Lung Development and Regeneration (Lung Biology in Health and Disease)

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LUNG DEVELOPMENT AND REGENERATION

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LUNG BIOLOGY IN HEALTH AND DISEASE Executive Editor Claude Lenfant Former Director, National Heart, Lung, and Blood Institute National Institutes of Health Bethesda, Maryland

1. Immunologic and Infectious Reactions in the Lung, edited by C. H. Kirkpatrick and H. Y. Reynolds 2. The Biochemical Basis of Pulmonary Function, edited by R. G. Crystal 3. Bioengineering Aspects of the Lung, edited by J. B. West 4. Metabolic Functions of the Lung, edited by Y. S. Bakhle and J. R. Vane 5. Respiratory Defense Mechanisms (in two parts), edited by J. D. Brain, D. F. Proctor, and L. M. Reid 6. Development of the Lung, edited by W. A. Hodson 7. Lung Water and Solute Exchange, edited by N. C. Staub 8. Extrapulmonary Manifestations of Respiratory Disease, edited by E. D. Robin 9. Chronic Obstructive Pulmonary Disease, edited by T. L. Petty 10. Pathogenesis and Therapy of Lung Cancer, edited by C. C. Harris 11. Genetic Determinants of Pulmonary Disease, edited by S. D. Litwin 12. The Lung in the Transition Between Health and Disease, edited by P. T. Macklem and S. Permutt 13. Evolution of Respiratory Processes: A Comparative Approach, edited by S. C. Wood and C. Lenfant 14. Pulmonary Vascular Diseases, edited by K. M. Moser 15. Physiology and Pharmacology of the Airways, edited by J. A. Nadel 16. Diagnostic Techniques in Pulmonary Disease (in two parts), edited by M. A. Sackner 17. Regulation of Breathing (in two parts), edited by T. F. Hornbein 18. Occupational Lung Diseases: Research Approaches and Methods, edited by H. Weill and M. Turner-Warwick 19. Immunopharmacology of the Lung, edited by H. H. Newball 20. Sarcoidosis and Other Granulomatous Diseases of the Lung, edited by B. L. Fanburg 21. Sleep and Breathing, edited by N. A. Saunders and C. E. Sullivan 22. Pneumocystis carinii Pneumonia: Pathogenesis, Diagnosis, and Treatment, edited by L. S. Young 23. Pulmonary Nuclear Medicine: Techniques in Diagnosis of Lung Disease, edited by H. L. Atkins 24. Acute Respiratory Failure, edited by W. M. Zapol and K. J. Falke 25. Gas Mixing and Distribution in the Lung, edited by L. A. Engel and M. Paiva 26. High-Frequency Ventilation in Intensive Care and During Surgery, edited by G. Carlon and W. S. Howland

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27. Pulmonary Development: Transition from Intrauterine to Extrauterine Life, edited by G. H. Nelson 28. Chronic Obstructive Pulmonary Disease: Second Edition, edited by T. L. Petty 29. The Thorax (in two parts), edited by C. Roussos and P. T. Macklem 30. The Pleura in Health and Disease, edited by J. Chrétien, J. Bignon, and A. Hirsch 31. Drug Therapy for Asthma: Research and Clinical Practice, edited by J. W. Jenne and S. Murphy 32. Pulmonary Endothelium in Health and Disease, edited by U. S. Ryan 33. The Airways: Neural Control in Health and Disease, edited by M. A. Kaliner and P. J. Barnes 34. Pathophysiology and Treatment of Inhalation Injuries, edited by J. Loke 35. Respiratory Function of the Upper Airway, edited by O. P. Mathew and G. Sant'Ambrogio 36. Chronic Obstructive Pulmonary Disease: A Behavioral Perspective, edited by A. J. McSweeny and I. Grant 37. Biology of Lung Cancer: Diagnosis and Treatment, edited by S. T. Rosen, J. L. Mulshine, F. Cuttitta, and P. G. Abrams 38. Pulmonary Vascular Physiology and Pathophysiology, edited by E. K. Weir and J. T. Reeves 39. Comparative Pulmonary Physiology: Current Concepts, edited by S. C. Wood 40. Respiratory Physiology: An Analytical Approach, edited by H. K. Chang and M. Paiva 41. Lung Cell Biology, edited by D. Massaro 42. Heart–Lung Interactions in Health and Disease, edited by S. M. Scharf and S. S. Cassidy 43. Clinical Epidemiology of Chronic Obstructive Pulmonary Disease, edited by M. J. Hensley and N. A. Saunders 44. Surgical Pathology of Lung Neoplasms, edited by A. M. Marchevsky 45. The Lung in Rheumatic Diseases, edited by G. W. Cannon and G. A. Zimmerman 46. Diagnostic Imaging of the Lung, edited by C. E. Putman 47. Models of Lung Disease: Microscopy and Structural Methods, edited by J. Gil 48. Electron Microscopy of the Lung, edited by D. E. Schraufnagel 49. Asthma: Its Pathology and Treatment, edited by M. A. Kaliner, P. J. Barnes, and C. G. A. Persson 50. Acute Respiratory Failure: Second Edition, edited by W. M. Zapol and F. Lemaire 51. Lung Disease in the Tropics, edited by O. P. Sharma 52. Exercise: Pulmonary Physiology and Pathophysiology, edited by B. J. Whipp and K. Wasserman 53. Developmental Neurobiology of Breathing, edited by G. G. Haddad and J. P. Farber 54. Mediators of Pulmonary Inflammation, edited by M. A. Bray and W. H. Anderson 55. The Airway Epithelium, edited by S. G. Farmer and D. Hay 56. Physiological Adaptations in Vertebrates: Respiration, Circulation, and Metabolism, edited by S. C. Wood, R. E. Weber, A. R. Hargens, and R. W. Millard 57. The Bronchial Circulation, edited by J. Butler 58. Lung Cancer Differentiation: Implications for Diagnosis and Treatment, edited by S. D. Bernal and P. J. Hesketh 59. Pulmonary Complications of Systemic Disease, edited by J. F. Murray

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60. Lung Vascular Injury: Molecular and Cellular Response, edited by A. Johnson and T. J. Ferro 61. Cytokines of the Lung, edited by J. Kelley 62. The Mast Cell in Health and Disease, edited by M. A. Kaliner and D. D. Metcalfe 63. Pulmonary Disease in the Elderly Patient, edited by D. A. Mahler 64. Cystic Fibrosis, edited by P. B. Davis 65. Signal Transduction in Lung Cells, edited by J. S. Brody, D. M. Center, and V. A. Tkachuk 66. Tuberculosis: A Comprehensive International Approach, edited by L. B. Reichman and E. S. Hershfield 67. Pharmacology of the Respiratory Tract: Experimental and Clinical Research, edited by K. F. Chung and P. J. Barnes 68. Prevention of Respiratory Diseases, edited by A. Hirsch, M. Goldberg, J. P. Martin, and R. Masse 69. Pneumocystis carinii Pneumonia: Second Edition, edited by P. D. Walzer 70. Fluid and Solute Transport in the Airspaces of the Lungs, edited by R. M. Effros and H. K. Chang 71. Sleep and Breathing: Second Edition, edited by N. A. Saunders and C. E. Sullivan 72. Airway Secretion: Physiological Bases for the Control of Mucous Hypersecretion, edited by T. Takishima and S. Shimura 73. Sarcoidosis and Other Granulomatous Disorders, edited by D. G. James 74. Epidemiology of Lung Cancer, edited by J. M. Samet 75. Pulmonary Embolism, edited by M. Morpurgo 76. Sports and Exercise Medicine, edited by S. C. Wood and R. C. Roach 77. Endotoxin and the Lungs, edited by K. L. Brigham 78. The Mesothelial Cell and Mesothelioma, edited by M. C. Jaurand and J. Bignon 79. Regulation of Breathing: Second Edition, edited by J. A. Dempsey and A. I. Pack 80. Pulmonary Fibrosis, edited by S. Hin. Phan and R. S. Thrall 81. Long-Term Oxygen Therapy: Scientific Basis and Clinical Application, edited by W. J. O'Donohue, Jr. 82. Ventral Brainstem Mechanisms and Control of Respiration and Blood Pressure, edited by C. O. Trouth, R. M. Millis, H. F. Kiwull-Schöne, and M. E. Schläfke 83. A History of Breathing Physiology, edited by D. F. Proctor 84. Surfactant Therapy for Lung Disease, edited by B. Robertson and H. W. Taeusch 85. The Thorax: Second Edition, Revised and Expanded (in three parts), edited by C. Roussos 86. Severe Asthma: Pathogenesis and Clinical Management, edited by S. J. Szefler and D. Y. M. Leung 87. Mycobacterium avium–Complex Infection: Progress in Research and Treatment, edited by J. A. Korvick and C. A. Benson 88. Alpha 1–Antitrypsin Deficiency: Biology • Pathogenesis • Clinical Manifestations • Therapy, edited by R. G. Crystal 89. Adhesion Molecules and the Lung, edited by P. A. Ward and J. C. Fantone 90. Respiratory Sensation, edited by L. Adams and A. Guz 91. Pulmonary Rehabilitation, edited by A. P. Fishman

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92. Acute Respiratory Failure in Chronic Obstructive Pulmonary Disease, edited by J. P. Derenne, W. A. Whitelaw, and T. Similowski 93. Environmental Impact on the Airways: From Injury to Repair, edited by J. Chrétien and D. Dusser 94. Inhalation Aerosols: Physical and Biological Basis for Therapy, edited by A. J. Hickey 95. Tissue Oxygen Deprivation: From Molecular to Integrated Function, edited by G. G. Haddad and G. Lister 96. The Genetics of Asthma, edited by S. B. Liggett and D. A. Meyers 97. Inhaled Glucocorticoids in Asthma: Mechanisms and Clinical Actions, edited by R. P. Schleimer, W. W. Busse, and P. M. O’Byrne 98. Nitric Oxide and the Lung, edited by W. M. Zapol and K. D. Bloch 99. Primary Pulmonary Hypertension, edited by L. J. Rubin and S. Rich 100. Lung Growth and Development, edited by J. A. McDonald 101. Parasitic Lung Diseases, edited by A. A. F. Mahmoud 102. Lung Macrophages and Dendritic Cells in Health and Disease, edited by M. F. Lipscomb and S. W. Russell 103. Pulmonary and Cardiac Imaging, edited by C. Chiles and C. E. Putman 104. Gene Therapy for Diseases of the Lung, edited by K. L. Brigham 105. Oxygen, Gene Expression, and Cellular Function, edited by L. Biadasz Clerch and D. J. Massaro 106. Beta2-Agonists in Asthma Treatment, edited by R. Pauwels and P. M. O’Byrne 107. Inhalation Delivery of Therapeutic Peptides and Proteins, edited by A. L. Adjei and P. K. Gupta 108. Asthma in the Elderly, edited by R. A. Barbee and J. W. Bloom 109. Treatment of the Hospitalized Cystic Fibrosis Patient, edited by D. M. Orenstein and R. C. Stern 110. Asthma and Immunological Diseases in Pregnancy and Early Infancy, edited by M. Schatz, R. S. Zeiger, and H. N. Claman 111. Dyspnea, edited by D. A. Mahler 112. Proinflammatory and Antiinflammatory Peptides, edited by S. I. Said 113. Self-Management of Asthma, edited by H. Kotses and A. Harver 114. Eicosanoids, Aspirin, and Asthma, edited by A. Szczeklik, R. J. Gryglewski, and J. R. Vane 115. Fatal Asthma, edited by A. L. Sheffer 116. Pulmonary Edema, edited by M. A. Matthay and D. H. Ingbar 117. Inflammatory Mechanisms in Asthma, edited by S. T. Holgate and W. W. Busse 118. Physiological Basis of Ventilatory Support, edited by J. J. Marini and A. S. Slutsky 119. Human Immunodeficiency Virus and the Lung, edited by M. J. Rosen and J. M. Beck 120. Five-Lipoxygenase Products in Asthma, edited by J. M. Drazen, S. E. Dahlén, and T. H. Lee 121. Complexity in Structure and Function of the Lung, edited by M. P. Hlastala and H. T. Robertson 122. Biology of Lung Cancer, edited by M. A. Kane and P. A. Bunn, Jr. 123. Rhinitis: Mechanisms and Management, edited by R. M. Naclerio, S. R. Durham, and N. Mygind

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124. Lung Tumors: Fundamental Biology and Clinical Management, edited by C. Brambilla and E. Brambilla 125. Interleukin-5: From Molecule to Drug Target for Asthma, edited by C. J. Sanderson 126. Pediatric Asthma, edited by S. Murphy and H. W. Kelly 127. Viral Infections of the Respiratory Tract, edited by R. Dolin and P. F. Wright 128. Air Pollutants and the Respiratory Tract, edited by D. L. Swift and W. M. Foster 129. Gastroesophageal Reflux Disease and Airway Disease, edited by M. R. Stein 130. Exercise-Induced Asthma, edited by E. R. McFadden, Jr. 131. LAM and Other Diseases Characterized by Smooth Muscle Proliferation, edited by J. Moss 132. The Lung at Depth, edited by C. E. G. Lundgren and J. N. Miller 133. Regulation of Sleep and Circadian Rhythms, edited by F. W. Turek and P. C. Zee 134. Anticholinergic Agents in the Upper and Lower Airways, edited by S. L. Spector 135. Control of Breathing in Health and Disease, edited by M. D. Altose and Y. Kawakami 136. Immunotherapy in Asthma, edited by J. Bousquet and H. Yssel 137. Chronic Lung Disease in Early Infancy, edited by R. D. Bland and J. J. Coalson 138. Asthma’s Impact on Society: The Social and Economic Burden, edited by K. B. Weiss, A. S. Buist, and S. D. Sullivan 139. New and Exploratory Therapeutic Agents for Asthma, edited by M. Yeadon and Z. Diamant 140. Multimodality Treatment of Lung Cancer, edited by A. T. Skarin 141. Cytokines in Pulmonary Disease: Infection and Inflammation, edited by S. Nelson and T. R. Martin 142. Diagnostic Pulmonary Pathology, edited by P. T. Cagle 143. Particle–Lung Interactions, edited by P. Gehr and J. Heyder 144. Tuberculosis: A Comprehensive International Approach, Second Edition, Revised and Expanded, edited by L. B. Reichman and E. S. Hershfield 145. Combination Therapy for Asthma and Chronic Obstructive Pulmonary Disease, edited by R. J. Martin and M. Kraft 146. Sleep Apnea: Implications in Cardiovascular and Cerebrovascular Disease, edited by T. D. Bradley and J. S. Floras 147. Sleep and Breathing in Children: A Developmental Approach, edited by G. M. Loughlin, J. L. Carroll, and C. L. Marcus 148. Pulmonary and Peripheral Gas Exchange in Health and Disease, edited by J. Roca, R. Rodriguez-Roisen, and P. D. Wagner 149. Lung Surfactants: Basic Science and Clinical Applications, R. H. Notter 150. Nosocomial Pneumonia, edited by W. R. Jarvis 151. Fetal Origins of Cardiovascular and Lung Disease, edited by David J. P. Barker 152. Long-Term Mechanical Ventilation, edited by N. S. Hill 153. Environmental Asthma, edited by R. K. Bush 154. Asthma and Respiratory Infections, edited by D. P. Skoner 155. Airway Remodeling, edited by P. H. Howarth, J. W. Wilson, J. Bousquet, S. Rak, and R. A. Pauwels 156. Genetic Models in Cardiorespiratory Biology, edited by G. G. Haddad and T. Xu

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157. Respiratory-Circulatory Interactions in Health and Disease, edited by S. M. Scharf, M. R. Pinsky, and S. Magder 158. Ventilator Management Strategies for Critical Care, edited by N. S. Hill and M. M. Levy 159. Severe Asthma: Pathogenesis and Clinical Management, Second Edition, Revised and Expanded, edited by S. J. Szefler and D. Y. M. Leung 160. Gravity and the Lung: Lessons from Microgravity, edited by G. K. Prisk, M. Paiva, and J. B. West 161. High Altitude: An Exploration of Human Adaptation, edited by T. F. Hornbein and R. B. Schoene 162. Drug Delivery to the Lung, edited by H. Bisgaard, C. O’Callaghan, and G. C. Smaldone 163. Inhaled Steroids in Asthma: Optimizing Effects in the Airways, edited by R. P. Schleimer, P. M. O’Byrne, S. J. Szefler, and R. Brattsand 164. IgE and Anti-IgE Therapy in Asthma and Allergic Disease, edited by R. B. Fick, Jr., and P. M. Jardieu 165. Clinical Management of Chronic Obstructive Pulmonary Disease, edited by T. Similowski, W. A. Whitelaw, and J. P. Derenne 166. Sleep Apnea: Pathogenesis, Diagnosis, and Treatment, edited by A. I. Pack 167. Biotherapeutic Approaches to Asthma, edited by J. Agosti and A. L. Sheffer 168. Proteoglycans in Lung Disease, edited by H. G. Garg, P. J. Roughley, and C. A. Hales 169. Gene Therapy in Lung Disease, edited by S. M. Albelda 170. Disease Markers in Exhaled Breath, edited by N. Marczin, S. A. Kharitonov, M. H. Yacoub, and P. J. Barnes 171. Sleep-Related Breathing Disorders: Experimental Models and Therapeutic Potential, edited by D. W. Carley and M. Radulovacki 172. Chemokines in the Lung, edited by R. M. Strieter, S. L. Kunkel, and T. J. Standiford 173. Respiratory Control and Disorders in the Newborn, edited by O. P. Mathew 174. The Immunological Basis of Asthma, edited by B. N. Lambrecht, H. C. Hoogsteden, and Z. Diamant 175. Oxygen Sensing: Responses and Adaptation to Hypoxia, edited by S. Lahiri, G. L. Semenza, and N. R. Prabhakar 176. Non-Neoplastic Advanced Lung Disease, edited by J. Maurer 177. Therapeutic Targets in Airway Inflammation, edited by N. T. Eissa and D. P. Huston 178. Respiratory Infections in Allergy and Asthma, edited by S. L. Johnston and N. G. Papadopoulos 179. Acute Respiratory Distress Syndrome, edited by M. A. Matthay 180. Venous Thromboembolism, edited by J. E. Dalen 181. Upper and Lower Respiratory Disease, edited by J. Corren, A. Togias, and J. Bousquet 182. Pharmacotherapy in Chronic Obstructive Pulmonary Disease, edited by B. R. Celli 183. Acute Exacerbations of Chronic Obstructive Pulmonary Disease, edited by N. M. Siafakas, N. R. Anthonisen, and D. Georgopoulos 184. Lung Volume Reduction Surgery for Emphysema, edited by H. E. Fessler, J. J. Reilly, Jr., and D. J. Sugarbaker 185. Idiopathic Pulmonary Fibrosis, edited by J. P. Lynch III 186. Pleural Disease, edited by D. Bouros

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187. Oxygen/Nitrogen Radicals: Lung Injury and Disease, edited by V. Vallyathan, V. Castranova, and X. Shi 188. Therapy for Mucus-Clearance Disorders, edited by B. K. Rubin and C. P. van der Schans 189. Interventional Pulmonary Medicine, edited by J. F. Beamis, Jr., P. N. Mathur, and A. C. Mehta 190. Lung Development and Regeneration, edited by D. J. Massaro, G. Massaro, and P. Chambon

ADDITIONAL VOLUMES IN PREPARATION Long-Term Intervention in Chronic Obstructive Pulmonary Disease, edited by R. Pauwels, D. S. Postma, and S. T. Weiss Sleep Deprivation: Basic Science, Physiology, and Behavior, edited by C. A. Kushida Sleep Deprivation: Clinical Issues, Pharmacology, and Sleep Loss Effects, edited by C. A. Kushida Pneumocystis Pneumonia: Third Edition, Revised and Expanded, edited by P. D. Walzer and M. Cushion Ion Channels in the Pulmonary Vasculature, edited by J. X.-J. Yuan Asthma Prevention, edited by W. W. Busse and R. F. Lemanske, Jr.

The opinions expressed in these volumes do not necessarily represent the views of the National Institutes of Health.

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LUNG DEVELOPMENT AND REGENERATION

Edited by

Donald J. Massaro Georgetown University School of Medicine, Washington, D.C., U.S.A.

Gloria DeCarlo Massaro Georgetown University School of Medicine, Washington, D.C., U.S.A.

Pierre Chambon Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France.

Marcel Dekker

New York

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Although great care has been taken to provide accurate and current information, neither the author(s) nor the publisher, nor anyone else associated with this publication, shall be liable for any loss, damage, or liability directly or indirectly caused or alleged to be caused by this book. The material contained herein is not intended to provide specific advice or recom-mendations for any specific situation. Trademark notice: Product or corporate names may be trademarks or registered trademarks and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress. ISBN: 0-8247-5439-5 This book is printed on acid-free paper. Headquarters Marcel Dekker, Inc., 270 Madison Avenue, New York, NY 10016, U.S.A. tel: 212-696-9000; fax: 212-685-4540 Distribution and Customer Service Marcel Dekker, Inc., Cimarron Road, Monticello, New York 12701, U.S.A. tel: 800-228-1160; fax: 845-796-1772 World Wide Web http://www.dekker.com The publisher offers discounts on this book when ordered in bulk quantities. For more infor-mation, write to Special Sales/Professional Marketing at the headquarters address above. Copyright © 2004 by Marcel Dekker, Inc. All Rights Reserved. Neither this book nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher. Current printing (last digit): 10 9 8 7 6 5 4 3 2 1 PRINTED IN THE UNITED STATES OF AMERICA

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Introduction Most often, advances in biology follow a very tortuous path. Lung biology illustrates this very well, perhaps better than any other system. Considerable research has been done not only to understand lung development, but also to uncover ways to accelerate it in the case of premature birth. Over the years, remarkable successes have been noted, as evidenced by the increasing survival of babies born well before their lungs are mature. This achievement is due in great part to the development of pharmacologic and instrumental interventions to accelerate lung maturation and to keep the immature lung functional until maturity is reached. On the other hand, the mature lung—the adult lung—is subject to injuries that, over time, lead to its structural alteration, a situation that culminates in emphysema. Most of the time, these alterations are the result of an excess of elastase due to exogenous factors such as cigarette smoke. Recently it has been shown that retinoic acid, a derivative of vitamin A, plays a role in the development of the lung and, conversely, that inhibition of retinoic acid results in elastase injury of the lung. The demonstration that exogenous retinoic acid can induce alveolar cell regeneration in an animal model of emphysema suggests the possibility that it could be used therapeutically in patients suffering from emphysema. Simply put, retinoic acid may be necessary not only for normal alveolar development but also for maintenance of the integrity of the alveolar surface. The mechanism of action of retinoic acid is not yet fully understood but, clearly, it somehow intervenes in the genetic machinery that leads to normal lung development. Whether or not that machinery is reactivated when retinoic acid is given to stimulate lung cell regeneration remains a major question. Bronchopulmonary dysplasia and chronic obstructive pulmonary disease are both defined by whether or not retinoic acid plays its presumed role, that is, alveolar cell generation in bronchopulmonary dysplasia or alveolar cell protection in emphysema. The public heath significance of this issue is huge, as both conditions are very prevalent in all regions of the world. This volume, edited by Donald Massaro and Gloria DeCarlo Massaro of the United States, and Pierre Chambon of France, gives the reader an opportunity to travel into the exciting field of the function and role of retinoic acid and its receptors. The editors have led the research on the role of retinoic receptors in the integrity and stability of alveolar cells in the perinatal and post-perinatal periods. They enrolled contributors who are experts highly recognized for their work in this area or other relevant fields of lung biology.

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As the Executive Editor of the series of monographs Lung Biology in Health and Disease, I highly value the opportunity to introduce this volume to the readership and thereby give readers a taste of a novel field of biology that has promising implications for clinical application.

Claude Lenfant, MD Gaithersburg, Maryland

Preface

The last decade has witnessed exponential growth of interest and work in the area of lung development, alveolar regeneration, and alveolar architectural stability. These events have led to new insights into mechanisms by which the lung develops and maintains its architecture. In particular, although lagging behind studies on development of conducting airways, interest in the formation of alveoli has grown. Old notions of the immutability of alveolar structure short of disease have given way to new concepts of alveolar plasticity, i.e., regeneration and nondisease-related loss. These ideas, supported by experiments, have given hope to the possibility that therapeutic alveolar regeneration and slowing of alveolar loss may be realistic expections over the next decade. The present volume was designed to focus on two diseases - bronchopulmonary dysplasia as an example of alveolar developmental failure, and chronic obstructive pulmonary disease, as an example of presently irremedial alveolar loss. Our aim was to present two important clinical entities and supportive experimental and technological means aimed at achieving their remediation. Donald Massaro Gloria DeCarlo Massaro Pierre Chambon v

Contents

Series Introduction Preface I.

Claude Lenfant

iii v

Diseases: Bronchopulmonary Dysplasia (BPD) and Chronic Obstructive Pulmonary Disease (COPD)

1. Arrested Alveolar Development in Bronchopulmonary Dysplasia Jacqueline J. Coalson

1

2. Bronchopulmonary Dysplasia in the Postsurfactant Era Richard D. Bland

21

3. What is Chronic Obstructive Pulmonary Disease? Robert M. Rogers

51

4. Small Airways Disease in Chronic Obstructive Pulmonary Disease James C. Hogg 5. Alveolar and Bronchiolar Inflammation in COPD Simonetta Baraldo, Graziella Turato, Bianca Beghe´, Renzo Zuin, and Marina Saetta 6. Pathophysiological Basis for the Treatment of Chronic Obstructive Pulmonary Disease Cynthia Brown and Robert A. Wise

67 99

115 vii

viii 7.

II.

8.

9.

Contents Clinical Approaches for Evaluating Retinoids as a Treatment for Human Emphysema Michael D. Roth, Jonathan G. Goldin, and Jenny T. Mao

149

Technological and Theoretical Foundations for Studies Aimed at Remedial Therapy Detecting Differentially Expressed Genes by Differential Display Yong-Jig Cho and Peng Liang

185

Expression Profiling as a Tool for Diagnosis and Pathway Discovery: Experimental Design and Technical Considerations Eric P. Hoffman, Donald Massaro, Gloria Massaro, Linda Clerch, and Yue Wang

197

10.

Plasticity of Circulating Adult Stem Cells Timothy R. Brazelton and Helen M. Blau

11.

The Mechanical and Cytoskeletal Basis of Lung Morphogenesis Eben Alsberg, Kimberly Moore, Sui Huang, Tom Polte, and Donald E. Ingber

217

247

III.

Comparative Lung Structure and Mechanics

12.

Morphogenesis of the Mammalian Lung: Aspects of Structure and Extracellular Matrix Johannes C. Schittny and Peter H. Burri

275

Structure and Function of Nonmammalian Vertebrate Lungs J.N. Maina

319

13.

IV.

Branching Morphogenesis

14.

Lung Branching Morphogenesis: Potential for Regeneration of Small Conducting Airways Minke van Tuyl, Veronica del Riccio, and Martin Post

355

Contents V.

The Gas-Exchange Region

15.

Apoptosis and Emphysema Norbert F. Voelkel, Laimute Taraseviciene-Stewart, and Rubin M. Tuder

16.

Genetic Analysis of Emphysema and Animal Models of COPD Steven D. Shapiro and Ravi Mahadeva

17.

Pulmonary Alveoli: Development, Structural Stability, and Regeneration Donald J. Massaro, Gloria Decarlo Massaro, and Linda Biadasz Clerch

ix

395

411

433

18.

Molecular Response to Pneumonectomy Leonard J. Landesberg and Ronald G. Crystal

19.

Pulmonary Limitations to Exercise Performance: The Effects of Healthy Ageing and COPD Jordan D. Miller and Jerome A. Dempsey

483

Pulmonary Adaptation to Sustained Changes in Metabolic Rate Jacopo P. Mortola

525

20.

Index

455

573

Contributors

Eben Alsberg Children’s Hospital, Harvard Medical School, Boston, Massachusetts, U.S.A. Simonetta Baraldo Bianca Beghe´

University of Padua, Padua, Italy

Richard D. Bland ifornia, U.S.A. Helen M. Blau nia, U.S.A.

University of Padua, Padua, Italy

Stanford University School of Medicine, Stanford, Cal-

Stanford University School of Medicine, Stanford, Califor-

Timothy R. Brazelton California, U.S.A.

Stanford University School of Medicine, Stanford,

Cynthia Brown Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A. Peter H. Burri

University of Bern, Bern, Switzerland

Yong-Jig Cho

Vanderbilt University, Nashville, Tennessee, U.S.A. xi

xii

Contributors

Linda Biadasz Clerch ington, D.C., U.S.A.

Georgetown University School of Medicine, Wash-

Jacqueline J. Coalson University of Texas Health Science Center, San Antonio, Texas, U.S.A. Ronald G. Crystal Weill Medical College of Cornell University, New York, New York, U.S.A. Jerome A. Dempsey sin, U.S.A.

University of Wisconsin–Madison, Madison, Wiscon-

Jonathan G. Goldin David Geffen School of Medicine at UCLA, Los Angeles, California, U.S.A. Eric P. Hoffman U.S.A. James C. Hogg bia, Canada

Children’s National Medical Center, Washington, D.C.,

University of British Columbia, Vancouver, British Colum-

Sui Huang Children’s Hospital, Harvard Medical School, Boston, Massachusetts, U.S.A. Donald E. Ingber Children’s Hospital, Harvard Medical School, Boston, Massachusetts, U.S.A. Leonard J. Landesberg Weill Medical College of Cornell University, New York, New York, U.S.A. Peng Liang

Vanderbilt University, Nashville, Tennessee, U.S.A.

Ravi Mahadeva University of Cambridge Institute for Medical Research, Cambridge, England J. N. Maina

University of the Witwatersrand, Johannesburg, South Africa

Jenny T. Mao David Geffen School of Medicine at UCLA, Los Angeles, California, U.S.A. Donald J. Massaro ton, D.C., U.S.A.

Georgetown University School of Medicine, Washing-

Contributors

xiii

Gloria Decarlo Massaro Georgetown University School of Medicine, Washington, D.C., U.S.A. University of Wisconsin–Madison, Madison, Wisconsin,

Jordan D. Miller U.S.A.

Kimberly Moore Children’s Hospital, Harvard Medical School, Boston, Massachusetts, U.S.A. Jacopo P. Mortola McGill University, Montreal, Quebec, Canada Tom Polte Children’s Hospital, Harvard Medical School, Boston, Massachusetts, U.S.A. Martin Post

University of Toronto, Toronto, Ontario, Canada

Veronica del Riccio

University of Toronto, Toronto, Ontario, Canada

Robert M. Rogers University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, U.S.A. Michael D. Roth David Geffen School of Medicine at UCLA, Los Angeles, California, U.S.A. Marina Saetta

University of Padua, Padua, Italy

Johannes C. Schittny

University of Bern, Bern, Switzerland

Steven D. Shapiro Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts, U.S.A. Laimute Taraseviciene-Stewart University of Colorado, Health Sciences Center, Denver, Colorado, U.S.A. Rubin M. Tuder

Johns Hopkins University, Baltimore, Maryland, U.S.A.

Graziella Turato

University of Padua, Padua, Italy

Minke van Tuyl

University of Toronto, Toronto, Ontario, Canada

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Contributors

Norbert F. Voelkel University of Colorado, Health Sciences Center, Denver, Colorado, U.S.A. Yue Wang Virginia Polytechnic Institute, Alexandria, VA, U.S.A. Robert A. Wise Johns Hopkins University School of Medicine, Baltimore, Maryland, U.S.A. Renzo Zuin University of Padua, Padua, Italy

1 Arrested Alveolar Development in Bronchopulmonary Dysplasia

JACQUELINE J. COALSON University of Texas Health Science Center San Antonio, Texas, U.S.A.

I. Evolution of BPD When bronchopulmonary dysplasia (BPD) was first described in premature infants in 1967, Northway et al. speculated that its pathogenesis was a prolongation of the healing phase of severe hyaline membrane disease (HMD) combined with generalized pulmonary oxygen toxicity. They recognized that endotracheal intubation and mechanical ventilation might have contributed to the development of the disease (1). The concept that BPD is primarily a disease that results from an arrest in alveolar development has only emerged over the last two decades. As better oxygenation and ventilatory strategies were developed and utilized, the classically described lung lesions of bronchial squamous metaplasia, alternating sites of emphysema (overinflation) and severe fibrosis, and the consequences of pulmonary vascular hypertension largely disappeared. These were replaced as the result of newer studies with descriptious of what were termed simplified lung (2); a ‘‘premature lung pattern (3); an interstitial form of BPD, characterized by arrested development of terminal air spaces (4); or acinar arrest’’ (5). Classic, or what is termed old BPD, pathological findings reflected primarily the consequences of elevated oxygen and ventilator-induced injury on a relatively immature and 1

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surfactant deficient lung. For example, Northway et al.’s study group had an average gestational age and birthweight of 32 weeks and 1893 gr, respectively. Even before the introduction of exogenous surfactant therapy, the better clinical management and technological advances had resulted in BPD occurring primarily in infants who were born <28 weeks’ gestation and weighed <1200 g. The more widespread use of prenatal steroids and postnatal exogenous surfactant has further influenced the incidence of BPD; infants now at most risk are 24–28 weeks’ gestation, and weigh 500–1000 g (6). It is not surprising that new descriptions of a simplified lung or acinar arrest reflect

Figure 1 A. A lung tissue section during the canalicular phase of lung development from an infant stillborn at 24 weeks’ gestation. The immersion-fixed lung specimen shows a terminal bronchiole (b) and its branches in the respiratory portion of the lung. The distal air spaces q [D] are rounded separated by intervening mesenchyme (ln) and little evidence of secondary crest formation. B. A lung tissue section during the alveolar phase of lung development from an infant stillborn at 38 week’s shows the remarkable maturation and differentiation the lung undergoes during prenatal development. Considerable thinning of the secondary crests (arrows) and a more thinned interstitium are evident throughout the lung. V, vessel (Hematoxylin & eosin, original magnification X110 and X106, respectively).

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Figure 1 Continued.

the present pathological findings in BPD, because these infants are born before their lungs have undergone alveolar development (Fig. 1). It is during the canalicular stage of lung development that alveolar and capillary growth and development start (Table 1), so the more extreme immaturity of the lung has become a dominant factor in the pathogenesis of BPD. II. Alveolization in Old and New BPD The pathology of old BPD has been detailed in an earlier volume of this series (7). Bonikas et al.’s detailed study described the histopathological changes in

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Table 1 Stages of Human Lung Development Embryonal First 7 weeks of gestation. Airways to level of bronchopulmonary segments develop. Lining epithelium is multilayered collection of spindle cells. Pseudoglandular 7–16 weeks’ gestation. Dichotomous branching of 16–25 generations of airways occurs to the level of the acinus. Differentiation of respiratory epithelial cells and cartilage. Angiogenetic development of preacinar vasculature completed. Canalicular 6–26 or 28 weeks’ gestation. Peripheral lining cells become cuboidal and distinct from bronchiolar epithelium. Differentiation of type 2 epithelial cells begins. Development of distal pulmonary circulation by vasculogenesis with capillaries present at 20 weeks. Interstitial tissue decreases, with thinning of the future gas-exchanging units. Saccular 26–28 to 32–36 weeks’ gestation. Marked decrease in interstitial space of saccular walls. Cylindrical saccules are subdivided by secondary crests (tissue projections into the air space). Crests contain a double capillary layer. Alveolar 32–36 weeks’ to term gestation Thinning of the secondary crests and fusion of capillaries form alveoli. Sources: From Refs. 32, 39, 67.

delivered infants who, with one exception, were older than 30 weeks gestation (8). This often-quoted study plus several others (9–17) resulted in the collection of pathological findings generally accepted for old BPD: an altered inflation pattern, with zones of overdistended fibrotic alveoli alternating with atelectatic and/or fibrotic zones; squamous metaplasia of airway epithelium; obliterative bronchiolitis; peribronchial fibrosis; airway smooth muscle hypertrophy and vascular hypertensive lesions (Fig. 2). Sobonya published a morphometric study of one infant born at 30 weeks’ gestation who developed BPD and experienced repeated episodes of apnea, bacterial pneumonias, and chronic cor pulmonale over a 13 month period before discharge (18). Following multiple admissions for pneumonia and progressive airway disease, the child died at 33 months of age. Lungs showed sites of large simple

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Figure 2 Lung tissue section obtained at autopsy from an infant with BPD who had been born at 34 weeks gestation and who died 4 months after birth. A. An altered inflation pattern of overexpanded, thinned sacular walls, separated from the atelectatic, thickened saccular walls by an interlobular septum that contains a vein (v). In a few of the overexpanded air spaces, rare alveolar duct septae are seen (arrow). B. The saccular walls in the atelectatic portion of the altered inflation pattern, although collapsed, show increased interstitial cellularity and fibroproliferation [ln]. Type 2 cell hyperplasia is evident (arrows). There are scattered small vessels (v), most of which are centrally located. C. The overexpanded air spaces (AS) show saccular walls that are thinned and fibrotic, with few capillaries. There are only a few short secondary crest/ alveolar eruptions along the fibrotic walls (arrows), as well as scattered blood-filled precapillary vessels (v) (hematoxylin & eosin, original magnification X46, X210, and X106, respectively).

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Figure 2

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Continued.

alveoli while other sites had well-formed alveoli similar to those in three agematched controls. The estimated number of alveoli was less by 6–10-fold and alveolar surface area by one-half compared to the three control lungs (18). So even when infants are born during the saccular stage of lung development, the stage of prominent secondary crest formation, and beginning alveolar formation, those with old BPD showed sites of enlarged simplified alveoli and lower number of alveoli. In reports that included both extremely small infants (less than 1000 g) and gestationally older and heavier infants, numbers of alveoli have been assessed morphometrically by several investigators. Hislop et al.’s study

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Figure 2 Continued.

included 20 infants (birthweights not given) with HMD, who were born at 25– 33 weeks’ gestation, mechanically ventilated for periods ranging from 3 days to 11 weeks, and died between 4 days and 14 months after birth (19). Shallow or variably shaped alveoli were evident at various study times, and a late-stage finding was a ‘‘generalised emphysema with overblown alveoli.’’ A seminal observation in this study was that children who had received mechanical ventilation with or without a history of HMD had reduced alveolar number and decreased internal surface area measurements. The lungs of control premature infants, who did not receive assisted ventilation, had normal alveolar counts and internal surface areas at death (19).

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In the Chambers and van Veizen study (2), 83 infants who died between 1983 and 1987 with birthweights ranging from 520 to 1658 g and gestational ages ranging from 23.5 to 30 weeks were studied. All infants who died after z10 days of mechanical ventilation had persistence of simple, evenly distributed terminal air spaces lined by undifferentiated cuboidal epithelium, and separated by evenly widened septa with hypercellular fibrous stroma and increased amounts of subepithelial elastic tissue. Arrested alveolar development was documented quantitatively by low Emery counts of the terminal respiratory units. In those infants who lived 17–156 days, airway changes, such as squamous metaplasia and peribronchial fibrosis, were infrequent and negligible. Margraf, et al. examined autopsy specimens of 8 infants who were born at gestational ages of 24–30 weeks (birthweights were not given), had HMD, and died at postnatal ages of 2–28 months (20). Their lungs showed varying amounts of bronchial and bronchiolar squamous metaplasia, marked simplification of acinar structure, variable but constant alveolar septal fibrosis, and abnormalities in elastic fiber architecture and arrangement. When compared to the lungs of six control infants, morphometric determinations showed that total alveolar number was decreased, and lung internal surface area was reduced. The surfactant-treated series reported by Husain et al. largely replicates the alveolar findings described for infants with BPD born just prior to the era of treatment with exogenous surfactant (5). Fourteen surfactant-treated infants with BPD, 8 non-surfactant-treated BPD patients, and 15 agematched controls who were autopsied from 1988 through 1994 were studied. The infants were treated with three doses of exogenous surfactant (either Survanta or Exosurf), given by intratracheal aerosolization during the first 24 h (note that aerosolization is an unusual method to administer surfactant). No history of prenatal steroid treatment was given. Gestational ages of the surfactant-treated BPD patients ranged from 24 to 32 weeks; birthweights were not stated. Gestational ages of the non-surfactant-treated BPD group ranged from 27 to 29 weeks. Eight of the 14 surfactant-treated infants lived for 1–6 weeks, while the other 6 survived for 12–413 weeks. Length of life in the non-surfactant-treated BPD group ranged from 2 to 71 weeks. Mild to moderate alveolar septal fibrosis was evident in 5 of the 14 surfactant-treated infants, while 7 of the 8 non-surfactant-treated infants had moderate to severe alveolar septal fibrosis of the type associated with the long-standing healed BPD changes described by Stocker in 1986 (21). No necrotizing bronchiolitis was evident in the surfactant-treated group, and in most cases a normalappearing capillary bed was noted. All (surfactant-treated or not) BPD patient specimens with a postconceptional age greater than 40 weeks, when compared to control specimens without BPD, showed acinar arrest (ratio of

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degree of alveolization and alveolar size). They concluded that the use of postnatal surfactant therapy did not alter the inhibition of acinar development that occurs in BPD infants. Not enough information is available in this study to determine how much surfactant was actually deposited in the more distal portions of the lungs; aerosolization of surfactant has not been shown to be beneficial in several recent human and animal studies (22–24). Because both old and new BPD both show decreased alveolization, it might be asked if there is really any difference between them. In regard to airway lesions, vascular changes, and degree of fibrosis in alveolar walls, infants with new BPD have considerably less disease. In the author’s experience, there is a more homogeneous pattern of simplication or arrest of alveoli in biopsy and autopsy material from infants with new BPD than in those with old BPD (Figs. 3, 4). The altered inflation pattern of atelectatic/ fibrosed sites vs. overexpanded air spaces is much rarer. The use of prenatal

Figure 3 Lung tissue section from an infant born at 28 weeks’ gestation and had a lung biopsy performed 2 weeks later. The terminal bronchiole (b) shows epithelial infolding, but no squamous metaplasia, and a thin muscular layer. The surrounding saccules (AS) are unevenly expanded and show little secondary crest formation (arrows), (hematoxylin & eosin, original magnification X110).

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Figure 4 Lung tissue section from an infant who was born at 26–27 weeks’ gestation and underwent an open lung biopsy 7 months after birth. There is considerable thinning of the distal air spaces (AS), but a lack of overall alveolarization persists (similar to pattern in Fig. 3). Note the variation in the interstitial fibroproliferation and the unevenly expanded air spaces. The bronchiole (b) branches into the alveolar duct area that shows a thickened alveolar septum (arrow) (hematoxylin & eosin, original magnification, X110).

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steroids and exogenous surfactant has no doubt influenced the more constant degree of lung inflation. In the surfactant-deficit lung of old BPD, Stocker suggested that the alternating sites of emphysema or overinflation and atelectasis were likely caused by small airway lesions, which, when obliterated, seemed to protect the alveoli distal to the small airway lesion. Obliteration of airways, however, was not documented consistently in old BPD reports. For example, Sobonya et al. reported alternating sites of simplified lung and nearnormal lung, but did not identify a small airway lesion, and it is not a feature of new BPD. In the 140 day baboon model of hyperoxia and positive pressure ventilation, following 7 days of 100% oxygen and then 14 days of 80% oxygen, a striking pattern of overinflated saccules with no alveoli alternated with underinflated and more normal looking saccular/alveolar structures. In another group of identically treated animals that survived to 33 weeks, lobectomy specimens showed that the overinflated, nonalveolated air spaces persisted, but the surrounding lung had alveoli, although fewer than the controls treated on an as-needed basis. Bronchiolitis obliterans was not present in either the 21 day or 33 week lung specimens. In new BPD, it is likely that the bulk of saccules or alveoli will be exposed more evenly to both the hyperoxic and volutrauma-induced injury, whatever their magnitude. The pathological findings described for both old and new BPD are representative of infants with the worst outcomes, because the information was invariably collected at postmortem examinations. Biopsy material is also suspect because the infant had not responded to therapy, and a biopsy was requested to define better the underlying lesion and/or complication. In the rare biopsies examined from known survivors, alveolar hypoplasia and an abnormal capillary configuration were evident, with minimal to no airway lesions. The most variable finding was the degree of cellularity and fibrosis in the simplified alveolar structures (7). What we do not know is what the lungs look like in the many infants with BPD who have left the hospital, require no supplemental oxygen, and are survivors. The majority of these infants were treated with prenatal steroids and exogenous surfactant, were ventilated with a low-volume ventilatory strategy, and received much less exposure to high oxygen concentrations over shorter periods of time. The lungs of these infants are increasing in size, but are they undergoing alveolization? An interesting observation is provided in Cherukupalli et al.’s study in which the lungs of 48 infants with HMD and BPD were studied (25). Using biochemical, clinical, and morphological methods, the authors categorized four patient groups based on four stages (I–IV) of acute lung injury, proliferation, early repair and late repair, respectively. Their group IV infants were ventilated for only 25% of their lives, and subjected to high oxygen levels for only 4% of their lives. They suggested that the reparative phase of BPD may be ‘‘almost completely successful’’ and cite that

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one of their group IV infants had ‘‘lungs that were almost entirely normal and could scarcely be distinguished from controls of the same age.’’ There are reports that describe so-called rebound or second-round growth of certain lung structures when the offending injury is removed (26– 28). Since the lung continues to form most of its alveoli in the first 2 years of life, might the possibility of new lung growth be expected in the less damaged lungs of patients with new BPD?

III. New Alveoli We now know that there is both pre- and postnatal alveolar development in the human. For many years it was thought that alveoli were not present at birth and only formed in the postnatal period. Although a few investigators had reported the presence of alveoli in lungs at birth (29–31), Langston et al.’s excellent study redefined the phases of intrauterine lung development in the human and clearly established a prenatal alveolar developmental period (32). They showed that at birth there were on average of 50  106 alveoli with a wide normal range. Hislop et al. have found an average of 150 million alveoli in term infants, which is about half the number expected by adulthood (33). However, infants born during the canalicular phase of lung development who develop BPD would largely skip the prenatal development of alveoli. If alveolar and associated capillary development could rebound following the milder lung injury in new BPD, the structural aspects of where alveoli are acquired postnatally is of interest. In 1923, Broman (34) demonstrated that terminal bronchioles did not cease division at birth and that new bronchioles and alveoli were produced postnatally for six or seven further generations of divisions. Using laborintensive serial sections and the Born wax-plate method, Boyden and Thompson reconstructed the distal airways and the more peripheral respiratory structures in a number of human lungs postnatally (35–38). In the newborn lung, a terminal bronchiole was seen to branch into two respiratory bronchioles that in turn branched into two or more additional orders of respiratory bronchioles, from which four very short transitional ducts arose. Following several dichotomous divisions, the transitional ducts gave rise to a cluster of 9–13 saccules (they thought adult-type alveoli developed after birth only). Over a 2 month period, larger alveoli were seen to develop in a centripetal direction: in a 2-month-old lung the most distal respiratory bronchiole had transformed into alveolar ducts, each containing 24–30 deep alveoli, ranging from 60 to 130 Am in diameter, and terminal bronchioles had converted into respiratory bronchioles. When Boyden examined the lungs of a 10-month-old infant, he observed that growth under compression, due to

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surrounding lung development, resulted in angulation of the alveolar ducts from which ‘‘herniations of alveoli or groups of alveoli’’ arose. In a lung specimen of a 3.7-year-old child, alveoli were seen to arise from the walls of the terminal bronchioles, which Boyden called ductular alveoli. In this way another means of transforming a terminal bronchiole into a first-order bronchiole was documented. His finding that older lungs had fewer nonrespiratory airway generations than did younger lungs further supported the concept of alveolization of the airways. Langston and Thurlbeck suggest that when alveoli are ‘‘well established’’ during late gestation, there are connective tissue areas immediately beneath the pleura and adjacent to bronchi and large vessels in which air spaces appear to form. The air spaces have double capillary walls, ‘‘while much of the rest of the lung showed well-developed alveoli, suggesting a peripheral component to alveolar acquisition at least during some periods of lung growth’’ (39). Zeltner and Burri also identified these sites in the human lung as prospective sites of late or renewed alveolar formation (40). In rodents, a critical period for alveolization has been identified (41). A comparable period has not been described for humans, but it would be suspected to be during the period of time that bulk alveolization occurs: up to 2 years of age. However, in an intriguing statement in a recent chapter Burri states that the bulk of alveolization may be completed by 6 months in the human infant (42). Morphometric studies of lungs within the first months of postnatal development support significant additional alveolar formation. Studying 10 children of unstated gender from childhood to 8 years of age, Dunnell found that the number of alveoli increased over the first 3 months from 24 million to between 73 and 86 million by 3 months, and that there was over a fivefold increase in alveoli over the first 13 months of postnatal life (43). Dunnill suggested that alveolar multiplication slowed after 4 years of age and stopped at 8 years of age. Thurlbeck and Angus also documented an increase in total number of alveoli in the first 4 years of life in 14 humans from birth to 19 years of age (44). A fourfold increase in alveoli in the first year and ninefold increase by puberty were reported by Emery and Mithal (29). However, in a large series of lungs from of 36 boys and 20 girls, Thurlbeck found that the bulk of rapid alveolar multiplication occurred during the first 2 years of life, with no increase or only a small increment in the total number of alveoli between the ages of 2–8 years of age. Assuming that the so-called arrest in alveolization might be reversible before 2 years of age, could the BPD survivors with milder disease show some increase in alveolar formation? The lack of significant airway lesions in new BPD would be expected to bode well for some potential alveolization but, as noted below, survivors with BPD are exhibiting airway dysfunction. Whether the hyperresponsiveness of the small airways

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in BPD survivors is structurally or biochemically based has not been determined. Long-term studies correlating the lung morphology and pulmonary function are needed, but follow-up lung biopsies in the human cannot be done ethically. However, long-term studies are underway in the baboon model of BPD to test this proposition.

IV. Long-Term Pulmonary Outcomes Reports regarding outcomes in old BPD included abnormal chest x-rays (45,46) and lung function tests (45,47,48), and episodes of recurrent bronchitis and pneumonia (45,49) during the first few years of life. Infants with severe BPD had mortality rates of 30–40%; most died during the first year of life secondary to repeated infections, respiratory failure, and cor pulmonale (50). In the more recent reports concerning outcomes of both extremely low birthweight and low-birthweight infants, recurrent cough and/or wheeze (51), airflow limitation (52–55), increased residual volume (55), decreased diffusing capacity (56,57), and increased hospital readmission rates have been described (51,58). However, lung function abnormalities have also been described in healthy preterm infants without BPD. These studies show that premature infants can have a moderately reduced functional residual capacity and a more pronounced impairment of gas mixing efficiency compared with infants born at term (59). The problem encountered with the published reports of lung pathology that likely reflect the more severely affected infants may also be evident in long-term pulmonary function reports. Some of the continuing trials, such as the EPICure population-based study of all births between 20 and 25 completed weeks gestation in the United Kingdom and Ireland, will no doubt yield reliable and very useful data on long-term pulmonary outcomes in the extremely immature infant. A declining number of premature infants are dying. In those who weigh 1000 g less at birth, causes of death were infection in about half of the infants, and respiratory distress syndrome/BPD and congenital defects in the remaining ones (60).

V. Treatment and Prevention Bronchopulmonary dysplasis has two major initiators: one is the developmental interruption or arrest of small airway, alveolar, and capillary development and differentiation; the other is the induction of inflammatory and reparative responses that compromise the ability of the lung to retain or regain its developmental program postnatally. To ameliorate the developmental initiator of BPD would require that preterm births be prevented. As

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Stahlman points out in her superb chapter on BPD prevention, maternal factors are dominant in this issue, and go beyond whether or not intrauterine infection is present (61). She makes a forceful argument that the demographic and socioeconomic circumstances of the women who deliver preterm need attention as well. In Bancalari’s recent review, he presents evidence supporting the major role inflammation is now thought to play in the pathogenesis of BPD (62). Ventilation with resultant volutrauma, oxygen therapy with free oxygen radical production, increased pulmonary blood flow due to a patent ductus arteriosus, and perinatal infections can induce the inflammatory response. The Miami neonatology group identified that, after prematurity, the main risk factors that predisposed infants to BPD were systemic infections and episodes of symptomatic patent duetus arteriosus in the postnatal period (63). Cesarean-delivered baboons show cytokine profiles, physiological findings, and pathological findings similar to the human with BPD. Based on our experience with the long-term baboon model of BPD, acquisition of postnatal infections dramatically affects the degree of alveolar wall fibrosis (64). Alveolization, per se, remains depressed in all the long term baboon studies examining the role of different ventilatory strategies [low volume–positive pressure ventilation, high frequency oscillatory ventilation (HFOV), and nasal continuous positive airway pressure (nCPAP)] on BPD pathogenesis in spite of differences in varying oxygen exposures, adequate nutritional support, and acquired nosocomial infections (64–66). Until the basic molecular developmental steps of alveolar and capillary development are discovered and specific therapies developed, a continued focus on how to ameliorate the injury-initiated insults that the immature lung suffers pre- and postnatally must continue.

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Coalson of alveoli: regulation and evidence for a critical period in rats. J Clin Invest 1985; 76:1297–1305. Burri P. Structural aspects of prenatal and postnatal development and growth of the lung. In: McDonald JA, ed. Lung Growth and Development. New York: Marcel Dekker, 1997:1–35. Dunnill MS. Postnatal growth of the lung. Thorax 1962; 17:329–333. Thurlbeck W, Angus G. Growth and aging of the normal human lung. Chest 1975; 67(suppl):3S–7S. Bryan MH, Hardie MJ, Reilly BJ, Swyer PR. Pulmonary function studies during the first year of life in infants recovering from the respiratory distress syndrome. Pediatrics 1973; 52:169–178. Edwards D, Dyer W, Northway W. Twelve years’ experience with bronchopulmonary dysplasia. Pediatrics 1977; 59:839–846. Coates A, Bergsteinsson H, Desmond K, Outerbridge E, Beaudry P. Long-term pulmonary sequelae of premature birth with and without idiopathic respiratory distress syndrome. J Pediatr 1977; 90:611–616. Stocks J, Godfrey S, Reynolds EOR. Airway resistance in infants after various treatments for hyaline membrane disease: special emphasis on prolonged high levels of inspired oxygen. Pediatrics 1978; 61:178–183. Kamper J. Long term prognosis of infants with severe idiopathic respiratory distress syndrome. II. Cardio-pulmonary outcome. Acta Paediatr Scand 1978; 67:71–76. Groothius J, Gutierrez K, Lauer B. Respiratory syncytial virus infection in children with bronchopulmonary dysplasia. Pediatrics 1988; 82:199–203. Greenough A, Giffin F, Yuksel B. Respiratory morbidity in preschool children born prematurely. Relationship to adverse neonatal events. Acta Paediatr 1996; 85:772–777. Parat S, Moriette M-F, Delaperche P, Escourrou P, Denjean A, Gaultier C. Long-term pulmonary outcome of bronchopulmonary dysplasia and premature birth. Pediatr Pulmonol 1995; 20:289–296. Pelkonen A, Hakulinen A, Turpeinen M. Bronchial lability and responsiveness in school children born very preterm. Am J Respir Crit Care Med 1997; 156:1178– 1184. Baralde E, Filippone M, Trevisanuto D, Zanardo V, Zacchello F. Pulmonary function until two years of life in infants with bronchopulmonary dysplasia. Am J Respir Crit Care Med 1997; 155:149–155. Jacob S, Coates A, Lands L, MacNeish C, Riley S, Hornby L, Outerbridge EW, Davis GM. Long-term pulmonary sequelae of severe bronchopulmonary dysplasia. J Pediatr 1998; 133:193–200. Hakulinen A, Jarvenpaa A-L, Turpeinen M, Sovijarvi A. Diffusing capacity of the lung in school-aged children born very preterm, with and without bronchopulmonary dysplasia. Pediatr Pulmonol 1996; 21:353–360. Mitchell S, Teague W. With technical assistance of Amy Robinson. Reduced gas transfer at rest and during exercise in school-age survivors of bronchopulmonary dysplasia. Am J Respir Crit Care Med 1998; 157:1406–1412.

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58. Chye J, Gray P. Rehospitalization and growth of infants with bronchopulmonary dysplasia: a matched control study. J Paediatr Child Health 1995; 31:105– 111. 59. Hjalmarson O, Sandburg K. Abnormal lung function in healthy preterm infants. Am J Respir Crit Care Med 2002; 165:83–87. 60. Barton L, Hodgman J, Pavlova Z. Causes of death in the extremely low birth weight infant. Pediatrics 1999; 103:446–451. 61. Stahlman M. The goal: prevention of BPD. In: Bland RB, Coalson JJ, eds. Chronic Lung Disease of Early Infancy. New York: Marcel Dekker, 2000:367– 376. 62. Bancalari E. Changes in the pathogenesis and prevention of chronic lung disease of prematurity. Am J Perinatology 2001; 18:1–9. 63. Gonzalez A, Sosenko I, Chandar J, Hummler H, Claure N, Bancalari E. Influence of infection on patent ductus arteriosus and chronic lung disease in premature infants weighing 1000 grams or less. J Pediatr 1996; 128:470–478. 64. Coalson J, Winter V, Siler-Khodr T, Yoder B. Neonatal chronic lung disease in extremely immature baboons. Am J Respir Crit Care Med 1999; 160:1333–1346. 65. Yoder B, Siler-Khodr T, Winter V, Coalson J. High-frequency oscillatory ventilation: effects on lung function, mechanics, and airway cytokines in the immature baboon model of neonatal CLD. Am J Respir Crit Care Med 2000; 162:1867–1876. 66. Coalson J, Thomson M, Yoder B, Winter V, Catland D, Martin H. Early nCPAP ventilation improves lung morphology and decreases lung infection in the baboon model of BPD. Am J Respir Crit Care Med 2002; 165:B39. 67. Roman J. Cell-cell and cell-matrix interactions in development of the lung vasculature. In: McDonald JA, ed. Lung Growth and Development. New York: Marcel Dekker, 1997:355–389.

2 Bronchopulmonary Dysplasia in the Postsurfactant Era

RICHARD D. BLAND Stanford University School of Medicine Stanford, California, U.S.A.

I. Introduction Infants who are born at a very early stage of development often experience respiratory failure because of their immature lungs, primitive respiratory drive, and susceptibility to infection. Survival of these infants has improved considerably in recent years owing to major advances in perinatal care, including widespread use of prenatal glucocorticoid treatment, postnatal surfactant replacement, and improved ventilatory and nutritional support. Yet the need for prolonged assisted ventilation in such infants frequently results in a chronic form of lung disease that was first described by Northway and associates as bronchopulmonary dysplasia (BPD) (1). The characteristic clinical, radiographic, and pathological features of this condition have changed during the past two decades, perhaps reflecting a progressive increase in survival of the tiniest premature infants with very immature lungs, as well as changes in the application of assisted ventilation and various supportive measures used in managing these infants. This paradigm of what is now described as the ‘‘new BPD’’ constitutes the most common cause of chronic lung disease (CLD) in early infancy (2). 21

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The reported incidence of this disease varies considerably among newborn care centers, reflecting not only differences in patient population and infant management practices but also different criteria used to define BPD. The most commonly accepted combination of criteria used to establish a diagnosis of BPD includes a history of early postnatal mechanical ventilation and subsequent oxygen dependency with characteristic pulmonary radiographic abnormalities (usually diffuse bilateral lung densities, often associated with areas of hyperinflation) that persist beyond 28 days of age. In some reports, the definition has been modified to include a need for supplemental oxygen and radiographic abnormalities beyond 36 weeks postconceptional age. This description underscores the importance of delayed healing of the initial injury to an incompletely developed lung. A recent workshop on BPD sponsored by the National Institutes of Health omitted abnormal radiographic findings as a diagnostic criterion (an interesting omission for a disease that was first described by a radiologist). It emphasized instead the persistent need for supplemental inspired oxygen for at least 28 days, with severity related to duration and magnitude of supplemental oxygen with or without assisted ventilation beyond 36 weeks postconceptional age (3). The incidence of this chronic respiratory ailment varies inversely as a function of birth weight and gestational age. It occurs in up to 75% of mechanically ventilated infants with a birth weight of 500–750 g. Most reports describe an incidence lower than 10% among ventilated infants with respiratory distress who weigh more than 1500 g at birth (4). Recent estimates indicate that there are 10,000–15,000 new cases of BPD in the United States each year, at least two-thirds of which occur in infants who weigh less than 1500 g at birth, with an estimated 10–20% mortality rate that is greater in males than in females (2,5). Although BPD is primarily a disease that evolves from lung injury after premature birth, it sometimes occurs in near-term infants who require prolonged mechanical ventilation for respiratory failure, usually because of pneumonia either from an infectious agent or from aspiration of foreign material, sepsis, lung hypoplasia, or cardiogenic pulmonary edema. As more and more infants survive the challenges of extreme prematurity and conditions associated with acute respiratory distress, the impact of BPD on health care resources during the newborn period and beyond has increased progressively over the past several years.

III. Changing Pattern of BPD In the years that preceded surfactant replacement and widespread use of prenatal glucocorticoid therapy, BPD occurred primarily in babies who were

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of sufficient size and maturity to survive the ravages of prolonged exposure to excessive oxygen and positive-pressure ventilation. These were predominantly babies born between 28 and 32 weeks of gestation and weighed between 1000 and 1500 g at birth. Their clinical course and lung pathology reflected the consequences of severe pulmonary oxygen toxicity and lung overexpansion. These often are manifest as extrapulmonary air leaks, protein-rich lung edema, extensive airway epithelial metaplasia, peribronchial fibrosis, and overgrowth of smooth muscle in airways and pulmonary arteries, sometimes leading to right heart failure (6–10). Mortality among these infants was high (greater than 50% in some series), and long-term ventilatordependent respiratory failure was common among survivors. With the advent of surfactant therapy to combat acute respiratory failure, coupled with widespread use of prenatal steroids to accelerate lung maturation before anticipated premature delivery, the epidemiological, clinical, and pathological picture of BPD changed considerably (3,11–15). Recent reports indicate that almost two-thirds of infants who acquire BPD weigh less than 1000 g and are less than 28 weeks of gestation at birth, and most cases of BPD now evolve without a prior history of severe respiratory distress syndrome (RDS) (4,16). In contrast to past experience, when pulmonary oxygen toxicity and lung overexpansion were considered major contributors to the development of chronic lung injury, these extremely small, immature infants often require little supplemental oxygen during their initial postnatal course. It is uncommon for them to receive mechanical ventilation with high inflation pressures or large tidal volumes. In many instances, ventilatory support for these infants is provided primarily because of apnea, weak inspiratory effort, and a compliant chest wall, all of which contribute to CO2 retention. Their respiratory status sometimes worsens as a result of pulmonary edema associated with a patent ductus arteriosus (PDA), or from sepsis and pneumonia (12), which in turn may increase the need for supplemental oxygen and positive pressure ventilation. Such infants tend to have a milder form of chronic respiratory failure than was described in the years that preceded surfactant replacement and prenatal glucocorticoid treatment. Recent reports indicate that the lung pathology of these extremely immature infants with BPD differs from the so-called classic form of BPD. There is less evidence of fibroproliferative airway damage and parenchymal fibrosis; the predominant pathological features of the new BPD are alveolar hypoplasia, arrest of acinar and associated vascular development, abundant smooth muscle in both the lung circulation and small airways, variable degrees of interstitial proliferation of extracellular matrix components, including elastin and collagen, as well as interstitial fluid accumulation (11,13,17–22). Although the pathogenesis of the new BPD remains unclear, there is much evidence to suggest that this form of chronic respiratory failure reflects

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abnormal growth and repair of the immature lung exposed to the continuous stress of repetitive inflation with O2-enriched gas in a setting of chronic inflammation, often aggravated by recurrent infection (23,24). Several studies have shown an increase in the number of neutrophils, macrophages, and neutrophil-derived elastase activity in liquid suctioned from the airways of infants with acute respiratory distress who later acquire BPD (25–28). These studies also showed that elastase inhibitory capacity and a1-protease inhibitor activity are reduced, as is secretory leukocyte protease inhibitor (29), in infants with evolving BPD compared to infants without BPD. Other studies indicate that antioxidant enzymes are deficient in the immature lung (30,31), which may further increase vulnerability to the oxidant stress associated with postnatal lung inflammation. The reported association of maternal chorioamnionitis and early lung inflammation in infants with subsequent BPD (32) led to the notion that BPD sometimes may have a prenatal inflammatory origin (14,33,34). The pivotal role of lung inflammation, either before or soon after birth, in the development of BPD has yet to be established, but remains a major thrust of many current investigations into the pathogenesis of BPD and the pursuit of effective therapy or prevention.

IV. Animal Models of Postsurfactant BPD Defining the pathophysiology of BPD in the postsurfactant era has been a formidable challenge and has relied to a large extent on the detailed observations made on authentic animal models of this condition. These have included chronic ventilation experiments conducted with prematurely delivered baboons at the Southwest Foundation in San Antonio, Texas (35–39), and similar studies with premature lambs mechanically ventilated for 3–4 weeks by the group in Utah (40–44). Both of these experimental models of BPD use animals that are delivered at a very immature stage of development, in which prolonged assisted ventilation with O2-enriched gas is essential to allow survival of sufficient duration for chronic lung injury to develop. The clinical condition and early postnatal management of these premature animals closely resemble the clinical history of infants with BPD (35,42): surfactant replacement is given at birth to reduce the need for supplemental O2 and high inflation pressures; complications associated with a patent ductus arteriosus are prevented with either pharmacological or surgical closure during the first few days after birth; cardiovascular instability is common, often requiring treatment with intravenous infusion of dopamine; recurrent infections develop that require protracted antimicrobial therapy; intravenous nutrition is provided beginning at birth, later supplemented with enteral feedings; and mechanical ventilation is applied using modest peak-inflation

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pressures and positive end-expiratory pressure, with sufficient supplemental oxygen and ventilatory support to maintain normal PaO2 and PaCO2 values. The most authentic animal homolog of the new BPD is the creation of Coalson and her colleagues, whose pioneering efforts with prolonged mechanical ventilation and hyperoxia of premature baboons began over 20 years ago in the presurfactant era (45–51). In recent years, this model has been modified to replicate almost all of the conditions that prevail in the development of the new BPD, including extreme prematurity, prenatal exposure to maternal glucocorticoid treatment, postnatal surfactant treatment, and assisted ventilation with modest inflation pressures and appropriate concentrations of inspired O2 (35). The only missing feature of this model of the new BPD is premature labor. As described in the initial report, baboons were delivered by cesarean section at about two-thirds of term gestation and then mechanically ventilated for at least 1–2 months. Airway secretions obtained from these animals showed evidence of lung inflammation. At autopsy their lungs had fewer alveoli and capillaries than did control term lungs. The respiratory units of the chronically ventilated preterm baboons were described as large simplified distal saccules, with walls that contained increased numbers of mesenchymal and mononuclear cells, and focal deposition of elastin and collagen fibers. Subsequent studies showed decreased pulmonary expression of vascular endothelial growth factor (VEGF) and two VEGF receptors (Flt-1 and TIE1) in the baboons with BPD, which may help to explain their reduced capillary volume density (38). A recent report also demonstrated reduced abundance of both endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS), coupled with decreased NOS activity in the lungs of these baboons. These findings might contribute to pulmonary vascular and airway dysfunction in infants with BPD (39). An ovine model of neonatal CLD has provided further insight into the pathophysiology of the new BPD. Lambs delivered by cesarean section at approximately 80% of term gestation and then mechanically ventilated for 3–4 weeks had persistent elevation of lung vascular and respiratory tract resistance compared to control lambs born at term. These physiological abnormalities were associated with increased abundance of smooth muscle and elastin in pulmonary arteries and airways (41,42). Studies of lung fluid balance showed a progressive increase in lung lymph flow and a consistent decrease in the lymph/plasma protein ratio, indicative of increased lung microvascular pressure rather than increased permeability. Postmortem histopathological examination revealed varying degrees of interstitial pulmonary edema (42). Subsequent studies showed evidence of lung vascular dysfunction, with loss of the pulmonary vasodilator response to inhaled nitric

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oxide. These findings were attributed to diminished abundance of eNOS and soluble guanylate cyclase (sGC) in the pulmonary circulation (43,44). These lambs also had fewer alveoli and lung microvessels than did control lambs born at term. There was a striking increase in tropoelastin gene expression in the lungs of the preterm lambs with CLD, and this was associated with excessive and disordered deposition of elastic fibers throughout the lung parenchyma (40). The abnormal abundance and distribution of elastin were especially notable in blunted secondary crests, where focal deposits of distally situated elastin normally define loci of future alveoli during lung development. The role that abnormal regulation of elastin may play in the pathogenesis of impaired alveolar and vascular development in BPD in unclear, but there is much evidence that cyclic stretch may induce tropoelastin expression in the developing lung (52–54), which in turn may yield increased elastin deposition. There is also evidence that the lung inflammation that accompanies acute and chronic neonatal respiratory failure is associated with increased elastase activity, which in turn may disrupt normal elastin deposition and alveolar formation (18,25,26,29,55). Understanding the mechanisms that regulate elastin distribution and abundance in the developing lung, and its dysregulation in neonatal lung injury and repair, is likely to provide important clues to the pathogenesis of impaired alveolar and lung vascular development in infants with BPD.

V. Impact of Surfactant Replacement on Development of BPD A recent commentary by Jobe (56) attempts to analyze the prevailing paradox that surfactant treatment at birth for very premature infants has greatly reduced the severity of acute RDS and thereby improved survival of these tiny infants without an apparent reduction in the incidence of BPD. As assessed by meta-analysis of multiple controlled clinical trials of the various surfactant preparations used to treat very premature infants, surfactant treatment has not decreased the incidence of BPD, irrespective of whether it is administered immediately after birth in the delivery room or after a brief period of observation designed to establish a diagnosis of RDS (57). Neither the magnitude of the dosage of surfactant nor the number of doses given had a significant effect on the incidence of BPD among tiny infants treated for RDS (58,59). Moreover, studies comparing different surfactant preparations found no difference in the incidence of BPD (60,61). A number of epidemiological studies comparing the incidence of BPD before and after the widespread use of surfactant treatment for very premature infants also showed no significant change in long-term respiratory outcome (62–64).

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This analysis is complicated by the fact that survival of very premature infants who are at greatest risk of BPD has improved by as much as 30% since the advent of surfactant treatment. It might be argued, therefore, that the proper denominator for comparing the incidence of BPD in the various surfactant trials should be the number of surviving infants rather than the number of randomized infants. At least one epidemiological study that took this tack, however, did not find a significant difference between the incidence of BPD among surfactant-treated and untreated survivors (62). Some neonatologists contend that the severity of BPD has diminished with use of surfactant. Unfortunately there are no data available from the clinical trials of surfactant to either support or refute this claim. Because of the many changes in newborn intensive care that have been implemented during the two decades since surfactant treatment became available, even if there has been a difference in the severity of BPD over this time, it would not be possible to ascribe an improved respiratory outcome to surfactant treatment alone without appropriate controls and legitimate criteria for assessing the severity of BPD. It is likely that other advances in patient management, such as widespread use of prenatal glucocorticoids, early closure of the ductus arteriosus, improved nutritional support, and gentler approaches to assisted ventilation, may have contributed to the perceived improvement in long-term respiratory outcome of tiny premature infants. There are several reasons why surfactant replacement soon after birth might not reduce the incidence, or even the severity, of BPD. First, because such treatment improves oxygenation of arterial blood and lends stability to the immature lung at end-expiration, it facilitates early postnatal management of the very tiny infants who are at greatest risk of acquiring BPD later in their course. Thus, surfactant treatment increases the pool of potential candidates for subsequent BPD. Although surfactant replacement clearly improves gas exchange in the surfactant-deficient lung, thereby allowing for less oxygen exposure and lower inflation pressures, these effects may be shortlived and fail to combat other key elements of extreme prematurity that are thought to increase vulnerability to BPD. These include: a poorly developed respiratory drive and weak chest wall that often lead to apnea and need for prolonged respiratory support; deficient host defense mechanisms that pave the way for infection and associated lung inflammation; persistent patency of the ductus arteriosus with resultant pulmonary edema; and inherent structural and chemical abnormalities in the lung, including fewer terminal air sacs and capillaries, and decreased abundance of antioxidants and protease inhibitors that render the lungs particularly susceptible to injury when exposed to even low concentrations of inspired oxygen and modest distending pressures. Moreover, nonuniform distribution of exogenous surfactant, or failure to reduce lung inflation pressures rapidly in response to improved lung

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mechanics from surfactant treatment, may lead to overdistention of terminal respiratory units, which in turn may contribute to the development of BPD. VI. Other Considerations in the Evolution of the New BPD and Its Treatment A. Prenatal Glucocorticoid Treatment

There is firm evidence that obstetrical management of fetal maturation with a single two-dose course of betamethasone given in anticipation of premature delivery has significantly reduced the incidence of RDS, intraventricular hemorrhage, and death of very premature infants. It also enhances the beneficial response to surfactant replacement in infants with RDS (65). There is also evidence that prenatal glucocorticoid treatment may lessen the incidence of persistent patency of the ductus arteriosus, a condition associated with an increased risk of BPD (66). Yet there is not convincing evidence that prenatal glucocorticoid treatment reduces the incidence of BPD (67–69), which again may reflect the fact that this therapy, like surfactant, significantly improves survival of tiny infants who are at greatest risk for BPD. The lack of clear benefit from either prenatal glucocorticoid therapy or postnatal surfactant replacement on the incidence of BPD is consistent with the view that the pathogenesis of the new BPD, in contrast to the old BPD, is not closely linked to the incidence and severity of RDS, which are reduced by both treatments. B. Assisted Ventilation Practices

Apart from these two life-saving advances in perinatal management, steroids before birth and surfactant after birth, several notable adjustments in newborn intensive care practices during the last two decades may have contributed to the evolution of clinical and pathological features that are now described as the new BPD. Perhaps the most heralded of these changes in patient care has been the emphasis on gentler approaches to assisted breathing, advocating early nasal application of constant airway-distending pressure, and limited use of positive-pressure mechanical ventilation, delivering much smaller tidal volumes than were used in the past. Rationale for this strategy sprang from a survey that compared the incidence of BPD at different institutions. The lowest incidence of the disease occurred in a neonatal unit that emphasized the use of continuous positive airway pressure without endotracheal intubation (70). Subsequent reports indicated that the incidence of BPD was greatest among very premature infants who had low PaCO2 values while receiving mechanical ventilation during the first few days after birth (71,72). These observations, coupled with studies showing that lung

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inflation with large tidal volumes soon after birth impaired responses to surfactant replacement and induced significant lung injury in preterm lambs (73,74), led to the notion that overzealous respiratory support and associated hypocapnia may predispose the incompletely developed lung to chronic injury and BPD. A recent report suggested that severe hypercapnia (PaCO2 95 F 5 mmHg) from CO2 added to the inspired gas may have attenuated lung injury in surfactant-treated preterm lambs that were mechanically ventilated for 6 h after 30 min of lung overinflation (75). Thus, many centers of newborn care recently have accepted a modest degree of carbon dioxide retention (PaCO2 45–60 mmHg, pH > 7.25) in order to minimize the adverse effects of prolonged, cyclic stretch on the fragile, immature lung. There is, however, only one published report of a randomized clinical trial designed to test the potential benefit of this approach in tiny (birth weight 854 F 163 g, gestation 26 F 1 weeks) preterm infants (76). This study compared a group of 24 infants who received mechanical ventilation designed to keep PaCO2 values between 45 and 55 mmHg and a group of 25 infants whose PaCO2 values were kept between 35 and 45 mmHg. The incidence of BPD was not significantly different between the two groups (43% in the permissive hypercapnia group, 64% in the normocapnia group). Thus, despite good rationale and compelling evidence that adult patients with ARDS may benefit from assisted ventilation with small tidal volumes and modest CO2 retention (77,78), it remains unclear whether this approach offers any advantage in preventing chronic lung injury in very premature infants. It is also important to ascertain whether intentional respiratory acidosis may have adverse consequences on the pulmonary and cerebral circulations of tiny newborn infants, who are predisposed to the complications of lung edema and intraventricular hemorrhage. Several reports have indicated that surfactant replacement followed by nasal application of continuous positive airway pressure (CPAP) may reduce the need for mechanical ventilation in premature infants with RDS (79–81). However, such treatment has not been shown to decrease the incidence of BPD. Additional multicenter randomized, controlled studies will be needed to assess the optimal respiratory management for this population of infants. C.

High-Frequency Mechanical Ventilation

After more than two decades of experimentation in both animals and human infants, there continues to be considerable controversy over the efficacy and safety of high-frequency mechanical ventilation in the management of very premature infants with respiratory failure. Initially developed as a method to reduce the cardiovascular effects of positive-pressure breathing and to prevent lung overdistention in experimental animals (82–84), high-frequency positive-

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pressure mechanical ventilation subsequently was used to treat newborn infants with severe respiratory distress (85). This approach, which used frequencies of 60–80 cycles/min (now considered to be conventional ventilation), was viewed as a logical means to reduce lung inflation volumes and pressures, with a resultant reduction in the incidence of extrapulmonary air leaks (86). In 1980, a research team in Toronto pioneered a new concept in respiratory support that would evolve over the next two decades: highfrequency oscillatory ventilation. These investigators demonstrated excellent respiratory gas exchange, first in dogs (87) and then in human infants with RDS (88), during prolonged application of vibratory ventilation delivered at frequencies of up to 1200 cycles/min. Several controlled, randomized trials of high-frequency mechanical ventilation using various devices designed to deliver tidal volumes as small as 1–2 ml at frequencies ranging from 3 to 16 Hz (180–960 cycles/min) have demonstrated effective treatment of RDS (89–100). Some of these studies have suggested that the incidence of BPD may be reduced with early application of high-frequency ventilation. Meta-analysis of multiple studies comparing high-frequency and conventional mechanical ventilation, however, has not yielded convincing evidence that early application of highfrequency oscillatory ventilation improves either survival or respiratory outcome of very premature infants (101,102). This view is consistent with the observation that high-frequency oscillatory ventilation, when compared with conventional mechanical ventilation, reduced markers of lung inflammation and improved measurements of lung mechanics, but did not prevent alveolar hypoplasia in the immature baboon model of neonatal chronic lung disease (37). Thus, optimal respiratory management for tiny infants with immature lungs remains an unsettled issue and a source of continuing impassioned debate between advocates and their adversaries. D. Oxygen

Although surfactant treatment at birth and early application of nasal CPAP have reduced the need for supplemental oxygen among premature infants at greatest risk for BPD, prolonged exposure to the toxic effects of supplemental oxygen in the face of a limited antioxidant defense system in such infants remains an important contributing condition in the pathogenesis of the new BPD. The role of long-term oxygen exposure in producing many of the features of neonatal lung injury observed in BPD is well documented in both newborn rodents and baboons (46,47,103). A recent study designed to examine the effects of supplemental oxygen on progression of retinopathy in premature infants showed a greater incidence of persistent lung disease in infants whose O2 saturations were maintained at 96–99% than in infants

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whose O2 saturations were maintained at 91–94%, again suggesting that prolonged delivery of excess oxygen can contribute to the development of the new BPD (104). Several studies have demonstrated the inhibitory effects of sustained hyperoxia on alveolar formation during the critical stage of lung development (51, 105–108). The reduced alveolar formation that occurs in the developing lung exposed to prolonged hyperoxia may reflect diminished pulmonary expression of tropoelastin mRNA (109,110), which in turn may yield abnormal lung elastin deposition and impaired alveolarization (111,112). Prolonged hyperoxia during a critical period of lung maturation also may cause impaired angiogenesis and hypertrophy of lung vascular smooth muscle, with resultant pulmonary hypertension (113,114). These oxygeninduced changes in the lung circulation have been attributed to decreased pulmonary expression of vascular endothelial growth factor (115) and increased lung protease activity (116) associated with prolonged oxygen exposure during development of the pulmonary circulation. A recent study showed that inhibition of angiogenesis by selective inhibition of a VEGF receptor in newborn rats was associated with arrested alveolar development (117). This observation, coupled with the recent report that VEGF may help to regulate surfactant production (118), supports the notion that VEGF signaling may serve important nonvascular as well as vascular functions to facilitate respiratory gas exchange after birth. Pulmonary expression of VEGF and its receptors is markedly reduced in both baboons and human infants with the new BPD (38,119). E.

Fluid and Salt Intake

Because pulmonary edema is a consistent feature of neonatal chronic lung disease (42,120), it has been suggested that early postnatal fluid and salt intake might contribute to the development of the new BPD (121). At least two prospective, randomized, controlled trials conducted during the postsurfactant era have examined the possible relationship between early postnatal fluid and salt intake and subsequent development of BPD. In a study that compared restricted daily fluid intake, ranging from 50 ml/kg soon after birth to 120 ml/kg at the end of the first week and up to 150 ml/kg thereafter, with a more liberal daily fluid regimen, ranging from 80 ml/kg soon after birth to 150 ml/kg at the end of the first week and up to 200 ml/kg thereafter, the incidence of BPD was significantly less and survival was greater among very premature infants who received the restricted fluid regimen (122). Another study compared early postnatal sodium restriction (no sodium supplements) to sodium administration of 3–4 mEq/kg daily for the first 3–5 days after birth in a group of very premature infants. It showed that sodium supplementation was associated with a significantly greater incidence of BPD (123). These

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observations, both of which are based on small numbers of patients enrolled in randomized, controlled clinical trials, support previous reports that high fluid intake during the first week of life may contribute to subsequent development of BPD in very premature infants (124,125). F.

Diuretics

Pulmonary edema that occurs in BPD is primarily the result of increased lung vascular filtration pressure, sometimes complicated by increased lung vascular permeability to protein associated with infection (42). Diuretics are the mainstay in the management of this type of pulmonary edema. Effective diuresis lowers pulmonary microvascular pressure and increases protein osmotic pressure in plasma. These two changes inhibit fluid filtration into the lungs and hasten the entry of water into the pulmonary microcirculation from the interstitium. In newborn lambs, intravenous furosemide caused an increase in plasma protein concentration and a decrease in pulmonary vascular pressures, with resultant reduction in lung fluid filtration, indicated by a decrease in lung lymph flow. These changes occurred both in the presence and absence of lung microvascular injury (126,127). Furosemide also has an effect on lung fluid balance independent of its diuretic action. In lambs without kidneys, intravenous furosemide consistently led to a small decrease in lung lymph flow, without any change in lung vascular pressures or plasma protein concentrations. This nondiuretic effect of furosemide may result from increased capacitance of systemic veins (128), leading to decreased pulmonary perfusion and an associated reduction in lung microvascular surface area for fluid exchange (126). The depressant effect of furosemide on cardiac output contraindicates its use in infants with respiratory distress and associated hypotension. In infants with BPD, however, effective diuresis with furosemide or other diuretic agents may have a beneficial effect on lung mechanics and respiratory gas exchange, thereby reducing the need for supplemental oxygen and assisted ventilation. Several studies have shown that short-term use of diuretics, either furosemide or thiazides and spironolactone, may improve lung mechanics and respiratory gas exchange in infants with BPD (129–134). Prolonged use of diuretics, however, requires careful attention to associated deficits of calcium, potassium, and chloride. Long-term treatment with furosemide has been associated with nephrocalcinosis and hearing impairment. One report showed that treatment with furosemide every other day, rather than daily, even when there was little or no increase in urine output, often was effective in reducing respiratory symptoms without causing harmful side effects (133). Other less potent diuretics, specifically thiazides and spironolactone, have

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been shown to improve pulmonary function and gas exchange in infants with BPD without apparent toxicity. G.

Retinol Treatment

Retinoids have been shown to have a profound influence on lung development and healing of lung injury in experimental animals (135–143). Because plasma concentrations of retinol are low in very premature infants compared to infants born at term, and particularly low in infants who acquire BPD (144,145), clinical trials of retinol treatment have been conducted in very premature infants with low plasma concentrations of retinol to determine the effects of retinol supplementation on the incidence of BPD (146,147). These randomized, controlled studies have shown that retinol treatment beginning soon after birth and continuing for 4 weeks thereafter leads to a modest but statistically significant reduction in the incidence of BPD, without apparent toxicity. Previous reports showed a beneficial effect of trans-retinoic acid treatment in attenuating both steroid-induced alveolar hypoplasia and oxygeninduced inhibition of lung septation in newborn rats (139,143). These observations, coupled with the aforementioned clinical trials of retinol treatment for premature infants, provided the basis for recent studies that examined the effects of daily, intramuscular retinol treatment (5000 IU/day) in lambs that were delivered prematurely and mechanically ventilated for 3 weeks. These were compared to lambs managed in an indentical manner except that they did not receive retinol (148,149). Lambs that received retinol had more alveoli, greater capillary surface density, and less elastin in their lungs than control lambs had. Immunoblot analysis of lung tissue harvested from these lambs showed greater expression of vascular endothelial growth factor (VEGF) and its receptor, Flk-1, in the lambs that received retinol. Northern analysis of peripheral lung tissue showed less expression of tropoelastin mRNA in the lungs of the retinol-treated lambs compared to controls. These new findings may provide important clues regarding the pathogenesis of BPD, perhaps implicating an association between excessive and disordered elastin accumulation and impaired development of alveoli and microvessels in the lung, as described in the premature lamb model of chronic lung injury (40–42) and in premature infants who have died with BPD (13,20,119,150). Elastin is known to have a pivotal role in normal mammalian lung development: deletion of the elastin gene in mice leads to neonatal death from cardiorespiratory failure associated with reduced terminal airway branching and impaired vasculogenesis in the lungs (151,152). Although the relationship between lung elastin and retinoic acid or retinol is less well established, the recent observation that mice bearing a deletion of retinoic

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acid receptors had reduced numbers of both alveoli and elastic fibers in their lungs raises the possibility that elastin’s role in alveolar formation may be regulated, at least in part, through retinoic acid signaling pathways (142). Disordered pulmonary elastin deposition in patients with BPD could be triggered by early postnatal elastolytic activity in the lung, which is known to occur in respiratory failure that is managed with assisted ventilation after premature birth (25,26). It also could result from prolonged, excessive lung stretch, which has been shown to increase tropoelastin gene expression in the developing lung of fetal sheep (54) and in cultured lung cells from fetal rats (52). The relationship between abnormal elastin accumulation and impaired alveolar and vascular development in the lungs of infants with BPD is intriguing and appears to warrant further exploration. H. Bronchodilators

Infants with BPD have increased pulmonary resistance, with marked limitation of forced expiratory gas flow through small airways (153–155). They typically manifest recurrent bronchoconstriction (156), and autopsy results on infants who have died with severe BPD show overgrowth of airway smooth muscle compared to control infants without lung disease (22). These structural and functional abnormalities of the respiratory tract in infants with BPD likely reflect the chronic airway inflammation characteristic of infants who receive long-term assisted ventilation with oxygen-enriched gas, sometimes complicated by pneumonia. Several studies have examined the acute respiratory response to shortterm inhalation of nebulized h2-adrenergic agonists in infants with BPD, showing transient improvement in lung mechanics and respiratory gas exchange (157–159). Short-term improvement in lung function also has been demonstrated with orally administered methylxanthines and h-adrenergic agonists given to infants with BPD (160–162). The high incidence of undesirable side effects and narrow therapeutic index, however, generally preclude prolonged use of these treatments (163). Studies of lung mechanics in chronically ventilated premature sheep and baboons have shown a reduction in respiratory tract resistance and improved lung compliance associated with long-term, continuous administration of inhaled nitric oxide (iNO) (164,165). Reports of clinical experience with iNO for infants with BPD suggest that this treatment may improve respiratory gas exchange and lead to a reduction in ventilatory support (166–168). The validity of these observations, however, awaits results of multicenter, randomized, controlled clinical trials, now underway, that have been designed to determine the potential efficacy and safety of low-dose iNO for treating preterm infants with BPD.

BPD in Postsurfactant Era I.

