction and some patients have been reported to faint following the first dose. Single oral doses of tadalafil (5 or 10  mg) produced dose-related reductions in standing blood pressure in men with ischemic heart disease at 2 h (i.e. at Tmax). Clinically significant longer-term reduction in blood pressure seems unlikely and certainly this drug class has no therapeutic role in systemic blood pressure reduction, or indeed angina [10]. Greater effects are observed on the pulmonary circulation, prompting claims that PDE5 inhibitors are selective for the pulmonary vascular bed [10]. Intravenous infusion of sildenafil (up to 40 mg over 60 min) produced small reductions in right atrial pressure (−28%), pulmonary arterial occluded pressure (−20%), pulmonary arterial pressure (−27%), and systolic (−6%) and diastolic (−10%) systemic arterial pressures at rest, but no change in heart rate, in patients with stable ischemic heart disease [35]. A small (−7%) reduction in cardiac output from the baseline was seen.

4.3.2 Cardiac Function The effects of single oral therapeutic and supratherapeutic doses of sildenafil (50 and 400  mg) and vardenafil (10 and 80  mg) on corrected QT interval have been examined in a

106  Phosphodiesterase Inhibitors in the Treatment of Pulmonary Hypertension

­ ell-designed study in healthy men [36]. Placebo-corrected w mean changes in both PDE5 inhibitors have no influence on absolute QT and only similar small increases were noted at the higher dose, with a shallow dose-response relationship. The effects of PDE5 inhibitors were considered clinically irrelevant.

4.3.3 Endothelial Function Single 50-mg oral doses of sildenafil prevented endothelial dysfunction induced by ischemia and reperfusion in healthy volunteers [37] and in patients with chronic heart failure [38, 39]. Tadalafil in a dose of 20 mg given on alternate days for 4  weeks improved endothelial function in men with increased cardiovascular risk (10-year cardiovascular risk greater than 20%) [40]. The effect of sildenafil was suggested to be the result of opening adenosine trisphosphate sensitive potassium channels [37].

4.3.4 Platelets PDE5 is abundant in platelets and PDE5 inhibition abrogates platelet aggregation [41, 42]. Following a single 100-mg oral dose of sildenafil, prolongation of platelet aggregation time in response to collagen has been detected at 1 h, but not at 4 h, in healthy volunteers [43]. No effect was seen with 50 mg. Sildenafil did not inhibit ADP-induced aggregation at either dose.

4.3.5 Endothelial Progenitor Cells Endothelial progenitor cells (EPCs) are typically thought to arise from mesodermal stem cells or hemangioblasts in the bone marrow. They are considered to have an important role in the formation of new blood vessels and maintaining vascular homeostasis and have been implicated both in the pathogenesis and in the treatment of pulmonary hypertension. A variety of cells have in fact been designated as EPCs, including haematopoietic cells of lymphocyte and monocyte lineage. This reflects the lack of specific markers as well as differences in the techniques used to identify and quantify cells and may account, at least in part, for the apparent variation between studies examining circulating EPCs and hematopoietic cells in PAH patients [44, 45]. Nonetheless, the treatment of idiopathic PAH patients with sildenafil has been associated with a dose-dependent increase in the abundance of circulating EPCs, defined by flow cytometry as CD34+/KDR+ and CD34+/CD133+/KDR+ cells [46]. Whether this effect is beneficial, promoting revascularization of the hypertensive lung, or contributes to the disease, by promoting pulmonary vascular remodeling, is unclear. It is also not known whether

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the only cells that display all the characteristics of progenitor cells committed to an endothelial lineage, so-called late outgrowth EPCs or endothelial colony-forming cells [47, 48], are affected by PDE5 inhibition in idiopathic PAH.

5 PDE5 Inhibitors in Pulmonary Hypertension 5.1 Preclinical Studies The primary rational for using the PDE5 inhibitors for PAH is based on the abundance of PDE5 in the pulmonary arterial wall, particularly smooth muscle cells [10]. In an awake lamb model of acute pulmonary hypertension induced by an intravenous infusion of the thromboxane analogue U46619, cumulative doses of sildenafil (12.5, 25, and 50 mg) decreased pulmonary arterial pressure by 21%, 28%, and 42%, respectively, and pulmonary vascular resistance by 19%, 23%, and 45%, respectively [49]. Systemic arterial pressure decreased 12% after the maximum cumulative sildenafil dose. Sildenafil, vardenafil, and tadalafil are equally efficacious in causing pulmonary artery relaxation induced by phenylephrine [50]. Further support for PDE5 as a therapeutic target in pulmonary hypertension is evident from chronic dosing studies in animal models of the condition. In chronic-hypoxia- and monocrotaline-induced rodent models, PDE5 inhibition attenuated the rise in pulmonary arterial pressure and vascular remodeling if given before exposure to hypoxia or ­monocrotaline (preventative strategy) and partially reversed the disease if given up to 2 weeks after inducing pulmonary hypertension (treatment strategy). The data also indicated a possible antiproliferative effect of PDE5 inhibition [51, 52]. In addition, right ventricular hypertrophy was reduced and survival improved. Studies in genetically manipulated mice revealed that both endothelial nitric oxide synthase and natriuretic peptides contribute to the pharmacological effects of PDE5 inhibition [53, 54]. PDE5 inhibition in combination with other treatments has also been examined. In the monocrotaline rat pulmonary hypertension model, oral gavage of sildenafil with beraprost, a prostacyclin analogue, produced additive effects on plasma cAMP and cGMP, pulmonary hemodynamics, and vascular remodeling [55]. This treatment combination also appeared to be superior in improving the survival rate (100%) at 6 weeks. The effects of sildenafil and bosentan combination have also been reported to be greater than those of either treatment alone in the monocrotaline-treated rat model [56]. In hypoxia-induced pulmonary hypertension, the combination of sildenafil with a neutral endopeptidase inhibitor, ecadotril (which increases endogenous natriuretic peptide levels),

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acted synergistically, in a cGMP-dependent manner, to reduce the pulmonary hypertension severity without significantly affecting systemic blood pressure [57].

5.2 Clinical Studies in PAH 5.2.1 Acute Effects In PAH patients, sildenafil reduces pulmonary vascular resistance when given as a single oral dose (50 or 75 mg) [58]. In an accumulative dosing study, the maximal effect of sildenafil was observed after 25 mg, with no further improvement with the following doses. Notably, the vasodilator effects are most pronounced in the pulmonary circulation, as compared with the systemic circulation [59]. The effect of sildenafil is comparable to the effect of inhaled NO at clinically relevant doses. Moreover, sildenafil acts synergistically with inhaled iloprost [60]. The acute response to sildenafil, tadalafil, and vardenafil has been compared [61]. Sixty consecutive PAH patients (New York Heart Association functional class II–IV) were assigned to oral administration of sildenafil (50 mg), vardenafil (10 or 20  mg), or tadalafil (20, 40, or 60  mg). Hemodynamics and changes in oxygenation were measured over a 120-min observation period. All three PDE5 inhibitors caused significant pulmonary vasorelaxation, with maximum effects obtained after 40–45  min (vardenafil), 60  min (sildenafil), and 75–90 min (tadalafil). Sildenafil and tadalafil, but not vardenafil, produced a significant reduction in the pulmonary to systemic vascular resistance ratio, supporting relative pulmonary vascular selectivity. Only sildenafil improved arterial oxygenation (equal to NO inhalation) significantly.

5.2.2 Chronic Treatment Sildenafil has been shown to be beneficial to patients with pulmonary hypertension, both idiopathic and associated with collagen vascular disease. In numerous studies, long-term sildenafil treatment of PAH patients has been demonstrated to reduce pulmonary vascular resistance, pulmonary arterial systolic pressure, right ventricular systolic pressure, and mean pulmonary arterial pressure [62–64]. In a randomized double-blinded study of 278 pulmonary hypertension patients (SUPER-1 study), sildenafil (20, 40, or 80 mg) administered orally three times (TID) daily for 12 weeks improved exercise capacity (up to 50 m after correction for the placebo in the 80-mg group), functional class, and hemodynamics when compared with placebo-treated patients, and was well tolerated [65]. The increase in the 6-min walk distance achieved at 3 months was maintained in those patients that continued on open-label treatment (80 mg TID) for 12 months. Largely

L. Zhao et al.

on the basis of these data, the FDA approved administration of sildenafil in a dosage of 20 mg TID (in 2005) for the treatment of patients with PAH, although there is more to be done to define the best dosing regimen for patients with pulmonary hypertension, as well as to determine the effect on survival. The dosing regimen for sildenafil is based on its plasma pharmacokinetics. Anecdotal data suggest that its pharmacodynamic half-life may be longer and perhaps even support once daily dosing, but this is not licensed. Tadalafil, with a longer plasma half-life, has the potential of once-daily administration. A recent multicenter study tested tadalafil at dosages of 2.5, 10, 20, or 40  mg daily for 16  weeks [66]. Tadalafil increased the 6-min walk distance in a dose-dependent manner. Only the 40-mg dose increased exercise capacity significantly as well as improved the quality-of-life measure and reduced clinical worsening. Tadalafil is now approved for the treatment of PAH. One other study compared sildenafil treatment with bosentan therapy using cardiac magnetic resonance based measurements of right ventricular mass as an end point (SERAPH study) [64]. Both treatments improved the 6-min walk distance, but only sildenafil reduced right ventricular mass significantly over the 4-month period of study. This effect was accompanied by a reduction in circulating B-type natriuretic peptide levels. Interestingly, sildenafil has been shown to improve right ventricular function in patients with heart failure complicated by secondary pulmonary hypertension [67]. A recent study has demonstrated induction of PDE5 expression in the hypertrophied right ventricle of patients with pulmonary hypertension [68]. An increase in myocardial contractility with PDE5 inhibition in myocardium that expresses the enzyme has also been reported, suggesting that PDE5 inhibition might improve directly right ventricular function in pulmonary hypertension. Sildenafil has been combined with inhaled iloprost both acutely [69] and as long-term therapy [70], showing beneficial additive effects without increasing the side effects of either agent. Sildenafil and bosentan in combination has been reported to be of benefit, rescuing patients from deterioration with one agent alone, in the short term, although concerns remain over pharmacokinetic interactions in which sildenafil blood levels are reduced [71, 72]. Combination of sildenafil with sitaxsentan or ambrisentan would have more predictable pharmacokinetics, as there is no clinically significant interaction, but no pharmacodynamic data from such combinations are currently available. Perhaps the combination of a PDE5 inhibitor with an agent which augments cGMP production may be therapeutically advantageous, but care must be taken to evaluate any additive effect on the systemic circulation [59]. Interestingly, sildenafil shows efficacy in treating patients with pulmonary hypertension associated with some other diseases, such as patients suffering from human immunodeficiency virus related pulmonary hypertension [73, 74] or patients with nonoperable chronic thromboembolic pulmonary hypertension [75, 76].

106  Phosphodiesterase Inhibitors in the Treatment of Pulmonary Hypertension

5.3 Studies in Hypoxia-Associated Pulmonary Hypertension An early study showed that a single oral dose of sildenafil (50  mg) inhibited the acute pressor response to hypoxia (11% inspired O2) in healthy volunteers [53]. To further address this, several studies have been conducted in subjects at altitude [77–79]. In 14 healthy mountaineers and trekkers, sildenafil (50 mg) treatment significantly reduced systolic pulmonary arterial pressure at rest and during exercise while increasing cardiac output and maximum workload at rest and during exercise. Treatment with sildenafil (40 mg TID) for 6 days in 12 subjects 6–8 h after arrival at high altitude (4,350  m) from sea level normalized the increased systolic pulmonary arterial pressure (measured by echocardiography) at high altitude before treatment (+29% versus sea level) [78]. Gas exchange was also improved at rest and during exercise. In patients with pulmonary hypertension from living above 2,500  m in the Kyrgyz Republic, treatment with sildenafil in a dosage of 25  mg TID for 3  months significantly reduced the mean pulmonary arterial pressure (−6.9  mmHg) compared with use of a placebo. A similar effect was seen with sildenafil in a dosage of 100  mg TID and both dosages improved the 6-min walk distance [80]. There is some evidence that PDE5 inhibitors may preserve ventilation–perfusion matching [81]. Alveolar oxygenation is an important determinant of local NO production and so of cGMP levels. In a randomized controlled, open-label trial, in 16 individuals with pulmonary hypertension secondary to lung fibrosis, a single oral 50-mg dose of sildenafil reduced the pulmonary vascular resistance index and improved ventilation– perfusion matching, resulting in a significant increase in the partial arterial pressure of oxygen. The results of these studies have stimulated further investigations to address the therapeutic potential of this drug in patients suffering from chronic hypoxic pulmonary hypertension associated with COPD, interstitial lung disease, and obstructive sleep apnea [82–86]. Recent work supports the possibility of effective treatment of pulmonary hypertension in patients suffering from advanced COPD [86]. Because of the significant impact of COPD on public health, further definitive studies in this field are warranted.

6 Summary Orally active PDE5 inhibitors are an exciting class of drugs, which have added another dimension to the treatment of pulmonary hypertension. Sildenafil is approved for the treatment of PAH. It demonstrates significant improvements in hemodynamic and exercise capacity over several

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months in patients with idiopathic PAH and pulmonary hypertension associated with connective tissue disease and life at high altitude. Mechanisms other than pulmonary vasodilatation may be responsible for the benefits of PDE5 inhibition. One important finding is that PDE5 is expressed in the hypertrophied right ventricle of patients with pulmonary hypertension. PDE5 inhibition may act directly on the right ventricle to enhance contractility and cardiac output and potentially improve right ventricular function. The effect of sildenafil on EPCs cannot be ignored. Another interesting aspect is the potential of PDE inhibition to reduce pulmonary vascular resistance without adversely affecting ventilation–perfusion matching, a property which would lend itself to the treatment of pulmonary hypertension associated with lung disease. Further studies are needed to examine these possibilities and there remains an important need for more information on survival from long-term PDE5 inhibition.

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69. Ghofrani HA, Wiedemann R, Rose F et  al (2002) Combination therapy with oral sildenafil and inhaled iloprost for severe pulmonary hypertension. Ann Intern Med 136:515–522 70. Ghofrani HA, Rose F, Schermuly RT et al (2003) Oral sildenafil as long-term adjunct therapy to inhaled iloprost in severe pulmonary arterial hypertension. J Am Coll Cardiol 42:158–164 71. Hoeper MM, Faulenbach C, Golpon H, Winkler J, Welte T, Niedermeyer J (2004) Combination therapy with bosentan and sildenafil in idiopathic pulmonary arterial hypertension. Eur Respir J 24:1007–1010 72. Mathai SC, Girgis RE, Fisher MR et al (2007) Addition of sildenafil to bosentan monotherapy in pulmonary arterial hypertension. Eur Respir J 29:469–475 73. Carlsen J, Kjeldsen K, Gerstoft J (2002) Sildenafil as a successful treatment of otherwise fatal HIV-related pulmonary hypertension. AIDS 16:1568–1569 74. Schumacher YO, Zdebik A, Huonker M, Kreisel W (2001) Sildenafil in HIV-related pulmonary hypertension. AIDS 15:1747–1748 75. Ghofrani HA, Schermuly RT, Rose F et al (2003) Sildenafil for long-term treatment of nonoperable chronic thromboembolic pulmonary hypertension. Am J Respir Crit Care Med 167:1139–1141 76. Reichenberger F, Voswinckel R, Enke B et  al (2007) Long-term treatment with sildenafil in chronic thromboembolic pulmonary hypertension. Eur Respir J 30:922–927 77. Ghofrani HA, Reichenberger F, Kohstall MG et al (2004) Sildenafil increased exercise capacity during hypoxia at low altitudes and at Mount Everest base camp: a randomized, double-blind, placebocontrolled crossover trial. Ann Intern Med 141:169–177 78. Richalet JP, Gratadour P, Robach P et al (2005) Sildenafil inhibits altitude-induced hypoxemia and pulmonary hypertension. Am J Respir Crit Care Med 171:275–281 79. Hsu AR, Barnholt KE, Grundmann NK, Lin JH, McCallum SW, Friedlander AL (2006) Sildenafil improves cardiac output and exercise performance during acute hypoxia, but not normoxia. J Appl Physiol 100:2031–2040 80. Aldashev AA, Kojonazarov BK, Amatov TA et  al (2005) Phosphodiesterase type 5 and high altitude pulmonary hypertension. Thorax 60:683–687 81. Ghofrani HA, Wiedemann R, Rose F et  al (2002) Sildenafil for treatment of lung fibrosis and pulmonary hypertension: a randomised controlled trial. Lancet 360:895–900 82. Naeije R (2005) Pulmonary hypertension and right heart failure in chronic obstructive pulmonary disease. Proc Am Thorac Soc 2:20–22 83. Higenbottam T (2005) Pulmonary hypertension and chronic obstructive pulmonary disease: a case for treatment. Proc Am Thorac Soc 2:12–19 84. Voelkel NF, Cool CD (2003) Pulmonary vascular involvement in chronic obstructive pulmonary disease. Eur Respir J 46:28s–32s 85. Barbera JA, Peinado VI, Santos S (2003) Pulmonary hypertension in chronic obstructive pulmonary disease. Eur Respir J 21:892–905 86. Alp S, Skrygan M, Schmidt WE, Bastian A (2006) Sildenafil improves hemodynamic parameters in COPD–an investigation of six patients. Pulm Pharmacol Ther 19:386–390

Chapter 107

Nitric Oxide for Children Judy L. Aschner, Candice D. Fike, Eric Austin, J. Donald Moore, and Frederick E. Barr

Abstract  The introduction of inhaled nitric oxide (iNO) for the treatment of hypoxemic respiratory failure in neonates ushered in a new era in neonatal intensive care. This inhalational therapeutic redefined the medical management of the infant with persistent pulmonary hypertension of the newborn (PPHN). With a selective pulmonary vasodilator in hand, a kinder, gentler approach to ventilation was embraced in many neonatal intensive care units, aggressive hyperventilation and alkaline therapy were abandoned, and the number of infants referred for extracorporeal membrane oxygenation (ECMO) declined. Yet, iNO is not a panacea. A significant number of infants fail to respond or sustain a response to iNO. Furthermore, iNO is being increasingly used off-label for populations in which its safety and efficacy have not been adequately studied. In this chapter on iNO in children, we will review the spectrum of approved and off-label uses of iNO in pediatrics, and address gaps in our knowledge regarding the optimal dosing, weaning, and patient selection. Keywords  Inhaled nitric oxide • Pediatric pulmonary hypertension • Neonatal intensive care • Persistent pulmonary hypertension of the newborn • Pulmonary vasodilation

1 Introduction The introduction of inhaled nitric oxide (iNO) for the treatment of hypoxemic respiratory failure in neonates ushered in a new era in neonatal intensive care. This inhalational therapeutic redefined the medical management of the infant with persistent pulmonary hypertension of the newborn (PPHN). With a selective pulmonary vasodilator in hand, a kinder, gentler approach to ventilation was embraced in many neonatal intensive care units, aggressive hyperventilation and alkaline therapy were abandoned, and the number of infants J.L. Aschner (*) Department of Pediatrics, The Monroe Carell Jr. Children’s Hospital, Vanderbilt University School of Medicine, Nashville, TN, USA e-mail: [email protected]

referred for extracorporeal membrane oxygenation (ECMO) declined. Yet, iNO is not a panacea. A significant number of infants fail to respond or sustain a response to iNO. Furthermore, iNO is being increasingly used off-label for populations in which its safety and efficacy have not been adequately studied. In this chapter on iNO in children, we will review the spectrum of approved and off-label uses of iNO in pediatrics, and address gaps in our knowledge regarding the optimal dosing, weaning, and patient selection.

2 iNO for PPHN In 1999, the US Food and Drug Administration (FDA) approved iNO for treatment of hypoxemic respiratory failure in newborn infants of at least 35 weeks’ gestation. This inhalation therapy represented the first evidence-based medical therapy for the treatment of infants with PPHN. The FDA approval of iNO was based on the results of two large, multicenter, randomized, placebo-controlled clinical trials conducted in term and late preterm neonates with hypoxemic respiratory failure that demonstrated improved outcomes in the infants treated with iNO. The Neonatal Inhaled Nitric Oxide Study Group (NINOS) trial showed that iNO reduced the need for ECMO [1], without increasing neurodevelopmental, behavioral, or medical morbidities at 2 years of age  [2]. The Clinical Inhaled Nitric Oxide Research Group (CINRGI) trial demonstrated that iNO reduced both the need for ECMO and the incidence of chronic lung disease [3]. There have been numerous subsequent clinical trials in the USA and in Europe that support the safety and efficacy of iNO in term and late preterm infants with hypoxemic respiratory failure and PPHN [4–9]. Individual clinical trials and several systematic reviews have shown that iNO improves outcomes by reducing the need for ECMO without adversely affecting the length of hospital stay or the number of ventilator days in neonates with PPHN [3, 10]. In centers where ECMO is available, iNO has not been shown to reduce mortality. Treatment with iNO results in an improvement in oxygenation and a lowering of the oxygenation index (OI; OI is

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mean airway pressure in centimeters of H2O × FiO2 × 100/ PaO2 in millimeters of Hg) in 50–60% of treated infants [10]. Long-term neurodevelopmental outcomes in infants treated with iNO are similar to those treated with a placebo [11]. Neonatologists now have considerable experience with the use of iNO in the treatment of PPHN in term and late preterm infants. Yet, gaps exist in our knowledge of the optimal dosing and weaning strategies as well as which patients with PPHN are most likely to benefit from this therapy. Efficacy varies with the severity of respiratory failure as well as with the underlying disease causing PPHN. Several published trials have addressed the severity of respiratory failure that should trigger use of iNO. Most infants enrolled in the NINOS and CINRGI trials had severe hypoxemic respiratory failure. The mean OI of the entire cohort was greater than 40, although the criterion for eligibility­ was an OI of 25 or greater [1, 3]. An OI of 25 is associated with a 50% risk of requiring ECMO or dying, whereas an OI of 40 is often used as a criterion to initiate ECMO therapy. iNO appears to reduce the likelihood of progression to ECMO in those infants with an OI between 25 and 40 and documented extrapulmonary right-to-left shunt. Currently acceptable indications for treatment with iNO include: (a) OI > 25 or PaO2 < 100 mmHg while receiving FiO2 of 1 with echocardiographic evidence of extrapulmonary right-to-left shunting. (b) Severe hypoxic respiratory failure with an OI > 40 with or without echocardiographic evidence of pulmonary hypertension. Two trials that enrolled less severely ill infants or infants at an earlier stage of their disease failed to demonstrate an effect on the primary outcome of death or ECMO. However, this may have been related to inadequate power as both trials were stopped before the targeted enrollments had been reached [9, 12]. There does not appear at this time to be a strong indication to use iNO in less severely ill infants. As shown by Konduri et al. [12], iNO improves oxygenation but does not reduce the incidence of death or need for ECMO in infants with an OI between 15 and 25. The underlying pulmonary disease clearly influences the likelihood of a positive response to iNO. More than 65% of infants with meconium aspiration syndrome, pneumonia, idiopathic PPHN, and respiratory distress syndrome will respond to iNO with improved oxygenation and survival without the need for ECMO. In contrast, less than 35% of infants with congenital diaphragmatic hernia (CDH) respond favorably to iNO or survive without ECMO [3]. The poor response to iNO in infants with CDH has been affirmed in numerous clinical trials; indeed, the likelihood of death or ECMO was higher in infants with CDH treated with iNO compared with those randomized to receive a placebo

J.L. Aschner et al.

[3, 13]. Worse outcomes in infants when treated with iNO was also shown in a trial that exclusively studied infants with CDH [14]. Despite lack of proven efficacy, it is not uncommon for a trial of iNO to be performed in infants with CDH and other forms of pulmonary hypoplasia prior to ECMO cannulation and after decannulation in those with persistently elevated pulmonary artery pressures [15]. Until more effective therapies for this patient population are developed, this non-evidence-based practice is unlikely to change.

2.1 Dosing of and Weaning from iNO in Infants with PPHN The recommended starting dose of iNO is 20 ppm (Fig. 1). This was the starting dose in both the NINOS [1] and the CINRGI [3] trials. The NINOS trial allowed escalation of dosing to 80 ppm in infants who failed to respond to lower doses; however, increasing the iNO dose to 80 ppm resulted in improved oxygenation in only 6% of infants. Increasing the dose to 40 or 80  ppm has not been shown to improve oxygenation in infants who fail to respond to a dose of 20 ppm [6, 9, 14, 16]. The effects of different doses of iNO were evaluated in a randomized, controlled, dose-response trial that compared the use of doses of 5, 20, and 80 ppm with that of a placebo [9]. Relative to the placebo, all doses of iNO improved oxygenation, and efficacy was similar among all iNO dosing groups. However, evidence of toxicity was seen at the highest dose of 80 ppm, with 35% of infants so treated developing methemoglobinemia. Methemoglobin, which is formed when nitric oxide combines with hemoglobin, causes tissue hypoxia when present at high levels in the circulation. Likewise, 19% of infants treated with 80  ppm experienced elevated levels of inspired nitrogen dioxide (NO2). NO2, the product of nitric oxide plus oxygen, can cause epithelial damage, airway reactivity, and pulmonary edema [17, 18]. Guthrie et al. [19] reviewed registry data on 476 patients from 36 centers that voluntarily reported outcomes of infants treated with iNO. The patients were divided into a low-dose group with starting doses less than 18  ppm, a mid-dose group with starting doses of 18–22 ppm, and a high-dose group with starting doses above 22  ppm. Treatment failure and ECMO referral were lowest in the low-dose group and highest in the high-dose group. Survival without need for supplemental oxygen at 28 days or at discharge was 93% in the low-dose group and 76% in the high-dose group, with no differences in mortality or length of hospital stay between the groups. Higher levels of

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Fig. 1  Flow diagram for initiation of inhaled nitric oxide (iNO) therapy in infants with persistent pulmonary hypertension of the newborn (PPHN) used in the neonatal intensive care unit at the Monroe Carell Jr. Children’s Hospital at Vanderbilt University. OI oxygenation index, SpO2 oxygen saturation per pulse oximetry, FiO2 fractional inspired

­oxygen concentration, PEEP positive end-expiratory pressure, PIP peak inspiratory pressure, Vt tidal volume, PSV pressure support ventilation, Paw mean airway pressure, pCO2 partial pressure of carbon dioxide, pO2 partial pressure of oxygen, BE base excess, ABG arterial blood gas, CRT/ RRT certified respiratory therapist/registered respiratory therapist

methemoglobin were seen in the high-dose group [19]. This study has a number of limitations. It was retrospective and the groups were not randomized. It is certainly possible, even likely, that infants deemed to be sicker were started on higher doses of iNO. Therefore, differences in the populations might explain the worse outcomes of infants in the

high-dose group. Nonetheless, to date no data suggest that doses higher than 20 ppm are more efficacious than lower doses and extensive data suggest that toxicity is more likely at doses of 80  ppm or higher. Thus, it is reasonable to consider 20 ppm both an appropriate starting dose and the maximal dose of iNO.

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Controversy remains about the lowest effective dose of iNO. One trial that evaluated a starting dose of 2 ppm suggested that this initial low starting dose impaired responses to subsequent higher doses [20]. The limitations of this study include a small sample size of 38 infants and an unblinded trial design. Current recommendations for dosing, therefore, include starting therapy at 20  ppm and weaning rapidly to

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5 ppm within 4–24 h (Figs. 1, 2). Response time, if response occurs, is typically rapid in infants with PPHN; a reduction in OI and an increase in PaO2 are typically seen within 30–60 min after commencing therapy. The duration of iNO therapy is up to 14 days or “until the underlying oxygen desaturation has resolved,” according to the package insert for INOmax®, matching the maximum

Fig. 2  Flow diagram for weaning infants with PPHN off iNO used in the neonatal intensive care unit at the Monroe Carell Jr. Children’s Hospital at Vanderbilt University

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duration of therapy in several early randomized, controlled trials [1, 4, 6, 9, 14]. The exception was the CINRGI trial, which allowed a maximum duration of therapy of 4 days [3]. Most infants in the reported trials were treated for 5 days or less, with the exception of infants with CDH, who, despite lack of efficacy, had typically been exposed to the drug for extended time periods [21, 22]. Unfortunately, there are no data to indicate the maximal safe duration of iNO therapy for treatment of PPHN. However, the need for iNO therapy beyond 5 days should trigger evaluation of causes of pulmonary­ hypertension that are less likely to respond to vasodilator therapy. These include pulmonary hypoplasia, alveolar capillary dysplasia, cardiovascular anomalies such as anomalous pulmonary venous drainage and cyanotic congenital heart disease (CHD), and genetic defects of surfactant biosynthesis or trafficking. Failure to demonstrate an immediate response to iNO should trigger careful assessment of lung volumes. When PPHN is associated with parenchymal lung disease, the ­combination of iNO plus a ventilation strategy that optimizes lung volume recruitment is more efficacious than iNO alone. High-frequency oscillatory ventilation (HFOV) has been shown to recruit lung volume and improve the response to iNO among infants with parenchymal lung ­disease and PPHN [5]. Infants with parenchymal lung ­disease were less likely to be referred for ECMO when treated with iNO ­combined with HFOV than when treated with either therapy alone. This was not true for infants with idiopathic pulmonary hypertension in the absence of parenchymal lung ­disease, where iNO was highly effective and the addition of HFOV was not synergetic. Likewise, surfactant decreased the use of ECMO in infants with respiratory distress ­syndrome, meconium aspiration syndrome, and sepsis, but not in infants with idiopathic PPHN [23]. It is thus clear that effective vasodilator therapy with iNO requires adequate lung inflation to optimize drug delivery and ventilation–perfusion matching. Conversely, suboptimal lung inflation may compromise the efficacy of iNO therapy for infants with PPHN. It has been suggested that poor lung inflation with inadequate alveolar recruitment is the most common cause of treatment failure [5]. An infant who fails to respond to iNO therapy should be evaluated by chest radiograph for airway obstruction and atelectasis. Lung volume recruitment strategies with therapies such as HFOV, optimal positive end-expiratory pressure, and surfactant therapy should be used to address the underlying parenchymal lung disease. Failure to respond to iNO is also an indication for evaluation of cardiac performance by echocardiogram. This is particularly important if there was a history of perinatal asphyxia or evidence of metabolic acidosis. Blood pressure and blood volume support to maximize cardiac output and systemic

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hemodynamics in combination with lung volume optimization and iNO to treat the pulmonary vascular disease are interrelated components of the optimal medical management of infants with PPHN. Information about appropriate weaning of patients off iNO therapy is scarce. It is well recognized that even in the absence of a clinical response, sudden discontinuation of iNO therapy can be associated with “rebound” pulmonary hypertension [24–27]. In part because the rebound hypoxemia can be severe, infants who are candidates for ECMO therapy should be started on iNO only in centers where ECMO is available or there is an ability to transport the infant while receiving iNO. Oxygen saturations should be carefully monitored during weaning from iNO. The mechanism for this rebound phenomenon is not certain but has been attributed to downregulation of endogenous nitric oxide and elevations in free radicals and endothelin-1 levels caused by iNO therapy [28–31]. Surprisingly, there are few published protocols that provide parameters to guide clinicians in weaning, dosing escalation, or reinitiation of iNO therapy. One such protocol developed for patients in the Neonatal Intensive Care Unit at the Monroe Carell Jr. Children’s Hospital at Vanderbilt University is provided as a template for adoption or modification to meet local needs (Figs. 1, 2). The weaning protocol for the CINRGI trial included a rapid wean from 20 to 5 ppm after 4 h of treatment in those patients who demonstrated improved oxygenation [3]. The NINOS trial [1] incorporated an iNO weaning attempt when the patient had an arterial PO2 greater than 50  Torr [32]. Davidson et  al. addressed the safety of withdrawing iNO therapy in infants with PPHN [26]. iNO weaning was initiated when the patient achieved an OI less than 10. These published studies support the safety of attempting to wean an infant off iNO within a few hours of initiating therapy if the infant has had an improvement in respiratory status after treatment with 20 ppm. The relative timing and advantages of reducing FiO2 ­versus weaning the infant off iNO have not been studied. There is some evidence that iNO, particularly at doses less than 80 ppm, has beneficial effects other than those attributed to lowering pulmonary vascular resistance [33]. For example, animal studies have shown that iNO reduces the accumulation of neutrophils in the lung and diminishes the inflammatory cascade associated with lung injury [13, 34]. The CINRGI trial showed an association between use of iNO and a decrease in the occurrence of chronic lung ­disease [3]. Therefore, an argument can be made to reduce FiO2 prior to weaning the infant off iNO. Supportive of this approach, clinical success has been reported using ­algorithms for reducing FiO2 to approximately 50–60% prior to reducing the dose of iNO [24, 26, 35]. Whether FiO2 or the dose of iNO is reduced first, it is important to note that the

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neonatal pulmonary vasculature is fully dilated when arterial oxygenation tension exceeds approximately 70–80  Torr [36–38]. There is no justification to maintain a higher arterial PO2. Instead, attempts to reduce FiO2 should be made once the infant is stable with an arterial PO2 greater than 50–80 Torr and within the first few hours of initiating iNO therapy (Figs. 1, 2). A variety of dose-reduction paradigms have been used to wean infants off iNO. Infants were weaned from 20 to 5 ppm with no intermediate reductions in iNO concentration in the CINRGI trial [3]. In the NINOS trial, the iNO concentration was reduced by 50% until a dose of 5 ppm was achieved [1]. Attempts were made to discontinue iNO therapy from 5 ppm, but if this strategy failed, the iNO concentration was reduced in 1-ppm decrements from 5 to 0 ppm [32]. Other published weaning strategies have incorporated either 5-ppm or 20% stepwise reductions in iNO concentration [24, 26, 35]. Unfortunately, there is no evidence to support any one particular weaning protocol over another. However, a number of studies have demonstrated a much greater success rate when discontinuing iNO therapy from 1 ppm than from 5 ppm or higher doses of iNO [24, 26, 32]. It therefore seems reasonable to reduce the iNO dose to 1 ppm before complete discontinuation is attempted. Some decline in arterial oxygenation should be anticipated and an increase in FiO2 of 10–20% considered reasonable when discontinuing iNO therapy (Fig. 2) iNO therapy should not be reinstated on the basis of a modest increase in required FiO2. An initial echocardiogram is important to rule out structural heart disease and can sometimes provide helpful information to explain a failure to respond to therapy. An example of the latter case is when left or right ventricular dysfunction is so severe that pulmonary vasodilation by itself is not ­sufficient to improve systemic oxygenation. Several trials demonstrating efficacy did not require the presence of ­clinical or echocardiographic evidence of pulmonary hypertension [1, 9, 39]. Infants without documented extrapulmonary rightto-left shunt may respond to iNO with increased oxygenation by improving ventilation–perfusion matching. None of the published clinical trials used resolution of echocardiographic evidence of pulmonary hypertension as a criterion to initiate weaning from or to discontinue iNO therapy. There is no evidence that an echocardiogram should be obtained prior to discontinuing iNO. An echocardiogram may be useful when an infant fails to wean from iNO within 5 days or demonstrates progressive clinical deterioration at any time during iNO therapy. The flow diagram shown in Fig. 2 is offered as a suggested strategy for weaning an infant from iNO and as a bedside tool to guide clinical decision making and track protocol adherence. Although other weaning approaches may be equally or more effective, the approach depicted in this flow diagram is based on the currently available evidence described above.

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2.2 Summary of iNO Use for Infants with PPHN When used in accordance with FDA guidelines, iNO appears to have a good overall safety profile in neonates. Its lack of effect on systemic hemodynamics has been well established. It is the only selective pulmonary vasodilator with established efficacy for infants with PPHN. Efficacy has generally been defined as a reduced need for ECMO, with the number needed to treat of 5.3 [10]. However, ECMO is itself a therapy of proven benefit in infants with respiratory failure. The clinical trials have allayed fears that a delay in the referral for ECMO or a decrease in ECMO usage would result in increased lung injury. In fact, iNO has been associated with a decrease in the incidence of chronic lung disease [3]. ECMO is an invasive and technologically sophisticated therapy with limited availability in many parts of the world. A large number of infants who would benefit from ECMO are not born in centers where this technology is available, necessitating an infant transport, often under less than ideal circumstances. ECMO is expensive and is associated with important complications. Thus, a drug with a satisfactory safety profile that reduces the need for ECMO is a valuable addition to our therapeutic repertoire. Administration of iNO must be accompanied by careful monitoring for toxicities, including methemoglobinemia and NO2 levels, as well as accurate monitoring of the delivered concentration of iNO. It cannot be overemphasized that rebound pulmonary hypertension can occur even in infants who fail to respond to initiation of iNO therapy. Deterioration of oxygenation upon discontinuation of iNO therapy in these infants has implications for the use of this drug in non-ECMO centers and for transport of infants between centers [40]. Reassuring information about longer-term neurodevelopmental outcomes has been provided by several studies [2, 41–43]. Among survivors seen in follow-up at 2 years of age, the findings of mental, motor, and audiology examinations as well as postdischarge pulmonary complications were similar in the iNO- and placebo-treated groups. Longer-term outcomes for these children at school age and beyond have not been reported. The American Academy of Pediatrics (AAP) issued practice guidelines for the use of iNO in 2000 and made several recommendations that remain valid today [40]. The AAP recommended that iNO therapy should be initiated in centers with ECMO capability. If iNO therapy is initiated in a center without ECMO capability, criteria and a procedure for transfer to an ECMO facility should be prospectively established between the two centers. Transfer must occur without interruption of iNO therapy. Centerspecific criteria for treatment failure should be developed to facilitate timely consideration of alternative therapies. Centers that provide iNO therapy should provide comprehensive

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long-term medical and neurodevelopment follow-up and should establish prospective data collection for treatment time course, toxic effects, treatment failure, and use of alternative therapies and outcomes.

3 iNO for the Management of Perioperative Pulmonary Hypertension in Infants with CHD As is true for many drugs used in pediatrics, iNO is not ­specifically approved by the US FDA to treat postoperative pulmonary hypertension in children undergoing repair of CHD. Nonetheless, off-label use for this purpose is commonplace in pediatric intensive care units. The clinical ­evidence supporting its use is derived primarily from small, single-center studies. Lack of equipoise among clinicians, the diversity of the patient population in terms of age, underlying cardiac defect, and surgical procedure, and the cost and difficulty of performing appropriately powered multicenter studies in this population make it unlikely that perioperative pulmonary hypertension in children undergoing repair of CHD will soon become an FDA-indicated use of iNO. One of the earliest clinical reports of the use of iNO after congenital cardiac surgery showed that iNO was an effective pulmonary vasodilator without significant effects on the systemic circulation in postoperative pediatric patients [44]. This 1993 publication was followed 2 years later by another nonrandomized report of iNO therapy in postoperative patients with severe refractory pulmonary hypertension ­unresponsive to conventional therapy [45]. Eleven of the 15 children had a good response to iNO at doses as low as 10–20 ppm. The four patients that did not respond clinically to iNO subsequently died. These two early clinical reports highlighted the need for randomized, placebo-controlled clinical trials to determine if iNO would improve postoperative outcomes in children with postoperative pulmonary hypertension. Several single-center, randomized, placebo-controlled clinical trials followed, all with fairly small sample sizes. The first randomized patients with documented preoperative pulmonary hypertension to a 20-min administration in the operating suite of either iNO at 80 ppm or nitrogen placebo [46]. Of the 35 patients studied, only 13 had documented postoperative pulmonary hypertension. Among these 13 patients, five received iNO and eight received the placebo gas. The response to iNO was fairly dramatic, with a rapid and sustained 20% reduction in pulmonary artery pressure compared with baseline values. Patients treated with the placebo had a small increase in pulmonary artery pressure over the 20-min study period that was not significant. In the remaining 22 patients who did not develop postoperative pulmonary hypertension there were no differences in pulmonary

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artery pressure between those who received iNO and those who received the placebo. Major limitations of the study include the very small sample size, which was compounded by the low incidence (37%) of postoperative pulmonary hypertension. A subsequent randomized clinical trial of iNO therapy targeted prevention of a postoperative pulmonary hypertensive crisis as the primary outcome [47]. Study entry was limited to patients with postoperative pulmonary hypertension, defined by a systolic pulmonary artery pressure greater than 50% of the systolic systemic artery pressure measured by indwelling pulmonary and systemic arterial catheters. Thirtyeight patients were randomized but the study was not blinded and control patients could cross over to receive iNO therapy if they had a sustained pulmonary hypertensive crisis that did not respond to conventional therapy. Hemodynamics before and 1  h after initiation of treatment showed no significant differences between the conventional treatment group and the iNO group. Four patients in the conventional therapy group and three patients in the iNO group developed pulmonary hypertensive crises. The conventional therapy patients who developed a pulmonary hypertensive crisis were all subsequently treated with iNO and had improvement in oxygenation and hemodynamics. The authors concluded that iNO did not significantly improve postoperative hemodynamics or prevent postoperative pulmonary hypertensive crises compared with conventional therapy but was effective as a rescue therapy for patients who failed conventional therapy. The largest study to date evaluating iNO therapy in the postoperative period was performed at the Royal Alexandra Hospital for Children in Sydney, Australia [48]. The authors enrolled 124 patients undergoing atrioventricular septal defect or unrestrictive ventricular septal defect repair who had preoperative evidence of high pulmonary artery pressure or flow. In this blinded study, patients were randomized to receive iNO (10 ppm) versus a placebo after surgery until extubation. Compared with the patients in the placebo group, the patients in the iNO group had a lower median pulmonary vascular resistance, 30% fewer pulmonary hypertensive crises, and a shorter median time to extubation readiness. The study was not powered to demonstrate a difference in the length of mechanical ventilation or mortality. A systematic review of the available data from ­randomized clinical trials was performed by Bizzarro and Gross [49]. Utilizing Cochrane database guidelines, the authors evaluated published studies of iNO therapy in the postoperative setting. Outcomes of interest included mortality, length of intensive care unit stay or hospitalization, neurodevelopmental outcome, and physiologic variables. Utilizing preestablished inclusion and exclusion criteria, they included only four randomized clinical trials in the analysis. However, because each of these studies reported different outcomes, the number of patients included in the subsequent meta-analysis

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of each outcome was very limited and did not add much to the previously published literature. For example, only the studies by Day et al. [47] and Miller et al. [48] reported mortality; thus, the subsequent meta-analysis was conducted on a cohort of 162 patients, hardly an adequate sample size in a population where postoperative mortality is a fairly rare event. Analysis of the incidence of postoperative pulmonary hypertensive crisis was limited to the study by Day et al. and thus only included data on 38 patients. The authors concluded that there were no differences in the use of iNO in the outcomes that were reviewed while acknowledging the limitations of the review due to the concerns over methodological quality, sample size, and heterogeneity of patients in the four studies. These limitations severely restrict any conclusions that can be drawn from this Cochrane review or about the efficacy of iNO for prevention or treatment of postoperative pulmonary hypertension.

4 Recommendations for Use of iNO in the Postoperative Setting On the basis of the available clinical trials, there is insufficient evidence to recommend the routine use of iNO for prevention or treatment of postoperative pulmonary hypertension in pediatric patients with CHD. The study by Miller et  al. [48] is the only study of sufficient size addressing relevant clinical outcomes, but this study was too small to address the important outcome of mortality. Nonetheless, it is acknowledged that this drug is often used in the pediatric cardiac intensive care unit and that a larger randomized, placebo-controlled trial in this patient population is unlikely owing to lack of equipoise and lack of funding for such a trial. Clinical recommendations for the use of iNO should provide guidance not only as to whether this therapy is useful but also how to discontinue the therapy. Optimally, iNO should be utilized when there is clear evidence of pulmonary hypertension by echocardiographic or pulmonary artery catheter data. It is acknowledged that pulmonary artery catheters are not routinely utilized in the postoperative period in most pediatric centers and echocardiographic data cannot always be obtained emergently. In those instances, if iNO is utilized, the physiologic response should be noted and echocardiographic measurements obtained. Once iNO therapy has been started, reasonable attempts to wean the infant off iNO should be made every 12–24 h utilizing an objective weaning protocol that measures the physiologic response to both the decrease in dose as well as the discontinuation of iNO therapy. The iNO weaning protocol utilized in the Pediatric Critical Care Unit at the Monroe Carell Jr. Children’s Hospital at Vanderbilt University is shown in Fig. 3.

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5 iNO in Premature Infants The FDA approved iNO therapy for infants with defined gestational age and disease restrictions. The initial randomized, controlled trials of iNO for PPHN restricted enrollment to infants of more than 34 weeks’ gestation [1, 3]. As of 2009, iNO is not approved by the FDA for infants born at lower gestational ages. Yet, hypoxemic respiratory failure associated with pulmonary hypertension occurs in infants born at younger gestational ages and off-label use of iNO is common in these circumstances. Animal data in preterm lambs treated with surfactant for hyaline membrane disease and either iNO or a placebo show that iNO can induce a sustained improvement in oxygenation and pulmonary hemodynamics [50]. Anecdotal reports and case series in human infants with severe hypoxemic respiratory failure and pulmonary hypertension also suggest some efficacy [51–54], but concerns about safety of iNO in this population were raised by the NICHD Neonatal Research Network trial [55]. A multicenter randomized, placebo-controlled trial of iNO in preterm infants with acute respiratory failure and echocardiographic evidence of pulmonary hypertension is needed to confirm efficacy and safety in this population of high-risk infants. The postnatal age restriction on the approved use of iNO to the newborn period is an unusual twist as most ­pharmaceuticals are studied and approved for use in adults. iNO is one of only a few drugs specifically approved for use in the neonatal population. Currently this drug does not have an FDA-approved indication in older children or adults. Most patients in the large randomized, controlled trials of iNO therapy were enrolled in the first few days of life, although several trials permitted enrollment of infants up to age 14 days. For example, the average age at enrollment in the NINOS study was 1.7 days [1]. In many centers, however, clinical use of iNO has been extended well beyond the first 2 weeks of life. Unfortunately, the efficacy of iNO in older infants and children with pulmonary hypertension has not been clearly demonstrated. Initiation or resumption of iNO therapy after ECMO in infants with persistence of pulmonary hypertension, and prolonged therapy for refractory pulmonary hypertension are well described [56], although not well supported by randomized, controlled trials. Despite limited evidence for the efficacy and safety of iNO beyond the neonatal period, this therapy has been widely adopted in neonatal and pediatric intensive care units for older infants and children with pulmonary hypertension caused by a wide range of pulmonary, cardiac, and systemic diseases [22, 38, 47, 48, 56–60]. Lack of equipoise has hampered the conduct of large, multicenter, appropriately powered trials for patients with CHD and pulmonary hypertension following cardiopulmonary bypass surgery as discussed earlier in this chapter.

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Fig. 3  Flow diagram for weaning infants and children with post-cardiac surgery pulmonary hypertension off iNO used in the pediatric critical care unit (PCCU) at the Monroe Carell Jr. Children’s Hospital at Vanderbilt University

6 iNO to Prevent or Treat Bronchopulmonary Dysplasia Reductions in lung surface area and abnormalities of vascular and alveolar structure are prominent features of bronchopulmonary dysplasia (BPD) in humans and in animal models of the disease [61–65]. There is biological plausibility and

great clinical interest in the use of iNO to reduce the incidence or severity of BPD in sick preterm infants. Studies in animal models have demonstrated that iNO improves lung angiogenesis and alveolarization via enhanced vascular endothelial growth factor (VEGF) signaling [66–69]. Long-term exposure to iNO has been reported to ameliorate the lung structural abnormalities in chronically ventilated preterm

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lambs [70] and baboons [71]. In preterm lambs ventilated for 3 weeks, radial alveolar counts were higher in the lambs that received iNO compared with those that received a placebo, although alveolar septation was not restored to the levels seen in term control lambs that were never ventilated [70]. In the ventilated preterm baboon, iNO (5 ppm over 14 days) normalized elastin deposition, stimulated ­secondary crest formation, and achieved rates of lung growth comparable to those in utero [71]. Two weeks of iNO therapy also improved postmortem pressure–volume curves in the baboon [71]. iNO is protective in several neonatal rat models of BPD [66–68]. Distal lung growth was enhanced by iNO exposure in newborn rats exposed to hyperoxia [67]. In rats treated with the VEGF inhibitor SU5416, iNO improved lung vascular growth and density [66]. In the endothelial nitric oxide synthase null mouse, exposure to mild hypoxia causes alveolar simplification; treatment with iNO restores lung structure in this transgenic animal model [68]. Despite consistent evidence of the beneficial impact of iNO in various animal models of BPD, clinical trials of iNO therapy in preterm infants have yielded inconsistent results [55, 72–74]. Possible reasons for the failure to translate the uniformly positive results in experimental models to the clinical arena include the more complex and less uniform pathogenesis of BPD in human infants which may include infection/inflammation, intermittent hypoxia and hyperoxia, free-radical generation, nonuniform approaches to fluid management and nutrition, and most likely a genetic component. The approach to the intervention also differed widely among the clinical trials published to date, with variations in dose, duration, and target population (see Fig. 4).

Fig. 4  Comparison of iNO dose and duration relative to postnatal age at the time of exposure for the largest randomized, controlled trials ­performed in preterm infants to prevent or treat evolving bronchopulmonary­ dysplasia. Trials are listed by the last name of first author followed by the mean gestational age (GA) in weeks of the study cohort. In the case of the Ballard et  al. trial, the GA at birth and the ­dosing regimen for the infants entered at the earliest time point allowed per study design (7 days) are shown in dark blue; this is followed by the mean GA and mean dosing regimen of the entire cohort in light blue. (Original figure by William Truog; modified with permission)

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In addition to differing inclusion criteria and treatment regimens, these trials have addressed very different outcome measures. One of the earliest publications of the use of nitric oxide in the context of BPD was a proof-of-concept, open-label, nonrandomized pilot study of nitric oxide as rescue therapy for infants with life-threatening, established BPD [57]. Eleven out of the 16 infants enrolled responded to iNO (20 ppm) with an immediate improvement in oxygenation. All infants were more than 4 weeks of age and still ventilator-dependent at the time of enrollment. Sustained improvements in oxygenation were seen in ten of the 11 responders, but mortality remained high in this population despite the clinical response. Although there have been no randomized trials of iNO in established BPD, off-label use of iNO is sometimes used as “rescue” therapy in the neonatal intensive care unit to treat infants with severe BPD, particularly when BPD is accompanied by documented pulmonary hypertension and cor pulmonale. These infants are often refractory to medical management and 40% or more die of cardiorespiratory failure [75]. The dismal prognosis for this group of patients drives the off-label use of iNO and other poorly studied vasodilator therapies, such as sildenafil, in these infants. There have been a number of randomized trials of iNO in preterm infants to prevent BPD, yielding somewhat dissimilar results [55, 72, 73, 76]. The first of these to be published was the single-site, double-blind, randomized, placebo-controlled study by Schreiber et  al. [72]. This study enrolled 207 premature infants of less than 34 weeks’ gestation with a birthweight less than 2,000  g in the first 72  h of life who were undergoing mechanical ventilation for respiratory distress syndrome. The infants were randomized to iNO (10 ppm on day 1, followed by 5 ppm for 6 days) or inhaled oxygen placebo for 7 days (Fig. 4). The primary outcome of death or BPD (defined as oxygen requirement at 36 weeks’ gestational age) was reduced from 64% in the placebo group to 49% in the iNO group (odds ratio 0.56; 95% confidence interval 0.32–097; p = 0.04). Surprisingly, iNO therapy reduced severe brain injury defined as cranial ultrasound evidence of intraventricular hemorrhage (IVH; grade 3 or 4) or periventricular leukomalacia (PVL) from 24% in the placebo group to 12% in the iNO group (odds ratio 0.45; 95% confidence interval 0.21–094; p = 0.03). Brain injury was included in the study design as a safety outcome as the platelet-inhibiting properties of iNO raised fears that this therapy would increase IVH; the reduction in IVH and PVL in the trial by Schreiber et al. was unanticipated. In 2005, the Chicago-based group published the results of the 2-year neurodevelopmental outcomes of this study cohort [77]. Consistent with the reduction in ultrasound evidence of brain injury, infants who received iNO had better neurodevelopmental outcomes, with a 47%

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decrease in the risk of cognitive impairment (Mental Developmental Index score of less than 70 on the Bayley II examination). With these promising results, the neonatology community anxiously awaited publication of the NICHD Neonatal Research Network trial of iNO in preterm infants [55]. This multicenter, randomized, blinded, placebo-controlled trial had a very different study design and enrolled infants who, on average, were much sicker than those in the Schreiber et al. trial. In fact, the infants in the study by Schreiber et al. had a mean OI of 7; those in the NICHD network study by Van Meurs et  al. had a mean OI of 17. The NICHD study randomized 240 infants who were less than 34 weeks, with birthweight between 401 and 1,500 g, and respiratory failure more than 4 h after surfactant treatment to iNO (5 ppm) or a placebo. Infants who responded with immediate improvement in oxygenation were weaned rapidly from the study drug according to the protocol. Those who failed to respond with improved oxygenation after 1  h had escalation of the iNO dose to 10 ppm for 1 h. Treatment with the study gas was discontinued in infants who did not have a physiological response after 1 h at 10 ppm (Fig. 4). In this population of extremely ill preterm infants, iNO failed to reduce the outcome of death or BPD or improve grade 3 or 4 IVH or PVL. In a preplanned post hoc analysis, those infants with birthweight below 1,000 g in the iNO group had a higher rate of death and grade 3/4 IVH or PVL than did infants with birthweight below 1,000  g in the control group. On the other hand, among infants with birthweight of 1,000  g of more, iNO reduced the outcome of death or BPD with no impact on IVH or PVL. The jury was clearly still out with regard to safety and efficacy of iNO to prevent BPD and the neonatal community awaited the results of the next large BPD prevention trial that was already under way. This NHLBI-sponsored trial included a longer duration of iNO therapy and enrolled less severely ill newborns than did the NICHD-sponsored study by Van Meurs et al. The NHLBI-sponsored study was published in 2006 [73]. The study enrolled 793 premature infants with birthweight between 500 and 1,250 g who required mechanical ventilation in the first 48 h after birth; randomization was stratified in 250-g increments by birthweight and iNO therapy (5 ppm) was continued for 21 days or until extubation (Fig. 4). In the entire cohort, iNO therapy failed to improve the outcome of death or BPD. However, in the subset of infants with birthweight between 1,000 and 1,250  g, iNO significantly reduced death or BPD from 64% in the control group to 39% in the iNO group (p < 0.004); this was primarily due to a 50% reduction in BPD from 60 to 30% in this largest birthweight subset of infants. Intriguingly, Kinsella et  al. also reported that iNO significantly reduced brain injury (grade 3/4 IVH, PVL or ventriculomegaly) in the entire cohort. However, when evaluated by subgroup, the brain-

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protective effects of iNO were mostly limited to the 750– 999-g birthweight subgroup. Unfortunately, there was no significant difference in 2-year neurodevelopmental outcomes in the two groups (presented in abstract form at the 2009 Pediatric Academic Societies meeting in Baltimore). All eyes were now on the European Nitric Oxide or “EUNO” trial to sort out the conflicting results of the previous trials and determine whether iNO as an early therapy (5 ppm for 7–21 days) in preterm infants requiring respiratory support could prevent BPD or improve neurodevelopmental outcomes (Fig. 4). This large multicenter randomized, blinded, placebocontrolled trial of 800 preterm infants with respiratory failure at birth failed to show a significant difference in survival without BPD; 60.8% in the control group versus 70.0% in the iNO-treated group survived without BPD (p = 0.08) [76]. There were no differences in the incidence of BPD or IVH in the two groups. This study seems to end the debate about the value of iNO to treat all preterm infants requiring respiratory support in the first days of life to prevent BPD development or provide neuroprotection. Another trial with a similar study design seems unlikely and iNO use for all ­preterm babies with respiratory distress at birth cannot be recommended. Keeping the debate about the usefulness of iNO for preterm infants alive is the study by Ballard et al. [74], which was published in 2006 in the same issue of the New England Journal of Medicine as the Kinsella et al. [73] article. The Ballard et al. study had a very different study design, choosing to focus on infants beyond the first week of life with evolving BPD. The multicenter NO CLD study, as it is often referred to, was a masked, placebo-controlled trial of 582 preterm infants with birthweight between 500 and 1,250 g, who were still requiring significant respiratory support on days of life 7–21; a time frame significantly later than for the previously described trials and a study design that targeted infants at highest risk of BPD. The starting dose of 20 ppm was higher than in the previous ­trials in preterm infants. iNO was reduced to 10 ppm at 72 h, then to 5 and 2 ppm at weekly intervals. The duration of therapy (24 days) was also longer than in other trials (Fig. 4). In this study, iNO improved the primary outcome of survival without BPD and was associated with shorter hospitalization and duration of supplemental oxygen use. No short-term adverse effects were noted, including no ­difference in the incidence of IVH or PVL. The greatest effect was seen in those infants enrolled between days 7 and 14 of life; infants enrolled after day 14 did not share in the benefit. Within the group enrolled in the second week of life, the effect of iNO was striking, with an improvement in survival without BPD from 28% in the control group to 49% in the iNO-treated group (p = 0.001). The respiratory benefit demonstrated in the parent study at 36 weeks (based on the most commonly used definition of

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BPD – oxygen requirement at 36 weeks) and also at 40 weeks (based on the predefined outcome of discharged or off all respiratory support at term equivalent) appeared to be sustained. In a follow-up article on the pulmonary outcomes of the entire study cohort at 1 year of age, use of bronchodilators, inhaled steroids, and systemic steroids was significantly lower in the iNO treatment group [78]. Likewise, diuretic use, any home oxygen use, and persistent need for oxygen at 1 year follow-up were lower in the iNO group compared with the control group. As expected on the basis of the cranial ultrasound data in the parent trial, there were no differences in neurodevelopmental outcomes in the two groups [79]. The approach taken by the NO CLD study, namely, targeting a high-risk group of infants who remain dependent on significant respiratory support beyond the acute phase of respiratory distress syndrome with a higher dose and longer duration of iNO therapy, merits further study (Fig.  4). Indeed, a second large trial with a similar study design and beneficial effect is required before the FDA will consider evolving BPD in preterm infants as an indication for iNO use. However, concerns about the safety profile for iNO use in preterm infants appear to be largely resolved with the aforementioned clinical trials, with the possible exception of infants with birthweight below 1,000  g with profound hypoxemic respiratory failure who might be at increased risk of IVH and death [55, 80]. Interestingly, this is the group of infants for which off-label use appears to be most common [51, 52, 54]. This is perhaps not so surprising in that hypoxemic respiratory failure in preterm infants is often fatal and largely resembles this condition in term and late preterm infants and is precisely the condition for which iNO is approved.

7 Conclusions iNO remains one of the few pharmaceuticals specially designed, studied and approved for use in the newborn population. Its efficacy and safety for infants beyond 34 weeks’ gestational age with PPHN has been demonstrated in multiple well-conducted randomized clinical trials and has been born out in extensive clinical use. Like many drugs approved for a narrow disease indication in a limited patient population, off-label use of iNO to treat acute respiratory failure, pulmonary hypertension of diverse causes, and post-cardiac surgery hypertensive crises in older children and adults is commonplace, despite a paucity of compelling evidence for efficacy or sufficiently powered trials to address meaningful clinical end points [59]. Likewise, iNO for vasodilator testing in the catheterization laboratory is commonplace and may exceed in volume its approved use in neonates. Noninvasive

J.L. Aschner et al.

iNO delivered for prolonged periods via nasal cannula in and out of the hospital setting has also been described [56, 81]. In the neonatal intensive care unit, off-label use in premature infants is increasingly common and has become an “accepted” practice in some units for infants with evolving BPD beyond the first week of life. Neurodevelopmental follow-up out to at least 2 years of age suggests an acceptable safety profile in newborns of all gestational ages. However, the cost of this drug, which is charged on an hourly basis, raises the urgency for cost-effectiveness analysis and for confirmatory clinical trials that demonstrate efficacy, refine the optimal treatment dose and duration, and further elucidate the subgroups of infants most likely to benefit from treatment. The current cost structure and cumbersome nature of drug delivery puts this therapy out of reach in most developing countries and should spur investigation into alternative therapies for neonatal, pediatric, and adult patients with pulmonary disease currently thought to be responsive to iNO.

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1499 31. Wedgwood S, McMullan DM, Bekker JM, Fineman JR, Black SM (2001) Role for endothelin-1-induced superoxide and peroxynitrite production in rebound pulmonary hypertension associated with inhaled nitric oxide therapy. Circ Res 89:357–364 32. Sokol GM, Fineberg NS, Wright LL, Ehrenkranz RA (2001) Changes in arterial oxygen tension when weaning neonates from inhaled nitric oxide. Pediatr Pulmonol 32:14–19 33. Gianetti J, Bevilacqua S, De CR (2002) Inhaled nitric oxide: more than a selective pulmonary vasodilator. Eur J Clin Invest 32:628–635 34. Guidot DM, Repine MJ, Hybertson BM, Repine JE (1995) Inhaled nitric oxide prevents neutrophil-mediated, oxygen radical-dependent leak in isolated rat lungs. Am J Physiol 269:L2–L5 35. Williams LJ, Shaffer TH, Greenspan JS (2004) Inhaled nitric oxide therapy in the near-term or term neonate with hypoxic respiratory failure. Neonatal Netw 23:5–13 36. Abman SH, Wolfe RR, Accurso FJ, Koops BL, Bowman CM, Wiggins JW Jr (1985) Pulmonary vascular response to oxygen in infants with severe bronchopulmonary dysplasia. Pediatrics 75:80–84 37. Fike CD, Hansen TN (1987) Hypoxic vasoconstriction increases with postnatal age in lungs from newborn rabbits. Circ Res 60:297–303 38. Mourani PM, Ivy DD, Gao D, Abman SH (2004) Pulmonary vascular effects of inhaled nitric oxide and oxygen tension in bronchopulmonary dysplasia. Am J Resp Crit Care Med 170:1006–1013 39. The Franco-Belgium Collaborative NO Trial Group (1999) Early compared with delayed inhaled nitric oxide in moderately hypoxaemic neonates with respiratory failure: a randomised controlled trial. Lancet 354:1066–1071. 40. American Academy of Pediatrics. Committee on Fetus and Newborn (2000) Use of inhaled nitric oxide. Pediatrics 106:344–345. 41. Ellington M Jr, O'Reilly D, Allred EN, McCormick MC, Wessel DL, Kourembanas S (2001) Child health status, neurodevelopmental outcome, and parental satisfaction in a randomized, controlled trial of nitric oxide for persistent pulmonary hypertension of the newborn. Pediatrics 107:1351–1356 42. Rosenberg AA, Kennaugh JM, Moreland SG et  al (1997) Longitudinal follow-up of a cohort of newborn infants treated with inhaled nitric oxide for persistent pulmonary hypertension. J Pediatr 131:70–75 43. Rosenberg AA (2002) Outcome in term infants treated with inhaled nitric oxide. J Pediatr 140:284–287 44. Wessel DL, Adatia I, Giglia TM, Thompson JE, Kulik TJ (1993) Use of inhaled nitric oxide and acetylcholine in the evaluation of pulmonary hypertension and endothelial function after cardiopulmonary bypass. Circulation 88:2128–2138 45. Curran RD, Mavroudis C, Backer CL, Sautel M, Zales VR, Wessel DL (1995) Inhaled nitric oxide for children with congenital heart disease and pulmonary hypertension. Ann Thorac Surg 60:1765–1771 46. Russell IA, Zwass MS, Fineman JR et  al (1998) The effects of inhaled nitric oxide on postoperative pulmonary hypertension in infants and children undergoing surgical repair of congenital heart disease. Anesth Analg 87:46–51 47. Day RW, Hawkins JA, McGough EC, Crezee KL, Orsmond GS (2000) Randomized controlled study of inhaled nitric oxide after operation for congenital heart disease. Ann Thorac Surg 69:1907–1912 48. Miller OI, Tang SF, Keech A, Pigott NB, Beller E, Celermajer DS (2000) Inhaled nitric oxide and prevention of pulmonary hypertension after congenital heart surgery: a randomised double-blind study. Lancet 28(356):1464–1469 49. Bizzarro M, Gross I (2005) Inhaled nitric oxide for the postoperative management of pulmonary hypertension in infants and children with congenital heart disease. Cochrane Database Syst Rev CD005055. 50. Kinsella JP, Ivy DD, Abman SH (1994) Inhaled nitric oxide improves gas exchange and lowers pulmonary vascular resistance in severe experimental hyaline membrane disease. Pediatr Res 36:402–408

1500 51. Aikio O, Saarela T, Pokela ML, Hallman M (2003) Nitric oxide treatment and acute pulmonary inflammatory response in very premature infants with intractable respiratory failure shortly after birth. Acta Paediatr 92:65–69 52. Uga N, Ishii T, Kawase Y, Arai H, Tada H (2004) Nitric oxide inhalation therapy in very low-birthweight infants with hypoplastic lung due to oligohydramnios. Pediatr Int 46:10–14 53. Chock VY, Van Meurs KP, Hintz SR et  al (2009) Inhaled nitric oxide for preterm premature rupture of membranes, oligohydramnios, and pulmonary hypoplasia. Am J Perinatol 26:317–322 54. Williams O, Hutchings G, Debieve F, Debauche C (2009) Contemporary neonatal outcome following rupture of membranes prior to 25 weeks with prolonged oligohydramnios. Early Hum Dev 85:273–277 55. Van Meurs KP, Wright LL, Ehrenkranz RA et  al (2005) Inhaled nitric oxide for premature infants with severe respiratory failure. N Engl J Med 353:13–22 56. Kinsella JP, Parker TA, Ivy DD, Abman SH (2003) Noninvasive delivery of inhaled nitric oxide therapy for late pulmonary hypertension in newborn infants with congenital diaphragmatic hernia. J Pediatr 142:397–401 57. Banks BA, Seri I, Ischiropoulos H, Merrill J, Rychik J, Ballard RA (1999) Changes in oxygenation with inhaled nitric oxide in severe bronchopulmonary dysplasia. Pediatrics 103:610–618 58. Channick RN, Newhart JW, Johnson FW et  al (1996) Pulsed delivery of inhaled nitric oxide to patients with primary pulmonary hypertension: an ambulatory delivery system and initial clinical tests. Chest 109:1545–1549 59. Dobyns EL, Cornfield DN, Anas NG et al (1999) Multicenter randomized controlled trial of the effects of inhaled nitric oxide therapy on gas exchange in children with acute hypoxemic respiratory failure. J Pediatr 134:406–412 60. Weiner DL, Hibberd PL, Betit P, Cooper AB, Botelho CA, Brugnara C (2003) Preliminary assessment of inhaled nitric oxide for acute vaso-occlusive crisis in pediatric patients with sickle cell disease. JAMA 289:1136–1142 61. Coalson JJ, Winter VT, Siler-Khodr T, Yoder BA (1999) Neonatal chronic lung disease in extremely immature baboons. Am J Respir Crit Care Med 160:1333–1346 62. Coalson JJ (1999) Pathology of chronic lung disease of early infancy. In: Bland RD, Coalson JJ (eds) Chronic lung disease in early infancy. Dekker, New York, pp 85–124 63. Jobe AJ (1999) The new BPD: an arrest of lung development. Pediatr Res 46:641–643 64. Jobe AH, Bancalari E (2001) Bronchopulmonary dysplasia. Am J Respir Crit Care Med 163:1723–1729 65. Thébaud B, Abman SH (2007) Bronchopulmonary dysplasia: where have all the vessels gone? Roles of angiogenic growth factors in chronic lung disease. Am J Respir Crit Care Med 175:978–985 66. Tang JR, Markham NE, Lin YJ et  al (2004) Inhaled nitric oxide attenuates pulmonary hypertension and improves lung growth in

J.L. Aschner et al. infant rats after neonatal treatment with a VEGF receptor inhibitor. Am J Physiol Lung Cell Mol Physiol 287:L344–L351 67. Lin YJ, Markham NE, Balasubramaniam V et  al (2005) Inhaled nitric oxide enhances distal lung growth after exposure to hyperoxia in neonatal rats. Pediatr Res 58:22–29 68. Balasubramaniam V, Maxey AM, Morgan DB, Markham NE, Abman SH (2006) Inhaled NO restores lung structure in eNOSdeficient mice recovering from neonatal hypoxia. Am J Physiol Lung Cell Mol Physiol 291:L119–L127 69. Thébaud B, Ladha F, Michelakis ED et al (2005) Vascular endothelial growth factor gene therapy increases survival, promotes lung angiogenesis, and prevents alveolar damage in hyperoxia-induced lung injury: evidence that angiogenesis participates in alveolarization. Circulation 112:2477–2486 70. Bland RD, Albertine KH, Carlton DP, MacRitchie AJ (2005) Inhaled nitric oxide effects on lung structure and function in chronically ventilated preterm lambs. Am J Respir Crit Care Med 172:899–906 71. McCurnin DC, Pierce RA, Chang LY et  al (2005) Inhaled NO improves early pulmonary function and modifies lung growth and elastin deposition in a baboon model of neonatal chronic lung disease. Am J Physiol Lung Cell Mol Physiol 288:L450–L459 72. Schreiber MD, Gin-Mestan K, Marks JD, Huo D, Lee G, Srisuparp P (2003) Inhaled nitric oxide in premature infants with the respiratory distress syndrome. N Engl J Med 349:2099–2107 73. Kinsella JP, Cutter GR, Walsh WF et al (2006) Early inhaled nitric oxide therapy in premature newborns with respiratory failure. N Engl J Med 355:354–364 74. Ballard RA, Truog WE, Cnaan A et al (2006) Inhaled nitric oxide in preterm infants undergoing mechanical ventilation. N Engl J Med 355:343–353 75. Mourani PM, Sontag MK, Ivy DD, Abman SH (2009) Effects of long-term sildenafil treatment for pulmonary hypertension in infants with chronic lung disease. J Pediatr 154:379–384 76. Mercier JC, Hummler H, Durrmeyer X, et al (2010) Inhaled nitric oxide for prevention of bronchopulmonary dysplasia in premature babies (EUNO): a randomised controlled trial. Lancet 376:346–354 77. Mestan KK, Marks JD, Hecox K, Huo D, Schreiber MD (2005) Neurodevelopmental outcomes of premature infants treated with inhaled nitric oxide. N Engl J Med 353:23–32 78. Hibbs AM, Walsh MC, Martin RJ et al (2008) One-year respiratory outcomes of preterm infants enrolled in the Nitric Oxide (to prevent) Chronic Lung Disease trial. J Pediatr 153:525–529 79. Keller RL, Walsh M, Vittinghoff E, Palermo L, Ballard PL, Ballard RA (2009) Response to inhaled nitric oxide (iNO) and neurodevelopmental impairment (NDI) in NO CLD. E-PAS2009:2155.7. 80. Barrington KJ, Finer NN (2007) Inhaled nitric oxide for preterm infants: a systematic review. Pediatrics 120:1088–1099 81. Ivy DD, Parker D, Doran A, Kinsella JP, Abman SH (2003) Acute hemodynamic effects and home therapy using a novel pulsed nasal nitric oxide delivery system in children and young adults with pulmonary hypertension. Am J Cardiol 92:886–890

Chapter 108

The Serotonin System as a Therapeutic Target in Pulmonary Hypertension Serge Adnot

Abstract  The effects of serotonin (5-hydroxytryptamine, 5-HT) on the pulmonary circulation attracted the interest of researchers when an increased risk of idiopathic pulmonary arterial hypertension (IPAH) was reported in patients who had used appetite suppressants that interact with 5-HT. An association between the anorexigen aminorex and IPAH was described in the 1960s. In the 1980s, fenfluramine use was shown to be associated with an epidemic of IPAH in France and Belgium. The 5-HT hypothesis received support from the observation that pulmonary hypertension (PH) develops spontaneously in fawn-hooded rats, which have an inherited platelet-storage defect. Early studies focused on circulating 5-HT and its potential effects on the pulmonary vascular bed. They showed that patients with IPAH had increased circulating 5-HT levels even after heart–lung transplantation. In addition to its vasoactive effects, 5-HT exerts mitogenic and comitogenic effects on pulmonary artery smooth muscle cells (PASMCs). In contrast to the constricting action of 5-HT on smooth muscle cells, which is mainly mediated by 5-HT receptors (5-HT1B/D, 5-HT2A, and 5-HT2B), the mitogenic and comitogenic effects of 5-HT require the internalization of indoleamine by the 5-HT transporter (5-HTT). Accordingly, drugs that competitively inhibit 5-HTT also block the mitogenic effects of 5-HT on smooth muscle cells. The appetite suppressants fenfluramine, d-fenfluramine, and aminorex differ from selective 5-HTT inhibitors in that they not only inhibit 5-HT reuptake, but also trigger indoleamine release and interact with 5-HTT and 5-HT receptors in a specific manner. Over the last few years, the role of the 5-HT pathway in the pathogenesis of PH has been extensively investigated, and severe alterations in several critical steps of this pathway have been found during the progression of PH. These alterations include changes in 5-HT synthesis and bioavailability, changes in 5-HT-mediating effects on target cells (PASMCs and fibroblasts), and alterations in intracellular signaling. These critical steps represent targets for future therapies and will be discussed individually. S. Adnot (*) Department of Physiology, Henri Mondor Hospital, Avenue du Marechal de Lattre de Tassigny, 94010 Créteil, France e-mail: [email protected]

Keywords  5-hydroxytryptamine • Serotonin receptor and transporter • Anorexigens • Fenfluramine • Aminorex • Serotonin uptake

1  Introduction The effects of serotonin (5-hydroxytryptamine, 5-HT) on the pulmonary circulation attracted the interest of researchers when an increased risk of idiopathic pulmonary arterial hypertension (IPAH), previously referred to as primary pulmonary hypertension, was reported in patients who had used appetite suppressants that interact with 5-HT [1]. An association between the anorexigen aminorex and IPAH was described in the 1960s. In the 1980s, fenfluramine use was shown to be associated with an epidemic of IPAH in France and Belgium [1]. The 5-HT hypothesis received support from the observation that pulmonary hypertension (PH) develops spontaneously in fawn-hooded rats, which have an inherited platelet-storage defect [2]. Early studies focused on circulating 5-HT and its potential effects on the pulmonary vascular bed. They showed that patients with IPAH had increased circulating 5-HT levels even after heart–lung transplantation [3]. In addition to its vasoactive effects, 5-HT exerts mitogenic and comitogenic effects on pulmonary artery smooth muscle cells (PASMCs). In contrast to the constricting action of 5-HT on smooth muscle cells, which is mainly mediated by 5-HT receptors (5-HT1B/D, 5-HT2A, and 5-HT2B) [2], the mitogenic and comitogenic effects of 5-HT require the internalization of indoleamine by the 5-HT transporter (5-HTT) [4, 5]. Accordingly, drugs that competitively inhibit 5-HTT also block the mitogenic effects of 5-HT on smooth muscle cells [6]. The appetite suppressants fenfluramine, d-fenfluramine, and aminorex differ from selective 5-HTT inhibitors in that they not only inhibit 5-HT reuptake, but also trigger indoleamine release and interact with 5-HTT and 5-HT receptors in a specific manner [6, 7] (Fig. 1). Over the last few years, the role of the 5-HT pathway in the pathogenesis of PH has been extensively investigated,

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_108, © Springer Science+Business Media, LLC 2011

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Fig. 1  Besides serotonin (5-HT), platelets contain three serotonergic components: the 5-HT2A receptor, the plasma membrane 5-HT-reuptake transporter (5-HTT), and vesicular monoamine transporter 2 (VMAT2). Intact platelets take up 5-HT by 5-HTT and store the monoamine in vesicles by VMAT2. Fenfluramine is 5-HTT-dependently transported into the cytoplasm and reverses the VMAT2 transport, increasing the cytoplasmic 5-HT concentration to levels that reverse the 5-HTT activity, resulting in a continuous slow release of 5-HT. In contrast, selective inhibitors of 5-HTT block the uptake of 5-HTT activity without affecting VMAT2 activity and, therefore, do not favor the release of 5-HT from platelet stores. Activation of 5-HT2A receptors potentiates the aggregation response to various platelet activators

and severe alterations in several critical steps of this pathway have been found during the progression of PH. These alterations include changes in 5-HT synthesis and bioavailability, changes in 5-HT-mediating effects on target cells (PASMCs and fibroblasts), and alterations in intracellular signaling. These critical steps represent targets for future therapies and will be discussed individually.

2 Role for 5-HT Synthesis in Pulmonary Hypertension: Is 5-HT Synthesis a Pharmacological Target in Pulmonary Hypertension? 5-HT production outside the central nervous system occurs chiefly in the enterochromaffin cells, from which indoleamine is released into the bloodstream before being taken up by circulating platelets. Thus, one hypothesis is that only 5-HT released from platelets acts on pulmonary vessels and that lung 5-HT bioavailability is independent from the rate of 5-HT synthesis (Fig. 1). However, in recent studies, we showed that 5-HT was also produced in the human lung by pulmonary endothelial cells [8]. The 5-HT synthesis rate was increased in endothelial cells from patients with IPAH, supporting a contribution to pulmonary vascular remodeling of increased 5-HT availability in the immediate vicinity of PASMCs.

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The functional impact of variations in 5-HT synthesis on PH development was recently examined in animal studies [9, 10]. The critical step in 5-HT biosynthesis is catalyzed by the rate-limiting enzyme tryptophan hydroxylase (Tph). Tph activity therefore serves as a marker for 5-HT synthesis. Tph, which was initially thought to derive from a single gene, exists as two isoforms, Tph1 and Tph2, encoded by separate genes. Tph1 is expressed mainly in the gut and pineal gland [11]. The recently identified Tph2 gene appears exclusively expressed in neurons and is known as the neuronal isoform [12]. Thus, 5-HT synthesis is thought to be controlled chiefly by Tph2 in the central nervous system and Tph1 in peripheral organs. As mentioned above, peripherally produced 5-HT is synthesized chiefly by enterochromaffin cells in the gut, but small amounts of 5-HT may also be formed at other sites, including endothelial cells in the systemic and pulmonary arteries [8, 13]. Human pulmonary microvascular endothelial cells produce 5-HT and express Tph1, and both 5-HT synthesis and Tph1 expression are increased in cells from patients with IPAH compared with cells from controls [8] (Fig. 2). In recent studies, we also found that the severity of hypoxic PH in mice was directly linked to the rate of 5-HT synthesis catalyzed by the Tph enzyme isoforms Tph1 and Tph2 [9, 10]. In mice lacking the Tph1 gene and therefore exhibiting marked reductions in 5-HT synthesis rates and contents in peripheral organs, hypoxic PH was far less severe than in wild-type mice. Treatment with the Tph inhibitor p-chlorophenylalanine further reduced PH severity in Tph1−/− mice, normalizing right ventricular systolic pressure and pulmonary artery muscularization, in keeping with persistence of some peripheral Tph activity in Tph1−/− mice [10]. Evidence for involvement of Tph2 was obtained in mice harboring a polymorphism of the Tph2 gene and showing differences in brain 5-HT synthesis rates according to their genotype. These animals also showed differences in the severity of hypoxic PH, which were abolished by p-chlorophenylalanine treatment, indicating a contribution for neuronal Tph2 activity to the severity of hypoxic PH. In addition, hypoxia was found to increase 5-HT synthesis in the forebrain and lung, although no changes were noted in either Tph1 or Tph2 messenger RNA (mRNA) levels. Although mRNA levels are not necessarily reflective of protein expression, this finding suggests that the hypoxia-induced increase in 5-HT synthesis may result from enhanced enzyme activity as opposed to increased gene expression. Hypoxia not only increases 5-HT synthesis, but also markedly diminishes lung 5-HT contents and blood 5-HT levels, a combination suggesting a sharp increase in the indoleamine turnover rate during hypoxia exposure and PH development [10]. Dysregulation of 5-HT synthesis in vivo therefore appears closely linked to the PH phenotype in mice. Moreover, variations in the rate of 5-HT synthesis seem to directly affect the

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Fig. 2  Cross talk between pulmonary artery endothelial cells (ECs) and smooth muscle cells (SMCs) in pulmonary hypertension. Human pulmonary ECs express tryptophan hydroxylase 1 (TPH1), which is the key enzyme controlling 5-HT synthesis. Both TPH1 expression and 5-HT production are increased in pulmonary ECs from patients with idiopathic pulmonary arterial hypertension. Pulmonary artery SMCs express 5-HTT and 5-HT1B, 5-HT2A, and 5-HT2B receptors. Internalization of 5-HT through 5-HTT is responsible for the mitogenic action of 5-HT on pulmonary artery SMCs

extent of pulmonary vascular remodeling, and, therefore, the severity of PH. Taken together with the observation that both 5-HT synthesis and Tph1 expression are increased in endothelial cells from patients with IPAH compared with cells from controls, these results suggest that Tph1 and possibly Tph2 may deserve consideration as candidate genes for human PH. However, targeting Tph activity does not seem to hold promise for treating human PH, as unacceptable side effects might occur.

3 5-HT-Mediated Effects on Target Cells: The 5-HTT and 5-HT Receptors as Potential Therapeutic Targets in Pulmonary Hypertension 3.1 Role of 5-HTT in Pulmonary Hypertension Accumulating evidence suggests that 5-HTT in the lung may be a key determinant of pulmonary vessel remodeling because of its effects on PASMC growth. 5-HTT is abundantly expressed in the lung, where it is predominantly located on PASMCs [14, 15] (Fig.  3). It is encoded by a single gene expressed in several cell types, such as neurons, platelets, and pulmonary vascular endothelial and smooth muscle cells. The level of 5-HTT expression in humans seems considerably higher in the lung than in the brain, suggesting that altered 5-HTT expression may have direct effects on PASMC function. The requirement for 5-HTT as a mediator for the mitogenic activity of 5-HT appears specific of PASMCs, since no such effect has been reported with other smooth muscle cell types. Direct evidence that 5-HTT plays a key

role in pulmonary vascular remodeling was recently provided by studies showing that mice with targeted 5-HTT gene disruption developed less severe hypoxic PH than did wild-type controls and that selective 5-HTT inhibitors attenuated hypoxia- and monocrotaline-induced PH [17, 18]. Conversely, increased 5-HTT expression was associated with increased severity of hypoxic PH [19]. To investigate whether 5-HTT overexpression in PASMCs was sufficient to produce PH, we generated transgenic mice overexpressing 5-HTT under the control of the SM22 promoter. We found that these transgenic mice with selective 5-HTT overexpression in smooth muscle cells spontaneously developed PH [18]. Importantly, PH in these SM22–5-HTT+ mice developed without any alterations in 5-HT bioavailability and, therefore, as a consequence solely of 5-HTT protein overexpression in smooth muscle cells. Taken together, these observations suggest a close correlation between 5-HTT expression and/or activity and the extent of pulmonary vascular remodeling during experimental PH.

3.1.1 Overexpression of 5-HTT and PASMC Hyperplasia in IPAH Evidence that 5-HTT plays an important role in the pathogenesis of human IPAH is now clearly established. 5-HTT expression was shown to be increased in platelets and lungs from patients with IPAH, where it predominated in the media of thickened pulmonary arteries and in onion-bulb lesions [15] (Fig. 3). Interestingly, the higher levels of 5-HTT protein and activity persisted in cultured smooth muscle cells from pulmonary arteries of patients with IPAH, compared with cells from controls [15]. Moreover, PASMCs from patients with IPAH grew faster than PASMCs from controls when stimulated by 5-HT or serum, as a consequence of

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Fig. 3  Immunolocalization of 5-HTT and 5-HT1B, 5-HT2A, and 5-HT2B receptors in lung sections from controls and patients with associated pulmonary arterial hypertension or idiopathic pulmonary arterial hypertension. 5-HTT and 5-HT2B immunoreactivities are located mainly in the

SMCs, but some immunostaining is also associated with pulmonary artery ECs. 5-HT1B and 5-HT2A immunoreactivity is mainly confined to SMCs of pulmonary arteries in both controls and patients with pulmonary hypertension. Scale bar 100 mm. (Modified from Marcos et al. [16])

increased expression of 5-HTT [15]. In the presence of 5-HTT inhibitors, the growth-stimulating effects of serum and 5-HT were markedly reduced, and the difference between growth of PASMCs from patients and controls was abolished. The proliferative response of PASMCs to various growth factors such as platelet-derived growth factor, epidermal growth factor, transforming growth factor b, acidic fibroblast growth factor, and insulin-like growth factor did not differ between patients with IPAH and controls [15]. It follows that 5-HTT overexpression and/or activity in PASMCs from patients with IPAH is responsible for the increased mitogenic response to 5-HT and to serum (which contains micromolar concentrations of 5-HT).

3.1.2 5-HTT Gene Polymorphism and Increased 5-HTT Expression in Pulmonary Arteries from Patients with IPAH That 5-HTT expression is genetically controlled has been convincingly demonstrated: a polymorphism in the promoter region of the human 5-HTT gene alters the level of transcription [20]. This polymorphism consists of two common alleles, a 44-bp insertion or deletion, designated the L and S allele, respectively. The L allele drives a two- to threefold higher level of 5-HTT gene transcription than does the S allele. In studies of PASMCs from controls, we found that cells from LL individuals had a twofold increase in 5-HTT mRNA

108  The Serotonin System as a Therapeutic Target in Pulmonary Hypertension

expression, compared with cells from SS subjects, and that LS individuals had an intermediate level of expression [15] (Fig. 3). Accordingly, the growth-stimulating effects of 5-HT or serum were more marked in LL cells than in LS or SS cells, indicating that the ability of PASMCs to proliferate in response to 5-HT or serum was directly linked to the functional polymorphism of the 5-HTT gene promoter. While studying a population of 89 patients with IPAH, we found that the L allelic variant of the 5-HTT gene promoter, associated with 5-HTT overexpression and increased PASMC growth, was present homozygously in 65% of patients and 27% of controls [15]. This finding suggested that the 5-HTT gene polymorphism might either convey susceptibility to PH or act as an important modifier of the PH phenotype. However, recent studies investigating larger populations of patients with IPAH, associated forms of pulmonary arterial hypertension (APAH), or familial pulmonary arterial hypertension did not confirm our previous results [21, 22]. This apparent discrepancy might be related to specific characteristics of our cohort, which included a relatively high percentage of lung-transplant recipients. Because patients referred for lung transplantation are selected on the basis of faster disease progression or poor responsiveness to conventional treatment, this may have introduced a bias in our cohort. Support for this possibility comes from our unpublished observation that among 50 patients with PH who underwent lung transplantation between 1998 and 2005 at the Marie Lannelongue Hospital (Le Plessis Robinson, France), 57% had the LL genotype [23]. Moreover, we previously reported a high frequency of the LL genotype among lung-transplant recipients with APAH [16]. Thus, one possible explanation for the differences between these studies may be related to differences in the disease progression profiles of the study populations. Therefore, the LL genotype may not deserve to be considered a susceptibility factor for the development of IPAH. The possibility that it may affect disease progression remains to be evaluated in a larger population in which transplantation and death are censored.

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support an association between maternal exposure to 5-HTT inhibitors and persistent PH in the newborn in the offspring, the underlying mechanism probably involves inhibition of lung development, as opposed to direct effects of the drugs on the pulmonary vasculature. Presently, the effects of 5-HTT inhibitors on the progression of human PH have not been assessed in a controlled study using appropriate drug doses. These drugs, however, have occasionally been given to patients with IPAH, in the usual recommended doses, with no major effects detectable in individual patients.

3.2 Role of the 5-HT Receptors in PH On release from platelets or nerve endings, 5-HT can activate up to 14 structurally distinct 5HT receptors, which are classified into seven families (5HT1–7). Of these, the 5HT2A, 5HT2B, and 5HT1B receptors have been investigated with respect to PH (Fig. 3).

3.2.1 5HT2A Receptor In most nonhuman mammals, the 5HT2A receptor mediates vasoconstriction in both the systemic and the pulmonary circulation. For example, 5HT2A-receptor-mediated vasoconstriction occurs in the mouse and rat lung under control conditions [25, 26]. The 5HT2A receptor antagonist ketanserin has proved clinically effective in the treatment of systemic hypertension, especially in the elderly [27]. Thus, ketanserin does not act specifically on the pulmonary circulation, and its systemic effects limit its use in both IPAH and secondary PH, where it fails to significantly improve pulmonary hemodynamics [27].

3.2.2 5HT2B Receptor 3.1.3 Potential Usefulness of 5-HTT Inhibitors in the Treatment of Patients with Pulmonary Arterial Hypertension 5-HTT inhibitors in the doses commonly used in depression may induce therapeutic effects in patients with IPAH. Inhibition of in vitro smooth muscle cell proliferation occurs with micromolar concentrations, which are at the lower end of the plasma level range obtained with the usual doses of these drugs. Increasing the dose within the authorized range would produce effective levels in the pulmonary circulation. 5-HTT inhibitors are considered safe, despite recent evidence of an increased risk of persistent PH in neonates born to mothers treated during pregnancy [24]. Although the data

Hypoxia does not induce PH in 5HT2B-receptor-knockout mice [28], and this receptor may control 5-HT plasma levels in mice [29]. However, caution is in order when considering blockade of this receptor as a treatment for PH. Indeed, the 5HT2B receptor may mediate pulmonary artery vasodilation [29], and loss of 5HT2B receptor function may predispose to fenfluramine-associated PH in humans [30].

3.2.3 5HT1B Receptor However, the 5HT1B receptor mediates constriction of human pulmonary arteries [26, 31]. There is also evidence supporting a role for the 5HT1B receptor in PH development [25, 32]:

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(1) Inhibition of 5HT1B activity, either by genetic knockout or by antagonism, reduces hypoxia-induced pulmonary vascular remodeling [25]. (2) 5-HT is required for the proliferative effect of the calcium-binding protein S100A4/Mts1, and the 5HT1B receptor and 5-HTT are codependent in regulating S100A4/Mts1-induced human PASMC proliferation [33]. (3) Cooperation occurs between the 5-HT1B receptor and 5-HTT in mediating pulmonary vascular contraction [34]. (4) The level of 5HT1B receptor is increased in mice overexpressing human 5-HTT and in the fawn-hooded rat, which also exhibits increased 5-HTT expression [34]; both models are particularly susceptible to hypoxia-induced pulmonary vascular remodeling. (5) The 5HT1B receptor and 5-HTT act cooperatively to facilitate 5-HT-induced proliferation and Rho kinase induced nuclear translocation of extracellular-regulated kinase (ERK) 1/ERK2 in PASMCs [35]. (6) The 5HT1B receptor plays a role in PH development in a pig model [36]. (7) Finally, remodeled pulmonary arteries from patients with PH overexpress the 5HT1B receptor [28]. These data suggest that 5-HT-induced activation of the 5HT1B receptor may be involved in pulmonary vascular remodeling and that cooperation exists between the 5-HT1B receptor and 5-HTT. As 5HT1B-receptor-mediated changes are specific of the pulmonary circulation, this receptor is an attractive therapeutic target for PH.

4 5-HT Signaling in PH As mentioned already, 5-HT transport within the cell via 5-HTT is involved in the proliferation of PASMCs and fibroblasts. How 5-HT internalization is linked to cell proliferation signaling remains unknown, although several intracellular signaling pathways are known to be involved. Reactive oxygen species (ROS) generation and ERK1/ERK2 activation have been suggested as potential mechanisms [37, 38]. ROS may be generated either via 5-HT breakdown by monoamine oxidase or via activation of NADPH oxidase. In bovine PASMCs, ROS may mediate the phosphorylation of ERK1/ ERK2 [38]. Phosphorylated ERK1/ERK2 may then be translocated to the nucleus, where it may increase the DNA binding of transcription factors such as GATA-4, Egr-1, and Elk-1, leading to the expression of proteins such as S1004/ Mts1 that play a major role in both human and experimental PH [33]. Most of these studies, however, did not specify the role for 5-HTT or 5-HT receptors in mediating the activation of these pathways. More recently, evidence has been provided that activation of the small G protein RhoA and its target Rho kinase may represent a major pathway involved in 5-HT-induced PASMC proliferation via 5-HTT [39]. First, it was shown that 5-HT internalized in platelets via 5-HTT was covalently linked to RhoA by

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intracellular type 2 transglutaminase, which led to constitutive RhoA activation [39]. This link was then demonstrated in vivo in pulmonary arteries of rats under hypoxic conditions, providing a connection between 5-HTT and RhoA activation [40]. Moreover, we recently provided evidence that RhoA activation by 5-HT occurred both in platelets and in PASMCs during PH progression and that this event contributed to PASMC proliferation [41]. In mice characterized by 5-HTT overexpression in smooth muscle cells and spontaneous development of PH, treatment with 5-HTT inhibitors limited both PH progression and RhoA/Rho kinase activation. Only partial limitation of PH progression, however, is observed after treatment with the Rho kinase inhibitor fasudil, suggesting that activation of the RhoA pathway may generate only part of the intracellular signals originating from internalization of 5-HT via 5-HTT. In conclusion, direct involvement of the 5-HTT/RhoA/ Rho kinase signaling pathway in 5-HT-mediated PASMC proliferation and platelet activation during PH progression identifies RhoA/Rho kinase signaling as a promising target for new treatments against PH.

5 Conclusions The data reviewed here support a crucial role for 5-HT synthesis and functional activities of 5-HT receptors and 5-HTT in the pathogenesis of pulmonary vascular remodeling. Agents targeting these molecular effectors deserve to be investigated as potential treatments for PH.

References 1. Abenhaim L, Moride Y, Brenot F et al (1996) Appetite-suppressant drugs and the risk of primary pulmonary hypertension. International Primary Pulmonary Hypertension Study Group. N Engl J Med 335:609–616 2. MacLean M, Herve P, Eddahibi S, Adnot S (2000) 5-Hydroxytryptamine and the pulmonary circulation: receptors, transporters and relevance to pulmonary arterial hypertension. Br J Pharmacol 131:161–168 3. Hervé P, Launay J-M, Scrobohaci M-L et  al (1995) Increased plasma serotonin in primary pulmonary hypertension. Am J Med 99:249–254 4. Lee SL, Wang WW, Moore BJ, Fanburg BL (1991) Dual effect of serotonin on growth of bovine pulmonary artery smooth muscle cells in culture. Circ Res 68:1362–1368 5. Eddahibi S, Fabre V, Boni C et  al (1999) Induction of serotonin transporter by hypoxia in pulmonary vascular smooth muscle cells. Relationship with the mitogenic action of serotonin. Circ Res 84:329–336 6. Eddahibi S, Adnot S (2002) Anorexigen-induced pulmonary hypertension and the serotonin (5-HT) hypothesis: lessons for the future in pathogenesis. Respir Res 3:9–13 7. Rothman RB, Ayestas MA, Dersch CM, Baumann MH (1999) Aminorex, fenfluramine, and chlorphentermine are serotonin

108  The Serotonin System as a Therapeutic Target in Pulmonary Hypertension transporter substrates. Implications for primary pulmonary hypertension. Circulation 100:869–875 8. Eddahibi S, Guignabert C, Barlier-Mur AM et al (2006) Cross talk between endothelial and smooth muscle cells in pulmonary hypertension: critical role for serotonin-induced smooth muscle hyperplasia. Circulation 113:1857–1864 9. Morecroft I, Dempsie Y, Bader M et al (2007) Effect of tryptophan hydroxylase 1 deficiency on the development of hypoxia-induced pulmonary hypertension. Hypertension 49:232–236 10. Izikki M, Hanoun N, Marcos E et al (2007) Tryptophan hydroxylase 1 knockout and tryptophan hydroxylase 2 polymorphism: effects on hypoxic pulmonary hypertension in mice. Am J Physiol Lung Cell Mol Physiol 293:L1045–L1052 11. Darmon MC, Guibert B, Leviel V, Ehret M, Maitre M, Mallet J (1988) Sequence of two mRNAs encoding active rat tryptophan hydroxylase. J Neurochem 51:312–316 12. Walther DJ, Peter JU, Bashammakh S et  al (2003) Synthesis of serotonin by a second tryptophan hydroxylase isoform. Science 299:76 13. Ni W, Geddes TJ, Priestley JR, Szasz T, Kuhn DM, Watts SW (2008) The existence of a local 5-hydroxytryptaminergic system in peripheral arteries. Br J Pharmacol 154:663–674 14. Fanburg B, Lee S-L (1997) A new role for an old molecule: serotonin as a mitogen. Am J Physiol 272:L795–L806 15. Eddahibi S, Humbert M, Fadel E et al (2001) Serotonin transporter overexpression is responsible for pulmonary artery smooth muscle hyperplasia in primary pulmonary hypertension. J Clin Invest 108:1141–1150 16. Marcos E, Fadel E, Sanchez O et  al (2004) Serotonin-induced smooth muscle hyperplasia in various forms of human pulmonary hypertension. Circ Res 94:1263–1270 17. Eddahibi S, Hanoun N, Lanfumey L et al (2000) Attenuated hypoxic pulmonary hypertension in mice lacking the 5-hydroxytryptamine transporter gene. J Clin Invest 105:1555–1562 18. Guignabert C, Raffestin B, Benferhat R et  al (2005) Serotonin transporter inhibition prevents and reverses monocrotalineinduced pulmonary hypertension in rats. Circulation 111: 2812–2819 19. MacLean MR, Deuchar GA, Hicks MN et al (2004) Overexpression of the 5-hydroxytryptamine transporter gene: effect on pulmonary hemodynamics and hypoxia-induced pulmonary hypertension. Circulation 109:2150–2155 20. Lesch KP, Wolozin BL, Estler HC, Murphy DL, Rieder P (1993) Isolation of a cDNA encoding the human brain serotonin transporter. J Neural Transm 91:67–72 21. Willers ED, Newman JH, Loyd JE et al (2006) Serotonin transporter polymorphisms in familial and idiopathic pulmonary arterial hypertension. Am J Respir Crit Care Med 173:798–802 22. Machado RD, Koehler R, Glissmeyer E et al (2006) Genetic association of the serotonin transporter in pulmonary arterial hypertension. Am J Respir Crit Care Med 173:793–797 23. Eddahibi S, Adnot S (2006) From functional to genetic studies of a candidate gene for pulmonary hypertension: any point? Am J Respir Crit Care Med 173:693–694 24. Chambers CD, Hernandez-Diaz S, Van Marter LJ et  al (2006) Selective serotonin-reuptake inhibitors and risk of persistent pulmonary hypertension of the newborn. N Engl J Med 354: 579–587

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25. Keegan A, Morecroft I, Smillie D, Hicks M, MacLean M (2001) Contribution of the 5-HT1B receptor to hypoxia-induced pulmonary hypertension. Circ Res 89:1231–1239 26. MacLean MR, Clayton RA, Templeton AG, Morecroft I (1996) Evidence for 5-HT1-like receptor-mediated vasoconstriction in human pulmonary artery. Br J Pharmacol 119:277–282 27. Frishman WH, Huberfeld S, Okin S, Wang YH, Kumar A, Shareef B (1995) Serotonin and serotonin antagonism in cardiovascular and non-cardiovascular disease. J Clin Pharmacol 35:541–572 28. Launay JM, Hervé P, Peoc’h K et al (2002) Function of the serotonin 5-hydroxytryptamine 2B receptor in pulmonary hypertension. Nat Med 8:1129–1135 29. Callebert J, Esteve JM, Hervé P et al (2006) Evidence for a control of plasma serotonin levels by 5-hydroxytryptamine2B receptors in mice. J Pharmacol Exp Ther 317:724–731 30. Blanpain C, Le Poul E, Parma J et  al (2003) Serotonin 5-HT2B receptor loss of function mutation in a patient with fenfluramineassociated primary pulmonary hypertension. Cardiovasc Res 60:518–528 31. Morecroft I, Heeley RP, Prentice HM, Kirk A, MacLean MR (1999) 5-Hydroxytryptamine receptors mediating contraction in human small muscular pulmonary arteries: importance of the 5-HT1B receptor. Br J Pharmacol 128:730–734 32. MacLean MR, Sweeney G, Baird M, McCulloch KM, Houslay M, Morecroft I (1996) 5-Hydroxytryptamine receptors mediating vasoconstriction in pulmonary arteries from control and pulmonary hypertensive rats. Br J Pharmacol 119:917–930 33. Lawrie A, Spiekerkoetter E, Martinez EC et al (2005) Interdependent serotonin transporter and receptor pathways regulate S100A4/Mts1, a gene associated with pulmonary vascular disease. Circ Res 97:227–235 34. Morecroft I, Loughlin L, Nilsen M et al (2005) Functional interactions between 5-hydroxytryptamine receptors and the serotonin transporter in pulmonary arteries. J Pharmacol Exp Ther 313: 539–548 35. Liu Y, Suzuki YJ, Day RM, Fanburg BL (2004) Rho kinase-induced nuclear translocation of ERK1/ERK2 in smooth muscle cell mitogenesis caused by serotonin. Circ Res 95:579–586 36. Rondelet B, Van Beneden R, Kerbaul F et al (2003) Expression of the serotonin 1b receptor in experimental pulmonary hypertension. Eur Respir J 22:408–412 37. Lee SL, Wang WW, Fanburg BL (1998) Superoxide as an intermediate signal for serotonin-induced mitogenesis. Free Radic Biol Med 24:855–858 38. Lee SL, Simon AR, Wang WW, Fanburg BL (2001) H2O2 signals 5-HT-induced ERK MAP kinase activation and mitogenesis of smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 281:L646–L652 39. Walther DJ, Peter JU, Winter S et al (2003) Serotonylation of small GTPases is a signal transduction pathway that triggers platelet a-granule release. Cell 115:851–862 40. Guilluy C, Rolli-Derkinderen M, Tharaux PL, Melino G, Pacaud P, Loirand G (2007) Transglutaminase-dependent RhoA activation and depletion by serotonin in vascular smooth muscle cells. J Biol Chem 282:2918–2928 41. Guilluy C, Eddahibi S, Agard C et al (2009) RhoA and Rho kinase activation in human pulmonary hypertension. Am J Respir Crit Care Med 179:1151–1158

Chapter 109

Combination Therapy for Pulmonary Arterial Hypertension Ioana R. Preston and Nicholas S. Hill

Abstract  Individual pharmacotherapies for pulmonary ­arterial hypertension (PAH) have significantly improved functional status, quality of life, and, in the case of epoprostenol (Epo), survival of PAH patients with the idiopathic form (IPAH), but these responses are usually partial and often temporary. In addition, these agents have potential adverse side effects and risks that may be dose-related. These limitations have stimulated interest in combining therapies to achieve more potent and durable therapeutic responses than with individual agents, and perhaps with less toxicity. An emerging literature in both experimental models of pulmonary hypertension (PH) and clinical settings is examining these possibilities. This chapter will discuss the rationale for and weigh the evidence supporting various combinations of agents. Although gradually accumulating, data on many of the possible combinations are deficient and our discussion will include emerging evidence as well as areas where additional study is needed. Keywords  Epoprostenol • Phosphodiesterase inhibitor • Combination use of drugs • Additive effect • Treatment of pulmonary hypertension

1  Introduction Individual pharmacotherapies for pulmonary arterial ­hypertension (PAH) have significantly improved functional status, quality of life, and, in the case of epoprostenol (Epo), survival of PAH patients with the idiopathic form (IPAH), but these responses are usually partial and often temporary. In addition, these agents have potential adverse side effects and risks that may be dose-related. These limitations have stimulated interest in combining therapies to achieve more I.R. Preston (*) Pulmonary Hypertension Center, Department of Pulmonary, Critical Care and Sleep Division, Tufts Medical Center, 800 Washington Street, 02111 Boston, MA, USA e-mail: [email protected]

potent and durable therapeutic responses than with ­individual agents, and perhaps with less toxicity. An emerging literature in both experimental models of pulmonary hypertension (PH) and clinical settings is examining these possibilities. This chapter will discuss the rationale for and weigh the evidence supporting various combinations of agents. Although gradually accumulating, data on many of the possible combinations are deficient and our discussion will include emerging evidence as well as areas where additional study is needed.

2 Rationale Currently, available pharmacotherapies for PAH that have been approved as “safe and effective” by the Food and Drug Administration (FDA) in the USA to treat PAH fall into three main therapeutic groups: prostacyclins [cyclic adenosine monophosphate (cAMP) pathway], phosphodiesterase (PDE) 5 inhibitors [nitric oxide (NO)–cyclic guanosine monophosphate (cGMP) pathway], and endothelin receptor antagonists [endothelin-1 (ET-1) pathway]. Similar to the approach with cancer chemotherapy or congestive heart failure, combining agents that work via different pathways might bring about ­additive ­benefits. Furthermore, considering that the pathogenesis­ of PAH is thought to include vasoconstriction as well as vascular remodeling, combining agents with vasodilator and antiproliferative actions has particular appeal. Conversely, these agents have dose-related toxicities that could be mitigated if agents administered at moderate doses are combined. Drugs such as calcium channel blockers and anticoagulants are commonly used to treat patients with PAH. Only a small minority of IPAH patients respond favorably to ­calcium channel blockers, but many others are treated with them for concomitant systemic hypertension or Raynaud’s phenomenon. Likewise, many patients with moderate to severe PAH are treated with warfarin purportedly to inhibit in situ thrombosis. No randomized, controlled trials have yet been ­performed to establish efficacy of either class of drugs.

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_109, © Springer Science+Business Media, LLC 2011

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Although combinations of these drugs with specific PAH drugs are quite common, data on such combinations are ­virtually nonexistent. The following discussion will focus on studies, both experimental and clinical, that have addressed the hypothesis that combinations of therapies have greater efficacy than single agents alone and it will highlight the benefits (or lack thereof) of various combinations of FDA-approved PAH agents.

I.R. Preston and N.S. Hill

Prostacyclins and PDE inhibitors make an appealing combination because, as shown in Fig. 1, they increase cAMP and cGMP levels and are likely to have additive vasodilating and

antiproliferative effects. Inhibitors of PDE3 and PDE4 ­potentiate the prostacyclin-induced increase in cAMP levels by blocking the degradation of cAMP. PDE5 selectively degrades cGMP; therefore, inhibitors of PDE5 augment intracellular cGMP levels, intensifying their vasodilator and antiproliferative effects. In addition, increases in cGMP levels may potentiate prostacyclin-induced increases in cAMP levels by inhibiting PDE3 and PDE4 via a mechanism referred to as “cross talk.” Schermuly et al. [1] have shown that combining prostacyclins with subthreshold doses of the specific PDE3/PDE4 dual inhibitor tolafentrine reduces pulmonary artery pressure (PAP) more in isolated rabbit lungs than either motapizone (PDE3-selective) or rolipram (PDE4-selective) alone. The same investigators have also shown that the PDE3 and PDE4 inhibitors zardaverine (dual-selective blocker) and zaprinast (PDE5 inhibitor) augment the pulmonary vasodilation induced by inhaled prostacyclin (Epo) in the U46619 (­thromboxane-induced) model of PH in isolated rabbit lungs [2]. These investigators also found, in another study using the same experimental model, that currently available nonspecific PDE inhibitors (theophylline, pentoxifylline, and dipyridamole) also potentiated the magnitude and

Fig. 1  The three main therapeutic pathways currently targeted in the treatment of pulmonary arterial hypertension (PAH). Plus signs show where increased administration of a substance can ameliorate pulmonary hypertension (PH), and minus signs show where inhibition might be beneficial. In the prostacyclin–cyclic adenosine monophosphate (cAMP) pathway, prostacyclin (PGI2) agonists act to stimulate cAMP production, and phosphodiesterase (PDE) 3 and PDE4 inhibitors block the degradation of cAMP, further reducing vasculature tone and inhibiting endothelial remodeling. In the NO–cyclic guanosine monophosphate (cGMP) pathway,

inhaled NO (iNO) activates soluble guanylate cyclase, stimulating cGMP, whereas PDE5 inhibitors prevent its degradation. In addition, PDE5 inhibitors act on the cAMP pathway by a “cross-talk” mechanism to inhibit PDE3 and PDE4. In the endothelin pathway, endothelin receptor antagonists either nonselectively block both ETA and ETB receptors or selectively block ETA receptors, inhibiting the vasoconstrictive and mitogenic actions of endothelin-1 (ET-1). By combining beneficial pharmacological actions in different pathways, one may achieve greater therapeutic effect. ANP atrial natriuretic peptide, BNP brain natriuretic peptide, Epo epoprostenol

3 Combinations of the Prostacyclin–cAMP and NO–cGMP Pathways 3.1  Prostacyclins with PDE Inhibitors 3.1.1 Acute Experimental Studies

109  Combination Therapy for Pulmonary Arterial Hypertension

d­ uration of prostacyclin-mediated vasodilator effects on the pulmonary circulation [3]. Combined prostacyclin and PDE inhibitors administered via the inhaled route significantly lowered the shunt fraction, indicating that combinations administered via this route can have salutary effects on gas exchange. These studies provide the experimental underpinnings for subsequent clinical studies.

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In an open-label study [4], 50 mg of sildenafil given to eight PAH patients on long-term Epo therapy acutely lowered mean PAP, cardiac output (CO), and pulmonary vascular resistance (PVR), indicating the potential for a greater hemodynamic benefit when sildenafil is added to chronic Epo therapy. An earlier study combined the dual-selective PDE3/ PDE4 inhibitor tolafentrine with iloprost in a study of 11 PAH patients, demonstrating prolongation of vasodilatation

(from 60 to 120 min) but no augmentation of the decrease in PVR compared with iloprost alone [5]. A more recent study found that the combination of PDE5 inhibition using ­sildenafil and iloprost in 30 patients with severe PH (ten with IPAH, six with connective-tissue-related PH, 13 with chronic thromboembolic PH, and one with aplasia of the left pulmonary artery) potentiated the pulmonary vasodilator response compared with iloprost alone [6] (Fig. 2). It also prolonged the duration of the vasodilator effect of iloprost to more than 3 h and augmented the increase in cardiac output, although PVR, systemic blood pressure, and heart rates were unchanged. These results suggest that less frequent dosing of iloprost may be applied in long-term treatment when combined with sildenafil. In an observational trial of 11 patients, most with IPAH, the dual-selective PDE3/PDE4 inhibitor tolafentrine was aerosolized 2 h after inhaled iloprost administration [5]. Two hours later, tolafentrine was given intravenously in seven patients and by aerosolization in five patients, followed by a second iloprost inhalation. The combination of

Fig.  2  Effects of iNO, inhaled iloprost, and increasing doses of orally administered sildenafil either alone or in combination with inhaled iloprost on pulmonary vascular resistance (PVR) over time. (a) Comparison of the effect of a low dose of sildenafil (12.5 mg) alone with inhaled NO and iloprost. (b) Comparison of the effect of a higher dose of sildenafil (50 mg) with NO and iloprost. (c) Comparison of inhaled NO and iloprost with

the combination of low-dose sildenafil (12.5 mg) and iloprost. (d) Comparison of the combination of high-dose sildenafil (50 mg) and iloprost. Notice the increased and prolonged benefit of combined therapy, as well as the improvement with the higher dose of sildenafil. Arrows indicate administration of therapy. Error bars indicate confidence intervals. (Adapted from [6] with permission from the American College of Physicians)

3.1.2 Acute Clinical Studies

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I.R. Preston and N.S. Hill

agents prolonged (60–120  min) but did not augment the magnitude of the decrease in PVR compared with iloprost alone. A Japanese study assessed the acute benefits of the orally active prostacyclin analog beraprost (40 mg) alone and in combination with sildenafil (25 mg) in six PAH patients with New York Heart Association (NYHA) class II or III [7]. The peak reduction in mean PAP was 15% lower than the baseline in the combination group, with an effect sustained for 4 h, whereas for beraprost alone the effect persisted for only 2 h. Mean systemic blood pressure was unchanged. These studies support the idea that the combination of PDE inhibitors and prostacyclins potentiates and prolongs acute vasodilator responses compared with each agent alone, suggesting that this combination is a very promising therapeutic strategy to improve hemodynamics in PAH.

3.1.3 Long-Term Experimental Studies Combinations of dual PDE3/PDE4 blockade using tolanfentine with intravenously administered iloprost [8] as well as sildenafil with orally administered beraprost [9] have reduced right ventricular systolic pressure, right ventricular hypertrophy, and pulmonary vascular remodeling more in the monocrotaline model of PH in rats than with either agent alone. In addition, administration of the combination of sildenafil and beraprost for 2 weeks beginning 28 days after monocrotaline administration when the PH is fully established reversed pulmonary hemodynamic abnormalities, right ventricular hypertrophy, and pulmonary vascular remodeling more than with either agent alone.

“rescue” therapy. When added to the inhaled iloprost, sildenafil significantly increased the 6-min walk distance (6MWD) and lowered the NYHA functional class, improvements that were sustained after 9–12 months of combination therapy [10]. Similar findings were reported when sildenafil was added to the regimen for nine PAH patients with NYHA functional class II–IV on stable doses of subcutaneously administered treprostinil [11]. More support for the idea that sildenafil could have a role as a rescue therapy for patients on prostacyclins derives from a 2-year study on 20 severe PAH patients who showed ­clinical deterioration on prostanoid therapy (eight subcutaneous, seven intravenous, and five inhaled [12]). After addition of sildenafil, 6MWD, NYHA functional class, and signs of heart failure all improved significantly at 1 and 2 years. In addition, echocardiography showed significant reductions in right ventricular end-diastolic diameter and left ventricular diastolic eccentricity index. Most notably, in a recently reported placebo-controlled randomized multicenter trial, 267 patients with PAH were given sildenafil (20, 40, or 80 mg thrice daily) for 16 weeks [13]. Significant improvements among patients receiving sildenafil compared with those receiving a placebo occurred not only in the primary end point, 6MWD (placebo-adjusted increase of 28.8 m, 95% confidence interval, 13.9–43.8 m), but also in pulmonary hemodynamics (3.8-mmHg decrease), quality of life, and clinical worsening (6% vs. 20% experiencing worsening events). These longer-term studies extend the findings of the acute studies, suggesting that the addition of sildenafil to prostacyclin is a promising therapeutic approach for PAH, and probably more effective than either agent alone.

3.1.4 Long-Term Clinical Studies Several small cohort studies suggest that the combination of sildenafil and prostacyclins is beneficial (Table 1). One study on 14 PAH patients clinically deteriorating while receiving inhaled iloprost alone suggests that sildenafil can serve as a

3.2 Prostacyclins with NO The combination of a prostacyclin (stimulator of cAMP production) and inhaled NO (iNO) (stimulator of cGMP

Table 1  Long-term combination trials of prostanoids plus phosphodiesterase 5 (PDE5) inhibitors Combination Study design Patients Dosing Results

p

Iloprost + sildenafil

Long-term OL

6MWD +90 m at 3 months

0.002

Treprostinil subcutaneously + sildenafil Prostanoids + sildenafil

Long-term OL

Treadmill time +42%

0.049

Long-term OL

  14

  20

Iloprost up to 9 times daily + sildenafil 25–50 mg tid Treprostinil 35–90 ng/kg/min + sildenafil 50 mg tid Prostanoids: intravenously, subcutaneously, or inhaled

6MWD +79 m (1 year) < 0.05 6MWD +105 m (2 years) NYHA functional class improved Epo + sildenafil paces 16-week RCT 267 Epo + sildenafil 20, 40, or 6MWD +26 m 0.0088 80 mg tid TCW delay 0.012 Epo epoprostenol, TCW time to clinical worsening, OL open label, RCT randomized controlled trial, tid three times a day, 6MWD 6-min walk distance, NYHA New York Heart Association

109  Combination Therapy for Pulmonary Arterial Hypertension

p­ roduction) should have additive benefits by intensifying the vasodilating and antiproliferative effects of both, similar to the combination of prostacyclins with sildenafil. However, few studies have examined the effects of this combination on pulmonary vascular responses. Hill and Pearl [14], using intact, anesthetized rats with established PH 4  weeks after monocrotaline injection, found that the combination of iNO and inhaled prostacyclin reduced PAP more than either agent alone. A retrospective case-control study on 24 infants and small children with PH following cardiac surgery [15] found that Epo (10 ng/kg/min intravenously) significantly reduced the likelihood of rebound side effects (5% of more decrease in oxygen saturation) after iNO withdrawal. These studies support the idea that the combination is beneficial, but because iNO is not available for long-term use, the findings are most relevant to the acute setting. As effective oral NO donors are introduced, long-term applications of this combination of agents will become more practical.

3.3 NO with PDE Inhibitors iNO has the advantages of high selectivity for pulmonary vessels, lack of systemic side effects owing to its immediate inactivation when combined with hemoglobin, and improvement of oxygenation via better ventilation/perfusion ­matching. Combining iNO with PDE5 inhibitors has particular­ appeal because one agent stimulates cGMP ­production (iNO), whereas the other slows its degradation (sildenafil), thus potentially augmenting and prolonging ­pulmonary vasodilatation (Fig.  3). Inhibitors of PDE3 and PDE4 would also be expected to have additive effects with iNO via their potentiation of the cAMP pathway. Because of the cumbersomeness and expense of iNO as well as lack of insurance coverage, no clinical data are available for longterm combinations of PDE inhibitors and iNO.

3.3.1 Acute Experimental Studies In anesthetized rats, the combination of the PDE5 inhibitor zaprinast and iNO abolished hypoxic pulmonary vasoconstriction, whereas each agent alone caused significantly less blunting [16]. In another study on fetal sheep, zaprinast had no effect on pulmonary hemodynamic responses to dissolved NO [17]. However, differences in age, species, and experimental techniques likely influenced these findings. Other PDE inhibitors also have additive effects when combined acutely with iNO in experimental models, including the PDE3 inhibitor milrinone [18] and the nonspecific PDE inhibitor dipyrimadole [19]. The combination of low-dose iNO with dipyridimole had additive effects on reductions in PVR and

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Fig. 3  Improvement in 6-min walk distances (6MWD) in response to iloprost and sildenafil therapy. Results were obtained at the baseline, after 3 months of inhaled iloprost therapy (Ilo 3 mon) following a mean treatment interval of 18 ± 4 months of therapy, and after clinical deterioration had occurred, repeat testing occurred (Pre-Sil). After 3, 6, and 9–12 months of combination sildenafil and inhaled iloprost (Sil–Ilo) therapy, 6MWD was measured. Plus signs with horizontal bars adjacent indicate a significant improvement in 6MWD with the addition of sildenafil to iloprost (via a Wilcoxon test with two-sided p valves). (Adapted from GombergMaitland et al. [11] with permission from Elsevier)

blunted the rebound PH seen after sudden ­withdrawal of iNO. Overall, these findings suggest that the combination of iNO with PDE inhibition allows the ­administration of a lower dose of iNO for a similar and prolonged hemodynamic effect and less risk of rebound upon iNO withdrawal.

3.3.2 Acute Clinical Studies In children with PH, iNO (20 ppm) and dipyridamole (0.6 mg/ kg) lowered PVR similarly, but iNO was more ­pulmonary selective [20]. Nearly half of the children had more than a 20% additional drop in PVR when iNO was added to dipyridimole, and in a separate group with severe PH following ­cardiac ­surgery, dipyridamole blunted the rebound rise in PAP after withdrawal of iNO. Case reports in the pediatric literature indicate that sildenafil has similar effects when combined with iNO [21, 22]. These reports are consistent with the ­findings from the animal models described earlier, but too few clinical data are available to draw firm conclusions.

3.4 Natriuretic Peptides with PDE Inhibitors Natriuretic peptides [atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and c-type natriuretic peptide]

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activate particulate guanylate cyclase via guanylate cyclase linked natriuretic peptide receptors (GC-A, GC-B), ­increasing cGMP production (Fig.  1). Thus, combining these agents with PDE inhibitors to slow cGMP degradation or potentiate the action of cAMP should have salutary effects.

3.4.1 Acute Experimental Studies Support for the notion that sildenafil and the natriuretic pathway have additive effects derives from a study of mice with a genetic deficiency of the natriuretic peptide A receptor, in which sildenafil caused less vasodilation than in wild-type mice with the receptor intact [23]. Another study on intact anesthetized rats observed additive effects of ANP and sildenafil on blunting of acute hypoxic vasoconstriction as well as on increasing cGMP levels [24]. In addition, systemic vascular resistance remained unchanged, indicating pulmonary selectivity. Another study on the combination of these two classes of drugs in perfused rabbit lungs found that urodilatin, a renally derived natriuretic peptide that stimulates guanylate cyclase, and a subthreshold dose of dipyridamole reduced mean PAP more than the natriuretic peptide alone, along with significant increases in plasma cGMP levels [25]. Unfortunately, significant reductions in systemic blood pressure were also seen with both agents, potentially limiting the utility of this combination as a therapy for PH.

3.4.2 Acute Clinical Studies In 13 adult patients with PAH, Klinger et  al. [26] infused BNP (2 mg/kg bolus, followed by 0.01 mg/kg/min infusion for 3 h) before and after a 100-mg oral dose of sildenafil. By itself, BNP had no significant hemodynamic effects, but the combination of BNP and sildenafil lowered PAP and PVR more than either drug alone. The authors concluded that the combination of BNP and a PDE5 inhibitor has favorable effects on pulmonary hemodynamics and has therapeutic potential, particularly in the acute situation. In addition, the lesser effect of the combination on systemic hemodynamics than with intravenously administered Epo might be an advantage. As of yet, no reports of long-term administration of natriuretic peptides to PAH patients have appeared, either alone or in combination with other drugs.

4 Combinations of Prostacyclins with Endothelin Receptor Antagonists This approach combines two distinct pathways that, singly, have proven favorable actions in PAH. Both pathways have

I.R. Preston and N.S. Hill

been shown to be adversely affected in PAH; prostacyclins via an imbalance in the production of the vasodilator prostaglandin prostacyclin and the vasoconstrictor prostaglandin thromboxane A2, favoring constriction [27], and endothelins, via the vasoconstrictive and mitogenic actions of the peptide ET-1, which has increased levels in the circulation and protein expression in plexiform lesions in patients with PAH [28]. In essence, the rationale is to stimulate the cAMP pathway while inhibiting the endothelin pathway, hoping that this approach will augment the beneficial effects. Because the actions of endothelin receptor antagonists are mainly long term, little attention has been paid to acute effects of this combination.

4.1 Long-Term Experimental Studies In rats with monocrotaline-induced PH, Ueno et  al. [29] showed that a combination of an orally administered prostacyclin (beraprost) with an orally administered ETA receptor antagonist (TA-0201) reduced pulmonary pressures by echocardiogram, medial wall thickening of the pulmonary arteries, right ventricular hypertrophy and the b/a ratio of myosin heavy chain messenger RNA (used as an index of the cardiac hypertrophic response) more than either agent alone. These experimental findings support the idea that this combination of agents is a promising therapeutic approach.

4.2 Long-Term Clinical Studies Earlier cohort studies yielded promising results for the combination of prostacyclins and endothelin receptor antagonists. In an open-label pilot study, Hoeper et al. [30] added the nonspecific endothelin receptor antagonist bosentan to inhaled iloprost (n = 9) or orally administered beraprost (n = 11) in patients on a stable dose of prostacyclin for at least 3  months. Maximal oxygen consumption (from 11.0 ± 2.3 to 13.8 ± 3.6 ml/kg/min), anaerobic threshold, oxygen pulse, peak systolic blood pressure, and minute ventilation/carbon dioxide production slope all increased after 3 months. The 6MWD increased by 58 ± 43 m, but this was not statistically significant. The combination was well tolerated, with only two patients developing transaminase level elevation (Table 2). Similarly, in an open-label trial, the addition of bosentan was studied in 16 PAH patients stable on either inhaled or intravenously administered iloprost, or orally administered beraprost [31]. The combination improved the average 6MWD significantly, as well as echocardiographic indices of right ventricular function (Tei). Nine patients had an improvement in functional class. Effects were sustained at over 6 months.

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Table 2  Long-term combination trials of prostanoids plus endothelin receptor antagonist Combination Study design Patients Dosing Bosentan + iloprost/ beraprost Bosentan + prostanoids

OL

  20

OL

  16

Bosentan + inhaled treprostinil

OL

  11

Subcutaneously administered treprostinil + bosentan Bosentan + Epo (BREATHE-2)

OL

  19

RCT 16 weeks

  33

Bosentan 125 mg bid +prostanoid, maximum tolerated dose Bosentan 125 mg bid + iloprost (intravenously or inhaled); or beraprost Bosentan 125 mg bid + inhaled treprostinil 30 or 45 mg qid for 12 weeks Subcutaneously administered treprostinil + bosentan if deteriorating on treprostinil (6 months) Bosentan 125 mg bid + Epo 12–16 ng/kg/min

Results

p

6MWD +45 m Exercise parameters 6MWD +42 m Tei index improved

<0.05 <0.05 <0.001 <0.001

6MWD + 67 m PAP – 10% PVR – 26% 6MWD Borg dyspnea PAP

0.01 0.041 0.052 0.001 0.02 <0.001

PVR – 36% vs. 23% 6MWD, NYHA functional class 6MWD, TCW NYHA functional class 6MWD +26 m delayed TCW 6MWD +20 m (median value)

NS NS

RCT 12 weeks   40 Bosentan 125 mg bid + iloprost NS Bosentan + iloprost (COMBI) 5 mg 6 times daily NS Bosentan + iloprost RCT 12 weeks   67 Bosentan 125 mg bid + iloprost 0.051 (STEP) 5 mg up to 6 times daily 0.022 0.0006 RCT 12 weeks 235 Bosentan 125 mg bid (165 Inhaled treprospatients), or sildenafil, any tinil + background dose (70 patients) oral therapy PAP pulmonary artery pressure, PVR pulmonary vascular resistance, Tei index echocardiographic index of right ventricular function, bid twice daily, qid four times daily, NS not significant

Another pilot study on 11 patients with symptomatic PAH despite bosentan monotherapy demonstrated significant improvements after addition of inhaled treprostinil at dosages of 30 or 45 mg four times daily [32]. After 12 weeks of therapy, 6MWD had improved by a mean of 67 m 1 h after inhalation (49  m at trough inhalation), NYHA functional class improved from III to II in nine of 11 patients and mean PAP and PVR fell by 10 and 26%, respectively. In a retrospective, single-center, open-label study [33], 38 patients with PH treated with subcutaneously administered treprostinil were followed for a mean duration of 984 ± 468 days (range, 165–1,847 days). Orally administered bosentan was added to the treprostinil regimen in 19 patients who remained in NYHA functional class III or II or had intolerable prostacyclin side effects that limited therapy. These patients experienced significant additional improvements in the PAP (p < 0.001), 6MWD (p = 0.001), and Borg dyspnea score (p = 0.020) compared with the baseline. In the first double-blind, placebo-controlled trial to study the efficacy and safety of the combination of a prostacyclin and an endothelin receptor antagonist (BREATHE-2) [34], 33 patients with NYHA class III or IV PAH were randomized 2 days after initiation of intravenous administration of Epo (2 ng/kg/min) to receive bosentan (62.5 mg orally twice daily) or a placebo (2:1 ratio). Two days later, patients had their Epo dose increased by 2  ng/kg/min with subsequent 2-week increments to a target dose of 12–16 ng/kg/min after 16 weeks. If liver functions permitted, the dosage of bosentan was also increased after the initial 4  weeks to a target

dosage of 125  mg twice daily. The combination tended to improve the primary end point (total pulmonary resistance) and other secondary hemodynamic end points, including mean PAP and PVR, but the differences were not statistically significant (Fig.  4). Furthermore, there were no significant differences between the two groups in the 6MWD (68 vs. 74 m median increase in the bosentan/Epo and placebo/Epo groups, respectively) and NYHA functional class. The combination was as well tolerated as placebo/Epo, with leg edema being the only side effect more commonly encountered in the group receiving bosentan (27% vs. 9%), and equal numbers of patients deteriorated and/or died in the two groups. The authors concluded that the combination of bosentan and Epo held promise as a therapeutic option for PAH, but because the study was underpowered, they called for larger prospective trials. The next double-blind, placebocontrolled trial to examine the combination of prostacyclins and endothelin receptor antagonists was the STEP trial (safety and pilot efficacy trial of iloprost inhalation solution in combination with bosentan for evaluation in PAH), which randomly assigned inhaled iloprost or inhaled placebo to 67 PAH patients already receiving bosentan [35]. After 12 weeks, patients in the bosentan plus iloprost group walked 26 m farther in 6 min than patients in the placebo plus bosentan group (p = 0.51). Combination therapy significantly improved other end points, including NYHA functional class (34% increased by at least one NYHA class in the combination group vs. 6% in controls), PAP, and rate of clinical worsening, and was well tolerated.

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I.R. Preston and N.S. Hill

At the trough (4 h after drug administration), it was 14 m and statistically significant. The study was not powered to analyze the subgroups of bosentan and sildenafil separately. Another randomized trial (FREEDOM-C) has compared the addition of oral administration of treprostinil to patients receiving sildenafil, an endothelin receptor antagonist, or both. Preliminary results have been reported in a press release, but not yet in a clinical abstract. Thus, although there are inconsistencies, on the whole, these data support the idea that combinations of prostacyclins and endothelin receptor antagonists have additive ­benefits for patients with PAH, but further study is necessary.

5 Combinations of Endothelin Receptor Antagonists with PDE Inhibitors

Fig. 4  Results of the BREATHE-2 study. Total pulmonary resistance (TPR) changes from the baseline are noted on the top graph (a) compared with a placebo plus Epo (n = 10) and on the bottom graph (b) with bosentan plus epoprostenol (n = 19). The isolated plus sign indicates median values for each group at the end of the 16-week study period. There was a trend for a greater reduction in mean TPR in the group receiving bosentan plus epoprostenol but the difference was not statistically significant (p = 0.08). (Adapted with permission from McLaughlin et  al. [35]. The official journal of the American Thoracic Society. Copyright American Thoracic Society)

Similar to the STEP trial, the COMBI multicenter trial randomized IPAH patients who were stable on bosentan to receive either inhaled iloprost or inhaled placebo as a second agent [36]. Assuming that iloprost would improve the 6MWD (the primary end point) to the same magnitude as if it were a monotherapy, the power analysis called for enrolling 72 patients. Unfortunately, it was terminated when an interim analysis performed after enrollment of the first 40 patients showed insufficient evidence of a benefit to justify continuation of the trial. Interestingly, as in the STEP trial, the three patients who showed significant deterioration in all objective outcomes were in the combination arm. Although no further randomized, double-blind studies specifically focused on the combination of prostacyclins and endothelin receptor antagonists have yet been published, one trial that added prostacyclin to oral therapies has been reported preliminarily. The TRIUMPH trial [37] tested the efficacy and safety of inhaled treprostinil in patients with PAH in combination with bosentan (165 patients) or sildenafil (70 patients). The median improvement in 6MWD (primary end point) 1 h after drug administration was 20 m (p < 0.0006).

The rationale for this combination is similar to that for the combination of prostacyclins and endothelin receptor antagonists; that agents working via two different pathways (the cGMP and endothelin pathways) should have additive effects. This combination is particularly attractive because the two therapies are administered orally, avoiding the inconveniences and potential dangers of infusion and inhaled therapies.

5.1 Long-Term Experimental Studies Rats injected with monocrotaline have reduced PAP, right ventricular hypertrophy, and mortality when treated with a combination of maximally effective doses of bosentan and sildenafil compared with either agent alone [38]. These findings support the idea that this combination might provide an additive benefit in clinical disease.

5.2 Long-Term Clinical Studies As of yet, relatively few clinical investigations on the combination of endothelin receptor antagonists and PDE inhibitors have been reported (Table 3). In a cohort of nine patients with severe IPAH, Hoeper et al. [39] observed a deterioration in 6MWD from 403 ± 80  m at initiation of bosentan therapy to 277 ± 80  m after 11 ± 5  months. They added sildenafil (25–50 mg three times daily) and after 3 months of combined therapy, 6MWD had increased to 392 ± 62 m (p = 0.007). This improvement was sustained throughout the remainder of the follow-up period (median of  9  months) (Fig.  5) and no adverse events, including liver function abnormalities, were observed.

109  Combination Therapy for Pulmonary Arterial Hypertension

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Table 3  Long-term trials of endothelin receptor antagonists plus PDE5 inhibitors Combination Study design Patients Dosing Bosentan + sildenafil

OL

    25

Bosentan + sildenafil

OL

    9

Bosentan 125 mg bid + sildenafil 20–100 mg tid

Bosentan 125 mg bid + sildenafil 25–50 mg tid Bosentan + sildenafil Postmarketing 4,996 Bosentan alone vs. surveillance bosentan + sildenafil VO2max maximal oxygen consumption, IPAH idiopathic pulmonary arterial hypertension

Fig.  5  The 6MWD of the nine patients with PAH. The open arrow demonstrates when bosentan therapy was started. Baseline 2 represents the average distance 11 ± 5 months later before the addition of sildenafil, during which time the 6MWD had declined. The closed arrow represents the time when sildenafil was started. The 6MWD was significantly improved at 3 months, an increase that was sustained for the 9-month follow-up period. (Adapted from [40] with permission from European Respiratory Journals Ltd.)

In another cohort of patients deteriorating on bosentan monotherapy, Mathai et  al. [40] found that in those with IPAH, 6MWD increased by an average of 47 m and five of 13 such patients improved by at least one NYHA class. In a subgroup of 12 patients with connective tissue disease associated PAH, 6MWD increased by only 7  m and only two improved their functional class. The authors concluded that the addition of sildenafil appears to benefit IPAH patients who are deteriorating on bosentan therapy, but this is not apparent in the connective tissue disease patients. The recently published EARLY trial, a randomized ­double-blind trial of bosentan in 185 class II PAH patients included 29 patients on baseline sildenafil therapy [41]. Among these patients on sildenafil therapy, bosentan lowered PVR by 20.4% (p < 0.05) and improved 6MWD by 17.3 m (p = 0.855) in the 15 patients randomized to bosentan as compared with the 14 patients randomized to the placebo. The drop in PVR raises the possibility that combining bosentan and sildenafil might be beneficial, but no large randomized trials testing this hypothesis have yet been published.

Results

p

6MWD +46 m in IPAH NYHA functional class improved in 5 of 13 IPAH patients 6MWD +115 m VO2max +3.4 ml/min/kg

0.05 NS

< 0.007 0.006

Safety reports were similar

The interaction between bosentan and sildenafil should be noted here, as well. Treatment with bosentan 62.5 mg twice daily was associated with a twofold increase in sildenafil clearance. Increasing the dosage of bosentan to 125 mg twice daily led to a further increase in sildenafil clearance, resulting in a decrease in the sildenafil plasma concentration in PAH patients by as much as 55% [42, 43]. In addition, sildenafil slowed bosentan metabolism, roughly doubling bosentan plasma levels [42]. Whether these interactions are clinically important has not been established, but the concern must be raised that when the drugs are combined, bosentan could blunt the clinical effects of sildenafil and sildenafil could potentiate not only the clinical efficacy but also the toxicity of bosentan. Some reassurance has been provided by a prospective, Internet-based, postmarketing surveillance study required by the European regulatory authorities for assessing the safety of bosentan in PAH patients [44]. Of almost 5,000 PAH patients treated with bosentan over a 30-month period, 218 received sildenafil in addition to bosentan. Those receiving the combination therapy tolerated it well and safety reports were similar to those for bosentan alone.

6 Unresolved Issues Regarding Combination Therapy 6.1 Add-On Therapy Versus Switching Studies of add-on therapy are not truly trials of combination therapy because there is no control for the baseline therapy; it cannot be known whether the results would have been just as good had the initial therapy been stopped when the new therapy was begun. A few studies have been performed to attempt the transition from infusion therapy to oral therapy and shed some light on this question of whether it is better to add on a new therapy or switch altogether. These studies were performed because many patients receiving infusion therapy are anxious to switch to an oral therapy because of the vastly greater convenience and

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t­ olerability. Suleman et  al. [45] transitioned 23 patients (WHO class II or III) stable on their current prostaglandin dose, without evidence of heart failure, and with primary or secondary PH from intravenous or subcutaneous prostacyclin therapy to, bosentan. Initially, patients were excluded if they received more than 50  ng/kg/min of prostacyclin, but that restriction was dropped as experience accrued. The transition occurred over two consecutive 4-week block periods according to the standard dosing regimen with bosentan, starting with 62.5 mg and followed by an increase to 125 mg, both twice daily, as long as liver function test results were acceptable. At the start of the second period, patients were instructed to reduce the prostacyclin infusion rate daily or every other day to taper off the medication within 4 weeks. Fifteen of the 23 patients (65%) were able to transition to orally administered bosentan, but four subsequently developed symptoms of worsening PH after another 7  weeks to 12  months, and were placed back on prostacyclin. Another two patients developed acute liver injury and were transitioned back to prostacyclin. Therefore, nine of the 23 patients (40%) were able to ­successfully transition to long-term bosentan therapy. The authors acknowledged that the study was small, nonrandomized, nonblinded, open-label, and had missing data that made statistical analysis unreliable. In a more recent multicenter trial, ten of 22 patients receiving prostacyclin infusions transitioned to orally administered bosentan, but three of the ten subsequently deteriorated and two of these died despite resumption of intravenous therapy [46]. A lower initial prostacyclin infusion rate and less severe PH at the baseline were associated with successful transitions. The 12 patients that failed the transition could not even tolerate lowering of the infusion rate. The authors concluded that although some patients can be transitioned successfully, careful patient selection and close monitoring during the transition are obligatory. In another transition study, 14 patients on long-term subcutaneous treprostinil therapy who wished to discontinue treatment because of injection-site pain were transitioned to sildenafil [47]. Ten of the 14 patients (71%), demonstrated stable NYHA class, 6MWD, and significantly improved quality-of-life measures at 3 months. Four patients discontinued the transition because of deterioration during treprostinil withdrawal, despite the introduction of sildenafil. These studies indicate that most patients on infusion prostacyclin therapy may be unable to switch entirely to an oral therapy of another class and because of the morbidity and mortality encountered in these studies, great caution is advised in attempting such therapeutic maneuvers. The problem is that even though a patient may be faring poorly and deteriorating on a given therapy, he might be even worse without the therapy. Our anecdotal experience has been that more often than not after withdrawing a therapy in such

I.R. Preston and N.S. Hill

patients doing poorly, they deteriorate even more rapidly. Although this could admittedly be a psychological reaction to the removal of a medication, our belief is that adding a medication to existing therapy is safer than and preferable to switching entirely.

6.2 Add-On Versus “Upfront” Combination Therapy Most studies on combination therapy, with the exception of the underpowered BREATHE-2 study, have examined the addition of a second agent to existing therapy (add-on ­therapy). An alternative approach would be to combine therapies upfront; equivalent to “induction” therapy frequently used in cancer treatment. Both approaches have theoretical advantages and disadvantages. In the first approach, physicians must follow their patients very closely to avoid a delay in initiating more aggressive therapy should patients not improve. Lack of improvement in functional class III or IV patients and development of class IV symptoms during treatment are associated with a very poor prognosis. In addition, it is not clear whether patients would benefit more from different class agents. On the other hand, employing the upfront combination therapy approach may expose patients to unnecessary drug–drug interactions, toxicities, and higher cost. Presently, not enough evidence is available to support one approach over the other, but since most studies have examined the add-on approach, it is the one most commonly used and most likely to be approved by insurance companies.

6.3 Goal-Directed Therapy An interesting study conducted in Europe adopted a rigorous algorithm to apply add-on therapy. In this “goal-directed” approach, Hoeper et  al. enrolled 123 consecutive PAH patients [48]. The goals of therapy were based on indicators of a favorable prognosis as determined in earlier studies and included improvement in 6MWD to over 380 m, peak systolic blood pressure above 120 mmHg during cardiopulmonary exercise testing, and a peak VO2 above 10.4 ml/min/kg. Patients were evaluated every 2–6  months. If the goals of therapy were not met, another therapy was added and the patient reassessed. Therapies were instituted in the following order: bosentan, sildenafil, inhaled iloprost, transition to intravenously administered iloprost, and urgent lung transplantation. Upon entry to the study, 98 patients were class III and 25 patients were class IV. Using this algorithm, survival was 93%, 83%, and 79.9% at 1, 2, and 3 years, respectively, an improvement compared with historical controls. It is

109  Combination Therapy for Pulmonary Arterial Hypertension

important to note that 43% of patients failed monotherapy. Sixteen percent of patients required three-drug combination therapy. Five percent failed triple therapy and were started on intravenously administered prostanoids. This systematic approach to add-on therapy holds promise but further study will be needed to address the question of which are the best end points to direct therapy.

6.4 Triple Therapy Very few trials have included patients on drugs from all three therapeutic pathways, yet such therapy is not uncommonly used. As noted earlier, 16% of patients in the goal-directed therapy trial received triple therapy and preliminary results of the REVEAL registry, the US registry of more than 3,000 PAH patients, revealed that 9% were receiving triple therapy (with another 36% receiving various double therapy ­combinations [49]). Patients are generally prescribed triple therapy in sequential add-on fashion because of an insufficient response to the initial agents, or deterioration after an initial response. Unfortunately, the efficacy of this approach is unsubstantiated and concerns must be raised regarding the potential addition of toxicities. Studies of add-on therapy suggest that the increase in 6MWD is less with the addition of one agent to another than when that agent is initiated as monotherapy. Whether the addition of a third agent yields an even smaller increase in 6MWD is unknown, but is a concern.

7 Conclusions Despite the introduction of a variety of effective pharmacotherapies for PAH in recent years, the therapeutic options remain suboptimal. Some of the therapies, such as intravenously administered Epo, are cumbersome to administer and entail potentially life-threatening complications. Others, such as subcutaneously administered treprostinil, are painful, and others, such as bosentan, incur the risk of liver ­toxicity. Accordingly, the need to transition from one therapy to another is a common occurrence. Considerable experience has accrued on certain transitions, such as from intravenously administered Epo to subcutaneously administered treprostinil or orally administered bosentan. These transitions can be accomplished safely if they are carried out with careful monitoring according to guidelines that are continually being refined as experience accrues. Even when therapies are well tolerated and effective, the response is usually partial, so interest is mounting in combination therapy. The idea is to combine therapies with different mechanisms of action that

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are likely to have additive or synergistic effects. For example, the combination of an agent that increases cAMP (prostacyclin) levels with one that increases cGMP (PDE5 inhibitor) levels may have more potent vasodilator and antiproliferative actions than either agent alone. Although studies in animal models show promising results for a variety of combinations, as of yet relatively few clinical studies have examined the efficacy of combination therapy for PAH. Nonetheless, combination therapy is likely to see increasing use as more agents become available. It is important to assess the various combinations with properly designed controlled trials so that we can determine which combinations are most beneficial.

References 1. Schermuly RT, Roehl A, Weissmann N et  al (2000) Subthreshold doses of specific phosphodiesterase type 3 and 4 inhibitors enhance the pulmonary vasodilatory response to nebulized prostacyclin with improvement in gas exchange. J Pharmacol Exp Ther 292:512–520 2. Schermuly RT, Ghofrani HA, Enke B et al (1999) Low-dose systemic phosphodiesterase inhibitors amplify the pulmonary vasodilatory response to inhaled prostacyclin in experimental pulmonary hypertension. Am J Respir Crit Care Med 160:1500–1506 3. Schermuly RT, Roehl A, Weissmann N et al (2001) Combination of nonspecific PDE inhibitors with inhaled prostacyclin in experimental pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 281:L1361–L1368 4. Kuhn KP, Wickersham NE, Robbins IM, Byrne DW (2004) Acute effects of sildenafil in patients with primary pulmonary hypertension receiving epoprostenol. Exp Lung Res 30:135–145 5. Ghofrani HA, Rose F, Schermuly RT et al (2002) Amplification of the pulmonary vasodilatory response to inhaled iloprost by subthreshold phosphodiesterase types 3 and 4 inhibition in severe pulmonary hypertension. Crit Care Med 30:2489–2492 6. Ghofrani HA, Wiedemann R, Rose F et  al (2002) Combination therapy with oral sildenafil and inhaled iloprost for severe pulmonary hypertension. Ann Intern Med 136:515–522 7. Ikeda D, Tsujino I, Ohira H et al (2005) Addition of oral sildenafil to beraprost is a safe and effective therapeutic option for patients with pulmonary hypertension. J Cardiovasc Pharmacol 45:286–289 8. Schermuly RT, Kreisselmeier KP, Ghofrani HA et  al (2004) Antiremodeling effects of iloprost and the dual-selective phosphodiesterase 3/4 inhibitor tolafentrine in chronic experimental pulmonary hypertension. Circ Res 94:1101–1108 9. Itoh T, Nagaya N, Fujii T et  al (2004) A combination of oral sildenafil­and beraprost ameliorates pulmonary hypertension in rats. Am J Respir Crit Care Med 169:34–38 10. Ghofrani HA, Rose F, Schermuly RT et al (2003) Oral sildenafil as long-term adjunct therapy to inhaled iloprost in severe pulmonary arterial hypertension. J Am Coll Cardiol 42:158–164 11. Gomberg-Maitland M, McLaughlin V, Gulati M, Rich S (2005) Efficacy and safety of sildenafil added to treprostinil in pulmonary hypertension. Am J Cardiol 96:1334–1336 12. Ruiz MJ, Escribano P, Delgado JF et al (2006) Efficacy of sildenafil as a rescue therapy for patients with severe pulmonary arterial hypertension and given long-term treatment with prostanoids: 2-year experience. J Heart Lung Transplant 25:1353–1357 13. Simonneau G, Rubin LJ, Galiè N et al (2008) Addition of sildenafil to long-term intravenous epoprostenol therapy in patients with pulmonary arterial hypertension: a randomized trial. Ann Intern Med 149:521–530

1520 14. Hill L, Pearl R (1999) Combined inhaled nitric oxide and inhaled prostacyclin during experimental chronic pulmonary hypertension. J Appl Physiol 86:1160–1164 15. Hermon M, Golej J, Burda G, Marx M, Trittenwein G, Pollak A (1999) Intravenous prostacyclin mitigates inhaled nitric oxide rebound effect: a case control study. Artif Organs 23:975–978 16. Nagamine J, Hill LL, Pearl R (2000) Combined therapy with zaprinast and inhaled nitric oxide abolishes hypoxic pulmonary hypertension. Crit Care Med 28:2420–2424 17. Skimming J, DeMarco V, Kadowitz P, Cassin S (1996) Effects of zaprinast and dissolved nitric oxide on the pulmonary circulation of fetal sheep. Pediatr Res 39:223–228 18. Deb B, Bradford K, Pearl R (2000) Additive effects of inhaled nitric oxide and intravenous milrinone in experimental pulmonary hypertension. Crit Care Med 28:795–799 19. Foubert L, De Wolf D, Mareels K et al (2002) Intravenous dipyridamole enhances the effects of inhaled nitric oxide and prevents rebound pulmonary hypertension in piglets. Pediatr Res 52:730–736 20. Ivy D, Ziegler J, Kinsella J, Wiggins J, Abman S (1998) Hemodynamic effects of dipyridamole and inhaled nitric oxide in pediatric patients with pulmonary hypertension. Chest 141:17S 21. Atz A, Lefler A, Fairbrother D, Uber W, Bradley S (2002) Sildenafil augments the effect of inhaled nitric oxide for postoperative pulmonary hypertensive crises. J Thorac Cardiovasc Surg 124:628–629 22. Mychaskiw G, Sachdev V, Heath BJ (2001) Sildenafil (viagra) facilitates weaning of inhaled nitric oxide following placement of a biventricular-assist device. J Clin Anesth 13:218–220 23. Zhao L, Mason NA, Strange JW, Walker H, Wilkins MR (2003) Beneficial effects of phosphodiesterase 5 inhibition in pulmonary hypertension are influenced by natriuretic peptide activity. Circulation 107:234–237 24. Preston IR, Hill NS, Gambardella LS, Warburton RR, Klinger JR (2004) Synergistic effects of ANP and sildenafil on cGMP levels and amelioration of acute hypoxic pulmonary hypertension. Exp Biol Med 229:920–925 25. Schermuly RT, Weissmann N, Enke B et  al (2001) Urodilatin, a natriuretic peptide stimulating particulate guanylate cyclase, and the phosphodiesterase 5 inhibitor dipyridamole attenuate experimental pulmonary hypertension: synergism upon coapplication. Am J Respir Cell Mol Biol 25:219–225 26. Klinger JR, Houtchens J, Thaker S, Hill NS, Farber H (2004) Acute cardiopulmonary hemodynamic effects of brain natriuretic peptide in patients with pulmonary arterial hypertension. Am J Respir Crit Care Med 169:A170 27. Christman BW, McPherson CD, Newman JH et  al (1992) An imbalance between the excretion of thromboxane and prostacyclin metabolites in pulmonary hypertension. N Engl J Med 327:70–75 28. Giaid A, Yanagisawa M, Langleben D et al (1993) Expression of endothelin-1 in the lungs of patients with pulmonary hypertension. N Engl J Med 328:1732–1739 29. Ueno M, Miyauchi T, Sakai S, Yamauchi-Kohno R, Goto K, Yamaguchi I (2002) A combination of oral endothelin-A receptor antagonist and oral prostacyclin analogue is superior to each drug alone in ameliorating pulmonary hypertension in rats. J Am Coll Cardiol 40:175–181 30. Hoeper MM, Taha N, Bekjarova A, Gatzke R, Spiekerkoetter E (2003) Bosentan treatment in patients with primary pulmonary hypertension receiving nonparenteral prostanoids. Eur Respir J 22:330–334 31. Seyfarth HJ, Pankau H, Hammerschmidt S, Schauer J, Wirtz H, Winkler J (2005) Bosentan improves exercise tolerance and Tei

I.R. Preston and N.S. Hill index in patients with pulmonary hypertension and prostanoid ­therapy. Chest 128:709–713 32. Channick RN, Olschewski H, Seeger W, Staub T, Voswinckel R, Rubin LJ (2006) Safety and efficacy of inhaled treprostinil as addon therapy to bosentan in pulmonary arterial hypertension. J Am Coll Cardiol 48:1433–1437 33. Benza RL, Rayburn BK, Tallaj JA, Pamboukian SV, Bourge RC (2008) Treprostinil-based therapy in the treatment of moderate-tosevere pulmonary arterial hypertension: long-term efficacy and combination with bosentan. Chest 134:139–145 34. Humbert M, Barst RJ, Robbins IM et  al (2004) Combination of bosentan with epoprostenol in pulmonary arterial hypertension: BREATHE-2. Eur Respir J 24:353–359 35. McLaughlin VV, Oudiz RJ, Frost A et al (2006) Randomized study of adding inhaled iloprost to existing bosentan in pulmonary arterial hypertension. Am J Respir Crit Care Med 174:1257–1263 36. Hoeper MM, Leuchte H, Halank M et al (2006) Combining inhaled iloprost with bosentan in patients with idiopathic pulmonary arterial hypertension. Eur Respir J 28:691–694 37. McLaughlin VV, Benza RL, Rubin LJ et al (2010) Addition of inhaled treprostinil to oral therapy for pulmonary arterial hypertension. J Am Cardiol Col 55:1915–1922 38. Clozel M, Hess P, Rey M, Iglarz M, Binkert C, Qiu C (2006) Bosentan, sildenafil, and their combination in the monocrotaline model of pulmonary hypertension in rats. Exp Biol Med (Maywood) 231:967–973 39. Hoeper MM, Faulenbach C, Golpon H, Winkler J, Welte T, Niedermeyer J (2004) Combination therapy with bosentan and sildenafil in idiopathic pulmonary arterial hypertension. Eur Respir J 24:1007–1010 40. Mathai SC, Girgis RE, Fisher MR et al (2007) Addition of sildenafil to bosentan monotherapy in pulmonary arterial hypertension. Eur Respir J 29:469–475 41. Galiè N, Rubin LJ, Hoeper M et  al (2008) Treatment of patients with mildly symptomatic pulmonary arterial hypertension with bosentan (EARLY study): a double-blind, randomised controlled trial. Lancet 371:2093–2100 42. Burgess G, Hoogkamer H, Collings L, Dingemanse J (2008) Mutual pharmacokinetic interactions between steady-state bosentan and sildenafil. Eur J Clin Pharmacol 64:43–50 43. Paul GA, Gibbs JS, Boobis AR, Abbas A, Wilkins MR (2005) Bosentan decreases the plasma concentration of sildenafil when coprescribed in pulmonary hypertension. Br J Clin Pharmacol 60:107–112 44. Humbert M, Segal ES, Kiely DG, Carlsen J, Schwierin B, Hoeper MM (2007) Results of European post-marketing surveillance of bosentan in pulmonary hypertension. Eur Respir J 30:338–344 45. Suleman N, Frost A (2004) Transition from epoprostenol and treprostinil to the oral endothelin receptor antagonist bosentan in patients with pulmonary hypertension. Chest 126:808–815 46. Steiner MK, Preston IR, Klinger JR et  al (2006) Conversion to bosentan from prostacyclin infusion therapy in pulmonary arterial hypertension: a pilot study. Chest 130:1471–1480 47. Keogh AM, Jabbour A, Weintraub R, Brown K, Hayward CS, Macdonald PS (2007) Safety and efficacy of transition from subcutaneous treprostinil to oral sildenafil in patients with pulmonary arterial hypertension. J Heart Lung Transplant 26:1079–1083 48. Hoeper MM, Markevych I, Spiekerkoetter E, Welte T, Niedermeyer  J (2005) Goal-oriented treatment and combination therapy for ­pulmonary arterial hypertension. Eur Respir J 26:858–863 49. McGoon MD, Barst RJ, Doyle RL, Liou TG, Miller D, Feldkircher K (2007) REVEAL registry: treatment history and treatment at baseline. Chest 132:631

Chapter 110

Thrombolytic and Anticoagulant Therapy for Pulmonary Embolism and Chronic Thromboembolic Pulmonary Hypertension Paul F. Currier and Charles A. Hales

Abstract  This chapter will focus on thrombolytic and anticoagulant therapy for pulmonary embolism (PE) and chronic thromboembolic pulmonary hypertension (CTEPH). It will examine the physiologic basis and pharmacologic aspects of various approaches and examine clinical data based on traditional, current, and emerging treatments of the diseases. Other pharmacologic treatments and surgical or procedurally based treatment for PE or CTEPH are explored elsewhere in this book. Keywords  Anticoagulation • Fibrinolysis • Thromboembolism • Platelet • Heparin

1 Introduction This chapter will focus on thrombolytic and anticoagulant therapy for pulmonary embolism (PE) and chronic thromboembolic pulmonary hypertension (CTEPH). It will examine the physiologic basis and pharmacologic aspects of various approaches and examine clinical data based on traditional, current, and emerging treatments of the diseases. Other pharmacologic treatments and surgical or procedurally based treatment for PE or CTEPH are explored elsewhere in this book.

or a systolic blood pressure less than 90 mmHg. Submassive PE refers to pulmonary emboli which are not associated with this degree of hemodynamic instability. Stratifying patients with a diagnosis of acute PE based on this classification to help guide therapy is rather crude in some respects. However, a significant portion of the literature on the treatment of acute PE uses this stratification. Therefore, although new ways of classifying patients may allow for more individualized treatment in the future, we believe this method of stratification provides at least a reasonable starting place for guiding therapy. Certainly, other clinical factors such as degree of hypoxemia, age, and comorbidities may play a significant role in decisions regarding the use of thrombolysis and the choice of anticoagulant. In patients with suspected PE, we begin treatment with anticoagulation even prior to confirmation of thrombus in the absence of contraindications. In those patients with massive PE, we favor treatment with thrombolytic therapy unless it is contraindicated followed by treatment with intravenously administered unfractionated heparin (UFH). Except in the most dire of clinical situations, thrombolysis should not be performed prior to confirmation of PE. Traditionally, UFH has been used to treat patients with submassive PE. However, data now support treatment with low molecular weight heparin (LMWH) in those patients for whom there is no contraindication.

2 Pulmonary Embolism 2.1 Principles of Treatment We favor stratifying treatment of acute PE on the basis of whether or not there is hemodynamic instability due to PE. Massive PE is defined as a PE which leads to a decrease in systolic blood pressure (BP) of 40 mmHg from the baseline P.F. Currier (*) Department of Medicine, Massachusetts General Hospital, Harvard Medical School, 55 Fruit Street, Bulfinch 148, Boston, MA 02114, USA e-mail: [email protected]

2.2 Treatment Agents 2.2.1 Thrombolytic Therapy Thrombolytic agents act to lyse clots by breaking up fibrin and fibrinogen. There are three thrombolytic agents presently approved for treatment of PE: streptokinase, urokinase, and tissue plasminogen activator (tPA). Dosing regimens for thrombolytic agents are detailed in Table .1. Streptokinase was the first thrombolytic agent reported to be used for PE, used in dogs and then in humans in 1962 [1]. It is derived from beta-hemolytic streptococci and acts to bind

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_110, © Springer Science+Business Media, LLC 2011

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P.F. Currier and C.A. Hales

Table 1  Choice of thrombolytic agents for pulmonary embolism Experimental Drug FDA-approved dosage Streptokinase

Urokinase

Tissue plasminogen activator (alteplase) Reteplase

250,000 units every 20 min 100,000 units/h every 24–72 h 4,400 units/kg bolus 4,400 units/kg/h every 12 h 100 mg every 2 h

1,500,000 units every 1 h

3,000,000 units every 2 h 0.6 mg/kg every 2–15 min

10 units twice, 30 min apart

plasminongen and activate plasmin, which lyses fibrin and fibrinogen. Because it is derived from streptococci, it has the potential to induce allergic reactions and hypotension when given. It also has a potential to be inactivated in individuals who already have circulating antibodies to streptococci [2]. Urokinase is derived from urine and also acts similarly by binding fibrinogen. It is expensive and has been rarely used for treatment of PE. tPA or alteplase is endogenously derived from endothelial cells. It binds fibrin directly, which stimulates plasminogen activation to lyse clots. Although streptokinase and urokinase have certainly demonstrated the ability to lyse clot, tPA has become the thrombolytic agent of choice for many. This is due to an ability to give the agent rapidly (over a period of 2 h), to avoid the potential allergic reactions of streptokinase, and on the basis of data in the use of tPA in myocardial infarction suggesting improved mortality when compared with streptokinase [3]. Given the potential for thrombolytic therapy to cause significant bleeding, an awareness of contraindications to treatment with these agents is important. The clearest contraindication to thrombolytic therapy is intracranial hemorrhage. Uncontrolled hypertension and age of more than 70 years are important risk factors for intracranial hemorrhage related to thrombolysis. Other significant contraindications to thrombolysis include other active bleeding, recent surgery or procedures, and intracranial, spinal, or ocular disease [4]. The decision to give tPA in the clinical context of contraindications being present must always be balanced with the potential benefits of the treatment.

Clinical Efficacy In unselected groups of patients with PE, multiple metaanalyses of existing trials have demonstrated no additional benefit to treatment with thrombolytic therapy over heparin alone and have suggested an increase in hemorrhagic complications [5–7].

However, there are data indicating that in massive PE, thrombolytic therapy is beneficial. In a meta-analysis by Wan et al., thrombolytic therapy in five trials (128 patients total) with massive PE was associated with a trend toward decreased mortality [odds ratio (OR) 0.47 (0.20–1.10)] and recurrent PE [OR 0.61 (0.23–1.62)]. When these end points were combined, there was a significant decrease in death or recurrent PE [OR 0.45 (0.22–0.92)]. There was a trend toward increased risk of major hemorrhage in the group that received thrombolytic therapy [OR 1.98 (1.00–3.92)] [5]. The use of thrombolytic therapy in patients without shock but with right ventricular dysfunction has been controversial. There is evidence to demonstrate a more rapid improvement in physiologic parameters with the use of thrombolytic agents, but the overall clinical implications of this are uncertain. For example, treatment with tPA improves pulmonary vascular perfusion at 24  h when compared with treatment with heparin [8]. In a study comparing thrombolysis with UFH, tPA improved signs of right ventricular overload and pulmonary hypertension acutely when compared with heparin. However, at 1 week after PE, there was no significant difference in the two groups in the physiologic parameters [9]. Because of the uncertainty of the physiologic parameters (other than systemic hypotension) in relation to long-term outcome, decisions on the use of the use of thrombolytic agents have generally not been based on these factors. In a recent study of patients with submassive PE and evidence of right ventricular dysfunction, patients were randomized to receive either heparin plus tPA or heparin plus a placebo. There was no significant difference in mortality (3.4% in the heparin-plus-alteplase group and 2.2% in the heparin-plus-placebo group, p = 0.71) or in significant bleeding [10]. The group receiving heparin plus a placebo was significantly more likely to meet the primary study end point of either mortality or escalation of treatment (e.g. the use of thrombolytic therapy or cathecholamine infusion), with p = 0.006. The study was criticized because of the use of a primary end point defined in part by a clinical decision to escalate treatment using, among other therapies, the agent being studied in the intervention arm of the study. Given the lack of true randomization to different therapies in each arm, it is difficult to draw significant conclusions from this trial regarding the efficacy of thrombolytic therapy in submassive PE with right ventricular dysfunction. Various biomarkers are also being used to help stratify risk with PE and to potentially help make decisions on the use of thrombolysis. Brain natriuretic peptide (BNP) or N-terminal pro-brain natriuretic peptide (NT-proBNP) is released in the context of myocardial stretch. In a meta-analysis of 16 studies of PE, mortality was increased sixfold [95% confidence interval (CI) 1.31–27.42, p = 0.0021] for BNP concentration greater than 100 pg/mL and 16.12-fold (95% CI 3.1–83.68, p = 0.001)

110  Thrombolytic and Anticoagulant Therapy for Pulmonary Embolism

for NT-proBNP concentration greater than 600 ng/L [11]. In another meta-analysis of 13 studies of patients with PE, patients with elevated levels of BNP or NT-proBNP were found to have a higher 30-day mortality (OR 7.6; 95% CI 3.4–17) and a 10% risk of dying [12]. Elevated levels of troponins may also help to predict death. In a meta-analysis of 20 studies, elevated troponin levels were significantly associated with increased short-term mortality (OR 5.24; 95% CI, 3.28–8.38) and death from PE (OR 9.4; 95% CI, 4.14–21.49) [13]. Combining NT-proBNP and troponin data may help to further predict mortality. In a study of 100 normotensive patients with acute PE, 40-day mortality in patients with NT-proBNP levels of 600 ng/L or above and troponin T levels of 0.07 mg/L or above reached 33%. There were no deaths in the group with NT-proBNP levels below 600 ng/L. When the NT-proBNP level was 600 ng/L or above and the troponin T level was below 0.07  mg/L, mortality was 3.7%. Echocardiographic data did not improve stratification [14]. New biomarkers promise to help further stratify the outcome for PE. Growth differentiation factor 15 is a cytokine induced by cardiac ischemia or heart failure. It has been shown to be an independent predictor for a complicated course due to PE and in multivariate logistic regression has been shown to enhance the ability of troponin T, NT-proBNP, and right ventricular dysfunction on echocardiogram to predict complications due to PE at 30 days [15]. However, such biomarkers alone should not be used to justify more invasive treatment strategies such as thrombolysis [12].

2.2.2 Unfractionated Heparin UFH binds to antithrombin to inactivate thrombin and factor Xa. By inactivating thrombin, heparin prevents fibrin formation and inhibits activation of platelets and factors V and VIII [16]. For treatment of PE, a bolus of 80 units/kg should be followed by an infusion of 18 units/kg/h in most patients. Its mean half-life of 1.5 h allows it to be cleared fairly rapidly upon cessation of dosing.

Clinical Efficacy In 1960, UFH was shown to decrease mortality when compared with a placebo in the treatment of PE [17]. It is the only anticoagulant to have been compared with a placebo in the treatment of PE and has therefore served as the standard with which other treatments have historically been compared. The anticoagulant effect of heparin varies significantly among patients. Therefore, the activated partial thromboplastin time (aPTT) must be monitored to adjust its dosing.

Table 2  Unfractionated heparin dosing scale Rate change aPTT (s) Bolus (units) Hold (min) [mL/h; (u/h)]

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Repeat aPTT in

<50 5,000 0 +1.5 (+150)   6 h 50–59 0 0 +1.0 (+100)   6 h 60–85 0 0 0 12 h 85–100 0 −1.0 (−100)   6 h >100 0 30 −1.5 (−150)   6 h Standard mix is 25,000 units in 250 mL. Bolus is 80 units/kg. Initial infusion is 1,200 units/h. The first activated partial thromboplastin time (aPTT) is 6 h after the bolus

An aPTT of 1.5–2.5 times the mean laboratory control should be achieved as quickly as possible. In an analysis of three randomized trials evaluating initial heparin treatment of proximal deep vein thrombosis (DVT), Hull et al. concluded that a failure to achieve a therapeutic aPTT by 24 h was associated with a 23.3% frequency of recurrent venous thromboembolism (VTE) versus 4–6% for those with an aPTT which was therapeutic by 24 h (p = 0.02) [18]. To rapidly achieve a target aPTT, the use of a nomogram is encouraged (Table 2). Certain individuals are considered “heparin-resistant” and may require high dosages of heparin (more than 35,000 units/ day) to achieve a therapeutic partial thromboplastin time (PTT). Being heparin-resistant has been associated with clinical factors such as antithrombin deficiency or elevations in the levels of factor VIII [19]. In patients requiring high dosages of heparin, it is recommended that the anti-Xa heparin level should be used to adjust the PTT. This is based on a trial that showed that in patients requiring more than 35,000 units per day of heparin, therepeutic anti-Xa levels (between 0.35 and 0.7 IU/mL) could be achieved with lower doses of heparin while the PTT was still subtherapeutic. There was no difference in clinical outcome in the patients in whom the anti-Xa level was targeted [20]. A potentially significant side effect of UFH is the development of heparin-induced thrombocytopenia (HIT). Clinically significant HIT is due to the development of antibodies to a heparin/platelet factor 4 complex. These antibodies activate platelets, which leads to thrombocytopenia, which can lead to thrombosis. The overall risk of the development of HIT in patients receiving UFH is about 2% [21]. Owing to the risk of HIT, platelet counts should be followed on a daily basis. On the basis of data detailed later, LMWH has largely replaced UFH as the treatment of choice for hemodynamically stable PE. However, UFH is the treatment of choice for submassive PE in patients in whom one may need to quickly stop therapy, such as in patients who are at risk of bleeding, those who may need an emergent procedure, or those who may need thrombolytic therapy. It is also used in patients in whom UFH is contraindicated, such as those with renal failure or morbid obesity.

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2.2.3 Low Molecular Weight Heparin LMWHs are derived from UFH and are approximately one third of its size. Because of their smaller size, they are unable to bind antithrombin and thrombin simultaneously and therefore have less ability to inactivate thrombin. However, they maintain their ability to inactivate factor Xa [16]. There is also evidence that LMWHs exert their effects via tissue factor pathway inhibitor, an important mediator of anticoagulant and antithrombotic effects [22]. LMWHs are primarily renally cleared. They seem to have a significantly lower rate of inducing HIT. In one meta-analysis, the average absolute risk for HIT with LMWH was 0.2% and with UFH the risk was 2.6% [23]. In most patients, no specific marker of anticoagulation activity is monitored. However, in patients with impaired renal function in whom there may be delayed clearance of LMWH or in obese patients in whom the absorption of subcutaneously injected LMWH may be uncertain, anti-factor Xa levels can be monitored if LMWH therapy is needed. A reasonable target range for anti-factor Xa levels is 0.6–1 IU/ mL. Levels should be measured approximately 4  h after dosing [19].

P.F. Currier and C.A. Hales Table  3   Low molecular weight heparin dosing for pulmonary embolism Drug FDA-approved Alternative dosing Adjustments Exoxaparin

1 mg/kg actual body weight every 12 h

Tinzaparin

175 IU/kg actual body weight once daily

Dalteparin

200 IU/kg actual body weight for 30 days, then 150 IU/ kg once daily

1.5 mg/kg actual body weight once daily

For creatinine clearance less than 30, 1 mg/ kg every 24 h Contraindicated creatinine clearance/ 60 or age over 90 years Maximum dose 18,000 IU, do use in those over 90 kg

Because it does not bind platelet factor 4, it is unlikely to cause HIT [25]. Fondaparinux is given subcutaneously once daily and is dosed as follows: 5.0  mg for patients weighing less than 50 kg, 7.5 mg for those weighing 50–100 kg, or 10.0 mg in patients weighing more than 100 kg [26].

Clinical Efficacy Clinical Efficacy When compared with treating acute PE with UFH, treating acute PE with LMWH seems to decrease mortality, decrease bleeding, and decrease recurrence of VTE. A recent Cochrane review examined 22 studies (8,867 patients) that compared LMWH and UFH [24]. Compared with UFH, LMWH showed (1) fewer thrombotic complications [3.6% vs. 5.4%; OR 0.68 (0.55, 0.84)] in 18 studies, (2) a lower rate of major hemorrhage [1.2% vs. 2.0%; OR 0.57 (0.39, 0.83)] in 19 studies, and (3) decreased mortality [4.5% vs. 6.0%; OR 0.76 (0.62, 0.92)]. These benefits also held when subgroups of patients with proximal thrombus were examined. In patients who are hemodynamically stable (i.e. for whom thrombolytic therapy is not planned), for whom it is not anticipated that anticoagulation will need to be quickly stopped (e.g. for patients undergoing procedures or at high risk of bleeding), and who do not have renal failure or are not morbidly obese, LMWH is an ideal choice for treating PE. Formulations of LMWH approved for the acute treatment of PE and their dosages are shown in Table 3.

2.2.4 Fondaparinux Fondaparinux is a synthetic anticoagulant that binds to antithrombin III and potentiates its inhibition of factor Xa.

There have been promising data on the use of fondaparinux to treat PE. A randomized, open-label trial of patients with submassive PE showed that fondaparinux dosed on the basis of weight was not inferior to UFH in mortality (5.2 vs. 4.4%), recurrent thromboembolism (3.5 vs. 5.0%), major bleeding complications (1.3 vs. 1.1%), and thrombocytopenia (0.9 vs. 1.2%) [26]. A randomized, double-blind study of 2,205 patients with acute symptomatic DVT comparing fondaparinux and enoxaparin for initial treatment showed no significant differences in mortality (3.8% vs. 3.0%), recurrent thromboembolism (3.9% vs. 4.1%), or major bleeding (1.1% vs. 1.2%). The authors concluded that fondaparinux once daily was not inferior to enoxaparin in the initial treatment of symptomatic DVT [27]. There has been no trial comparing fondaparinux with LMWH in the treatment of PE. Although there are certainly fewer data on the efficacy of fondaparinux compared with either UFH or LMWH, the presumed low likelihood of inducing HIT makes it an appealing choice as an anticoagulant. In fact, there have been case reports of successful treatment of HIT with fondaparinux without the development of recurrent thrombotic or bleeding events [28]. In the open-label trial of fondaparinux versus UFH in submassive PE, thrombocytopenia occurred in only ten of 1,103 patients, one of whom had an associated clot with a myocardial infarction. None of these patients had antiplatelet antibodies [26].

110  Thrombolytic and Anticoagulant Therapy for Pulmonary Embolism

2.2.5 Warfarin Warfarin remains the only currently approved oral anticoagulant. It acts by inhibiting the vitamin K dependent clotting factors II, VII, IX, and X and by impairing the function of the anticoagulants activated proteins C and S [29]. Warfarin therapy can be started concurrently with or after the initiation of intravenous or subcutaneous therapy for treatment of PE. It should not be started as the only initial therapy for acute PE. Patients with proximal DVT who received only the vitamin K antagonist acenocoumarol, similar to coumadin, were shown to have a greater rate of symptomatic and asymptomatic thrombotic events when compared with patients treated with intravenously administered heparin and acenocoumarol together [30]. The lack of sufficient efficacy of vitamin K antagonists as single agents for anticoagulation relates to its mechanism of action. The prothrombin time, used to monitor the effect of warfarin, is prolonged initially (often within 24–36 h) owing to the depletion of factor VII, which has a half-life of only about 6  h. However, the anticoagulant effects of warfarin seem significantly related to the inhibition of prothrombin, which has a half-life of 60 h [29]. This argues for overlapping treatment with warfarin with an additional anticoagulant for at least 4 days. Overlapping of anticoagulation with coumadin with intravenous or subcutaneous UFH or LMWH therapy is also recommended to help prevent warfarin-induced skin necrosis. The mechanism of this rare but devastating complication is thought to be related to depletion of activated protein C early after initiation of therapy with warfarin. Because coumadin’s anticoagulant effects are not present early after the initiation of therapy, the depletion of activated protein C induces a relative hypercoagulable state which results in microvascular occlusion and skin necrosis. Concurrent therapy with heparin, LMWH, or fondaparinux will prevent this from occurring by counterbalancing this tendency of hypercoagulability [31]. Warfarin therapy should be started at a dosage of between 5 and 10 mg once daily for the first 2 days or less than 5 mg once daily for elderly patients or those with concurrent liver disease or on medications which act to increase warfarin’s effect. The prothrombin time and international normalized ratio (INR) should be monitored after 2–3 doses [32].