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Postnatal Glucocorticoid Treatment

Treatment of infants with BPD with high doses of corticosteroids is based on observations that BPD is a chronic inflammatory condition that usually occurs in the context of incomplete lung development, and that glucocorticoids can accelerate lung maturation during early development and reduce inflammation in a variety of acute and chronic diseases. Reports that ventilator-dependent infants with BPD might benefit from pharmacological doses of dexamethasone appeared in the literature two decades ago (169,170). These studies showed that dexamethasone could improve pulmonary function and facilitate successful removal from assisted ventilation, but did not increase survival of infants with BPD. A subsequent study showed that prolonged treatment (6 weeks) with high doses of dexamethasone could reduce the duration of mechanical ventilation, the need for supplemental oxygen, and the length of hospitalization of infants with BPD (171). Such treatment was associated with a high incidence of serious side effects, including hypertension, myocardial hypertrophy, and adrenal suppression (172). Frequent improvement in lung function and reduction in ventilator support after initiation of steroids led to widespread use of this treatment during the 1990s, often beginning as early as the first week after birth in tiny, immature infants at greatest risk of BPD. A multicenter randomized trial of twice-daily dexamethasone treatment for 4 weeks beginning on postnatal day 1, compared to placebo controls, showed a significant reduction in the incidence of BPD among infants whose birth weight was 1500 g or less (173). In this study, dexamethasone treatment was associated with less early postnatal lung inflammation, as assessed by cell counts, protein concentration, and inflammatory mediators in airway secretions. There was, however, an increased incidence of bacterial sepsis in the steroid-treated infants, with no significant difference in overall mortality compared to controls. A recent meta-analysis of several randomized, controlled trials of postnatal treatment with corticosteroids, begun either soon after birth or after a delay of several days to a few weeks, showed a significant reduction in the incidence of BPD, defined as oxygen dependence at 36 weeks postconceptional age, with no significant decrease in mortality (174). Proliferation of steroid use in the management of tiny, very premature infants took place in the shadow of prior experimental evidence that such treatment during early mammalian development can impair normal growth of the lungs (175–177), as well as the brain (178). Earlier efforts to treat RDS with postnatal corticosteroids failed to show benefit (179). Subsequent evaluation showed a high incidence of intraventricular hemorrhage (180) and neurological abnormalities (181) among infants who had been treated

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with steroids beginning at birth. These ominous findings, made years earlier in both newborn animals and human infants, were a portent of grim consequences that would emerge from critical follow-up evaluation of infants who had been treated with high doses of corticosteroids for prolonged periods to treat or prevent BPD. Enthusiasm for glucocorticoid treatment of premature infants quickly waned with the alarming news that such treatment appeared to be associated with an increased risk of abnormal neurological and developmental outcome (182–184). A 6 week course of dexamethasone begun 2–4 weeks after birth in ventilator-dependent preterm infants was associated with a 25% incidence of cerebral palsy compared to a 7% incidence in the placebo-treated control group (184). A large multicenter trial of dexamethasone treatment beginning either 2 or 4 weeks after birth showed diminished weight gain and head growth for both groups of infants during the period of steroid administration, with an increased risk of bacteremia and hyperglycemia in the early treatment group, without a difference in ventilation requirements or incidence of BPD (182). The toxic effects of high doses of corticosteroids on early postnatal brain development have been documented in recent animal studies (185,186) and in imaging studies of premature newborn infants treated with dexamethasone for BPD (187). Based on the accumulating evidence that systemic corticosteroid treatment has serious adverse effects in premature infants, the American Academy of Pediatrics and Canadian Paediatric Society published a joint statement warning against the routine use of systemic corticosteroids to treat or prevent BPD in such infants (188). To avoid the adverse effects of systemic steroids, several studies have been conducted to evaluate the efficacy and safety of inhaled glucocorticoids on subsequent development of BPD. A meta-analysis of seven trials of inhaled corticosteroid treatment found no reduction in the incidence of BPD in treated compared with placebo control infants (189), a finding that could be related to ineffective drug delivery to the lung (190). Failure of corticosteroid therapy to improve long-term respiratory outcome of very premature infants without considerable risk of serious and permanent adverse consequences underscores the need to define the specific inflammatory pathways that contribute to the development of BPD. The expectation is that such knowledge will help in the design and delivery of more specific anti-inflammatory agents than are now available.

VII. Epilogue: The New BPD The history of BPD and its treatment is a revealing saga of how newborn intensive care evolved from a primitive curiosity to a modern miracle mill

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during the last third of the 20th century. BPD did not exist until infants with respiratory failure could be rescued with oxygen-enriched gas delivered to the lungs with sufficient pressure to disrupt the delicate structure of air sacs and their capillaries still under construction. The pioneers of this noble and challenging mission, using simple respirators adapted from machines designed for adult anesthesia, soon learned that saving infants whose lungs were not fully developed often led to chronic respiratory failure and subsequent death or disability that required long-term and sometimes recurrent hospitalization. Highly skilled nursing care coupled with a host of technological advances, including high-risk obstetrical management and prenatal steroid treatment, application of continuous positive airway pressure, availability of better respirators designed specifically for infants, accurate and constant monitoring of blood pressure to combat shock, effective delivery of intravenous nutrition, and early detection and treatment of sepsis and pneumonia served to increase survival of extremely premature infants whose lungs are especially vulnerable to the adverse effects of high concentrations of inspired oxygen and long-term ventilator support. During the past two decades, widespread use of surfactant replacement after premature birth has facilitated management of even the tiniest infants, reducing their needs for supplemental oxygen and aggressive respiratory support. Increased survival of these so-called micropremies, whose respiratory drive frequently is impaired and whose host defenses are modest at best, often results in a protracted clinical course complicated by severe apnea and recurrent infection that calls for prolonged treatment with oxygen and assisted ventilation, culminating in the new BPD. The disease sometimes starts before birth, triggered by fetal inflammation associated with maternal chorioamnionitis. Thus, the clinical, radiographic, and pathological features of this condition are considerably different from the welldefined progression of lung disease described by Northway and associates almost four decades ago. The challenge now is to improve understanding of the molecular mechanisms that regulate normal lung growth and development, and to clarify the dysregulation that occurs with injury and subsequent repair so that specific measures can be devised to treat effectively, or even prevent, what is more aptly described today as neonatal chronic lung disease.

Acknowledgments Dr Bland gratefully acknowledges the support of the National Heart, Lung and Blood Institute (Grants HL-62512 and HL-56401) that provided funds for much of the research that is reported in this chapter.

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108. Warner BB, Stuart LA, Papes RA, et al. Functional and pathological effects of prolonged hyperoxia in neonatal mice. Am J Physiol 1998; 275:L110–117. 109. Bruce MC, Bruce EN, Janiga K, et al. Hyperoxic exposure of developing rat lung decreases tropoelastin mRNA levels that rebound postexposure. Am J Physiol 1993; 265:L293–300. 110. Bruce MC, Honaker C, Karathanasis P. Postnatal age at onset of hyperoxic exposure influences developmentally regulated tropoelastin gene expression in the neonatal rat lung. Am J Respir Cell Mol Biol 1996; 14:177–185. 111. Bruce MC, Pawlowski R, Tomashefski JF Jr, Changes in lung elastic fiber structure and concentration associated with hyperoxic exposure in the developing rat lung. Am Rev Respir Dis 1989; 140:1067–1074. 112. Mariani TJ, Sandefur S, Pierce RA. Elastin in lung development. Exp Lung Res 1997; 23:131–145. 113. Roberts RJ, Weesner KM, Bucher JR. Oxygen-induced alterations in lung vascular development in the newborn rat. Pediatr Res 1983; 17:368–375. 114. Wilson WL, Mullen M, Olley PM, et al. Hyperoxia-induced pulmonary vascular and lung abnormalities in young rats and potential for recovery. Pediatr Res 1985; 19:1059–1067. 115. Maniscalco WM, Watkins RH, D’Angio CT, et al. Hyperoxic injury decreases alveolar epithelial cell expression of vascular endothelial growth factor (VEGF) in neonatal rabbit lung. Am J Respir Cell Mol Biol 1997; 16:557–567. 116. Koppel R, Han RN, Cox D, et al. Alpha 1-antitrypsin protects neonatal rats from pulmonary vascular and parenchymal effects of oxygen toxicity. Pediatr Res 1994; 36:763–770. 117. Jakkula M, Le Cras TD, Gebb S, et al. Inhibition of angiogenesis decreases alveolarization in the developing rat lung. Am J Physiol Lung Cell Mol Physiol 2000; 279:L600–607. 118. Compernolle V, Brusselmans K, Acker T, et al. Loss of HIF-2a and inhibition of VEGF impair fetal lung maturation, whereas treatment with VEGF prevents fatal respiratory distress in premature mice. Nat Med 2002; 8:702–710. 119. Bhatt AJ, Pryhuber GS, Huyck H, et al. Disrupted pulmonary vasculature and decreased vascular endothelial growth factor, Flt-1, and TIE-2 in human infants dying with bronchopulmonary dysplasia. Am J Respir Crit Care Med 2001; 164:1971–1980. 120. Bland RD, Carlton DP. Pulmonary edema after premature birth. Bland RD, Coalson JJ, eds. Chronic Lung Disease in Early Infancy. Vol. 137. New York: Marcel Dekker, 2000:711–747. 121. Van Marter LJ, Pagano M, Allred EN, et al. Rate of bronchopulmonary dysplasia as a function of neonatal intensive care practices. J Pediatr 1992; 120: 938–946. 122. Tammela OK, Koivisto ME. Fluid restriction for preventing bronchopulmonary dysplasia? Reduced fluid intake during the first weeks of life improves the outcome of low-birth-weight infants. Acta Paediatr 1992; 81:207–212. 123. Costarino AT Jr, Gruskay JA, Corcoran L, et al. Sodium restriction versus daily maintenance replacement in very low birth weight premature neonates: a randomized, blind therapeutic trial. J Pediatr 1992; 120:99–106.

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124. Brown ER, Stark A, Sosenko I, et al. Bronchopulmonary dysplasia: possible relationship to pulmonary edema. J Pediatr 1978; 92:982–984. 125. Van Marter LJ, Leviton A, Allred EN, et al. Hydration during the first days of life and the risk of bronchopulmonary dysplasia in low birth weight infants. J Pediatr 1990; 116:942–949. 126. Bland RD, McMillan DD, Bressack MA. Decreased pulmonary transvascular fluid filtration in awake newborn lambs after intravenous furosemide. J Clin Invest 1978; 62:601–609. 127. Berner ME, Teague WG Jr, Scheerer RG, et al. Furosemide reduces lung fluid filtration in lambs with lung microvascular injury from air emboli. J Appl Physiol 1989; 67:1990–1996. 128. Dikshit K, Vyden JK, Forrester JS, et al. Renal and extrarenal hemodynamic effects of furosemide in congestive heart failure after acute myocardial infarction. N Engl J Med 1973; 288:1087–1090. 129. Kao LC, Warburton D, Cheng MH, et al. Effect of oral diuretics on pulmonary mechanics in infants with chronic bronchopulmonary dysplasia: results of a double-blind crossover sequential trial. Pediatrics 1984; 74:37–44. 130. McCann EM, Lewis K, Deming DD, et al. Controlled trial of furosemide therapy in infants with chronic lung disease. J Pediatr 1985; 106:957–962. 131. Engelhardt B, Elliott S, Hazinski TA. Short- and long-term effects of furosemide on lung function in infants with bronchopulmonary dysplasia. J Pediatr 1986; 109:1034–1039. 132. Albersheim SG, Solimano AJ, Sharma AK, et al. Randomized, double-blind, controlled trial of long-term diuretic therapy for bronchopulmonary dysplasia. J Pediatr 1989; 115:615–620. 133. Rush MG, Engelhardt B, Parker RA, et al. Double-blind, placebo-controlled trial of alternate-day furosemide therapy in infants with chronic bronchopulmonary dysplasia. J Pediatr 1990; 117:112–118. 134. Kao LC, Durand DJ, McCrea RC, et al. Randomized trial of long-term diuretic therapy for infants with oxygen-dependent bronchopulmonary dysplasia. J Pediatr 1994; 124:772–781. 135. Chytil F. The lungs and vitamin A. Am J Physiol 1992; 262:L517–527. 136. Fraslon C, Bourbon JR. Retinoids control surfactant phospholipid biosynthesis in fetal rat lung. Am J Physiol 1994; 266:L705–712. 137. Zachman RD. Role of vitamin A in lung development. J Nutr 1995; 125:1634S– 1638S. 138. Chytil F. Retinoids in lung development. FASEB J 1996; 10:986–992. 139. Massaro GD, Massaro D. Formation of pulmonary alveoli and gas-exchange surface area: quantitation and regulation. Annu Rev Physiol 1996; 58:73–92. 140. Massaro GD, Massaro D. Retinoic acid treatment abrogates elastase-induced pulmonary emphysema in rats. Nat Med 1997; 3:675–677. 141. Massaro GD, Massaro D, Chan WY, et al. Retinoic acid receptor-beta: an endogenous inhibitor of the perinatal formation of pulmonary alveoli. Physiol Genomics 2000; 4:51–57. 142. McGowan S, Jackson SK, Jenkins-Moore M, et al. Mice bearing deletions of

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retinoic acid receptors demonstrate reduced lung elastin and alveolar numbers. Am J Respir Cell Mol Biol 2000; 23:162–167. Veness-Meehan KA, Pierce RA, Moats-Staats BM, et al. Retinoic acid attenuates O2-induced inhibition of lung septation. Am J Physiol Lung Cell Mol Physiol 2002; 283:L971–980. Shenai JP, Chytil F, Jhaveri A, et al. Plasma vitamin A and retinol-binding protein in premature and term neonates. J Pediatr 1981; 99:302–305. Shenai JP, Chytil F, Stahlman MT. Vitamin A status of neonates with bronchopulmonary dysplasia. Pediatr Res 1985; 19:185–188. Shenai JP, Kennedy KA, Chytil F, et al. Clinical trial of vitamin A supplementation in infants susceptible to bronchopulmonary dysplasia. J Pediatr 1987; 111:269–277. Tyson JE, Wright LL, Oh W, et al. Vitamin A supplementation for extremelylow-birth-weight infants. NICHD Neonatal Research Network. N Engl J Med 1999; 340:1962–1968. Albertine KH, Jiancheng S, Dahl MJ, et al. Retinol treatment from birth increases expression of vascular endothelial growth factor (VEGF) and its receptor, fetal liver kinase (FLK-1), and is associated with greater lung capillary surface density in chronically ventilated preterm lambs. Pediatr Res 2002; 51:60A abstract #350. Bland RD, Albertine KH, Pierce RA, et al. Impaired alveolar development and abnormal lung elastin in preterm lambs with chronic lung injury: potential benefits of retinol treatment. Biol Neonate 2003; 84:101–102. Thibeault DW, Mabry SM, Ekekezie II, et al. Lung elastic tissue maturation and perturbations during the evolution of chronic lung disease. Pediatrics 2000; 106:1452–1459. Li DY, Brooke B, Davis EC, et al. Elastin is an essential determinant of arterial morphogenesis. Nature 1998; 393:276–280. Wendel DP, Taylor DG, Albertine KH, et al. Impaired distal airway development in mice lacking elastin. Am J Respir Cell Mol Biol 2000; 23:320– 326. Morray JP, Fox WW, Kettrick RG, et al. Improvement in lung mechanics as a function of age in the infant with severe bronchopulmonary dysplasia. Pediatr Res 1982; 16:290–294. Tepper RS, Morgan WJ, Cota K, et al. Expiratory flow limitation in infants with bronchopulmonary dysplasia. J Pediatr 1986; 109:1040–1046. Gerhardt T, Hehre D, Feller R, et al. Serial determination of pulmonary function in infants with chronic lung disease. J Pediatr 1987; 110:448–456. Motoyama EK, Fort MD, Klesh KW, et al. Early onset of airway reactivity in premature infants with bronchopulmonary dysplasia. Am Rev Respir Dis 1987; 136:50–57. Gomez-Del Rio M, Gerhardt T, Hehre D, et al. Effect of a beta-agonist nebulization on lung function in neonates with increased pulmonary resistance. Pediatr Pulmonol 1986; 2:287–291. Rotschild A, Solimano A, Puterman M, et al. Increased compliance in response

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Bland to salbutamol in premature infants with developing bronchopulmonary dysplasia. J Pediatr 1989; 115:984–991. Denjean A, Guimaraes H, Migdal M, et al. Dose-related bronchodilator response to aerosolized salbutamol (albuterol) in ventilator-dependent premature infants. J Pediatr 1992; 120:974–979. Rooklin AR, Moomjian AS, Shutack JG, et al. Theophylline therapy in bronchopulmonary dysplasia. J Pediatr 1979; 95:882–888. Kao LC, Durand DJ, Phillips BL, et al. Oral theophylline and diuretics improve pulmonary mechanics in infants with bronchopulmonary dysplasia. J Pediatr 1987; 111:439–444. Stefano JL, Bhutani VK, Fox WW. A randomized placebo-controlled study to evaluate the effects of oral albuterol on pulmonary mechanics in ventilatordependent infants at risk of BPD developing. Pediatr Pulmonol 1991; 10:183– 190. Hazinski TA. Drug treatment for BPD established. Bland RD, Coalson JJ, eds. Chronic Lung Disease in Early Infancy. Vol. 137. New York: Marcel Dekker, 2000:257–283. Bland RD, Albertine KH, Carlton DP, et al. Continuous inhalation of nitric oxide from birth decreases airway resistance and bronchiolar smooth muscle in chronically ventilated preterm lambs. Pediatr Res 1998; 43:275A (abstract #1613). Ballard PL, Gonzales LW, Godinez M, et al. Inhaled nitric oxide in the baboon model of chronic lung disease improves compliance and modifies surfactant. Pediatr Res 2003; 53:390A (abstract #2208). Banks BA, Seri I, Ischiropoulos H, et al. Changes in oxygenation with inhaled nitric oxide in severe bronchopulmonary dysplasia. Pediatrics 1999; 103:610– 618. Kinsella JP, Walsh WF, Bose CL, et al. Inhaled nitric oxide in premature neonates with severe hypoxaemic respiratory failure: a randomised controlled trial. Lancet 1999; 354:1061–1065. Early compared with delayed inhaled nitric oxide in moderately hypoxaemic neonates with respiratory failure: a randomised controlled trial. The FrancoBelgium Collaborative NO Trial Group. Lancet 1999; 354:1066–1071. Mammel MC, Green TP, Johnson DE, et al. Controlled trial of dexamethasone therapy in infants with bronchopulmonary dysplasia. Lancet 1983; 1:1356– 1358. Avery GB, Fletcher AB, Kaplan M, et al. Controlled trial of dexamethasone in respirator-dependent infants with bronchopulmonary dysplasia. Pediatrics 1985; 75:106–111. Cummings JJ, D’Eugenio DB, Gross SJ. A controlled trial of dexamethasone in preterm infants at high risk for bronchopulmonary dysplasia. N Engl J Med 1989; 320:1505–1510. Bloomfield FH, Knight DB, Harding JE. Side effects of two different dexamethasone courses for preterm infants at risk of chronic lung disease: a randomized trial. J Pediatr 1998; 133:395–400. Yeh TF, Lin YJ, Hsieh WS, et al. Early postnatal dexamethasone therapy for

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the prevention of chronic lung disease in preterm infants with respiratory distress syndrome: a multicenter clinical trial. Pediatrics 1997; 100(e3):1–8. Grier DG, Halliday HL. Corticosteroids in the prevention and management of bronchopulmonary dysplasia. Semin Neonatol 2003; 8:83–91. Massaro D, Teich N, Maxwell S, et al. Postnatal development of alveoli. Regulation and evidence for a critical period in rats. J Clin Invest 1985; 76: 1297–1305. Blanco LN, Massaro GD, Massaro D. Alveolar dimensions and number: developmental and hormonal regulation. Am J Physiol 1989; 257:L240–247. Ellington B, McBride JT, Stokes DC. Effects of corticosteroids on postnatal lung and airway growth in the ferret. J Appl Physiol 1990; 68:2029–2033. Howard E. Reductions in size and total DNA of cerebrum and cerebellum in adult mice after corticosterone treatment in infancy. Exp Neurol 1968; 22:191–208. Baden M, Bauer CR, Colle E, et al. A controlled trial of hydrocortisone therapy in infants with respiratory distress syndrome. Pediatrics 1972; 50:526–534. Taeusch HW Jr, Wang NS, Baden N, et al. A controlled trial of hydrocortisone therapy in infants with respiratory distress syndrome: II Pathology. Pediatrics 1973; 52:850–854. Fitzhardinge PM, Eisen A, Lejtenyi C, et al. Sequelae of early steroid administration to the newborn infant. Pediatrics 1974; 53:877–883. Papile LA, Tyson JE, Stoll BJ, et al. A multicenter trial of two dexamethasone regimens in ventilator-dependent premature infants. N Engl J Med 1998; 338: 1112–1118. Yeh TF, Lin YJ, Huang CC, et al. Early dexamethasone therapy in preterm infants: A follow-up study. Pediatrics 1998; 101(e7):1–8. O’Shea TM, Kothadia JM, Klinepeter KL, et al. Randomized placebocontrolled trial of a 42-day tapering course of dexamethasone to reduce the duration of ventilator dependency in very low birth weight infants: outcome of study participants at 1-year adjusted age. Pediatrics 1999; 104:15–21. Flagel SB, Vazquez DM, Watson SJ Jr, et al. Effects of tapering neonatal dexamethasone on rat growth, neurodevelopment, and stress response. Am J Physiol Regul Integr Comp Physiol 2002; 282:R55–63. Edwards HE, Burnham WM. The impact of corticosteroids on the developing animal. Pediatr Res 2001; 50:433–440. Murphy BP, Inder TE, Huppi PS, et al. Impaired cerebral cortical gray matter growth after treatment with dexamethasone for neonatal chronic lung disease. Pediatrics 2001; 107:217–221. American Academy of Pediatrics and Canadian Pediatric Society. Postnatal corticosteroids to treat or prevent chronic lung disease in preterm infants. Pediatrics 2002; 109:330–338. Shah PS. Current perspectives on the prevention and management of chronic lung disease in preterm infants. Paediatr Drugs 2003; 5:463–480. Cole CH, Colton T, Shah BL, et al. Early inhaled glucocorticoid therapy to prevent bronchopulmonary dysplasia. N Engl J Med 1999; 340:1005–1010.

3 What Is Chronic Obstructive Pulmonary Disease?

ROBERT M. ROGERS University of Pittsburgh School of Medicine Pittsburgh, Pennsylvania, U.S.A.

I. Introduction Throughout history, in their efforts to understand disease and treat patients effectively, physicians have noted that similar symptoms are likely to be caused by similar precipitating factors. During the 1950s in America and Britain, physicians noted that patients with a history of smoking often presented with symptoms that amounted to ‘‘having difficulty breathing.’’ In the United Kingdom, physicians tended to diagnose the condition as chronic bronchitis, while in the United States the same symptoms would probably have resulted in a diagnosis of emphysema. On both sides of the Atlantic, clinicians would also find their diagnoses complicated by patients presenting with asthma alone or in conjunction with other lung impairments. Compounding the problem of definition and nomenclature, the UK population in general (not just smokers) tended to experience more respiratory symptoms because of environmental factors. Investigators from the Netherlands further complicated the issue with their theory, the so-called Dutch hypothesis, that the patients who had mild asthma and smoked were more prone to develop significant airflow limitation. Thus, clinicians and laypersons alike on each side of the Atlantic tended to view chronic obstructive 51

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pulmonary disease (COPD) differently This undoubtedly slowed the research and understanding of the diseases themselves, as well as their subsequently established connection to precipitating—and preventable—factors such as smoking. This chapter will be written from the perspective of a pulmonologist also trained in pulmonary physiology, and will reflect experience and insights into the development of the terminology and the evolution of our understanding of the disease process over the last three to four decades.

II. The Early Years In the past pulmonary medicine had an infectious disease orientation, with a focus on diagnosis and treatment of tuberculosis. Many of the early leaders in the field had active tuberculosis, spent time in a sanatoria as patients, and participated in the successful treatment of tuberculosis with antibiotics. While they had a knowledge of antibiotics and chest radiology, they did not understand the physiological processes as well. Early chest physicians were extremely facile in interpreting the chest radiology of tuberculosis, but that skill was of little or no help in the diagnosis of COPD. For these early pulmonary physicians, and certainly for all generalists and family physicians, physiology seemed to be a foreign language that was difficult, if not impossible, to understand. During the 1940s great strides were made toward understanding the pathology of emphysema, a pathological entity first described by Laennec, including the recognition of different types: panlobular, centrilobular, and irregular emphysema. During the 1950s and 1960s, great progress was made in understanding the pathology of emphysema, including a sentinel paper by Mclean (1) on the pathogenesis of emphysema that was the first to link inflammation to centrilobular emphysema. The work of many outstanding pathologists including Gough (2), Kleinman , Dunnill, Reid, Thurlbeck (3,4), and Hogg educated clinicians about the destructive process called emphysema, and graphically demonstrated the lesions in fixed whole lung sections. About this time, Thurlbeck and others began to work on the pathogenesis and pathophysiology of what is now called COPD. To quote him: ‘‘Already by 1960, most of the descriptive pathology of emphysema and chronic bronchitis had been written and the main thrust was morphologic–physiologic correlations’’ (5,6). In the 1960s, an excellent textbook called Respiratory Function in Disease was written with the goal of bridging the gap between clinical medicine and physiology, with a strong emphasis on pathological correlation. While the subject index of this text does not even list the term COPD (7), it devotes 20–30 pages to bronchitis (chronic) and 50 pages of discussion to

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53

various aspects of emphysema. In contrast, by the 1974 edition of the book, the term COPD does appear in the index. The following quotation from the 1974 version of the book outlines the issues of nomenclature and diagnosis: During the past fifteen years a great deal of attention has been devoted to the vexing and controversial problem of the definition of chronic respiratory disease. It cannot be said yet that these efforts have yet been wholly successful, nor have the systems of differentiation and classification proposed by any investigator or group of investigators proven universally acceptable.

The author goes on to detail the diagnostic dilemma: It has been generally believed that pulmonary emphysema can be realistically defined only in morphological terms, and that chronic bronchitis is best defined on clinical criteria at least in the first instance. In Britain and America the difference between spasmodic asthma from these two entities does not seem to present a special problem, but in countries where it is taught that there are considerable allergic components to chronic bronchitis the two diseases are not regarded as sharply distinct.

Most interesting about this particular textbook is that the authors also posed three questions that remain relevant today: 1. Can the clinician differentiate between irreversible airways obstruction due to chronic bronchitis alone and that due to morphological emphysema? 2. Should the same term (chronic bronchitis) be used for the clinical diagnosis based on a history of chronic productive cough, and the severe irreversible airways obstruction syndrome that may lead to abnormal arterial blood–gas tensions and cor pulmonale in the absence of morphological emphysema? 3. To what extent is severe V/Q imbalance produced by the morphological changes of emphysema and the lesions of chronic bronchitis? The questions above remain relevant today, but by the early 1960s diagnostic criteria were defined and the term chronic obstructive pulmonary disease became recognize on both sides of the Atlantic to describe the condition of patients whose symptoms derive from either chronic bronchitis or emphysema or both. The American Thoracic Society officially adopted the term COPD in 1962, following a symposium (8). The published proceedings of the symposium became the standard reference for researchers and clinical specialists interested in lung disease. Chronic bronchitis was defined as: ‘‘symptoms (cough and sputum production) occurring on most days for at least three months of the year for a two-year period.’’ The term COPD is not used to describe the condition of patients with asthma alone. In addition, a

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diagnosis of emphysema is based on anatomical changes and a diagnosis of chronic bronchitis is made on clinical grounds. Before the term COPD was widely accepted, other terms such as chronic airflow obstruction (CAO), chronic obstructive lung disease (COLD), chronic airflow limitation (CAL), chronic obstructive airway disease (COAD) were used. By the 1980s the term COPD was commonly used by clinicians and scientific investigators, and during the 1990s was given a great boost by two initiatives begun during that decade, The National Lung Health Education Program and the Global Initiative for Chronic Obstructive Lung Disease (9). A good indication of acceptance for the term COPD was established with the creation of an International Classification of Diseases, 9th rev., (ICD9) code for COPD with and without exacerbation, introduced in the early 1980s and commonly used today.

III. A Difficult Diagnosis The whole lung sections presented in the 1950s and 1960s graphically demonstrated the destruction of lung parenchyma, the hyperinflation of the lung , the reason for V/Q mismatch, and decreased tethering of the airway. Unfortunately, very little of this information could be applied to patient care. Thus, getting the attention of nonpulmonary specialists whose memory of physiology from medical school was not positive represented a significant challenge. The issue was further complicated by the fact that many leaders in the field emphasized either emphysema or chronic bronchitis, depending on their research interests, which left clinicians confused about the term COPD. Although most pulmonary specialists were aware of the adverse effects of cigarette smoking on lung function, it was also true that severe pulmonary dysfunction occurred only in some smokers, and most smokers failed to show much in the way of symptoms or signs. Patients with chronic bronchitis often denied or minimized their symptoms and without pulmonary function tests, emphysema was likewise difficult to diagnose definitively in a living patient. Compounding the difficulty in diagnosis was the lack of a globally accepted term for the disease(s). Pulmonary physiologists and pathologists were making great advances academically and scientifically, but the specialty of pulmonary medicine, as we know it today was in its infancy. It was at about this time that many clinical physiologists began to use a pedagogical device to assist in teaching the recognition of symptoms of COPD. Two extreme caricatures of patients with COPD were labeled: the ‘‘pink puffer,’’ and the ‘‘blue bloater.’’ The pink puffer described a patient with severe emphysema, who was not cyanotic but was gasping for breath while

What is COPD?

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leaning forward with the elbows on the knees in the classic tripod position. The blue bloater was characterized as cyanotic, obese, with peripheral edema, cor pulmonale, and frequently had an elevated partial pressure of CO2. These stereotypic clinical presentations gained wide acceptance among those of us who taught pulmonary disease and physiology, and also had great appeal to our students because of their simplicity. However, while these stereotypical patients gave us some dramatic fodder from which to teach, it also gave the students an unrealistic picture of the typical patient with COPD (Fig. 1). Epidemiological studies of smokers gave us the important insight that the clinical spectrum of COPD was extremely broad, ranging from asymptomatic or mildly symptomatic to severely disabled patients. Most patients referred to a specialist for evaluation had moderate to severe symptoms, while many smokers had only mild or moderate symptoms. This latter group never saw a pulmonoligist then: this still holds true today. In the early years family physicians rarely advised smokers to quit, partly because there was no clear therapeutic strategy for smoking cessation at that time. Today this has dramatically changed, with multiple behavioral and pharmacological interventions available. However, advising asymptomatic patients to quit smoking can still be a hard sell for many practioners.

Figure 1 This cartoon by my son Rob Rogers portrays the clinical dilemma of using the designation of ‘‘Pink Puffer’’ and ‘‘Blue Bloater,’’ i.e., most patients are neither and many are asymptomatic.

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Rogers IV. Tobacco and COPD

The first Surgeon General’s Report linking smoking with lung and other disease was published in 1964. Even with the overwhelming evidence presented in this report, the precise connection among cigarette smoking, emphysema, and chronic bronchitis was not universally accepted. Despite advances in understanding the disease physiology during the 1960s and 1970s, translating the knowledge to clinical practice continued to be a monumental task. Relevant issues included a high incidence of smoking in the general and physician population, lack of clinical physiology laboratories in any but the major teaching hospitals, a paucity of symptoms and signs experienced by patients until the disease was advanced, and the intense promotion of cigarettes by cigarette companies. Compounding the problem, the causative role of cigarette smoking in lung disease was being hotly debated and vigorously opposed by the tobacco industry. It has recently been shown that the tobacco industry was probably aware of the carcinogenic effect of their product and deliberately obfuscated that evidence, based on a sinister and, it has lately been adjured, criminal misrepresentation of facts. Although the Lung Health Study (10) demonstrated that smoking cessation is the primary therapeutic intervention, and this is now widely accepted by the medical community, practicing physicians report that many smokers deny the compelling nature of their habit and its eventual consequences on their health. Many smokers rationalize their addiction by saying ‘‘We’ve all got to die sometime anyway,’’ or may refer to an aged person of their acquaintance who ‘‘smoked all their life’’ and lived, supposedly symptomfree, to a ripe old age. It is clear that these asymptomatic smokers have no inkling of the true facts of COPD as a long-term, debilitating disease process that is painful to witness, painful to experience, and frustrating to treat. Clinicians are encouraged to point out that death from smoking-related illness is neither quick nor painless. It is, however, completely avoidable.

V. Physiology of COPD After much heated debate over several decades, the complex physiology of emphysema and chronic bronchitis moved from the basic science laboratory into the mainstream medicine, greatly assisting in the diagnosis and treatment of both diseases. The clinical physiologist examined numerous perturbations of the forced expiratory maneuver such as the flow-volume loop, forced expiratory volume in 5s (FEV 5) and FEV 3 to mention only three. It soon became clear that the FEV 1 was the most reliable and reproducible measure of airflow obstruction, and one of the easiest measurements to perform.

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However, because airflow limitation has a variety of causes such as airway inflammation, mucus gland hypertrophy and airway mucus, smooth muscle contraction, or loss of airway tethering due to lung destruction and loss of elastic recoil, the FEV 1 can identify airway obstruction but cannot tell us what mechanism causes it. One or all of these mechanisms may be operative in any one or group of patients. Discovering which mechanism plays the major role is difficult and requires sophisticated studies such as measurement of lung compliance, lung volumes, Dlco, and arterial blood gas at rest and during exercise. Performing these tests on a large number of subjects is expensive, time-consuming, and impractical for epidemiological studies and in the everyday practice of clinical medicine. So, although we have a clear indication for the presence of COPD, how do we determine the presence or absence of emphysema? It is by measuring the lung’s carbon monoxidediffusing capacity (Dlco). A low Dlco indicates the presence of anatomical emphysema and has been used for many years to determine the presence of absence of emphysema in smokers with airflow limitation. However, since the Dlco has a wide normal range (at least 80–120% of predicted) one can only say that emphysema is present if the Dlco is less then 80% of predicted. However, if the patient started with a very high Dlco (120% of predicted), he or she may have had a decrease in Dlco due to emphysema but values may still be in the normal range.

VI. Predicting Who Will Develop Severe Disease Starting in the 1970s, the medical community became aware that some patients developed severe disease while others did not. This fact continues to be a problem for physicians who wish to make the case for smoking cessation. It provoked the investigative community to search for a test that would predict the development of significant airflow limitation in smokers before their FEV1 diminished. The research conducted during the 1970s and early 1980s was triggered by an important observation by Hogg et. al., who performed physiological studies on postmortem lungs of patients with COPD. Unlike the lungs of normal subjects in which central airways resistance exceeds peripheral resistance, in the lungs from patients with COPD the reverse was true. Hogg’s group coined the phrase ‘‘small airway disease,’’ since the principal site of airflow obstruction was in the distal small airways (11). Investigators postulated that the early lesion of emphysemabased COPD was in the small airways where obstruction occurred before the FEV1 was impaired. It was hypothesized that if clinicians could detect small airways obstruction, smokers at high risk of developing COPD could be identified before they experienced significant impairment. The hypothesis

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stimulated a decade or more of research, which included development of specific tests to detect small airway obstruction in smokers who had a normal FEV1. After much excellent work, however, it became apparent that these tests failed to predict which patients would develop airflow limitation and the research was largely abandoned. Although these research efforts failed to produce a physiological test to predict severe airflow limitation, it did stimulate many interesting epidemiological studies from which we have gained a better understanding of the spectrum of COPD. VII. A Reproducible Standard for Establishing Airflow Limitation The forced vital capacity (FVC), FEV1, and FEV1/FVC ratio became the standard for screening for the presence of airflow obstruction and since the 1980s has been available in almost all hospitals and large clinics throughout the United States. Spirometry became the basic test used in many epidemiological and treatment trials such as the lung health study (10,12), the IPPB Trial (13), and others. Screening spirometry allows the physician to identify patients who have slight airflow limitation. This group, or a subpopulation of this group, appears to be at high risk for developing significant airflow limitation and/or emphysema. Although 20–30% of smokers have a rapid decline in their FEV1 (Lung–Heart Study; LHS) (10,12), there is evidence that 100% of smokers develop some degree of bronchitis. It is believed, but not yet proven, that the group with rapid decline in FEV1 has a significant risk of developing emphysema and may have a genetic predisposition to do so. The tendency for only a small percentage of heavy smokers to experience significant airflow obstruction is illustrated in Table 1, in which data from the SPORE lung cancer-screening program is plotted using the Global Initiative for Chronic Obstructive Lung Disease (GOLD) criteria. Table 1 shows the preliminary data from the University of Pittsburgh Spore Lung

Table 1 Tendency to Airflow Obstruction in Smokers GOLD stages

997 (%)

0: 1: 2: 3:

536 144 306 11

At risk Mild Moderate Severe

(54) (14) (31) (1)

FEV1 mean (liters)

FVC mean (liters)

FEV1/ FVC%

Average pack-years

2.71 2.78 1.56 0.61

3.54 4.26 2.88 1.82

76 65 54 34

48 54 60 62

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Cancer Screening program, which documents that the majority of smokers (68%) with similar smoking histories do not have severe airflow limitation. Screening smokers with spirometry does identify patients with airflow limitation, and should help to predict those at highest risk of developing accelerated decline. However, it does not necessarily help us to gain insight into the early lesions of COPD. VIII. Early Diagnosis and Improved Outcome There are two major initiatives to encourage physicians to make early diagnosis of COPD utilizing spirometry: the National Health Lung Education Program (NHLEP) (14) and the GOLD initiative (15–17). It is hoped that both will bring the facts of COPD to the attention of the lay public, general internists, and family physicians. Because the vast majority of patients with early COPD are rarely seen by the pulmonary specialist, the opportunities for providing treatments that may significantly slow, prevent, or even reverse further deterioration are limited. To promote the early identification of at-risk patients and the subsequent cost avoidance in both health-care and human misery, lung specialists must recruit generalists and family physicians to the cause. These clinicians are in the best position to see the asymptomatic or minimally symptomatic patients whom they may be evaluating for other illnesses. The following procedures for primary care physicians are therefore recommended: 1. Record an accurate and consistent smoking history as another vital sign in all patients, including those age 11–18, when many begin smoking. 2. Perform spirometry for all smokers: there are many good to excellent inexpensive office spirometers that meet the standards of the American Thoracic Society.

IX. Recognition of COPD Cynics for years have taken a pessimistic view of COPD: because it is perceived as a self-inflicted disease, a lack of sympathy for patients may have been in a factor in the relatively low amount of research monies awarded for studying COPD. Funds have instead been allocated to other diseases and basic research. Today much has changed with the worldwide recognition that COPD is a pandemic of major proportions. Currently COPD is the 12th largest disease burden in the world, and is currently predicted to rank 5th by the 2020. In the United States alone it is estimated that more than 16 million

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people have COPD, but this is probably a gross underestimate. It is clear from both the trend predictions and, in many cases, anecdotal evidence from clinicians that the incidence of COPD is on the rise. While the term COPD is widely used today and accepted on a worldwide basis by medical personnel, the layperson seems to be more familiar and fearful of the term emphysema. This could be because the patient with severe emphysema can easily be recognized from across the room even by the casual observer. As noted, one challenge in promoting awareness of COPD is that many patients are asymptomatic or have minimal symptoms until the disease is well advanced and significant, irreversible damage and impairment are apparent. X. COPD Now Clinicians are now equipped with better tools to make the diagnosis of COPD. An effective campaign against smoking over the past 20 years has been waged by many strong organizations on many fronts; there is no smoking in government buildings, no smoking on airlines, and, in some areas, no smoking in restaurants. Former smokers have successfully sued tobacco companies for damage to their health and, in today’s health and legislative climate, even the tobacco companies concede that smoking is dangerous by printing a warning on their product and funding antismoking commercials on television. Increasing patient awareness of the consequences of smoking, a ‘‘vital sign’’ approach to monitoring smokers’ lung function, and the ready availability of pulmonary physiology laboratories and office spirometry will reduce the incidence of COPD in a patient population. XI. Improving Diagnosis One of the most important new imaging techniques is computed tomography (CT) of the chest. The images produced by modern multidetector CT scanners have vastly improved quality, speed of acquisition, and digital imaging that make assessments more detailed and accurate. With each new advance we are able to come closer to duplicating the gross anatomical features of the lung previously only available at postmortem or with biopsy. It is hoped that we can quantify changes in the airways and lung parenchyma; many investigators are working on such an analysis. In collaboration with colleagues in Vancouver, B. C., we have created a methodology to allow the quantification of the amount and localization of emphysema (18,19). These studies use quantitative histopathological methods to examine the resected lungs of patients who undergo lung resection for pulmonary nodules, or undergo lung volume

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reduction surgery and had recent CT studies of the thorax. The technique called quantitative computed tomographic morphometry (CTM) allows investigators to examine the CT scan of the thorax and determine the amount of both normal lung and of moderate and severely affected lung. The latter determinations are based on histopathological correlation. In addition, CTM allows us to estimate tissue and gas volume. From these data a formula for estimating surface area was also calculated. The study examined three different populations of heavy smokers: one group had normal pulmonary function, one had decreased FEV1 and normal Dlco, and the third group had severe pulmonary dysfunction (i.e., a severe decrease in FEV1 and Dlco). We labeled these groups as having normal, moderate, and severe emphysema. The first sign of emphysema (moderate) was an increase in lung volume as measured by the CT scan (see Table 2). Although the sample size was small, the data strongly suggest that the decreased FEV1 may not be the first abnormality in subjects with COPD (18), and supports the finding of increased peripheral resistance in the small airways reported by Hogg et al. (20). It also suggests an explanation for the clinical disparity between the mucus gland hyperplasia seen by pathologists in

Table 2 Quantitative Analysis of the Lung in Emphysema Control Total lung volume (ml) Air space volume (ml) Tissue volume (ml) Lung weight (g) Measured surface area/ volume (cm2/ml) Predicted surface area/ volume (cm2/ml) Measured surface area (cm2) Predicted surface area (cm2)

Moderate emphysema

Severe emphysema

4772 +/

223

6232 +/

410a

6725 +/

384b

3815 +/

194

5195 +/

388a

5964 +/

353b

957 +/

34

1037 +/

33

760 +/

35b

1019 +/ 256 +/

37 24

1104 +/ 165 +/

35 23a

810 +/ 43 +/

37b 6b

300 +/

9

212 +/

12a

138 +/

7b

118 +/

11

97 +/

128 +/

5

119 +/

Values are given as mean +/ S.E.M. a Different from control, p <0.05. b Different from control and mild emphysema, p <0.005.

8 3

30 +/ 60 +/

5b 3b

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patients who smoked but die of other causes and have no history of cough or other respiratory symptoms. If the mucus is in the peripheral airways, rather then the central airways, expectoration of sputum would be more difficult. This is interesting, since it has recently been emphasized that the dyspnea associated with COPD does not correlate with the FEV1 but does correlate with the presence of hyperinflation (21–23). XII. The Future A. Genetics

We know from many observations that the effects of cigarette smoking are variable; only 20–30% of smokers exhibit decline in lung function (10). Therefore, it is seems reasonable to hypothesize that a genetic predisposition combined with the effects of smoking causes COPD. If a genetically predisposed, asymptomatic patient could be identified, the physician could make a clear and compelling case for immediate smoking cessation. Recently investigators have identified some genetic patterns in subjects in the LHS (24) who showed a rapid decline in lung function. Newer techniques for studying gene expression provide hope for a future in which at-risk patients can be quickly identified and treated accordingly. B. Growth of New Alveoli

New research suggests that rodents who have had emphysematous changes induced in their lungs are able to grow new alveoli if given retinoic acid (25). These findings have been confirmed by several investigators (26) but a similar study in guinea pigs has shown no effect (27). The research has been extended to humans in a preliminary drug safety study, the results of which have not yet been published. If clinical trials prove successful there is the exciting possibility that some of the destructive changes of emphysema can be reversed (28). C. Understanding the Inflammatory Response in COPD

Recent work has shown that the inflammatory response in the small airways is distinct for patients with COPD. Understanding this concept may lead to the development of new and specific acting anti-inflammatory medications (29). D. Newer Modalities of Lung Imaging

The computed tomographic image of the chest is an important clinical and research technique that provides insight into the pathology of the lung during

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life. With the continuing improvement in our imaging techniques, the possibility that we may image smaller airways and look at the lung in threedimensional reconstruction is very promising. Other imaging techniques also show promise but are not available clinically at this time. E.

Treatment for Nicotine Addiction

Much research has been done on nicotine addiction and replacement therapy has had modest success. Antidepressant medication also seems to be helpful, indicating perhaps that the psychological component of cigarette smoking is at least as strong as the physiological addiction. Perhaps we will see newer drugs that combine treatment of nicotine addiction with a treatment for the psychological addiction to the act of smoking reported by many smokers. F.

Future Pandemic

With the world beginning to recognize the dangers of tobacco, there have been attempts to decrease cigarette smoking in countries with heavy consumption. A commitment to decrease the consumption of tobacco has been accepted by many countries and the World Heath Organization. With wider acceptance of the NHLEP and GOLD initiatives, we are optimistic that progress in containing this pandemic will occur. G.

Lung Volume Reduction Surgery

The medical community has just received the first report on the multicenter study of lung volume reduction surgery (National Emphysema Treatment Trial) (30, 31). The study and all its ramifications will take more time to digest, but it has confirmed that some patients, but not all, experience improvement in their quality of life after the surgical procedure but there appears to be no improvement in survival compared to the control group. These data should help us to select patients who are most likely to obtain benefit from surgery. Some work in animals suggests that the procedure may be performed endobronchially through a bronchoscope, which could make it more widely available and reduce its risk. XIII. Summary and Conclusions The future holds hope and promise for patients with COPD and the physicians who treat them. With the new emphasis on early spirometric screening from the NHLEP and GOLD initiatives, smokers with airflow limitation will be identified early in the course of their disease and may benefit from early intervention to prevent progression. We also expect significant

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breakthroughs in the genetic identification of susceptible subjects as well as more data on gene expression leading to pharmacological intervention for treating or even reversing the disease. New information about the type and localization of the inflammatory process should also lead to new insights into pathogenetic mechanisms. These insights should also allow development of new pharmacological agents. However, many of the questions that Bates and his colleagues noted in 1972 still need to be answered. The history of medicine over the past 100 years is an unprecedented success story by any standards, strongly suggesting that these questions will eventually be answered. Once the questions are answered, it is more likely that treatments and prevention strategies may almost eliminate the problem and concomitant costs in both health care and human suffering. However, timing is important; if we do not find the answers soon, current predictions for the incidence of COPD suggest that it may become one of the five leading causes of death by the 2020. If we fail to decrease cigarette smoking, COPD could become the leading cause of death in the world.

References 1. 2.

Mclean K. Pathogenesis of pulmonary emphysema. Am J Med 1958; 25:62–74. Gough J, Wentworth JE. The use of thin sections of entire organs in morbid anatomical studies. J R Microsc Soc 1949; 69:231–235. 3. Thurlbeck WM, Dunnill MS, Hartung W, Heard BE, Heppleston AG, Ryder RC. A comparison of three methods of measuring emphysema. Hum Pathol 1970; 1:169–178. 4. Thurlbeck WM. Aspects of chronic airflow obstruction. Chest 1977; 72(3):341– 349. 5. Thurlbeck WM, Wright JL. Thurlbeck’s Chronic Airflow Obstruction. 2d ed. Hamilton, Ont., Canada: B.C. Decker, 1999. 6. Thurlbeck WM. Chronic airflow obstruction in lung disease. Philadelphia: WB Saunders, 1976. 7. Bates DV, Christie RV. Respiratory function in disease; an introduction to the integrated study of the lung. Philadelphia: WB Saunders, 1964. 8. Ciba Foundation Guest Symposium. Terminology, definitions, and classification of chronic pulmonary emphysema and related conditions. Thorax 1959; 14:286–299. 9. Gelb AF, Gold WM, Nadel JA. Mechanisms limiting airflow in bullous lung disease. Am Rev Respir Dis 1973; 107(4):571–578. 10. Anthonisen NR. Prognosis in chronic obstructive pulmonary disease: results from multicenter clinical trials. Am Rev Respir Dis 1989; 140(3 Pt 2):S95–S99. 11. Hogg JC, Macklem PT, Thurlbeck WM. Site and nature of airway obstruction in chronic obstructive lung. N Engl J Med 1968; 278(25):1355–1360.

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12. Anthonisen NR, Connett JE, Kiley JP, Altose MD, Bailey WC, Buist AS, Conway WAJ, Enright PL, Kanner RE, O’Hara P, et al. Effects of smoking intervention and the use of an inhaled anticholinergic. JAMA 1994; 272(19): 1497–1505. 13. Wilson DO, Rogers RM, Wright EC, Anthonisen NR. Body weight in chronic obstructive pulmonary disease. The National Institutes of Health Intermittent Positive-Pressure Breathing Trial. Am Rev Respir Dis 1989; 139(6):1435– 1438. 14. Petty TL. The National Lung Health Education Program. A new healthcare initiative for America. J Cardiopulm Rehab 2001; 21(3):149–151. 15. Pauwels RA, Buist AS, Calverley PM, Jenkins CR, Hurd SS. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. NHLBI/WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD) Workshop summary. Am J Respir Crit Care Med 2001; 163(5):1256– 1276. 16. Siafakas NM, Vermeire P, Pride NB, Paoletti P, Gibson J, Howard P, Yernault JC, Decramer M, Higenbottam T, Postma DS, et al. Optimal assessment and management of chronic obstructive pulmonary disease (COPD). The European Respiratory Society Task Force. Eur Respir J 1995; 8(8):1398–1420. 17. American Thoracic Society. Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1995; 152(5 Pt 2):S77–S121. 18. Coxson HO, Rogers RM, Whittall KP, D’Yachkova Y, Pare PD, Sciurba FC, Hogg JC. A quantification of the lung surface area in emphysema using computed tomography. Am J Respir Crit Care Med 1999; 159(3):851–856. 19. Rogers RM, Coxson HO, Sciurba FC, Keenan RJ, Whittall KP, Hogg JC. Preoperative severity of emphysema predictive of improvement after lung volume reduction surgery, use of CT morphometry. CHEST 2000; 118(r):1240– 1247. 20. Hogg JC, Macklem PT, Thurlbeck WM. Site and nature of airway obstruction in chronic obstructive lung disease. N Engl J Med 1968; 278(25):1355–1360. 21. O’Donnell DE, Lam M, Webb KA. Spirometric correlates of improvement in exercise performance after anticholinergic therapy in chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1999; 160(2):542–549. 22. O’Donnell DE, Webb KA, Bertley JC, Chau LK, Conlan AA. Mechanisms of relief of exertional breathlessness following unilateral bullectomy and lung volume reduction surgery in emphysema. Chest 1996; 110(1):18–27. 23. O’Donnell DE, Webb KA. Exertional breathlessness in patients with chronic airflow limitation. The role of lung hyperinflation. Am Rev Respir Dis 1993; 148(5):1351–1357. 24. Sandford AJ, Chagani T, Weir TD, Connett JE, Anthonisen NR, Pare PD. Susceptibility genes for rapid decline of lung function in the lung health study. Am J Respir Crit Care Med 2001; 163(2):469–473. 25. Massaro GD, Massaro D. Retinoic acid treatment abrogates elastase-induced pulmonary emphysema in rats. Nat Med 1997; 3:675–677.

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26. Belloni PN, Garvin L, Mao CP, Bailey-Healy I, Leaffer D. Effects of all-transretinoic acid in promoting alveolar repair. Chest 2000; 117(5 suppl 1):235S–241S. 27. Meshi B, Vitalis TZ, Ionescu D, Elliott WM, Liu C, Wang XD, Hayashi S, Hogg JC. Emphysematous lung destruction by cigarette smoke. The effects of latent adenoviral infection on the lung inflammatory response. Am J Respir Cell Mol Biol 2002; 26(1):52–57. 28. Massaro GD, Massaro D. Retinoic acid treatment abrogates elastase-induced pulmonary emphysema in rats. Nat Med 1997; 3(6):675–677. 29. Hogg JC, Chu F, Utokaparch S, Elliott M, Cherniak RM, Rogers RM, Coxson HC, Sciurba F, Pare PD. Small airway remodeling and the mucosal immune response in chronic obstructive pulmonary disease. Submitted 2004. 30. NETT, T. N. E. T. T. R. G. Rationale and design of The National Emphysema Treatment Trial: a prospective randomized trial of lung volume reduction surgery. Chest 1999; 116(6):1750–1761. 31. Fishman A, Martinez F, Naunheim K, Piantadosi S, Wise R, Ries A, Weinmann G, Wood DE. A randomized trial comparing lung-volume-reduction surgery with medical therapy for severe emphysema. N Engl J Med 2003; 348(21):2059– 2073.

4 Small Airways Disease in Chronic Obstructive Pulmonary Disease

JAMES C. HOGG University of British Columbia Vancouver, British Columbia, Canada

I. Introduction This chapter reviews the nature of the pathology in the small airways of human lungs from patients with chronic obstructive pulmonary disease (COPD). It begins with a brief description of the anatomy of the airways below the larynx as a background to a review of the current literature on the response of these airways to the major risk factors for COPD. This is followed by a review of inflammatory traffic in the peripheral lung and a review of the innate and adaptive immune response in order to discuss the lesions described in the small airways in terms of these fundamental mechanisms. The observations made on the lower airways of patients with COPD are reviewed in relation to what is known about the inflammatory cell traffic and the innate and acquired immune response of the host. Figure 1 illustrates that when the trachea is assigned generation 0, the small conducting airways (less than 2 mm in internal diameter) are found between the 4th and 14th generation of the airway branching system depending on the length of the pathway followed (1,2). These airways continue to divide into smaller airways, but Figure 1C shows that the greatly increased number of smaller airways collectively provides a much larger total cross 67

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Figure 1 A. Bronchogram from a normal human lung shows the branching system of the tracheobronchial tree. B. When the trachea is designated 0, the number of branches to the terminal bronchioles varies from as few as 8 to as many as 24 depending on the pathway followed. C. The small conducting airways (less than 2 mm in diameter) are found from the 4th to the 14th generation of branching in the tracheobronchial tree. D. Remarkable increase in cross-sectional area occurs beyond the small airways. (B,C,D modified from Refs. 1 and 2.)

section than that provided by the smaller number of central airways larger than 2 mm in diameter (1). The purely conducting airways end in many thousand terminal bronchioles, each of which supplies a unit of lung termed the acinus (3). The diameter of the terminal bronchiole supplying each acinus ranges from 400 to 600 Am and the average volume of each acinus is 189 F 79 mm3 (3). The first intra-acinar airways are the respiratory bronchioles that are defined by the presence of alveolar openings in their walls (Fig. 2). The numbers of alveolar openings in the airway wall increase as the respiratory bronchioles continue to branch until the bronchiolar epithelium is completely replaced by alveolar openings in the alveolar ducts. These ducts branch several more times until they end blindly as alveolar sacs (3). The distance from

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Figure 2 A. Photograph of the pleural surface of the lung: the boundary of a secondary lobule is outlined by connective tissue septa (unlabeled arrow). Several terminal bronchioles (TB) can be seen and the first generation of respiratory bronchioles (RB). B. Photomicrograph of a terminal bronchiole supplying respiratory bronchiole. (Magnification A  1; B  20.)

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the first respiratory bronchiole to the alveolar ducts is approximately 9 mm and the number of generations of branching ranges from 6 to 12 over this distance (3). Figure 2A shows a secondary lung lobule defined by the connective tissue septa that surrounds it. This unit contains several terminal bronchioles, each of which supplies a single acinus. Although the connective tissue surrounding the secondary lobule is clearly visible on this photograph of the pleural surface, it becomes less distinct and may disappear near the hilum of the lung (1).

II. Site of Airway Obstruction The total resistance to airflow of a healthy person breathing through the mouth varies with lung volume and is approximately 2cm H2O/L/s at functional residual capacity (FRC) (4). The resistance to airflow through the airways below the larynx accounts for about 50% of total respiratory resistance at FRC (5,6). Most agree that the small airways account for approximately 20% of the resistance below the larynx and 10% of total airway resistance because their large total cross-sectional area offers very little resistance to airflow (1,6,7). For this reason the small airways have been referred to as the lung’s silent zone because their resistance must increase substantially before it affects total airway resistance (8). This knowledge led to the attractive hypothesis that COPD had a long subclinical course during which it might be detected and reversed before the standard tests for airway obstruction showed abnormal results. This concept was subsequently challenged for several reasons. First, a separate report on the distribution of resistance in the lung suggested that although the small airways were the major site of resistance in COPD, they accounted for a larger proportion of the total resistance in the normal lung (9). Although this discrepancy can be explained by the way in which the new measurements were made (i.e., at very low frequencies where tissue viscance adds a pressure in phase with flow and artificially elevates small airway resistance), there were other problems with the theory. More important was that it became apparent that everyone who smokes cigarettes experiences lower airway inflammation, whereas only a minority of heavy smokers go on to develop COPD (10,11). This meant that to be of any value the specialized small airway tests that detected peripheral airway dysfunction in so-called healthy smokers would also have to identify the minority of smokers who are destined to develop COPD. This hypothesis is difficult to test because the tests of small airway function are not easy to apply to large longitudinal population-based studies. As a result the maximum volume of air that can be forcibly expired in 1 s (FEV1) has remained the standard for both diagnosing airway obstruction and following the natural history of COPD.

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This test has the important advantages that it has been widely used, has good prediction values, and is multivalent: it is influenced by both small airways obstruction (6) and the emphysematous lung destruction that reduces the elastic recoil pressure that drives air out of the lung (12).

III. Pathology of Airway Disease Postmortem studies of the lung performed in many laboratories in the midportion of the last century defined the nature of emphysema and provided the insight that the inflammatory process is a key to the pathogenesis of COPD (13–18). Physiological measurements made initially on postmortem specimens and later in living patients identified the small conducting airways as the major site of obstruction in COPD (6,7,9). These direct measurements of peripheral lung pressures and flows established that the major cause of the fixed airway obstruction was disease in the wall and lumen of the small airways. The marked decrease in flow seen during forced expiration was determined to be due to dynamic compression of airways downstream from this fixed airway obstruction (6). Although the resistance of the small airways may also be increased as a result of emphysematous destruction or reduced recoil in the alveolar support of the small airways, the direct measurements of small airway resistance suggested that this is less important than the disease in their wall and lumen (6). Dunnill (19) recognized that the inflammatory process in the small airways of emphysematous lungs was predominantly in the adventitial space of the outer wall of the small airways. The inflammatory cells consisted of a mixture of mononuclear and polymorphonuclear leukocytes. Matsuba and Thurlbeck (20) also reported marked connective tissue deposition in the adventitial space around the small airways of patients with severe emphysema. The Denver group (21–26) had remarkable success reproducing measurements of lung function made during life in the postmortem room to provide a better understanding of airway structure and function. The difficulties in obtaining suitable specimens as autopsy rates fell and lung transplantation became more common has virtually stopped this line of investigation. The work on postmortem lungs was followed by studies of lobes and sometimes entire lungs that had been surgically resected to remove tumors (27–34). This type of study made it possible to examine lungs that were free of postmortem artifact and relate the findings to measurements of lung function made in the immediate preoperative period. The severity of pathological findings in the small airways was estimated in histological sections using a picture grading system (31). These estimates were reproducible both between observers and within the same observer (27). The results showed that

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there was a progressive worsening of the disease in the small conducting airways and respiratory bronchioles as FEV1 declined (27–29,32–34). They also showed that there was inflammatory disease in the small airways when the FEV1 was normal and the functional tests for small airways disease were abnormal (27,33,34). These estimates were encouraging because they showed that small airways inflammation could be detected using specialized tests for peripheral lung function when the FEV1 was normal. They could not, however, answer the larger question of whether these abnormalities were useful in predicting the relatively small percentage of smokers who would go on to develop COPD.

IV. Chronic Bronchitis and Small Airway Obstruction The considerable body of work summarized in the initial CIBA symposium, during which experts defined chronic bronchitis as recurrent excessive mucus secretion in the tracheobronchial tree, suggested that chronic cough and sputum production were the causes of airflow obstruction (35). The early studies of Lynne Reid correlating the clinical symptoms of mucus hypersecretion with the size of the mucus glands led to the later criticism of the use of the term bronchitis, because very little inflammation was thought to be associated with an increase in gland size (36–38). Mullen et al. (28) reexamined this question in a quantitative study of surgically resected lungs. They established that chronic bronchitis was associated with an inflammatory process involving the glands, gland ducts, and luminal epithelium of the central airways (Fig. 3). They also found that small airways disease and airway obstruction could be present in the absence of chronic bronchitis. Saetta et al. (39) confirmed and extended this work by showing the nature of the cells involved in the inflammatory response in the mucus-secreting apparatus of the central airways. Mullen’s findings were consistent with the early epidemiological studies of Fletcher, Peto, and their colleagues (10,40) who reported that mucus hypersecretion did not predict the development of chronic airflow limitation, and with the more recent study on a Copenhagen city cohort of patients (41). The effect of small airway luminal content on lung function was further examined by Cosio et al. in surgically resected specimens of lung from patients with mild to moderate COPD (27). Although they were able to show that these estimates of the severity of the lumen occlusion were reproducible within the same observer and between two observers, the results failed to correlate with either the measurements of small airway function or the decline in FEV1. The results of all of these studies suggest that the occlusion of the small airway lumen by an exudate containing mucus is not an important cause of airway

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Figure 3 Low-power photomicrograph of the bronchial wall shows demonstrating a normal mucus-producing epithelial gland with its duct connecting to the epithelial surface of the bronchial lumen. The symptoms that define chronic bronchitis are associated with an inflammatory response involving the glands, gland ducts, and the surface epithelium covering the bronchial lumen (28,29). (Magnification  20.) (Courtesy of the late Professor William M. Thurlbeck.)

obstruction in mild to moderate COPD. However, a separate report from the Copenhagen group showed that the presence of chronic bronchitis carried an increased risk of hospitalization in patients with moderate to severe COPD (42). This suggests that mucus hypersecretion is a more important issue: in more advanced disease it might predispose to infection and exacerbation of COPD.

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The introduction of fiberoptic bronchoscopy with biopsy of the epithelium and subepithelial tissue lining the central airways, and the study of induced sputum, has provided new information about the pathology of chronic bronchitis (43–54). These studies have shown that the epithelium of the central airways is normally intact in patients with COPD but it is affected by both squamous and goblet cell metaplasia. There have also been reports of increased numbers of neutrophils and eosinophils in chronic bronchitis (43), particularly during exacerbations of COPD (44). The thickness of the reticular basement membrane remains within the normal range, but some cases overlap the lower end of the distribution in patients with asthma in whom the reticular basement membrane thickness is increased. The epithelium and subepithelial tissue contain increased numbers of mononuclear cells including mast cells (45) and lymphocytes (46–49). Saetta et al. (46,47) have shown these cells include CD3 (T-lymphocytes), including CD25-positive cells (indicating early activation), VLA-1 positive cells (indicating late activation), and macrophages. O’Shaughenessey et al. (48) have shown there were greater numbers of CD8 than CD4 cells and reported an inverse association between CD8positive T lymphocytes in central airway biopsies and a decline in FEV1 (33). Because the biopsy specimens that can be obtained using the fiberoptic bronchoscope are nowhere near the site of obstruction in COPD, this conclusion is based on the underlying assumption that the disease at the site of obstruction in the small airways is similar to that at the biopsy site in the central airways. Although it is difficult to be certain where the inflammatory cells found in induced sputum actually come from, this type of study has become a popular method of assessing airway inflammation (50,51). Some investigators have used the induced sputum quite successfully to show changes in cytokines and chemokines suggesting that inflammatory response may be amplified in COPD (54,55). One pioneering study involving a 15-year follow-up of a single group of patients reported that an accelerated decline in lung function was associated with an increased number of neutrophils in the airway lumen (56). This same study showed that in subjects with a more rapid decline in FEV1, the neutrophils exhibited an increased expression of the adhesion molecule CD11b/CD18, the ligand for ICAM-1, suggesting that the adhesion molecules responsible for polymorphonuclear leukocyte (PMN) migration are upregulated in patients with COPD (57). Ratemales et al. (58) conducted a detailed study of inflammatory response in the peripheral lung beyond the conducting airways based on tissue obtained by lung volume reduction surgery. They showed that the number of inflammatory cells present in the tissue is amplified in emphysema. It was interesting that the distribution of inflammatory cell types was similar to that reported in the biopsy studies of the central airways. This similarity suggests

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that the small conducting airways between these two sites contain a similar population of inflammatory cells; studies of the small airways in surgically resected peripheral lung tissue tend to support this view. Bosken et al. (59) found more B lymphocytes in the adventitial compartment of the peripheral airways in patients with mild to moderate COPD and showed that the increase in PMN in the epithelium and subepithelial compartment correlated with the amount smoked. Her study is important in that it separated the inner and outer aspect of the small airway wall because they are supplied by separate sets of microvessels. Comparing her work to the earlier work of Dunnill (19) and Matsuba and Thurlbeck (20) supports the notion that there is an important inflammatory process in the adventitial space. Furthermore, the increases in B cells noted by Bosken et al. suggest a response to antigens introduced into the lower airways that we will return to later in this chapter. The major risk factor for COPD is the chronic inhalation of toxic particles and gases as a result of tobacco smoking (60). This risk is increased by the atmospheric pollution produced by coal-burning power generators and the internal combustion engines that power automobiles and trucks. Localized forms of air pollution, such as those found in homes that use wood burning fires to cook and by a variety of exposures that are specific to the work place, add to the risk of smoking. Lower respiratory tract infections represent a separate source of risk for COPD. The upper airways and oropharynx are permanently infected with micro-organisms that exist in a symbiotic relationship with the host, but the airways below the larynx remain sterile in health. Microbes gain entry into the lower respiratory tract as a result of aspiration, which is a relatively frequent event in normal healthy people particularly during sleep (61). The inflammatory response generated by tissue damage and the innate and acquired defense mechanisms of the host are able to maintain sterility below the larynx in spite of aspiration unless they are suppressed by the influence of alcohol, consciousness-depressing drugs, or anesthetic agents. Childhood infections of the lower respiratory tract increase the risk of COPD in adult life (60) and infections of the lower respiratory tract account for about one-third of acute exacerbations of COPD. The micro-organisms that cause disease differ from those that live in symbiosis with the host in that they are capable of invading the natural tissue barriers and resisting both the innate and adaptive defense mechanisms of the host.

V. Leukocyte Traffic in the Lung Our current knowledge about the lower airway inflammatory response in humans has been obtained from autopsies, surgically resected tissue, biopsies, induced sputum, and lung lavage. The bulk of the reports based on sputum

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and lavage fluid measure of the severity of the inflammatory response from the number and type of inflammatory cells present in the sample. Studies based on tissue used either a picture grading system or counts of the number of cell profiles per square millimeter of tissue in histological sections. These two approaches to quantitative histology are unsatisfactory on theoretical grounds (62), and even if correct provide only a tiny snapshot of a dynamic system at a single moment in time. The number of inflammatory cells present in any tissue is dependent on their rate of production in the marrow, their distribution between the circulating and marginated pools in the blood, their rate of delivery to the tissue, their migration out the vascular space, and the time they spend in the tissue. Chronic cigarette smoking produces a 20–25% increase in the peripheral blood leukocyte count and a neutrophilia associated with an increase in band cells, indicating that there has been increased production and early release from the bone marrow (63). A similar marrow response has been observed during an acute natural exposure to atmospheric pollution by smoke from forest fires in the southeast Asian haze of 1997 (64,65). Animal experiments have confirmed that these exposures increase the production and release of PMN from the marrow and that cytokines generated by alveolar macrophages as they phagocytose atmospheric particulates are responsible for the marrow stimulation (66). The newly released PMN behave differently than those already in the circulation: they marginate within the circulation to a greater degree than fully mature PMN cells and migrate out of the vascular space into the tissue less readily than mature cells (67). There is also preliminary evidence suggesting that breathing in an atmosphere with low particulate contamination lowers the circulating leukocyte count even in those who continue to smoke cigarettes (68). The inflammatory cells are delivered to the peripheral lung by both the systemic and pulmonary circulations (69). Those in the systemic blood flow arrive through a network of bronchial arteries on the outer wall of the conducting airways. These arteries send penetrating branches through the airway wall to supply one microvascular bed located in the lamina propria beneath the epithelium and a second microvascular bed of slightly larger vessels in the adventitia (70). Both sets of microvessels contain arterioles, capillaries, and venules and they are connected to each other by vessels that pass through the muscle layer (Fig. 4). Therefore an important feature of the microvascular arrangement in the walls of the conducting airways is that an inflammatory response can be mounted on the microvessels in the lamina propria when the stimulus is delivered to the small airway epithium or in the adventitia when the stimulus arrives in from the alveolar surface. The blood from the larger bronchi drains centrally through the azygous and hemiazygous venous systems into the right atrium. The systemic venous blood from the small conducting airways drains through the pulmonary venous system into the left

Figure 4 A. Diseased small airway in cross section: one microvascular bed can be seen beneath the reticular basement membrane and a second in the adventitial space outside the muscle layer. The two separate sets of microvessels both contain arterioles, capillaries, and venules and they are connected by the epithelial reticular basement membrane. A second microvascular bed can be seen outside the muscle. These two microvascular beds are connected by vessels (CV) that penetrate the muscle layer. B. Electron micrograph of the liquid layer lining the airway surface in a guinea pig lung. Note that the lining fluid is divided into an electrondense surface layer and an electronlucent subsurface layer. (Courtesy of Dr. David Walker.) C. Guinea pig airway surface epithelium damaged by cigarette smoke: the inflammatory cells are migrating through the epithelium in a file rather than in random order between two epithelial cells and entering the subsurface layer. D. Tracheal surface of a guinea pig lung following more severe cigarette-smoke-induced damage. The surface layer has been increased by an exudation of fluid and cells. Note the sloughed epithelial cells floating on the liquid surface. (From Ref. 98.)

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atrium. There are connections between the bronchial and pulmonary vascular systems at the arterial, capillary, and venous level. The bronchial venous blood that drains into the pulmonary veins without passing through the pulmonary capillaries represents part of the physiological shunt through the lung. The blood flow through the connections that join these two systems is small in health but can increase substantially in disease. The margination of PMNs in microvessels and their migration out of the vascular space is a multistep process regulated by a cascade of molecular events involving several families of adhesion proteins. Adams and Shaw (71) have reviewed the evidence for the hypothesis that this process begins with a selectin-mediated rolling of the PMNs on the endothelial surface of the postcapillary venules followed by a chemokine-induced triggering of a stronger, integrin-mediated adherence between the leukocyte and the endothelial surface that occurs in preparation for migration. The data that support this hypothesis are impressive, particularly for the systemic vessels where both margination and migration occur in postcapillary venules. However, there are compelling reasons to believe that, in the pulmonary circulation, the circulating cells slow down, adhere, and migrate through the endothelium of the capillaries, and that only a fraction of the PMNs passing through an inflammatory site become significantly activated to migrate out of the vascular space (72). The progressive activation of PMNs is most easily investigated in vitro. Studies using a prototypic agonist of neutrophils, f-met-leu-phe (FMLP), have shown that low concentrations (10 12 – 10 10 M) are associated with reorganization of the cytoskeleton to a configuration that allows the cells to move independently (73–75). As the FMLP concentration is increased, the specific granules release CD 11b/CD18 onto the surface and L-selectin is shed (76). These events occur in association with increased intracellular levels of inositol trisphosphate and calcium (77). However, it is only at very high concentrations (10 7 – 10 6 M) that the major cytotoxic responses of azurophilic granule release and superoxide production are demonstrable (76). Other agonists that use structurally similar receptors (interleukin 8, platelet aggregating factor, C5a) have similar dose-dependent responses but these agonists can also prime the cell and amplify its cytotoxic response (78,79). Priming refers to an enhanced response that results from the sequential addition of priming agent and agonist (79). Priming of the superoxide response usually occurs at intermediate dosages sufficient to cause increases in intracellular calcium and specific granule release, but just at or below the dosages necessary to release superoxide (78,79). Other mediators such as the cytokines [interleukin 1, granulolyte–macrophage colony-stimulating factor, (GMCSF) tumor necrosis factor] and endotoxin are incapable of producing a full repertoire of neutrophil responses.

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The relationship between the graded neutrophil response in vitro and their behavior in vivo has only been partially determined. Their response within the vascular space where agonist concentrations are low is probably limited to the assembly of actin, a reduction in cell deformability, upregulation of CD1lb/CD18, and a loss of L-selectin from the cell surface. Once adherent to the endothelium, the cells move along chemotactic or haptotactic gradients centered at the site of injury. This allows the PMNs to encounter increasing agonist concentrations that first prime and then trigger a full cytotoxic response. Any abnormality in this sequence that allows large numbers of PMNs to become fully activated before leaving the vascular space puts the lung tissue itself at risk of injury. At a cardiac output of 6 L/min approximately 8640 L blood pass through the pulmonary circulation of an adult human in 24 h. Since each liter of blood contains 4.5  109 PMNs, about 3.8  1013 PMNs pass through the pulmonary microvessels each day. The human PMN has a similar diameter to, but a larger volume than, the erythrocytes (80). The pulmonary capillary bed is formed by an interconnecting network of approximately 1011 short segments with an average diameter of 7.5 F 2.3 Am and an average length of 14.4 (F 5.8) Am (80–82). The distributions of PMN diameters (6.8 F 0.8 Am) overlap that of the capillary segments by approximately 38%, which means that circulating cells encounter segments that restrict their passage on each transit through the lung (82). During this encounter the PMNs deform and come into close contact with the capillary endothelial surface (82). Because the postcapillary venules have much larger diameters and a total endothelial surface area of only 5m2, compared with the >100 m2 available in capillaries, there is a much greater opportunity for interactions to occur between PMNs and the endothelium in the capillaries than in the postcapillary venules of the lung (83). Direct observations of the pleural surface (84) and indirect measurements from deeper lung regions (81) have shown that it takes leukocytes much longer than the erythrocytes to travel through pulmonary capillaries. The slower movement of the PMNs compared with the erythrocytes is a result of the fact that they are about 300 times less deformable than erythrocytes and are held up for longer periods in narrow segments (82,85). The interconnecting network of segments that forms the capillary bed allows the erythrocytes to have median transit times of 1s compared with 120 s for PMNs (81). The concentration of PMNs with respect to erythrocytes that results from this difference in speed can be influenced by mechanical events affecting either the vessels or the circulating cells. Studies of patients undergoing simultaneous right and left cardiac catheterization have shown that an immediate arteriovenous difference for PMNs occurred when the lung capillaries were compressed by raising alveolar pressure and shifting lung from zone III to zone II

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conditions (86). Animal studies subsequently showed that the arteriovenous difference produced in this way disappears with time as a new equilibrium is established at the longer PMN transit times (87). PMNs rapidly accumulate in the lung when stimulated by the infusion of zymosan-activated plasma (88). This accumulation is a two-step process in which reduced PMN deformability dominates the initial retention and CD11/CD 18-mediated adherence plays a secondary role (89). It is interesting that the massive PMN margination produced by the infusion of activated plasma causes only minor changes in the epithelial permeability, with no increase in extravascular lung water or protein (90). This suggests that the plasma-derived stimuli that massively increase PMN margination fail to activate the cells to a point at which they are capable of injuring lung tissue. Downey et al. (91) showed that in the lung PMNs migrate out of capillaries rather than postcapillary venules. Doerschuk et a1. (92) showed that this migration could be CD-18-independent in the lung but not in the systemic circulation (92) and that only 1–2% of the cells delivered to an area of pneumonia actually migrate out into the airspaces (72) compared with the 60–80% migration rate observed in vitro (93). Elegant studies by Walker and associates (93–95) have shown that the PMN seeks out areas on the thick side of the capillaries where gaps in the endothelium form at the corners where three endothelial cells meet (Fig. 5). The PMN then migrate into the interstitial space through these gaps and come into contact with interstitial fibroblasts. Careful reconstruction of the interstitial space of the alveolar wall by Walker et al. have shown that these fibroblasts span the alveolar wall interstitial space and send extensions through the basement membranes to interact with epithelial and endothelial cells (94). They also showed that surface of the fibroblast provides a pathway for the migration of PMN from the point where they enter the interstitium to their point of exit into the airspace (95). The kinetics of leukocyte behavior in the bronchial circulation has not received the same attention, but indicator dilution studies have shown that PMN are not delayed to the same degree in the bronchial as in the pulmonary circulation (96). Walker et al. (97) have also shown that a network of fibroblasts in the airway wall interstitial matrix guide inflammatory cells migrating from the subepithelial microvasculature to defects in the basement membrane where they gain entry to the subepithelial space (96), and migrate through the epithelium in a nonrandom fashion (98) (Fig. 4). They probably use a similar mechanism to migrate out of the microvascular bed in the adventitia of the small airways, although this has not yet been studied. The fact that the migrating inflammatory cells come into direct contact with the fibroblasts as they pass through the interstitial space suggests that they might influence the synthesis of interstitial matrix. This influence could be a key factor controlling connective tissue matrix deposition in the adventitia

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Figure 5 A. A single alveolar wall on face; the interconnecting network of short capillary segments can be clearly seen. B. Diagram of a single capillary segment cut in cross section demonstrates the gap in the tight junctions where three endothelial cells meet. C. Location of the endothelial exit points at the junction between the thick and thin portions of the alveolar wall. D. Pathway taken by PMNs as they migrate out of the capillary lumen, across the interstitial space, to the junction between the type 1 and type 2 epithelial cells, which they migrate through to reach the alveolar airspace. Note that the migrating PMN uses the surface of the fibroblast (F) as a guide to cross the interstial space of the alveolar wall.

surrounding the small airways disease in patients with COPD and the disappearance of the alveolar wall tissue in those with emphysema. This brief review of leukocyte traffic in the lung has concentrated on the kinetics of PMN because they are the easiest inflammatory cell to document; they do not divide after they leave the bone marrow, remain inside the vascular space except at inflammatory sites, and do not re-enter the circulation after they leave. The monocyte–macrophage cell line is more difficult to monitor because they divide in the tissue. Lymphocyte traffic is the most difficult of

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all because they continuously migrate from the blood into the lymphatic tissue, where they can divide rapidly in response to antigens presented to them before they re-enter the blood and home to specific tissue sites. In all cases our knowledge of the behavior of these cells in humans in vivo is very largely based on studies that have counted the number of profiles present per square millimeter of tissue obtained by biopsy, surgical resection, or autopsy. The snapshot provided by this approach puts us in the position of a movie critic trying to evaluate a three hour movie from a single frame. VI. Innate and Adaptive Immune Response The innate host response is the first line of defense against infection (99), but there is increasing evidence that it also responds to the deposition of atmospheric particulates in the lung. The components of the innate host response listed in Table 1 include the mucus-producing and -clearance system of the airways that physically removes material deposited on the airway surface

Table 1 The Innate Host Response Innate Defense Mucus clearance Epithelium Migrating leukocytes PMN Macrophage Natural killer cells Dendritic cells Co-operating Molecules Plasma Coagulation system Complement system (alternate pathway) Acute-phase proteins Mannose-binding protein Creactive protein Locally produced Lysozyme Lactoferrin Surfactant protein A, C, B Defensins

Mechanism Physical removal Mechanical barrier Phagocytosis and killing

Assist with isolation, phagocytosis, and killing

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(100); the protective barrier provided by the epithelium and the tight junctions between epithelial cells (99); the migration of polymorphonuclear neutrophils, alveolar macrophages (AM), and natural killer (NK) cells into the damaged site and a population of dentritic cells that process and transport antigen to the lymphatic tissue (99).Molecules produced by cells at the site of injury (lysozyme, lactoferrin, the surfactant protein A and D, and the defensins) (101–103) and produced in the liver and delivered to the site in the plasma ( c-reactive protein, mannose-binding protein, and complement protein C-3) cooperate with the migrating cells to enhance phagocytosis and killing of the microbes (99). The innate response is controlled by cytokines that have local (autocrine and paracrine) functions that activate the endothelium and epithelium to produce the chemokines and adhesion molecules that control the migrating cells (Table 2). Some of these cytokines tumor neurons factor alpha (TNFa), interleukin 1 beta (IL-1h) activate the endothelium and epithelium to control the migration of leukocytes. They also have endocrine functions that stimulate the hypothalamus to initiate fever (TNFa, IL-1h), activate the synthesis of acute phase proteins in the liver (TNFa, IL-1hand IL-6), and activate the production and release of leukocytes and platelets from the bone marrow (TNFa, GM-CSF IL-6). Other studies have shown that the production of IL-12 by macrophages stimulates the NK cells to produce interferon (INF) g that stimulates macrophages to enhance their respiratory burst and promotes major histocompatibility complex (MHC) class 1 and class 11 expression on surrounding cells. INF g also promotes the proliferation of antigen-stimulated T cells to help initiate both the cellular and humoral components of the adaptive immune response. Recent evidence suggests that an innate response is also stimulated by the inhalation of the atmospheric particles (64) because phagoctosis of these particles by alveolar macrophages resulted in the production of TNFa, IL-6, GM-CSF, macrophage inhibitory protein (MIP)1a, and IL-1h (65). Other studies showed that phagocytosis of atmospheric particles by bronchial epithelial cells resulted in the production of leukemia inhibitory factor (LIF), GM-CSF, IL-1a, and IL-8 (103). These results show that the deposition of particulates in the lower airways as a result of atmospheric pollution stimulates the innate immune system. The adaptive immune response is initiated by the interaction of foreign antigens with mature B and T lymphocytes. This requires T cells and B cells with the same antigen specificity to interact, an event that would be extremely rare if it were not for the organization of the lymphatic tissue, because only 1 in every 105 or 106 circulating lymphocytes demonstrates such specificity. The peripheral lymphatic tissue greatly improves the chance for such a meeting because the lymphocytes leave the blood and enter the lymph in peripheral lymph nodes and antigens are transported to the nodes from the site of tissue

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Table 2 Cytokines in the Innate Response Principal cell source

Principal cellular targets and biological effects

TNF

Macrophages, T cells

IL-1

Macrophages, endothelial cells, some epithelial cells

Chemokines

Macrophages, endothelial cells, T cells, fibroblasts, platelets Macrophages, dendritic cells

Endothelial cells: activation (inflammation, coagulation) Neutrophils: activation Hypothalamus: fever Liver: synthesis of acute-phase proteins Muscle, fat: catabolism (cachexia) Many cell types: apoptosis Endothelial cells: activation (inflammation, coagulation) Hypothalamus: fever Liver: synthesis of acute-phase proteins Leukocytes: chemotaxis, activation

Cytokine

IL-12

Type 1 IFNs (IFN-a, IFN-h)

IFN-a: macrophages IFN-h: fibroblasts

IL-10

Macrophages, T cells (mainly TH2)

IL-6

Macrophages, endothelial cells, T cells

IL-15

Macrophages, others

Source: Modified from Ref. 99.

NK cells and T cells: IFN-g synthesis, increased cytoloytic activity T cells: TH1 differentiation All cells: antiviral state, increased class I MHC expression NK cells: activation Macrophages: inhibition of IL-12 production, expression of costimulators and class II MHC molecules B cells: proliferation Live: synthesis of acute-phase proteins B cells: proliferation of antibody-producing cells NK cells: proliferation T cells: proliferation

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damage. The arteriole supplying a peripheral lymph node enters at the hilum and forms a microvascular bed around the lymphoid follicles that drain to the venous system and exit at the hilum. Lymphocytes are able to leave the blood by migrating out of venules lined by specialized endothelial cells and percolate through the lymph in the lymph node. This allows the B cells to become associated with the follicles and T cells with the tissue surrounding the follicles. These lymphocytes eventually leave the node in the efferent lymphatic duct and are returned to the blood as the right lymphatic and thoracic ducts drain into the central veins (105). Foreign material that penetrates the innate defense system is picked up by dendritic cells located in the epithelium and lamina propria and processed as they are transported to the lymph nodes. They enter the lymph node in the afferent lymphatics that penetrate the capsule of the lymph node and deliver the lymph to the underlying marginal sinus. The lymph flows from the marginal sinus through the paracortical tissues between and beneath the follicles located in the cortex of the node before leaving the efferent lymphatic. This provides the dendritic cells with the opportunity to present antigen both to B cells that have accumulated at the edge of the lymph follicles and to the T cells that they meet in the paracortical regions of the node. The CD4 T cells activated by antigen presented to them in the paracortical tissue migrate to the edge of the B-cell-enriched follicles, where their chance of meeting a B cell that has been activated by the same antigen is much improved. The B cells that receive help signals from the CD4 T cells proliferate and migrate into the germinal center of the lymphoid follicle, where they produce antibody of varying affinity and display it on their surface where it can bind to antigen presented to a different set of follicular dendritic cells. The B cells that produce low-affinity antibody receive a death signal and undergo apoptosis; those that present high-affinity antibody continue to mature into memory cells and mature plasma cells. The major function of the humoral component of the adaptive immune response is to generate mature B cells. These produce antibodies that protect the host against microbes that remain in the extra cellular space by neutralizing their toxins and binding to the micro-organisms’ surface to initiate a much more efficient opsonization and phagocytic process than can be mounted by the innate system. Antibodies that attach to the surface of microorganisms also bind the C1q component of the large multimeric C1 complement molecule circulating in the plasma and initiate the complement cascade by the classic complement pathway. Table 3 lists the cytokines that influence acquired immune system, their cellular source, principal targets, and biological effects. Interleukin 2 is produced by T lymphocytes and stimulates the proliferation of T cells, B cells, and natural killer cells. A subset of T cells referred to as the Th 2 subset of CD4-positive lymphocytes and mast cells secrete both IL-4 that initiates antibody isotype switching to IgE in B cells and IL-5

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Table 3 Cytokines in the Adaptive Immune Response Cytokine

Principal cellular sources

IL-2

T cells

IL-4

CD4+ T cells (TH2), mast cells

IL-5

CD4+ T cells (TH2)

IFN-g

T cells (TH1, CD8+ T cells), NK cells

TGF-h

T cells, macrophages, other cell types

Lymphotoxin

T cells

IL-13

CD4+ T cells (TH2)

Source: Modified from Ref. 105 and 106.