2.3 Duration of Treatment with Medical Therapy The duration of treatment is related to clinical and underlying predisposing factors. In a meta-analysis of trials comparing treatment of symptomatic VTE with vitamin K antagonists,

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ongoing treatment reduces the risk of recurrent VTE throughout its use. However, since the risk of recurrent VTE declines with time since the initial event and since the risk of bleeding from ongoing therapy remains the same, much effort has been made to determine the ideal time to stop anticoagulation [33]. Most trials have included a combination of patients with DVT and PE, both of which are included here. Full anticoagulation for treatment of VTE is recommended on the basis of studies demonstrating higher rates of recurrence of VTE with less intensive treatment [34, 35]. For example, for treatment of DVT, low-dose subcutaneously administered UFH (5,000  units twice daily) was shown to lead to significantly greater rates of recurrence of VTE than treatment with warfarin (p = 0.001). Risk of bleeding was also higher for patients receiving warfarin [35].

2.3.1 Transient Risk Factor For VTE in the context of a transient risk factor (e.g. surgery, immobilization), treatment is recommended for 3 months as opposed to shorter time periods [34]. In a small study, treating for 1 month versus 3 months in patients with a transient risk factor for VTE resulted in a nonsignificant increase in recurrent VTE with no major bleeds in either group [34].

2.3.2 Unprovoked VTE Unprovoked VTE occurs in the absence of any underlying malignancy, thrombotic disorder, or transient risk factor [36]. For a first VTE which is unprovoked, anticoagulation for at least 3–6 months is recommended, with evaluation at the end of that period as to the risks and benefits of further anticoagulation. For a first proximal DVT, long-term anticoagulation is recommended in the absence of contraindications [34]. Treatment for less than 3 months for unprovoked VTE seems inadequate. In a study of patients with first VTE without congenital deficiencies of antithrombin, protein C, or protein S, treatment with anticoagulation for 6 months as opposed to treatment for 6 weeks significantly reduced the risk for recurrent VTE by about half without any increase in bleeding or mortality [37]. In a trial which just looked at patients with PE, there was found to be no significant benefit to continuing anticoagulation beyond 3 months to either 6 months or 1 year in preventing recurrent VTE [36]. However, another trial including patients with DVT and PE which compared patients receiving 3 months of anticoagulation or extended anticoagulation was stopped early (after 10 months) owing to a significantly decreased rate of recurrent VTE in patients receiving extended anticoagulation [38].

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Conflicting data on the duration of anticoagulation beyond 3 months makes it difficult to suggest a definitive time point for stopping anticoagulation. In an attempt to address this issue, one study checked D-dimer levels 1 month after completing 3 months of anticoagualation following a first unprovoked DVT or PE. Patients with a normal D-dimer level did not resume anticoagulation and had a rate of recurrent VTE of 6.2% at average follow-up of 1.4 years. Patients with an abnormal D-dimer level were divided into groups that stopped or continued anticoagulation. They had rates of recurrent VTE of 15 and 2.9%, respectively [4.26 (1.23, 14.6), p = 0.02]. The adjusted hazard ratio for recurrent VTE for those who stopped anticoagulation with an abnormal D-dimer level compared with those who had a normal D-dimer level was 2.27 [(1.15, 4.46), p = 0.02] [39]. Another study ­combined D-dimer with factor VIII levels approximately 1 month following cessation of anticoagulation following the first episode of idiopathic proximal DVT of the lower extremities. At 2 years, recurrent VTE was found in 6% of patients with negative D-dimer levels and factor VIII levels below the 75th percentile and in 20% of those with negative D-dimer levels and factor VIII levels above the 75% percentile. Recurrent VTE was found in 29% of patients with D-dimer levels above 500 ng/mL, and in over 50% of patients with D-dimer levels above 500 ng/mL and factor VIII levels above the 75th percentile [40]. Although these studies do not completely help to clarify the length of duration of anticoagulation (particularly in patients with a normal D-dimer level), it does take a step toward helping to further stratify patient risk. It also provides a model for future work in the use of various biological markers to help stratify risk and treatment.

2.3.3 Recurrent VTE For patients with a second episode of VTE, long-term anticoagulation is recommended [34]. In a multicenter trial involving patients with a second episode of VTE, there was a significantly lower rate of recurrent VTE (3%) in a group receiving ongoing anticoagulation at 4 years compared with a group in which anticoagulation had been stopped at 6 months [41]. Both coumadin and LWMH appear to be safe for longterm treatment of PE. A meta-analysis of seven studies showed no statistically significant difference in rates of recurrent VTE. There appears to be at least a trend toward a decreased risk of bleeding with the use of LMWH [42].

2.3.4 Malignancy For patients with malignancy and VTE, treatment with LMWH for the first 3–6 months followed with long-term

P.F. Currier and C.A. Hales

treatment with either LMWH or warfarin is recommended over treatment only with warfarin [34]. A meta-analysis concluded that treatment with LMWH compared with vitamin K antagonists did not improve survival [hazard ratio 0.96 (0.81, 1.14)] but did decrease recurrent VTE [hazard ratio 0.47 (0.32, 0.71)] [43]. In one multicenter trial, patients with cancer who had either proximal DVT, PE, or both were randomized to receive dalteparin or vitamin K antagonists for 6 months following initial treatment with dalteparin in both groups. The probability of recurrent thromboembolism at 6 months was 17% in the group on oral anticoagulants and was significantly decreased to 9% in the group receiving dalteparin (hazard ratio 0.48, p = 0.002). There was no significant difference in bleeding or in mortality between the two groups [44]. On the basis of these data, dalteparin is now FDA-approved to treat VTE with a dosage of 200  IU/kg subcutaneously once per day for the first month followed by 150  IU/kg subcutaneously once per day for the subsequent 5 months, not to exceed a daily dose of 18,000 IU. Certainly, any benefits derived from the use of LWMH versus warfarin in treatment must also be balanced with the issue of giving a medicine by subcutaneous injection versus the necessity of periodic blood draws for monitoring of the INR when the patient is receiving coumadin. There are no routine blood draws recommended while the patient is on long-term therapy with LMWH for VTE.

2.4 Emerging Therapies Therapies for VTE that are currently being investigated include direct thrombin inhibitors and inhibitors of factor Xa [45]. The factor Xa inhibitors block the generation of thrombin. Thrombin inhibitors block activity of existing thrombin. Ximelagatran is a direct thrombin inhibitor which demonstrated good efficacy and safety in treatment for VTE. It was withdrawn from the market owing to concerns over its hepatotoxicity [45]. The following describes therapies currently under development for potential treatment of VTE.

2.4.1 Factor Xa Inhibitors Idraparinux is a hypermethylated derivative of fondaparinux. Owing to its high binding affinity, it has a half-life of 80 h and can be given once weekly; it has no antidote [45]. One study showed it to be similar in prevention of recurrent VTE when compared with conventional therapy (LMWH or UFH followed by vitamin K antagonists), but inferior in treating PE [46]. Compared with a placebo, idraparinux significantly decreased recurrent VTE in the period from 6 to 12 months

110  Thrombolytic and Anticoagulant Therapy for Pulmonary Embolism

after the initial VTE. However, there was a significantly increased risk of bleeding with idraparinux compared with a placebo (including three fatal intracranial hemorrhages) [47]. This risk of bleeding has led to further development of the drug being stopped [45]. SSR 126517 is a derivative of idraparinux that has an additional biotin moiety. It has a similar half-life of 80  h and can be given once weekly. Because of its biotin moiety, avidin can be given as an antidote which binds SSR 126517 and can rapidly clear the drug. Trials are currently under way and the ability to reverse the effects of SSR 126517 has led to focus on this instead of idraparinux as a next-generation subcutaneous therapy. Rivoaroxaban is an oral agent which inhibits factor Xa reversibly and competitively. It has more than 80% bioavailability, with a half-life between 5 and 9 h in the young and 11–13 h in the elderly. It is cleared in the kidneys and gastrointestinal tract and metabolized by the liver. Phase I and phase II trials comparing rivoaroxaban with treatment with LMWH followed by vitamin K antagonists have shown similar rates of recurrent VTE without an increase in major bleeding [36, 45]. Apixaban is a reversible oral inhibitor of factor Xa with a bioavailability of 50%. Its half-life is between 9 and 14 h. Like rivaroxaban, it is excreted in both the kidneys and the gastrointestinal tract and is metabolized by the liver. On the basis of preliminary data showing favorable results comparing apixiban with LMWH or fondaparinux followed by vitamin K antagonists, phase III trials using two different doses of apixaban are planned.

2.4.2 Thrombin Inhibitors Dabigatran is an oral direct thrombin inhibitor. Its bioavailability is only 6%, dictating that high doses of the drug are given to obtain adequate drug levels. Better absorption is obtained via decreased acid in the stomach and small intestine, prompting its dosing in tartaric acid containing capsules and in conjunction with proton pump inhibitors to raise the pH. Dabigatran is primarily excreted via the kidneys, making it contraindicated in patients with renal failure. On the basis of the results of a phase II trial of dabigatran in stroke prevention in atrial fibrillation, a phase III trial is under way to evaluate the efficacy of dabigatran in treating VTE [45].

3 Chronic Thromboembolic Pulmonary Hypertension The pathogenic mechanisms in the development of CTEPH are not particularly well understood. It is commonly accepted

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that this disease process is the result of initial venous thromboembolic events that are not treated or are improperly treated followed by the development of changes in the pulmonary vasculature leading to pulmonary hypertension [48]. However, alternate hypotheses in the development of CTEPH include a primary pulmonary arteriopathy followed by in situ thrombosis [49]. In the absence of treatment, CTEPH seems to progress in virtually all cases [50]. Although surgical treatment is generally recommended as first-line treatment in cases amenable to operative intervention, there are some data to suggest that anticoagulation may be beneficial in some cases. Twenty-nine patients with newly diagnosed CTEPH who did not undergo surgical endarterectomy for a variety of reasons (patient refusal, presence of concomitant peripheral embolism, mild pulmonary hypertension) were prospectively studied [51]. Patients were anticoagulated with warfarin for a target INR of 2–3. Patients were followed for 1 year; 26 of 29 patients survived to 1 year. The three patients who died had the highest pulmonary systolic pressures of the entire group (120, 127, and 133 mmHg). In 12 of 26 survivors, pulmonary pressures decreased to within normal during the study period. Although the degree to which ongoing thrombosis plays a role in the disease process is unclear and although there are minimal data on its efficacy, we recommend anticoagulation as standard therapy for patients who have CTEPH. We recommend anticoagulation in patients prior to and after their undergoing thromboendarterectomy in those patients who will not undergo surgical treatment.

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1528 9. Konstantinides S, Tiede N, Geibel A, Olschewski M, Just H, Kasper W (1998) A comparison of alteplase versus heparin for resolution of major pulmonary embolism. Am J Cardiol 82:966–970 10. Konstantinides SK, Geibel A, Heusel G et al (2002) Heparin plus alteplase compared withheparin alone in patients with submassive pulmonary embolism. N Engl J Med 349:1143–1150 11. Cavallazzi R, Nair A, Vasu T, Marik PE (2008) Natriuretic peptides in acute pulmonary embolism: a systematic review. Intensive Care Med 34:2147–2156 12. Klok FA, Mos IC, Huisman MV (2008) Brain-type natriuretic peptide levels in the prediction of adverse outcome in patients with pulmonary embolism. Am J Respir Crit Care Med 178:425–430 13. Becattini C, Vedovati MC, Agnelli G (2007) Prognostic value of troponins in acute pulmonary embolism, a meta-analysis. Circulation 116:427–433 14. Kostrubiec M, Pruszczyk P, Bochowicz A et al (2005) Biomarkerbased risk assessment model in acute pulmonary embolism. Eur Heart J 26:2166–2172 15. Lankeit M, Kempf T, Dellas C et al (2008) Growth differentiation factor-15 for prognostic assessment of patients with acute pulmonary embolism. Am J Respir Crit Care Med 177:1018–1025 16. Hirsh J, Anand SS, Halperin JL, Fuster V (2001) Mechanism of action and pharmacology of unfractionated heparin. Arterioscler Thromb Vasc Biol 21:1094–1096 17. Barritt DW, Jordan SC (1960) Anticoagulant drugs in the treatment of pulmonary embolism. A controlled trial. Lancet 1:1309–1312 18. Hull RD, Raskob GE, Brant RF, Pineo GF, Valentine KA (1997) Relation between the time to achieve the lower limit of the aPTT therapeutic range and recurrent venous thromboembolism during heparin treatment for deep vein thrombosis. Arch Intern Med 157:2562–2568 19. Hirsh J, Warkentin TE, Shaughnessy SG et  al (2001) Heparin and low-molecular-weight heparin mechanisms of action, pharmacokinetics, dosing, monitoring, efficacy, and safety. Chest 119: 64S–94S 20. Levine MN, Hirsh J, Gent M et al (1994) A randomized trial comparing activated thromboplastin time with heparin assay in patients with acute venous thromboembolism requiring large daily doses of heparin. Arch Intern Med 154:49–56 21. Fabris F, Luzzatto G, Stefani PM, Girolami B, Cella G, Girolami A  (2000) Heparin-induced thrombocytopenia. Haematologica 85: 72–81 22. Hoppensteadt DA, Walenga JM, Fasanell A, Jeske W, Fareed J (1995) TFPI antigen levels in normal human volunteers after intravenous and subcutaneous administration of unfractionated heparin and a low molecular weight heparin. Thromb Res 77:175–185 23. Martel N, Lee J, Wells PS (2005) Risk for heparin-induced thrombocytopenia with unfractionated and low molecular-weight heparin thromboprophylaxis: a meta-analysis. Blood 106:2710–2715 24. Van Dongen CJJ, van den Belt AG, Prins MH, Lensing AWA (2004) Fixed dose subcutaneous low molecular weight heparins versus adjusted dose unfractionated heparin for venous thromboembolism. Cochrane Database Syst Rev 4:CD001100 25. Dager WE, Andersen J, Nutescu E (2004) Special considerations with fondaparinux therapy: heparin induced thrombocytopenia and wound healing. Pharmacotherapy 24:88S–94S 26. Büller HR, Davidson BL, Decousus H et  al (2003) Subcutaneous fondaparinux versus intravenous unfractionated heparin in the initial treatment of pulmonary embolism. N Eng J Med 349:1695–1702 27. Büller HR, Davidson BL, Decousus H et al (2004) Fondaparinux or enoxaparin for the initial treatment of symptomatic deep venous thrombosis. Ann Intern Med 40:867–873 28. Spyropoulos AC, Magnuson S, Koh SK (2008) The use of fondapainux for the treatment of venous thromboembolism in a patient with heparin-induced thrombocytopenia and thrombosis caused by heparin flushes. Ther Clin Risk Manag 4:653–657

P.F. Currier and C.A. Hales 29. Hirsh J, Dalen JE, Deykin D, Poller L, Bussey H (1995) Oral anticoagulants: mechanism of action, clinical effectiveness, and optimal therapeutic range. Chest 108:231S–246S 30. Brandjes DP, Heijboer H, Büller HR, de Rijk M, Jagt H, ten Cate JW (1992) Acenocoumarol and heparin compared with acenocoumarol alone in the initial treatment of proximal-vein thrombosis. N Engl J Med 327:1485–1489 31. Chan YC, Valenti D, Mansfield AO, Stansby G (2000) Warfarin induced skin necrosis. Br J Surg 87:266–272 32. Ansell J, Hirsh J, Hylek E et al (2008) Pharmacology and management of the vitamin K antagonists: American College of chest physicians evidence-based clinical practice guidelines (8th edition). Chest 133:160S–198S 33. Hutten BA, Prins MH (2006) Duration of treatment with vitamin K antagonists in symptomatic venous thromboemboism. Cochrane Rev 1:1–25 34. Kearon C, Kahn SR, Agnelli G et al (2008) Antithrombotic therapy for venous thromboembolic disease: American College of chest physicians evidence-based clinical practice guidelines (8th edition). Chest 133:454S–545S 35. Hull R, Delmore T, Genton E et al (1979) Warfarin sodium versus low-dose heparin in the long-term treatment of venous thrombosis. N Engl J Med 301:855–858 36. Agnelli G, Gallus A, Goldhaber SZ et al (2007) Treatment of proximal deep-vein thrombosis with the oral direct factor Xa inhibitor rivaroxaban (BAY 59-7939): the ODIXa-DVT (Oral Direct Factor Xa Inhibitor BAY 59-7939 in Patients With Acute Symptomatic Deep-Vein Thrombosis) study. Circulation 116:180–187 37. Schulman S, Rhedin AS, Lindmarker P et al (1995) A comparison of six weeks with six months of oral anticoagulant therapy after a  first episode of venous thromboembolism. Duration of Anticoagulation Trial Study Group. N Engl J Med 332:1661–1665 38. Kearon C, Gent M, Hirsh J (1999) A comparison of three months of anticoagulation with extended anticoagulation for a first episode of idiopathic venous thromboembolism. N Engl J Med 340:901–907 39. Palareti G, Cosmi B, Legnani C (2006) D-dimer testing to determine the duration of anticoagualation therapy. N Engl J Med 355: 1780–1789 40. Cosmi B, Legnani C, Cini M, Favaretto E, Palareti G (2008) D-dimer and factor VIII are independent risk factors for recurrence after anticoagulation withdrawal for a first idiopathic deep vein thrombosis. Thromb Res 122:610–617 41. Schulman S, Granqvist S, Holmström M et al (1997) The duration of oral anticoagulant therapy after a second episode of venous thromboembolism. N Eng J Med 336:393–398 42. van der Heijden JF, Hutten BA, Büller HR, Prins MH (2002) Vitamin K antagonists or low-molecular-weight heparin for the long term treatment of symptomatic venous thromboembolism. Cochrane Database Syst Rev 1:CD002001 43. Akl E, Barba M, Rohilla S et al (2008) Anticoagulation for the long term treatment of venous thromboembolism in patients with cancer. Cochrane Database Syst Rev 2:CD006650 44. Lee AY, Levine MN, Baker RI et al (2003) Low-molecular-weight heparin versus a coumarin for the prevention of recurrent venous thromboembolism in patients with cancer. N Engl J Med 349:146–153 45. Gross PL, Weitz JI (2008) New anticoagulants for the treatment of venous thromboembolism. Arteroscler Thromb Vasc Biol 28:380–386 46. Büller HR, Cohen AT, Davidson B et al (2007) Idraparinux versus standard therapy for venous thromboembolic disease. N Engl J Med 357:1094–1104 47. Büller HR, Cohen AT, Davidson B et al (2007) Extended prophylaxis of venous thromboembolism with idraparinux. N Engl J Med 357:1105–1112 48. Fedullo PF, Rubin LJ, Kerr KM, Auger WR, Channick RN (2000) The natural history of acute and chronic thromboembolic disease: the search for the missing link. Eur Respir J 15:435–437

110  Thrombolytic and Anticoagulant Therapy for Pulmonary Embolism 49. Egermayer P, Peacock AJ (2000) Is pulmonary embolism a common cause of chronic pulmonary hypertension? Limitations of the embolic hypothesis. Eur Respir J 15:440–448 50. Kunieda T, Nakanishi N, Satoh T, Kyotani S, Okano Y, Nagaya N (1999) Prognoses of primary pulmonary hypertension and chronic major vessel thromboembolic pulmonary hypertension

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determined from cumulative survival curves. Intern Med 38: 543–546 51. Romaszkiewicz R, Lewczuk J, Piszko P et al (2006) Results of oneyear anticoagulation in patients with newly detected chronic thromboembolic pulmonary hypertension not treated with pulmonary endarterectomy. Kardiol Pol 64:1196–1202

Chapter 111

Nursing Care of Patients with Pulmonary Arterial Hypertension Christine Archer-Chicko

Abstract  Patients with pulmonary arterial hypertension (PAH) ultimately develop symptoms of right-sided heart failure as a result of low cardiac output and arterial hypoxemia. Patients typically manifest dyspnea with exertion or fatigue as the initial symptoms of PAH. In our clinic, patients report they have noticed changes in their breathing or exercise tolerance for periods ranging from several weeks to several months or even years before seeking evaluation by a physician. They tend to minimize the significance of their symptoms by relating them to increasing age, being overworked, not sleeping well, being out of shape, or being overweight As time passes, these symptoms persist and in many instances progress. Patients may experience any of the following symptoms with PAH (either at rest or upon exertion): fatigue, shortness of breath, edema, chest discomfort, palpitations, dizziness, presyncope, and syncope. Once diagnosed, patients should be referred to an experienced pulmonary hypertension (PH) center for further evaluation and management. PH program nurses are important in managing the care of patients. There are four specific roles that nurses assume: coordination of care, patient advocacy, patient assessment, and patient education. Patients need to understand PH as a disease process, the signs and symptoms that signify worsening, and the various treatment options. In addition, nurses provide ongoing emotional support to help patients, their families, and support persons to cope with living with this unpredictable chronic disease and to achieve the best quality of life possible. The benefits of good nursing care from an experienced PH center nurse are similar to those from advanced practice nurses managing congestive heart failure patients and they include improved quality of life, decreased number of hospital admissions, and prolonged survival. This chapter will focus specifically on the nursing care of PAH patients. It will highlight practical issues including assessing patients, developing a treatment plan, supporting patients

C. Archer-Chicko (*) Penn Presbyterian Medical Center, 51 N. 39th St. – Suite 120 PHI Bldg, Philadelphia, PA 19104, USA e-mail: [email protected]

and families/caregivers, handling special circumstances, and managing insurance issues. Keywords  Coordination of care • Patient advocacy • Patient care • Clinical worsening • Treatment option • Caregiver

1  Introduction Pulmonary arterial hypertension (PAH) is a progressive and debilitating disease characterized by smooth muscle cell proliferation, intimal fibrosis, and in situ thrombosis of the small pulmonary arteries [1]. PAH patients also have alterations in the balance of vasoactive substances that regulate vasoconstriction and vasodilation of the pulmonary vascular bed [2]. The resulting effect is a vasculopathy in the pulmonary vascular bed, where the walls of the small pulmonary vessels are hypertrophied and the lumens have narrowed. In some cases, the vessel lumen may become completely obliterated by the uncontrolled cellular proliferation and in situ microthrombi [2]. As a result of the pathologic changes remodeling the pulmonary vascular bed, it becomes less distensible and unable to accommodate the increases of cardiac output needed for daily activity or exercise. This loss of accommodation causes increased resistance to blood flow and results in an increase in pressure within the pulmonary vascular bed. Over time, right ventricular hypertrophy and dilation occur as a result of increased pulmonary vascular resistance; this compromises the ability of the right ventricle to maintain cardiac output especially when demand is increased. In addition, these pathologic changes may alter ventilation–perfusion matching, contributing to arterial hypoxemia. Patients ultimately develop symptoms of right-sided heart failure as a result of low cardiac output and arterial hypoxemia [3]. Patients typically manifest dyspnea with exertion or fatigue as the initial symptoms of PAH. In our clinic, patients report they have noticed changes in their breathing or exercise tolerance for periods ranging from several weeks to ­several months

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or even years before seeking evaluation by a physician [4]. They tend to minimize the significance of their symptoms by relating them to increasing age, being overworked, not sleeping well, being out of shape, or being ­overweight As time passes, these symptoms persist and in many instances progress. Patients may experience any of the following symptoms with PAH (either at rest or upon exertion): fatigue, shortness of breath, edema, chest discomfort, palpitations, dizziness, presyncope, and syncope. Because of the vague and nonspecific nature of symptoms, most patients are not readily diagnosed early in their disease course. Data from the National Institutes of Health Primary Pulmonary Hypertension Registry indicate that the average time from onset of symptoms to diagnosis was approximately 2 years [4]. The delay in diagnosis places patients at risk as PAH is a progressive disease, especially if untreated. Once diagnosed, patients should be referred to an experienced pulmonary hypertension (PH) center for further evaluation and management. PH program nurses are important in managing the care of patients. There are four specific roles that nurses assume: coordination of care, patient advocacy, patient assessment, and patient education. First, nurses are directly involved in all aspects of patient care from the initial patient visit, through the diagnostic evaluation, to the initiation of therapy and then following patients on a long-term basis. PH program nurses do not care for these patients alone; they collaborate with a multidisciplinary team including physicians, bedside clinical nurses, nursing assistants, social workers, nutritionists, ­physical therapists, respiratory therapists, financial ­coordinators, insurance case managers, other physician specialists, and families and other patient-support persons. PH nurses serve as the team coordinator interfacing with the other health care professionals to provide quality care. Second, nurses approach each patient individually when considering treatment options, and knowing the patients, they advocate for them. Patients differ in the specific diagnosis of PAH, associated medical conditions, their social support system, and their motivation. Nurses are accustomed to take these factors into account when considering treatment options for a specific patient. Third, PH nurses possess advanced assessment skills that enable them to monitor how a patient is responding to therapy. Nurses can identify patients who are failing medical therapy with disease progression or who are not adhering to the treatment plan. In these instances, it is vital to recognize the problem and initiate a change in the treatment plan. The fourth role that PH center nurses undertake is to provide extensive patient education and emotional support. Patients need to understand PH as a disease process, the signs and symptoms that signify worsening, and the various treatment options. In addition, nurses provide ongoing emotional support to help patients, their families, and support persons to cope with ­living with this unpredictable chronic disease and to achieve the best quality of life

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possible. The benefits of good nursing care from an experienced PH center nurse are similar to those from advanced practice nurses managing congestive heart failure patients and they include improved quality of life, decreased number of hospital admissions, and prolonged survival [5, 6]. This chapter will focus specifically on the nursing care of PAH patients. It will highlight practical issues including assessing patients, developing a treatment plan, supporting patients and families/caregivers, handling special circumstances, and managing insurance issues.

2  Assessing Patients Starting at the initial visit, PAH patients should be evaluated for signs and symptoms of right-sided heart failure. A complete and thorough history should be obtained and a complete and thorough physical examination should be performed. In addition, there are key questions nurses should ask that will yield more specific clinical information. Assessing for the level of dyspnea includes asking patients what activities make them short of breath and how has this affected their usual daily routine. • • • • • •

• • •

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Are they short of breath at rest? Are they short of breath with activity? How far can they walk before they have to stop? Can they walk a normal pace or do they have to walk at a slow pace? Can they walk up an incline? Can they climb a flight of stairs? How many steps can they climb before they have to stop because they feel short of breath? If they climb a flight of stairs, do they have to stop immediately at the top to catch their breath and recover? Have they had to modify their job or stop working because of their shortness of breath? Have they had to reduce or give up activities at home? Ask what a typical day used to be like for the patient, and how it is now. Do they require assistance from family members for completing activities of daily living (ADLs) such as bathing/ showering, dressing, etc.?

In addition, it is important to assess if they are using ­supplemental oxygen. Notice if the patient is currently wearing a nasal cannula with oxygen. • Ask if they have ever been prescribed supplemental oxygen. How has the oxygen been prescribed? (With activity or exertion? Continuously? For sleep?) • How are they using their oxygen? Do they ever increase the flow rate?

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• Ask if they have ever been prescribed continuous positive airway pressure (CPAP) or bilevel positive airway pressure (BiPAP) therapy. • If the patient is not using the oxygen or CPAP/BiPAP therapy as prescribed, ask patient why he/she is not doing so. Is the patient having any difficulties managing the oxygen or the oxygen system? Is the patient having problems with nasal passage dryness and nosebleeds? Is the patient’s tank too heavy or inconvenient? Does the patient have an ambulatory system? Ask how the patient feels about using oxygen: Does the patient feel embarrassed using oxygen in public? Is the patient having difficulty using the CPAP/BiPAP system?

need a more urgent workup or reevaluation. It is helpful to consider where a specific patient fits in the World Health Organization (WHO) functional classification system. Ideally, the patients who have had a rapid progression of symptoms or those patients who are very symptomatic (WHO functional class III or IV) should be evaluated and started on therapy in an expedited manner to prevent complications or an untoward outcome [7]. The diagnostic evaluation for assessing right-sided heart failure in PH patients includes history and physical examination, bloodwork, 6-min walk testing, serial echocardiograms, and cardiac catheterization. (Note, the following is a discussion of evaluating a patient for the presence and the extent of right-sided heart failure in PAH patients. It is not a discussion of the complete diagnostic workup for PAH. Refer to Chaps. 99–102 regarding diagnostic approaches.) Taking a thorough history and performing a thorough physical examination are important steps in the initial evaluation of PAH. In the National Institutes of Health study of patients with primary PH, dyspnea and fatigue were the most frequently reported symptoms. Other common symptoms include chest pain, palpitations, lower-extremity edema, presyncope, and syncope [8]. Less common symptoms include cough, hemoptysis, and hoarseness. Interestingly, the hoarseness may be caused by compression of the left laryngeal nerve when the left pulmonary artery dilates. This is known as Ortner’s syndrome [3]. When taking a detailed history, it is important to include questions regarding other conditions or exposures associated with PAH. Examples include:

Assessing for cardiac signs and symptoms includes asking about chest discomfort, irregular heartbeats, and dizziness. • Does the patient feel any chest discomfort? Ask the patient to describe the chest discomfort in specific detail. Prompt the patient to identify quality, location, intensity, frequency, timing, duration, any radiation of pain, any associated symptoms, etc. • Does the patient experience or is the patient aware of any palpitations, rapid heart rate, irregular heartbeats, or skipped or extra heart beats? • Does the patient experience any light-headedness or dizziness? Does the light-headedness occur at rest or with activity? • Has the patient ever felt like he/she was going to pass out? • Has the patient ever passed out and lost consciousness? What was the patient doing immediately before this? It is also important to assess for edema as an indication of heart failure. • Has the patient experienced any swelling or edema? Where has the patient noticed the swelling (feet/ankles, legs, abdomen)? Is the swelling intermittent or continuous? • Has their body weight or abdominal girth increased? What is their usual body weight? Does the patient weigh himself/herself regularly? • How much fluid does the patient drink in a day? Assessing functional level is important so that the health care provider can begin to get an idea of how functionally limited a patient has become as a result of the PH. Patients and family/support persons can be asked directly about their daily activities at home or at work. Has their physical activity level changed? How rapidly? For a more objective assessment, a 6-min walk test can be performed. The 6-min walk test will be more fully described in the next section. We have found it helpful in our practice to consider how rapidly a patient is progressing and how functionally limited a patient has become so that we can identify which patients

• A history of Raynaud’s phenomenon (fingers turning white, blue, painful when exposed to cold temperatures), possibly indicating a collagen vascular disorder • A history of liver disease • A history of anorectic drug use • A history of illicit drug use (especially cocaine, amphetamine, or methamphetamine use) • A history of multiple sexual partners or illicit sexual practices possibly indicating a risk of HIV-associated PH • A history of snoring, daytime somnolence, witnessed apneic events, indicating possible sleep apnea It is also important to explore family history. Ask if the patient has any family members diagnosed with PH. If the patient describes family members who have died as a result of unspecified cardiopulmonary disease (particularly at a younger age), the records of those family members should be reviewed to determine if PAH (diagnosed or previously undiagnosed) was present. The physical examination is one of the most important components in evaluating a patient with PH. In general, patients with more advanced disease will demonstrate more obvious and severe signs of cor pulmonale. The patient may present to clinic appearing pale and fatigued and ambulating

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into the examination room may provoke dyspnea, tachypnea, or desaturation. Typically, the patient does not appear dyspneic or distressed when at rest, although some patients become dyspneic when speaking for a period of time. Vital signs may show normal or low blood pressure with a normal or rapid heart rate. Jugular venous distension may be present. Lung sounds are usually clear unless there is an underlying lung disorder. For example, if the patient has collagen vascular disease with interstitial lung involvement, crackles may be present. The cardiac examination may reveal a palpable right ventricular lift at the lower left sternal border. Upon auscultation, an accentuated pulmonic component of the second heart sound can be heard as the pulmonic valve closes forcefully because of the increased pulmonary artery pressures. Other ausculatory findings include narrow splitting of the second heart sound, a mid-systolic ejection murmur, a holosystolic tricuspid regurgitant murmur, a diastolic pulmonary insufficiency murmur, and a right ventricular S3 or S4 gallop [3]. The abdomen may reveal hepatomegaly and/or ascites from hepatic congestion in advanced disease. The extremities may show pale or dusky nail beds and peripheral edema. Bloodwork used to evaluate these patients includes a basic metabolic panel (sodium, potassium, chloride, carbon dioxide, blood urea nitrogen, creatinine, and glucose), complete blood count (CBC), and B-type natriuretic peptide (BNP) level or N-terminal proBNP (NT-proBNP) level. A basic metabolic panel is useful to evaluate a patient’s diuretic therapy and to ensure that critical electrolytes are within normal limits and to monitor for compromise of renal function with diuresis. A CBC will demonstrate if a patient has anemia which may affect his/her level of fatigue and dyspnea, or if he/she has secondary polycythemia related to hypoxemia. The BNP level and the NT-proBNP may be used as a plasma biomarker to indicate when the patient is experiencing right ventricular strain and worsened heart failure. These natriuretic peptides are generated by the cardiac atria and ventricles when the myocytes are stretched, indicating the heart is failing. Elevated BNP or NT-proBNP levels may identify worsening heart failure. Serial levels may be helpful to trend progression of a patient’s PH [9, 10]. The 6-min walk test is a simple test reflecting a patient’s exercise capacity. The test involves an unencouraged patient walking at his/her own pace on a hard, flat surface for 6 min [11]. Patients should wear their usual supplemental oxygen for this test. The patient’s walking speed, distance, and oxygen saturation are measured. The 6-min walk test has been found to correlate with mean right atrial pressure, cardiac output, pulmonary vascular resistance, and New York Heart Association functional class [12]. Serial measurements can be helpful in monitoring for changes in a patient’s functional capacity and/or oxygenation. The 6-min walk test may predict disease progression and demonstrate a patient’s response to

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therapy [13]. If the patient uses supplemental oxygen ­chronically, the oxygen should be used during the walk test at the same flow rate. Oxygen therapy may be uptitrated for desaturations observed during the study to prevent hypoxemia. This observation may be used to modify the patient’s oxygen prescription. The transthoracic echocardiogram is a noninvasive test which provides ultrasound images of the heart which may be useful when evaluating PH. This test is widely available and is frequently used as a screening test to identify which patients should proceed to cardiac catheterization to formally diagnose PAH. Patients cannot be diagnosed with PAH on the basis of an echocardiogram alone [14]. An echocardiogram can estimate pulmonary artery pressures based on the Doppler tricuspid jet velocity. In addition, an echocardiogram can provide information on right atrial and right ventricular characteristics (hypertrophy, dilation, and contractility), left atrial and left ventricular size and function, valvular function, and wall motion (including the septum) [15]. For patients suspected of having left-to-right shunting, a bolus of agitated saline (“bubble”) can be injected intravenously to assess if a shunt is present. Echocardiograms may be performed on a periodic basis to help the PH physician and nurse monitor a patient’s clinical status on medical therapy. An echocardiogram can provide information predicting prognosis, such as worsening right ventricular size and function, or detecting a pleural effusion which is associated with a worsened prognosis [16]. Forfia et al. demonstrated the degree of tricuspid annular displacement [tricuspid annular plane systolic excursion (TAPSE)] detected on an echocardiogram is a useful measure of right ventricular function and has prognostic significance [17]. A TAPSE of less than 1.8 cm was associated with greater right ventricular systolic dysfunction, remodeling of the right side of the heart and right ventricle–left ventricle disproportion as compared with a TAPSE of 1.8  cm or greater. In patients with PAH, survival estimates at 1 and 2 years were 94% and 88% in those with a TAPSE of 1.8 cm or greater compared with 60% and 50% in those with a TAPSE less than 1.8  cm [17]. Although echocardiography is a useful diagnostic tool, there are some disadvantages, which include cost, technician error, difficulty imaging patients with chronic obstructive lung disease or obesity, and errors in interpretation of pressures [15]. Stress echocardiograms are performed with imaging of the patient at rest and then after exercise to evaluate the effect of exertion (with increased cardiac output) on pulmonary pressures and cardiac function. They are typically used to evaluate patients who have resting echocardiogram findings that are only, at most, mildly abnormal and who describe feeling symptomatic only with activity. Some patients seen are early in the PAH disease process; they have no PH at rest, but rather they have exercise-associated PH [18]. There is no universally accepted protocol or exercise technique ­(treadmill

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or cycle ergometry) for performing a stress echocardiogram, nor are there clear definitions as to what defines exerciseassociated PH. The goal is to have the patient exercise to his/ her perceived maximal exercise level and then quickly image the heart. Similarly to transthoracic echocardiograms, there are several difficulties inherent in this testing. There may be difficulties obtaining adequate cardiac images quickly after exercise and in interpretation because of coexisting disease processes and patient conditioning [15]. Right-sided heart catheterization remains the gold standard for confirming the diagnosis of PAH, evaluating the severity of the disease, and in aiding in the selection of appropriate medical therapies [14, 19]. In addition to standard right-sided heart pressure and flow measurements, each patient may have an individualized cardiac catheterization procedure based upon his or her specific clinical situation and diagnosis. For example, a patient with congenital heart disease may require sequential oxygen saturation measurements to evaluate the direction and magnitude of intracardiac shunting. Likewise, a patient with a suspected concomitant left-sided heart dysfunction may also require a left-sided heart catheterization to evaluate the coronary arteries, function of the left side of the heart, and to directly measure the left ventricular end-diastolic pressure. During right-sided heart catheterization, many right-sided hemodynamic parameters should be assessed: right atrial pressure, right ventricular pressure, pulmonary artery pressures (systolic, diastolic, and mean), pulmonary capillary wedge pressure, mixed venous oxygen saturation, cardiac output, cardiac index, and pulmonary vascular resistance. Left-sided hemodynamics (left ventricular end-diastolic pressure, the extent and location of coronary artery disease, and evaluation of intracardiac shunting through a patent foramen ovale or as a consequence of congenital heart disease) measurements may be indicated in some patients. Patients who demonstrate normal pulmonary hemodynamics at rest may need to be exercised with the catheter in the right side of the heart to assess hemodynamics with activity. Arm exercises (i.e. lifting saline bags), serial leg raises, and recumbent and upright stationary bicycle riding are forms of exercise that can be used during the right-sided heart catheterization. Pulmonary vasoreactivity testing should also be performed at the time of right-sided heart catheterization. Testing with a short-acting vasodilator (intravenously administered ­adenosine, intravenously administered prostacyclin, inhaled nitric oxide, or inhaled prostacyclin) remains a crucial step in the evaluation of PAH and especially of idiopathic PAH (IPAH). The current definition of acute vasoreactivity is a fall of mean pulmonary artery pressure by at least 10 mmHg to less than 40 mmHg with an increased or unchanged cardiac output [20]. Fewer than 10% of patients with PAH will demonstrate acute vasoreactivity and these are most commonly patients with IPAH. Only those patients who demon-

strate an acute response to short-acting vasodilators during a right-sided heart catheterization (i.e. “responders”) are appropriate for treatment with oral calcium channel blockers. Although these represent a minority of patients seen at PH centers, their treatment regimens and prognoses may differ dramatically from those of “nonresponders” [21]. These patients require close ongoing monitoring to be sure they maintain this clinical response over time. Right-sided heart catheterizations may be repeated on a periodic basis or at the discretion of the physician when there has been a change in the patient’s clinical status, is a need to evaluate a patient’s response to interventions, or is a need to optimize medical therapies. Through a detailed assessment and diagnostic testing, physicians and nurses can evaluate the functional level of a patient and begin to formulate an individualized treatment plan. In addition, nurses may begin to identify areas of teaching needed (i.e. fluid intake, sodium intake, daily weights, etc.), how compliant the patient has been with current medical therapies, and the patient’s motivation.

3 Developing a Treatment Plan PH patients require an individualized treatment plan based on their specific PAH diagnosis, the severity of their disease, their motivation, and their support systems. Following the initial patient visit and assessment, PH physicians and nurses work together with patients and their families to develop an individualized treatment plan. Because of the level of complexity and the cost of some PH therapies, there needs to be an extensive discussion about therapies with patients and their families. They need to understand the proposed treatment plan and the expectation of the PH team that the patient will be compliant with therapy. Patients should demonstrate motivation and commitment to adhering to the treatment plan before therapy is initiated. In regard to the treatment plan, PH nurses have a major impact in patient care. Not only can they provide extensive education for patients and their families, but they can also follow patients clinically to assess if they are having any issues or difficulties managing the treatment plan. Because nurses are frequently more accessible to patients and their families (via telephone calls, e-mail, or outpatient office visits) than the PH physicians, they are often more attuned to how a patient is managing his/her specific treatment plan. If there is a problem with adhering to the original treatment plan or the patient is not improving clinically, nurses may intervene to alter the plan or to suggest switching entirely to another treatment plan. Generally, we have found in our practice patients will do better in adhering to the treatment plan if they are educated

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about why certain therapies are being used and how they can potentially benefit from them. We spend time teaching patients about several key areas: • The pathophysiology of PAH – the specific pathologic changes that affect the pulmonary arteries and how these affect the heart to produce symptoms of right-sided heart strain and failure • Signs and symptoms of PAH – to know their current symptoms and how to monitor themselves for changes that may identify a problem or disease progression • Their specific treatment plan: • Medications – understand why they are taking each medication, know the correct dosing, be aware of potential side effects, need for compliance with therapy (no underuse or overuse), possible interactions with other medications, need for routine bloodwork to monitor therapy • Oxygen therapy – understand why supplemental oxygen is used, following prescribed dosing, safety precautions, use of portable systems • Physical exercise – understand how physical exercise helps promote pulmonary conditioning, know how to safely exercise • Routine bloodwork – understand the importance of bloodwork as a way to monitor medical therapies • Diet recommendations – know what constitutes a lowsodium diet, be aware of which foods are high in sodium, importance of complying with diet restrictions, importance of working toward their optimal weight • Fluid management – understand why it is important to monitor their weight, know when to report changes in weight, know how to avoid excessive fluid intake • Sleep – know that lack of sleep impacts how they feel, be aware of good sleep hygiene practices • Routine diagnostic testing – understand the importance of periodic diagnostic testing to monitor their PAH and their response to medical therapy. Because of the importance of the treatment plan, each specific area will be discussed in detail, beginning with medications.

3.1 Medications 3.1.1 Diuretic Therapy Diuretics have long been used in the management of PAH and right-sided heart failure to control intravascular volume as a way to reduce edema and right-sided heart dilatation and strain. Appropriate use of diuretics can dramatically improve a patient’s symptoms, functional class, and probably survival. Patients need to take their diuretics regularly and

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r­ outine bloodwork needs to be performed on a periodic basis to monitor their therapy. Nurses are well suited to help patients manage their diuretic therapy. (Specific details of fluid management will be discussed later in this section.) Patients should be instructed to call their PH nurse before attempting to alter their diuretic regimen. There are various types of diuretics available today, including loop, thiazide, and potassium-sparing diuretics. A loop diuretic, such as furosemide (Lasix®), bumetanide (Bumex®), or torsemide (Demadex®), is typically one of the first diuretic agents used. Because this type of diuretic does not spare potassium, the patient should also be given a prescription for potassium supplementation to prevent hypokalemia. For mild cases of edema, a thiazide diuretic, such as hydrochlorothiazide (Hydrodiuril®), may be appropriate. Spironolactone (Aldactone®) is also a commonly used mild diuretic choice for several reasons. First, it may spare the patient from requiring a potassium supplement to treat diuretic-associated hypokalemia. Second, spironolactone has been shown to be efficacious in reducing both the risk of death and morbidity among patients with severe heart failure (left ventricular ejection fraction less than 35%) [22]. The risk of death was reduced by 30% in patients taking spironolactone (as compared with the placebo group) and this lowered risk of death was attributed to death from both progressive heart failure and sudden death from cardiac causes. There was a 35% reduction in the frequency of hospitalization for worsening heart failure for patients taking spironolactone. In addition, patients in the spironolactone group had a significant improvement in the symptoms of heart failure, based on the New York Heart Association functional classification system [22]. Dosing of diuretics is very individualized, with the goal being to maintain the patient at an optimal (dry) weight. Common side effects of diuretics include electrolyte imbalance, nocturia, polyuria, volume depletion, dehydration, hypotension, renal insufficiency, dizziness, abdominal discomfort, nausea, vomiting, constipation, muscle spasm, and weakness [23]. When patients decompensate clinically with increased edema and right-sided heart failure, we alter their diuretic therapy, often using higher doses of furosemide (Lasix®) or adding an additional diuretic agent such as metolazone (Zaroxolyn®), or we may suggest both changes in therapy. These patients will then require close monitoring by telephone or outpatient office appointment and more frequent bloodwork. Careful attention must be paid to avoid overdiuresis, which can cause hypotension, renal insufficiency, electrolyte imbalance, orthostasis, or syncope [20]. If the patient does not diurese adequately with oral therapy, we may need to admit him/her to the hospital for more ­aggressive intravenous diuretic therapy and close monitoring of electrolytes and renal function.

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3.1.2  Anticoagulation Anticoagulants are a second class of medications commonly used for PAH patients. Warfarin (Coumadin®) has been associated in several studies with improved survival in PH patients [20, 24]. It is believed that warfarin reduces the formation of in situ microthrombi within small pulmonary vessels. There is a lack of consensus regarding if warfarin should be used only in IPAH or, in a similar manner, in other forms of PAH. There is also no agreement on what pulmonary artery pressure warrants initiating anticoagulation [25]. There have been no studies evaluating the role of other anticoagulant agents such as any of the other heparins, antiplatelet agents, or use of combination therapy [25]. Warfarin dosing is individualized and monitored by prothrombin time (PT) and international normalized ratio (INR). For prophylactic anticoagulation in PAH, the usual target INR is 1.5–2.5, but this may vary among PH centers. There are clearly patients who are not appropriate for prophylactic anticoagulation because of coexisting medical conditions, functional status, or their social circumstance. Patients with a history of atrial fibrillation, cardiac valve replacement surgery, deep vein thrombosis, or pulmonary emboli may have a higher INR as their target. Nurses provide support to patients by educating them on warfarin therapy and helping to monitor the therapy. Bloodwork can be performed on patients every 4–8 weeks if they have demonstrated stable INRs and more frequently if patients have fluctuating INRs. Nurses can review the ­bloodwork results and in collaboration with the physician adjust the warfarin therapy. Patients require education about several important points to be safe with their warfarin therapy [26]: • Patients are instructed to monitor themselves for unusual or excessive bleeding such as prolonged nosebleeds, bleeding from gums, blood in urine or red or black/tarry stools, easy bruising, prolonged bleeding from venipuncture sites, heavy menstrual periods, and coughing or vomiting blood and to immediately report such problems to the PH center nurse. • Patients need to notify the PH nurse if they experience a fall or injury (such as a motor vehicle accident) while on warfarin therapy. • Patients are continually reminded about the importance of scheduled bloodwork for PT and INR to ensure adequate and safe dosing of warfarin. • Diet recommendations: Some PH centers instruct their patients to avoid foods containing vitamin K (i.e. green leafy vegetables, fish, canola oil, soybean oil, etc.) because these foods can alter the warfarin requirements. Other PH centers encourage their patients to eat a balanced diet and to be consistent in their intake of vegetables (and the other

•

•

•

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foods listed above); then the warfarin dosing will be adjusted as needed. Numerous medications and herbal supplements may interact with warfarin therapy. Nurses must remind patients to notify the office of any changes to their medication regimen (including courses of antibiotics) or before they begin taking new vitamins or dietary supplements. Patients are instructed to avoid aspirin (acetylsalicylic acid) or aspirin-containing products, such as Excedrin® or Pepto-Bismol®, or nonsteroidal anti-inflammatory agents, such as Motrin® or Aleve®, which can contribute to bleeding when taken while on warfarin therapy. Patients are instructed to stop taking warfarin if undergoing dental work (including dental cleaning), surgical procedures, or other procedures such as colonoscopy or endoscopy. Patients should contact the PH nurse for specific directions regarding dosing of warfarin when such procedures are scheduled. It is recommended that patients wear a MedicAlert bracelet when receiving warfarin therapy.

3.1.3 Inotropic Therapy – Digoxin Digoxin (Lanoxin®) is an inotropic agent used by many PH centers particularly when the right ventricle is dilated or exhibits depressed function. Digoxin improves left ventricular function in patients with left-sided congestive heart failure. It is felt that digoxin is also an inotrope for the right ventricle and will benefit patients with PAH by supporting right ventricular function [20]. Dosing is usually 0.125–0.25 mg by mouth daily and is monitored periodically with serum digoxin levels. Digoxin is metabolized through the kidney and therefore patients with renal insufficiency may require a lower dosage. It is prudent that the digoxin levels of these patients are checked more frequently to avoid toxicity. Side effects of digoxin include nausea, anorexia, diarrhea, abdominal pain, headache, confusion, hallucinations, depression, changes in vision (yellow–green halos), and bradycardia or other disturbances in heart rhythm [27].

3.1.4 Calcium Channel Antagonists Calcium channel antagonists, one of the earliest classes of PAH medications, are used in carefully selected PAH patients (predominantly IPAH patients) for their vasodilatory effects which reduce pulmonary vascular resistance and pulmonary artery pressures. Calcium channel antagonists are indicated only in patients who have demonstrated a significant vasoreactivity response to short-acting vasodilators given during right-sided heart catheterization. Patients with right-side heart failure (cardiac index below 2.0  l/min/m2) are not

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c­ andidates for these agents because of potential cardiodepressant effects. The most frequently used calcium channel antagonist agents include nifedipine (Procardia XL®), diltiazem (Cardizem CD®) and amlodipine (Norvasc®). The choice is often based on the patient’s heart rate; those patients with relative bradycardia often receive nifedipine, whereas those with relative tachycardia receive diltiazem. Calcium channel antagonists with a significant negative inotropic effect, such as verapamil (Calan®), should be avoided [20]. Although patients typically require higher doses for PAH management than those used for systemic hypertension, most physicians initiate calcium channel antagonists cautiously in responders (at low dose) and titrate upward gradually. Long-acting forms of these agents are used frequently to help eliminate the risk of rebound symptoms if a dose is late or missed, and the total daily dose is often divided into long-acting pills taken twice or three times a day to minimize the side effects of peak serum levels. Patients treated with calcium channel antagonists should have a repeat right-sided heart catheterization after several months of therapy to confirm that the pulmonary hemodynamics are significantly improved. If the pulmonary hemodynamics are suboptimal at the time of repeat right-sided heart catheterization, an acute vasodilator trial should be repeated to assess for additional reactivity and the potential benefit of a higher dose of calcium channel antagonist therapy. Patients need to be educated to take calcium channel antagonists on a regular schedule and not to stop therapy abruptly. Missing doses or abrupt discontinuation in a ­vasoreactive patient can result in rebound pulmonary ­vasoconstriction, increased symptoms, and possibly syncope or death. Calcium channel antagonists may also be used in PAH patients to help control symptomatic Raynaud’s phenomenon, systemic hypertension, and for rate control for patients with atrial fibrillation, although, when used in these situations, these agents should not be viewed as treatment for PAH and patients should also be treated with PAH-specific therapeutic agents (see later).

3.1.5 Endothelin Receptor Antagonist Therapy Endothelin-1 is a potent vasoconstrictor and smooth muscle mitogen found to play an important role in the development of PAH [28]. Patients with PAH have a higher level of endothelin than the general population and this level correlates with disease severity and progression [29, 30]. Once this was recognized, researchers worked to develop drug therapy targeted at reducing the deleterious effects of the elevated endothelin levels.

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Bosentan (Tracleer®) Bosentan was the first oral endothelin receptor antagonist approved by the US Food and Drug Administration (FDA). It was approved in November 2001 for PAH patients with WHO functional class III or IV symptoms. More recently, bosentan has proved beneficial in patients with mild symptoms of PAH (WHO functional class II) with a reduction in pulmonary vascular resistance and an improvement in 6-min walk distance [31]. Bosentan works as a dual receptor antagonist; it inhibits both endothelin-A and endothelin-B receptors. Bosentan improves hemodynamics and exercise capacity in patients with PAH, and slows the rate of clinical deterioration [32]. Overall it is generally well tolerated by patients. Side effects observed in a 1-year follow-up safety study included low rates of headache, upper respiratory tract infection, dyspnea, chest pain, sinusitis, bronchitis, palpitations, dizziness, fatigue, cough, arthralgia, dyspepsia, epistaxis, influenza-like illness, nausea, and lower limb edema [33]. In our practice, fatigue and mild peripheral edema have been the most troubling side effects. Many patients report the fatigue resolves spontaneously after a few weeks on therapy. The mild edema is managed by adding or adjusting diuretic therapy. Bosentan has the potential to alter liver function test (LFT) levels and may be teratogenic. Because of these possible side effects, before therapy is started, baseline LFT levels must be drawn and recorded. In addition, women of childbearing age must have a negative serum pregnancy test ­documented and must be counseled regarding contraception (see later). Bosentan is initiated at 62.5 mg by mouth every 12 h. LFT levels are drawn after 1 month and, if normal, the dosage of bosentan is increased to 125  mg by mouth every 12  h. Thereafter, this dosage is maintained and liver function is monitored monthly and the CBC is checked every 3 months. If the levels of liver transaminases become elevated, they are either rechecked for confirmation and a dosage adjustment is made or bosentan therapy is discontinued depending upon the severity of the abnormality [34]. The manufacturer, Actelion Pharmaceuticals, has provided guidelines for managing elevated levels of transaminases (Table  1). Elevated LFT levels up to 3 times normal have occurred transiently in up to 12.7% of patients studied; higher elevations occur much less frequently [34]. In our practice we have seen patients experience elevations in LFT levels because of taking acetaminophen (i.e. for headaches or orthopedic strains), drinking alcoholic beverages, or after a change in their hyperlipidemia medication regimen. When these elevations in LFT levels occur, the PH nurse can counsel patients, adjust dosing as needed, and recheck the LFT levels to ensure a return to normal baseline values. Some patients may experience a modest decrease in hemoglobin values (an average of 0.9 g/dl), usually during the

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Table  1  Recommended action for abnormal liver function with bosentan therapy LFTs: ALT/AST levels Action

class II or III symptoms. Ambrisentan was approved by the FDA in June 2007. Similar to bosentan (see earlier), patients are started on ambrisentan after baseline LFT levels, CBC, and serum pregnancy test results (for women of childbearing age) have been documented. The initial dosage is 5 mg by mouth once daily. The dosage may be increased to 10  mg daily after 1  month (or later) if the LFT levels are within the normal range and if the dose increase is clinically appropriate. LFT levels are monitored every month and the CBC is checked every 3  months. Ambrisentan has demonstrated an advantage over bosentan regarding the incidence of elevations of LFT levels. In the initial clinical trials, no patients receiving ambrisentan developed elevations of LFT levels over 3 times the upper level of normal [36]. In a 2-year extension study of ambrisentan, the Kaplan–Meier analysis showed the rate of elevations of LFT levels over 3 times the upper level of normal was 4.2% [37]. Similar to bosentan, ambrisentan may cause a modest decrease in hemoglobin levels. For patients experiencing a more severe decrease in hemoglobin levels, an anemia workup should be performed. Ambrisentan also carries a pregnancy category X warning from the FDA. Women must be cautioned not to become pregnant while taking ambrisentan because of the potential for fetal defects. Monthly serum pregnancy tests are mandatory for women of childbearing age. Women are counseled to use two reliable means of contraception; at least one should be a nonhormonal barrier form of contraception [38]. Ambrisentan differs from bosentan in that it is not metabolized by the cytochrome P-450 system and therefore does not have many potential pharmacokinetic interactions. No significant interaction has been noted when ambrisentan is given concomitantly with warfarin therapy. In clinical trials, ambrisentan improved 6-min walk distance and delayed clinical worsening [36]. In the 2-year data from the extension study of the initial clinical trials, survival was 88% and the probability of no clinical worsening (defined as death, lung transplantation, hospitalization for PH, atrial septostomy, addition of prostanoid therapy, or study withdrawal due to addition of other PAH drugs) was 72% [36]. Side effects of ambrisentan include peripheral edema, nasal congestion, sinusitis, flushing, palpitations, nasopharyngitis, abdominal pain, constipation, dyspnea, and headache [38]. In the US clinical trial comparing 5- and 10-mg doses of ambrisentan with a placebo, the rate of edema peripheral edema was 26.9% (5-mg dose) and 28.4% (10-mg dose) [36]. In our practice, we have noticed a higher percentage of our older patients develop peripheral edema. This edema is occasionally quite significant and requires the addition of or adjustment of diuretic therapy. Although this side effect is not life-threatening, it does warrant providing specific education to patients. We emphasize to patients starting

>3 times to Repeat LFTs to confirm £5 times ULN If confirmed, reduce the dose or interrupt therapy; repeat LFTs every 2 weeks Restart after return to normal values ³5 times to Repeat LFTs to confirm £8 times ULN If confirmed, stop therapy and monitor every 2 weeks Consider restart after return to normal values >8 times ULN Stop therapy Do not consider reintroduction of therapy Reproduced with permission [35] LFT liver function tests, ALT alanine aminotransferase, AST aspartate aminotransferase, ULN upper limits of normal

first few weeks of treatment. This is felt to be a dilutional effect as patients may retain a small amount of fluid. For patients who experience a more significant anemia, we counsel them to pursue a diagnostic workup through their primary care provider, as we cannot assume that it is medication-related. Women of childbearing age must be informed that bosentan can potentially have teratogenic effects on the unborn fetus (FDA category X) and they must have monthly serum pregnancy tests. These women must be counseled to use adequate protection to avoid pregnancy. The efficacy of hormonal contraceptives may be altered when they are taken in conjunction with bosentan, so women using hormonal ­contraceptive methods are advised they must use two forms of birth control. At least one form of contraception should be a nonhormonal type (i.e. a barrier method, such as a condom or a diaphragm) [35]. While reinforcing the importance of avoiding pregnancy, PH program nurses may find that female patients ask questions regarding acceptable options for contraception. These nurses may provide their patients with contraception information and counseling or they may choose to refer their patients back to their local gynecologist. Another practical issue regarding bosentan is that it is metabolized by the cytochrome P-450 system and it has several potential pharmacokinetic interactions. It is contraindicated to coadminister bosentan with cyclosporine or glyburide [35]. In addition, we have found in our practice that coadministration of bosentan with warfarin may result in modestly lowered PT and INR values, necessitating the upward adjustment of warfarin dosing to maintain therapeutic levels.

Ambrisentan (Letairis®) Ambrisentan is an oral endothelin receptor antagonist thought to be more selective for endothelin-A receptors. It is indicated for the treatment of PAH in patients with WHO functional

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therapy that it is crucial that they monitor their weight daily and report the development of any weight gain or edema.

Sitaxsentan (Thelin®) Sitaxsentan, a selective endothelin-A receptor antagonist, had been in clinical trial at the time of writing this chapter. It was recently removed from the market and all clinical ­trials have been stopped because of the serious potential of liver toxicity.

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quality-of-life measures, and delayed time to clinical worsening. Tadalafil was well tolerated with headache (in 32% of patients) as the most common adverse event. Discontinuation due to adverse events was low [43]. The FDA is currently reviewing the data regarding tadalafil for treatment of PAH. Additional studies are needed to compare the efficacy of phosphodiesterase inhibitors with that of other therapies and to assess combination therapy.

3.1.7 Prostanoid Therapy 3.1.6 Phosphodiesterase Inhibitor Therapy

Inhaled Iloprost (Ventavis®)

Sildenafil Citrate (Revatio®)

Inhaled iloprost was approved by the FDA in December 2004 for patients with PAH who have WHO functional class III or IV symptoms [44]. Iloprost is a synthetic analog of ­prostacyclin that dilates the systemic and pulmonary vascular beds. It also inhibits platelet aggregation. Iloprost is available as a clear solution at 10  mg/ml for inhalation via the I-neb adaptive aerosol delivery (AAD) system. Each glass ampule contains 2  ml (20  mg) of iloprost, and a single treatment is set to deliver a dose of either 2.5 or 5.0  mg to the patient. (The remaining drug is “lost” in the delivery system.) The patient is instructed to draw the iloprost solution into a small pipette and to place the medication into the medication chamber of the I-neb AAD system. The patient then engages the device and takes the treatment holding the I-neb device in a horizontal position. Each inhalational treatment last 6–12  min while the patient breathes with steady and regular breaths. Specialty pharmacies (i.e. Accredo Therapeutics and Caremark) are available for providing education and support to patients, nurses, and respiratory therapists on this inhalational therapy. The initial treatment dose is 2.5 mg, which if tolerated, is then increased and maintained at 5.0 mg per treatment. The serum half-life of iloprost is 20–25  min. Because of this short half-life, six to nine inhaled treatments are required each day during waking hours [20, 45]. A patient may receive a maximum dosage of 45  mg/day. Because inhaled iloprost is administered on an intermittent basis, patients are instructed to administer a morning treatment immediately upon awakening (before performing any activities) to prevent syncope or other symptoms. Patients taking bosentan or sildenafil may have an apparent prolongation of the iloprost duration of action and may be able to get by with fewer doses per day [46]. Before iloprost therapy can be initiated, approval must be obtained from the patient’s insurance company. To begin this process, the PH nurse must have the patient sign a consent form releasing their insurance information and clinical

Sildenafil citrate is a phosphodiesterase-5 inhibitor that inhibits the breakdown of cyclic guanosine monophosphate, which promotes the relaxation of smooth muscle cells of the vascular bed, thus resulting in vasodilation [39]. It was approved by the FDA for patients with PAH in June 2005. In a clinical trial, sildenafil improved pulmonary hemodynamics and exercise capacity in patients with WHO functional class II and III symptoms, and 20-mg tablets (marketed as Revatio®) taken three times a day is the approved dosage [40]. Sildenafil was well tolerated at that dosage, with few minor side effects: headache, back pain, dyspepsia, flushing, diarrhea, limb pain, myalgia, and cough [40]. Optimal dosing of sildenafil is still unclear as patients have been studied at a variety of dosages (20, 40, or 80 mg three times a day or 50 or 100 mg two times a day). The current strategy at most PH centers today is to start patients at 20  mg by mouth three times a day and increase this if patient does not appear to benefit or if the patient’s disease progresses [41]. Sildenafil has vasodilator properties and may result in a lower blood pressure. Before starting therapy, PH physicians and nurses should consider each patient for medical conditions that could be worsened by a mild decrease in blood pressure. Sildenafil may not be coadministered with nitrates [42].

Tadalafil (Adcirca®) Tadalafil (Adcirca®), a longer-acting phosphodiesterase-5 inhibitor, is currently being studied for efficacy in treating PAH. In a 16-week clinical trial, tadalafil 40 mg by mouth once daily as monotherapy or added to bosentan therapy demonstrated beneficial effects [43]. PAH patients treated with tadalafil showed significant improvements in 6-min walk distance, hemodynamics, WHO functional class,

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information to one of the specialty pharmacies. The clinical information needed is documentation of the specific ­diagnosis of PAH, the WHO functional class, and the PAH diagnostic testing results. These test results include echocardiogram, cardiac catheterization, ventilation–perfusion lung scan, computed tomography scan of the chest, pulmonary function studies, and relevant collagen vascular disease serologic findings. The specialty pharmacy will then work with the patient’s insurance company to obtain the necessary approval. Coverage for inhaled iloprost falls under the patient’s medical insurance, not under the pharmacy coverage. Iloprost therapy may be initiated in the outpatient clinic or in the hospital, with vital signs monitored closely. Inhaled iloprost should not be started for a patient who exhibits relative hypotension (systolic blood pressure below 85  mmHg) [47]. Physicians and nurses should be aware of concomitant conditions or medications that may increase the risk of hypotension or syncope. When inhaled iloprost is initiated, patients should be watched closely for signs of pulmonary edema. If these occur, the treatment should be stopped immediately, because this may be a sign of pulmonary venous hypertension (i.e. pulmonary venoocclusive disease) [47]. In a long-term study, inhaled iloprost treatments were well tolerated. Mild coughing, headache, and jaw pain occurred in some patients [45]. Patients may develop prostanoid side effects such as, headache, chest pain, flushing, dizziness, jaw pain, nausea, vomiting, and diarrhea if dosing is excessive. Reducing the dose of iloprost to 2.5  mg per treatment or decreasing the number of treatments per day may help patients in this instance. We instruct patients to notify us immediately should they develop any adverse effects. Patients receive individualized teaching on the inhaled iloprost therapy by clinical nurses, specialty pharmacy nurses, and/or respiratory therapists. Patients are taught about the medication itself, how to administer the inhalational treatments using slow and regular breaths, how to clean and care for the I-neb AAD device, and symptoms to report to the PH center nurse. Additional points emphasized during patient teaching include:

• Patients must follow the specific cleaning protocol designed by the manufacturer to maintain optimal functioning of the I-neb AAD device. Noncompliance may result in improper dosing and long treatment times. • When taking a treatment, other persons (especially babies and pregnant women) should not be exposed to the iloprost. • If iloprost therapy is initiated in the hospital setting, the patient is discharged when clinically stable and independent in self-administering the treatments. A follow-up nursing visit from the specialty pharmacy is scheduled within 1 week of discharge to reinforce teaching and to evaluate the patient’s status.

• Iloprost solution should not come in contact with skin or eyes. Iloprost should not be taken orally. • Patients should rinse their mouths after each inhaled treatment to prevent glossitis from any iloprost that may have come in contact with the tongue. • The I-neb AAD device was specifically designed to deliver accurate dosing of the iloprost inhalational solution. This device is not a nebulizer. Patients should not mix any other medications with the iloprost. Similarly, other inhaled medications may not be administered with the I-neb AAD device.

On a practical level, we have found that patients who have been on inhaled iloprost therapy chronically require continued nursing support and education. One issue affecting these patients is the time commitment. In our outpatient clinic patients complain they become tired of performing the iloprost treatments every 3 h. They express frustration with the time commitment saying the treatment administration times are taking longer than expected. In these instances, we ask a nurse from the specialty pharmacy to make a home visit to reassess how patients are self-administering the iloprost treatments. In many instances we have found patients are incorrectly following the treatment protocol. In one instance, the patient was not focusing solely on administering the inhalational treatment, but rather trying to perform other household activities simultaneously. The I-neb AAD device is designed to sense the patient’s breathing pattern and then dispense the iloprost upon inhalation. Therefore, the I-neb AAD device will only administer the medication if the patient is breathing in a regular steady breathing pattern. In another situation, a patient with progressive PAH disease who was deconditioned became fatigued performing the treatments and had difficulty breathing in a regular steady pattern. Our solution was to teach her to pause her I-neb AAD device and to take a short break mid-treatment for rest. A second issue affecting these patients is managing the screens within the I-neb AAD device which are critical for aerosolization of the iloprost solution. Patients have experienced problems with the screens in the I-neb AAD device becoming clogged and thereby making treatment times excessive. The specialty pharmacies have worked closely with the manufacturer to supply patients with additional screens and have developed a revised cleaning protocol. Our patients report this has helped them to reduce treatment times back toward reasonable levels. To summarize, we feel it is important to listen to the concerns of the patients even if they have been on therapy on a long-term basis. If they feel they are supported and they are being helped to work through the practical problems, they will continue to comply with therapy. Patients need continued collaborative support from the PH center, the specialty pharmacies, and the manufacturers.

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Subcutaneously Administered Treprostinil(Remodulin®) Treprostinil sodium is a prostacyclin analog developed as an alternative to epoprostenol. It was approved for continuous subcutaneous administration by the FDA in May 2002 for PAH patients with WHO functional class II, III, and IV. Similar to epoprostenol, it is a potent pulmonary and ­systemic vasodilator and inhibitor of platelet aggregation. Continuous subcutaneous treprostinil therapy is typically used for patients who are not candidates for or who have failed oral therapies or in patients on intravenous epoprostenol therapy who have experienced recurrent intravenous line infections [48]. Treprostinil has several pharmacologic properties that are potential advantages when compared with epoprostenol. First, it has a half-life of 3–4 h when administered subcutaneously; the longer half-life can be viewed as a safety issue should there be an inadvertent interruption in therapy. Second, it is stable at room temperature, which means it does not need to be kept cold with ice packs. Third, it is stable at neutral pH. Because of these properties, treprostinil may be administered subcutaneously as a continuous infusion using a small syringe pump. Treprostinil is dispensed in a 30-ml multidose vial in four concentrations. Each vial is very expensive and contains approximately a 2-week supply of medication. Many hospitals will not purchase and stock the treprostinil; they expect patients will bring their own supply when admitted to the hospital. Treprostinil is available from three specialty pharmacies (Accredo Therapeutics, Curascript, and Caremark). In a manner similar to inhaled iloprost, the patient must sign a consent form allowing his/her insurance information and clinical data to be reviewed by the specialty pharmacy. The specialty pharmacy will work with the patient’s insurer to obtain clearance for the medication. Treprostinil therapy is covered under the patient’s medical insurance rather than under the pharmacy coverage. Once the insurance clearance has been obtained, patients may begin therapy. Patients may be started on subcutaneous treprostinil therapy in the outpatient clinic or in the hospital setting. Because of its potent vasodilatory effects, ­treprostinil should be started in a monitored setting with emergency equipment and staff readily available. Patients need to learn how to be independent in caring for the treprostinil infusion. They must learn how to operate the small portable syringe pump; it is the same system used by some diabetic patients for continuous subcutaneous insulin delivery (MiniMed, Medtronic). Patients must learn how to fill a syringe with treprostinil, set the pump, attach tubing, and insert a small cannula into a subcutaneous site (Fig. 1). The whole system is changed every 3–7 days or more ­depending

Fig. 1  Placement of a subcutaneous catheter for treprostinil therapy. (Reproduced with permission of the Pulmonary Hypertension Association)

on the condition of the injection site. Patients receive extensive training on the system, including safety measures such as how to respond to pump alarms, pump malfunctions, and injection site problems. The specialty pharmacies have a hotline telephone number for patients to call to speak to experienced nurses 24  h a day in the event of an emergency. It is recommended that dosing begin at 1.25  ng/kg/min and is increased each week [48]. Most PH centers titrate the dosing up as quickly as the patient can tolerate to help the patient achieve clinical improvement. Patients who achieved a dosage of 13 ng/kg/min or more by the end of a 12-week clinical trial were able to walk significantly further than those at lower dosages [49]. Common treprostinil side effects include headache, diarrhea, jaw pain, nausea, rash, and infusion site pain. Infusion site pain and reaction is the most challenging problem with subcutaneous treprostinil infusion. This ­problem affects most patients. Although this suggests slower dosing increases, it is generally agreed that there is little or no relationship between the dosage of treprostinil and infusion site pain. Patients may experience severe site pain at low doses [49]. There are several strategies for relieving site pain, ranging from varying the size and shape of the insertion needle, rotating insertion sites less frequently, to use of various medications from oral analgesics to narcotics. Patients starting treprostinil therapy require a lot of emotional support to continue the therapy during the first few months. Some patients experience an improvement in the site pain after 3–6  months, whereas in others the pain remains significant.

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A long-term outcome study in PAH patients treated with subcutaneous treprostinil therapy for up to 4 years revealed 68–87% survival, although most patients were not on subcutaneous treprostinil monotherapy [50]. Of the 860 patients in the study, 57% of the patients discontinued subcutaneous treprostinil therapy during the follow-up: 23% discontinued therapy owing to adverse events, 16% died, 14% discontinued therapy owing to deterioration, 3% withdrew consent, and 1% underwent transplantation. Furthermore, another 26% required a change of therapy: 11% switched from subcutaneously administered treprostinil to another prostanoid, 12% added bosentan, and 3% added sildenafil. The safety profile was consistent with the safety profiles of the previous short-term trials. Infusion site pain (affecting 92% of patients) and infusion site reaction (erythema, swelling, induration or rash affecting 81% of patients) remained as significant problems for patients on this therapy.

Epoprostenol sodium is available as a lyophilized white powder in glass vials of 0.5  mg (blue-top vials) or 1.5  mg (red-top vials). It must be reconstituted with a specially formulated alkaline diluent into a medication cassette (most commonly 100  ml). Epoprostenol may not be mixed or reconstituted with any other parenteral medications or solutions before or during administration [51]. It is relatively unstable in solution and must be kept cold (with the use of small ice packs) to remain effective; however, even when kept cold, once reconstituted, it will only remain stable with predictable potency for 48 h. Epoprostenol has an extremely short half-life (only 2–3 min) and thus requires administration by a continuous intravenous infusion without interruption. If drug delivery is interrupted or stopped, the patient may rapidly lose therapeutic effects and there is a risk of rebound PH, clinical deterioration, and even death [3, 20]. Epoprostenol is administered as a continuous infusion with a CADD-Legacy ambulatory pump through a central venous catheter [such as a Hickman catheter, a Broviac catheter, or a peripherally inserted central catheter (PICC)] (Fig.  2). It must be prepared daily and kept cold throughout the day with ice packs. Patients receive extensive education regarding the pharmacokinetics of the medication and safety measures to avoid interruptions in therapy. Before therapy with epoprostenol is initiated, careful ­consideration must be given to patient selection. Deciding to use intravenous epoprostenol therapy requires attention to both medical and psychosocial issues. From a medical standpoint, patients typically fit into two scenarios.

Intravenously Administered Epoprostenol(Flolan®) Epoprostenol sodium (Flolan®) was approved by the FDA in 1996 for the treatment of primary PH (now classified as IPAH) patients with WHO class III or IV symptoms; it was subsequently approved for PAH in the scleroderma spectrum of diseases. It is a potent pulmonary and systemic vasodilator. Epoprostenol inhibits platelet aggregation and may also have significant inotropic effects on the right side of the heart [20].

Fig. 2  Central line catheter placement. (Reproduced with permission of the Pulmonary Hypertension Association)

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The first are patients who are failing traditional oral and inhaled therapy. They are experiencing progressive signs of cor pulmonale (deteriorating exercise capacity, intractable edema, worsening oxygenation) and worsening hemodynamics. The second are patients who come to medical attention emergently in severe right-sided heart failure (cardiac index typically 2.0 l/min/m2, right atrial pressure more than 20  mmHg, episodes of syncope, generalized edema) [52]. Judging a patient’s capacity to manage this complicated therapy is a critical issue. It is sometimes difficult to assess how a patient will adapt to ­managing a complex therapy when he/she is actively experiencing a medical crisis situation (i.e. acute cor pulmonale). In addition, in acute crisis situations, it may not be possible to accurately evaluate the patient’s family and social support system. The PH center’s nurse needs to collaborate closely with the PH physician to evaluate physical and psychosocial issues in determining whether continuous intravenous therapy with a central venous catheter is safe and appropriate for the patient [53]. Considerations include: • Has the patient demonstrated compliance with medical therapy in the past? • Does the patient independently perform self-care activities or does he/she depend on others? • Is the patient motivated to prepare the epoprostenol infusion daily and to care for a central venous catheter safely? • Is the patient willing to accept placement of a central venous catheter? • Does the patient have a history of substance abuse or mental illness that may make caring for such a complex system risky on a long-term basis? • Does the patient have a stable home environment (telephone service, electrical service, etc.)? • Does the patient possess the cognitive skills to follow a sterile protocol and to troubleshoot alarms? • Does the patient have physical limitations (loss of digital mobility from collagen vascular disease, loss of hearing, poor eyesight) that would make handling the infusion pump or its supplies difficult? • Will the patient be able to manipulate small drug vials, needles, and syringes in a sterile fashion to prepare the infusion? • Does the patient have a social support system (spouse, significant other, family, or close friend) committed to assisting with care of the infusion when the patient is unable? It is important to discuss the issues pertaining to the care of the epoprostenol infusion therapy with both the patient and ­family/ support persons before initiating therapy. All ­individuals involved need to make a commitment to caring for the infusion. A lack of such ability or commitment may result in a life-threatening complication after therapy is initiated.

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When a patient has been identified as appropriate for intravenous epoprostenol therapy, it is best to initiate the process of obtaining insurance approval immediately. Generally, epoprostenol is covered while the patient is an inpatient, but before discharge, insurance approval must be secured for the outpatient aspect of ongoing care. The outpatient insurance approval covers all necessary supplies such as epoprostenol medication vials, sterile diluent, needles, syringes, medication cassettes, tubing, central line dressing kits, rental of two CADD-Legacy infusion pumps, and in-home nursing visits. Epoprostenol therapy is billed under the home infusion benefit of a patient’s medical insurance policy. For patients who may have inadequate insurance coverage, an expedited application for state or federal coverage may need to be filed. A hospital social worker may be helpful to assist with submitting this paperwork. PH nurses seeking to obtain insurance approval can make a referral to Accredo Therapeutics, currently the sole specialty pharmacy that provides Flolan® brand epoprostenol therapy. The specialty pharmacy will need the patient’s demographic and insurance information. In addition, it will need clinical data that confirm the diagnosis of PAH (heart catheterization, echocardiogram, 12-lead electrocardiogram, ventilation–perfusion scan, chest radiograph, chest computed tomography scan, antinuclear antibody results, and pulmonary function testing results). Documentation regarding the patient’s WHO functional class and the result of vasoreactivity testing and response to oral vasodilator agents (if any) are required (i.e. “The patient had worsening symptoms and progression of disease despite being on nifedipine.”). Such clinical data support the medical necessity of epoprostenol therapy in the event the insurer questions the need. The specialty pharmacy will contact the patient’s insurer and assist in obtaining approval for the patient. Epoprostenol therapy is initiated on an inpatient basis with close clinical monitoring because of its potential for severe side effects, including systemic hypotension and pulmonary edema. Dosing may be approached differently at various PH centers, but in general, an intravenous epoprostenol infusion is initiated at 2  ng/kg/min and gradually increased, usually over several days, until a balance is achieved between improved symptoms (i.e. dyspnea, poor oxygenation, reduced exercise tolerance, etc.) and toleration of side effects. We refer to this as the “acute dosing phase.” The most common adverse effects that may occur during the acute dosing phase include jaw pain, headache, diarrhea, systemic hypotension, nausea, vomiting, flushing, rash, and photosensitivity [52]. The jaw pain (trismus) is an usual symptom that affects almost all patients as they take their first bites of food at mealtimes. Patients describe it as a muscle spasm or dull pain in the mandibular area. As they continue to chew their food, the pain gradually subsides. We have found it helpful to warn patients ahead of time that

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they may experience this pain with acute dosing, thereby reducing their anxiety when it occurs. Some of our patients have experienced this sensation in response to the smell of food, leading us to wonder if it may relate to salivary glands and not the chewing activity. Chronic side effects of epoprostenol may include anorexia, weight loss, ascites, thrombocytopenia, pancytopenia, leg and foot pain, and high cardiac output failure (if the dosing is excessive) [51]. Complications may occur related to the central venous catheter, including local catheter site infections, catheter tract infections, bacteremia and/or sepsis, thrombosis, catheter line malfunction (hole or tear), obstruction, and catheter line displacement. Complications associated with an infusion pump include incorrect programming, battery failure, pump not turned on, improperly attached medication cassette, and catheter clamp not opened [52]. Once the patient has demonstrated some clinical improvement with the initiation of an epoprostenol infusion, we begin to focus on teaching the patient and family or support persons how to care for the infusion at home. Many PH centers have developed written materials for patients to teach them how to care for the epoprostenol infusion. These materials are helpful for patients and family or support persons to review what they have learned after the teaching session has ended. It is important to initiate epoprostenol teaching slowly so as not to overwhelm the patients. Typically these patients are beginning to recover from cor pulmonale; they may still be somewhat dyspneic, needing high flow oxygen, and may be fatigued, anxious, and overwhelmed. Most patients are unfamiliar with sterile technique and have never worked with needles and syringes, and are frightened to handle them. They are often clumsy in handling the supplies required to prepare the infusion. Some patients are weak and deconditioned and have difficulty working the syringe plunger. It is best to watch over them closely and to guide them to avoid mistakes such as their poking themselves with the needle. To help patients reduce their anxiety, we divide the teaching into several sections:

Patients learn in different ways and this is clearly evident when teaching a complex therapy such as managing intravenous epoprostenol therapy. PH nurses know it is very important to match their teaching technique to their patient’s learning style. For instance, some patients learn by a hands-on approach and are easily distracted if trying to refer to written materials. These patients learn better by following or mimicking a demonstration of preparing the infusion and handling the supplies. Other patients learn by following step-by-step written instructions similar to following a recipe. Nurses who can assess a patient’s style of learning and adjust their teaching technique accordingly will provide the most effective education. It is also helpful to have only one or two designated nurses teach a patient. When too many nurses teach a patient, each may use different terminology or present a different style to a task and the patient can easily become confused. Consistency with technique makes teaching go more smoothly. Accredo Therapeutics offers inpatient nursing support to assist with teaching patients and family or support persons regarding intravenous epoprostenol therapy. It is important to include the patient’s family or support persons in the teaching sessions. A trained family member or support person (the “backup person”) is essential in case a situation occurs where the patient is unable to mix or manage the epoprostenol infusion independently. In addition to learning while watching the patient, the support person can assist by providing emotional support. Individualized teaching sessions, a slow and repetitive pace of teaching, and emotional support are vital to a successful outcome. A central venous catheter is placed after insurance clearance has been secured and while the patient is focused on learning. We consult either the general surgery or the interventional radiology department for placement of an adultsize single-lumen Hickman catheter. Typically the catheter is inserted through a small incision near the clavicle and tunneled under the skin to an exit site near the sternum. The whole procedure takes approximately 30  min. Other PH centers use a smaller catheter, such as a pediatric-size Hickman catheter or a Broviac catheter. We avoid placement of multilumen catheters, which require routine flushing of the extra ports, as this may increase the risk of infection. A patient should be discharged only when independent and comfortable caring for the epoprostenol infusion. Following discharge, each patient should have a follow-up visit by a nurse from the specialty pharmacy (Accredo Therapeutics for intravenous Flolan® therapy). This home visit is important to support the patient’s transition to home. The specialty pharmacy nurse can review and reinforce epoprostenol therapy techniques, teach the patient and family how to stock and order additional supplies, review central venous catheter care, and review emergency procedures.

1. A brief overview of epoprostenol therapy (including the pharmacokinetics of the medication – short half-life, keep cold, etc.) 2. How to mix the epoprostenol medication 3. How to attach the medication cassette to the infusion pump and how to set the pump 4. How to care for the central venous catheter 5. What to do in an emergency situation By focusing on one section at a time, patients can gradually build self-confidence. Generally, it takes about 4–7 days of hands-on training for most patients to become proficient with epoprostenol therapy and to become comfortable with the equipment.

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If there are unexpected problems, the specialty pharmacy nurse may notify the PH center nurse to intervene before complications become of a severer nature. Epoprostenol dosing is very individualized. Once a patient has been discharged, we begin the chronic dosing phase. Upon discharge, the patient is given a schedule of small dose increases for their epoprostenol infusion; we keep the dose steps small to try to minimize side effects. Once patients have completed this schedule, they are to call the office for further instructions. Patients are instructed to call their PH nurse if they feel the dose is too low (and they are having dyspnea, fatigue, or fluid retention) or too high (and they are experiencing signs such as excessive diarrhea, nausea, headaches, tachycardia, or skin flushing). The goal is to increase the intravenous epoprostenol dose in a manner to prevent or reduce the symptoms of PAH and to keep the patient comfortable from excessive side effects. Over time, patients become proficient at evaluating how they feel and recommending how their infusion should be adjusted. There are two common problems that patients on intravenous epoprostenol therapy encounter: Hickman catheter line displacement and Hickman catheter infections. Hickman catheters will last for several years if they are properly maintained. Unfortunately, patients may inadvertently pull on their catheter and tubing during daily activities. In these instances, we have taught patients to call our office and describe the event. If the catheter was pulled significantly and there is a chance it has become displaced, we instruct them to call 911 for an EMS squad to insert a peripheral intravenous catheter or a peripheral heparin lock and for the patient to restart the infusion immediately via that peripheral site. The patient will then be transported to the local hospital for assessment of the catheter, and for replacement of the Hickman catheter or insertion of a percutaneous inserted central catheter (PICC line) if it has been displaced. If the catheter was pulled gently, we make sure the patient is not experiencing any PH symptoms and ask the patient to get a chest X-ray locally to confirm the position of the catheter. Some PH centers obtain an annual chest X-ray to verify Hickman catheter tip placement to ensure that the catheter has not moved out of optimal positioning. Hickman catheter site infections should be addressed quickly or they may progress to a bacteremia and sepsis. Patients are taught to recognize and report signs of infection: redness, tenderness, swelling, drainage, fevers, and chills. For local Hickman site infections, broad-spectrum oral antibiotics may be appropriate as the initial therapy. If the patient experiences recurrent site infections, consideration should be given to removal of the Hickman catheter line. If the patient exhibits signs of fevers or bacteremia, peripheral blood cultures should be obtained. Positive blood cultures warrant admission for initiation of a 14-day course

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of intravenous antibiotic therapy, Hickman catheter line removal, and PICC line insertion. Our PH center utilizes a double-lumen PICC line in this instance to infuse both the intravenous antibiotic therapy and the epoprostenol therapy. Patients must complete the intravenous antibiotic therapy and have negative follow-up blood cultures before a Hickman catheter will be replaced.

Intravenously Administered Generic Epoprostenol In April 2008 the FDA approved a generic epoprostenol marketed by TEVA Pharmaceutical Industries. It is claimed to be bioequivalent to GlaxoSmithKline’s brand Flolan®. Generic epoprostenol is available in the 0.5-and 1.5-mg vial strengths and utilizes an equivalent sterile diluent. The advantage is that this generic formulation is less expensive than Flolan®; however, to date, no clinical trials on humans have been performed. A second epoprostenol was approved by the FDA in June 2008. It is to be marketed by Actelion Pharmaceuticals. It is not identical to Flolan®, and at the time of the writing of this chapter it is not yet clinically available. It will be available as a 1.5-mg vial of epoprostenol that may be reconstituted with water or sodium chloride for injection. No special diluent is required. Once reconstituted, it is stable at room temperature for 24 h.

Intravenously Administered Treprostinil(Remodulin®) Treprostinil, in its intravenous form, is a prostacyclin analog similar to epoprostenol. It was approved by the FDA for intravenous use in November 2004. It is a potent pulmonary and systemic vasodilator and inhibits platelet aggregation and it may have some advantages over epoprostenol. It has a half-life of approximately 2 h, which provides a larger safety margin in case of an infusion interruption. Treprostinil is stable at neutral pH and at room temperature and thus does not require the use of ice packs [48]. Furthermore, because the drug is more stable in solution, treprostinil can last up to 48 h before a change of medication cassette is needed. These advantages make caring for this infusion system easier for patients. The principles of obtaining intravenously administered treprostinil from a specialty pharmacy, selecting appropriate patients, initiating therapy, and patient teaching are all similar to those of intravenous epoprostenol therapy. Dosing is also similar with the goal of relieving patients’ PH symptoms but not causing adverse side effects by titrating up too rapidly or in too big dosing increments. In our experience, patients have required higher doses of intravenously ­administered treprostinil (more than 3  times higher) as

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c­ ompared with intravenously administered epoprostenol. In addition, the cost of intravenous treprostinil therapy is higher than that of intravenous epoprostenol therapy. Less is known regarding the long-term efficacy of intravenous treprostinil therapy than that of epoprostenol therapy. In September 2006 the Centers for Disease Control and Prevention was notified of a suspected increase in the incidence of Gram-negative bloodstream infections (BSIs) among PAH patients treated with intravenous treprostinil therapy [54]. A retrospective study of patients receiving intravenous prostanoid therapy (either epoprostenol or treprostinil) from January 1, 2004 through late 2006 was then performed [55]. Chart reviews were conducted at two large PH centers and a survey of infection control practices was conducted at one center. In this study, 224 PAH patients had been given intravenous prostanoid treatment, corresponding to 146,093 treatment days during the study. Overall, there were 0.55 cases of BSI and 0.18 cases of BSI from Gram-negative organisms per 1,000 treatment days. BSI rates were higher in patients who received intravenous treprostinil therapy than for patients who received intravenous epoprostenol therapy (1.13 vs. 0.42 BSIs per 1,000 treatment days). Furthermore, the rates of BSIs from Gram-negative organisms were much higher in the patients receiving intravenous treprostinil therapy (0.81 vs. 0.04 per 1,000 treatment days) [55]. The survey did not identify any significant differences in medication-related infection control practices [55]. PH nurses and physicians caring for PAH patients on intravenous prostanoid therapy need to be aware of the risk for development of Gram-negative infections and to consider choosing appropriate empiric antibiotic therapy when a catheter-associated infection is suspected. In addition, guidelines for prevention of BSIs with prostanoid therapy were developed and published by a group of PH health care professionals and the Scientific Leadership Council of the Pulmonary Hypertension Association [56].

Patients can be reluctant when told they need supplemental oxygen. They may feel embarrassed to be seen wearing the nasal cannula, or they do not want to deal with handling the oxygen cylinders or tanks. We emphasize to patients that they must consider oxygen therapy as a vital medication. Patients must qualify for oxygen therapy with a low oxyhemoglobin saturation (below 88%) recorded by a health care professional. It requires a prescription from a physician or nurse practitioner which details flow rates in liters per minute and directions for use (i.e. with activity, during sleep, or continuously, etc.). We expect patients to use the oxygen therapy as ordered since it is an essential part of the treatment plan. Likewise, patients who have been diagnosed with sleep apnea for whom CPAP or BiPAP therapy has been prescribed should demonstrate compliance with therapy. In our practice we generally will not evaluate additional medical therapies to manage PAH unless a patient has been using prescribed oxygen therapy consistently or CPAP/BiPAP therapy reliably for a minimum of 3 months. It is important that the supplemental oxygen system meet the patient’s needs. For patients who require oxygen at night, we recommend a concentrator. For patients who require oxygen with activity, we prefer an ambulatory system that is lightweight and refillable so that patients can get out of the house and remain as physically active as possible. A conserving device may also be helpful to make the patient’s portable oxygen supply last longer. (Patients must be evaluated on the conserving device to ensure they can safely maintain adequate oxyhemoglobin saturations to qualify for this type of equipment.) If patients report they are experiencing nosebleeds and dry nasal passages, we recommend use of humidification. A humidification bottle can be added to the concentrator system or the patient can set up a room humidifier unit in his/her bedroom. (Patients must be reminded to follow the manufacturers’ recommendations for cleaning the unit to avoid the growth of mold.) Patients may also use a topical normal saline spray (such as OCEAN® nasal spray) to moisten nasal passages and ease dryness. Patients should be educated regarding necessary safety precautions required for the use of supplemental oxygen therapy. It should never be used near an open flame, such as that of a gas cook stove. Supplemental oxygen should never be used when a patient is actively smoking or near others who are smoking. Although oxygen is not explosive, it does potentiate and accelerate combustion. Unfortunately, we have seen patients who have used their oxygen improperly and suffered severe (commonly facial) burns. It is important to periodically reevaluate each patient’s oxygen requirements because the clinical condition may change. We check oxyhemoglobin saturation at each outpatient clinic visit. An oxygen desaturation exercise test or a 6-min walk test may be required to titrate oxygen requirements during activity.

3.2  Oxygen Therapy The first line of medical therapy for PAH is supplemental oxygen. Patients who are hypoxemic increase their pulmonary vascular resistance and may manifest hypoxic pulmonary vasoconstriction, which elevates pulmonary artery pressures and thereby increases symptoms and the risk of right-sided heart failure [20]. We generally attempt to maintain an oxyhemoglobin saturation of over 90%. However, in patients with congenital heart disease (i.e. Eisenmenger syndrome), a patent foramen ovale, or interstitial lung disease, this may not be feasible with routinely achievable oxygen flow rates.

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For patients with nocturnal desaturations or sleep apnea, an overnight record of oximetry or a formal sleep study may be needed to assess requirements during sleep. Most insurers require an annual evaluation of oxygen needs for patients to requalify for coverage of oxygen therapy.

3.3  Physical Exercise For many years, there was a general belief that physical activity or exercise may be harmful to PAH patients. It was believed that with physical activity, PAH patients may have a higher risk of sudden cardiac death, worsened right-sided heart failure, and progression of their disease. Therefore, many physicians counseled their PAH patients (especially those patients with advanced PAH disease) to avoid physical exercise [57]. In a small clinical trial, Mereles et al. followed chronically ill PAH patients through a 15-week respiratory and exercise training program and found that PAH patients were able to safely perform low levels of exercise without adverse effects. The training consisted of bicycle ergometry, walking, dumbbell training with low weights, and respiratory training with stretching and breathing techniques. The training, supervised by physicians and physical therapists, was limited by a heart rate less than 120 beats per minute, oxygen saturation above 85%, and subjective physical exertion. At the end of the training program, these PAH patients demonstrated significant improvement in exercise capacity, 6-min walk distance, ­quality of life, WHO functional class, and peak oxygen ­consumption. Although this study was limited by the small number of patients, it does suggest that PAH patients may safely perform low levels of exercise with substantial clinical benefits [57]. Supporting the concept that regular physical exercise is beneficial to PAH patients, the Pulmonary Hypertension Association’s Scientific Leadership Council developed a consensus statement on recommendations for exercise [58]. For patients with PAH, it is recommended that they stay physically active to promote physical conditioning of muscles, but they should avoid vigorous activity to the point of exhaustion. ADLs and light to moderate aerobic activities such as walking, bicycling, swimming, and light-resistance training of small muscle groups such as lifting hand weights are examples of acceptable levels of physical exercise; PAH patients should not be lifting heavy weights [58]. Patients should discuss how much exercise is reasonable with their PH physician and nurse before beginning to exercise regularly. Patients should be instructed to pace themselves with exercise and to rest when short of breath or fatigued. They should stop exercise if they feel light-headed or experience severe dyspnea or chest discomfort [58].

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Patients should be reminded to use their supplemental ­oxygen when exercising (as prescribed) to avoid oxyhemoglobin desaturations. PAH patients also gain psychological benefit from being physically active. When we evaluate patients that have become too sedentary and are deconditioned, we enroll them in a formal pulmonary rehabilitation program for pulmonary conditioning and muscle strengthening. The benefit of exercise helps them not only become more independent with ADLs, but they also have social interaction with other patients. They become more motivated to reach identified exercise goals to achieve a better quality of life.

3.4 Routine Bloodwork Patients must understand that part of their treatment plan involves routine bloodwork so the PH nurse and physician can assess how well the therapies are helping the patient and to meet FDA requirements for endothelin receptor antagonist therapy. Diuretic therapy can be assessed with a basic metabolic panel. Anticoagulation with warfarin requires a PT and INR test approximately every 4–6 weeks, more frequently if there are significant variations in levels, less frequently when the levels are stable. Patients on endothelin receptor antagonists are required to have routine bloodwork by the FDA: LFTs and serum pregnancy testing monthly and CBCs every third month. Other bloodwork may be needed to assess a patient’s specific concurrent conditions (i.e. BNP level, anemia studies, thyroid function studies, etc.). Patients must be compliant with having bloodwork for their safety. We advise patients to call their insurance company and inquire about the correct protocol for having outpatient bloodwork. Some patients are mandated to specific outpatient laboratories by their insurance company. Patients must comply with their insurer’s policies or risk being not covered for the costs of the bloodwork.

3.5 Diet Recommendations PAH patients should consume a healthy well-balanced diet. Most patients consume three meals a day, but some benefit from eating four or five smaller-portioned meals because it prevents them from feeling too full and uncomfortable. Overeating may result in abdominal distension (and decreased diaphragm excursion), which may cause dyspnea in some patients. Patients should be encouraged to consume a no-addedsalt diet, including refraining from using table salt and avoiding foods high in sodium, such as pickles, olives, pickled foods, processed meats (hot dogs, salami, sausage, pepperoni,

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smoked meats, dried beef), cheese, salty snack foods, salted crackers, canned soups, and processed frozen foods/dinners. We remind them that condiments such as ketchup, steak sauce, relish, and soy sauce are also high in sodium. We recommend they read food labels carefully when at the grocery store and aim for a diet with no more than 1,500  mg of sodium per day. For patients on warfarin therapy, the PH program protocol should be checked. Some physicians tell their patients to avoid foods containing vitamin K (e.g. leafy green vegetables, fish, canola oil, and soybean oil), because they can alter warfarin requirements. Other physicians advise patients to eat a healthy diet consistently and warfarin dosing will be adjusted accordingly. Patients with other health concerns such as diabetes, obesity, or hyperlipidemia should also follow the specific diet recommendations as indicated by their health care team.

3.6 Weight Patients should be instructed to weigh themselves daily in the morning before breakfast and to identify edema (swelling of their feet, legs, and abdomen). They should be instructed to call their PH nurse if their weight has increased by 3–4 lb rather than after they have retained larger amounts of fluid. PH nurses can alter the diuretic regimen over the telephone and have patients call back in a few days to report progress with diuresis. It is important to explain to patients that ­diuretics work by creating a degree of intravascular volume depletion and so they will probably feel more thirst. Patients should be reminded to resist the urge to drink more fluid. We generally recommend that patients restrict their fluid intake to five 8-oz glasses a day (approximately 2  l/day). This restriction includes all liquids (coffee, tea, soda, water, sports drinks, milk, juice, etc.) and ice chips. For patients with severe edema we place them on a more stringent fluid restriction of 1.2–1.5 l/day. Using sugarless hard candies, such as lemon drops, for the sensation of a dry mouth, may reduce their desire to drink more fluid. Nurses can also send the patient for bloodwork to monitor electrolytes and replace them as needed and to monitor kidney function (blood urea nitrogen and creatinine).

3.7 Sleep In our practice we often hear patients report that they cannot get a good night of sleep. Although there is no known link between insomnia and PH, it is important that patients have a full night of sleep.

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We encourage patients to use good sleep hygiene practices: • Patients should remain as physically active as possible during the day and avoid taking naps. If patients must nap, they should keep the nap to 1 h or less. • Patients should try to adhere to a regular schedule (arise in morning, eat meals, take medications, perform ADLs, retire to sleep) on a daily basis. • Patients should avoid eating a heavy meal or drinking caffeine close to bedtime. • Patients should use their bedroom for sleep. They should not use a computer, watch television, or eat in their bedroom. • Bedrooms should be conducive to relaxation. They should be dark, quiet, and kept at a comfortable temperature. • Patients should establish a bedtime routine that promotes relaxation at the end of the day. They should take a warm bath, write in a journal, read a book/magazine, or perform other quiet activity. • For patients who still have insomnia despite good sleep hygiene practices, we may prescribe temazepam (Restoril®) or zolpidem (Ambien®), starting at the lowest possible dose and titrating as needed.

3.8  Serial Diagnostic Studies After a patient has been started on therapies for PAH, the PH center physician will consider the appropriate timing for obtaining follow-up diagnostic studies such as an ­echocardiogram or a cardiac catheterization to evaluate if the patient has had any improvement or deterioration. On the basis of the results of those studies and the patient’s clinical status, the PH physician may chose to continue the present therapy, add to the therapy, or alter the therapy.

4 Patient and Family Support 4.1 Providing Support Providing support to the patient and his/her family is as important as choosing appropriate medical therapy. Patients often come to our clinic for the first visit frightened about their diagnosis after having read survival statistics on the Internet. Most survival data on the Internet are derived from older research dating back to the mid-1980s before any PAHspecific drug therapies were available. In that first clinic visit we spend time with the patient and his/her family and provide them with a general overview of PAH. Patients need to be educated about the disease process of PAH, how this

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d­ isease is evaluated, what the presently available treatment options are, and about their life expectancy. We spend time discussing what therapeutic options the patient may choose on the basis of his/her symptoms and motivations. Together we make a plan for diagnostic testing and/or making a change in the medical regimen on the basis of the patient’s wishes and the PH physician’s and nurse’s recommendations. We allow patients ample time to ask all their questions and express their concerns so that they leave the clinic visit feeling satisfied that they now have some realistic knowledge about PAH and they know the upcoming plan. We set the plan in motion before the patient leaves – either scheduling the diagnostic testing or ordering a change in medical therapy. As the patient leaves, we provide him/her with our telephone number to call if he/she thinks of any further questions or has any further concerns.

4.2 Communication Communication is a key element of providing support to the patient and family. As mentioned already, we provide our telephone number at the first visit so the patient can have access to the PH nurse. We encourage patients to call with questions or concerns so they can be reassured about their condition or treatment plan. Often, patients feel more comfortable asking a nurse what they perceive as a “stupid” question. It is these patient calls that a nurse can use as an opportunity to get to know the patient and family on a more personal level. Telephone calls are a time for providing information to patients, reinforcing teaching and instructions, reviewing testing results, assessing clinical changes in a patient’s condition, providing emotional support, and clarifying the medical regimen.

4.3 Dealing with the Chronic Nature of PAH/Periods of Exacerbation PAH is a chronic and disabling disease where patients may experience episodes of symptom exacerbation (shortness of breath, fatigue, and edema) that make it difficult to perform ADLs. When these episodes occur, patients need help from family or support persons with practical issues of daily living and with emotional/psychological issues. On a practical level, symptomatic patients need help to perform daily activities, such as bathing, dressing, preparation of meals, grocery shopping, housekeeping, and transportation. Often we hear from our sicker patients that just bathing and dressing uses all of their energy in the morning. These patients express frustration that they cannot care for

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themselves and must rely on others. For these patients, if deconditioning is a part of the problem, we will arrange to enroll them into a local pulmonary rehabilitation program for pulmonary conditioning and muscle strengthening. In some circumstances, in-home physical therapy is used as an initial stage before progression to a pulmonary rehabilitation program. For other patients, we need to teach them to pace themselves with their activities; we encourage them to use their energy for quality time such as family dinners, time with grandchildren, and taking car rides, etc. If a patient has limited family support, we investigate community services that may meet some of the patient’s needs such as Meals on Wheels, community transport services, and housekeeping services. Patients also need support on an emotional and psychological level. Coping with such a serious illness causes PAH patients to be depressed, anxious, angry, and to become socially isolated. Many of these patients are young to middle-aged with a family and it is difficult for them to refocus their lives to handle the unpredictable nature and chronicity of this disease. Patients may need medications (such as antidepressants and anxiolytics) and professional counseling to help them deal with psychological issues. PAH patients may experience an episode of significant clinical deterioration or of a serious complication requiring hospitalization. Some patients experience recurrent episodes of heart failure with intractable edema necessitating admission for intravenous diuresis, frequent bloodwork, and possibly a change in medical therapy. Others may develop a community-acquired pneumonia causing them to be so hypoxemic they require mechanical ventilation and a ­prolonged hospital stay. There are many scenarios that can occur resulting in a PAH patient needing hospitalization. Supporting patients and their family through these setbacks is important, but can be very challenging. When the patient is an inpatient, providing both the patient and the family with a daily update on the patient’s condition and the treatment plan is helpful. Identifying treatment and personal goals and showing patinets their progress toward the goals gives them inspiration and hope. Keeping a patient’s family apprised of the patient’s condition helps them to provide additional support and encouragement to the patient. With a prolonged hospital stay, patients become depressed, discouraged, and anxious that they will not recover to their previous level of functioning. Managing their anxiety or depression or lack of sleep with appropriate medications is important. Providing continued emotional support is also important. When planning for discharge, involving social work, nurse case managers, and family is essential. Patients will have practical needs upon discharge (transportation, picking up medications, meals, assistance with ADLs, housekeeping, etc.) and together a plan can be formulated to cover these needs.

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4.4 Supporting the Caregivers Family members are the traditional type of caregiver; however, we have seen other types of caregivers (boyfriend/girlfriend, cousin, sister-in-law, coworker, neighbor, church member, etc.) supporting our patients with PAH. Irrespective of the nature of the caregiver, we as health care providers need to remember to take time to support the caregiver too. Often we, as health care providers, become so involved in caring for the patient’s numerous physical and emotional needs, we forget to spend time with the caregiver. It is important to consider the caregiver’s needs and emotions as an extension of the patient. An overwhelmed, exhausted caregiver cannot be expected to provide care and support for himself/herself or for the patient. The following are some suggestions for supporting caregivers: • When the patient is an inpatient, the PH nurse and physician should take time to speak with the caregiver(s) directly. Update them regularly (and realistically) on the patient’s clinical condition. Allow them time to voice their own concerns and questions as this may help to relieve some of their anxieties and fears. • For exhausted caregivers, identify a time when the patient is relatively stable so the caregiver can stay home and rest. Reassure them it is okay to take a break for themselves and that if anything should change in the patient’s condition, the clinical nurses will notify them. • Allow the caregiver to participate in the patient’s care while the patient is in the hospital so the caregiver can feel more confident and comfortable when it is time for discharge; the caregiver cannot be expected to learn all aspects of a new therapy on the morning of discharge. • Involve the caregiver in both treatment planning and discharge planning. Many times caregivers can provide valuable insights into the home situation that are helpful in making the transition from hospital to home. • Once the patient has been discharged, encourage caregivers to call the PH nurse if they feel the patient is having a problem, or they have questions, or they are experiencing difficulty following the treatment plan. • In clinic follow-up appointments, the PH nurse and physician should inquire how the patient and caregiver are managing at home. If the patient and caregiver are struggling, are there community services that they may qualify for? • Continue to remind and support caregivers to take breaks for themselves. Recommend that they ask for and accept help from others (family, friends, neighbors, work colleagues, church members, etc.) Suggest a friend stay with the patient while the spouse takes a few hours away from the house.

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• Support them to be realistic about participating in social activities. If the patient must cancel a friend’s visit to his/her home because he/she is overfatigued and not feeling well, provide assurance that this is okay and perhaps the visit can be rescheduled for another day. If the patient and spouse cannot host their usual big holiday dinner because of recent hospitalization, this is okay. Remind them it is reasonable to put themselves first.

4.5 Outside Resources There are several outside resources where patients and family can find support. First, there are support groups such as the Pulmonary Hypertension Association, the Scleroderma Foundation and local PH support groups. The Pulmonary Hypertension Association deserves special mention. It is an organization founded by PAH patients dedicated to awareness, advocacy, and support of PAH patients and their families. It has a quarterly newsletter (“Pathlight”), a peer-reviewed journal (Advances in Pulmonary Hypertension), a patient education manual entitled “A Patient’s Survival Guide” [59], a biannual international patient conference, a telephone support helpline, a physician registry, a nursing and allied health network (Pulmonary Hypertension Resource Network), a Web site (http://www.phassociation.org) with the most current PAH news/information and resources, and they support local PH support groups. A second resource is the Internet. Patients can find Web sites committed to PH such as that of the Pulmonary Hypertension Association and PHCentral, both of which provide patients with a wealth of information. The Internet can also help connect patients to other patients via Web chat rooms, or to help them find where research studies are conducted, or to find information regarding specific medications or oxygen systems. There is a list of informative Web sites and Web pages following the reference list. A third resource for PAH patients to utilize is the ACCESS® (Advocating for Chronic Conditions, Entitlements, and Social Services) program. The ACCESS® program is a service of Accredo Health, Hemophilia Health Services, and Accredo Therapeutics, and its purpose is to assist patients with chronic conditions to find solutions to social and economic problems. The service can help patients navigate through state and federal entitlement programs and other health insurance programs. It can be reached at a toll-free telephone number (888-7007010). A fourth resource is the local church or synagogue. Many religious congregations have members willing to devote time to help others with practical issues such as housekeeping, meal preparation, transportation, and running errands, etc.

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4.6 Future Planning Another way to support patients and their families is to promote future care planning. Ideally, this discussion should take place before the patient is in a medical crisis situation. We recommend patients consider discussing and signing an advance directive (a living will or a durable power of attorney) and clearly communicating their wishes for care to their family. While many patients balk at the idea of holding this discussion, it should be approached as a method of empowering patients in that their directions for care will be followed. It also gives family members satisfaction that they are following the patient’s wishes. PH physicians and nurses can support patients and their families to make an advanced directive decision by providing them with information about the PAH condition, what setbacks may occur if there is a clinical deterioration, and clearly explaining what constitutes “life-saving measures” such as cardiopulmonary resuscitation, defibrillation, mechanical ventilation, etc. Ideally, this important conversation should occur in a quiet and thoughtful setting without interruptions from others. Asking the patient what his/her wishes are regarding how aggressive he/she wants the health care team to be may help to foster the conversation. For PAH patients near the end of life who are debilitated and require ongoing nursing support at home, we consider prehospice care. We have found that his level of service provides patients and their families with a higher level of inhome patient support services as compared with traditional home nursing services. Prehospice patients receive the maximum level of nursing services allowable, in-home blood draws, and ancillary support services such as physical therapy, occupational therapy, and home health aid support. Then, when the patient’s condition progresses, he/she and the family may choose to move to a hospice level of care, where patient comfort is the main focus.

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of primary care issues as they arise (i.e. colds/upper ­respiratory tract infections, episodes of flu, etc.) Communication between the primary care physician and the PH center is key to a successful collaboration. Other outpatient health care team members who should also be kept informed of a patient’s clinical status are those team members involved directly in a patient’s care: visiting nurses, physical therapists, oxygen distributors, specialty pharmacy distributor nurses and pharmacists, and pulmonary rehabilitation therapists. Keeping an open line of communication not only helps them care more efficiently for the patient, but they will also feel more comfortable reporting clinical changes to the PH center nurse.

5.2 Inpatient Collaboration Inpatient collaboration of PAH patients is similar. Keeping all team members abreast of a patient’s clinical condition and plan of care is crucial to providing efficient care. All team members (bedside nursing, medical staff, physician consultants, respiratory therapists, physical therapists, nutritionists, social workers, nurse case managers) must be aware of the patient’s current treatment plan, including goals that will make the patient eligible for discharge. Nursing, again, is in a prime position to facilitate communication between the services to coordinate the care. We have found it is best that the nurse/physician PH team serve as the leaders directing care. Frequently patients may hear opinions and considerations from other health care team members during the course of their inpatient stay. We tell our PAH patients that we alone coordinate and direct their inpatient care and unless they hear directions specifically from us, it is only an opinion.

6 Special Circumstances 5 Collaborating with Other Health Care Professionals to Support Patient Care

6.1  Counseling on Issues of Pregnancy and Contraception

5.1  Outpatient Collaboration

6.1.1 Pregnancy

Because management of PAH is specialized, it is important to keep other members of the patient’s multidisciplinary team up to date on the patient’s clinical condition. As mentioned earlier, nurses are in the best position to coordinate keeping other health care professionals apprised of a patient’s condition. One of the most important team members to keep up to date is the primary care physician. If he/she is updated, he/she will feel more comfortable assisting with management

Women with PAH are strongly advised to avoid pregnancy because the rate of maternal mortality is excessively high at 30–50%. For this reason, pregnancy is contraindicated for PAH patients as stated by the Scientific Leadership Council of the Pulmonary Hypertension Association [60]. Another reason why pregnancy is not advised is the physiological changes that occur normally during pregnancy may exacerbate PAH. With the progression of a pregnancy, blood

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volume and cardiac output increase by at least 40%, which can adversely affect women with PAH by causing volume overload and right-sided heart failure. With the onset of labor, uterine contractions may cause additional hemodynamic changes. Pain and the effort of labor may increase blood pressure, cardiac output, and right atrial pressure. In addition, during delivery, there is an autotransfusion of blood and fluid from the uteroplacental unit (of approximately 500  ml), which further increases maternal blood volume. These physiological changes of pregnancy (intravascular volume shifts, elevated catecholamine levels, and hypoxemia), which occur normally in the peripartum period, increase right ventricular volume and decrease cardiac output, worsening PAH and right-sided heart failure [61, 62]. Maternal death typically occurs during the third trimester (as a result of the right side of the heart failing) during delivery ,or in the peripartum period. The highest risk of death occurs during the first 10 days after delivery [63]. In addition to maternal risk, there are risks to the neonate. Premature delivery occurs in more than 50% of cases of women with PAH [62]. There is a reduced survival rate for neonates born to women with PAH (87–89%). Maternal hypoxemia is the main risk to the fetus and can lead to growth retardation, premature delivery, and stillbirth [64]. Considering both the maternal and the neonate risk, women with PAH who are early in pregnancy should be counseled to consider termination of the pregnancy. For women with PAH who choose to proceed with the pregnancy, a joint effort between the high-risk fetal/maternal medicine team and the PH team should be undertaken to coordinate care and follow the patient through pregnancy, delivery, and postpartum phases of care.

use of birth control pills because of the concern over the possible role of estrogen in worsening PAH [60]. Progesterone, taken orally, can be another effective method of contraception for PAH patients if it is taken consistently every day at the same time of day. Intramuscular injections of progesterone (Depo-Provera®) may also be used to prevent pregnancy for PAH patients. Progesterone (by intramuscular injection) may be associated with fluid retention and possibly irregular heavy menstrual cycles when taken concomitantly with warfarin [59]. As mentioned previously, PAH patients on hormonal therapies for contraception and taking an endothelin receptor antagonist must be aware that the hormonal therapy will not be as effective; these women must use an additional nonhormonal method to avoid pregnancy. Intrauterine devices (IUDs) are another method of contraception. The traditional IUDs may be associated with excessive bleeding during the menstrual cycle, especially when used in combination with warfarin. If excessive bleeding does occur, the IUD should be removed and another contraceptive method should be considered. A newer IUD that releases progesterone (Mirena®) has a lower risk of bleeding and may be an effective option [60]. PH nurses are well suited to provide education to women with PAH by reinforcing the exceptional high risk of maternal mortality, highlighting the importance of avoiding pregnancy (especially for those patients receiving an endothelin receptor antagonist agent) and providing a general overview of acceptable methods of contraception. Additional gynecologic ­counseling (for specifics on the various methods of ­contraception) can be obtained by referring the patient back to the local gynecologic physician. For PAH patients who feel desperate to have a family of their own, counseling about hiring a surrogate mother, adoption, and foster parenting may bring them some comfort and hope.

6.1.2 Contraception There is no consensus among PH physicians about which method of birth control is best. The two safest methods of contraception are (1) a vasectomy in men whose female partner has PAH (in a monogamous relationship) and (2) the barrier method, which may include condoms in men and/or a diaphragm with use of spermicide in women [60]. Another method of contraception is a tubal ligation. Since this method involves a surgical procedure with use of anesthesia, it may only be appropriate for PAH patients who are not in a state of severe heart failure. Hormonal therapies may also be considered as options to prevent pregnancy for PAH patients. Birth control pills containing estrogen are felt to be an acceptable form of contraception by many PH physicians if the patients are also anticoagulated with warfarin. Birth control pills containing the least amount of estrogen are recommended. However, there are still some PH physicians who advise against the

6.2 Patients Needing Surgery for Other Conditions As patients are living longer on PAH-specific therapies, we find that over time they may become affected by other chronic medical conditions that are not related to their PAH. Some examples of other chronic medical conditions are back pain related to degenerative disk disease, knee pain related to prior knee injury/surgeries, and foot pain related to advanced bunions. On occasion, our PAH patients require elective surgery to correct these medical conditions and to relieve pain or restore their functional level. On other occasions, our PAH patients may develop an acute medical problem that requires urgent surgical intervention such as an incarcerated hernia or an inflamed gallbladder. Generally, the patient or the ­surgeon’s

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office may contact the PH physician asking for clearance for surgery. In all cases, before surgery clearance can be given, careful consideration is given to each individual patient as to the level of the PAH, the type of surgery needed, where the surgery is being performed, the type of anesthesia required, potential operative complications, and postoperative care. In our practice, for elective cases, we ask the surgeon’s office to provide us with written details about the proposed surgery. The PH physician will then review the patient’s chart, including the last echocardiogram and/or cardiac catheterization. If needed, a more recent echocardiogram may be obtained for the PH physician to assess the current status of the patient’s PH. Then the PH physician will consider the details of the surgery and give his/her opinion regarding the safety of surgery. For the more urgent cases, the opinion for surgery may be granted over the telephone. In our practice, if one of our PH patients lives in a rural setting and needs a complicated surgery, our PH physician may recommend the patient be transferred into an academic setting as the surgical and anesthesia teams will be more familiar with managing the postoperative care of PAH patients. For all PAH patients undergoing general anesthesia, careful attention should be paid to avoiding either dehydration or overhydration with intravenous fluids. It is often preferable that vasoactive agents be used (instead of intravenous fluids) to maintain blood pressure. In addition, for patients who have sleep apnea, they should be able to use their BiPAP or CPAP equipment for sleep and naps while they are in the hospital postoperatively. Judicious pain management should also be employed to avoid oversedation in the postoperative period.

6.3 Dealing with Emergencies or Hospitalizations In our practice we instruct PAH patients and their family/ support persons that when there is a significant change in the medical condition, it is safest to seek medical attention at the nearest emergency room where they can be quickly assessed and stabilized. Then the treating emergency room physician can contact our PH center to discuss the patient’s clinical status and consideration for transfer will be discussed at that time. Patients with worsening right-sided heart failure or complications of PAH therapies (such as Hickman catheter malfunction, sepsis related to an infected Hickman catheter, etc.) are appropriate for transfer into the PH center for more aggressive PH management. Other medical conditions (such as episodes of bronchitis or pneumonia) where the PAH patient is relatively stable and the issue is routine and

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u­ nrelated to the PH may be appropriately managed by the local emergency room and/or hospital.

6.4  Patients Failing Medical Therapy There are some PAH patients who despite aggressive medical therapy continue to show clinical signs of progressive rightsided heart failure. Signs of poor prognosis include [7, 65, 66]: • Worsened hemodynamics: increased right atrial pressure, decreased cardiac index, increased pulmonary vascular resistance, low mixed venous oxygen saturation • Worsened echocardiogram findings: enlargement of the right atrium, dilation of the right ventricle, presence of pericardial effusion, reduced TAPSE (TAPSE measuring right ventricular function), increased Tei index (a measure of right ventricular function – isovolumetric contraction and relaxation times divided by the right ventricular ejection time) • Poor exercise tolerance: functional class III or IV despite aggressive medical therapy, low 6-min walk distance (less than 150 m) • Elevated BNP or NT-proBNP levels • Pleural effusion • Hyponatremia (Na+ less than or equal to 136 mEq/l) For PAH patients who continue to show signs of clinical deterioration despite maximal medical therapies, there are two potential surgical options that may be considered: atrial septostomy and lung transplantation. (For more complete discussions of atrial septostomy refer to Chap. 112 and for lung transplantation refer to Chaps. 116–118.) Atrial septostomy is used as a palliative measure or as a bridge to lung transplantation in select PAH patients with refractory right-sided heart failure or persistent syncope despite aggressive medical therapy [67, 68]. It involves creating a small intra-atrial opening which would allow right-toleft shunting, thereby increasing preload to the left ventricle and increasing cardiac output. Also the atrial septal defect would decompress the right atrium and ventricle and may reduce right-sided heart failure. As a consequence of creating this intra-atrial opening, the patient may have a reduction in systemic arterial oxygenation; however, the benefit of improved hemodynamics outweighs the reduction in oxygenation. Atrial septostomy should only be performed in an experienced PH center as the procedure carries substantial risk. Lung transplantation is another surgical option considered for PAH patients failing medical therapy. It is often difficult for PH physicians and nurses, to determine the best “window” of time for transplantation. The optimal time is when the advanced PAH patient is no longer maintaining an adequate quality of life balanced with the fact that the PH

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patient cannot be too debilitated to tolerate the rigors of lung transplantation surgery. Lung transplantation is a major decision for a PAH patient and his/her family/support persons. PH nurses can help to educate patients considering lung transplantation [69, 70]:

International Society for Heart and Lung Transplantation statistics, in 1990 IPAH accounted for 10% of transplantation procedures. More recent reports show that IPAH accounted for only 3.9% of transplantation procedures. • Most patients who have a lung transplantation have a sufficient improvement in exercise capacity within 1  year that they are able to resume an active lifestyle. Long-term benefit from a lung transplantation depends on the number and quality of comorbidities and on the development and the degree of chronic rejection.

• Lung transplantation is not cure or a simple fix. These patients will be changing the chronic disease of PAH for the chronic condition of being a solid organ transplant recipient. Patients who have had a transplantation require close monitoring through bloodwork, outpatient clinic visits, diagnostic studies (such as chest X-rays, pulmonary function testing, exercise testing, etc.). Patients who have had a transplantation must follow the lung transplantation program’s postoperative protocols, including complex medication regimens and exercise programs. • PAH patients considering lung transplantation will need to go through extensive diagnostic testing (an evaluation) and meet the transplant team members (social worker, financial counselor, pulmonary rehabilitation team, nutritionist, etc.) before being considered as a candidate. • Most lung transplant centers perform a double-lung transplantation for PAH patients. For patients with complex congenital heart disease, a heart–lung transplantation may be performed. • In May 2005, the United Network of Organ Sharing enacted new guidelines for lung transplantation. A lung allocation score is calculated from each patient’s medical condition on the basis of the severity of illness and the chance of survival. This lung allocation score determines how available organs are assigned for transplantation. • For patients with PAH, lung transplantation results in immediate and sustained improvements in pulmonary vascular resistance, pulmonary artery pressures, and cardiac output. Over time there is a gradual remodeling of the right ventricle, with a decrease in the right ventricular wall thickness. • Postoperative complications include primary graft dysfunction, anastomotic complications, acute rejection, chronic rejection, infection, and complications from immunosuppressive therapy (osteoporosis, renal insufficiency, systemic hypertension, hypercholesterolemia, gastroparesis, and esophageal reflux disease). • Kaplan–Meier survival for PAH patients from the International Society for Heart and Lung Transplantation statistics for adult lung transplantations performed between January 1990 and June 2006 were: • One-year survival, 67.2% • Three-year survival, 57% • Five-year survival, 48.5%. • Because medical therapies for PAH have improved over the past several years, the number of PAH patients who have had a transplantation has declined. According to the

7 Insurance Issues Most PAH patients become overwhelmed easily when dealing with insurance issues. PH nurses can provide support to the patient and family by offering some guidance when it comes to managing insurance issues: • Patients need to understand the basics of their insurance coverage (including medical insurance, supplemental insurance, and pharmacy coverage): • Which services the insurance plan covers and the amount of coverage. • The amount of co-payments required for services (i.e. physician office visits, emergency room visits, medications, etc.). • They need to follow the insurance plan rules for securing precertification or referrals before testing or procedures are performed. • Some PH-specific medications (such as the endothelin receptor antagonists and the phosphodiesterase inhibitors) require precertification because of their expense. Patients need to be aware of this rule and to call the PH center nurse before the authorization is due or they may risk having an interruption in their drug therapy. • For patients with high co-payments for medications, there are some patient-assistance programs available (Caring Voice Coalition, Pfizer Patient Assistance Program, Tracleer Bridge Program). It is best to speak with the PH program nurse as to the current availability of patientassistance programs. These programs will require documentation of financial need and the patient will have to qualify by meeting the program’s requirements. • Patients need to notify their physician’s office of any changes in their insurance when they receive their new insurance cards (some patients may need to notify other health care services, such as oxygen suppliers, and specialty pharmacy distributors). • At any point, if patients are unsure of who to call, we tell our patients to call and speak with the PH nurse for direction on how to manage their insurance matter.

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• There are additional resources to assist patients with insurance issues: insurance company representatives or case managers, hospital social workers, hospital financial coordinators, specialty pharmacy distributors, the ACCESS program, and the Internet are a few.

8 Summary In summary PAH is a progressive and debilitating disease causing patients to suffer with episodic and chronic right-sided heart failure. Patients are best treated at a dedicated PH center with experienced nurses and physicians who can provide current therapies and ongoing assessment to achieve the best quality of life possible. PH nurses are a vital component of the PH team as they coordinate, advocate, educate, and assess patients in an ongoing fashion. Nurses help patients and their families/support persons to navigate the overwhelming issues that come with this unpredictable and chronic disease.

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37. Oudiz R (2008) Ambrisentan therapy in patients with pulmonary arterial hypertension: 2 year outcome. Abstract AS2244. Presented at CHEST 2008, Philadelphia 38. Gilead Sciences (2007) Package insert: Letairis (ambrisentan). Gilead Sciences, Foster City 39. Stiebellehner L, Petkov V, Vonbank K et al (2003) Long-term treatment with oral sildenafil in addition to continuous IV epoprostenol in patients with pulmonary arterial hypertension. Chest 123:1293–1295 40. Galiè N, Ghofrani HA, Torbicki A et al (2005) Sildenafil use in pulmonary hypertension (SUPER) study group: sildenafil citrate therapy for pulmonary arterial hypertension. N Engl J Med 353:2148–2157 41. Klinger JR (2007) The nitric oxide/cGMP signaling pathway in pulmonary hypertension. In: Palevsky HI (ed) Pulmonary arterial hypertension. Clinics in chest medicine. Saunders Elsevier, Philadelphia, pp 143–167 42. Revatio US Physician Prescribing Info, www.pfizer.com/products/ rx/rx_product_revatio.jsp, Pfizer Labs, New York, NY 43. Galiè N, Brundage B, Ghofrani H et al (2008) Tadalafil therapy in pulmonary arterial hypertension: results of a randomizes, doubleblind, placebo controlled, phase III study. Eur Heart J 29:519 44. Olschewski H, Simonneau G, Galiè N et al (2002) Inhaled iloprost for severe pulmonary hypertension. N Engl J Med 347:322–329 45. Hoeper MM, Schwarze M, Ehlerding S et al (2000) Long-tern treatment of primary pulmonary hypertension with aerosolized iloprost, a prostacyclin analogue. N Eng J Med 342:1866–1870 46. Hoeper MM, Taha N, Bekjarova A et al (2003) Bosentan treatment in patients with primary pulmonary hypertension receiving nonparenteral prostanoids. Eur Respir J 22:330–334 47. Actelion Pharmaceuticals US (2010) Package insert Iloprost (Ventavis) inhalational solution, www.4ventavis.com/pdf/Vent_ PromoPIUpdate6_10_vv6334.pdf, Actelion Pharmaceuticals US, Inc., South San Francisco, CA 48. United Therapeutics (2008) Package insert: treprostinil sodium (Remodulin) injection. United Therapeutics, Research Triangle Park 49. Simonneau G, Barst RJ, Galiè N et al (2002) Continuous subcutaneous infusion of treprostinil, a prostacyclin analogue, in patients with pulmonary arterial hypertension: a double-blind, randomized, placebo-controlled trial. Am J Respir Crit Care Med 165:800–804 50. Barst RJ, Galiè N, Naeiji R et al (2006) Long-term outcome in pulmonary arterial hypertension patients treated with subcutaneous treprostinil. Eur Respir J 28:1195–1203 51. Flolan (Epoprostenol sodium)-Official FDA information, side effects and uses. Drugs.com-Drug Information Online, www.drugs. com/pro/flolan.html 52. Fortin TA, Tapson VF (2004) Intravenous prostacyclin for pulmonary arterial hypertension. In: Peacock AJ, Rubin LJ (eds) Pulmonary circulation: diseases and their treatment, 2nd edn. Oxford University Press, London, pp 255–267 53. Severson CJ, McGoon MD (2002) Continuous intravenous epoprostenol for pulmonary arterial hypertension: highlighting practical issues, special considerations. Adv Pulm Hypertens 1:4–8 54. Centers for Disease Control and Prevention (CDC) (2007) Bloodstream infections among patients treated with intravenous epoprostenol or intravenous treprostinil for pulmonary arterial hypertension – seven sites, United States 2003–2006. Morb Mortal Wkly Rep 56:170–172 55. Kallen AJ, Kederman E, Balaji A et al (2008) Bloodstream infections in patients given treatment with intravenous prostanoids. Infect Control Hosp Epidemiol 29:342–349 56. Doran AK, Ivy D, Barst RJ et al (2008) Guidelines for the prevention of central venous catheter related bloodstream infections with prostanoid therapy for pulmonary arterial hypertension. Int J Clin Pract 62:5–9 57. Mereles D, Ehlken N, Kreuscher S et al (2006) Exercise and respiratory improve exercise capacity and quality of life in patients with severe chronic pulmonary hypertension. Circulation 114:1482–1489

58. Pulmonary Hypertension Association (2010) Recommendations for exercise in patients with pulmonary arterial hypertension. http:// www.phassociation.org/Learn/Consensus-Statements 59. Hayes GB (2008) Pulmonary hypertension: a patient’s survival guide, 3rd edn. Pulmonary Hypertension Association, Silver Spring 60. Pulmonary Hypertension Association (2010) Birth control and hormonal therapy in pulmonary arterial hypertension. http://www. phassociation.org/Learn/Consensus-Statements 61. Bildrici I, Shumway JB (2004) Intravenous and inhaled epoprostenol for primary pulmonary hypertension during pregnancy and delivery. Obstetrics Gynecol 103:1102–1105 62. Huang S, Desantis ER (2007) Treatment of pulmonary arterial hypertension in pregnancy. Am J Health Syst Pharm 64:1922–1925 63. Wong PS, Constantinides S, Kanellopoulos V et al (2001) Primary pulmonary hypertension in pregnancy. J R Soc Med 94:523–526 64. Bonnin M, Mercier FJ, Sitbon O et al (2005) Severe pulmonary hypertension during pregnancy; mode of delivery and anesthetic management of 15 consecutive cases. Anesthesiology 102:1133–1137 65. Friedman E, Palevsky H, Taichman D (2006) Classification and prognosis of pulmonary arterial hypertension. In: Mandel J, Taichman D (eds) Pulmonary vascular disease. Saunders Elsevier, Philadelphia, pp 66–79 66. Forfia PR, Mathai SC, Fisher MR et  al (2008) Hyponatremia ­predicts right heart failure and poor survival in pulmonary arterial hypertension. Am J Respir Crit Care Med 177:1364–1369 67. Sandoval J, Rothman A, Pulido T (2001) Atrial septostomy for ­pulmonary hypertension. Clin Chest Med 22:547–560 68. Sandoval J, Gaspar J, Pulido T et al (1998) Graded balloon dilation atrial septostomy in severe primary pulmonary hypertension. J Am Coll Cardiol 32:297–304 69. Sager J, Ahaya VN (2007) Surgical therapies for pulmonary arterial hypertension. In: Palevsky HI (ed) Pulmonary arterial hypertension. Clinics in chest medicine. Saunders Elsevier, Philadelphia, pp 187–202 70. Christie JD, Edwards LB, Aurora P et  al (2008) Registry of the international society for heart and lung transplantation: twenty-fifth official adult lung and heart/lung report 2008. J Heart Lung Transplant 27:957–969

Web Sites and Web Pages for Additional References Centers for Medicare and Medicaid Services (CMS): www.cms.gov, Accessed December 30, 2010. Center for Medicine and Medicaid Services, Baltimore, Maryland. Clinical Trials - A service of the U.S. National Institutes of Health: http://clinicaltrials.gov, Accessed December 30, 2010. Medicare Approved Lung and Heart-Lung Transplant Centers: http:// www.cms.hhs.gov/ApprovedTransplantCenters/, Accessed December 30, 2010. Centers for Medicare and Medicaid Services, Baltimore, Maryland. National Institutes of Health: http://www.nih.gov. National Institutes of Health - Turning Discovery into Health: www. nih.gov, Accessed December 30, 2010. National Institutes of Health, Bethesda, Maryland. National Organization for Rare Disorders (NORD): http://www.rarediseases.org, Accessed December 30, 2010. 2009 National Organization for Rare Disorders, Inc. National Registry for Familial Primary Pulmonary Hypertension: www.mc.vanderbilt.edu/root/vumc.php?site=vupphstudy, Accessed December 30, 2010. 2010 Vanderbilt University Medical Center, Nashville, Tennessee.

1558 Pulmonary Hypertension Association - Empowered by Hope: www. phassociation.org, Accessed December 30, 2010. 2010 Pulmonary Hypertension Association, Silver Spring, Maryland. Pulmonary Hypertension Central - Pulmonary Hypertension Information, Support and Advocacy, since 1999: www.PHCentral. org, Accessed December 30, 2010. 1998–2010 PH Central. Scleroderma Foundation - Support, Education, Research: www. scleroderma.org. Accessed December 30, 2010. 2001-2010 Scleroderma Doundation, Danvers, Massachusetts.

C. Archer-Chicko Second Wind Lung Transplant Association, Inc.: www.2ndwind.org, Accessed December 30, 2010. 1996–2008 Second Wind Lung Transplant Association, Inc. Transplant Recipients International Organization (TRIO) - Your Voice of the Transplant Community: www.trioweb.org, Accessed December 30, 2010. United Network for Organ Sharing (UNOS): www.unos.org, Accessed December 30, 2010.

Chapter 112

Atrial Septostomy Julio Sandoval, Jorge Gaspar, and Héctor Peña

Abstract  Atrial septostomy (AS) represents an additional strategy for the treatment of right ventricular failure (RVF) from severe pulmonary arterial hypertension (PAH) and several reasons still justify its use in this setting: (1) the deleterious impact of RVF on survival of patients, (2) the unpredictable response to medical treatment, (3) the limited access to lung transplantation, and (4) the disparity in the availability of these treatments throughout the world. In this chapter, we review the role of AS as an interventional therapy for the management of RVF from severe PAH. Keywords  Treatment of right ventricular failure • Cardiac intervention • Heart function

1 Introduction Atrial septostomy (AS) represents an additional strategy for the treatment of right ventricular failure (RVF) from severe pulmonary arterial hypertension (PAH) and several reasons still justify its use in this setting: (1) the deleterious impact of RVF on survival of patients [1], (2) the unpredictable response to medical treatment [2–4], (3) the limited access to lung transplantation, and (4) the disparity in the availability of these treatments throughout the world [5]. In this chapter, we review the role of AS as an interventional therapy for the management of RVF from severe PAH.

2 Background and Rationale Several experimental as well as clinical observations have suggested that an interatrial defect may be of benefit in severe PAH. In 1964, Austen et al. [6] were the first to suggest and

J. Sandoval (*) Instituto Nacional de Cardiología Ignacio Chávez, Juan Badiano No. 1, Col. Seccion XVI, Tlalpan 14080, Mexico City, DF, Mexico e-mail: [email protected]

demonstrate the potential benefit of an AS in PAH treatment. Working in a canine model of acute and chronic right ventricular hypertension, these authors showed that the surgical creation of an atrial septal defect in this setting produced beneficial hemodynamic effects, particularly during exercise, as well as a favorable effect on the survival of the animals. From the clinical point of view, patients with PAH and with a patent foramen ovale had a better survival than those without a patent foramen ovale [7]. Likewise, patients with Eisenmenger syndrome have a better survival than patients with PAH [8, 9]. Thus, the rationale for the use of AS in PAH relies on the fact that deterioration of symptoms and death in PAH are associated with obstruction to systemic output and the ­development of RVF. An AS in this setting would allow right-to-left shunting to increase systemic output, which, in spite of the fall in systemic arterial oxygen saturation (SaO2), will produce an increase in systemic oxygen transport (SOT) [10, 11]. In addition, the shunt would allow decompression of the heart, resulting in alleviation of RVF.

3 Experience with Atrial Septostomy in Pulmonary Hypertension The first use of blade AS for the treatment of refractory PAH was unsuccessful as the patient died 24 h later as a result of pulmonary edema and refractory hypoxemia [12]. Subsequent studies [10, 11], however, demonstrated that blade–balloon AS (BBAS) could be successfully performed in patients with advanced PAH and result in significant clinical and hemodynamic improvement. Graded balloon-dilation AS (BDAS), a variant of BBAS introduced by Hausknecht et  al. [13] and Rothman et  al. [14], is the technique most used in recent series [15–25]. The precise role of AS in the treatment of PAH remains uncertain because most of the knowledge regarding its use comes from small series or case reports. The limitations of these studies include the following: (1) the series were all  uncontrolled, (2) the indication for performing the

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p­ rocedures varied between studies (3), the cause of the ­pulmonary vascular disease was not the same in all patients, and (4) the medical treatment has changed over the past two decades [26]. Despite these limitations, AS appears to have a place as a therapeutic modality for advanced PAH; in fact, the performance of the BDAS procedure has steadily increased over the years [27]. The indications for AS in the setting of PAH, the potential benefits, and the recommendations to reduce the risk for procedure-related mortality have been addressed in the most recent guidelines [28–31]. The knowledge has now expanded with reports of new cases in the last few years [15–25, 32–39].

4 Worldwide Collective Experience Of the 223 cases reported to date [27] (age 27.7 ± 17 years), most of them (69.6%) are women and severe idiopathic PAH has been the etiologic indication for AS in 175 of them (81.4%). Other causes have included PAH associated with surgically corrected congenital heart disease in 18 cases (8.3%), PAH associated with collagen vascular disease in ten cases (4.6%), peripheral (distal) chronic thromboembolic pulmonary hypertension not susceptible to surgical treatment in six cases (2.8%), and other less frequent causes (2.8%). Congestive heart failure (42.5%), syncope (38%), or both (19.4%) have been the main symptomatic indications for the procedure. The mean New York Heart Association (NYHA) functional class for the group was 3.56 ± 0.4. As many as 96 patients who have undergone AS have been regarded as nonresponsive to maximal medical treatment, including long-term intravenous prostacyclin infusion (n = 57), bosentan (n = 18), sildenafil (n = 8), beraprost (n = 6), subcutaneously administered treprostinil (n = 4), and inhaled iloprost (n = 3). In ten of these patients, different combinations of these drugs were used. The simultaneous use of these drugs and AS, as well as the evidence for the safe administration of intravenously administered epoprostenol, subcutaneously administered treprostinil, or bosentan in the setting of PAH associated with congenital heart disease [40–42] supports the safety of this potential combined therapy. Finally, AS has been used as a bridge to transplantation in 31 of the reported patients.

5 Procedures Two types of AS, BBAS (n = 71; 31.8%) and BDAS (n = 152; 68.2%), have been used in the treatment of PAH. The basic difference between the two procedures is that, in contrast to

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BBAS, in BDAS, the Park blade septostomy catheter is not used and the interatrial orifice is created by puncture with a Brockenbrough needle and use of progressively larger balloon catheters in a step-by-step fashion [43] (Fig. 1). A 10% decrease in SaO2 and an increase in left ventricular end-diastolic pressure approaching 18 mmHg preclude further dilatation. A prospective evaluation of potential differences between the two procedures in regard to hemodynamic results and risk of complications has not been done. The election to perform BBAS or BDAS should be a decision taken in each center on the basis of institutional expertise; however, regardless of the procedure, it is advisable to follow the recommendations to minimize the risk of death during the procedure (Table 1) and the procedures should be performed only in centers experienced in both interventional cardiology and pulmonary hypertension [28–31].

6 Immediate Outcome After Atrial Septostomy In most of the reported series, AS was performed in the setting of severe PAH and RVF. Accordingly, there was an inherent risk of complications and death during the procedure. Since recommendations to minimize this risk have been established [29–31], procedure-related mortality appears to be decreasing. In the analysis of the 223 cases reported [27], there are 16 immediate (24  h) procedurerelated deaths (7.1%), and 1-month mortality occurred in 33 patients (14.8%). Immediate procedure-related mortality in patients with the BDAS technique was lower (approximately 5%) [27]. The causes of immediate death in 33 of the 223 cases reported so far were refractory hypoxemia (n = 20), progressive right-sided heart failure (n = 6), procedure complications (n = 4), multiple organ failure (n = 1), hemoptysis (n = 1), and unrelated causes (voluntary dialysis withdrawal, n = 1). Factors associated with procedure-related mortality (at 1 month) in the whole group are shown in Table 2. As in the prior analysis of the worldwide experience [29], a mean right atrial pressure (RAP) above 20 mmHg remains the most significant risk factor for procedure-related death (risk rate 30.5; p < 0.001). Higher NYHA functional class and congestive heart failure, as an indication for AS, tended to be associated with an increased risk of death, whereas syncope had a protective effect against such a risk. Finally, a higher SaO2 after the procedure also had a protective effect against the risk of death at 1 month [27]. Symptoms and signs of RVF are improved immediately after AS in most surviving patients (i.e. syncope and systemic venous congestion either disappear or decrease in frequency or intensity) [29, 43]. From a total of 186 surviving

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Fig.  1  Balloon-dilation atrial septostomy technique. The procedure involves a standard right-sided and left-sided heart catheterization. Trans-septal puncture is performed with the Mullins introducer (without the sheath) and the Brockenbrough needle using standard techniques. Once the correct and stable position of the Mullins introducer in the left atrium has been confirmed, the needle is withdrawn and a Inoue circular-end guidewire is passed to the left atrium. Over this guidewire, septostomy dilatation is done, in a graded step-by-step

approach, beginning at 4-mm diameter with the Inoue septostomy dilator and completed using peripheral balloons of successive diameters. (a) The circular-end guidewire is shown positioned in the left atrium. (b) A 4-mm initial dilation of the atrial septum is done with the Inoue dilator, and is concluded with a 6-mm balloon (c, d). Inflation of the balloon with contrast medium is increased just enough to eliminate the balloon waist (arrow) and is repeated at least twice to counter elastic recoil

patients in the current worldwide experience, 163 patients (87.6%) were reported as improved and 23 patients (12.4%) as not improved. Thirty-one of the 186 surviving patients (16.6%) had transplants. The NYHA functional class of the surviving patients decreased from 3.49 ± 0.58 to 2.1 ± 0.67 (p < 0.001). Finally, exercise endurance, as assessed by the 6-min walk, improved in the three studies evaluating this parameter; from 110 to 220 m [15], from 230 to 380 m [18], and from 210 to 260 m [21].

7 Spontaneous Closure of Atrial Septostomy In most of the reported series, subsequent closure of the defect was a relatively frequent (27/223 = 12.1%) and undesirable outcome of BDAS. The reason for this remains unknown. In our experience, there have not been significant differences in age, hemodynamic profile, or septostomy size between patients with spontaneous closure and those in

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Table 1  Recommendations to minimize the risk of procedure-related mortality of atrial septostomy 1.

Atrial Septostomy should be attempted only by those institutions with an established track record in the treatment of advanced pulmonary hypertension where atrial septostomy is performed with low morbidity 2. Atrial Septostomy should not be performed in the patient with impending death and severe right ventricular failure on maximal cardiorespiratory support. A mRAP > 20 mmHg, a PVRI > 55 U/m2, and a predicted 1 year-survival <40% are all significant predictors of procedure-related death 3. Before cardiac catheterization, it is important to confirm an acceptable baseline systemic oxygen saturation (>90% in room air) as well as to optimize cardiac function (adequate right heart filling pressure, additional inotropic support if needed) 4. During cardiac catheterization is mandatory: a. Supplemental oxygen if needed b. Mild and appropriate sedation to prevent anxiety c. Careful monitoring of variables (LAP, SaO2%, and mRAP) d. To attempt a step-by-step procedure 5. After atrial septostomy, it is important to optimize oxygen delivery. Transfusion of packed red blood cells or erythropoietin (prior to and following the procedure if needed) may be necessary to increase oxygen content mRAP mean right atrial pressure, PVRI pulmonary vascular resistance index, LAP left atrial pressure, SaO2% arterial oxygen saturation Reprinted from [29] with permission from Elsevier

Table 2  Variables associated with procedure-related mortality (1 month) HR (95% confidence Variable interval) p value Age, years 0.99 (0.96–1.03) 0.966 Age >18 years old 1.12 (0.29–4.34) 0.865 Gender, female 0.73 (0.18–2.8) 0.635 NYHA functional class 8.53 (0.89–81.2) 0.062 Diagnosis of CVD 3.18 (0.67–14.9) 0.143 Syncope 0.14 (0.03–0.66) 0.013 RHF 5.97 (0.75–47.2) 0.089 Septostomy type, blade 1.19 (0.30–4.6) 0.800 mRAP (mmHg) 1.19 (1.1–1.29) 0.000 RAP > 20 mmHg 30.5 (3.8–244) 0.001 mPAP (mmHg) 1.01(0.98–1.05) 0.321 mLAP (mmHg) 1.11(0.86–1.43) 0.420 Baseline CI (L/min/m2) 0.38 (0.09–1.6) 0.189 Baseline PVRI (U/m2) 1.04 (0.98–1.09) 0.148 Baseline SaO2% 0.97 (0.83–1.14) 0.773 SaO2% after procedure 0.90 (0.84–0.96) 0.001 mSAP (mmHg) 0.96 (0.92–1.01) 0.148 CVD collagen vascular disease, RHF right-sided heart failure, RAP right atrial pressure, mPAP mean pulmonary artery pressure, mLAP mean left atrial pressure, CI cardiac index, mSAP mean systemic artery pressure, RHF right-sided heart failure, HR hazard ratio, NYHA New York Heart Association. From [27]

whom the septostomy remained open. To solve the problem of closure, we have elected to repeat septostomy as many times as necessary, and achieved this without complications. Recently, however, this problem has been approached differently by other investigators. Micheletti et  al. [24] placed a custom-made fenestrated atrial septal device at the end of the procedure to maintain the septostomy open. By doing this in seven out of 20 children, they successfully avoided the ­spontaneous closure of the defect. This approach has been followed by other investigators [37–39]. These additional interventions may help in reducing the risk of the subsequent closure of the defect. Presently, however, it is difficult to anticipate the long-term risk/benefit of this approach. In fact,

in a recent communication, Lammers et  al. reported the occlusion of the fenestration in four of nine patients, after a follow-up of 10 months, despite the concomitant use of aspirin or warfarin [44].

8 Immediate Hemodynamic Effects Individual patient information regarding the hemodynamic parameters before and after AS has been described in only 117 cases of the worldwide experience. In the group as a whole, there was a significant (p < 0.000) decrease in mean RAP (from 14.6 ± 8 to 11.6 ± 6.3  mmHg) and SaO2 (from 93.3 ± 4.1 to 83 ± 8.5%); this was accompanied by an also significant (p < 0.000) increase in mean left atrial pressure (from 5.7 ± 3.3 to 8.10 ± 3.98  mmHg) and cardiac index (CI; from 2.04 ± 0.69 to 2.62 ± 0.84 L/min/m2). The magnitude of hemodynamic changes is not the same for all patients and it depends on the level of the baseline mean RAP [28, 29, 43]. As noted in Table 3, the hemodynamic changes after AS are progressively more important as the mean RAP is higher. In patients with a mean RAP less than 10  mmHg, the decrease in RAP was not significant (10.6% from the baseline), yet there was a significant increase (22.5%) in CI. In patients with a mean RAP more than 20  mmHg, RAP and SaO2 decreased by 25 and 14.5%, respectively, whereas CI increased by 38% from the baseline. Patients with a baseline mean RAP between 11 and 20 mmHg had an intermediate response; RAP decreased by almost 20% and CI increased by 30%. It is important to stress the fact that it is in the group of patients with a RAP more than 20 mmHg where the risk of procedure-related death is elevated. Likewise, it is important to note that although the decrease in RAP was not significant in the group of patients with RAP less than 10  mmHg

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Table 3  Hemodynamic effects of atrial septostomy according to baseline mRAP [27] RAP < 10 mmHg (n = 42) RAP = 11–20 mmHg (n = 49) Variable Before After p< Before After p< mRAP (mmHg) mPAP (mmHg) mLAP (mmHg) SaO2% CI (L/min/m2) mSAP (mmHg) NYHA functional class From [27]

6.6 ± 2.4 62 ± 16 5.0 ± 2.7 93.8 ± 4 2.36 ± 0.58 83 ± 15 3.25 ± 0.64

5.9 ± 3.2 64 ± 19 6.8 ± 2.4 85.8 ± 7 2.89 ± 0.72 83 ± 13 2.00 ± 0.65

0.214 0.329 0.005 0.001 0.001 0.931 0.001

14.8 ± 2.8 66.4 ± 17 5.3 ± 3.6 93.0 ± 4.0 2.04 ± 0.71 84.5 ± 14 3.63 ± 0.49

(perhaps because it was already low), the changes in CI and NYHA functional class were significant and clinically relevant. Although the immediate hemodynamic changes after septostomy seem only moderate (particularly in the group with baseline mRAP less than 10 mmHg), it has to be stressed that these measurements represent only the resting state. The net hemodynamic effect of septostomy is likely to be different during exercise, as shown in dogs with right ventricular hypertension [6] where an increase in cardiac output in the face of minimal elevation of right ventricular end-diastolic pressure occurred only in dogs with an atrial septal defect. This might explain the increase in exercise endurance after AS reported in patients with PAH [15, 18, 21]; however, the net hemodynamic effects of AS during exercise in PAH have not been established. The effects of AS on other hemodynamic variables such as pulmonary blood flow and, therefore, on pulmonary vascular resistance (PVR) are difficult to assess as in most of the studies reported so far, a direct measurement of pulmonary blood flow was not done. In recent work by Kurzyna et al. [32], a significant increase in PVR was seen in some of the patients. This increase in PVR correlated with the level of mixed venous PO2 after the procedure and it was though to be responsible in part for the refractory hypoxemia following AS. In their study, they were able to manage refractory hypoxemia with inhaled iloprost [32]. This most interesting finding deserves future investigation. Several mechanisms may account for the immediate hemodynamic and the observed beneficial clinical effects after AS. These include decompression of right ventricular chambers at rest [45], prevention of further right ventricular dilation and dysfunction during exercise, and a increase in cardiac output and SOT both at rest and during exercise (via right-to-left shunt) [6]. The increase in SOT and delivery might also produce beneficial effects on peripheral oxygen utilization and may also be responsible for the improvement in the exercise capacity of the patients [29, 43]. Other mechanisms for improvement after AS are also likely. It has been shown that PAH patients have an increase in sympathetic nervous activity [46], which may, in fact, be

11.9 ± 3.5 66.4 ± 16 7.8 ± 4.5 82.8 ± 7.2 2.65 ± 0.96 88.8 ± 15 2.21 ± 0.78

0.001 1.000 0.001 0.001 0.001 0.065 0.001

RAP > 20 mmHg (n = 26) Before After

p<

26.6 ± 4.4 63.4 ± 20 7.9 ± 3.0 93.1 ± 4.3 1.55 ± 0.49 78 ± 20 3.71 ± 0.49

0.001 0.102 0.029 0.001 0.001 0.254 0.001

19.9 ± 3.8 67.5 ± 20 10.9 ± 4.0 78.6 ± 10.3 2.14 ± 0.56 81 ± 18 2.00 ± 0.0

one of the pathophysiologic mechanisms leading to RVF [43]. In a recent study, Ciarka et al. [33] demonstrated a significant decrease in muscle sympathetic nerve activity after septostomy in patients with PAH. By decreasing sympathetic overdrive, AS might also improve right ventricular function. In the study of Ciarka et al. the decrease in muscle sympathetic nerve activity correlated with the decrease in RAP after septostomy [33], which is in favor of the decompression phenomenon of the right side of the heart after AS [45]. In this regard, it is interesting that in a recent study of patients with PAH, O’Byrne et  al. [47] demonstrated a decrease in the levels of brain-type natriuretic peptide after septostomy.

9 Long-Term Effects Very little information exists with regard to the long-term hemodynamic effects of AS. Only two studies have evaluated the long-term hemodynamic status after the procedure [11, 34]. Both studies found an improvement in right ventricular function (higher CI and lower RAP) over time in patients who had repeat catheterization after a mean of about 2 years after septostomy. Echocardiography studies performed before and about 6 months after AS in patients with PAH by Espínola-Zavaleta et al. [45] suggest that AS may also exert beneficial effects on right-sided heart structure and function. One of the findings in this study was a significant decrease in right atrial and right ventricular systolic and diastolic areas after septostomy as a reflection of less right-sided heart dilation after the procedure. This simple decompression effect (decrease in radius) reduces wall stress and may improve right ventricular performance via the Laplace relationship [48, 49]. From the clinical point of view, not all patients improve after the procedure. Rothman et  al. [16] showed that the long-term clinical outcome after septostomy depends on the immediate hemodynamic response to the procedure, particularly in terms of the change in CI and SOT after AS.

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Compared with patients with no clinical improvement after the procedure, those with significant clinical benefit had a higher and significant increase in CI and SOT immediately after the procedure [16, 43].

10 Long-Term Survival After Atrial Septostomy The true impact of AS on survival of patients with PAH has not been established in prospective and controlled studies. Most reported series, however, have suggested a short-term beneficial effect on the survival of these very sick patients [11, 34]. Long-term survival is limited by late deaths, ­primarily as a result of progression of the pulmonary vascular disease [29]. From the current worldwide experience [27], we have analyzed the survival characteristics of 106 patients in whom complete follow-up and outcome data after septostomy were available. The mean survival time was 63.1 months (95% confidence interval 50–76.3 months). Mortality after septostomy was associated with increasing age [hazard ratio (HR) 1.04; p < 0.001), scleroderma diagnosis (HR 8.32; p < 0.004), NYHA class after septostomy (HR 4.71; p < 0.000), and NYHA functional class 3 and 4 after septostomy (HR 6.24; p < 0.009)]. Increased CI after septostomy (HR 0.179; p < 0.002), higher left atrial pressure after septostomy (HR 0.737; p < 0.005), and increased SOT after septostomy (HR 0.99; p < 0.002) all had a protective effect against the risk of death [27].

11 Summary AS is as an additional, promising strategy in the treatment of severe PAH. Experience with this procedure is limited in part owing to the relative availability and success of the new forms of pharmacological interventions. The definitive role of AS in the management of PAH is uncertain owing to the lack of a prospective and controlled study. However, on the basis of analyses of the worldwide experience, several general conclusions can be made: 1. AS can be performed successfully in selected patients with advanced pulmonary vascular disease. 2. In patients with PAH who have undergone successful AS, the procedure has resulted in a significant clinical improvement, beneficial and long-lasting hemodynamic effects at rest, and a trend toward improved survival. Although not prospectively assessed, the impact of AS on the survival of patients with severe PAH is beneficial and, in the midterm, is at least comparable to that of other current pharmacological therapeutic interventions.

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3. The procedure-related mortality is still high but appears to be decreasing. Operator experience and strict adherence to WHO recommendations account for a low fatality rate. 4. Because the disease process in PAH is unaffected by the procedure itself (late deaths), the long-term effects of an AS must be considered to be palliative. Indications for the procedure include (1) failure of maximal medical therapy (including oral calcium channel blockers, prostacyclin analogues, bosentan, and phosphodiesterase-5 inhibitors, alone or combined) with persisting RVF and/or recurrent syncope, (2) as a bridge to transplantation, and (3) when no other therapeutic options exist [27–31].

12 Final Remarks The current practice regarding the management of PAH is to consider AS for treatment only after maximal medical ­(pharmacologic) therapy has failed. The advanced stage of PAH and RVF of the patients undergoing the procedure in the ­analysis of the current worldwide experience is in support of this. Procedure-related mortality is clearly associated with the advanced stage of the disease. Accordingly, we should attempt AS in an earlier stage of the disease. Likewise, the potential benefit of an early combination of AS and PAHspecific drug therapies is appealing and, in our opinion, worthwhile evaluating further [50].

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1565 hypertension. ACCP evidence-based clinical practice guidelines. Chest 126:63S–71S 31. Klepetko W, Mayer E, Sandoval J et al (2004) Interventional and surgical modalities of treatment for pulmonary arterial hypertension. J Am Coll Cardiol 43:73S–80S 32. Kurzyna M, Dabrowski M, Bielecki D et al (2007) Atrial septostomy in treatment of end-stage right heart failure in patients with pulmonary hypertension. Chest 131:947–948 33. Ciarka A, Vachiery JL, Houssiere A et al (2007) Atrial septostomy decreases sympathetic overactivity in pulmonary arterial hypertension. Chest 131:1831–1837 34. Law MA, Grifka RG, Mullins CE, Nihill MR (2007) Atrial septostomy improves survival in select patients with pulmonary hypertension. Am Heart J 153:779–784 35. Wawrzynska L, Remiszewski P, Kurzyna M et al (2006) A case of a patient with idiopathic pulmonary arterial hypertension treated with lung transplantation: a bumpy road to success. Pol Arch Med Wewn 115:565–571 36. Rogan MP, Walsh KP, Gaine SP (2007) Migraine with aura following atrial septostomy for pulmonary arterial hypertension. Nat Clin Pract Cardiovasc Med 4:55–58 37. Fraisse A, Chetaille P, Amin Z et al (2006) Use of Amplatzer fenestrated atrial septal defect device in a child with familial pulmonary hypertension. Pediatr Cardiol 27:759–762 38. Prieto LR, Latson LA, Jennings C (2006) Atrial septostomy using a butterfly stent in a patient with severe pulmonary arterial hypertension. Catheter Cardiovasc Interv 68:642–647 39. O’Loughlin AJ, Keogh A, Muller DW (2006) Insertion of a fenestrated Amplatzer atrial septostomy device for severe pulmonary hypertension. Heart Lung Circ 15:275–277 40. Rosenzweig EB, Kerstein D, Barst RJ (1999) Long-term prostacyclin for pulmonary hypertension with associated congenital heart defects. Circulation 99:1858–1865 41. Simonneau G, Barst RJ, Galiè N et al (2002) Continuous subcutaneous infusion of treprostinil, a prostacyclin analogue, in patients with pulmonary arterial hypertension. Am J Respir Crit Care Med 165:800–804 42. Galiè N, Beghetti M, Gatzoulis MA et al (2006) Bosentan therapy in patients with Eisenmenger syndrome: a multicenter, double-blind, randomized, placebo-controlled study. Circulation 114:48–54 43. Sandoval J, Gaspar J (2004) Atrial septostomy. In: Peacock AJ, Rubin LJ (eds) Pulmonary circulation, 2nd edn. Arnold, London, pp 319–333 44. Lammers AE, Derrick G, Haworth SG et  al (2007) Efficacy and long-term patency of fenestrated Amplatzer devices in children. Catheter Cardiovasc Interv 70:578–584 45. Espínola-Zavaleta N, Vargas-Barrón J, Tazar JI et  al (1999) Echocardiographic evaluation of patients with pulmonary hypertension before and after atrial septostomy. Echocardiography 16:625 46. Velez Roa S, Ciarka A, Najem B et al (2004) Increased sympathetic nerve activity in pulmonary artery hypertension. Circulation 110:1308–1312 47. O’Byrne ML, Berman-Rosenzweig ES, Barst RJ (2007) The effect of atrial septostomy on the concentration of brain-type natriuretic peptide in patients with idiopathic pulmonary arterial hypertension. Cardiol Young 17:557–559 48. Bristow MR, Zisman LS, Lances BD et  al (1998) The pressureoverloaded right ventricle in pulmonary hypertension. Chest 114:101S–106S 49. Chin KM, Kim NHS, Rubin LJ (2005) The right ventricle in pulmonary hypertension. Coron Artery Dis 16:13–18 50. Sandoval J, Gaspar J, Peña H, et al. Effect of atrial septostomy on the survival of patients with severe pulmonary arterial hypertension. The benefit of combining strategies. Editorial review (ERJ)

Chapter 113

Evaluation of Patients with Chronic Thromboembolic Pulmonary Hypertension for Pulmonary Endarterectomy William R. Auger, Peter F. Fedullo, and Kim M. Kerr

Abstract  The evaluation of patients with suspected chronic thromboembolic disease is meant to establish the degree of existing pulmonary hypertension and cardiac compromise, to confirm the diagnosis of chronic thromboembolic disease, to estimate the extent of coexisting small vessel pulmonary vascular disease, and to establish the surgical accessibility of the chronic thromboembolic residua. These are the critical components in determining whether or not a patient is a candidate for pulmonary endarterectomy (PEA). This chapter outlines an approach which can assist the clinician in achieving these diagnostic goals. Keywords  Surgical treatment • Pulmonary thromboendar­ terectomy • Inoperative • Preoperative and postoperative hemodynamics

1  Introduction The evaluation of patients with suspected chronic thromboembolic disease is meant to establish the degree of existing pulmonary hypertension and cardiac compromise, to confirm the diagnosis of chronic thromboembolic disease, to estimate the extent of coexisting small vessel pulmonary vascular disease, and to establish the surgical accessibility of the chronic thromboembolic residua. These are the critical components in determining whether or not a patient is a candidate for pulmonary endarterectomy (PEA). This chapter outlines an approach which can assist the clinician in achieving these diagnostic goals.

W.R. Auger (*) Division of Pulmonary Critical Care Medicine, University of California, San Diego, 9300 Campus Point Drive, MC 7381, San Diego, CA 92037, USA e-mail: [email protected]

2 Physical Examination Physical examination findings reflect the stage at which a patient with chronic thromboembolic pulmonary hypertension (CTEPH) presents for evaluation. In the absence of significant right-sided heart dysfunction, physical findings in CTEPH patients may be unremarkable. Even in the setting of severe pulmonary hypertension, patients can appear relatively well. The examination findings of pulmonary hypertension such as a right ventricular lift, a pronounced pulmonic component of the second heart sound, a right ventricular S4 gallop, and a tricuspid regurgitation murmur should be carefully sought after. It is not until the development of rightsided heart failure will jugular venous distention, a right ventricular S3 gallop, severe tricuspid regurgitation, hepatomegaly, ascites, and/or peripheral edema become evident. In the absence of coexisting parenchymal lung disease or airflow obstruction, the lung examination findings of the CTEPH patient can be unexceptional. In approximately 30% of patients with CTEPH, pulmonary flow murmurs can be appreciated [1]. These bruits, which appear to result from turbulent flow across narrowed or partially obstructed pulmonary vessels, are high pitched in quality. They are auscultated over the lung fields rather than the precordium, can accentuate following inspiration, and may require the patient to breath-hold to be clearly heard. However, this examination finding is not specific to chronic thromboembolic disease, and can be heard in other diseases which feature large pulmonary vessel narrowing, such as congenital pulmonary branch stenosis and major-vessel pulmonary arteritis. Their importance when discovered in the pulmonary hypertensive patient is that they have not been described in patients with idiopathic or small-vessel pulmonary arterial disease. Further examination findings in the CTEPH patient might include peripheral cyanosis, alerting the clinician to the possibility of a right-to-left shunt through a patent foramen ovale. Examination of the lower extremities may disclose superficial varicosities and venous stasis skin discoloration in those individuals who have experienced prior venous thrombosis.

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3 Preliminary Testing Information to be gathered from laboratory testing has utility in defining the severity of disease in patients suffering from CTEPH. For those with severe right ventricular dysfunction and coexisting liver congestion, elevation of transaminase and bilirubin levels can be expected. It is this same subgroup of patients where renal blood flow and glomerular perfusion may be compromised, either from a low cardiac output or from the use of diuretics (or both). Elevation of serum creatinine and blood urea nitrogen levels may occur as a result. Important in this patient population is the identification of a procoagulant state [2, 3]. Antithrombin III, protein C, and protein S deficiencies, as well as factor II and factor V Leiden mutations are among the hereditary thrombophilic states which should be pursued in the evaluation of the CTEPH patient. Elevated levels of factor VIII and antiphospholipid antibodies (anticardiolipin antibodies, lupus anticoagulant) have also been shown to be risk factors in the development of chronic thromboembolic disease [2, 4]. In the absence of heparin anticoagulation, an elevated activated partial thromboplastin time may be the first indication that a lupus anticoagulant is present. It is particularly important to detect antiphospholipid antibodies in those CTEPH patients undergoing PEA as they appear to be at higher risk for postoperative thrombosis. As a result, more aggressive post-PEA anticoagulation may be warranted. Chest radiography findings in patients with CTEPH may be deceptively unremarkable in the early stages of the disease process. However, with disease progression, several radiographic abnormalities may become apparent. With significant pulmonary hypertension, enlargement of the proximal pulmonary vascular bed typically occurs. If chronic thromboemboli involve the main or lobar pulmonary arteries, this central pulmonary artery enlargement can be asymmetric, an appearance not expected in those patients with small-vessel disease [5]. As the right ventricle adapts to the rise in pulmonary vascular resistance, radiographic signs of chamber enlargement, such as obliteration of the retrosternal space and prominence of the border of the right side of the heart, can be seen. Without coexisting parenchymal lung disease, interstitial–alveolar markings within the lung fields are atypical. However, relatively avascular lung regions can be appreciated if a large organized thrombus is compromising blood flow to that area. In these poorly perfused lung regions, the sequelae of lung injury such as peripheral alveolar opacities, linear scarlike lesions, and pleural thickening may be found. Pulmonary function testing is most useful in evaluating a patient for coexisting parenchymal lung disease or airflow obstruction. CTEPH by itself will not significantly impact spirometric or lung volume measurements. For those patients with parenchymal scarring from prior lung ­infarction, a mild

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to moderate restrictive defect may be detected [6]. Similarly, a modest reduction in single breath diffusing capacity for carbon monoxide (DLco) may be present in some CTEPH patients, although a normal value does not exclude the diagnosis [7]. A severe reduction in DLco should raise concerns that the distal pulmonary vascular bed is significantly compromised, making it imperative that an alternative diagnosis other than CTEPH be considered. This is relevant in that experience has shown that post-PEA outcomes are poor if a CTEPH patient also presents with a severely reduced DLco. CTEPH patients will frequently exhibit normal resting arterial oxygenation (PaO2), although if measured, dead-space ventilation may be elevated. With exercise, patients with extensive chronic thromboembolic disease will often experience a decline in PaO2 and an inappropriate rise in dead-space ventilation. These findings reflect the ventilation–perfusion (V/Q) mismatch in CTEPH and an inadequate cardiac output response to exercise resulting in a low mixed venous oxygen saturation [8]. Hypoxemia at rest implies severe right-sided heart dysfunction or the presence of a considerable right-toleft shunt, such as through a patent foramen ovale.

4 Echocardiography Transthoracic echocardiography has become an extremely valuable noninvasive tool in the evaluation of the pulmonary hypertensive patient. Available technology allows for estimates of pulmonary artery systolic pressure (using Doppler analysis of the degree of tricuspid regurgitation), along with cardiac output and right ventricular performance [9]. Enlargement of the chambers of the right side of the heart, tricuspid regurgitation as a result of this chamber enlargement, flattening or paradoxical motion of the interventricular septum, encroachment of an enlarged right ventricle on the left ventricular cavity, and impaired left ventricular diastolic filling that it is not the result of primary left ventricular diastolic dysfunction or valvular heart disease are findings in patients with significant pulmonary hypertension [10, 11]. Contrast echocardiography using intravenously administered agitated saline can detect the presence of an intracardiac shunt, such as a patent foramen ovale or a previously undetected septal defect. Identification preoperatively is important as the intracardiac shunt can be surgically repaired at the time of an endarterectomy. Should an echocardiogram obtained at rest demonstrate minimally elevated pulmonary artery pressures or only modest right ventricular compromise in a patient experiencing cardiopulmonary symptoms with exertion, a study with exercise may be able to document a substantial rise in pulmonary artery pressures with dilatation of the right ventricle.

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Fig. 1  Lung ventilation (xenon)–perfusion scan in a patient with chronic thromboembolic pulmonary hypertension (CTEPH)

5 Ventilation–Perfusion Lung Scan Although V/Q scintigraphy has seen a diminishing role in the evaluation of suspected acute pulmonary embolic disease, it still provides essential information in the pulmonary hypertensive patient. In these patients, the V/Q scan often is the first indication that chronic thromboembolic disease should be considered. Interpretation of an abnormal perfusion pattern can provide a differentiation between disorders involving the central or proximal vascular bed from those primarily affecting the peripheral pulmonary circulation. This distinction thereby allows a more focused approach for subsequent diagnostic tests. In chronic thromboembolic disease, at least one, and more commonly several segmental or larger mismatched perfusion defect are present (Fig.  1). For those patients with small-vessel pulmonary vascular disease, perfusion scans either are normal or exhibit a “mottled” appearance characterized by nonsegmental defects [12, 13]. Possible exceptions to this include cases of pulmonary venoocclusive disease or pulmonary capillary hemangiomatosis in which multiple, larger mismatched defects have been reported [14, 15]. Experience has shown, however, that a relatively normal perfusion pattern on V/Q scan excludes the diagnosis of surgically accessible chronic thromboembolic disease. Although it is true that patients with operable CTEPH will exhibit unmatched perfusion defects on a V/Q study, the magnitude of these defects may understate the degree of pulmonary vascular obstruction determined by angiographically or at surgery [16]. The likely explanation for this observation is that during the process of thrombus organization, proximal

vessel thromboemboli may recanalize, or narrow the vessel in such a manner that radiolabeled macroaggregated albumin may traverse the area of partial obstruction, creating gray zones or regions of relative hypoperfusion on the perfusion scan. Therefore, chronic thromboembolic disease should be considered even if a V/Q scan demonstrates a single, mismatched segmental perfusion defect or hypoperfused lung region in a patient with pulmonary hypertension.

6 Right-Sided Heart Catheterization Right-sided heart catheterization in the evaluation of patients with suspected CTEPH objectively defines the severity of pulmonary hypertension and the degree of cardiac dysfunction at rest. This hemodynamic assessment is important in discussions with patients as to perioperative risks should they prove to be surgical candidates. For example, available data suggest that patients with severe pulmonary hypertension (pulmonary vascular resistance above 1,000 dyn s cm−5) bear a greater perioperative mortality risk [17, 18]. And although unsubstantiated to date, for those patients with significant preoperative right ventricular dysfunction, there may be benefit in “medical stabilization” prior to surgery in an effort to improve postoperative outcomes [19]. For symptomatic CTEPH patients with modest pulmonary hypertension at rest, exercise hemodynamic measurements may be obtained. In these cases it is likely the normal compensatory mechanisms of recruitment and dilation of the

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pulmonary vasculature have been overcome, and with ­exercise, a linear elevation in pulmonary artery pressure as cardiac output increases can be observed. This hemodynamic information provides objective evidence to explain an individual’s symptoms, and likely reflects a clinically relevant stage in the development of severe CTEPH in which there are coexisting small-vessel hypertensive changes. Furthermore, right-sided heart catheterization has the potential to provide objective data in analyzing the degree of this small vessel disease, information which may help predict post-PEA outcomes. As reviewed by Nick Kim in Chap. 89, partitioning the different elements (proximal vs. distal) of the pulmonary vascular resistance in CTEPH have been investigated. In a small series of 26 CTEPH patients, Kim et  al. utilizing pulmonary artery occlusion waveform analysis, demonstrated excellent inverse correlation between the percent upstream resistance and postoperative mean pulmonary artery pressure and pulmonary vascular resistance. In addition, all four deaths in this series occurred in patients in whom the upstream resistance was less than 60% [20]. If future investigations validate these preliminary observations, this information may identify a subgroup of CTEPH patients who should be excluded from surgical consideration.

7 Conventional Pulmonary Angiography Once the possibility of CTEPH has been suggested by preliminary studies, both confirmation of the diagnosis and assessment of the proximal extent of disease are essential in evaluating patients for PEA. Often considered the “gold standard” for achieving these diagnostic goals, conventional pulmonary angiography can be safely performed, when taking proper precautions, even in severely pulmonary hypertensive patients [21]. In terms of technique, multiple selective injections are not required. A single injection of nonionic contrast into both proximal pulmonary arteries, with the volume and injection rate adjusted on the basis of cardiac output, appears to be sufficient. As little as 15–20 ml of contrast may be required for each pulmonary artery injection. Ideally, biplane acquisition provides optimal anatomic detail, the lateral projection providing more definition of lobar and segmental anatomy than can be achieved with an anterior–posterior view alone. Under essentially all circumstances, a properly performed biplane angiogram will provide sufficient information on which to base a decision regarding chronic thrombus location, and as a result, surgical accessibility. The angiographic appearance of chronic thromboembolic disease bears little resemblance to that of the well-defined, intraluminal filling defects of acute pulmonary embolism. Instead, the angiographic patterns encountered in chronic thromboembolic disease reflect the complex patterns of

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o­ rganization and recanalization that occur following an acute thromboembolic event. Several angiographic patterns have been described in chronic thromboembolic disease which correlate with the material removed at the time of surgery [22]. These include “pouch defects,” pulmonary artery webs or bands, intimal irregularities, abrupt, frequently angular narrowing of the major pulmonary arteries, and complete obstruction of main, lobar, or segmental vessels at their point of origin (Figs. 2–4). In most CTEPH patients, two or more of these angiographic findings are present, typically involving both lungs.