Principal cellular targets and biological effects T cells: proliferation, increased cytokine synthesis; potentiates Fas-mediated apoptosis NK cells: proliferation, activation B cells: proliferation, antibody synthesis B cells: isotype switching to IgE T cells: TH2 differentiation, proliferation Mast cells: proliferation Eosinophils: activation, increased production B cells: proliferation, IgA production Macrophages: activation (increased microbicidal functions) Endothelial cells: activation Various cells: increased expression of Class I and Class II MHC molecules, increased antigen processing and presentation to T cells T cells: inhibition of proliferation and effector functions B cells: inhibition of proliferation, IgA production Macrophages: inhibition Recruitment and activation of neutrophils Lymphoid organogenesis Inhibition of macrophage activation

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that stimulates eosinophil production and activation. Il-5 also cooperates with TGFh to switch B cells to the production of an IgA antibody isotype. Without these stimuli the B cells produce the more common IgM, IgG, and its subclasses. The major function of the cellular component of the adaptive immune system is to protect the host from microbes that survive within phagocytes or infect nonphagocytic cells. The acquired immune response to microbes residing within the phagosomes of phagocytes is mediated by T lymphocytes of the CD-4 Th1 sub type as well as CD 8 T cells. Both these cells are able to recognize antigens displayed on the surface of the phagocyte and secrete cytokines that activate the macrophages to kill the organisms. The CD-8positive lymphocytes are also able to recognize nonphagocytic cells that are infected by intracellular pathogens and destroy them in three steps. The first is a recognition step where the cytotoxic lymphocyte uses the T-cell receptor to bind to foreign material displayed on the surface of the target cell with the human leukocyte antigen (HLA) self molecule. The second step is the use of the molecule perforin to create holes that connect the cytotoxic T cell to its target and allow granzyme to enter the target cell. The third is the induction of apoptosis in the target cell as granzyme activates the target cell caspases. The summary in Tables 2 and 3 indicates that the cellular component of the adaptive immune response is induced by IFN g and IL-12 from the innate response that promote naı¨ ve CD-4 cells to the TH1 subset and inhibit the proliferation of Th 2 cells. On the other hand, IL -4 and IL-13 produced by the Th2 subset act with IL-10 from the innate response to inhibit the macrophage activation induced by Th1 lymphocytes. TGF h, which is produced by many cells, acts with IL-10 from the innate system to inhibit the immune and inflammatory response and initiate the repair process. The proliferation of the secondary lymphatic tissue acts to form lymphoid collections and is stimulated by lymphotoxin. In addition to the lymph nodes present in the lung, there are also peripheral lymphoid collections that differ sharply from fully formed lymph nodes in that they have no capsule and no afferent lymphatics. Those associated with small conducting airways are referred to as either bronchialassociated lymphoid tissue (BALT) or mucosal-associated lymphoid tissue (MALT). They receive antigen transported directly across the epithelium by specialized M cells in the epithelium covering the follicle. The cuff of lymphocytes that surrounds a B-cell-rich germinal center of the follicles located in the small airways can extend to the epithelium of both the conducting airways and the alveoli, suggesting that they receive antigen from both the alveolar and small airway surface. The dominant class of antibody produced in a mucosal immune response are IgA and the major stimuli for the switching of antibody isotype from IgM to IgA during this response are TGF-h and IL-5

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(Table 3) (105). The B cells that mature in the germinal centers of the mucosal lymphatics home back to the lamina propria below the epithelial basement membrane after they enter the circulation and secrete their IgA as a dimer held together by a J chain. This complex binds to poly-IG receptors at the base of the epithelial cells and is actively transported to the airway luminal surface, where it is released into the lumen by proteolytic cleavage. The role of the mucosal immune response in small airways disease is not particularly well understood, but the use of descriptive terms like follicular bronchiolitis probably represent situations in which this system is activated.

VII. Summary and Interpretation Physiological studies conducted both in vitro and in vivo have established that the small airways are the site of increased obstruction in COPD (6,7,9) and that this increase in resistance combines with the loss on lung elastic recoil to reduce the FEV1 (12). The major risk factors involved in the pathogenesis of airway obstruction in COPD are well known, but the precise mechanisms of tissue damage caused by risk factors such as chronic cigarette smoking are only partially understood. Recent studies in both intact humans (63–65) and cultured human cells (65,66,104) indicate that the phagocytosis of atmospheric particles by lung cells initiates both an inflammatory and innate immune response in the lung. The cytokines generated by lung cells circulate in the blood where they activate the bone marrow to elevate the circulating white blood cell count and the liver to increase the production of acute phase proteins. The airway biopsy data (44–49) provide important information about the types of inflammatory cells present in the central airway tissue supplied by the subepithelial bronchial microvessels. These data show that the epithelium lining the central airways of cigarette smokers remains intact, that it sits on a relatively normal reticular basement membrane, and is infiltrated with inflammatory cells that include mast cells, PMNs, macrophages, CD 4 and CD-8 lymphocytes, and sometimes eosinophils. The examination of autopsy and surgically resected tissue has provided more direct evidence that small airways mount an inflammatory reaction on both the submucosal and the adventitial vessels in the outer wall of the small airways (19,20,59). One of these studies suggests that the PMN response in the epithelium is related to the amount smoked and that an increase in B cells associated with follicle formation in the outer walls of the airway correlates to a decline in FEV (59). The presence of mucosal lymphoid follicles is rare in nonsmokers and more common in smokers (107). Recent studies from our laboratory suggest that that there is a further increase in these follicles with progression of COPD. The fact that the inflammatory response in the outer walls of the small airways

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is an important feature of small airway obstruction is consistent with an older report from Matsuba and Thurlbeck, who found that peribronchiolar connective tissue deposition was a feature of small airway obstruction in advanced emphysema (20). When taken collectively, these data are consistent with the hypothesis that cigarette smoking produces a chronic inflammatory response in the small airway tissue supplied by the subepithelial bronchial vessels where the presence of PMNs correlates with the amount smoked. This inflammatory response is blended with an innate response initiated by the phagocytosis of foreign particles deposited on the surface of the small airways and distal lung tissue by both smoking and/or atmospheric pollution. The innate system initiates the systemic response that results in elevation of the circulating leukocytes, the production of acute phase proteins, and activation of the acquired immune response. This results in the increase in both the T lymphocytes observed in the biopsy studies and the B lymphocytes observed in relation to the formation of lymphoid follicles in the adventitial compartment of the airway wall. The source of antigen driving the acquired immune response is not clear but probably involves both the inhalation of antigens attached to the surface of atmospheric particles and microbial colonization and infection of the lower respiratory tract. O’Shaughnessy et al. (48) have argued that the CD-8 cell is the key to the pathogenesis of airflow limitation in patients with COPD and support this argument with data showing that the decline in FEV1 correlates with the number of these cells in bronchial biopsy. This hypothesis needs further examination in relation to what is known about the contribution of the cellular component of the acquired immune response to COPD risk factors to determine the exact mechanism by which CD-8 cells initiate the small airway remodeling and increase small airway resistance. An alternative hypothesis is that TGFh production that dampens the immune response may also initiate an excessive repair process that remodels the small airway wall and lumen to produce obstruction to airflow. Some very interesting animal studies are beginning to provide insight into this problem. For example, studies performed in transgenic animals by Lee et al. have shown that overexpression of IL-13 resulted in TGFh1 production and activation by pathways involving both plasmin and matrix metallo-proteinase (MMP)9, and causes massive connective tissue deposition in the walls of the peripheral airways and an increase in the formation of lymphoid follicles (108). This finding is certainly provocative with respect to what we know about small airways disease in humans. However, the hypothesis that IL-13 overexpression is a cause of human COPD requires further examination in human tissue. Other studies by Meshi et al. in guinea pigs suggest that the presence of latent adenoviral infection amplifies the inflammatory response to cigarette smoke and increases the amount of emphysema (109). Studies of human emphysema

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also show evidence that latent adenoviral gene expression correlates with the severity of the lung inflammation in patients with advanced emphysema (58). Because infection of the lower respiratory tract in children increases the risk of developing COPD, the possibility that a latent adenoviral infection from an episode of childhood bronchiolitis might drive the inflammatory process produced by cigarette smoking to produce COPD is interesting. We know that only minorities of smokers develop COPD; what remains uncertain is what determines who will develop COPD and latent viral infections might explain some of these cases. The current hypothesis that susceptibility to the effect of cigarette smoking is a result of differences in the genetic make up of the host or to an as yet unidentified gene environment interaction is being actively investigated. New knowledge in these areas, as well as a much better understanding of the initiation and progression of the remodeling process that causes airway obstruction, should be of great assistance to those attempting to develop better treatments for COPD.

Acknowledgments This work was supported by the National Heart Lung and Blood Institute (# 5 ROI HL 6117-04) and Canadian Institutes for Health Research (CIHR) (# 7246).

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5 Alveolar and Bronchiolar Inflammation in COPD

SIMONETTA BARALDO, GRAZIELLA TURATO, BIANCA BEGHE´, RENZO ZUIN, and MARINA SAETTA University of Padua Padua, Italy

Chronic obstructive pulmonary disease (COPD) is a public health problem worldwide, being a major cause of chronic morbidity and mortality. The World Health Organization (WHO) estimates COPD is currently the fourth leading cause of death in the world, and further increases in its prevalence and mortality can be predicted in the coming decades (1). According to the most recent guidelines, COPD is defined as a disease state characterized by not fully reversible airflow limitation that is usually both progressive and associated with an abnormal inflammatory response of the lungs to noxious particles or gases (1). A number of studies in the past demonstrated that a chronic inflammation is present throughout the airways, parenchyma, and pulmonary vasculature in patients with COPD and that this inflammatory response has an important role in the development of chronic airflow limitation (2–4). The recognition that inflammatory cells play a key role in the pathogenesis of COPD is now so widespread that, for the first time, it has led to the inclusion of the terms ‘‘abnormal inflammatory response’’ in the disease definition. It has long been recognized that smoking is the most important risk factor for the development of COPD and that exposure to cigarette smoke can elicit an inflammatory response in the lung (5–7). However, only a minority of 99

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cigarette smokers experience overt airflow obstruction. The susceptibility factors are still poorly understood and may involve several components, including genetic predisposition (i.e., polymorphisms in proinflammatory cytokines and genetic control of the balance of helper and cytotoxic T lymphocytes) or environmental conditions triggering or maintaining the disease (i.e., viral infections and pollutants). Although the precise mechanisms of individual susceptibility to the development of COPD are unknown, it has been hypothesized that, in genetically predisposed individuals, the inflammation initiated by cigarette smoke is likely to be responsible for most of the pathological abnormalities associated with the establishment of airflow obstruction and its subsequent progression (8). In the first part of this chapter we will summarize the current knowledge on the inflammatory response present in peripheral airways and lung parenchyma of smokers with established COPD. In the second part we will review the few studies that have examined how this inflammatory process evolves when the disease progresses. In the last part we will address the issue of the potential reversibility of the inflammatory response in patients with COPD.

I. Inflammatory Changes in Established COPD A. Bronchiolar Inflammation

It is now well accepted that cigarette smoking can induce an inflammatory reaction involving the entire tracheobronchial tree, even in the absence of an established airflow obstruction. T lymphocytes and macrophages are the predominant cells infiltrating the central airways in smokers (9), whereas neutrophils, which are scanty in the airway wall, are increased in the lumen (10). This discrepancy led to the hypothesis that the inflammation in the lumen may be different from that in the bronchial walls of smokers. However, these differences could reflect differences in traffiking of these cells, so that, at any given time, more neutrophils are present in the bronchial lumen than in the bronchial wall. Niewoehner and co-workers (5) were the first to demonstrate that an inflammatory reaction is already present in the peripheral airways of young smokers who experienced sudden death outside the hospital, supporting the idea that early structural changes may occur in peripheral airways of smokers before COPD is established. These early lesions included an inflammatory infiltrate in the airway wall consisting predominantly of mononuclear cells and clusters of macrophages in the respiratory bronchioles. The authors reported that these lesions were present in the absence of noteworthy tissue destruction and fibrosis, and suggested that this stage of the disease could still be largely reversible.

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This early inflammatory infiltrate in smokers’ airways, could represent a nonspecific response of airways to injury in general. It would seem, therefore, that the majority of smokers would develop a chronic, nonspecific inflammation in the airway and lung parenchyma. However, for unknown reasons, only some smokers develop structural abnormalities that eventually become clinical COPD. The remainder of the smokers will still harbor the nonspecific inflammatory infiltrate but with otherwise normal airways and lung parenchyma, and only what are termed mild functional changes that never become clinically relevant (11). In smokers, the establishment of COPD is associated with a further increase in this inflammatory response, which is paralleled by the development of structural abnormalities in both airway wall and lung parenchyma. These events are reflected by the progressive deterioration of pulmonary function. In central airways the development of airflow obstruction is associated with an increase of macrophages and T lymphocytes in the airway wall, and of neutrophils in the airway lumen (2,9,10). Although the mechanism of neutrophil accumulation into the airway lumen in smokers with COPD is not entirely clear, an imbalance between pro and anti-inflammatory cytokines may play a role. Interleukin-10 (IL-10), a cytokine that reduces inflammatory responses, is decreased in the airway lumen of smokers with COPD (12). IL-8, a cytokine that promotes neutrophil chemotaxis, and tumor necrosis factor (TNF-a), a cytokine that activates adhesion molecules, are increased (10). The observation of an upregulation of the adhesion molecules E-selectin and intercellular adhesion molecule 1 (ICAM-1) on submucosal vessels and on bronchial epithelium of smokers with COPD (13) suggests a mechanism for recruitment of neutrophils from the circulation and for their migration into the airway lumen through the epithelium. The finding of an increased number of neutrophils in the bronchial epithelium of smokers with COPD supports this hypothesis (14). Neutrophils are also increased in the bronchial glands of these subjects (14), and this location may be crucial for the development of mucus hypersecretion in COPD, because neutrophil elastase is a remarkably potent secretagogue. Although for many years mucus hypersecretion has been considered to be irrelevant to the development of chronic airflow obstruction in smokers (15), a more recent study has shown that chronic sputum production was significantly associated with both an excess of forced expiratory volume in 1s (FEV1) decline and an increased risk of subsequent hospitalization because of COPD, supporting a role for mucus hypersecretion in the development of chronic airflow obstruction (16). Mucus hypersecretion is a feature of COPD that can have important functional consequences in peripheral airways as well. In fact, in smokers with COPD, an increased number of mucus-secreting goblet cells associated with

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an increased number of epithelial neutrophils has been demonstrated (17). An increased number of goblet cells in the epithelium of peripheral airways can potentially contribute to the development of smoking-induced airflow obstruction in at least two ways. The first is by producing excess mucus that could alter the surface tension of the airway lining fluid, rendering the peripheral airways unstable and facilitating their closure (18). The second is by inducing luminal occlusion through the formation of mucus plugs in peripheral airways (19). Studies examining peripheral airways in smokers are particularly relevant because, as elegantly shown by the pioneering work of Hogg and co-workers, peripheral airways are the major site of increased resistance in smokers (20), and therefore lesions in this region of the lung may have important functional consequences. Pathological lesions in peripheral airways that are associated with the development of airflow obstruction include airway remodeling (fibrosis and smooth muscle hypertrophy) and an increased number of inflammatory cells, particularly of CD8+ T lymphocytes (19,21). CD8+ T lymphocytes, which are increased not only in peripheral but also in central airways and in lung parenchyma (2,4), seem to play a crucial role in the pathophysiology of COPD. However, the cytokine profile of these T lymphocytes and their chemokine receptor expression has not been fully investigated. A current paradigm in immunology is that the nature of an immune response to an antigenic stimulus is determined largely by the pattern of cytokines produced by activated T cells (22). Type-1 T cells express cytokines, such as interferon g (IFNg), crucial in the activation of macrophages and in the response to viral and bacterial infections, whereas type-2 T cells express cytokines, such as interleukin (IL)-4 and IL-5, involved in Ig-E mediated responses and eosinophilia characteristic of allergic diseases. It has recently been shown that the CD8+ T cells infiltrating the peripheral airways in COPD produce IFNg and express CXCR3 (23), a chemokine receptor known to be preferentially expressed on type 1 cells (24). Moreover, CXCR3 expression is paralleled by a strong epithelial expression of its ligand CXCL10, suggesting that the CXCR3/CXCL10 axis may be involved in the recruitment of type 1 cells in peripheral airways of smokers with COPD. As we have seen, type 1 inflammatory cells and their cytokines are present in peripheral airway wall of smokers with COPD. The analysis of inflammatory cell localization within the airway wall could help to clarify possible mechanisms for the development of airflow obstruction. In fact, if the distribution of the inflammatory infiltrate varies within the different compartments of the airway wall, local gradients of inflammatory mediators could occur that might influence the response to a phlogistic environment. Therefore regional differences in inflammatory cell density could have important

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implications for the pathogenesis of COPD, because they can contribute to airway obstruction through different mechanisms. Smokers with chronic airflow obstruction have an increased number of inflammatory cells in the inner submucosal layer compared with the outer adventitial region, and this greater cell density is correlated with reduced expiratory flow (17). This regional difference in inflammatory cell density has not been observed in smokers with normal lung function, nor in nonsmoking subjects. This suggests that this inner vs. outer pattern is not part of a nonspecific inflammatory response, but may rather be related to the disease. It is conceivable that the increased cellular density in the submucosa would promote airway constriction by amplifying the effect of airway smooth muscle shortening on the caliber of the airways (25). Inflammation, fibrosis, and smooth muscle hypertrophy, by increasing the thickness of the airway wall, may facilitate uncoupling between airways and parenchyma, therefore promoting airway closure. In addition, airway wall inflammation could contribute to the destruction of alveolar attachments (i.e., the alveolar walls directly attached to the airway wall), allowing the airway wall to deform and thus narrowing the airway lumen. This hypothesis is supported by the observation that, in smokers, the destruction of alveolar attachments is correlated with the degree of inflammation in peripheral airways (26). This finding suggests a pathogenetic role for airway inflammation in inducing destruction of alveolar attachments. It is possible that mediators released by inflammatory cells may weaken the alveolar tissue and facilitate its rupture, particularly at the point were the attachments join the airway wall, where the mechanical stress is maximal. B.

Alveolar Inflammation

We will now focus on inflammatory changes in alveolar tissue to highlight their relationship with the destruction of alveolar walls. We will therefore review the studies on lung tissue pathology, while studies on bronchoalveolar lavage will not be reviewed. One of the most important morphological hallmarks of COPD is emphysema. Emphysema is defined anatomically as a condition of the lung characterized by permanent abnormal enlargement of the respiratory air spaces, accompanied by destruction of their walls without obvious fibrosis (27). Emphysema can contribute to the development of airflow obstruction by reducing the elastic recoil of the lung, which decreases the intra-alveolar pressure that drives exhalation, and by diminishing the number of alveolar attachments to conducting airway. Smokers can develop two main morphological forms of emphysema, which can be identified according to the region of the acinus involved (28).

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Centriacinar (or centrilobular) emphysema is characterized by focal destruction restricted to respiratory bronchioli and to the central portions of the acinus, surrounded by areas of grossly normal lung parenchyma. In this form pathological lesions occur more frequently in the upper lobes of the lung. Panacinar emphysema is characteristic of patients who develop emphysema relatively early in life. It is usually associated with deficiency of alpha1antitrypsin, which normally protects the respiratory region by forming a highly effective antielastase screen. This form of emphysema, in contrast to the centriacinar form, occurs more frequently in the lower lobes than in the upper ones and involves destruction of the alveolar walls in a fairly uniform manner. The most common type of parenchymal destruction in smokers is centriacinar emphysema, but also the panacinar form can be observed. Especially in the severe stage of the disease, classic examples of centriacinar emphysema are uncommon, atypical examples predominate, and their interpretation may be difficult. The two major morphological forms of emphysema are thought to have distinct functional properties and distinct peripheral airway involvement (28– 30): a higher compliance in the panacinar form, and a higher degree of hyperreactivity and airway inflammation in the centriacinar form. The inflammatory process present in peripheral airways of smokers could favor centriacinar destruction and the consequent development of airflow obstruction observed in centriacinar emphysema. By contrast, in panacinar emphysema, airflow obstruction seems to be due mainly to loss of elastic recoil and to have little relation to peripheral airway inflammation. In both centriacinar and panacinar emphysema the destructive process can be detected microscopically in the alveolar walls even when there is no evidence of airspace enlargement. The microscopic measurement of this parenchymal destruction [destructive index (DI)] can, therefore, allow an early identification of the disease, at a time when emphysema is not detectable macroscopically. The functional significance of such early destruction is demonstrated by its correlation with indices of airflow obstruction and loss of elastic recoil of the lung (31). The pathogenesis of parenchymal destruction remains unknown, although a mechanism involving a proteases–antiproteases imbalance is widely supported. This hypothesis is based on the observation that activated inflammatory cells release proteases that, by overwhelming local antiprotease activity, can destroy lung parenchyma. Smoking promotes an inflammatory reaction characterized by an increase of neutrophils and macrophages, which are potential sources of proteases such as leukocyte elastase and matrix metalloproteases, which can damage lung cells and degrade the interstitium (e.g., elastin, collagen, proteoglycans). However, because many cigarette smokers and patients with other inflammatory lung parenchymal diseases (such as pneumonia and adult respiratory distress syndrome) do not develop significant lung destruction despite a

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striking inflammatory process, this hypothesis may not fully explain the loss of lung tissue in emphysema induced by cigarette smoking. The parenchymal destruction is associated with the presence of an inflammatory process in the alveolar walls (32,33), which consists predominantly of CD8+ T-lymphocytes (4,34). The major activity of CD8+ cytotoxic T lymphocytes has been considered the rapid resolution of acute viral infections, and viral infections are a frequent occurrence in patients with COPD. The observation that people with frequent respiratory infections in childhood are more prone to develop COPD supports the role of viral infections in this disease (35). It is conceivable that, in response to repeated viral infections, an excessive recruitment of CD8+ T lymphocytes may occur and damage the lung in susceptible smokers, possibly through the release of TNFa (36). On the other hand, it is also possible that CD8+ T lymphocytes are able to damage the lung even in the absence of a stimulus such as viral infection, as shown by Enelow and co-workers (37). They clearly demonstrated that recognition of a lung autoantigen by Tcytotoxic cells may directly produce a marked lung injury. Taking into account these findings, it can be hypothesized that the CD8+ cytotoxic Tcell accumulation observed in patients with COPD could be a response to an autoantigenic stimulus originating in the lung and induced by cigarette smoking. The observation that CD8+ T cells are increased in lung parenchyma in smokers who develop COPD and showed a significant correlation with the degree of airflow obstruction is intriguing and supports the notion that tissue injury may be dependent on T-cell activity (4,11). One of the most important consequences of the effects of cytotoxic CD8+ T lymphocytes is the apoptosis of target cells. It would be not surprising if apoptosis plays a role in the destruction of lung tissue in patients with emphysema. Majo and co-workers (35) have reported that, in smokers with emphysema, both the degree of apoptosis and the number of CD8+ T cells in the alveolar walls increased in parallel with the amount of cigarette smoke inhaled. Moreover, Kasahara and co-workers (38) demonstrated that the destruction of lung tissue in patients with emphysema may involve accelerated apoptosis of endothelial and epithelial cells through a mechanism dependent on vascular endothelial growth factor (VEGF). It can therefore be hypothesized that the proliferation of cytotoxic CD8+ T lymphocytes induced by smoking may participate in the destruction of the lungs by inducing apoptosis of structural cells.

II. Inflammatory Changes in Severe COPD COPD is a progressive disease that in a minority of subjects may become very severe. Among patients with COPD the rate of decline in FEV1 can vary from

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apparently normal rates of decline to greater than 150 ml/year, despite similar smoking histories and levels of initial FEV1 (39). Such striking variation in the rate of decline in FEV1 among individuals suggests unknown genetic or environmental factors may be important determinants of the course of the disease. Smoking is the main recognized factor that accelerates the decline of lung function in patients with COPD, and smoking cessation is the only measure shown to decrease the rate of decline of FEV1 in the disease. Recently Donaldson and co-workers demonstrated for the first time that another factor contributing to long-term decline in lung function in patients with COPD is the frequency of acute exacerbations (i.e. worsening of symptoms) (40). The authors showed patients with COPD who experienced frequent exacerbations experienced a significantly greater decline in FEV1 than those with infrequent exacerbations (40). Although exacerbations typically punctuate the progression of COPD, a standardized definition of COPD exacerbations is still lacking. A mild exacerbation of COPD may be defined on clinical grounds as increased dyspnea, cough, and sputum production that cause subjects to seek medical attention. COPD exacerbations may be defined as severe when they are associated with acute respiratory failure (41). It is noteworthy that not all exacerbations are characterized by large spirometric changes. Exacerbations of COPD are a major cause of morbidity and mortality and are associated with a significant health and economic burden because of increased rates of hospital admission and absence from work. Patients who survived hospitalization had a substantial risk of requiring care in a nursing home or readmission to the hospital in the following months. They were likely to be dependent on others for one or more activities of daily living, and commonly considered their quality of life to be poor (42,43). Despite the fact that exacerbations represent an important feature of the clinical manifestations and natural history of COPD, they are not included in the definition of the disease. The cause of COPD exacerbations is not established, although there is increased evidence that both bacterial and viral infections may play a role (44– 49). The precise mechanism by which these respiratory infections may induce COPD exacerbations is poorly understood. This may be due to the fact that only a few pathological studies have examined patients with COPD during an exacerbation and, in particular, the pathology of bronchiolar and alveolar walls has not been extensively investigated. Examination of patients with exacerbations of COPD by collecting bronchial biopsies, bronchoalveolar lavage, and, more recently, spontaneous or induced sputum showed increased airway inflammation and elevated levels of airway inflammatory cytokines (45–47,50–52). In particular, subjects with exacerbations of COPD are characterized by a marked recruitment of neutrophils associated with an increased expression of IL-8, myeloperoxidase, and TNF-a (46,47,50). IL-8 is

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a potent neutrophil chemoattractant, myeloperoxidase is a marker of neutrophil activation, and TNF-a is a cytokine known to increase expression of adhesion molecules on endothelial cells, thus facilitating leukocyte influx in the airway tissue. The presence of this neutrophilic inflammatory reaction and the fact that IL-8 correlated with the bacterial counts (44) suggest a role for bacterial infection in the exacerbations of COPD. This hypothesis is consistent with the finding by Stockley and co-workers who demonstrated that sputum purulence is associated with the detection of a bacterial pathogen at exacerbation (48). In bronchial biopsy specimens of subjects with mild exacerbations of COPD the neutrophilia was paralleled by a marked eosinophilia, which was associated with up-regulation of the eosinophil chemoattractants eotaxin and regulated upon activation normal T-cell expressed and secreted (RANTES) (50–52). Although bacteria may also play a role, viral infections may be the most likely cause since respiratory viruses are able to stimulate the production of both eotaxin and RANTES. There is also evidence that RANTES may act synergistically with CD8+ cells to enhance apoptosis of virally infected cells. Thus, as hypothesised by Zhu et al. (52), when CD8+ cells predominate, as in patients with stable COPD, exacerbations and increased RANTES may promote CD8+ cell-mediated tissue damage. Increased frequency of viral exacerbations may thus destroy airway and alveolar tissue directly, encouraging the development of small airway disease and microscopic emphysema. In this way, repeated exacerbations due to viral infections may accelerate a decline in lung function in smokers whose CD8+ T-cell numbers are already increased. This hypothesis is supported by the recent observation that, as mentioned above, exacerbation frequency is an important determinant of FEV1 decline in patients with COPD (40). In patients with COPD, as airflow obstruction progressively worsens, the lung inflammation induced by cigarette smoking increases, as shown by the few studies that investigated the lung pathology in patients with end-stage COPD (53–55). Investigating these patients may be of interest because a better characterization of their lung pathology may help clarify why, among patients with a similar smoking history, only a minority develop a severe disease. Furthermore, even if patients with severe COPD represent only a small percentage of smokers, they require an enormous amount of health care resources. Lung specimens from patients undergoing lung volume reduction surgery (LVRS) or lung transplantation for treatment of severe emphysema represent a unique opportunity to examine lung pathology in living patients at a severe stage of the disease. Moreover, the fact that these patients perform pulmonary function tests before surgery allows investigation of the relationship between measurements of lung pathology and pulmonary function. The pioneering study by Retamales and co-workers was the first to examine and quantify the lung pathology in living patients undergoing LVRS

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for treatment of severe emphysema (53). The authors demonstrated that there is an increase in the intensity of the inflammatory response in the alveolar walls and alveolar spaces of these patients and concluded that the lung inflammation induced by cigarette smoking is amplified in severe emphysema. In a recent study we extended these findings by demonstrating that, when the disease progresses, there is an amplification of the inflammatory response even in the peripheral airways. This enhanced airway inflammatory process is correlated with the degrees of airflow limitation, lung hyperinflation, CO diffusion impairment, and radiological emphysema, suggesting a role for this inflammatory response in the clinical progression of the disease (54). Among the different inflammatory cells types contributing to the inflammatory response in peripheral airways of smokers with severe COPD, CD8+ and CD4+ T lymphocytes in the airway wall and macrophages in the airway epithelium appear to be the most relevant. Conversely, in the lung parenchyma, all the inflammatory cells, including neutrophils, appear to be increased (53). A prominent neutrophilia, which was correlated with the degree of airflow obstruction, has also been reported in central airways of patients with severe COPD (55). Taken together, these findings suggest a role for neutrophils in the progression of the disease. There are surprisingly few autopsy studies on subjects with COPD. The largest study, performed by Nagay and colleagues (56), showed that these subjects had both emphysema and peripheral airway abnormalities. Although the relative contribution of each of these pathological lesions to the development of airflow obstruction is difficult to establish, the contribution of each may vary according to the stage of the disease. In mild COPD the degree of airflow obstruction correlates with bronchiolar inflammation. In more severe patients the degree of airflow limitation correlates with parenchymal destruction but not with bronchiolar inflammation. These findings may indicate that, when emphysema is severe, loss of elastic recoil assumes overwhelming importance in causing airflow obstruction, thus masking the effects of peripheral airway abnormalities. By contrast, when emphysema is mild, peripheral airway abnormalities do appear to play a role in causing airflow obstruction (57).

III. Potential Reversibility of Inflammatory Changes in COPD Although smoking cessation is the only intervention proven to modify the progressive development of airflow obstruction, the role of pharmacological interventions in modifying the natural history of COPD is still debated. Long acting h-agonists improve lung function and health status in patients with COPD and reduce exacerbations (58,59). Recent studies have shown that in

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patients with COPD inhaled corticosteroids have no influence on long-term decline in lung function (60), although they can reduce the incidence of exacerbations (61). A possible explanation for the effectiveness of corticosteroids in the exacerbations of COPD is the finding that the pattern of bronchial inflammation changes during an exacerbation of the disease, showing a prominent airway eosinophilia (50). The idea that eosinophilic inflammation is a marker for responsiveness to corticosteroids is supported by the observation that airway eosinophilia is present in a subgroup of patients with COPD whose pulmonary function improves in response to a short course of steroids (62,63). Moreover, a recent report showed that corticosteroids determined a reduction of the number of mast cells in biopsies of patients with COPD, and that this reduction was paralleled by a decrease in the exacerbation incidence (64). These findings suggest the presence of a subgroup of patients with COPD characterized by so-called asthmatic features (i.e., eosinophil and mast cell infiltration) who are responsive to corticosteroids. It is therefore conceivable that corticosteroids may have effects on the pattern of inflammation characteristic of asthma, driven by CD4 T-lymphocytes, eosinophils, and mast cells, but not on that characteristic of COPD, driven by CD8 T-lymphocytes, macrophages, and neutrophils. An alternative explanation of the lack of effect of inhaled steroids in the majority of patients with COPD is that the devices used may be unable to deliver the corticosteroid to the more distal airways and the lung parenchyma, which are the sites responsible for the development of airflow obstruction in this disease. Moreover, it would seem reasonable to think that, although lesions such as airway inflammation could be reversible, lesions such as parenchymal destruction and fibrosis may not be easily reversible. In recent years there has been renewed interest in strategies directed at the restoration of the lost alveolar surface area in patients with emphysema. Retinoids have received considerable attention as alveolar morphogens and as potential therapeutic agents, especially because retinoic acid was able to reverse the emphysematous lesions induced by intratracheal administration of elastase in rats (65). Nevertheless, given the different mechanisms of lung development in rats and humans, more studies are needed to establish the clinical utility of retinoids in the treatment of human emphysema.

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6 Pathophysiological Basis for the Treatment of Chronic Obstructive Pulmonary Disease

CYNTHIA BROWN and ROBERT A. WISE Johns Hopkins University School of Medicine Baltimore, Maryland, U.S.A.

I. Introduction This chapter reviews the treatment of chronic obstructive pulmonary disease (COPD) based on the pathophysiological alterations that occur in this disease. We do not intend for this chapter to substitute for formal, expertconsensus evidence-based guidelines for the treatment of COPD such as those that have been produced by the National Heart, Lung and Blood Institute (NHLBI)/World Health Organization (WHO) Global Initiative for Chronic Obstructive Lung Disease (GOLD) Workshop (1) or the excellent systematic evidence-based reviews of the Cochrane Collaboration (2). Our intent is instead to organize the approach to the therapy of COPD based on an understanding of the biological and physiological abnormalities that characterize the disease. Therefore, the chapter addresses the use of bronchodilators in the context of airways reactivity, oxygen supplementation in the context of gas exchange abnormalities, antibiotics and the role of infection, and antiinflammatory treatments in the context of what is known about inflammatory changes in COPD. We point out important gaps in our knowledge to focus future research directions for the treatment of COPD. 115

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COPD has been considered a disease of irreversible airway obstruction due to decreased elastic recoil and airway inflammation usually resulting from smoking tobacco (3). However, nonspecific airway hyperreactivity (AHR) can be demonstrated in up to 70% of patients with COPD (4), even in those with mild obstruction and minimal symptoms. The best tests to identify airway hyperreactivity in individuals with COPD are provocative tests with methacholine or histamine because these correlate best with symptoms and disease progression. In contrast, there is little correlation between reversibility testing with bronchodilators and response to provocative testing (5). In addition, bronchodilator response has not consistently been shown to have prognostic significance in COPD and does not affect long-term symptomatic response to treatment with bronchodilators (6,7). The role of airway hyperreactivity in the pathogenesis of COPD is controversial, reflecting two viewpoints. Originally proposed by Orie et al. in 1961, the Dutch hypothesis theorizes that airway hyperreactivity represents a constitutional state that can lead to accelerated decline in pulmonary function in individuals exposed to cigarette smoke or other environmental factors (8). As an alternative, airway hyperreactivity may reflect changes in lung dynamics that have already occurred as a result of inflammation and remodeling of the lung caused by cigarette smoke. Regardless of the cause of AHR, it has prognostic importance in patients with COPD. In several large studies, the magnitude of airway reactivity is an independent risk factor for accelerated decline in lung function (9–12). On average, an additional decline in forced expiratory volume in 1s (FEV1) of 10 ml/year can be expected in patients exhibiting airway hyperreactivity (6). In addition, the effects appear to be greater in elderly populations, with women showing a greater prevalence of increased airway reactivity (11,13). AHR also predicts a group of patients with COPD more likely to exhibit respiratory symptoms. In a large cohort of nonasthmatic patients with AHR followed for 24 years, Xu et al, showed that people with AHR were more likely to complain of chronic cough, chronic sputum production, dyspnea, and wheezing (14). This cohort was also less likely to note remission of symptoms. Recently, AHR has been linked to increased mortality in COPD (15). In more than 2000 patients evaluated for 24 years, 21 deaths were attributed to COPD as a primary cause, with COPD being a secondary cause in 39 other deaths, accounting for a total 11.4% of all deaths. However, in the cohort with AHR, the percentage of deaths attributable to COPD increased significantly to 19.4% of the total (15). The degree of histamine responsiveness also correlated with mortality (15). The group with AHR at the lowest dosage (1 mg/ml) of histamine had a relative risk of death from COPD almost five

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times (15.8 vs. 3.83) greater than the risk at the highest dosage that attributed any increased risk (32 mg/ml). In contrast, there was no increased mortality risk attributed to AHR in patients with cardiovascular disease or lung cancer. The pathological mechanism underlying AHR in COPD is unclear. As discussed later in this chapter, COPD is characterized by an inflammatory infiltrate in the terminal airways (16). In normal lungs, no mucous glands are found in these airways; in COPD, the epithelium undergoes mucous metaplasia with secretion of mucus into the bronchioles (17,18). In addition, the terminal airways develop smooth muscle hypertrophy and increased mural edema (19). Taken cumulatively, these changes may cause increased tendency for the airways to constrict: the narrow lumen and thickened airway wall will tend to close more easily for the same magnitude of smooth muscle tone (20). As an alternative, increased reactivity in the airways may reflect the emphysematous changes in the lung parenchyma. As alveolar destruction progresses, bronchiolar attachments to the airway perimeter are lost (18). This contributes to the loss of elastic recoil and subsequent distortion of airway integrity, thus causing early airway closure (21). A final possibility to consider is that the relationship between AHR and COPD is an artifact of the way in which AHR is measured. When calculating degree of airway reactivity, the absolute value of change in lung function in response to provocative challenge is compared to a lower baseline and therefore represents a higher percentage of the baseline than in people with normal lung function. Patients with COPD tend to have increases in AHR over time, which is correlated with the reduction in baseline pulmonary function. Smoking cessation decreases this progression, which is attributable largely to the beneficial effect on FEV1 (22). Long-term management of patients with stable COPD incorporates the use of bronchodilating agents regardless of whether AHR is present on provocative testing. Small amounts of bronchoconstriction and air trapping can cause significant dyspnea; in contrast, minimal bronchodilation can produce significant relief of symptoms. Several classes of bronchodilators are available including anticholinergic medications, beta-2 agonists, and methylxanthines. All available bronchodilators increase FEV1 for a period of time after their administration, but do not alter the decline in lung function or mortality associated with COPD (3). Inhaled medications are given most effectively by metered-dose inhaler (MDI) through a spacer device. Proper technique is essential to drug delivery, and patient instruction in inhaler technique should be routine. Treatment with nebulized bronchodilators is usually necessary during acute exacerbations, although no difference in outcome is seen when compared to MDIs (23). Although more expensive and less convenient than MDIs, nebulization may be required as long-term therapy in some patients who are unable to get symptomatic relief with MDIs or drypowder inhalers (DPIs) (24). Treatment is usually initiated with an inhaled

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short-acting medication on an as-needed basis, with a stepwise increase to scheduled therapy if significant dyspnea persists. In most patients, additional classes of bronchodilators are necessary, with evidence supporting better symptom relief with combined therapy (25). Ipratropium bromide (IB) is the only anticholinergic medication currently available in the United States. It inhibits vagal stimulation of the airways, preventing bronchoconstriction. FEV1 is increased after treatment with IB with an effect that lasts for 4–6 h (26). Because of its relatively few side effects and lack of development of tolerance, it is often selected as the initial therapy. Systemic side effects are uncommon even when it is administered at high dosages. The most common side effects are dry mouth and cough. With long-term use, there may be a slight increase in atrial tachyarrhythmias (27). Tiotropium is an inhaled anticholinergic medication that should soon be available in the United States. It selectively targets M1 and M3 receptors with high affinity and has a long duration of action, allowing oncedaily administration (28). The next class of bronchodilators is the beta-2 adrenergic agonists. This class of medication directly stimulates bronchodilation through its effects at the beta-2 sympathetic receptor. Short-acting beta-2 agonists have long been used for acute symptomatic relief of dyspnea. Exercise capacity can be increased when beta-2 agonists are used just prior to exertion, which is the result of reversal of gas trapping as well as increases in airway caliber. Significant side effects can limit the usefulness of short-acting beta agonist in some individuals; tremor, tachycardia, anxiety, and hypokalemia can occur. In addition, the effectiveness of short-acting beta-agonists can decline with frequent use, which is not found with anticholinergic agents (29,30). Salmeterol and formoterol are the two long-acting beta-agonists currently available for treatment of COPD. FEV1 increases after administration of long-acting beta agonists, lasting for approximately 12 h (31). These agents have an important role in the treatment of COPD: they both have been shown to cause an improvement in quality of life with minimal side effects (31–33). Long-term effects of long-acting beta agonists on decline in lung function are unknown. Side effects with long-acting beta-agonists are minimal although there is concern that overuse may lead to cardiac arrhythmias (31). Combining anticholinergic and beta-agonist medications leads to a greater increase in FEV1 than either medication alone (32,34,35). A combination preparation of ipratropium bromide and albuterol is currently available. This may aid in compliance by simplifying the medication regimen and joining the more rapid onset of beta-agonist treatment to the slightly longer duration of benefit from ipratropium. The final class of bronchodilators used in the treatment of COPD is the methylxanthines. Theophylline is currently the only commonly used medica-

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tion from this class. It is taken as an oral preparation once or twice daily. There is a relatively narrow therapeutic window, and some authors recommend frequent serum levels (36). Side effects such as tremor, nausea, vomiting, and irritability are common, but do not correlate with serum levels. Use of theophylline has declined as other long-acting bronchodilators have become available, but it is still an effective and inexpensive treatment for COPD (37). Although theophylline does cause an increase in FEV1, it also has effects beyond those related to bronchodilation (38). Some of these include improvement in diaphragm function, prevention of respiratory muscle fatigue, increased ventilatory drive, increased mucociliary clearance, suppression of leukocyte activation, and inhibition of mast cell release (39). The antiinflammatory properties of low-dosage theophylline in the treatment of COPD have been documented, which may prove to be the reason that this agent is perceived by some patients as having benefit even in the absence of bronchodilation (40). During acute exacerbations, increased frequency of bronchodilating medications is required. Although inhaled short-acting beta-agonists are used most frequently, there is no proven benefit over anticholinergic medications (23). Once the maximum dosage of one class of bronchodilator is reached, additional benefit can be realized with the addition of a second class (23). Nebulization of bronchodilator solutions is the most commonly used delivery system. However, a recent review of the evidence shows no clear benefit of nebulization over MDIs used with a spacer device (23). Parenteral administration of aminophylline is controversial and is not often used. Small changes in lung function are frequently offset by the cardiac toxicity as well as potential worsening of ventilation/perfusion (V/Q) inequality (23,39,41).

III. Hypoxemia and Oxygen Supplementation Correction of severe hypoxemia through oxygen therapy is the only intervention that clearly improves survival in COPD (42,43). Hypoxemia at rest occurs as a consequence of V/Q mismatching. Emphysematous changes in the lungs cause dilation of terminal airspaces as well as destruction of the capillary bed supplying these regions. Imbalance in ventilation and perfusion is thus caused by increasing physiological dead space through continued ventilation of areas with decreased perfusion. By definition, this pattern of V/ Q mismatch causes a high V/Q ratio. In contrast, patients with symptoms primarily of chronic bronchitis exhibit a different pattern of V/Q mismatch, in which normal blood flow continues to areas of the lung that are poorly ventilated. The morbid anatomy of chronic bronchitis shows fibrotic and tortuous airways with increased mucus production and subsequent partial or

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complete obstruction of small airways. This translates physiologically to areas with low ventilation to perfusion ratios. These two gas-exchange patterns in COPD frequently overlap, causing a mixed pattern of V/Q abnormalities. Wagner et al. first confirmed this in 1977 when he described the character of V/Q mismatch in a series of patients by the multiple inert gas elimination technique (44). He studied eight patients who had changes primarily of emphysema (type A) with overdistention of their lungs, mild hypoxemia, and hypercarbia; another 12 patients had more prominent symptoms of chronic bronchitis (type B) with copious sputum production, severe hypoxemia, and retention of CO2. Three patients exhibited mixed features (44). Seven of the eight patients categorized as having type A disease were found to have distribution patterns consistent with high V/Q ratios; in contrast, the type B patients were quite heterogeneous, with one-third of the group showing high V/Q ratios, one-third low, and one-third with a mixed pattern (3). Hypoxemia during COPD exacerbations appears to be due to worsening of V/Q inequality caused by greater perfusion of poorly ventilated lung areas (45). In pathophysiological terms, this is likely related to increased airway inflammation with subsequent bronchospasm and increased mucus production. The greater work of breathing increases metabolic demands and can cause a fall in mixed venous oxygen saturation, thus amplifying hypoxemia caused by V/Q mismatch (45). The pathophysiology of hypoxemia that occurs with exercise is less well understood. The response to exercise in COPD is complex in that PaO2 may rise, fall, or be unaffected, and the same is true of PaCO2 as well. Oxygen extraction rises during exercise with a concordant drop in mixed venous oxygen content. The magnitude of the fall in mixed venous oxygen content is buffered by the extent that cardiac output can rise in concert with oxygen consumption. In patients with low V/Q regions, the fall in mixed venous oxygen content exaggerates the concomitant fall in arterial oxygen content. Patients with COPD may also have decreased ability to increase their cardiac output in response to exercise due to concurrent ischemic heart disease or cor pulmonale, augmenting the fall in mixed venous oxygen tension. The increased CO2 production is eliminated through increased alveolar ventilation by increasing tidal volume and/or respiratory rate. In patients with obstructive lung disease, there is less reserve in ventilatory capacity. If alveolar ventilation cannot rise to meet the demands of increased CO2 production, the arterial PCO2 rises with a reciprocal fall in PO2. There is also the possibility of hypoxemia caused by limitation of diffusion of oxygen across the alveolar– capillary membrane. It is surprising that, in the few studies done, the amount of ventilation–perfusion inequality does not predict hypoxemia with exercise; instead, the best predictor of exercise-induced hypoxemia is a low diffusing capacity for carbon monoxide (46). In one recent study, a lung diffusing

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capacity for carbon monoxide (DLCO) below 62% of the predicted value had a specificity and sensitivity of 75% for desaturation with exercise (47). Thus, it appears that the cause of oxygen desaturation with exercise is multifactorial, including changes in V/Q distribution, decreased mixed venous oxygen content, alveolar hypoventilation, and diffusion limitation. A number of physiological changes occur with sleep that, in individuals without COPD, cause no drop in arterial oxygen content. However, these same changes can cause marked hypoxemia in patients with COPD and worsens during rapid eye movement (REM) sleep. During all stages of sleep, central respiratory drive and the peripheral responsiveness to central stimuli are diminished. This effect is greatest on the accessory muscles, which can cause hypoventilation and decrease in functional residual capacity. In the patient with COPD, the fall in FRC causes worsening V/Q mismatch with a fall in oxygen saturation (48). Furthermore, concurrent sleep apnea occurs in as many as 10–20% of patients with COPD with resultant hypoxemia that is greater than can be expected with either diagnosis alone (49,50). Exercise oxygen desaturation does not appear to predict which patients with COPD are more likely to desaturate at night, but obesity and awake hypercapnia are risk factors for nocturnal desaturation (51–53). Treatment with oxygen has been shown to prolong survival in a subset of patients with COPD with severe daytime hypoxemia (42,43). In 1980, the Nocturnal Oxygen Therapy Trial (NOTT) enrolled 203 patients with a baseline PaO2 of less than 55 mmHg or PaO2 less than 59 mmHg with evidence of cor pulmonale or polycythemia (42). These patients were randomized to receive continuous oxygen therapy or nocturnal therapy for 12 h nightly. At the end of 12 months, the mortality in the nocturnal group was almost twice that of the group receiving continuous therapy (20.6% vs. 11.9%). The results remained true at 24 months as well (40.8% vs. 22.4%). In subgroup analysis, the patients with poorer baseline pulmonary function [(PCO2 > 43 mmHg, pH < 7.40, low nocturnal oxygen saturation, and decreased forced vital capacity (FVC)] showed more benefit from continuous oxygen therapy. However, patients with evidence of established cor pulmonale (i.e., higher pulmonary vascular resistance or decreased exercise capacity) showed less benefit from oxygen. This trial was corroborated by an oxygen therapy trial of the Medical Research Council in Great Britain in 1981 (43). Patients with similar baseline values of PaO2 and PCO2 were randomized to receive oxygen therapy for at least 15 h daily or no oxygen. Over 5 years of follow up, 30 of 45 control patients died compared to 19 of 42 patients receiving oxygen therapy (Fig. 1). These two studies lead to the current recommendations for initiation of oxygen therapy (Table 1). In general, oxygen supplied by nasal cannula with either liquid oxygen or concentrator devices is suitable for longterm therapy.

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Figure 1 Survival curves plotted from the NOTT Trial (42) and the Medical Research Council Oxygen Trial (43). There is a survival benefit of 15–24 h continuous oxygen/day. Twelve hours per day of oxygen is not clearly superior to no oxygen.

Although never formally studied, controlled oxygen therapy is considered essential for treating hypoxemia in exacerbations of COPD. The goal for treating patients with exacerbations is to use the minimum amount of supplemental oxygen required to keep the oxygen saturation above 90%. Higher oxygen saturation can cause alveolar hypoventilation with consequent rise in PCO2. Venturi masks deliver oxygen at a more constant fraction of inspired oxygen (FiO2) independent of minute ventilation and are used for patients with unstable disease. Nasal cannula systems are more comfortable for patients; however, they deliver variable concentrations of oxygen depending upon the patient’s minute ventilation (54). The value of oxygen therapy in other situations is not clear. Few studies have investigated the benefit of oxygen supplementation in patients with an intermediate degree of hypoxemia (PaO2 between 56 and 69 mmHg). Gorecka

Table 1 Indications for Oxygen Therapy Indications for continuous supplemental oxygen PaO2 V 55 mmHg or SaO2 V 88% in a patient on optimal stable medical therapy PaO2 = 55–59 mmHg or SaO2 V 89% for a patient with evidence of cor pulmonale, erythrocytosis, or neuropsychological impairment Elective indications for continuous supplemental oxygen: PaO2 V 55 mmHg or SaO2 V 88% during exercise or sleep but daytime values are higher

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et al. showed no improvement in survival in a group of 135 patients with a baseline PaO2 of 56–65 mmHg who were prescribed oxygen for 17 h daily (55). However, compliance in this trial was poor as patients only used their oxygen for 13.5 h daily and the sample size was adequate only to detect very large survival differences. In patients who develop hypoxemia during exercise, supplemental oxygen increases oxygen delivery to muscles and reduces minute ventilation and respiratory rate for a given workload (56,57). Other benefits supporting the use of oxygen therapy in exercise-induced hypoxemia include increased endurance, decreased dyspnea, and delayed onset of respiratory muscle fatigue (58). However, even patients who do not have exercise hypoxemia may improve exercise capacity with supplemental oxygen, probably because of the reduction in minute ventilation and reduced air trapping (59,60). There are no studies to suggest that treatment of exerciseinduced hypoxemia changes quality of life, morbidity, or mortality. One recent study by Garrod et al. randomized a small group of patients with severe COPD in a pulmonary rehabilitation program to training with oxygen or air (61). In this group of 25 patients, 11 were receiving long-term oxygen therapy; five of these were randomized to receive only air while training. The only significant difference between the groups was improvement in dyspnea among the subjects receiving oxygen. There were no differences noted in distance walked or quality of life, including ability to perform activities of daily living. The long-term prognostic implication of nocturnal oxygen desaturation is unknown. Fletcher et al. reported that patients with nocturnal oxygen desaturation associated with REM sleep had more disturbance in their pulmonary hemodynamics than those without nocturnal oxygen desaturation and predicted a subset of patients more likely to progress to chronic hypoxemia (62,63). In addition, one retrospective study showed increased mortality in a group of patients with REM-associated nocturnal desaturation compared to a group without nocturnal oxygen desaturation (64). Randomized trials of oxygen therapy for nocturnal hypoxemia have also been inconclusive. In a recent trial, nocturnal oxygen treatment was not helpful in decreasing mortality, although the sample size was not large (65). In addition, a follow-up study showed no increased risk of development of pulmonary hypertension or worsening of daytime blood gases in patients with nocturnal hypoxemia (66). In a small study by Fletcher et al. the patients with REM-associated nocturnal desaturation who received oxygen therapy had a fall in their mean pulmonary artery pressure compared with sham-treated controls (67). However, there was no change in the pulmonary vascular resistance between groups. The number of patients in the study was too small to evaluate for any mortality benefits. Thus, it remains inconclusive whether nocturnal oxygen therapy has benefit for patients with COPD who exhibit nocturnal oxygen desaturation.

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Brown and Wise IV. Airway Inflammation and Corticosteroid Therapy

The mechanisms by which cigarette smoke causes COPD is not known, but has long been thought to be mediated by an inflammatory response that causes an imbalance between proteases and antiproteases within the lung. A complex mixture of more than 4700 chemicals, free radicals, and other oxidants, cigarette smoke stimulates an intense inflammatory response in the terminal airways (68). In some individuals, this response becomes sustained resulting in destruction of lung tissue with progressive airway enlargement and increased sputum production. Increases in neutrophils, eosinophils, macrophages, and lymphocytes have all been noted in the lungs of cigarette smokers as well as increased amounts of a multitude of inflammatory cytokines and proteases produced by these cells. Much of the initial research into the pathophysiology of emphysema and chronic bronchitis was directed toward the neutrophil. In the 1960s, the genetic deficiency of alpha-1 antitrypsin (A1AT) was noted to be associated with early onset of emphysema and chronic bronchitis particularly in smokers. Alpha-1 antitrypsin was found to inactivate neutrophil elastase, thus focusing research on the neutrophil. Since that time, significant research has examined the role of the neutrophil in the pathogenesis of COPD. Although neutrophils form only a small fraction of the total numbers of inflammatory cells recovered by bronchoalveolar lavage in smokers with emphysema, their numbers have been noted by some investigators to be increased when compared to nonsmokers (69–72). This remains controversial, however, as other investigators show no increase in neutrophil numbers (73–75). Contained within the primary azurophilic granules of neutrophils, neutrophil elastase, cathepsin G, latent cathepsin C, and other serine proteases can destroy the elastin elements of the lung parenchyma. In murine models, progressive airway enlargement mimicking emphysema can be induced by instilling leukocyte and pancreatic elastase into the lung parenchyma (76). The levels of some chemoattractants for neutrophils such as interleukin-8 and leukotriene B4 are elevated in patients with COPD and some smokers without COPD, implying a stimulus for neutrophil recruitment (69,70). As neutrophils migrate through connective tissue, they release the contents of their primary granules. Even in genetically normal hosts: the concentration of neutrophil elastase (NE) will temporarily be supraphysiological: alpha-1 antitrypsin and other antiproteases will be overwhelmed, causing localized tissue destruction. In most normal hosts, this damage is repaired with no long-term consequences; however, in A1AT deficiency as well as perhaps in some smokers, there continues to be an imbalance between proteases and antiproteases such that destruction of elastic elements of the lung continues (77–79). However, NE has not been shown to have any increased activity in lung tissue of patients with COPD; in addition, there is no correlation between

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neutrophil numbers and disease symptoms (74,80). Betsuyaku et al. identified the 92 kD protein-labeled matrix metalloproteinase 9 (MMP9) or gelatinase B present in both neutrophils and macrophages (81). MMP-9 degrades several types of collagen and was shown to be present in its active form only from neutrophil-derived sources, suggesting a novel role of the neutrophil in COPD. Neutrophil elastase is likely to have a prominent role in the symptoms of chronic bronchitis (82). NE causes goblet cell metaplasia. In addition, neutrophil elastase (as well as cathepsin G) is a potent secretagogue for goblet cells, causing maximal degranulation within 4 h of neutrophil infiltration (82,83). Other effects of neutrophil elastase include decreased ciliary movement and epithelial damage. More recently, research has focused on the alveolar macrophage as the primary inflammatory cell involved in the pathogenesis of COPD. Alveolar macrophages are the most prevalent cell found in the bronchoalveolar lavage (BAL) fluid of smokers as well as patients with emphysema, making up 95–98 % of all cells recovered (75). Further evidence supporting the role of the macrophage in the origins of COPD is found by examining histological specimens of lungs from patients with emphysema: macrophages are located primarily in the respiratory bronchioles and centroacinar areas where emphysematous changes are first seen. Monocytes, the circulating precursors to macrophages, share with neutrophils the ability to generate neutrophil elastase and cathepsin G. When monocytes differentiate into tissue macrophages, they lose their ability to generate serine proteases, but gain their own complement of matrix metalloproteinases and cysteine proteases (75). The cysteine proteases, cathepsins L, S, and K, have elastolytic ability but are packaged tightly in lysosomes, indicating a primarily intracellular function. However, if they are secreted in an acidic environment, they could cause significant lung destruction. The variety of matrix metalloproteinases produced by the macrophages has shown more promise as the pathogenic enzymes causing destruction of alveolar tissue. One protease, matrix metalloelastase (MMP-12), has received considerable attention because it can solubilize many matrix proteins including elastin. In murine models, mice that have been genetically altered so that they are unable to produce MMP-12 do not develop emphysema after exposure to high levels of cigarette smoke (84). Macrophage recruitment was also markedly decreased in the MMP-12 knockout mice, indicating a role for MMP-12 not only as a protease but also as a chemokine for further macrophage recruitment (84). However, after stimulating macrophage recruitment by intrapulmonary instillation of a chemoattractant, MMP-12 knockout mice still develop no increase in alveolar size despite tissue infiltration of similar levels of macrophages to controls (84). MMP-12 is the primary matrix metalloproteinase in the mouse; however, humans have a much larger number of matrix metalloproteases. In

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human studies, MMP-12 expression is upregulated in all smokers regardless of whether emphysema develops (74). Imai et al. recently showed lack of MMP-12 expression in the lung tissue from patients with emphysema, but these subjects had stopped smoking for at least 3 months prior to procurement of the specimens, and some of the smoking-related changes may have been reversed (80). Other matrix metalloproteinases that primarily destroy the collagenous framework of the lung have been implicated in the pathogenesis of COPD as well, many of which are secreted by the macrophage. As alluded to in the discussion of neutrophils above, gelatinase B (MMP-9) can degrade many different types of collagen. This protease is present in both neutrophils and macrophages, although the primary source is still unclear. Finlay et al. showed significantly increased amounts of MMP-9 in both its active and latent forms in 10 patients with emphysema compared to 10 control patients, of whom 4 were active smokers (74). There were measurable levels of MMP-9 in the control group, but it was of the latent rather than the active type. Betsuyaku found similar increases in MMP-9 in neutrophils of emphysema compared to smokers without emphysema (81). Some research has emphasized the role of collagenase (MMP-1) in human emphysema. Finlay noted a significant correlation between interstitial, or human, collagenase (MMP-1) expression by alveolar macrophages in patients with emphysema compared to controls (74). In addition, MMP-1 activity did not differ with smoking status in patients with emphysema. A recent study examined tissue expression of MMP-1 in specimens from patients with emphysema who had quit smoking for at least 3 months prior to specimen acquisition (81). It was interesting that MMP-1 expression was found to be localized to type II pneumocytes in addition to resident macrophages. Tissue specimens from smokers without emphysema and normal controls showed no staining for MMP-1 in type II pneumocytes, although resident macrophages were positive (81). The pathogenesis of emphysema and chronic bronchitis requires a complex interaction among multiple inflammatory cells, cytokines, and a balance of proteases and antiproteases. In addition to the neutrophil and macrophages, eosinophils and CD8 lymphocytes are increased in the lung parenchyma of patients with emphysema, although their role is obscure (85,86). The complete story of the interactions that lead to COPD in susceptible cigarette smokers continues to be discovered. Because inflammation in the terminal airways is implicated as the cause of COPD, the use of corticosteroids to mediate the inflammatory response has been logical in COPD. Several corticosteroids are now available in inhaled formulations, and several studies have examined their effects on both the inflammatory response and clinical outcomes. Despite much research in this area the results have been variable, with most studies showing minimal or

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absent response. The effects of corticosteroids on markers of airway inflammation are uncertain. Some studies have shown that use of corticosteroids causes visible decreases in the amount of inflammation seen during bronchoscopy as well as in the number of inflammatory cells recovered in BAL fluid (87–89). In addition, a study by Llewellyn-Jones et al. found a decrease in chemotactic activity and an increase in neutrophil elastase inhibitory activity with corticosteroid treatment (90). Contradictory results were reported by two other studies, with no changes demonstrated in the number of inflammatory cells after treatment with corticosteroids (91,92). In the study by Culpitt et al., levels of MMP-1, MMP-9, secretory leukoprotease inhibitor, and tissue inhibitor of metalloproteinase were all unaffected by treatment (91). Further studies have shown that isolated alveolar macrophages from patients with COPD are not inhibited from releasing IL-8 by dexamethasone in comparison with macrophages from smokers without COPD (93). It is postulated that this is the consequence of inhibition of histone deacetylase by cigarette smoke, which is an important intermediary in the suppression of inflammation by corticosteroids (94). The reasons for the variable results are not clear. Although all studies used high-dosage corticosteroids, the duration of treatment was only 2–4 weeks in the studies demonstrating no benefit (88,89), compared to 6–8 weeks in the other studies (91,92). Whatever the cause of the disparate results, it is clear that the inflammatory response in COPD is far less susceptible to corticosteroid inhibition than allergic or asthmatic inflammatory lung responses. Regardless of the effect of corticosteroids on inflammatory markers, the role of corticosteroids in treatment of COPD is primarily determined by whether they exhibit clinical efficacy. Several large randomized, placebocontrolled trials that cumulatively enrolled over 3000 patients have recently been published regarding the long-term use of inhaled corticosteroids in COPD (95–98). None of these trials demonstrated a beneficial effect of corticosteroids in the ability to alter the decline in FEV1. At this time, the effect of these medications on mortality is unknown. The studies mentioned above did not show a survival advantage with corticosteroid treatment, although the patients enrolled had mild to moderate disease. Therefore, the average follow-up time of 3 years is unlikely to be sufficient to make a definitive conclusion. However, one recent retrospective analysis of a large group patients with severe COPD who were prescribed inhaled corticosteroids at hospital discharge documented a 29% reduction in all-cause mortality (99). In addition, the patients who received inhaled corticosteroids at hospital discharge had a 24% reduction in need for repeat hospitalization. Inhaled corticosteroids may also have an effect on other clinical endpoints, although these results were variable among the studies mentioned above. The Lung Health Study reported significantly less dyspnea in patients treated with inhaled corticosteroids, with fewer new respiratory symptoms developing in this group

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as well (98). Corticosteroids may also reduce the need to utilize health care resources. In one study, the number of unscheduled physician visits was reduced (98) while another study reported a 25% reduction in the number of COPD exacerbations experienced (95). No improvement, however, was demonstrated in overall quality of life although the rate of decline in quality of life may be altered by treatment (95). The incidence of side effects was greater in patients treated with inhaled corticosteroids than in those not receiving inhaled corticosteroid in all studies to date. Patients frequently report increased bruising. A significant decrease in bone mineral density has been shown in one study after 3 years of treatment with inhaled triamcinolone (98). There also appears to be an increase in risk of hip fracture in elderly users of inhaled corticosteroids (100). Current guidelines regarding the use of inhaled corticosteroids in patients with stable COPD recommend using these medications only in patients with a documented response to treatment or moderate to severe COPD (FEV1 < 50% predicted) with frequent exacerbations requiring oral corticosteroids (1). A trial of inhaled corticosteroids for 6–12 weeks may be useful to identify individuals who have a component of reversible airflow limitation. If a patient experiences an increase in FEV1 greater than 200 ml or 15%, the guidelines recommend continued therapy with inhaled corticosteroids. Others have recommended a 2 week trial of oral glucocorticoids to determine if a patient is responsive to steroid treatment. Recently, however, several studies have shown that this is a poor predictor of long-term steroid response (95,101). Because of the significant toxicity associated with oral corticosteroids administration, no prospective trials have assessed whether treatment alters the decline in pulmonary function. Some retrospective analyses have suggested that long-term oral steroid treatment may slow the progression of disease, but the evidence is not strong in comparison with the well-defined adverse effects of such treatment (102). The most significant side effect in patients with COPD is steroid myopathy, which can contribute to decreased functional status and decreased respiratory muscle function (103). Other side effects include weight gain, osteoporosis, cataracts, increased bruising, and hyperglycemia. Treatment with oral corticosteroids is only recommended during acute exacerbations. In this setting, oral corticosteroids significantly improve FEV1 within the first day of treatment, decrease relapse rate, and shorten recovery time (23,104). The initial improvement in FEV1 is still evident after 2–3 days of treatment, but was not significant at 2 weeks. The optimal dosage and length of treatment have not been determined, although one study has shown no difference between an 8 week and a 2 week regimen (104). The current consensus recommendation is to treat with 40 mg prednisolone daily for 10

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days (1,105). However, many physicians prefer to initiate higher dosages and taper over 9–14 days or longer depending upon the severity of the exacerbation. In patients who have received prolonged treatment with oral corticosteroids, tapering the dosage at the equivalent of 5 mg prednisone per week can be safely done in most cases without adverse consequences, with the aim of avoiding the long-term sequelae of such treatment (106). In the future, we may be able to target specific anti-inflammatory treatments to the key components of the inflammatory network that promotes the progression of COPD. Among candidate targets are leukotriene B4 antagonists, IL-8 antagonists, chemokine antagonists, antioxidants, phosphodiesterase-4 inhibitors, nuclear factor-kappaB and MAP kinase inhibitors, inhibitors of neutrophil elastase, cathepsins, or matrix metalloproteinases (107,108).

V. Infection and the Use of Antimicrobials in COPD Bacterial and viral infections have long been known to cause exacerbations in COPD, but the role of infectious agents in the pathogenesis of COPD is unproven. There is evidence that latent viral infection as well as chronic bacterial colonization can promote inflammation in association with cigarette smoking, providing intriguing theories about why only a small percentage of smokers develop COPD. Most viral lower respiratory tract infections (LRTIs) are caused by RNA viruses such as respiratory syncytial virus, influenza, and parainfluenza (109). However, adenovirus, a DNA virus, accounts for 14% of viral LRTIs and can persist in the host genome by integrating into host DNA or by forming an extrachromosomal plasmid (110). Persistence of adenoviral DNA proteins, specifically the E1A protein, has been shown in airway epithelial cells and type II alveolar cells. The function of E1A protein in adenoviral infection is to produce activation proteins aiding in viral replication (111). In latent infection, the proposed role of E1A protein in promoting inflammation involves amplifying the transcription of host genes expressed when exposed to cigarette smoke, leading to increased inflammation and migration of inflammatory cells (110). In a guinea pig model of adenovirus 5 infection, the expression of E1A protein in airway epithelial cells was associated with increased markers of lung inflammation including increased CD8 cells in the airway wall and increased B cells, macrophages, CD8, and CD4 cells in the lung parenchyma that persisted after viral replication had ceased (112,113). When these same animals were exposed to cigarette smoke, the inflammatory response was enhanced and led to emphysematous lung changes at 13 weeks (114). Expression of E1A protein in human tissue also appears to be associated with increased lung inflammation and increased

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incidence of COPD compared to age- and gender-matched controls with similar smoking histories (115). In addition, a human lung epithelial cell line transfected to express E1A protein has been shown in vitro to produce more IL-8 and ICAM-1 when exposed to cigarette smoke (116). Preliminary data from comparison of resected lung tissue from patients with severe obstructive disease compared to that of patients with mild disease show increased expression of E1A protein (117). This body of data has supported the novel hypothesis that latent adenoviral infection is a component of the susceptibility of some smokers to COPD. Bacterial colonization has also been postulated to have a role in perpetuating inflammation in patients with COPD and thus contributing to the progression of disease. Twenty to 40% of patients with COPD have evidence of bacterial colonization documented by bronchoscopy, with nontypable H. influenzae being the most commonly isolated bacteria (118,119). Cigarette smoking has been identified as the most common predisposing factor to bacterial colonization (118,119). In the so-called vicious circle hypothesis proposed by Sethi and Murphy, inflammation initiated by cigarette smoke or irritants causes impaired lung defenses by impairing mucociliary clearance through both increased production of mucus and decreased ciliary function (120). This allows bacterial overgrowth in the lower airways and further stimulates airway inflammation and injury. However, there is currently no convincing evidence that increased inflammation due to bacterial colonization leads to progressive decline in FEV1 (118,121). It has been suggested, however, that repeated exacerbations from which recovery is incomplete can augment the long-term fall in FEV1 (122). The role of viral and bacterial infections in exacerbations of COPD is more clearly understood. Infectious causes are thought to be the most common precipitants of COPD exacerbations, although no identifiable cause is found in up to one-third of exacerbations (1). Viral infections can be identified in 20–50% of cases when cultures and serological results are obtained (123). Influenza vaccination is recommended for all patients with COPD and can reduce the incidence of serious illness and death due to influenza by 50% (1,124). A recent review by the Cochrane Database concluded that the number of exacerbations occurring more than 3 weeks after vaccination was significantly reduced (125). At present, there are no recommendations for treatment of patients with antiviral therapy for influenza; however, if influenza infection is proven by nasopharyngeal aspirate, or a compatible illness occurs during an influenza outbreak, treatment with neuraminidase inhibitors is reasonable (126). A pneumococcal vaccine containing 23 virulent serotypes is currently available and widely used in patients with COPD. The effectiveness of the

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vaccine in COPD is unknown (1). One meta-analysis found no evidence of benefit in adults at high risk of pneumococcal disease (127), although a subsequent study had contradictory results (128). Some, 20% of healthy elderly adults do not develop an antibody response to the vaccine (129). The side effects of the vaccine are limited primarily to pain at the injection site; therefore, in most patients, the potential for benefit, although unproven, outweighs the risks of the vaccination. The use and choice of antibacterial therapy in patients with exacerbations of COPD continue to be debated in the literature. Clinical guidelines today recommend treatment with antibiotics when patients with an exacerbation of COPD experience increased dyspnea with increased sputum production and purulence (1). This recommendation is based largely on data reported by Anthonisen et al. (130) In this trial, patients were randomized to receive antibiotics or placebo, and severity of exacerbations was graded by whether the patients had the following symptoms: increased dyspnea, increased sputum production, or increased sputum clearance (130). The largest benefit was seen in the patient group with all three of the above symptoms, with 63% reporting significant recovery. Of note, however, even in the group with the highest severity, 43% improved with placebo alone (130). No significant difference was found if the patient had only one of three symptoms. However, in patients with multiple exacerbations, antibiotic therapy decreased the length of exacerbations by 2.2 days (130). In addition, a recent meta-analysis of nine placebo-controlled trials reports a small but significant benefit from antibiotic therapy in duration of illness and change in peak flow (131). Patients with more severe exacerbations are also more likely to benefit from antibiotic therapy (23,130). Appropriate choice and duration of antibiotic therapy remain unclear. The study by Anthonisen used amoxicillin, trimethoprim–sulfamethoxazole, or doxycyline as first-line antibiotic therapy (130). However, this study was performed before there was marked concern about the emergence of resistant bacteria, particularly penicillin-resistant S. pneumoniae. Many experts continue to recommend the above antibiotics as first-line therapy in patients with mild disease or minimal symptoms (23). However, in patients with severe COPD or a history of frequent exacerbations requiring treatment with corticosteroids, there is controversy about which antibiotics should be chosen as first-line therapy. The British, European, and American guidelines suggest no change in antibiotic choice; the Canadian, Latin American, and Asia– Pacific guidelines suggest using antibiotics that are effective against resistant organisms such as fluoroquinolones or beta-lactamase inhibitors (121). The studies of antibiotic use in patients with COPD typically have chosen duration of treatment between 3 and 14 days, but only a single retrospective study addressed the appropriate duration of antibiotic therapy. It found that a

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favorable response was seen in 70% of patients who received 6–10 days of therapy (23). The use of continuous antibiotics in the treatment of COPD is not recommended. To date, there have been nine prospective, placebo-controlled, randomized trials to address whether continuous antibiotic therapy reduces the number of exacerbations in COPD (132). The data have been conflicting, with most studies showing no decrease in the number of exacerbations suffered by individuals with COPD (132). Some studies have suggested that time lost from work can be decreased with continuous antibiotics (121). However, given the appropriate concern over the emergence of resistant strains of bacteria, the potential benefit of continuous antibiotic therapy does not warrant this risk.

VI. Cigarette Smoking and Nicotine Dependence Cigarette smoking is the single most important risk factor for the development of COPD and is also the one most amenable to change. Even among smokers with established obstructive lung disease, smoking cessation will slow the rate of decline in lung function to that of age-matched peers (3). However, cigarette smoking alone is insufficient for the development of COPD: only 10–15% of all smokers and 26% of heavy smokers will develop clinically relevant COPD (3). Other factors that interact with cigarette smoke to increase the likelihood of developing COPD include genetic susceptibility and environmental exposures. Smoking cessation is the only intervention shown to attenuate the rate to decline in lung function. In normal adults, the FEV1 declines an average of 30 ml/year. The Lung Health Study was designed to study the effect of an intense smoking cessation program on quit rates as well as rate of decline in FEV1 in a cohort of patients with mild to moderate COPD over 5 years. Regardless of when during the study a patient quit smoking, the FEV1 showed an average increase of 47 ml in the first year after smoking cessation, followed by a fall in FEV1 thereafter of 32 ml/year (equivalent to that of nonsmokers). Participants who continued to smoke, by contrast, had a rate of decline in FEV1 of 62 ml/year, with intermittent quitters showing a intermediate decline in FEV1 of 47 ml/year. Factors predicting a more precipitous decline in percentage predicted FEV1 include lower FEV1 at baseline, airway hyperreactivity, and age (3) (Fig. 2). Despite what is known about the relationship of cigarette smoking to COPD, cardiovascular disease, and multiple cancers, smoking cessation therapies are largely ineffective with a best long-term quit rate of 20–30% (133). The difficulty in smoking cessation is due primarily to the addictive properties

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Figure 2 Effect of smoking cessation on subsequent decline in FEV1 from the Lung Health Study. The initial cohort comprised 5887 persons with mild to moderate COPD. The figure represents only those individuals who were free of smoking for 5 years (quitters) or those who were smokers (smokers) during 5 years. The smokers are designated by the hatched circles and dotted line. The size of the circle is proportional to the total number of participants in each group. The quitters maintain nearly constant lung function for their age, gender, and height while the continuing smokers show progressive decline. Of the total group, only 22% of those with intensive smoking intervention and only 4% of those with usual care were able to sustain smoking cessation. (Adapted from Ref. 3.)

of nicotine. Nicotine is a freely available and highly addictive drug and causes physical addiction and psychological dependence. Nicotine acts physiologically on both central and peripheral nicotinic receptors to cause nerve depolarization. In the central nervous system (CNS), depolarization of nicotinic receptors on neurons in the nucleus accumbens stimulates release of dopamine, producing euphoria and a sense of arousal (134). Similar actions to increase CNS dopamine levels are seen with other addictive drugs, including opioids and cocaine. Nicotine also has multiple other psychological effects including an antidepressant effect, increased attention, and a possible increase in task performance (135,136). Nicotine also causes peripheral stimulation of sympathetic and parasympathetic ganglia. This leads to increased vascular tone, increased heart rate, and increased bowel motility (134,137). In addition to the euphoria and other psychological effects, nicotine dependence is further reinforced by an intense withdrawal syndrome. Symptoms

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of irritability and anxiety begin within hours of last tobacco use, peaking in 1–2 days (138). Other symptoms include sleep disturbance, restlessness, hunger, weight gain, impaired concentration, and depression (139). Most symptoms resolve after 3–6 weeks, but the craving for tobacco may continue for months. Success in smoking cessation is also limited by personal and social factors. Personal characteristics, such as coping skills, influence an individual smoker’s ability to stop smoking. Smoking is often a conditioned behavior with multiple stimuli occurring throughout daily activities that reinforce the desire to smoke. The presence of other smokers in the household or work environment and lack of social support are common barriers to successful smoking cessation. Finally, recent research has also demonstrated the insidious effect of advertising and media portrayal of tobacco use on smoking behaviors, especially among adolescents (140–142). In the United States today approximately 23% of adults smoke cigarettes (143). Most smokers acknowledge that tobacco use is harmful to their health, with 70% reporting that they wish to stop smoking (144). In addition, more than 66% of smokers visit a physician each year (145), giving physicians a unique opportunity to offer intervention in smoking cessation. Although most smokers do not seek help, 33% will attempt to quit each year, with fewer than 10% reporting success without additional aid through counseling or drug therapy. Most patients require more than one attempt at smoking cessation before achieving long-term abstinence (144). The most effective smoking cessation therapies incorporate both behavioral and pharmacological therapy. Physicians should not underestimate their influence on smoking cessation; simple encouragement and counseling regarding the benefit of smoking cessation in a physician’s office has been shown to improve quit rates (1). Studies show that 1.9% of individuals quit smoking as a direct result of physician advice and sustained this rate at 1 year (146). Therefore, the amount of tobacco use and efforts to quit smoking should be recorded at every visit, leading to the idea of tobacco use as what has been termed the fifth vital sign (145,147). The Agency for Health Care Policy and Research published clinical practice guidelines for treating tobacco dependence in 2000 and recommended a five step intervention (5 As) aimed at helping health care practitioners aid their patients in smoking cessation. (Table 2) (145). Individual or group counseling should also be considered. A review of multiple trials comparing individual counseling, group therapy, or minimal intervention showed an increased likelihood of successful smoking cessation among smokers who underwent individual or group counseling over minimal intervention including self-help materials. There was no difference in quit rates between those undergoing individual or group therapies (148).

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Table 2 The Five As of Smoking Cessation Ask about tobacco use Advise to quit smoking Assess willingness Assist in cessation attempt Arrange follow-up

Substantial evidence exists to support the finding that nicotine replacement therapy in any form increases long-term smoking cessation rates. Nicotine can be given in the form of transdermal patches, intranasal spray, oral lozenges, chewable gum, and as an inhalant. The choice of which form of nicotine replacement depends largely on patient preference: no form of nicotine replacement shows better outcomes than any other (149). The starting dosage should be individualized based on the daily number of cigarettes smoked. The goal of nicotine replacement is to keep blood nicotine concentration at a level that minimizes withdrawal symptoms; use of an immediate release form of nicotine in addition to the transdermal system may help to maintain levels on blood nicotine levels at the appropriate level and thereby increase smoking cessation rates (150–152). Some studies have suggested that higher doses of nicotine or longer duration of treatment may improve smoking cessation rates (145,152). Bupropion is a newly available antidepressant that increases rates of smoking cessation alone or in combination with nicotine replacement therapy. It inhibits neuronal reuptake of dopamine, serotonin, and norepinephrine and is thought to diminish nicotine craving by exerting its effect on dopaminergic neurons. Care should be taken when prescribing bupropion because it decreases the seizure threshold and can worsen eating disorders. When used for 7–12 weeks, smoking cessation rates can be increased from 8 to 21% (153). Adding bupropion to nicotine replacement therapy increases the quit rate compared to use of either therapy alone (154). Second-line oral agents for smoking cessation are nortriptyline and clonidine. In two trials, nortriptyline has been shown to be nearly three times more effective than placebo in long-term smoking cessation (153,155). Therapy should be continued at dosages of 75–100 mg per day for 7–12 weeks. Clonidine, a centrally acting alpha-agonist, can help to attenuate the symptoms of nicotine withdrawal. Given either by the oral route or via transdermal patch, clonidine should be initiated 1 week before the patient’s stop date and continued for up to 10 weeks (156). Its usefulness, however, has been limited by side effects including dry mouth and sedation.

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Alternative therapies have been tried to improve smoking cessation. Acupuncture was first reported to have benefit in treating withdrawal symptoms from opiates in 1973; subsequent trials have looked at acupuncture as an alternative therapy for multiple addictions. The Cochrane Collaboration reviewed 18 trials of acupuncture as a treatment for nicotine dependence. At no time was active acupuncture better than sham acupuncture in improving smoking cessation (157). Hypnotherapy, which is poorly standardized as a treatment, has also been tried by some individuals to improve rates of smoking cessation. A review of published trials showed significant heterogeneity with no clear effectiveness of therapy (158). Other therapies include exercise programs and meditation therapy. While none of these shows any significant increased rate of smoking cessation over conventional therapies, they are also not harmful and could be considered if a patient expresses a specific interest. VII. Summary and Conclusions Despite our ability to recognize COPD early with noninvasive testing (spirometry) and to identify the major risk factor for development of the disease (cigarette smoking), the treatment of COPD remains largely symptomatic. The only treatment that can delay the progression of mild to moderate disease is smoking cessation, and the only treatment that can prolong survival in advanced disease is oxygen therapy. All other treatments are directed toward symptomatic relief and to the prevention and treatment of complications. Improved understanding of the pathogenesis of the disease at the cellular and biochemical level, coupled with well-designed clinical trials, will, we hope, lead to treatments that can truly modify the course of the disease. References 1.

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7 Clinical Approaches for Evaluating Retinoids as a Treatment for Human Emphysema

MICHAEL D. ROTH, JONATHAN G. GOLDIN, and JENNY T. MAO David Geffen School of Medicine at UCLA Los Angeles, California, U.S.A.

I. Introduction Emphysema affects over 2 million Americans and is one of the most serious respiratory complications of cigarette smoking (1,2). The pathogenesis of this disease is complex, but an imbalance between pulmonary protease and antiprotease activity appears to be centrally involved (3,4). Protease-induced tissue destruction leads to the rupture of alveolar septa and the progressive enlargement of terminal airspaces: the pathological hallmark of emphysema (5,6). This tissue disruption directly reduces the surface area of the lung available for gas exchange and reduces inherent tissue elasticity. These anatomical and physiological changes lead over time to hyperinflation of the lungs, airflow obstruction, ventilation/perfusion mismatching, inadequate gas exchange, increased pulmonary vascular resistance, and right heart dysfunction (Fig. 1). The adult lung does not spontaneously regenerate and the destructive effects of emphysema have heretofore been considered progressive and irreversible. Current therapies primarily focus on reducing the rate of injury, reducing complications, or controlling and ameliorating associated symptoms such as cough, sputum, or bronchospasm (7,8). Lung transplantation is 149

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Figure 1 The complex pathophysiology of emphysema. An imbalance between pulmonary protease activity (favoring tissue injury) and antiprotease activity (providing tissue protection), in combination with chronic inflammation, leads to destruction of alveolar septa and a complex network of functional complications that contribute to the clinical manifestations of emphysema.

one exception to this rule, but the tremendous cost, limited supply of organs, and high morbidity and mortality keep this procedure from becoming a viable standard of care (9). Since the initial report by Cooper and associates (10), lung volume reduction surgery has attracted wide attention as a treatment modality. However, questions about its efficacy, safety, durability, cost, and risk/benefit ratio remain to be answered in clinical trials now underway. Patients with advanced and homogeneous lung disease are at a particularly high risk for surgical complications (11). Even when clinically successful, the beneficial effects of lung volume reduction decline at an accelerated rate, with 50% of patients returning to pretreatment lung function by 2–3 years after operation (12). As of this date, no available treatment directly addresses the goal of restoring protease/antiprotease balance or regenerating the structure and function of damaged lung tissue. Given the lack of effective therapy for emphysema at present, innovative research programs are needed to explore new frontiers and therapeutic options for this debilitating disease. In 1997, Massaro and Massaro reported that the administration of all trans-retinoic acid (ATRA) reversed anatomical and physiological consequences of elastase-induced emphysema in a rat model (13). This was the first suggestion that lung injury resulting from emphysema might be reversible. Similar in vivo findings were soon replicated by Belloni and colleagues (14)

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using either ATRA or 9-cis retinoic acid. Retinoids are known to activate genes involved in lung development and promote alveolar septation in the pre- and postnatal period (15–17). In the study by Massaro and Massaro (13), elastase was instilled into the lungs of adult rats to produce changes characteristic of emphysema, including enlarged terminal air spaces with ruptured septa, a decrease in alveolar number, loss of elastic tissue, and resulting hyperinflation of the lungs. After allowing time for the destructive changes to stabilize, a 12 day treatment with ATRA resulted in tissue regeneration with a significant reversal of the anatomical and physiological manifestations of the disease. No spontaneous regeneration was observed in the absence of retinoid therapy. Although findings from these animal models are exciting, caution is warranted when comparing elastase-induced emphysema with the complex pathogenesis of human emphysema (Table 1). Unlike the elastase-treated rat, in which unaffected lung tissue appeared normal, the remaining lung tissue in patients with emphysema is functionally and structurally altered. The tissue shows changes in basement membrane, pulmonary blood flow, epithelial cells, endothelial cells, and inflammatory cells (18,19). Structural and functional changes in the human lung are also heterogeneously distributed, with some areas appearing relatively normal while other areas range from minimally to severely involved with disease (20). This poses a complex challenge in

Table 1 Comparison Between Elastase-Induced Emphysema Model and Human Emphysema

Similarities

Differences

Conditions

Rat elastase model

Human emphysema

Enlarged distal airspaces Reduced alveolar number Hyperinflation Reduced gas exchange Pathogenesis Duration of injury Ongoing injury Tissue alterations Heterogeneity of injury Presence of comorbidity Age Lung developmental period Potential for toxicity

Yes Yes Yes Yes Simple Short No Limited Limited No Young adult 2 weeks Limited

Yes Yes Yes Yes Complex Lifetime Yes Extensive Extensive Frequent Elderly Many years Extensive

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measuring the disease and monitoring its response to therapy. Comorbid conditions are also common in patients with emphysema, including chronic bronchitis and bullous lung disease, as well as precancerous changes, heart disease, and other chronic medical conditions (21). Medications that are tolerated for the short term in animals may produce considerable side effects when used to treat this population of patients for extended periods of time. Interspecies and age-related differences in gene regulation also likely exist, as well as considerable differences in the normal rate of lung maturation and the duration of disease (22,23). Therefore, although animal models provide essential information and a strong motivation to treat patients, understanding the capacity for retinoids to stimulate repair in the mature human lung will require carefully designed and well-controlled human clinical trials. This chapter reviews the rationale for using retinoids to treat human emphysema, describes a strategy for investigating their therapeutic use, and summarizes results from early clinical investigations. II. Using Retinoids to Treat Emphysema Retinoids constitute a group of pleotropic regulatory molecules that mediate their effects by binding to heterodimeric receptor complexes composed of retinoic acid receptors (RAR), retinoid X receptors (RXR), and a variety of other steroidlike receptor proteins including vitamin D, estrogen, and thyroid receptors (24). Under physiologic conditions, retinoids are probably derived from intracellular oxidation of plasma retinol and carptenes (such as h-carotene) absorbed from the gastrointestinal GI tract (25). Intracellular isomerases further convert these compounds into their biologically active forms including 9-cis-, 11-cis-, or 13-cis-retinoid acid (26,27). Each cell type seems to produce its own pool of retinoids, which normally function as what are termed intracrine or paracrine mediators. With respect to the lung, the accumulation of retinoids, and activation of their receptors, leads to transcriptional regulation of genes involved in epithelial cell proliferation, differentiation, and morphogenesis (28,29); elaboration of matrix proteins including collagen and elastin (30,31); production and balance of both proteases and antiproteases (32–34); inflammatory cell activation (35); cytokine and chemokine production (35–37); and secretion of mucus and surfactant (38–40). All of these tissues, cells, and processes are altered in the emphysematous lung, providing a biological rationale for considering retinoids as a potential therapy. Developmental studies, focusing on postnatal lung development, document that both endogenous and exogenous retinoids can regulate these events in a coordinated manner to produce functional alveolar gas-exchange units (17). There is a clear temporal and spatial

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accumulation of retinoids, retinoid-binding proteins, and retinoic acid receptors in the developing lung during the time of maximal alveolar septation (15,16,22). Administration of exogenous retinoic acid to newborn rats promotes development in the lungs of a larger number of smaller, more efficient alveoli and overcomes the suppressive effects that glucocorticoids have on septation and alveolar maturation (17). More recently, the use of specific RAR and/or RXR knockout mice has demonstrated that activation of some retinoid receptors, namely RARh, can inhibit alveolarization, while activation of other receptors, such as RARg, promote it (41,42). Although the coordinated regulation of lung growth by retinoids is well documented for the developing lung, recent work by Massaro and Massaro (13) suggested for the first time that administration of exogenous retinoids can recapitulate these events in the mature, but damaged, lung.

III. Clinical Availability and Experience An important factor in considering retinoids as a treatment for human emphysema is their track record in treating other medical problems. Retinoids have been used to treat a variety of human diseases ranging from dermatological disorders to malignancies. Perhaps the best examples of this are the Food and Drug Administration’s (FDA) approved treatments using 13-cis retinoic acid (cRA) for acne (43,44) and ATRA for acute promyelocytic leukemia (45,46) (Table 2). There is also considerable evidence suggesting that retinoids may have a role as chemotherapeutic or chemopreventive agents for

Table 2 Retinoids Approved for Human Use FDA-approved indications

Recommended dosages

Duration of treatment

13-cis retinoic acid (isotretinoin)

Severe recalcitrant nodular acne

Maximum 15–20 weeks, may be repeated after 2 months

Stable, first-order kinetics

All-trans retinoic acid (tretinoin)

Induction therapy for acute promyelocytic leukemia

0.5–2.0 mg/kg/day in divided doses twice daily 45 mg/m2/day in divided doses twice daily

Maximum 90 days

Induces own metabolism with levels reduced by 2/3 after 1 week

Metabolism

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malignancy. Multiple phase I and phase II trials with both ATRA and cRA have evaluated their efficacy in a variety of malignant or premalignant states, as well as their safety profile and pharmacokinetics (47–54). In general, side effects produced by the clinical use of ATRA and cRA are frequent, but generally mild in degree and manageable (Table 3). The most frequently reported adverse events are similar to those resulting from high dosages of vitamin A (55). Headache occurring several hours after ingestion of the drug is very common (52,56). Mild analgesics generally suffice for control, and tolerance develops with continued use. Pseudotumor cerebri is a very rare complication to be considered in the setting of persistent or very severe headaches. Dry skin, itching, flaking, xerostomia, and cheilitis are also common, occurring in most treated patients. These reactions of skin and mucous membranes can be managed with topical lubricants or adjustments in dosing and rarely require interruption of drug therapy. Nasal stuffiness, clogged ears, and cervical or tonsillar lymphadenopathy have also been encountered. Fatigue, bone pain, and arthralgia may occur in 20–30 % of patients. Like headache, these effects tend to occur early during treatment and remit with continued therapy. Significant hypertriglyceridemia and hypercholesterolemia also occur, with hyperlipidemia being one of the dosage-limiting toxicities (47,53). Adverse consequences of this hyperlipidemia have not yet been described, but using these agent long-term in elderly patients at risk for diabetes and heart disease could increase the clinical importance of this side effect. Transient increases in levels of serum aminotransferases, alkaline phosphatase, and bilirubin have also been recorded in patients using retinoids, but permanent liver damage has not been reported (53). As a group, the retinoids are exceptionally potent teratogens that lead to marked craniofacial and limb deformities (57). Their use must be actively avoided in women likely to become pregnant. There is also concern regarding the administration of retinoids to individuals at risk for lung cancer if they continue to smoke. Several studies have indicated that active smokers treated with retinoids, including hcarotene and cRA, experience a higher incidence of new lung cancers than do placebo-treated controls (50,58). Perhaps the most pronounced toxicity

Table 3 Common Side Effects Associated with cRA and ATRA Therapy . . . . . .