8 Computerized Tomographic Scanning Although computed tomographic (CT) angiography has assumed an increasingly important role in the diagnostic algorithm for acute pulmonary embolism, its place in the evaluation of chronic thromboembolic disease has yet to be clearly defined. There are a number of CT findings which have been described in patients with chronic thromboembolic disease. These include mosaic perfusion of the lung parenchyma, enlargement of the central pulmonary arteries and chambers of the right side of the heart, variability in the size of lobar and segmental-level vessels with a reduction in vessel caliber of those involved with chronic thrombi, peripheral, scarlike lesions in poorly perfused lung regions, and the presence of mediastinal collateral vessels arising from the systemic arterial circulation (aorta, coronary vessels). With appropriate contrast enhancement of the pulmonary vasculature during CT imaging, organized thrombus can be seen to line the pulmonary vessels, often in an eccentric manner. Associated narrowing of pulmonary arteries, web strictures, “pouch defects,” and other irregularities of the intima may also be appreciated [23, 24] (Fig. 5). As is the case with conventional pulmonary angiography, these CT findings are distinct from the intraluminal filling defects of acute thromboemboli and primary pulmonary vascular tumors [25]. What remains unvalidated is the utility of CT angiography in determining operability in certain subgroups of CTEPH patients. Growing experience has demonstrated that the absence of lining thrombus or thickened intima of the central vessels on computed tomography does not exclude the diagnosis of chronic thromboembolic disease or the possibility of surgical intervention. This clinical observation is supported by a recent series where V/Q scintigraphy was shown to be more sensitive (96 vs. 51%) in detecting chronic thromboembolic disease than multidetector CT pulmonary angiography [26]. It has also been demonstrated that central thrombi detected by computed tomography have been described in processes other than CTEPH such as idiopathic pulmonary hypertension, Eisenmenger syndrome, and chronic obstructive pulmonary disease [27–29]. Furthermore, studies directly comparing computed tomography with pulmonary angiography are

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Fig.  2  (a) Pulmonary arteriogram demonstrating a rounded appearance (white arrow) to the distal aspect of the right descending pulmonary artery (“pouch defect”). (b) On lateral view of the

same pulmonary arteriogram, proximal narrowing of a segmental vessel to the right lower lobe at the base of the “pouch” (black arrow)

Fig. 3  Intimal irregularities along the lateral aspect of the right descending pulmonary artery consistent with organized thrombus lining the vessel (white arrows). Proximal narrowing and vessel attenuation from underfilling of the right middle lobe artery (black arrow)

Fig.  4  Lateral view of a right-sided pulmonary arteriogram. Focal narrowing or “band” (indicated by the arrow) with poststenotic dilatation of a lower lobe vessel

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limited in number. In one such study, computed tomography and digital angiography were nearly equivalent in terms of identifying complete vessel occlusion at the segmental level. However, for nonocclusive changes, computed tomography was significantly inferior to angiography [30]. The accuracy of CT scanning has improved with technological advances and the introduction of 64-detector row scanners. In a preliminary study involving 27 patients with suspected CTEPH, the sensitivity of 64-detector row computed tomography was 98.3% at the main and lobar levels and 94% at the segmental level when compared with digital subtraction angiography [31]. There is considerable value for computed tomography in detecting disorders of the pulmonary parenchyma and medi-

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astinum. For CTEPH patients with coexisting interstitial lung disease or emphysema, computed tomography will be able to define the extent and location of the parenchymal lung process. Reperfusion of diseased lung parenchyma with a PEA may result in an undesirable postoperative outcome, and thereby exclude a patient from surgical consideration. For those patients whose V/Q scan demonstrates absence or near complete absence of perfusion to an entire lung, computed tomography is an essential study to rule out extrinsic pulmonary vascular compression from mediastinal adenopathy, fibrosis [32], or neoplasm [33].

9 Magnetic Resonance Imaging

Fig. 5  Computed tomographic angiogram of a CTEPH patient. Right descending pulmonary artery involved with a vascular web (thick white arrow); left descending pulmonary artery with lining thrombus, nearly occluding the entire vessel (thin white arrow)

There is expanding experience using magnetic resonance imaging (MRI) and magnetic resonance angiography (MRA) to visualize the pulmonary vascular system in patients with CTEPH [34]. For centers where conventional pulmonary angiography is either unavailable or felt to be too risky to perform, the evolving information on MRI as an alternative means to determine surgical candidacy for CTEPH patients is encouraging. Kreitner et  al. have shown that contrastenhanced MRA is able to demonstrate the vascular changes typical for chronic thromboembolic disease. In a study of 34 CTEPH patients, wall-adherent thromboembolic material involving the central pulmonary arteries down to the segmental level could be demonstrated; intraluminal webs and bands, as well as abnormal vessel tapering and “cutoffs” were also detected (Fig. 6). Further, they showed that MRA was superior to digital subtraction angiography in determining

Fig. 6  Magnetic resonance angiogram demonstrating large vessel narrowing and “bands” (arrow) consistent with chronic thrombotic lesion. (a) Maximal intensity projection (MIP) reconstruction of preoperative contrast

enhanced MRA. (b) Reconstruction demonstrating intralumalinal band (indicated by the arrow) in the pulmonary artery. (Reproduced with kind permission of Springer Science + Business Media [34])

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the proximal location of resectable chronic thromboem­ bolic material [35]. An additional study comparing magnetic resonance techniques with conventional contrast angiography involved 29 patients with either CTEPH or idiopathic pulmonary arterial hypertension. Nikolaou et al. showed that the combined interpretation of the findings from magnetic resonance perfusion imaging and MRA led to a correct diagnosis of idiopathic pulmonary arterial hypertension or CTEPH in 26 (90%) of 29 patients when compared with the reference diagnosis based on V/Q scintigraphy, digital subtraction angiography, or CT angiography. The interpretation of the findings from MRA alone had a sensitivity of 71% for wall-adherent thrombi, 50% for webs and bands, and between 83 and 86% for detection of complete vessel obstruction and free-floating thrombi when compared with digital subtraction angiography or CT angiography [36]. There are other features of MRI that can be useful in the evaluation of CTEPH patients. Cine imaging allows an assessment of right ventricular and left ventricular function, providing data on end-systolic and end-diastolic volumes, ejection fraction, and muscle mass [37, 38]. Furthermore, phase contrast imaging may be used to measure cardiac output, along with pulmonary and systemic arterial flow. In CTEPH patients undergoing PEA, this technique has been used to measure changes in aortic and pulmonary arterial blood flow before and after surgery [35, 39].

10 Summary The goals in the evaluation of patients with CTEPH are to first establish whether or not PEA is feasible, and then to determine whether or not surgery is advisable. Feasibility issues center around the technical aspects of surgery; that is, are the clots surgically accessible? Although there are training and interpretive skills involved (e.g. recognizing the angiographic patterns or magnetic resonance findings that are consistent with operable chronic thromboembolic disease), this is the more objective part in determining if a patient is a candidate for surgery. Harder to predict are the factors that influence perioperative mortality risks and postoperative outcomes. The impact of an individual’s advancing age, obesity, and/or comorbid medical conditions on surgical risk is always difficult to assess. Therefore, for an individual patient, the level of cardiopulmonary difficulties and the functional limitations experienced by him or her are important considerations in the decision to proceed with surgery and in his or her acceptance of associated risks. The anticipation that a hemodynamic benefit will result from an endarterectomy is critical to the decision to undergo surgery. If a meaningful reduction in pulmonary pressures seems unlikely with an attempted endarterectomy, proceeding with surgery is ill-

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advised. However, the prediction of a hemodynamic benefit is based primarily on the degree of coexisting small-vessel disease which will not be remedied with an endarterectomy. To date this assessment is clearly subjective, making it increasingly important to develop evaluative techniques to make it less so. With experience, the clinician soon appreciates that the most difficult decision to make in evaluating CTEPH patients for endarterectomy is the one which excludes them from this potential life-saving surgery. Ongoing research and careful clinical observations are required to make this decision as precise as possible.

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1574 hemangiomatosis: a rare disorder causing primary pulmonary hypertension. Clin Nucl Med 16:913–917 16. Ryan KL, Fedullo PF, Davis GB, Vasquez TE, Moser KM (1988) Perfusion scan findings understate the severity of angiographic and hemodynamic compromise in chronic thromboembolic pulmonary hypertension. Chest 93:1180–1185 17. Thistlethwaite P, Kemp A, Du L, Madani MM, Jamieson SW (2006) Outcomes of pulmonary thromboendarterectomy for treatment of extreme thromboembolic pulmonary hypertension. J Thorac Cardiovasc Surg 131:307–313 18. Hartz RS, Byme JG, Levitsky S, Park J, Rich S (1996) Predictors of mortality in pulmonary thromboendarterectomy. Ann Thorac Surg 62:1255–1260 19. Nagaya N, Sasaki N, Ando M et  al (2003) Prostacyclin therapy before pulmonary thromboendarterectomy in patients with chronic thromboembolic pulmonary hypertension. Chest 123:338–343 20. Kim NH, Fesler P, Channick RN et al (2004) Preoperative partitioning of pulmonary vascular resistance correlates with early outcome after thromboendarterectomy for chronic thromboembolic pulmonary hypertension. Circulation 109:18–22 21. Pitton MB, Duber C, Mayer E, Thelen M, Düber C (1996) Hemodynamic effects of nonionic contrast bolus injection and oxygen inhalation during pulmonary angiography in patients with chronic major-vessel thromboembolic pulmonary hypertension. Circulation 94:2485–2491 22. Auger WR, Fedullo PF, Moser KM, Buchbinder M, Peterson KL (1992) Chronic major-vessel chronic thromboembolic pulmonary artery obstruction: appearance at angiography. Radiology 183:393–398 23. King MA, Bergin CJ, Yeung D et  al (1994) Chronic pulmonary thromboembolism: detection of regional hypoperfusion with CT. Radiology 191:359–363 24. Schwickert HC, Schweden F, Schild HH et al (1994) Pulmonary arteries and lung parenchyma in chronic pulmonary embolism: preoperative and postoperative CT findings. Radiology 191: 351–357 25. Kerr KM (2005) Pulmonary artery sarcoma masquerading as chronic thromboembolic pulmonary hypertension. Nat Clin Pract Cardiovasc Med 2:108–112 26. Tunariu N, Gibbs SJR, Win Z et  al (2007) Ventilation-perfusion scintigraphy is more sensitive than multidetector CTPA in detecting chronic thromboembolic pulmonary disease as a treatable cause of pulmonary hypertension. J Nucl Med 48:680–684

W.R. Auger et al. 27. Moser KM, Fedullo PF, Finkbeiner WE, Golden J (1995) Do patients with primary pulmonary hypertension develop extensive central thrombi? Circulation 91:741–745 28. Silversides CK, Granton JT, Konen E, Hart MA, Webb GD, Therrien J (2003) Pulmonary thrombosis in adults with Eisenmenger syndrome. J Am Coll Cardiol 42:1982–1987 29. Russo A, De Luca M, Vigna C et al (1999) Central pulmonary artery lesions in chronic obstructive pulmonary disease. A transesophageal echocardiography study. Circulation 100:1808–1815 30. Pitton MB, Kemmerich G, Herber S, Schweden F, Mayer E, Thelen M (2002) Chronic thromboembolic pulmonary hypertension: diagnostic impact of multislice CT and selective pulmonary DSA. Rofo 174:474–479 31. Reichelt A, Hoeper MM, Galanski M, Keberle M (2009) Chronic thromboembolic pulmonary hypertension: evaluation with 64-detector row versus digital subtraction angiography. Eur J Radiol 71(1):49–54 32. Rossi SE, McAdams HP, Rosado-de-Christenson ML, Franks TJ, Galvin JR (2001) Fibrosing mediastinitis. Radiographics 21:737–757 33. Shields JJ, Cho KJ, Geisinger KR (1980) Pulmonary artery constriction by mediastinal lymphoma simulating pulmonary embolus. Am J Roentgenol 135:147–150 34. Kreitner K-F, Kunz RP, Ley S et al (2007) Chronic thromboembolic pulmonary hypertension – assessment by magnetic resonance imaging. Eur Radiol 17:11–21 35. Krietner K-F, Ley S, Kauczor H-U et al (2004) Chronic thromboembolic pulmonary hypertension: pre- and postoperative assessment with breath-hold magnetic resonance techniques. Radiology 232:535–543 36. Nikolaou K, Schoenberg SO, Attenberger U et al (2005) Pulmonary arterial hypertension: diagnosis with fast perfusion imaging and high-spatial-resolution MR angiography – preliminary experience. Radiology 236:694–703 37. Alfakih K, Reid S, Jones T, Sivananthan M (2004) Assessment of ventricular function and mass by cardiac magnetic resonance imaging. Eur Radiol 14:1813–1822 38. Beygui F, Furber A, Delepine S et al (2004) Routine breath-hold gradient echo MRI-derived right ventricular mass, volumes and function: accuracy, reproducibility and coherence study. Int J Cardiovasc Imaging 20:509–516 39. Miller FNAC, Coulden RA, Sonnex E et al. (2003) The use of MR flow mapping in the assessment of pulmonary artery blood flow following pulmonary thrombo-endarterectomy. Radiology, RSNA Proceedings, p. 462

Chapter 114

Pulmonary Endarterectomy Michael M. Madani and Stuart W. Jamieson

Abstract  Pulmonary thromboendarterectomy (PTE), or pulmonary endarterectomy, is the definitive treatment for chronic thromboembolic pulmonary hypertension (CTEPH). There is curative role for medical management for patients with CTEPH, and surgery remains the only option. In fact, once the mean pulmonary pressure in patients with thromboembolic disease reaches 50 mmHg or more, the 3-year mortality approaches 90%. Surgical options are dependent on both the primary disease process and the reversibility of the pulmonary hypertension. With the exception of thromboembolic pulmonary hypertension, lung transplantation is the only effective therapy for patients with idiopathic pulmonary arterial hypertension. Pulmonary transplantation is also still used in some centers as the treatment of choice for those with thromboembolic disease. However, a true assessment of the effectiveness of any therapy should take into account the total mortality once the patient has been accepted and put on the waiting list. Thus, the mortality for transplantation (and especially double-lung or heart-lung transplantation) as a therapeutic strategy is much higher than is generally appreciated because of the significant loss of patients awaiting donors. Considering, in addition, the long-term use of antirejection medications with their associated side effects, the higher operative morbidity and mortality, the long waiting time, and inferior prognosis even after transplantation, transplantation is clearly an inferior option to pulmonary thromboendarterectomy. We consider it to be inappropriate therapy for this disease, leaving pulmonary thromboendarterectomy as the only curative option for patients with chronic thromboembolic pulmonary hypertension. Keywords  Pulmonary thromboendarterectomy • Chronic thromboembolic pulmonary hypertension • Pulmonary embolism • Surgical treatment

M.M. Madani (*) Department of Surgery, University of California, 200 W. Arbor Drive, MC 8892, San Diego, CA 92105, USA e-mail: [email protected]

1 Introduction Pulmonary thromboendarterectomy is the definitive treatment for chronic pulmonary hypertension as the result of thromboembolic disease. Although pulmonary embolism is one of the more common cardiovascular diseases affecting Americans, pulmonary thromboendarterectomy remains an uncommon procedure, primarily because this form of chronic pulmonary hypertension remains an underdiagnosed condition. Patients affected by chronic thromboembolic pulmonary hypertension may present with a variety of debilitating cardiopulmonary symptoms. However, once it has been diagnosed, there is no curative role for medical management, and surgery remains the only option. Pulmonary thromboembolism is a significant cause of morbidity and mortality in the USA and worldwide. The exact incidence of pulmonary embolism remains unknown, but there are some valid estimates. The estimated incidence of acute pulmonary embolism is approximately 63 per 100,000 patients in the USA on the basis of clinical and radiographic data [1] and is related to approximately 235,000 deaths per year on the basis of autopsy data. Acute pulmonary embolism occurs half as often as acute myocardial infarction and is 3 times as common as cerebrovascular accident. It is the third most common cause of death (after heart disease and cancer) [2]. Estimates of the incidence of acute pulmonary embolism, however, are generally thought to be low, because in 70–80% of patients in whom the primary cause of death was pulmonary embolism, the diagnosis was unsuspected before death [3, 4]. The prognosis for patients with pulmonary hypertension is poor, and it is worse for those who do not have intracardiac shunts. Thus, patients with primary pulmonary hypertension and those with pulmonary hypertension due to pulmonary emboli fall into a higher-risk category than those with Eisenmenger syndrome and encounter a higher mortality rate. In fact, once the mean pulmonary pressure in patients with thromboembolic disease reaches 50 mmHg or more, the 3-year mortality approaches 90% [5]. Surgical options are dependent on both the primary ­disease process and the reversibility of the pulmonary

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_114, © Springer Science+Business Media, LLC 2011

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h­ ypertension. With the exception of thromboembolic ­pulmonary hypertension, lung transplantation is the only effective therapy for patients with pulmonary hypertension. Pulmonary transplantation is also still used in some centers as the treatment of choice for those with thromboembolic disease. However, a true assessment of the effectiveness of any therapy should take into account the total mortality once the patient has been accepted and put on the waiting list. Thus, the mortality for transplantation (and especially double-lung or heart-lung transplantation) as a therapeutic strategy is much higher than is generally appreciated because of the significant loss of patients awaiting donors. Considering, in addition, the long-term use of antirejection medications with their associated side effects, the higher operative morbidity and mortality, the long waiting time, and inferior prognosis even after transplantation, transplantation is clearly an inferior option to pulmonary thromboendarterectomy. We consider it to be inappropriate therapy for this disease, leaving pulmonary thromboendarterectomy as the only curative option for patients with chronic thromboembolic pulmonary hypertension.

2 Natural History For most episodes of pulmonary embolism, the natural history is generally total embolic resolution, or resolution leaving minimal residua, but with restoration of a normal hemodynamic status. However, for unknown reasons, embolic resolution is incomplete in a small subset of patients. If the acute emboli are not lysed in 1–2 weeks, the embolic material becomes attached to the pulmonary arterial wall at the main pulmonary artery, lobar, segmental, or subsegmental levels [6]. With time, the initial embolic material progressively becomes converted into connective and elastic tissue. Often visualization of the pulmonary arteries by angioscopy a few weeks after unresolved pulmonary embolism reveals vessel narrowing at the site of embolic incorporation. In some patients, recanalization of some of the pulmonary arterial branches occurs, with the formation of fibrous tissue in the form of bands and webs [7]. By a mechanism that is poorly understood, this chronic obstructive disease may lead to a small-vessel arteriolar vasculopathy characterized by excessive smooth muscle cell proliferation around small arterioles in the pulmonary circulation [8]. This small-vessel vasculopathy is seen in the remaining open vessels, which are subjected to long exposure at high flow. Pulmonary hypertension results when the capacitance of the remaining open bed cannot absorb the cardiac output, because of either the degree of primary obstruction by embolus or the combination of a fixed obstructive lesion and secondary small-vessel vasculopathy.

M.M. Madani and S.W. Jamieson

Once pulmonary hypertension has developed as the result of chronic thromboembolic material, patients will be in need of expeditious treatment. These patients do not generally respond to medical management as the underlying cause of the disease is vascular obstruction. The only curative option is to proceed with surgical removal of the scarred thromboembolic material by means of endarterectomy. Unfortunately, in the last few years we have started to see a delay in referral of these patients for appropriate therapy. There seems to be a developing trend to start pulmonary vasodilator therapy before referring patients for surgical management, and in some cases this comes at a cost of significant delay in referral. We need to be very clear that the appropriate treatment of chronic thromboembolic pulmonary hypertension is the surgical removal of the disease, and vasodilator therapy will not add any benefit for most patients. Of course, in a rare situation one may encounter a patient with long-standing disease and significant right-sided heart failure secon­dary to significant pulmonary hypertension who only has a mild to moderate degree of obstructive disease. Such a patient may temporarily benefit from a trial of vasodilator therapy (e.g. epoprostenol) prior to surgery, but the benefit is generally temporary and should not delay referral for surgery. Without surgical intervention, the survival of patients with chronic thromboembolic pulmonary hypertension is poor and is inversely related to the degree of pulmonary hypertension at the time of diagnosis. Riedel et al [5]. found a 5-year survival rate of 30% among patients with a mean pulmonary artery pressure greater than 40 mmHg at the time of diagnosis and 10% in those whose pressure exceeded 50 mmHg. In another study a mean pulmonary artery pressure as low as 30  mmHg was identified as a threshold for poor prognosis [9].

3 Incidence The exact incidence of pulmonary hypertension caused by chronic pulmonary embolism is difficult to determine and remains unknown. There are more than 500,000 survivors of symptomatic episodes of acute pulmonary embolism per year [10]. The exact incidence of chronic thrombotic occlusion or stenosis in the population depends on what percentage of patients fail to resolve acute embolic material. One estimate is that chronic thromboembolic disease develops in only 3.8% of patients with a clinically recognized acute pulmonary embolism [5]. If these figures are correct and only patients with symptomatic acute pulmonary emboli are counted, approximately 19,000 individuals would progress to chronic thromboembolic pulmonary hypertension in the USA each year. However, because many (if not most) patients

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diagnosed with chronic thromboembolic disease have no antecedent history of acute embolism, the true incidence of this disorder is probably much higher. Regardless of the exact incidence, it is clear that acute embolism and its chronic relation, fixed chronic thromboembolic occlusive disease, are both much more common than is generally appreciated and are seriously underdiagnosed. In 1963, Houk et al. [11] reviewed the literature of 240 cases of chronic thromboembolic obstruction of major pulmonary arteries, but found that only six cases had been diagnosed correctly before death. Calculations extrapolated from mortality rates and the random incidence of major thrombotic occlusion found at autopsy supported the postulate that more than 100,000 people in the USA currently have pulmonary hypertension that could be relieved by operation. A recent autopsy analysis of 13,216 patients showed pulmonary thromboembolism in 5.5% of autopsies, and up to 31.3% in the elderly [12].

4 Etiological Factors Chronic thromboembolic pulmonary hypertension is a disease characterized by initial thromboembolism to the pulmonary arterial tree that does not resolve itself. Whether this is due to an endothelial insult, preexisting prothrombotic state, or endothelial progenitor cell trapping and remodeling is the subject of debate. No clear cause has been defined for most patients who develop chronic thromboembolic pulmonary hypertension. Lupus anticoagulant may be detected in approximately 10% of chronic thromboembolic patients [13] and 20% carry anticardiolipin antibodies, lupus anticoagulant, or both. A recent study has demonstrated that the plasma level of factor VIII, a protein that is associated with both primary and recurrent venous thromboembolism, is elevated in 39% of patients with chronic thromboembolic pulmonary hypertension. Analyses of plasma proteins in patients with chronic thromboembolic disease have shown that fibrin from these patients is resistant to thrombolysis in vitro. In this study, the fibrin b chain N-terminus was particularly resistant to thrombolysis, suggesting that it could be responsible for thrombus nonresolution [14]. In addition, there are limited data on the role of thrombomodulin, an endothelial cell membrane surface protein that binds ­thrombin and acts as a cofactor for the conversion of protein C to activated protein C, a natural anticoagulant in this disease. One group has shown a negative correlation between thrombomodulin plasma concentrations and mean pulmonary artery pressure and pulmonary vascular resistance (PVR) in chronic thromboembolic patients compared with acute venous thromboembolism and control patients [15]. In patients who underwent pulmonary ­thromboendarterectomy,

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thrombomodulin concentration increased significantly, implying endothelial cell dysfunction in addition to a prothrombotic state in the pre-pulmonary thromboendarterectomy patients. Case reports and small series have suggested links between chronic thromboembolism and previous splenectomy, permanent intravenous catheters, and ventriculoatrial shunts for the treatment of hydrocephalus or chronic inflammatory conditions. In addition to these observations, associations with sickle cell disease, hereditary stomatocytosis, and Klippel–Trenaunay syndrome have been described [16]. However, the vast majority of cases of chronic thromboembolic pulmonary hypertension are not linked with a specific coagulation defect or underlying medical condition as described above.

5 Pathology and Pathogenesis Although most individuals with chronic pulmonary thromboembolic disease are unaware of a past thromboembolic event and give no history of deep venous thrombosis, the origin of most cases of unresolved pulmonary emboli is from acute embolic episodes. Why some patients have unresolved emboli is not certain, but a variety of factors must play a role, alone or in combination. The volume of acute embolic material may simply overwhelm the lytic mechanisms. The total occlusion of a major arterial branch may prevent lytic material from reaching, and therefore dissolving, the embolus completely. Repetitive emboli may not be able to be resolved. The emboli may be made of substances that cannot be resolved by normal mechanisms (already well-organized fibrous thrombus, fat, or tumor). The lytic mechanisms themselves may be abnormal, or some patients may actually have a propensity for thrombus or a hypercoaguable state. After the clot becomes wedged in the pulmonary artery, one of two processes occurs [17]: 1. The organization of the clot proceeds to canalization, producing multiple small endothelialized channels separated by fibrous septa (i.e. bands and webs). 2. Complete fibrous organization of the fibrin clot without canalization may result, leading to a solid mass of dense fibrous connective tissue totally obstructing the arterial lumen. In addition, there are other special circumstances. Chronic indwelling central venous catheters and pacemaker leads are sometimes associated with pulmonary emboli. Rarer causes include tumor emboli; tumor fragments from stomach, breast, and kidney malignancies have also been demonstrated to cause chronic pulmonary arterial occlusion. Right atrial myxomas may also fragment and embolize.

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As previously described and discussed, in addition to the embolic material, a propensity for thrombosis or a hypercoaguable state may be present in a few patients. This abnormality may result in spontaneous thrombosis within the pulmonary vascular bed, encourage embolization, or be responsible for proximal propagation of thrombus after an embolus. But, whatever the predisposing factors for residual thrombus within the vessels, the final genesis of the resultant pulmonary vascular hypertension may be complex. With the passage of time, the increased pressure and flow as a result of redirected pulmonary blood flow in the previously normal pulmonary vascular bed can create a vasculopathy in the small precapillary blood vessels similar to Eisenmenger syndrome. Factors other than the simple hemodynamic consequences of redirected blood flow are probably also involved in this process. For example, after a pneumonectomy, 100% of the right ventricular output flows to one lung, yet little increase in pulmonary pressure occurs, even with follow-up to 11 years [18]. In patients with thromboembolic disease, however, we frequently detect pulmonary hypertension even when less than 50% of the vascular bed is occluded by thrombus. It thus appears that sympathetic neural connections, hormonal changes, or both might initiate pulmonary hypertension in the initially unaffected pulmonary vascular bed. This process can occur with the initial occlusion being either in the same or in the contralateral lung. Regardless of the cause, the evolution of pulmonary hypertension as a result of changes in the previously unobstructed bed is serious, because this process may lead to an inoperable situation. Consequently, with our accumulating experience in patients with thrombotic pulmonary hypertension, we have increasingly been inclined toward early operation so as to avoid these changes.

5.1 Clinical Presentation Chronic thromboembolic pulmonary hypertension is an uncommon and frequently underrecognized, but treatable cause of pulmonary hypertension. There are no signs or symptoms specific for chronic thromboembolism. The most common symptom associated with thromboembolic pulmonary hypertension, as with all other causes of pulmonary hypertension, is exertional dyspnea. This dyspnea is out of proportion to any abnormalities found on clinical examination. Like complaints of easy fatigability, dyspnea that initially occurs only with exertion is often attributed to anxiety or being “out of shape.” Syncope, or presyncope (light-headedness during exertion), is another common symptom in pulmonary hypertension. Generally, it occurs in patients with more advanced disease and higher pulmonary artery pressures.

M.M. Madani and S.W. Jamieson

Nonspecific chest pains or tightness occur in approximately 50% of patients with severer pulmonary hypertension. Hemoptysis can occur in all forms of pulmonary hypertension and probably results from abnormally dilated vessels distended by increased intravascular pressures. Peripheral edema, early satiety, and epigastric or right upper quadrant fullness or discomfort may develop as the right side of the heart fails (cor pulmonale). Some patients with chronic pulmonary thromboembolic disease present after a small acute pulmonary embolus that may produce acute symptoms of right-sided heart failure. Careful history taking brings out symptoms of dyspnea on minimal exertion, easy fatigability, diminishing activities, and episodes or angina-like pain or light-headedness. Further examination reveals the signs of pulmonary hypertension. Sometimes hemoptysis occurs. The physical signs of pulmonary hypertension are the same no matter what the underlying disorder. Initially, the jugular venous pulse is characterized by a large A-wave. As the right side of the heart fails, the V-wave becomes predominant. The right ventricle is usually palpable near the lower left sternal border, and pulmonary valve closure may be audible in the second intercostal space. Occasional patients with advanced disease are hypoxic and slightly cyanotic. Clubbing is an uncommon finding. The second heart sound is often narrowly split and varies normally with respiration; P2 is accentuated. A sharp systolic ejection click may be heard over the pulmonary artery. As the right side of the heart fails, a right atrial gallop is usually present, and tricuspid insufficiency develops. Because of the large pressure gradient across the tricuspid valve in pulmonary hypertension, the murmur is high-pitched and may not exhibit respiratory variation. These findings are quite different from those usually observed in tricuspid valvular disease. A murmur of pulmonic regurgitation may also be detected. Pulmonary function tests reveal minimal changes in lung volume and ventilation; patients generally have normal or slightly restricted pulmonary mechanics. Single-breath diffusing capacity for carbon monoxide (DLCO) is often reduced and may be the only abnormality on pulmonary function testing. Pulmonary artery pressures are elevated and suprasystemic pulmonary pressures are not uncommon. Resting cardiac outputs are lower than the normal range, and pulmonary artery oxygen saturations are reduced. Most patients are hypoxic; room air arterial oxygen tension ranges between 50 and 83 Torr, the average being 65 Torr [19]. CO2 tension is slightly reduced and is compensated by reduced bicarbonate. Dead-space ventilation is increased. Ventilation–perfusion studies show moderate mismatch with some heterogeneity among various respirator units within the lung and the findings correlate poorly with the degree of pulmonary obstruction [20].

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5.2 Diagnostic Criteria and Evaluation of Chronic Thromboembolic Pulmonary Hypertension To ensure diagnosis in patients with chronic pulmonary thromboembolism, a standardized evaluation is recommended for all patients who present with unexplained pulmonary hypertension. Pulmonary vascular disease must always be considered in the differential diagnosis of unexplained dyspnea. The diagnostic evaluation serves three purposes: to establish the presence and severity of pulmonary hypertension; to determine its cause; and, if thromboembolic disease is present, to determine whether it is surgically correctable. Chest radiography is often unrevealing in the early stages of chronic thromboembolic pulmonary hypertension. As the disease progresses, several radiographic abnormalities may be found. These include peripheral lung opacities suggestive of scarring from previous infarction, cardiomegaly with dilation and hypertrophy of the right-sided chambers, and dilation of the central pulmonary arteries. Pulmonary function tests are often performed in the evaluation of dyspnea and serve to exclude the presence of obstructive airways or parenchymal lung disease. There are no characteristic spirometric changes diagnostic of chronic thromboembolic pulmonary hypertension. DLCO may be moderately reduced, and it has been reported that 20% of patients have a mild to moderate restrictive defect caused by parenchymal scarring [21]. Arterial blood oxygen levels may be normal even in the setting of significant pulmonary hypertension. Most patients, however, experience a decline in PO2 with exertion. Transthoracic echocardiography is the first study to provide objective evidence of the presence of pulmonary hypertension. An estimate of pulmonary artery pressure can be provided by Doppler evaluation of the tricuspid regurgitant envelope. Additional echocardiographic findings vary depending on the stage of the disease and include right ventricular enlargement, leftward displacement of the interventricular septum, and encroachment of the enlarged right ventricle on the left ventricular cavity with abnormal systolic and diastolic function of the left ventricle [22]. Contrast echocardiography may demonstrate a persistent foramen ovale, the result of high right atrial pressures opening the previously closed intra-atrial communication. Once the diagnosis of pulmonary hypertension has been established, distinguishing between major-vessel obstruction and small-vessel pulmonary vascular disease is the next ­cri­tical step. Radioisotope ventilation–perfusion lung scanning is the essential test for establishing the diagnosis of unresolved pulmonary thromboembolism. The ventilation–perfusion scan typically demonstrates one or more mismatched segmental defects caused by obstructive thromboembolism. This is in contrast to the normal or “mottled” perfusion scan seen in patients with primary pulmonary hypertension or other

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small-vessel forms of pulmonary hypertension [23]. It is important to note that ventilation–perfusion scanning may underestimate the magnitude of perfusion defects with chronic thromboembolic pulmonary hypertension, as partial recanalization of the vessel lumen can occur, resulting in some perfusion, although with significant obstruction to flow [24]. Cardiac catheterization provides essential information in the evaluation of patients with suspected thromboem­ bolic pulmonary hypertension. Right ventricular catheterization allows for the quantification of the severity of pulmonary hypertension and assessment of cardiac function. Measurement of oxygen saturations in the vena cava, right ventricular chambers, and the pulmonary artery may document previously undetected left-to-right shunting. Coronary angiography and left-sided heart catheterization provide additional information about patients at risk for coronary artery or valvular disease and establish baseline measurements for cardiac output and left ventricular function. This information is crucial in the preoperative risk assessment of patients deemed candidates for pulmonary endarterectomy. Pulmonary angiography is the gold standard for defining pulmonary vascular anatomy and is performed to identify whether chronic thromboembolic obstruction is present, to determine its location and surgical accessibility, and to rule out other diagnostic possibilities. In angiographic imaging, thrombi appear as unusual filling defects, pouches, webs, or bands, or completely thrombosed vessels that may resemble congenital absence of a vessel. Organized material along a vascular wall produces a scalloped or serrated luminal edge [25]. Despite concerns regarding the safety of performing pulmonary angiography in patients with pulmonary hypertension, with careful monitoring, pulmonary angiography can be performed safely even in patients with severe pulmonary hypertension [26]. Biplane imaging is preferred, offering the advantage of lateral views that provide greater anatomical detail compared with the overlapped and obscured vessel images often seen with the anterior–posterior view. Maturation, organization, and recanalization of clot produces angiographic patterns of the following: (1) pouch defects, (2) webs or bands, (3) intimal irregularities, (4) abrupt narrowing of major vessels, and (5) obstruction of main, lobar, or segmental pulmonary vessels [27]. More recently, helical computed tomography scanning [28], single-photon-emission computed tomography–computed tomography fusion imaging [29], and magnetic resonance ­angiography [30] have been used to screen patients with suspected thromboembolic disease. Features of chronic thromboembolic disease seen by these modalities include evidence of organized thrombus lining the pulmonary vessels in an eccentric fashion, enlargement of the right ventricle and central pulmonary arteries, variation in the size of segmental arteries (relatively smaller in affected segments compared with uninvolved areas), and parenchymal changes compatible with pulmonary infarction.

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In approximately 10% of cases, the differential diagnosis between primary pulmonary hypertension and distal and smallvessel pulmonary thromboembolic disease remains unclear and hard to establish. In these patients, pulmonary angioscopy is often helpful. The pulmonary angioscope is a fiber-optic telescope that is placed through a central line into the pulmonary artery. The tip contains a balloon that is then filled with saline and pushed against the vessel wall. A bloodless field can thus be obtained to view the pulmonary artery wall. The classic appearance of chronic pulmonary thromboembolic disease by angioscopy consists of intimal thickening, with intimal irregularity and scarring, and webs across small vessels. These webs are thought to be the residue of resolved occluding thrombi of small vessels, but are important diagnostic findings. The presence of embolic disease, occlusion of vessels, or the presence of thrombotic material is diagnostic.

6 Pulmonary Thromboendarterectomy Although there were previous attempts, Allison [31] performed the first successful pulmonary “thromboendarterectomy” through a sternotomy using surface hypothermia, but only fresh clots were removed. The operation was 12 days after a thigh injury that led to pulmonary embolism, and there was no endarterectomy. Since then, there have been many occasional surgical reports of the surgical treatment of chronic pulmonary thromboembolism, but most of the surgical experience in pulmonary endarterectomy has been reported from the University of California, San Diego (UCSD) Medical Center. Braunwald commenced the UCSD experience with this operation in 1970, which now totals more than 2,400 cases. The operation described later, using deep hypothermia and circulatory arrest, is the standard procedure.

6.1 Indications When the diagnosis of thromboembolic pulmonary hypertension has been firmly established, the decision for operation is made on the basis of the severity of symptoms and the general condition of the patient. Early in the pulmonary endarterectomy experience, Moser et al. [32] pointed out that there were three major reasons for considering thromboendarterectomy: hemodynamic, alveolorespiratory, and prophylactic. The hemodynamic goal is to prevent or ameliorate right ventricular compromise caused by pulmonary hypertension. The respiratory objective is to improve respiratory function by the removal of a large ventilated but unperfused physiologic dead space, regardless of the severity of pulmonary hypertension. The prophylactic goal is to prevent progressive right ventricular dysfunction or retrograde extension of the

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obstruction, which might result in further cardiorespiratory deterioration or death. Our subsequent experience has added another prophylactic goal: the prevention of secondary arteriopathic changes in the remaining patent vessels. Most patients who undergo operation are within New York Heart Association (NYHA) class III or class IV. The ages of the patients in our series have ranged from 7 to 85 years. A typical patient will have a severely elevated PVR at rest, the absence of significant comorbid disease unrelated to right-sided heart failure, and the appearances of chronic thrombi on angiogram that appear to be relatively in balance with the measured PVR. Exceptions to this general rule, of course, occur. Although most patients have a PVR of around 800 dyn s/cm5 and pulmonary artery pressures less than systemic levels, the hypertrophy of the right ventricle that occurs over time makes pulmonary hypertension to suprasystemic levels possible. Therefore, many patients (approximately 20% in our practice) have a PVR in excess of 1,000 dyn s/cm5 and suprasystemic pulmonary artery pressures. There is no upper limit of PVR, pulmonary artery pressure, or degree of right ventricular dysfunction that excludes patients from operation. We have become increasingly aware of the changes that can occur in the remaining patent (unaffected by clot) pulmonary vascular bed subjected to the higher pressures and flow that result from obstruction in other areas. Therefore, with the increasing experience and safety of the operation, we are tending to offer surgery to symptomatic patients whenever the angiogram demonstrates thromboembolic disease. A rare patient might have a PVR that is normal at rest, although elevated with minimal exercise. This is usually a young patient with total unilateral pulmonary artery occlusion and unacceptable exertional dyspnea because of an elevation in deadspace ventilation. Operation in this circumstance is performed not only to reperfuse lung tissue, but also to reestablish a more normal ventilation–perfusion relationship (thereby reducing minute ventilatory requirements during rest and exercise), and also to preserve the integrity of the contralateral circulation and prevent chronic arterial changes associated with long-term exposure to pulmonary hypertension. If not previously implanted, an inferior vena caval filter is routinely placed several days in advance of the operation.

6.2 Operation 6.2.1 Principles There are several guiding principles for the operation. Surgical treatment and endarterectomy must be bilateral because this is a bilateral disease in the vast majority of our patients. Furthermore, for pulmonary hypertension to be a major factor, both pulmonary vasculatures must be substantially

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involved. The only reasonable approach to both pulmonary arteries is through a median sternotomy incision. Historically, there were many reports of unilateral operation, and occasionally this is still performed, in inexperienced centers, through a thoracotomy. However, the unilateral approach ignores the disease on the contralateral side, subjects the patient to hemodynamic jeopardy during the clamping of the pulmonary artery, does not allow good visibility because of the continued presence of bronchial blood flow, and exposes the patient to a repeat operation on the contralateral side. In addition, collateral channels develop in chronic thrombotic hypertension not only through the bronchial arteries but also from diaphragmatic, intercostal, and pleural vessels. The dissection of the lung in the pleural space via a thoracotomy incision can therefore be extremely bloody. The median sternotomy incision, apart from providing bilateral access, avoids entry into the pleural cavities, and allows the ready institution of cardiopulmonary bypass. Cardiopulmonary bypass is essential to ensure cardiovascular stability when the operation is performed and to cool the patient to allow circulatory arrest. Excellent visibility is required, in a bloodless field, to define an adequate endar­ terectomy plane and to then follow the pulmonary endarterectomy specimen deep into the subsegmental vessels. Because of the copious bronchial blood flow usually present in these cases, periods of circulatory arrest are necessary to ensure perfect visibility. Again, there have been sporadic reports of the performance of this operation without circulatory arrest. However, it should be emphasized that although endarterectomy is possible without circulatory arrest, a complete endarterectomy is not. We always initiate the procedure without circulatory arrest, and a variable amount of dissection is possible before the circulation is stopped, but never complete dissection. The circulatory arrest periods are limited to 20 min, with restoration of flow between each arrest. With experience, the endarterectomy usually can be performed with a single period of circulatory arrest on each side. A true endarterectomy in the plane of the media must be accomplished. It is essential to appreciate that the removal of visible thrombus is largely incidental to this operation. Indeed, in most patients, no free thrombus is present; and on initial direct examination, the pulmonary vascular bed may appear normal. The early literature on this procedure indicates that thrombectomy was often performed without endarterectomy, and in these cases the pulmonary artery pressures did not improve, often with the resultant death of the patient.

There is usually severe right ventricular hypertrophy, and with critical degrees of obstruction, the patient’s condition may become unstable with the manipulation of the heart. Anticoagulation is achieved with the use of beef-lung heparin sodium (400  units/kg, intravenously) administered to prolong the activated clotting time beyond 400 s. Full cardiopulmonary bypass is instituted with high ascending aortic cannulation and two caval cannulae. These cannulae must be inserted into the superior and inferior venae cavae sufficiently to enable subsequent opening of the right atrium. The heart is emptied on bypass, and a temporary pulmonary artery vent is placed in the midline of the main pulmonary artery 1 cm distal to the pulmonary valve. This will mark the beginning of the left pulmonary arteriotomy. When cardiopulmonary bypass is initiated, surface cooling with both the head jacket and the cooling blanket is begun. The blood is cooled with the pump-oxygenator. During cooling, a 10°C gradient between arterial blood and bladder or rectal temperature is maintained [33]; cooling generally takes 45–60 min. When ventricular fibrillation occurs, an additional vent is placed in the left atrium through the right superior pulmonary vein (Fig. 1). This prevents atrial and ventricular distension from the large amount of bronchial arterial blood flow that is common with these patients. It is most convenient for the primary surgeon to stand ­initially on the patient’s left side (Fig. 1). During the cooling period, some preliminary dissection can be performed, with full mobilization of the right pulmonary artery from the ascending aorta. The superior vena cava is also fully mobilized.

6.2.2 Surgical Technique

Fig. 1  Intraoperative setup for pulmonary endarterectomy. Initially the surgeon stands on the left side of the patient. In addition to cannulae in both inferior and superior venae cavae, pulmonary artery and left atrial vents are also placed. The left atrial vent is placed via the right superior pulmonary artery and directed into the left ventricle while the patient is cooling and the heart fibrillating. SVC superior vena cava, IVC, inferior vena cava, PA pulmonary artery, LA left atrium

After a median sternotomy incision has been made, the pericardium is incised longitudinally and attached to the wound edges. Typically the right side of the heart is enlarged, with a tense right atrium and a variable degree of tricuspid regurgitation.

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Fig. 2  Surgical approach to the right pulmonary artery and endarterectomy. The right pulmonary artery is approached between the aorta and the superior vena cava (a, b), and not lateral to the superior vena cava. This gives better exposure of the more distal

branches. As depicted in a, the plane of dissection is raised posteriorly initially and then a complete endarterectomy is performed. SVC superior vena cava, PA pulmonary artery, RUL right upper lobe, RLL right lower lobe

The approach to the right pulmonary artery is made medially, not laterally, to the superior vena cava (Fig. 2). All dissection of the pulmonary arteries takes place intrapericardially, and neither pleural cavity should be entered. An incision is then made in the right pulmonary artery from beneath the ascending aorta out under the superior vena cava and entering the lower lobe branch of the pulmonary artery just after the takeoff of the middle lobe artery (Fig. 2). It is important that the incision stays in the center of the vessel and continues into the lower, rather than the middle lobe artery. Any loose thrombus, if present, is now removed. This is necessary to obtain good visualization. It is most important to recognize, however, that first, an embolectomy without subsequent endarterectomy is quite ineffective and, second, that in most patients with chronic thromboembolic hypertension, direct examination of the pulmonary vascular bed at operation generally shows no obvious embolic material. Therefore, to the inexperienced or cursory glance, the pulmonary vascular bed may well appear normal even in patients with severe chronic embolic pulmonary hypertension. If the bronchial circulation is not excessive, the endarterectomy plane can be found during this early dissection. However, although a small amount of dissection can be performed before the initiation of circulatory arrest, it is unwise to proceed unless perfect visibility is obtained because the development of a correct plane is essential. There are four broad types of pulmonary occlusive disease related to thrombus that can be appreciated, and we use the following classification [27, 34, 35]. Type I disease

(approximately 38% of cases of thromboembolic pulmonary hypertension; Fig. 3) refers to the situation in which majorvessel clot is present and readily visible on the opening of the pulmonary arteries. As mentioned earlier, all central thrombotic material has to be completely removed before the endarterectomy. In type II disease (approximately 42% of cases; Fig. 4), no major-vessel thrombus can be appreciated. In these cases only thickened intima can be seen, occasionally with webs, and the endarterectomy plane is raised in the main, lobar, or segmental vessels. Type III disease (approximately 18% of cases; Fig. 5) presents the most challenging surgical situation. The disease is very distal and confined to the segmental and subsegmental branches. No occlusion of vessels can be seen initially. The endarterectomy plane must be carefully and painstakingly raised in each segmental and subsegmental branch. Type III disease is most often associated with presumed repetitive thrombi from indwelling catheters (such as pacemaker wires) or ventriculoatrial shunts. Type IV disease does not represent primary thromboembolic pulmonary hypertension and is inoperable. In this entity, there is intrinsic small-vessel disease, although secondary thrombus may occur as a result of stasis. Small-vessel disease may be unrelated to thromboembolic events (“primary” pulmonary hypertension) or may occur in relation to thromboembolic hypertension as a result of a high-flow or high-pressure state in previously unaffected vessels similar to the generation of Eisenmenger syndrome. We believe that there may also be sympathetic “cross talk” from an affected contralateral side or stenotic areas in the same lung.

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Fig. 3  Surgical specimen removed from a patient showing evidence of some fresh and some old thrombus in the main pulmonary artery and the right and left pulmonary arteries. It is important to note that simple removal of the gross disease initially encountered upon pulmonary arteriotomy will not be therapeutic, and will certainly result in patient’s demise unless a full endarterectomy is performed Fig. 5  Specimen removed from a patient with type III disease. Note that in this group of patients the disease is more distal, and the plane of dissection has to be raised individually at each segmental level

Fig.  4  Specimen removed in a patient with type II disease. Both pulmonary arteries have evidence of chronic thromboembolic material, but there is no evidence of fresh thromboembolic material. Note the distal tails of the specimen in each branch. Full resolution of pulmonary hypertension is dependent on complete removal of all the distal tails

When the patient’s temperature reaches 20°C, the aorta is cross-clamped and a single dose of cold cardioplegic solution (1  L) is administered. Additional myocardial protection is obtained by the use of a cooling jacket. The entire procedure is now performed with a single aortic cross-clamp period with no further administration of cardioplegic solution. A modified cerebellar retractor is placed between the aorta and superior vena cava. When blood obscures direct vision of the pulmonary vascular bed, thiopental is administered (0.5–1 g) until the electroencephalogram becomes isoelectric. Circulatory arrest is then initiated, and the patient undergoes exsanguination. All monitoring lines to the patient are turned off to prevent the aspiration of air. Snares are tightened around the cannulae in the superior and inferior venae cavae. It is rare that one 20-min period for each side is

exceeded. Although retrograde cerebral perfusion has been advocated for total circulatory arrest in other procedures, it is not helpful in this operation because it does not allow a completely bloodless field, and with the short arrest times that can be achieved with experience, it is not necessary. Any residual loose, thrombotic debris encountered is removed. Then, a microtome knife is used to develop the endarterectomy plane posteriorly, because any inadvertent egress in this site could be repaired readily, or simply left alone (Fig.  2). Dissection in the correct plane is critical because if the plane is too deep, the pulmonary artery may perforate, with fatal results, and if the dissection plane is not deep enough, inadequate amounts of the chronically thromboembolic material will be removed. When the proper plane is entered, the layer will strip easily, and the material left with the outer layers of the pulmonary artery will appear somewhat yellow. The ideal layer is marked with a pearly white plane, which strips easily. There should be no residual yellow plaque. If the dissection is too deep, a reddish or pinkish color indicates the adventitia has been reached. A more superficial plane should be sought immediately. Once the plane has been correctly developed, a full-thickness layer is left in the region of the incision to ease subsequent repair. The endarterectomy is then performed with an eversion technique. Because the vessel is everted and subsegmental branches are being worked on, a perforation here will become completely inaccessible and invisible later. This is why the absolute visualization in a completely bloodless field provided by circulatory arrest is essential. It is important that each subsegmental branch is followed and freed individually until it ends in a “tail,” beyond which there is no further obstruction.

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Residual material should never be cut free; the entire specimen should “tail off” and come free spontaneously. Once the right-sided endarterectomy has been completed, circulation is restarted, and the arteriotomy is repaired with a continuous 6-0 polypropylene suture. The hemostatic nature of this closure is aided by the nature of the initial dissection, with the full thickness of the pulmonary artery being preserved immediately adjacent to the incision. After the completion of the repair of the right arteriotomy, the surgeon moves to the patient’s right side. The pulmonary vent catheter is withdrawn, and an arteriotomy is made from the site of the pulmonary vent hole laterally to the pericardial reflection, avoiding entry into the left pleural space (Fig. 6). Additional lateral dissection does not enhance intraluminal visibility, may endanger the left phrenic nerve, and makes subsequent repair of the left pulmonary artery more difficult (Fig. 6). The left-sided dissection is virtually analogous in all respects to that accomplished on the right. The duration of

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circulatory arrest intervals during the performance of the left-sided dissection is subject to the same restriction as the for the right-sided dissection. After the completion of the endarterectomy, cardiopulmonary bypass is reinstituted and warming is commenced. Methylprednisolone (500  mg, intravenously) and mannitol (12.5 g, intravenously) are administered, and during warming a 10°C temperature gradient is maintained between the perfusate and body temperature, with a maximum perfusate temperature of 37°C. If the systemic vascular resistance level is high, nitroprusside is administered to promote vasodilatation and warming. The rewarming period generally takes approximately 90–120 min but varies according to the body mass of the patient. When the left pulmonary arteriotomy has been repaired, the pulmonary artery vent is replaced at the top of the incision. The heart is retracted upwards and to the left, and a posterior pericardial window is made, between the aorta and the left phrenic nerve. Alternatively, prior to closure, a posterior pericardial drain can be placed, and can be removed once drainage has substantially decreased. The right atrium is then opened and examined. Any intra-atrial communication is closed. Although tricuspid valve regurgitation is invariable in these patients and is often severe, tricuspid valve repair is not performed. Right ventricular remodeling occurs within a few days, with the return of tricuspid competence. If other cardiac procedures are required, such as coronary artery or mitral or aortic valve surgery, these are conveniently performed during the systemic rewarming period. Myocardial cooling is discontinued once all cardiac procedures have been concluded. The left atrial vent is removed, and the vent site is repaired. All air is removed from the heart, and the aortic cross-clamp is removed. When the patient has rewarmed, cardiopulmonary bypass is discontinued. Dopamine hydrochloride is routinely administered at renal doses, and other inotropic agents and vasodilators are titrated as necessary to sustain acceptable hemodynamics. The cardiac output is generally high, with a low systemic vascular resistance. Temporary atrial and ventricular epicardial pacing wires are placed. Despite the duration of extracorporeal circulation, hemostasis is readily achieved, and the administration of platelets or coagulation factors is generally unnecessary. Wound closure is routine. A vigorous diuresis is usual for the next few hours, also a result of the previous systemic hypothermia.

7 Adverse Effects Fig. 6  Surgical approach to the left pulmonary artery. The heart is retracted medially using a mesh retractor and the left pulmonary artery is exposed. The arteriotomy is then directed toward the descending pulmonary artery past the upper lobe takeoff. Again the endarterectomy is initiated by raising the correct plane over the posterior aspect of the vessel

Patients are subject to all complications associated with open-heart and major lung surgery (arrhythmias, atelectasis, wound infection, pneumonia, mediastinal bleeding, etc.)

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but  also may develop complications specific to this operation. These include persistent pulmonary hypertension, ­reperfusion pulmonary response, and very rarely disorders related to deep hypothermia and total circulatory arrest.

7.1 Persistent Pulmonary Hypertension The decrease in PVR usually results in an immediate and sustained restoration of pulmonary artery pressures to normal levels, with a marked increase in cardiac output. In a few patients, an immediately normal pulmonary vascular tone is not achieved, but an additional substantial reduction may occur over the next few days because of the subsequent relaxation of small vessels and the resolution of intraoperative factors such as pulmonary edema. In such patients, it is usual to see a large pulmonary artery pulse pressure, the low diastolic pressure indicating good runoff, yet persistent pulmonary arterial inflexibility still resulting in a high systolic pressure. There are a few patients in whom the pulmonary artery pressures do not resolve substantially. We do operate on some patients with severe pulmonary hypertension but equivocal embolic disease. Despite the considerable risk of attempted endarterectomy in these patients, since transplantation is the only other avenue of therapy, there may be a point when it is unlikely that a patient will survive until a donor is found. In our most recent 500 patients, more than one third of perioperative deaths were directly attributable to the problem of inadequate relief of pulmonary arterial hypertension. This was a diagnostic rather than an operative technical problem. Attempts at pharmacological manipulation of high residual PVRs with sodium nitroprusside, epoprostenol sodium, or inhaled nitric oxide are generally not effective. Because the residual hypertensive defect is fixed, it is not appropriate to use mechanical circulatory support or extracorporeal membrane oxygenation in these patients if they deteriorate subsequently.

7.2 The “Reperfusion Response” A specific complication that occurs in most patients to some degree is localized pulmonary edema, or the “reperfusion response.” Reperfusion response or reperfusion injury is defined as a radiologic opacity seen in the lungs within 72 h of pulmonary endarterectomy. This unfortunately loose definition may therefore encompass many causes, such as fluid overload and infection. True reperfusion injury that directly and adversely impacts the clinical course of the patient now occurs in less than 10% of patients. In its most dramatic form, it occurs soon after operation

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(within a few hours) and is associated with profound desaturation. Edema-like fluid, sometimes with a bloody tinge, is suctioned from the endotracheal tube [36]. Frank blood from the endotracheal tube, however, signifies a mechanical violation of the blood airway barrier that has occurred at operation and stems from a technical error. This complication should be managed, if possible, by identification of the affected area by bronchoscopy and balloon occlusion of the affected lobe until coagulation can be normalized. One common cause of the reperfusion pulmonary edema is persistent high pulmonary artery pressures after operation when a thorough endarterectomy has been performed in certain areas, but there remains a large part of the pulmonary vascular bed affected by type IV change. However, the reperfusion phenomenon is often encountered in patients after a seemingly technically perfect operation with complete resolution of high pulmonary artery pressures. In these cases the response may be one of reactive hyperemia, after the revascularization of segments of the pulmonary arterial bed that have long experienced no flow. Other contributing factors may include perioperative pulmonary ischemia and conditions associated with high permeability lung injury in the area of the now denuded endothelium. Fortunately, the incidence of this complication is much less common now in our series, probably as a result of the more complete and expeditious removal of the endarterectomy specimen that has come with the large experience over the last decade.

7.3 Management of the “Reperfusion Response” Early measures should be taken to minimize the development of pulmonary edema with diuresis, maintenance of the hematocrit levels, and the early use of peak end-expiratory pressure. Once the capillary leak has been established, treatment is supportive because reperfusion pulmonary edema will eventually resolve if satisfactory hemodynamics and oxygenation can be maintained. Careful management of ventilation and fluid balance is required. The hematocrit is kept high (32–36%), and the patient undergoes aggressive diuresis, even if this requires ultrafiltration. The patient’s ventilatory status may be dramatically position sensitive. The FiO2 level is kept as low as is compatible with an oxygen saturation of 90%. A careful titration of positive end-expiratory pressure is carried out, with a progressive transition from volume-limited to pressure-limited inverse ratio ventilation and the  acceptance of moderate hypercapnia [36]. The use of steroids is discouraged because they are generally ineffective and may lead to infection. Infrequently, inhaled nitric oxide at 20–40 ppm can improve the gas exchange. On occasion, we have used extracorporeal perfusion support (extracorporeal

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membrane oxygenator or extracorporeal carbon dioxide removal) until ventilation can be resumed satisfactorily, usually after 7–10 days. However, its use is limited to patients who have benefited from hemodynamic improvement, but are suffering from significant reperfusion response. Extracorporeal devices should not be used if there is no evidence or hope of subsequent hemodynamic improvement, since their use carries mortality close to 100%, and will not play a role in improving irreversible pulmonary pressures.

7.4 Pericardial Effusion Probably because of the lymphatic tissue that is encountered during the dissection of the hilum and the mobilization of the superior vena cava, possibly combined with the diminution of cardiac size that occurs immediately after the operation, we have encountered significant pericardial effusions in several patients. It is now our practice to either create a posterior pericardial window at the end of the operation, or place a posterior pericardial drain which we usually keep longer. These techniques have essentially eliminated the problem, and in general it is much easier to treat the pleural effusion on the left side in the occasional patient who may develop this complication.

8 Prognosis and Results Although pulmonary endarterectomy is now performed at several major cardiovascular centers throughout the world, most experience with this operation has been at UCSD, where the technique of this operation was pioneered and

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refined. More than 2,400 pulmonary endarterectomy operations have been performed at UCSD since 1970, whereas the entire reported world’s literature on this operation (exclusive of UCSD) is approximately 1,500 cases. The mean patient age in the last 10 years was 51.6 years, with a range of 8.9–84.8 years. There is a slight male predominance, reflecting disease predilection, surgical bias, or both. In 40% of cases, at least one additional cardiac procedure was performed at the time of operation. Most commonly, the adjunct procedure was closure of a persistent foramen ovale or atrial septal defect (18.9%) or coronary artery bypass grafting (7.9%) [37]. With this operation, a reduction in pulmonary pressures and resistance to normal levels and corresponding improvement in pulmonary blood flow and cardiac output are generally immediate and sustained [38, 39]. These changes are permanent [40]. Table 1 lists the most current statistics for the last 1,100 patients who underwent pulmonary endarterectomy at UCSD with respect to hemodynamic improvement stratified for thromboembolic disease classification group. Whereas before the operation more than 78.7% of the patients were in NYHA functional class III or IV in this series, at 1 year after operation, 97% of patients were reclassified as NYHA functional class I or II. In addition, echocardiographic studies have demonstrated that, with the elimination of chronic pressure overload, right ventricular geometry rapidly reverts toward normal [41]. Right atrial and right ventricular hypertrophy and dilatation regresses. Tricuspid valve function returns to normal within a few days as a result of restoration of tricuspid annular geometry after the remodeling of the right ventricle; therefore, tricuspid valve repair is not performed with this operation [42]. In addition to the UCSD experience, centers from around the world are now presenting results of mid-sized surgical series of pulmonary endarterectomy operations. Table  2 summarizes the survival and hemodynamic outcome after

Table 1  University of California, San Diego (UCSD) thromboembolic disease classification – pulmonary endarterectomy hemodynamic results Type IV Type III Type II Type I All patients (n = 24; 2.2%) (n = 192; 17.5%) (n = 469; 42.6%) (n = 415; 37.7%) (n = 1,100; 100%) (preoperative/ (preoperative/ (preoperative/ (preoperative/ (preoperative/ postoperative) postoperative) postoperative) postoperative) postoperative) Variables PVR (dyn s/cm5)

859.4 ± 439.5 924.2 ± 450.4 799.9 ± 417.2 863.2 ± 454.6 884.6 ± 412.3 290.4 ± 195.7 269.8 ± 176.6 270.5 ± 191.3 350.8 ± 183.3 595.2 ± 360.2 CO (L/min) 3.9 ± 1.3 3.7 ± 1.4 4.1 ± 1.3 4.0 ± 1.5 3.8 ± 1.2 5.4 ± 1.5 5.5 ± 1.5 5.5 ± 1.5 5.2 ± 1.4 4.5 ± 1.1 Systolic PAP (mmHg) 75.9 ± 18.6 76.8 ± 18.7 75.0 ± 19.5 75.8 ± 16.4 78.4 ± 15.6 46.4 ± 16.6 44.4 ± 15.1 44.5 ± 15.0 52.7 ± 17.1 73.8 ± 32.1 Diastolic PAP (mmHg) 28.5 ± 9.7 29.8 ± 9.6 27.3 ± 10.0 28.3 ± 8.8 32.3 ± 9.5 18.5 ± 7.2 17.7 ± 6.5 17.9 ± 6.8 20.6 ± 7.9 27.3 ± 12.8 Mean PAP (mmHg) 46.2 ± 11.3 47.0 ± 11.4 45.2 ± 11.6 46.5 ± 10.3 50.2 ± 10.5 28.4 ± 9.6 27.2 ± 8.7 27.5 ± 9.1 31.8 ± 10.1 42.4 ± 15.5 Mortality (%) 52 (4.7) 16 (3.9) 22 (4.7) 12 (6.3) 4 (16.7) Data are shown as means ± the standard deviation or a number (percentage). Upper numbers are preoperative values and lower numbers are postoperative values obtained just prior to removal of the Swan–Ganz catheter. PVR pulmonary vascular resistance, CO cardiac output, PAP, pulmonary artery pressure

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Table  2  Mid-sized pulmonary endarterectomy series of more than 20 patients (2005–2008) Preoperative Date of Dates No. of Perioperative mean PAP (mmHg) Authors publication of study patients deaths

Postoperative Preoperative mean PVR mean PAP (dyn s/cm5) (mm/Hg)

Condliffe et al. [64] 2008 2001–2006 236 37 48.3 26.8 Corsico et al. [40] 2008 1994–2006 157 18 47.6 23.9 Freed et al. [65] 2008 1997–2006 229 0 47.0 25.0 Mikus et al. [43] 2008 2004–2007   40 2 48.0 25.9 Thomson et al. [44] 2008 2003–2006 150 22 52.0 29.0 Yoshimi et al. [66] 2008 1999–2006   40 3 NR NR Bonderman et al. [58] 2007 1992–2006 181 12 45.8 25.4 D’Armini et al. [22] 2007 2000–2003   45 1 48 22 Hardziyenka et al. [59] 2007 2002–2005   58 6 44.9 NR Maeba et al. [60] 2007 NR   51 0 NR NR Reesink et al. [61] 2007 2003–2005   42 0 48 NR Rubens et al. [62] 2007 1995–2006 116 10 47 28 Suntharalingam et al. [63] 2007 2004–2006 111 15 47.6 28.6 Mellemkjaer et al. [53] 2006 1994–2004   50 12 50 37 Lindner et al. [50] 2006 2004–2006   21 1 54.8 NR Macchiarini et al. [51] 2006 2004–2005   30 1 57 25 Matsuda et al. [52] 2006 1995–2005 102 8 46 21 Ogino et al. [54] 2006 1995–2004   88 7 45.2 18.6 Piovella et al. [55] 2006 1994–2006 134 13 47 25 Reesink et al. [56] 2006 NR   27 3 44 24 Tanabe et al. [57] 2006 1992–2004   95 3 42.8 NR Heinrich et al. [47] 2005 1996–2002   60 NR 47 NR D’Armini et al. [46] 2005 1999–2005 134 13 47 25 Kramm et al. [48] 2005 2001–2002   22 1 45 36 Puis et al. [49] 2005 1999–2003   40 NR 50 38 Total/mean a 1992–2007 2,259 188+b 47.8 26.9 a  Total number of worldwide reported cases except UCSD’s experience with corresponding mean values b  Total number of deaths likely higher as this was not reported in some studies PAP pulmonary artery pressure, PVR pulmonary vascular resistance, NR not recorded

pulmonary endarterectomy from this growing body of surgical experience [40, 43–66]. Severe reperfusion injury is the single most frequent complication after pulmonary endarterectomy, historically occurring in approximately 15% of patients, but now in a much smaller percentage of our patients (about 5%). Of patients with reperfusion injury, most resolve the problem with a short period of ventilatory support and aggressive diuresis. A minority of patients with severe lung reperfusion injury require prolonged periods of ventilatory support, whereas extreme cases require venovenous extracorporeal support for oxygenation and blood carbon dioxide removal [67]. Neurologic complications from circulatory arrest have mostly been eliminated by shorter circulatory arrest periods and the use of a direct cooling jacket placed around the head, which provides even cooling to the surface of the cranium. Perioperative confusion and stroke rates for pulmonary endarterectomy are similar to those seen with conventional open-heart surgery. In the last 10 years at UCSD, reexploration for bleeding occurred at a rate of 3.3%, and 29.1% of patients required intraoperative or postoperative blood transfusion. Despite an average duration of surgery of 6.5 h, wound infection occurred only in 1.3% of patients.

1,091 1,140 800 791 740 NR 586 1,143 847 NR 976 810 1,016 819 NR 1,110 1,072 1,028 1,149 837 876 906 1,149 768 1,246 950

Postoperative mean PVR (dyn s/cm5) 464 327 244 280 336 NR 269 296 NR NR NR 215 496 373 NR 279 386 320 322 415 NR 290 322 503 515 350.4

The single greatest risk factor for operation remains the severity of PVR and the ability to lower it to a normal range at operation. Those patients with high PVR with minimal vascular obstruction on angiogram (type IV small-vessel vasculopathy indistinguishable from idiopathic pulmonary arterial hypertension) have the worst prognosis, and surgery does not correct pulmonary hypertension in this population. Arteriolar–capillary vasculopathy without larger-vessel thromboembolic disease was not influenced by blind endarterectomy of the proximal pulmonary arterial tree. Most early deaths after this operation are in this subgroup, and efforts are directed at better determining who these patients are in the preoperative setting to avoid unnecessary operation. In the UCSD experience, overall perioperative mortality was 9% for the entire cohort of patients, encompassing a time span of more than 30 years. In the last 1,000 cases, surgical mortality for pulmonary endarterectomy was 4.7%. In the last 2 years, the operative mortality for isolated pulmonary endarterectomy cases at UCSD has been about 2.5%, despite a patient population with higher risk factors. This reflects the learning curve for safely performing this operation and the refinements in surgical technique that enhance patient outcome.

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A survey of surviving patients who underwent pulmonary endarterectomy between 1970 and 1995 at UCSD has formally evaluated long-term outcome from this operation [68]. Questionnaires were mailed to 420 patients who were more than 1 year after operation, and responses were obtained from 308 patients. Survival, functional status, quality of life, and the subsequent use of medical assistance were assessed. Survival after pulmonary endarterectomy was found to be 75% at 6 years or more. This survival exceeds single-lung or double-lung transplantation survival for thromboembolic pulmonary hypertension. Ninety-three percent of the patients were found to be in NYHA class I or II, compared with about 95% of the patients being in NYHA class III or IV preoperatively. Of the working population, 62% of patients who were unemployed before operation returned to work. Patients who had undergone pulmonary endarterectomy scored several quality-of-life components slightly lower than normal individuals, but significantly higher than the patients before operation. Only 10% of patients used oxygen after surgery. In response to the question “How do you feel about the quality of your life since your surgery?”, 77% replied “much improved,” and 20% replied “improved.” These data appear to confirm that pulmonary endarterectomy offers substantial improvement in survival, function, and quality of life.

9 Summary Pulmonary hypertension due to chronic pulmonary emboli is a condition that is underrecognized and carries a poor prognosis. Medical therapy for this condition is ineffective, and only transiently improves symptoms. The only therapeutic alternative to pulmonary endarterectomy is lung transplantation. The advantages of pulmonary endarterectomy include a lower operative mortality, better long-term results with respect to survival and quality of life, and the avoidance of chronic immunosuppressive treatment and allograft rejection. Currently, mortality rates for pulmonary endarterectomy are in the range of 2.5–4.7%, and the operation allows for sustained clinical benefit. These results make it the treatment of choice over transplantation for thromboembolic disease to the lung both in the short term and in the long term. Although pulmonary endarterectomy is a technically demanding operation, excellent results can be achieved. Improvements in operative technique developed over the last four decades allow pulmonary endarterectomy to be offered to patients with an acceptable mortality rate and anticipation of clinical improvement. With the increasing recognition of patients who have thromboembolic pulmonary hypertension and the realization that pulmonary endarterectomy is a safe and effective operation for this condition, it is anticipated that this will be an expanding area of surgical therapy in the future.

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114  Pulmonary Endarterectomy 21. Morris TA, Auger WR, Ysrael MZ et al (1996) Parenchymal scarring is associated with restrictive spirometric defects in patients with chronic thromboembolic pulmonary hypertension. Chest 110:399–403 22. D’Armini AM, Zanotti G, Ghio S et al (2007) Reverse right ventricular remodeling after pulmonary endarterectomy. J Thorac Cardiovasc Surg 133:162–168 23. Tunariu N, Gibbs SJ, Win Z et al (2007) Ventilation-perfusion scintigraphy is more sensitive than multidetector CTPA in detecting chronic thromboembolic pulmonary disease as a treatable cause of pulmonary hypertension. J Nucl Med 48:680–684 24. Ryan KL, Fedullo PF, Davis GB, Vasquez TE, Moser KM (1988) Perfusion scan findings understate the severity of angiographic and hemodynamic compromise in chronic thromboembolic pulmonary hypertension. Chest 93:1130–1185 25. Coulden R (2006) State-of-the-art imaging techniques in chronic thromboembolic pulmonary hypertension. Proc Am Thorac Soc 3:577–583 26. Auger WR, Kim NH, Kerr KM, Test VJ, Fedullo PF (2007) Chronic thromboembolic pulmonary hypertension. Clin Chest Med 28:255–269 27. Jamieson SW, Kapelanski DP (2000) Pulmonary endarterectomy. Curr Probl Surg 37:165–252 28. Reichelt A, Hoeper MM, Laganski M, Keberle M (2009) Chronic thromboembolic pulmonary hypertension: evaluation with 64-detector row CT versus digital subtraction angiography. Eur J Radiol 71:41–54 29. Suga K, Kawakami Y, Hayashi N, Hayashi N, Seto A, Matsunaga N (2006) Comprehensive assessment of lung CT attenuation alteration at perfusion defects of acute pulmonary thromboembolism with breath-hold SPECT-CT fusion images. J Comput Assist Tomogr 30:83–91 30. Nikolaou K, Schoenberg SO, Attenberger U et al (2005) Pulmonary arterial hypertension: diagnosis with fast perfusion MR imaging and high-spatial-resolution MR angiography – preliminary experience. Radiology 236:694–703 31. Allison PR, Dunnill MS, Marshall R (1960) Pulmonary embolism. Thorax 15:273–283 32. Moser KM, Houk VN, Jones RC, Hufnagel CC (1965) Chronic, massive thrombotic obstruction of the pulmonary arteries: analysis of four operated cases. Circulation 32:377–385 33. Winkler MH, Rohrer CH, Ratty SC et  al (1990) Perfusion techniques of profound hypothermia and circulatory arrest for pulmonary thromboendarterectomy. J Extra Corpor Technol 22:57–60 34. Madani MM, Jamieson SW (2007) Pulmonary embolism and thromboendarterectomy. In: Cohn LH (ed) Cardiac surgery in the adult, vol 3. McGraw-Hill, New York, pp 1309–1331 35. Jamieson SW (1998) Pulmonary thromboendarterectomy. In: Franco KL, Putnam JB (eds) Advanced therapy in thoracic surgery. Decker, Hamilton, pp 310–318 36. Levinson RM, Shure D, Moser KM (1986) Reperfusion pulmonary edema after pulmonary artery thromboendarterectomy. Am Rev Respir Dis 134:1241–1245 37. Thistlethwaite PA, Auger WR, Madani MM, Pradhan S, Kapelanski DP, Jamieson SW (2001) Pulmonary thromboendarterectomy combined with other cardiac operations: indications, surgical approach, and outcome. Ann Thorac Surg 72:13–17 38. Menzel T, Kramm T, Mohr-Kahaly S, Mayer E, Oelert H, Meyer J (2002) Assessment of cardiac performance using Tei indices in patients undergoing pulmonary thromboendarterectomy. Ann Thorac Surg 73:762–766 39. Thistlethwaite PA, Madani MM, Jamieson SW (2006) Outcomes of pulmonary endarterectomy surgery. Semin Thorac Cardiovasc Surg 18:257–264 40. Corsico AG, D’Armini AM, Cerveri I et al (2008) Long-term outcome after pulmonary endarterectomy. Am J Respir Crit Care Med 178:419–424

1589 41. Iino M, Dymarkowski S, Chaothawee L, Delcroix M, Bogaert J (2008) Time course of reversed remodeling after pulmonary endarterectomy in patients with chronic pulmonary thromboembolism. Eur Radiol 18:792–799 42. Thistlethwaite PA, Jamieson SW (2003) Tricuspid valvular disease in the patient with chronic pulmonary thromboembolic disease. Curr Opin Cardiol 18:111–116 43. Mikus PM, Mikus E, Martin-Suàrez S et  al (2008) Pulmonary endarterectomy: an alternative to circulatory arrest and deep hypothermia: mid-term results. Eur J Cardiothorac Surg 34:159–163 44. Thomson B, Tsui SS, Dunning J et al (2008) Pulmonary endarterectomy is possible and effective without the use of complete circulatory arrest – the UK experience in over 150 patients. Eur J Cardiothorac Surg 33:157–163 45. Ji B, Liu J, Wu Y et al (2006) Perfusion techniques for pulmonary thromboendarterectomy under deep hypothermia circulatory arrest: a case series. J Extra Corpor Technol 38:302–306 46. D’Armini AM, Zanotti G, Vigano M (2005) Pulmonary endarterectomy: the treatment of choice for chronic thromboembolic pulmonary hypertension. Ital Heart J 6:861–868 47. Heinrich M, Uder M, Tscholl D, Grgic A, Kramann B, Schäfers HJ (2005) CT scan findings in chronic thromboembolic pulmonary hypertension: predictors of hemodynamic improvement after pulmonary thromboendarterectomy. Chest 127: 1606–1613 48. Kramm T, Eberle B, Guth S, Mayer E (2005) Inhaled iloprost to control residual pulmonary hypertension following pulmonary endarterectomy. Eur J Cardiothorac Surg 28:882–888 49. Puis L, Vandezanonade E, Vercaemst L et  al (2005) Pulmonary thromboendarterectomy for chronic thromboembolic pulmonary hypertension. Perfusion 20:101–108 50. Lindner J, Jansa P, Kunstyr J et al (2006) Implementation of a new programme for the surgical treatment of CTEPH in the Czech Republic–pulmonary endarterectomy. Thorac Cardiovasc Surg 54:528–531 51. Macchiarini P, Kamiya H, Hagl C et al (2006) Pulmonary endarterectomy for chronic thromboembolic pulmonary hypertension: is deep hypothermia required? Eur J Cardiothorac Surg 30:237–241 52. Matsuda H, Ogino H, Minatoya K et al (2006) Long-term recovery of exercise ability after pulmonary endarterectomy for chronic thromboembolic pulmonary hypertension. Ann Thorac Surg 82:1338–1343 53. Mellemkjaer S, Ilkjaer LB, Klaaborg KE et  al (2006) Pulmonary endarterectomy for chronic thromboembolic pulmonary hypertension. Ten years experience in Denmark. Scand Cardiovasc J 40: 49–53 54. Ogino H, Ando M, Matsuda H et al (2006) Japanese single-center experience of surgery for chronic thromboembolic pulmonary hypertension. Ann Thorac Surg 82:630–636 55. Piovella F, D’Armini AM, Barone M, Tapson VF (2006) Chronic thromboembolic pulmonary hypertension. Semin Thromb Hemost 32:848–855 56. Reesink HJ, Meijer RC, Lutter R et al (2006) Hemodynamic and clinical correlates of endothelin-1 in chronic thromboembolic pulmonary hypertension. Circ J 70:1058–1063 57. Tanabe N, Amano S, Tatsumi K et al (2006) Angiotensin-converting enzyme gene polymorphisms and prognosis in chronic thromboembolic pulmonary hypertension. Circ J 70:1174–1179 58. Bonderman D, Skoro-Sajer N, Jakowitsch J et al (2007) Predictors of outcome in chronic thromboembolic pulmonary hypertension. Circulation 115:2153–2158 59. Hardziyenka M, Reesink HJ, Bouma BJ et  al (2007) A novel echocardiographic predictor of in-hospital mortality and mid-term haemodynamic improvement after pulmonary endarterectomy for

1590 chronic thrombo-embolic pulmonary hypertension. Eur Heart J 28:842–849 60. Maeba H, Nakatani S, Sugawara M et  al (2007) Different time course of changes in tricuspid regurgitant pressure gradient and pulmonary artery flow acceleration after pulmonary thromboendarterectomy. Circ J 71:1771–1775 61. Reesink HJ, van der Plas MN, Verhey NE, van Steenwijk RP, Kloek JJ, Bresser P (2007) Six-minute walk distance as parameter of functional outcome after pulmonary endarterectomy for chronic thromboembolic pulmonary hypertension. J Thorac Cardiovasc Surg 133:510–516 62. Rubens FD, Bourke M, Hynes M et al (2007) Surgery for chronic thromboembolic pulmonary hypertension–inclusive experience from a national referral center. Ann Thorac Surg 83:1075–1081 63. Suntharalingam J, Goldsmith K, Toshner M et  al (2007) Role of NT-proBNP and 6MWD in chronic thromboembolic pulmonary hypertension. Respir Med 101:2254–2262

M.M. Madani and S.W. Jamieson 64. Condliffe R, Kiely DG, Gibbs JS et al (2008) Improved outcomes in medically and surgically treated chronic thromboembolic pulmonary hypertension. Am J Respir Crit Care Med 177: 1122–1127 65. Freed DH, Thomson BM, Tsui SS et al (2008) Functional and haemodynamic outcome 1 year after pulmonary thromboendarterectomy. Eur J Cardiothorac Surg 34:525–529 66. Yoshimi S, Tanabe N, Masuda M et al (2008) Survival and quality of life for patients with peripheral type chronic thromboembolic pulmonary hypertension. Circ J 72:958–965 67. Thistlethwaite PA, Madani MM, Kemp AD, Hartley M, Auger WR, Jamieson SW (2006) Venovenous extracorporeal life support after pulmonary endarterectomy: indications, techniques, and outcomes. Ann Thorac Surg 82:2139–2145 68. Archibald CJ, Auger WR, Fedullo PF et al (1999) Long-term outcome after pulmonary thromboendarterectomy. Am J Respir Crit Care Med 160:523–528

Chapter 115

Evaluation of Patients with Pulmonary Hypertension for Lung Transplantation Gordon L. Yung

Abstract  The process of lung transplantation is complex, time-consuming, and varies considerably among transplant centers. In part because a donated organ is considered a gift from society and is a limited resource, physicians involved in transplant evaluation have the responsibility beyond giving patients a “second chance.” Putting it in another way, it is not enough to offer a transplant to a patient simply because the patient would die without one. At the level of societal interests, transplantations should be distributed in a way that the largest number of patients can benefit for the longest duration. Because of the shortage of available organs, there is a conflict with the traditional expectations of individual physicians and patients, when the primary obligation of a physician is to do the best for his/her individual patients. For example, if the only treatment available has only 10% chance of curing a dying patient, it is reasonable for the physician to give the drug to the patient, when the alternative is death. In the transplant community, the question may be: Is it right to give the organ to a patient with 10% chance of living after surgery, or should another dying patient with 80% chance of posttransplant survival receive that organ instead? As long as there is a shortage of organs, transplant physicians need to balance the needs of society with individual interests. For the same reasons, transplant evaluation is more extensive and detailed than most other surgeries. Typically, a transplant evaluation includes assessment of medical risks and benefits, psychosocial suitability (e.g. compliance), and financial conditions (whether the patient can afford costly ongoing treatment and monitoring after transplant surgery to ensure long-term success). It is clear that the presence of pulmonary hypertension, primary or secondary, has significant implications for transplantation and its outcome, and should be a factor in deciding on transplant suitability.

G.L. Yung (*) Division of Pulmonary and Critical Care Medicine, University of California, 200 West Arbor Drive, MC 8373, 92103 San Diego, CA, USA e-mail: [email protected]

Keywords  Lung transplant • Posttransplant survival • Donated organ • Complication • Treatment of pulmonary hypertension

1 Introduction The process of lung transplantation is complex, time-consuming, and varies considerably among transplant centers. In part because a donated organ is considered a gift from society and is a limited resource, physicians involved in transplant evaluation have the responsibility beyond giving patients a “second chance.” Putting it in another way, it is not enough to offer a transplant to a patient simply because the patient would die without one. At the level of societal interests, transplantations should be distributed in a way that the largest number of patients can benefit for the longest duration. Because of the shortage of available organs, there is a conflict with the traditional expectations of individual physicians and patients, when the primary obligation of a physician is to do the best for his/her individual patients. For example, if the only treatment available has only 10% chance of curing a dying patient, it is reasonable for the physician to give the drug to the patient, when the alternative is death. In the transplant community, the question may be: Is it right to give the organ to a patient with 10% chance of living after surgery, or should another dying patient with 80% chance of posttransplant survival receive that organ instead? Assuming that 1,000 lung transplant surgeries take place in the USA in 1 year, a 10% 1-year survival would mean that 100 patients would be alive after 1 year, compared with 800 patients if only those with a “reasonable” chance of survival had transplantations. Thus, as long as there is a shortage of organs, transplant physicians need to balance the needs of society with individual interests. For the same reasons, transplant evaluation is more extensive and detailed than most other surgeries. Typically, a transplant evaluation includes assessment of medical risks and benefits, psychosocial suitability (e.g. compliance), and financial conditions (whether the patient can afford costly ongoing treatment and monitoring

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6_115, © Springer Science+Business Media, LLC 2011

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1592 Table  1  Potential impact of pulmonary hypertension on lung transplantation Suitability and choice of transplant surgery Timing of transplant Risk of transplantation Intraoperative and postoperative management Outcome of surgery

after transplant surgery to ensure long-term success). It is clear that the presence of ­pulmonary hypertension, primary or secondary, has significant implications for transplantation and its outcome, and should be a factor in deciding on transplant suitability (Table  1). Despite this understanding, there is no consensus on the best practice to evaluate these patients.

2 Initial Evaluation The primary purpose of an initial evaluation is to determine whether a patient would be a candidate for transplanta­ tion [1–4]. There are published guidelines for the general selection of patients for lung transplantation. These criteria are based on medical “evidence” that is often incomplete or inconclusive, and an individual center’s experience may significantly affect the decision [5–9]. In general, an evaluation consists of three components: medical evaluation, psychological evaluation, and financial considerations.

2.1 Medical Evaluation The presence of certain conditions is generally considered a contraindication for surgery (Table 2). In general, significant (irreversible) extrapulmonary organ failure, active or recent malignancies (except some skin cancers), HIV disease, significant thoracic cage abnormality, and uncontrolled or untreatable extrathoracic infections are factors that would preclude transplantation. The presence of other factors may significantly increase the risk of transplants, such as mechanical ventilation, high-dose (chronic) steroid use, symptomatic osteoporosis, active or carriers of hepatitis B and hepatitis C, and significant pleural disease or pleurodesis. On the basis of data on age and survival outcome, the upper age limit for lung transplantation was somewhat arbitrarily set at 55 years for heart-lung transplantation, 60 years for single lung transplantation, and 65 years for “double” lung transplantation. However, it is recognized that many patients older that these age “limits” can do well, at least in the short term. Each patient should,

G.L. Yung Table 2  “Traditional” contraindications for lung transplantation Absolute Relative Recent/ongoing malignancy Irreversible or significant extrapulmonary organ failure Significant active extrapulmonary infections Significant thoracic cage abnormality Poor compliance or inability to follow posttransplant care

Old age “Too sick” (hemodynamic instability, poor mobility, etc.) Pulmonary infection with resistant or difficult to treat organisms Extreme body weight Mechanical ventilator support

therefore, be considered individually, since “chronologic” age may be different from “biologic” age. For example, a 67-year-old patient with an active lifestyle and no other medical problem would likely have lower surgical risks than a 55-year-old patient who is morbidly obese, on chronic steroids, with diabetes and severely deconditioned. In addition, many “conventional wisdoms” are being challenged continually. The cause of pulmonary hypertension may significantly impact the decision for transplantation at multiple levels. For instance, patients with HIV disease are traditionally not considered for transplantation, selected cases of long-term successful outcomes have now been reported in different organ transplantations. Portopulmonary hypertension is an interesting condition in which pulmonary hypertension is associated with portal hypertension in 2–10% of patients with liver cirrhosis [10]. In this instance, liver transplantation may lead to resolution of pulmonary hypertension and lung transplantation may be inappropriate and risky in this condition, whereas untreated pulmonary hypertension (mean pulmonary artery pressure of more than 40 mmHg) has been associated with high post-liver-transplantation mortality [11]. In this condition, it may be more important to look at transplant risks in terms of the pulmonary vascular resistance than pulmonary arterial pressure. Many patients with liver disease have very high cardiac output, and a mildly elevated pulmonary vascular resistance may result in relatively high pulmonary artery pressure. Survival of scleroderma patients with pulmonary hypertension is often significantly worse than that of those with idiopathic pulmonary arterial hypertension or pulmonary hypertension associated with congenital heart disease, and the condition may also be associated with higher posttransplant mortality [12]. Because the reason for the outcome difference is likely due to extrapulmonary involvement, many centers would still consider the diagnosis on a case-tocase basis. Individual centers should therefore look at all factors together and decide on transplant suitability on the basis of both scientific evidence and experience.

115  Evaluation of Patients with Pulmonary Hypertension for Lung Transplantation

2.2 Psychosocial Evaluation Success in lung transplantation should be defined not only on the basis of patients able to survive the surgery, but also on the basis of long-term survival and morbidity, and it is important to select patients who are compliant and able to follow complex instructions. Transplantation may be more appropriately considered an “exchange” of another disease, than simply a cure of one condition. Most transplant patients face various long-term problems associated with immunosuppression, such as diabetes, hypertension, rejection, infections, malignancies, and chronic renal insufficiency (possible dialysis) and patients should be made aware of these potential issues prior to surgery. A good social support network is also necessary to help patients get through these issues and may be associated with better outcomes [13]. Substance abuse, including tobacco and alcohol addiction, is often considered a risk factor for potential compliance issues and poor long-term outcomes [14]. Thus, patients with pulmonary hypertension associated with methamphetamine use should be evaluated carefully for active drug use prior to transplantation. Similarly, it is generally agreed that patients should be tobacco-free for at least 6 months prior to listing them for transplantation, even though there may not be strong data to suggest a poor outcome. Finally, patients should be evaluated for their willingness to accept another person’s body parts. A typical example is that of Jehovah’s Witnesses; these patients do not, in general, accept blood transfusions but some may consider transplantation acceptable if the surgery only utilizes autologous transfusion or “cell-saver” techniques. In these cases, involvement of the patients’ support group and religious establishment may help to resolve the conflicts.