Skin rash, drying, pruritius Xerostomia, cheilitis, mucositis Headache Hypertriglyceridemia Fatigue Bone pain, arthralgia

. . . . . .

Nausea, abdominal pain Liver function abnormalities Hypercholesterolemia Visual disturbances Reports of depression Category D teratogen

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associated with the clinical use of ATRA has been the acute promyelocytic leukemia (APL)–retinoic acid syndrome (59). This potentially life threatening complication results in respiratory insufficiency and acute respiratory distress syndrome (ARDS), and is characterized by fever, dyspnea, weight gain, pulmonary infiltrates, pleural effusion, and/or pericardial effusion. This syndrome has occasionally been accompanied by impaired myocardial contractility, episodic hypotension, and multiorgan failure. Cotreatment with corticosteroids reduces the incidence considerably. The pathogenesis of this complication is specific for acute promyelocytic leukemia and is likely related to toxic degranulation by responding leukemic cells. Although both ATRA and cRA can cause minor leukocytosis or increases in red cell mass in any patient, the APL–retinoic acid syndrome occurs exclusively in patients with leukemia and should not be an issue when used for other indications. Of more potential importance to patients with emphysema is that retinoids have been associated with depression and bronchospasm (60,61). However, the relationship of these complications to drug administration is unclear and their applicability to elderly patients with emphysema is currently unknown. A wealth of knowledge exists regarding the potential complications of retinoids, their pharmacokinetics, and the dosages that can be safely given for sustained periods of time. This information provides a rationale for selecting specific retinoid dosages, for screening patients for risk factors, and monitoring their responses to avoid toxicity.

IV. Evaluating Different Retinoic Acid Derivatives Both 13-cis retinoic acid and ATRA are approved by the FDA for clinical use and are commercially available, which facilitates their evaluation in earlystage trials for emphysema. cRA is a biologically active isomer of ATRA: up to 30% of orally administered cRA spontaneously isomerizes to ATRA (62). There are several reasons to consider it as an alternative agent to ATRA. Although cRA shares many of the biological effects of ATRA, its clinical pharmacology is very different. ATRA is more rapidly cleared from plasma following oral administration, reducing its bioavailability. ATRA also autoinduces its own oxidative catabolism by hepatic P450 enzymes, resulting in progressively declining levels when administered continuously over time (63,64). Plasma concentrations of ATRA decrease by an average of 60– 70% when given continuously for even one week (Table 2). This may be clinically significant when given long-term to patients and may be responsible for what is termed the retinoic acid resistance syndrome associated with clinical relapses in patients treated for acute promyelocytic leukemia (63). This autoinduction of metabolizing enzymes does not occur with cRA, which

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is a potentially important difference. In addition to pharmacokinetic advantages, cRA is also more readily available (marketed as Accutane). Both retinoids are known to exert their effects via retinoid receptors, although they interact with different affinities to the various receptor subtypes (65). It is interesting that pretreatment with cRA was reported to convert an ATRAunresponsive cancer cell line to an ATRA-responsive one by upregulating expression of retinoic acid receptors. Thus, treating with cRA may potentiate the effects of other retinoids, including ATRA (66). These favorable characteristics support the choice of cRA as an alternative retinoid to evaluate for the treatment of emphysema. cRA and ATRA are both considered nonselective retinoids, binding to all three major RAR subtypes: a, h, and g. Studies with selective agonists and antagonists, or in receptor subtype knockout mice, suggest that simultaneous activation of RARh and RARg might limit alveolar formation compared to stimulation of RARg alone (41,42). RARg is also preferentially expressed in skin and lung, while RARh is preferentially expressed in liver, suggesting that receptor-selective ligands might have advantages in limiting toxicity (67). Activation of RXR receptors may also play a role in lung morphogenesis, as demonstrated in both knockout mice (42) and in rodent models evaluating the utility of 9-cis retinoic acid, a RXR-selective retinoid, in promoting alveolar reconstitution in elastase-treated rats (14). As information is gained from in vitro studies now underway and from animal models, and experience is gained from initial clinical investigations with cRA and ATRA, custom-designed retinoids with different patterns of receptor selectivity may become increasingly attractive as potential therapeutic agents. There is also considerable interest in inhaled retinoids for delivering high local drug concentrations while limiting their systemic effects (68–70). Animal models have already demonstrated the potential feasibility of this approach as chemoprevention for lung cancer (68). However, there are still obstacles to be overcome including the ability to deliver medication selectively to alveolar tissue, while limiting exposure in the oropharynx and conducting airways. Bronchospasm, epithelial changes, and mucus hypersecretion are all potential consequences of airway delivery that may pose problems in the treatment of patients with chronic obstructive pulmonary disease (38,60,71). Advances in ultrafine particle preparations, currently being tested for the delivery of inhaled corticosteroids, may overcome some of these concerns. Retinoids are also extremely teratogenic, leading to speculation that bystanders might be exposed to higher than acceptable secondary exposure to inhaled medications. Again, as with receptor-selective retinoids, there is a clear rationale for initial human studies to focus on previously tested compounds with easily controlled routes of delivery. Once potential treatment obstacles

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are identified and benefits defined, there will likely be indications to evaluate alternative preparations and routes of delivery.

V. Early-Phase Clinical Trials: Inclusion and Exclusion Criteria Early-phase studies require a significant focus on safety and tolerability. It is therefore necessary to establish eligibility criteria that will minimize potential adverse events and facilitate completion of clinical trials. At the same time, in order to document a benefit, the extent of disease needs to be accurately measured and reproducibly evaluated over time. Patients’ age, comorbid conditions, disease type and severity, and potential risk for drug toxicity all need to be carefully considered when one is establishing inclusion and exclusion criteria. Symptomatic emphysema is a disease of the elderly, rarely occurring before the age of 50 and with the majority of patients greater than 65 years old (72). Even in individuals with alpha-1-antitrypsin deficiency, the majority of symptomatic patients are still over age 50 (73). Establishing a lower age limit of 45–50 would therefore encompass the majority of eligible patients while reducing the medication-associated risk to individuals of reproductive potential. However, women less than 5 years postmenopausal should be enrolled only after reproductive counseling and implementation of effective birth control. Primary inclusion criteria should focus on validated measures for identifying emphysema (Table 4). In the majority of patients, emphysema coexists to variable degrees with chronic bronchitis, small airways disease, and reactive airways disease (6,7). These concurrent conditions affect symptoms, alter measured pulmonary function, and may lead to therapy-associated side effects, but are not expected to respond to retinoids. Although spirometry is the most widely used tool for diagnosing and measuring chronic obstructive pulmonary disease (COPD) (8), it does not focus on the destruction of alveolar septa and the enlargement of terminal airspaces, which are the hallmarks of emphysema (5). A moderate obstructive pattern on spirometry [forced expiratory volume in 1s (FEV1)<80% with an FEV1/forced vital capacity (FVC) ratio <70] provides an important screening tool, but should be combined with an abnormal lung diffusing capacity (DLCO<75%) in order to improve diagnostic accuracy for emphysema (74). Computer tomography (CT) of the chest has also rapidly emerged as a mechanism for identifying and measuring emphysematous tissue destruction, as well as identifying unsuspected comorbidity such as pulmonary nodules, cancers,

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Table 4 Recommended Inclusion and Exclusion Criteria to Consider for EarlyStage Trials of Retinoids for the Treatment of Emphysema Inclusion criteria Men and women age z45–50 years

Symptoms of dyspnea on exertion Postbronchodilator FEV1 <80% predicted in combination with an FEV1/FVC ratio <70 DLCO <75% predicted

Evidence of emphysema on CT scan

History of tobacco use or alpha-1-antitrypsin deficiency

Exclusion criteria Concurrent pregnancy or women <5 years postmenopausal unwilling to use physician-prescribed birth control Smoking with past 6 months Chronic use of corticosteroids or use within past 2 months Three or more exacerbations of lower respiratory disease in the past year or hospitalization for respiratory disease within past 6 months History of lung volume reduction surgery, lung transplantation, pneumonectomy, giant bullae, bronchiectasis, or other active lung disease Medical conditions that would hinder participation or increase risk for complications including unstable heart disease, liver disease, alcohol abuse, infection, or cancer Pulmonary nodules requiring medical evaluation Hyperlipidemia History of depression or suicide Concurrent use of vitamin A

infections, fibrosis, bronchiectasis, and bullous lung disease (75,76). Combined inclusion criteria using findings from spirometry, DLCO, and chest CT may therefore be optimal for detecting and categorizing patients with emphysema. In the absence of additional information, it is currently impossible to know whether the cause of the emphysema, the duration, or the extent of disease will have an impact on the response to retinoid therapy. As such, early-phase trials should be encouraged to enroll patients with both smokingrelated and alpha-1 antitrypsin-related emphysema, and with a range of severity until more information is known. Several potential exclusion criteria are specific to trials employing retinoids, including smoking status, corticosteroid use, hyperlipidemia, and a history of depression or suicide (Table 4). There are at least two important reasons to exclude active smokers from early-phase testing. First, the rate of

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decline in lung function is directly related to smoking status (77,78). On average, FEV1 declines at a rate of 62 mL/year in active smokers compared to only 31 mL/year in exsmokers, suggesting that continued smoking might negate improvements in lung function resulting from retinoid therapy. In addition, the combination of smoking and retinoid therapy increased the incidence of lung cancer in several large clinical trials, an effect that was not observed in exsmokers (50,58). Until there are proven benefits, these particular risks warrant significant caution in considering the use of retinoids in active smokers. Systemic corticosteroids are frequently administered to patients with COPD, although there is no clear evidence of benefit and some suggestion of long-term adverse consequences (79,80). At the same time, their impact on the developing lung is well-documented, suppressing septation and limiting the positive effects of retinoids on lung maturation in the postnatal period (17). This antagonistic interaction should be avoided. Hyperlipidemia is a dosage-limiting toxicity for both ATRA and cRA (47,53). This effect may be especially important in elderly patients in whom pre-existing hyperlipidemia, hypertension, diabetes, and peripheral and cardiovascular disease are frequent comorbidities (21,56). To reduce potential risk, the detection and treatment of hyperlipidemia should be considered before starting either ATRA or cRA. While the exact interaction between retinoids and depression remains to be defined, it would be prudent to exclude individuals with active depression or a history of severe depressive disorders. VI. Clinical Trial Goals and Outcome Measures In addition to monitoring for toxicity and tolerability, early-phase studies may provide important insight into the capacity of retinoids to reverse symptomatic, physiological, anatomical, and/or biological manifestations of emphysema. Five distinct categories of outcome measures should therefore be considered in designing trials for emphysema: toxicity, quality of life measures, pulmonary function testing, quantitative chest CT analysis, and biological monitoring using samples collected from the blood and/or lung microenvironment. A.

Monitoring Toxicity

Retinoid receptors are expressed in most organs and tissues and are responsible for retinoid-associated side effects involving the central nervous system, skin and mucosal surfaces, lipid metabolism, muscles and bones, gastrointestinal tract, liver, hair, and vision (53,56). The National Cancer Institute Clinical Toxicity Criteria (NCI CTC) provide a standardized approach to monitoring multiorgan toxicity that can be easily adapted for

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retinoid trials (52,56,81,82). The severity of blood test abnormalities, physiological determinations, objective findings, and symptomatic reporting for most organ systems are classified from 1 to 4, allowing rapid assessment and triage. Dosage-limiting toxicity is usually considered when the NCI CTC for a given parameter reaches grade 3. With specific respect to the known toxicity profile of retinoids, slight modifications to the hyperlipidemia and skin toxicity scales have been suggested by some investigators to reduce long-term risk in the setting of comorbidity from underlying conditions or other therapy (47,52,53). Whenever retinoids are administered for more than a few weeks, the FDA also mandates that depression be carefully monitored as a toxicity measure. Short depression inventories such as the Beck Depression Inventory or the Center for Epidemiological Studies Depression Scale have been previously used to identify depression in patients with COPD, providing simple and validated tools for this purpose (83,84). B. Impact on Quality of Life

An important goal for any therapy is to improve the symptoms and well-being of patients, outcomes which do not always correlate with changes in tissue structure or lung function. For example, pulmonary rehabilitation has no disease-modifying impact on lung physiology yet produces significant improvements in functional capacity and perceived quality of life (85). At the other end of the spectrum, lung volume reduction surgery produces temporary changes in hyperinflation, elastic recoil, and airflow obstruction that also correspond to improvements in quality of life (12,86,87). Both general health-related and disease-specific quality-of-life measures provide valuable outcomes in patients with COPD (88). General instruments have the advantage of monitoring overall well-being and the integrated impact of the disease and its treatment on life in general, but may be less responsive to changes in respiratory function or symptoms. Disease-specific instruments measure dimensions such as dyspnea and cough and tend to be more directly relevant to the pathophysiology of emphysema and to disease-modifying interventions (89). The Medical Outcomes Study Short Form-36 (SF-36) has been extensively studied as a general health instrument and validated for patients with COPD (90,91). It contains only 36 self-administered questions and provides feedback on 9 domains including physical functioning, social functioning, role limitations due to physical or emotional problems, a mental health index, a pain index, and measures of vitality, general health perception, and health transition. The St. George’s Respiratory Questionnaire is a disease-specific questionnaire that has also been extensively studied and validated for use in patients with COPD and emphysema (92). It is also self-completed and provides information in four different domains including a total score, symptoms score, activity score, and disease impact score. The

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Chronic Respiratory Disease Questionnaire is another disease-specific instrument that is well validated for this population of patients (89,93). C.

Monitoring Physiological Responses

Global physiological responses can be measured by serial pulmonary function tests (PFTs) including spirometry, lung volume by body plethysmography, pressure–volume curves, diffusing capacity, maximal inspiratory and expiratory pressures, and arterial blood gases (7,94). These tests provide a state-ofthe-art evaluation of resting whole lung function. Classic emphysema is defined as the destruction of alveolar walls leading to irreversible dilation of air spaces distal to the terminal bronchioles. Physiological consequences include a reduction in the alveolar–capillary surface area available for gas exchange and an increase in lung compliance due to destruction of lung elastic tissue and collagen. Expiratory airflow obstruction develops secondary to the decrease in lung elastic recoil and a reduction in peribronchial lung recoil, promoting increased collapsibility of the airway due to loss of tethering by surrounding lung parenchyma. These changes lead to hyperinflation and air trapping, resulting in high lung volumes and impairment of diaphragm and respiratory muscle functions (5,95). In the context of these physiological derangements, PFTs should detect the effects of retinoids on tissue remodeling and restoration of the elastic properties. Spirometry allows for quantitative measurements of airflow obstruction; lung volumes (by whole-body plethysmography) provide evidence for the degree of hyperinflation and air trapping; diffusing capacity measurements reflect the presence/loss of alveolar–capillary surface area available for gas exchange; resting blood gases directly measure the efficiency of gas transfer; maximal inspiratory and expiratory pressures monitor respiratory muscle strength; and static pressure–volume curves reflect lung elasticity and recoil (compliance). Although PFTs directly address the pathophysiology of this disease, there are limitations in their use for monitoring subtle changes in lung function over time. Their reproducibility is complicated by variability in patient effort, technique, bronchospasm, anemia, and other factors (7,94). As much as a 10–20% change in FEV1, total lung capacity (TLC), or DLCO might be required before improvement can be definitively documented (96,97). Serial tests should be performed on the same equipment and with the same technician to reduce variability. In addition, emphysema tends to be heterogeneous in its distribution and severity. Each PFT measurement provides a single global measure of entire lung function that may be insensitive to regional changes in tissue structure or function. With an average decline in FEV1 of only 30 ml/year in exsmokers (74,77), a large clinical trial would be required to confirm modest improvements in PFT measures of lung function over short periods of time. These considerations support the use of

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alternative outcome measures to evaluate responsiveness in early-phase trials until conditions are established that will result in significant changes in overall pulmonary physiology. D. Radiographic Imaging

Lung biopsy is the gold standard for evaluating the extent and severity of emphysema (6). However, the invasive nature of this procedure and the heterogeneity of lung disease make serial biopsies both impractical and inherently unreliable. In its place, high-resolution chest CT (HRCT) has become an anatomical surrogate that allows repetitive evaluation of tissue structure with a resolution in the range of 200–300 Am (98–100). Dynamic imaging, in which serial images are rapidly acquired at the same location during a single respiratory cycle, provides information about structure– function relationships that can be more informative than spirometry (101– 104). Computer-driven quantitative image analysis (QIA) allows even greater discrimination than can be determined by visual impression alone. One of the key features of CT imaging is its ability to evaluate lung structure at the regional level. Emphysema is a heterogeneous process with areas of mild, moderate, and severe disease often coexisting in the same patient. CT allows each site to be individually evaluated, reducing the likelihood that subtle changes at one site will be masked by a lack of response at another. When it comes to evaluating the impact of retinoids, CT imaging may provide one of the most sensitive and valuable measures of drug response. This section will review quantitative CT techniques available for assessing emphysema and their potential application to clinical trials. Imaging Protocols

The best imaging protocol for acquiring CT data in patients with emphysema remains to be determined. One routinely chooses between two different approaches to image acquisition sampling the lungs with high-resolution thin sections at selected intervals (HRCT) or a volume acquisition technique to acquire a three-dimensional CT data set for the entire lung during a single breathhold (spiral or multidetector CT). In the past, thick sections (5–10 mm) were necessary to ensure a contiguous acquisition of the entire lung in the same breathhold. As a consequence, subtle abnormalities in lung texture and density due to emphysema or air trapping were difficult to detect. For this reason, thin-section sampling at intervals through the lung was chosen to allow detection of greater lung detail. However, as multidetector and subsecond scanners begin to replace earlier equipment, it will be possible to obtain full-volume datasets in a single breathhold, essentially allowing HRCT of the entire lung.

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Standardization of the lung volume to either TLC, residual volume (RV), or another predetermined volume at the time of acquisition is critical to ensure both the validity and reproducibility of quantitative measures. This is most accurately achieved with spirometric gating, in which the CT scan is triggered, and airflow mechanically inhibited, at a predetermined userselected level of breathhold (105). However, commercial apparatus for spirometric gating is currently scarce and, thus, most quantitative studies are performed without such methods. In this setting, it is possible to obtain reproducible lung volumes by using a simple incentive spirometer to guide breathhold volumes for the patient. In addition to volume-triggered scanning, the ability to acquire or reconstruct images at subsecond intervals can be used to provide real-time sequences of the lung during the respiratory cycle (103,106). Images of 1–3 mm collimation, obtained at 300–500 ms intervals, can also be acquired as sequences that are triggered or simultaneously monitored by spirometry. For most applications, sequences acquired through the upper, mid, and lower lungs provide an adequate sample of the lungs. By integrating the spirometric and imaging data, changes in lung attenuation for isolated regions of interest can be measured as functions of time, airflow, and lung volume. This yields information about regional lung function, airway disease, and parenchymal features that cannot be determined by spirometry alone (Fig. 2). This

Figure 2 Spirometry-gated CT combined with computerized image analysis detects regional changes in lung function. Serial HRCT scans obtained at the same lung level (A) were obtained at up to 500 ms intervals in a patient with emphysema with simultaneous spirometric gating. The right and left lungs were identified as individual regions of interest and the distribution of tissue attenuation readings in each ROI measured (B). The patient was then coached to perform a maximal forced expiratory maneuver starting from TLC. The average attenuation value in each lung plotted over time as measured on serial HRCT scans (C). Significant differences in the rate of tissue attenuation over time can be detected when the right and left lungs are compared, despite the absence of obvious visual differences.

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approach has been used to monitor independent lung function in patients following single-lung transplantation and to detect bronchodilator responses at the regional level when standard pulmonary function fails to identify response for whole-lung function (103,107). Quantitative Image Analysis

The quantitative information obtained with HRCT may advance our understanding of pulmonary pathophysiology and offer insights into the possible mechanisms involved in the progression of emphysema or its response to treatment. There has been an explosion in investigations to provide quantitative structural and functional information from digitally acquired image data. These methods include visual quantitation scoring systems (108), image display (such as multiplanar reformations, surface shading for three-dimensional and volume rendering) (109–111), anatomical image quantitation (e.g., area and volume of airways and lungs) (101,103,112–114), and regional characterization of lung tissue (analyzing attenuation, changes in attenuation and texture patterns in the imaged lung) (75,103). Visual evaluations utilize scoring systems that categorize different parenchymal patterns and attempt to quantitate their extent and severity by assessing the amount of lung involved. In designing a visual scoring system it is important to score separately the extent of different CT patterns since these different patterns most likely have different functional effects. Several visual scoring approaches have been developed for grading emphysema severity (20,108). In most studies, a final score is calculated either by agreement between two reviewers at a joint reviewing session or by obtaining the mean of the reading scores for the two reviewers. For the most part, they are relatively subjective and lack reproducibility with larger inter- and intrareader variation. Thus, attention has turned to computer-aided detection and quantitative techniques. For computer-aided techniques, several approaches and software packages have been developed. These packages consist of varying sets of image segmentation and analysis tools written to answer specific thoracic clinical and research questions (110, 115–117). For the most part, the segmentation process is fully automated but allows manual correction if needed. Three basic types of measurements are made on a segmented region of interest (ROI). The first category includes measures relating to the size and shape of the ROI. Lung tissue can be accurately differentiated from airways, blood vessels, and other structures, allowing lung volumes to be accurately calculated in patients with emphysema (Fig. 3) (118,119). The second category relates to attenuation (gray level) statistics and the third to image texture within the ROI. Much of the lung consists of

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Figure 3 Total lung volumes were determined in a cohort of 20 patients with moderate to severe emphysema by performing QIA on CT scans obtained at TLC and compared to lung volumes as measured by plethysmyography. This yielded an excellent correlation and reproducibility on repeated measurements.

intrathoracic gas. Changes in attenuation of the x-ray can be used to assess changes in the relative proportion of gas. An increase in blood volume, fluid content (cellular or interstitial), or parenchymal tissue volume results in increased attenuation of pixels relative to air. The greater the leftward shift of what is termed the lung attenuation curve (e.g., reflecting a greater proportion of pixels with low attenuation), the more extensive the lung destruction due to emphysema and/or expiratory airflow obstruction. Conversely, a rightward shift of the curve (to a greater proportion of high attenuation) reflects an increase in blood flow or in the interstitial, cellular, or fluid content of the lung parenchyma. In addition, texture measurements take into account the pattern of local attenuation differences between adjacent pixels, including both the distance and direction of these differences, to allow computerized pattern recognition of emphysematous vs. nonemphysematous tissue (75,117,120). Taken collectively, these three categories of computerized CT data analysis have been applied to identify normal and severely emphysematous patients on either a global basis (entire lung section) or regional basis (75); healthy nonsmokers, smokers, and smokers with abnormal lung function (COPD) on either a global or regional basis (117); airway reactivity and treatment response (103); and other patient populations such as those with interstitial lung disease [i.e., idiopathic pulmonary fibrosis (IPF) and sarcoidosis, asbestosis, and cystic fibrosis] (120,121). QIA can also be used to

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measure responses to therapy due to their ability to detect subtle regional changes not measurable by conventional lung function tests (103). Applying QIA to Evaluate and Monitor Emphysema

CT images obtained in patients with emphysema have been studied extensively using QIA techniques. In 1988, Muller and colleagues correlated CT image data with pathological results from surgically resected lung specimens using the density mask program commercially available on CT scanners (122). CT scans obtained at total lung capacity using 10-mm collimation (thick section) contiguous scanning techniques were obtained of the entire thorax in that study. The density mask program calculated the amount of emphysematous lung based on the finding that normal lung had an attenuation greater than 910 Hounsfield units (HU) and emphysematous lung had an attenuation less than 910 HU. The same density mask approach was used by Kinsella and co-workers (123) to compare CT images with pulmonary function tests. There was a good correlation between mean lung density and the volume of emphysema present, as well as CT-determined total lung volumes and measures of percent predicted for FEV1, FEV1/FVC, FRC, RV, and TLC. A good correlation was also demonstrated between mean lung density and volume of emphysema with measures of % DLCO and % DLCO/VA. An important feature of density mask analysis is that regional variations can be captured and independently evaluated. In a recent study, Goldin and co-workers evaluated a group of 20 patients with moderate to severe emphysema and performed regional density mask QIA (56). As presented in Figure 4, average density mask values for the whole lung demonstrated a normal distribution of diseased tissue. However, when upper lung images were compared to lower lung images, more severe disease was observed in the upper lung zones, consistent with the known pattern for tobacco-associated emphysema. When regional analysis is used to study changes in lung attenuation before and after retinoid treatment, it is likely that it will provide the most information about lung remodeling. Regional analysis is one of the clear advantages of CT analysis over standard PFTs. In addition to QIA performed at total lung capacity, analysis at end expiration may provide additional or perhaps more sensitive information. Knudson and colleagues (124) reported that expiratory scans were superior to inspiratory CT scans in quantitating the amount of emphysema. In that study, normal lung attenuation was assumed to be 600 to 900 HU and emphysematous lung was assumed to have an attenuation greater than 900 HU. The amount of emphysema present on the expiratory/inspiratory scans correlated with the measures of pulmonary function including % FEV1, % FEV1/FVC, % RV/TLC, % DLCO, and % DLCO/VA, with better

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Figure 4 A cohort of 20 subjects with moderate to severe emphysema underwent baseline density mask analysis to determine the distribution of lung tissue with minimal (<10%) to severe (up to 70%) involvement by emphysema as determined by the percentage of voxels < 910 HU. Although integrated measurements for the entire lung suggested a uniform distribution of disease, QIA at the regional level demonstrated a higher frequency of severe disease in the upper lung fields than the lower lung fields. This heterogeneity of disease is consistent with the known pathophysiology of smoking-related emphysema.

correlation to the expiratory scan data. The expiratory scans also correlated significantly with a measure of elastic recoil of lung (k parameter) while the inspiratory scan data did not. Other investigators have reported similar results including a good correlation with physiological measures of lung function, including maximum workload achieved during exercise and maximum oxygen utilization (125,126). Results from QIA assessments have successfully identified patterns of emphysema that correlate with improved outcomes following lung volume reduction surgery. Gierada and associates (127) compared quantitative CT scan analysis, preoperative physiological assessment, and outcome measures in 46 patients who underwent lung volume reduction surgery for treatment of severe emphysema. The postoperative outcome was better when the mean preoperative lung attenuation was greater than 900 HU. It was also better when 75% or more of upper lung fell below 900 HU (emphysema index) and when more than 25% of the lung fell below 960 HU (surveyor emphysema index). Postoperative outcome was better when the ratio of the upper to lower lung emphysema indices was 1.5 or higher. They also noted a better outcome in patients who had more than 1L normal lung (defined as lung with an attenuation between 850 and 701 HU). There were often two- to threefold differences in outcome measures between groups that were stratified using quantitative CT values. In contrast to the results from QIA, which relied heavily on regional analysis, there were few correlations between preoperative PFT values and outcome measures.

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In addition to measuring structural damage at static lung volumes, analysis of the rate of change in HU during an FVC maneuver can give additional functional information. The plot of HU/time curves for normal lungs demonstrates that maximum lung attenuation is achieved within the first 2–4 s forced expiration. In patients with obstructed airflow, such as severe emphysema, airflow is considerably decreased and there is minimal change in lung attenuation over time, resulting in marked flattening of the HU/time curve. Despite similar structural damage as identified by visual analysis, HU/ time and lung attenuation curves can show marked differences in airflow and airtrapping that provide further insights into the pathophysiology of emphysema (Fig. 2). The application of these CT data analysis techniques holds great promise for identifying and quantitating regional changes in lung structure and function over time. The feasibility of using these approaches to follow patients with emphysema during treatments was recently demonstrated in alpha-1 antitrypsin-treated patients undergoing replacement therapy and in a preliminary study in which patients with emphysema received ATRA (56,128). E.

Cellular and Molecular Biomarkers of Lung Remodeling

The pathophysiology of emphysema is complex and develops over decades of tobacco exposure. There is currently no information on the rate at which human lung tissue might regenerate or the length, or dosage, of medication that might be required before functional changes can be observed. It is reasonable to speculate that a year or more of continuous therapy might be required. Some of the methods used to evaluate outcome, such as PFTs, provide global assessment of function that may not be sensitive enough to detect small responses to treatment, or significant responses that are limited to certain microenvironments. This notion is supported by studies of the airway and alveolar microenvironment in young smokers of marijuana and tobacco (129–133). In these studies, bronchoscopy was used to examine the lungs of smokers who had entirely normal pulmonary function. However, the samples recovered from their lungs demonstrated a marked inflammatory cell infiltrate (133), alterations in oxidative burst and antimicrobial properties of alveolar macrophages (131,132), altered cytokine levels in the airways and from alveolar macrophages (129,131), and selective defects in nitric oxide production by cells recovered from marijuana and cocaine users but not from tobacco smokers (131). These observations from the lung microenvironment correlate with the known progression of emphysema and chronic bronchitis in tobacco smokers and with the development of bronchitis and pulmonary infections, but not emphysema, in marijuana smokers (134). At the molecular

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level, we observed a very high incidence of molecular dysregulation in the airway epithelium of tobacco and marijuana smokers, including overexpression of epidermal growth factor receptor and upregulation of the Ki-67 nuclear proliferative antigen (130). It was quite striking that the bronchi of some smokers were intensely inflamed, even to the point of near obstruction (129). Despite these striking changes, subjects reported few symptoms and had essentially normal lung function as measured by standard spirometry and DLCO. In the same manner, it is very possible that the response to retinoids will be initially compartmentalized so that it does not register on standard measurements of global pulmonary function. Use of HRCT and dynamic CT imaging may circumvent part of this problem by allowing assessment and comparison of regional variations in lung tissue and dynamics that are missed by clinical studies. Monitoring for biological markers of lung remodeling should likewise allow detection of retinoid-induced changes in the lung microenvironment that predate the onset of clinical changes. The formation of functional lung tissue is a complex process. A therapeutic agent must be able to activate molecular and cellular mechanisms and reconstitute the lung parenchyma in a coordinated fashion. Experimental data suggest that retinoids may be capable of orchestrating this intricate process. Retinoids are capable of acting on the alveolar epithelium, the endothelium, inflammatory cells, and the extracellular matrix. Many of the retinoid-related effects on these targets, including the induction of RAR expression, changes in collagen production and secretion, increases in tropoelastin and elastin tissue content, upregulation of surfactant proteins, and regulation of MMP, are directly related to the process of lung remodeling and alveolar regrowth. Based on current knowledge regarding the pathogenesis of emphysema, it should be possible to evaluate the in vivo effects of retinoids on lung remodeling by obtaining specimens from bronchoalveolar lavage (BAL), peripheral bronchiolar brushing (PBB), and possibly from blood. RAR Receptor Expression as Indicator of Early Lung Activation

Retinoids participate in the process of tracheal and bronchopulmonary tree formation, postnatal growth of the lung, and repair after injury. The tight regulation of RAR, which are increased in the early postnatal lung but downregulated in the adult lung, supports this hypothesis (22,135). Coordinated activation of RARh and RARg has been associated with regulation of tropoelastin gene expression, lung branching, septation, and alveolar formation in neonatal rats (13,41,42). Exogenous administration of retinoids can regulate RAR expression via activation of RARE sites in their respective promoters. Therefore, measuring changes in the expression of genes encoding for the various RAR receptors (RARa, RARh, RARg) in alveolar macro-

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phages and bronchoalveolar epithelial cells recovered at bronchoscopy should provide a useful index of retinoid-induced activation in lung samples from treated patients. Evaluation of Turnover of Lung Extracellular Matrix

Elastin, initially secreted as tropoelastin, is one of the major components of the extracellular matrix. Under the influence of extracellular and intracellular factors, tropoelastin molecules are synthesized and deposited into the extracellular space and aligned on a so-called scaffold of microfibrils consisting of a number of proteins, including latent TGF-h-binding proteins. In the extracellular space, lysyl oxidase cross-links tropoelastin monomers to form elastin. The lysine-derived cross-links are known as desmosine and are unique to elastin. Desmosines can be measured to quantify elastin in tissue and used as markers of elastin degradation in body fluids. Under normal circumstances, elastin synthesis in the lung begins in the late fetal period, peaks during early postnatal development, continues to a much lesser degree through adolescence (paralleling lung growth), and stops in adult life. Elastic fibers in the lung normally last a lifetime (136) and there is virtually no synthesis in the normal adult lung. If retinoids are capable of regenerating lung tissue in humans, one would expect increased production of elastin or its major building block tropoelastin, and changes in levels of desmosine or elastin fragments. Since the 1960s, elastases and elastin destruction have dominated much of our understanding of the development of emphysema. Over the past decade, however, as our knowledge about the pathogenesis of emphysema expands, it has become increasingly clear that alveolar septal collagen destruction and aberrant collagen repair also contribute to pathogenesis. Not much is known, however, about the turnover of these extracellular matrix components in human lungs affected by COPD. Theoretically, proteases such as MMPs secreted by inflammatory cells in the lung (neutrophils and macrophages) contribute to the break down of collagen in the extracellular matrix. Supporting this hypothesis is the fact that cigarette smoking leads to neutrophil retention in the pulmonary microcirculation and deposition in the lung parenchyma, and causes activation and marked accumulation of alveolar macrophages, combined with the recent observation that increased number of alveolar macrophages correlates with the severity of alveolar destruction. Previous studies demonstrate that activated alveolar macrophages produce several matrix MMPs that are capable of degrading the alveolar matrix. Macrophages derived from patients with emphysema express interstitial collagenase (MMP-1), gelatinase B (MMP-9), and macrophage metalloelastase (MME). Furthermore, numerous in vitro and in vivo studies

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have demonstrated that retinoids can regulate the expression of MMPs. Inhibition of the MMPs is thought to occur, at least in part, at the level of gene transcription by suppression of the AP-1 transcription site. As in the case with elastin, if retinoids are effective at stimulating new lung growth, one may expect to see modulation of the MMP as well as collagen synthesis and degradation. Evaluation of Airway Epithelium

Several lines of evidence indicate that retinoids may also modulate gene expression in airway epithelial cells and type II cells, which are responsible for the production of surfactant. Surfactant (Sp) lowers the surface tension at the air–tissue interface and is critical to the proper functioning of the alveoli. Although there are no clinical data on the level of these proteins in patients with emphysema, they are likely to be reduced, due to the decrease in alveolar surface area and loss of type II pneumocytes. Rats exposed to cigarette smoke were found to develop Sp-B deficiency. In addition, retinoids have been reported to activate surfactant protein B gene transcription in respiratory epithelial cells (40). Retinoids have also been shown to induce proliferation of lung alveolar epithelial cells, associated with a decrease in the expression of the insulin-like growth factor (IGF) system (28). Surfactants (Sp-B, Sp-C) are among the most readily detected protein in BAL. Hence, increase in Sp-B and /or Sp-C in BAL fluid from retinoid-treated patients may represent the effect of retinoids on the surfactant promoters and/or their effects on regenerating alveoli and increasing the number of type II cells. In summary, analyzing biological samples recovered from the lung environment provides a unique opportunity to perform biological monitoring in patients treated with retinoids. Not only will we learn more about the pathogenesis of emphysema through this approach, and about the role of retinoids in its repair, but we will also have an extremely sensitive tool for evaluating early clinical responses that might otherwise be missed. VII. Initial Clinical Trials The striking results obtained with ATRA in animal models (13,14), combined with the availability of ATRA for human use, provided the basis for proceeding with initial human clinical studies. In November, 1998, a phase II, placebo-controlled study to determine the feasibility of ATRA as a medical treatment for advanced emphysema was initiated (56). Primary objectives for the University of California at Los Angeles (UCLA) ATRA Emphysema Study were to determine safety in elderly patients with moderate to severe emphysema; evaluate the stability of drug levels using an intermittent dosing

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regimen; and ascertain if 3 months of ATRA therapy improved physiological, anatomical, and/or symptomatic changes as measured by pulmonary function test, chest CT scans, and Quality of Life questionnaires. After only limited advertising, thousands of phone calls were received from interested patients and 20 individuals with moderate to severe emphysema were ultimately enrolled (Fig. 5). Participants included 16 male and 4 female former smokers, mean age of 66, average smoking history of 75 packyears (pky), and two with alpha-1 antitrypsin deficiency. The average FEV1 was 1.24 L, FEV1/FVC ratio was 31%, TLC was 7.95 L, RV was 3.86 L, DLCOcorr was 11.2 ml/min/mmHg. Patients were treated according to a double-blind, placebo-controlled design with either 3 months of ATRA (50 mg/m2/day) or 3 months of placebo, followed by a 3 month crossover phase. Plasma drug levels were evaluated and outcome measures included serial pulmonary function tests, blood gases, lung compliance, CT imaging, and quality of life questionnaires.

Figure 5

UCLA ATRA Emphysema Study: screening and enrollment flow diagram.

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There were several reasons for choosing a crossover design. First, all patients received treatment, an approach that maximized treatment sample size and facilitated subject recruitment. Most patients with emphysema realize that they have a progressive and currently irreversible disease and are highly motivated to try experimental therapies. Second, the serial assessment of endpoints during both the placebo and active treatment phases in a blinded manner allowed us to examine outcome measures for reproducibility, stability, and therapy-associated changes. In other words, the subjects acted as their own statistical control, thereby eliminating the problems with intersubject variability. In addition, the study arm that received ATRA first also provided a mechanism to evaluate patients for any residual benefit or risk that may occur after cessation of active treatment. The 3-month treatment course was chosen based on past experience with administration of ATRA, the expected stability of patient population over this time period, and experience with using pulmonary physiology and CT measurements to monitor changes in lung function over time (as in patients with pulmonary fibrosis undergoing treatment). The reason for choosing the orally administered, 50 mg/m2/day dosage of ATRA stems from the fact that this is a clinically accepted dosage for the treatment of APL. At this dosage, ATRA is capable of promoting the differentiation/maturation of leukemic cells while its toxicity is limited. In addition, the dosage is quite close to that used in the rat elastase model (f500 Ag/kg/day via intraperitoneal injection). Based on existing knowledge of the pharmacokinetics of ATRA, an intermittent dosing regimen (only 4 days of drug/week) was selected to minimize the problem of autoinduction of its own metabolism when given on a continuous daily basis. Drug levels were monitored throughout the study to provide feedback regarding this administration strategy. In general, treatment with ATRA at 50 mg/m2/day was well tolerated and was associated with frequent but mild side effects including skin changes, transient headache, hyperlipidemia, transaminitis, and musculoskeletal pains (56). Pretreatment of hyperlipidemia, a common baseline comorbidity in these patients, might have reduced the incidence of this dosage-limiting toxicity and could be used as a strategy to allow higher dosages of ATRA, or longer treatment, to be considered in the future. Plasma drug levels varied considerably between subjects and decreased significantly over time in 35% of the participants despite the use of an intermittent dosing schedule. Physiological and CT density mask values did not change appreciably in response to this brief (3 month) therapy (56). However, there was a trend suggesting improvement in the Quality of Life questionnaire (SGRQ) scores. Six of the 10 subjects who received ATRA first, and were followed for an additional 3 months after treatment, reported significant improvements in both total score

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subscores evaluated by the SGRQ (Fig. 6). We conclude that ATRA is well tolerated in patients with emphysema, and trials evaluating higher doses, longer treatment, or different dosing schedules are feasible. Although this initial pilot study failed to demonstrate definite efficacy of ATRA as a therapeutic agent for emphysema, the promising results from animal models, the improvement in symptoms reported by our patients, and the demonstrated feasibility of human clinical investigations in this field provide important impetus for moving forward. In this respect, the National Heart, Lung and Blood Institute/National Institutes of Health is sponsoring a multicenter pilot study entitled Feasibility of Retinoid Therapy for Emphysema (FORTE). The FORTE study will address many of the issues raised by this preliminary trial and was designed to include a broader range of outcome measures, such as biological monitoring and regional CT analysis. FORTE is a multicenter, randomized, double-blind, placebo-controlled clinical trial, enrolling nonsmoking persons with emphysema at five participating clinical centers (Boston University, Columbia University, University of California at Los Angeles, University of California at San Diego, and the University of Pittsburgh). Subject accrual began in January, 2000, and ended

Figure 6 A disease-specific quality of life survey suggested delayed improvement in response to treatment with ATRA. Participants were administered the St. George’s Respiratory Questionnaire (SGRQ) at enrollment and at 3-month intervals corresponding to the 3 month treatment phases with either ATRA followed by placebo (left Figure) or placebo followed by ATRA (right Figure). Answers to the questionnaire were transformed into scores for symptoms (.), activity (x), impact (n) and total score (E), with a score of 100 designating poor health and a score of 0 denoting excellent health. Values represent averages F SEM with 10 subjects in each group. By the end of the study, 6 of 10 patients treated with ATRA first and then placebo were scored as improved according to total score (average improvement 18 points, range 6.3–49 points). Only 2 of 10 participants receiving placebo first and then ATRA had improved findings (average improvement, 4.5 points).

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in July, 2002. Study participants were randomly assigned to one of three retinoid treatment arms (high-dosage ATRA: 2 mg/kg/day; low-dosage ATRA: 1/mg/kg/day; cRA: 1 mg/kg/day). Within each study arm, participants were randomized to receive active treatment or to a matched placebo. After 6 months, subjects were switched to a 3 month crossover period in which those initially assigned to active treatment would begin taking the matched placebo, and those originally assigned placebo would begin taking the active treatment. There were several reasons for this rather intricate FORTE study design. First, preliminary experience with ATRA suggested that 3 months of therapy was well tolerated but insufficient to reverse the full effects of emphysema. This provided a rationale for extending the length of treatment to 6 months, as well as for evaluating a higher (double) dosage of ATRA. Second, the pharmacokinetic instability of ATRA, despite an intermittent dosing regimen, prompted the addition of cRA as a treatment arm. Third, the 6 vs. 3 months crossover treatment interval, in addition to the aforementioned advantages of a crossover design, provided the opportunity to compare 3 month and 6 month treatment outcomes. Lastly, the design allowed for a primary 6 month endpoint according to a standard placebo-controlled design. Serial measurements of PFT, CT, quality of life determinants, and retinoid-induced biological changes are being monitored at different time points. A major component of FORTE is in the biological monitoring of treatment responses to retinoids. FORTE was uniquely designed to allow establishment of a biological repository with serial procurements of blood samples (buffy coat and plasma), and bronchoscopy specimens (BAL, PBB, and alveolar macrophage RNA) from participants during the course of treatment. If retinoids are indeed capable of treating emphysema, then favorable alterations of relevant biomarkers known to be involved in the pathogenesis of emphysema should be evident. Although this is a complex disease, even if retinoids are only capable of altering the pathogenic forces in the lung microenvironment and preventing the lungs from further deterioration, retinoid therapy may represent a significant new approach to the management of emphysema.

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8 Detecting Differentially Expressed Genes by Differential Display

YONG-JIG CHO and PENG LIANG Vanderbilt University Nashville, Tennessee, U.S.A.

I. Introduction Of the estimated 30,000–50,000 genes encoded by the human genome, only 10–15% are expressed as proteins by the average cell in bodies. The choice of which subset of genes to be activated is a major determinant of a cell’s properties. In addition to the abnormal expression of genes in pathological processes, the differentially expressed gene (either by induction or repression) controls the normal developmental process of organisms, such as lung development. Two-dimensional protein electrophoresis, which was developed in late 1970s, is the first method developed for comparative studies of gene products and cellular protein species (1). Owing to the problems of sensitivity and difficulty in recovery of protein species of interest for further molecular analysis, this method gave way to the newer methodologies developed in the early 1980s. These methods included differential screening and substractive hybridization, which focused on analysis of the gene messenger, mRNA. This can be copied into cDNA and then cloned into plasmids or bacterial phage vectors for propagation and analysis (2). 185

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Since the advent of polymerase chain reaction PCR, differential display (DD) methodology was invented in 1992 (3). Since then, this method has been widely used to identify differentially expressed genes, because of its simplicity, sensitivity, and ability to compare multiple mRNA samples simultaneously for the identification of both up- and downregulated genes without prior knowledge of their sequences (4). Recently, fluorescent differential display (FDD) was optimized to have a similar sensitivity, with the aim of enhancing the efficiency of isotope-labeled differential display (5). The principle of this method is very similar to the conventional radioactive differential display. Combined with robotics and digital data analysis, the automated fluorescent differential display was shown to be accurate and have high throughput (6). Computer programs were developed to allow positive band identification automatically from a fluorescent DD image (7,8). Differential display has been used successfully in a wide range of biological systems from lower eukaryotes, such as yeast, to high eukaryotes such as flies, frog, fish, plants, and higher mammals. This chapter focuses on the principle and practice of differential display, and introduces the fluorescent differential display.

II. Principles of Differential Display Differential display amplifies systemically messenger RNA 3V termini using a pair of specially designed primers (Fig. 1) (9). First, mRNA are converted to cDNAs using three individual one-base anchored oligo-dT primers that differ from each other at the last 3V non-T base. The use of one-base anchored primers allows for the homogeneous initiation of cDNA synthesis at the beginning of the poly(A) tail for any given mRNA. The three subpopulations of cDNAs are amplified and labeled by incorporation of radiolabeled nucleotides (33P) or fluorescent one-base anchored primer during PCR in the presence of a set of arbitrary primers, which are short (10–13 bases in length) and arbitrary in sequence. The length of arbitrary primer is so designed that by probability each will recognize 50–100 mRNAs under a given PCR condition. The amplified cDNAs are separated on denaturing polyacrylamide gel. Side-by-side comparison of mRNA species from two or more related samples allows identification of both up- and downregulated genes of interest. By changing primers from both directions, the statistical probability is that most of the expressed genes in a cell may be visualized by this method. Differentially expressed cDNA bands can be retrieved from the denaturing polyacrylamide gel, cloned, and sequenced for further molecular characterization. DNA sequence of these fragments can be identified in a BLAST search of the Genbank (http://www.ncbi.nlm.nih.gov/BLAST).

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Figure 1 Schematic representation of differential display. (Illustration courtesy of GenHunter Corporation, Nashville, TN.)

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Northern blot analysis is essential not only for confirmation of the obtained cDNA fragment but also for information about the size of the gene.

III. Materials and Methods A. Materials

The following materials are used: 1. 5
reverse transcription (RT) buffer: 125 mM Tris-Cl, pH 8.3, 188 mMKCl, 7.5 mM MgC12, 25 mM dithiothreitol. 2. Moloney murine leukemia virus (MMLV) reverse transcriptase (100 units/ml) 3. dNTP (250 AM) 4. 5V-AAGCTTTTTTTTTTTG-3V (2 AM) 5. 5V-AAGCTTTTTTTTTTTA-3V (2 AM) 6. 5V-AAGCTTTTTTTTTTTC-3V (2 AM) 7. 5V-RAAGCTTTTTTTTTTTG-3V (2 AM), (R; rhodamine-labeled) 8. 5V-RAAGCTTTTTTTTTTTA-3V (2 AM), (R; rhodamine-labeled) 9. 5V-RAAGCTTTTTTTTTTTC-3V (2 AM), (R; rhodamine-labeled) 10. Arbitrary 13-mers (2 AM) 11. 10
polymerase chain reaction (PCR) buffer 12. dNTP (25 AM) 13. dNTP (2.5 mM) 14. Glycogen (10 mg/ml) 15. Distilled H2O 16. Loading dye 17. Taq DNA polymerase (5 units/Al, Qiagen) 18. [a-33P]dATP (2000 Ci/mmol) 19. RNase-free DNase I (10 units/Al) 20. Thermocycler 21. DNA sequencing apparatus 22. RNApure Reagent (GenHunter, Nashville, TN) 23. MessageClean Kit (GenHunter, Nashville, TN) 24. RNAimage Kit (GenHunter, Nashville, TN) 25. RNAspectra Red Kit (GenHunter, Nashville, TN) 26. HotPrime DNA labeling Kit (GenHunter, Nashville, TN) 27. pCR-TRAP cloning Kit (GenHunter, Nashville, TN) Although individual components may be purchased separately from various suppliers, most of them can be obtained in kit forms from GenHunter Corporation (Nashville, TN).

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B. Methods RNA Isolation from Cell Cultures

Total cellular RNAs can be easily isolated with a one-step acid–phenol extraction method such as RNApure reagent (GenHunter Corporation). The procedure is as follows: 1. After removal of the cell culture medium, rinse the cells with 10–20 ml prechilled PBS buffer. Add 2 ml RNApure reagent per 150 mm tissue culture plate, and lyse the cells by equally distributing the solution with gentle shaking. Let sit on ice for 10 min and transfer the lysates into two 1.5 ml tubes and add 150 Al chloroform per ml of lysate. Vortex for 10 s and put on ice for 10 min. Freeze the tubes at 80jC or proceed to next step. 2. Centrifuge the tubes at 4jC for 10 min and carefully transfer the upper phase into clean tubes. 3. Add equal volume of isopropanol and put on ice for 10 min. 4. Centrifuge for 10 min at 4jC. Rinse the RNA pellet with 1 ml prechilled 70% ethanol and spin for 2 min. Discard the ethanol, spin briefly, and remove the residual liquid with a P200 pipette. 5. Dissolve the RNA in 50 Al DEPC-H2O and measure concentration at 260 nm (1 OD at 260 nm = 40 Ag RNA). Store RNA at 80jC until use. 6. Check the integrity (18S and 28S rRNA bands) of RNA samples by running 1–2 Ag each RNA on 1% agarose gel with 7% formaldehyde.

DNase I Treatment of Total RNA

Purification of polyadenylated RNAs is neither necessary nor helpful for differential display. The major pitfall of using the purified mRNAs is the frequent contamination of the chromosomal DNA. This contamination usually yields high background smearing in the display gel and results in difficulty in determining the accurate concentration and assessing the integrity of RNA templates. Therefore, removal of all contaminating chromosomal DNA from RNA samples is essential before carrying out differential display. The MessageClean Kit is specifically designed for the complete digestion of single- and double-stranded DNA. The procedure for its use is as follows: 1. Incubate 10–100 Ag total cellular RNA with 10 units DNase I (RNase free) in 10 mM Tris-Cl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2 for 30 min at 37jC.

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Cho and Liang 2. Inactivate DNase I by adding an equal volume of phenol/chloroform (3:1) to the sample. 3. Mix by vortex and leave the sample on ice for 10 min. Centrifuge the sample for 5 min at 4jC in an Eppendorf centrifuge. 4. Save the supernatant. Ethanol precipitate the RNA by adding 3 volumes ethanol in the presence of 0.3 M NaOAC. Incubate at 80jC for 30 min. 5. Pellet the RNA by centrifuging at 4jC for 10 min. Rinse the RNA pellet with 0.5 ml 70% ethanol (made with DEPC-H2O) and dissolve the RNA in 20 Al DEPC-treated H2O. 6. Measure the RNA concentration at OD260 with a spectrophotometer. Check the integrity of the RNA samples before and after cleaning with DNase I by running 1–3 Ag each of RNA on a 7% formaldehyde agarose gel. Store the RNA sample at a concentration higher than 1 Ag/Al at 80jC before using for differential display. RNA sample with a lower concentration is more likely to be degraded than a higher concentration. Reverse Transcription of mRNA

The procedure is as follows: 1. Set up three reverse transcription reactions for each RNA sample in three microfuge tubes (0.5 ml size), each containing one of the three different one-base anchored oligo-dT primers as follows: For 20 Al final volume, add 9.4 Al dH2O, 4 Al 5
RT buffer, 1.6 Al dNTP (250 AM), 2 Al (0.1 Ag/Al, freshly diluted) total RNA (DNA-free), and 2 Al (M can be either G, A, or C) AAGCT11M (2 AM). 2. Program your thermocycler to 65jC, 5 min ! 37jC, 60 min !75jC, 5 min ! 4 jC. 3. Add 1 Al MMLV reverse transcriptase to each tube 10 min after 37C and mix well quickly. Continue incubation. At the end of the reverse transcription reaction, spin the tube briefly to collect condensation. Set tubes on ice for PCR or store at 80jC for later use. Polymerase Chain Reaction for Differential Display Conventional Isotope-Labeled Differential Display

The RNAimage Kit can be used for this step as well as the described above RT reactions. 1. Set up PCR reactions on ice as follows. For 20 Al final volume for each primer set combination, add 10 Al dH2O, 2 Al 10X PCR buffer,

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1.6 Al dNTP(250 AM), 2 Al arbitrary 13mer (2 AM), 2 Al AAGCT11M (2 AM), 2 Al RT-mix from above, 0.2 Al a-[33P]-dATP (2000 Ci/mmole), and 0.2 Al Taq DNA polymerase (5 units/Al, Qiagen). Make core mixes whenever possible to avoid pipetting errors. 2. Run the PCR at 94jC, 30 s ! 40jC, 2 min ! 72jC, 1 min. for 40 cycles ! 72jC, 5 min and store at 20jC until the gel run. Fluorescent Differential Display

The RNAspectraTM Red Kit can be used for this step as well as the RT reactions described above. 1. Set up PCR reactions on ice as follows. For 20 Al final volume for each primer set combination, add 4.2 Al dH2O, 2 Al of 10X PCR buffer, 1.6 Al dNTP (2.5 mM), 8 Al arbitrary 13mer (2 AM), 2 Al R-AAGCT11M (2 AM, red color), 2 Al RT-mix from above, and 0.2 Al Taq DNA polymerase (5 units/Al, Qiagen). Make core mixes whenever possible to avoid pipetting errors. 2. PCR condition for 40 cycles are as follows: at 94jC, 30 s ! 40jC, 2 min ! 72jC, 1 min. Followed by 72jC for 5 min and store at 20jC until the gel run.

Separating PCR Products by Denaturing Polyacrylamide Gel Electrophoresis Isotope-Labeled Differential Display

The procedure is as follows: 1. Prepare a 6% denaturing polyacrylamide gel in TBE buffer. Let it polymerize for more than 2 h before using. Prerun the gel for 30 min. It is crucial that the urea in the wells be completely flushed out right samples are loaded. For best resolution, flush every four to six wells each time during sample loading while trying not to disturb the samples already loaded. 2. Mix each PCR tube with 8 Al loading dye and incubate at 8jC for 5 min immediately before loading onto a 6% DNA sequencing gel. 3. Electrophorese for about 3.5 h at 60 W constant power (with voltage not to exceed 1700 v) until the xylene dye (the slower moving dye) reaches the bottom. 4. Turn off the power supply and blot the gel onto a piece of 3 M paper. Cover the gel with plastic wrap and dry it at 80jC for 1 h (do not fix the gel with methanol/acetic acid). Orient the autoradiogram and

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Cho and Liang dried gel with radioactive ink or needle punches before exposing to x-ray film. 5. After developing the film (overnight–72 h exposure), orient the autoradiogram with the gel. 6. Locate bands of interest either by marking with a clean pencil from underneath the film or cutting through the film with a razor blade. Another way found to work very well is to punch through the film with a needle at the four corners of each band of interest. (Handle the dried gel with gloves and save it between two sheets of clean paper). Cut out the located band with a clean razor blade.

Fluorescent Differential Display

The procedure is as follows: 1. Also usable is a 6% denaturing polyacrylamide gel in TBE buffer, as for isotope-labeled differential display. 2. Mix each PCR reaction with 8 Al FDD loading dye and incubation for 3 min before loading. 3. Electrophorese for 2 h at 60 W until the xylene dye reaches the bottom. 4. After the gel run, scan the gel without drying using FMBIO II fluorescent laser scan with FMBIO Read Image version 1.5 software. 5. Cut out the bands of interest after analysis of the digital FDD Image using FMBIO data analysis software. Figure 2 shows a representative fluorescent differential display (FDD) of RNA from tetracycline-regulated wild-type p53 induction. Reamplification of cDNA Bands

The procedure is as follows: 1. 2. 3. 4. 5.

Soak the gel slice in 1 ml dH2O for 5 min. Remove the water and add new 100 Al dH2O. Boil the tube with tightly closed cap for 20 min. Spin for 2 min and transfer the supernatant to a new microfuge tube. Reamplification should be done using the same primer set and PCR conditions, except with no isotopes added. A 40 Al reaction volume is recommended. 6. For 40 Al final volume for each primer set combination, add 20.4 Al dH2O, 4 Al 10X PCR buffer, 3.2 Al dNTP(250 AM), 4 Al arbitrary 13mer (2 AM), 4 Al AAGCT11M (2AM), 4 Al cDNA template from RT, and 0.4 Al Taq DNA polymerase (5 units/Al, Qiagen).

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Figure 2 A representative fluorescent differential display (FDD) analysis of p53 induction. (a) FDD analysis of p53 induction tetracycline-regulated wild-type p53 gene expression in A2 DLD-1 colon cancer cell line (13). P53 was induced by the removal of tetracycline from the culture media at different time points (8h -tet and 12h -tet). Four RNA samples from 8 and 12 h with tetracycline (no p53 induction) and without (p53 induced) were compared with arbitrary 13mer in combination with fluorescent-labeled G-anchored oligo-dT primer. p53 was identified by comprehensive FDD screening. (b) The confirmation of p53 induction by Northern blot rRNA was used as control for equal loading of each total RNA (each lane 10 Al total RNA). (c) Western blot analysis of p53 induction. The induction of p53 was confirmed by western blot using a polyclonal antibody against p53. As control for equivalent protein loading, anti-actin antibody was used.

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Cho and Liang 7. Run 10–15 Al PCR sample on a 1.5% agarose gel to check the size of reamplified PCR products, which is consistent with their size on the denaturing polyacrylamide gel. 8. Subclone PCR products into the vectors using a method such as the pCR-TRAP cloning system (GenHunter, TN). PCR samples can also be run as a duplicate to perform the reverse Northern blot to screen differentially expressed cDNAs before cloning. Confirmation of Differentially Expressed cDNA by Northern Blot

Northern blot analysis of differentially expressed gene, which was found by the differential display, is very important because it provides not only information about the size of the gene but also the confirmation of cDNA fragment. The HotPrime DNA labeling kit, following the standard procedure, can be used to perform Northern blot analysis. IV. Discussion One of the most frequently asked questions by those who wish to use differential display is what the false positive rate is. There is no clear answer to this very complex question, because it depends on so many factors (both intrinsic and extrinsic) of the differential display method (Table 1) (10,11).

Table 1 Factors Affecting The Quality and Reproducibility of Differential Display Intrinsic factors Primer designs Anchored primers (one bases vs. two bases) Length of arbitrary primers (short vs. long) Quality of reagents (enzymes, dNTP, and primers) Extrinsic factors Experimental design (controls) Quality and quantity of RNA (integrity and contamination of DNA) PCR tubes (thickness) Isotopes (a-33P-dATP) Experimental set-up (core mixes, pipetting errors) Thermocyclers (calibration) Quality of the denaturing polyacrylamide gel electrophoresis Criteria for picking bands Experience of the researcher

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The extrinsic factors that can contribute to the rate of false-positive results include the systems being compared, experimental designs, appropriate internal controls, criteria for choosing bands, reaction set-up, type of PCR reaction tubes, type of thermocyclers, and researchers’ training and experience. Taking one extrinsic factor (the system being compared) as an example, serves to illustrate some of the potential causes of false-positive results. If one is to compare gene expression between rat liver and rat brain, where it is known that nearly 50% of the genes expressed are brain-specific, the number of false-positive results isolated is likely to be very low because the difference between samples compared is very large. Great effort has since been made to improve the method itself (intrinsic factors) to enhance the efficiency of cDNA amplification, the coverage of genes, and the downstream screening process (12). Most of the key factors intrinsic to DD have been dealt with at length recently. One intrinsic factor (the optimal length of arbitrary primers) is determined by statistical consideration that each primer will recognize 50–100 mRNA species. To do so, these primers must hybridize six to eight mers. In practice, arbitrary primers of 10 bases or longer are more commonly used because they can hybridize in a degenerate fashion at a lower annealing temperature, while providing better reproducibility. Differential display itself can be perfectly reproducible if there are no intrinsic and extrinsic problems. Acknowledgments This work was supported in part by grants from the National Institutes of Health (CA76969 and CA 74067 to P.L.). We thank GenHunter Corporation for permission to adapt part of its protocol of RNAimage kit and RNASpectra Kit for differential display and for use of the fluorescent laser scanner. References 1. 2.

3.

O’Farrell PH. High resolution two-dimensional electrophoresis of proteins. J Chem 1975; 250(10):4007–4021. Littman DR, Thomas Y, Maddon PJ, Chess L, Axel R. The isolation and sequence of the gene encoding T8: a molecule defining functioning classes of T lymphocytes. Cell 1985; 40(2):237–246. Liang P, Pardee AB. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science 1992; 257(5072):967–971.

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Cho and Liang Liang P, Bauer D, Averboukh L, Warthoe P, Rohrwild M, Muller H, Strauss M, Pardee AB. Analysis of altered gene expression by differential display. Methods Enzymol 1995; 254:304–321. Cho YJ, Meade JD, Walden JC, Chen X, Guo Z, Liang P. Multicolor fluorescent differential display. Biotechniques 2001; 30(3):562–572. Liang P. Gene discovery using differential display. Genet Eng News 2000; 20:37. Aittokallio T, Ojala P, Nevalainen TJ, Nevalainen O. Analysis of similarity of electrophoretic patterns in mRNA differential display. Electrophoresis 2000; 21(14):2947–2956. Aittokallio T, Ojala P, Nevalainen TJ, Nevalainen O. Automated detection of differently expressed fragments in mRNA differential display. Electrophoresis 2001; 22(10):1935–1945. Liang P, Zhu W, Zhang X, Guo Z, O’Connell RP, Averboukh L, Wang F, Pardee AB. Differential display using one-base anchored oligo-dT primers. Nucleic Acids Res 1994; 22(25):5763–5764. Liang P, Pardee AB. Recent advances in differential display. Curr Opin Immunol 1995; 7(2):274–280. Liang P. Factors ensuring successful use of differential display. Method 1998; 16(4):361–364. Cho YJ, Prezioso VR, Liang P. Systematic analysis of intrinsic factors affecting differential display. Biotechniques 2002; 32(4):762–766. Yu J, Zhang L, Hwang PM, Rago C, Kinzler KW, Vogelstein B. Identification and classification of p53-regulated genes. Proc Natl Acad Sci USA 1999; 96(25):14517–14522.

9 Expression Profiling as a Tool for Diagnosis and Pathway Discovery: Experimental Design and Technical Considerations

ERIC P. HOFFMAN Children’s National Medical Center Washington, D.C., U.S.A.

YUE WANG

DONALD MASSARO, GLORIA MASSARO, and LINDA CLERCH Georgetown University Medical Center Washington, D.C., U.S.A.

Virginia Polytechnic Institute Alexandria, VA, U.S.A.

I. Introduction Expression profiling using microarrays is a widely used technique to scan for differences or changes in the steady-state levels of messenger ribonucleic acids (mRNAs) in cells or tissues. The majority of the genes of a number of organisms can be assayed for mRNA transcripts using 25 mer oligonucleotide probe sets (f10–40 oligonucleotides per gene) [human, mouse, rat, plant (Arabidopsis), Drosophila, worm (C. elegans), yeast, bacteria (E. coli, P. aeruginosa)]. In addition, 60–75 mer oligonucleotide microarrays (1 oligo/ gene) are becoming commercially available. The major technical advance in microarrays has been the ability to conduct highly parallel data generation (f1 million oligonucleotide hybridizations assayed per human sample). The ability to generate enormous amounts of data quickly has come with liabilities. Is expression profiling simply an enormous descriptive experiment providing no mechanistic insights? Is the technology standardized to a sufficient degree to allow comparison of data sets from different laboratories? Are there sufficient quality control methods to ensure accuracy, and 197

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data transportability? Are there any experimental designs that help move away from the so-called descriptive snapshot, to more mechanistic pathway data? This chapter describes some of the debates surrounding experimental platforms (oligonucleotides, cDNA arrays, Affymetrix arrays), image analysis, methods for determining mRNA levels from microarray data (signal detection), statistical methods, and efforts at data dissemination and data standardization. We present a publicly accessible time series study of lung remodeling (alveolar degeneration and regeneration) following sequential caloric restriction and refeeding (1). We end with a description of a public access data warehouse of the type that should facilitate mRNA pathway research worldwide.

II. Experimental Platform: To Spot or Not to Spot? Two fundamentally different experimental platforms are used for expression profiling: spotted microarrays and Affymetrix oligonucleotide microarrays. There are clear differences in how these two types of microarrays are produced, and also in how the resulting fluorescent images are acquired and interpreted. Spotted microarrays are generated through the mechanical spotting of solutions of DNA probes on glass slides or filters. Robotic spotters track and spot up to 100,000 solutions per slide, with a different DNA probe in each solution. Relatively common types of DNA spotted on these arrays are cloned DNA (plasmid), polymerase chain reaction (PCR) products from inserts in plasmids (either expressed sequence tags [ESTs] or full length cDNA clones), or synthesized oligonucleotides (typically 70 mer oligos). The amount of solution spotted on each spot is difficult to control. Likewise, the concentration of probe in the solution can be difficult to standardize. Thus, a profile generated and analyzed in one institution may be difficult to compare to a similar array in another institution. The variability intrinsic to spotted microarrays requires the cohybridization of an experimental solution of RNA (one color) with a control solution from another source (labeled with a different color). The ratio of the intensities of the two fluorescent signals hybridized to the same spot gives an estimation of the relative concentrations of mRNA in the two solutions applied to the microarray. Unless a common control RNA solution is used between different experiments, and different laboratories, it becomes inherently difficult to compare data across experiments and sites. Databasing is also problematic, given the generally nonstandardized nature of the technology. The major advantages of this experimental platform are the flexibility in printing whatever is desired

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(e.g., customization of arrays), and the relatively low cost of producing and processing spotted arrays. The second platform is a technology marketed by Affymetrix (San Jose, CA, USA). This technology involves the light-activated synthesis of oligonucleotides in situ on glass substrates, using photolithography overlays to add the correct base sequentially at each spot on the array. Currently available microarrays have 500,000 twenty-five base oligonucleotides synthesized in a 1.2 cm2 area of glass (2). An advantage of this approach is that it is largely bioinformatic-driven, so that there are typically fewer human errors in the production of the arrays. The platform is also redundant, with multiple measurements per gene. Currently available human microarrays use 22 distinct oligonucleotides to query each mRNA, comprised of 11 pairs of perfect match and mismatch probes (the latter having a single mutation in the center of the 25 base probe, as a control for nonspecific hybridization). Both the manufacturing process and the redundancy of measurements lead to a stand alone platform, in which only one RNA solution is placed on an array, with the levels of each transcript normalized to the total array fluorescence. This non-ratio-based method should allow array data generated in one facility to be directly compared to data generated in another lab. This depends on quality control metrics and use of standard operating procedures; these are only beginning to emerge. We have developed a public access data warehouse of 1,500 Affymetrix profiles that adhere to specific quality control protocols (see http://pepr.cnmcresearch.org) (3). As mentioned above, the methods used for data acquisition and data interpretation are different for spotted microarrays and Affymetrix arrays. Spotted microarrays generate a ratio for each spot between an experimental and control solution (e.g., drug-treated cells vs. non-drug-treated cells). This ratio is relevant only to the specific experiment conducted, and samples can be compared only when the same control solutions are used. Spotted oligonucleotide arrays have more control over the amount of nucleic acid spotted on the slide, although most still output a ratio rather than an absolute value for the amount of a specific RNA in a solution. The remainder of this review focuses on Affymetrix arrays. This platform is becoming increasingly popular, and has the potential to become something of a standard (2). Development of a standard has pros and cons; a true standard would greatly facilitate data sharing and the creation of powerful data warehouses. However, the transportability of a standard is accompanied by liabilities, such as limitations in improving and customizing methods. Quick adherence to a specific standard may limit further technological advances. So as to not become entangled in a detailed debate on experimental platforms and interpretation methods, we use the Affymetrix

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platform as a forum for description of experimental design, data generation, and data interpretation.

III. Experimental Design: Where Are the Variables? The goal of expression profiling is to define gene expression changes (measured as steady-state levels of a single mRNA in a complex solution) that reflect some biological variable under study. Key in any experimental design is a good handle on the controlled, and uncontrolled, variables. Experimental design should allow for maximizing sensitivity for the biological variable under study, while minimizing the effect of confounding noise on data interpretation. For example, in one well-designed microarray study, the investigators wished to study the targets of the transcription factor, MyoD (4). An expression construct containing the MyoD transcription factor under the control of the inducible estrogen promoter was transfected into cells that had the endogenous MyoD gene knocked out. MyoD was quickly induced by addition of estrogen to the cultured transfected cells, and expression profiling done to determine the genes induced by MyoD expression. One possible source of noise in this analysis (confounding variable) is the possibility of downstream expression changes not directly due to MyoD binding; for example, MyoD can induce transcription factor Z, and transcription Factor Z can then activate its downstream targets. The latter would also be detected as a MyoD-induced gene by expression profiling, but would not represent true direct targets of MyoD binding. The authors recognized this mitigating variable, so all translation was blocked using cycloheximide. Thus, factor Z protein was unable to be produced, and thus unable to initiate changes downstream of MyoD. This relatively clean in vitro system can be contrasted to a different study of subjects with acute lung injury (ALI). Subjects are recruited upon hospital admission for trauma or pneumonia, and blood samples taken as a function of time after admission. The goal is to identify gene transcription changes that predict progression into multiorgan failure and death or recovery. Expression profiling can be done on whole-blood lysates, and statistical tests used to compare the profiles of recovering subjects and those progressing to death. This experimental design, while clinically extremely important, is fraught with uncontrolled confounding variables. What is the variation in expression profiles caused by the age, gender, or ethnic background of the subject? What of the type of initiating insult, and the timing of collection postinsult? Blood as a tissue source is quite unstable. The proportion of different mononuclear cell populations is highly variable both between subjects and within a subject, depending on many factors. In the end, will

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this experiment accomplish the stated goal, or simply result in a fancy differential cell count? The many potential confounding variables in expression profiling studies (particularly those of clinical relevance), coupled with the control vs experimental snap shot design, have led to many scientists viewing expression profiles as the ultimate descriptive experiment, with little basic scientific value. Many pharmaceutical companies are implementing microarrays as one endpoint in drug trials, and they will say that they are unconcerned about biological significance: they are simply interested in an endpoint measure. If the endpoint measure from whole-blood profiling provides an elaborate differential cell count, and if this differential cell count proves important in monitoring drug effects and side effects, then this is a perfectly valid experimental design. One would have to agree with this point of view, particularly if whole blood is the only accessible tissue from subjects in a clinical trial (there is simply no other choice). With these two extreme experimental designs as a backdrop, we next describe two emerging designs that are proving particularly informative: the supervised training/test design, and the time series study (temporal cause/ effect).

IV. Supervised Training/Test Study Expression profiling is becoming increasingly important in cancer research, both for the molecular phenotyping of tumor subtypes (5,6), for assaying response to chemotherapy (7), and for defining the biology behind tumor progression (8). Most experimental designs include what is termed a training set, namely a series of tumors carrying a defined histological diagnosis. These known tumors are then expression profiled, and supervised clustering done to define the so-called expression fingerprint characteristic of each subtype of tumor (training set). To determine the sensitivity and specificity of these defined fingerprints, a set of blinded tumors (test or validation set) is expression profiled, and then clustered using the discriminatory gene fingerprints defined in the training set. The code is then broken, and the success of the expression profile in providing a diagnosis is assessed. Key to this approach is what is termed the supervised nature of the analysis. In array terms, supervised means that the computational programs are told which tumors/profiles belong to which diagnostic groups. This is very different from unsupervised approaches, in which the computer is allowed to make its own decisions as to how many tumor groups there are, and which tumor belongs to each group. Unsupervised clustering is a suitable way to determining if what is termed the biological variable of interest (e.g., different lung cancer subtypes) is the

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dominant variable, or if this biological variable is obscured by uncontrolled mitigating variables. A more clinically relevant application of the supervised two-cohort study involves predicting the stage of the tumor (e.g., metastatic medulloblastoma vs. nonmetastatic disease) (8). It may be highly relevant to patient care to determine if the tumor has metastasized or not. A carefully designed study would include a training set of tumors (primary site tumors ultimately known to have metastasized or not), with a validation set, followed by implementation in prospective clinical trials of the discriminatory expression fingerprint. Many studies have used expression profiles not so much for diagnostic purposes but for uncovering pathogenetic pathways involved in disease or physiological states. Such snap shot comparisons (often between what are termed normal and disease cohort) can prove highly informative; they may set up models for disease pathogenesis, or define a tissue response to a stimulus. For example, we have published a number of papers on expression profiling in muscle, where models can be built for inflammatory disease (9), muscular dystrophy (10,11), and limb muscle vs. extraocular muscle (12). These encyclopedic compendia of expression differences can serve as the first step in defining the nature of a cell type or tissue, and the biochemical players in homeostasis and disease.

V. Time Series Study (Temporal Cause/Effect) Expression profiling data have a high degree of dimensionality, and one of these is time. As given in the MyoD transcription factor example above, induction of MyoD in cultured cells can define potential downstream gene targets of this activating transcription factor, but some of these targets may themselves be transcription factors. Conducting a time series study can begin to help resolve what is directly the result of the stimulus, as differentiated from an indirect downstream event. Direct effects should occur earlier in a time series than indirect effects. Thus, conducting expression profiling time series can go far in establishing cause and effect in a biological pathways. By controlling for the dimension of time, the experimentalist has a much better grasp on pathways. One would prefer a time series that is every few seconds over a time frame of days and weeks; this would give very fine resolution to the study. This is clearly impractical, in terms of both effort and cost. The necessary resolution will depend on the variable being studied. To provide an example, lung alveolae have been recently documented to show substantial remodeling following calorie restriction and refeeding (1). A two-thirds reduction in

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caloric intake for 2 weeks leads to a 55% reduction in alveolar number and 25% reduction in surface area. This coincides with a 25% reduction in lung DNA content, and histological evidence of cell apoptosis. Refeeding brings all measures back to control levels within 72 h. This experiment suggests that murine lungs have the ability to regenerate their alveolae after damage. The molecular pathways responsible for both loss and gain of alveolae are therefore of great interest. To begin to dissect the molecular pathways responsible for this process, the process of alveolar degeneration/apoptosis was first studied using an expression profiling time series. Mice were calorie restricted, and lungs harvested at eight time points following caloric restriction (0, 2 h, 4 h, 12 h, 24 h, 48 h, 72 h, 96 h). Two different mice were used for replicates at each time point (each microarray corresponded to RNA from a different mouse) (13). This data set can then be queried for any of the 12,000 genes studied, in this case via an interactive web database (http://microarray.cnmcresearch. org), and the time series data can be graphed. An example output of the webbased Single Gene Query Tool (http://microarray.cnmcresearch.org/SingleGeneMain.asp) is shown in Figure 1. In this case, the probe set corresponding to Kruppel-like factor 2 (lung) was searched (also called KLF2, or LKLF). This gene was originally identified in lung, and has been found to be expressed in vascular endothelial cells where it is highly sensitive to fluid shear stress (14), is negatively regulated by tumor necrosis factor alpha (TNF-a), and has been shown to inhibit differentiation of adipocytes (15). The expression profiles show that KLF2 was induced by calorie restriction, with steady increases in expression until 48 h, and then a leveling off (Fig. 1). Mouse overs of each data point and average provide a summary of the GeneChip data associated with the datapoint (see Fig. 1 inset for 72 h data point). A download function from this web site permits local download of all data associated with the graph (Table 1). From Table 1 one sees that three of the four profiles at time 0 showed an Absent call, where the mismatch signals of the probe set were not sufficiently different from the perfect match to provide a detection p value above background. By 24 h, the signal had increased, and the detection probe set p values declined to significant levels, so that both replicates (and all further time point data points) were deemed as present calls (Table 1). Note from Table 1 that there is not a complete correlation between signal intensity and present/absent call. This is due to variations in hybridization to the mismatch probe sets. However, visual inspection of the graph (Fig. 1) places relatively high confidence on the data, despite many absent calls in the early time points. An additional time series available on this web site is for muscle regeneration: two different histologically matched mice were harvested at 27 time points following induction of regeneration (16,17). Query of this 54

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Figure 1 Example of a time series study in lung remodeling following caloric restriction: output of the web-based data warehouse query tool (found at http:// microarray.cnmcresearch.org/SingleGeneMain.asp). In the example shown, ‘‘lung’’ was searched within this eight time point series (two mice per time point), and the probe set for ‘‘Kruppel-like factor 2’’ selected. The gene is shown to be markedly induced by calorie restriction, with replicates showing highly concordant data.

profile time series via the Single Gene Query Tool web site (http://microarray. cnmcresearch.org/SingleGeneMain.asp), with myogenin gives the graph shown in Figure 2. Myogenin is a transcription factor, and this temporal profiling series can be used to identify potential downstream targets of this factor. As shown in Figure 2, myogenin is induced between 2 and 3 days following induction of regeneration. Any potential downstream targets of this transcription factor should be induced at or after the 3 day time point. One problem with in vivo time series data of whole tissue as shown in Figure 1 (lung) and Figure 2 (muscle) is that the expression profiles are an average of the transcriptomes of the constituent cell types (e.g., myofiber, connective tissue, vasculature, inflammatory cells, etc.). Although in vitro data in cultured cells can provide much better control over cell types and stimuli, the in vitro models suffer from questionable relevance to the in vivo state. We have recently described a method of using the in vivo data shown in

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Table 1 Data Associated with Graph in Figure 1: Affymetrix Probeset: 96109_at Name PGA-MCR-ctr-0h-1aAv2-s1 PGA-MCR-ctr-0h-1bAv2-s1 PGA-MCR-ctr-0h-2aAv2-s1 PGA-MCR-ctr-0h-2bAv2-s1 PGA-MCR-CR-2h-1aAv2-s1 PGA-MCR-CR-2h-1bAv2-s1 PGA-MCR-CR-4h-1aAv2-s1 PGA-MCR-CR-4h-1bAv2-s1 PGA-MCR-CR-12h-1aAv2-s1 PGA-MCR-CR-12h-1bAv2-s1 PGA-MCR-CR-24h-1aAv2-s1 PGA-MCR-CR-24h-1bAv2-s1 PGA-MCR-CR-48h-1aAv2-s1 PGA-MCR-CR-48h-1bAv2-s1 PGA-MCR-CR-72h-1aAv2-s1 PGA-MCR-CR-72h-1bAv2-s1 PGA-MCR-CR-96h-1aAv2-s1 PGA-MCR-CR-96h-1bAv2-s1

Time (h)

A/P

SIGNAL

Average

AVG Fold Change

0 0 0 0 2 2 4 4 12 12 24 24 48 48 72 72 96 96

A A A P A A P M A A P P P P P P P P

899.8287 343.1313 676.5944 922.8417 848.7089 630.502 1077.241 733.4751 1250.713 956.1664 1498.281 1660.163 2734.515 2518.404 1599.38 1962.543 1754.89 2288.14

710.599 710.599 710.599 710.599 739.604 739.605 905.357 905.355 1103.44 1103.44 1579.22 1579.22 2626.45 2626.45 1780.96 1780.96 2021.51 2021.51

1 1 1 1 1.04082 1.04082 1.274077 1.274077 1.55283 1.55283 2.222381 2.222381 3.69612 3.69612 2.506282 2.506282 2.844804 2.844804

Figure 2 as a relevance filter for in vitro transcription factor induction of MyoD (6). It is likely that this approach, in which both in vitro and in vivo experiments are compared to define transcriptional cascades in a sensitive and specific manner, is likely to become commonplace in the near future. Repeated sampling of tissues from the same human research subject is an excellent method to control for the considerable individual variability in humans due to polymorphic variation. Thus, a serial sampling in humans, in which each person serves as his or her own control, would be ideal. Blood sampling can typically be done sequentially, and a number of studies are underway with either whole blood RNA or specific isolated cell types in a number of conditions (drug studies, acute lung injury, asthma; see http:// www.hopkins-genomics.org/). We recently described a microarray study in which subjects with features of metabolic syndrome were subjected to a regimented exercise program, and muscle biopsies taken at study entry, after 9 months of exercise, and at 2 detraining time points (96 h, 2 weeks). The ability to use each subject as his or her own control greatly increased the sensitivity of the study. Using only three subjects, we were able to conclude that muscle produces large amounts of fibrinolytic proteins during exercise, and that it appears to play a major role in setting the systemic fibrinolytic state (18).

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Figure 2 Single gene query tool analysis of myogenin during in vivo muscle regeneration (27 time points). In this time series study, myogenin is seen strongly induced between 2 days and 3 days following induction of muscle degeneration using cardiotoxin (16,17). Expression then quickly falls off. Downstream targets of myogenin would be expected to be induced on or after the 3 day time point. (From http://microarray.cnmcresearch. org, using the ‘‘Single Gene Query Tool’’; http:// microarray.cnmcresearch.org/Single GeneMain.asp).

VI. Data Warehouse Design One can envision that time series microarray data of ever-increasing resolution and sensitivity will allow the definition of many transcriptional and biochemical pathways. It is also easy to imagine that the ability to compare pathways across experimental platforms will lead to increased understanding of cell-specific pathways specific for certain stimuli, and others that are shared between many different cell types and stimuli. The creation of large cancer profile data warehouses should likewise eventually allow one to take an expression profile of an unknown tumor type, and have it diagnosed by providing a best match in the tumor data warehouse. Such warehouses would include information on the intrinsic variability of each human gene between individuals, specificity of gene changes for disease state and tissue type, and diagnostic fingerprints of certain expression patterns known to be informative for the questions under study.

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Any data warehouse that hopes to build effective and powerful user interfaces and analytical tools must have a standardized data format. The problem facing RNA expression profiling is that it is a field in transition, with a plethora of experimental platforms in which to generate data. These include (in historical order): differential display, clone frequency in libraries (virtual northerns), serial analysis of gene expression (SAGE), spotted cDNA clone arrays, Affymetrix probe set oligonucleotide arrays, and spotted oligonucleotide arrays. Additional experimental platforms are also in development, including Illumina fiber optic bead arrays, Nanogen electronic chip arrays, and others. Given the current heterogeneity in experimental platform, there are different approaches emerging to developing databases for expression profile data. The most developed resource is the National Library of Medicines GEO resource (Gene Expression Omnibus) (http:\\www.ncbi.nih.gov/geo). A key advantage of the NCBI GEO database is the flexibility in data input, permitting data from many experimental platforms. This design is inclusive in nature, permitting participation of nearly all laboratories generating expression profile data. In addition, a specific gene in genome browsers is already linked back to profiles containing this gene http://www.genome.ucsc. edu). We designed and implemented an Affymetrix data conversion utility (collaboratively with NCBI’s Alex Lash, Director of GEO resource), through which all data are automatically copublished to our web data warehouse (see below), and the GEO database, where links are retained back to the original data files and interpretations in our integrated internal/web LIMS environment. A clear disadvantage of the NCBI open access format is that it is difficult to generate cross-project data mining tools, due to the inherent heterogeneity in data format. Moreover, much of the statistical power of certain platforms is lost. For example, all the information regarding individual probe features and probe sets inherent in Affymetrix data is not transferred to GEO, resulting in a single average number of hybridization for each gene. To use this single issue as an example, there are many different software packages and methods available for image interpretation and probe set interpretation for Affymetrix profiles; users wishing to use these different methods are unable to do within GEO. However, our conversion software retains links back to our data warehouse with original image (.dat) and processed image (.cel) files, if users wish to download and reanalyze these. Finally, the lack of a experimental standard makes it difficult to impossible to apply quality control standards; thus, there are no available quality measures of data submitted to data repositories. To begin to circumvent some of the problems with a completely flexible data entry format, a consortium of array manufacturers and users was established approximately 4 years ago to define a standardized data format for

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array data. In anticipation of a standard, the European Community (specifically the EMBL) established a team of 20 computer scientists and bioinformaticists to build a public access database that was to use this standard (ArrayExpress; http://www.ebi.ac.uk/arrayexpress/). The conclusion of the deliberations was the MIAME data format (19). Affymetrix and other manufacturers of microarrays have developed routines to reformat data into MIAME-compliant files that can then be databased and compared. The advantage of the ArrayExpress format is that it still retains some flexibility: it can accept both spotted microarray (cDNA and oligonucleotides and Affymetrix oligonucleotide array data, and all data must contain a large number of queriable fields that contain substantial amounts of experimental information (e.g., time series data, organism, strain, cell type, array type, etc.). This should make it possible to query the database rapidly to identify the profiles of interest, and begin data mining of these. However, there remains the major limitation that the nonstandardized nature of the experimental platform results in ArrayExpress being limited to more of a data repository, rather than a true integrated database. In brief, the ratio output of the spotted array systems (ratio of control vs. experimental samples for a single spot) is fundamentally different in character from the single gene/probe-set normalized signal of the Affymetrix platform. This makes it difficult if not impossible to compare different experiments in different laboratories: the controls (denominator for the ratio) are different (not standardized), so that the numbers are noncomparable. There is an emerging consensus regarding intrinsic advantages of the Affymetrix experimental platform relative to the spotted cDNA and oligonucleotide platforms (2). The Affymetrix arrays are built with high degree of redundancy of measurements, with 22–120 specific oligonucleotides querying each gene (the probe set). This enables accurate and reproducible measurements of gene expression. The majority of genes in the genome are assayed, with the genome-anchored human U133A,B array set currently querying 44,000 genes and expressed sequence tags (ESTs), and the recently released genome-anchored murine and rat arrays similarly covering these rodent genomes. The bioinformatic-driven design and production of Affymetrix arrays enables refined cross-species orthologue mapping, and refined algorithms have recently been developed that have dramatically increased the informativeness (nonoverlapping, improved performance) of the probe sets. The stand-alone nature of the profiles makes them intrinsically comparable to single profiles generated in any other laboratory if similar methods and standard operating procedures and quality control are followed. The normalization to whole-chip intensities involving f500,000 oligonucleotide features provides target intensities that facilitate comparability between labs. Finally, improvements in GeneChip manufacturing processes at Affymetrix have led to a relatively dramatic improvement in chip performance (in our experience, up

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to 60% of chips were defective 4 years ago compared with only 2–5% today, with much greater reproducibility between arrays and production lots). Given the above points, the Affymetrix platform should be amenable to development of a standardized database. Crucial to database development is defined quality control (QC) and standard operating procedures (SOP) that ensure the comparability of arrays. We have developed QC/SOP protocols that provide quality assurance for expression profiles, and ensure the comparability of different profiles from different experiments (2,20,21), and these are posted on our web site (http://microarray.cnmcresearch.org/pgaoutline-qcofsamples. asp). The database-driven, genome-anchoring of the genes/ESTs permits direct interface with genome browsers, and avoids many of the ambiguities often associated with spotted array platforms (probe identification, contamination). It is anticipated that a number of integrated databases for specific experimental platforms will be created over the next few years. As each resource populates the database with profiles, and builds web analysis tools, the power of cross-analysis of data from different projects becomes more and more tangible. Moreover, the release of these standardized resources to the general scientific public will serve to parallelize research efforts, and thus greatly speed progress.