2.3 Financial Considerations In principle, one can argue that no one in the USA should be denied a life-saving treatment because of financial reasons. In reality, transplantation is expensive and since none of the immunosuppression treatments have been approved by the Food and Drug Administration, patients can run into financial difficulties trying to pay for the “best” treatments. Inability to pay for proper posttransplant care would affect graft survival and financial hardship may force patients to be noncompliant with expensive treatment. Some states in the USA do not have lung transplant centers, which means that patients in those areas will have to be able to travel to another state and possibly stay there for extended periods of time before and after the transplantation (especially if the caregiver is also the one financially supporting the family). With

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an average posttransplant survival of 4–5 years only, some patients may also be reluctant to spend their life savings, depriving their family of future financial support for the possibility of additional survival. Needless to say, the issue can be very emotional, and unfortunately, very few transplant centers can afford to pay for long-term care of these patients without government or insurance involvement.

3 Evaluation: Timing of Transplant Surgery Prior to May 2005, patients in the USA waiting for lung transplantation were allocated organs on the basis of the time they had been placed on a national transplant waiting list. Since then, patients have been prioritized on the waiting list according to a complex set of criteria, in an attempt to optimize organ use (Table 3). The system is based on the principle that, in general, sicker patients should have priority over those who not as sick. At the same time, it is recognized that as patients become sicker, there comes a point where the risks of transplantation may increase significantly, resulting in death and consequent “wasting” of organs that might have benefited another patient. A “score” is then calculated on the basis of these criteria according to past statistical analysis of how each factor affects either the likelihood of death within 1 year without transplantation (the “needs”) or the likelihood of death within 1 year after transplantation (the “benefits”). With better data and more public input, this set of criteria is constantly being updated. Some of these criteria may only be applied to certain diagnoses in the calculation of the lung allocation score, such as the use of absolute serum bilirubin level and the change in the serum bilirubin level to determine the lung allocation score in pulmonary hypertension patients. In addition, the waiting time may be affected by other factors, such as blood type, donor-recipient lung size matching, type of transplantation, and preexisting HLA antibodies. Organ availability may vary significantly in different

Table 3  Criteria determining the lung allocation score Diagnosis of lung disease Age Body mass index Presence or absence of diabetes Requirement of mechanical ventilator support Supplemental oxygen requirements Force vital capacity Pulmonary hemodynamics (pulmonary artery pressure, capillary wedge pressure) Functional status (assistance with daily activities) Arterial blood gas (PaCO2) Serum bilirubin level

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regions of the country and since organs cannot be shared nationally because of limitation in cold ischemic time, the waiting time may differ in different transplant centers. Finally, because the availability of organs is unpredictable, patients have to live within a reasonable distance from transplant centers, and this may have significant practical implications as to where and when patients should be evaluated. There are significant implications when employing a waiting list that is primarily based on “needs.” In the past, when both the median survival of primary pulmonary hypertension patients and the average waiting time for lung transplantation were 2–3 years, patients were often placed on the transplant list as soon as the diagnosis was made. In theory, the new system allows physicians to more accurately predict organ availability, and with the assurance that, if a patient’s condition worsened, his or her lung allocation score will increase, thus improving the chance of transplantation. The use of “average survival” has little meaning to individual pulmonary hypertension patients when physicians have to decide on the timing for them to undergo transplantation. The first step in evaluating a pulmonary hypertension patient for lung transplantation is therefore to determine whether the patient is sick enough to affect transplantation. In patients where pulmonary hypertension is the reason for transplantation, the decision is primarily based on disease severity and the rate of disease progression. When pulmonary hypertension is secondary (such as from idiopathic pulmonary fibrosis or COPD), each case has to be evaluated to see if the degree of pulmonary hypertension may affect transplant surgery. For example, over 50% of transplant surgeries for COPD or emphysema are single lung transplantations but double lung transplantations may be more appropriate if there is significant pulmonary arterial hypertension [14]. Advances in medical management in the past two decades have dramatically changed the role of lung transplantation for pulmonary hypertension patients. Prior to the availability of effective therapies, the prognosis of pulmonary hypertension patients was almost uniformly fatal within a short time, and lung transplantation was considered in most pulmonary hypertension patients with significant or worsening symptoms. Often, patients with relatively mild disease might also undergo transplantation, because of the concern for future worsening and unpredictable waiting time. Given the complexity of pulmonary hypertension treatment and the constantly changing landscape of transplantation, the timing of evaluation of patients, as well as the actual timing of listing patients for lung transplantation, is best determined by those familiar with the diseases and with the outcome of transplantation (Fig. 1). In general, a 1-year survival of about 80% and a 5-year survival of about 50% are expected after transplantation [15]. The risk of individual patients,

G.L. Yung

Fig. 1  Survival of idiopathic pulmonary arterial hypertension patients after diagnosis, with and without medical treatment

however, can vary substantially. Thus, a 65-year-old pulmonary hypertension patient with florid right-sided heart failure, in renal and liver failure, and who has been wheelchair-bound with deconditioning, is obviously more in need of transplantation than another pulmonary hypertension patient who may be very symptomatic but hemodynamically well compensated. On the other hand, the chance of posttransplant long-term survival of the first patient is also significantly worse. Because of the complexity of these patient medical issues, there are no clear-cut criteria for timing of transplantation. In general, pulmonary hypertension patients should be considered for transplantation if they remain significantly symptomatic (New York Heart Association functional class III or IV), supported by objective evidence of cardiac decompensation (elevated right atrial pressure and/or low cardiac index) despite maximal medical therapies. Other recommendations have included using 6-min walk tests but, because of the inherent limitations of the test, it is still recommended that the 6-min walk test be a “screening” test, and that the severity of illness be confirmed by right-sided heart catheterization before making the decision for transplantation. Most pulmonary hypertension experts agree that intravenously administered prostanoids should be considered in patients prior to making a decision for transplantation. Because there are symptomatic patients who can remain stable for years, the rate of progression of disease should also be taken into consideration in determining the timing of transplantation. Thus, a patient whose condition is worsening rapidly should be considered for early referral.

4 Evaluation: Type of Lung Transplantation After determining that a patient has no contraindications to transplantation, and that the condition is severe enough to require transplantation, the evaluation is then focused on

115  Evaluation of Patients with Pulmonary Hypertension for Lung Transplantation Table 4  Pros and cons of different transplant surgeries in pulmonary hypertension Type of transplantation Advantages Single lung Double lung Heart-lung

Living lobar

Short operation time Shorter waiting time Fewer perioperative risks Longer survival Technically simpler Less hemodynamic instability “Elective” surgery HLA matching

Disadvantages Reperfusion injury Hemodynamic instability Longer waiting time Use two organs Lower long-term survival Use three organs Risks to two healthy donors Lower long-term survival

choosing the appropriate transplant surgeries. Four types of transplantation have been performed on patients with pulmonary hypertension or bilateral sequential lung, heart-lung, and living (related) lobar lung. Each of these surgeries has unique benefits and drawbacks (Table 4). In general, heart-lung transplantations are technically easier, thus offering shorter ischemic time [for the donor organ(s)], and surgery time (for the recipient). Although the first transplantation for primary pulmonary hypertension was a heart-lung transplantation, this procedure is rarely done nowadays (20–50 cases per year in the USA) and should probably be best performed in centers experienced in this option. By replacement of both the failing heart and the highresistance lungs, the chance of perioperative hemodynamic instabilities and reperfusion injuries is minimized. However, with three organs involved, the chance of primary graft dysfunction increases correspondingly, and overall short-term and long-term survival in these patients remain somewhat lower than in lung transplantation alone. In addition, the ethical issue of using three organs for one single patient, when potentially three patients could each have a transplantation, should be considered. Heart-lung transplantation is therefore generally reserved for pulmonary hypertension patients who have uncorrectable cardiac conditions (coexisting cardiomyopathy, some cases of large ventricular septal defect, or complex congenital heart disease), or in rare cases of pulmonary hypertension when cardiac recovery is not expected. Most transplant centers now favor double lung transplantations for patients with significant pulmonary hypertension and because these cases are now performed by sequentially replacing the two lungs, the procedure is more appropriately referred to as bilateral sequential single lung transplantation. The major benefits of replacing two lungs appear to be lower risks of reperfusion injury and primary graft failure in the immediate postoperative period. More recently, bilateral thoracotomies, sometimes performed

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with “mini-incisions,” have been used without the need for sternotomy. Although these patients tend to have less postoperative pain, data on long-term outcomes are still lacking and whether these translate to long-term survival benefits are not clear. In the USA, most patients who undergo lung transplantation because of pulmonary hypertension would now be considered for double lung transplantation. Single lung transplantation remains a reasonable choice of transplantation for selected patients with pulmonary hypertension. Because it is a shorter surgery, the “ischemic time” of the allograft is shorter and the patients are under general anesthesia for a shorter period as well. Postoperative pain is also generally less and pulmonary toilet and effective cough are generally better in single lung transplantations. In addition, offering patients both single and double lung transplantations may “widen” the potential donor pool and shorten the waiting time for some patients. Previous thoracic surgery may also affect the choice of transplant surgery. The traditional use of the “clamshell” approach for double lung transplantation may not be feasible in patients with previous surgeries that involved sternotomy. Single lung transplantation is still possible in these patients using unilateral thoracotomy. With the increasing use of bilateral thoracotomies, double lung transplantation may still be possible in these patients but the tissue exposure of this newer technique is less optimal than that of the clamshell approach, and difficulties may be encountered with scar tissues in the mediastinum. In single lung transplantation, since high pulmonary vascular resistance persists in the native lung, most of the right sided cardiac output will be directed to the lower-resistance allograft after single lung transplantation. This highflow state may predispose patients to ischemic–reperfusion lung injury and hemodynamic instability, and may explain why single lung transplantation for pulmonary hypertension patients has been associated with more complications in the immediate postoperative period, including a higher incidence of reperfusion lung injury, a longer initial intubation period, and possibly a longer hospital stay. All these are factors that need to be evaluated, in conjunction with the patient’s condition. In summary, lung transplantation remains a viable and acceptable option for pulmonary hypertension patients who failed medical therapies. The process of transplantation evaluation is complex and time-consuming. Medical, psychosocial, and financial considerations are all important factors in considering which patients will most likely benefit from the surgery. Transplantation is much more than surgery and patients need to be aware of the problems facing them after a successful operation. To optimize the use of organs, physicians should work closely with transplant centers to chose the right patients and to perform transplantations at the right time.

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References 1. Orens JB, Estenne M, Arcasoy S et al (2006) International guidelines for the selection of lung transplant candidates: 2006 update – a consensus report from the Pulmonary Scientific Council of the International Society for Heart and Lung Transplantation. J Heart Lung Transplant 25:745–755 2. Kreider M, Kotloff RM (2009) Selection of candidates for lung transplantation. Proc Am Thorac Soc 15:20–27 3. Nathan SD (2005) Lung transplantation: disease-specific considerations for referral. Chest 127:1006–1016 4. Glanville AR, Estenne M (2003) Indications, patient selection and timing of referral for lung transplantation. Eur Respir J 22: 845–852 5. Hertz MI, Aurora P, Christie JD et  al (2008) Registry of the International Society for Heart and Lung Transplantation: a quarter century of thoracic transplantation. J Heart Lung Transplant 27:937–942 6. Taylor DO, Edwards LB, Aurora P et  al (2008) Registry of the International Society for Heart and Lung Transplantation: twentyfifth Official Adult Heart Transplant Report – 2008. J Heart Lung Transplant 27:943–956 7. Christie JD, Edwards LB, Aurora P et  al (2008) Registry of the International Society for Heart and Lung Transplantation: twentyfifth Official Adult Lung and Heart/Lung Transplantation Report – 2008. J Heart Lung Transplant 27:957–969

G.L. Yung 8. Kirk R, Edwards LB, Aurora P et  al (2008) Registry of the International Society for Heart and Lung Transplantation: eleventh Official Pediatric Heart Transplantation Report – 2008. J Heart Lung Transplant 27:970–977 9. Aurora P, Edwards LB, Christie J et  al (2008) Registry of the International Society for Heart and Lung Transplantation: eleventh Official Pediatric Lung and Heart/Lung Transplantation Report – 2008. J Heart Lung Transplant 27:978–983 10. Hoeper MM, Krowka MJ, Strassburg CP (2004) Portopulmonary hypertension and hepatopulmonary syndrome. Lancet 363:1461 11. DeWolf AM, Scott VL, Gasior T, Kang Y (1993) Pulmonary hypertension and liver transplantation. Anesthesiology 78:213–214 12. Schachna L, Medsger TA Jr, Dauber JH et  al (2006) Lung transplantation in scleroderma compared with idiopathic pulmonary fibrosis and idiopathic pulmonary arterial hypertension. Arthritis Rheum 54:3954–3961 13. Dobbels F, Vanhaecke J, Dupont L et al (2009) Pretransplant predictors of posttransplant adherence and clinical outcome: an evidence base for pretransplant psychosocial screening. Transplantation 87:1497–1504 14. Dobbels F, Verleden G, Dupont L, Vanhaecke J, De Geest S (2006) To transplant or not? The importance of psychosocial and behavioural factors before lung transplantation. Chron Respir Dis 3:39–47 15. The International Society for Heart and Lung Transplantation Heart/ lung transplant registry. http://www.ishlt.org/registries/heartLungRegistry.asp. Accessed 10 Jul 2009

Chapter 116

Lung Transplantation for Pulmonary Hypertension Ashish S. Shah and John V. Conte

Abstract  Lung transplantation is an effective although imperfect therapy for end-stage lung disease. As medical therapeis improve the prognosis of patients with pulmonary hypertension, use of lung transplantation for primary pulmonary hypertension has declined. Nonetheless, it remains the last life-saving option for properly selected candidates. This chapter will review the surgical aspects of lung transplantation for pulmonary hypertension.

for ­pulmonary hypertension [1, 2]. Bilateral sequential lung transplantation was utilized by 1990 and currently is the standard of care for patients with pulmonary hypertension [3].

Keywords  Lung transplant • Surgical procedure • Surgical treatment of pulmonary hypertension

3.1 Absolute Contraindication

1  Introduction Lung transplantation is an effective although imperfect therapy for end-stage lung disease. As medical therapies improve the prognosis of patients with pulmonary hypertension, use of lung transplantation for primary pulmonary hypertension has declined. Nonetheless, it remains the last life-saving option for properly selected candidates. This chapter will review the surgical aspects of lung transplantation for pulmonary hypertension.

2 History Combined heart-lung transplantation was performed by Reitz et  al. in 1981 for pulmonary hypertension and was the primary treatment until the Toronto Lung Transplant Group successfully introduced isolated lung transplantation

A.S. Shah (*) Division of Cardiac Surgery, The Johns Hopkins Hospital, 600 North Wolfe Street, Blalock 618, 21287 Baltimore, MD, USA e-mail: [email protected]

3 Indications

There are a number of absolute contraindication to lung transplantation as defined by the International Society for Heart and Lung Transplantation [4]. Except for certain skin cancers, patients cannot have had a malignancy in the last 2 years. In patients with a history of cancer, a disease-free interval of at least 5 years is recommended. A second contraindication is untreatable end-organ dysfunction. This would include uncorrectable coronary artery disease, significant left ventricular dysfunction, and end-stage liver disease. Patients with renal failure or significant insufficiency should not be considered unless lung transplantation is combined with kidney transplantation. Active viral infections are a contraindication and include hepatitis B, hepatitis C, and HIV. Patients who are actively using tobacco or abusing other drugs or alcohol should not be considered for transplantation and should be abstinent for at least 6 months. Finally, significant psychosocial problems preclude transplantation.

3.2 Relative Contraindications As results have improved, several centers have been emboldened to offer lung transplantation to a wider spectrum of patients. As a result, there are several relative contraindications to lung transplantation and each center must decide on

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the importance of each. Initially an age cutoff for lung transplantation was proposed; however, several centers have performed transplantations on patients over the age of 70 years with reasonable results [5]. Nonetheless, the international guidelines have set 65 years as the upper limit and advanced age as a relative contraindication. Severely limited functional status and obesity lead to poor outcomes and patients should be actively participating in rehabilitation programs if possible and maintaining a BMI below 30 kg/m2. Long-term steroid use after transplantation makes severe osteoporosis a relative contraindication [4].

3.3 Specific Indications for Pulmonary Hypertension All patients with pulmonary hypertension should have been optimized on current medical therapy before referral. Primary pulmonary hypertension is unique in that the specific indications for lung transplantation center around hemodynamic deterioration. First, on a functional level, it is clear that patients with New York Heart Association class IV symptoms have a poor prognosis with medical management and should be referred for transplantation. Objective measures begin with the 6-min walk test (6MWT). Miyamoto et  al. demonstrated the correlation between 6MWT distance and survival in primary pulmonary hypertension. Patients with a 6MWT distance less than 332  m had a 1-year survival of approximately 50% and therefore the International Society for Heart and Lung Transplantation has defined a 6MWT distance of less than 350 m as an indication for lung transplantation [4, 6]. Invasive hemodynamic tests are the next step. Patients with a cardiac index less than 2.0 L/min/m2 and/or a right atrial pressure greater than 15 mmHg have a poor prognosis and represent failures of medical management. Interestingly, absolute pulmonary artery pressure or changes in pulmonary artery pressure over time are not currently an indication for transplantation. Rarely, patients will develop hemopytsis and they too should be referred for transplantation. Table 1 lists specific indications for lung transplantation in patients with primary pulmonary hypertension.

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4 Single Versus Double Lung Transplantation For nonsuppurative lung diseases, both single and double lung transplantation have been used effectively to save lives. Increasingly, double lung transplantation has been adopted over single lung transplantation in the USA [7]. Patients with double lung transplantations are simpler to care for postoperatively since the pulmonary compliance and vascular resistance are uniform. There may also be long-term advantages of improved survival and freedom from chronic rejection [8]. Nonetheless, single lung transplantation has been used successfully in pulmonary hypertension [9]. It does create unique problems. The normally functioning allograft will have lower pulmonary vascular resistance than the abnormal contralateral lung. This may lead to preferential flow through the allograft and a risk for overcirculation and primary graft dysfunction. Our preference at Johns Hopkins Hospital is to utilize double lung transplantation in all patients with pulmonary hypertension.

5 Combined Heart and Lung Transplantation Though rare, there may still be value in combined heart and lung transplantation for pulmonary hypertension. Patients with severe right ventricular failure that are inotrope-dependent or patients with biventricular failure may be better served by combined heart and lung transplantation. Organ scarcity and the nature of multiorgan allocation in the USA make this choice difficult. Toyoda et  al. reviewed the University of Pittsburgh experience with heart and lung transplantation for pulmonary hypertension and found very acceptable short-term and long-term outcomes. Importantly, in the modern era survival was not different from that for isolated lung transplantation [10].

6 Surgical Technique 6.1 Donor Organ Recovery

Table 1  Specific indications for lung transplantation in patients with pulmonary hypertension Persistent New York Heart Association class III or IV on maximal medical therapy 6-min walk test distance <350 m Cardiac index <2.0 L/min/m2 Right atrial pressure >15 mmHg

Since recipients with pulmonary hypertension are at high risk for primary graft dysfunction, high-quality donors are preferable. Lungs are recovered using antegrade and retrograde flush with low-potassium dextran solution and prostaglandin at most centers. A small degree of experience exists

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with using non-heart-beating donors with satisfactory initial results [11]. If a single lung is to be recovered, it is critically important that the potential allograft has excellent function in terms of gas exchange and compliance.

6.2 Recipient Preparation Pulmonary hypertension patients can have significant hemodynamic problems during induction of anesthesia and the surgical team should be present. Intubation may be with a single- or a double-lumen tube. We prefer double-lumen intubation as it allows for hilar dissection without cardiopulmonary bypass and gentle ventilation of the first lung while the second is being implanted. A Swan–Ganz catheter and volume lines are placed as well as radial and femoral arterial lines for blood pressure monitoring. A generous bilateral anterolateral thoracotomy with transverse sternotomy is then performed with ligation of both internal mammary arteries. Some centers utilize sternal sparing techniques, but we have not experienced significant sternal complications that would offset the exposure that a true clamshell approach affords (Fig. 1). If the patient will tolerate it, the pulmonary veins and artery are isolated using sharp and blunt dissection prior to cardiopulmonary bypass. Care is taken to dissect around the pulmonary arteries as they are very large and fragile. The patient is heparinized for full cardiopulmonary bypass and cannulated via the ascending aorta and right atrium. If a large patent foramen ovale is present, bicaval cannulation is

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performed and the defect closed. Most of these patients will have tricuspid regurgitation, and valve repair may be performed at this time without cardioplegic arrest. Care is taken to preserve the phrenic and vagus nerves. When the donor organs arrive, the right lung is removed. The hilar structures are then mobilized into the pericardium. The pulmonary artery is dissected under the superior vena cava and the pulmonary veins are mobilized completely off the posterior pericardium and onto Sondergaard’s groove anteriorly. The bronchus is transected with a no. 10 blade and two posterior chest tubes are placed. The allograft is then sewn in the bronchus first using 4-0 Prolene (although PDS or Maxon may be used), then the pulmonary artery, then the pulmonary veins as an atrial cuff. The allograft is deaired through the veins by leaving volume in the heart and reducing pump flows. The left lung is removed and the allograft is sewn in a similar manner. We give induction immune suppression at the time of antibiotic infusion and solumedrol during each atrial anastamosis (500 mg each side). Both lungs are then ventilated using a pressure control mode of ventilation and positive end-expiratory pressure of 8–12  cmH2O. The goal tidal volumes are 8  cm3/kg using room air and nitric oxide at 20 ppm. Separation from bypass begins the process of reperfusion. We wean the patient slowly over 10–15 min maintaining a mean pulmonary artery pressure less than 15–20  mmHg during this time. Some centers have utilized a specific enriched reperfusion solution. These solutions contain glutamate, aspartate, lidocaine, and other free-radical scavengers [10, 12]. Inotropes are also necessary to support the right ventricle and minimize volume resuscitation. Regardless of allograft function, we utilize a period of modified ultrafiltration after separation from bypass. For significant allograft dysfunction, we extend this period for as long as the patient hemodynamically tolerates fluid removal. Once satisfactory hemodynamics and pulmonary function have been achieved, the heparinization is reversed and the wound closed. If the patient requires an inspired oxygen fraction greater than 60%, we utilize venovenous (V/V) extracorporeal membrane oxygenation (ECMO) for allograft support. We utilize venoarterial (V/A) ECMO for severe right ventricular failure.

7 Extracorporeal Membrane Oxygenation Fig. 1  Bilateral anterolateral thoracotomy with transverse sternotomy. The patient is cannulated centrally. The right lung is the newly implanted allograft and the left lung is the native lung in a patient with pulmonary hypertension and sarcoidosis

ECMO is a closed-circuit system that incorporates a pump and oxygenator. It differs from conventional cardiopulmonary bypass in that there is no reservoir and no control of venous

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return but may be utilized in a variety of ways to support both cardiac and pulmonary function. When placed in a V/V mode with cannulas in the superior vena cava and inferior vena cava, it provides pure respiratory support. When cannulas are placed in a vein and an artery (V/A), it provides both cardiac and respiratory support. Our institutional preference is to utilize V/V ECMO where possible, although several groups have moved to V/A ECMO for postoperative support. The Vienna Lung Transplant Group reported an experience using V/A ECMO intraoperatively prior to and during implantation and then terminated some time postoperatively when the high-risk period of reperfusion injury has passed [13]. Cannulation may be done centrally or peripherally. We prefer central cannulation when using V/A ECMO and peripheral cannulation when using V/V ECMO. V/V ECMO is easily discontinued in the intensive care unit.

8 Postoperative Considerations Lung transplantation for pulmonary hypertension is a high-risk enterprise. The patients may be very deconditioned, the right ventricular function is poor, and their allograft gas exchange or compliance may be marginal. Protection of the allograft is the most important postoperative consideration. We utilize a ­protective ventilation strategy starting with maintaining the positive end-expiratory pressure at 8–12 cmH2O initially and we minimize fluid administration. The inspired oxygen is kept to a minimum (usually room air) and we use nitric oxide liberally. Diuretics are also given to minimize intravascular fluid and total lung water. Occasionally we will place patients on continuous V/V ultrafiltration for aggressive fluid removal. Patients should also be sedated appropriately (we use propofol and avoid paralytics) and weaning from the ventilator is begun only when the allograft function is stable. These patients typically remain intubated for 24–48 h. Pain control is critically important and we place an epidural catheter postoperatively and prior to extubation. Right ventricular function will take several weeks to recover and patients may require inotropes for a prolonged period. We transition patients to dopamine within 24 h after surgery to minimize end-organ injury and then wean patients from it slowly.

9 Conclusion Lung transplantation for pulmonary hypertension is an acceptable therapy for select patients. Either single or double lung transplantation may be utilized, although double lung

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transplantation is the commoner method. Intraoperative management centers on using cardiopulmonary bypass, minimizing blood transfusions, and controlled reperfusion. Postoperatively, patients require close attention to both right ventricular and pulmonary allograft function. Although upfront mortality is higher, long-term outcomes are comparable to other pulmonary transplantation indications with excellent quality of life.

References 1. Reitz BA, Wallwork JL, Hunt SA et al (1982) Heart-lung transplantation: successful therapy for patients with pulmonary vascular disease. N Engl J Med 306:557–564 2. Fremes SE, Patterson GA, Williams WG, Goldman BS, Todd TR, Maurer J (1990) Single lung transplantation and closure of patent ductus arteriosis for Eisenmenger’s syndrome; Toronto Lung Transplant Group. J Thorac Cardiovasc Surg 100:1–5 3. Pasque MK, Cooper JD, Kaiser DA, Haydock DA, Triantafillou TEP (1990) Improved technique for bilateral lung transplantation: rationale and initial clinical experience. Ann Thorac Surg 49: 785–791 4. Orens JB, Estenne M, Arascoy S et al (2006) International guidelines for the selection of lung transplant candidates; 2006 update – a consensus report from the pulmonary scientific council of the International Society of Heart and Lung Transplantation. J Heart Lung Transplant 25:745–755 5. Nwakanma LU, Simpkins CE, Williams JA et al (2007) Impact of bilateral versus single lung transplantation on survival in recipients 60 years of age and older: analysis of the United Network for Organ Sharing database. J Thorac Cardiovasc Surg 133: 541–547 6. Miyamoto S, Nagaya N, Satoh T et al (2000) Clinical correlates and prognostic significance of six minute walk test in patients with primary pulmonary hypertension. Am J Respir Crit Care Med 161:487–492 7. Mulligan MS, Shearon TH, Weill D, Pagani FD, Moore J, Murray S (2008) Heart and lung transplantation in the United States, 1997–2006. Am J Transplant 8:977–987 8. Hadjiliadis D, Chaparro C, Guitierrez C et  al (2006) Impact of lung transplant operation on bronchiolotis obliterans syndrome in patients with chronic obstructive lung disease. Am J Transplant 6:183–189 9. Gammie JS, Pham SM, Colson YL et  al (1998) Single versus double lung transplantation for pulmonary hypertension. J Thorac Cardiovasc Surg 115:397–402 10. Toyoda Y, Thacker J, Santos R et al (2008) Long-term outcome of lung and heart-lung transplantation for idiopathic pulmonary arterial hypertension. Ann Thorac Surg 86:1116–1122 11. Mason DP, Thuita L, Alster JM et al (2008) Should lung transplantation be performed using donation after cardiac death? The United States experience. J Thorac Cardiovasc Surg 136:1061–1066 12. Schnickel GT, Ross DJ, Beygui R et al (2006) Modified reperfusion in clinical lung transplantation: the results of 100 consecutive cases. J Thorac Cardiovasc Surg 131:218–223 13. Aigner C, Wisser W, Taghavi S et al (2007) Institutional experience with extracorporeal membrane oxygenation in lung transplantation. Eur J Cardiothorac Surg 31:468–473

Chapter 117

Living-Donor Lobar Lung Transplantation for Pulmonary Arterial Hypertension Hiroshi Date and Takahiro Oto

Abstract  Living-donor lobar lung transplantation (LDLLT) was developed in the University of Southern California (USC) to offset the mismatch between supply and demand for those patients awaiting cadaveric lung transplantation (CLT). A single donor was used in the beginning and successful living-donor single lobe transplantation was reported. However, the following experience of single lobe transplantation was not satisfactory. It is for this reason that the USC group developed a bilateral LDLLT in which two healthy donors donate their right or left lower lobes. Because only two lobes are transplanted, LDLLT seems to be best suited for children and small adults, and was applied most exclusively to cystic fibrosis patients in the beginning. Pulmonary arterial hypertension (PAH) is defined as a group of diseases characterized by a progressive increase in pulmonary vascular resistance. Until the introduction of intravenous epoprostenol therapy in the 1990s, the disorder was rapidly progressive, leading to death in a median of 2.8 years from diagnosis. Epoprostenol, a potent shortacting vasodilator and inhibitor of platelet aggregation, was a therapeutic breakthrough that brought new hope to patients with PAH. In long-term follow-up studies of patients with idiopathic PAH (IPAH) receiving intravenous epoprostenol therapy, 3-year survival ranged from 62 to 88%. Bilateral CLT remains the treatment of last resort when medical therapy has failed. However, compared with other conditions for which CLT is performed, patients with IPAH had the highest risk of death within the first year of CLT. The 3-year survival rate after CLT was reported to be less than 60%, which was worse than that of patients receiving epoprostenol treatment. We and others have expanded the indications for LDLLT to include both pediatric and adult IPAH patients. As of 2008, LDLLT had been performed in approximately 300 patients with various lung diseases worldwide. In this chapter, the current status of LDLLT for PAH is summarized. Keywords  Bilateral lung transplant • Surgical treatment of pulmonary hypertension • Lower lobes of the lung H. Date (*) Department of Thoracic Surgery, Kyoto University Hospital, 54 Shogoin Kawahara-cho, 606-8507 Sakyo-ku, Kyoto, Japan e-mail: [email protected]

1 Introduction  

Living-donor lobar lung transplantation (LDLLT) was developed at the University of Southern California (USC) to offset the mismatch between supply and demand for those patients awaiting cadaveric lung transplantation (CLT) [1]. A single donor was used in the beginning and successful living-donor single lobe transplantation was reported. However, the following experience of single lobe transplantation was not satisfactory [2]. It is for this reason that the USC group developed a bilateral LDLLT in which two healthy donors donate their right or left lower lobes [3, 4]. Because only two lobes are transplanted, LDLLT seems to be best suited for children and small adults, and was applied most exclusively to cystic fibrosis patients in the beginning [3]. Pulmonary arterial hypertension (PAH) is defined as a group of diseases characterized by a progressive increase in pulmonary vascular resistance [5]. Until the introduction of intravenous epoprostenol therapy in the 1990s [6], the disorder was rapidly progressive, leading to death in a median of 2.8 years from diagnosis [7]. Epoprostenol, a potent shortacting vasodilator and inhibitor of platelet aggregation, was a therapeutic breakthrough that brought new hope to patients with PAH. In long-term follow-up studies of patients with idiopathic PAH (IPAH) receiving intravenous epoprostenol therapy, 3-year survival ranged from 62 to 88% [8–11]. Bilateral CLT remains the treatment of last resort when medical therapy has failed [12]. However, compared with other conditions for which CLT is performed, patients with IPAH had the highest risk of death within the first year of CLT [13]. The 3-year survival rate after CLT was reported to be less than 60%, which was worse than that of patients receiving epoprostenol treatment [12, 13]. We and others have expanded the indications for LDLLT to include both pediatric [14, 15] and adult [16] IPAH patients. As of 2008, LDLLT had been performed in approximately 300 patients with various lung diseases worldwide. In this chapter, the current status of LDLLT for PAH is summarized.

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2 Basic Principle  

In this relatively new procedure, two healthy donors donate their right or left lower lobes and these two lobes are implanted in the recipient after bilateral pneumonectomy (Fig. 1). Advantages and disadvantages of LDLLT compared with CLT are summarized in Table 1. The current availability of cadaveric donor lungs has not been sufficient to meet the increasing demand of potential recipients in most countries. The use of LDLLT has decreased in the USA, and the recent change by the Organ Procurement and Transplantation Network to an urgency/benefit allocation system for cadaveric donor lungs in patients 12 years and older may further reduce the demand in the USA. In contrast, the average waiting time for a cadaveric lung is more than 2 years in Japan. LDLLT can be scheduled and performed within a few weeks, during which time donor evaluation and informed consent can be completed. In general, the ischemic time for LDLLT is much shorter than that for CLT. Despite improved preservation methods, severe primary graft dysfunction occurs in 10–20% of CLT recipients [17]. Although only two lobes are transplanted,

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LDLLT seems to be associated with less frequent primary graft dysfunction. “A small but perfect graft” is a great advantage in LDLLT. Experienced centers have recently reported the incidence of bronchial complications in CLT to be about 5% [18]. Contraindications to CLT include current high-dose systemic corticosteroid therapy because it may increase airway complications, although low-dose pretransplantation corticosteroid therapy (20 mg of prednisone or less per day) is acceptable. We have accepted high-dose systemic corticosteroid therapy, as high as 50 mg/day of prednisone, in LDLLT [19]. Among 91 anastomoses, excellent bronchial healing was observed in 89 (98%) anastomoses. Various factors, such as short donor bronchial length, high blood flow in the small grafts implanted, and well-preserved lung parenchyma with short ischemic time, may contribute to better oxygen supply to the donor bronchus, thus resulting in excellent bronchial healing in LDLLT. Bronchiolitis obliterans syndrome (BOS) has been the major obstacle after CLT. The USC group suggested that LDLLT was associated with a lower incidence of BOS particularly in pediatric patients [20]. They also indicated that the shorter ischemic time in LDLLT could explain the reduced incidence of BOS [21]. In our 40 LDLLT recipients who survived longer than 6 months, ten recipients (25%) developed BOS. Interestingly, seven of the ten recipients developed unilateral BOS and their forced expiratory volume in 1 s (FEV1) decline stopped within 9 months. Transplanting two lobes obtained from two different donors appears to be beneficial in the long term because the contralateral unaffected lung may function as a reservoir organ in the event of unilateral BOS.

3 Indication 3.1 Recipient Selection Fig. 1  Bilateral living-donor lobar lung transplantation Table 1  Comparison between living-donor lobar lung transplantation (LDLLT) and cadaveric lung transplantation (CLT) LDLLT CLT Waiting time Short Long Schedule Controllable Uncontrollable Ischemic time Short Long Graft size Small Full Primary graft Infrequent 10–20% dysfunction Number of teams 3 2 Bronchial complication Rare 5% Chronic rejection Often unilateral Major cause of death LDLLT living-donor lobar lung transplantation, CLT cadaveric lung transplantation

Because of the remarkable improvements in medical treatment for PAH during the past decade, determining the indications and timing for transplantation as a PAH treatment is a difficult challenge [22]. The Pulmonary Scientific Council of the International Society for Heart and Lung Transplantation recently updated the guidelines for referral and transplantation [23] (Table 2). Patients being considered for LDLLT should meet the criteria for conventional bilateral CLT. Because LDLLT subjects healthy donors to a lower lobectomy procedure associated with potentially serious complications [24, 25], hospitalization, discomfort, and loss of work, as well as the irreversible reduction in lung function, we have accepted only critically ill IPAH patients who failed maximal medical treatment including epoprostenol therapy (Table 3). Although

117  Living-Donor Lobar Lung Transplantation for Pulmonary Arterial Hypertension Table  2  Guideline for CLT for patients with pulmonary arterial hypertension (PAH) Guideline for referral •  NYHA functional class III or IV, irrespective of ongoing therapy •  Rapidly progressive disease Guideline for transplantation •  Persistent NYHA class III or IV on maximal medical therapy •  Low (<350 m) or declining 6MWT distance • Failing therapy with intravenously administered epoprostenol, or equivalent •  Cardiac index of less than 2 L/min/m2 •  Right atrial pressure exceeding 15 mmHg Data from Orens et al. [23] Table 3  Guideline for LDLLT for patients with PAH Patients who fulfill the guideline for cadaveric lung transplantation (see Table 2) Critically ill patients who fail maximal medical treatment, including epoprostenol therapy Patients who would die or become unsuitable recipients before cadaveric lungs become available

knowledge of the predictors of survival in patients with PAH is helpful, ultimately the timetable must be set by the unique situation of each patient. In our LDLLT experience, all PAH patients were on high-dose intravenous epoprostenol therapy (average, 89  ng/kg/min) with inotropic support except for one patient. Most of them were in WHO class IV and were bed-bound.

3.2 Donor Selection Although immediate family members (relatives within the second or third degree or a spouse) have been the only donors in our institution, other institutions have accepted extended family members and unrelated individuals. An international conference of transplant physicians, surgeons, and allied health professionals was held in Vancouver, Canada, in 2005 to address the care of the live lung, liver, pancreas, and intestine organ donor [26]. The Vancouver Forum Lung Group proposed eligibility criteria for living lobar donation (Table 4). Potential donors should be mentally competent, willing to donate free of coercion, medically and psychosocially suitable, fully informed of the risks and benefits as a donor, and fully informed of the risks, benefits, and alternative treatment available to the recipient. In our institutions, potential donors are interviewed by three physicians with an observer to safeguard against coercion and to ensure donor comprehension of the procedure. The interview is performed at least three times to provide potential donors with multiple opportunities to question, reconsider, or withdraw their offer to be a donor.

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Table 4  The eligibility criteria for living lobar donation Age 18–60 years and able to give informed consent No active tobacco smoking or a significant smoking history No active lung disease/previous ipsilateral thoracic surgery • No identifiable risk for familial lung disease (i.e. familial forms of IPF of PAH) • No cachexia (BMI <18 kg/m2) or obesity (BMI ³30 kg/m2) ABO blood type compatibility with recipient Donor lobe size compatible with recipient Normal pulmonary function and arterial blood gas results No conditions that significantly increase the risk of general anesthesia, surgery, and postoperative recovery No psychosocial, ethical issues, or concerns about donor motivation Not pregnant No active malignancy No active significant infection (HIV, hepatitis, acute CMV) IPF idiopathic pulmonary fibrosis, BMI body mass index Data from Barr et al. [26]

3.3 Size Matching Appropriate size matching between donor and recipient is important in LDLLT. It is often inevitable that small grafts are implanted in LDLLT, in which only two lobes are implanted. Excessively small grafts may cause high pulmonary artery pressure, resulting in lung edema [27]. A pleural space problem may increase the risk of empyema. Overexpansion of the donor lobes may contribute to obstructive disease by early closure of small airways [28]. We have previously proposed a formula to estimate the graft forced vital capacity (FVC) based on the donor’s measured FVC and the number of pulmonary segments implanted [29]. Given that the right lower lobe consists of five segments, the left lower lobe consists of four segments, and the whole lung consists of 19 segments, the total FVC of the two grafts is estimated by the following equation: Total FVC of the two grafts = measured FVC of the right donor × 5/19 + measured FVC of the left donor × 4/19. When the total FVC of the two grafts is more than 45% of the predicted FVC of the recipient (calculated from knowledge of height, age, and sex), we accept the size disparity. For PAH patients, the ratio should be more than 50%. The total FVC of the grafts in our PAH patients was estimated to range from 51.4 to 103.0% (average 70.1%) of the predicted FVC of the recipient. We compared the graft FVC estimated by the equation with the recipient’s measured FVC at 6 months [30]. The 6-month FVC was used because the risk of bronchiolitis obliterans after this point would make the analysis more complicated. There was a very close correlation between the two parameters. In contrast, there was no significant correlation between the recipient’s predicted FVC and the recipient’s measured FVC. These results indicate that the recipient’s

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FVC is determined by the amount of lung tissue implanted and is not dependent on the recipient’s factors.

4 Operative Technique The recipient and the right-side donor are brought to separate operating rooms at the same time. The left-side donor is brought to the third operating room 30 min later. Three surgical teams are required and they communicate closely to minimize graft ischemic time.

4.1 Donor Operation Epidural catheters for postoperative analgesia are placed routinely the day before the surgery to avoid complications related to heparinization during the donor lobectomies. After induction of general anesthesia, donors are intubated with a left-sided double-lumen endotracheal tube. Fiber-optic bronchoscopy is performed to determine if lower lobectomy is feasible and to leave adequate tissue length for closure on the donor bronchus and anastomosis in the recipient. The donors are placed in the lateral decubitus position and a posterolateral thoracotomy is performed through the fifth intercostal space. Fissures are developed using linear stapling devices. The pericardium surrounding the inferior pulmonary vein is opened circumferentially. Dissection in the fissure is carried out to isolate the pulmonary artery to the lower lobe, and to define the anatomy of the pulmonary arteries to the middle lobe in the right-side donor and to the lingular segment in the left-side donor. If the branches of middle lobe artery and lingular artery are small, they are ligated and divided. Prostaglandin E1 is administered intravenously to decrease the systolic blood pressure by 10–20 mmHg. Five thousand units of heparin and 500 mg of methylprednisolone are administered intravenously. After vascular clamps have been placed in appropriate positions, divisions of the pulmonary artery (Fig. 2a), the pulmonary vein (Fig. 2b), and the bronchus (Fig.  2c) are carried out in that order. Vascular stumps are oversewn with a 5-0 Prolene continuous suture. The bronchial stump is closed with 3-0 Prolene interrupted sutures. Then, each bronchial closure is covered with pedicled pericardial fat tissue. Heparinization is reversed by administering protamine. On the back table, the lobes are flushed with 1 L of preservation solution both antegradely and retrogradely from a bag about 50  cm above the table. We use an extracellular solution, such as ET-Kyoto solution [31], for preservation. Lobes are gently ventilated with room air during the flush.

Fig. 2  Right lower lobectomy of the donor. Dissection and division of the right pulmonary artery (a), the inferior pulmonary vein (b), and the right lower bronchus (c). RUL right upper lobe, RML right middle lobe, RLL right lower lobe

4.2 Recipient Operation Patients are anesthetized and intubated with a single-lumen endotracheal tube in children and with a left-sided doublelumen endotracheal tube in adults. When the patient’s hemodynamic instability is remarkable, partial cardiopulmonary bypass is initiated using the femoral vessels under local anesthesia. Then, the recipient is anesthetized and intubated. A Swan–Ganz catheter is placed. Intraoperative transesophageal echocardiography is employed routinely. The “clamshell” incision is used and the sternum is transected. The sternum is notched at the level of transection by aiming the sternal saw at a 45° angle and cutting toward the midpoint to facilitate postoperative sternal adaptation. Pleural and hilar dissection is carried out before heparinization to reduce blood loss. The ascending aorta and the right atrium are cannulated after heparinization and patients are placed on standard cardiopulmonary bypass. After bilateral pneumonectomy, the right lower lobe implantation is performed, followed by the left lower lobe implantation. The implanted right graft is packed in iced saline and slush while the left graft is implanted. The anastomosis sequence of the recipient is the bronchus (Fig.  3a), vein (Fig.  3b), and artery (Fig.  3c). The bronchial anastomosis is begun with a running 4-0 polydioxanone suture for the membranous portion and is completed with simple interrupted sutures for the cartilaginous portion. We use end-to-end anastomosis when the bronchial size is equivalent, and use a telescoping technique when the discrepancy in bronchial size is obvious. Bronchial wrapping is not employed except in patients on high-dose

117  Living-Donor Lobar Lung Transplantation for Pulmonary Arterial Hypertension

Fig.  3  Right graft implantation in the recipient. The right bronchial anastomosis (a), the pulmonary venous anastomosis (b), and the pulmonary artery anastomosis (c)

steroid therapy. Pedicled pericardial fat tissue is used to cover the bronchial anastomosis if necessary. The venous anastomosis is placed between the donor’s right inferior pulmonary vein and the recipient’s right upper pulmonary vein using a running 6-0 Prolene suture. Finally, the pulmonary artery anastomosis is performed in an end-to-end fashion using a running Prolene suture. In PAH patients, pulmonary artery anastomosis is often challenging owing to marked size discrepancy. Just before completing the bilateral implantations, 0.5–1 g of methylprednisolone is given intravenously and nitric oxide inhalation is initiated at 10–20  ppm. A left atrial line is placed through the left appendage and left atrial pressure is carefully monitored. After both lungs have been reperfused and ventilated, the patient is gradually weaned off cardiopulmonary bypass and then it is removed. All blood accumulated in the chest cavity during the operation is discarded to avoid infection. Two chest tubes are placed in each chest cavity and the chest is closed in the standard fashion.

5 Postoperative Management The patient is kept intubated at a positive end-expiratory pressure of 5 cmH2O for at least 3 days to maintain optimal expansion of the small implanted lobes. Weaning from the ventilator is intentionally slow, and a tracheostomy is performed when patients show any signs of sputum retention [32]. Suction of the chest drainage tubes starts at 10 cmH2O and gradually decreases to water seal in a couple of days. Fiberoptic bronchoscopy is performed every 12 h while the patient

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is intubated to assess donor airway viability and to suction any retained secretions. An intensive program of chest physiotherapy is given every day and bedside postoperative pulmonary rehabilitation is initiated as soon as possible. Cytomegalovirus prophylaxis with ganciclovir is given to all recipients for the first 3 months. Postoperative immunosuppression is a triple-drug therapy consisting of cyclosporine (CSA) or tacrolimus (FK), azathioprine (AZA) or mycophenolate mofetil (MMF), and corticosteroids. Induction therapy with monoclonal or polyclonal antibodies is not used. The combination of CSA, AZA, and steroid is chosen for patients with infectious lung diseases and pediatric patients. The combination of FK, MMF, and steroid is chosen for patients with noninfectious lung diseases. CSA (100 mg) or FK (2 mg) delivery begins during the first few postoperative hours via a nasal feeding tube inserted in the proximal jejunum. The dose is adjusted to maintain trough levels in the target range listed in Table 5. These drugs often induce a significant increase in the level of serum creatinine, necessitating a reduction in their dose. AZA (2 mg/kg) or MMF (500 mg) is given orally preoperatively and then given via the nasal feeding tube after transplantation. The dose must be adjusted to maintain a white blood cell count in excess of 3,500. MMF often causes significant diarrhea, necessitating dose reduction. We have adopted the use of moderate-dose corticosteroids during the early postoperative period. Intravenous administration of methylprednisolone is used before reperfusion and during the first 3 days. Then we initiate prednisone delivery via the nasal feeding tube on day 4. In CLT, transbronchial lung biopsy offers a safe and accurate means of diagnosis of acute rejection and has emerged as the procedure of choice. However, the risk of pneumothorax and bleeding from transbronchial lung biopsy may be higher in LDLLT because the small grafts are receiving high blood flow. It is for this reason that we judge acute rejection on the basis of radiographic and clinical findings. Early acute rejection episodes are characterized by dyspnea, low-grade fever, leukocytosis, hypoxemia, and diffuse interstitial infiltrate on chest radiographs. Because two lobes are donated by different donors, acute rejection is usually seen unilaterally. A trial bolus dose of 500 mg of methylprednisolone is administered and various clinical signs are carefully observed. If acute rejection is indeed the problem, two additional daily bolus doses of methylprednisolone are given. If acute rejection is encountered more than three times, the combination of CSA and AZA is switched to FK and MMF. When all these treatments fail, OKT3 is used. Three months after LDLLT, patients are allowed to return to their home town. They are asked to keep a diary which includes daily pulmonary function, digital saturation, body temperature, body weight, blood pressure, and heart rate.

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H. Date and T. Oto Table 5  Immunosuppressive protocol for LDLLT Cyclosporine Before transplantation None After transplantation Dose adjusted to maintain trough level at 3 months after transplantation:    250–350 ng/dL 6 months after transplantation:    200–300 ng/dL 12 months after transplantation:   150–250 ng/dL Tacrolimus Before transplantation None After transplantation Dose adjusted to maintain trough level at 3 months after transplantation:    10–20 ng/dL 6 months after transplantation:    10–15 ng/dL 12 months after transplantation:   8–12 ng/dL Azathioprine Before transplantation 2 mg/kg After transplantation 2 mg/kg/day Mycophenolate mofetil Before transplantation 500 mg After transplantation 500–1,000 mg twice daily Corticosteroids Before reperfusion Methylprednosilone (intravenously) After transplantation 3 days after transplantation Methylprednosilone (intravenously) 6 months after transplantation Prednisolone (orally) 12 months after transplantation Prednisolone (orally)

The diary is sent to a lung transplant coordinator every month. Routine full postoperative assessment is performed at 6 months, 12 months, and then annually.

6 Complications 6.1 Complications in the Donor There has been a relatively low incidence of donor morbidity in our experience and all donors have returned to their previous lifestyles. However, serious complications, such as bronchial fistula and pulmonary embolism, have been reported [24, 25]. The Vancouver Forum Lung Group summarized the world experience on approximately 550 living lung donors [26]. Sixty percent of the live lung donors have been male, 76% have been related to the recipient, and 24% were unrelated. There has been no reported perioperative mortality of a lung donor. Approximately 4% of live lung donors have experienced an intraoperative complication that included the necessity of a right middle lobe sacrifice. Approximately 5% of them have experienced complications requiring surgical or bronchoscopic intervention. The USC group reported that right-side donors were more likely to have a perioperative complication than left-side donors, probably secondary to right lower and middle lobe anatomy [25].

500–1,000 mg 125 mg/day 0.4 mg/kg/day 0.2 mg/kg/2 days

6.2 Complications in the Recipient The operative morbidity of the recipients is very high; therefore, meticulous postoperative management is mandatory. We have encountered various operative complications (Table 6) and some of them were unique to LDLLT. Because the graft ischemic time is short in LDLLT, primary graft dysfunction is infrequently encountered as compared with conventional CLT. However, three of the first six recipients with PAH experienced transient lung edema associated with left ventricular dysfunction within a month after LDLLT. Clinical findings demonstrated by echocardiography helped establish the diagnosis of left ventricular dysfunction. Contrary to the early right ventricular function recovery, the impaired left ventricular function persisted at 2 months despite findings that left ventricular geometry was restored earlier after reversal of pulmonary hypertension (PH). Chronic preoperative preload reduction may adversely affect left ventricular compliance and muscle stiffness. All three patients responded to therapy including steroid pulse, inotropic drugs, afterload reduction with vasodilators, and nitric oxide inhalation. The last ten PAH patients were kept on a ventilator for at least 1  week and were weaned off very slowly along with elective tracheostomy. A left atrial line was placed through the left appendage at the time of transplantation and left atrial pressure was carefully monitored. As a result, none of the last ten PAH patients developed lung edema.

117  Living-Donor Lobar Lung Transplantation for Pulmonary Arterial Hypertension Table 6  Operative complications in the 16 recipients with PAH after LDLLT Complication No. of patients Lung edema associated with left ventricular dysfunction Steroid-resistant acute rejection requiring OKT3 Hemorrhage necessitating rethoracotomy Kinking of pulmonary artery Cardiac tamponade Peroneal nerve palsy Renal failure requiring artificial dialysis Heart failure during pleural dissection Massive hemoptysis Bronchial stenosis Right phrenic nerve palsy Left recurrent nerve palsy Decubitus PAH pulmonary arterial hypertension, donor lobar lung transplantation

3 (19%) 3 (19%) 3 (19%) 2 (13%) 2 (13%) 2 (13%) 2 (13%) 1 (6%) 1 (6%) 1 (6%) 1 (6%) 1 (6%) 1 (6%) LDLLT living-

In PAH patients, the pulmonary artery anastomosis is often challenging owing to a marked size discrepancy. The pulmonary artery anastomosis was seen to be severely kinked in two patients. After placement of several tacking stitches in both cases, there was immediate improvement in gas exchange and pulmonary artery pressure. Life-threatening massive hemoptysis was encountered in one patient, which was successfully treated with extracorporeal membrane oxygenation [33]. Although the cause of the bleeding was unknown, it could be related to increased blood flow in the small grafts implanted. Excellent bronchial healing was observed in all but one anastomosis. Various factors, such as short donor bronchial length, high blood flow in the small grafts implanted, and well-preserved lung parenchyma with short ischemic time, may contribute to better oxygen supply to the donor bronchus, resulting in excellent bronchial healing in LDLLT. One patient developed a right bronchial stenosis requiring repeated balloon dilatation. The donor pulmonary venous branch from the superior segment of the right lower lobe was accidentally sacrificed at the time of donor lobectomy. We speculate that poor venous drainage from the superior segment of the right graft was the cause of bronchial stenosis in this particular case.

7 Outcome and Prognosis Only three groups have reported a summary of recipient outcome. The USC group recently published their 10-year experi-

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ence on 123 LDLLT recipients, including five patients with PH [21]. In their series, retransplantation and mechanical ventilation were identified as risk factors for mortality. One, 3-, and 5-year survival were 70%, 54%, and 45%, respectively. The St. Louis group reported similar results in 38 pediatric LDLLT recipients [34]. Only 2% of their recipients had PH. The authors and their colleagues performed LDLLT in 47 patients with various lung diseases at Okayama University between 1998 and 2007. Sixteen of them (34%) were diagnosed with PH. Final pathologic diagnoses of the excised lungs were IPAH in 11 patients, pulmonary veno-occlusive disease in two patients, pulmonary capillary hemangiomatosis in one patient, diffuse peripheral pulmonary arterial stenosis in one patient, and Eisenmenger syndrome with atrial septal defect in one patient. There were 12 females and four males, with ages ranging from 8 to 43 years (average 23.3 years). Six of the patients were children and ten were adults. Height ranged from 122 to 175 cm (average 154 cm) and weight ranged from 20 to 60 kg (average 38.8 kg). All patients were hospital-bound and were dependent on continuous oxygen inhalation. Except for the patient with Eisenmenger syndrome, 15 patients were on high-dose intravenous epoprostenol therapy (average 105 ng/kg/min) along with inotropic support. Two patients were about to be intubated and required a noninvasive positive pressure ventilator with 100% inspired oxygen. All patients showed severely elevated mean pulmonary artery pressure (average 62 mmHg) and depressed cardiac index (average 2.1 L/min/m2). One patient with diffuse peripheral pulmonary arterial stenosis and a past history of empyema developed acute heart failure during pleural dissection. The donors had not been anesthetized and the transplant procedure was canceled. Bilateral LDLLT was performed in 14 patients and right-sided single LDLLT was performed in a 10-year-old boy with IPAH because his mother was the only available donor [15]. Cardiopulmonary bypass was used in all cases. In two cases, partial cardiopulmonary bypass was initiated using the femoral vessels under local anesthesia because of the recipient’s poor preoperative condition. Among 29 living donors, nine were the fathers of recipients, eight were mothers, seven were brothers, three were husbands, and two were sisters. The total FVC of the grafts was estimated to range from 51.4 to 103.0% (average 70.9%) of the predicted FVC of the recipient. Cardiopulmonary bypass was successfully removed from all patients at the end of the procedure. The mean ischemic time of the right graft was 161 min and that of the left graft was 120 min. During the first month, acute rejection occurred at an average rate of 1.7 episodes per patient. Three patients required OKT3 treatment owing to steroid-resistant acute rejection. The average duration of mechanical ventilation required was 15.6 days, the average intensive care unit stay was 31.1 days,

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H. Date and T. Oto

Fig. 4  A 14-year-old male with idiopathic pulmonary arterial hypertension. (a) Before transplantation. (b) Six months after receiving living-donor lobar lung transplantation

and the average hospital stay was 74.4 days. All 15 operative survivors were discharged without oxygen inhalation therapy. Chest X-rays showed dramatic improvement of cardiomegaly (Fig. 4). Mean pulmonary artery pressure was normal (average 15 mmHg) by the time of discharge. Pulmonary hemodynamics continued to be excellent at 3 years (Fig. 5) after transplantation. During the average follow-up time of 68 months, four of 15 operative survivors (27%) developed BOS. Interestingly, three of the four recipients developed unilateral BOS and their FEV1 decline stopped within 9 months. One recipient developed bilateral BOS and died at 65 months. Transplanting two lobes obtained from two different donors appears to be beneficial in the long term because the contralateral unaffected lung may function as a reserve organ in the event of unilateral BOS. Survival after LDLLT was excellent (Fig. 6). The 5-year survival rate was 93.8% for PH patients (n = 16) and was 86.4% for non-PH patients (n = 31). We recently reported that the survival was better in IPAH patients who received LDLLT than in those in whom medical treatment was continued [35]. All PH survivors have returned to normal lifestyles and are able to carry out daily activities without restriction. Although the outcomes have been well investigated in the recipient, long-term outcomes of live donors have not been well documented. The Massachusetts General Hospital group reported that mean FVC decreased by 14 ± 4% [36]. Despite preservation of lung function within the normal range, some donors also experienced a subjective decline in exercise tolerance. Donors reported positive feelings about donation, but wish to be recognized and valued by the transplant team and the recipient. In our experience, all donors have returned to their previous lifestyles.

Fig. 5  Changes of mean pulmonary artery pressure and cardiac index before and after living-donor lobar lung transplantation

Fig.  6  Survival after living-donor lobar lung transplantation. The 5-year survival rate was 93.8% for pulmonary hypertension (PH) patients (n = 16) and was 86.4% for non-PH patients (n = 31)

117  Living-Donor Lobar Lung Transplantation for Pulmonary Arterial Hypertension

8 Summary LDLLT is a viable option in patients with PAH who would die soon otherwise.

References 1. Starnes VA, Lewiston NJ, Luikart H et al (1992) Current trends in lung transplantation: lobar transplantation and expanded use of single lungs. J Thorac Cardiovasc Surg 104:1060–1068 2. Starnes VA, Barr ML, Cohen RG (1994) Lobar transplantation: indications, technique, and outcome. J Thorac Cardiovasc Surg 108:403–411 3. Starnes VA, Barr ML, Cohen RG et al (1996) Living-donor lobar lung transplantation experience: intermediate results. J Thorac Cardiovasc Surg 112:1284–1291 4. Cohen RG, Barr ML, Schenkel FA et al (1994) Living-related donor lobectomy for bilateral lobar transplantation in patients with cystic fibrosis. Ann Thorac Surg 57:1423–1428 5. Simonneau G, Galiè N, Rubin LJ et al (2004) Clinical classification of pulmonary hypertension. J Am Coll Cardiol 43:5S–12S 6. Barst RJ, Rubin LJ, Long WA et al (1996) A comparison of continuous intravenous epoprostenol (prostacyclin) with conventional therapy for primary pulmonary hypertension. N Engl J Med 334:296–301 7. D’Alonzo GE, Barst RJ, Ayres SM et al (1991) Survival in patients with primary pulmonary hypertension: results from a national prospective registry. Ann Intern Med 115:343–349 8. Sitbon O, Humbert M, Nunes H et al (2002) Long-term intravenous epoprostenol infusion in primary pulmonary hypertension: prognostic factors and survival. J Am Coll Cardiol 40:780–788 9. McLaughlin VV, Shillington A, Rich S (2002) Survival in primary pulmonary hypertension: the impact of epoprostenol therapy. Circulation 106:1477–1482 10. Kuhn KP, Byrne DW, Arbogast PG et al (2003) Outcome in 91 consecutive patients with pulmonary arterial hypertension receiving epoprostenol. Am J Respir Crit Care Med 167:580–586 11. Yung D, Widlitz AC, Rosenzweig EB, Kerstein D, Maislin G, Barst RJ (2004) Outcomes in children with idiopathic pulmonary arterial hypertension. Circulation 110:660–665 12. Doyle RL, McCrory D, Channick RN et al (2004) Surgical treatments/interventions for pulmonary arterial hypertension: ACCP evidence-based clinical practice guidelines. Chest 126:63S–71S 13. Trulock EP, Christie JD, Edwards LB et al (2007) Registry of the International Society for Heart and Lung Transplantation; twentyfourth official adult lung and heart-lung transplantation report-2007. J Heart Lung Transplant 26:782–795 14. Starnes VA, Barr ML, Schenkel FA, Horn MV, Cohen RG, Hagen JA, Wells WJ (1997) Experience with living-donor lobar transplantation for indications other than cystic fibrosis. J Thorac Cardiovasc Surg 114:917–922 15. Date H, Sano Y, Aoe M, Matsubara H, Kusano K, Goto K, Tedoriya T, Shimizu N (2002) Living-donor single lobe lung transplantation for primary pulmonary hypertension in a child. J Thorac Cardiovasc Surg 123:1211–1213 16. Date H, Nagahiro I, Aoe M, Matsubara H, Kusano K, Goto K, Shimizu N (2001) Living-donor lobar lung transplantation for primary pulmonary hypertension in an adult. J Thorac Cardiovasc Surg 122:817–818 17. Christie JD, Carby M, Bag R et  al (2005) Report of the ISHLT working group on primary lung graft dysfunction part II: definition.

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A consensus statement of International Society for Heart and Lung Transplantation. J Heart Lung Transplant 24:1454–1459 18. Date H, Trulock EP, Arcidi JM et al (1995) Improved airway healing after lung transplantation. An analysis of 348 bronchial anastomoses. J Thorac Cardiovasc Surg 110:1424–1433 19. Date H, Tanimoto Y, Yamadori I et al (2005) A new treatment strategy for advanced idiopathic interstitial pneumonia: living-donor lobar lung transplantation. Chest 128:1364–1370 20. Starnes VA, Woo MS, MacLaughlin EF et al (1999) Comparison of outcomes between living donor and cadaveric lung transplantation in children. Ann Thorac Surg 68:2279–2284 21. Starnes VA, Bowdish ME, Woo MS et al (2004) A decade of living lobar lung transplantation. Recipient outcomes. J Thorac Cardiovasc Surg 127:114–122 22. Conte JV, Gaine SP, Orens JB et al (1998) The influence of continuous intravenous prostacyclin therapy for primary pulmonary hypertension on the timing and outcome of transplantation. J Heart Lung Transplant 17:679–685 23. Orens JB, Estenne M, Arcasoy S et al (2006) International guidelines for the selection of lung transplant candidates: 2006 update – a consensus report from the pulmonary scientific council of the International Society for Heart and Lung Transplantation. J Heart Lung Transplant 25:745–755 24. Battafarano RJ, Anderson RC, Meyers BF et al (2000) Perioperative complications after living donor lobectomy. J Thorac Cardiovasc Surg 120:909–915 25. Bowdish ME, Barr ML, Schenkel FA et al (2004) A decade of living lobar lung transplantation: perioperative complications after 253 donor lobectomies. Am J Transplant 4:1283–1288 26. Barr ML, Belghiti J, Villamil FG et  al (2006) A report of the Vancouver Forum on the care of the live organ donor. Lung, liver, pancreas, and intestine data and medical guidelines. Transplantation 81:1372–1387 27. Fujita T, Date H, Ueda K et al (2002) Experimental study on size matching in a canine living-donor lobar lung transplant model. J Thorac Cardiovasc Surg 123:104–109 28. Haddy SM, Bremner RM, Moore-Jefferies EW et  al (2002) Hyperinflation resulting in hemodynamic collapse following living donor lobar transplantation. Anesthesiology 95:1315–1317 29. Date H, Aoe M, Nagahiro I et  al (2003) Living-donor lobar lung transplantation for various lung diseases. J Thorac Cardiovasc Surg 126:476–481 30. Date H, Aoe M, Nagahiro I et al (2004) How to predict forced vital capacity after living-donor lobar-lung transplantation. J Heart Lung Transplant 23:547–551 31. Omasa M, Hasegawa S, Bando T et  al (2004) Application of ET-Kyoto solution in clinical lung transplantation. Ann Thorac Surg 77:338–339 32. Date H, Aoe M, Sano Y et al (2004) Improved survival after livingdonor lobar lung transplantation. J Thorac Cardiovasc Surg 128:933–940 33. Kotani K, Ichiba S, Andou M et  al (2002) Extracorporeal membrane oxygenation with nafamostat mesilate as an anticoagulant for massive pulmonary hemorrhage after living-donor lobar lung transplantation. J Thorac Cardiovasc Surg 124:626–627 34. Sweet SC (2006) Pediatric living donor lobar lung transplantation. Pediatr Transplant 10:861–868 35. Date H, Kusano KF, Matsubara H et al (2007) Living-donor lobar lung transplantation for pulmonary arterial hypertension after failure of epoprostenol therapy. J Am Coll Cardiol 50:523–527 36. Prager LM, Wain JC, Roberts DH et al (2006) Medical and psychologic outcome of living lobar lung transplant donors. J Heart Lung Transplant 25:206–212

Chapter 118

Results of Lung Transplantation Janet R. Maurer

Abstract  Idiopathic pulmonary arterial hypertension or pulmonary hypertension secondary to congenital cardiac defects has became the main recipients of heart-lung transplantations. In 1983 the first successful isolated lung transplantation was performed in a patient with pulmonary fibrosis. That initiated the era of both unilateral and bilateral isolated lung grafts that have since become the principal options both for patients with pulmonary hypertension and for other patients with various advanced lung diseases. In 1995 when the FDA approved epoprostenol, the first prostanoid, for idiopathic pulmonary arterial hypertension treatment. Initially considered primarily a bridge to transplantation, clinicians soon observed dramatic improvements in the status of some patients placed on this drug and a few reports of improved survival were also published. Concomitant with these reports, the proportion of patients with a pulmonary hypertension diagnosis undergoing transplantation relative to other diagnoses fell. In addition to epoprostenol, several new drugs (e.g., endothelin receptor antagonists, phosphodiesterase inhibitors) and oral prostacyclin analogues have been developed for treatment of pulmonary hypertension. This menu of different options now used to medically treat pulmonary hypertension appears to have delayed the rate at which patients are presenting for transplantation. The impact of medical therapy on patient survival is still under study, although a few studies have suggested a survival benefit for both prostanoids and endothelin receptor antagonists. Thus, lung or heart–lung transplantation remains a final therapeutic option for these patients. This chapter will discuss selection of appropriate transplant candidates, various peritransplant issues, survival, intermediate and long-term follow-up, complications, and long-term function and quality of life. Keywords  Surgical treatment of pulmonary hypertension • Prognosis • Postoperative outcome • Survival • Complication J.R. Maurer (*) Department of Provider Services and Coaching Effectiveness, Health Dialog Service Corporation, 38732 N. 10th St., Desert Hills, AZ 85086, USA e-mail: [email protected]

1  Introduction On March 9, 1981, Bruce Reitz from Stanford University performed the first successful heart–lung transplantation. The recipient was a 45-year-old woman from Yuma (AZ, USA), who had advanced idiopathic pulmonary arterial hypertension [1]. The woman lived for more than 5 years. Just prior to this transplantation, the US Food and Drug Administration (FDA) had approved cyclosporine A, the first calcineurin inhibitor, for immunosuppression. That drug approval would transform solid organ transplantation and, in particular, make possible long-term survival for patients receiving lung or heart–lung transplantations. In the following few years, end-stage patients with either idiopathic pulmonary arterial hypertension or pulmonary hypertension secondary to congenital cardiac defects became the main recipients of heart– lung transplantations [2]. In 1983 the first successful isolated lung transplantation was performed in a patient with pulmonary fibrosis [3]. That initiated the era of both unilateral and bilateral isolated lung grafts that have since become the principal options both for patients with pulmonary hypertension and for other patients with various advanced lung diseases. In 1981 the understanding of the pathogenesis of and the treatment for idiopathic pulmonary arterial hypertension was rudimentary. Typically, patients were treated with calcium channel blockers or other contemporary vasodilators such as hydralazine; however, a small minority of patients responded to these approaches and the outcomes were dismal. The National Heart, Lung, and Blood Institute Primary Pulmonary Hypertension Patient Registry results published in 1991 [4] documented a mean survival of 2.8 years from diagnosis. With those poor survivals and publicity around the success of the initial successful heart–lung transplantation in 1981, many more patients were referred to emerging lung transplant centers than there were available donor organs. The treatment landscape changed again in 1995 when the FDA approved epoprostenol, the first prostanoid, for idiopathic pulmonary arterial hypertension treatment. Initially considered primarily a bridge to transplantation, clinicians soon observed dramatic improvements in the status of some

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patients placed on this drug and a few reports of improved survival were also published [5]. Concomitant with these reports, the proportion of patients with a pulmonary hypertension diagnosis undergoing transplantation relative to other diagnoses fell. Epoprostenol, however, had (and has) several drawbacks as a treatment: it is administered intravenously and requires a continuous-infusion pump, it is very expensive and patients tend to become tachyphylactic to the drug and require increasing doses over time, and it has all of the potential complications of a continuous-infusion drug, e.g. unintended stoppage and infections. Epoprostenol has been followed – and to some degree replaced – by other, somewhat easier to administer, prostanoids and more recently by other oral drugs that have a different mechanism of action. These include the endothelin receptor antagonists (e.g. bosentan) and phosphodiesterase inhibitors (e.g. sildenafil). This menu of different options now used to medically treat pulmonary hypertension, both primary and secondary, has led to the use of a variety of combination therapies and, at least temporary, improvements in symptoms. This appears to have delayed the rate at which patients are presenting for transplantation [6]. The impact of medical therapy on patient survival is still under study [7–9], although a few studies have suggested a survival benefit for both prostanoids and endothelin receptor antagonists [10]. Thus, lung or heart–lung transplantation remains a final therapeutic option for these patients. This chapter will discuss selection of appropriate transplant candidates, various peritransplant issues, survival, intermediate and long-term followup, complications, and long-term function and quality of life.

2 Role of Transplantation in Management of Pulmonary Hypertension As noted already, lung or heart–lung transplantation is rarely, if ever, a first-line treatment for either idiopathic pulmonary arterial or secondary pulmonary hypertension as most patients respond for a variable period of time to one or more of the available medical therapies. Patients who have New York Heart Association (NYHA) class II or class III symptoms are frequently started on oral therapy. A small number of patients – less than 20% – will respond to traditional therapy with calcium channel blockers and may have prolonged survival without need for lung transplantation [9]. Those who do not respond to this approach are started on endothelin receptor antagonists or less frequently on a phosphodiesterase inhibitor; those with NYHA class IV symptoms may start therapy on a prostanoid, typically intravenously administered epoprostenol or subcutaneously administered treprostinil [9]. Patients who worsen on the initial oral drug therapy are usually given a trial of combination therapy with the addition of a phosphodiesterase inhibitor or endothelin

J.R. Maurer

receptor antagonist [11] or a more easily administered prostanoid such as iloprost. Combination therapy may also be tried in patients with severe symptoms to increase the likelihood of response. These therapies are used in both idiopathic pulmonary arterial hypertension and in secondary disease such as that found in scleroderma or other connective tissue diseases, congenital heart disease, or liver disease [12–14]. Pediatric patients with idiopathic pulmonary arterial hypertension are approached in much the same way as adults [15]. The availability of oral agents has greatly simplified treatment in this age group and appears to be effective at least over the medium term [16]. Medical management for secondary pulmonary hypertension is also used effectively in diseases such as congenital cardiac defects and sickle cell [17, 18]. Despite initial symptom relief, however, a number of these children will ultimately deteriorate and present for transplantation [6].

3 Selection of Patients for Transplantation A number of factors must be considered in selecting patients for transplantation. These include not only the characteristics and progression of the underlying disease, but also the general medical condition of the patient. This latter aspect is very important because of the extensive immunosuppressive regimens that are required to maintain the lung or heart–lung grafts. These regimens include multiple drugs with side effects potentially detrimental to the patient’s overall health. In July 2006, the Pulmonary Scientific Council of the International Society for Heart and Lung Transplantation published a consensus report updating the selection guidelines for lung transplant recipients [19]. These are generally followed by all lung transplant programs. The guidelines emphasize that, given the limited donor pool and the importance of patient quality of life, only patients with a realistic chance of long-term outcomes should be offered the transplant option. Heart–lung transplant candidates must adhere to these guidelines as well; however, potential cardiac contraindications to isolated lung transplantation – unless those contraindications represent a systemic illness – do not apply.

3.1 General Medical Concerns The following absolute contraindications are listed in the guidelines: • Malignancy, except for common skin cancers, within the past 2 years. A 5-year interval from any other type of cancer to potential transplantation is considered prudent.

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• Untreatable, advanced dysfunction of another major organ system. This would include coronary artery disease or disease not amenable to surgical intervention or left ventricular dysfunction unless the patient is being considered for heart–lung transplantation. • Incurable, chronic extrapulmonary infection. Examples include chronic active viral hepatitis and human immunodeficiency virus. • Significant chest wall or spinal deformity. • Documented nonadherence or inability to follow through with medical therapy or office visits. • Untreatable psychiatric or psychologic issues associated with inability to comply with medical therapy. • Absence of a consistent or reliable social support system. • Substance addiction, e.g. alcohol, tobacco, or narcotics, within the previous 6 months. In addition to the absolute contraindications noted above, the following relative contraindications are also noted: • Age greater than 65 years. Although the guidelines do not support an absolute upper age limit, they note that the outcomes for patients in the upper age range are poorer than for younger patients. Of particular concern is older age combined with other relative contraindications [19]. • Critical or unstable clinical condition (e.g. shock), acutely ill on mechanical ventilation, extracorporeal membrane oxygenation. • Severely limited functional capacity with limited rehabilitation potential. • Colonization with highly resistant or highly virulent organisms. • Severe obesity defined as a body mass index of 30 kg/m2 or greater. • Severe or symptomatic osteoporosis. • Mechanical ventilation. This is limited to selected candidates who are able to participate in a meaningful rehabilitation program. • Other medical illnesses but without end-organ damage (e.g. diabetes). Patients with coronary artery disease are acceptable if they have disease amenable to intervention either prior to or concurrent with lung transplantation. Coronary artery disease, of course, is not a contraindication to heart–lung transplantation.

3.2 Disease-Specific Selection Criteria It is important to think of referring patients for consideration for transplantation well before they are ready for transplantation. There are several reasons for this. First, the patient can learn about all the pros and cons of transplantation and the detailed medical care that is associated with good outcomes.

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This helps them make a choice, in a nonpressurized environment, about whether they want to eventually go down this path. Second, it allows the transplant team to closely follow the patient and to make an appropriate decision about when the patient should formally be listed for transplantation. Other decisions, such as what type of graft should be used and whether comorbidities can be adequately managed, can also be made in a carefully considered way and with time to achieve optimal results. The guidelines recommend referral of patients with pulmonary arterial hypertension who (1) are NYHA functional class III or IV whether or not they are receiving ongoing medical therapy or (2) have rapidly progressive disease. Features that have been shown to be particularly predictive of survival in advanced-stage patients are functional status as measured by 6-min walk distance [20] and hemodynamic status as measured by cardiac index and right atrial pressures [4, 21]. On the basis of those published studies, the guidelines suggest that patients should be formally listed for transplantation when they meet the following criteria: • Persistent NYHA functional class III or IV level while on maximal medical therapy • A 6-min walk distance of less than 350 m or a declining 6-min walk distance • Failing therapy on intravenously administered epoprostenol or equivalent therapy • Cardiac index of less than 2 l/min/m2 • Right atrial pressure greater than 15 mmHg Presumably the functional capacity and hemodynamic values must meet the threshold values while the patient is on optimal medical therapy.

3.3 Selection Criteria for Patients with Secondary Pulmonary Hypertension The bulk of the published literature that gives prognostic data for patients with pulmonary hypertension reports on patients with idiopathic pulmonary arterial hypertension. Secondary pulmonary hypertension occurs in many other diseases, including other primary pulmonary disorders such as idiopathic pulmonary fibrosis, pulmonary Langerhans cell histiocytosis, and sarcoidosis, autoimmune diseases, and congenital heart diseases. Recent studies have reported that in both pulmonary fibrosis [22] and sarcoidosis [23], the finding of significant pulmonary hypertension has a negative impact on prognosis. Thus, patients with these diseases who have pulmonary hypertension should probably be preferentially considered for transplantation. Most of the data accumulated on patients with autoimmune or collagen vascular disease and pulmonary hypertension

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have been in scleroderma patients. This group of patients is routinely treated with medical therapy for their pulmonary hypertension. Unfortunately, these patients have a poorer prognosis even when treated with epoprostenol [24, 25]. From 1995 through June 2007, 153 collagen vascular disease patients were reported to the International Society for Heart and Lung Transplantation Registry [6]. This represented 0.8% of all transplant recipients reported during that period to the registry. Another, smaller group of patients are those with chronic thromboembolic pulmonary hypertension that do not have disease amenable to pulmonary thromboendarterectomy. Generally, similar referral and listing criteria are used for all of these groups of patients as for patients with idiopathic pulmonary arterial hypertension. Occasional patients with portopulmonary hypertension have undergone either combined lung–liver transplantation or sequential liver and lung transplantation [26]. No specific criteria have been published to identify appropriate candidates; however, presumably candidates would need to meet the criteria for each individual organ. In addition, moderate pulmonary hypertension has been reported to respond to liver transplantation alone. Patients who develop pulmonary arterial hypertension (Eisenmenger syndrome) secondary to congenital cardiac abnormalities (shunts) appear to have a somewhat different clinical course from those who develop disease related to primary pulmonary conditions. Despite typical severe hypoxemia unresponsive to supplemental oxygen, these patients historically have a higher survival while awaiting transplantation than patients with idiopathic pulmonary arterial hypertension. Hemodynamic variables, in particular, tend to be different from those of patients with primary disease. Hopkins et al. reported that despite having similar or slightly higher pulmonary artery pressures, patients with Eisenmenger syndrome tend to have higher cardiac indices and lower mean right atrial pressures [27]. Thus, it is probably important to perform serial hemodynamic measurements – with attention paid to falling cardiac indices and rising right atrial pressures – to identify the appropriate time to list these patients for transplantation. Patients with pulmonary veno-occlusive disease have rarely had transplantations [28]. These patients are not amenable to medical therapies and should probably be referred for transplantation as soon as their condition is diagnosed.

3.4 Selection Criteria for Pediatric Patients Primary and secondary pulmonary arterial hypertension is second only to cystic fibrosis as an indication for lung or heart–lung transplantation in the pediatric age group. Specific selection criteria have been difficult to define [29] in part

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because of the small numbers of patients involved and improved survival rates in recent years because of the good res­ ponse to prostanoids and other medical treatment advances [30]. In general, pediatric patients are listed for transplantation when they experience progressive functional deterioration in spite of medical therapy. Specific hemodynamic and functional parameters that correlate with poor prognosis while the patient is on maximal medical therapy have not been published. In a recent study, B-type natriuretic peptide (BNP) levels were measured in 50 children, about half of whom had primary and half secondary pulmonary hypertension. They were followed for a mean of 14 ± 7.5 months, during which 14 patients either died or required transplantation. The authors found that a BNP level of more than 130 pg/ml predicted a poor prognosis; however, the sensitivity was only 57%, so more study is required to determine the value of this marker [31].

4 Transplantation Options for Pulmonary Hypertension Patients As noted in Sect. 118.1, the initial transplantation approach for patients with pulmonary hypertension was heart–lung transplantation. This type of transplantation is rarely done now unless a patient has not only advanced lung disease, but also advanced cardiac disease that is not amenable to surgical intervention [6]. As soon as isolated lung transplantations became feasible and were shown to be a reasonable approach for these patients, they became the preferred type of lung graft. Heart–lung grafts were largely abandoned because separating the heart and lungs allowed more than one candidate to receive an organ from a single donor. Both unilateral and bilateral grafts have been successfully used for idiopathic primary arterial pulmonary hypertension and for secondary pulmonary hypertension; however, the number of bilateral grafts greatly exceed the number of unilateral grafts. In the most recently published report of the International Society for Heart and Lung Transplantation International Registry, between January 1995 and June 2007, bilateral transplantations were reported in 622 patients compared with unilateral transplantations in 67 patients. In disease secondary to Eisenmenger syndrome, there were 17 unilateral transplantations and 138 bilateral grafts [6]. There are several reasons for the dominance of bilateral grafts. First, many transplant teams have felt that the functional capacity conferred by two lungs, particularly if chronic rejection (or bronchiolitis obliterans) were to develop, would allow a longer survival and higher quality of life. In pulmonary hypertension, however, historically there was another important perceived reason. Transplant teams were concerned that it would be difficult for an already severely impaired right

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side of the heart (almost universal in severe pulmonary hypertension) to function if the entire blood flow were forced through a single lung [32]. In fact, pulmonary hypertensive transplant recipients did and do tend to have very stormy early perioperative courses characterized by rapid onset, but fluctuating, pulmonary infiltrates more consistent with transient pulmonary edema than primary graft failure. However, these perioperative infiltrates were reported in both unilateral and bilateral grafts, thus not supporting the theory about too much blood flow through a single lung [33]. In the more recent era, the peritransplant course of these patients is more easily managed with careful attention to fluid administration, use of nitric oxide, prostanoids, and other agents to control fluctuating pulmonary pressures, and early support of right ventricular decompensation [34]. Living lobar transplantations are, in some circumstances, also an option for patients with pulmonary hypertension. Typically, living lobar donor transplantations consist of the transplantation of a left and right lower lobe from each of two compatible donors. Selection criteria usually include compatible blood type, size and age of the donor, and a donor with no chronic illness. Although pioneered in the USA, living lobar transplantation is the most common type of lung transplantation in Japan, where centers have focused on candidates with pulmonary hypertension. Because of issues around brain death, there are very few cadaveric donors in Japan, and this has forced transplant teams to use living-donor lobar transplantations. A Japanese group recently reported a series of 11 living-donor lobar transplantations performed in patients with pulmonary hypertension. Remarkably, all 11 were alive at 5 years [35]. The only registry dedicated to living-donor lung transplantation is a Japanese registry which first reported on 87 living-donor transplantations early in 2008 [36]. Combined organ transplantations, particularly combined liver and lung transplantation, have been reported in cystic fibrosis patients and in patients with portopulmonary hypertension. These patients are relatively uncommon, however, and criteria to determine whether a patient can do well with a liver graft alone as opposed to a liver–lung transplantation are not clear.

4.1 Pediatric Transplantation Options Heart–lung, unilateral lung, bilateral lung, and living-donor lobar lung transplantations are all options for pediatric patients with pulmonary hypertension. As with adult patients, bilateral lung transplantations are much more common [6]. In children, especially in the USA, the principal indications for heart–lung transplantation are primary and secondary hypertension, whereas in Europe cystic fibrosis is

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the ­principal indication. Heart–lung ­transplantations are typically reserved for those patients with Eisenmenger ­syndrome and uncorrectable congenital heart disease. However, since 2001 fewer than 15 pediatric heart–lung transplantations per year have been reported to the registry compared with between 58 and 83 isolated lung transplantations per year [6]. Most pediatric transplantations are performed in the adolescent age group and the fewest are performed in infants.

5 Donor Organ Allocation: The Lung Algorithm Prior to 2005, lung grafts were allocated to candidates on the basis of compatible blood group, size, and waiting time. Essentially, those who had waited the longest received the grafts regardless of whether someone else on the list was more likely to die sooner without a transplantation. In 1998, the US Department of Health and Human Services published the Organ Procurement and Transplantation Network final rule that required organ allocation policies to meet several rules. Among those rules was a requirement to place more emphasis on medical criteria and urgency in organ distribution and less on waiting time [37]. This ruling led to the creation of an organ allocation model based on data from various sources that provided information on historical waiting-list survival and posttransplant survival. The model incorporates the concept of “net transplant benefit” that essentially predicts the additional survival the patient might expect if he/ she has a transplantation compared with if he/she did not receive a transplantation. The model includes patient demographics, diagnosis, percent predicted forced vital capacity (FVC), functional status, whether diabetes is present and diabetic medication requirements, gas exchange including amounts of supplemental oxygen and whether assisted ventilation is used, pulmonary artery and capillary wedge pressures, and serum creatinine level [38]. These values are entered into the model and a score, the lung allocation score, is generated. The higher the score, the higher the patient is placed on the waiting list. This system is used for patients older than 12 years and scores must be updated every 6 months by the listing center. A particular patient’s place in line may be always changing depending on new patients coming on the list or the recalculation of scores of patients already on the list or the recalculation of the patient’s own score. Since the allocation system was implemented in May 2005, several papers have documented a shift in the diagnoses of patients who have undergone transplantation. Generally, patients with idiopathic pulmonary fibrosis have more often been recipients of organs and those with emphysema and ­cystic fibrosis have been less often been recipients compared

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with the old system [39, 40]. A significant change in the number of pulmonary hypertensive patients achieving scores high enough to receive grafts has not been documented.

6 Survival Following Lung Transplantation 6.1 Heart–Lung Transplantation: Adult Although fewer than 50 heart–lung transplantations a year are now reported to the International Registry, the bulk of those continue to be performed for idiopathic pulmonary arterial hypertension and secondary pulmonary hypertension secondary to congenital heart disease. The survival curve of all adult heart–lung patients reported to the International Registry between January 1982 and June 2006 shows that only 50% of patients survive longer than 3.5 years. Most of the deaths occur in the first year after transplantation and particularly in the perioperative period. Thus, the conditional half-life (50% survival of those who survive the first year) is 9.1 years [6]. This survival has somewhat improved in the modern era. Since 2000, the 50% overall survival has improved to about 6 years. This predominantly reflects improved perioperative survival rates. For the idiopathic pulmonary arterial hypertensive patients and Eisenmenger syndrome patients, the conditional half-life of patients who received transplantations between January 1990 and June 2006 is somewhat better at 9.7 and 10.6 years, respectively; the overall half-life is 4.3 and 5.8 years, respectively. Early deaths are attributed primarily to infection, graft failure, and technical issues and late deaths are attributed to chronic rejection, graft failure, and chronic rejection [6]. A recent single-center report of 46 heart–lung and five bilateral lung transplantations in what the authors termed “grown up congenital heart disease” reported 30 mortality rates of only 11.8% and longer term survivals of 80% at 1 year, 69% at 5 years, and 53% at 10 years. These were similar to survival rates of patients with other indications at their center [41]. This suggests that particular centers with very experienced teams may be able to achieve somewhat better survivals.