VII. Technical Variables: Where is the Noise? One of the more challenging aspects of research using microarrays is the careful consideration of all the variables that are controlled, and uncontrolled. Many of these variables are obvious (difference between a spleen and a muscle) but many are less obvious (variability intrinsic to cell culture experiments) (20). One method to visualize overall variation between expression profiles is unsupervised hierarchical clustering. Hierarchical clustering is a data visualization method in which the performance of each gene in studied across a series of profiles, and if the gene changes in the same direction and magnitude in two profiles, then that relationship becomes linked. The strongest linkages are then visualized as a branch on a dendrogram. The method generally does not contain any statistical measures of the significance of the linkages established, and different algorithms for establishing linkage will give very different results (22). Hierarchical clustering is a very popular method for visualizing the relationship between samples (Fig. 3), or the relationship of gene expression changes within those samples (Fig. 4). While not statistically driven, hierarchical clustering typically finds clusters of genes with similar regulation patterns according to user-defined variables, and the results are often concordant with correlation coefficient studies of nucleated clusters. We have recently described the use of hierarchical clustering to investigate different

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Figure 3 Unsupervised hierarchical clustering to view relationship between murine expression profiles: a series of expression profiles from cultured C2C12 cells (VSM samples), murine spleen (KNagaraju samples), and skeletal muscle (FBooth samples). The branch sites of the dendrogram provide an estimate of the relatedness of the profiles to each other. In this case, the cell culture, spleen, and muscle are clearly differentiated (major branches). However, the different spleen samples appear more closely related to each other than the different cell cultures, based on the height of the branch points. This diagram also can show that the desired biological variable is dominant in the analysis over confounding variables: the Fbooth mouse muscle samples show that the controls (CON samples) are more closely related than the muscular dystrophy samples (MDX). (From Ref. 21.)

types of probe set analyses and identify the appropriate signal/noise ratio (23). Future developments in hierarchical clustering will likely include weights given to specific genes that show the most robust (23). It is important to point out that each expression profiling project has its unique biological variables, and set of confounding uncontrolled variables. For example, inbred mice have very little expression profile variation between individuals of the same inbred strain, but substantial variability between strains. Transgenic mice, in which exogenous genes are added to the mouse

Figure 4 Supervised clustering of a temporal series in muscle regeneration groups together genes involved in regeneration. Shown is hierarchical clustering with a defined time period after injection of cardiotoxin to induce muscle degeneration/regeneration. The inset to the lower left shows a gene cluster involved in differentiation of myogenic cells (late up regulated), the center panel shows macrophage associated genes (early up regulated), and the right panel shows myofibrillar genes involved in myofiber structure/function (early down-regulated). (From Ref. 16,17.)

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genome, are created on a specific inbred mouse background, and thus there are ideal inbred controls. Knock-out or knock-in mice are very different. Chimeras are produced using ES cells of one strain, and blastocysts of another strain, with subsequent interstrain crosses to identify germ-line transmission of the transgene. Good parental or background strains are more difficult to obtain in knock-out or knock-in mice, due to the interstrain breeding of the technique. Thus, interindividual noise in mouse transgenic studies are different, dependent on the precise technique used. Rats and humans are generally outbred, leading to substantial interindividual variability that can greatly complicate the interpretation of microarray data. Techniques used to circumvent this problem includes the use of large numbers of individuals (replicates), or longitudinal studies (see section on time series studies, above). In the latter case, each individual serves as their own control, thereby effectively canceling out interindividual variation as a confounding variable. We have found that a major source of uncontrolled variability is tissue heterogeneity. In our studies of muscle biopsies, the variation of cell content and histopathology within a single biopsy can be quite large (21). Indeed, we found muscle biopsy tissue heterogeneity to often be a greater confounding variable than interindividual variation. One method we often use to mitigate tissue heterogeneity is to profile two independent regions of the same biopsy (3,20). This approach leads to two profiles from each subject, each derived from a different region of tissue. Another approach is to use laser capture microscopy (LCM) to obtain specific cell types or regions of a tissue. The next year should bring many reports of expression profiling applied to LCM samples from pathological tissue. The technical experimental aspects of generating expression profile data, such as RNA isolation and labeling, and chip hybridization, are relatively minor confounding variables (20). Key to generating good microarray data is adherence to strict quality control criteria, such as integrity of RNA, and in vitro transcription cRNA-labeling efficiencies. These are all explained in detail on a public web site (http://microarray.cnmcresearch.org/pgaoutlineqcofsamples.asp). Use of poorly preserved RNA (as from autopsies) typically makes it impossible to achieve subsequent quality control steps.

VIII. Data Visualization and Statistical Analyses There are many methods for data visualization and statistical analysis of array data, and the reader is referred to more systematic reviews of this topic elsewhere (see 24,25). As mentioned above, some of the most popular are visualization methods that serve to reduce the dimensionality and complexity of a set of microarray data so that variables can be visualized in a single two-

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dimensional picture. If noise can be from differentiated from signal, then a data set can be reduced to a more manageable size, while retaining most or all of the informative data points. However, most noise filters only reduce the number of genes studies by 50% (from 12,000 to 6000 when using an Affymetrix U74A mouse array and a 20% present call noise filter). Commonly used visualization techniques go much further, by reducing data to just a single graphed data point for each array (principal component analysis), or to a user-defined number of branches on a dendrogram (see Fig. 3 for one branch per array, and Fig. 4 for one branch per gene) (26). Methods that reduce dimensionality to a single point per array (multidimensional scaling, principal component analyses, singular value decomposition) (27–29) lose considerable information, and are best applied after a more statistically based method first selects those genes strongly associated with the biological variables under study. One recent method that combines statistical methods to reduce dimensionality, and then present principal components is VISDA (30,31). This ap-

Figure 5 Use of VISDA to reduce the dimensionality of a 27 time point microarray data set in muscle regeneration. The data set included two profiles per time point (54 U74A expression profiles) derived from distinct muscles. Methods for inducing degeneration/regeneration are the same as in Figure 5, although the time series was extended from 0–6 days (6 time points; Figure 5) to 0–40 days (27 time points). VISDA found evidence for 18 temporally defined clusters of genes, each with a membership of about 200–600 genes (from 12,000 genes profiled). (From Ref. 16.)

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proach uses soft bayesian statistics to evaluate the probability for certain numbers of clusters or components within a set of data, then selects the optimal number of clusters and presents visual representations of data based upon the best reduction in dimensionality. The appropriate visualization and statistical methods ultimately depend on both the desired outcome of the analysis and the integrity (and amount) of data being fed into the analysis. To provide one example, given a 27-timepoint time series data set with replicates at each data point for muscle regeneration, VISDA found support for 18 temporal clusters of genes, with membership of 200–600 genes (from 12,000 profiled, prior to noise filtering) (Fig. 5). These temporal clusters correspond to macrophage infiltration, muscle regeneration, and other groups evident in the smaller time series shown in Figure 4. If the number of replicates were increased, from two per time point, to five mice per time point, the ability of VISDA to identify support for clusters would increase, and more clusters would be identified. On the other hand, use of the smaller six time point study (Fig. 4) would lead to identification of far fewer clusters. This single example shows how increasing the resolution of the study six time points to 27 time points), and the replicates (statistical power) will change the data obtained from the expression analysis experiment. This same effect would be expected to be seen on all visualization and statistical methods. IX. Conclusion Expression profiling is a critical new tool to define biologically relevant pathways by assaying the steady-state mRNA levels of most genes in the genome. The power of the technology will be fully realized with very large, quality controlled data sets, with a focus on dense temporal series and longitudinal studies. These studies are only beginning to emerge in humans and rodent models. Key to realizing the potential of the technology is the development of public access databases with web analysis tools. Expression profiling should be considered a relatively insensitive tool for biochemical pathway construction, because the bulk of biological regulation takes place at the protein level. Highly parallel technologies for protein analysis are many years from reaching the current state of the art available for mRNA and DNA analyses. References 1.

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10 Plasticity of Circulating Adult Stem Cells

TIMOTHY R. BRAZELTON and HELEN M. BLAU Stanford University School of Medicine Stanford, California, U.S.A.

The discovery that bone-marrow-derived cells can contribute to adult tissue is fundamentally exciting in that it demonstrates a previously unrecognized plasticity of cell fate in adulthood. In addition, if sufficiently robust, these findings could lead to novel therapeutic strategies to treat a wide variety of diseases and injuries. The focus of this chapter is on bone-marrow-derived stem cells (BMDC), which include all cell types that are in the marrow including hematopoietic stem cells (HSC), marrow stromal cells (MSC), and other subsets of cells whose identity is unknown or less clearly defined. The vagueness of this term is intentional because it reflects the fact that very little is known about the identifying characteristics of the BMDC that have been shown to contribute to nonhematopietic tissues in vivo. In this chapter, we first critically discuss several classic adult stem cell concepts, some of which may need to be re-evaluated in light of recent findings. We then discuss evidence for plasticity in differentiated adult cells (i.e., the existing evidence for changes in cell fate via transdifferentiation, fusion in vitro, and in local sites of regeneration in adult tissues). A brief but current review of tissue-associated stem cells in mammals in provided along with a discussion about methods currently used to identify these cells. 217

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In subsequent sections, we describe criteria for establishing that adult bone-marrow-derived cells are capable of cell fate changes. Evidence for plasticity of BMDC and their contribution to diverse adult tissues is summarized. Finally, we discuss two major mechanisms by which BMDC could exhibit such plasticity: a de novo cell fate change in response to the tissue microenvironment, and fusion to pre-existing cells (in vivo heterokaryons). Both mechanisms could be required for tissue maintenance or repair and could result in reprogramming of BMDC gene expression. These early mechanistic data suggest the novel and exciting possibility that BMDC are a back-up reservoir for tissue-associated stem cells in tissue regeneration following injury.

I. Classic Stem Cell Concepts Stem cells have long been regarded as cells capable of proliferation, selfrenewal, production of a large number of differentiated progeny, regeneration of tissue after injury, and flexibility in the use of these options (1). This functional definition has stood the test of time and continues to be supported by emerging research. However, over the past three decades stem cell research has resulted in the formulation of a number of additional concepts regarding adult stem cells: (1) they are tissue, specific; (2) they give rise to progeny through linear, irreversible differentiation pathways; and (3) they are undifferentiated (i.e., unspecialized), quiescent cells with distinct identities. Here we revisit these concepts in light of recent discoveries regarding the multi-tissue potential, or apparent plasticity, of adult stem cells. A. Are All Adult Stem Cells Tissue-Specific?

The evidence is clear that various tissues in adults contain stem cells that participate in tissue regeneration by replacing differentiated cells lost to physiological turnover or injury. Thus, it was believed for decades that each tissue has its own type of stem cell, generally referred to as a ‘‘tissue-specific’’ stem cell, that resides in that tissue and has regenerative potential limited to only that type of tissue (2). Examples include hematopoietic stem cells in the bone marrow that reconstitute the blood, satellite cells that give rise to muscle, stratum basalis keratinocytes that generate epidermis, and stem cells in crypts of Lieberku¨hn that give rise to intestinal epithelial cells. However, recent evidence suggests that cells distinct from tissue specific stem cells can also contribute to differentiated tissues.

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B. Can Stem Cells Give Rise to Progeny Only Through Linear, Irreversible Differentiation Pathways?

Much research has been dedicated to the elucidation of differentiation pathways for various types of stem cells. Limitations on lineage restriction and self renewal have classically been thought to increase as cells progress down differentiation pathways. The best documented example of this is the hematopoietic system (Fig. 1) (3–5). The HSC with long-term reconstituting capacity (LT-HSC) give rise to HSC with short-term reconstituting capacity (ST-HSC). The repopulating ability of ST-HSC appears identical to that of LT-HSC in the near term. However, transplanted populations of ST-HSC are able to generate sufficient number of blood cells for only limited periods of time (usually a few months) and, thus, the self-renewal abilities of ST-HSC appear to be limited. ST-HSC, in turn, generate multipotent progenitor cells which are unable to self-renew but are still capable of generating cells of all blood lineages. Multipotent progenitors give rise to committed progenitors that can give rise to only a limited range of cell fates. Examples include

Figure 1 Conceptual scheme represents the classic hematopoietic differentiation pathways in which mature cells arise from progenitors through a series of linear, irreversible steps.

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myeloid or lymphoid progenitors. As typically described, this process of increasing specialization and fate restriction progresses as hematopoietic cells become differentiated (Fig. 1). Furthermore, each of these steps has typically been characterized as irreversible, resulting in a cell with an increasingly limited capacity for self-renewal and a restricted repertoire of potential fates. These classic restrictions on cell behavior accurately describe the normal physiological expansion and specialization of hematopoietic progenitor cell populations in healthy adults, but recent data suggest that, in the context of severe cell loss or tissue damage, these restrictions on cell behavior may not be upheld. Thus, while the classic view provides a reasonable model of average cell behavior in healthy adults, it may need to be modified and expanded to accommodate the flexibility in individual cell behavior hypothesized to occur during disease or injury. The recent plasticity of cell fate observed in many experiments supports this hypothesis. C. Are Adult Stem Cells Undifferentiated (i.e., Unspecialized), Quiescent Cells with Distinct Identities that Allow Them to be Identified and Isolated?

It is important to note that the formal presence of stem cells is detected ‘‘after the fact’’ by the demonstration of stem cell function (i.e., repopulating ability) within a given cell population. Various surrogate methods for identifying stem cells prospectively have been devised including the expression and/or absence of specific membrane proteins, dye exclusion properties, or morphological characteristics. In most cases, these methods identify populations of cells enriched for stem cells. They have not usually been shown to be capable of prospectively isolating a particular stem cell. In other words, with the possible exception of LT-HSC (discussed below), at this time it is not possible to predict whether any given individual cell is in fact a stem cell based solely on its static characteristics. A more critical point is that although populations enriched for stem cell capacity can clearly be selected, these populations typically fail to capture all of the stem cell capacity. In other words, cells outside of the selected population are also able to carry out stem cell functions, suggesting that not all stem cells from a given tissue compartment necessarily have the same static characteristics. This has typically been attributed to contamination of the non-stem cell population with true stem cells during the cell sorting procedure, but this supposition has not been directly demonstrated experimentally. Indeed, little evidence exists favoring the homogeneity of stem cell populations, whereas several reports document heterogeneity in the population of cells containing regenerative capacity (6–9).

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II. Plasticity of Differentiated Adult Cells Other than BMDC A. Plasticity Resulting from Transdifferentiation

That adult cells can change their fate has been known for decades. In rare cases, transdifferentiation occurs spontaneously in vertebrates, for instance, when melanin-producing iris cells become crystallin-producing lens cells in newts (10,11), or when mouse esophageal smooth muscle converts to skeletal muscle (12). More often, experimental manipulations, such as cell fusion or transplantation, have been used to reveal this otherwise concealed potential for cellular plasticity. In Drosophila, serial transplantation of imaginal discs to alternative body sites led to the generation of diverse structures in which discs destined to become genital structures instead first generated leg, head, or wing structures (13,14). Interestingly, the sequence of transformations was not random, but instead precisely specified, suggesting that a progression through diverse differentiated states was necessary. Furthermore, this progression was always precisely specified and could be mimicked with mutations in homeotic genes (15). B. Plasticity Demonstrated by Cloning

Cloning experiments in amphibia first demonstrated that cells from differentiated adult tissues could yield nuclei that upon transplantation into enucleated eggs could give rise to entire organisms (16). More than three decades later, cloning was achieved in mammals, leading to Dolly the sheep (17), and more recently cloned mice (18,19). These studies provided the first evidence that in most cells the entire genome is maintained, even when only a subset of genes is expressed upon differentiation. Nonetheless, it was unclear from such cloning experiments as to whether progression through the egg was required to allow the cell to adopt a new fate. C. Plasticity Resulting from Cell Fusion in Vitro: Heterokaryons

Experimentally induced fusion of two distinct types of differentiated cells to form stable heterokaryons demonstrated that exposure to a novel cytoplasmic environment alone was sufficient to reprogram the genome of a terminally differentiated cell. For example, when differentiated mouse muscle cells were fused with human primary diploid cells isolated from all three embryonic lineages (hepatocytes, keratinocytes, and fibroblasts), muscle gene expression in the nonmuscle nuclei was rapidly induced (20–23). Moreover, this expression of previously silent genes did not require cell division or DNA replication and the changes in chromatin structure associated with these events (24).

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Similarly, in heterokaryons formed from the fusion of fibroblastic cells with erythroid cells, hepatocytes, or pancreatic cells, expression of differentiationspecific genes characteristic of the fusion partner was observed (25–27). Differences among cell types, in the frequency and rate of gene activation and in the ratio of activator to responder nuclei required, suggested that the underlying molecular mechanisms differed (22–24). However, a unifying principle emerged. These experiments demonstrated that the differentiated state in adult mammalian cells was not fixed and irreversible, but was regulated by a dynamic active process requiring continuous regulation (28,29). At any given time, te differentiated state was dictated by the balance of regulators present in the cell, which was a function both of that cell’s history and its environment. D. Plasticity Associated with Regeneration

Another unexpected finding was the observation that the terminal differentiation of cells, such as multinucleate skeletal muscle cells, appears to be reversible. It has long been known that amphibians such as urodeles are capable of regenerating entire limbs following amputation. Recently a protein factor was defined in extract from regenerating newt limbs that was capable of inducing the fragmentation of multinucleate myofibers into mononucleate myoblasts that, in turn, were capable of regenerating skeletal muscle (30). Another recently discovered factor, myoseverin, obtained from a random screen of peptides, had a similar effect on a myogenic cell line, C2C12 (31). Most remarkable was the demonstration that retroviral expression of the homeobox gene, msx-1, caused multinucleate nondividing myotubes to become mononucleate mitotic cells capable of differentiation into fat, chondrocytes, and osteoblasts in the appropriate media (32). Furthermore, msx-1 was found to be expressed naturally at sites of regeneration in newts and mice (33,34). It remains to be shown whether a similar reversal of differentiated muscle fibers into mononucleate cells occurs in the living mammal.

III. Identification of Hematopoietic and Other Types of Stem Cells The best-characterized stem cells are the long-term hematopoietic stem cells (LT-HSC), which have the ability to reconstitute the hematopoietic system. Many of the methods currently being used to identify stem cells were first established to study hematopoietic stem cells. The majority of HSC enrichment protocols rely on fluorescent-activated cell sorting (FACS), which

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allows cells to be selected based on the expression of a set of membrane proteins. Most protocols use lineage depletion panels (Lin( )) to exclude cells expressing a protein characteristic of a mature cell. Antibodies frequently included in Lin( ) panels include those against CD3e, CD4, CD5, CD7, CD8a, CD10, CD11b/Mac1, CD14, CD15, CD16, CD19, CD20, B220, Gr-1 (Ly-6G), and/or TER-119. The difference between negative and low expression of many of these proteins can be subtle and, as a result, the cells obtained by lineage depletion can be somewhat investigator-dependent. This has resulted, in some cases, in disagreements about whether LT-HSC express low levels of some lineage markers or lack them altogether. Despite considerable effort, there remains a paucity of markers that positively identify LT-HSC, which are the best characterized stem cells to date. Proteins that have been used to identify human LT-HSC include CD34, AC133, and Thy-1 and those used to identify murine LT-HSC include Sca-1, c-kit, Thy-1.1, and CD34. Even the most rigorous isolation protocols currently available result in heterogeneous populations that are enriched for HSC but in which many of the cells fail to demonstrate multipotency and/ or long-term reconstituting ability. An alternative method to identify both murine and human HSC was recently developed based on the differential retention of the DNA-binding Hoechst 33342 dye in the presence of the drug verapamil, which blocks dye efflux (35). A cell population capable of increased efflux in the absence of drug has been found in multiple tissues including bone marrow, muscle, neural, and epidermal basal cells (36–38). Termed the side population (SP), these cells are characterized by their pattern of fluorescence when observed in both far red ( > 675 nm) and blue (450 nm) emission channels after excitation at 351 nm. The SP population has been isolated from mouse and human bone marrow and contains LT-HSC expressing completely absent or low levels of CD34 (39). Additional techniques are emerging to identify stem cells from large populations of tissue-derived cells. For example, following proteolytic dissociation of brain tissue, populations enriched for neural stem cells can be obtained based on differential density in a sedimentation gradient (40). New markers may be identified through microarray techniques and the increasing use of differential display of peptide libraries to find proteins that bind to cell surface epitopes, as well as through continuing attempts to identify molecular components that induce pluripotent cells to differentiate or self-renew. Clearly, new markers are needed that identify stem cells and distinguish them from other cells. On the other hand, depending on the definition of stem cells, specific markers may not be constitutively expressed, making their prospective isolation difficult.

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That so-called ‘‘tissue-specific’’ stem cells (which are not necessarily tissuerestricted and are therefore referred to in this review as ‘‘tissue-associated’’) reside in many adult tissues has been clearly documented even though their specific identities are, in most cases, not characterized sufficiently to allow prospective isolation. These cells are responsible for regenerating damaged tissue and maintaining tissue homeostasis, for example, physiological replenishment of skin and blood cells. In some cases, such as hematopoiesis, the stem cells can be highly enriched and markers that distinguish these cells have been well characterized for that purpose (3). In other cases in which extensive regenerative capacity of the stem cells has been documented, such as in the liver, stem cell markers are virtually nonexistent. The following sections summarize data on selected samples of these tissue-associated stem cells complete reviews of each exist. The goal is to highlight areas that warrant further research and to put these findings in the context of the new non-tissueassociated stem cell findings described below. A. Hematopoietic Stem Cells

The best characterized tissue-associated stem cells in adults are undoubtedly the multipotent HSC, which have the ability to reconstitute all cells of the blood (3,41). Two classes of HSC have been defined—ST-HSC and LTHSC—that are capable of reconstituting the blood of mice for 2 and greater than 6 months, respectively (42,43). FACS enrichment protocols for LT-HSC allow the isolation of stem cell populations in which more than 80% of the cells have the potential to reconstitute the blood (41). Some investigators have claimed the prospective isolation of LT-HSC but these findings are generally complicated by the reliance on a statistical argument to interpret experimental results: If a single, isolated cell is in fact an LT-HSC, then by definition it should be able to reconstitute fully the entire hematopoietic system of a mouse in which the marrow was ablated. In fact, only one out of every 10–30 mice injected with a single cell survives (44,45). The explanation is that approximately only one in ten LT-HSC injected in vivo reach the bone marrow compartment and are thus able to participate in hematopoiesis. Cell trafficking studies of intravascularly injected cells support this explanation; nonetheless this limitation poses a technical barrier to proving definitively the prospective isolation of LT-HSC. Moreover, since most of the protein markers used to identify HSC are not essential to stem cell function, the expression of these proteins may not directly correlate with stem cell potential. For example, C34 expression on LT-HSC has been found to be reversible and dependent on not only the activation state of the cells but also the developmental age of the donor (46–

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49). Nonetheless, the hematopoietic stem cell clearly can be highly enriched up to 10,000-fold, delivered to marrow-ablated recipients, and fully reconstitute the blood of experimental animals and humans (50). B. Neural Stem Cells

The discovery that stem cells exist in the adult brain was quite unexpected and required years of investigation to become widely accepted. It was long thought that damage to the brain could not be repaired, since adult central nervous system (CNS) cells were not thought to divide. A series of studies in rats and in songbirds first revealed that somatic cells from adult brains could be formed anew (51,52). Additional studies by a number of investigators have now confirmed that mammalian adult neuronal progenitors are capable of extensive cell division and self-renewal (53–56). Moreover, neural progenitors can migrate and home to specific sites of damage or regeneration, for instance, to the olfactory bulb of rodents (57,58) the hippocampus of humans, (55,59) and sites of tumors such as gliomas (60). Thus, stem cells from the CNS provide a second source of wellcharacterized tissue-associated stem cells. That they are indeed stem cells is clear from the fact that in tissue culture, as well as following direct injection into brains, clones from the neural stem cell (NSC) population give rise to all three major types of cells characteristic of the CNS: neurons, astrocytes, and oligodendrocytes (55,56). These NSC progeny have typical morphologies, characteristic patterns of protein expression, and exhibit physiological evidence of function (61). Nonetheless, despite the extensive characterization of these cells, existing surface markers allow for only a 40-fold enrichment of neural stem cells from embryonic brain (62). C. Hepatic Stem Cells

Following a partial hepatectomy, in which two-thirds of the liver of a rat is removed, the remaining lobes enlarge over 5–7 days until the liver reaches its original mass. In contrast to bone marrow or skin, in which a relatively small population of cells undergoes massive expansion to support regeneration, liver regeneration following partial hepatectomy involves a modest proliferation by a variety of differentiated cells including hepatocytes, biliary epithelial cells, fenestrated endothelial cells, and hepatic stellate cells. Furthermore, serial transplantation of typical hepatocytes from adult donors indicates that these cells have a tremendous proliferative potential. In one study, hepatocytes gave rise to a minimum of 69 cell doubling or a 7.3  1020-fold expansion (63). However, following some types of injury that compromise hepatocytes, a smaller portion of stem-like cells from the bile ducts gave rise to a proliferation of oval cells that subsequently generated hepatocytes and

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ductular cells (64–66). Thus, in the context of generalized hepatocyte injury, the presence of a small population with substantial regenerative potential was revealed. It has also been observed that ductal epithelium is required for oval cell proliferation (67). These data, taken together with morphological studies of hepatocellular regeneration following injury, have led some to argue that the true liver stem cells with multilineage potential reside in or near the terminal bile ductules (canals of Herring) (68,69) and that these cells give rise to oval cells. However, the possibility that the ductal epithelium is not the source of oval precursors, but is instead required to play a supportive or inductive role, cannot be ruled out. Yet another source of hepatic stem cells is the islets of Langerhans of the pancreas, which it is presumed, due to their common embryonic origin, can give rise to hepatocytes as well as the endocrine and exocrine cells of the pancreas (70). D. Skeletal Muscle Stem Cells

The satellite cell is defined as a quiescent mononucleate cell ensheathed in a membrane juxtaposed to a multinucleate muscle fiber. Such cells were thought to constitute a reserve for muscle regeneration (71). Numerous studies showed that satellite cells could be activated, induced to proliferate, and contribute to intact skeletal muscle fibers (22,72–75). Moreover, these cells could be separated from single fibers, plated in culture, and induced to divide (76). In a genetically deficient animal (the mouse equivalent of Duchenne muscular dystrophy), the satellite cells were usurped early in life. In addition, the Pax 7 knock-out mouse, which reportedly lacks satellite cells, is capable of regenerating muscle damage and is thought to have other Pax7/ cells that can act as satellite cells (77). Evidence is also accumulating that satellite cells are heterogeneous in the genes they express and consequently the markers of such cells are not consistent (78). Thus, it is clear that tissue-associated stem cells exist in skeletal muscle that can contribute to muscle growth and repair, decline in number and proliferative capacity with age, and are depleted in patients with chronic muscle degenerative diseases such as Duchenne muscular dystrophy (79,80). Markers for the prospective isolation of proliferative myoblasts as well as for at least a subset of satellite cells are well characterized by contrast with other tissues (78,81–84). Thus, muscle is an advantageous tissue for study since tissue-associated stem cells can be prospectively isolated and their progression to mature myofibers can be monitored in vitro and in vivo (83,84). E. Pancreatic Stem Cells

Endocrine cells of the rat pancreas, including h-islet cells, turn over every 40– 50 days. Until recently, replacement of endocrine cells was believed to derive solely from proliferation and differentiation of ductal epithelial progenitor

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cells (85). However, a subset of cells in the islets of Langerhans that express nestin in adult rats has now been found to be able to give rise to cells of the endocrine and exocrine pancreas as well as to cells expressing hepatocyte specific genes (70). Even after long periods of culture and multiple clonal passages, these nestin-positive cells maintain their multipotentiality (70). F. Skin Stem Cells

Both the epidermis and hair follicle require stem cells to support high rates of epithelial turnover. Many considered the epidermis and hair follicle to be distinct tissue compartments, each with their own stem cell (86). However, it now appears that a single stem cell can give rise to both. Studies of severe burn injury revealed that keratinocytes can migrate from the hair follicles to regenerate the epidermis (87). Recent data suggest that the keratinocyte stem cells for both epidermis and hair follicles reside in a specific portion of the follicular epithelium, the bulge zone, where they cycle slowly as what are termed label-retaining cells, express keratin K5 and K14, and generate progeny to replenish the epidermal basal layer (88,89). These slow cycling bulge cells can be selected by flow cytometry based on high expression of a6 integrin and low to null expression of CD71, the transferrin receptor (90). However, in some areas of skin that lack hair follicles (palms, soles), the epidermal stem cells reside directly in the epidermis. Debate persists regarding the keratinocyte stem cells in such nonhairy skin (88). Thus, recent data suggest that in hairy skin, a single group of stem cells, the bulge cells, regenerate both the epidermis and follicular epithelium. G. Summary

Stem cells within various tissues have been extensively studied and, in some cases, a great deal is known about their capacities to participate in the regeneration of their tissue of origin in adults. However, emerging data indicate that adult stem cells may not be limited to performing regenerative roles in only one tissue. Furthermore, many of the concepts developed to understand stem cells within a single tissue compartment may need to be expanded to accommodate recent discoveries that at least some adult stem cells exhibit a surprising degree of plasticity.

V. Criteria for Establishing Cell Fate Changes Although many types of cell fate conversions have recently been described, here we focus particularly on the evidence that bone marrow cells can contribute to nonhematopoietic tissues. The criteria used to determine if a cell has actually changed its fate are critical to the interpretation of such experiments.

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Tracking of cells typically involves the use of certain markers such as the Y-chromosome when male cells are introduced into female animals, green fluorescent protein (GFP), or beta-galactosidase (h-gal). But even the expression of genetic markers may require cautious interpretation. For example, in experiments using ROSA mice (a common bone marrow transplant donor mouse strain that ubiquitously but weakly expresses h-gal), pH changes that enhance the bacterial ROSA h-gal signal also enhance the signal from endogenous mammalian h-gal. Thus, it is preferable to use markers that have no endogenous counterparts, such as the Y-chromosome or GFP. Several levels of scrutiny are required to prove conclusively that a cell fate transition has occurred. The lowest level of scrutiny, but the easiest to achieve experimentally, is the demonstration that one or more previously silent genes believed to be indicative of a specific cell type are newly expressed in the cell of interest. The expression of multiple proteins indicative of a distinct cell type is clearly more convincing than the expression of only one identifying protein. Taken even further, a true cell fate transition should result not only in the gain of new proteins characteristic of the new cell identify but also, the loss of proteins associated with the previous cell identity, since this observation suggests ‘‘reprogramming’’ or in other words, that a program of regulated gene expression has been activated. A. Physiological Relevance

When identifying cells based on protein expression, the context of the cell fate transition is important. Cells respond to the environment in dynamic ways and, in addition, cells with distinct phenotypes often make use of conserved signaling elements. Given this premise, how difficult is it to change the protein expression pattern of a cell if it is placed in an abnormal environment? The environment need only be unusual for that particular cell type. As a result, placement of a cell in an unusual tissue compartment in vivo, or by exposure to unexpected or superphysiological levels of intra- or extracellular signaling molecules in vitro, may suffice. The actual propensity of a cell to express abnormal proteins that could be interpreted as a cell fate transition is most likely dependent on several factors, including the cell itself, the environmental signals, and the proteins being assayed. As a consequence, assessment of such results needs to occur on a case-by-case basis. B. Colocalization of Markers

In order to demonstrate a cell fate conversion it is essential to document definitively that two or more molecule are co-expressed in the same cell. This can be achieved, for example, using a cell tracking marker such as GFP and an identifying protein such as myosin. Typically one or more antibodies are used in conjunction with appropriate microscopic methods or fluorescence-acti-

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vated cell sorting (FACS). The advantage of microscopy is that cell morphology can be evaluated whereas, the advantage of the FACS is that large cell populations can be analyzed and quantified rapidly. Special microscopic techniques are required to demonstrate protein coexpression in a single cell in a field of similar cells, since it is essential that the uncommon occurrence of cells that overlay or wrap around one another not be mistaken as evidence for protein co-localization. This is particularly important in the adult stem cell field because cell fate transitions can be extremely rare events. Thus, the use of laser scanning confocal or deconvolution microscopic methods that resolve optical sections with a depth of less than 1 Am are required. C. Integration into Tissue

A third criterion is that the cells need to appear well integrated into the tissue. That a cell has arrived at its destination, was incorporated into the tissue structure, is morphologically indistinguishable from its neighbors, and expresses new proteins typical of other cells in its microenvironment provides strong evidence of a cell fate conversion. D. Demonstration of Function

The fourth and most stringent criterion for demonstrating a change in cell fate is a functional assay. An example is the production of a missing enzyme or other molecule specific to a particular organ in a genetically deficient animal that rescues it from lethality or ameliorate a deficit that is disease-related. A demonstration of function in the heart would constitute showing that the BMDC not only express cardiac-specific proteins but also contract in a cardiodynamically beneficial manner and possibly even improve cardiac function following an injury such as a myocardial infarction. For neurons of the CNS, the ability of a stem cell to participate actively in a neuronal circuit by generating action potentials in response to appropriate signals and producing synaptic potentials indicative of connectivity with other neurons would provide strong evidence that these cells can functionally repopulate the adult brain. This last requirement (electrophysiological evidence and function in the intact brain) has only been reported for neural stem cells (61); for BMDC such a demonstration awaits development of more efficient methods for identifying BMDC-expressing neuronal proteins in intact CNS tissue.

VI. Plasticity: Bone Marrow-Derived Cells Contributing to Adult Tissues In Vivo Although the evidence for lineages of tissue-associated stem cells remains strong, recent studies indicate that the potential of at least some stem cells may

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not be limited to generating cells of the tissue compartment in which they reside. In particular, it appears that bone marrow contains cells that can circulate in the blood and are capable of previously unrecognized plasticity. The results documented below provide clear evidence that BMDC can give rise to cells typical of other tissues. Thus, the accumulating evidence suggests the existence of stem cell populations that are not tissue-specific but instead have the capacity to generate multiple cell types that exist in diverse tissues. In the bone marrow, stem cell populations capable of generating various cells types can be derived from either the hematopoietic or stromal compartments. Given that the blood has access to and is essential to all tissues of the body, it is not surprising that the circulation appears central to stem cell movement to disparate tissues. That such cell fate transitions may be an ongoing physiological process in adults is suggested by the fact that most of the studies cited below utilized direct transplantation of adult cells not exposed to tissue culture. In all cases, the results suggest that BM-derived stem cells can undergo a process entailing migration, conversion to a new phenotype, and expression of functions characteristic of the new tissue in which they reside. A. General Transplantation Protocol

The recently reported studies that demonstrate bone marrow giving rise to cell types typical of other tissues in muscle, brain, heart, epithelium, and liver have generally used a similar protocol. Bone marrow cells from a genetically marked adult mouse are delivered intravascularly into isogeneic lethally irradiated normal adult hosts. The bone marrow cells were either taken from transgenic donor mice that constitutively expressed either GFP or h-gal in all of their cells or from a male mouse, which, following the cells’ transplantation into female mice, could be detected based on the Y-chromosome. Following an ablation procedure (often lethal irradiation) to remove endogenous bone marrow cells from the recipient, injected BMDC were able to rescue the recipients by reconstituting all the cell lineages of blood. The success of a bone marrow transplant can be therefore be ascertained by survival of the animal and the degree of chimerism in the blood: the proportion of the cells in the circulation of the recipient that express the genetic marker of the donor as determined by microscopy, FACS, or fluorescence in situ hybridization. Four to eight weeks are usually required to reconstitute the blood in adult mice (8– 10 weeks of age) with donor cells, and detection in the tissue of interest typically requires several additional weeks or months. B. Skeletal Muscle

In the first demonstration that BMDC could contribute to skeletal muscle, whole bone marrow cells from a transgenic donor mouse line were used in

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which a lacZ gene encoding a nuclear h-gal under the control of the musclespecific myosin light chain-3F promoter was used (91). Following a bone marrow transplant with cells from this donor, h-gal was detected histochemically in chemically damaged muscle fibers. Subsequent reports (37,92) showed that genetic damage characterized by chronic muscle degeneration also resulted in BMDC that contributed to host muscle fibers. Following transplantation of male marrow into irradiated female mdx mice (the mouse model of Duchenne muscular dystrophy), male muscle cells produced musclespecific transcription factors myf5 and myogenin, and the missing dystrophin protein as shown by fluorescence or laser confocal microscopy (37,92). Thus, in response either to chemically or genetically induced damage, the BMDC were capable of migrating, dividing, committing to the myogenic lineage, and fusing with host myofibers. Recent work by LaBarge and Blau (84) extended these observations by demonstrating for the first time (for any tissue) that BMDC generate the mature, differentiated cells of a nonhematopoietic tissue by first directly contributing to the tissue’s own stem cell population. They demonstrated that BMDC generated satellite cells (the stem cell of skeletal muscle) and that these bone-marrow-derived satellite cells were capable of contributing to mature skeletal myofibers. Critical to the participation of BMDC in each of these steps were two sequential tissue damaging injuries. It remains to be determined whether BMDC will follow known stem cell pathways for tissue regeneration in other tissues as well. Whether all BMDC replenish tissuespecific stem cells en route to tissues remains to be determined. C. CNS

Two studies have shown that intravascularly delivered BMDC could give rise to cells with neuronal characteristics in the CNS of mice (93,94). In one case, GFP-labeled BMDC were detected primarily in the olfactory bulb of adult mice (a site of extensive regeneration), suggesting that homing to this site was physiological. These cells integrated into the tissue of the CNS and co-expressed GFP as well as one more neural-specific markers characteristic of the brain (NeuN, class III beta tubulin, and 200 kilodalton neurofilament) as determined by laser confocal microscopy using sub-micron-thick optical sections. The second study used as recipients PU.1 knockout mice that are unable to survive for more than a week after birth without a bone marrow transplant. Male-derived BMDC introduced intravascularly into neonatal female mice rescued these animals from death and resulted in Y-chromosomelabeled neural cells expressing NeuN and a neuronal-specific enolase in the hippocampus of the brain. Subsequent work has demonstrated that BMDC are able to contribute to a specific type of neuron, the Purkinje, in both mice and humans (95–97). The underlying mechanism is as yet unclear.

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Although lacking tissue-associated stem cells, evidence has been reported for the contribution of BMDC to the myocardium via either the circulation or direct intracardiac injection (92,98,99). However, a controversy exists as to the frequency with which this occurs. Orlic et al. (98) reported that following experimentally induced myocardial infarction in mice, direct injection of BMDC into the peri-infarcted left ventricle resulted in replacement of dead myocardial tissue by BMDC that subsequently expressed of a number of cardiac muscle-specific proteins. To date, others have been unsuccessful in replacing large portions of the ventricular wall with BMDC. In contrast, a number of investigators have observed rare cardiomyocytes (0.01–0.05%) of noncardiac origin following bone marrow or heart transplantation (92,99,100). Notably, following human cardiac transplantation, three groups have shown the presence of cardiomyocytes of recipient origin indicating that the mouse models are likely to be predictive of human biology in this regard. However, in contrast to these publications, two other reports indicated that no cardiomyocytes of recipient origin were present, although one of these papers was successful in identifying recipient-derived smooth muscle cells in the graft (101,102). Thus, considerable debate exists regarding this phenomenon. Several additional studies question whether BMDC can contribute to cardiomyocytes in vivo, based on the evidence that new cardiomyocytes are not formed in adulthood in mammals. Although studies have found that cardiomyocytes do divide in adulthood, this event was found to be exceedingly rare (0.0005% in normal myocardium, 0.008% in injured) (103). Other publications have questioned even these rare divisions by demonstrating that while cardiomyocytes do rarely synthesize new DNA detectable by markers of division such as the incorporation of Brd-U, this often results only in an increase in nuclear ploidy but not a true cell division event (104). Although cardiomyocyte division may be a very rare event in normal hearts, this may not be the case following injury. In the borders of infarcts in human hearts, it was recently demonstrated that up to 4% of cardiomyocytes express Ki-67, a marker of proliferation, and mitotic spindles, indicating an unexpected degree of cardiomyocyte proliferation following cardiac injury (105). Furthermore, following ventricular resection in a nonmammalian organism, the zebrafish, proliferation of up to 32% of cardiomyocytes in the border of the injury site was sufficient to repair the damaged myocardium (106). Learning the reasons for these distinct biological responses may prove useful in developing strategies to enhance cardiomyocyte regeneration in mammals. Irrespective of the above controversy, multiple groups have observed dramatic and convincing improvements following either intravascular or

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direct intracardiac injection of BMDC after myocardial infarction (107–113). Although these functional improvements were initially attributed to regeneration of the myocardium by BMDC, several recent papers have provided convincing evidence that the functional improvements resulted from improved angiogenesis in the cardiac lesion (109,111,113–115). The response was likely directly mediated by BMDC, which were found to produce several angiogenic factors in the lesion site including fibroblast growth factor-2, vascular endothelial growth factor, angiopoietin-1, interleukin-1h, and tumor necrosis factor-a (109,110). In fact, the degree and organization of the angiogenic response to administered BMDC were as good or better than those of many angiogenic therapies currently under clinical investigation. Thus, intramyocardial or intracoronary administration of isolated BMDC may be an advantageous means to induce therapeutic angiogenesis following myocardial infarction. E. Liver

HSC were found to contribute to progenitor oval cells in the liver. This is not surprising, since oval cells share several characteristics with HSC (116–119). Various investigators tested whether HSC could contribute to liver regeneration. When mice that had received a gender- or strain-mismatched bone marrow transplant were treated with drugs that both block hepatocyte proliferation and induce hepatic injury, BMDC Thy-1(+) cells that were presumably oval cells appeared by 9 days after liver injury. By day 13, BMDC hepatocytes expressing the mature, hepatocyte-specific markers H4 and CCAM were present (120). Perhaps the most robust and well-defined demonstration of bone marrow regeneration of any nonhematopoietic tissue to date derives from studies in which HSC not only regenerated large portions of liver but functioned in their new site, rescuing mice from death due to genetic liver disease. Lagasse and co-workers (121), using a different experimental paradigm that resulted in sustained selective pressure for wild-type hepatocytes, transplanted wild-type HSC into lethally irradiated mice deficient in fumarylacetoacetate hydrolase, a model of fatal hereditary tyrosinemia type I liver disease. Under this selective pressure, BMDC hepatocytes accounted for 30– 50% of the liver mass by 7 months after transplantation, and multiple liver functions including the missing liver hydrolase were restored to near wildtype levels, leading to survival (121). Although injury to the tissue probably played a role in the robustness of the response and generation of large numbers of marrow-derived hepatocytes, liver repopulation by marrow-derived cells occurred even in the absence of injury, albeit at a lower frequency (120). In humans, marrow-derived cells also gave rise to substantial numbers of both

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hepatocytes and ductular cells, which are evident in livers from patients who received gender-mismatched bone marrow or liver transplants (122,123). Thus, liver regeneration by marrow-derived cells occurred with minimal injury or even during normal physiological maintenance (i.e., in the absence of strong selective pressure) in both mice and humans, providing a source of hepatic cells not currently available in tissue culture. F. Epithelium

Bone marrow transplantation experiments have provided clear evidence that BMDC migrate to and reside in various types of epithelium. In addition, several groups have reported that some of these epithelially located BMDC express characteristic epithelial proteins, particularly cytokeratins. In the lung, BMDC develop a morphology and protein expression pattern typical of respiratory epithelial cells in the alveoli and bronchi (124– 126). Of particular interest was the finding that type II pneumocytes, identified by their production of surfactant B, were particularly likely to have come from BMDC. Early reports of BMDC contributions to gastrointestinal epithelium in mice have been followed by reports of this same phenomena in humans (127,128). Okamota et al. (127) demonstrated in a group of female patients who had received male bone marrow that male BMDC expressed cytokeratin, indicative of their epithelial identify, but did not express CD45, a marker of blood cells, in the esophagus, stomach, and small and large intestines. Two reports, one in mice and one in humans, have observed that BMDC are found in the follicles and dermis of the skin where they expressed multiple cytokeratins but lacked expression of lymphocyte or macrophage markers (125,128), suggesting that they had adopted an epidermal fate. Cells isolated from adult rat liver, when cultured in a high-sugar medium, differentiate into cells with the essential features of pancreatic islet cells, including the production of a large number of characteristic proteins (including insulin) and the ability to reverse hyperglycemia after injection into NOD/scid mice (129). Thus, if liver cells can generate pancreatic islet cells and BMDC can generate liver cells, it is tempting to speculate that under the right conditions pancreatic islet cells could be derived from BMDC. To date this has not been demonstrated, but several laboratories are pursuing this exciting possibility. G. Marrow Stromal Cells

In addition to circulating HSC and possibly other yet-uncharacterized BMDC, there is a noncirculating bone-marrow-derived cell population with remarkable plasticity: the MSCs. MSCs line the bone marrow cavity and

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support hematopoiesis by providing critical growth factors. MSCs have recently been shown both in culture and following injection into particular tissues in mammals to give rise to a range of mesenchymal cell types, including chondrocytes, osteoblasts, adipocytes, cardiac, skeletal and smooth muscle cells (130,131), as well as cells typical of the CNS (132–134). MSC have been extensively reviewed elsewhere (135,136) and are not included in our review.

VII. Potential Mechanisms of Stem Cell Plasticity It is not difficult to imagine a variety of mechanisms by which adult BMDC could contribute to various nonhematopoietic tissues. The two most discussed mechanisms are de novo cell fate transitions and cell fusion. Cell fate transitions termed de novo are simply those that are cell inherent, relying only on the cell’s responses to extracellular signals to elicit a change in the pattern of gene regulation and cell identity. The two major hypothesized types of de novo cell fate transitions are transdifferentiation and dedifferentiation. Transdifferentiation, strictly defined, describes the direct conversion of one differentiated cell type into another without an intermediate state that lacks most characteristics of either differentiated state. An alternative to transdifferentiation has been loosely termed dedifferentiation, by which most investigators mean that a cell with a mature differentiated phenotype loses these characteristics, becoming undifferentiated or more stem-cell-like before entering a standard differentiation pathway to assume a new cell fate. Whether a hypothesized cell fate transition is best described as de- or transdifferentiation is often subjective: in reality, there may be a spectrum of cell fate transitions between these alternative descriptions. At this time, few experimental data exist to substantiate either hypothesized transition mechanism for the contribution of BMDC to adult tissues. Indeed, a distinct possibility is that cells within the bone marrow are not predestined or specialized to regenerate a specific tissue and may comprise the quintessential pre-tissue-specific adult stem cell. A. De Novo Cell Fate Change

Recent work by LaBarge and Blau (84) provides the first direct evidence that de novo mechanisms are involved in at least one cell fate transition in vivo: the contribution of BMDC to skeletal muscle. Following bone marrow transplantation with GFP-expressing bone marrow, GFP-expressing (and thus bone-marrow-derived) satellite cells were observed. Following isolation of satellite cells from skeletal muscles in these mice, a karyotypic analysis of the chromosome numbers in individual GFP-expressing satellite cells revealed

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that the population of cultured satellite cells was diploid. This important observation indicates that a reprogramming of cell fate occurred in response to the microenvironment or niche following damage induced by irradiation. Thus, their paper provides data that support a de novo mechanism of adult stem cell plasticity in skeletal muscle. B. Cell Fusion

Evidence for the other major potential mechanism of cell fate transitions, cell fusion, has also received support recently. The first direct reports of fusion demonstrated only that embryonic stem cells fused at very low frequency with either adult bone marrow or neural progenitor cells in culture under strong selective pressure. Results were intriguing but their applicability to adult stem cells in vivo remained unknown (137,138). Recently it was found that most liver hepatocytes with a genetic contribution from a BMDC had a chromosomal make up most consistent with a fusion-mediated mechanism of cell fate change (139,140). While studying the human CNS, Weimann and Charlton et al. (95) documented a Purkinje neuron in a human female recipient of male bone marrow that contained one Y and two X chromosomes. Because the thin paraffin sections they evaluated included less than half of the total volume of the Purkinje nuclei, this finding is indicative of a mechanism by which a BMDC fused with an existing Purkinje cell. Indeed, since each Purkinje cell has, with millions of connections to other neurons in the human adult brain, it would seem to be a cell type with such complex morphology that de novo regeneration in adult life would be prohibitive. Accordingly, a cell rescue mechanism could be envisioned by which fusion may be an advantageous mechanism for maintaining and repairing damaged cells that are morphologically complex or otherwise difficult to replace. According to this hypothesis, cell fusion might yield in vivo heterokaryons that allow the damaged cell to recover and continue to function (141). Thus, the current data suggest that at least two mechanisms of cell fate transition may occur, dependent upon the specific cells and fate transitions involved. This also suggests a complex system of cellular regeneration and rescue that remains to be elucidated. VIII. What is a Stem Cell and How Do BMDC Expand Classic Stem Cell Concepts? Since cells from a single tissue compartment, the bone marrow, can contribute to various types of tissues, it is tempting to speculate that a universal stem cell exists in adults. As discussed above, BMDC have been reported to give rise to

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several cell types including neurons, astrocytes, hepatocytes, epithelium, and skeletal and cardiac muscle (37,91,93,94,98,121,125,133,142). Given these findings, does a universal stem cell exist? If the stem cells in a given tissue have identical features, it should be possible to isolate these cells prospectively. This has proven difficult with LTHSC, and even among tissue-specific stem cells there is often heterogeneity within populations of stem cells. For example, recent evidence in skeletal muscle suggests that satellite cells, the mononucleate stem cells characteristic of muscle, are heterogeneous with respect to the proteins they express (78). While few data exist that directly demonstrate the relative heterogeneity of the cells in a stem cell population, the data demonstrating the homogeneity of a stem cell population are equally inconclusive. Thus, whether the cells compromising a given stem cell population express similar sets of proteins remains an open question. It is also possible that a compartment of dedicated stem cells may not exist in perpetuity, as cells capable of becoming stem cells may perform other functions in the interim. A striking example of cells that appear to be well along a differentiation pathway from which they can turn back are oligodendrocyte precursor cells (OPC). OPCs routinely give rise to one of the three major neuronal phenotypes in brain, but committed OPCs can be isolated and induced to dedifferentiate into stem cells under one set of culture conditions. Under another set of conditions they can be reversed to differentiate once again into cells of the three main CNS lineages: neurons, astrocytes, and oligodendrocytes (143,144). Another example is the potential for differentiated multinucleate myotubes to give rise to mononucleate proliferative myoblasts, a property first discovered in newts (145). Such examples demonstrate a remarkable plasticity of ‘‘committed’’ or ‘‘differentiated’’ cells and how they can be induced to give rise to cells with more stem-cell-like properties. The accumulating evidence raises the possibility that many types of cells from distinct tissues displaying varying degrees of differentiation could potentially be recruited to function as stem cells, particularly in times of increased regenerative need such as following injury. Thus, the ability to act as a stem cell may be a cellular function shared by numerous cell types expressing diverse sets of genes. Just as most cell types in the body are able to engage in an apoptotic program in response to specific types of damage, many diverse cell types in the body may be able to engage in stem cell functions. Consistent with this suggestion, it is not necessary that the potential for stem cell function be equal among all cells. The propensity of a cell to initiate stem cell functions is likely to decrease as cells mature. Routine physiological processes would be expected to enlist cells with higher stem cell propensities. This may explain the ease with which bone-marrow-derived cells are able to reconstitute cells not only of the blood but also other organs. However, in the context of damage or

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physiological perturbation, atypical levels of environmental signals may allow cells with lower stem cell propensities to be recruited to function as tissue-regenerating stem cells. Thus, we suggest that the conceptual view of a stem cell may need to re-evaluated to include the possibility that the concept refers not to an entity but to a function that can be carried out by various cell types.

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11 The Mechanical and Cytoskeletal Basis of Lung Morphogenesis

EBEN ALSBERG, KIMBERLY MOORE, SUI HUANG, TOM POLTE, and DONALD E. INGBER Children’s Hospital, Harvard Medical School Boston, Massachusetts, U.S.A.

I. Introduction The development of complex tissues with characteristic three-dimensional (3D) morphology is made possible through tight temporal and spatial coordination of cell proliferation. This occurs within an embryonic environment filled with various types of soluble growth factors and insoluble extracellular matrix (ECM) molecules, in addition to being mechanically active. For example, formation of branching epithelium in the lung (1) and many other tissues (2) is driven by local differentials in cell growth that promote outward tissue expansion (i.e., epithelial bud formation) relative to neighboring quiescent regions of the same tissue. Localized production of growth factors may promote directional branching in the developing lung by influencing the general site where new branches will form (3). However, the sharp growth differentials responsible for tissue patterning often have abrupt boundaries, with growth being activated in an all-or-none manner within neighboring groups of cells only separated by a few micrometers. Moreover, normal branching morphogenesis in the lung requires the presence of ECM as well as growth factors (4). Gradients of soluble mitogens alone therefore cannot account for how these critical growth differentials are established. 247

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Figure 1 The importance of local control mechanisms for tissue development. Although soluble growth factors drive lung development, they can diffuse over large distances and multiple factors are likely to coexist in the same microenvironment. Given that conflicting cell behaviors, including growth, differentiation (and quiescence), and apoptosis, all can be observed in the same microenvironment, cells must have evolved a local mechanism to control their responses to soluble stimuli. We explore the possibility that this local control is exerted by ECM and mechanical forces.

Neighboring cells within the same tissue microenvironment (and hence exposed to the same cytokines and growth factors) also can simultaneously undergo differentiation, motility, apoptosis, and growth (5) (Fig. 1). Thus, to create normal tissue patterns, there must be a mechanism for local ECMdependent control of cellular responses to diffusible chemical stimuli. In this chapter, we first review our current understanding of the molecules that contribute to control of lung development. We then place these molecules in a physical context by considering the potential contribution of structural as well as mechanical cues to this local control mechanism, including ECMdependent changes in cell shape and the cytoskeleton (CSK). Finally, we will review recent work that demonstrates the key role that tensional forces generated in the CSK play in cell and developmental control, with a particular focus on lung morphogenesis.

II. Embryonic Development of the Lung Embryonic lung develops through a process of branching morphogenesis (Fig. 2) similar to that observed in other organs, including prostate, pancreas,

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Figure 2 Diagramatic representation of epithelial pattern formation during lung development. Left. Branching morphogenesis is made possible through the establishment of increases in BM turnover that involve both accelerated degradation and new synthesis within localized regions of the growing lung epithelium (black regions). E, epithelium; BM, basement membrane; M, mesenchyme. Middle. BM extension is accompanied by cell growth (mitotic figures) limited specifically to these regions of accelerated ECM remodeling. Right. Continued BM extension (net ECM accumulation) coupled with local increases in cell proliferation result in outward expansion of the epithelium, and hence formation of new epithelial buds separated by residual clefts.

kidney, tooth, salivary gland, mammary gland, and submandibular gland (6,7). Lung buds initiate in the foregut epithelium and protrude into the surrounding mesenchyme. The specialized epithelial ECM or basement membrane (BM) deposited by the epithelial cells physically separates the epithelium from the mesenchyme. Lung buds continue to grow in length, with a proximal stalk and a distal spherical bud that proceeds to form a concave notch (8). A branch point forms during epithelial expansion when a deep notch or cleft develops between two new buds; the cleft is characterized by increased accumulation of BM molecules as well as interstitial collagen bundles. These lung buds then continue to branch in a dichotomous manner in 3D (9) via the recurring development of new clefts and lobules using similar rules (2) (Fig. 3). Epithelial branch formation is driven by local differentials in epithelial cell growth rates: cells at the tips of the growing buds proliferate more rapidly than their neighbors and, hence, this localized region expands outwards (2). The growth differentials that drive tissue patterning are established through a complicated interplay among soluble, insoluble, and physical signals from the local microenvironment. Below we review these regulatory factors and describe how cellular responses to growth stimuli are controlled locally.

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Figure 3 Photomicrographs of embryonic mouse lung explanted on day 12 (0 h) and maintained for 48 h in whole organ culture. Note that branching morphogenesis occurs through the repeated process of bud growth followed by cleft formation at the bud’s tip and initiation of two or three new buds (original magnification, 40x).

A. Role of Soluble Growth Factors

Soluble growth factors, such as fibroblast growth factor-10 (FGF-10), play a key role in embryonic lung development, and the 3D pattern of FGF-10 production within the mesenchyme may help to establish the location where new buds form (10). This hypothesis is supported by the finding that lung explants cultured in vitro in the presence of FGF-10-coated beads form new buds preferentially oriented towards the beads (11). FGF-10 also promotes epithelial proliferation and new bronchial bud formation (9), whereas deletion or mutation of FGF-10 (12–14) or its receptor FGFR-2 (15) results in inhibition of lung branching. Moreover, bone morphogenetic protein-4 (BMP-4) (16, 17), transforming growth factor-h1 (TGF-h1) (18) and sonic hedgehog (Shh) (19), which are all produced by lung epithelial cells, both inhibit FGF-10 production in the mesenchyme and suppress epithelial cell growth (10,20). Thus, the presence of these factors in such highly proliferative regions may feed back to shut down growth, induce quiescence, and promote bud maturation. Continued FGF-10 expression in smaller focal areas on either side of these newly formed buds may promote continued growth and branching in these regions (21). At the same time, TGF-h1 promotes deposition of ECM components, such as type I collagen, in the clefts during branching morphogenesis (22). Type I collagen protects BM from degradation (23) and hence it likely stabilizes tissue structure in these static regions of the growing gland. There is extensive literature characterizing the effects of these and other [e.g., epidermal growth factor (EGF)] soluble regulatory factors on lung development (21,24–27). Yet it is still not clearly understood how these soluble molecules control pattern formation. For example, although the localized production of chemoattractants may help to establish the pattern of future branch formation, diffusible growth factors will act over long distances.

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Although addition of large amounts of growth factor to culture medium (i.e., where there is no local concentration gradient) can promote branching morphogenesis in isolated embryonic lung epithelium, it only does so if exogenous BM matrix is provided (4). Thus, the formation of localized buds that exhibit abrupt growth differentials across only a few cell diameters cannot be explained solely through local regulation by soluble signals (28) (Fig. 1). These observations suggest that although growth factors drive lung development, the responses of cells to these soluble cues must be controlled locally in order to establish the spatial differentials in cell growth that drive epitheliogenesis. We must therefore search for other sources of local control. B. Role of Epithelial–Mesenchymal Interactions

Active and dynamic interactions between neighboring epithelium and mesenchyme are critical for the correct presentation and interpretation of environmental growth signals during lung morphogenesis. The presence of lung mesoderm is necessary for proper lung development (29), and exchange of tracheal mesenchyme for bronchial bud mesenchyme inhibits bronchial branching and promotes tracheal budding (30,31). In fact, analysis of the development of various embryonic epithelia consistently shows that cytodifferentiation is epithelium-dependent, whereas histodifferentiation (the establishment of tissue-specific patterns) is mesenchyme-dependent (32). The mesenchyme controls the 3D structure of developing salivary epithelium (8), for example, by determining sites of BM degradation, whereas the epithelium is primarily responsible for continuing BM synthesis. Yet BM synthesis must outpace degradation within the localized regions that exhibit highest ECM turnover in order to promote lateral BM extension and epithelial expansion (bud formation) in these regions. In the developing lung, the epithelium alone appears to be sufficient to establish these local differentials in ECM remodeling and growth as isolated epithelium can undergo branching morphogenesis if provided with soluble growth factors and the exogenous insoluble BM matrix (Matrigel) (4). Isolated endothelial cells are similarly capable of autonomous morphogenesis if provided with growth factors and exogenous ECM during capillary development (33–35). Matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) are two of the key regulatory elements governing ECM turnover. Cleavage of ECM components during the development of branching structures may release bioactive ECM protein fragments, in addition to making new space available for tissue expansion (36). ECM remodeling also may influence cell growth and function by releasing bound growth factors (37), unmasking specific functional sites on bioactive molecules (e.g., arginineglycine-aspartic acid-serine (RGDS) and arginine-glutamic acid-aspartic

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acid-valine (REDV) on fibronectin (38)), or altering cell–cell interactions through the cleavage of cell surface adhesions molecules (e.g., cadherins, selectins) (39). It is important that ECM degradation also alters the mechanical properties of lung tissue (40,41), as discussed in greater detail below. Multiple MMPs are involved in lung branching morphogenesis (42), and the expression of specific MMPs is likely dependent on the ECM microenvironment and the stage of development as observed during ureteric bud branching (43). The expression and activity of MMPs and TIMPs during lung development are also tightly regulated by growth factors. For example, mice lacking EGF exhibit abnormal MMP expression as well as aberrant lung branching (44). Abnormal lung development also can result from treatment with exogenous EGF or TGF-a, which increases MMP activity (45). Tumor necrosis factor, which promotes epithelial branching in mammary gland, similarly stimulates MMP-9 production (46). However, the mechanism by which lung mesenchyme determines exactly where to increase ECM remodeling remains unknown. One possibility is that integrin receptors may exist in different functional states depending on whether they are exposed to intact or fragmented ECM molecules (47). Alterations in integrin signaling may, in turn, regulate MMP expression (47) such that proteolytic enzyme expression may be reduced when cells are attached to intact BM. This mechanism might facilitate ECM turnover once initiated; however, it cannot explain how the initial site of degradation is selected or how pattern is determined. The cytokine TGF-h influences various lung cell functions related to ECM turnover, including integrin expression (48–50), synthesis of collagen types I, III, and V (51), fibronectin (52), and some proteoglycans (53), production of proteolytic enzymes and MMP inhibitors (54), as well as cell proliferation (54). The possibility that proteins of the TGF-h family may contribute to spatial control of ECM remodeling is supported by the finding that there is a distinct spatial and temporal pattern of expression for these proteins (55–57) and their associated receptors (58–61) during embryonic development. In fact, TGF-h1 is found mainly colocalized with fibronectin, collagen, and glycosaminoglycans (GAGs) in stalks and clefts (22) and hence, it could play an important role in branching morphogenesis by promoting the accumulation of ECM in these regions. Localized degradation of ECM also may release TGF-h or activate latent TGF-h locally (62) and thus allow for precision spatial regulation of signaling. In addition, a feedback mechanism may exist: some ECM molecules can influence the bioactivity of TGF-h. For example, the proteoglycans betaglycan, biglycan, and decorin bind TGF-h and control its activity (63–65). It is not known whether TGF-h’s primary signaling function is based in the epithelium (66) or the mesenchyme (67). However, TGF-h inhibits cell proliferation as well as tissue branching (66,68) in lung bud cultures, in addition to altering surfactant production (69). It is

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interesting that homozygous mice lacking TGF-h1 develop normal branching patterns (70), but this may only indicate that there is a certain degree of redundancy during branching morphogenesis. C. Role of Extracellular Matrix and Integrins

Insoluble ECM molecules differentially expressed at specific locations during the various stages of lung embryogenesis actively contribute to developmental control and convey distinct signals from those elicited by soluble growth factors. The epithelial BM and interstitial ECM of the mesenchyme are composed of several different types of collagens, proteoglycans (e.g., heparan sulfate-containing), GAGs (e.g., hyaluronan), glycoproteins (e.g., laminin, fibronectin, tenascin-C), and elastin fibers. The major difference is that the BM contains types IV and V collagens that self-assemble with laminin and other molecules to form a planar array, whereas the interstitial collagens (e.g., types I and III) form linear bundles that self-organize into fibrillar networks. These ECM components are deposited in a specific temporal and spatial pattern during lung morphogenesis (71–74), and all have been implicated in the process of lung branching. Lung branching morphogenesis can be inhibited or reduced in cultured embryonic lung explants by addition of collagen synthesis inhibitors (75,76), collagenase (77), inhibitors of proteoglycan synthesis (72), reagents that inhibit fibronectin assembly (78), and blocking antibodies directed against laminin (79) or tenascin-C (74). Furthermore, isolated embryonic lung epithelium will undergo branching morphogenesis in the absence of mesenchyme if exposed to FGF in the presence of exogenous BM matrix, but not in the presence of a type I collagen gel (4). Thus, the specific ECM microenvironment is critical for pattern formation in the lung. The ECM exerts its control over branching through multiple overlapping mechanisms all tied back to a finely tuned balance of ECM production and degradation, as described above. The epithelial BM may serve as a barrier and permit epithelial–mesenchymal contact only at bud tips where the BM thins and occasional small gaps form (80). These gaps may enhance local diffusion of soluble signaling molecules (e.g., growth factors) and facilitate cell–cell (81) contact between the mesenchyme and the epithelium. ECM molecules also may filter regulatory molecules, sequester soluble components, enhance cell binding to growth factors (80), and/or release growth factors (e.g., FGF) as a result of BM degradation (37). Although local changes in cytokines and growth factors may promote branching morphogenesis, studies with cultured cells suggest that the presence of these factors alone is not sufficient to promote cell growth. For example, epithelial cells exhibit what is termed anchorage-dependence and hence

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will not proliferate in response to mitogenic stimuli unless they adhere to ECM (82) and receive distinct signals through transmembrane integrin receptors that mediate ECM adhesion (83). In fact, cell–ECM interactions mediated by integrins play an important role not only during development (i.e., lung branching) but also in many other biological processes including wound healing, cancer, and thrombosis (84). Integrins represent a family of cell surface receptors comprised of interacting heterodimers of a and h subunits (84); there are over 20 different pairing combinations of these subunits (85). Integrins recognize specific peptide sequences (e.g., RGD) in ECM molecules, bind to these ligands, and cluster together within specialized anchoring complexes known as focal adhesions. In the focal adhesion, the cytoplasmic domains of the transmembrane integrin receptors physically associate with actin-associated proteins (e.g., talin, vinculin, a-actinin, paxillin) and thereby form a molecular bridge between the ECM and the actin CSK. In addition, the cytoskeletal backbone of the focal adhesion orients many of the molecules of the cell’s signaling transduction machinery, including various protein kinases (e.g., Focal adhesion kinase (FAK), src), small GTPases, heterotrimeric G proteins, inositol lipid kinases, ion channels, and even cell surface receptors, including FGF receptors (86,87). It is important that occupancy and clustering of cell surface integrin receptors can activate these same intracellular signaling cascades (e.g., extracellular signal-related kinase (ERK), FAK, STAT, cAMP) (88) and thereby exert control over cell growth and function, independently of growth factor receptors. Focal adhesions also represent sites where mechanical signals are preferentially transferred across the cell surface by integrins (89,90), as well as transduced into a chemical signaling response (91,92) in a process known as mechanotransduction (93). Thus, integrins may play a central role in developmental control. D. Importance of ECM Micromechanics

All cells of the epithelium remain in direct contact with BM throughout morphogenesis, and thus adhesion to ECM and integrin binding alone cannot explain its effects on pattern formation. ECM differs from soluble stimuli in that it conveys different signals depending on its ability to resist cell traction forces and thereby promotes changes in cell shape and cytoskeletal organization. For example, ECM can switch various types of cultured cells among growth, differentiation, and apoptosis, as well as control the direction of motility, based on its ability to support or prevent tension-dependent changes in cell spreading (33,34,94–97). Rigid ECM substrates tend to promote proliferation and motility, whereas highly flexible substrates (e.g., Matrigel, floating collagen gels) that can retract in response to cell traction forces inhib-

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it growth and movement, and induce differentiation (98–106). These local effects of ECM mechanics on cell behavior may be based, in part, on the ability of integrins to elicit different chemical signals within the focal adhesion depending on the level of stress applied to integrins (93). In addition, changes in the level of stress applied to integrins, either from the extracellular environment or from the internal CSK, also feed back to control focal adhesion assembly (107–110), and hence intracellular signaling. The observation that changes in the balance of mechanical forces between cells and the ECM can influence growth as well as other behaviors that are critical for development is important because cells experience both macroscale and microscale forces within the developing lung. Lung is subject to large-scale forces caused by breathing movements and alterations in lung fluid volumes and pressures in the developing fetus (111,112). These external forces have a dramatic impact on fetal lung growth and maturation in experimental animal models (113,114). It has been difficult, however, to separate the effects of forces caused by cyclic stretching due to breathing movements and those resulting from changes in lung fluid volumes (115). Nevertheless, it is clear that disruption of these normal movements, for example, by production of a diaphragmatic hernia in the embryo, can severely inhibit lung morphogenesis and produce lung hypoplasia (116), as is observed in human patients with this developmental defect (117). The response of fetal lung cell populations to defined mechanical force regimens has been more fully studied in vitro. Mechanical stretching of fetal lung cells produces pronounced effects on cell proliferation (118–121), ECM turnover (118,122–125), cell differentiation (126,127), production of soluble growth factors (128,129), and intracellular signaling (130,131). Physical distortion of cultured pulmonary epithelial cells also triggers secretion of surfactant by the cells (132), much as is observed in response to the first breath in the newborn. Lung cells also increase their release and activation of MMPs in response to cyclic mechanical stretch in vitro (133). ECM-degrading enzymes may be similarly upregulated or activated by changes in the mechanical properties of the ECM or CSK (134) that are produced by stresses induced by injury or abnormal insufflation of the lung (135). Changes in the mechanical compliance of the ECM or in the level of isometric tension in the CSK also can influence ECM production by altering expression of genes that encode ECM proteins (e.g., tenascin-C) (136) or by promoting fibronectin fibril assembly (99,137). In addition, cytoskeletal reorganization can induce cells to concentrate and activate MMPs at focal contacts (138). Thus, induction of MMP secretion in lung cells by mechanical loading changes with ECM compliance and associated cell shapes changes (139,140), as well as the ECM’s ability to provide stress shielding (141). Physical distortion of individual ECM molecules caused by cell traction forces (i.e., cytoskeletal tension) (142) or overall

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ECM deformation at specific tissue sites also may promote ECM remodeling by exposing specific bioactive sequences. In this context, TGF-h may indirectly influence cell shape and growth in local regions of the developing lung (e.g., in clefts) by modulating ECM accumulation and thereby altering its mechanical stiffness. Finally, it is interesting to note that the mesenchyme of different tissues that produce different organotypic branching patterns also exhibit different mechanical properties (e.g., contractile function) (143). E. Role of Cell Shape and the Cytoskeleton

Taken together, these observations suggest that mechanical signals may be as important for control of lung morphogenesis as soluble growth factors and insoluble ECM components. Cells experience macroscale forces that produce tissue distortion and alter tissue development through their adhesions to ECM and to other cells. Microscale forces generated within the contractile apparatus of epithelial and mesenchymal cells are similarly sensed through these same adhesion sites that link the internal CSK to external support scaffolds (i.e., ECM and other cells). This is important because it suggests that cells may experience forces differently depending on the local physical properties of the ECM on which they anchor. Embryonic and adult tissues, such as lung, are tensionally prestressed (i.e., exist in a state of isometric tension) due to an internal balance of mechanical forces. Expansion (outward-directed) forces in the developing lung result from traction forces exerted on the BM by surrounding mesenchyme, from increases in epithelial cell growth (i.e., due to compaction of increased numbers of adherent cells on the same area of BM), and from inflation pressures in later stages when the lumen fills with amniotic fluid. These forces are matched by inward-directed contractile forces exerted on the BM by the epithelium and by the mechanical stiffness of this ECM scaffold. Thus, cells that adhere to more compliant (flexible) regions of the tensed ECM, such as in the thinned portions of the BM at the tips of growing buds (42), will experience greater strain (distortion) than cells attached to the thicker and hence stiffer regions of the BM within the neighboring clefts (Fig. 4). A simple analogy is to envision this localized region of thinning as a run in a nylon stocking that will stretch out more than the rest of the material in response to a constant distending force. Another example is how a weak spot in a balloon will inflate (bud) more than the surrounding surface when pneumatically tensed. Thus, both macroscale and microscale forces may be focused and concentrated locally on integrins within particular subsets of epithelial cells through establishment of spatial differentials in ECM structure and mechanics (Fig. 5). Because individual cells generate mechanical forces within actomyosin filaments in their CSK, they are also in a prestressed state prior to the appli-

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Figure 4 An engineering view of morphogenesis illustrates theoretical strain distributions within the BM of the developing lung epithelium. Below each strain diagram (top) are photomicrographs of individual buds showing the sequential structural alterations that mediate morphogenesis in the mouse lung from 0 to 48 h of culture (a–f). The strain diagrams show a corresponding small region of the tissue in which strain field lines are drawn on the surface of the epithelium. Increased spacing between the lines indicates regions where the BM loses it mechanical stiffness, thins, and experiences increased mechanical strain (distortion). Note that regions of increased strain correlate precisely with regions of epithelial expansion and new bud (B) formation. Increased BM deposition results in decreased strain in the proximal stalks (S) of newly formed buds and in the intervening clefts (C).

cation of any exogenous load (89). When cells attach to ECM ligands through integrin receptors, an attempt is made to transfer a portion of the internal cellular mechanical prestress to the ECM. The degree to which internal mechanical stress may be imparted to the matrix over integrins is influenced by the ECM’s adhesive and mechanical properties. Cells attempt to contract a substrate to reach a specific amount of force per cell, instead of a specific amount of displacement per cell (144), and thus the mechanical properties of the ECM directly influence cell shape. Highly flexible substrates cannot resist

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Figure 5 Schematic model for local mechanical control of epithelial bud formation by ECM. 1. A small region of embryonic lung epithelium (E) separated from the underlying mesenchyme (M) by the continuous basement membrane (BM). Tissue morphology is stable because contractile forces generated within all cells are balanced by the mechanical stiffness of the ECM and, hence, the tissue is maintained in a state of isometric tension. 2. Localized secretion of proteolytic enzymes increases BM degradation within a small area and leads to a decrease in the mechanical stiffness of the BM. 3. The increase in BM flexibility results in BM extension and thinning due to residual stresses in the tissue. 4. Associated cell distortion supports local cell growth in response to growth factors that may be present over larger areas within the tissue, as well as a concomitant increase in new BM deposition. The localized increase in cell number coupled with a net increase in BM accumulation results in epithelial expansion in these local regions. 3a. If tissue tension is important for local morphogenetic control, then dissipation of this tensile prestress may prevent BM thinning and epithelial branching. Recent studies suggest that this is the case (see Fig. 6).

cell-generated forces or sustain isometric tension in the CSK; cells usually assume a rounded or retracted morphology on these substrates. In contrast, stiffer ECM substrates that resist cell-generated forces and maintain cytoskeletal prestress promote cell spreading (89). As described above, these tension-dependent alterations in cell shape produce important changes in cytoskeletal organization and alter various cell functions. When cultured on a flexible ECM or a poorly adhesive substrate

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that cannot resist cell tractional forces, various cell types become quiescent (100,102–104), differentiate (105,145), increase synthesis of differentiationspecific proteins (106), lose stress fibers (99), and decrease fibronectin fibril assembly (137,146). In contrast, when cultured on rigid ECMs, highly adhesive ECM-coated dishes, or flexible ECM substrates that are placed under tension, the same cells will proliferate (99–101), increase synthesis of ECM proteins (147,148), and form both large actin microfilament bundles and fibronectin fibrils (137,146). The sensitivity of cells to growth factor stimulation also has been shown to be directly related to both substrate mechanics and cell morphology (149). Our laboratory has developed a method that allows us to investigate the effects of cell distortion on cell behavior, independently of signals elicited by direct integrin binding and growth factor stimulation. Using microfabrication technology (microcontact printing and soft lithography) originally developed for the microelectronics industry, adhesive islands coated with a saturating density of ECM molecules were produced with micrometer-scale control over their shape, size, and position (95); these islands were surrounded by nonadhesive regions that resist protein absorption. When plated on these culture surfaces, cells attach and spread over the ECM-coated adhesive islands until they reach the nonadhesive boundary regions (95,96,150). Thus, the cells literally take on the shape of their container: cells adherent to square ECM islands exhibit 90 degree corners. In this system, the ECM density and concentration of soluble growth factors are held constant, and therefore changes in cell behavior may be directly attributed to changes in cell extension induced by ECM substrates that differ only in size and shape. These studies revealed that epithelial cells, such as primary rat hepatocytes, spread and increased their growth in parallel when they adhered to large laminin-coated adhesive islands (100  100 Am squares), whereas on smaller (40  40 Am) islands that inhibited cell spreading, growth was inhibited and differentiation was induced (95). Capillary endothelial cells exhibited similar behavior when plated on different-sized islands coated with fibronectin (96). Dramatically decreasing the ability of these cells to spread not only inhibited cell proliferation but also turned on programmed cell death (apoptosis) in cells that remained adherent to ECM despite the presence of soluble growth factors, such as FGF. In addition, linear adhesive substrates that permitted only a modest amount of cell spreading and inhibited both growth and apoptosis induced differentiation (capillary tube formation) in these cells (34). Later studies confirmed that the smallest islands supported similar integrin receptor signaling to the larger islands (140). Experiments also were carried out with substrates comprised of multiple smaller (3–5 Am diameter) adhesive islands that allowed cell spreading while preserving the total cell-ECM contact area at a level comparable to that on a single small island. Again, these

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spread cells proliferated, verifying that cell distortion is the critical local determinant of whether cells will grow or die (96). More recent studies demonstrate that ECM-induced changes in cell shape and cytoskeletal organization that polarize focal adhesions (i.e., sites of stress application to ECM) to particular regions of the cell, such as corners in square cells, also can influence the direction of cell motility; lamellipodia, filopodia, and microspikes only extend from these corner regions (97). Analysis of the mechanism of shape-dependent control of cell function revealed that the ability of spread cells to undergo cell cycle progression, suppress apoptosis, and move is dependent on signals conveyed by an intact CSK, in addition to those elicited directly by binding of integrins and growth factor receptors (151,152). Growth-factor-stimulated capillary cells spread on ECM can be blocked in late G1 phase of the cell cycle by disrupting the actin CSK using cytochalasin D or latrunculin B, or by inhibiting cytoskeletal tension using pharmacological (150) or genetic techniques (153). Spread cells also can be induced to undergo apoptosis when still adherent to ECM in the presence of mitogens by disrupting the actin or microtubule CSK using cytochalasin D or nocodazole, respectively (152). Moreover, extension of lamellipodia also ceases when cytoskeletal tension is dissipated (97). These findings suggest that localized variations in ECM compliance that result from spatial differentials in ECM turnover and that may alter cell shape or CSK structure could have a profound affect on cell behavior during lung development. In fact, the differentiated phenotype of cultured pulmonary cells has been shown to vary depending on the flexibility of the ECM substrate (154): rigid substrates that promote cell spreading prompt alveolar type II cells to assume a type I phenotype, whereas flexible substrates lead to both cell rounding and a differentiated type II cell phenotype. The finding that CSKdisrupting drugs, such as cytochalasins, also inhibit embryonic lung branching in vivo (1) supports this concept that cell shape and the CSK play a central role in tissue morphogenesis.

III. Mechanochemical Model of Lung Development A. Mechanochemical Mechanism

Taken together, these observations support the possibility that lung may utilize a mechanochemical mechanism for control of tissue morphogenesis (28,155,156) (Fig. 5). This mechanism may function in the following way. Because cells transfer a portion of their CSK-based mechanical prestress to the ECM through their integrin linkages, the ECM also exists in a state of isometric tension (156). As discussed earlier, regions of the BM at the tips of growing buds in the embryonic lung experience a high rate of ECM turnover.

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Proteolytic enzymes can decrease the mechanical properties (e.g., stiffness) of lung tissue by degrading specific ECM components (157,158). These local BM regions become thinned because their compliance is increased and they are unable to balance tensional forces applied by adherent epithelium and mesenchyme. BM distention would, in turn, alter the force balance within adherent epithelial cells and thereby produce changes in cell shape and cytoskeletal organization. In vitro studies suggest that cell distortion will make the cells more sensitive to mitogenic signals in their environment and hence, the distorted cells at the tips of the buds may proliferate more rapidly than their neighbors. In fact, local differentials in cell proliferation are observed in these regions after lung bud outgrowth is initiated (159). Fine coupling between cell division and BM expansion in these regions would result in outward budding of the epithelium without compromising tissue integrity. In this manner, tissue patterning (Fig. 2) may be controlled through local alterations in micromechanics (Fig. 4) manifested through spatial control of ECM remodeling (Fig. 5). B. Cytoskeletal Tension as a Critical Control Element During Lung Development

We recently tested the hypothesis that cell-generated tension contributes to the local control of cell growth and ECM remodeling during embryonic lung development. CSK tension is generated in contractile cells through calciumdependent activation of myosin light chain (MLC) kinase (160,161). More recently an alternative pathway has been identified that involves the small GTPase Rho, which promotes MLC phosphorylation and stimulates CSK contraction through activation of its downstream target, Rho-associated kinase (ROCK) (162,163). Rho is a critical regulator of early-stage morphogenesis in Xenopus embryos, chicks, and mice (164,165). Rho and ROCK also mediate the effects of altering mechanical force transfer across integrins on tension-dependent changes in focal adhesion assembly and CSK structure in cultured cells (109,166–168). To determine whether Rho-dependent tension generation is a significant factor during lung morphogenesis, embryonic mouse lungs were explanted on embryonic day 12 (E12) and treated for 48 h with the Rho activator CNF-1. Increasing cytoskeletal tension by treatment with moderate amounts of the CNF-1 significantly accelerated lung branching within the observation period of 48 h (169). Histological analysis revealed that individual glands were more highly developed at a low dose of CNF-1, whereas a high dosage produced gland contraction. In more recent studies, we found that similar treatment of glands with the ROCK inhibitor Y27632 suppressed epithelial bud formation in a dose-dependent and reversible manner, but

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without toxicity or global inhibition of cell proliferation (Moore, Huang, Polte, Alsberg and Ingber, in preparation). Similar results were obtained using other pharmacological agents that interfere with cytoskeletal tension generation through the actin–myosin contractility pathway. An interesting finding was that inhibition of tension generation with Y27632 also resulted in the loss of normal spatial differentials in ECM remodeling. Although untreated control glands exhibited basement membrane thinning in regions of the most rapid epithelial expansion, lungs treated with Y27632 had a relatively thick BM beneath the entire epithelium (Fig. 6). Biochemical quantitation of MLC phosphorylation revealed a dose-dependent increase in contraction in lungs treated with CNF-1 and complete suppression of MLC phosphorylation in Y27632-treated lungs. Rho activity levels increased with the dosage of CNF-1, but saturated at moderate dosages. These findings show that Rho is critical for both regulation of tissue tension and developmental control during lung development, and that lung branching morphogenesis can be accelerated or slowed by respectively increasing or decreasing cytoskeletal tension. In addition, these findings suggest that changes in cell tension also feed back to modulate ECM remodeling. This may explain, in part, how the lung epithelium alone may be sufficient to create local differentials in ECM turnover and hence support branching morphogenesis in the absence of surrounding mesenchyme.

Figure 6 Control of lung branching morphogenesis and BM remodeling by cytoskeletal tension. Immunofluorescence images of embryonic mouse lungs cultured in the absence (a) or presence (b) of the ROCK-inhibitor Y27632 (40 AM) for 48 h and stained for the BM protein laminin. a. Although BM continuity is maintained during normal lung morphogenesis, BM thinning can be clearly seen in regions of new bud formation (arrows) relative to clefts. b. Inhibition of cytoskeletal tension using Y27632 prevented local BM thinning and completely inhibited new branch formation. L, epithelial lumen; M, mesenchyme.

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IV. Conclusion The majority of work to date investigating the mechanism of tissue pattern formation during lung morphogenesis has focused on the role of soluble growth factors. However, various studies show that growth factors alone are not sufficient to explain pattern formation; local changes in ECM remodeling are also critical for tissue development. Although past work has identified large-scale mechanical stresses as important contributors to lung development, the mechanism is unknown and the potential impact of local differences in ECM micromechanics has been largely overlooked. We have examined the possibility that local changes in ECM turnover, and hence flexibility, may contribute actively to morphogenetic control. Epithelial cells adherent to thinned regions of the BM that are more compliant will deform more than their neighbors due to the action of cell-generated tensional forces within the developing tissue. In vitro studies with various cell types show that changes in ECM mechanical compliance or adhesivity that produce cell deformation and changes in the CSK have dramatic effects on cell behaviors, including growth, differentiation, apoptosis, and motility. Thus, local changes in ECM remodeling that alter ECM micromechanics could produce local cell growth differentials through stress-dependent changes in cell shape and cytoskeletal organization. This hypothesis that cell-generated tissue tension controls branching morphogenesis by translating the diffuse mitogenic activity of growth factors and local changes of ECM compliance into highly localized cell proliferation is supported by recent studies with embryonic lung. These studies demonstrate that chemical modulators of cytoskeletal tension can selectively inhibit or stimulate branching morphogenesis. This finding raises the possibility that growth factors may contribute to the control of tissue branching by promoting local changes in ECM remodeling or mechanics, in addition to directly activating growth signaling pathways. However, the key point is that the cell’s ability to integrate simultaneously the various chemical and mechanical stimuli responsible for control of its growth depends directly on physical interactions between the CSK and ECM. Further elucidation of this mechanochemical mechanism of branching morphogenesis will require development of new microtechnologies that will permit manipulation and quantitation of local mechanical properties within specific tissue microdomains, as well as modulation of chemical factors within developing organs in vivo.

Acknowledgments This work was supported by a grant from The National Institutes of Health (P01-HL 67669). We also thank Dr. M. Sunday and members of her lab-

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12 Morphogenesis of the Mammalian Lung: Aspects of Structure and Extracellular Matrix

JOHANNES C. SCHITTNY and PETER H. BURRI University of Bern Bern, Switzerland

I. Introduction The lung is designed to provide a large gas exchange surface area where capillary blood very efficiently comes into close contact to the inspired air. This goal is achieved by a sequence of different developmental processes. Organogenesis starts with a ventral outpouching of the foregut resulting in the appearance of the lung buds (for the timing of lung development in different species see Table 1). The following development of the airways and the gas exchange area requires two quite different steps. First, the conducting and parts of the respiratory airways are formed by continuous cycles of branching and grow into the surrounding mesenchyme starting at the lung buds (branching morphogenesis). Most of this development takes place during the pseudoglandular stage. Second, during the alveolar stage the distal part of the bronchial tree is further enlarged by a lifting off of new, secondary septa from existing primary septa (septation/alveolarization). The canalicular and saccular stages may be considered as intermediate stages, occurring between pseudoglandular and alveolar stage. Very important, during the canalicular stage the first functional gas exchange surface (air–blood barrier) is formed. During the saccular stage the switch from branching to septation occurs. In 275

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Table 1 Stages and Duration of Lung Development Period

Stage

Embryonic

Embryonic

Fetal

Pseudoglandular

Duration Rabbit: n.d.–E18 Sheep: E17–E30 Human: E26–E49 (4–7 weeks) Mouse: E9.5–E12 Rat: E11–E13 Rabbit: E18–E24 Sheep: E30–E85 Human: E35–E119 (5–17 weeks) Mouse: E12–E16.5 Rat:E13–E18.5

Canalicular

Rabbit: E23–E27 Sheep: E80–E120 Human: E112–E182 (16–26 weeks) Mouse: E16.5–E17.5 Rat: E18.5–E20

Saccular or terminal sac

Rabbit: E27–E30 Sheep: E110–E140 Human: E168–E266 (24 weeks–term) Mouse: E17.5–P4 Rat: E21–P4 Rabbit: E30-term (E31) Sheep: E120-term (E145) Human: E252 (36 weeks preterm)-1–2 years Mouse: P4–P14 Rat: P4–P14 Rabbit: unknown Sheep: unknown Human: 0–3 years Mouse: P14–P21 Rat: P14–P21 Rabbit: Birth– adulthood

Alveolar

Postnatal

Microvascular maturation

Normal Growth

Characteristics Start of organogenesis; formation of major airways

Formation of bronchial tree and large parts of prospective respiratory parenchyma; birth of the acinus Completion of conducting airways; epithelial differentiation; first air-blood barrier; appearance of surfactant Expansion of air spaces

Alveolarization by formation of secondary septa (septation)

Remodeling and maturation of interalveolar septa and of the capillary bed normal growth of the lungs

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Table 1 Continued Period

Stage

Postnatal

Normal growth

Duration Sheep: Birth– adulthood Human: f2nd year–adulthood Mouse: 4 weeks– adulthood Rat: 4 weeks– adulthood

Characteristics Normal growth of the lungs

The described duration of the stages represents the time during which the bulk of a particular developmental alteration takes place. Stages are overlapping, in particular the alveolar stage and the stage of microvascular maturation. In addition, regional differences are common: especially between central and peripheral regions. Litter size and nutrition also influence the exact timing of development (69,114,128). E, embryonic day (days post coitum): n.d., not determined: P, postnatal day. Source: From Refs. 129,130.

order to optimize gas exchange after bulk alveolarization is completed, the interalveolar septa and their capillary networks are remodeled during the phase of microvascular maturation. At this point lung development is viewed as finished and normal growth of the organ follows. Relative to lung development, the time point of birth differs between mammals. In humans, birth happens at the beginning of the alveolar stage. The current review describes the morphogenesis of the human lung in comparison to some important laboratory mammals. Particular attention will be given to the structural influence of some important extracellular matrix components on lung development. Table 1 gives a comparative overview of the phases of lung development for human, sheep, rabbit, rat, and mouse. Table 2 summarizes the ultimate goal of human lung development: It shows the dimensions of the adult lung.