6.2 Heart–Lung Transplantation: Pediatric The proportion of pediatric patients receiving heart–lung grafts for either primary or secondary pulmonary hypertension compared with other indications is greater in the USA than in Europe and other areas of the world. Survival of

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p­ ediatric patients reported to the International Registry and who received transplantations between January 1990 and June 2006 shows a half-life for primary pulmonary hypertensive patients of 3.3 years and for Eisenmenger syndrome patients of 2.6 years. The conditional half-life of these patients, however, is significantly better – more than twice the unconditional values. This, again, is because of perioperative survival issues. When looked at by era, there is minor improvement in survival rates over time, but there continues to be a high perioperative death rate. Early perioperative mortality is particularly high for infants [6].

6.3 Unilateral Lung Transplantation: Adult In general, the overall and conditional half-life of unilateral transplantations is less than that of bilateral transplantations. For unilateral transplantation, overall survival is 4.5 years compared with 6.2 years for bilateral transplantation and conditional survival is 6.4 years compared with 8.8 years [6]. Between January 1995 and June 2007, only 84 patients (less than 1%) were reported as having undergone unilateral transplantations for either primary or secondary pulmonary hypertension [6]. A few other patients with connective tissue disease or other pulmonary processes received a unilateral transplantation because of pulmonary hypertension or had concomitant pulmonary hypertension, but the data on these patients are not separated out in formal registry reports. However, a study of these patients using data from the registry concluded that the presence of secondary pulmonary hypertension did not negatively impact outcome in unilateral transplantations [42]. It is of interest that in the early 1990s, the bulk of transplantations performed in idiopathic pulmonary arterial hypertension patients were unilateral transplantations, but this mix shifted in the mid-1990s, and since 1995 an average 90% or more of grafts are bilateral [6]. The International Registry reports the survival rate for the unilateral recipients to be about 40% at 5 years and 25% at 10 years.

6.4 Unilateral Lung Transplantation: Pediatric Less than 10% of pediatric transplantations reported have been unilateral transplantations. Unilateral pediatric transplantations have poorer survival than bilateral grafts, with a half-life of 2.2 years compared with 4.6 years, and data for unilateral grafts for specific diagnoses are not reported by the International Registry [6]. Survival of isolated lungs overall will be discussed in Sect. 118.?.

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6.5 Bilateral Lung Transplantation: Adult Although bilateral lung transplantation is the most common type of transplantation for pulmonary hypertensive patients, between January 1995 and June 2007, these patients made up only 7% (n = 760) of the 10,775 bilateral transplantations reported to the International Registry [6]. Overall survival of these patients up to 5 years is exceeded slightly by that of a1-antitrypsin-deficient recipients and cystic fibrosis recipients. The bulk of the difference in survival occurs in the first 3 months after transplantation [6]. The conditional half-life (for those patients surviving the first 3 months) is 8.3 years for idiopathic pulmonary arterial hypertension compared with 7.9 years for cystic fibrosis, 5.6 years for COPD, and 5.4 years for idiopathic pulmonary fibrosis [6]. Overall, the 5-year survival for bilateral recipients with primary disease is 48.5%. The half-life for Eisenmenger syndrome patients varies depending on whether the cardiac disorder is an atrial septal defect (ASD) or a ventricular septal defect (VSD). For patients reported between January 1990 and June 2006, the ASD patients (n = 100) had a half-life of 3.9 years and the VSD patients (n = 56) had a half-life of 1.1 years [6].

6.6 Bilateral Lung Transplantation: Pediatric Although pulmonary hypertension is the second most common presenting condition for bilateral transplantation in children, it accounts for a small minority of procedures, with the bulk of procedures performed for cystic fibrosis. Thus, survival is generally not separated out by diagnosis, with the exception of congenital disease (see later). Survival rates are generally age-related. Interestingly, in isolated bilateral transplantations, infants have the highest half-life (6.4 years) but also the highest perioperative mortality. The half-life of other age groups is 5.9 years for ages 1–11 years and 4 years for ages 12–17 years. The conditional half-life (for those patients surviving 1 year) even more dramatically points out the early mortality of infants. This half-life is 12.1 years for infants, 10.5 years for ages 1–11 years, and 6.1 years for ages 12–17 years. Going forward, the half-life is likely to be significantly improved since early survival rates in the era from 2002 through June 2006 were improved over those of previous eras [6]. Congenital diagnosis survival rates are reported separately. Patients with Eisenmenger syndrome compared with patients with other congenital heart diseases appear to have a lower perioperative survival. However, the numbers are small

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and the combined 5-year survival rates appear to be slightly higher than 50% [6].

6.7 Unilateral and Bilateral Transplantation in Pulmonary Disease with Secondary Pulmonary Hypertension The International Registry does not directly address the impact of pulmonary hypertension in end-stage pulmonary parenchymal disease on transplant outcomes. A recent single-center report compared the outcomes of 45 patients with secondary pulmonary hypertension with those of 42 patients who did not have secondary pulmonary hypertension. There were bilateral and unilateral graft recipients in both groups. Although an increased rate of reperfusion injury in the patients with secondary pulmonary hypertension was noted, no difference in outcomes at 4 years between the two groups was found [42].

6.8 Living-Donor Lobar Transplantation Living-donor lobar transplantation was initially performed primarily in patients with cystic fibrosis but later was applied to a few patients with pulmonary hypertension from the group that pioneered this approach at the University of Southern California [43, 44]. The pulmonary hypertension patients in this group were reported to have good intermediate outcomes. More recently (see earlier), large numbers of living lobar transplantations have been reported by the Japanese [36]. The principal indication for living lobar transplantation in this registry is primary pulmonary hypertension. It reports a 9-year experience, but for most patients the follow-up was relatively short. Nevertheless, survival rates appear to be at least a good as those for cadaveric donors in both pediatric and adult recipients. Longer-term data will need to be accumulated to better define the role of livingdonor transplantation in pulmonary hypertensive patients.

6.9 Liver–Lung Transplantation A small number of patients with portopulmonary hypertension have undergone combined transplantation. A recent report of 13 cases of combined liver–lung transplantation reports survival rates of 69%, 62%, and 49% at 1, 3, and 5 years, respectively [26].

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7 Early Postoperative Issues There are a number of early postoperative issues that have been reported in pulmonary hypertensive as well as other types of lung transplant recipients. In addition to the early hemodynamic/pulmonary edema/reperfusion injury (mentioned earlier) that may be more common in patients with elevated pulmonary pressures [45], anastomotic venous thrombosis [46] and airway complications [47] may occur. These latter two issues appear to have decreased in incidence and/or morbidity as transplant centers have become more sophisticated both in technique and in early detection and management when these issues do occur. Airway complications are now reported in 10–15% of patients and range from complete dehiscence (rare), which may result in death, to mild to moderate stenosis [47]. Recently identified risk factors include prolonged donor ventilation, tall recipients, and telescoping anastomosis [48]. Mild to moderate stenosis may require no treatment or dilation alone; severer stenosis often requires repeated dilation and stenting [48, 49]. Stents typically remain in place indefinitely and may themselves be complicated by ingrowth of granulation tissue or a predisposition to airway colonization by fungi. Atrial fibrillation that occurs within a few days of transplantation has been reported in a higher percentage of lung recipients with pulmonary hypertension than in the overall lung transplant population [50]. The atrial fibrillation can be difficult to control and may require multiple therapeutic modalities.

8 Acute and Chronic Rejection Immunosuppression may be initiated with antilymphocytic drugs – IL-2 receptor blockers, monoclonal antibodies directed against cell-surface markers, or less selective antilymphocyte/antithymocyte preparations. Controversy remains about the best approach in terms of the type of induction to use or even if induction is necessary [6, 51–53]. Maintenance immunosuppression most typically includes three drugs (at least initially) that include a calcineurin inhibitor, either azathioprine or mycophenolate mofetil, and a steroid. The exact combination of drugs differs among centers and in some centers use of steroids is discontinued after a period of time. Recently, studies have suggested that tacrolimus may be the preferred calcineurin inhibitor, although a number of centers continue to use cyclosporine preparations [6, 54, 55]. Acute rejection, which is characterized by perivascular and interstitial mononuclear infiltrates, is graded from 0 to 4 depending on the severity of the infiltrates [56]. When acute rejection is present, notation is also made of the grade of airway inflammation which is often also present. Diagnosis of acute ­rejection is typically made by transbronchial biopsy and

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occurs most often in the first 3 months after transplantation. From approximately 40 to 60% of patients will experience at least one episode of acute rejection [57]. Although untreated acute rejection can destroy a graft, the signs and symptoms are easily recognized by experienced transplant physicians and it is rare for a patient to lose his/her graft to this process. Treatment is focused on bolus steroids that may be followed by enhanced maintenance immunosuppression; for the 15% or so of patients who experience multiple recurrences of acute rejection or persistent acute rejection, more aggressive immunosuppression may be used. In fact, the numbers, severity, and timing of acute rejection episodes that a patient experiences are the most predictive risk factors for the development of chronic rejection or bronchiolitis obliterans [called bronchiolitis obliterans syndrome (BOS) when compatible pulmonary function abnormalities exist in the absence of definitive pathological diagnosis]. Lymphocytic bronchiolitis and bronchitis are also significant risk factors as are some common viral infections; however, the evidence implicating cytomegalovirus (CMV) infections (except for invasive disease) and HLA mismatching is less impressive [58–60]. Recently, two other risk factors have been identified and are being further ­studied: primary graft dysfunction and gastroesophageal reflux ­disease (GERD) with aspiration [61, 62]. BOS is characterized by a progressive fall in the forced expiratory volume in the first second (FEV1) on pulmonary function studies ultimately resulting in a loss of functional capacity and, in some patients, death. Pathologically, the airway is progressively and circatricially obliterated with scar tissue secondary to a chronic inflammatory process. There is no evidence that the pretransplant diagnosis significantly influences the development of BOS. If, however, preoperatively a patient has a specific risk factor, e.g. GERD, he/she may be at higher risk of BOS. BOS is graded as a percent loss in function from the highest FEV1 obtained after transplantation as determined by at least two measurements obtained at least 3 weeks apart. Impending BOS (stage 0) is identified by a sustained fall in FEV1 of up to 20% or in midexpiratory flows of at least 25% from the baseline. Successive stages are defined as follows: stage 1 – fall in FEV1 to between 66 and 80% of the baseline; stage 2 – fall in FEV1 to between 51 and 65% of the baseline; and stage 3 – fall in FEV1 to less than 50% of the baseline [63]. From the time of transplantation onward, BOS affects a progressive number of lung and heart–lung transplantations. By 5 years after transplantation, at least 50% of patients have developed some degree of BOS. At 10 years after transplantation, this number approaches 70% [6]. It is the primary intermediate and late-term cause of death [6]. Research has focused on multiple biomarkers and other approaches such as early radiological changes that would signal impending BOS. Finding signs or markers of early inflammatory changes

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is critical since by the time initial pulmonary function changes occur, the patient already has relatively late disease that is poorly responsive to treatment [63]. Unfortunately, to date no reliable, i.e. sensitive and specific, early diagnostic methods have been discovered. Approaches to treatment have also been disappointing. Early-era approaches consisted primarily of enhancing immunosuppression, which, paradoxically, often increased the susceptibility of patients to opportunistic infection without significant improvement in pulmonary function. More recently a new class of immunosuppressive drugs, mammalian target of rapamycin inhibitors (e.g. sirolimus, everolimus) and macrolide antibiotics have been tried with limited success in maintaining and improving pulmonary function once BOS is present and are being studied as a prophylactic approach [64, 65]. BOS occurs in pediatric patients at approximately the same rate as in adults and is a primary cause of death in those who survive the early posttransplant period [6]. Approaches to management are similar to those used in adults.

9 Infection and Malignancy in Transplant Recipients From the immediate posttransplant period onward, transplant recipients are at risk of both opportunistic and more garden variety infections. To prevent early infectious deaths, patients usually receive antibiotic prophylaxis at least during the most intense period of immunosuppression. Prophylaxis against Pneumocystis jiroveci with sulfonamide–trimethoprim combinations is virtually universal and usually continued indefinitely. Antibiotic prophylaxis against primary or recurrent CMV infection is also often used [66]. Another approach in patients who are at moderate risk of developing CMV disease is to follow prospectively the levels of CMV antigen in the blood or the number of viral copies (viremia) and treat the patient only when threshold levels are reached. To date, the risk of CMV disease and disease complications is similar with prophylaxis or preemptive monitoring [67]. In patients at high risk of infection and invasive disease – those who have not previously been exposed to CMV (antibody-negative) but who receive a graft that has been exposed to CMV (antibody-positive) – anti-CMV immunoglobulin might also be given, although this approach is somewhat controversial [68, 69]. Early postoperative prophylaxis may also include antibacterial and antifungal antibiotics especially if the patient is colonized preoperatively. Another indication for fungal prophylaxis is the presence of a perioperative airway problem as the presence of a foreign body such as a stent or a poor healing area has been reported to frequently result in airway colonization or even bronchitis [70].

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With these aggressive approaches to prophylaxis, death rates from CMV and P. jiroveci have fallen dramatically, yet infection – often from unusual and aggressive opportunistic fungal, viral, bacterial, and mycobacterial organisms [71] – remains a major cause of death particularly in the first year after transplantation [6]. When BOS develops, patients again become very susceptible to infections, particularly pseudomonas infections, which are often present in patients who die from this complication [72]. Immunosuppression also predisposes to malignancies. The most common malignancies seen after transplantation are skin cancers, but much more deadly are posttransplant lymphoproliferative disorders (PTLD) and a variety of other cancers [6]. For most of these malignancies, the risk increases with increased time from transplantation; however, PTLD occurs primarily in the first year after transplantation and affects less than 5% of adult patients [6]. The risk for PTLD is generally related to the replication of Epstein–Barr virus. To monitor patients for the level of risk, the Epstein–Barr DNA viral load can be followed [73]. Despite early detection of increased Epstein–Barr replications, however, definitive management of resulting lymphoproliferative processes has been somewhat disappointing and preemptive therapies have been lacking. Traditional chemotherapy is usually reserved for late-occurring disease (at least 1 year after transplantation) primarily in adults and that is less likely related to Epstein–Barr virus. The first line of treatment for other patients with Epstein–Barr virus related disease is usually some reduction in immunosuppression and rituximab. Median survival with this approach, however, is limited [74]. PTLD is a particular risk in pediatric patients. As many as 10–15% of pediatric lung or heart–lung recipients will develop PTLD – at least twice the rate reported in the adult population [6]. Although most patients are Epstein–Barr virus negative and receive a seropositive graft, PTLD also occurs in seropositive patients [75]. Management and survival rates are similar to those of adult patients.

10 Intermediate and Long-Term Medical Complications On one hand, immunosuppressive drugs are life-saving, but on the other they – particularly the calcineurin inhibitors – have toxic effects on practically every other organ system. Probably the most important of these is their impact on renal function. Virtually every patient on calcineurin inhibitors experiences a significant decline in renal function that begins to occur very early after transplantation. By 5 years after transplantation, almost 40% of patients have abnormal creatinine levels and almost 4% are on dialysis or have had renal

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transplantations [6]. Correlated with the diminished renal function is hypertension recorded in more than 85% of patients at 5 years and nearly 100% of patients at 10 years [6]. A large single-center study reported more dismal results, with 7.3% of patients developing end-stage renal disease. This same study noted that aggressive control of hypertension and use of tacrolimus instead of cyclosporine were renoprotective and tended to increase the time to end-stage disease [76]. Pediatric patients also experience renal impairment, but to a lesser degree. By 5 years about 23% have abnormal creatinine levels and about 65% have hypertension; by 7 years, 36% have abnormal creatinine levels and 75% have hypertension [6]. New onset of diabetes is also a common complication. Two immunosuppressive agents, corticosteroids and tacrolimus, are particularly prone to result in new onset of glucose intolerance. Other contributing factors are likely obesity and possibly the use of sirolimus [77]. The impact of tacrolimus can be seen by comparing the rates of new-onset diabetes to the pre-2000 transplant era, when tacrolimus was used in fewer patients than in the post-2000 era: the International Registry reports that by 1 year after transplantation, the rates were 17.1 and 30.6%, respectively, in those eras [6]. By 5 years after transplantation, 35.5% of lung transplant recipients have diabetes. The rates in pediatric patients mirror those of adults. The alarming rate of diabetes in lung and heart–lung transplant recipients (and other solid organ transplants) has been addressed with a guideline for management that recommends relatively stringent management similar to that recommended for nontransplant patients with diabetes [78]. Hyperlipidemia is also a common complication of immunosuppressive agents occurring in more than half of adult patients by 5 years [6]. This effect of immunosuppressives, however, has been reported in less than 10% of pediatric patients [6]. Aggressive management of hyperlipidemia is particularly important in patients with diabetes as hyperlipidemic diabetics are at high risk for cardiovascular events. Osteoporosis has been reported as frequent in patients with a variety of end-stage diseases coming to transplantation including patients with idiopathic pulmonary arterial hypertension [79–81]. Following transplantation, ­particularly in the first 6 months after transplantation, recipients are reported to have a dramatic fall in bone mineral density and to be at risk for fracture regardless of preoperative bone status [82]. This dramatic loss in bone mineral is thought to be related to both corticosteroid and calcineurin inhibitor use and probably is exacerbated by the relative inactivity following transplant surgery. Bisphosphonates are typically used for treatment/prophylaxis which is often initiated in highrisk patients preoperatively [83, 84]. Venous thromboembolic disease has been reported in 12–29% of lung transplant patients [85]. Most episodes occur within the first year and 20% occur within the first

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month, although a significant number also occur between 2 and 5 years after transplantation. A significant risk factor for early venous thromboembolic disease is use of cardiopulmonary bypass [85]. Intra-abdominal complications are reported in approximately 20% of lung transplant recipients [86]. These complications can include a wide variety of illnesses from acute cholecystitis to appendicitis to diverticulitis and many require surgical intervention. Unfortunately surgical intervention is often delayed because immunosuppressed patients often present with subtler symptoms than are typically seen in patients with surgical abdomens. These events can occur early or late after transplantation and those who are rapidly identified for surgical intervention have better survival. Impaired gastric motility and GERD have been identified as very common in both adult and pediatric patients and GERD has been identified as a significant risk for bronchiolitis obliterans [62, 87, 88]. Aggressive treatment, including Nissen fundoplication in both populations, has been advocated to reduce the risk of BOS [89]. Neurological complications are often neglected, but can be very varied and are not rare in lung transplant recipients. They are primarily related to calcineurin inhibitors and can vary from severe headache to seizures to cognitive dysfunction to strokelike syndromes to peripheral neuropathy. Central nervous system imaging typically reveals diffuse white matter changes [90, 91].

11 Pregnancy in Posttransplant Patients A number of pregnancies have been reported in thoracic organ transplant recipients. Many of the outcomes are successful, but the women are at high risk for infection, hypertension, preeclampsia, preterm delivery, and low-birth-weight infants. The risk of rejection has been reported as significant and it is important to closely monitor immunosuppressive levels [92]. The impact of pregnancy on patient survival is not clear; however, patients considering pregnancy should have a thorough understanding of their predicted survival and the medical ­complications and limitations of long-term survivors. A National Transplantation Pregnancy Registry is kept at Thomas Jefferson University in Philadelphia (PA, USA). Any pregnancy and outcome should be reported to the registry [93].

12 Functional Outcomes and Quality of Life Patients who do not experience significant pulmonary complications perioperatively typically reach maximal lung function in the first year after transplantation [62].

118  Results of Lung Transplantation

Single lung transplant recipients achieve FVCs of 50–60% of those predicted and bilateral transplant recipients achieve essentially normal or near normal predicted FVCs [94]. Heart–lung transplant recipients typically only achieve a FVC of about 60% of that predicted and have similarly limited maximal exercise capacity [95]. Bilateral living-donor lobar transplant recipients who survive at least 3 months have been reported to have intermediate and long-term pulmonary function and exercise tolerance similar to those of bilateral transplant recipients [96]. Pediatric living-donor transplant recipients are reported to have similar outcomes to those of adults [97]. Functionally, both single and bilateral lung transplant recipients have similar 6-min walk distances and achieve similarly on graded exercise testing, achieving between 40 and 60% of predicted values [94, 98]. A number of theories have been proposed for the poorer than normal exercise performance particularly in bilateral lung and heart–lung recipients. These theories center on impaired muscle function, e.g. focusing on impaired mitochondrial function, possibly as the result of the immunosuppressive drugs, since these patients’ exercise capacity does not appear to be either cardiac- or pulmonary-limited [99]. However, recently a subgroup of idiopathic pulmonary arterial hypertensive patients who received bilateral lung transplantations have been shown to have elevated pulmonary artery pressures on exercise [100]. Despite their somewhat limited maximal exercise capacities, lung and heart–lung transplant recipients can function easily doing normal activities of daily living. Nevertheless, only about 30% ever return to full-time work [6]. A variety of quality-of-life reports have been published on lung transplant recipients. Several studies have been published by Vermeulen et al. from the Netherlands. In one study that surveyed patients surviving at least 55 months from transplantation, these researchers found that in the first 3.5 years after transplantation, patients improved on both functional scales and on scales measuring anxiety, depression, and well-being [101]. But as they got farther than that from transplantation, patients began to experience increasing anxiety, dyspnea, and a lower sense of well-being. This was especially true in patients who developed BOS, kidney dysfunction, insulin-dependent diabetes, and other medical complications. Other studies have documented that the onset and progression of BOS in particular is associated with both increased scores on depression scales and lowered quality of life in addition to the obvious functional decline [102, 103]. A small study (n = 28) of 10-year survivors of both single and bilateral transplantation using the SF-36 instrument showed that these patients had lower scores on physical function, role-physical/emotional, and general health – but not mental health and bodily pain – than the general population and a population of chronically ill patients. These 10-year

1621

survivors had a plethora of complications ranging from BOS to end-stage renal disease [104].

13  Summary Remarkable improvements in the medical management of pulmonary hypertension have improved quality of life in patients with both primary and secondary disease and have lengthened the time between diagnosis and the need for transplantation. Transplantation offers a therapeutic option to those patients who fail medical therapy or have become resistant to pharmacological treatment. The aggressive immunosuppressive regimens that result in many medical complications and the frequent development of chronic rejection or bronchiolitis obliterans despite these regimens significantly limit the long-term quality of life of surviving patients. Ongoing improvements in medical management approaches will hopefully obviate the need for transplantation in these patients.

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118  Results of Lung Transplantation after heart transplantation: a non-inferiority trial. J Heart Lung Transplant 26:258–263 53. Ailawadi G, Smith PW, Oka T et  al (2008) Effects of induction immunosuppression regimen on acute rejection, bronchiolitis obliterans, and survival after lung transplantation. J Thorac Cardiovasc Surg 135:194–206 54. Treede H, Klepetko W, Reichenspurner H et al (2001) Tacrolimus versus cyclosporine after lung transplantation: a prospective, open, randomized two-center trial comparing two different immunosuppressive protocols. J Heart Lung Transplant 20:511–517 55. Hachem RR, Yusen RD, Chakinala MM et al (2007) A randomized controlled trial of tacrolimus versus cyclosporine after lung transplantation. J Heart Lung Transplant 26:1012–1018 56. Stewart S, Fishbein MC, Snell GI et al (2007) Revision of the 1996 working formulation for the standardization of nomenclature in the diagnosis of lung rejection. J Heart Lung Transplant 26: 1229–1242 57. Zuckermann A, Reichenspurner H, Birsan T et  al (2003) Cyclosporine A versus tacrolimus in combination with mycophenolate mofetil and steroids as primary immunosuppression after lung transplantation: one-year results of a 2-center prospective randomized trial. J Thorac Cardiovasc Surg 125:891–900 58. Sharples LD, McNeil K, Stewart S, Wallwork J (2002) Risk factors for bronchiolitis obliterans: a systematic review of recent publications. J Heart Lung Transplant 21:271–281 59. Scott AI, Sharples LD, Stewart S (2005) Bronchiolitis obliterans syndrome: risk factors and therapeutic strategies. Drugs 65:761–771 60. Billings JL, Hertz MI, Savik K, Wendt CH (2002) Respiratory viruses and chronic rejection in lung transplant recipients. J Heart Lung Transplant 21:559–566 61. Daud SA, Yusen RD, Meyers BF et al (2007) Impact of immediate primary lung allograft dysfunction on bronchiolitis obliterans syndrome. Am J Respir Crit Care Med 175:207–213 62. D’Ovidio F, Mura M, Tsang M et al (2005) Bile acid aspiration and the development of bronchiolitis obliterans after lung transplantation. J Thorac Cardiovasc Surg 129:1144–1152 63. Estenne M, Maurer JR, Boehler A et al (2002) Bronchiolitis obliterans syndrome 2001: an update of the diagnostic criteria. J Heart Lung Transplant 21:297–310 64. Gottlieb J, Szangolies J, Koehnlein T et  al (2008) Long-term azithromycin for bronchiolitis obliterans syndrome after lung transplantation. Transplantation 85:36–41 65. Snell GI, Valentine VG, Vitulo P et al (2006) Everolimus versus azathioprine in maintenance lung transplant recipients: an international, randomized, double-blind clinical trial. Am J Transplant 6:169–177 66. Hodson EM, Craig JC, Strippoli GF, Webster AC (2008) Antiviral medications for preventing cytomegalovirus disease in solid organ transplant recipients. Cochrane Database Syst Rev 2:CD003774 67. Strippoli GF, Hodson EM, Jones C, Craig JC (2006) Preemptive treatment for cytomegalovirus viremia to prevent cytomegalovirus disease in solid organ transplant recipients. Transplantation 81:139–145 68. Hodson EM, Jones CA, Strippoli GF, Webster AC, Craig JC (2007) Immunoglobulins, vaccines or interferon for preventing cytomegalovirus disease in solid organ transplant recipients. Cochrane Database Syst Rev 2:CD005129 69. Bonaros N, Mayer B, Schachner T, Laufer G, Kocher A (2008) CMV-hyperimmune globulin for preventing cytomegalovirus infection and disease in solid organ transplant recipients: a metaanalysis. Clin Transplant 22:89–97 70. Dummer JS, Lazariashvilli N, Barnes J et al (2004) A survey of anti-fungal management in lung transplantation. J Heart Lung Transplant 23:1376–1381

1623 71. Valentine VG, Bonvillain RW, Gupta MR et al (2008) Infections in lung allograft recipients: ganciclovir era. J Heart Lung Transplant 27:528–535 72. Botha P, Archer L, Anderson RL, Lordan J et  al (2008) Pseudomonas aeruginosa colonization of the allograft after lung transplantation and the risk of bronchiolitis obliterans syndrome. Transplantation 85:771–774 73. Bakker NA, Verschuuren EA, Veeger NJ et al (2008) Quantification of Epstein-Barr virus-DNA load in lung transplant recipients: a comparison of plasma versus whole blood. J Heart Lung Transplant 27:7–10 74. Blaes AH, Peterson BA, Bartlett N et al (2005) Rituximab therapy is effective for posttransplant lymphoproliferative disorders after solid organ transplantation: results of a phase II trial. Cancer 104:1661–1667 75. Allen UD, Farkas G, Hébert D et al (2005) Risk factors for posttransplant lymphoproliferative disorder in pediatric patients: a case-control study. Pediatr Transplant 9:450–455 76. Ishani A, Erturk S, Hertz MI et al (2002) Predictors of renal function following lung or heart-lung transplantation. Kidney Int 61:2228–2234 77. Bodziak KA, Hricik DE (2009) New-onset diabetes mellitus after solid organ transplantation. Transpl Int 22(5):519–530 78. Wilkinson A, Davidson J, Dotta F et al (2005) Guidelines for the treatment and management of new-onset diabetes after transplantation. Clin Transplant 19:291–298 79. Jørgensen NR, Schwarz P, Holme I et al (2007) The prevalence of osteoporosis in patients with chronic obstructive pulmonary disease: a cross sectional study. Respir Med 101:177–185 80. Shane E, Silverberg SJ, Donovan D et al (1996) Osteoporosis in lung transplantation candidates with end-stage pulmonary disease. Am J Med 101:262–269 81. Tschopp O, Schmid C, Speich R et al (2006) Pretransplant bone disease in patients with primary pulmonary hypertension. Chest 129:1002–1008 82. Spira A, Gutierrez C, Chaparro C et al (2000) Osteoporosis and lung transplantation: a prospective study. Chest 117:476–481 83. Trombetti A, Gerbase MW, Spiliopoulos A et al (2000) Bone mineral density in lung-transplant recipients before and after graft: prevention of lumbar spine post-transplantation-accelerated bone loss by pamidronate. J Heart Lung Transplant 19:736–743 84. Cahill BC, O’Rourke MK, Parker S et  al (2001) Prevention of bone loss and fracture after lung transplantation: a pilot study. Transplantation 72:1251–1255 85. Kahan ES, Petersen G, Gaughan JP, Criner GJ (2007) High ­incidence of venous thromboembolic events in lung transplant recipients. J Heart Lung Transplant 26:339–344 86. Miller CB, Malaisrie SC, Patel J et al (2006) Intraabdominal complications after lung transplantation. J Am Coll Surg 203:653–660 87. Kasimir MT, Mereles D, Aigner C, Jaksch P, Benz A, Kreuscher S, Klepetko W, Grünig E, Young LR, Hadjiliadis D, Davis RD, Palmer SM (2003) Lung transplantation exacerbates gastroesophageal reflux disease. Chest 124:1689–1693 88. Benden C, Aurora P, Curry J et al (2005) High prevalence of gastroesophageal reflux in children after lung transplantation. Pediatr Pulmonol 40:68–71 89. Cantu E 3rd, Appel JZ 3rd, Hartwig MG et al (2004) J. Maxwell Chamberlain Memorial Paper. Early fundoplication prevents chronic allograft dysfunction in patients with gastroesophageal reflux disease. Ann Thorac Surg 78:1142–1151 90. Edwards LL, Wszolek ZK, Normand MM (1996) Neurophysiologic evaluation of cyclosporine toxicity associated with bone marrow transplantation. Acta Neurol Scand 94:358–364 91. Patchell RA (1994) Neurological complications of organ transplantation. Ann Neurol 36:688–703

1624 92. Wu DW, Wilt J, Restaino S (2007) Pregnancy after thoracic organ transplantation. Semin Perinatol 31:354–362 93. Armenti VT, Daller JA, Constantinescu S, et al. Report from the National Transplantation Pregnancy Registry: outcomes of pregnancy after transplantation. Clin Transpl 2006:57–70 94. Orens JB, Becker FS, Lynch JP 3rd et al (1995) Cardiopulmonary exercise testing following allogeneic lung transplantation for different underlying disease states. Chest 107:144–149 95. Ambrosino N, Bruschi C, Callegari G et al (1996) Time course of exercise capacity, skeletal and respiratory muscle performance after heart-lung transplantation. Eur Respir J 9:1508–1514 96. Bowdish ME, Pessotto R, Barbers RG et al (2005) Long-term pulmonary function after living-donor lobar lung transplantation in adults. Ann Thorac Surg 79:418–425 97. Starnes VA, Bowdish ME, Woo MS et al (2004) A decade of living lobar lung transplantation: recipient outcomes. J Thorac Cardiovasc Surg 127:114–122 98. Levy RD, Ernst P, Levine SM et al (1993) Exercise performance after lung transplantation. J Heart Lung Transplant 12:27–33

J.R. Maurer 99. Lands LC, Smountas AA, Mesiano G et al (1999) Maximal exercise capacity and peripheral skeletal muscle function following lung transplantation. J Heart Lung Transplant 18:113–120 100. Kasimir MT, Mereles D, Aigner C et al (2008) Assessment of pulmonary artery systolic pressures by stress Doppler echocardiography after bilateral lung transplantation. Heart Lung Transplant 27:66–71 101. Vermeulen KM, Ouwens JP, van der Bij W et al (2003) Long term quality of life in patients surviving at least 55 months after lung transplantation. Gen Hosp Psychiatry 25:95–102 102. Vermeulen KM, Groen H, van der Bij W et al (2004) The effect of bronchiolitis obliterans syndrome on health-related quality of life. Clin Transplant 18:377–383 103. van Den Berg JW, Geertsma A, van Der Bij W et  al (2000) Bronchiolitis obliterans syndrome after lung transplantation and health-related quality of life. Am J Respir Crit Care Med 161:1937–1941 104. Rutherford RM, Fisher AJ, Hilton C et al (2005) Functional status and quality of life in patients surviving 10 years after lung transplantation. Am J Transplant 5:1099–1104

Index

A Accompanying vessels (AVs), 101 Accutase, enzymatic passaging, 616 Acinus, developmental stages, 43, 44 Acquired pulmonary arterial hypertension (APAH), 752 ACT–064992 (Actelion-1), 1473 Activated protein C, 402 Activating protein 1, 719 Activating protein-1 (AP-1), 341–342 Acute hypoxic regulation capacitative Ca2+ entry (CCE), 689–690 cellular mechanisms, 688–689 TRPC, 689–690 VDCC antagonists, 689 Acute lung injury (ALI), 381–382. See also Statins animal models, statins in, 895–896 BAL fluid, pathologic findings, 889 barrier integrity mechanism activated protein C, 211 adenosine triphosphate, 210 hepatocyte growth factor, 210–211 methylnaltrexone, 212 MLCK inhibitors, 208 oxidized phospholipids, 211–212 PBEF neutralizing antibodies, 212 simvastatin, 209–210 SIP, 208–209 therapeutic agents, 207 cytoskeleton role actin, 199 adhesive protein-cytoskeleton linkages, 200–201 endothelial cell, 198–199 intermediate filaments, 200 microtubules, 199–200 myosin, 199 development, 889 endothelial barrier, 197 paracellular and transcellular transport, 198 permeability mediators agonists, 204–205 mechanical stress and vascular barrier function, 203–204 oxidative stress, 204 vascular integrity, 201–202 vascular permeability, regulation, 202–203 statin protection, in murine model, 896 statins, clinical trials of, 896 vascular biomarkers IL–8 receptor, 213–214 intercellular adhesion molecule-1, 215 interleukin-6, 215

plasminogen activator inhibitor-1, 214–215 pre-B-cell colony-enhancing factor, 215–216 protein C pathway, 214 S1P3 receptor/tyrosine-nitrated, 213 sphingosine 1-phosphate, 208–209 ADAMTS–13, 376 Adcirca®. See Tadalafil Add-on therapy vs. combination therapy, PAH switching, 1517–1518 upfront combination therapy, 1518 Adenoviral-mediated transduction, 781 Adipocyte differentiation, 632 Adrenomedullin, 843–844 Adult respiratory distress syndrome, 751–752 Adult right ventricular hypertrophy, 1309–1310 Adult stem and progenitor cells application, 635 categories of, 621 cell-surface-marker expression combinations, 625–626 fluorescence-activated cell sorting, 628 immunofluorescence imaging, 628–629 immunohistochemistry, 626–628 single-cell clonogenic assay, 629 telomeres, 629–630 endothelial acetylated low density lipoprotein (LDL), 630–631 formation, 631–632 postnatal vasculogenesis mechanisms, 623–624 vascular growth factor, 622–623 hematopoietic, 630 limitations, 635 mesenchymal, differentiation adipocyte, 632 chondrocyte, 633 myocyte, 633 osteocyte, 633 minimal criteria, mesenchymal, 622 resident tissue, 624 side population (SP) cells, 622 Adventitial fibroblast phenotype chemokines and cytokines, 771 control of vascular tone and structure, 770 in vitro experimental studies, 769–770 Aerosolized prostacyclin, 1100–1101 Aglucerase, 1049 Air after hyperoxia distal pulmonary vessel and capillary networks, 744–746 hemodynamic measurements, 742 loss of perivascular cells, 743–744, 747 perivascular cell development, 742–743, 745

J.X.-J. Yuan et al. (eds.), Textbook of Pulmonary Vascular Disease, DOI 10.1007/978-0-387-87429-6, © Springer Science+Business Media, LLC 2011

1625

1626 Air pollution, pulmonary vasculature clinical effects particulate, 972–974 tropospheric ozone, 974 diagnosis, 974 etiology and pathogenetic mechanisms computational models for particles, 967 dosimetry concepts and terminology, 965–966 methodological constraints, 964 ozone, 965–967 particulate air pollution, 965 regulatory framework, 964 prognosis, 974 systemic and molecular pathophysiology pulmonary hypertension, 968–970 vasoactive mediator transcription, 970–971 ventilation/perfusion mismatch and impaired gas exchange, 971 treatment and prevention, 974 a-Actin, 490 Alveolar-capillary dysplasia, 51 Alveolar capillary networks functional adaptation of zone I, 21 zone II, 21–22 zone III, 22–23 Alveolar-capillary sheet model, 93 Alveolar epithelial fluid transport anesthesia, 866 cytokines, 867 distal airway epithelia ion transport, 862–863 hypoxia, 866 lung fluid absorption catecholamine regulation, 864–865 non-catecholamine regulation, 865–866 tight junction, 861–862 transepithelial fluid movement, 862 pathologic conditions, 867 reactive oxygen species, 866–867 Alveolar fluid clearance, 880–881 Alveolarization stage, 138 Alveolar oxygen tension cellular and molecular mechanisms of perivascular cell development and loss, 746, 748–750 of vessel and capillary loss, 750 of vessel and capillary restoration, 750–751 Alveolar vessels, pulmonary circulation, 9 Alveolocapillary model, 94 Ambrisentan adverse effects, safety, and drug interactions, 1473 clinical trials, 1472–1473 metabolism, 1472 Amiloride-sensitive epithelial Na+ channel (ENaC), 236 Anesthesia, 866 Angiogenesis, 137 Angiography computerised tomography (CTA), 513 magnetic resonance (MRA), 515–516 Angiotensin-converting enzyme (ACE), 106 Animal models lung vascular permeability cross-talk mechanisms between endothelial barrier enhancement and disruption, 478 edema fluid formation, 472 endotoxin and polymicrobial, 476–477 evaluation, 478–481

Index fluid accumulation in lungs, 472 histamine, 474 ideal model, 472 imaging methods, 480 mechanical ventilation, 477 oleic acid (OA), 477 oxidant generation and ischemia/ reperfusion injury, 474–475 thrombin, 472 vascular endothelial growth factor (VEGF), 475–476 pulmonary hypertension (PH) chronic hypoxia-induced PH, 455 classification, 456–457 endothelin–1 (ET–1), 456 genetic models, 455–456 left-to-right shunt, 455 monocrotaline-induced PH, 453–454 newborn, 455 other conditions, 456 pulmonary embolism, 455 Anorectic agents aminorex chemical structures of, 1043, 1045 survival rate, 1045 appetite suppressants, 1047 fenfluramines, 1045–1047 ANOVA, 660–661 Antiaortic endothelial cell antibodies (AAECAs), 1334 Anticoagulant pathways activation, of protein C, 363 antithrombin function and structure, 364–365 APC anticoagulant function, 363–364 endothelial cell protein C receptor (ECPR), 362 heparin and heparan sulfate role, 366 protein C, 361 protein C/APC, 362 protein S, 362–363 thrombin inhibition, 365–366 thrombomodulin, 362 tissue factor pathway inhibitor (TFPI), 360 Anticoagulants elaborated by vascular endothelium, 405–406 membrane-associated, 406–407 Anticoagulant therapy. See Thrombolytic and anticoagulant therapy Anticoagulation, 1537 Antiphospholipid antibodies, 853 Antiremodeling Gaucher’s disease type I, 1067 PLCH, 1072–1073 sarcoidosis, 1076–1077 splenectomy-induced PAH, 1069 Antiretrovirals, 1039 Antisense-olignucleotide-mediated gene silencing concept and mechanism, 557–558 design and synthesis, 558 limitations in therapeutic application, 558 Antithrombin, 407 Anti-TNF therapy, 1339 Apoptosis, vascular smooth muscle and endothelial cells cell execution and clearance, 353–354 cell-signaling pathway extrinsic, 349–350 intrinsic, 348–349 definition, 347–348 effector proteins, apoptic Bcl–2 superfamily, 351–352 caspases, 350–351

Index inhibitors, 351 p53 and DNA damage, 352 endoplasmic reticulum, 352–353 ion channels, 352 pulmonary arterial hypertension, 354–355 Apparent viscosity, 92 Aquaporins, 239 Arachidonic acid metabolites production and action, 176–178 regulation, 178–179 Arginine, 1099 Arterial and precapillary vessel pathology adventitial lesions, 1417 intimal lesions, 1416–1417 medial lesions, 1417 Arterial spin labeling (ASL), 517 Arterial tree, cast model, 76, 77 Arterioles, 18 AS. See Atrial septostomy Atherosclerosis, 797 Atrial septal defects (ASDs), 1147 Atrial septostomy (AS) immediate hemodynamic effects, 1562–1563 immediate outcome after, 1560–1561 long-term effects, 1563–1564 long-term survival, 1564 in PH, 1559–1560 procedures, 1560 rationale for, 1559 recommendations, 1562 spontaneous closure of, 1561–1562 worldwide collective experience, 1560 Automated patch clamp techniques, 497–501 cell positioning, 502 glass pipette interfaces, 502 planar-array-based approaches, 502–504 planar electrodes, 501–502 surface electrogenic event reader (SURFE2R), 505 Axial artery, 18 B Balloon-dilation atrial septostomy technique, 1561 Basic fibroblast growth factor (bFGF), 1300 Bcl–2 superfamily, 351–352 Behçet's disease, 1340 BENEFIT trial, 1470 Benfluorex, 1048 Beraprost, 1456 Bernheim effect, 1184 b-actin, 490 Bilateral lung transplantation, 1617 Bilirubin/biliverdin, actions, 782–783 Binary data diagnostic tests, 664–665 logistic regression, 666–667 proportions, 663–664 receiver operating characteristic curves, 665–666 Bioinformatics complete genome sequencing, 577–578 data analysis dataset identification, 574–576 subset effects, 575 DNA sequence variation HapMap database, 572–573 human gene mutation database (HGMD), 572

1627 OMIM database, 572 epigenetics, 578 gene expression analysis, 571–572 genetic mapping/meiotvapping computational methods, 570–571 data analysis, 569 genome-wide association (GWA), 568–569 linkage disequilibrium (LD), 568, 570 variations identification, 568 genetic research, 567–568 genome data integration, 578 heritable factors, 568 patients data analysis analyses packages, 576 dataset identification, 574 GDS504 record link, 574 GSE identifier, 575–576 heatmap, 575 inflammatory pathways, 576 probe identifiers to gene names, 576 thresholds box, 576 protein expression analysis, 571–572 protein structure, 574 resources, 574 resources expression analysis, 573 protein structure and proteomics, 574 translational genomic studies, 578 Biological gases, chemistry carbon monoxide biological function, 294 biosynthesis, 293 chemical properties, 293 with nitric oxide interactions, 294 hydrogen sulfide anti-inflammatory effects, 296–297 biochemical properties, 295–296 biosynthesis, 295 blood pressure control, 296 catabolism, 295 cytoprotective effects, 297 hemorrhagic shock, 297 hypoxia tolerance, 297 hypoxic pulmonary vasoconstriction, 296 pulmonary hypertension, 296 reactive hydrogen sulfide species, 296 suspended animation, 296 nitric oxide biosynthesis, 290 hemoglobin, 292 with metals, 291 oxygen relationship, 292 reactions with oxygen, 290–291 reactive nitrogen species, 290–292 with thiols, 291 tyrosine nitration, 292 oxygen hydrogen peroxide, 288–289 hydroxyl radical, 289 physiology, 289 reactive oxygen species, 287–288 superoxide, 288 Bladder fluke. See Schistosoma haematobium Blood distribution, pulmonary. See Magnetic resonance imaging technology; Multidetector-row CT (MDCT) technology Blood pool agents, 518–519

1628 Blood pressure measurement, 6–7 Blotting northern, 528–529 southern, 526–527 western, 533–534 BMP pathway PH models BMPR2 Function, 467 BMPR2 knockout and hypomorph, 466 BMPR2 siRNA and Cre–loxP, 466 inducible BMPR2 mutants, 466–467 BMP receptor polymorphisms, 550–553 BMPR signaling animal studies, 831–832 cellular studies, 832 development, 832 mutations, 831 overview, 829–831 BMPR vs. notch signaling pathways, 833 Bone morphogenetic protein receptor–2 (BMPR2), 429–431, 1142 Bone morphogenetic proteins (BMPs) BMPRII and PAH, 126 BMPRII/TGF-b receptors, 125–126 functions, 125 in hypoxia-mediated PH, 704–705 reduced disease penetrance, mutation carriers, 126 serine/threonine kinases receptor, 254 Bosentan, 1038, 1206, 1371 adverse effects, safety, and drug interactions, 1470 BENEFIT trial, 1470 clinical trials, 1467–1468 connective tissue disease, 1469 functional class II patients, 1469 HIV and, 1470 oral dose administration, 1466–1467 pediatric and congenital heart disease (CHD) patients, 1469–1470 survival with, 1468–1469 Brain natriuretic peptide (BNP), 1234 Breeding, 463 Bronchial and pleural circulation, 15 Bronchial arteries, 15 Bronchial circulatory system, human anatomy arterial system, 440 bronchopulmonary connections, 440–441 capillary beds, 440–441 venous system, 441 blood flow measurement Berry and Daly technique, 443 dimethyl ether (DME), 444 laser Doppler velocimetry, 444 inhaled drugs effect, bronchial vasculature bronchodilators, 447 corticosteroids, 447 drug interactions, 447 lung diseases asthma, 444 bronchiectasis and tuberculosis, 445 chronic bronchitis and emphysema, 445 pulmonary edema, 445 tissue repair, 445 tumors, 445 physiological functions air conditioning, 441–442 autonomic and nonautonomic regulation, 442–443 bronchial mucosa nourishment, 441 gas exchange, 442

Index vascular changes drug absorption and metabolism, 446 heat and water exchange, 446 mucociliary clearance, 446 mucosal thickness, influence, 445–446 noninvasive marker, inflammation, 446–447 Bronchial veins, 15 Bronchoalveolar lavage (BAL) characteristics, early HAPE, 879 chest radiograph with, 873 for lung vascular permeability, 481 Bronchodilators, 447 Bronchopulmonary dysplasia (BPD) clinical presentation, 1090 compromised lung vascular development, 52 iNO to prevent, 1495–1498 management, 1091–1092 pathogenesis, 1089–1090 patient management, NICU discharge, 1092–1093 PH assessment, 1090–1091 C Ca2+ and ion channels, pathogenic roles abnormal homeostasis, 683 acute hypoxic regulation capacitative Ca2+ entry (CCE), 689–690 cellular mechanisms, 688–689 TRPC, 689–690 VDCC antagonists, 689 caveolin and caveolae, colocalization, 691 chronic hypoxic regulation K+ channels downregulation, 690 TRPCs upregulation, 690–691 cytosolic concentration, regulation calcium homeostasis, 685–686 store-operated Ca2+ channels, 687 voltage-dependent Ca2+ channels (VDCC), 686–687 membrane potential, regulation K+ channels, 687–688 molecular composition and structure, 688 Nernst and Goldman–Hodgkin–Katz equations, 687 PASMCs, 1212 pulmonary vasoconstriction and proliferation CaM complex propels progression, 684–685 myosin light chain kinase (MLCK) activation, 684 Cadaveric lung transplantation (CLT), 1602–1603. See also Livingdonor lobar lung transplantation (LDLLT) Caging, 463 Calcitonin, 262 Calcium-binding gene, 468 Calcium channel antagonists, 1537–1538 Calcium channel antagonists (CCBs), 985, 987–988 Calcium channel blockers, 1371 Calcium channel blockers (CCBs) for HAPH, 1218 for HIV-PAH, 1037 for PAH cellular mechanisms, 1447 chronic vasoconstriction, 1448 drugs, 1448–1449 initial clinical studies, 1447–1448 without acute vasoreactivity, 1450 Calcium channels. See Ion channels and transporters Calcium entry receptor-operated calcium channels, 268 receptor tyrosine kinases, 265, 266

Index store-operated channel (SOC), 266–267 transient receptor potential (TRP), 266 T-type voltage-gated channel, 268 Calcium homeostasis blood blood calcium level regulation, 262 calcitonin, 262 filteration, kidney, 262 paracellular mechanism, 262 transcellular mechanism, 262 cell calcium-binding proteins, 263–264 calcium entry, 265–269 calcium mobilization, 264–265 calcium transporters, 264 intracellular calcium compartments, 264 Calcium role, signaling Drosophila melanogaster, 266 homeostasis, blood blood calcium level regulation, 262 calcitonin, 262 filteration, kidney, 262 paracellular mechanism, 262 transcellular mechanism, 262 homeostasis, cell calcium-binding proteins, 263–264 calcium entry, 265–269 calcium mobilization, 264–265 calcium transporters, 264 intracellular calcium compartments, 264 InsP3 receptor, 265 membrane skeleton, 267 origins, 261 physiological functions, 269 receptor-operated calcium channels, 268 receptor tyrosine kinases, 265, 266 store-operated channel (SOC), 266–267 transient receptor potential (TRP), 266 T-type voltage-gated channel, 268 Calcium (Ca2+) role, smooth muscle gene expression and proliferation genes alteration BMP–2, 342–343 hypoxia exposure, 343–344 microarray analysis, 342 phenotypical switching, 335 physiological function, 335 sources classic pathway, 335–336 intracellular stores, 337 IP3 and ryanodine receptors (RyRs), 336–337 K+ channels, 338 PMCA and SERCA pumps, 337–338 voltage-dependent channels, 336 transcription factors regulation activating protein–1 (AP–1), 341–342 cyclic AMP response element binding protein, 341 nuclear factor of activated T cells (NFAT), 338–339 serum response factor (SRF), 339–340 Calcium sensitization, 679–680 Calcium sources, 677–678 Calveolin–1, 583 Ca2+ mobilization entry pathways Ca2+ removal, 150 plasmalemmal Ca2+-ATPase (PMCA), 150–151 voltage-dependent entry, 148–149 voltage-independent entry, 149–150

1629 integration of Ca2+ signaling pulmonary artery smooth muscle cell (PASMC), 154 waves and oscillations, 153–154 intracellular Ca2+ stores lysosomes, 152–153 mitochondria, 153 sarcoplasmic reticulum, 151–152 Canalicular stage, 138 Cancer of the pulmonary artery. See Idiopathic pulmonary arterial hypertension Canonical transient receptor potential 6 channel (TRPC6) siRNA downregulation of, 564 generation of recombinant adenovirus, 562–563 history, 562 synthesis and selection of, 562 Capillary network discrete tubules, 93–95 sheet model, 92–93 Capillary stress failure, 871 Carbon monoxide biological function, 294 biosynthesis, 293 chemical properties, 293 heme oxygenase–1 and cytoprotection, 781–782 with nitric oxide interactions, 294 Cardiac catheterization, 8–9 noninvasive imaging, role of, 1387–1388 pulmonary angiography anatomy, 1394–1395 angioscopy, 1398–1400 findings, patterns of, 1395–1398 significance, in CTEPH patients, 1398 PVR partitioning correlations, 1392 PAP occlusion waveforms, 1391 RHC, exercise, 1392–1393 vasodilator challenge, 1393–1394 right-sided heart (RHC), 1387–1391 safety consideration, 1388–1389 Cardiac hypertrophy, 1306 Cardiac imaging, right ventricular assessment, 516–517 Cardiopulmonary system, 3, 4 CART. See Classification and regression trees Caspases, 350–351 Catecholamine regulation, 864–865 Caveolae, 790, 840–841 Cav–1-/- and Cavin-/- mice, 274–275 function, 273–274 signaling Ca2+ handling, 277 caveolin–1, 275–276 eNOS/Akt, 277 G-protein-coupled receptors, 276–277 receptor tyrosin kinases, 276–277 structure, 273–274 vascular function regulation EC and SMC, 278 endocytosis vs. transcytosis, 278–279 mechanosensation and shear stress, 280 paracellular permeability, 279–280 SMC mitogenic signaling, 281 vascular tone, 280 Caveolin and caveolae, colocalization, 691 caveolin–1 and eNOS, 840–841 vasoreactivity, 464 Caveolin–1, 274–275

1630 Caveosome, 274 CCBs. See Calcium channel antagonists (CCBs) CD39, 406–407 Cell isolation protocols, 643 Cell positioning, 502 Cell proliferation, 767 Cell recruitment, 737–739 Cell-signaling pathway, apoptosis extrinsic, 349–350 intrinsic Apaf–1, 349 signals, 348 voltage-dependent anion channel (VDAC), 348–349 Cell sorting, 644 Cell subculture and storage, 489–490 Cell-surface-marker expression combinations, 625–626 fluorescence-activated cell sorting, 628 immunofluorescence imaging, 628–629 immunohistochemistry, 626–628 single-cell clonogenic assay, 629 telomeres, 629–630 Cell thawing and reseeding, 490 cGMP pathway, phosphodiesterase inhibitors, 1478 Channelopathy, 495. See also Patch clamp techniques and recordings Chemical fixation, 14 Chemokine receptor 4 nonmuscle myosin II A, 902 PubMatrix citations, 902 Chemokines, 846 Chemokine signaling, 819–820 Chemotherapy-related drugs for acute lymphoblastic leukemia, 1051–1053 chemical structures of, 1049 malignant tumors and PAH, 1049, 1050 for myelofibrosis and PVOD, 1051–1053 solid malignant tumor and PVOD, mitomycin C for, 1049–1050 for solid malignant tumors and PVOD, 1051–1052 thalidomide and PAH, 1049 Chest radiography, CTEPH, 1568 Chest X-ray, 1318, 1319 posterior–anterior and lateral, 1403 pulmonary emboli findings, 1404 Childhood interstitial lung disease (ChILD), 1095 Chimeras construction, 545 Chloride channels. See Ion channels and transporters Chondrocyte differentiation, 633 Chronic hypoxia-induced pulmonary hypertension animal model of, 455 BMPs in, 704–705 changes in pulmonary endothelium, 698–699 endothelial progenitor cells, 705–706 endothelium-derived mitogenic signals FGF2, 703 PDGF, 703 SMCs, 703 VEGF, 703–704 endothelium-derived vasoactive mediators of endothelin–1, 700–701 nitric oxide, 699–700 prostacyclin, 701–702 serotonin, 702 mechanisms of, 706 pulmonary vascular remodeling, 697–698 pulmonary vascular responses, 696–697 Chronic hypoxic pulmonary hypertension, 582–583

Index Chronic hypoxic regulation K+ channels downregulation, 690 TRPCs upregulation, 690–691 Chronic lung disorders (CLD) pulmonary hypertension assessment, 1094–1096 clinical presentation, 1094 management, 1096 Chronic mountain sickness (CMS), 1211, 1213, 1214, 1216–1218 Chronic obstructive pulmonary disease (COPD), 66, 954 Chronic obstructive pulmonary diseases PH and cor pulmonale, history, 1189–1190 pulmonary hypertension (PH) clinical presentation, 1190–1192 and cor pulmonale, 1193–1194 diagnosis, 1192 pathobiology, 1192–1193 pathophysiology, 1192 therapy, 1194–1195 Chronic thromboembolic plumonary hypertension (CTEPH), 390, 392 left ventricular compression vs. left ventricular underfilling, 1184 Chronic thromboembolic pulmonary hypertension (CTEPH) alternative therapy, 1247–1249 clinical presentation, 1240–1241 computerized tomographic scanning, 1570–1572 conventional pulmonary angiography, 1570 diagnosis angiographic patterns, 1243 chest radiography, 1242, 1243 CT scanning, 1244 magnetic resonance (MR) imaging and angiography, 1243–1244 transthoracic echocardiography, 1241 echocardiography, 1568 epidemiologic features, 955 goals, patient evaluation, 1573 magnetic resonance imaging, 1572–1573 occurence, 1239 physical examination, 1567 preliminary testing, 1568 progression, 1240 prostanoids management, 1459 PTE patients, outcomes, 1247 pulmonary angiogram, 1243, 1244 reperfusion injury, 1247 right-sided heart catheterization, 1569–1570 risk factors antiphospholipid antibodies, 1256 cancer survivors, 1256 coagulation factor VIII level (see von Willebrand factor plasma levels) congenital vascular malformations, 1257 female gender and HLA polymorphisms, 1257 fibrinogen Aa Thr312Ala polymorphism, 1257 hereditary, 1253 infected ventriculoatrial shunts and pacemaker leads, 1254 inflammatory bowel disease, 1257 non-O blood groups, elevated, 1256 odds ratios (ORs), 1253, 1254 splenectomy, 1254–1255 thyroid disease, 1255 venous thromboembolism, 1255 small-vessel arteriopathy evaluation case studies, 1263–1264 PAH medical therapy role, 1265–1266 persistent PH, after PTE, 1261–1262

Index preoperative tests and assessment, 1264–1265 types and mechanism, 1262 surgery, 1246–1247 surgical selection, 1245–1246 thrombolytic and anticoagulant therapy, 1527 vascular remodeling in, 858–859 ventilation-perfusion lung scan, 1569 ventilation–perfusion scan, 1242 Circulating tissue factor, 404 Circulating vascular progenitors pulmonary vascular remodeling animal study, 816–818 human study, 818–819 systemic vascular remodeling angiogenesis, 815 contribution, 814 fibrocytes, 815 Citrulline therapy, 1100 Classification and regression trees (CART) advantage, 667 contingency tables, 667–668 definition, 667 c2 test, 668 Fisher’s exact test, 668 Clinical data analysis. See Statistical data analysis Coagulation. See Pulmonary embolism Coagulation cascade anticoagulant pathways activation, of protein C, 363 antithrombin function and structure, 364–365 APC anticoagulant function, 363–364 endothelial cell protein C receptor (ECPR), 362 heparin and heparan sulfate role, 366 protein C, 361 protein C/APC, 362 protein S, 362–363 thrombin inhibition, 365–366 thrombomodulin, 362 tissue factor pathway inhibitor (TFPI), 361 hemostasis, 357 plasma clotting factors epidermal growth factor (EGF) like domains, 359–360 FV and FVIII clotting cofactors, 360 Gla domains, 359 serine proteases domains, 360 process, 357–359 thrombin exosites and cofactors, 368 hemostatic substrates, 367–368 roles, 366–367 Cocaine, 1054 Coinfection, 1290 Collagen, 81 Collagen pathway, 403 Collagen vascular diseases (CVDs) mixed connective tissue disease (MCTD), 1015 patients, survival of, 1018 primary Sjogren’s syndrome (pSS), 1015–1016 rheumatoid arthritis (RA), 1015 systemic lupus erythematosus (SLE), 1015 therapy anticoagulation, 1018 anti-inflammatory drugs, 1018 combination therapy, 1017 endothelin receptor antagonists, 1016–1017 lung transplantation, 1018

1631 phosphodiesterase inhibitors, 1017 prostaglandins, 1016 tyrosine kinase inhibitors, 1018 Combination therapy, IPAH PACES trial, 991 STEP trial, 992 Combination therapy, PAH add-on therapy vs. switching, 1517–1518 upfront combination therapy, 1518 endothelin receptor antagonists with PDE inhibitors long-term clinical studies, 1516–1517 long-term experimental studies, 1516 goal-directed therapy, 1518–1519 natriuretic peptides with PDE inhibitors, 1513–1514 NO with PDE inhibitors, 1513 prostacyclins with endothelin receptor antagonists long-term clinical studies, 1514–1515 long-term experimental studies, 1514 prostacyclins with NO, 1512–1513 prostacyclins with PDE inhibitors acute clinical studies, 1511–1512 acute experimental studies, 1510–1511 long-term clinical studies, 1512 long-term experimental studies, 1512 rationale, 1509–1510 triple therapy, 1519 Compliance, 76 Compuertised tomography (CT) technology. See Multidetector-row CT (MDCT) technology Computerized tomographic scanning, CTEPH, 1570–1572 Conductance vessel remodeling animal models, 760 bovine large elastic vessels, 762 hypoxia-induced cell proliferation, 761, 762 stiffness, neonatal calf main PA, 762, 763 TGF-b receptor, 761 Conducting arteries, 16–18 Congenital diaphragmatic hernia (CDH) clinical presentation, 1087 management, 1088 pathophysiology, 1086–1087 patient management, NICU discharge, 1089 PH assessment, 1088 Congenital heart defects at high altitude. See Pulmonary hypertension, congenital heart defects at high altitude Congenital heart disease (CHD) assessment, 1097–1098 PH presentation, 1098 pulmonary circulation cardiac structural lesions, 1125–1130 clinical management, 1132–1134 mechanical forces, 1131 microarchitecture, 1121–1222 pathology and physiology, 1120 pulmonary vascular resistance (PVR), 1119–1120 reversibility, 1222–1225 Congenital-heart-disease-associated pulmonary hypertension (CHD-PAH), surgical evaluation anesthetic considerations cardiac surgery, 1165–1166 noncardiac surgery, 1165 preoperative assessment, 1165 combined heart and lung transplantation, evaluation, 1164–1165 considerations, CHD lesions repair, 1158–1159 description, 1153

1632 Congenital-heart-disease-associated pulmonary hypertension (CHD-PAH), surgical evaluation (cont.) Eisenmenger patients, 1161 interventional lung assist, 1165 lung transplantation and CHD repair, 1164 mitral valve disease, 1159–1160 occurence, 1153 operability evaluation, investigations cardiac imaging, 1155–1157 clinical assessment, 1154–1155 invasive hemodynamic assessment, 1157 open-lung biopsy, 1155 PVR reversibility studies, 1157–1158 temporary shunt occlusion, 1158 paradigm, shunt closure, 1163 shunt closure, 1162–1163 surgery, 1162 therapy, 1160–1161 transplantation, 1163–1164 univentricular circulations, 1160 Congenital systemic-to-pulmonary (left-to-right) shunts atrial septal defects (ASDs), 1147 development, 1139 drug therapy, 1147–1148 pathogenic mechanisms endothelin production, 1141 gene mutations, 1142 pulmonary vascular endothelium damage, 1140 shear- and stretch-induced cytoskeletal reorganization, 1140 pathology, 1142–1143 pulmonary hemodynamics, clinical assessment, 1143–1145 treatment, 1145–1147 Connective tissue continuum extraalveolar arteries and, 16 role of, in relation to capillaries, 15–16 Conventional pulmonary angiography, CTEPH, 1570 Conventional supportive therapy, PVOD, 1176 COPD. See Chronic obstructive pulmonary diseases Corner capillaries, 19–20 Corner pleats, 20–21 Cor pulmonale classification, 1355–1363 clinical presentation, 1363 COPD, associated PH, COPD-associated heart disease, 1193–1194 COPD, PH and, 1190 definition, 1305, 1355–1356 diagnostic evaluation biomarkers, 1369 cardiovascular magnetic resonance imaging (CMR), 1366–1369 chest radiography, 1363 electrocardiography, 1363 radionuclide ventriculography, 1369 right-sided heart catheterization (RHC), 1369–1370 transthoracic echocardiography (TTE), 1363–1366 epidemiology, 1357–1358 etiology, 1356–1357 heart in, 1358 histopathology lung, 1358–1359 right ventricle (RV), 1358 lung histology, 1358–1359 pathophysiology catecholamines role, 1359–1360 inflammation, 1362 Kv pathway, 1360–1361

Index lung mechanics, abnormal, 1361–1362 mechanism, 1359 polycythemia, 1362 pulmonary vascular remodeling, 1360 prognosis chronic obstructive pulmonary disease (COPD), 1370 interstitial lung disease, 1370 pulmonary artery pressure (PAP) assessment, 1365 estimation, 1365, 1366 right (RV) dysfunction, 1362–1363 treatment COPD treatment, 1373 lung transplant, 1372 oxygen, 1371 pulmonary vasodilators, 1371–1372 symptomatic therapies, 1372 Corticosteroids, 447 Creep, 79 Cre–loxP system, 460–461 CT angiography (CTA), 513 CTEPH. See Chronic thromboembolic pulmonary hypertension CT pulmonary angiograms advantages, 1407–1408 pulmonary arterial, 1405–1406 CT technology. See Multidetector-row CT (MDCT) technology Cystic fibrosis (CF), 1095 Cytokines, 402, 844–845, 867 CytoPatch (Cytocentrics), 504 Cytoprotection, lung adenoviral-mediated transduction, 781 bilirubin/biliverdin, actions, 782–783 carbon monoxide, actions, 781–782 dinucleotide repeat polymorphism, 781 protective effects, 783–784 therapeutic implications, 784 upstream enhancers, 781 Cytosolic concentration, regulation calcium homeostasis, 685–686 store-operated Ca2+ channels, 687 voltage-dependent Ca2+ channels (VDCC), 686–687 D Dana point classification chronic hemolytic anemia, 941 clinical, updated, 939, 940 congenital heart diseases, 940 heritable PAH, 939 PVOD and PCH, 941 risk factors and drug- and toxin-induced PAH, 939–940 schistosomiasis, 940 Deep vein thrombosis, 851–852 Deep vein thrombosis (DVT). See Venous thromboembolism (VTE) Dichloroacetate (DCA), 1325 Diethylpropion, 1047 Diffuse alveolr hemorrhage (DAH), 378 Diffusion-weighting imaging, 518 Digoxin therapy, 1372 PAH, 1537 in RVF, 1323 Dinucleotide repeat polymorphism, 781 Distal airway epithelia ion transport, 862–863 Distal muscular PA remodeling BMPR2, 765 growth-resistant differentiated SMC, 764

Index hypoxia-induced perivascular remodeling, 763 myofibroblast, 764 plexiform lesions, 765 Distal pulmonary vessels and capillaries loss of, 740–741, 743, 744 loss of perivascular cells, 743–744, 747 perivascular cell development cell proliferation, 767 cell recruitment, 737–739 neomuscularization, 739–740 restoration of, 744–746, 748 Diuretic therapy, 1372 Diuretic therapy, PAH, 1536 DNA quantification cell signaling components, 525–526 southern blotting DNA isolation, 526 gel electrophoresis, 527 probe hybridization, 527 uses, 526 Docking proteins, 253 Doppler echocardiography (DE) anatomy and physiology, 1425–1427 exercise echocardiography, 1441–1442 hemodynamic assessment cardiac output, 1434 pulmonary vascular resistance, 1434 RV-PA interaction, 1434–1435 initial diagnostic assessment, DE examination integration, 1440–1441 pressure assessment, 1427–1429 PVD, findings acute pulmonary embolism, 1437–1438 chronic pulmonary embolism, 1437 pulmonary arterial hypertension, 1435–1437 pulmonary venous hypertension, 1438–1440 systemic-to-pulmonary shunts (SPS), 1438 RV systolic function assessment direct measures, 1429–1431 indirect measures, 1431–1433 Doppler echocardiography, PH, 1185 Doxycycline (or tetracycline)-inducible systems, 462 Dual-energy CT, 512–513 Ductus arteriosus, 729 Ductus arteriosus (DA) control, 142–143 Dynaflow®HT, 504 Dynamically perfused capillary surface area (DPCSA), 108, 110, 111 E EC cytoskeleton, 890 Echocardiography CTEP, 1568 PAH, 1534–1535 RVF, 1318–1320 Ectoenzymes, 106 Edema fluid formation, 472 Effector proteins, apoptic Bcl–2 superfamily, 351–352 caspases, 350–351 inhibitors, 351 p53 and DNA damage, 352 Eicosonoids, 123 Eisenmenger syndrome, 1140 Elastin, 80–81 Electrocardiography, RVF, 1318–1319

1633 Embolectomy catheter-based, 1236 inferior vena cava (IVC) filters, 1236 surgical, 1236 Embolism, pulmonary, 519–520 Embryonic stem (ES) cells applications, 646–647 cell isolation protocols, 643 characteristics of, 638 differentiation through genetic modulation human, 645 mouse, 645–646 differentiation to vascular cell lineages, 638–639 ECs derived from mouse, 642 human ES cell differentiation, 642–643 human ES-derived SMCs, 640–641 human vs. mouse, 639 limitations, 647–648 mouse, extracellular matrix proteins and growth factors application, 640 protocol, 639–640 PECAM1+ cell isolation, 644 shear-stress-induced EC differentiation, 641–642 VE-cadherin and cell sorting, 644 Emphysema. See Chronic obstructive pulmonary diseases Endarterectomy. See Pulmonary thromboendarterectomy (PTE) Endogenous plasmatic system, 854–855 Endoplasmic reticulum, cellular apoptosis, 352–353 Endothelial apoptosis and repair apoptosome, 426 biochemical events, 427 BMPR2 signaling haploinsufficiency, 431 pulmonary SMCs, 429 pulmonary vascular ECs, 429–431 caspases, 426 defenition, 425 endothelial dysfunction, PAH endothelin, 428 nitric oxide, 427–428 prostaglandins, 427 EPC circulation EC precursors, 432–433 endothelial colony-forming cells (ECFCs), 432 hypothesis, 433 genetics, PAH, 428 growth and survival experimental models, PAH, 431–432 history, 426 proliferation, 432 therapeutic implications, 433 Endothelial cells (ECs) analysis, 585–586 cessation of flow, 793 pulmonary blood flow and forces, 788–789 shear-dependent gene regulation, 791 shear stress response element, 791 vs. smooth muscle cell, 278 surface area, 105 Endothelial-derived constricting/proliferative factors endothelin–1 receptors, in PA, 119–120 synthesis, 119–120 serotonin receptors, 120–121 serotonin transporter (SERT), 121–122 synthesis, 122

1634 Endothelial–mesenchymal transition (EnMT), 765 Endothelial monocyte activating polypeptide II (EMAPII), 38 Endothelial progenitor cells (EPCs) acetylated low density lipoprotein (LDL), 630 formation, 631–632 hypoxia-mediated PH, 705–706 PDE5 inhibitors, clinical pharmacology, 1481 postnatal vasculogenesis mechanisms, 623–624 vascular growth factor, 622–623 Endothelial regulation arachidonic acid metabolites production and action, 176–178 regulation, 178–179 carbon monoxide, 183 endothelin production and action, 179–180 regulation, 180–181 endothelium-derived hyperpolarizing factor (EDHF), 181–182 epoxyeicosatetraenoic acids (EET), 181–182 hydrogen sulfide, 183 leukotrienes, 182–183 nitric oxide exogenous stimuli, 174–176 production and action, 171–172 regulation, 172–174 perinatal changes, pulmonary vascular tone arachidonic acid metabolites, 185 endothelin (ET), 184–186 fetus, 183–184 neonatal, 185 NO pathway, 185 prostacyclin (PGI), 185 pulmonary arterial hypertension endothelin dysregulation, 186 histopathological changes, 186 leukotriene production, 186–187 NO pathway, 186 vascular effectors dysregulation, 186 pulmonary vascular tone, 168 upstream exogenous stimuli humoral mechanisms, 169 hypoxic mechanisms, 170 mechanical factors, 169–170 neural mechanisms, 168–169 vascular smooth muscle cell (VSMC), 167 vasoactive intestinal peptide (VIP), 183 Endothelial, smooth muscle, and fibroblast/myofibroblast circulating SPCs, 812 fibrocytes differentiation, 813 hematopoietic lineage markers, 811 orgin, 812 side population, 814 vascular potential, 814 vasculogenic zone, 813 Endothelial surface enzymes. See Ectoenzymes Endothelin production and action, 179–180 regulation, 180–181 Endothelin–1 (ET–1), 456, 464–465 clearance, 109–110, 112–114 direct effects of HIV, 1034–1035 extraction and permeability surface product, 113, 114 isoforms of, 700 murine models, 701 PH patients, studies in, 112–114 plasma concentrations, 107 primary receptors for, 700

Index properties, 106 as vasoactive peptide, 1309–1310 Endothelin-converting enzyme (ECE), 106 Endothelin receptor antagonists, 1279 Endothelin receptor antagonist therapy, PAH ambrisentan, 1539–1540 bosentan, 1538–1539 sitaxsentan, 1540 Endothelins (ETs) receptor antagonists, PAH ACT–064992 (Actelion–1), 1473 ambrisentan adverse effects, safety, and drug interactions, 1473 clinical trials, 1472–1473 metabolism, 1472 bosentan adverse effects, safety, and drug interactions, 1470 BENEFIT trial, 1470 clinical trials, 1467–1468 connective tissue disease, 1469 functional class II patients, 1469 HIV and, 1470 oral dose administration, 1466–1467 pediatric and congenital heart disease (CHD) patients, 1469–1470 survival with, 1468–1469 clinical practice, 1474 with PDE inhibitors long-term clinical studies, 1516–1517 long-term experimental studies, 1516 pharmacology, 1465–1466 sitaxsentan adverse effects, safety, and drug interactions, 1471–1472 clinical trials, 1471 connective tissue disease, 1471 metabolism, 1471 Endothelium adhesion molecules, 846–847 adrenomedullin, 843–844 caveolae, 840–841 caveolins, 840–841 chemokines, 846 cytokines, 844–845 eNOS, 841 reactive oxygen species, 843 serotonin, 842–843 tetrahydrobiopterin, 841 thrombin, 844 treatments, 847 vascular growth factor, 842 vasculature, 837–838 vasoactive mediators principal, 839–840 prostacyclin, 840 thromboxane, 840 Endothelium-derived hyperpolarizing factor (EDHF), 142 Endothelium-derived vasoactive mediators, hypoxic PH endothelin–1 isoforms of, 700 murine models, 701 primary receptors for, 700 nitric oxide alveolar hypoxia, 699–700 high-altitude living conditions, 700 role, 700 synthesis, 699 prostacyclin., 701–702 serotonin, 702

Index Endothelium-derived vasodilators/antiproliferative factors cAMP, cGMP, and PDEs cAMP and cGMP, 124 phosphodiesterases, 124–125 nitric oxide (NO) effects on pulmonary vasculature, 122–123 isoforms, 122 for PAH treatment, 123 prostacyclin eicosonoids, 123 PGI2 receptor, 123 Endotoxin model, 476–477 eNOS, 841 Enzymatic dissociation, 616 Enzymatic passaging with collagenase IV and accutase, 616–617 Enzymatic treatment, 610 Enzymes for tissue dissociation and cell harvesting, 485–486 EphB4, 38–39 Ephrin B2, 38–39 Epidermal growth factor (EGF) like domains, 359–360 Epigenetics, 578 Epithelial apical sodium channel (ENaC), 880 Epoprostenol, 988 for HIV-PAH, 1038 intravenously administered catheter placement, 1543 common problems, 1546 considerations, 1544 dosing of, 1544–1545 FDA approval, 1546 generic, 1546 Hickman catheter, 1546 insurance policy, 1544 patient teaching, 1545 side effects, 1545 Epoprostenol, intravenously administered prostanoids, 1454–1455 Epoxyeicosatetraenoic acids (EET), 181–182 Erythropoietin (EPO), 715 ES. See Embryonic stem cells E-selectin, 393–394 ESI-TOF mass spectrometry, 599–600 ETA-selective endothelin receptor blockade ambrisentan adverse effects, safety, and drug interactions, 1473 clinical trials, 1472–1473 metabolism, 1472 sitaxsentan adverse effects, safety, and drug interactions, 1471–1472 clinical trials, 1471 connective tissue disease, 1471 metabolism, 1471 ET receptors, 251 Evian classification clinical, 937, 938 PH, thrombotic or embolic diseases, 938 pulmonary arterial hypertension (PAH), 937–938 pulmonary vasculature disorders, PH, 938 pulmonary venous hypertension, 938 respiratory system disorders or hypoxemia, PH, 938 Excitation–contraction coupling actin polymerization, 158–159 Ca2+ desensitization, 157–158 Ca2+ mobilization entry pathways, 148–151

1635 integration of Ca2+ signaling, 153–154 intracellular Ca2+ stores, 151–153 Ca2+ sensitization activation of rho, 156 inhibition of myosin phosphatase, 154–156 rho kinase, activation, 156 src-family kinases, 156–157 focal adhesions, 158–159 mechanotransduction, 158–159 Exercise testing, 1320 Experimental therapies, IPAH, 993 Extra-alveolar vessels, pulmonary circulation, 9 Extracellular matrix (ECM) collagen, 807 elafin, EVE inhibitor, 802–804 elastase activity, 801–802 elastin assembly genes, 804–806 fibronectin, 807 hyaluronic acid, 808 tenascin-C and matrix metalloproteinases, 806–807 Extracorporeal membrane oxygenation (ECMO), 1114, 1116, 1492–1493, 1599–1600 Extravascular lung water (EVLW) content, 478 F Factor Xa inhibitors, 1526–1527 Fahraeus–Lindqvist effect, 92 Familial and idiopathic pulmonary arterial hypertension (PAH) BMPR2 mutations distributions in gene, 1001 haploinsufficiency, 1002 importance, 1000–1001 in IPAH, 1002 “multiple hits,”; 1002–1003 nonsense-mediated decay (NMD), 1002 prevalence, in HPAH, 1001 in PVOD, 1005 BMP signaling pathway, 1004–1005 clinical evaluation and therapy, 1006 clinical transmission patterns and expression genetic anticipation, 1000 incomplete penetrance, 998–1000 onset, variable Age of, 999, 1000 skewed gender ratio, 1000 epidemiology, 998 genetic modifiers, 1003–1004 hereditary hemorrhagic telangiectasia (see Rendu–Osler–Weber syndrome) history, 997 screening and counseling, 1006–1007 Familial PAH (FPAH), 997, 998, 1001, 1004 Feeder dishes preparation, 615 Feeder-free culture, hESCs, 617 Fetal and neonatal pulmonary circulation perinatal, developmental physiology ductus arteriosus (DA) control, 142–143 fetal pulmonary circulation, 139–142 pulmonary vasodilation at birth, mechanism, 143 vascular growth in developing lung embryonic period, 136 fetal stages, 138 hemodynamic forces, 139 O2 tension role, 138–139 postnatal period, 138 stages, 136 vascular morphogenesis, 136–137

1636 Fetal pulmonary circulation, physiology CO role, 142 eNOS role, 140 ET–1 role, 142 NO-cGMP cascade, 141 NO release, 141 PVR increase, 139–140 ROCK, 141–142 Fibrin B chain N-terminus (B knob), 391, 394 Fibrin degradation products (FDPs), 853 Fibrinogen Aa Thr312Ala polymorphism, 1257 Fibrinogen, pulmonary thromboemboli fibrin B chain N-terminus (B knob), 391, 394 resistance, fibrinolysis and CTEPH, 392 structure, 390–391 Fick principle, 7 Ficoll-paque density gradient centrifugation, 630 Fisher’s exact test, 668 Flolan®. See Epoprostenol, intravenously administered Flow sensors caveolae, 790 cytoskeleton, 790 integrins, 790 ion channels, 789–790 Fluid accumulation in lungs, 472, 478–480 Fluid dynamics, 76–78 Fluorescence-activated cell sorting, 628 Fluorescent phosphoprotein stains, 608–609 [18F]Fluorodeoxyglucose ([18F]FDG) PET, 1338 Fluoxetine, 1053–1054 Focal adhesion kinase (FAK), 330 Fondaparinux, 1235, 1524 Fondaparinx, 394–395 Fourier transform ion cyclotron resonance (FT-ICR), 599 FPAH. See Familial PAH (FPAH) Functional proteomics identification of protein complexes by antibody-based immunoprecipitation, 606 protein microarrays, 606–607 recombinant protein-based affinity pull-down, 604–606 Y2H system, 607–608 identification of protein phosphorylation sites and phosphoproteomics gel-based techniques, 608–609 gel-free techniques, 609–610 FV and FVIII clotting cofactors, 360 G Galen’s scheme, 3–4 Gas exchange, 23 GATA–4, 1309 Gaucher’s disease type I anticoagulation, 1067 antiremodeling and vasodilating agents, 1067 clinical presentation and evaluation, 1066 diagnostic testing, 1066 history and background, 1064 key concepts, 1067–1068 management, 1066–1067 pathophysiology, 1065–1066 prevalence of, 1064–1065 Gel-based techniques, 590–594 functional proteomics antibody-based recognition of phosphoproteins, 609 fluorescent phosphoprotein stains, 608–609

Index phosphatase dephosphorylation assays, 609 32 P labeling, 608 multidimensional separation of protein mixtures, 594–596 proteomics method 1D electrophoresis, 593 2D electrophoresis analysis, 594 differential in-gel electrophoresis (DIGE), 595–596 silver staining, 595 Gel-free techniques multidimensional protein mixture separation, 596–597 protein phosphorylation sites and phosphoproteomics, 609–610 Gene cloning computer resources, 540 expression vectors and cell systems, 541 gene delivery methods heterologous overexpression, 543 stable transfected cells, 542 transient transfection, 541–542 viral vectors, 542–543 gene mutation, 539–540 library screening, 540–541 PCR, 540 Gene delivery methods heterologous overexpression, 543 stable transfected cells, 542 transient transfection, 541–542 viral vectors, 542–543 Gene encodin, 583 Gene expression analysis, 571–572 Gene expression manipulation antisense-olignucleotide-mediated gene silencing concept and mechanism, 557–558 design and synthesis, 558 limitations in therapeutic application, 558 RNAi-mediated gene silencing concept and mechanism, 558–559 limitations in therapeutic application, 561–562 siRNA delivery, 560–561 siRNA design and synthesis, 559–560 siRNA application downregulation of TRPC6, 564 generation of recombinant adenovirus, 562–563 history, 562 synthesis and selection, 562 traditional, 557 Gene expression profiling lung patterns, 583 peripheral blood monocytes, microarray, 583–584 progenitor cells, 585 pulmonary artery smooth muscle cells, 584 of right ventricle, 585 utilizing genetically engineered animal models, 586–587 Gene silencing antisense-olignucleotide-mediated concept and mechanism, 557–558 design and synthesis, 558 limitations in therapeutic application, 558 RNAi-mediated concept and mechanism, 558–559 limitations in therapeutic application, 561–562 siRNA delivery, 560–561 siRNA design and synthesis, 559–560 Genetic models, pulmonary hypertension, 455–456 Genomic analysis, components, 588 Genomics, acute lung injury gene and biomarkers

Index c-Met (hepatocyte growth factor receptor), 906 growth arrest DNA damage inducible a (GADD45a), 907–908 macrophage migration inhibitory factor (MIF), 905 myosin light chain kinase (MLCK), 904–905 pre-B cell colony enhancing factor (PBEF), 906–907 new genes identification and biomarkers gene polymorphisms, 904 module/ontology categories, 904 novel approach, 902–903 therapeutics pre-B cell colony enhancing factor blockage, 909 sphingosine 1-phosphate, 908 vascular barrier regulatory cytokines angiotensin-converting enzyme (ACE), 900 chemokine receptor 4, 902 interleukin–6 (IL–6), 901 tumor necrosis factor (TNF), 900–901 vascular endothelial growth factor (VEGF), 901 Genomics studies, 587–588 Genotyping, 463 Giant cell arteritis (GCA). See Takayasu’s arteritis (TA) Gigaohm seal formation, 495, 497 Gla domains, 359 Glass pipette interfaces, 502 Glycogen storage disease type I clinical presentation and evaluation, 1063 diagnostic testing, 1063 history, 1062 incidence, 1062 key concepts, 1064 management and therapy, 1063–1064 pathophysiology, 1062–1063 prevalence of, 1062 Goal-directed therapy, PAH, 1518–1519 Gp120, 1034–1035 GpIb complex, 375–376 G-protein-coupled receptors caveolae and signaling, 276 G-protein-coupled receptors (GPCR) adrenergic receptors, 249–250 endothelial barrier function, 252 ET receptors, 251 GTPase activity, 246 5-HT receptors, 251 muscarinic cholinergic receptors, 250 prostanoid receptors, 251–252 protein subunit, 246 pulmonary arterial vasodilation, 247–248 pulmonary vascular cells, 249 purinergic receptors, 250–251 stoichiometric relationship, 246–247 vasoconstriction and vascular cell proliferation, 248–249 Granuloma, 1286 Gravimetric quantification, pulmonary extravascular fluid accumulation, 478 H HAPE. See High-altitude pulmonary edema HapMap database, 572–573 Heart catheterization, right-sided CTEPH, 1569–1570 RVF, 1320 Heart failure with a preserved ejection fraction (HFpEF) diagnosis of PH, 1185 heart failure syndrome, differential diagnoses, 1184–1185

1637 Kaplan–Meier survival curves, 1185 mechanism, 1183–1184 prevalence, PH, 1184 Heart-lung transplantation, 1616 Heme oxygenase–1 cytoprotection adenoviral-mediated transduction, 781 bilirubin/biliverdin, actions, 782–783 carbon monoxide, actions, 781–782 dinucleotide repeat polymorphism, 781 protective effects, 783–784 therapeutic implications, 784 upstream enhancers, 781 molecular mechanisms, 779–781 Hemodynamic effects, AS, 1562–1563 Hemodynamics NO and endothelin 1 (ET–1), 876 primary pulmonary hypertension (PPH), 877 right-sided heart catheterization, 873–874 susceptibility characteristics, HAPE, 874 uneven regional HPV, 877 Hemolytic-anemia-associated pulmonary hypertension disorders, 1269, 1270 sickle cell disease (see also Sickle cell disease-associated pulmonary hypertension) diagnostic evaluation, 1275 echocardiography, 1276–1277 epidemiology, 1270–1272 laboratory analysis, 1275–1276 pathogenesis, 1272–1275 pulmonary function tests, 1276 recommendations, diagnostic screening, 1277–1278 right-sided heart catheterization, 1277 treatment, 1278–1280 thalassemia, 1270 Hemorrhagic shock, 297 Hemostasis definition, 372 Heparan sulfate proteoglycans, 407 Heparin, 394–395 Hepatic urea cycle, 1100 Herb toxins, 1054–1055 Hereditary hemorrhagic telangiectasia (HHT). See Osler–Rendu– Weber syndrome Hereditary telangiectasia, 1459 Heritable factors, 568 Heritable PAH (HPAH), 997–1003 Heterologous overexpression, 543 Hexagonal capillary network, 14, 18–19 High-altitude pulmonary edema (HAPE) capillary permeability increase, mechanism dynamic alterations, 881–882 stress failure, 881 clinical presentation diagnosis and evaluation, 873 epidemiology, 872–873 incidence rate, 872 pathophysiology alveolar fluid clearance, 880–881 hemodynamics, 873–878 inflammation, 878–880 prevention ascent rates and activity level, 882 pharmacological prophylaxis, 883–884 susceptibility prediction, 882–883 pulmonary artery pressure (PAP), 874

1638 High-altitude pulmonary edema (HAPE) (cont.) subclinical, 872 susceptibility, 872 treatment, 884 High-altitude pulmonary hypertension (HAPH) clinical manifestations and complications, 1216 diagnosis, 1216–1217 etiology, 1212–1213 in newborns and children, 1217–1218 pathophysiology, 1215 prognosis, 1218 risk factors and genetics, 1213–1215 treatment, 1218 High-frequency oscillatory ventilation (HFOV), 1115 Histamine model, 474 Histone deacetylase 7 overexpression application, 646 protocol, 645–646 HIV-associated pulmonary arterial hypertension (HIV-PAH) clinical presentation and diagnostic evaluation, 1036–1037 vs. idiopathic pulmonary arterial hypertension, 1036 key observations, 1034 pathobiology direct effects of HIV, 1034–1035 host response and factors, 1035–1036 pathology, 1034 risk factors for, 1036 symptoms, 1036 treatment of antiretrovirals, 1039 bosentan, 1038 calcium channel blockers, 1037 epoprostenol, 1038 sildenafil, 1038–1039 Homeostasis, 239. See also Calcium homeostasis Homeostatic Oxygen-sensing System carotid body, 314 ductus arteriosus, 315 Hormone replacement therapy, 1048 HPAH. See Heritable PAH (HPAH) HPV. See Hypoxic pulmonary vasoconstriction (HPV) 5-HTT and 5-HT receptor, 768 Hughes–Stovin syndrome, 1340 Human embryonic stem cells (hESCs) characterization, 617–618 cultures initiation cell density, 615 generating MEF conditioned medium, 615 preparing feeder dishes, 615 thawing, 615–616 derivation, 614 derived SMC, 640–641 differentiation into ECs, by VEGF expression, 645 into SMCs, adherent monolayer culture, 642–643 differentiation via EBs, 618–619 feeder-free culture, 617 feeding, 616 freezing, 617 passaging enzymatic, 616–617 mechanical, 616 propagating, 614–615 reagents, 619–620 Human gene mutation database (HGMD), 572 Human herpesvirus 8 (HHV8), 586

Index Human pulmonary circulation alveolar and extra-alveolar vessels, 9 blood pressure measurement, 6–7 cardiac catheterization, 8–9 discovery, 5 Galen’s scheme, 3–4 hypoxic pulmonary vasoconstriction, 11 longitudinal distribution of vascular resistance, 10–11 nongravitational factors, 10 nonrespiratory functions, 11 pulmonary blood flow measurement, 7–8 pulmonary capillaries, discovery of, 5–6 Starling resistor effect, 9 topographical distribution, 9–10 transit of blood, 4–5 Human vs. mouse ES cells, 639 Husbandry breeding, 463 caging, 463 genotyping, 463 3-Hydroxy–3-methylglutaryl-coenzymeA (HMG-CoA) reductase inhibitors, 889 5-Hydroxytryptamine (5-HT) receptors, 1505–1506 signaling, 1506 transporter (5-HTT) gene polymorphism, 1504–1505 immunolocalization, 1503, 1504 overexpression, 1503–1504 potential usefulness, 1505 tryptophan hydroxylase (Tph), 1502–1503 5-Hydroxytryptamine. See Serotonin Hyperoxia-induced PH angiograms, 735, 736 distal pulmonary vessels and capillaries, loss of, 740–741, 743, 744 hemodynamic measurements, 735 perivascular cell development, 736–741 Hyperpolarized 3He imaging, 517–518 Hypertension, pulmonary, 520. See also Pulmonary hypertension arterial and precapillary vessel pathology adventitial lesions, 1417 intimal lesions, 1416–1417 medial lesions, 1417 capillary pathology, 1417 characteristic pathologic appearances chronic thrombotic and embolic disease, 1421 IPAH and familial PAH, 1419 left-sided heart disease, 1420–1421 PAH-related diseases, 1419–1420 PCH and VOD, 1420 respiratory system and hypoxemia, 1421 sarcoidosis, 1421 components capillaries, 1415 pathology vs. physiology, 1415 pulmonary arteries and precapillary vessels, 1414–1415 venules and veins, 1415 inflammation, 1417–1418 parenchyma pathology, 1419 specimens preparation, 1413–1414 systematic diagnostic approach, 1415–1416 thromboembolic disease, 1418–1419 venous pathology, 1417 Hypothesis testing one-sample vs. two-sample, 653–654 p values, 653

Index Hypoxia, 866 carotid body sensing, 676–677 vascular responses, 677 Hypoxia-inducible factor (HIF) erythropoietin (EPO), 715 isoforms, 714–715 oxygendependent- degradation-domain (ODD), 715 target genes, 715 Hypoxia-inducible factor 1a modulates kca channel expression, 729–731 Hypoxia-mediated pulmonary hypertension Ca2+ and ion channels, pathogenic roles abnormal homeostasis, 683 acute hypoxic regulation, 688–690 chronic hypoxic regulation, 690–691 cytosolic concentration, regulation, 685–687 membrane potential, regulation, 687–688 microdomains and organized signaling, 691–692 Hypoxia tolerance, 297 Hypoxic pulmonary vasoconstriction, 11, 296 Hypoxic pulmonary vasoconstriction (HPV), 66, 147–148, 520–521, 871, 873–878 calcium sensitization, 679–680 calcium sources, 677–678 carotid body sensing, 676–677 characteristics, 313 diffusible mediator, 313 effector protein, 313 K+ channels, 318–319 membrane potential vs sarcoplasmic reticulum (SR), 678–679 oxygen sensing examples, 676 properties, 315–316 pulmonary circulation animal models, 316 properties,humans, 315–316 redox mechanism mitochondria, 316–317 NOX isoforms, 317–318 redox model, 314 redox signaling, 318 redox theory, 314 ROS role ETC signals and inhibitor, 320 hemodynamic response, hypoxia, 320 hypoxia decreases ROS generation, 319 hypoxia increases ROS generation, 319–320 vascular responses, 677 Hypoxic ventilatory response (HVR), 1224 Hypoxic ventilatory responsiveness (HVR), 874 I Idiopathic pulmonary arterial hypertension (IPAH), 751, 997, 998, 1002–1004 caveats, 948 diagnosis acute vasodilator testing, 985 signs, 983 symptoms, 983 testing, 983–985 echocardiographic assessment, 946 epidemiology, 979 impact of therapy, 948–950 pathogenesis, 981–983 pathology and pathophysiology, 980–981 patient distribution, 945 prognostic factors, 946–948

1639 role of TRPC6 siRNA downregulation of, 564 generation of recombinant adenovirus, 562–563 history, 562 synthesis and selection of, 562 treatment and prognosis altitude avoidance, 985 calcium channel antagonists, 987–988 combination therapy, 991–992 diuretics, 987 endothelin receptor antagonists, 990 endothelium-derived mediators, imbalance offset, 988 exercise and physical activity, 985 experimental therapies, 993 lung transplantation, 992, 993 phosphodiesterase–5 inhibitors, 990–991 pregnancy avoidance, 986 prostacyclin analogues, 988–989 supplemental oxygen, 987–988 survival, 993 vasoreactivity test, 992 warfarin, 986 WHO functional classification, 945, 946 ILD. See Interstitial lung disease (ILD) Iloprost combination with, 1457–1458 inhaled, 1456–1457 intravenous, 1455 IMAC. See Immobilized metal affinity chromatography Imaging chest X-ray posterior–anterior and lateral, 1403 pulmonary emboli findings, 1404 CT pulmonary angiograms advantages, 1407–1408 pulmonary arterial, 1405–1406 magnetic resonance angiography (MRA), 1408 pulmonary angiography, 1408–1410 radionuclide lung scans pulmonary emboli, multiple large and small, 1405, 1406 pulmonary scintigraphy, 1404 usage, purpose of, 1403 Immobilized metal affinity chromatography (IMAC), 609 Immunofluorescence imaging, 628–629 Immunohistochemistry, 626–628 Increased lung vascular permeability. See Lung vascular permeability Induced pluripotent stem cells. See Human embryonic stem cells (hESCs) Inferior vena cava (IVC) filters, 1236 In-gel digestion, protein identification, 602–603 Inhaled iloprost dosage, 1540 FDA approval, 1540 patient experience, 1541 patient teaching, 1541 side effects, 1541 Inhaled nitric oxide, 1371–1372 Inhaled nitric oxide (iNO), 1099 bronchopulmonary dysplasia (BPD), 1495–1498 perioperative pulmonary hypertension, CHD, 1493–1494 PPHN clinical trial, 1487–1488 dosing and weaning, 1488–1492 ECMO, 1492–1493 premature infants, 1494–1495 recommendations, postoperative setting, 1494

1640 Inhaled nitric oxide (iNO) therapy, 1114 Inositol 1,4,5-trisphosphate (IP3), 336–337 Integrins, 790 Interferon-a2, 1049 Interleukin–6 (IL–6), 901 Interleukin–10 (IL–10), 395 Intermediate-sized arteries and microcirculation, 81–83 Interstitial lung disease (ILD) causes, 1199 PH clinical manifestation, 1202–1203 complications and prognosis, 1207 diagnosis, 1203–1204 epidemiology of, 1199 etiology and pathogenic mechanisms, 1199–1201 pathology, 1201–1202 treatment, 1204–1207 types, 1197 Intrapulmonary shunt, 1113 Intravenous vasodilator drug therapy, 1114 Ion channel currents macroscopic currents channel gating properties, 508 channel permeation and ion selectivity, 507–508 passive cell membrane properties, 507 unitary currents channel conductance, selectivity, and current amplitude, 506 channel gating, 506 channel open probability, 506 Ion channels and transporters aquaporins, 239 calcium channels cytosolic Ca2+ concentration regulation, 226–231 PASMC contraction and proliferation, 224–226 chloride channels calcium-activated chloride channels, 237–238 voltage-gated Cl- channels, 239 volume-sensitive chloride channels, 239 cytosolic electrolyte concentrations, 224 electrical and chemical gradients, 224 homeostasis and disease, 239 plasmalemmal K+ channels, 240 potassium channels ATP-sensitive K+ channels, 233 Ca2+-activated K+ (KCa) channels, 233 KCNQ channels, 234 K2P channels, 234 Na+-K+-ATPase, 234–235 structure and function, 232 voltage-gated K+ channels, 231–233 sodium channels amiloride-sensitive epithelial Na+ channel, 236 expression and function, 236 Na+-H+ exchanger, 236–237 Na+-Mg2+ exchanger, 237 structure and function, 235 voltage-gated Na+ channels, 235–236 IonWorks HT system, 503 IPAH. See Idiopathic pulmonary arterial hypertension Ischemia, 474–475 K K+ and Ca2+ homeostasis, 768–769 Kaplan–Meier curves, 661–662

Index K+ channels classes, 126–127 hypoxic pulmonary vasoconstriction, 318–319 and hypoxic pulmonary vasoconstriction (HPV), 127 and proliferation, 127 Knockouts and knock-ins technology, 460 L Laminar shear stress, 788, 791, 796, 797 Large vessel pulmonary arteritis. See Takayasu’s arteritis (TA) Leflunomide, 1048 Left-to-right shunt, pulmonary hypertension, 455 Left ventricular compression vs. underfilling, 1184 Left ventricular diastolic heart function. See Pulmonary hypertension (PH) Levonorgestrel, 1382 Lipopolysaccharide (LPS), 476–477 Living-donor lobar lung transplantation (LDLLT) complications in donor, 1606 recipient, 1606–1607 indication donor selection, 1603 recipient selection, 1602–1603 size matching, 1603–1604 operative technique donor operation, 1604 recipient operation, 1604–1605 outcome and prognosis, 1607–1608 postoperative management, 1605–1606 principle, 1602 Logrank test, 662 Low molecular weight heparin (LMWH), 1524 Low molecular weight heparins (LMWHs), 1235 L-Tryptophan, 1056 Lung functional and anatomic aspects (see Microcirculation) transit of blood, 4–5 vascular development, 25–30 vascular morphogenesis, 30–42 vascular remodeling (see Pulmonary vascular remodeling) vascular restriction acquired/idiopathic PH, 752–753 in adult respiratory distress syndrome, 751–752 in neonatal respiratory distress, 753 vascular templates in adult, 48–49 fetal lung, 42–45 neonatal and child, 46–48 Lung diseases bronchial circulation, human asthma, 444 bronchiectasis and tuberculosis, 445 chronic bronchitis and emphysema, 445 pulmonary edema, 445 tissue repair, 445 tumors, 445 Lung fluid absorption catecholamine regulation, 864–865 non-catecholamine regulation, 865–866 tight junction, 861–862 transepithelial fluid movement, 862 Lung injury, acute. See Acute lung injury Lung transplantation absolute contraindication, 1597

Index acute and chronic rejection, 1618–1619 bilateral adult, 1617 pediatric, 1617 secondary pulmonary hypertension, 1617 donor organ allocation, 1615–1616 donor organ recovery, 1598–1599 early postoperative issues, 1618 epoprostenol, 1611–1612 evaluation financial considerations, 1593 medical evaluation, 1592 psychosocial evaluation, 1593 transplant surgery timing, 1593–1594 type, 1594–1595 extracorporeal membrane oxygenation (ECMO), 1599–1600 functional outcomes and quality life, 1620–1621 heart-lung transplantation adult, 1616 pediatric, 1616 heart transplantation, combination, 1598 history, 1597 infection and malignancy, 1619 intermediate and long-term medical complications, 1619–1620 liver, 1617 living-donor lobar transplantation, 1617 management, role of, 1612 options for pediatric, 1615 unilateral and bilateral grafts, 1614–1615 patients selection criteria disease-specific, 1613 general medical concerns, 1612–1613 pediatric patients, 1614 secondary pulmonary hypertension, 1613–1614 postoperative considerations, 1600 pregnancy, posttransplant patients, 1620 recipient preparation, 1599 relative contraindications, 1597–1598 single vs. double, 1598 specific indications, 1598 unilateral adult, 1616 pediatric, 1616 secondary pulmonary hypertension, 1617 Lung Transplantation, IPAH, 992, 993 Lung vascular development alveolar-capillary dysplasia, 51 bronchopulmonary dysplasia, 52 dysanaptic growth, 49–50 fetal and postnatal lung development, stages, 26, 28 human lung development, 25, 27 induced postnatal growth, 52–53 laws, 26, 27 main pulmonary artery branches development, 25–26, 28 morphogenesis, regulation of assembly, prespecification patterns, 31–32 vessel morphogenesis, 32–42 pulmonary bed, 25, 26 temporal-spatial stages, mouse lung, 53 vascular restriction and wall remodeling, 51–52 vascular templates adult lung, 48–49 fetal lung, 42–45 neonatal and child lung, 46–48 vascular units development failure, 50

1641 Lung vascular dysfunction, ventilator-induced mechanical stress barotrauma and biotrauma, 916 bioactive molecular interactions coagulation, thrombin, 925–926 GTPases, vascular permeability, 926 hepatocyte growth factor (HGF), 925 vascular endothelial growth factor (VEGF), 925 cellular mechanisms ion channels, 917 mechanosensors, 916–917 cyclic stretch effects, pulmonary vascular pathophysiological responses apoptosis, 923 endothelial permeability, 922 inflammation, 922–923 mechanosensing and mechanotransduction, pulmonary vasculature caveolae, 919 cell-cell adhesions, 918 cell-matrix contacts, 917–918 heterotrimeric G proteins, 918–919 nonreceptor protein tyrosine kinases, 920 phosphatidylinositol 3-kinase/Akt signaling, 921 reactive oxygen species, 921–922 receptor tyrosine kinases, 918 small GTPases, 920 stress-activated kinases, 919–920 plasma membrane reparation mechanisms, 916 pulmonary circulation capillary stress failure, 915 physiologic and pathologic lung stretch regimen, 915 vascular wall and hypertension, 914 pulmonary vasculature, lung injury magnitude-dependent effects, 924–925 two-hit model, 923 treatment b-adrenergic agonists, cyclic AMP levels, 928 FTY720, 926–927 hepatocyte growth factor, 928 oxidized synthetic 1-palmitoyl–2-arachidonoyl-sn-glycero–3phosphorylcholine, 927–928 Rho kinase inhibitors, 927 sphingosine 1-phosphate, 926 vascular wall, mechanical forces shear stress, 914 strain, 914 Lung vascular permeability cross-talk mechanisms between endothelial barrier enhancement and disruption, 478 edema fluid formation, 472 evaluation bronchoalveolar lavage, 481 gravimetric quantification, 478 noninvasive quantification, 478–480 protein, 481 vascular permeability measurements, 480–481 fluid accumulation in lungs, 472 ideal model, 472 imaging methods, 480 models endotoxin and polymicrobial, 476–477 histamine, 474 mechanical ventilation, 477 oleic acid (OA), 477 oxidant generation and ischemia/ reperfusion injury, 474–475 thrombin, 472 vascular endothelial growth factor (VEGF), 475–476

1642 Lung vessel morphogenesis arterial and venous specification and differentiation, 38–39 endothelial tube formation, 32–38 vessel and capillary network formation, 40–42 vessel and capillary wall maturation, 39–40 Lung volume reduction surgery ( LVRS), 1372 Lymphatics, 15 M Macroaggregated albumin (MAA), 1404 Macrophage migration inhibitory factor (MIF), 905 Macroscopic ion channel currents channel gating properties, 508 channel permeation and ion selectivity, 507–508 passive cell membrane properties, 507 Magnetic resonance angiography (MRA), 1408 Magnetic resonance imaging (MRI) CTEPH, 1572–1573 RVF, 1320 Magnetic resonance imaging (MRI) technology angiography, 515–516 arterial spin labeling (ASL), 517 blood pool agents, 518–519 cardiac imaging for right ventricular assessment, 516–517 diagnosis, pulmonary vascular disease embolism, 519–520 emphysema, 521–522 hypertension, 520 hypoxic pulmonary vasoconstriction (HPV), 520–521 hyperpolarized 3He imaging, 517–518 MALDI-TOF mass spectrometry, 599 Manson’s blood fluke, 1284 Marfan syndrome, 86 Mazindol, 1047 Mean pulmonary artery pressure (mPAP), 1198, 1199, 1202–1204 individual variation nonresponders, 1214 sea level and high-altitude residents, 1216 variation age function, 1214 altitude function, 1214 Mechanical dissociation, 616 Mechanical-ventilation-induced lung injury. See Ventilator-induced lung injury (VILI) Mechanosensing, pulmonary vasculature caveolae, 919 cell-cell adhesions, 918 cell-matrix contacts, 917–918 heterotrimeric G proteins, 918–919 nonreceptor protein tyrosine kinases, 920 phosphatidylinositol 3-kinase/Akt signaling, 921 reactive oxygen species, 921–922 receptor tyrosine kinases, 918 small GTPases, 920 stress-activated kinases, 919–920 Mechanosensory complexes, 790 Mechanotransduction, 787 Mechanotransduction, pulmonary vasculature caveolae, 919 cell-cell adhesions, 918 cell-matrix contacts, 917–918 heterotrimeric G proteins, 918–919 nonreceptor protein tyrosine kinases, 920 phosphatidylinositol 3-kinase/Akt signaling, 921 reactive oxygen species, 921–922

Index receptor tyrosine kinases, 918 small GTPases, 920 stress-activated kinases, 919–920 Medications, PAH anticoagulation, 1537 calcium channel antagonists, 1537–1538 digoxin therapy, 1537 diuretic therapy, 1536 endothelin receptor antagonist therapy ambrisentan, 1539–1540 bosentan, 1538–1539 sitaxsentan, 1540 phosphodiesterase inhibitor therapy, 1540 prostanoid therapy inhaled iloprost, 1540–1541 intravenously administered epoprostenol, 1543–1546 intravenously administered generic epoprostenol, 1546 intravenously administered treprostinil, 1546–1547 subcutaneously administered treprostinil, 1542–1543 Membrane-associated anticoagulants, 406–407 Membrane depolarization, 793 Membrane potential, regulation K+ channels, 687–688 molecular composition and structure, 688 Nernst and Goldman–Hodgkin–Katz equations, 687 Membrane potential vs sarcoplasmic reticulum (SR), 678–679 Mesenchymal, differentiation adipocyte, 632 chondrocyte, 633 myocyte, 633 osteocyte, 633 MetaCore, software package, 577 Methamphetamine, 1054 Microarray gene expression, peripheral blood monocytes, 583–584 Microcirculation alveolar capillary networks to respiratory movements, 21–23 bronchial and pleural circulation, 15 conducting arteries, 16–18 connective tissue continuum, role of, 15–16 extraalveolar arteries and connective tissue continuum, 16 gas exchange, 23 static capillary alveolar microcirculation corner capillaries, 19–20 corner pleats, 20–21 hexagonal capillary network, 14, 18–19 primary vs. secondary alveolar walls, 19 systemic component of, 15 technical fixation problems, 14–15 vascular perfusion, 14–15 MicroRNA species, 588 Microvascular remodeling lung breathing air after high oxygen, 754 lung breathing high oxygen, 753 Mirena®, 1383 Mitogen-activated protein kinase (MAPK), 253 Mixed connective tissue disease (MCTD), 1015 MLCK inhibitors, 208 Monocrotaline-induced pulmonary hypertension, 453–454 Mouse embryonic fibroblasts (MEFs) generation, 615 Mouse thrombosis model of conditional fetal liver kinase–1 deletion, 857–858 with Staphylococcal infection, 857 MRI technology. See Magnetic resonance imaging technology Multidetector-row CT (MDCT) technology, 96, 97 angiography, 513 blood pool agents, 518–519

Index diagnosis, pulmonary vascular disease embolism, 519–520 emphysema, 521–522 hypertension, 520 hypoxic pulmonary vasoconstriction (HPV), 520–521 dual-energy CT, 512–513 four-dimensional X-Ray CT perfusion measurements, 513–514 quantitative assessment of volumetric chest images, 513 scanners development, 512 Multiplex ligation-dependent probe amplification (MLPA), 1001 Mutagenesis pulmonary hypertension BMP receptor polymorphisms, 550–553 oxygen-sensitive potassium channel, Kv1.5, 546–550 recombinant mutant clones, design chimeras construction, 545 large deletions, 544–545 single base pair change, 545 Myelofibrosis, 381 Myeloproliferative disorders clinical presentation and evaluation, 1079 diagnostic testing and management, 1079 key concepts, 1079–1080 pathophysiology, 1078–1079 prevalence of, 1077–1078 syndromes, 1077 Myocyte differentiation, 633 Myosin light chain (MLC), 890 phosphorylation and dephosphorylation, 154–156 Myosin light chain kinase (MLCK), 148, 890, 904–905 Myosin light chain phosphatase (MLCP), 148 N Nanion, Port-a-Patch, 504 Natriuretic peptides with PDE inhibitors, 1513–1514 Near-normal perfusion, 387 Neomuscularization, 739–740 Neonatal intensive care unit (NICU), 1084, 1089, 1092 Neonatal respiratory distress, 753 Nernst and Goldman–Hodgkin–Katz equations, 687 Newborn, pulmonary hypertension, 455 New York heart association (NYHA)/WHO functional class, 1037, 1038 Nitric oxide, 699–700 anticoagulants elaborated by vascular endothelium, 405 exogenous stimuli, 174–176 production and action, 171–172 regulation, 172–174 vasoactive mediators, 402 Nitric oxide (NO) effects on pulmonary vasculature, 122–123 isoforms, 122 for PAH treatment, 123 with PDE inhibitors, 1513 Nitric oxide synthases, 464 Noninvasive quantification, pulmonary extravascular fluid accumulation, 478–480 Nonmuscular alveolar wall vessels, 765–766 Nonselective (dual) endothelin receptor blockade. See Bosentan Nonsense-mediated decay (NMD), 1002 Nonspecific interstitial pneumonia (NSIP), 1198 Nonsteroidal anti-inflammatory drugs, 1053 Notch3 signaling arterial smooth muscle cell, 826–827 development, 828–829

1643 extracellular domain, 826 feature, 826 N-terminal pro-brain natriuretic peptide (NT-proBNP), 1234 Nuclear factor kappa B, 717–718 Nuclear factor of activated T cells (NFAT), 338–339 Nursing care, PAH characteristics, 1531 health care professionals, collaboration with, 1552 insurance issues, 1555–1556 patient and family support caregivers, 1551 chronic nature of PAH, 1550 communication, 1550 future planning, 1552 outside resources, 1551 patient assessment bloodwork, 1534 for cardiac signs and symptoms, 1533 common symptoms, 1533 for dyspnea, 1532 for functional level, 1533 pulmonary vasoreactivity testing, 1535 right-sided heart catheterization, 1535 for right-sided heart failure, 1533 for supplemental oxygen, 1532–1533 transthoracic echocardiogram, 1534 walk test, 1534 role in, 1532 special circumstances contraception, 1553 emergencies or hospitalizations, 1554 patients failing medical therapy, 1554–1555 pregnancy, 1552–1553 surgery, 1553–1554 symptoms, 1532 treatment plan diet recommendations, 1548–1549 medications, 1536–1547 oxygen therapy, 1547–1548 patient education, 1535–1536 physical exercise, 1548 routine bloodwork, 1548 serial diagnostic studies, 1549 sleep, 1549 weight, 1549 O Obstructive sleep apnea (OSA), 1095–1096 Odds ratios (ORs), 1253, 1254 Oleic acid (OA) model, 477 OLS regression, 660 Online Mendelian Inheritance in Man (OMIM) database, 572 Operability, 1143–1145 Oral contraceptives, 1048 Osler–Rendu–Weber syndrome, 1301 Osler’s disease. See Hereditary telangiectasia Osteocyte differentiation, 633 Oxidant generation, 474–475 Oxygendependent- degradation-domain (ODD), 715 Oxygen sensing examples, 676 Oxygen-sensitive potassium channel, Kv1.5, 546–550 Oxygen-sensitive transcription factors activated T cells, nuclear factor, 719–720 activating protein 1, 719 cyclic AMP response element binding protein, 720

1644 Oxygen-sensitive transcription factors (cont.) hypoxia-inducible factor (HIF) erythropoietin (EPO), 715 isoforms, 714–715 target genes, 715 level of, 714 nuclear factor kappa B, 717–718 PPAR gamma, 718 pulmonary hypertension, 718 transcription factor growth-inducing, 719 zinc finger, 718–719 Oxygen therapy, PAH, 1547–1548 Ozone, pulmonary vasculature Bohr model, 966 coarse mode and fine mode, 965 inhaled vs. exhaled dose, lung, 966 uptake rate, 967 P PA banding, 1133 PAH. See Pulmonary arterial hypertension Particulate air pollution, pulmonary vasculature activation, coagulation system, 974 coarse and fine mode, 965 epidemiology, 972 excess risk estimates, 972–973 surface chemistry, 965 total mortality, 972 PASMC proliferation, factors calcium and smooth muscle proliferation, 327–328 growth factors receptor tyrosine kinases (RTKs), 326 transforming growth factor b superfamily, 326–327 hypoxia, 329–330 inflammation, 330–331 mechanical stress, 330 vasoactive substances angiotensin II, 329 endothelin (ET), 329 serotonin, 328 thromboxane A2 (TXA2), 329 VSMC proliferation, 325 PASMCs. See Pulmonary artery SMCs Patch clamp techniques and recordings automated technologies, 497–501 cell positioning, 502 glass pipette interfaces, 502 planar-array-based approaches, 502–504 planar electrodes, 501–502 surface electrogenic event reader (SURFE2R), 505 basic techniques configurations, 497, 499–500 gigaohm seal formation, 495 troubleshooting and limitations, 500–501 electrophysiology first century: 1900s–2000, 495–496 new millennium improvements, 496–497 ion channel currents macroscopic currents, 506–508 unitary currents, 505–506 PatchXpress (Molecular Devices), 504 Patent ductus arteriosus (PDA), 1224 PBEF. See Pre-B-cell colony-enhancing factor (PBEF) PCR cloning, 540

Index PDE5 inhibitors acute effects, 1482 chronic treatment, 1482 clinical pharmacology blood pressure, 1480 cardiac function, 1480–1481 endothelial function, 1481 endothelial progenitor cells (EPCs), 1481 platelets, 1481 hypoxia-associated pulmonary hypertension, 1483 pharmacokinetics and pharmacodynamics absorption, 1479–1480 drug interactions and elimination, 1480 metabolism, 1480 pharmacology, 1479 preclinical studies, 1481–1482 PECAM1+ cell isolation, 644 Pediatric pulmonary hypertension aim, 1083 assessment cardiac catheterization, 1085–1086 echocardiography, 1085 bronchopulmonary dysplasia (BPD) clinical presentation, 1090 management, 1091–1092 pathogenesis, 1089–1090 patient management, NICU discharge, 1092–1093 PH assessment, 1090–1091 comprehensive program, 1103 congenital diaphragmatic hernia (CDH) clinical presentation, 1087 management, 1088 pathophysiology, 1086–1087 patient management, NICU discharge, 1089 PH assessment, 1088 congenital heart disease (CHD) assessment, 1097–1098 PH presentation, 1098 definition, 1084–1085 developmental age and disease spectrum, 1083, 1084 guidelines, 1101–1102 interventions, 1102 neonatal presentation, 1083, 1084 perioperative PH assessment, 1098–1099 clinical presentation, 1098 management, 1099 PH management, repaired CHD setting, 1101 persistence, neonatal period age and disease processes, 1093 assessment, CLD patients, 1094–1096 clinical presentation, CLD patients, 1094 management, CLD patients, 1096 prevalence and severity, 1094 progressive PH, 1086 Peptide mass fingerprinting (PMF), 600 Pergolide, 1048 Periovular granulomas, 1286 Peripheral PA stenosis, 1130 Perivascular cell development cellular and molecular mechanisms of, 742, 744, 745 in distal vessels and capillaries cell proliferation, 737, 738 cell recruitment, 737–739 neomuscularization, 739–741 Peroxisome-proliferator-activated receptor a (PPARa), 893

Index Persistent pulmonary hypertension of newborn (PPHN) vascular units development, 51–52 Persistent pulmonary hypertension of the newborn (PPHN) chest X-rays, 1111 clinical aspects, 1111–1112 clinical presentation and evaluation, 1112–1113 clinical trial, 1487–1488 dosing and weaning, 1488–1492 ECMO, 1492–1493 experimental, pathophysiology of, 1109–1111 NO-cyclic GMP cascade, abnormalities, 1109, 1110 treatment cyclic AMP (cAMP) levels, 1114, 1116 extracorporeal membrane oxygenation (ECMO), 1116 high-frequency oscillatory ventilation (HFOV), 1115 inhaled nitric oxide (iNO) therapy, 1114 intravenous vasodilator drug therapy, 1114 management, 1113 mechanical ventilation, 1114 sildenafil, 1116 PH. See Pulmonary hypertension (PH) Phendimetrazine, 1047 Phenformin, 1048 Phentermine, 1047 Phenylpropanolamine, 1047 Phosphatase dephosphorylation assays, 609 Phosphodiesterase inhibitors, 1325–1326 Phosphodiesterase inhibitors, PAH cGMP pathway, 1478 in lungs cGMP pathway and PDE5, 1479 phosphodiesterase type 1, 1478 phosphodiesterase type 5, 1478 PDE5 inhibitors acute effects, clinical studies, 1482 chronic treatment, clinical studies, 1482 clinical pharmacology, 1480–1481 hypoxia-associated pulmonary hypertension, 1483 pharmacokinetics and pharmacodynamics, 1479–1480 pharmacology, 1479 preclinical studies, 1481–1482 Phosphodiesterase inhibitor therapy, PAH, 1540 Phosphopeptide enrichment strategies, 609–610 Phosphospecific antibodies, 609 32 P labeling, 608 Planar-array-based approaches, 502–504 Planar electrodes, 501–502 Plasma clotting factors structure epidermal growth factor (EGF) like domains, 359–360 FV and FVIII clotting cofactors, 360 Gla domains, 359 serine proteases domains, 360 Plasminogen activator inhibitors functions, 418–419 vascular wall, 419 Plasminogen system interaction, vessel wall components, 412 plasminogen activator inhibitors functions, 418–419 vascular wall, 419 plasminogen and plasmin interaction, with ECs, 414–415 receptors and modulation, 413–414 regions, 412–413 role, 415

1645 tissue plasminogen activator (tPA) receptors interaction, with ECs, 416 roles, 416 structure and binding, 415–416 uninjuired vessel wall, 419 urokinase plasminogen activator ECs, 417–418 functions, 418 receptor, 416–417 Platelet-derived growth factor (PDGF), 127–128, 746, 748–750, 1300 Platelets coagulation factor interactions, 372–373 hemostasis and thrombosis, 372 leukocyte interactions, 373 pathology hermorrhagic interplay, 378 thrombotic interplay, 378–382 production, 371–372 pulmonary vascular anatomy and rheology, 374 secretion, 373 thrombopoiesis, 372 thrombosis and inflammation, 373–374 Virchow’s triad blood, 375–377 blood flow, 374 vessel, 377–378 Platelet tethering and activation collagen pathway, 403 tissue factor pathway, 403–404 Plexiform lesions, 765 Plexogenesis PH models S100A4, 468 vascular endothelial growth factor A (VEGFA), 468 Polymicrobial model, 476–477 POPH. See Portopulmonary hypertension (POPH) Portohypertension, 1290 Portopulmonary hypertension (POPH), 951 diagnostic criteria, 1023, 1024 etiology and pathogenetic mechanisms, 1024 history, 1023–1024 IV epoprostenol effect, 1028 long-term survival, 1029 mild, autopsy specimen, 1024 pressure-flow relationships, 1027 prevalence and clinical manifestations, 1024–1025 prognosis, 1029 screening and diagnostic criteria, 1026–1027 setting, liver transplantation outcomes, 1028 treatment, 1027–1029 Postcapillary wedge pressure (PCWP), 1389 Potassium channels. See Ion channels and transporters Power and sample size determination analyses, 654–655 compute, 655 error types, 652 sample size vs. effect size vs. power, 653–654 PPAR gamma, 718 Pre-B-cell colony-enhancing factor (PBEF), 906–907, 212 Pregnancy and contraception, PAH estrogen and progesterone pills, 1382 Federal Drug Administration (FDA) drug ratings advanced PAH drugs, 1381, 1382 general, 1381, 1382 levonorgestrel, 1382 mortality rate, 1378 permanent sterilization, 1383

1646 Pregnancy and contraception, PAH (cont.) physiological changes hemodynamic, 1378 serial hemodynamics, single pregnanacy, 1378 recommendations, 1383 survival, 1379 therapies, adverse effects, 1381–1382 treatment anticoagulation, 1380 diagnosis and monitoring, 1380–1381 inhaled prostanoids and NO, 1379 labor and delivery, 1381 prostacyclin, intravenous, 1379 Primary Sjogren’s syndrome (pSS), 1015–1016 Primary vs. secondary alveolar walls, 19 Procoagulation factors, pulmonary endothelium-leukocyte interaction circulating tissue factor, 404 platelet recruitment and thrombus growth, 404–405 platelet tethering and activation collagen pathway, 403 tissue factor pathway, 403–404 Progenitor cells, 585 characterization, 634 chemokine signaling, 819–820 contribution pulmonary vascular remodeling, 816–819 systemic vascular remodeling, 814–815 endothelial acetylated low density lipoprotein (LDL), 630 formation, 631–632 postnatal vasculogenesis mechanisms, 623–624 vascular growth factor, 622–623 endothelial, smooth muscle, and fibroblast/myofibroblast circulating SPCs, 812 fibrocytes differentiation, 813 hematopoietic lineage markers, 811 orgin, 812 side population, 814 vascular potential, 814 vasculogenic zone, 813 identification, 633–634 resident tissue, 624 Proinflammatory factors, pulmonary endothelium-leukocyte interaction activated protein C, 402 adhesion molecules, 401 cytokines, 402 pulmonary-endothelium-derived vasoactive mediators, 402 Prominent clubbing of digits, 1173 Propylhexedrine, 1047 Prostacyclin, 176–179, 405–406, 701–702 eicosonoids, 123 with endothelin receptor antagonists long-term clinical studies, 1514–1515 long-term experimental studies, 1514 with NO, 1512–1513 with PDE inhibitors acute clinical studies, 1511–1512 acute experimental studies, 1510–1511 long-term clinical studies, 1512 long-term experimental studies, 1512 PGI2 receptor, 123 Prostacyclin (epoprostenol), 1016 Prostacyclins, 1371 Prostaglandin, definition, 1451. See also Prostanoids Prostaglandins, 1279

Index Prostanoids beraprost, 1456 epoprostenol, 1454–1455 iloprost combination with, 1457–1458 inhaled, 1456–1457 intravenous, 1455 management chronic thromboembolic pulmonary hypertension (CTEPH), 1459 daily management issues, 1461 hereditary telangiectasia (Osler’s disease), 1459 liver diseases, 1459 lung diseases, 1459 lung transplantation, 1460–1461 prostanoid therapy, 1458 pulmonary veno-occlusive disease, 1458–1459 targeted PAH therapies, transition, 1459–1460 treatment, starter, 1459 properties differences, 1452–1453 hemodynamic effects, 1453–1454 prostaglandin, definition, 1451 treprostinil inhaled, 1458 intravenous, 1455 subcutaneous, 1455–1456 Prostanoid therapy, PAH inhaled iloprost, 1540–1541 intravenously administered epoprostenol, 1543–1546 intravenously administered generic epoprostenol, 1546 intravenously administered treprostinil, 1546–1547 subcutaneously administered treprostinil, 1542–1543 Protease-associated receptor–1 (PAR–1), 473 Protein C, 361, 362 Protein C and Protein S, 407 Protein C receptor, 406 Protein expression analysis, 571–572 Protein fingerprinting. See Peptide mass fingerprinting Protein interacting partner, detection of by antibody-based immunoprecipitation, 606 by recombinant protein-based affinity pull-down, 604–606 Y2H system for, 607–608 Protein microarrays, 606–607 Protein/peptide visualization, 597 Protein quantification gel electrophoresis, 534 isolation, 534 probe hybridization, 534–536 western blotting, 533–534 Protein S, 362–363 Proteins identification and characterization ESI-TOF mass spectrometry, 599–600 Fourier transform ion cyclotron resonance, 599 by in-gel digestion, 602–603 ionization, 598 MALDI-TOF mass spectrometry, 599 MS data analysis, 601–602 peptide mass fingerprinting, 600 quadrupole mass analyzers, 599 tandem MS, 600–601 TOF analyzer, 598–599 mixtures, multidimensional separation extraction and solubilization, 592–593 gel-based methods, 594–596

Index gel-free methods, 596–597 hybrid approaches, 597–598 Protein, vascular junctional permeability determination, 481 Proteomics four main areas, 591–592 vs. genomics, 591 methods identification and characterization of proteins, 598–603 multidimensional separation of protein mixtures, 592–598 P-selectin, 393–394 Pseudoglandular period, 138 Pulmonary angiography, 1408–1410 anatomy, 1394–1395 angioscopy, 1398–1400 findings, patterns of, 1395–1398 significance, in CTEPH patients, 1398 Pulmonary arterial hypertension apoptosis, 354–355 BMPR signaling animal studies, 831–832 cellular studies, 832 development, 832 mutations, 831 overview, 829–831 BMPR vs. notch signaling pathways, 828–829 endothelium-specific changes endothelin dysregulation, 186 histopathological changes, 184 leukotriene production, 186–187 NO pathway, 184 vascular effectors dysregulation, 186 notch3 signaling arterial smooth muscle cell, 826–827 development, 828–829 extracellular domain, 826 feature, 826 Pulmonary arterial hypertension (PAH), 983. See also Pulmonary hypertension atrial septostomy in (see Atrial septostomy) collagen vascular diseases (CVDs)-related, 1015–1019 (see also Collagen vascular diseases (CVDs)) drugs aglucerase, 1049 anorectic agents, 1043–1047 benfluorex, 1048 chemotherapy-related, 1049–1053 fluoxetine, 1043–1054 interferon-a2, 1049 leflunomide, 1048 nonsteroidal anti-inflammatory, 1053 oral contraceptives and hormone replacement therapy, 1048 pergolide, 1048 phenformin, 1048 endothelial apoptosis and repair apoptosome, 426 biochemical events, 427 BMPR2 signaling, 429–431 caspases, 426 defenition, 425 endothelial dysfunction, 426–428 EPC circulation, 432–433 genetics, 428 growth and survival experimental models, 431–432 history, 426 proliferation, 432 therapeutic implications, 433

1647 etiology of coinfection, 1290 hypothesis, 1288 immunological and inflammatory role, 1288–1290 portohypertension, 1290 familial and idiopathic, genetics of BMPR2 mutations, 1000–1003, 1005 BMP signaling pathway, 1004–1005 clinical evaluation and therapy, 1006 clinical transmission patterns and expression, 998–1000 epidemiology, 998 genetic modifiers, 1003–1004 hereditary hemorrhagic telangiectasia (see Rendu–Osler–Weber syndrome) history, 997 screening and counseling, 1006–1007 in Gaucher’s disease type I anticoagulation, 1067 antiremodeling and vasodilating agents, 1067 clinical presentation and evaluation, 1066 diagnostic testing, 1066 history and background, 1064 key concepts, 1067–1068 management, 1066–1067 pathophysiology, 1065–1066 prevalence of, 1064–1065 in glycogen storage disease type I clinical presentation and evaluation, 1063 diagnostic testing, 1063 history, 1062 incidence, 1062 key concepts, 1064 management and therapy, 1063–1064 pathophysiology, 1062–1063 prevalence of, 1062 microvascular remodeling lung breathing air after high oxygen, 754 lung breathing high oxygen, 753 in myeloproliferative disorders clinical presentation and evaluation, 1079 diagnostic testing and management, 1079 key concepts, 1079–1080 pathophysiology, 1078–1079 prevalence of, 1077–1078 syndromes, 1077 PLCH antiremodeling, and vasodilator agents, 1072–1073 clinical presentation and evaluation, 1072 diagnostic testing, 1072 epidemiologic risk factor, 1070 key concepts, 1073 management, 1072–1073 pathophysiology, 1070–1072 prevalence of, 1070 pregnancy and contraception estrogen and progesterone pills, 1382 Federal Drug Administration (FDA) drug ratings, 1381–1382 levonorgestrel, 1382 mortality rate, 1378 permanent sterilization, 1383 physiological changes, 1377–1378 recommendations, 1383 survival, 1379 therapies, adverse effects, 1381–1382 treatment, 1379–1381 right ventricular dysfunction in (see Right ventricular failure)

1648 Pulmonary arterial hypertension (PAH), 983. See also Pulmonary hypertension (cont.) in sarcoidosis antiremodeling and vasodilating agents, 1076–1077 clinical presentation and evaluation, 1075 diagnostic testing, 1075–1076 history and background, 1073 key concepts, 1077 management of, 1076 pathophysiology, 1073–1075 prevalence of, 1073 by schistosomiasis clinical presentations, 1292–1293 complications, 1283 definition, 1283 etiology, 1288–1290 immunological changes, 1286 issues and problems, 1293 life cycle of, 1284–1286 management of, 1293 parasite, 1284 pathological changes, 1286–1288 prevalence of, 1290–1291 scleroderma-related, 1012–1015 shear stress, pulmonary vasculature, 788–789 splenectomy-induced antiremodeling and vasodilating agents, 1069 clinical presentation, and evaluation, 1069 diagnostic testing, 1069 key concepts, 1070 management and therapy of, 1069 pathophysiology, 1068–1069 prevalence of, 1068 substance abuse, 1054 toxins herb toxins, 1054–1055 L-tryptophan, 1056 toxic rapeseed oil, 1055 Pulmonary arterial hypertension (PAH), epidemiology anorectic and stimulant agents, 952–953 classification, 943–945 collagen vascular diseases, 950–951 familial, 950 hemoglobinopathies, 953 human immunodeficiency virus, 951 idiopathic caveats, 948 echocardiographic assessment, 946 impact of therapy, 948–950 patient distribution, 945 prognostic factors, 946–948 WHO functional classification, 945, 946 non-PAH forms chronic respiratory disease, 954–955 chronic thromboembolic, 955 portal hypertension, 951–952 pulmonary veno-occlusive disease (PVOD), 954 risk factors, 944–945 schistosomiasis, 954 Pulmonary arterial sarcoma (PAS), 1344–1347 Pulmonary arterial tree perfusion models subject-specific, 96–97 summary, 95–96 Pulmonary arteries (PAs) types, 117 vascular tone regulation, 117–119 vasoactive influences, distribution of, 117, 118

Index Pulmonary artery (PA), 871, 873, 882 Pulmonary artery endothelial (PAECs) cell isolation theory, 485–486 cell storage, 489–490 cell thawing and reseeding, 490 functional characteristics, 491–492 morphological characteristics and identification, 489 primary culture preparation, 487–489 subculturing cells, 489 tissue collection, 487–489 Pulmonary artery endothelial cell (PAEC) calcium-sensitive channels, 727–728 consequences, 727 cytosolic calcium, 726 nitric oxide production, 727 properties, 725–726 role, 725 Pulmonary artery endothelial cells (PAECs), 1212 Pulmonary artery pressure (PAP), 874, 1154, 1155, 1157–1159, 1162, 1166, 1211, 1213–1219 Pulmonary artery SMCs (PASMCs), 325–331 Pulmonary artery smooth muscle cell (PASMC), 154 intracellular Ca2+ role calmodulin, 225 cell proliferation, 225 muscle contraction, 225 regulation, 225–226 plasmalemmal K+ channels role, 240 Pulmonary artery smooth muscle cells (PASMCs), 582, 981–982, 1212 cell isolation theory, 485–486 cell storage, 489–490 cell thawing and reseeding, 490 morphological characteristics and identification, 490 primary culture preparation, 487–489 subculturing cells, 489 tissue collection, 487–489 Pulmonary bed arteriograms and polymer cast, 25, 26 fetal and postnatal development, 40, 41 Pulmonary blood flow measurement of, 7–8 nongravitational factors affecting the distribution of, 10 topographical distribution of, 9–10 Pulmonary blood flow measurement, 7–8 Pulmonary capillary endothelium-bound (PCEB) ACE activity, 108, 110–112 Pulmonary capillary hemangiomatosis characteristics, 1348–1349 clinical features, 1349 imaging, 1349 pathologic findings and histopathologic differential diagnosis, 1349–1350 treatment and prognosis, 1350 Pulmonary capillary hemangiomatosis (PCH) characterization, 1297 clinical features and pathology capillary proliferation, lung biopsy, 1298 PVOD, 1298–1299 symptoms, 1298 differential diagnosis, 1299 management, 1299 mechanisms angiogenic factors, 1300 endothelial NO synthase (eNOS), 1301 hypoxia, 1299–1300

Index matrix proteases, 1300 Osler–Rendu–Weber syndrome, 1301 risk factors, 1299 Pulmonary capillary sheet model, 82 Pulmonary circulation. See also Human pulmonary circulation fetal and neonatal perinatal, developmental physiology, 139–143 vascular growth in developing lung, 136–139 functional state, 61 hypoxic pulmonary vasoconstriction animal models, 316 properties, humans, 315–316 time constanct, 68–69 Pulmonary circulation, CHD cardiac structural lesions complex lesions, 1128–1129 increased PA pressure and flow, 1126–1127 PA pressure, increase in, 1126 pulmonary blood flow (PBF) increase, 1126–1127 right-sided obstructive lesions, 1129–1130 clinical management prevention, 1132 surgical intervention, 1132–1133 therapy, 1133–1134 mechanical forces and vascular remodeling, 1131–1132 and vasoconstriction, 1131 microarchitecture morphometric insights, 1122 observations, 1121 pathological remodeling, angiographic assessment, 1122 pathology and physiology, 1120 pulmonary vascular resistance (PVR), 1119–1120 reversibility, 1122–1225 Pulmonary embolism, 455 mouse thrombosis model conditional fetal liver kinase–1 deletion, 853 with Staphylococcal infection, 852–853 nonresolving thrombosis model, 858–859 thrombosis and thrombus resolution complex vascular remodeling process, 853–856 plasmatic thrombosis, 852–853 vena cava stenosis model, 856–858 VTE, spectrum of epidemiology, 851, 852 risk factors, 852 varicose veins and deep vein thrombosis, 851–852 Pulmonary embolism (PE), 378 factor Xa inhibitors, 1526–1527 fondaparinux, 1524 low molecular weight heparin (LMWH), 1524 malignancy, 1526 recurrent VTE, 1526 thrombin inhibitors, 1527 thrombolytic therapy, 1521–1523 transient risk factor, 1525 treatment principles, 1521 unfractionated heparin, 1523 unprovoked VTE, 1525–1526 warfarin, 1525 Pulmonary embolism (PV). See Venous thromboembolism (VTE) Pulmonary emphysema, 521–522 Pulmonary endarterectomy, CTEPH for. See Chronic thromboembolic pulmonary hypertension Pulmonary endothelial ACE activity acute lung abnormalities, 111–112

1649 chronic lung abnormalities, 112 human coronary circulation, 112 Pulmonary endothelial clearance assessment carrier proteins, 109 ET–1 clearance, 109–110 Pulmonary endothelium. See also Endothelial regulation anticoagulation via, 407 derived vasoactive mediators, 402 leukocytes interaction with procoagulation factors, 403–405 proinflammatory factors, 400–402 modulation of coagulation anticoagulants elaborated by vascular endothelium, 405–406 anticoagulation via circulating factors, 407 membrane-associated anticoagulants, 406–407 Pulmonary fibrosis, 379–380 Pulmonary flow bruits, 1241 Pulmonary hemodynamics pulsatile-flow pressure and flow wave morphology, 69–70 time constant, pulmonary circulation, 68–69 vacular impedance, 67–68 steady-flow active hypoxic regulation, 65–67 passive regulation, 64–65 pressure-flow diagram, 63 vascular resistance, 61–62 Pulmonary hypertension (PH), 380–381. See also Chronic obstructive pulmonary diseases air pollution histology and vessel morphometry, 968 isolated vessel function, 969 right-sided heart function, 969–970 animal models chronic hypoxia-induced PH, 455 classification, 456–457 endothelin–1 (ET–1), 456 genetic models, 455–456 left-to-right shunt, 455 monocrotaline-induced PH, 453–454 newborn, 455 other conditions, 456 pulmonary embolism, 455 BMP receptor polymorphisms, 550–553 cardiac catheterization noninvasive imaging, role of, 1387–1388 pulmonary angiography, 1394–1400 PVR partitioning, 1391–1394 right-sided heart (RHC), 1387–1391 safety consideration, 1388–1389 chronic hypoxic, 582–583 chronic obstructive pulmonary diseases (COPD) clinical presentation, 1190–1192 cor pulmonale, 1193–1194 diagnosis, 1192 history, 1189–1190 pathobiology, 1192–1193 pathophysiology, 1192 therapy, 1194–1195 classification diagnostic, 937–941 functional class, 941 pathologic findings, 94 clinical classification, 1355, 1356 congenital heart defects at high altitude adaptation, 1224–1225 high altitude vs. sea level, distribution, 1226

1650 Pulmonary hypertension (PH), 380–381. See also Chronic obstructive pulmonary diseases (cont.) history, 1224 hypoxia, 1225 systolic pulmonary arterial pressure (SPAP), 1226, 1227 trisomy 21 syndrome (T21), 1227–1228 ventricular septal defect (VSD) surgery, 1226, 1227 congenital-heart-disease-associated (CHD-PAH) anesthetic considerations, 1165–1166 combined heart and lung transplantation, evaluation, 1164–1165 considerations, CHD lesions repair, 1158–1159 description, 1153 Eisenmenger patients, 1161 interventional lung assist, 1165 lung transplantation and CHD repair, 1164 mitral valve disease, 1159–1160 occurence, 1153 operability evaluation, investigations, 1154–1158 paradigm, shunt closure, 1163 shunt closure, 1162–1163 surgery, 1162 therapy, 1160–1161 transplantation, 1163–1164 univentricular circulations, 1160 congenital systemic-to-pulmonary (left-to-right) shunts atrial septal defects (ASDs), 1147 development, 1139 drug therapy, 1147–1148 pathogenic mechanisms, 1140–1142 pathology, 1142–1143 pulmonary hemodynamics, clinical assessment, 1143–1145 treatment, 1145–1147 consortium members, 588 endothelial cell analysis, 585–586 endothelium, role adhesion molecules, 846–847 adrenomedullin, 843–844 caveolae, 840 caveolins, 840–841 chemokines, 846 cytokines, 844–845 eNOS, 841 reactive oxygen species, 843 serotonin, 842–843 tetrahydrobiopterin, 841 thrombin, 844 treatments, 847 vascular growth factor, 842 vasculature, 837–838 vasoactive mediators, 838–840 extracellular matrix (ECM) collagen, 807 elafin, EVE inhibitor, 802–804 elastase activity, 801–802 elastin assembly genes, 804–806 fibronectin, 807 hyaluronic acid, 808 tenascin-C and matrix metalloproteinases, 806–807 gene expression profiling lung patterns, 583 peripheral blood monocytes, microarray, 583–584 progenitor cells, 585 pulmonary artery smooth muscle cells, 584 of right ventricle, 585 utilizing genetically engineered animal models, 586–587

Index genomics studies, 587–588 goal, consortium, 588 hypothesis-generating tool, 588–589 interstitial lung disease (see also Interstitial lung disease (ILD)) clinical classification, revised, 1198 clinical manifestation, 1202–1203 complications and prognosis, 1207 diagnosis, 1203–1204 epidemiology, 1199 etiology and pathogenic mechanisms, 1199–1201 pathology, 1201–1202 treatment, 1204–1207 left ventricular diastolic heart function Doppler echocardiography, 1185 evaluation, 1184–1186 guidelines, 1187 heart failure with a preserved ejection fraction (HFpEF), 1183 left ventricular compression vs. underfilling, 1184 mechanism, 1183–1184 pathology, 1183 treatment, 1186–1187 microvascular endothelial surface, metabolic and clearance function ACE activity, 110–112 ectoenzymes, 106 endothelial cells (ECs), 105 ET–1, pulmonary endothelial clearance of, 112 functions, 105, 106 indicator-dilution techniques, 107–110 metabolic and clearance functions, assessment, 107 metabolic properties, 105–106 PAH, pathological features, 106–107 PH patients, studies in, 112–114 transpulmonary gradient assessment, 107 oxygen-sensitive potassium channel, Kv1.5, 546–550 pathogenesis of, 581 pediatric (see also Pediatric pulmonary hypertension) aim, 1083 assessment, 1085–1086 bronchopulmonary dysplasia (BPD), 1089–1093 comprehensive program, 1103 congenital diaphragmatic hernia (CDH), 1086–1089 congenital heart disease (CHD), 1096 definition, 1084–1085 developmental age and disease spectrum, 1083, 1084 guidelines, 1101–1102 interventions, 1102 neonatal presentation, 1083, 1084 perioperative PH, 1099–1101 persistence, neonatal period, 1093–1096 progressive PH, 1086 respiratory system, diseases of, 1356 transgenic and gene-targeted models BMP pathway models, 465–467 generating methods, 459–462 plexogenesis models, 467–468 vasoreactivity-related models, 463–465 vasoactive mediators bone morphogenetic protein (see transforming growth factor b) endothelial-derived constricting/proliferative factors, 118–122 endothelium-derived vasodilators/ antiproliferative factors, 122–125 K+ channels, 126–127 platelet-derived growth factor (PDGF), 127–128 Pulmonary hypertension breakthrough initiative research network, 588 Pulmonary input impedance spectrum, 84

Index Pulmonary Langerhans cell histiocytosis (PLCH), 1197–1202 antiremodeling, and vasodilator agents, 1072–1073 clinical presentation and evaluation, 1072 diagnostic testing, 1072 epidemiologic risk factor, 1070 key concepts, 1073 management, 1072–1073 pathophysiology, 1070–1072 prevalence of, 1070 Pulmonary metabolism assessment ACE activity parameters, calculations, 108–109 ACE synthetic substrates, 108 Pulmonary microcirculation models capillary network, discrete tubules, 93–95 capillary network, sheet model, 92–93 Fahraeus–Lindqvist effect, 92 Pulmonary microvascular endothelial surface ACE activity nondiseased lung, 110–111 under pathological conditions, 111–112 ectoenzymes, 106 endothelial cells (ECs), 105 ET–1, pulmonary endothelial clearance of, 112 functions, 105, 106 indicator-dilution techniques clearance assessment, 109–110 metabolism assessment, 108–109 metabolic and clearance functions, assessment, 107 metabolic properties, 105–106 PAH, pathological features, 106–107 PH patients, studies in, 112–114 transpulmonary gradient assessment, 107 Pulmonary obstructive venopathy. See Pulmonary veno-occlusive disease (PVOD) Pulmonary thromboemboli components, 385 CTEPH, 390 fibrinogen fibrin B chain N-terminus (B knob), 391 resistance, fibrinolysis and CTEPH, 392 structure, 390–391 fibrinolysis, pharmacologically enhanced, 389–390 fibrinolytic enzymes, 390 incomplete perfusion recovery, clinical importance, 389 inflammation and remodeling clinical condition, 392–393 E-selectin and P-selectin, 393–394 heparin and fondaparinx, 394–395 interleukin–10 (IL–10), 395 VE-cadherin, 393 normal perfusion recovery, 385–387 relative perfusion recovery, 388 Pulmonary thromboendarterectomy (PTE), 1239–1240, 1244–1248, 1261–1265 adverse effects management, reperfusion response, 1585–1586 pericardial effusion, 1586 persistent pulmonary hypertension, 1585 reperfusion response, 1585 etiological factors, 1577 incidence, 1576–1577 indications, 1580 natural history, 1576 operation principles, 1580–1581 surgical technique, 1581–1584

1651 pathology and pathogenesis clinical presentation, 1578 diagnostic criteria and evaluation, 1579–1580 prognosis and results, 1586–1588 Pulmonary thromboendarterectomy specimens, 1246 Pulmonary vascular bed sarcomas arteries, primary sarcomas characteristics, 1344 clinical manifestations, 1344 differential diagnosis, 1346 epidemiology, 1344 imaging, 1344–1345 pathologic findings, 1345–1346 postulated cell of origin and molecular pathogenesis, 1347 treatment and prognostic factors, 1346–1347 categories, 1343 pulmonary capillary hemangiomatosis characteristics, 1348–1349 clinical features, 1349 imaging, 1349 pathologic findings and histopathologic differential diagnosis, 1349–1350 treatment and prognosis, 1350 pulmonary vasculature, secondary tumor involvement, 1350–1351 veins, primary sarcomas characteristics, 1347 clinical manifestations, 1348 epidemiology, 1347–1348 imaging, 1348 pathologic findings, 1348 treatment and prognosis, 1348 Pulmonary vascular development. See Lung vascular development Pulmonary vascular function blood flow and vascular pressures, limits, 62 effects of exercise, 64 right ventriculoarterial coupling, 70–71 steady-flow pulmonary hemodynamics active hypoxic regulation, 65–67 passive regulation, 64–65 pressure-flow diagram, 63 pressure-flow relationships, 62–64 pulsatile-flow, 67–68 vascular resistance, 61–62 Pulmonary vascular impedance (PVZ), 67–68 Pulmonary vascular mechanics effect of disease, 86–87 intermediate-sized arteries and microcirculation, 81–83 large pulmonary arteries, mechanics of, 79–81 mechanical behavior blood vessel constituents, 78–79 blood vessels, 74–76 fluid dynamics, 76–78 pulmonary vasculature and airways, interactions, 83–84 strain stiffening, 74 vascular impedance, 84–86 Pulmonary vascular oxygen sensing artery endothelial cell calcium-sensitive channels, 727–728 consequences, 727 cytosolic calcium, 726 nitric oxide production, 727 properties, 725–726 role, 725 ductus arteriosus, 725 hypoxia-inducible factor 1a modulates kca channel expression, 729–731 ontogeny of, 728–729

1652 Pulmonary vascular remodeling, 67–698 in acute PE, 855–856 animal study circulating fibrocytes, 816 c-Kit, 816 green fluorescent protein (GFP)-bone marrow transplant chimeric mice, 816 potential of, 818 vascular endothelial growth factor (VEGF), 816 in CTEPH, 858–859 by high oxygen air after hyperoxia, 741–746 alveolar oxygen tension, 746–751 hyperoxia-induced PH, 734–744 injures, 734 lung vascular restriction, 751–753 in PH, 753–754 photosynthesis, 733 in vivo models, 735 human study CD133+ cell increase, 819 cell fusion, 819 hallmark of, 819 longitudinal axis of pulmonary circulation conductance vessels, 760–762 distal muscular PAs, 762–765 nonmuscular alveolar wall vessels, 765–766 role of inflammation, progenitor cells, and vasa vasorum in, 771–772 shear stress, cell signaling, and cellular response, 793–795 disease manifestation, 795–797 mechanotransduction, 787 physiological response to, 789–793 pulmonary vasculature, 788–789 vascular cell phenotype adventitial fibroblast phenotype, 769–771 endothelial cell phenotype, 766–767 5-HTT and 5-HT receptor, 768 K+ and Ca2+ homeostasis, 768–769 RhoA/ROCK signaling, 769 SMC phenotype, 767–768 Pulmonary vascular resistance (PVR), 167, 1153–1159, 1240, 1245, 1247–1249 Pulmonary vascular resistance (PVR) partitioning correlations, 1392 PAP occlusion waveforms, 1391 RHC, exercise, 1392–1393 vasodilator challenge, 1393–1394 Pulmonary vascular smooth muscle cell proliferation cell cycle, 325–327 PASMC proliferation, factors calcium and smooth muscle proliferation, 327–328 growth factors, 326–327 hypoxia, 329–330 inflammation, 330–331 mechanical stress, 330 vasoactive substances, 328–329 VSMC proliferation, 325 pulmonary vasculature, 325 vasoactive factors, balance of, 331 Pulmonary vascular structure measurement. See Magnetic resonance imaging technology; Multidetector-row CT (MDCT) technology Pulmonary vascular tone. See Endothelial regulation Pulmonary vasculature, 325

Index Pulmonary vasculature modeling arterial tree, perfusion models, 95–97 gravity and vascular structure, contributions, 97–101 microcirculation models capillary network, discrete tubules, 93–95 capillary network, sheet model, 92–93 Fahraeus–Lindqvist effect, 92 model geometry, 91 perfusion, 91 supernumerary vasculature function, 101 Pulmonary vasoconstriction, 1393–1394 Pulmonary vasoconstriction and proliferation CaM complex propels progression, 684–685 myosin light chain kinase (MLCK) activation, 684 Pulmonary vasodilators, 1279–1280 Pulmonary vein sarcomas (PVSs), 1343, 1347–1348 Pulmonary veno-occlusive disease (PVOD), 954, 980, 984 bronchial arterial system engorgement, 1175 clinical manifestations cardiac catheterization, 1174–1175 prominent clubbing of digits, 1173 radiographic features, 1174 description, 1169 diagnosis, 1175 epidemiology, 1169–1170 etiology autoimmune disorders, 1173 genetic susceptibility, 1171–1172 infectious agents, 1172 thrombophilia, 1172–1173 toxic exposures, 1172 mitomycin c containing chemotherapy for, 1049–1050 pathology, 1170–1171 prognosis and treatment calcium channel antagonists, 1176 conventional supportive therapy, 1176 epoprostenol, 1176 experimental therapies, 1177 immunosuppressive agents, 1177 lung transplantation, 1177 related to chemotherapy treating acute lymphoblastic leukemia/myelofibrosis, 1051–1053 lymphoma, 1051 solid malignant neoplasms, 1050 Pulmonary venoocclusive disease (VOD), 381 Pulsatile-flow pulmonary hemodynamics pressure and flow wave morphology, 69–70 time constant, pulmonary circulation, 68–69 vacular impedance, 67–68 PVOD. See Pulmonary veno-occlusive disease (PVOD) PVZ. See Pulmonary vascular impedance Q QPatch (Sophion), 504 Quadrupole ion trap, 599 Quadrupole mass analyzers, 599 R Radionuclide scans pulmonary emboli, multiple large and small, 1405, 1406 pulmonary scintigraphy, 1404 Radionuclide ventilation–perfusion (V/Q) scanning, 1387 Radionuclide ventriculography, 1369

Index RANTES, 1334 Reactive hydrogen sulfide species, 296 Reactive nitrogen species, 290–292 Reactive oxygen-derived species (ROS) cytochrome P450, 304 gp91phox (Nox2), 304 hypoxia, 301 metabolic control, 304–305 metabolic interactions, 305–306 mitochondria, 303–304 NAD(P)H oxidases (Nox), 303 nitric oxide synthase, 304 oxidases activating processes, 302 pathophysiological, signaling, 309–310 pulmonary vasculature, 302 signaling pathways control autocoids and endothelium, 307 vascular smooth muscle force, 307–309 superoxide dismutase and NO, 304 vascular function, 301–302 vascular tone effects, 301 xanthine oxidase, 304 Reactive oxygen species (ROS), 287–288, 843 Reagents, 619–620 Real-time PCR general protocol, 531–532 normalization, 532–533 probe choice, 531 reaction efficiency, 532 Receptor tyrosine kinases (RTKs), 326 Receptor tyrosin kinases caveolae and signaling, 276 Recombinant mutant clones chimeras construction, 545 large deletions, 544–545 single base pair change, 545 Red blood cells, 376–377 Redox-controlled mechanisms cyclic GMP signaling, 308 cytosolic NADP(H) redox, 307–308 ion channels, 308–309 protein kinases, vasoconstriction, 309 Relative perfusion recovery, 388 Remodulin®. See Treprostinil Remodulinr, 989 Rendu–Osler–Weber syndrome, 1005–1006 Reperfusion injury, 474–475 Respiratory alkalosis, 1099 Resynchronization therapy, 1322, 1324 Revatio®. See Sildenafil citrate Reverse-transcription PCR methods, 529 primer design, 529–530 products quantification, 530 programmable thermal cycles, 530 reagents and buffers, 530 uses, 529 Rheumatoid arthritis (RA), 1015 RhoA/ROCK Signaling, 769 Rho kinase (ROCK), 141–142 Right-sided heart (RHC) catheterization and noninvasive imaging Role, 1387–1388 postcapillary wedge pressure (PCWP) determination, 1389 pulmonary hemodynamic abnormalities, 1391 techniques, 1389

1653 Right ventricular failure (RVF) acute, 1325 causes and mechanisms of, 1314 chronic, 1322–1325 definition, 1314 digoxin therapy, 1323 history and physical examination chest X-ray, 1319 echocardiography, 1319–1320 electrocardiography, 1318–1319 exercise testing, 1320 laboratory evaluation, 1319 magnetic resonance imaging, 1320 right-sided heart catheterization, 1320 key physiological concepts, 1316–1318 management, 1320–1322 pathophysiology of, 1314–1316 patient evaluation, 1316 pearls and pitfalls, 1326–1327 postulated molecular mechanisms of, 1315 resynchronization therapy, 1322, 1324 therapies for, 1325–1326 Right ventricular hypertrophy adult right ventricular hypertrophy, mechanisms, 1309–1310 cardiac hypertrophy and dilation, 1306 cor pulmonale, definition, 1305 mammalian heart development, 1307, 1308 right side heart adult, electrophysiological and contractile properties, 1307–1309 development, 1306–1307 right vs. left ventricle, 1305, 1306 ventricular muscle cells morphology, 1306, 1307 Right ventriculoarterial coupling, 70–71 Right vs. left ventricle, 1305, 1306 Primary pulmonary hypertension. See Idiopathic pulmonary arterial hypertension (IPAH) RNAi-mediated gene silencing concept and mechanism, 558–559 limitations in therapeutic application, 561–562 siRNA delivery, 560–561 siRNA design and synthesis, 559–560 RNA quantification isolation, 527–528 northern blotting gel electrophoresis, 528 probe hybridization, 528–529 real-time PCR general protocol, 531–532 normalization, 532–533 probe choice, 531 reaction efficiency, 532 reverse-transcription PCR methods, 529 primer design, 529–530 products quantification, 530 programmable thermal cycles, 530 reagents and buffers, 530 uses, 529 RVF. See Right ventricular failure Ryanodine receptors (RyRs), 151, 336–337 S S100A4, 468 Saccular stage, 138

1654 Sarcoidosis, 1421 antiremodeling and vasodilating agents, 1076–1077 clinical presentation and evaluation, 1075 diagnostic testing, 1075–1076 history and background, 1073 key concepts, 1077 management of, 1076 pathophysiology, 1073–1075 prevalence of, 1073 Sarcoplasmic reticulum, 151–152 Scanners development, CT imaging, 512 Schistosoma life cycle of, 1284–1286 parasite, 1284 Schistosoma haematobium, 1284 Schistosoma japonicum, 1284 Schistosoma mansoni, 1284 Schistosoma mekongi, 1284 Schistosomiasis clinical presentations, 1292–1293 complications, 1283 definition, 1283 early symptoms of, 1292 etiology, PH coinfection, 1290 hypothesis, 1288 immunological and inflammatory role, 1288–1290 portohypertension, 1290 immunological changes, 1286 issues and problems, 1293 life cycle of, 1284–1286 management of, 1293 parasite, 1284 pathological changes granuloma, 1286 liver pathology, 1286–1287 lung pathology, 1287–1288 prevalence of, 1290–1291 Scleroderma (SSc), 86 Scleroderma-related PAH collagen vascular diseases (CVDs) mixed connective tissue disease (MCTD), 1015 patients, survival of, 1018 primary Sjogren’s syndrome (pSS), 1015–1016 rheumatoid arthritis (RA), 1015 systemic lupus erythematosus (SLE), 1015 therapy, 1016–1018 etiology and pathogenic mechanisms genetic factors, 1013 SSc-related PAH, 1012–1015 systemic sclerosis (SSc), 1011 Serine proteases domains, 360 Serine/threonine kinases receptor bone morphogenetic protein, 254 pulmonary circulation, 255 transforming growth factor, 254 Serotonin, 465, 702, 842–843, 1310. See also 5-Hydroxytryptamine (5-HT) Serotonin transporter (SERT), 121–122 Serum response factor (SRF), 339–340 Shear-stress-induced EC differentiation, 641–642 Shear stress, pulmonary vasculature cellular response to cell proliferation, 795 endothelial signaling cascade, 795 generation of ROS, 793–794

Index intracellular Ca2+, 794 membrane depolarization, 793 NO generation, 794 phosphorylation signaling cascades, 794 transcription factors, activation of, 794–795 disease manifestation atherosclerosis, 797 disturbed flow and cell proliferation, 796 inflammatory responses, 796 pulmonary hypertension, 796–797 rarefaction, 796 endothelial cells, blood flow laminar, pulsatile, and disturbed flow, 788 shear stress values, 788–789 physiological response flow and development, adaptation to, 791 flow sensors and mechanosensing, 789–790 immediate response to shear, 790–791 signaling associated with altered shear, 792–793 Sickle cell disease (SCD), 379, 1095 Sickle cell disease-associated pulmonary hypertension diagnostic evaluation history and physical examination, 1275 six-minute walk test, 1275 echocardiography, 1276–1277 epidemiology pediatric sickle cell disease patients, epidemiology, 1272 right-sided heart catheterization findings, 1271–1272 laboratory analysis hemolysis markers, 1275 PH markers, 1276 systemic vasculopathy markers, 1275–1276 pathogenesis altered redox biology, 1273–1274 coagulation, alterations in, 1274–1275 hemolysis and nitric oxide bioavailability, 1272–1273 inflammation role, in vasculopathy, 1274 PH epidemiology, 1274 PH histopathology, 1272 pulmonary function tests, 1276 recommendations, diagnostic screening, 1277–1278 right-sided heart catheterization, 1277 treatment general, 1278 pulmonary vasodilators, 1279–1280 specific, 1278–1279 Signal transduction and cell signaling G-protein-coupled receptors (GPCR) adrenergic receptors, 249–250 endothelial barrier function, 252 ET receptors, 251 GTPase activity, 246 5-HT receptors, 251 muscarinic cholinergic receptors, 250 prostanoid receptors, 251–252 protein subunit, 246 pulmonary arterial vasodilation, 247–248 pulmonary vascular cells, 249 purinergic receptors, 250–251 stoichiometric relationship, 246–247 vasoconstriction and vascular cell proliferation, 248–249 receptor cross talk, 255–256 serine/threonine kinases receptor bone morphogenetic protein, 254 pulmonary circulation, 255 transforming growth factor, 254

Index tyrosine kinases receptor dimers, 253 pulmonary circulation, 253–254 structure, 253 Sildenafil, 1017, 1038–1039, 1161, 1206, 1279, 1371, 1382 Sildenafil (Revatio), 990 Sildenafil citrate, 1533–1534 Sildenafil therapy, 1100 Sildenafil use in pulmonary arterial hypertension (SUPER–1), 991 Single beat method, 70 Single-cell clonogenic assay, 629 Single-channel openings, 497 Single-nucleotide polymorphisms (SNPs), 588 Sitaxsentan, 1206 adverse effects, safety, and drug interactions, 1471–1472 clinical trials, 1471 connective tissue disease, 1471 metabolism, 1471 Small interfering RNA (siRNA) delivery, 560–561 design, 559 synthesis, 560 TRPC6, 562–564 Small-vessel arteriopathy evaluation case studies high-risk patient, 1263 large PTE specimen, 1263 small PTE specimen, 1263, 1264 PAH medical therapy role, 1265–1266 persistent PH, after PTE, 1261–1262 preoperative tests and assessment, 1264–1265 types and mechanism, 1262 Smooth muscle cell (SMC) vs. endothelial cell, 278 mitogenic signaling, 281 Smooth muscle cells (SMCs), 29–30 SNP. See Single-nucleotide polymorphisms (SNPs) Sodium channels. See Ion channels and transporters Sphingosine 1-phosphate (S1P), 478–479 Sphingosine 1-phosphate receptor 1, 905–906 Sphingosylphosphorylcholine (SPC), 150 Spin EB formation, 619 Splenectomy-induced PAH antiremodeling and vasodilating agents, 1069 clinical presentation and evaluation, 1069 diagnostic testing, 1069 key concepts, 1070 management and therapy of, 1069 pathophysiology, 1068–1069 prevalence of, 1068 SSc. See Systemic sclerosis SSc-related PAH autoantibodies, 1012–1013 clinical features, 1013–1014 diagnosis, 1014 genetic factors, 1013 organ involvement, 1014 pathophysiology, 1012 prognosis, 1014–1015 Stable transfected cells, 542 Starling resistor effect, 9 Starling resistor model, 62, 63 Static capillary alveolar microcirculation corner capillaries, 19–20 corner pleats, 20–21

1655 hexagonal capillary network, 14, 18–19 primary vs. secondary alveolar walls, 19 Statins action mechanism, 889–890 in animal models, ALI, 895–896 anti-inflammatory properties, 893–894 clinical trials, in ALI, 896 differential endothelial gene expression, 894–895 endothelial cytoskeletal rearrangement, 890–891 and endothelial NADPH oxidase, 892–893 and endothelial NO, 891–892 NO bioavailability, 886 protection, in murine model, 896 Statistical data analysis ANOVA, 660–661 binary data diagnostic tests, 664–665 logistic regression, 666–667 proportions, 663–664 receiver operating characteristic curves, 665–666 CART advantage, 667 contingency tables, 667–668 definition, 667 c2 test, 668 Fisher’s exact test, 668 categorical or qualitative data, 657 controlling type I error interim analyses, 669–670 multiple comparisons, 669 correlation, 660 experimental design/model building/ causality design, 655 types, 652 hypothesis testing one-sample vs. two-sample, 653–654 p values, 653 measures of variability, 656 OLS regression, 660 power and sample size determination analyses, 654–655 compute, 655 error types, 652 sample size vs. effect size vs. power, 655 standard error, 656 survival analysis clustered data, 663 competing risks, 663 Kaplan-Meier curves, 661–662 Logrank test, 662 proportional hazards regression, 662–663 visual displays box plots, bar plots and histograms, 657–658 scatter plots, 658–659 time series plots, 659–660 Steady-flow pulmonary hemodynamics active hypoxic regulation, 65–67 passive regulation gravity, 64–65 left atrial pressure, 64 lung volume, 64 pressure-flow diagram, 63 vascular resistance, 61–62 Store-operated Ca2+ entry (SOCE), 492 Strahler system, fluid dynamics, 78 Strain stiffening, 74

1656 Stress relaxation, 79 Stress-strain behavior elastin and collagen fibres, 79 materials, 74 viscoelastic materials, 79 Stromal interaction molecule 1 (STIM1), 337 Subacute mountain sickness, 877 Substance abuse, 1054 SUPER–1. See Sildenafil use in pulmonary arterial hypertension Supernumerary arteries, 18 Supernumerary vessels (SVs), 101 Supernumerary vessels (SVs) vs. accompanying vessels (AVs), 101 Superoxide dismutases (SOD), 465 Supportive therapies, IPAH altitude avoidance, 985 calcium channel antagonists, 987–988 diuretics, 987 exercise and physical activity, 985 pregnancy avoidance, 986 supplemental oxygen, 987–988 warfarin, 986 Surface electrogenic event reader (SURFE2R), 505 SURFE2R. See Surface electrogenic event reader Survival analysis clustered data, 663 competing risks, 663 Kaplan-Meier curves, 661–662 Logrank test, 662 proportional hazards regression, 662–663 Swamp fever. See Schistosoma mansoni Swiss HIV cohort study, 1037 Systemic lupus erythematosus (SLE), 1015 Systemic sclerosis (SSc), 1011 Systemic-to-pulmonary shunts (SPS), 1438 Systolic pulmonary artery pressure (sPAP), 1199, 1204, 1206, 1255, 1227 T Tadalafil, 1540 Takayasu’s arteritis (TA) anti-TNF therapy, 1339 Behçet's disease, 1340 clinical manifestations, 1334–1335 diagnosis clinical criteria, 1335–1336 imaging, 1336–1338 laboratory findings, 1336 pathology, 1338–1339 epidemiology, 1334 etiology and pathogenic mechanisms, 1333–1334 giant cell arteritis (GCA), 1340 granulomatous vascular involvement, 1340 history, 1333 Hughes–Stovin syndrome, 1340 prognosis, 1339–1340 pulmonary artery involvement, 1335 treatment medical therapy, 1339 vascular interventions, 1339 Tandem mass spectrometry (MS), 600–601 Targeted therapies, IPAH endothelin receptor antagonists, 990 endothelium-derived mediators, imbalance offset, 988 phosphodiesterase–5 inhibitors, 990–991 prostacyclin analogues, 988–989 Telokin, 158

Index Telomeres, 629–630 Terotogenicity, 1381 Tetrahydrobiopterin, 841 Tetralogy of Fallot, 1129 Thalassemia-associated pulmonary hypertension, 1270. See also Hemolytic-anemia-associated pulmonary hypertension Thermo-dye dilution method, 478–479 Th–1 proinflammatory mediators, 1289 Th–2 proinflammatory mediators, 1289–1290 Thrombin, 844 exosites and cofactors, 368 hemostatic substrates, 367–368 inhibition, 365–366 roles, 366–367 Thrombin model, 472–473 Thrombolytic and anticoagulant therapy chronic thromboembolic pulmonary hypertension (CTEPH), 1527 pulmonary embolism (PE) factor Xa inhibitors, 1526–1527 fondaparinux, 1524 low molecular weight heparin (LMWH), 1524 malignancy, 1526 recurrent VTE, 1526 thrombin inhibitors, 1527 thrombolytic therapy, 1521–1523 transient risk factor, 1525 treatment principles, 1521 unfractionated heparin, 1523 unprovoked VTE, 1525–1526 warfarin, 1525 Thrombolytic therapy, 1234 Thrombomodulin, 362, 406 Thrombophilia, 852–853, 1172–1173 Thrombosis plasmatic, 852–853 vascular remodeling process in acute PE, 855–856 endogenous plasmatic and tissular fibrinolytic system, 854–855 Thrombotic microangiopathies, 378–379 Thromboxane, 176–179 Thromboxane A2 (TXA2), 329 Tissue factor pathway, 403–404 Tissue factor pathway inhibitor (TFPI), 406 anticoagulant pathway, 361 Tissue plasminogen activator, 406 Tissue plasminogen activator (tPA) receptors interaction, with ECs, 416 roles, 416 structure and binding, 415–416 Tissular fibrinolytic system, 854–855 Titanium dioxide affinity, 609 TOF analyzer, 598–599 Toxic rapeseed oil, 1055 Toxins herb toxins, 1054–1055 L-tryptophan, 1056 toxic rapeseed oil, 1055 Transcription factor growth-inducing, 719 zinc finger, 718–719 Transepithelial fluid movement, 862 Transforming growth factor (TGF)-b, 125–126, 254 Transgenic and gene-targeted models BMP pathway models BMPR2 Function, 467 BMPR2 knockout and hypomorph, 466

Index BMPR2 siRNA and Cre-loxP, 466 inducible BMPR2 mutants, 466–467 generating methods Cre–loxP system, 460–461 doxycycline (or tetracycline)-inducible systems, 462 knockouts and knock-ins technology, 460 transgenics, 461 husbandry breeding, 463 caging, 463 genotyping, 463 plexogenesis models S100A4, 468 vascular endothelial growth factor A (VEGFA), 468 vasoreactivity-related models caveolin, 464 endothelin–1 (ET–1), 464–465 nitric oxide synthases, 464 serotonin, 465 superoxide dismutases (SOD), 465 Transgenics, 461 Transient receptor potential (TRP), 150 Transient receptor potential channels (TRPCs), 337 Transient transfection. See Gene delivery methods Transpulmonary gradient assessment, 107 Transthoracic echocardiography (TTE), 1363–1366. See also Echocardiography Treprostinil, 1016 inhaled, 1458 intravenous, 1455 intravenously administered, 1546–1547 subcutaneous, 1455–1456 subcutaneously administered dosage, 1542 FDA approval, 1542 long-term outcome study, 1543 pain and reaction, 1542 patient training, 1542 pharmacologic properties, 1542 Treprostinil sodium. See Remodulinr Tricuspid regurgitant (TR) jet velocity, 1085 Trisomy 21 syndrome (T21), 1227–1228 Tryptophan hydroxylase (Tph), 122, 1502–1503 Tyrosine kinases receptor dimers, 253 pulmonary circulation, 253–254 structure, 253 Tyrosine nitration, 292 U Unfractionated heparin (UFH), 1235 Unilateral lung transplantation adult, 1616 pediatric, 1616 secondary pulmonary hypertension, 1617 Unitary ion channel currents channel conductance, selectivity, and current amplitude, 506 channel gating, 506 channel open probability, 506 Upfront combination therapy, PAH, 1518 Urokinase in pulmonary embolism trial (UPET), 389 Urokinase plasminogen activator ECs, 417–418 functions, 418 receptor, 416–417

1657 V Varicose veins, 851–852 Vasa vasorum, 771–772 Vascular barrier dysfunction. See Genomics, acute lung injury Vascular biomarkers IL–8 receptor, 213–214 intercellular adhesion molecule–1, 215 interleukin–6, 215 plasminogen activator inhibitor–1, 214–215 pre-B-cell colony-enhancing factor, 215–216 protein C pathway, 214 sphingosine 1-phosphate, 210–211 S1P3 receptor/tyrosine-nitrated, 213 Vascular endothelial growth factor (VEGF), 475–476, 1090, 1300 in chronic-hypoxia-mediated PH, 703–704 expression, 645 Vascular endothelial growth factor A (VEGFA), 468 Vascular impedance, 84–86 Vascular perfusion, 14–15 Vascular permeability measurements, 480–481 Vascular smooth muscle cells (vSMCs), 78–79, 323–329, 330. See also Excitation–contraction coupling proliferation, 325 Vascular-space tissue ratio (VSTR), 82–83 Vascular templates adult lung, 48–49 fetal lung, 42–45 neonatal and child lung, 46–48 Vasculogenesis, 136 Vasoactive factors autocoids and endothelium-derived endothelin, 307 nitric oxide, 307 prostaglandins, 307 Vasoactive mediators. See also Endothelial regulation arachidonic acid metabolites production and action, 176–178 regulation, 178–179 carbon monoxide, 183 endothelial-derived constricting/proliferative factors endothelin–1, 119–120 serotonin, 120–122 endothelin production and action, 179–180 regulation, 181 endothelium-derived hyperpolarizing factor (EDHF), 181–182 endothelium-derived vasodilators/antiproliferative factors cAMP, cGMP, and PDEs, 124–125 nitric oxide (NO), 122–123 prostacyclin, 123 epoxyeicosatetraenoic acids (EET), 181–182 hydrogen sulfide, 183 K+ channels, 126–127 leukotrienes, 182–183 nitric oxide exogenous stimuli, 174–176 production and action, 171–172 regulation, 172–174 platelet-derived growth factor (PDGF), 127–128 principal vasoconstrictor, 839–840 vasodilator, 838–839 prostacyclin, 840 thromboxane, 840 vasoactive intestinal peptide (VIP), 183

1658 Vasodilating agents Gaucher’s disease type I, 1067 PLCH, 1072–1073 sarcoidosis, 1076–1077 splenectomy-induced PAH, 1069 Vasodilator testing, acute, 985 Vasoreactivity-related PH models caveolin, 464 endothelin–1 (ET–1), 464–465 nitric oxide synthases, 464 serotonin, 465 superoxide dismutases, 465 VE-Cadherin, 644 VE-cadherin, 393 Vena cava stenosis model conditional fetal liver kinase–1 deletion, 857–858 staphylococcal infection, 857 Venice classification, 939 Venous thromboembolism (VTE) current treatment embolectomy, 1235–1236 medical management, 1234–1235 epidemiology, 851, 852 history, 1231–1232 incidence and epidemiology, 1232–1233 pathogenesis, 1232 risk factors, 852, 1233 varicose veins and deep vein thrombosis, 851–852 Ventavis®. See Inhaled iloprost Ventilation-perfusion lung scan, CTEPH, 1569

Index Ventilation-perfusion mismatch. See Intrapulmonary shunt Ventilator-induced lung injury (VILI), 477 Ventricular septal defect (VSD), 1139 Ventriculoatrial (VA) shunts, 1254 Viral vectors, 542–543 Virchow’s triad, platelets blood ADAMTS–13, 376 coreceptors, 376 platelet GpIb complex, 375–376 red blood cells, 376–377 von Willebrand factor, 375 blood flow, 374 vessel, 377–378 Visual displays box plots, bar plots and histograms, 657–658 scatter plots, 658–659 time series plots, 659–660 Voltage-dependent anion channel (VDAC), 348–349 Volume distensibility coefficient, 76 von Willebrand factor, 375 von Willebrand factor plasma levels, 1256 vSMC. See Vascular smooth muscle cells VTE. See Venous thromboembolism W Warfarin, 986, 1206, 1235, 1525 Whole-body scintigraphy, 479 Williams syndrome, 86