II. Prenatal Lung Development: Embryonic Period (1–7 Weeks) A.

Lung Anlage

Between 4 and 8 weeks after fertilization the anlage of all organs is laid down. The lung anlage appears at day 26 as a ventral bud of the foregut at the caudal end of the laryngotracheal sulci (Fig. 1, Table 1). It gives rise to the left and

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Table 2 Average Dimensions of the Adult Human Lung Body weight (kg) Number of generations of branches Number of alveoli Mean diameter of one alveolus (Am) Alveolar surface area (m2) Alveolar surface area of capillaries (m2) Lung volume (L) Parenchymal volume (L) Volume of parenchymal airspaces (L) Fraction of airspace in the parenchyma (%) Parenchymal capillary blood (ml) Diameter of capillaries (Am) Fraction of capillary volume in total septal volume (%) Daily total blood flow through the lung (L) Arithmetic mean barrier thickness (Am) Harmonic mean barrier thickness (Am)

75 20–28 >300 million 250 140 120 4.3 3.9 3.4 90 230 6–7 >50 7000–8000 2.2 0.62

Figure 1 Early development of the human lung. At day 26 post coitum (p.c.)(E26) the respiratory diverticulum is formed by an outpouching of the foregut a. The prospective trachea forms by a distal-to-proximal segregation. a + b. A dichotomous branching that happens in parallel to the outpouching gives rise to the prospective main bronchi of the lungs at day E32 b. Further branching results in the formation of the lobar bronchi at day E37 (c) and later at day E41 of the segmental bronchi (d). u, upper lobe; m, middle lobe; l, lower lobe. (Modified from Ref. 8.)

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Figure 2 Lung organogenesis. Freshly explanted mouse lung is shown at days E11.5 (a) and E12.5 (b). Towards the end of the embryonic period (E11.5/a) the visceral pleura and the main bronchi of the lung lobes are formed. In mice the right lung consists of four lobes (cranial, middle, caudal, and accessory) and the left lung of only one. At the beginning of the pseudoglandular stage (E12.5/b) the lungs are already subdivided into definitive lobes. As seen in the left lung (LL) the branching pattern of the bronchial tree is not strictly dichotomous in mice. LL, left lung; LR, right lung. Bar = 0.5 mm.

right lung buds. Details of how the two buds are formed are still the subject of debate. They may be the result of the first branching as shown in Figure 1, or they may be formed by two independent outpouchings of the foregut (1,2). Nevertheless, both lung buds elongate, grow into the surrounding mesenchyme, and give rise to the left and right main bronchi (day 32). Branching and growth of the terminal buds continue in humans during the canalicular stage or most likely up to the saccular stage. In average it is equal to 23 generations of airways (3,4). By day E37 the future conducting airways are preformed to the lobar, by day E41 to the segmental, and by day E48 to the subsegmental bronchi (Figs. 1, 2) (2,5). Because the lungs form by a budding from the foregut, the epithelium of the newly formed tubules is derived from the endoderm. This high columnar epithelium is rich in glycogen, which serves as energy source for its proliferation. Branching morphogenesis of the epithelial tubules is strongly governed by epithelial–mesenchymal interactions (see section below on pseudoglandular stage). B.

Development of Esophagus and Trachea

In parallel to the appearance of the first airway branches, the laryngotracheal sulci are deepening and joining. As a result the foregut divides into the prospective trachea and the esophagus (Fig. 1). The formation of the trachea appears to be independent of the formation of the lung buds, because Fgf10

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null mice develop a trachea, but no lung buds (6). Toward the end of the embryonic period, mesenchymal cells surrounding the trachea condense focally and differentiate into cartilage precursors. At the end of week 7 the precursor cells are well differentiated into embryonic cartilage. Cartilage formation moves distally along the future airways until it reaches its completion around the smallest bronchi (week 25) (2,5).

C. Pleura and Formation of Lobes

When the lung buds start to grow, they expand in caudolateral direction into the coelomic cavity. This rather narrow space consists of two pericardioperitoneal canals located at each side of the foregut and is gradually filled by the growing lungs. The pleural cavities are separated from the pericardial cavity by the pleuropericardial folds during week 5. These folds originate along the lateral body wall, grow between the heart and the developing lungs, and finally meet and fuse with the foregut mesenchyme. Caudally the pleural cavities are closed by a pair of horizontal pleuroperitoneal membranes growing from the posterior body wall to meet and fuse with the posterior edge of the septum transversum (weeks 5–7) (7). The splanchic mesoderm covering the outside of the lung will become the visceral pleura. The somatic mesoderm layer covering the inner surface of the body wall becomes the parietal pleura (8). The visceral pleura deeply separates the lobar bronchi, giving rise to the lobar fissure and to the separated lung lobes. Little is known about the mechanisms involved. It appears however, that the basement membrane of the visceral pleura plays a role, because mice lacking the laminin-a5 chain or the nidogen-binding domain of the laminin-g1 chain show defective lobar septation and visceral pleura basement membrane formation (9,10).

D. Development of the Pulmonary Vasculature

During organogenesis the development of the pulmonary vasculature occurs closely related to the development of the primitive systemic blood vessels and the heart. The pulmonary arteries branch off from the sixth pair of aortic arches and descend to the newly formed lung buds (Fig. 3a). They form a vascular plexus that first connects to systemic veins around the trachea and esophagus before it contacts with the pulmonary veins. In a next step the ventral (proximal) part of the right aortic arch is incorporated into the right pulmonary artery and the dorsal (distal) part disappears. The dorsal part of the left arch is maintained and eventually forms the ductus arteriosus (Botallo’s duct: connection between the pulmonary artery and the aortic

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Figure 3 Origin and fate of pulmonary arteries. a. During arterial tree development paired ventral (dark shading) and dorsal aortae (light shading) are interconnected by six pairs of aortic arches (1–6), which are, however, never simultaneously present. b. Fate of the arches: Dark shading represents preserved segments of the ventral aortae and light shading those of the dorsal aortae. Dashed lines show eliminated segments of both aortae. a, aorta; da, ductus arteriosus; do, dorsal aorta; ec, external carotid artery; ic, internal carotid artery; pa, pulmonary artery; pt, pulmonary trunk; sa, subclavian artery; ve, ventral aortae. (From Ref. 71.)

arch). Due to remodeling and differential growth of the branches, the ventral part of the sixth aortic arches will form a common pulmonary trunk that emerges from the right ventricle of the heart. The heart has now evolved to its definitive configuration with paired atria and ventricles (Fig. 3b). During week 5 the pulmonary veins start to develop as a single short evagination in the sinoatrial portion of the heart. This bud elongates, grows dorsad, divides several times, and finally connects to the pulmonary plexus (5). III. Prenatal Lung Development: Fetal Lung Development (5 Weeks to Term) The fetal period starts roughly during week 5 and ends at term. Based on purely morphological criteria fetal lung development is classified into three

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consecutive, but overlapping stages: pseudoglandular, canalicular, and saccular. Because the speed of development differs between cranial and caudal lung lobes and between central and peripheral regions the transition between stages is not abrupt but gradual (11,12). The same is true for the overlap between embryonic and fetal period. A. Pseudoglandular Stage (5–17 Weeks)

At the beginning of the pseudoglandular stage the lung looks like a primitive gland (for the timing, see Table 1). The terminal epithelial buds undergo multiple cycles of dichotomous branching and outgrowth into the surrounding mesenchyme (Figs. 4a, 5a). During this stage most of the branching takes place. Epithelial differentiation starts and the future bronchi and bronchioli begin to acquire their smooth muscle layer. Branching Morphogenesis-Epithelial–Mesenchymal Induction

Branching is governed by an epithelial–mesenchymal induction. Classic transplantation experiments have shown that a cross-talk between the endodermal epithelium and the mesodermal mesenchyme is needed for the control of branching morphogenesis and cytodifferentiation. For example, after removal of the mesenchyme at the growing tip further branching of the epithelial tubules is prevented. When the mesenchyme of the growing tip is transplanted next to the prospective trachea, an abnormal outgrowth of bronchial branches was observed in this region (1,13,14). Today some of the mechanisms involved are known. The epithelium of the growing terminal bud and its basement membrane differ from the more proximal epithelium and the epithelium of the forming cleft. The epithelial proliferation rate is significantly elevated at the growing tip compared to the epithelium directly proximal to it. The basement membrane of the outgrowing

Figure 4 Light microscopic images of human lung during prenatal lung development. The pseudoglandular stage (a: 103 mm crown–rump length, approx. 15th week of gestation) is characterized by a glandlike appearance of the developing lung and an intensive branching of the epithelial tubules (open arrow, branching point). During the canalicular stage (b: early canalicular stage, 140 mm crown–rump length, approx. 19th week) capillaries of the future lung parenchyma multiply and the future airways are widening (black asterisks). At the transition of the canalicular stage to the saccular stage (c: 225 mm crown–rump length, approx. 26th week) the peripheral airways form typical terminal clusters of widened airspaces called saccules (white asterisk). Parts of the parenchyma appear to be collapsed (arrow). Br, bronchiolus. Bar, 100 Am. (From Ref. 124.)

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Figure 5 Lung in the pseudoglandular, canalicular, and saccular stage. During the pseudoglandular stage (a) the epithelial tubules branch constantly and penetrate into the surrounding mesenchyme (open arrow, branching point). A loose three-dimensional capillary network is located in the mesenchyme. The canalicular stage (b) is characterized by a widening of the future airways, a differentiation of the epithelium into type I and type II cells, a multiplication of the capillaries and their first close contacts to the epithelium, and the formation of first air–blood barriers. Throughout the saccular stage (c) the mesenchyme condenses in order to form thick interairway septa that contain a capillary layer on either side of the septum. The widened terminal ends of the bronchial tree are recognized as sacculi (asterisks). Aw, future airway; ca, capillary; me, mesenchyme. (Modified from Ref. 87.)

terminal buds is lacking nidogen-1 [also known as entactin-1 (15)], collagen IV, and fibronectin (16), but it contains tenascin-C (17). In addition, a switching of the laminin isoforms takes place between the basement membrane of the terminal bud and the immediately proximal parts of the epithelial tubules (18). The behavior of the epithelial cells is strongly influenced by a large number of factors, which are produced in both the epithelium and mesenchyme. The set of factors secreted into the mesenchyme represents a major contribution to the epithelial–mesenchymal cross-talk. It includes thyroid transcription factor 1 (TTF-1), Gli2, and Gli3; as well as growth factors including fibroblast growth factor 10 (FGF-10), transforming growth factor beta (TGF-h), bone morphogenetic protein (BMP-4), sonic hedgehog (Shh), epithelial growth factor (EGF), and vascular endothelial growth factor and their respective receptors: FGFRs, EGFR, and ‘‘patched’’ (19). Also involved are extracellular matrix proteins including collagens, elastin, laminin–isoforms, tenasin-C, fibrillins, and nidogens, as well as their receptors including integrins and dystroglycan (20,21).

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Branching Morphogenesis-Influence of Growth Factors

Fibroblast growth factor-10 may be one of the most important factors during branching morphogenesis (22–26). It takes part in the determination of the pattern of airway branching; without this factor, no branching takes place (6). FGF-10 is expressed in the mesenchyme and controls the expression of BMP4 at the growing terminal epithelial bud. BMP4 itself belongs to the TGF-h superfamily and takes part in the control of cell proliferation at the epithelial buds (27). Stimulation of growth of the epithelial tubules involves factors such as EGF/TGFa, hepatocyte growth factor, and FGF-7. TGF-h1 opposes these effects. Its signaling is thought to prevent local budding by suppressing epithelial cell proliferation and promoting synthesis of extracellular matrix proteins around the proximal airways (28,29). For further details on the action of growth and transcription factors see Chapter 13 of this volume. Branching Morphogenesis-Influence of Extracellular Matrix and Its Receptors

The interactions between the epithelial cells of the future airways and their underlying basement membrane appear to be another key event in epithelial– mesenchymal induction. Basement membranes are characterized by their typical layered morphological appearance and by a number of distinct glycoproteins and proteoglycans. A well-defined sheetlike matrix, consisting of a laminin and a collagen type IV polymer, provides the structural scaffold. Nidogens and other basement membrane components serve as bridging molecules between these two interwoven polymers. As well as their structural role, basement membranes influence cell differentiation and migration, as well as cellular life and death (30). The biological activity of basement membranes depends not only on their components but also on the three-dimensional architecture of their building blocks. As a further regulatory tool this architecture may be modified (31–33). As are any other extracellular matrix components, basement membrane proteins are specifically recognized by cellular receptors such as the integrins or the a-dystroglycan. Integrins represent a diverse family of integral membrane glycoproteins that form noncovalently associated ah heterodimers and play critical roles in both cell–matrix and cell–cell adhesion. These may be more generally referred to as cell migration, proliferation, and differentiation, as well as receptor-mediated laminin self-assembly (34). Laminins are composed of three chains (a, h, and g) encoded by separate genes, which assemble to form a cross-shaped or Y-shaped heterotrimer. To date five a, three h, and three g chains have been identified that may combine to form up to 15 different isoforms (35,36). The developmental role of laminins and their receptors were studied in perturbation experiments using

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functional antibodies in fetal lung organ cultures. Antibodies against the Cterminal domain of laminin-a1 chain, laminin-a5 chain, dystroglycan, and integrin subunits a6 and h1 resulted in an inhibition of branching and of organ growth. (18,25,37–39). Knockout experiments of a3 and a6 integrins revealed a synergistic role of a3 and a6 integrins during prenatal lung development. Both integrins contribute to branching morphogenesis and basement membrane assembly (25,40). A large number of, but not all, basement membrane proteins are synthesized by the overlaying epithelium (18,41). Nidogen-1 [also known as entactin-1 (15)] is synthesized by mesenchymal cells. It binds to the laminin-g1 chain and to collagen IV and serves as a bridging protein between these two networks of the basement membrane, (42). The interruption of the binding between laminin-g1 chain and nidogen leads to an inhibition of branching and growth in fetal lung organ cultures (22). In transgenic animals it leads in addition to disrupted basement membranes, resulting in abnormal air–blood barriers (10). Tenascin-C (TN-C) represents a large hexamer forming an extracellular matrix protein with adhesive and antiadhesive properties. It is only transiently found in the basement membrane at the tips of the branching and growing airways. Perturbation with antitenascin-C antibodies or with tenascin-C peptides causes a branching defect and larger terminal endbuds, but no inhibition of the growth of lung explants (24). These in vitro data were confirmed in vivo using TN-C deficient mice. In these mice branching, but not the growth of the lung, was reduced. This phenotype represents the first developmental phenotype of the tenascin-C null mouse strain (43). The same phenotype was observed in a8 integrin-deficient mice. However, the correlation to TN-C remains open, because it binds to fibronectin, vitronectin, osteopontin, and nephronectin in addition to tenascin-C (44). Secreted protein, acidic and rich in cysteine (SPARC) represents another protein possessing antiadhesive properties. It appears also to be involved in the regulation of branching, as shown by perturbation of fetal lung cultures with antibodies and synthetic SPARC peptides (45). A general interference with the collagen synthesis and secretion leads also to a reduction of branching in fetal lung organ cultures (26). However, which collagens are responsible for the described effect remains to be determined. Most likely it is not collagen type I, because branching is not affected in transgenic animals missing this collagen (46). Collagen type XVIII is likely to be involved. This collagen contains a so-called frizzled domain in its longest isoform, which implicates a role in Wnt signaling (47). Endostatin, a proteolytic fragment of collagen XVIII, possesses antiangiogenic activities (48). During branching morphogenesis collagen XVIII is expressed in the

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basement membrane zone at the growing tip of the bronchial tree. Blocking antibodies against collagen XVIII caused a reduction of Wnt2 expression and of branching in lung explants, but not in kidney explants (49). The extracellular matrix proteins fibrillin 1 and fibrillin 2 form microfibrils that take part in the formation of elastic fibers (50). Throughout branching morphogenesis, both proteins appear mostly in a subepithelial zone of the bronchial tree, with the exception of the matrix surrounding the growing terminal buds (51). Because the fibrillins appear earlier than elastin (52), an elastin-independent role of the fibrillins has to be postulated at least during early lung development. This was shown for both proteins: fibrillin 1 contributes to fetal lung development by the control of the activation of TGF-h (53), and a silencing of fibrillin 2 causes reduced branching and collapsed future conducting airways (54). Toward the end of branching morphogenesis, elastin is expressed in the mesenchyme surrounding the developing airways. In elastin-deficient mice early branching was unaltered, but late branching was stopped. The branching defect is accompanied by fewer but dilated terminal sacculi. Therefore, the fibrillins and elastin possess partly independent roles during branching morphogenesis. In addition, these proteins have structural and functional roles during alveolarization and in the mature lung (21). Not only growth factors but also extracellular matrix proteins contribute to the epithelial–mesenchymal induction processes, both including their receptors. The list of known factors taking part in the epithelial–mesenchymal cross-talk is not complete by far. Further details are found in various reviews (19,55–57). Epithelial and Smooth Muscle Cell Differentiation

Differentiation of the epithelium of the proximal airways proceeds centrifugally. In humans, basal cells are found as early as week 10. Ciliated, nonciliated, and goblet cells appear already by week 13 (58). At the end of the pseudoglandular stage the larger future airways are lined by a high columnar epithelium. The most distal epithelium is always maintained in a cuboidal undifferentiated state until branching morphogenesis is completed. These caps of cuboidal epithelial cells represent the growing and branching tips of the bronchial tree (59). The proximal larger future airways possess a continuous outer layer of contractile a-smooth muscle actin-positive cells. Because morphologically they are not yet fully differentiated, they are defined as smooth muscle cell precursors (60). More distally this layer of contractile cell becomes discontinuous and ends in front of the terminal buds (61). Until birth these con-

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tractile cells perform spontaneous contractions that start centrally and travel like a peristaltic wave into the periphery, pushing the intertubular liquid into the terminal buds. As a result the terminal buds are extended rhythmically. It was postulated that this mechanical signal stimulates branching via mechanotransduction (62,63). Arteries and Veins

The arteries develop simultaneously with the bronchial tree. As a general rule, arterial branches closely accompany the airway branches. In addition to these so-called conventional arteries, supernumerary arteries branch off between conventional ones and transport supplementary blood to regions along the airways. As first shown around week 14, both the conventional and supernumerary branches develop simultaneously throughout airway branching morphogenesis (64). The veins develop quite differently. They run interaxially in mesenchymal septa separating the lungs in segments and subsegments. Verbeken et al. (65) showed that venous branches systematically follow the connective tissue septa, extending in a plane between each generation of dichotomous airway branching. In the central areas this rule is broken: The large venous branches join the arteries and airways in order to reach the hilum. Currently it is estimated that approximately five-sixths of the three pulmonary trees (airway, arteries, and veins) are preformed at the end of the pseudoglandular stage. However, due to the exponential growth and branching (2n), this number is only true for the number of generations. In a first approximation, the number of branches, or the total length and total volume of these structures roughly double with every additional generation. Zones of Prenatal Lung Development

Based on morphological observation during rat lung development, Burri and Moschopulos (59) established a concept of zonal development. Zone I forms a superficial mantle around the lobes and the future acini. Consisting of primitive mesenchymal cells, it represents a zone of growth and branching of the epithelial tubules, which disappears with the onset of the saccular stage when branching is completed. Zone II is mainly a zone of differentiation. In electron micrographs its interstitium stains intensely due to a dense population of dark cells. Zones III and IV contain the elements of the airway tree and vascular system; zone IV corresponds to the most proximal generations with an adventitial layer. For all differentiation processes, a centrifugal directionality is manifested (Fig. 6). As well as its morphological aspect, the zone concept is also interesting for molecular aspects, because the expression of many factors correlates with

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Figure 6 Zonal prenatal development of the lung. Schematic drawing of the zone concept. A. Early pseudoglandular stage is characterized by the growing and branching epithelial tubules and by the absence of zone IV. B. Late pseudoglandular stage. All four zones are now present. Parts of zone III are transformed into zone IV. As a result of the recurrent growth of zone II (see arrow and asterisk in A), remnants of zone I form a thin sheet around structures of zone IV (central airways and vessels). These parts of zone I represent the peribronchial and perivascular growth regions of the bronchial tree. C. Canalicular stage. The airway lumina of zone III and the air spaces of zone II have widened. The terminal buds of epithelial tubules are still lined with cuboidal epithelium. D. Saccular stage. Zone I has completely disappeared. Airway lumina and airspaces have undergone a marked widening. (From Ref. 59.)

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the zones: FGF-10, the Gils, Sprouty 4, and hepatocyte nuclear factor 3h are expressed in zone I, and hepatocyte nuclear factor/forkhead homologue 4 in zone II. B. Canalicular Stage (16–26 Weeks)

At the beginning of the canalicular stage, large portions of the future gasexchanging parenchyma are already formed in the periphery of the human lung. Assuming that on average the lung contains 23 generations of airways (4), the last few generations formed by active branching are added at the beginning of the canalicular stage (3). Additional air space generations are likely to be added later by septation. Epithelial Differentiation: Formation of the Air–Blood Barrier

To achieve an operational parenchyma and to give premature babies a chance to survive [currently this is during the canalicular stage roughly around week 22 postcoital or week 24 postmenstrual (66)], epithelial differentiation and capillary proliferation are needed during this stage. The cuboidal epithelium of the bronchial tree differentiates first into type II epithelial cells, which start to produce surfactant; and next into type 1 epithelial cells, which cover most of the internal surface and contribute to the formation of the first areas providing thin air–blood barriers. The formation of the air–blood barrier requires a process that originally gave the canalicular stage its name. The lung parenchyma becomes canalized by capillaries, resulting in a dense capillarization of the primitive interstitium. The epithelial tubules distal of the conductive zone not only grow in length but also widen at the expense of the mesenchyme (Fig. 4b). The widened structures are called canaliculi. In rats the reduction of the mesenchymal volume includes a peak of programmed cell death (apoptosis) (67). The cellular death indicates that not only condensation of the mesenchyme but also a reduction of the cell number takes place. At the same time large parts of the cuboidal epithelium reduce their height and develop sheetlike extensions that cover most of the internal surface of the parenchyma. Finally, the type 1 epithelial cells are born. During the pseudoglandular stage the capillaries form a loose threedimensional network inside the mesenchyme (Fig. 5a). During the canalicular stage the (micro) vasculature of the mesenchyme proliferates strongly and capillaries come to lie closer to the epithelial tubules, forming a pericanalicular network (Fig. 5b). Underneath the regions covered by the thin epithelium, the capillaries form the air–blood barrier by an intimate contact to the squamous cells (Fig. 7). Both cells are only separated by one basement membrane, consisting morphologically of one central lamina densa and two laminae lucidae: one underneath the epithelium and the second underneath the endothelium.

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Figure 7 Formation of the air–blood barrier/canalicular stage. Although the most terminal part of the bronchial tree still contains a cuboidal, glycogenrich epithelium (closed arrow head), the epithelium of the more proximal parts begins to flatten out (open arrowhead) and to form thin air–blood barriers. During the latter process, capillaries located inside the mesenchyme (closed arrow) move towards the epithelium (open arrow). Bar, 100 Am.

Currently, it is only poorly understood what governs the formation of the air–blood barrier. It was hypothesized that again an interaction between the mesodermally derived endothelium and the endodermally derived epithelium was controlling this developmental step (68). First evidence was given by a transgenic mouse in which the sequence coding for the nidogen-binding site, g1III4, within the laminin-g1 chain (Lamc1 gene) was selectively deleted by gene targeting. These mice die directly after birth, most likely due to a failure of the gas exchange in the lungs. In large parts of the air–blood barriers the basement membranes are disrupted or missing, and epithelial and endothelial cells do not form close contacts (10). The cuboidal epithelium also gives rise to the type II epithelial cells. These cells start to accumulate lamellar bodies containing components of surfactant. Soon after this differentiation, surface-active material appears in the lung liquid, which is actively secreted by type II cells. In contrast to most species, in which surfactant appears late in gestation (at 80–85% of total

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duration of gestation), it is already present in small amounts at about weeks 22–24 (60% of gestation) in the human fetus (69). Before the lungs fully mature, surfactant appears to be more abundant in apical regions compared to basal regions of the lungs (70). This uneven distribution may explain the clinical observation that in some premature infants the development of hyaline membrane disease is more pronounced in basal than in apical parts of the lung. C. Saccular Stage (24–38 Weeks)

During the saccular stage the lung acquires its aerated or alveolar appearance, even if there are no alveoli present yet (Figs. 4c, 8a). The distal airways expand in length and width, acquire a saccular shape at the expense of the mesenchyme, and end in thick-walled termina saccules. Since these terminal saccules can be further subdivided, they may be transformed into ducts. Therefore it was suggested that the saccular airspaces be named transitory airspaces or transitory saccules (71). Capillary Network

The remarkable expansion of the future respiratory airspaces leads to a significant reduction of the fraction of interstitial tissue. This has important consequences for the arrangement of the capillaries. The capillary network forms a capillary layer around each future airway. Because the airways approach each other, a capillary double layer is formed inside the intersaccular septa (Fig. 5b,c). The interstitium in these septa contains numerous interstitial cells, but only a delicate network of collagen fibers. During the course of the saccular stage, a network of elastic fibers is deposited throughout the interstitium by smooth muscle cell precursors. This network of elastic fibers prefigures the appearance of the future interalveolar walls. As shown in experiments using transgenic mice, the combination of the presence of these smooth muscle cell precursors and of elastic fibers is a prerequisite for the formation of new septa during septation (21,72).

Figure 8 Rat lung during alveolarization and microvascular maturation. Postnatal lung development is shown at days P4 (a: end of the saccular stage), P10 (b: alveolarization), and P16 (c: microvascular maturation). Due to the formation of new septa, the mean size of an individual terminal air space decreases from days P4 to P10. During microvascular maturation the thickness of the alveolar septa decreases in parallel to the maturation of the alveolar capillary network. (Modified from Ref. 125.)

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In late fetal life the arteries, which are accompanying the airways, already possess the same type as in adult lungs. From the heart to the periphery the structure of their walls changes from elastic to muscular and finally to partially muscular. Not so the intrapulmonary veins. Depending on their diameter they possess very little or no smooth muscle during most of the fetal period. Throughout the saccular stage they start to develop a thin circular muscle layer resembling that of mature lung vessels (69). Time of Birth

In mammals the time point of birth varies relative to lung development. The marsupial quokka wallaby (Setonix brachyurus) is born in the canalicular stage and represents the mammal possessing the immaturest lung at birth so far documented (73). Insessorial mammals such as mice and rats are born during the saccular stage; humans are born in the early alveolar stage; precocial mammals such as sheep are born during late alveolar stage. In the latter mammals microvascular maturation occurs predominantly postnatally (Table 1). In humans the number of alveoli was estimated to be between 0 and 50 millions at birth (69). The exact number is almost irrelevant, because with a range of 212–605 million most alveoli are formed after birth (4,11).

IV. Postnatal Lung Development At birth the maturity of the lung varies very much between different mammals (see above). Although the onset of respiration represents a radical environmental change for the lung, it is more a functional than a structural event. Although lung liquid is rapidly replaced by air, this change has little influence on the pulmonary tissue framework. For technical reasons most of our knowledge about postnatal lung development is based on investigations of rats (74–77). However, it has been well established by comparative studies that the main characteristics of human and rat postnatal lung development are highly congruent, except for the absolute timing (11,78,79). In both species, postnatal lung development includes two stages—alveolarization and microvascular maturation—before normal growth occurs. The exact timing of these stages is still a matter of debate, because these slowly changing developmental processes do partly overlap. The transition from the alveolar stage to the stage of microvascular maturation especially occurs gradually (Table 1).

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Alveolar Stage (36 Weeks to 1–2 Years)

At the end of the saccular stage the lung goes though a short phase of expansion without any alteration of the parenchymal complexity. The distal airways consist of smooth-walled channels and saccules. The septa of the parenchyma are thick and contain a double capillary network. They are called primary septa (Fig. 8a). Septation: Alveolarization

During the next, alveolar, stage (humans: week 36, rats: postnatal day 4; (Table 1), the alveoli are formed by the lifting off of new tissue ridges from the existing primary septa. In light microscopic sections a large number of small buds appear along the primary septa (Figs. 8b, 9a). In three dimensions these buds correspond to low ridges and they represent newly forming septa (Fig. 10a). Soon these low ridges increase in height and subdivide the airspaces into smaller units: the alveoli (Fig. 10b). The newly formed septa have been termed secondary septa to differentiate them from the primary septa originally present at birth. The hypothesis has been put forward that the combination of the three components—smooth muscle cells, elastic fibers, and collagen fibrils—provides the critical driving force for septation (Fig. 11). Inside the primary septa, platelet-derived growth factor (PDGF)-receptor-positive smooth muscle cell precursors proliferate and move to the locations where the secondary septa will be formed. There, they deposit a network of mainly elastic fibers, but also of interstitial collagens. During lifting off of the new ridges the so-called ropes of this network stay at the tip of the newly forming septa. It is believed that these ropes take up mechanical forces as indicated by the mechanical stresssensitive expression of tenascin-C at the septal tip (Fig. 11) (24,72,80–84). The alveolar smooth muscle cells (or myofibro-blasts) are required for septation, because in PDGF-A-deficient mice, where these cells do not appear at their normal position and do not deposit the network of elastic fibers, no alveolarization takes place (84). However, it is not known if smooth muscle cells are only required for the production of elastic fibers, or if they also exert tractile forces. The investigation of elastin-null mice did not answer this question, because they do not survive beyond P3.5 and, therefore, do not enter the alveolar stage (21). Folding of Capillary Network

The ropes consisting of the smooth muscle cells, elastic fibers, and collagen fibrils are connected to the capillary layer underlying the septal surface where the new ridge will be formed. During lifting off, they pull this capillary layer

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Figure 9 Postnatal lung development of human lung. At 30 days after birth the lung parenchyma is characterized by thick alveolar septa (arrowheads) and numerous newly forming secondary septa (a: alveolarization; arrows). At 17 months the parenchyma consists mostly of thin septa and larger alveoli (asterisks). Newly forming secondary septa are barely detectable. Bar, 150 Am. (From Ref. 124.)

into the new septum. As a result, this leaflet of the double capillary network inside the primary septum folds up and gives rise to the double capillary network within the secondary septa (Fig. 12) (68,80). Both kinds of septa, primary and secondary, now contain two capillary layers, one on each side of a central layer of interstitium. This kind of thick septum is called immature or primitive to differentiate it from the thin mature septa of adult lungs that contain only a single capillary network. According to the model of lung alveolarization described by Burri (74), septation can only occur in the parenchyma as long as these double-capillary networks are present or when the capillary layer covers a sheet of connective tissue, as in the outermost periphery (peripleural, peribronchial, and perivascular air spaces) (85). In the described model of septation the capillary network has to grow in accord with the increasing surface area. This hypothesis was tested by the application of drugs inhibiting angiogenesis during the alveolar stage. In rats the application of fumagillin, thalidomide, and Su-5416 decreased alveolarization up to 22% and the pulmonary arterial density up to 36% (86). This finding emphasizes the importance of the capillary growth during postnatal lung development.

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Figure 10 Alveolarization as illustrated in scanning electron micrographs: rat lungs at days 4 (a) and 10 (b). Toward the end of the saccular stage the branches of the bronchial tree end in smooth-walled sacculi. At day 4 (a) at the start of alveolarization very low ridges start to appear, indicating the sites of secondary septa formation (arrow, a). At day 10 (b) the formation of the secondary septa (interalveolar walls, arrowheads) results in an incomplete subdivision of the sacculi into numerous shallow cups. The latter represent the forming alveoli. a, alveoli; s, saccules. Bar, 50 Am. (From Ref. 69.)

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Figure 11 Formation of secondary septa: septal structures. Smooth muscle cell precursors, or myofibroblasts, respectively, cause an accumulation of elastic fibers and tenascin-C (asterisk) at sites where low ridges start to form in the primary septa (closed arrow, a; b). The ridges grow into secondary septa (open arrow, c), which still contain a bilayered capillary network. (Modified from Ref. 71.)

Capillary Growth/Intussusceptive Growth and Remodeling

Capillary growth is important not only during alveolarization but also durig lung growth. In humans the lung volume increases about 23 times between birth and adulthood. The alveolar and capillary surface areas expand about 20 times and the capillary volume about 35 times during the same period (78,79). In principle, angiogenesis occurs by means of two distinctly different mechanisms: sprouting angiogenesis and intussusceptive angiogenesis (87,88). The latter has first been described in lung. Based on investigations

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Figure 12 Formation of secondary septa: capillary layers. Two flat perforated sheets represent the two capillary layers of a primary septum. The string represents elastic fibers, (a). b. Lifting off of the secondary septa. Note that the capillary double layer of the newly formed secondary septum is only interconnected at the tip of the septum. c. Scanning electron micrograph of a vascular cast (Mercox) showing the lifting off of a capillary layer during alveolarization. The dotted line labels the location, where elastic fibers, smooth muscle cells and tenascin-C were located. (Modified from Ref. 68.)

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of the microvasculature during lung development, Short postulated in 1950 (89) and our group independently in 1986 (87) that the capillary network of intervalveolar walls could grow by the addition of new meshes. These new meshes may be formed by the insertion of tiny transcapillary tissue posts or pillars <2.5 mm in diameter. Once the posts are formed, they are able to grow to full-size capillary meshes (Figs. 13, 14). Later, our group showed that intussusceptive microvascular growth is not limited to lung, but represents a ubiquitous mechanism of capillary network growth (90). Furthermore, it is also encountered during vascular tree formation and branching remodeling as part of an adaptive development of vascular systems (91–93). Expansion and Septation of Alveoli

Based on quantitative analysis of three-dimensional reconstructions of rat lungs, Randell et al. (77) postulated that newly formed alveoli may expand and be further subdivided by new septa. They proposed that successive rounds of alveolar expansion and septation must occur to explain the large increase in the number of alveoli in rats between birth and day 21. This would mean that additional septa would arise from still immature primary and secondary septa. This hypothesis is supported by the investigation of marsupials such as the quokka wallaby (94), in which a very large portion of the alveolar surface is formed by septation and not by branching. In the quokka wallaby the amount of septation is only conceivable if secondary septa may give rise to additional septa, and so on. Furthermore, such a multistep process of alveolarization may also explain the formation of complex clusters of alveoli as observed in casts of peripheral airways in humans (95). The proposed model also implies that not only the alveoli are formed by septation but also the last generations of the airway tree. Therefore, at least parts of the alveolar duct generations and the last terminal alveolar sacs may be formed by septation. The partitioning of the air spaces by septation resulted not only in a much larger parenchymal complexity but also in a strong increase in the alveolar surface (Sa). As shown by morphometry in rats, the alveolar surface increased with lung volume to the power of 1.6 during alveolarization. This represents a significantly larger increase than an assumed simple expansion of the airspaces, which would yield only an exponent of 0.67 (75). Completion of Alveolarization

In rats and mice bulk alveolar formation appears to be substantially complete at postnatal day 14 (81). Based on an estimation of the number of alveoli in three-dimensional reconstructions of rat lungs, it was postulated that additional alveoli are formed later (late alveolarization). Massaro and Massaro

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Figure 13 Microvascular maturation observed on vascular casts of rat lungs. Scanning electron micrographs of vascular casts (Mercox) of rat lungs are shown at postnatal days 4 (a) and 44 (b). The immature septa contain a double capillary network (open arrow, a). During maturation the capillaries rearrange and form, in most parts of the septa, one central single-layered capillary network (closed arrows, b). These capillary networks grow mainly by intussusceptive growth. Slender transcapillary posts (holes <2 Am in diameter, open arrowhead (a) are introduced into the capillaries and grow out to capillary meshes (closed arrowhead, a). Bar, 50 Am. (From Ref. 69.)

estimated that the number of alveoli in rats is increasing by at least a factor of 2 after bulk alveolarization has been completed (96). Sery et al. in rabbits (97) found formation of alveoli after the age of 12 weeks, which is far beyond the stage of initial alveolarization. Assessing alveolar numbers remains problematic even after the application of refined stereological counting methods, because of the difficulty in unequivocally defining an alveolus (85,98). Therefore,

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Figure 14 Intussusceptive capillary growth. During intussusceptive capillary growth the number of segments, the surface area, and the volume of an existing capillary network increase by the insertion of new transluminar tissue pillars (a–c). In the next step the formed pillars enlarge to form a new mesh (d). (Based on Ref. 126.)

the amount and also the mechanism of the postulated late alveolarization remain incompletely understood. In humans, the timing of alveolarization is not as well defined. Most likely the bulk of the alveoli occurs very rapidly after birth. Langston et al. (11) reported that alveolarization might be completed within 12–24 months. Previously it was suggested that alveoli may be formed up to the age of 8 years, but according to Langston et al. after the age of 2 years this may only happen at a very reduced pace. Cell Proliferation and Stem Cells

As shown for rats, the formation of secondary septa involves cell proliferation. Mesodermally derived interstitial and endothelial cells were identified by autoradiography to be the first cells to enter DNA synthesis. They were followed by type II epithelial cells, which showed the highest [3H]thymidine uptake at day 7. Usually uptake was higher at the base and in the immediate vicinity of the forming crests in these cell populations. Unlike in type II cells, no labels were detected on type I epithelial cells (99). This results confirmed that type I cells differentiate from type II cells and that the latter represent a stem cell population for the alveolar epithelial cells in the growing lung. In addition, type II cells serve also as stem cells during adult lung repair (100,

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101). Recently it has been shown in adult mice that circulating bone marrow cells may also be recruited to the lung (102). Influence of Glucocorticoids and Retinoids

Several studies have showed a strong influence of glucocorticoids on the process of alveolar formation. Massaro et al. (76,103,104) reported a very strong suppression of alveolarization in rats treated with dexamethasone from postnatal days 4–13. Repeating the experiments of Massaro and Massaro (104), Tschanz et al. (105) confirmed the results and found evidence that the failure of alveolarization may be due to a premature maturation of the interalveolar septa including their capillary layers. They hypothesized that in areas where the double capillary layers were prematurely reduced to single ones, the formation of secondary septa was hampered and the number of alveoli formed was decreased. Because postnatal dexamethasone treatment for 10 days induced permanent structural changes of the lung parenchyma in adult rats (104–106), we studied the consequences of an early and short high-dosage treatment of rats at postnatal days 1–4 (end of saccular stage, just before alveolarization starts). In these experiments the same premature maturation of the septa was manifest. This time we observed a delay and not a suppression of alveolarization, followed by a complete rescue of the structural changes (106,107). We were able to show that a peak of cell proliferation around postnatal day 4 and a peak of programmed cell death at postnatal days 19–21 were reduced to baseline (108). Furthermore, we found that the suppression of cell proliferation was caused by a transient downregulation of the cell cycle machinery. At day 4 cyclin/cyclin-dependent kinase complex activities were downregulated by an upregulation of the cyclin-dependent kinase inhibitors p21CIP1 and p27KIP1 (109). Massaro and Massaro showed that the glucocorticoid-induced permanent structural alterations of the lung parenchyma may be rescued by a treatment with retinoids (all-trans retinoic acid) following the glucocorticoid treatment. In the same experiments they showed that induction of a late alveolarization (formation of new secondary septa) with retinoids is also possible in control animals that did not receive any glucocorticoids (110,111). Alveolarization, microvascular maturation, and normal growth of the lung may also be altered by many other factors: for example mechanical ventilation, starvation, obesity, and changes in pO2. B.

Stage of Microvascular Maturation (Birth to 2–3 Years)

During approximately the third postnatal week, the stage of microvascular maturation takes place in rats and mice. In humans this stage starts a few

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months after birth and lasts for 2–3 years. Independent of the species, a large overlap between the alveolar stage and the stage of microvascular maturation has been observed. Therefore, no sharp limits of this stages may be given (112). Septal Maturation

In functional terms, septal maturation represents an optimization of the gas exchange apparatus. Morphometric measurements in rats revealed that the absolute mass of interstitial tissue decreases during septal maturation despite an overall volume gain of the lung by 25%. The loss of interstitium is due to a significant thinning of the alveolar septa. In parallel to the thinning, most of the double capillary networks are reduced to one central capillary layer (see below and Fig. 15). The former central layer of connective tissue is reduced to a thin fibrous meshwork interwoven with the capillaries. The capillaries are located on either side of the layer of connective tissue. As a result, most of the capillaries of the central microvascular network come in close contact with both surfaces of the septum. By this means the side possessing the thinner air–blood barrier will evidently contribute more to the gas exchange than the one containing the thicker barrier (Fig. 15) (74,75). After maturation of the septa development is completed and normal growth of the lung follows. Capillary Fusion and Differential Growth

Based on electron microscopic observations, Burri and co-workers (74,88, 113) postulated that the reduction of the double-capillary network to a single one involves two different processes. They posited that capillary fusion and differential capillary growth are employed in parallel and are equally important. An alternative possible mechanism would be the partial degradation of one of the two capillary leaflets. However, no evidence supporting this third hypothetical mechanism has been given so far. In the mature septum the layer of connective tissue is interwoven with the capillaries. If during microvascular maturation local growth of the capillary network occurs only on one side of the connective tissue layer, a single capillary layer will be formed in this part of the septum. As an alternative, the same result is obtained if growth occurs mainly in an area where fusion of capillary layers has already taken place. In both cases a septum, as shown in Figure 15, will be formed. So far, evidence has been obtained for the latter of these mechanisms. Serial sections of immature alveolar septa of rats at postnatal days 7 and 13 revealed very close contacts as well as fused lumina between the two capillary layers (113). Because every microscopic investigation of sections can only

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Figure 15 Septal maturation during the phase of microvascular maturation. Immature septa contain two capillary layers underneath both surfaces (arrowheads), which are separated by a layer of connective tissue (a, schematic drawing; b, electron micrograph of a human lung 30 days after birth). Upon maturation the connective tissue layer condenses to a septum that is interwoven with a now single-layered capillary network (c, schematic drawing; d, electron micrograph of a human lung 17 months after birth). One capillary of a mature septum is shown in an electron micrograph (e). Its extremely thin air–blood barrier is visible at its left upper and the connective tissue layer at its lower edge. Bm, basement membrane; ca, capillary; ct, connective tissue; ec, erythrocyte; fb, fibroblast/fibrocyte; ep I, type I alveolar epithelium; en, endothelium; lc, leukocyte; nu, nucleus; pl blood plasma. Bar, 5 Am. (a–d from Ref. 124; e from Ref. 127.)

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Figure 16 Formation of interalveolar pores of Kohn. Interalveolar pores start after the respective part of the septum matured (a, b, f ). Pores may be formed with (f–i) or without (b–d) the involvement of type II epithelial cells. a. Immature interalveolar septum with double capillary network. b. Cross-section of a part of an interalveolar septum covered by type I epithelial cells (ep I) and containing two capillaries. c. Due to a thinning of the septum in the region of a capillary mesh, the connective tissue disappears in a spotlike area and permits contact between the type I cells resting on both surfaces of the septum. After rupture (arrowhead) the cell margins withdraw (arrows) and only one leaflet of one of the type I cells remains. d. The same process happens again (arrowhead and arrows), resulting in the formation of an interalveolar pore (i). e. Type II epithelial cells may also be involved in the formation of interalveolar pores. In the first step a type II cell makes contact with a type I cell resting on the opposite side of the septum. f. The type II cell also becomes integrated into the epithelium opposite its original side. g. Parallel to the retraction of the type II cell, the two leaflets of the type I cells move toward each other. h. While forming the type I cell–cell

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provide a static picture of a dynamic process, it cannot be definitively determined in which direction—splitting or fusion—the capillary contacts will develop. However, in comparing immature and mature alveolar septa it is evident that, as a sum, capillary fusion drives the septum to maturity and not splitting. Therefore, Burri and co-workers (88,113) concluded that capillary fusion contributes to septal maturation together with differential capillary growth. Programmed Cell Death

The thinning of the alveolar septa does results in a reduction of the absolute mass of the interstitial tissue, the absolute number of fibroblasts by 10–20%, as well as of epithelial cells by more than 10% in rats (99). It was shown, first by Schittny et al. (114) and then by Bruce et al. (115), that toward the end of microvascular maturation (third postnatal week in rats) the surplus of fibroblasts is eliminated by classic apoptosis. Apoptosis is structurally defined by a typical pattern of morphological changes including ultimate fragmentation of the cell into membrane-enclosed vesicles (apoptotic bodies)(116). While this happens in fibroblasts, the surplus of epithelial cells, mainly of type II cells, is eliminated by programmed cell death without the appearance of apoptotic bodies. Most likely apoptotic type II epithelial cells are phagocytosed by alveolar macrophages in an early stage of programmed cell death before apoptotic bodies are formed. Both cell types are eliminated without an inflammatory reaction (114). The elimination of the cells differentiates programmed cell death and apoptosis from necrosis, in which cells disintegrate and inflammation is induced by cellular lysis (117). As expected, the cell proliferation index is low during microvascular maturation. The discrepancy between the rapid growth in alveolar surface area and lack of cell proliferation could be explained by an expansion of the type I epithelial cells, meaning that every single cell covers a larger surface area. Because the type II epithelial cells cover less than 10% of the alveolar surface, a reduction in these cells is only important in terms of surfactant production and reduction of the epithelial stem cells. Until the stage of microvascular maturation, a surplus of type II cells seems to exist that now may be

junction, the type II cell retracts completely and gives rise to a new pore. i. The type II cell is shifted along the septum and thus leaves the immediate vicinity of the pore. As an alternative, it may stay or differentiate into a type I cell. Cellular junction runs all around the pore (open arrowhead). c, capillary; ep I, type I epithelial cells; ep II, type II epithelial cells; ic, interstitial cell; p, pore. (Based on Ref. 118.)

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removed. However, type II cells that are forming type I cells are most likely not included in this process. Interalveolar Pores (Pores of Kohn)

Maturation of the alveolar septa includes the formation of interalveolar pores (arrowheads in Fig. 17b). Weiss and Burri (118) proposed that a local thinning of the alveolar septa leads to a transseptal contact of the epithelial cells. The formation of junctional complexes, the reorganization of the cell–cell contacts, and the retraction of epithelial cells finally lead to the formation of pores. Both type I–type I and type II–type I cell contacts may be formed (for details see Fig. 16). Because of the cuboidal shape of the type II cells, type II– type I contacts contribute more frequently to the formation of a pore than type I–type I cell contacts. In mice a first peak of pore formation appears in the third postnatal week during the stage of microvascular maturation. A second peak was described during the 6th–10th postnatal week (119–121). Interalveolar pores may also be formed later, perhaps even up to an older age, because septal thinning continues with further aging of the lung (85). Under normal physiological conditions the pores are bridged by surfactant. The pores may serve interalveolar exchange of alveolar liquid, surfactant components, and macrophages. Tubular myelin may be stored in the pores without increasing the gas diffusion pathway thickness in the alveolar subphase itself. It is believed that interalveolar pores are not used as pathways for collateral ventilation during normal breathing (122,123). V. Growth of the Lung After maturation of the interalveolar walls, lung development is completed and normal lung growth starts. In analogy to earlier steps of lung development, this transition happens smoothly. As shown in Figure 17 normal lung growth is characterized by an overall size increase of the organ without major structural changes. The compartments of the lung are now growing in a highly proportionate fashion. This means that at the start of this phase of growth the lung appears to be a miniaturized version of the adult one and that total lung volume increases linearly with body mass. If we take a closer, morphometric look, it appears that a stable lung morphology with a quantitatively constant proportion of the tissue framework does not exist. In our view, development blends unnoticeably into growth and growth into aging. Rat lung may serve as an example for this statement. At day 21 the rat lung has largely reached maturity and normal growth seems to follow. The lung volume increases steadily at roughly the

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Figure 17 Normal lung growth. Scanning electron microscopic images of a juvenile (a: 17 months) and adult human lung (b). During normal lung growth, the size of the alveoli but not their number increases. Interalveolar pores (Kohn) are present in the adult lung, but not in the juvenile one (arrowhead). Macrophages are labeled with an arrow. Bar, 100 Am. (From Ref. 124.)

three-quarters power of the body weight. However, in comparing the growth rate of different compartments such as air space, tissue, and capillary volumes, it appears that they do not grow proportionally to each other. Between days 21 and 131 tissue volume increases by a factor of 2.6, air spaces by a factor of 6.2, and capillary volume by a factor of 7.6 (75). These data clearly demonstrate that the adult lung does not possess a fixed composition of its structures, but that with aging it becomes more aerated and capillarization of

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the septa increases. Expansion of the capillary volume is achieved by intussusceptive capillary growth. As a result, not only the capillary surface increases but also the complexity of the capillary network: the number of segments and capillary meshes per area increases. The latter will result in a larger gas exchange surface area. The questions of to what extent late alveolarization is involved in dimensional alterations and to what extent the described differences are caused by differential growth remain open (85).

Acknowledgments We thank Barbara Krieger for expert technical assistance. The support of the Swiss National Science Foundation (grants number 0031.55895.98 and 0031.068256.02) is gratefully acknowledged.

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Schittny and Burri natal lung development in the quokka wallaby (Setonix brachyurus): a light microscopic study. Respir Physiol Neurobiol 2003; 134:43–55. Haefeli Bleuer B, Weibel ER. Morphometry of the human pulmonary acinus. Anat Rec 1988; 220:401–414. Massaro GD, Massaro D. Formation of pulmonary alveoli and gas-exchange surface area: quantitation and regulation. Annu Rev Physiol 1996; 58:73–92. Sery Z, Keprt E, Obrucnik M. Morphometric analysis of late adaptation of the residual lung following pneumonectomy in young and adult rabbits. J Thorac Cardiovasc Surg 1969; 57:549–557. Hansen JE, Ampaya EP. Lung morphometry: a fallacy in the use of the counting principle. J Physiol 1974; 37:951–954. Kauffman SL, Burri PH, Weibel ER. The postnatal growth of the rat lung II. autoradiography. Anat Rec 1974; 180:63–76. Bachofen M, Weibel ER. Basic pattern of tissue repair in human lungs following unspecific injury. Chest 1974; 65(suppl):14S–19S.: Suppl-19S. Evans MJ, Cabral LJ, Stephens RJ, Freeman G. Transformation of alveolar type 2 cells to type 1 cells following exposure to NO2. Exp Mol Pathol 1975; 22:142–150. Kotton DN, Ma BY, Cardoso WV, Sanderson EA, Summer RS, Williams MC, et al. Bone marrow-derived cells as progenitors of lung alveolar epithelium. Development 2001; 128:5181–5188. Blanco LN, Massaro GD, Massaro D. Alveolar dimensions and number: developmental and hormonal regulation. Am J Physiol 1989; 257:L240–L247. Massaro D, Massaro GD. Dexamethasone accelerates postnatal alveolar wall thinning and alters wall composition. Am J Physiol 1986; 251:R218–R224. Tschanz SA, Damke BM, Burri PH. Influence of postnatally administered glucocorticoids on rat lung growth. Biol Neonate 1995; 68:229–245. Schwyter M, Burri PH, Tschanz SA. Geometric properties of the lung parenchyma after postnatal glucocorticoid treatment in rats. Biol Neonate 2003; 83: 57–64. Tschanz SA, Makanya AN, Haenni B, Burri PH. Effects of neonatal high-dose short-term glucocorticoid treatment on the lung: a morphologic and morphometric study in the rat. Pediatr Res 2003; 53:72–80. Luyet C, Burri PH, Schittny JC. Suppression of cell proliferation and programmed cell death by dexamethasone during postnatal lung development. Am J Physiol Lung Cell Mol Physiol 2002; 282:L477–L483. Corroyer S, Schittny JC, Djonov V, Burri PH, Clement A. Impairment of rat postnatal lung alveolar development by glucocorticoids: involvement of the p21CIP1 and p27KIP1 cyclin-dependent kinase inhibitors. Pediatr Res 2002; 51:169–176. Massaro GD, Massaro D. Retinoic acid treatment partially rescues failed septation in rats and in mice. Am J Physiol Lung Cell Mol Physiol 2000; 278:L955–L960. Massaro GD, Massaro D. Postnatal treatment with retinoic acid increases the number of pulmonary alveoli in rats. Am J Physiol 1996; 270:L305–L310.

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112. Burri PH. Fetal and postnatal development of the lung. Annu Rev Physiol 1984; 46:617–628. 113. Caduff JH, Fischer LC, Burri PH. Scanning electron microscopic study of the developing microvasculature in the postnatal rat lung. Anat Rec 1986; 216:154– 164. 114. Schittny JC, Djonov V, Fine A, Burri PH. Programmed cell death contributes to postnatal lung development. Am J Respir Cell Mol Biol 1998; 18:786–793. 115. Bruce MC, Honaker CE, Cross RJ. Lung fibroblasts undergo apoptosis following alveolarization. Am J Respir Cell Mol Biol 1999; 20:228–236. 116. Wyllie AH, Kerr JF, Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol 1980; 68:251–305. 117. Lockshin RA, Williams CM. Programmed cell death: cytology of degeneration in the intersegmental muscles of the silkmoth. J Insect Physiol 1965; 11:123–133. 118. Weiss M, Burri PH. Formation of interalveolar pores in the rat lung. Anat Rec 1996; 244:481–489. 119. Amy RWM, Bowes D, Burri PH, Haines J, Thurlbeck WM. Postnatal growth of the mouse lung. J Anat 1977; 124:131–151. 120. Ranga V, Kleinerman J. Interalveolar pores in mouse lung. Regional distribution and alterations with age. Am Rev Respir Dis 1980; 122:477–481. 121. Kawakami M, Paul JL, Thurlbeck WM. The effect of age on lung structure in male BALB/cNNia inbred mice. Am J Anat 1984; 170:1–21. 122. Bastacky J, Goerke J. Pores of Kohn are filled in normal lungs: low-temperature scanning electron microscopy. J Appl Physiol 1992; 73:88–95. 123. Peao MN, Aguas AP, de Sa CM, Grande NR. Morphological evidence for migration of particle-laden macrophages through the interalveolar pores of Kohn in the murine lung. Acta Anat (Basel) 1993; 147:227–232. 124. Tschanz SA, Burri PH. Pra¨- und postnatale Entwicklung und Wachstum der Lunge. In: Rieger C, von der Hardt H, Sennhauser FH, Wahn U, Zach M, eds. Pa¨diatrische Pneumologie. Heidelberg: Springer Verlag, 1999:3–16. 125. Schittny JC, Luyet C, Burri PH. Suppression of a peak of programmed cell death by dexamethasone in postnatal developing lung. Ann Anat 1999; 181:307. 126. Kurz H, Burri PH, Djonov VG. Angiogenesis an vascular remodeling by intussusception: from form to function. News Physiol Sci 2003; 18:65–70. 127. Schittny JC, Burri PH. Anatomie des Respirationstraktes. In: Rieger C, von der Hardt H, Sennhauser FH, Wahn U, Zach M, eds. Pa¨diatrische Pneumologie. Heidelberg: Springer Verlag, 1999:17–28. 128. Ten Have-Opbroek AAW. Lung development in the mouse embryo. Exp Lung Res 1991; 17:111–130. 129. Miettinen PJ, Warburton D, Bu D, Zhao JS, Berger JE, Minoo P, et al. Impaired lung branching morphogenesis in the absence of functional EGF receptor. Dev Biol 1997; 186:224–236. 130. Bryden MM, Evans H, Binns W. Embryology of the sheep. 3. The respiratory system, mesenteries and celom in the fourteen to thirty-four day embryo. Anat Rec 1973; 175:725–735.

13 Structure and Function of Nonmammalian Vertebrate Lungs

J. N. MAINA University of the Witwatersrand Johannesburg, South Africa

I. Introduction

The respiratory system of any animal species has its own characteristics, and there are more than a million species. The physiological diversity parallels the phenotypic diversity. From the body plan and from the main characteristics of the respiratory system, some simplifying models may be constructed that lead to clarification of the main general dispositions and to their possible working mechanism. (1)

Respiratory organs provide the first interface between molecular oxygen in the ambient environment and the aerobic machinery of the body. Their design and construction (i.e., the assembly and size of their constitutive parts) influence the effectiveness of their transfer of oxygen and, consequently, of the generation of energy through oxidative phosphorylation. Therefore, the oxidative capacity of an animal is greatly affected by the efficiency of its respiratory organs/structures. Body size, habitat occupied, respiratory medium utilized, phylogenetic status reached, and lifestyles pursued determine the 319

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structure and function of the respiratory organs. Because the physics of the respiratory gases is determined by the immutable laws that govern their behavior, the basic principles of gas exchange should fundamentally be the same in all animals irrespective of their various phylogenetic levels of development. At the gas exchange level, lungs have certain common structural attributes. These include vast surface area, large capillary blood volume, and thin partitioning between air and blood. These features confer a high diffusing capacity for the respiratory gases. An extensive respiratory surface area is produced through internal subdivision and branching of the airways; a large pulmonary capillary blood volume by intense vascularization of the surfaces of the terminal gas exchange units; and a thin tissue barrier by remodeling of tissue and cellular components over the blood–gas barrier (2,3). At all levels of organization of biological entities from cellular through organic to organismal levels, morphology is the outward display of the form, size, and arrangement of the constitutive elements. Function is the expression of calculated performances of the various formative parts. One inevitably affects the other. It is now unequivocally clear that for optimal function of an organ, the two attributes of structure and function must be inextricably interrelated (4,5). In the purposeful (function-oriented) biological engineering through evolution and adaptation, the task of developing optimal (i.e., costeffective) structures has not been a cheap one. About 99.99% of all animal species ever evolved in the 4 billion years that life has existed on Earth are now extinct (6). Regardless of the factor(s) that directly or indirectly precipitated their demise, from the perspective of their having been inadequately prepared for life, such animals can circumspectively be considered to have been failed evolutionary experiments. As equally well pertains to human structural engineering, in biology only certain designs and constructions perform best under a given set of conditions. To perpetuate fitness for survival, when it has been established, novel states are severely defended and subsequently genomically conserved. Notwithstanding the great morphological diversity that exists in modern respiratory organs, a common cellular mechanism may have evolved to overcome the fundamental problems associated with the capacity for acquisition of oxygen by air breathing. Indeed, at their most elementary level of operation, the designs of vertebrate lungs are remarkably structurally and functionally similar. Under a partial pressure gradient created by the ventilation and perfusion of a respiratory organ, flux of respiratory gases (oxygen and carbon dioxide) occurs by passive diffusion across a thin trilaminar tissue barrier. In extreme cases, a barrier that measures only a fraction of a micron separates air and blood. In birds (2,3), for example, the blood–gas (tissue) barrier is comprised of highly attenuated epithelial and endothelial cells separated by a thin common basement membrane. Functionally, at that level, the respiratory medium utilized, complexity, composition, and organization of the tissue

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elements are inconsequential. For example, at a partial pressure gradient equivalent to that of air at sea level, air-breathing animals such as rats, cats, and dogs have been experimentally kept alive while being mechanically ventilated with isotonic liquids (particularly perfluorocarbons) enriched with oxygen (7). When death occurs, it does not result from asphyxia. It is due to an acid–base imbalance (acidosis) that arises from accumulation of carbon dioxide that cannot be efficiently eliminated from the body owing to the higher viscosity of the liquid compared with air. The vertebrate lung is estimated to consume as much as 10% of the total body’s oxygen consumption (8). By default, compared with other organs, the respiratory organs are unique in that functionally a conflict among oxygen uptake, utilization, and delivery to the tissues can occur. For optimal performance, as much as possible of the oxygen procured by a respiratory organ must be transferred to the body’s tissues/cells for aerobic metabolism, with the organ itself consuming as little of it as possible. To obviate a conflict, the minimal quantity of tissue/cells should be used in the construction of the respiratory organs without compromising their functional integrity. Although best known for their gas exchange function, respiratory organs are fundamentally multifunctional. Their nonrespiratory roles include synthesis, metabolism, and regulation of concentrations of pharmacologically active agents and lipids such as pulmonary surfactant (9). Although gas exchange efficiency should be enhanced by use of minimal structural tissue, a critical tissue/cell mass is necessary for the performance of nonrespiratory roles. The design requirements for respiratory and the nonrespiratory functions therefore conflict. As a consequence, in developmental terms, the ultimate designs of respiratory organs provoke certain trade-offs and compromises. The morphologies and physiologies of the respiratory organs should be examined judiciously from a holistic perspective: from their inclusive functions and not from the better-known, narrow aspect merely of gas exchange. This chapter will succinctly review the functional design of nonmammalian adult vertebrate lungs. Dipnoan, amphibian, reptilian, and avian lungs are considered. We briefly underscore the essence of the various designs of lungs in animal taxa that have adapted to different habitats, attained different phylogenetic statuses, and pursued various lifestyles.

II. Lungfish (Dipnoi) Lungs The discovery (almost simultaneously in the 1830s) of the South American lungfish, Lepidosiren paradoxa, and the African lungfish, Protopterus, and 150 years ago (10) of the Australian lungfish, Neoceratodus forsteri, stimulated intense interest and controversy that was only to be later rivaled by the discovery of the archaic coelecanth, Latimeria chalumnae (termed a living

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fossil) in the 1920s (11). The lungfishes (Order Dipnoi) are considered to be a monophyletic group (12). There are only 3 genera and 6 extant species of an estimated 55 extinct genera and 112 species. The central systematic position of the dipnoans, especially regarding the emergence of the tetrapods and their fascinating natural history, makes them ideal models in studies of most evolutionary and adaptive processes in vertebrate biology. The transitional mode of breathing typical of the lungfishes (Lepidosiren and Protopterus being obligate air-breathers and Neoceratodus a facultative air-breather) presents an important prototype for understanding the motivation and the drive for switching from water to air breathing (13). The consequential impact of the capacity for air breathing on the adaptive radiation of terrestrial animal life is of profound interest to evolutionary biologists, ecologists, systematists, morphologists, and physiologists. The transition from water to land, one of the most important events in the evolution of animal life, presents an archetypal event for understanding elaborate transactions in the change from gill to pulmonary respiration and the founding of intricate neural control process necessary for the coordination of respiration with circulation. The attainment of air breathing was a decisive event in a sequence of calculated preadaptations for terrestrial habitation. With rise in the levels of atmospheric oxygen, the shift from water to air breathing was an imperative. The archaic fish (e.g., lungfishes and the bichirs) and three-quarters of modern amphibious fish living in tropical and subtropical regions breathe air to various extents (14,15). This indicates that the factors leading to air breathing may have been most severe in such regions of the Earth. High environmental temperatures in the tropical regions caused low oxygen solubility in water; increased the putrefaction of organic plant matter; and caused the shallow, extensive continental shelves to dry up. This resulted in overcrowding and competition for finite resources. Increase in salinity and turbidity may have acted as further compelling factors for organisms to leave the water. A compounding of different events provoked a hypoxic crisis exacerbated by hypercapnia. In the Silurian–Devonian periods (16), for example, the levels of oxygen dropped to about 10% compared with the modern ones (21%). Environmental Hypoxia is invariably associated with some degree of hypercapnia, especially in standing, plant-infested waters of the tropical swamps (17). Hypoxia, particularly when accompanied by hypercapnia, constitutes a very strong driving force that induces air breathing (18). With their over 350 million years of evolution (12,19), by evolving a capacity for breathing air, the lungfish faced and surmounted severe environmental conditions. The natural history of lungfish has been studied (17,20). The structure and function of the lungs of L. paradoxa were studied (21–23), those of Protopterus (24–29), and those of N. forsteri (21,30–33). The diving physiology of lungfish has also been reviewed (34). Except for Neoceratodus (an obligate water breather) in which secondary lamellae occur in all the gill

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arches (35), the gills of Lepidosiren and Protopterus (facultative water breathers) are devoid of secondary lamellae (36). The gills of Lepidosiren and Protopterus are predominantly involved in the clearance of carbon dioxide (37). The lungs of Protopterus aethiopicus are greatly internally subdivided by hierarchically arranged septa that delineate air cells (faveoli) (Figs. 1–3) . The faveoli open into an eccentrically placed air duct (Fig. 1). Compartmentalization of the lung greatly increases the respiratory surface area (29). The septa support blood capillaries exposed to air only on one side (Figs. 3, 4),

Figure 1 Scanning electron micrograph of a transverse section a lung of the lungfish, P. aethiopicus, shows an eccentrically located air duct (d) that opens into peripherally placed air spaces—faveoli (c)—that are separated by septa (s). Arrowheads (a a ), arrows (—a a ), and stars (x) show the elaborate pleural coverings of the lung. Scale bar = 1.3 mm.

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Figure 2 Scanning electron micrograph of the internal surface of a lung of the lungfish, P. aethiopicus, shows hierarchical septa (s) that divide the lung into small air spaces: faveoli. Scale bar = 0.5 mm.

constituting what is termed a double capillary arrangement. The perikarya of undifferentiated pneumocytes (Figs. 5, 6) occur in shallow depressions between loops of blood capillaries that project into the air space (Figs. 4, 5). The lungs of lungfish contain a surfactantlike material (22,29) that contains both surfactant A and surfactant B-like proteins (33). III. Amphibian Lung The modern amphibians occupy a pivotal position in our understanding of the evolution of the tetrapod lung and the progressive development of

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Figure 3 Histological section of the internal surface of a lung of the lungfish, P. aethiopicus, shows respiratory air spaces—faveoli (a)—separated by septa (stars). Arrowheads, blood capillaries found on opposite sides of the interfaveolar septae; arrows, smooth muscle tissue. Scale bar = 1 mm.

preadaptations required for transition from water to land. Dual subsistence in water and land has compelled unique physiological and morphological adaptations in amphibians. The multiplicity of respiratory structures corresponds with the diversity of the environments that amphibians occupy, the various lifestyles they pursue, and their uncharacteristic interrupted pattern of development (38). In amphibians, gills are transitory respiratory organs that develop in the embryo. They serve as the principal gas exchangers during aquatic larval existence and disappear at metamorphosis, when lungs take over during semiterrestrial or fully terrestrial adult life. Among the tetrapods, the amphibian lungs are the simplest in structural terms (39). Pulmonary vascularization correlates with the extent of terrestriality, behavior, and

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Figure 4 Scanning electron micrograph of an interfaveolar septum (s) of a lung of the lungfish, P. aethiopicus, shows blood capillaries (c) bulging from opposite sides of the interfaveolar septae. Arrows, red blood cells. Scale bar = 30 Am.

tolerance to dehydration (40). In predominantly aquatic species, the skin is the foremost respiratory pathway while in the more terrestrial ones it has been relegated to a lesser role or totally rendered redundant. In such groups, the lung serves as the primary respiratory organ. Although amphibians generally live in water and humidic habitats, a few exceptional species have adapted very well for life in highly dessicated areas (40). Terrestrial anurans such as Chiromantis xerampelina have developed an impermeable skin (41) and ureotelism (42) for water conservation. The two species can endure water loss in excess of 60% of their total body mass (43). The highly xerophilic African tree frog, Chiromantis petersi, leads a characteristically unamphibian lifestyle: it prefers direct solar radiation and temperatures of 40–42jC (44). The need to balance water conservation with gas exchange may explain why there are no large modern amphibians. Among the contemporary amphibians there are three Orders: These are the Gymnophiona (= Apoda = caecilians), Salentia (= Anura), and

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Figure 5 Scanning electron micrograph of the surface of an interfaveolar septum of a lung of the lungfish, P. aethiopicus, shows blood capillaries (c) containing red blood cells (e). g, Nuclei of undifferentiated pneumoctes; arrowheads, secretory material; arrows, blood–gas barrier. Scale bar = 5 Am.

Caudata (= Urodela). The highly elusive (largely fossorial or aquatic), vermiform, tropical caecilians are the least studied. They are in evolutionary terms a monophyletic distant group that has been isolated from the other two other orders for at least 70 million years (45). Compared with other airbreathing vertebrates, amphibians have manifestly simple lungs with low diffusing capacities for oxygen. Notwithstanding their simplicity, such lungs are adequate in a taxon that has typically low aerobic metabolism (46,47) and multiple respiratory sites (39). The lungs of Necturus and Cryptobranchus are thin-walled, transparent, poorly vascularized, and nonseptate (39,47). The lungs of anurans and apodans are morphologically and morphometrically more advanced than those of the urodeles (48,49). In amphibians, the anurans have the most complex lungs, which are intensely subdivided: a central air

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Figure 6 Transmission electron micrograph of a pneumocyte of a lung of the lungfish, P. aethiopicus, shows osmiophilic lamellated bodies (asterisks). n, Nucleus; arrowheads, microvilli; arrows, intercellular junctions; m, mitochondrion; r, rough endoplasmic reticulum; v, multivesicular body. Scale bar = 0.5 Am.

duct opens into peripheral stratified air spaces (50,51) (Fig. 7). The internal morphology of such more elaborate lungs corresponds to that of the lungs of lungfish (Dipnoi) (28,52). On average, the thickness of the blood–gas barrier in urodeles is 2.59 Am, 2.35 Am in apodans, and 1.89 Am in anurans (48). Some regions of the blood– gas barrier of the lungs of caecilians, Chthonerpoton indistinctum and Ichthyophis paucesulcus, may, however, be only 1 Am thick (52) while in the tree frog, Hyla arborea, the barrier may be as thin as 0.6 Am (48). The lungs of the highly terrestrial amphibian species such as the toad, Bufo marinus (49); the tree frogs, H. arborea (50); and C. petersi (53), are markedly elaborate. A series of hierarchical septa delineate air cells that range in diameter from 1.45 mm in Rana pipiens to 2.3 mm in B. marinus and Rana catesbeiana (54). The respiratory surface area in the lungs of more terrestrial amphibian species is greater than that in the lungs of the more aquatic ones (54). The caecilians have simple, long, tubular, internally divided lungs. In some species such as the African caecilian, Boulengerula taitanus, the left lung is very small (55). However, in the aquatic Typhlonectes compressicauda, as

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Figure 7 Transmission electron micrograph of the lung of the tree frog, C. petersi, shows a respiratory air space—faveolus (a)—and blood capillaries (c) containing red blood cells (e). g, Granular (type II) pneumocyte; arrows, intercellular junctions; b, osmiophilic lamellated bodies; stars, blood–gas barrier; s, nucleus of a type I pneumocyte; p, nucleus of a pericyte; n, nucleus of an endothelial cell. Scale bar = 10 Am.

many as three lungs develop (56). The lungs of B. taitanus are supported by two conspicuous diametrically placed trabeculae onto which septae that delineate respiratory air spaces insert (55). The lungs are comparable in structural terms to those of the almost limbless, large aquatic salamanders such as Amphiuma and Siren (57). The long caecilian lung may undergo ventilatory limitations during locomotion from compression by the trunk muscles. As has been reported in a running lizard (58), an animal with correspondingly long lungs and elongate body shape, temporal dissociation between breathing and locomotion may occur in caecilians. Regarding functional and structural adaptations for respiration, parameters such as high hemoglobin concentration, small numerous erythrocytes, large blood volume, vascularization, and internal subdivision of the lungs occur in amphibians (59). These features correspond with the metabolic

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requirements of individual species. The newt, Triturus alpestris, with characteristically low metabolic rate has smooth-surfaced lungs (60) with 569 capillary meshes per square centimeter (61). On the other hand, the relatively more metabolically active tree frog, H. arborea (50), has more elaborate lungs with 652 capillary meshes per square centimeter (60). Plethodontid salamanders, a taxon that constitutes the largest family among the Caudata, acquire all their oxygen requirements across a highly vascularized skin from the cold, well-oxygenated water they subsist in. In lungless amphibians, the length of the skin blood capillaries constitutes 90% of all blood vessels on the respiratory surfaces, with the rest occurring in the buccal cavity (60). To enhance oxygen uptake, the epithelial lining of the buccal cavity is very thin (60–62). Caudates such as Salamandra, Amphiuma, Megalobatrachus, and Siren species, which mainly use lungs for gas exchange, have well internally subdivided lungs (56,63): the skin is poorly vascularized and the epidermis is very thick (47–110 Am) (60,62). In two species of Salentia that live in welloxygenated high mountain lakes, Telmatobius and Batrachophrynus, the lungs are very small, the body is very well vascularized, and the epidermis is very thin (57). In the lungs of most amphibian species, smooth muscle tissue is

Figure 8 Scanning electron micrograph of a surface macrophage (m) of the lung of the tree frog, C. petersi. Arrows, filopodia. Scale bar = 2 Am.

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predominant (53,55,64). That may explain the high mechanical compliance of the amphibian lung (63,64). In Amphiuma, during expiration, the lung virtually collapses, producing an almost 100% turnover of ventilated air (63). The lungs of Pipa pipa and Xenopus laevis are reinforced with septal cartilages that maintain the patency of the air passages (65,66). Welldifferentiated pneumocytes (Fig. 7) as well as dust cells (free = surface phagoctes) (Fig. 8) occur on the pulmonary surfaces of certain amphibian lungs (55,67).

IV. Reptilian Lung Reptiles were the first vertebrates to be adequately adapted for terrestrial habitation. Switch from ammonia to urea/uric acid elimination of nitrogenous endproducts of metabolism and development of an impermeable body

Figure 9 Scanning electron micrograph of the internal aspect of the lung of the black mamba, Dendroaspis polylepis, shows interfaveolar septa (s) that separate faveoli (f ). Arrows, faveoli running from a central air duct to the pleural aspect; t, trabeculae supporting interfaveolar septa. Scale bar = 0.1 mm.

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cover curtailed water loss, averting risk of dessication on land. In a trade-off, the skin was relinquished as a respiratory pathway, with the reptiles becoming lung (= air) breathers. The respiratory organs of reptiles show marked structural diversity (68,69). This correlates with the multiplicity of the habitats the taxon occupies and the lifestyles they pursue. There is no model reptilian lung (69). In the more advanced snakes (e.g., Colubridae, Viperidae, and Elapidae), the left lung is remarkably small and is in some cases entirely lacking. In the primitive species of snakes (e.g., boas and pythons), the left lung is present (70–72). In the Amphisbenia, the right lung is very small (73). In the order Squamata, single-chambered lungs predominate especially in families such as Teiidae (68), Scindae (74), Lacertidae (75), and Gekkonidae (76). Extremely simple lungs occur in the family Angioidea (73). Because the lungs of the more primitive species of reptiles are more homogeneous, it would appear that pulmonary morphological heterogeneity imparts a distinct functional advantage. Based on degrees of internal subdivision, various classifications of the reptilian lungs have been proposed (77,78). The multicameral lungs (e.g.,

Figure 10 Scanning electron micrograph of the internal aspect of the lung of the black mamba, D. polylepis, shows openings into faveoli (f ) separated by septa (s). t, Trabeculae supporting interfaveolar septa and delineating air ducts. Scale bar = 0.2 mm.

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those of turtles, monitor lizards, crocodiles, and snakes) (71,79–81) (Figs. 9–12) are intensely subdivided. The paucicameral ones (chameleons and the iguanids) are less elaborate. The unicameral ones (teju lizard, Tupinambis nigropunctatus) are simple, saccular, smooth-walled, and transparent (74,82). The land-based chelonians have paucicameral lungs: the lungs have two or three peripheral compartments that open into a central air space. Such lungs lack an intrapulmonary bronchus. Marine reptiles have multichambered subdivided into bronchioles lungs (83,84). The elongated lungs of the Ophidia (snakes) and Amphisbaenids are divided structurally into two zones: the anterior respiratory region is highly vascularized while the posterior one is saccular and avascular (71,72,74,85,86). In the crocodilian lung, where much of the parenchyma (the gas-exchange tissue) is found in the anterior twothirds of the lung, blood makes up 38–50% of the total volume of the region (79). The posterior, less vascularized part of the lung is assumed to store air

Figure 11 Scanning electron micrograph of the internal aspect of the lung of the monitor lizard, Varanus exanthematicus, shows air spaces—faveoli (stars)— separated by septa (s). Scale bar = 0.3 mm.

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Figure 12 Scanning electron micrograph of a cast of the lung of the pancake tortoise, Malacochersus tornieri, shows faveoli (asterisks). Arrows, interfaveolar septa; arrowheads, peripheral subdivision of the faveoli into smaller air spaces. Scale bar = 0.2 mm.

(87), serve a hydrostatic role (88), and mechanically ventilate the exchange tissue in the anterior region of the lung. The current morphological classification of the reptilian lung based on the intensity of internal compartmentalization is oversimplistic: transitional forms and gradations occur. The simple lungs in Sphenodontia correspond in their level of development to those of amphibians. Such lungs have a central air duct that opens into peripherally located, shallow respiratory air spaces that are poorly vascularized. The brochoalveolar lung of mammals and the parabronchial one of birds are thought to have evolved from transformation of the reptilian multicameral lung (68,89–92). It has been speculated that inherent structural inadequacies in the design of the reptilian lungs may have hindered reptiles from realizing endothermy–homeothermy (68,69), relegating their aerobic capacities to less than those of birds and mammals. Varanids (monitor lizards) present the greatest degree of pulmonary developmental

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complexity in the suborder Sauria. V. exanthematicus and the pancake tortoise, M. tornieri, have multichambered lungs with bifurcated intrapulmonary bronchi and profuse internal subdivision (81,93) (Figs. 11, 12). Singlechambered lungs require less energy to ventilate. They occur in those animals with low metabolic rates (72). For animals of equivalent body mass, a reptile has a lung volume seven times greater than that of a mammal (94,95) but its diffusing capacity for oxygen is relatively low (94). The aerobic capacities of reptiles are lower than those of mammals: at a temperature of 37jC, a 1 kg lizard consumes 122 ml O2/h, which constitutes only 18% of the oxygen consumption of an equivalent-sized mammal (96). The upper air passages of the reptilian lungs are lined by ciliated and mucus-secreting epithelial cells (Fig. 13). In the more advanced species, the pulmonary epithelial cells are distinctly differentiated into types I (squamous : smooth), II (cuboidal : granular) (70,71,76,97) (Fig. 14), and III (brush) pneumocytes (98). A rare mitochondria-rich cell has been described in the lung of the turtle, Pseudemys scripta (99). It is conceivable that differentiation

Figure 13 Scanning electron micrograph of the trachea of the black mamba, D. polylepis, shows ciliated cells (c) and nonciliated cells (s). Some of the nonciliated cells (m) have microvilli on their free surface. Arrows, intercellular junctions; arrowheads, secretory substances. Scale bar = 10 Am.

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Figure 14 Transmission electron micrograph of a type II cell of the lung of the black mamba, D. polylepis, shows osmiophilic lamellated bodies (a). v, Vesicular bodies; arrow, Golgi body; stars, mitochondria; n, nucleus; c, collagen fibers; p, pericytes; arrowheads, microvilli. Scale bar = 0.5 Am.

of pulmonary pneumocytes, as has occurred in mammals, birds, and certain reptiles and amphibians, may adaptively enhance respiratory efficiency. This may occur through a reduction in numerical density of the metabolically active surfactant-producing type II cells and by stretching and attenuation of the type I cells (metabolically inert cells that largely lack cell organelles) in order to cover the areas vacated by the type II cells (Figs. 15, 16). Delivery of oxygen to the tissue cells is promoted by reduction of oxygen consumption by the respiratory organ itself and by development of a thin blood–gas (tissue) barrier. Dust cells (surface macrophages) occur in reptilian lungs, such as, in the turtle: Testudo graeca (98). What is termed a double capillary arrangement, in which pulmonary blood capillaries are exposed to air only on one side, prevails in the reptilian lungs (Fig. 15). Reptilian lungs have a preponderance of smooth muscle tissue. In the lungs of the tegu and monitor lizards, smooth muscle tissue constitutes 7.4% and 1.3%, respectively, of the nontrabecular tissue (83,100). Smooth muscle tissue is involved in promoting intrapulmonary convective air movement (80,101). Compliance of the garter snake lung, Thamnophis sirtalis, of 0.042

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Figure 15 Transmission electron micrograph of blood capillaries (c) of the lung of the monitor lizard, V. exanthematicus. Arrows, smooth muscle; w, white blood cell; e, erythrocyte; arrowheads, blood–gas barrier. Scale bar = 10 Am.

ml/cmH2O/g is 50 times that of the lung of a mouse, a mammal of equivalent body mass (79,102). Compliance of crocodile lung (0.7 ml/cmH2O/g) is over four times that of the body wall (79). The overall compliance of the reptilian lung is determined by the contractile elements of the lung: the smooth muscle and elastic tissue, the saccular nature of the lung (103), and a very efficient pulmonary surfactant (93). Combined with their characteristic irregular pattern of breathing, the contractile properties may constitute an energysaving system in the reptilian lung (104). The volume-specific lung compliance of the multicameral crocodile lung is comparable to that in the much simpler lung of the gecko (76,79). This suggests that lung compliance in reptiles may be an attribute of parenchymal structure rather than the lung type. V. Avian Lung Among the air-breathing vertebrates, birds have the most complex and functionaly efficient respiratory system (105,106). The capacity of the lung– air sac system to provide the large amounts of oxygen required for flight at

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Figure 16 Transmission electron micrograph of the lung of the black mamba, D. polylepis, shows axonal profiles (stars) close to the blood–gas barrier. a, Air space; p, epithelial cell; arrows, collagen fibers; c, endothelial cell, e, red blood cell. Scale bar = 5 Am.

high speeds, over immense distances, and at great heights is exceptional by mammalian standards. In birds, the practically rigid, virtually nonexpansile lungs (mainly arising from their firm attachment to the ribs) have been structurally and functionally uncoupled from the air sacs. During respiration, the volume of the avian lungs changes by a mere 1.4% (107). The lungs are continuously and unidirectionally ventilated by synchronized action of totally avascular air sacs that play no direct role in gas exchange itself (108). Lung rigidity has allowed intense subdivision of the gas exchange tissue (parenchyma) to occur because surface tension was not a greatly constraining factor in determining the ultimate size of the terminal respiratory units: the air capillaries. The much smaller avian lung has a greater respiratory surface area than that in the relatively much larger one of mammals (109,110). The air

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capillaries (Figs. 17–19; see Figs. 21–23) are very narrow. They range in diameter from 8 to 20 Am (111,112). In comparison, the smallest alveoli (30 Am) occur in the bat lung (113). The exceptional functional efficiency of the avian lung is produced by synergism of various structural properties and functional parameters. The foremost ones include the cross-current arrangement formed between the air flow in the parabronchial lumen and the venous blood (Figs. 17a, Fig. 20); the countercurrent arrangement between the air capillaries that arise from the parabronchial lumen across the atria and the infundibulae (Fig. 21a) and the blood capillaries that are the terminal branches of the intraparabronchial arteries (Figs. 21b–23); large tidal volume afforded by capacious air sacs; large cardiac output resulting from a large heart; unidirectional and continuous ventilation of the parabronchial tissue by synchronized action of the air sacs;

Figure 17 Light micrograph of the lung of an ostrich, Struthio camelus. p, Parabronchial lumen; arrows, atrial muscles; arrowheads, atria; star, interparabronchial blood vessel; v, intraparabronchial blood vessels; a, parabronchial (gas exchange) tissue that is largely comprised of air and blood capillaries. Scale bars = 1 mm.

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Figure 18 Scanning electron micrographs of a cast of a parabronchus from the lung of the domestic fowl, Gallus gallus var. domesticus, shows interatrial septa (white arrowheads) separating atria. Black arrowheads, infundibula arising from atria; a, air capillaries; arrows, blood capillaries that interdigitate with the air capillaries. Scale bar a = 3 mm; scale bar b = 1.5 mm.

short pulmonary circulatory time; and superior morphometric parameters (109,110). The total volume of the respiratory system in birds (i.e., the combined volume of the lungs, air sacs, and connected pneumatic spaces) constitutes about 20% of the total body volume, with the value being as high as 34% in the mute swan, Cygnus olor (114). The combined volume of the lung and the air sacs is three to five times greater than that of the lungs of mammals and twice that of the reptilian lungs. The total volume of blood in the avian lung constitutes as much as 36% of the lung volume, with 58–80% of it occurring in the blood capillaries (111). The pulmonary capillary blood volume in birds

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Figure 19 Transmission electron micrograph of the lung of the black-headed gull, Larus ridibundus, shows air capillaries (a) and blood capillaries (c) containing red blood cells (e). arrowheads, blood–gas barrier. Scale bar = 5 Am.

is 2.5–3 times greater than in mammals, where only 20% of it is found in the alveolar capillaries (115). The parenchyma (gas-exchange tissue) of the bird lung constitutes only about 46% of the volume of the lung, while in the mammalian lung it constitutes about 90% (116). The surface density of the blood–gas barrier (i.e., the surface area per unit volume of parenchyma) in the avian lung ranges from 172 mm2/mm 3 in the domestic fowl, G. gallus var. domesticus (109,117) to 389 mm2/mm3 in the hummingbird, Colibri coruscans (118). In the mammalian lung, the values are about one-tenth those of birds. It is important to stress that in birds a relatively greater respiratory surface area has developed through intense subdivision of the parenchyma within existing constraints of a small lung with low parenchymal volume density. The terminal gas exchange components (the air and blood capillaries) (Figs. 17–19,

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Figure 20 Scanning electron micrograph of a cast of the lung of the domestic fowl, G. gallus var. domesticus, shows an intraparabronchial blood vessel (stars) giving rise to blood capillaries (c) that intertwine with the air capillaries (arrowheads). Scale bar = 40 Am.

21b, 22) interdigitate very closely. The extent of the epithelial cell lining of the air capillaries corresponds to that of the capillary endothelium (Fig. 19). This provides optimal exposure of the pulmonary capillary blood to air (Fig. 19), enhacing the lung’s diffusing capacity of the lung. Considering the large number of extant species of birds (about 9000 species) (119), the remarkable diversity of the habitats in which they live, and the various lifestyles they pursue, in morphological terms the avian lung is remarkably homogeneous. Subtle differences, particularly in the development of the tertiary bronchi (parabrochi), arrangement of secondary bronchi, and size and location of the air sacs, however, exist. An interesting difference in the morphology of avian lungs is that of the arrangement, extent of development, and location of the parabronchi. Two sets, the paleopulmonic and the neopulmonic occur. The main structural and functional differences between paleopulmonic and neopulmonic parabronchi are that the paleopulmonic

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Figure 21 Scanning electron micrographs of the lung of the domestic fowl, G. gallus var. domesticus. Stars, interatrial septa; arrows, infundibula; c, blood capillaries; a, air capillaries; e, red blood cells. Scale bar a = 40 Am; scale bar b = 20 Am.

parabronchi are located on the dorsocranial region of the lung while the neopulmonic set is located ventrocaudally; the paleopulmonic parabronchi are arranged as parallel stacks while the neopulmonic ones form a dense network; the air flow in the paleopulmo is continuous and unidirectional while that in the neopulmo changes with the phase of respiration; and the paleopulmonic parabronchi develop before the neopulmonic ones. In the lungs of primitive birds such as the kiwi and the penguin, only paleopulmonic parabronchi occur. In the more advanced species (e.g., passerines) the neopulmonic parabronchi are well developed and may make up as much as onethird of the total lung volume. The evolutionary and adaptive significance of the development of the neopulmo is not clear. There are no morphological or

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Figure 22 Scanning electron micrograph of a cast of the lung of the domestic fowl, G. gallus var. domesticus, shows blood capillaries (c) that intertwine with the air capillaries (a). Scale bar = 6 Am.

morphometric differences in the structure of the air and blood capillaries in the paleo- and the neopulmonic regions of the avian lung (120). The bidirectionally ventilated neopulmonic parabronchi may, however, serve as a site of carbon dioxide cycling for minimizing respiratory alkalosis from excess carbon dioxide washout across the undirectionally ventilated paleopulmonic parabronchi. This may be particularly important when a bird pants under thermal stress. The ostrich, for example, is known to hyperventilate continually for as long as 8 h without experiencing acid–base imbalance (121). Compared with those of the large and less energetic species, the lungs of small metabolically active birds have unique morphometric specializations (122,123). The highest mass-specific respiratory surface area (about 90 cm2/g)

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Figure 23 Scanning electron micrograph of a cast of the lung of the domestic fowl, G. gallus var. domesticus, shows a blood capillary containing compacted red blood cells (e). The blood capillary is literally suspended in air (a). Arrows, blood–gas barrier. Scale bar = 3 Am.

occurs in the small, highly energetic violet-eared hummingbird, C. coruscans (118), and the African rock martin, Hirundo fuligula (122). The value is substantially greater than that of 43 cm2/g in the shrews, Crocidura flavescens and Sorex spp. (124). An extremely thin blood–gas barrier (harmonic mean thickness) of 0.090 Am has been reported in H. fuligula (122) and C. coruscans (118); the thickness of the barrier in the shrew (0.334 Am) is relatively much thicker (124). The flightless galliform birds, such as, the domestic fowl, G. gallus var. domesticus (117), and the guinea fowl, Numida meleagris (125), have low pulmonary morphometric diffusing capacities. Among birds, the

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Figure 24 Lung of the domestic fowl, G. gallus var. domesticus. a. Scanning electron micrograph shows an atrium. Star, atrial muscle; arrows, surface macrophages; arrowhead, red blood cells being absorbed by a phagocytic surface epithelium; i, infundibulum. Scale bar = 0.5 mm. b. Scanning electron micrograph shows the floor of an infundibulum. Arrows, filopodia of surface macrophages; n, nuclei of the surface macrophages; star, a type II (granular) pneumocyte. Scale bar = 6 Am. c. Transmission electron micrograph shows a surface macrophage. Arrows, filopodia; arrowheads, electrondense lysosomal bodies; r, rough endoplasmic reticulum; n, nucleus; v, vesicular body. Scale bar = 0.5 Am. d. Transmission electron micrograph shows epithelial cells of the trachea. Arrows, vesicular bodies. n, nucleus; arrowheads, cilia. Scale bar = 15 Am.

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lowest values occur in the emu, Dromaius novaehollandiae (126), a large bird that in its natural habitat (Australia) is exposed to few predators. The Humboldt penguin, Spheniscus humboldti, an excellent diver, and the ostrich, S. camelus, the largest extant bird, have among birds the thickest blood–gas barriers: 0.530 (127) and 0.690 Am, respectively (112). In the penguin, it is conceivable that a thick blood–gas (tissue) barrier may allow the lung to resist collapse under hydrostatic pressure during dives. The avian lung has evolved an intricate defense system (128,129). This is particularly necessary since birds generally have a relatively more extensive respiratory surface area and a thin blood–gas (tissue) barrier (109,110,130). These structural features, although favoring gas exchange, predispose lungs to infection by pathogenic micro-organisms and deleterious particulate matter. Robust free surface macrophages, intravascular macrophages, and highly autolytic respiratory epithelium form part of a complex defense arsenal (Fig. 24).

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101. Carrier DR. Lung ventilation in running lizards. Am Zool 1984; 24:84A. 102. Bartlett D, Mortola JP, Doll EJ. Respiratory mechanics and control of the ventilatory cycle in the garter snake. Respir Physiol 1986; 64:13–27. 103. Craig P. Respiration and body weight in the reptilian genus Lacerta: a physiological, anatomical and morphometric study, PhD thesis, University of Bristol, England, 1975. 104. Milsom WK. The interrelationship between pulmonary mechanics and spontaneous breathing pattern in the tokay lizard Gekko gecko. J Exp Biol 1984; 113:203–214. 105. Scheid P. Mechanisms of gas exchange in bird lungs. Rev Physiol Biochem Pharmacol 1979; 86:137–186. 106. Maina JN. Principles of the structure and function of birds. In: Rosskopff E, Woepel P, eds. Petrak’s Diseases of Cage and Aviary Birds. New York: Lea & Febiger, 1996:167–256. 107. Jones JH, Effmann EL, Schmidt-Nielsen K. Lung volume changes during respiration in ducks. Respir Physiol 1985; 59:15–25. 108. Magnussen H, Willmer H, Scheid P. Gas exchange in the air sacs: contribution to respiratory gas exchange in ducks. Respir Physiol 1976; 26:129–146. 109. Maina JN. Morphometrics of the avian lung. In: King AS, McLelland J, eds. Form and Function in Birds. Vol 4. London: Academic Press, 1989:307–368. 110. Maina JN, King AS, Settle G. An allometric study of the pulmonary morphometric parameters in birds, with mammalian comparison. Phil Trans R Soc Lond 1989; 326B:1–57. 111. Duncker H-R. Structure of the avian respiratory tract. Respir Physiol 1974; 22:1–34. 112. Maina JN, Nathaniel C. A qualitative and quantitative study of the lung of an ostrich, Struthio camelus. J Exp Biol 2001; 204:2313–2330. 113. Tenney SM, Remmers JE. Comparative morphology of the lung: diffusing area. Nature Lond 1963; 197:54–56. 114. Duncker HR, Guntert M. The quantitative design of the avian respiratory system: from hummingbird to the mute swan. In: Nachtigall W, ed. BIONA Report No. 3. Stuttgart: Gustav-Fischer Verlag, 1985:361–378. 115. Weibel ER. Morphometry of the Human Lung. Berlin: Springer Verlag, 1963. 116. Maina JN, King AS. The structural functional correlation in the design of the bat lung. A morphometric study. J Exp Biol 1984; 111:43–63. 117. Abdalla MA, Maina JN, King AS, King DZ, Henry J. Morphometrics of the avian lung. 1. The domestic fowl Gallus gallus variant domesticus. Respir Physiol 1982; 47:267–278. 118. Dubach M. Quantitative analysis of the respiratory system of the house sparrow, budgerigar, and violet-eared hummingbird. Respir Physiol 1981; 46:43–60. 119. Gruson ES. Checklist of Birds of the World. London: William Collins, 1976. 120. Maina JN. A stereological analysis of the paleopulmo and neopulmo respiratory regions of the avian lung (Streptopelia decaocto). Int Res Commun Syst (Biochem) 1982; 10:328. 121. Schmidt-Nielsen K, Kanwisher J, Lasiewski RC, Cohn JE, Bretz WL.

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14 Lung Branching Morphogenesis: Potential for Regeneration of Small Conducting Airways

MINKE VAN TUYL, VERONICA DEL RICCIO, and MARTIN POST University of Toronto Toronto, Ontario, Canada

Lung development can be subdivided into five distinct stages (1,2). The early stages of lung development are embryonic and pseudoglandular. During the pseudoglandular period the primitive airway epithelium starts to differentiate and neuroendocrine, ciliated, and goblet cells appear while mesenchymal cells have begun to form cartilage and smooth muscle cells. In the subsequent canalicular period, the airway branching pattern is completed and the prospective gas-exchange region starts to develop. During this period respiratory bronchioli appear, interstitial tissue decreases, vascularization of peripheral mesenchyme increases, and distal cuboidal epithelium differentiates into type I and type II cells. In the saccular (terminal sac) period, the growth of the pulmonary parenchyma, the thinning of the connective tissue between the airspaces, and maturation of the surfactant system are the most important steps towards extrauterine life. During the alveolar period, which is a predominantly postnatal process in human and rodents, alveoli are formed through a septation process that greatly increases the gas exchange surface area and the capillaries will fuse to form a single layer (2). If we assume that repair after injury recapitulates some of the developmental pathways used during lung development, understanding the underlying molecular and cellular mechanisms might be relevant for the clinical management of end355

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stage lung diseases such as chronic obstructive pulmonary disease (emphysema and chronic bronchitis), pulmonary fibrosis, cystic fibrosis, lung cancer, as well as the newborn airway disease bronchopulmonary dysplasia (BPD). Regeneration of small airways in order to restore normal gas exchange function could be a valuable therapeutic option for these lung diseases. Besides lung transplantation, at present there is no way to regenerate functional lung units damaged by these processes. Restoration of functional lung requires regeneration of both the vascular and epithelial components organized with a proper alveolar architecture. Epithelial airways are rendered useless without adjacent capillaries, while supportive fibroblasts and mesenchyme are crucial to keep the lung structure together. It would be ideal if all appropriate types of cells should proliferate and differentiate to restructure the lung. Therefore, it is important to identify the angiogenesis and branching morphogenetic factors guiding small airway formation during normal development.

I. Early Lung Development Lung development starts as an endodermal outgrowth of the ventral foregut around the fourth week of human development. This foregut mass rapidly elongates into a single tube that divides into a ventral esophagus and a dorsal trachea, which in turn bifurcates into a right and a left primary lung bud. This process is modified in the mouse: the respiratory system develops from paired endodermal buds in the ventral half of the primitive foregut, just anterior to the developing stomach at day 9.5 of gestation. In humans, the left lung bud will give rise to two main stem bronchi, while the right lung bud gives rise to three main stem bronchi. In the mouse, the right lung characteristically has four stem bronchi whereas the left lung consists of one stem bronchus. The main bronchi will branch and rebranch, in a process called branching morphogenesis, and eventually form the airway tree. Endoderm-derived epithelial cells will line the airways, while the surrounding mesenchyme will provide the elastic tissue, smooth muscles, cartilage, vascular system, and other connective tissues. The formation of the bronchial tree is finished at 16 days of gestation in the mouse and 16 weeks of gestation in humans. At this stage of development, the tracheobronchial tree from the trachea to the terminal bronchioles resembles a system of branching tubules that terminates in exocrine glandlike structures (2). Recent genetic studies have implicated several transcription factors and morphogens, including peptide growth factors and their cognate receptors, in specifying the morphogenetic progenitor field of the lung along the foregut axis. One important transcription factor in this process is hepatocyte nuclear

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factor 3h (Hnf3h), which recently has been renamed Forkhead Box (Fox)a2 (3,4). Targeted ablation of Foxa2 in mice led to embryonic death between embryonic day (E) 6.5 and E9.5, which is before lung formation (5,6). However, chimeras rescued for the embryonic–extraembryonic constriction showed that Foxa2 was essential for foregut and lung formation (7). In the lung, Foxa2 is expressed in epithelial cells from the onset of lung development and continues to be expressed in bronchiolar–alveolar type II cells after birth (8,9). Overexpression of Foxa2 in distal pulmonary epithelial cells using the human surfactant protein C (SP-C) promoter arrested lung development in the pseudoglandular stage and markedly disrupted branching morphogenesis and vasculogenesis (10). Fibroblast growth factor (Fgf) 10 is a member of the large family of fibroblast growth factors involved in multiple processes during embryonic development (11–13). In the murine lung, Fgf10 mRNA is dynamically expressed in the distal mesenchyme adjacent to the primitive lung buds, where it is proposed to act as a chemoattractant for the developing epithelium (14,15). The importance of Fgf10 for lung development was shown in Fgf10deficient mice that die at birth with complete lung agenesis: lung development had stopped after the formation of the trachea (16,17). Fgfs bind to and signal via Fgf tyrosine kinase receptors (Fgfr) (11,12,18). The Fgf10 receptor Fgfr2IIIb, an Fgfr2 splice variant, is expressed in lung epithelium while Fgf10 is expressed in the mesenchyme, indicative of an epithelial–mesenchymal signaling loop during lung development (19). Fgfr2-IIIb is also capable of binding Fgf1 and Fgf7, which have been implicated in lung development as well (11,14,20). The null phenotype of Fgfr2 is lethal around E8.5–E11.5 (21,22), while defective Fgfr2-IIIb signaling resulted in a similar phenotype as in Fgf10 deficiency: only a trachea was formed without any or minimal further pulmonary branching (23,24). Altogether, these data indicate that Fgf10 signaling via the Fgfr2-IIIb isoform of Fgfr2 plays a crucial role in the initiation of lung bud formation. Sonic hedgehog (Shh) is a secreted signaling molecule involved in many fundamental processes during embryonic development (25). Shh is expressed in early pulmonary epithelium at the tips of developing lung branches (26,27). Shh signals via the mesenchymally located patched (Ptc) receptor, suggesting a signaling loop between pulmonary epithelium and mesenchyme during lung development (27). Shh-null mutant mice have lung buds consisting of only one lobe on each side of the trachea that show an almost complete failure of branching morphogenesis (28,29). Overexpression of Shh in distal pulmonary epithelial cells resulted in postnatal death due to respiratory distress (27). The lungs showed increased mesenchymal and epithelial cell proliferation, causing a disproportionate increase in pulmonary mesenchyme and postnatal lack of alveoli (27). These results clearly show a profound role for Shh in pulmonary

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cell growth and proliferation during development. Shh binds to Ptc, and this releases the basal repression that Ptc exerts on Smoothen (Smo), a transmembrane-spanning protein that has homology to G-protein-coupled receptors. Inside the cell, Smo activation modifies the activity of cubitus interruptus (Ci) or Gli family of transcriptional regulators (25,30). Three Gli genes have been described in mice: Gli1, Gli2 and Gli3, all of which are expressed in early pulmonary mesenchyme (31). In comparison to Shh-null mutants, an even more dramatic phenotype was observed in mice lacking both Gli2 and Gli3. These Gli2-/-; Gli3-/- mutant mice have no lung, trachea, or esophagus and die early in gestation (32), suggesting that combined Gli2 and Gli3 signaling is essential for the initiation of lung bud formation. All-trans-retinoic acid (RA) is the active form of vitamin A (retinol) that plays a crucial role during development and is involved in the developmental process of almost every organ (33–35). Both a deficiency and an excess of RA cause congenital defects during human development in a variety of organs (33,34). RA exerts its effect via the RAR and Retinoid X-Receptor (RXR) tyrosine kinase receptors, which function as transcriptional regulators of target genes. The RAR family is composed of three genes, which produce several isoforms: (RARa1,2, h1
4 and g1,2), all activated by both all-trans RA and 9-cis RA, whereas the three isoforms from the RXR family: (RXRa, h, g) are activated only by 9-cis RA (33). Mice deficient for only one of the isoforms showed a less severe phenotype than expected on the basis of their expression patterns, indicating a high degree of redundancy among the RA receptors (33). In contrast, compound mutant mice had similar congenital defects as seen with fetal vitamin A deficiency (33,34). RARa-/-; h2-/- double mutant mice die soon after birth with agenesis of the left lung and hypoplasia of the right lung (36,37). Lung hypoplasia was also reported in RARa1-/-; h-/- and RXRa-/-; RARa-/- double mutants (37,38). Furthermore, RA may regulate Hox genes (39–42). Hox genes form a large family of homeobox-containing transcription factors expressed in clusters along the anterior–posterior axis of the developing body (43). Genes from the 3V Hoxb cluster are predominantly expressed in early pulmonary mesenchyme in a proximal–distal gradient, suggesting a role for Hoxb genes in specifying proximal from distal pulmonary mesenchyme (41,44–48). Single mutant mice for Hox genes are generally normal, most likely because of redundancy. However, compound Hoxa1-/-; Hoxb1-/mutants have severe lung hypoplasia ranging from five hypoplastic lung lobes to only two lung lobes (49). Hoxa-5-/- mice die perinatally with laryngotracheal malformations, a reduced tracheal lumen, and lung hypoplasia (50). Another homeodomain transcription factor expressed at the onset of lung morphogenesis is thyroid transcription factor-1 (Ttf-1), also known as Nkx2.1 (51–53). Expression of Ttf-1 localizes to epithelial cells of the developing pulmonary tubules and decreases in more proximal conducting

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airways with advancing gestation (8,54,55). Ttf-1 continues to be expressed in adult bronchiolar and alveolar epithelial type II cells, where it plays an important role in the regulation of Clara-cell-secreted protein (CCSP) and surfactant protein (SP) synthesis (56). Targeted disruption of Ttf-1 resulted in severe hypoplasia of the thyroid and lung, with a developmental arrest at the pseudoglandular stage of lung development and a lack of distal epithelial cell differentiation (53,57).

II. Branching Morphogenesis Proper branching and differentiation of the lung buds are tightly controlled by epithelial–mesenchymal tissue interactions (58,59). The mesenchymal component dictates the branching pattern of the epithelium (60–63). Some progress has been made in elucidating the complex mixture of transcription factors and morphogens that guide proper lung branching. Several growth factors have been shown to regulate lung branching morphogenesis (64,65). Fgfs are generally produced by the pulmonary mesenchyme while their receptors are present in the lung epithelium. Exceptions are Fgfr1 and Fgf2, which have been found to be expressed in both fetal pulmonary epithelium and mesenchyme (66–68). Also the splice variant bek (IIIc) from the Fgfr2 is expressed in pulmonary mesenchyme, while the Kgfr (IIIb), which is another splice variant from Fgfr2, localizes to the epithelium (19,69,70). Transcripts for Fgf7 [also known as keratinocyte growth factor (kgf)] are detected in embryonic and adult lung mesenchyme at sites of active branching morphogenesis (71,72). Exogenous recombinant human (rh) Fgf7 inhibited rat lung branching morphogenesis in vitro (73) but stimulated proliferation of rat pulmonary type II cells in vitro (74) and in vivo (75,76). Mice bearing a null-mutation of the Fgf7 gene had no obvious lung abnormalities (77). Fgf1, which binds to Fgfr1 and both Fgfr2 splice variants Fgfr2IIIb and Fgfr2-IIIc, is crucial for branching of embryonic mouse epithelium in mesenchyme-free culture (11,78). Fgf2, which binds to Fgfr1 and Fgfr2-IIIc, but hardly to Fgfr2-IIIb, did not affect epithelial branching in these cultures. This suggests that the effect of Fgf1 on epithelial branching is mediated via Fgfr2-IIIb, which is the only receptor that binds Fgf7 (11,70,78). Fgf10 also binds to Fgfr2-IIIb and both null mutants for Fgfr2-IIIb and Fgf10 showed complete lung agenesis (16,17,23). Fgf1, Fgf7, and Fgf10 all induced epithelial expansion in E11.5 mouse lung explants and in mesenchyme-free distal lung buds in Matrigel culture, while Fgf1 and Fgf10, but never Fgf7, also induced epithelial branching (14,15,79). In contrast, epithelial proliferation was induced with Fgf7, but not with Fgf10 (15). An interesting finding was that embryonic lung mesenchymal cells cultured without epithelium showed a

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decreased expression of Fgf7 mRNA while the expression of Fgf10 mRNA was dramatically increased (80). Taken together, these results indicate different functions for Fgf1, Fgf7, and Fgf10 during lung development. Fgf7 seems to be involved in lung bud expansion and pulmonary type II cell proliferation, but not branching and depends on the presence of lung epithelium for its expression in the mesenchyme. Fgf1 and Fgf10, on the other hand, are able to induce branching morphogenesis and mesenchymal Fgf10 expression appears to be inhibited by pulmonary epithelium. The fact that Fgf1, Fgf7, and Fgf10 are transduced in such different physiological responses may explain, for example, why Fgf7 cannot compensate for loss of Fgf10 in vivo (16,17). Another member of the Fgf family, Fgf9, is expressed in pulmonary mesothelium and epithelium in early development and later only in the pleural mesothelium (19,81). Targeted deletion of Fgf9 resulted in severe lung hypoplasia and immediate postnatal death (81). Analysis of the lungs revealed decreased branching morphogenesis and a lack of alveoli; however, the number of lung lobes and primary bronchi were normal (81). Due to their profound influence on lung development and branching morphogenesis, Fgfs are excellent candidates to guide regeneration of airways following injury. The high postnatal expression of both the receptors and ligands further indicates a role in postnatal alveolarization and lung homeostasis (68,82,83). In this regard, important data will be coming from the conditional overexpression of Fgfs during pre- and postnatal lung development (84). Conditional overexpression of Fgf7 in proximal airways of the mouse using the rat CCSP promoter induced cystadenomatoid malformations when overexpressed in the fetal lung, but epithelial cell hyperplasia and type II cell differentiation when induced and overexpressed postnatally (85). Similar results were obtained with the overexpression of Fgf10 (86). In the embryonic lung this resulted in adenomatoid hyperplasia and marked hyperplasia of epithelial cells in small conducting airways, while overexpression of Fgf10 in the postnatal lung caused the formation of multifocal tumors with type II cell differentiation (86). Other studies showed that intratracheal installation of rhFgf7 in adult rat lung caused diffuse alveolar cell hyperplasia and a transient increase in surfactant protein mRNA expression (76,87). It was also found that rats receiving intratracheal rhFgf7 exhibited a dramatically reduced mortality after exposure to hyperoxia. Both intra-alveolar hemorrhage and exudation were greatly reduced as a result of rhFgf7 administration (75). Regulated overexpression of Fgf3 in distal epithelial cells of the adult lung using the human SP-C promoter resulted in an inflammation reaction consisting of an influx of alveolar macrophages and an upregulation of interleukin (IL) -7 and granulocyte–macrophage colonystimulating factor (GM-CSF) together with an intense increase in alveolar type II cell proliferation, including increased expression levels of Ttf-1 and

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surfactant protein mRNA (88). Both inflammation and subsequent proliferation recapitulate regeneration after lung injury, suggesting a role for Fgf3 in this process. Taken together, these results demonstrate the powerful capacity of Fgfs to induce type II cell proliferation, which places them high on the list of potential tools in pulmonary regeneration. It is important to realize that the conditional overexpression systems allow re-expression of embryonic growth and transcription factors in the adult lung. The challenge will now be to use these systems in disease models such as BPD and emphysema to investigate what is termed the rescue potential of embryonic developmental factors in airway repair and regeneration. Transforming growth factor (Tgf) h belongs to a superfamily that includes activin, bone morphogenic protein (Bmp), and Tgfh1, 2, and 3 (89). These peptides can exert a variety of biological effects including regulation of cell growth and differentiation and expression of a variety of proteins. However, during lung development, Tgfh1 plays an inhibitory role (90–94). Tgfh1 mRNA and protein are found in both pulmonary mesenchyme (mRNA and protein) and epithelium (protein) (95–97). Both addition of exogenous Tgfh1 to cultured embryonic mouse lung explants and the in vivo overexpression of Tgfh1 in distal lung epithelial cells resulted in decreased branching morphogenesis (90,93). Overexpression of Tgfh1 in distal pulmonary mouse epithelium in vivo arrested fetal lung development in the pseudoglandular stage of development with inhibited epithelial and vascular development and differentiation, resulting in postnatal death (94). Most, if not all, biological activities of Tgfh are transmitted via transmembrane Ser/ Thr kinase receptors, known as Tgfh receptors (Tgfhr) type I and type II (98). Signal transduction requires the formation of a heteromeric complex of TgfhrI and TgfhrII. In line with the negative influence of Tgfh1 on lung branching, inhibition of TgfhrII signaling stimulated lung morphogenesis in whole lung explants in vitro (99). Smad proteins function as downstream effectors in Tgfh signaling (100). Smad1–3 proteins are expressed in distal lung epithelium, while Smad4 is expressed in both distal lung epithelium and mesenchyme (101,102). Downregulation of Smad2/3 and Smad4 expression increased branching morphogenesis in cultured lung explants (102). The latter finding underlines the negative effects of Tgfh1 signaling on pulmonary branching morphogenesis. Of interest, Tgfh1 was increased in bronchiolar alveolar lavage (BAL) fluid form from infants with respiratory distress syndrome (RDS) who went on to develop chronic lung disease compared to children with RDS who did not develop chronic lung disease. This indicates that Tgfh1 may contribute to the fibrotic response observed in the lungs of infants with chronic lung disease (103,104). Hepatocyte growth factor/scatter factor (Hgf) has been shown to be a major regulatory factor in postnatal compensatory lung growth. Hgf mRNA

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is expressed in pulmonary mesenchyme, while its receptor, c-met/Hgfr, is expressed in adjacent epithelium, consistent with mesenchymal–epithelial signaling (105). The prenatal influence of Hgf on lung development is somewhat unclear. Hgf null mice die around E14.5 in utero, with no reported abnormalities of the lungs (106,107). Exogenous Hgf had no effect on branching morphogenesis of E14.5 rat lung explants in one study (73), but stimulated growth and branching morphogenesis of lung explants in another (105). Also, both the addition of antisense oligonucleotides (ODNs) targeted against Hgf or anti-Hgf antibodies inhibited branching morphogenesis of embryonic rat lung in vitro (105). Similarly to Fgf7, Hgf stimulated DNA synthesis and proliferation of adult rat alveolar type II cells in primary culture (74). Postnatal dexamethasone therapy to reduce inflammation and subsequent development of BPD in premature infants reduced Hgf levels in tracheal aspirates (108). Taking into account Hgf ’s positive effect on lung growth, reduced levels of it may explain the suppressive effect of dexamethasone on lung development (108). For additional information on Fgf7 and Hgf in lung injury, the reader is referred to a recent review by Ware et al. (109).

III. Epithelial Differentiation As branching proceeds, numerous different cell phenotypes are formed along the anterior–posterior axis of the developing epithelial tubules and associated mesenchymal components, each with different morphologies and patterns of gene expression. This patterning of differentiated lung cells is also controlled by epithelial–mesenchymal interactions (63,110). Over the last decade some regulatory molecules involved in epithelial morphogenic patterning in the lung have been identified. Pulmonary neuroendocrine cells (PNECs) are the first cells to differentiate in humans and animals (111,112). The development of PNECs seems to be dependent on the expression of Mash-1, since Mash-1-deficient mice failed to develop PNECs (113). Mash-1 is a bHLH gene, expressed in neural precursor cells, directing terminal neural differentiation (114). On the other hand, Hes-1 represses neural differentiation by suppression of proneural bHLH factors such as Mash-1. Indeed Hes-1-deficient embryos had increased pulmonary Mash-1 mRNA expression and increased numbers of PNECs (114–117). In the lung, Mash-1 is expressed in clusters or single progenitor PNECs (113,116), while Hes-1 is expressed in pulmonary epithelial cells other than PNECs (116). These results indicate an essential role for both transcription factors in the differentiation of PNECs, but not in pulmonary development per se, because gross lung morphology and differentiation in both mice appear to be unaffected (113,116). Hes-1 has been implicated in the Notch

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signaling pathway, which is involved in cell–cell interactions, creating differences between neighboring cells and determining cell fate (118,119). Factors of the Notch signaling pathway have recently been localized in the developing lung; however, the functional role of the Notch signaling pathway in lung development remains to be elucidated (116,120). Epithelial transcription factors such as Foxa2, Gata-6, and Ttf-1 (Nkx2.1) have been shown to influence lung epithelial specification. In both fetal mouse and human lung, the temporal–spatial distribution of Ttf-1 follows the pattern of expression of surfactant proteins (8,55). It has been shown that Ttf-1 regulates the transcription of SP-A, B, and C (121–125) and CCSP (121,126). As a consequence, Ttf-1 null mutants lack distal epithelial cell differentiation (53), while a proximal epithelial cell marker Foxj1 (Hfh4) was unaffected (127). Taken together, these data underline the importance of Ttf-1 for the establishment of the distal epithelial cell phenotype. Also, Gata-6 transactivates SP-A and Ttf-1 (128,129) and it has recently been shown that Gata-6 acts synergistically with Ttf-1 to influence the activity of the SP-C promoter (130). In some respiratory epithelial cells, Ttf-1 is coexpressed with members of the Fox family of transcription factors. Transcripts for Foxa1 and Foxa2 are detected in foregut cells forming the embryonic lung bud, and later in the distal epithelium of the developing and mature lung (8,131). Like Ttf-1, Foxa1 and Foxa2 modulate the expression of SP-B and CCSP (121,131,132). Foxa1 and Foxa2 are likely upstream regulators of Ttf-1 (133) and it is possible that both members of the Fox family confer lung-specific gene expression in the primitive foregut through Ttf-1 as the intermediate. Secreted morphogens such as Shh appear not to be involved in regulating proximal–distal epithelial specification, since SP-C and CCSP are expressed in Shh-deficient mice (29). In Drosophila, Hedgehog may regulate the expression of Decapentaplegic (Dpp), the Drosophila counterpart of Bmp (25). In the murine lung, Bmp4 is implicated in lung epithelial specification. Bmp4 is expressed in early distal lung tips and, at lower levels, in the mesenchyme adjacent to the distal lung buds (134,135). Overexpression of Bmp4 in the distal epithelium in vivo resulted in hypoplastic lungs with grossly dilated terminal lung buds separated by abundant mesenchyme (134). Distal epithelial differentiation was abnormal with decreased SP-C expression, while proximal differentiation (CCSP expression) was unaffected (134). On the other hand, exogenous Bmp4 clearly enhanced peripheral lung epithelial branching morphogenesis and SP-C expression in vitro (136), while inhibition of Bmp4 signaling also resulted in a severe reduction in distal epithelial cell types and an increase in proximal cell types (135,136). Further studies have confirmed a role for Bmp4 in proximal–distal epithelial cell differentiation during lung development (135–137).

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The beauty of alveolar structures lies in the fact that their surface area is tremendously larger than what would be expected from the size of the lung. Since the alveoli are the only structures in the human body that can supply oxygen, a decrease in alveolar surface area (the number of alveoli) will directly affect oxygen supply to the whole body. Both BPD and emphysema are examples of such conditions. Alveolar simplification (or reduction) is one of the hallmarks of the new BPD seen in premature infants (24–26 weeks) who are ventilated and exposed to O2. Thinning of alveolar walls and abnormal expansion of alveoli characterize emphysema. In humans this lung disease is strongly associated with smoking. The main characteristic of the disease is destruction of alveolar ducts, leading to diminished alveolar surface. Alveolarization is the last step in lung development (2). Alveoli are formed by septation of the pulmonary saccules that form the immature lung. In both humans and rodents, alveolarization occurs predominantly after birth. In the first 2 weeks after birth, alveolar formation, measured as proliferation, occurs in both central and peripheral areas of the murine lung, while afterwards alveolar formation occurs mainly in peripheral areas of the lung (138). Proliferation in alveolar septa is greatly diminished in adult lungs (138). A key player in the alveolarization process appears to be one of the three isoforms of the platelet-derived growth factors (Pdgf), namely PdgfA (139). PdgfA mRNA and protein are expressed in early pulmonary epithelium (140–142). The receptor Pdgfra is expressed in the mesenchyme adjacent to the epithelium that expresses PdgfA, suggesting a paracrine signaling loop between epithelium and mesenchyme (140,142–144). In vitro inhibition of PdgfA or Pdgfra in embryonic rat lung explants decreased both lung size and number of terminal buds, indicating a role for PdgfA-Pdgfra signaling in early lung development (142,145). On the other hand, in vivo overexpression of PdgfA in distal airway epithelium doubled lung size and increased distal branching morphogenesis (146). However, lung morphology was arrested in the canalicular stage of lung development with abundant mesenchyme and a lack of air spaces, resulting in neonatal death (146). These results indicate that PdgfA is a potent growth factor for mesenchymal cells in the developing lung. In mice, absence of PdgfA resulted in pre- and postnatal death (143,144). Postnatal deaths were characterized by emphysematous lungs with areas of atelectasis, without any formation of septa and/or alveoli and only dilated pre-alveolar saccules were found (143,144). In normal mice, alveolar septa contain smooth muscle cell a-actin (a-sma) positive myofibroblasts. The postnatal PdgfA null mutant lungs lacked alveolar staining for a-sma, indicating a lack of alveolar myofibroblasts. In addition, they were almost completely devoid of parenchymal elastic fibers, which most likely contrib-

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uted to the failure of alveolar formation (143,144). Myofibroblasts surrounding vessels and bronchioles appeared normal and were tropoelastin-positive, suggesting a different developmental lineage (144). Moreover, Pdgfr a-positive cells were specifically missing from lungs of PdgfA-null mutants and it has been proposed that these cells are progenitor cells for tropoelastinpositive alveolar myofibroblasts (143,144). Pdgfra-deficient mice die in utero with severe skeletal malformations and incomplete cephalic closure (147). Pdgfra-deficient lungs were hypoplastic; however, primary branching and structure were not affected (148,149). Postnatal alveolar formation could not be examined in these mice. Together the data suggest that PdgfA is needed for the development of alveolar myofibroblast that produce elastin, which in turn is crucial for alveolar septation and formation. The critical value of elastin in the development of proper alveolar structures was demonstrated in mice lacking tropoelastin, the soluble component of the elastic fiber. Tropoelastin-null mice exhibited a severe reduction in alveolar formation and decreased terminal airway branching, leading to almost immediate postnatal death (150). The tropoelastin-deficient mouse, however, shows alveolar destruction much earlier and more severely than the PdgfA-deficient mouse, suggesting that PdgfA is not the only regulator of elastin (143,144,150). During late gestation and early postnatal life, rodent pulmonary fibroblasts contain a considerable amount of vitamin A (151–153). Before birth, pulmonary fibroblasts contain retinyl esters, which around birth are converted into retinol and RA, the active components of vitamin A (152). Both endogenous (154) and exogenous RA (155) levels of tropoelastin mRNA increased almost threefold in neonatal rat lung fibroblasts, while inhibition of the production of RA decreased tropoelastin gene expression in postnatal rat lung fibroblast (154). Two enzymes, Aldh-1 (aldehyde dehydrogenase) and Raldh-2 (retinal dehydrogenase), are rate-limiting in the conversion of retinal to RA (35). Both are expressed at high levels in the immediate postnatal mouse lung, the time of maximal alveolarization, and at lower, more adult-like, levels 2 weeks after birth (138). Aldh-1 is expressed in central regions and alveolar septa, while Raldh-2 is expressed in central and subpleural regions (138). Further evidence for RA’s role in lung elastin maintenance and alveolarization was provided by genetic manipulation of RAR and RXR in mice. Compound mice homozygous for a RARg and heterozygous for a RXRa deletion had a reduced number of alveoli and less elastic fiber in their alveolar walls (156). RARh, on the other hand, appears to be an endogenous inhibitor of septation and as a result the RARh-null mutant shows early onset septation resulting in twice as many alveoli in the null mutant lungs compared to wildtype lungs (157). Most significant is that RA administration has been shown to increase the number of alveoli in

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postnatal rats and even to abrogate decreased alveolarization seen after the experimental use of dexamethasone or elastase that decrease alveolar formation, making RA the most powerful potential factor in so-called alveolar neogenesis after lung injury (158–162). These results show the importance of RA and tropoelastin as key regulators of alveolar development and maintenance, and hence their use in alveolar regeneration should be investigated. Other factors implicated in postnatal alveolarization include Fgfs, Ttf1, and Tgfa. Both Fgfr3 and Fgfr4 are expressed in postnatal pulmonary mesenchyme, while their ligands are expressed in pulmonary epithelial cells (163). Although a null mutation of either Fgfr3 or Fgfr4 caused no obvious lung defects, silencing of both receptors resulted in severe overall body growth retardation and a failure of postnatal alveolar formation (163). Despite the large dilated saccules without any proper alveolar septation, differentiation (including a-sma-positive myofibroblasts) and proliferation proceeded normally (163). Besides its role in prenatal lung development, Ttf-1 also regulates postnatal lung development and homeostasis. Although Ttf-1 expression decreases dramatically after birth, it remains detectable in adult alveolar type II cells (8,55,164). Overexpression of Ttf-1 in distal lung epithelial cells, using the SP-C promoter, did not affect prenatal lung development but perturbed postnatal alveolarization and led to emphysema, severe inflammation, and fibrosis (165). In human cases of pulmonary hypoplasia, Ttf-1 was found to be upregulated in proximal airways (bronchi and bronchioles) (54). It has been suggested that a sustained high expression of Ttf-1 may lead to pulmonary hypoplasia (54). On the other hand, another study reported decreased Ttf-1 and Foxa2 expression in inflamed and atelectic areas of lungs in humans with BPD. Ttf-1, Foxa2, and surfactant proteins, however, reappeared in regions of regeneration, supporting the possibility that Ttf-1 may be a critical factor in the restoration of alveolar structures after neonatal lung injury (9,55). Tgfa is a member of the epidermal growth factor (Egf) family that signals via the Egf receptor (Egfr) and both have been shown to be expressed and involved in pre- and postnatal lung development (166). The Egfr-null mutant dies soon after birth with immature lung morphology, impaired alveologenesis, and surfactant protein deficiency, which is a phenotype that resembles human RDS (167,168). The Tgfa-null mutant on the other hand, survived into adulthood without reported lung abnormalities, indicating that other factors than Tgfa that signal via the Egfr are important in lung development (169). However, overexpression of Tgfa in distal pulmonary epithelial cells using the human SP-C promoter resulted in disruption of postnatal alveolarization causing lung emphysema and fibrosis (170). It increased proliferation of alveolar epithelial cells, including SP-C-expressing type II cells, without causing inflammation (171). Elastin fibers were shorter

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and blunter in the bronchiolar regions and deficient in alveolar septa, most likely contributing to the emphysematic lesion (170). Tgfa is upregulated in patients with a number of lung disorders including RDS, BPD (166), and idiopathic pulmonary fibrosis (172). Tgfa-deficient mice are protected from lung fibrosis after bleomycin instillation, implying that Tgfa contributes to the pathogenesis of lung fibrosis after acute lung injury (173). These studies clearly indicate that increased proliferation of alveolar type II cells does not always result in a better lung. It is clear that both lung development and small airway regeneration are fine balances among too much, too little, and just the right amount of stimulatory and inhibitory regulating factors.

V. Vascular Development The lung is composed of a complex network of airways and vessels. Although much has been learned regarding the mechanisms controlling lung bud formation and airway branching, the mechanisms involved in vascular formation during lung development remain obscure. Three processes are believed to control pulmonary vascular development: angiogenesis, which is defined as sprouting of new vessels from pre-existing ones giving rise to the central vessels; vasculogenesis, which is de novo synthesis of blood vessels from blood lakes in the periphery of the lung; and the fusion of proximal and peripheral vessels to form the pulmonary circulation (174,175). More recently it was shown that even in the early stages of lung development vascular connections are well established and that vascular development takes place during all stages of lung development, with completion of a single capillary network during the alveolar period (2,176). The molecular mechanisms involved in pulmonary vascular formation are relatively unknown. Members of the vascular endothelial growth factor (Vegf) (177–179), angiopoietin (180,181), and ephrin family (182) have all been implicated in controlling vascularization of the pulmonary system. Vegf is a potent mitogen for endothelial cells, influencing angiogenesis and vasculogenesis (183). It is essential for embryonic development and haploinsufficiency of Vegf is enough to cause embryonic death (184,185). In the embryonic lung, Vegf mRNA is mainly detected in lung epithelium. Its expression increases only prior to birth and remains high in the adult lung (186–190). Vegf signals via two high-affinity tyrosine kinase receptors: Vegfr1 (Flt-1) and Vegfr2 (Kdr/Flk-1). Both are localized in the embryonic lung mesenchyme (187,188). The adjacent expression patterns of Vegf and the two Vegf receptors suggest a paracrine mode of action on the formation of the pulmonary vascular system, which may influence lung branching morphogenesis (191). Alternative splicing of the Vegf gene produces multiple species

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of mRNA. These encode different Vegf protein isoforms that vary from 120 to 205 amino acids. Overexpression of the Vegf 164 isoform in distal airway epithelium of mice resulted in perinatal death (192). The lungs appeared abnormal, with dilated respiratory tubules and saccules and a decreased number of terminal buds as well as a lack of alveolar type I cell differentiation (192). On the other hand, mice that lacked the Vegf 164 and 188 isoforms and only expressed the Vegf 120 isoform showed a decrease in peripheral vascular development, with fewer air–blood barriers and a general delay in lung development, but normal type I cell differentiation (193). Neonatal mice treated with a soluble decoy receptor for Vegfr1 to block endogenous Vegf signaling likewise exhibited an overall dramatic decrease in body and organ growth and died within 4–6 days after birth (194). The lungs of these mice were immature, with simplification of the alveolar region and a decrease in Vegfr2 expression (194). Inhibition of Vegfr signaling using the Vegfr blocker Su-5416 either before or after birth also resulted in reduced pulmonary vascularization and alveolarization, accompanied by increased apoptosis in alveolar septae, leading to an emphysemalike phenotype (195,196). An interesting finding in this study was that early inhibition of vascular development caused long-term effects on alveolarization as well as pulmonary hypertension (196). The development of emphysema was inhibited when a caspase inhibitor was injected simultaneously, indicating that alveolar septal cell apoptosis contributes to the pathogenesis of emphysema (195). Taken together, these results again suggest a role for Vegf itself, or vascular development, in alveolar formation. Another factor implicated in pulmonary vascular development is Foxf1, also known as Hfh8 or Fraec1. Foxf1-null mutant mice die in utero due to defects in mesodermal differentiation and cell adhesion (197). In the embryonic lung, Foxf1 expression is restricted to the pulmonary mesenchyme, while in the adult lung Foxf1 is expressed in smooth muscle cells surrounding bronchioles and in endothelium and fibroblasts of the alveolar sacs (198,199). Heterozygous mutant mice carrying a disruption of Foxf1 gene, in which Foxf1 levels are reduced by 80%, displayed a 55% postnatal mortality with lung hemorrhaging. Analysis of the lungs revealed abnormalities in alveolar formation and pulmonary vasculature (199). Injury to the lung with butylated hydroxytoluene (BHT) results in extensive damage to distal airway epithelial and endothelial cells, followed by cellular proliferation. This process is associated with a 65% reduction in Foxf1 levels in control mice (200). Surviving Foxf1-heterozygote mice were not able to sustain this injury and died within 7 days after the injury with massive pulmonary hemorrhaging, while wildtype mice usually survived the injury. This study indicates the importance of wildtype levels of Foxf1 during lung injury and repair (200). If

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applied in general, it is possible that susceptibility to certain diseases such as fibrosis and emphysema are the result of a failing repair mechanism due to suboptimal levels of expression of certain genes that are good enough for normal lung function but not for a challenged lung. Taken together, these studies suggest that a disruption in pulmonary vascular development leads to impaired branching morphogenesis and lung hypoplasia. This concept is supported by data on BPD and hyperoxia-induced lung injury. BPD is characterized by remarkably decreased numbers of distal respiratory units and abnormal microvessels (201,202). Infants dying of BPD have decreased levels of pulmonary Vegf and Pecam (endothelial marker) protein and gene expression compared to infants who do not develop BPD (203). It was likewise shown that preterm infants who go on to develop BPD had lower Vegf levels in their tracheal aspirates during the first 10 postnatal days than those preterms who did not develop BPD (204). Also, hyperoxia induced 2 weeks after birth reduced alveolarization in the neonatal rat lung (205), and these abnormalities continued even after recovery in room air (206). In the neonatal and adult rabbit lung, hyperoxic injury decreased Vegf expression, which, however, did increase again with recovery in room air (207). The initial decrease in Vegf expression seen in both studies might well be responsible for the vascular abnormalities seen with hyperoxic injury. The above-mentioned findings suggest that stimulating vascular development could be a potential tool for alveolar regeneration. Exogenous Vegf has been shown to stimulate airway epithelial cell proliferation and differentiation in human fetal lung in vitro (208). Further evidence for a potential use of Vegf for stimulating alveolar development was provided by a recent study on hypoxia-inducible transcription factors (209). The hypoxia-inducible transcription factors, Hif-1a and Hif-2a, are upregulated in response to hypoxia and in turn upregulate expression of oxygen-sensitive target genes such as Vegf (210,211). Hif-1a-null mutants die early in gestation, while Hif2a-deficient mice die after birth with symptoms resembling RDS (209,212). Compared to control mice, lungs of Hif-2a-deficient mice have decreased Vegf protein levels. Histological analysis of the Hif-2a deficient lungs revealed normal numbers of alveoli, but alveolar septa were abnormally thick with scattered and abnormal alveolar capillaries. Type II cells were immature and produced less surfactant phospholipids and proteins (209). Exogenous Vegf was injected either intra-amniotically or intratracheally 1 day before preterm delivery significantly improved lung maturation and decreased mortality due to RDS in the Hif-2a-deficient mice. Based on these intriguing observations, it is evident that therapeutic Vegf in a clinical setting needs immediate attention, if only for the possibility of reducing corticosteroid usage or oxygen levels in the treatment of RDS (209).

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Probably one of the best-established models for airway regeneration is the adult dog pneumonectomy model, in which either the left or the right lung is removed, which causes compensatory lung growth, including alveolar growth (213,214), in the remaining lung. The exact mechanism responsible for the compensatory lung growth, however, remains largely unknown, especially at the molecular level. Mechanical lung strain is thought to be a major player in compensatory lung growth. Implanting an inflatable prosthesis in the empty thorax after pneumonectomy prevented mediastinal shift but only partially prevented compensatory lung growth, indicating that other signals are implicated in compensatory lung growth (215). The observation that left pneumonectomy performed at different gestation times on pregnant rats enhanced fetal lung growth without affecting maturation led to the suggestion that a lung-specific growth factor is released into the circulation after pneumonectomy, which is responsible for compensatory lung growth (216). Recent studies have identified several potential factors responsible for postnatal compensatory lung growth. In left-side pneumonectomized 3 week old rats, compensatory lung growth was blocked by receptor decoy inhibition of the native Pdgfh-receptor using truncated soluble Pdgfh-receptors, indicating a role for Pdgf-B signaling via the Pdgfh receptor in compensatory lung growth (217). Tropoelastin and type I procollagen mRNA increased dramatically in the alveolar walls of postpneumonectomy rat lungs compared to sham-treated controls (218). This is an intriguing finding considering the role of tropoelastin in normal alveolar formation and maintenance (218). Fgf7 has recently been shown to enhance compensatory lung growth significantly after left pneumonectomy in adult rats. Compared to untreated pneumonectomized rats, weekly systemic injections of Fgf7 increased lung weight and lung volume indexes as well as the total volume of the alveolar region and the alveolar surface area per unit volume (219). Moreover, Fgf7 treatment greatly enhances postpneumonectomy alveolar BrdU incorporation, indicating a role in alveolar proliferation during compensatory lung growth (219). Hgf expression is also upregulated in the remaining lung after left pneumonectomy in mice (220). Hgf mRNA levels were transiently upregulated in the liver and kidney as well, suggesting the existence of a systemic factor or response after pneumonectomy. The increase in Hgf expression was accompanied by a short but dramatic increase in the expression of the Hgf receptor, c-met/Hgfr, 3 days after left pneumonectomy (220). A neutralizing antibody against Hgf significantly attenuated DNA synthesis as measured by BrdU labeling in the remaining lungs 3 and 5 days after surgery, while systemic treatment with Hgf increased pulmonary BrdU labeling 3 days after surgery (220). On the other hand, Hgf mRNA is rapidly induced in adult rat lungs following hepatic or

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renal injury (221). These results favor a universal role for Hgf in organ regeneration (222). In human patients, it was similarly shown that Hgf serum levels were upregulated after lung resection compared to patients undergoing mastectomy (223) and in patients with various lung diseases (224), indicating a role for Hgf in lung regeneration or compensatory lung growth in humans. This is further supported by the unique characteristics of Hgf, being a mitogen (225), morphogen (226), and inducer of pulmonary blood vessel formation in vivo (227,228). Endothelial nitric oxide synthetase (eNOS) protein is upregulated 3 days after left pneumonectomy, together with an increase in right lung weight and volume, indicating a role for it in compensatory lung growth (229). Indeed, eNOS-null mice failed to upregulate lung weight and volume and showed no signs of alveolar cell proliferation after left pneumonectomy, indicating an impairment of compensatory alveolar growth in these mice (229). Similar results were obtained when mice were treated with a NOS inhibitor (L-NAME) after pneumonectomy (229). Since Vegf is known to upregulate eNOS, an intriguing explanation for the experimental results would be that when vascular development is impaired, compensatory alveolar growth is prevented (229). Again, it underscores the need to study whether coordinated stimulation of vascularization and airway growth should be attempted as a tool for proper airway regeneration.

VII. Stem Cells and Airway Regeneration Lung regeneration in situ is likely to require a combined stimulation of angiogenesis and alveologenesis via induction of branching from existing small airways. This might be achieved by gene delivery of angiogenesis and/or branching morphogenesis factors via tissue-specific or multipotent stem cells (230). Type II, Clara, and neuroendocrine (PNEC) cells have all been pursued as potential tissue-born stem cells. The alveolar type II cells have been found to have an unlimited potential to proliferate (231). They serve as the putative stem cells for type I cells; having an excess of type II cells during development will ensure that enough type I cells are formed during alveolarization (231). Type I cells are crucial in the maturation of the air–blood barrier during alveolarization and are solely responsible for gas exchange in human lungs (2,232). The pulmonary type II cell is geared for self-maintenance, terminal differentiation (including surfactant production), and source for type I cells, all of which are characteristics of a tissue-derived stem cell (231). In other words, the type II cell acts as what can be termed the caretaker of the alveolar compartment. When vulnerable type I cells are injured, type II cells react by

Egf(r) Tgfa

PdgfA Pdgfra

Hgf

Tgfh

Fgfs

Factor

Hgf-/-: embryonic lethal with normal lung development Exogenous Hgf in vitro: z branching? In vitro antisense inhibition: # branching PdgfA-/-: embryonic lethal before lung development PdgfA-/-: embryonic lethal; lung hypoplesia Overexpression PdgfA: z lung size; arrest in canalicular stage of lung development (lack of airspaces); postnatal respiratory insufficiency ! death Egf-/-: death; immature lungs Egfr-/-: dies at birth with immature lung morphology; RDS-like symptoms Tgfa-/-: no reported lung abnormalities

Overexpression: Fgf7 and Fgf10: cystadenomatoid malformations; respiratory failure at birth ! death Overexpression Tgfh: arrest in pseudoglandular stage of lung development, postnatal death In vitro inhibition of TgfhrII: z branching

Prenatal

Fgfr2-IIIb : lung agenesis Fgf10-/-: lung agenesis Fgf7-/-: no lung abnormalities Fgf9-/-: lung hypoplasia; lack of alveoli

-/-

z levels of Tgfa in RDS, BPD and fibrosis; Tgfa deficiency protects against fibrosis ! inhibition of Tgfa therapeutic tool in regeneration? Signaling via Egfr by more than one ligand important in development and perhaps regeneration.

PdgfA-/-: no alveolar myofibroblasts or elastin production; failure of alveolar separation ! death with emphysematous lungs

Exogenous Hgf in vitro: z type II cell proliferation

Tgfa-/-: protected from fibrosis after lung injury Overexpression Tgfa: z alveolar epithelial cells, emphysema and fibrosis but no inflammation

Injury and regeneration Fgfs are expressed in the pre- and postnatal lung and type II cell proliferation ! powerful tool in regeneration. Role of z Fgf7 in lung disease models and pneumonectomy suggest an endogenous regenerative function. z levels of Tgfh1 in BPD ! dysregulation of Tgfh signaling involved in abnormal repair response to injury? Inhibition of Tgfh1 therapeutic tool in regeneration? z levels of Hgf after lung injury (i.e. pneumonectomy); has characteristics of a mitogen, morphogen and vascular inducer key intrinsic factor for lung regeneration. PdgfA is a growth factor during lung development and critical for elastin production in alveolar walls ! key regulator in alveolar septation ! necessary for airway remodeling.

Postnatal/Adult Overexpression: Fgf3, Fgf7 and Fgf10: epithelial type II cell proliferation and differation Fgfr3-/-/Fgfr4-/-: no postnatal alveolar formation

Table 1 Potential Morphogenetic Factors for Lung Regeneration

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Foxa2-/-: lethal before lung development Overexpression Foxa2: arrest in pseudoglandular stage of lung development

Foxa2 (HNF -3h)

Overexpression Ttf-1: emphysema; inflammation; fibrosis

Exogenous RA in vitro: in alveolar number

Postnatal inhibition of Vegfr in rats # alveolarization and vascular formation ! emphysema

Ttf-1 anf Foxa2 are expressed in adult type II cells, where involved in SP and CCSP expression. They are # during injury (BPD) but z in areas of regeneration. Perhaps this recapitulates developmental transcriptional signaling.

RA signaling is key regulator in alveolar septation ! proven factor in regeneration.

RA experimentally prevents elastase- and corticosteroid-induced emphysema.

Shh z cell growth and proliferation ! role in regeneration?

Vegf # in BPD and z during lung repair. Intra-amniotic/tracheally Vegf z lung maturation and # mortality due to experimental RDS. Together with the potential to induce cell proliferation, Vegf/vascular growth is a promising tool for alveolar regeneration.

z, increase; #, decrease; SP, surfactant proteins; CCSP, Clara-cell-secreted protein; RDS, respiratory distress syndrome; BPD, bronchopulmonary dysplasia.

Ttf1

RA

Shh

Vegf-/-: embryonic lethal Vegf164/188-/-: # pulmonary vascular and airway development Overexpression Vegf164 in vivo: perinatal death; z pulmonary vascularization; impaired lung maturation with premature sacculation, lack of alveolar type I cells exogenous Vegf in vitro: z airway epithelial cell proliferation and differentiation Shh-/-: lung hypoplasia, only two single lobes; lack of branching morphogenesis Overexpression Shh: perinatal death; lungs with z epithelial and mesechymal proliferation; disproportionate increase in pulmonary mesenchyme and lack of alveoli RARa-/-/h2-/-,RARa1-/-/h2-/-,RXRa-/-/RARa-/-: die soon after birth; left lung agenesis; right lung hypoplasia RARg-/-/RXRa-/+: # in alveoli number and elastic fibers in alveolar walls RARh-/-: early-onset septation; # in alveolar number Tft1-/-: failure of early branching, pseudoglandular arrest of lung development Overexpression Ttf-1: no effect before birth

Vegf

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extensive proliferation, acting as a progenitor cell for both type I and type II cells (231). During the resolution phase of the repair process, type II cells undergo extensive apoptosis (233) to re-establish a functional air–blood barrier (234). Administration of rhFgf7, a potent mitogen of alveolar epithelial type II cells, resulted in type II hyperplasia (76,235). Restoration of normal alveolar epithelium after instillation of Fgf7 is accomplished by terminal differentiation into type I cells and apoptosis of hyperplastic alveolar epithelial type II cells in vivo. Taken together, these results indicate that pulmonary type II cells may serve as the putative stem cells for the alveolar region during regeneration. Due to their overlapping localization during lung development and regeneration, Clara cells and PNEC have been thought to be potential airway stem cell (236). Naphthalene-induced lung injury denudes the proximal and terminal bronchioles selectively of their nonciliated CCSP, positive epithelium (i.e., Clara cells) (237,238). Naphthalene is cytotoxic to Clara cells, because the latter contain the cytochrome P-450 2F2 isozyme that metabolizes naphthalene to a toxic substance (239). Regeneration after naphthaleneinduced injury involves reoccurrence of CCSP-positive cells predominantly at bronchiolar bifurcations (238). On the other hand, PNECs were unaffected by naphthalene and showed extensive proliferation in the repair phase of naphthalene-induced injury, resulting in PNEC hyperplasia (240). Some socalled variant Clara cells that appeared to lack detectable cytochrome P450 2F2 isozyme protein survived and proliferated after naphthalene-induced lung injury in close apposition to hyperplastic PNECs in neuroendocrine bodies (NEBs) located at bronchiolar branch points (241,242). These results support a role for NEBs in the maintenance of multiple progenitor cell population in the mature airway. However, other studies do not support this concept. Transgenic CCtk mice allow the timed and selective ablation of CCSP- positive cells (243). In these mice, treatment with ganciclovir, which renders the thymidine kinase (tk) cytotoxic by phosphorylation, resulted in a complete ablation of CCSP-positive cells, while proliferation and hyperplasia of calcitonin gene-related peptide (cGRP) positive PNECs were unaffected (243). Regeneration of CCSP-positive cells that normally occurs in the regeneration phase after naphthalene-induced lung injury was absent in these CCtk mice, indicating that PNECs are unable to differentiate into CCSPpositive cells (242). Ciliated bronchiolar epithelial cells similarly disappeared and did not regenerate in these transgenic mice (243). Moreover, Mash-1 null mutant lungs have no PNECs; however, type II and Clara cell markers were expressed normally (113). These results indicate a role for CCSP-positive cells for their own regeneration and the maintenance of ciliated bronchiolar epithelial cells, but CCSP-positive cells are not important for the proliferation

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of PNECs (243). Furthermore, it suggests that PNECs merely function as a self-renewing population (242). Kotton et al. (244) provided some interesting information on the use of multipotent bone marrow stem cells in lung repair. Cultured fibroblast-like GTRosa26 bone-marrow-derived stem cells were injected into the tail vein of either normal or intratracheally bleomycin-injured mice. LacZ positive cells were found 30 days after injection in subpleural regions of control lungs. In the injured lung, considerably more LacZ positive cells were found, predominantly as clusters in the alveolar regions of the lung. These cells appeared to be type I cells as demonstrated by their morphological features and positive immunohistochemical staining for type I cell markers. These results suggest that cultured bone marrow cells can serve as type I cell precursors and that

Figure 1 Stem cells in lung regeneration. BV, blood vessel; NEB, neuroendocrine bodies; PNECs, pulmonary neuroendocrine cells.

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their engraftment is enhanced when the lung is injured (244). Recently it has been reported that type II alveolar epithelial cells can be derived from murine embryonic stem cells in vitro (245). Yet another study strongly argues against the transdifferentiation of hematopoietic stem cells into nonhematopoietic tissue-specific cells making up the lung. In contrast to the previous study, injected hematopoietic green fluorescent protein (GFP) positive cells mainly localized to the hematopoietic system, except for a few cells in the liver and a single cell in the brain. Tissue injury did not push transdifferentiation, since irradiation of the intestine did not result in the accumulation of GFP-positive intestinal-specific cells (246). Also, long-term joining of the circulatory systems of a GFP positive and a GFP negative mouse (parabiotic system) did not result in GFP-positive cells in GFP-negative organs other than the hematopoietic system (246). Thus, whether multipotent bone-marrow-derived stem cells can be used to deliver gene therapy to lung epithelium remains to be determined. VIII. Conclusion A variety of pathological processes including chronic obstructive pulmonary disease, pulmonary fibrosis, and cystic fibrosis, lead to substantial loss of functional respiratory units. In situ regeneration of these functional lung units will be a major clinical advancement. Restoration of functional lung requires regeneration of both vascular and epithelial components in a proper structural organization. In the future, this may be achieved by induction of vascular and epithelial branching from existing small airways using gene delivery of pulmonary angiogenesis and/or branching morphogenesis factors (see Table 1) via lung-specific or multipotent stem cells (see Fig. 1). References 1. 2.

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15 Apoptosis and Emphysema

NORBERT F. VOELKEL and LAIMUTE TARASEVICIENE-STEWART University of Colorado Health Sciences Center Denver, Colorado, U.S.A.

RUBIN M. TUDER Johns Hopkins University Baltimore, Maryland, U.S.A.

I. Introduction Cigarette smoking has increased worldwide the incidence of chronic obstructive lung diseases of which chronic bronchitis, the involvement of microscopically small airways, and emphysema are manifestations (1,2). Emphysema is anatomically defined as airspace enlargement due to loss of alveolar gas exchange units, that is, loss of alveolar septal cells. Since the discovery of an association of the genetically determined deficiency of an antiproteolytic protein, a1-antitrypsin, and development of emphysema in relatively young individuals who also smoked cigarettes (3), the so-called protease-antiprotease hypothesis has dominated investigators’ thinking about the pathogenesis of emphysema. In large measure the proteolysis concept has become popular because airway instillation of either neutrophil or pancreatic elastase causes predictable inflammation and tissue destruction in rodent models (4–6). This hypothesis also receives support from the idea that mediators of inflammation act to establish chemotactic gradients in the lung tissue. These activate neutrophils and macrophages with the end result of release into their immediate vicinity of various proteolytic enzymes and otherwise cell-damaging molecules. Thus, concepts 395

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of cell–cell interactions and of proteolytic activity gradients emanating from activated inflammatory cells (neutrophils, macrophages, and mast cells) all have been brought to bear on the protease–antiprotease hypothesis. Furthermore, the presence of a variety of proteolytic enzymes, metalloproteinases, including gelatinases, and cathepsins, has been documented either in lung tissue or sputum from patients with chronic obstructive lung disease (COPD) (7–9). Lastly, genetic manipulations in mice directed either to delete proteases or antiproteases (10–12) also support the protease–antiprotease pathogenetic concept of emphysema development. In parallel with the studies of protease–antiprotease mechanisms in mice, a number of other rodent models of emphysema have been investigated, including the tight skin mouse, which carries a mutation of the fibrillin-1 gene (13,14); the klotho mouse, which displays an element of endothelial cell dysfunction and premature aging (15); and the pallid mouse, (16), which also has a platelet storage pool deficiency. The more recent discovery of airspace enlargement in genetically engineered mice, in particular in mice overexpressing interferon g (17), interleukin 13 (18), and those with a deletion of the surfactant protein D (19), teaches us that there are a number of control factors, perhaps control systems, which participate in the maintenance or (alternatively) destabilization of the cells that build the alveolar spaces. Therefore, at least in animal models, cytokines, surfactant proteins, oxidative stress (20,21) and growth factors (22,23) enter the stage, join the proteases and antiproteases, and need to be integrated in a larger pathobiological scheme of lung health and structure maintenance. In the following sections we propose that a final common pathway may exist to explain the emphysematous destruction of the airspaces and that fundamental cell biology principles such as apoptosis and phagocytosis are at work.

II. Apoptosis Programmed cell death or apoptosis is fundamental to the development, growth, repair, and organ structure maintenance of all cellular organisms (24,25). Recently it has been recognized that the removal of apoptosed cells either by professional phagocytes or by neighboring cells, including epithelial and endothelial cells, is an important mechanism contributing to the control of inflammation (26–28), affecting the local immune response (29) and even stimulating new cell growth in the vicinity (30). Apoptosis can be a caspasedependent or caspase-independent process, by which cell membrane events,

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Figure 1 Oversimplified schematic of cell survival and programmed cell death. Proapoptotic and antiapoptotic signals converge on the mitochondrion.

mitochondrial enzyme activity, and nuclear events are synchronized in largely unknown complicated signaling pathways (31–35) (Fig. 1). Apoptosis can be triggered by myriad mechanisms including cytokines, oxidative stress, proteases, cytotoxic T cells, radiation, and chemotherapeutic agents (34). There are pro- and antiapoptotic proteins (35–39) and apoptosed cells, if not phagocytosed, can undergo necrosis. Necrotic cells generate a proinflammatory environment (40). During necrosis there is cell swelling, enlargement of mitochondria, rupture of the cell membrane, and release of cytoplasmic contents; as stated, this often generates an inflammatory response. During apoptosis the cell shrinks, the chromatin is being condensed, the cell membrane develops blebs and exposes phosphatidylserine on its outer surface, but it does not rupture and the cell does not release cellular contents into the environment. In vivo, as in vitro, apoptosed cells are recognized, phagocytosed, and thus removed. Bcl-2 and caspases (cysteinyl aspartate-specific proteases) have been identified as regulators and effectors of apoptosis. The members of the caspase cysteine protease family are classified as apoptosis initiators and effectors. Caspase-3-like proteins are activated during most, but not all, apoptotic processes by proteolytic processing at specific aspartic residues resulting in the removal of an N-terminal prodomain. The activation of these proteolytic enzymes can either be accomplished by themselves or by the upstream caspases. It is now being recognized that mitochondrial damage, increased

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generation of reactive oxygen species, and protease activation are intricately linked during apoptosis and lead to DNA fragmentation, which can be observed as DNA laddering. III.

Is Emphysema a Vascular Disease?

Pathologists in the 19th century demonstrated a loss of lung capillaries in human emphysematous lungs, which likely originated from nonsmokers (41). Liebow examined the microvessels of lungs from patients with emphysema and stated: Loss of septa among distal air spaces, with ultimate confluence of increasing moieties of pulmonary substance has already been considered. Whether the process is indicated by airtrapping or the result of atrophy otherwise induced, the effect is that the rich vascular beds vanish with the tissues that they once supplied. The loss of vascularity of alveoli in emphysema noted so many years ago is still not certainly explained. (42)

A few years later radiologists described the vascular destruction of emphysema lungs based on angiography (43). A dramatic decrease in the diffusion capacity has been observed in many patients with emphysema, supporting the association of alveolar and microvascular gas exchange surface area loss in emphysematous lungs. New concepts and mechanisms explaining how alveolar septal microvascular cells are lost during emphysema development can now be reviewed. IV.

Lung Structure Maintenance Program (Fig. 2)

We postulate that growth factors and regulators of apoptosis that are instrumental during lung development and maturation also play a role as maintenance factors in the adult lung. Specifically we postulate that the vascular endothelial growth factor (VEGF) pathway plays an extraordinarily important and central role in the maintenance of the adult lung structure. We were surprised when we and others (44–46) found a large amount of VEGF mRNA and protein in adult lung tissue and when immunohistochemistry of adult lung tissue demonstrated a ubiquitous presence of VEGF in bronchial epithelial cells, vascular smooth muscle cells, and alveolar macrophages. The question arose: Why does the adult lung express so much endothelial cell growth factor? We now believe that VEGF is an obligatory survival factor for pulmonary microvascular endothelial cells, as has been shown for endothelial cells in culture and at other sites (47–49). We likewise believe that impairment of VEGF receptor-initiated signal transduction causes lung vascular endo-

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Figure 2 Conceptual schematic shows elements of the lung structure maintenance program.

thelial cell apoptosis (50,51). The VEGF receptor VEGF-RII/KDR (Kinase Domain-Containing Receptor/Flk (total liver kinase)) is expressed by endothelial cells and also by alveolar type II cells. In addition, VEGF appears to bind to the VEGF-RI/Flt (tms-like tyrosine kinase) (52) on monocytes and macrophages to facilitate phagocytosis of apoptosed cells and thus to limit inflammation (Fig. 3). A phosphatidylserine receptor expressed on phagocytes recognizes on the exterior cell membrane of cells undergoing apoptosis exposed phosphatidyserine; this receptor and subsequent signal transduction downregulate inflammatory responses (27,31,53). Not only apoptosis but also cellular senescence may jeopardize the lung structure maintenance program. Senescent cells have lost their ability to replicate and cellular senescence is likely also related to mitochondrial dysfunction. It is characterized by altered expression of the proteins p16, p21, and p53 (54), which play important roles in cell-cycle control. Preliminary studies of human emphysematous lung tissue indicate that the cellular senescence-related protein p16 is increased in its expression in alveolar septal cells when compared to nonemphysematous normal lungs (54). V. Alveolar Septal Cell Apoptosis in Human Lungs and Experimental Animals Independently the research group in Mexico City (9) and our own (23) looked for apoptosis in lungs from patients with emphysema and found alveolar

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Figure 3 VEGF acts on endothelial cells and on macrophages. VEGF, released either from the extracellular matrix (ECM) or from macrophages activates the VEGF-RII. Increased expression of superoxide anion dismutase (SOD) and the antiapoptotic Bcl-2, and increased production of nitric oxide and prostacyclin are the consequence. VEGF can also activate the VEGF-RI on macrophages and affect macrophage functions.

septal cells with fragmented DNA, capillary endothelial cells and endothelial cells lining precapillary arterioles, and also epithelial cells that stained positive with the Tdt-mediated dUTP Nick End Labeling (TUNEL) technique (Fig. 4(C and D)). In addition, Kasahara and co-workers demonstrated diminished expression of the genes encoding VEGF and its receptor VEGF-RII. In the lung from patients with severe emphysema likewise there was a decreased expression of the VEGF and VEGF RII proteins (23). This was surprising since the patients whose lungs were examined were hypoxemic and hypoxia is usually a strong inducer of VEGF and VEGF-RII gene expression (45) promoted by the transcription factor HIF-1-a (55). Therefore, in severe human emphysema there is an association between alveolar septal cell apoptosis and diminished VEGF and VEGF receptor protein expression. If indeed VEGF and intact signal transduction via VEGF-RII and Akt were necessary for the maintenance and survival of pulmonary capillary endothelial cells, then blockade of VEGF receptors should cause endothelial (and perhaps also alveolar epithelia) cell apoptosis and emphysema in experimental animals. Indeed, Kasahara et al. showed in rats that chronic VEGF-R blockade caused alveolar septal cell apoptosis, a dramatic loss of the pulmonary capillary network and emphysema (56). If newborn mice or rats are subjected to VEGF receptor blockade, alveolarization is impaired (57,58). Adult rats treated as neonates with a VEGF receptor blocker continue to have fewer alveoli compared with untreated controls. These animals also develop a

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greater degree of pulmonary hypertension when exposed to chronic hypoxia (58). Recently, Wagner and associates produced emphysema in adult mice using a conditional VEGF-R knockout strategy (59). Taraseviciene-Stewart et al. found that intraperitoneal injection of human umbilical vein endothelial cells (HUVECs), but not of vascular smooth muscle cells, caused emphysema in rats within weeks (60). Serum from these HUVEC-injected animals inhibits the growth of cultured endothelial cells; when injected into naı¨ ve rats it causes endothelial cell apoptosis and emphysema. Thus antibodies directed against unknown endothelial cell epitopes can cause pulmonary emphysema. It is worth emphasizing that all of these experimental strategies are themselves noninflammatory. Taken together these data indicate, first, that VEGF is an obligatory maintenance factor for pulmonary capillary endothelial cells; and, second, that antibodies targeting pulmonary microvascular endothelial cells cause emphysema. In contrast, VEGF receptor blockade and HUVEC immunization of animals did not cause apparent structural damage in other examined organs. We thus wondered whether lung microvascular endothelial cells, which appear to be so extraordinarily dependent on VEGF for their survival, synthesize and release VEGF. Indeed, when compared with macrovascular pulmonary endothelial cells, microvascular pulmonary endothelial cells, even without stimulation, produce about 20-fold more VEGF. It now becomes clear that the alveolar space is a VEGF-rich environment where this growth and maintenance factor is produced and released by endothelial cells, epithelial cells, and alveolar macrophages; in addition, VEGF can be released intravenously from storage sites in platelets (61). Although the pulmonary microvascular and alveolar septal endothelial cells may be highly dependent on the trophic VEGF-receptor-transmitted signals, this may to some degree also be true for alveolar type II cells. However, the relative contribution of VEGF-RII and VEGF-RI signals, and how they interact, are presently incompletely understood (62–64). In all likelihood there is also a symbiotic interdependence of endothelial cells and alveolar septal epithelial cells, that is, apoptosis of endothelial cells may lead also to apoptosis of epithelial cells. Apoptosis of alveolar septal cells may be the critical and final event that leads to airspace enlargement, whether the result of growth and maintenance factor withdrawal or caused by other mechanisms. Activation of caspases may be indeed sufficient to cause emphysema, as has recently been shown by Aoshiba et al (65). They instilled caspase 3 intratracheally in mice and found airspace enlargement and increased proteolytic activity in the lungs of these mice within a few hours after instillation of the caspase. In this context, it is worth mentioning that caspases do double duty; they also have elastolytic activity.

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Emphysema, Oxidative Stress, and A1-Antitrypsin

It is, however, also of great importance that chronic VEGF receptor blockade, which leads to emphysema, is associated with oxidative stress as shown by lung tissue immunohistochemistry. Figure 4(E and F) shows expression of 8-OH-guanosine in VEGF-receptor-blocked rat lungs. It also shows that chronic treatment of the VEGF-R blocked animals with a superoxide dismutase (SOD) mimetic prevents caspase activation, alveolar septal cell apoptosis, and emphysema (21) (Fig. 4). VEGF-R blockadeinduced emphysema can also be prevented by intravenous administration of a1-antitrypsin (66), indicating that active proteases are also somehow involved in this VEGF receptor blockade emphysema animal model. One concept might be that a1-antitrypsin protects against the cleavage of cell surface receptors: including cleavage of VEGF receptors and/or protects against oxidative stress. It is already known that VEGF increases the expression of SOD in endothelial cells (67,68). Taken together, there may be a step-by-step sequence of events leading from VEGF receptor signal transduction interruption (perhaps following proteolytic VEGF-RII cleavage) to uncontrolled oxygen radical production and activation of apoptosis mechanisms in the mitochondria. We know that lung microvascular endothelial cells are highly metabolically active cells; they are exposed to higher concentrations of oxygen than any other capillary network in the body. Perhaps one of the functions of VEGF in the adult lung is to protect the capillary endothelial cells against oxidative stress.

VII.

Steroid Hormone-Induced Emphysema

A different model of emphysema in rats is based on daily administration of methylprednisolone; animals so treated develop emphysema within 3–4 weeks (69). Analysis of the lung tissue demonstrates evidence of activation of caspase 6 since the lungs stain positive for lamin A, a cleavage product of caspase 6. Zymography of lung tissue identifies activation of matrix metalloproteinase 9/gelatinase B which also has been identified in the bronchoalveolar lavage fluid from patients with COPD (9). Treatment of adult rats with the combination of methylprednisolone and GM6001, a broad-spectrum inhibitor of metalloproteinases, prevents development of emphysema in these animals. Thus, as in the thymus gland, steroids cause apoptosis of lung parenchyma cells, and activated gelatinase B is involved in the development of emphysema (69). Whether methylprednisolone treatment also generates oxidative stress in the lungs is yet unknown. Here, in paradoxical fashion, an anti-inflammatory drug causes emphysema in healthy adult rats. Whether

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Figure 4 A. Emphysematous morphology of SU5416-treated lung, which is prevented by a manganese superoxide dismutase M40419 (B). C, D. TUNEL demonstration of apoptotic cells in SU5416-treated and SU-5416 + M40419 lungs. Note the M40419 causes a normalization of lung alveolar cell apoptotsis caused by VEGF receptor blockade (immunofluorescence, rhodamine). E, F. Immunohistochemical detection of the oxidative stress markers 8-hydro-2’ deoxyguanosine (8HG) in SU5416-treated and SU5416 + M40419 treated lungs. SU5416 treatment results in marked increase in oxidative stress, which is more prominent in the centrilobular than peripheral lung tissue. M40419 treatment results in normalization of markers of oxidative stress in SU5416-treated lungs (immunohistochemistry, alkaline phosphatase, fast red detection product).

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chronic steroid treatment in susceptible patients with obstructive lung diseases contributes to the development of emphysema is unknown. VIII.

Summary and Conclusion

Most likely there is a genetic condition that makes cigarette smokers susceptible to and ill-defended against inhaled particles, oxidants, and carcinogens. Whether this form of genetic weakness is in the area of antiprotease or antioxidant activity remains unclear. Emphysema investigators of the last 150 years have produced libraries of data and a large number of pathoetiological concepts rooted in the contemporary biological framework. This once was mechanical and now is grounded in cellular and molecular biology. Concepts of emphysema as atrophy of the lung tissue or of aging of the lung can now be explored using the new microscopes of signal transduction, functional genomics and proteomics. New animal models of emphysema challenge us. We are asked to integrate and synthesize new information and to explore which aspect(s) of a particular model is indeed operative in the condition of the genetically susceptible human smoker. It appears to us that nonsmoking models of emphysema are as important as those based on chronic cigarette smoke inhalation. They do not deal with the xenobiotic challenge of the lung cells, but they provide new insights into and a better understanding of the various components and workings of the adult lung structure maintenance program. Although activated inflammatory cells, cytokines, and mediators of inflammation are a reality of chronic obstructive disease, it is now clear that noninflammatory triggers can jeopardize the alveolar septal structures. A number of different danger signals triggered in alveolar cells by cigarette smoke inhalation, or by other noxious stimuli, may cause mitochondrial stress and eventually apoptosis of alveolar structure cells. Gene expression profiling of lungs from patients with severe emphysema reveals a pattern of expression characterized by decreased expression of genes encoding various growth factors and a decreased expression of a number of genes encoding ribosomal proteins and mitochondrial enzymes, including genes encoding subunits of the mitochondrial electron chain enzyme cytochrome c oxidase (70). To some extent, similar alterations in lung tissue gene expression can be found in the VEGF receptor blockade emphysema model (71). The search for the loci of genetic susceptibility in human smokers should include the examination of mitochondrial genes. New therapeutic targets may include not only signal transducers of inflammation but also mitochondrial stabilizers and antiapoptotic and antioxidant proteins.

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16 Genetic Analysis of Emphysema and Animal Models of COPD

STEVEN D. SHAPIRO

RAVI MAHADEVA

Brigham and Women’s Hospital and Harvard Medical School Boston, Massachusetts, U.S.A.

University of Cambridge Institute for Medical Research Cambridge, England

I. Introduction Although hypotheses and mechanisms of emphysema can be addressed in the test tube and cell culture, they need to be tested in mammals in order to dissect the complex pathophysiology of the disease. Since Gross reported nearly 40 years ago that instillation of the plant proteinase papain into rat lungs resulted in emphysema (1), animal studies have been critical in shaping contemporary views regarding the pathogenesis of the disease. Indeed, that study, in combination with the nearly simultaneous discovery that humans with alpha-1-antitrypsin deficiency are at an increased risk of emphysema (2), engendered the elastase–antielastase hypothesis, which remains to this day the prevailing theory for the development of emphysema. Since then, various animal models of the disease (including exogenous administration of proteinases, chemicals, and particulates, and exposure to cigarette smoke and proapoptotic agents) have produced results that mimic some aspect of human COPD (3). Transgenic mice and gene-targeted mice in which individual proteins are overexpressed or deleted respectively allow investigators to perform highly controlled experiments in mice and thus specifically dissect pathogenetic pathways in mammals. Genetic manipula411

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tion itself can result in air space enlargement during development and throughout life. No single model precisely replicates the human condition, but each has its own merits and limitations, and is discussed below. II. Traditional Animal Models of Emphysema A. Elastase-Induced Emphysema

Since Gross’s initial experiments, investigators have instilled a variety of proteinases into the lungs of many small and large animals. Administration of porcine pancreatic elastase (PPE, 1–4 mg/kg) has produced the most consistent and impressive air space enlargement in rodents, guinea pigs, dogs, and primates (4). Emphysemaoccursfollowing instillation of other elastases, including neutrophil elastase (5–7) and proteinase 3 (8,9), but not nonelastolytic enzymes such as bacterial collagenase. Instillation of PPE results in rapid and significant air space enlargement followed acutely by neutrophil recruitment with subacute macrophage accumulation in the lungs. Air space enlargement continues over the first month and then stabilizes. Elastin content initially decreases, but appreciable elastin mRNA expression and elastic fiber deposition, albeit disorganized, are observed within weeks. In situ hybridization demonstrates that elastin mRNA is strongly expressed in pleura, blood vessels, and airways following PPE administration. Within the alveoli, expression is observed primarily at the free margins of alveolar septa, with minimal expression in walls of air spaces (10). Extracellular matrix (ECM) repair must be partially effective since lesions are much more severe with coincident application of beta-aminopropionitrile (BAPN), which prevents elastin and collagen cross-linking (11). Despite significant air space enlargement, experimental animals generally survive. Elastase-induced emphysema remains a useful model of emphysema since it is relatively simple to perform and replicates many aspects of the disease. It has also been instrumental in assessing the efficacy of many therapeutic agents. For example, Cantor found that hyaluronidase, when instilled into hamster lungs, interacts with and protects elastin from subsequent elastase-induced emphysema (12), while Massaro demonstrated that retinoic acid has the capacity to promote alveolarization and lung repair in adult male rats previously exposed to PPE (13). However, extrapolating findings directly from elastase administration to the slowly developing, complex changes that occur in response to cigarette smoking is not straightforward; additional mediators are likely to be involved. B. Cigarette Smoke Exposure

A major advantage of studying COPD is that the primary inciting agent— cigarette smoke—is known. There must be additional genetic and/or envi-

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ronmental factors that predispose an individual to COPD since not all smokers develop the clinical disease, but it remains true that the vast majority of patients with COPD are current or former smokers. Therefore, cigarette smoke exposure seems a logical foundation for the development of an animal model of the disease. Indeed, a variety of animals (including dogs, rabbits, guinea pigs, and rodents) have been exposed to cigarette smoke over the years (3). Each species demonstrates a somewhat unique inflammatory response and a variable capacity for air space enlargement, but guinea pigs are perhaps the most susceptible species, developing profound neutrophilia and significant emphysema after only a few months of exposure (14). Latent adenoviral infection combined with cigarette-smoke exposure potentiates CD8+ cell influx and emphysema in guinea pigs (15). Although rats appear to be more resistant to emphysema, Ofulo has used the species to confirm the importance of macrophage elastases in the development of emphysema (16). His group treated rats with antibodies directed against polymorphonuclear leukocytes (PMN) or monocytes/macrophages (MoMac) while subjecting them to daily smoke exposure for 2 months. As expected, cigarette smoke exposure induced lung elastin breakdown and emphysema in non-antibody-treated controls. While such changes were still present in the lungs of anti-PMN antibody-treated smoke-exposed rats, they were prevented in lungs of anti-MoMac antibody-treated smokeexposed rats. Emphysema susceptibility in mice in response to cigarette smoke is strain-dependent, providing a unique opportunity to determine COPD susceptibility genes. Moreover, application of gene-targeted mice to COPD models allows investigators to determine the relative contribution of a particular protein to the disease. Given the capacity to manipulate the mouse genome, this most useful species will be described in detail below. C. Exposure to Other Chemicals and Particulates Cadmium Chloride

A chemical constituent of cigarette smoke, cadmium chloride has been used extensively to generate air space enlargement. However, air space enlargement in this model appears to be secondary to fibrosis, with subsequent tethering and enlargement of air spaces (3). Coadministration of the lathyritic agent BAPN with cadmium made the resulting air space enlargement more severe (17). In the latter experiments, depleting animals of neutrophils did not worsen emphysema, and it was still not clear whether elastin degradation occurred. Although this model may not produce classic COPD, we now appreciate that centrilobular emphysema is also characterized by small airway fibrosis, and that the relationship between emphysema and fibrosis adds to the complexity of COPD.

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Several events are related to oxidative stress and important in the pathogenesis of COPD. These include epithelial cell injury and increased neutrophil inflammation secondary to gene expression of proinflammatory mediators through redox-sensitive transcription factors such as NF-nB and AP-1. Oxidative inactivation of antiproteinases occurs in vitro, but has been difficult to convincingly demonstrate in vivo. Nitrogen dioxide and ozone produce similar toxic effects including epithelial injury, edema, and airway and parenchymal infiltration by inflammatory cells. Repeated administration of nitrogen dioxide results in mild focal emphysema, while ozone results in fibrosis (18). Inorganic Dusts

Epidemiological studies have established that exposure to inhaled particulates, particularly coal dust and silicates, is a risk factor for the development of emphysema. PM10 particles induce oxidant stress, causing inflammation and injury to the airway epithelium (19). Silica and coal dust exposure results in neutrophil-mediated injury with connective tissue breakdown and focal emphysema (20). Smoke from solid fuel used for cooking or heating, particularly in unventilated rooms, is thought to cause chronic lung disease in developing countries. Intermittent exposure to combustion from wood resulted in mild bronchiolitis, hyperplasia, and hypertrophy of bronchiolar epithelial lining cells (21). These changes were associated with mild nonprogressive emphysema in experimental animals. Severe Starvation

In rats severe starvation results in increased air space size without obvious connective tissue turnover. Refeeding adult rats starved to 25% of their initial body weight restored lung volumes and air- and saline-filled specific compliance (22). The relevance of this model, except in conditions of severe starvation, is not clinically obvious, but mechanistically it raises important issues such as the requirement for matrix injury and capacity for repair associated with air space enlargement. D. Apoptosis-Induced Emphysema

Noninflammatory models of emphysema have recently been generated with alveolar septal cell apoptosis as the primary event. Vascular endothelial growth factor receptor (VEGFR) is required for endothelial cell survival and normal blood vessel development, and its absence leads to endothelial cell apoptosis. Chronic receptor blockade with SU5416 caused alveolar septal cell

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apoptosis, pruning of the pulmonary arterial tree, and air space enlargement (23). There was no evidence of inflammation, and the air space enlargement and apoptosis could be prevented by treatment with the caspase inhibitor ZAsp-CH(2)-DCB. This mechanism of air space enlargement revisits the previous so-called vascular hypothesis of the disappearing alveolar cells in patients with emphysema (24). Yet one can also generate emphysema by directly targeting epithelial cells for apoptosis (and sparing endothelium) by instilling caspases (with Chariot for cell uptake) (25). Traditional theories suggest that inflammatory cell proteinases degrade lung extracellular matrix as the primary event in the pathogenesis of emphy-

Figure 1 Mechanisms of air space enlargement in pulmonary emphysema. The traditional inflammatory cell hypothesis stipulates that cigarette smoke results in accumulation of inflammatory cells that release proteinases [neutrophil elastase (NE), macrophage elastase (MMP-12), and cysteine proteinases], disrupting extracellular matrix and basement membrane. Loss of cell–matrix attachment leads to apoptosis with loss of the entire alveolar unit. An alternative hypothesis suggests that cell death is the primary stimulus, with subsequent loss of matrix components resulting in loss of the alveolar unit.

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sema. These studies suggest that the primary event in emphysema could be noninflammatory, structural cell death (Fig. 1). Although this has not been investigated, it is likely that these structural cells release proteinases since one must dissolve the extracellular matrix for loss of an entire alveolar unit. Whether these mechanisms play a role in human COPD is unknown; however, it has been shown that there is increased septal cell death associated with reduced lung expression of VEGF and VEGFR-2 (KDR/Flk-1) in human emphysematous lungs (26).

III. Genetic Engineering and Mouse Models of Emphysema A. Genetic Engineering in Mice

We now know the sequence of all human genes. However, the functions of the proteins encoded by most of these genes remain a mystery. Transgenic and gene-targeted mice are powerful tools in determining protein function in vivo. Gain-of-function models may be achieved by overexpression of proteins in transgenic mice, and loss-of-function models by targeted mutagenesis or gene targeting. These techniques allow investigators to change single variables and perform controlled experiments in mammals. Genetically engineered mice may help to determine the physiological functions of proteins as well as dissect mechanisms of disease. With respect to COPD, genetically engineered mice (in most cases applied to disease models) have helped to confirm previous notions of pathogenesis while pointing the field in new directions. Transgenic mice are generated by injecting DNA into the pronucleus of individual egg cells soon after fertilization. Inducible and cell- or lung-specific (conditional) transgenic mice can also be generated using cell-specific promoters such as Clara cell protein 10 (CC10) for small airway expression or surfactant protein C (SP-C) for alveolar type II cell expression. Once can also achieve expression at particular times using inducible systems such as the tetracycline-activating system. As an alternative, models of underexpression or gene deletion can be generated by inserting mutant DNA into embryonic stem (ES) cells and screening for clones that undergo homologous recombination. These cells are then inserted into blasotcysts and the mutation is transmitted to the germline, resulting in a null mutation often referred to as a knockout (27). Using ES technology, investigators can essentially place (or knock in) any gene under the control of any other. Conditional gene deletion is also possible using Cre-recombinase-lox-P technology. The main advantage of using mice is that one can isolate and manipulate their embryonic stem cells and reintroduce them into blastocysts without abrogating the cells’ capacity to contribute to the germline of

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offspring. So far, this appears to be a characteristic unique to mice. Among other advantages are that more is known about the mouse genome than any other animal, and cDNA and antibody probes are abundant. Mice also offer the practical benefits of short breeding times, large litter sizes, and relatively cheap housing. The main issue confounding the use of mice and other animals as a template for human disease is that we do not know how accurately they reflect human biology and pathology. Mice and humans clearly share many basic physiological processes, but the details regarding species-specific lung structure and function are largely unknown. Thus, direct extrapolation of the importance of individual proteins from gene-altered mice to humans should be avoided until these similarities and differences are more clearly defined. One must also recognize the limitations of gene-targeted mice: Loss of a protein from the blastocyst stage onward might alter complex biological processes leading to what is commonly referred to as compensation; due to redundancy, mutation of a gene may not unmask the true biological function of the protein it encodes; despite the comments above, mice are not humans, and direct translation from mouse to human biology requires knowledge of similarities and differences between these species; and one must also be aware of potential strain differences and the possibility of what is termed a neighborhood knockout effect (the inhibited expression of physically linked genes) likely due to the retention of the phosphorylglyceral kinase (PGK) promoter used to drive selectable markers (28). Transgenic mice that overexpress the protein of interest are easier to derive than gene-targeted mice that lack it. However, because the random introduction of a gene into the genome may interfere with other host functions, investigators should establish multiple transgenic founders. For both techniques one must be cognizant of strain differences between mice, keeping in mind that obtaining or breeding genetically engineered mice into a pure background is advised. Differentiating between transgenic overexpression and gene deletion deserves comment. If transgenic overexpression of a particular protein results in emphysema, one may conclude that this protein is capable of causing a lesion similar to the actual disease state. If such a protein is also expressed in the actual disease state, it is likely, but unproven, that it contributes to the disease. A more direct approach to understanding the disease process is to apply gene-targeted null mutant mice to an appropriate disease model. For example, exposure of mice to cigarette smoke, which is the causative agent in COPD, leads to emphysema. If mice that are identical except for the absence of a single protein fail to develop a lesion when exposed to cigarette smoke, then one can directly infer that this protein participates in the pathogenesis of disease, at least in this model.

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When a transgenic or gene-targeted mouse spontaneously develops air space enlargement, it is necessary to determine the cause. Air space enlargement can result from impaired alveogenesis (a process that takes place during the first 2 weeks postnatally) or from alveolar destruction following normal maturation. The former implies a developmental abnormality; the latter suggests a disease state consistent with pulmonary emphysema. Often it is difficult to differentiate one from the other. Indeed, stress-induced destruction of an abnormal substrate might be relevant to development of emphysema in humans. Developmental abnormalities occurring in a knockout background suggest that the deleted protein was critical to lung development, while transgenic overexpression of an ectopic protein is only significant if the protein is also overexpressed in clinical pathological circumstances. Even though abnormal alveogenesis is fundamentally different from pulmonary emphysema, understanding the process of alveogenesis will lead to insights into abnormal repair in emphysema and to strategies to repair emphysematous lung tissue.

IV. Transgenic/Gain-of-Function Models of Air Space Enlargement Mice overexpressing collagenase (MMP-1), driven by the presumed liverspecific haptoglobin promoter, somehow show transgene expression in the lung and have resultant air space enlargement (29). Whether this interesting phenotype is due to the destruction of collagen in mature lungs or to interference with lung growth and development is unclear. In either case, emphysema appears progressive, suggesting a component of destruction, perhaps on an abnormal developmental substrate. The role of collagen in emphysema remains of great interest. Clearly there is increased collagen turnover in emphysema, but overall collagen levels are higher, particularly in the small airways. Overexpression of platelet-derived growth factor A (PDGF-A) led to air space enlargement that could be secondary to fibrosis and airway tethering, although other explanations are possible (30). It was a surprising finding that lung-specific expression of IL-11 led to enlarged air spaces, elegantly shown to be developmental in origin since inducible expression of IL-11 in adult mice did not alter normal alveolar architecture (31,32). In contrast, inducible, lungspecific, transgenic expression of the Th2 cytokine IL-13 (33) results in inflammation with expression of MMP-12, MMP-9, and cysteine proteinases. Depending upon the degree of IL-13 expression, emphysema results within weeks or months. In addition, these mice have mucous gland hypertrophy and small airway fibrosis. Backcrossing them to MMP-12
/
mice inhibits inflammation and emphysema, while backcrossing them to MMP-9
/
mice

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abrogates both air space enlargement and subepithelial fibrosis (goblet cell hypertrophy remains), despite continued inflammation. Taken together, these data suggest that MMP-12 is responsible for inflammation, while MMP-9 may cause matrix destruction and induce fibrosis. The latter may be due to dual effects on elastin degradation and transforming growth factor beta (TGFh) release in the small airways with collagen accumulation. Inducible, lung-specific overexpression of the Th1 cytokine interferon gamma (IFNg) also results in more subtle inflammation and air space enlargement (34).

V. Loss-of-Function Models of Air Space Enlargement A. Natural Mutant Mice with Air Space Enlargement

Several inbred strains of mice develop emphysema spontaneously due to genetic mutations (Table 1). Such abnormalities are usually developmental in origin, but they tend to be progressive over the lifespan of the mouse. Tight skin (Tsk+/
) mice (the homozygous null mutation is embryonic lethal) have a mutation in fibrillin-1, which is involved in elastic fiber assembly (35*ndash;37). These mice experience abnormal air space development and progressive alveolar enlargement with age. The effect of fibrillin on elastogenesis and lung development is plausible, yet the degree and severity of emphysema in patients with the human equivalent, Marfan’s syndrome, are controversial. Pallid mice (pa/pa) develop mild emphysema late in life (38). The genetic abnormality in pallid mice has been linked to the syntaxin 13 gene (39), but the function of the protein encoded and how it leads to air space enlargement are not known. Blotchy mice have enlarged air spaces believed to be due to abnormal connective tissue or cross-linking (40,41). Recently this defect has been attributed to abnormal RNA processing of the Menke gene on the X chromosome mottled locus (42), but its relationship to emphysema is unclear. Beige mice (bg) have a defect in the formation of primary granules (43,44), but it remains controversial whether they produce normal levels of serine proteinases and have the capacity to develop emphysema. Emphysema in some of these lines, particularly the pallid and others in a C57BL/6 background, may at least in part be attributed to lower alpha-1-antitrypsin (a1AT) levels. However, in none of these inbred strains do a1AT levels dip below the 15% threshold required for protection in humans. B. Gene-Targeted Mice with Spontaneous Air Space Enlargement

Several transcription-factor-null mutant mice possess various abnormalities of lung developmental described in detail elsewhere (45,46) and in other chapters of this volume (Table 2). Members of the large family of fibroblast

Duplication of Fibrillin 1 (Fbn-1) Tsk Fbn-1 copolymerizes with wild-type Fbn-1 rendering it susceptible to proteolysis Fbn-1 main component of microfibrils which act as a scaffold for tropoelastin 5-kb deletion in Lyst (causes Chediak-Higashi syndrome) Defective intracellular transport Lysosomal missorting of proteins (including serine proteinases) X-linked Abnormal RNA splicing of Atp7A (Menke gene), a copper ion transporting ATPase, Impaired lysyl oxidase and abnormal collagen and elastin cross linking

Tight skin mouse (Tsk)/ Marfan’s syndrome

Blotchy mouse/ Menkes disease

Beige mouse (Bg)/ Chediak-Higashi syndrome (CHS)

Genetic/physiological defect

Mouse/human disease

Panlobular emphysema develops between 4 days and is progressive

Abnormal alveolarization

Enlarged air spaces Disruption of elastic fibers

Generalized immunodeficiency with abnormal function of NK cells, CTL

Aortic aneurysms, reduced skin tensile, develop osteoarthrosis

Lung phenotype

Tight skin, subcutaneous connective tissues hyperplasia, increased growth of cartilage and bone, and small tendons with hyperplasia of the tendon sheaths

Phenotype

Table 1 Naturally Occurring Mouse Strains with Air Space Enlargement

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Macrophage colony stimulating factor deficiency

Osteopetrotic (Op/Op)

CTL = cytotoxic T lymphocytes; NK = Natural Killer cells.

Nonsense mutation at codon 69 in Pallidin which interacts with syntaxin 13, a tSNARE protein; important in vesicle-docking and fusion A platelet storage pool deficiency Defect in organelle biosynthesis

Pallid (Pa)

Osteopetrosis

Prolonged bleeding time, pigment dilution, kidney lysosomal enzyme elevation, abnormal otolith formation, serum a1-antitrypsin activity deficiency Normal at 1 month, progressive development of emphysema Increased elastase and decreased elastin in the alveolar intestitium Increased intracytoplasmic crystalloid inclusions related to collagen degradation in pulmonary macrophages Young mice have reduced numbers of lung macrophages (which return to normal as they age) Macrophages release more MMP’s z BAL IL-3 Emphysema

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Spontaneous emphysema: developmental

Emphysema Characterization

Forkhead Box F1 (Foxf1) +/


Retinoic acid receptor (RAR) g
/


Elastin 1
/


Fibulin-5/DANCE
/


Platelet derived growth factor A (PDGF-A)
/
Fibroblast Growth Factor Receptor (FGFR)
3 and 4
/


Gene

Table 2 Gene-Targeted Mice with Air Space Enlargement

Lack of myofibroblasts, the precursors of tropoelastin-positive smooth muscle cells Mice lacking FGFR 3 and 4 (but not FGFR 4 alone) are normal at birth do not form secondary septae and therefore do not form alveoli Integrin ligand for avh3, avh5 and a9h1 anchoring cells and elastic fibres enabling their correct organization Abnormal airway branching and alveogenesis Tortuous aortaand cutis laxa Arrested terminal airway development, fewer and dilated distal air sacs Die perinatally with obstructive arteropathy (aortic root subendothelial cell and smooth muscle. proliferation) Embryonic lethality. Increased alveolar size worsened by co-deletion of retinoid X receptor-a Decreased tropoelastin mRNA and whole lung elastic tissue Severity proportional to Foxf1 mRNA levels Defects in alveolarization and vasculogenesis mice with relatively low levels of expression, those with lowest levels have lung hemorrhage with disruption of the mesenchymal-epithelial cell interfaces in the alveolar and bronchiolar regions

Phenotype

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Progressive air space enlargement: developmental and destructive

Tissue inhibitor of metalloproteinases (TIMP) -3
/


Surfactant Protein D (SP-D)
/


POD-1 (Tcf21, capsulin, epicardin)
/


Tumor necrosis factor- a converting enzyme (TACE/ADAM-17)
/


Air space enlargement at 2 weeks and progressive

Transcription factor deficiency associated with # expression of vascular endothelial growth factor (VEGF), the VEGF receptor 2 (Flk-1), bone morphogenetic protein 4 (Bmp-4), transcription factors of the Brachyury T-Box family (Tbx2Tbx5) and Lung Kruppel-like Factor. Transmembrane metalloprotease-disintegrin cleaves cell surface proteins including cytokines and growth factors which are proteolytically shed. Impaired branching morphogenesis, defects in epithelial cell proliferation and differentiation and delayed vasculogenesis. Lungs fail to form normal saccular structures with fewer peripheral epithelial sacs, deficient septation and thick-walled mesenchyme Basic-helix-loop-helix transcription factor expressed at sites of mesenchymal-epithelial interaction in the lung, kidney, intestine and pancreas. Hypoplastic lungs, abnormal lung branching lacking an alveolar region Progressive pulmonary emphysema starting from 3 weeks of age Activated, large, lipid-laden alveolar macrophages with increased oxidant production (activates NFkappa-B) and MMP expression.

Animal Models of Emphysema and COPD 423


/
= null mutant mice.

Normal lung structure: emphysema after exogenous challenge

Emphysema Characterization

Table 2 Continued

Interleukin-1 (IL1)
/
+ TNFa
/


Macrophage Elastase (MMP-12)
/


Beta-6 integrin
/


Gene

Non-inflammatory increased collagen turnover, net decreased collagen with disorganized collagen fibrils in the alveolar interstitium cell infiltration Die at 13 months of age avh6 deficiency, lack of TGFh activation, macrophage infiltration and MMP-12 production, progressive emphysema over time. Normal spontaneous lung development Protected from cigarette smoke-induced emphysema Compound mutant (not individual knockouts) protected from pancreatic elastase-induced emphysema

Phenotype

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growth factors (FGF) mediate epithelial–mesenchymal interactions and are essential for several stages of mammalian lung development. FGF-10 null mutant mice fail to undergo airway branching distal to the trachea, but mice homozygous for a targeted disruption of FGF-9 exhibit lung hypoplasia and early postnatal death (47). Taken together, these studies suggest that FGF-9 signaling from the epithelium and reciprocal FGF-10 signaling from the mesenchyme coordinately regulate epithelial airway branching and organ size during lung embryogenesis. Lungs of gene-targeted mice lacking receptors for both FGF receptors FGFR-3 and -4, but not either individually, fail to undergo alveogenesis and do not form secondary septa to delimit alveoli (48). Other transcription-factor-null mutant mice with abnormal airway/air space development include thyroidtranscriptionfactor1 (TTF1), GATA6(49), and sonic hedgehog (Shh). Retinoic acid signaling is essential for normal embryonic development. Deletions of retinoic acid receptor isoforms have varied effects on morphogenesis (50). Retinoic acid receptor-g-deficient mice display air space enlargement potentiated by codeletion of retinoid X receptora (51). Transcription-factor-null mutant mice with altered lung vascular development include Forkhead Box F1 (Foxf1) (52) and POD-1 (53). Disruption of elastic fibers and other extracellular matrix components would be expected to impair the structural integrity of the lung. In support of this concept, mice deficient in elastin (54) and fibulin-5 (a microfibrillar component) (55) exhibit air space enlargement. Platelet-derived-growthfactor-A- (PDGF-A) null mutant mice lack myofibroblasts (a key source of tropoelastin), which are required for alveolar septation (56). It is surprising that deletion of individual matrix-degrading proteinases has rarely resulted in abnormal lung development. Yet gene targeting of two membrane-bound disintregrin metalloproteinases—tumor necrosis factor-a converting enzyme (TACE/ADAM-17) (57) and ADAM-19—leads to abnormal alveolar development. Gene targeting of TIMP-3 (58), but not TIMP1, or –2, results in early air space enlargement with subsequent progression over time, suggesting both developmental and acquired proteolysis-mediated abnormalities. While the so-called klotho mouse (59) is technically transgenic, its phenotype results not from the inserted transgene but from the loss of function at the locus (termed the klotho locus) into which the transgene was inserted. These mice have many systemic abnormalities consistent with premature ageing. The lung appears to develop abnormally, and emphysema progresses quickly. Mice deficient in either surfactant protein D (SP-D
/
) (60) or the integrin beta 6 (h6
/
) (61) appear to experience normal lung development followed by progressive macrophage-mediated destructive air space enlargement with age. SP-D gene targeting resulted in a lipidosis with abnormal,

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lipid-laden, activated macrophages that produce excessive MMP-9, MMP12, and oxidants. Air space enlargement was noted, particularly in areas associated with these macrophage clusters. h6 gene targeting results in mice deficient in avh6. These mice were known to have spontaneous lung macrophage accumulation. Affymetrix expression profiling comparing h6
/
with h6+/+ lung tissue uncovered MMP-12 as the most differentially regulated gene, with a greater than 100-fold increase in h6
/
lung tissue. Further study demonstrated that the mice developed progressive air space enlargement throughout life following attainment of normal adult alveolar dimensions. Backcrossing h6
/
to MMP-12
/
mice abrogated air space enlargement. In addition, backcrossing h6
/
mice to those overexpressing TGFh also inhibited air space enlargement. These findings suggest that TGFh serves to inhibit MMP-12 production under normal circumstances and that, in the absence of the h6 integrin, there is lack of active TGFh, and thus enhanced MMP-12 catalysis leading to emphysema. C. Gene-Targeted Mice that are Protected from Emphysema Upon Challenge

Some mice have normal lungs under normal conditions and also are protected from injury following exposure to models of emphysema. These proteins therefore are required for the development of emphysema. As discussed above, the most relevant model of COPD involves response to long-term cigarette smoke exposure. Murine Model of Cigarette-Smoke-Related COPD

Mice are able to tolerate at least two cigarettes per day for a year with nontoxic carboxyhemoglobin levels, and minimal effects on body weight (62). Despite the fact they are obligate nose-breathers they tend not to have extensive cilia, which means that they inefficiently filter tobacco smoke. Other anatomical differences from humans include a paucity of submucosal glands (limited to the trachea) and fewer Clara cells. Goblet cell hypertrophy has also been described in this model. They also have less extensive airway branching and lack respiratory bronchioles. Neutrophil recruitment occurs following the first cigarette, which is followed by a more gradual T-cell and macrophage accumulation. The early neutrophil influx in the lungs is accompanied by a measurable increase in collagen and elastin degradation products. At this very early stage both the bronchoalveolar lavage (BAL) neutrophilia and the connective tissue breakdown are preventable by pretreating the mice with an antibody to neutrophils or alpha-1 antitrypsin (63). After a few months of exposure there is a reduction in the number of ciliated

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epithelial cells, followed by occasional obstruction of small airways with inflammatory cells and debris and a loss of alveolar attachments (Fig. 2). An increased alveolar duct area and enlarged alveolar spaces are clearly seen after 3–6 months of exposure to cigarette smoke. The effect on physiological parameters following prolonged cigarette smoke exposure is not known. Further studies are also required to quantify the effect of cigarette smoke inhalation on collagen and elastin metabolism. Exposure of Gene-Targeted Mice to Cigarette Smoke

MMP-12 null mutant mice develop normally (64). In contrast to wild-type mice, the MMP-12
/
mice fail to develop emphysema following long-term exposure to cigarette smoke. MMP-12
/
mice also did not experience macrophage accumulation in response to cigarette smoke (65). This effect may be related to MMP-12 generation of elastin fragments that are chemotactic for monocytes (66,67). Whether human emphysema is also dependent on this single MMP is uncertain and unlikely. This study demonstrates a critical role of macrophages in the development of emphysema, shows that macrophage matrix metalloproteinases have the capacity to cause air space

Figure 2 Emphysema in response to long-term cigarette smoke exposure in mice. Adult C57BL/6J female mice were exposed to the smoke from two cigarettes for 9 months. Scanning electron microscopy (400X) demonstrates air space destruction and enlargement in cigarette-exposed mice (left) compared with non-smoke-exposed age-matched littermates (right).

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enlargement in response to cigarette smoke, and unmasks a proteinasedependent mechanism of inflammatory cell recruitment that may have broader biological implications. Serine proteinases, particularly neutrophil elastase (NE), are also involved. NE
/
mice developed only 40% as much air space enlargement as wild-type mice (Shapiro, unpublished findings). Several interactions between NE and MMPs were also uncovered. MMPs degrade a1-AT and NE degrades tissue inhibitor of metalloproteinases (TIMPs), potentiating each other’s proteinase activity. NE mediates monocyte migration, and MMP-12 might influence acute neutrophil accumulation via TNFa-shedding (Churg and Shapiro, unpublished findings). These findings reinforce the concept that many cells and proteinases are likely to be important in the development of emphysema and that they interact to augment injury. It has recently been shown that mice deficient in both IL-1h type-1 receptor and type1 and type 2 TNF-a receptors are protected from the development of emphysema following intratracheal challenge with PPE. They exhibited reduced inflammation and increased apoptosis. However, there was no such protection in the individual null mutant strains (68).

VI. Conclusions and Future Directions Synthesis of our knowledge on the pathogenesis of emphysema, largely obtained from animal models, suggests that cigarette smoking leads to acute neutrophilia within the lung, followed by subacute accumulation of other immune and inflammatory cells, most notably macrophages. These inflammatory cells (and perhaps resident cells) release elastolytic proteinases in excess of inhibitors in local microenvironments, causing damage the extracellular matrix of the lung. Ineffective repair of alveoli and elastic fibers and perhaps other extracellular matrix components results in air space enlargement that defines pulmonary emphysema. As discussed, alternative explanations such as apoptosis as a primary event are also possible. In comparison to emphysema, our understanding of airway disease in COPD is much less advanced. In addition, other fundamental questions remain to be addressed: How does smoking cause pulmonary inflammation and how is inflammation maintained when cigarette smoking is discontinued? Why do only a minority of cigarette smokers get emphysema? What are the factors that control normal lung development, and how can we restore normal alveolar tissue? We now have the exciting opportunity to combine proteomics and gene microarray technology with animal models. Therefore, rather than focusing solely on individual mediators, we can begin to determine both the cellular interactions that lead to emphysema and the factors that govern normal

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alveolar development. This will, it is hoped, identify novel strategies to achieve the ultimate goal of a cure for this devastating disease. References 1. 2. 3. 4. 5.

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35. Kielty CM, et al. The tight skin mouse: demonstration of mutant fibrillin-1 production and assembly into abnormal microfibrils. J Cell Biol 1998; 140(5):1159–1166. 36. Green MC, Sweet HO, Bunker LE. Tight-skin, a new mutation of the mouse causing excessive growth of connective tissue and skeleton. Am J Pathol 1976; 82(3):493–512. 37. Siracusa LD, McGrath R, Ma Q, Moskow JJ, Manne J, Christner PJ, Buchberg AM, Jimenez SA. A tandem duplication within the fibrillin 1 gene is associated with the mouse tight skin mutation. Genome Res 1996; 6(4):300–313. 38. Keil M, Lungarella G, Cavarra E, van Even, P, Martorana PA. A scanning electron microscopic investigation of genetic emphysema in tight-skin, pallid, and beige mice, three different C57 BL/6J mutants. Lab Invest 1996; 74(2):353–362. 39. Huang L, Kuo YM, Gitschier J. The pallid gene encodes a novel, syntaxin 13interacting protein involved in platelet storage pool deficiency. Nat Genet 1999; 23(3):329–332. 40. Fisk DE, Kuhn C. Emphysema-like changes in the lungs of the blotchy mouse. Am Rev Respir Dis 1976; 113(6):787–797. 41. Mechanic GL, Farb RM, Henmi M, Ranga V, Bromberg PA, Yamauchi M. Structural crosslinking of lung connective tissue collagen in the blotchy mouse. Exp Lung Res 1987; 12(2):109–117. 42. Mercer JF, Grimes A, Ambrosini L, Lockhart P, Paynter JA, Dierick H, Glover TW. Mutations in the murine homologue of the Menkes gene in dappled and blotchy mice. Nat Genet 1994; 6(4):374–378. 43. Nagle DL, et al. Identification and mutation analysis of the complete gene for Chediak-Higashi syndrome. Nat Genet 1996; 14(3):307–311. 44. Perou CM, et al. Complementation of the beige mutation in cultured cells by episomally replicating murine yeast artificial chromosomes. Proc Natl Acad Sci USA 1996; 93(12):5905–5909. 45. Warburton D, et al. The molecular basis of lung morphogenesis. Mech Dev 2000; 92(1):55–81. 46. Costa RH, Kalinichenko VV, Lim L. Transcription factors in mouse lung development and function. Am J Physiol Lung Cell Mol Physiol 2001; 280(5):L823–L838. 47. Colvin J, et al. Lung hypoplasia and neonatal death in Fgf9-null mice identify this gene as an essential regulator of lung mesenchyme. Development Supp 2001; 128:2095–2106. 48. Weinstein M, et al. FGFR-3 and FGFR-4 function cooperatively to direct alveogenesis in the murine lung. Development 1998; 125(18):3615–3623. 49. Keijzer R, et al. The transcription factor GATA6 is essential for branching morphogenesis and epithelial cell differentiation during fetal pulmonary development. Development Supp 2001; 128:503–511. 50. Luo J, et al. Compound mutants for retinoic acid receptor (RAR) beta and RAR alpha 1 reveal developmental functions for multiple RAR beta isoforms. Mech Dev 1996; 55(1):33–44. 51. McGowan S, et al. Mice bearing deletions of retinoic acid receptors demonstrate

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17 Pulmonary Alveoli: Development, Structural Stability, and Regeneration

DONALD J. MASSARO, GLORIA DECARLO MASSARO, and LINDA BIADASZ CLERCH Georgetown University School of Medicine Washington, D.C., U.S.A.

I. Introduction The lung’s only known essential function is to meet the organism’s need for oxygen. Because CO2 is much more soluble in water than O2, alveolar architecture that allows sufficient O2 uptake provides adequate elimination of CO2. The interesting and important relationship between O2 need and alveolar surface area is discussed fully and with great insight by Mortola in this volume. The tight match between O2 need and alveolar architecture may not be met if alveoli fail to develop normally, as can occur in prematurely born babies (1–4). Even if alveoli have developed normally, the link between O2 need and the size, number, and surface area of alveoli may be broken if alveoli are destroyed; this occurs most commonly in patients with chronic obstructive pulmonary disease (5,6) and, with age, even in individuals who do not have lung disease and who may or may not be, or ever have been, smokers (see below). The loss of alveoli and lung function due to age alone is substantial and, to our knowledge, rarely considered as contributing to the burden of those with underlying cardiopulmonary disease. Because alveoli are lost as well as formed, even in the absence of disease, we have adopted the notion of alveolar turnover. However, unlike a popu433

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lation of individual molecules, which are recognized as being in a continuous simultaneous state of synthesis and degradation (i.e., turning over), it is not known if in a population of alveoli some are being formed while others are lost. The present evidence suggests that there is a period during which alveoli are formed and when, or shortly after the period of alveolus formation ends, alveolar loss begins and continues for the rest of one’s life. This chapter will review alveolar turnover, mainly, but not exclusively, based on work with which we have direct experience.

II. Quantitating Alveoli and Alveolar Dimensions The measurement of alveolar surface area (Sa) by intersection and point counting is efficient, easy, and free of restrictive assumptions (7,8). Estimating alveolar size using the mean chord length (Lm) obtained from single histological sections, also easy, is flawed for the following three reasons. First, alveolar air spaces cannot be reliably differentiated from alveolar duct air spaces on single histological sections; therefore, Lm determined from single histological sections represents, to an unknown degree, the sum of alveolar air space and alveolar duct air space. Second, the inability to differentiate between these spaces, whose volume can vary independently during normal development, and can be differently affected by experimental manipulations (9,10), decreases the sensitivity of Lm as an index of alveolar size. Finally, estimates of alveolar size made from single histological sections overestimate large alveoli, underestimate small alveoli, and are influenced by alveolar shape and distribution (i.e., they are biased methods). More recent assessments of alveolar dimensions, with methods developed (11–13) in lung pioneered by others (14) have used an analysis of serially sectioned lung to differentiate alveolar from alveolar duct air space (9,10,15– 23). Analysis of serial lung sections, which is very labor-intensive, is required to make the distinction reliably. Having identified alveoli, the selector (13) or disector (11) methods can be used to choose alveoli for analysis in an unbiased manner: uninfluenced by their size, shape, or orientation. Use of the pointsampled intercept method (12) allows determination of the volume of individual alveoli without the inclusion of alveolar duct air space. Based on the average volume of individual alveoli, total lung volume at a fixed transpulmonary pressure (easily measured by volume displacement) (24), and the volume of alveolar air calculated from the fraction of total lung volume that is alveolar air space (determined by point counting), the number of alveoli can be calculated (14–23). This approach has, as we show in this chapter, led to interesting new insights into the regulation of the turnover (formation and loss) of alveoli.

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III. Alveolus Formation Following Burri et al. (25,26), we call the gas-exchange structures of the architecturally immature mammalian lung alveolar saccules; their subdivision into smaller gas-exchange units (alveoli) septation; and the time during development when subdivision of alveolar saccules occurs the period of septation. The term period of septation is useful because it has come to define a time in development when the alveolar saccules are subdivided. The period of septation may occur prenatally, as in guinea pig (27), or postnatally, as in most strains of rat and mouse (25,26,28). Humans seem to be the only species reported that subdivide alveolar saccules pre- and postnatally (29–31). However, although useful as a shorthand way of denoting a stage of development, the term period of septation should not be taken to mean that it is the only time alveolar septa are formed. Considering the appearance of an alveolus, formation of a septum seems to be the only way an alveolus could form from a single-layered epithelial sheet. Therefore, because alveoli continue to be made after the period of septation, septa continue to form. We find it useful in thinking about the regulation of alveolus formation to differentiate between the eruption of septum from the epithelium of alveolar saccules present in the immature lung and the eruption of septa from epithelial sheets that did not line the original gas-exchange saccules. We think that the formation of alveoli from these different sites is, at least in part, under the control of different regulators (20). It is clear from the important measurements made by Burri et al. (25,26) that the size of alveoli, as assessed by measuring the distance between alveolar walls (Lm), decreases in rats during the period of septation. These measurements of Lm (26), plus a morphological assessment (25), have led to the current notion that septation begins in rats about postnatal day 4 and ends about postnatal day 14. However, about 70% of the total decrease in Lm between postnatal day 2 and 14 occurs by postnatal day 7(26), suggesting that most alveoli formed by subdivision of alveolar saccules are made in the first 7 days after birth. Because Lm is not a very sensitive measure of the size of an alveolus, the onset and end of septation of the large saccules present at birth are probably not sufficiently well identified for studies that hope to use global gene expression analysis as a means of precisely linking molecular and morphological events. However, morphometric methods (9–22), equal in power to microarray analysis, are now available to define and correlate precisely the architectural and cellular timetables of alveolus formation. Serial section analysis of lung, used by Randell et al. (14), in important studies on rats, demonstrated that septation of alveolar saccules present at birth accounts for only about one-third of alveoli formed between birth and postnatal day 6 (14). Subsequent work examined the entire period of septation

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with similar results: i.e., only about one-third of alveoli are formed by subdivision of the gas-exchange saccules present at birth (32). Thus, during the period of septation, there are at least two precursor structures to mature alveoli: the alveolar saccules present at the onset of the period of septation and, we believe, saccules that develop as lung volume increases. The latter we think occurs at the periphery of lung (16). Because alveoli continue to form after septation of the saccules present at birth has ended, it is likely that these two sites of alveolus formation are under different controllers. It is clear that septation of the original gas-exchange saccules, which accounts for about one-third of alveoli formed during the period of septation, occurs diffusely throughout the lung (25,26). The evidence, albeit meager, suggests that the other site of alveolus formation is in the subpleural region (16). In male rats (32) and mice (33), alveoli continue to form after the period of septation until about age 40 days. There are insufficient data about female rodents to determine the age at which the number of alveoli in their lungs stops increasing. However, the number of alveoli in female rats and mice continues to increase at an age when male rats have begun to lose alveoli (21). It is usually said that alveoli in humans increase in number up to about age 8 years. However, analysis of the data available (34–36) might be interpreted to indicate that alveoli increase in number until body height stops increasing (37). If alveoli do continue to form in humans until vertical body growth stops, alveolus formation would end at about the same time that gas-exchange function such as diffusing capacity (38), lung tissue recoil (39–47), and resting and maximum VO2 (48) reach a maximum. That is, there would be a development-associated tight link between alveolar structure and function and oxygen need. The signals integrating these events are poorly understood but might include corticosteroids, retinoids, and, because it regulates closure of the epiphyses of long bones (48a), estrogen.

IV. Glucocorticosteroid Hormones and Alveolus Formation Analysis of the regulation of the formation of alveoli based on comparative physiology indicates that the period of septation, whether prenatal (27) or postnatal (25,26,28), seems to occur at a time in development when there is a trough in the serum concentration of the species major corticosteroid hormone (49,50). This concurrence of events suggested that endogenous corticosteroids might be inhibitors of septation, their decreased serum concentration thereby fostering gene expression that initiates the onset of septation and their subsequent increased serum concentration resulting in changes in gene expression that end septation. A further clue that cortico-

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steroids are involved in the regulation of alveolus formation came from a consideration that the eruption and elongation of structures such as alveolar septa are brought about by folding epithelium into ridges. This process requires replication of epithelial and other cells (see Alsberg et al., this volume). Because glucocorticosteroids inhibit cell division in several tissues including lung (51–53), it seemed that they might inhibit septation. Experiments tested these ideas and showed that treatment of rat pups with a corticosteroid hormone during the period of septation inhibits septation in a dose-dependent manner (53). Corticosteroid treatment also impairs the formation of the pulmonary arterial tree (53a). Cessation of dexamethasone treatment at age 14 days, which is the end of septation in rats, is not followed by spontaneous septation assessed at age 60 (9,53) and 90 days (54). In addition, corticosteroid treatment of newborn rat results in pulmonary hypertension in the adult animals (53a). Thus, there is a critical period during which septation must occur. The molecular and cellular basis for this critical period is unknown; it might reflect intrapulmonary conditions, systemic conditions, or both.

V. Retinoids and Alveolus Formation: Guilt by Association A. Developmental Formation of Alveoli

Several lines of evidence have indicated that retinoids might be an important regulator (inducer) of alveolus formation. This evidence came, in part, from seminal work by Brody et al. (55–59), who demonstrated that fibroblasts rich in vitamin A (retinol) storage granules (60), designated lipid interstitial cells (LICs), form a large fraction of the alveolar wall when septa are being formed. Burri found that during the period of septation LICs are concentrated at what are termed septal junctions: sites from which several septa emanate (25). Chytil and colleagues provided evidence of substantial lung metabolism of retinoids at about the time that septation occurs in rats (61–64). McGowan et al. reported the presence of retinoid receptors in LICs (64a) and the ability of LICs to synthesize elastin (64b), a key structural (64c), and perhaps informative, protein in the alveolus. These considerations, the importance of retinoids as regulatory signals for general developmental events (65), and the evidence suggesting that all-trans retinoic acid (ATRA) is a morphogen (66,67) led to the hypothesis that ATRA could induce alveolus formation. This was tested in otherwise untreated rat pups. ATRA caused a 50% increase in the number of alveoli but resulted in smaller alveoli, without a concomitant increase in lung in volume or alveolar surface area (17). The latter finding, and the absence of an effect of ATRA on organismal O2 consumption (68), suggest

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the presence of a regulatory mechanism to prevent the formation of unneeded alveolar surface area (17). More recent work has explicated the influence on alveolus formation of specific retinoic acid receptors (RARs) and retinoid X receptors (RXRs). The absence of RARa does not alter alveolus formation during the perinatal period, but its absence diminishes alveolus formation after the perinatal period (69). RARh is an endogenous inhibitor of alveolus formation during the perinatal period but not afterwards (20). RARg and RXRa are required for postnatal alveolus formation but it is not clear if they are required only during the period of septation or during the entire time during which alveoli are formed (70). The spatiotemporal link between LICs and sites of alveolus formation suggests that LICs are the cells responsible for the release of ATRA that initiates and increases alveolus formation. This suggestion is strongly supported by the following findings. In cultured rat LICs, ATRA increases retinol storage granules. LICs release ATRA and its release, but not the release of retinol, is diminished 50% by dexamethasone, which is a potent inhibitor of alveolus formation (70a). The pulmonary microvascular endothelial cell (PMVC) in culture responds to exogenous ATRA with an increase of CRBP-1 mRNA. Medium previously conditioned by LICs rich in retinol storage granules upregulated CRBP-1 mRNA in PMVCs; medium conditioned by LICs with few or no retinol storage granules did not alter expression of CRBP-1 mRNA in PMVCs. Finally, action of medium conditioned by LICs with retinol storage granules is decreased by a RAR panantagonist and by a RXR panantagonist. These findings, in concert with the spatiotemporal concurrence of LICs and alveolus formation (16,25), in our view strongly suggest that LICs produce retinoid signals that induce alveolus formation. Put differently, perhaps LICs are the alveolar equivalent of the SpemannMangold organizing center in blastocysts (73a). B. ATRA Maintains Alveolus Formation and Alveolar Forming Ability Under Conditions that Inhibit Alveolus Formation

Treatment of rat pups with ATRA prevents the inhibition of septation by dexamethasone (17). More recent work confirmed and extended these findings in rats by demonstrating that ATRA has a similar effect in newborn mice treated with dexamethasone (71). Premature birth, and the medical therapy (supplemental O2 and mechanical ventilation) often required for the survival of premature babies, inhibit alveolus formation (1–4,72) (Coalson, this volume). This clinical problem led to studies showing that treatment with retinol diminishes the impaired formation of alveoli produced in premature lambs by prolonged mechanical ventilation (73).

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Exposure of rat pups (74), as in nonhuman primates and humans (1– 4,72), to hyperoxia impairs septation. Treatment of rat pups with ATRA during exposure to hyperoxia does not attenuate the inhibition of septation by O2 (75,76). However, those treated with ATRA while exposed to hyperoxia exhibit spontaneous post hoc septation. Pups examined several weeks after being removed from hyperoxia, and not having received posthyperoxia treatment with ATRA, were found to have septate alveolar saccules (75,76). The mechanism by which treatment with ATRA achieves this is completely unclear. C. All-trans Retinoic Acid Partially Rescues Failed Alveolus Formation

ATRA administered to rats weeks after septation has been prevented by treatment with dexamethasone during the period of septation partially rescues failed septation. (19). Tight-skin mice exhibit a genetic defect in the formation of fibrillin, a key molecule for laying down elastin, and fail to septate. Treatment of adult Tsk mice with ATRA partially rescues this failed septation(19). D. ATRA and Regeneration of Destroyed or Excised Alveoli

The demonstration that ATRA increases the formation of alveoli in rat pups (17) led to the obvious experiment to determine if ATRA induces the formation of alveoli in adult animals not spontaneously forming alveoli. In these animals alveolar destruction, loss of elastic recoil, and diminished volume-corrected surface area had previously been produced by the intratracheal administration of elastase. ATRA induces the formation of alveoli, and returns alveolar size, number, volume-corrected surface area, and tissue elastic recoil to values present in same-sized rats not treated with intratracheal elastase (18). This work was repeated and confirmed in rats and in mice (77,78). Furthermore, ATRA treatment diminishes alveolar destruction and Lm in mice with cigarette smoke-induced emphysema (79,80). Unlike its reversal of emphysema in rats and mice, ATRA does not reverse cigarette-smoke-induced emphysema in guinea pigs (81). The reason for this failure is unclear. The notion that ATRA works in adult mice and rats because their epiphyses remain open and they continue to grow, but does not induce alveolus formation in guinea pigs because they stop growing, is neither compelling nor based on available information. Male rats (32) and mice (33) stop making alveoli by about age 40 days. The studies that showed ATRA reversing elastase-induced emphysema were carried out in rats much older than 40 days (18). A potential, but also not too convincing explanation for the failure of ATRA to reverse emphysema in smoking guinea pigs is that

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exposure of guinea pigs to cigarette smoke results in an approximately eightfold increase in the level of retinol in the lung (82). It is therefore possible that the effect of ATRA would have been blunted by high levels of endogenous levels of retinoids in guinea pigs exposed to cigarette smoke that were not given exogenous ATRA. This possibility is consistent with the demonstration that cigarette smoke does not cause an elevation of retinol in the lungs of mice (82a) and that ATRA reverses cigarette smoke-induced emphysema in mice (78). In contrast to the demonstration that ATRA augments alveolus formation in newborn rats (17) and induces alveolus formation in adult rats (18), ATRA treatment diminishes the gain of lung function that occurs in dogs after unilateral pneumonectomy (83). The basis for this inhibitory effect is completely unknown but important to understand.

VI. Age and Gender Matter in Pulmonary Alveolar Architecture, Turnover, and the Concomitant Decline of Lung Function Chronic obstructive pulmonary disease (COPD), a combination of chronic bronchitis, loss of bronchioles, and alveolar destruction (emphysema), is due mainly to cigarette smoking and is a major cause of disability, suffering, death (84,85), and economic loss (86). Dwarfed by the devastating effect of cigarette-smoking-induced emphysema, little attention seems to be paid to the loss of lung function due to age alone, or to the impact this loss may have on the aged, in particular those with COPD or other cardiopulmonary diseases. For example, a decline in the timed forced expiratory volume (FEV) is often used as an index of progression of COPD. Yet aging alone in persons who have never smoked causes a greater loss of timed FEV in a general population than does cigarette smoking (87,88). Loss of lung function due to age alone begins in the third decade (38). Furthermore, and perhaps most telling and important men with mild to moderate emphysema determined spirometrically (89) lose FEV1 only about 35% faster than men who have never smoked (87,88,90–95). Put differently, as with cigarette smoking, age accounts for about two-third of the yearly loss of FEV1 in men with mild to moderate COPD. A. Elastic Recoil

Elastic recoil reflects, in part, the lung’s connective tissue, in particular elastin at mid to lower lung volumes and collagen at higher lung volumes (96). Although there is some disagreement, the evidence overwhelmingly indicates

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that lung tissue elastic recoil is lost with age. The loss begins by age 30–40 years, is more rapid in men than in women, and seems to occur among most racial and ethnic groups; as in humans, lung recoil also falls with age in animals (39–47). B. Timed Expiratory Volume

In individuals without bronchospasm, the timed FEV is determined largely by the lung’s elastic recoil and by attachments of alveoli to conducting airways (96–99). The age-related loss of recoil and of airway attachments, as might be expected to occur with the age-related loss of alveoli (100,101), cause excessive airway narrowing during expiration and diminish the timed FEV (97–99). The loss of FEV1 begins about age 25 years, and the effect of age on timed FEV is similar among patients of different races, countries, and ethnic groups (87,88,90–95). Male patients who have never smoked have a greater rate of loss of timed FEV than women who have never smoked (87), and aging in men who have never smoked causes as great a loss of FEV1 as the combination of age and cigarette smoking in women. C. Nitrogen Washout

Nitrogen (N2) washout is an index of the degree of asynchronous emptying of lung units and the equality of distribution of inspiratory air; abnormalities in either reflect disease of small conducting airways and of alveoli. In a general survey conducted in three North American cities, aging had a much greater detrimental effect on N2 washout than smoking (102). D. Diffusing Capacity

Diffusing capacity (DL), corrected for alveolar volume (VA), falls with age. The fall begins early (age 30–39 years) and is more rapid in men than in women until after menopause, when the rate of decline of DL/VA in women accelerates and matches that of men (38). In this study, smoking history was not provided, so it is possible that the more rapid decline of DL/VA in men was due to the likelihood that they were heavier smokers than women. In a more recent study, the distribution of ventilation, which affects DL, became increasingly abnormal with age (103,104). In a relatively small number of postmenopausal women who had never smoked the distribution of ventilation was strikingly abnormal compared to values in premenopausal women (105). However, there is a question that constricting corsetry, used by the older women but not the younger women, adversely affected the performance of the lung function test (105).

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Unlike in patients with cardiovascular disease, in whom exercise improves function and diminishes risk (106–108), longitudinal studies demonstrate that regular physical exercise in healthy, fit, elderly people does not modify either the loss of lung function with age or the higher work of breathing that occurs during exercise in the elderly compared to younger people (109–111). Furthermore, even among those who have never smoked, a decline of timed FEV is a risk factor for mortality and poor outcomes in the elderly (87,88,112–120). F. Human Aging-Related Loss of Alveoli and Alveolar Surface Area

In humans without anatomical evidence of lung disease, morphometric studies clearly show that alveoli enlarge and both Sa and the number of alveoli decline with age (93,94). These observations are supported by an exponential analysis of static pressure–volume relationships in life (120). To illustrate the age and gender dependence of the loss of Sa, one may take previously published tabular data (94), separate the data of men from those of women, and estimate a correlation coefficient. The graph would reveal the early onset of loss of Sa in both genders and the more rapid rate of decline in men than in women. Because lung volume increases with age due to a loss of elastic recoil, the fall in Sa, in association with an increase in lung volume, reflects a loss of alveoli with age. This interpretation is supported by morphometnc data demonstrating an early onset of progressive alveolar loss with age in humans (100). G. Early Decline in the Number of Alveoli in Male Rats and Continued Formation of Alveoli in Female Rats

In animals of species in which the thorax does not enlarge during adulthood, alveolar surface area diminishes with age even though lung recoil diminishes and alveolar size increases (121,122). This means that there is a loss of alveoli. In rats, which continue to grow as adults, lung volume and the volume of individual alveoli increase with age but the number of alveoli falls (32). In male rats between age 60 and 95 days, the volume of an average alveolus increases 20%, the number of alveoli falls 11%, and Sa does not change even though the lung volume rises. By contrast, in female rats, the size of alveoli does not increase between age 60 and 95 days but there is a marked increase in the number of alveoli, which results in a statistically significant increase in alveolar surface area. Thus, there are clear gender-related differences in agebased alveolar turnover in rats that begin between age 60 and 95 days.

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VII. Sexual Alveolar Architectural Dimorphism Small mammals have a much greater 02 need per body mass than large organism (123). However, all mammals have a lung the volume of which, relative to body mass, is about the same. This indicates, as pointed out by Tenney and Remmers (123), that the lung matches its alveolar architecture to the organism’s oxygen consumption (VO2) by forming smaller, more numerous alveoli (i.e., alveoli with a higher surface-to-volume ratio), not by having a lung that is disproportionately large compared to the organism’s body mass. Thus, there is a direct linear relationship between resting VO2 and Sa, an indirect linear relationship between VO2/body mass and alveolar size, but lung volume is usually 20% of body mass. In the short-term unsteady state (e.g., exercise), the lung has sufficient Sa to sustain a 10-fold increase of VO2. However, it responds to smaller but more sustained (weeks to months) elevations of VO2, by increasing its Sa and hence its capacity for gas exchange. Pregnancy and lactation are concurrent long periods during which VO2 is elevated. For example, in rats gestation is about 22 days and on the 22nd day of gestation the VO2 in pregnant rats is twice that in same-aged virgin female rats (22). On postnatal day 14, the VO2 of lactating females is also twice that of virgin same-aged female rats. Although an elevated VO2 during pregnancy and lactation would be sustained for about 6 weeks, Sa was the same in same-aged virgin female and 22 day pregnant rats (22). Virgin female rats and same-aged rats at the 14th day of lactation (10 pups/litter) likewise had the same Sa (22). Thus, unlike the intestine, which adapts to the increased energy needs of reproduction with elevations in its mass and absorptive capacity (124), the lung does not increase its Sa to meet the metabolic cost of reproduction. However, at about the onset of sexual maturity, virgin female rats and mice have a higher body-mass-specific Sa than same-aged males of the same species (22), although body-mass-specific VO2 was the same in males and females. Furthermore, alveoli were more numerous and 30% and 50% smaller in female rats and mice, respectively, than in males of the same species (22); thus, alveoli in female rats and mice have a higher surface-to-volume ratio than alveoli in males. We suggest, based on these findings, the overall energy cost of reproduction, and the encroachment on the thoracic volume by an enlarging uterus, that smaller, more numerous, alveoli in females and their greater body-massspecific Sa were selected for in evolutionary terms. This preparation for reproduction helps females to meet the enormous energy needs of reproduction without adding the energy cost of forming additional lung. Ovariectomy at age 21 days results in larger adult rats with bigger lungs. The differences in body weight (21) and lung volume are abrogated by estrogen replacement. Furthermore, ovariectomy leads to larger alveoli and

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a slower rate of increase of the number. alveoli; estrogen prevents these effects of ovariectomy (21). An interesting finding is that estrogen administration to sham-ovariectomized rats results in a greater, more rapid increase of the number of alveoli than in ovariectomized-estrogen treated rats. We do not know the basis for the greater effect of estrogen in rats undergoing sham ovariectomy compared to those undergoing ovariectomy. It may be that in the former there are two sources of estrogen—endogenous and exogenous— and hence a higher concentration. As an alternative, and of sufficient potential therapeutic importance and biological interest to test, other ovarian hormones or ovarian-dependent hormones may augment the effect of estrogen on alveolar size and number.

VIII. Endogenous Programs of Alveolar Destruction and Regeneration The liver is the most complex regenerating organs in mammals; it regenerates complex architectural units to match systemic functional need (125). However, the evidence that liver size diminishes to match functional demand is not as convincing (125,126). The lung of mammals replaces lost units (127), probably including conducting airways (128), after pneumonectomy but it is not known if lung regeneration occurs in humans following pneumonectomy. Several findings, taken together, have suggested that lungs of mammals lose and regenerate gas-exchange units to match structure to functional need. Alveolar surface area is proportional to the organism’s VO2 across the entire range of mammalian body mass (123). Calorie restriction, which is common in nature (124,129) and even now a constant problem among humans (130), diminishes VO2 (131), thereby lessening the need for gasexchange surface area. Calorie restriction doubles the rate of proteolysis in lung (132), indicating that lung tissue is destroyed; destruction requires energy but also saves energy by avoiding the energy cost of maintaining unneeded tissue, and simultaneously provides substrate for gluconeogenesis needed to provide glucose to maintain brain metabolism. Refeeding after calorie restriction increases VO2 (131), and therefore more gas-exchange surface is required. Finally, calorie restriction of adult rodents increases the distance between alveolar walls and diminishes the increase of alveolar surface area (133–136). Because these anatomical changes are present in cases of human emphysema, the findings in calorie-restricted rodents were considered to reflect a lung abnormality designated nutritional emphysema (133–136). The return of the distance between alveolar walls (Lm) and Sa to usual values upon ad libitum consumption of food following calorie

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restriction was thought to represent catch-up lung growth in association with the increase in body mass (133,134). More recent studies using morphometric techniques that avoid the ambiguity of the Lm measurement demonstrated that within 72 h of the onset of calorie restriction by two-third there is destruction of alveoli and a fall in lung DNA; due at least in part to apoptosis of alveolar wall cells. Within 72 h of ad libitum refeeding after calorie restriction, lung DNA increases in association with increased lung cell replication (23) and alveolar regeneration occurs (23). These studies led to the concept that adult mammals have endogenous programs of alveolar destruction and regeneration. The former would conserve energy and provide substrate for gluconeogenesis, and both would match alveolar architecture to functional need (23). A recent case report of emphysema in a 34-year-old woman who had never smoked, with severe calorie restriction due to anorexia nervosa (137), and the evidence from the tragedy of the starvation in the Warsaw Ghetto (130) suggest that adult humans have calorie-related endogenous program(s) of alveolar destruction. It will be of great interest to learn if adult humans also have an endogenous program of alveolar regeneration induced by refeeding after severe calorie restriction. The implications of humans having highly conserved calorie related programs of alveolar destruction and regeneration are interesting and may be very important to our knowledge about diseases associated with loss of alveoli and to the therapy of diseases in which there are too few alveoli for adequate gas exchange. The presence of a calorie-related endogenous program of alveolar destruction raises the possibility it could be inappropriately activated, unrelated to calorie restriction. For example, the program might be inappropriately activated by the alveolar environment in some smokers, leading to the inexorable loss of gas-exchange function present in patients with COPD. In support of this, recent publications show, in human emphysema, that alveolar walls are lost in an all-or-nothing fashion (5,6). All-or-nothing loss of alveoli seems counter to the long-standing paradigm of focal destruction of alveolar septa by the quantal release of proteases by neutrophils or macrophages that act over a very limited distance (much smaller than an alveolar septum). However, the all-or-nothing loss of alveolar septa does not exclude the possibility that very focal damage due to the quantal release of a protease, so elegantly shown by Campbell et. al., (138–141), triggers a programmed all-or-nothing destruction of an entire alveolar septum. Understanding the molecular and cellular basis for these calorie-related endogenous programs of alveolar destruction and alveolar regeneration may lead to much needed therapies to slow or prevent alveolar loss and induce alveolar regeneration in patients with COPD and other diseases associated with alveolar destruction.

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126. Kawasaki S, Makuuchi M, Ishizone S, Matsunami H, Terada M, Kawarazaki H. Liver regeneration in recipients and donors after transplantation. Lancet 1992; 339:580–581. 127. Hsia CC, Wu EY, Wagner E, Weibel ER. Preventing mediastinal shift after pneumonectomy impairs regenerative alveolar tissue growth. Am J Physiol Lung Cell Mol Physiol 2001; 281:L1279–L1287. 128. Hsia CC, Zhou XS, Bellotto DJ, Hagler HK. Regenerative growth of respiratory bronchioles in dogs. Am J Physiol Lung Cell Mol Physiol 2000; 279:L136–L142. 129. Wikelski M, Thom C. Marine iguanas shrink to survive El Nino. Nature 2000; 403:37–38. 130. Winick M. Hunger disease. Studies by the Jewish Physicians in the Warsaw Ghetto. New York: Wiley, 1979. 131. Munch IC, Markussen NH, Oritsland NA. Resting oxygen consumption in rats during food restriction, starvation and refeeding. Acta Physiol Scand 1993; 148:335–340. 132. Thet LA, Delaney MD, Gregorio CA, Massaro D. Protein metabolism by rat lung: influence of fasting, glucose, and insulin. J Appl Physiol 1977; 43:463–467. 133. Sahebjami H, Wirman JA. Emphysema-like changes in lungs of starved rats. Am Rev Respir Dis 1981; 124:619–624. 134. Harkema JR, Mauderly JL, Gregory RE, Pickerell JA. A comparison of starvation and elastase models of emphysema in the rat. Am Rev Respir Dis 1984; 129:584–591. 135. Kerr JS, Riley DJ, Lanza-Jacoby S, Berg RA, Spilker WC, Yu SY, Edelman NH. Nutritional emphysema in the rat. Influence of protein depletion and impaired lung growth. Am Rev Respir Dis 1985; 131:644–650. 136. Karlinsky JB, Goldstein RH, Ojserkis B, Snider GL. Lung mechanics and connective tissue levels in starvation-induced emphysema in hamsters. Am J Physiol Regul Integr Comp Physiol 1986; 251:R282–R288. 137. Cook VJ, Corson HD, Mason AG, Bai TR. Bullae, bronchiectosis and nutritional emphysema in severe anorexia nervosa. Can Respir J 2001; 8:361–365. 138. Campbell EJ, Campbell MA, Boukedes SS, Owen CA. Quantum proteolysis by neutrophils: implications for pulmonary emphysema in alpha-1-antitrypsin deficiency. J Clin Invest 1999; 104:337–344. 139. Liou TG, Campbell EJ. Non-isotropic enzyme-inhibitor interactions: a novel non-oxidative mechanism for quantum proteolysis by human neutrophils. Biochemistry 1995; 34:16171–16177. 140. Liou TG, Campbell EJ. Quantum proteolysis resulting from release of single granules by neutrophils: a novel, non-oxidative mechanism of extracellular proteolytic activity. J Immunol 1996; 157:2624–2631. 141. Campbell EJ, Campbell MA. Pericellular proteolysis by neutrophils in the presence of proteinase inhibitors: effects of substrate oxidation. J Cell Biol 1988; 106:667–676. 142. Krogh A. Comparative Physiology of Respiratory Mechanisms. Philadelphia, PA: University of Pennsylvania, 1942.

18 Molecular Response to Pneumonectomy

LEONARD J. LANDESBERG and RONALD G. CRYSTAL Weill Medical College of Cornell University New York, New York, U.S.A.

I. Introduction Postpneumonectomy compensatory lung growth refers to a phenomenon of rapid restoration of lung parenchyma following the resection of significant amounts of lung tissue. Whether this growth response represents a recapitulation of normal so-called pre- or postnatal developmental lung growth, and whether it represents a hypertrophic or hyperplastic response with addition of new alveoli, have been debated (1,2). Furthermore, although postpneumonectomy lung growth has been well characterized morphologically and physiologically in experimental models of several species of higher animals, its application to humans, both scientifically and clinically, is less clear (3,4). Nevertheless, with recent scientific advances allowing study of physiological mechanisms at the molecular level, new data have provided valuable clues to understanding the stimuli, genetic and cellular responses, and physiological adaption to lung growth following pneumonectomy. A number of genes, including specific transcription factors, have been identified as participants in lung morphogenesis and alveolarization (5–7). This chapter will review the historical studies defining the timing, pattern, and morphological response to pneumonectomy in animal models. It will then summarize the current 455

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understanding of postpneumonectomy compensation at the molecular level, including recent studies that have attempted to use modern techniques to manipulate the system to augment or inhibit lung growth.

II. Background and Mechanisms for Inducing Postpneumonectomy Compensatory Lung Growth Postpneumonectomy compensatory lung growth has been studied for more than a century and has been described in multiple model systems (8). In several species of small mammals the lung has the ability to generate a significant growth response after surgical removal of substantial amounts of functional tissue. This has usually been accomplished via either right or left pneumonectomy, but several studies have utilized strategies involving single or multiple lobectomy (9–18). The rat has been the most common model system utilized, but studies have also been carried out in the mouse, ferret, rabbit, dog, lamb, and pig (14,15,19–33). The dog is the largest animal to be studied rigorously (34–42). In most, but not all, studies, the postpneumonectomy response results in a final lung volume and weight close to the corresponding values in both lungs of intact control animals (with or without sham surgery). The completeness of response may depend on the age and size of the animal, the ability of the mediastinum to move within the thoracic cage to accommodate changes in pulmonary dimensions, and the continuity of lung growth paralleling somatic growth over the course of the period of study for each animal (e.g., the rat continues to grow through adulthood, while larger animals, including humans, do not) (1). The impact of age and size has been evaluated in the rat, rabbit, and dog. In the rat, although the overall compensation is complete in terms of lung weight and volume, younger rats achieve the response more rapidly and may acquire new alveoli, while older animals respond with a hypertrophic response only (43–45). Cagle et al. (22) studied white rabbits at three ages corresponding to infancy, prematurity, and full maturity and demonstrated that the postpneumonectomy increase in alveolar surface area was less robust morphologically in the oldest group (22). In a comparison of young and adult beagles, although younger animals had a larger increase in lung weight, volume, and alveolar surface area, none of the groups were noted to have achieved a complete response (46). Subsequent studies by Takeda et al. (38–41) have noted a more vigorous compensation in immature dogs subjected to removal of more than 50% of their preoperative lung parenchyma. Limited data are available on postresection compensation in humans (2). Longitudinal follow-up of patients undergoing lobectomy or pneumonectomy

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suggests that the capacity for complete response is greater in children than adults and that the compensatory lung growth may be some combination of hypertrophy (i.e., hyperinflation) and hyperplasia (3,4). Taken together, the available data suggest that the capacity for complete postpneumonectomy lung growth is greatest in younger, smaller animals still in an active phase of somatic growth (Table 1). The capacity for postpneumonectomy lung growth among animal models of varying size and maturity has been well-documented, but the stimuli and physiological mechanisms responsible for initiating the response are still unclear. The acute effects of hypoxia, augmented blood flow to a single lung, and mechanical forces leading to radical changes thoracic proportion and lung inflation have been investigated. Our interpretation of the literature on the influence of each of these postulated regulatory factors suggests that mechanical stretch, hormonal effects, and a variety of molecular factors dominate (Table 1). It is known that mice raised at high altitudes have larger lungs and more alveoli than their counterparts at sea level (47). The earliest animal thoracotomy studies were performed without the assistance of small-animal ventilators (9,48) and, thus, perioperative changes in gas exchange would likely be profound and could stimulate the remaining lung to grow. However, in this type of experiment, perioperative hypoxemia is generally mild, inconsistent, and short-lived; minimizing this hypoxemia through positive-pressure ventilation produces no change in the overall compensatory lung growth (45,49). Thus hypoxia is unlikely to account for the prolonged phase of lung growth occurring over an interval of several weeks. In the rat, left pneumonectomy accounts for a loss of approximately 35% of total lung volume and vasculature. Thus, the contralateral lung would acutely accept this added proportion of the cardiac output, thereby increasing blood flow to that lung by 50% (44). Landesberg et al. (25) found a transient upregulation of several stress-response transcription factors, including early

Table 1 Factors Modulating Postpneumonectomy Compensatory Lung Growth Factor Age/growth Hypoxemia Increased blood flow (shear stress?) Mechanical forces/stretch Hormonal influence Molecular factors

Impact on postpneumonectomy models ++ + + +++ +++ +++?

Results are based on the authors’ interpretation of the available literature; scale + to +++.

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growth response-1 (Egr-1), in the remaining lung of mice 2 h after pneumonectomy. Egr-1 is a zinc-finger transcription factor induced in a wide range of cell types in response to a variety of stimuli, including fluid shear stress on endothelial cells (50). These shear forces may come into play early after pneumonectomy and provide some of the impetus for lung growth. Several experiments have directly studied the effect of pulmonary arterial blood flow on postpneumonectomy compensation. Romanova et al. (51) showed that pulmonary artery ligation did not induce significant accumulation of RNA or DNA, nor did it result in a significant elevation of the mitotic index over sham-operated controls. Tartter and Goss (52) evaluated four models of compensatory lung growth in rats and found that although ligation of the complete right pulmonary artery alone did result in some compensatory growth of the left lung, the impact was much less profound than that of pneumonectomy or a model of isolated mainstem bronchial occlusion. McBride et al. (53) demonstrated that prior ligation of the pulmonary artery in the lower lobe of the left lung of the ferret did not affect lung growth in either lobe after right pneumonectomy. Thus, while the effects of transient and abrupt changes in pulmonary arterial blood flow may induce molecular responses and result in some lung growth, they are clearly not the sole factors in postpneumonectomy compensation. The most compelling information about the dominant mechanism in postpneumonectomy lung growth is derived from studies evaluating the effects of early tissue distortion or stretch applied to the remaining lung after removal of substantial lung tissue from a relatively rigid thoracic cage. The earliest documentation of this phenomenon was by Cohn (9), who showed that, after left upper lobectomy, rats kept at lower than normal ambient pressures had an augmented compensatory growth response. This growth could be inhibited by plombage: replacement of the empty thoracic space with material to prevent any shift of the mediastinum. Brody et al. (20) demonstrated that DNA synthesis and therefore growth could be inhibited by similar plombage after pneumonectomy in mice. Cowan and Crystal (23) likewise showed that postpneumonectomy collagen synthesis was attenuated by wax plombage in the rabbit. Recent experiments in adult foxhounds have shown that plombage attenuates the postpneumonectomy volume response as well as the ability to recruit diffusing capacity during exercise (36,37,42). It has also been shown that sham thoracotomy, which is the traditional control in postpneumonectomy lung growth experiments, leads to at least transient lung collapse and does induce some compensatory response in the contralateral lung (54). The tissue distortion or stress may also be applied externally via mechanical ventilators able to cause hyperinflation with varying amounts of positive pressure. The lungs of open-chest rabbits ventilated with high

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positive end-expiratory pressure (PEEP) for 4 h demonstrated an upregulation of the mRNA of multiple genes involved in pulmonary growth and development, including a1(III) and a2(IV) procollagens, fibronectin, basic fibroblast growth factor (FGF), and transforming growth factor (TGF)-h1 (6,7,55). In rats, as early as 30 min after pneumonectomy and after isolated lung inflation in vitro, two early response genes, the transcription factors c-fos, and junB, were likewise upregulated at the mRNA level (56). It therefore appears that mechanical forces leading to early distention of the lung must play an important role in initiating or potentiating compensatory lung growth after pneumonectomy. The possible contributing roles of humoral and lung transcription factors will be addressed below in the section on molecular response to pneumonectomy.

III. Biochemical and Morphological Analysis Since it has been well documented that postpneumonectomy compensatory growth occurs, the logical question to be pursued is whether the response is hypertrophic, implying an enlargement of existing structures, hyperplastic, with generation of new airways and gas exchange units to account for the added volume, or simply hyperinflationary by enlarging existing structures without added tissue mass. In reviewing the available data, it is clear that the postpneumonectomy response is not simply hyperinflationary. Whether the response is hypertrophic or hyperplastic is of critical importance since it addresses the ultimate question of whether new alveoli can be generated by stresses applied at a time after the initial period of alveolarization during development has been completed. The answer to this question is not clear, with many studies supporting either side (2,22). As discussed in Section IV, most of the current studies have left this question aside and have focused on mechanisms occurring at the genetic level. However, it has been established that postpneumonectomy lung growth occurs as a result of a cellular response in which tissue mass is added, and that this response can be quantified by available biochemical, morphological, and physiological techniques. The earliest documentation of a biochemical confirmation of the cellular response to pneumonectomy was by Addis in 1928 (48), who performed left pneumonectomy in rats and noted a 45% increase in the nitrogen content of the remaining lung after sacrifice at 60 days. Subsequent studies confirmed that the added tissue mass was cellular by demonstrating significant increases in total lung DNA, RNA, and total protein that paralleled lung growth throughout the study interval. These data have been documented in several species including mouse, rat, rabbit, and pig (15,20,24,57,58). Cowan and Crystal (23) provided evidence that collagen

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stuctural protein is also added to accompany the accrual of nucleic acids. In the context that the rat has been the most commonly utilized model system, the remaining discussion will focus on the rodent model (although one cannot necessarily make cross-species comparisons). DNA synthesis has been measured in various studies according to several quantitative techniques including counting of mitoses on histological specimens; autoradiography after incorporation of [3H]thymidine; and most recently, by immunostaining after incorporation of 5V-bromo-2V-deoxyuridine (BrdU) (14,20,58). In general, these studies have shown that DNA synthesis (mitotic index) begins to rise significantly by the third day after pneumonectomy, peaks by the end of the first week, and then returns to baseline control levels thereafter. Accumulation of total lung DNA, RNA, protein, collagen, and glycosaminoglycans continues throughout the lung growth period until completion by approximately 2 weeks (16,57,59,60; Fig. 1). Taken together, these data clearly indicate that postpneumonectomy compensatory lung growth at least occurs by hypertrophy with a cellular response characterized by increases in DNA, RNA, and protein synthesis. As mentioned above, the issue of whether the cellular compensatory response results in the formation of new or simply enlarged alveoli with larger surface area has been controversial. Most studies seeking to resolve this question have attempted to utilize modern morphometric techniques to derive conclusions about alveolar size, geometry, and number. It is beyond the scope of this chapter to review or critique the methods of all of these studies, but data from a few are worth highlighting. Thet and Law (61) performed morphometric analysis on the remaining lungs of adult rats 1 week following left pneumonectomy, which is the maximal period of lung growth in this

Figure 1 Postpneumonectomy compensatory lung growth may be classified into three phases, presented diagrammatically in rows according to morphological, biochemical, and molecular biological response criteria. This schema is based on the chronology of the rat rodent model. In the early phase (day 0–3), mechanical and other stimulatory effects predominate, leading to stretch and alveolar/ductal distention. This phase is followed by a cell proliferation phase (day 3–8) in which the early growth stimuli promote nucleic acid and protein synthesis, including collagen and glycosaminoglycans, and morphologically result in hyperplasia and septal thickening. Finally, in the late phase, cellular redistribution and tissue remodeling occur, leading to morphologically normal lung tissue. In the molecular response row, the time course of representative gene expression is presented as best estimate curves based on published time points along the period of growth measured in each study. For the purposes of this schematic representation, both mouse and rat data are presented together. (Adapted from Refs. 1, 2.)

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rodent model. Their data demonstrated that, relative to sham-operated controls, the number of type II and capillary endothelial cells increased in concert and in proportion to lung growth by weight and DNA. The number of type I or interstitial cells did not change, but the surface area of type I and II cells, as well as the total airspace volume, increased significantly (61). These data complement earlier work in mice showing a progression of cellular labeling by [3H]thymidine incorporation from pleura on day 1, to interstitial cells, endothelium, and peripheral alveoli by day 3, and then to the remainder of parenchymal alveoli by day 6. Type II alveolar cells made up the bulk of labeled alveolar cells and the data suggested that some type II cells differentiated to type I cells by the end of the growth period (20). Taken together, these studies imply a coordinated cellular response, in which an initial proliferation of interstitial, endothelial, and type II cells precedes a redistribution of cell populations in order to restore a more normal parenchymal architecture by the end of the growth response period. The most detailed set of morphological observations in the rodent model has been made by Burri and colleagues (11,12) in a model of right bilobectomy using both light and scanning electron microscopic techniques. Their data also suggest a coordinated progression of morphological, cellular changes leading to a complete response with ultimate normalization of all parenchymal and nonparenchymal structures. In this proposed schema, following resection of lung tissue, the remaining lobes first undergo an early ductal and airspace dilatation, followed by cellular proliferation manifested by thickened septa at the end of the first week. The complete response is accomplished by rearrangement of tissue leading to normal morphology (10– 12,18). This data parallels the biochemical response and cellular responses detailed above (Fig. 1). The work of Hsia and colleagues (34–41) deserves mention since it includes the most comprehensive morphological and physiological observations in a large-animal model by utilizing a variety of complementary techniques including standard morphometry, computed tomography (CT), and exercise testing. In both immature and adult male foxhounds following right pneumonectomy, they demonstrate that, although the volume response is incomplete compared with controls, the remaining lung does undergo a vigorous hyperplastic growth. This leads ultimately to normal restored morphology, total diffusing capacity and surface area, septal tissue volume and thickness, pulmonary blood flow, and cardiopulmonary exercise parameters. As described in the rodent model, and suggested in the available data in humans, immature foxhounds at 2 months of age present a more vigorous response than their adult (2–4 years) counterparts (34–42). They also suggest a coordinated cellular response of initial hyperplasia and septal thickening followed by a period leading to normal morphology (35). Although these data

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are the most complete and closest comparisons available to the human pneumonectomy model, their applicability to humans is not clear since the canine mediastinum is capable of complete contralateral displacement by the growing lung (39). In summary, the available biochemical and morphological data confirm a coordinated cellular proliferative compensatory response to significant lung resection. Although the issue of alveolar multiplication is still controversial, it is clear that, in the animal models described above, DNA, RNA, and protein synthesis parallel the period of maximal growth, and eventually result in morphologically normal lung tissue. IV. Molecular Responses to Pneumonectomy For the purposes of this review, it is convenient to classify postpneumonectomy compensatory lung growth into three phases corresponding to the identified morphological and bi-chemical phenomena described above (Fig. 1) (1). In the early phase (day 0–3), mechanical and other stimulatory effects predominate, leading to stretch and alveolar/ductal distention. This phase is followed by a cell proliferative phase (day 3–8) in which the prior early growth stimuli promote nucleic acid and protein synthesis and result morphologically in hyperplasia and septal thickening. Finally, in the late phase, cellular redistribution and tissue remodeling occur leading to morphologically normal lung tissue, and, in general, result in a complete response. A number of studies have aimed to identify specific molecular responses occurring in the lung at either the messenger RNA or protein levels, including potential serum-derived growth factors. To place these results into the context of the complete growth model described above, data pertaining to the timing of each growth phase will be grouped together (Table 2). A. Early Phase

The early phase of postpneumonectomy compensatory lung growth is characterized physiologically by a rapid increase in the volume of blood flow through the pulmonary arterial circulation of the remaining lung and a sudden change in available thoracic volume leading to mechanical stretch forces. At the end of this phase, it is characterized histologically by ductal and alveolar dilatation with thin septa representing an early volume response (18) (Fig. 1). Immediate early genes are nuclear transcription factors that have had documented roles in differentiation and development, and have been associated with the early growth period in the liver compensatory growth model (50,62). As mentioned above, Gilbert and Rannels (56) describe a rapid and

Quantify protein expression in lung of enzymes involved in glycogenolysis and metabolism

Quantify lung L-ornithine decarboxylase activity Quantify lung glycosaminoglycan content

Mouse, left upper lobectomy

Rat, right upper trilobectomy

Quantify total lung hydroxyproline as surrogate for collagen Quantify [14C]proline as a measure of collagen synthesis Measure 3thymidine uptake in type II cells in vitro

Measured molecular variables

Rat, left pneumonectomy

Rabbit, left pneumonectomy, serum drawn day 9 and 21

Rabbit, left pneumonectomy

Experimental model

Table 2 Molecular Response to Pneumonectomy (listed chronologically)

Total lung collagen increased significantly Collagen synthesis peaks day 7–14, baseline day 2 8 Wax plombage attenuates response DNA synthesis occurs in type II cells but not in lung/skin fibroblasts Positive dose–response relationship Serum from sham controls does not have same effect Increased levels of adenylate cyclase, phosphodiesterase, and protein kinase and elevated total lung cAMP preceding time period of maximum lung growth Early significant peak in expression by 4 h in lobe with maximal growth response Total glycosaminoglycan increased throughout study (14 days) Hyaluronate, chondroitin sulfate, dermatan sulfate increased over study Levels/mg dry lung peak at 4 days and return to baseline by 14 days

Findings (relative to controls)

16

17

68

89

23

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Quantify activity of lung thymidine kinase and DNA polymerase Quantify lung calmodulin content and activity

Quantify TNF-a

Rat, left pneumonectomy

Rat, left pneumonectomy

Rat, left pneumonectomy-BAL, serum, and in vitro lung perfusate

Rat, left pneumonectomy

Quantify stimulation of fibroblasts in vitro Measure BAL IGF-1

Rat, left pneumonectomy, BAL fluid

Measure binding of TNF-a to lung immunoblots Quantify tropoelastin and type I procollagen expression

Quantify lung cAMP content and protein kinase activity

Rat, left pneumonectomy

Lung homogenates bind increasing amounts TNF-a through day 14 Lung collagen and elastin increased significantly

Peak in both variables at day 4, to baseline day 7 Results similar in perfused lungs in vitro with applied CPAP BAL from day 2 and 6 animals caused significant fibroblast replication Response attenuated by IGF-1 antibody BAL IGF-1 elevated in day 2 and 6 DNA polymerase and thymidine kinase increased day 1 and 7 DNA polymerase alpha most active isomer Calmodulin content and activity increased early (day 1,2) Treatment with trifluoperazine decreased lung calmodulin activity and inhibited postpneumonectomy increase in lung mass and DNA accumulation TNF-a elevated significantly and peaked in serum and BAL day 7

59

64

66

69

90

67

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Pig, left upper lobectomy

Quantify lung IGF-1 expression

Neonatal lamb, left pneumonectomy Mouse, left pneumonectomy Identify upregulated transcription factors by array analysis Quantify degree upregulation relative to controls Quantify epidermal growth factor receptor expression by Western blot

Identify expression zinc-finger transcription factors

Quantify desmosine (elastin) and hydroxyproline (collagen) Quantify immediate early gene expression

Localize expression by in situ hybridization

Measured molecular variables

Rat, left pneumonectomy

Rat, left pneumonectomy

Experimental model

Table 2 Continued

Northern analysis shows elevation (day 3) and peak (day 7) tropoelastin and type I procollagen expression over 14-day time course In situ shows expression in alveolar walls Northern analysis shows peak c-fos, jun B expression within 30 min Similar results produced with in vitro perfusion and inflation under constant pressure Demonstrated differential expression with downregulation of several genes Small elevation of IGF-1 mRNA expression at 21 days Identified elevated expression of Egr1, Nurr-77, tristetraproline, LRG21, GKLF, InB-a mRNA 2 h after pneumonectomy Twofold elevation at 2 weeks, declines to baseline by 3 mo

Findings (relative to controls)

13

25

29

65

56

Reference

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transient upregulation of the mRNA of two immediate early genes, c-fos and junB, within 30 min after pneumonectomy in rats. Time course analysis was limited to 6 h. Within this time period, c-fos mRNA was upregulated sevenfold over unoperated controls and rapidly returned to baseline by 2 h. junB mRNA was upregulated almost fourfold, declined by 2 h, but remained elevated over baseline through the 6-h time period. The sham-operated control animals demonstrated only minimal changes in the expression pattern. Similar significant upregulation of c-fos and junB was maintained in lungs perfused in vitro and ventilated with applied positive airway pressures (56). Landesberg et al. (25) utilized a cDNA expression array to screen for upregulated transcription factors in the remaining lungs of mice 2 h after pneumonectomy (with the animals recovered from anesthesia and back to normal activity) compared to unoperated controls. Using array technology to make comparisons of the expression of multiple genes within a single experiment, this study was successful in identifying six known transcription factors upregulated at the mRNA level compared with controls: Egr-1, nurr77, LRG-21, tristetraproline (TTP), GKLF, and InB-a. Northern blot analysis of a 24-h time course confirmed the transient nature of the upregulation and demonstrated a rapid return toward baseline by 6 h. Sham surgery also caused some upregulation of these genes; in one case, InB-a, the sham surgery was identical to the pneumonectomy group. This result is not unexpected, since lung collapse, as would occur transiently after thoracotomy, is known to mimic some of the stimuli leading to compensatory growth of the contralateral lung (52,54). Two of the identified genes in the Landesberg et al. study (25) warrant additional discussion. Egr-1 is a zinc-finger transcription factor induced in a wide range of cell types in response to a variety of stimuli including fluid shear stress on endothelial cells (50). These shear forces may come into play early after pneumonectomy as the pulmonary arterial blood flow is rapidly shifted to the remaining lung.TTP is another zinc-finger transcription factor that functions as a feedback antagonist to tumor necrosis factor (TNF)-a by destabilizing its mRNA (63). TNF-a is an alveolar macrophage-derived cytokine with known effects on cell proliferation and cellular metabolism. Dubaybo et al. (64) measured serum and bronchoalveolar lavage (BAL) fluid levels over the time course of rat postpneumonectomy lung growth. They found significant increases in TNF-a protein in both fluids that peaked by the end of the first week and declined thereafter. Quantitative immunoblots made from homogenates of the remaining lungs over the 2-week study range demonstrated increasing TNF-a binding compared with sham controls. This suggested a possible requirement for this humorally mediated cytokine during compensatory growth as well as as a possible autoregulatory link between TTP and TNF-a expression.

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Despite that fact that Egr-1 and TTP represent zinc-finger transcription factors upregulated at the mRNA level during early postpneumonectomy lung growth, several other zinc-finger transcription factors were found to be specifically downregulated 1 day after pneumonectomy (65). In another study of the early postpneumonectomy period, Tata´r-Kiss et al. (17) demonstrated upregulation of expression of L-ornithine decarboxylase (ODC) protein in the remaining lungs of mice after left upper lobectomy. An interesting finding was that ODC peaked at 4 h and declined thereafter. It also was maximal in the lobe that grew the most. ODC has been shown to have increased activity in the regenerating liver and compensatory growing kidney (17). Ofulue et al. (66) studied calmodulin (CaM) protein expression and activity over a 7-day period in the rat model. CaM is known to modulate the activities of adenylate cyclase and cAMP-phosphodiesterase in the lung (for a further discussion of these enzymes in the context of lung growth, see below). CaM was shown to be maximally elevated during days 1 and 2 after pneumonectomy. This elevation predates the subsequent rise of lung cAMP levels that peaks by day 3 postpneumonectomy. An in vitro study of isolated perfused lungs with applied continuous positive airway pressure (CPAP) demonstrated a similar upregulation of cAMP, implying an inductive role for mechanical forces (67). Inhibition of CaM by trifluoperazine-attenuated lung CaM activity and decreased lung growth in terms of tissue mass and total DNA, suggesting that CaM plays a vital role as an autocrine factor regulating cAMP and subsequent early protein metabolism in postpneumonectomy compensation (66). Additional supporting evidence for the role of cAMP metabolism at this stage in postpneumonectomy lung growth is provided by Nijjar and Thurlbeck (68), who measured the relative lung content of adenlyate cyclase, cAMP-phosphodiesterase, cAMP-dependent protein kinase, phosphorylase, and glucose-6-phosphatase (G-6-PD) activity over the 14-day time course in the rat model. As expected, the level of adenylate cyclase increased by day 1, peaked by day 3, and ultimately declined to control levels by the end of the study period. The ratio of adenylate cyclase to phosphodiesterase activity also rose and fell in parallel with the lung levels of cAMP. cAMP-dependent protein kinase activity likewise rose to a peak by days 3–5 and returned to baseline levels thereafter. Phosphorylase and G-6-PD activities were also elevated in sham controls, but the activity of G-6-PD was significantly higher in the pneumonectomy group at the critical early time points during the first week. In summary, the available data for the early phase of postpneumonectomy compensatory lung growth document a rapid but transient upregulation of several transcription factors involved in cell proliferation and differenti-

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ation as well as a coordinated upregulation of enzymes responsible for cAMP accumulation and metabolism prior to the beginning of DNA synthesis. B. Cell Proliferative Phase

The cell proliferative phase is characterized physiologically by cellular hyperplasia and DNA synthesis. In morphological terms, septal thickening accompanies cell proliferation and protein synthesis (Fig. 1). Although DNA synthesis has been documented to occur during this phase of compensatory lung growth, Kuboi et al. (69) reported additional evidence of upregulation of DNA polymerase a and thymidine kinase activity in rats that peaked at 7 days. Koh et al. (59) documented upregulation of expression of both tropoelastin and type I procollagen mRNA beginning at day 3, peaking at the end of the first week, and remaining elevated until completion. In situ hybridization data localized this expression specifically to the alveolar walls. These studies confirm early data and provide further strengthen the correlation between molecular events and the morphological appearance during this phase (23). Two recent studies provide strong evidence of the molecular signaling that affects both epithelial and endothelial cell differentiation and growth during this period. Hepatocyte growth factor (HGF) is a transcription factor with potent mitogenic and morphogenic activities on respiratory epithelia. In the rat lung, it is produced by alveolar macrophages and vascular endothelial cells and functions via the c-Met/HGF receptor. During lung development, HGF is an important mediator in epithelial–mesenchymal interactions leading to morphogenesis. Sakamaki et al. (30) studied the expression of HGF and its receptor following left pneumonectomy in mice. HGF protein expression was elevated in both the plasma and lung tissue by day 1 and 5, respectively, and remained elevated over sham controls throughout the 10 day study period. In lung tissue, HGF mRNA was likewise upregulated by day 3, peaked at day 5, and declined thereafter. The expression of the c-Met/HGF receptor was significantly upregulated by day 3 and rapidly returned to baseline by day 5. Administration of intraperitoneal recombinant HGF led to a significant augmentation of DNA synthesis as measured by BrdU immunostaining as well as a greater-than-expected lung weight by days 3 and 5. Administration of intraperitoneal anti-HGF antibody, however, attentuated both DNA synthesis and the expected lung weight at day 3 and 5 compared to standard pneumonectomy. Nitric oxide (NO) has been shown to be a potent stimulus for endothelial cell proliferation and migration. It is produced by three nitric oxide synthase enzymes. Endothelial nitric oxide synthetase (eNOS) is constitutively expressed on vascular endothelial cells. NO mediates its action on endothelial cells via the

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mitogenic effects of vascular endothelial growth factor (VEGF). VEGF is a secreted protein that is one of the most important mediators of angiogenesis (70,71). In this regard, NO should be a critical mediator in the expansion of endothelial cells occurring during postpneumonectomy lung growth. To test this hypothesis, Leuwerke et al. (27) demonstrated an upregulation of eNOS protein in the remaining lungs of wild-type C57Bl/6 mice after left pneumonectomy. Quantitative analysis by Western analysis demonstrated a significant elevation of eNOS by day 3, a peak at day 7, with decreasing levels thereafter. With eNOS / mice, a severe inhibition of the expected weight and volume response after pneumonectomy was observed. DNA synthesis was depressed to normal sham control levels. Treatment of wildtype animals with NG-nitro-Larginine methyl ester, a nitric oxide synthase inhibitor, produced the same effect as was observed in the eNOS-deficient group. Given the basic premise that a coordinated postpneumonectomy response must account for parallel expansions of both epithelial and endothelial components in order to ensure proper ventilation–perfusion matching and gas exchange, the studies discussed above provide elegant examples demonstrating known genetic pathways involved in both epithelial cell proliferation and angiogenesis during the cell proliferation period of compensatory lung growth. C. Late Phase

If, as suggested by the available data, postpneumonectomy lung growth is well coordinated at the genetic and molecular levels, there must be significant and specific signaling that governs the halt of cell proliferation and promotes completion of the response as the histologically normal lung fills the remaining available volume (Fig. 1). However, there is a paucity of data available describing the molecular events occurring during the late phase of postpneumonectomy lung growth. As described above, nucleic acid, collagen, and noncollagen protein accumulation continue throughout this period, but each have passed their peaks during the first week (16,57,59,60). V. Models to Augment or Inhibit the Growth Response As the model of coordinated compensatory growth is being defined, the next logical step is to utilize current molecular techniques to attempt to augment or inhibit the response. Data derived from such experiments are useful in helping to explore specific mechanisms and pathways, and help to shed light on the coordinated pattern of molecular signaling responsible for post-pneumonectomy compensation. The clinical implications of being able to enhance or reinitiate postnatal lung growth would be crucial to the care of patients with endstage lung disease.

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A number of earlier studies have documented critical parameters, including the effects of mechanical stretch, hypoxia/hyperoxia, sodium balance, and the adrenal hormone pathway, which lead to modification of the growth response in rodent models (9,72–76). Five recent studies have utilized powerful molecular techniques to document critical pathways involved in postpneumonectomy lung growth. Two of these, involving the use of recombinant proteins, specific inhibitory antibodies, and knockout animals, were discussed above in the section on molecular response (27,30) (Table 3). Epidermal growth factor (EGF) is a transcription factor with known roles in fetal lung morphogenesis. It acts via the EGF receptor (EGF-R), which is known to be present on alveolar cells (6,7). Kaza et al. (77) investigated the effect of exogenously administered EGF on the rat model of postpneumonectomy lung growth. This group has also reported an upregulation of EGF-R at the protein level in the remaining left lower lobe of pigs 2 weeks following left upper lobectomy. Following intraperitoneal injection of recombinant EGF, the remaining lungs of rats following left pneumonectomy demonstrated both an accelerated and augmented growth response. Compared with results after standard pneumonectomy, the lungs of the EGF-treated group were heavier and larger over time and ended with significantly greater lung mass and volume at the conclusion of the 3-week study period. Western analysis demonstrated elevated EGF-R expression in the EGF-treated animals, suggesting that the augmented and accelerated growth effect may be mediated via an autoregulatory process leading to upregulation of this receptor. Retinoic acid (RA) has also been recognized to be critical for lung growth and differentiation and for maintaining the integrity of the respiratory epithelium (78). In a similar study to that with EGF, Kaza et al. (14) investigated the role of RA in the rat model of postpneumonectomy lung growth. Intraperitoneal administration of RA led to a significantly accelerated and augmented compensatory response over the 21-day period of study. Even at the 3-week timepoint, the cell proliferation index (measure by BrdU immunostaining) was significantly elevated in the RA-treated group. Western analysis demonstrated a significant upregulation of EGF-R in the RA-treated group. Consistent with this data, prior work has described an interaction between RA and EGF-R in lung development (79). Platelet-derived growth factor (PDGF)-BB, functioning through the PDGF receptor (PDGF-R), is known to play a role in lung development and repair (80–82). In vitro studies suggest that PDGF-BB mediates lung cell proliferation in response to mechanical strain (83). Yuan et al. (84) studied the effect in Wistar rat pups following left pneumonectomy. Although the level of expression of PDGF-BB mRNA in the postpneumonectomy lungs remained constant, inhibition of PDGF using a truncated, soluble PDGF-R/Fc chimera protein led to an attenuation of DNA synthesis and total lung DNA

Low atmospheric pressure augments growth; decreasing intrathoracic volume attenuates rate and extent of response Growth hormone excess and pneumonectomy are additive for lung growth in terms of cell size and alveolar volume, but no additional alveoli. Hypophysectomy inhibits response Adrenalectomy leads to accelerated hyperplastic response with overcompensation vs. pneumonectomy alone. Overcompensation inhibited by corticosteroids. Early steroid (0–7 days) blocked only early acceleration; late steroid (7–14 days) blocked late acceleration. Sodium deficiency attenuates both somatic and postpneumonectomy compensation Hypoxia augments response; hyperoxia attenuates response

Rat, left upper lobectomy/ hypobaric conditions vs. wax plombage

Rat, left pneumonectomy/hypoxia or hyperoxia

Rat, left pneumonectomy/sodium deficiency

Rat, left pneumonectomy/ adrenalectomy

Rat, left pneumonectomy/growth hormone excess via MtTF4 tumor implantation or deficit via hypophysectomy

Effect on compensatory lung growth

Experimental model/intervention

Postpneumonectomy hypoxia has not been demonstrated to be consistent or significant in experimental animals (45)

Endocrine factor. Has temporal effect on growth curve.

9

Makes an early case for the importance of stretch and mechanical forces on postresection lung growth Endocrine factors play a role in compensation

76

74

72,75

73

Reference

Comment

Table 3 Models to Augment or Inhibit Postpneumonectomy Compensatory Lung Growth (listed chronologically)

472 Landesberg and Crystal

Mouse, left pneumonectomy/ intraperitoneal HGF and antiHGF antibody

Mouse, left pneumonectomy/ eNOS / deficient mice

Rat, left pneumonectomy/ intraperitoneal retinoic acid (RA)

Rat, left pneumonectomy/ Intraperitoneal epidermal growth factor (EGF)

Expression of c-met/HGF receptor by immunohistochemistry increased day 3, preceding time period of maximum cellular proliferation

Exogenous EGF leads to accelerated and augmented compensatory growth response. Induces its own receptor expression in lung Exogenous RA leads to accelerated and augmented compensatory growth response by weight, volume, cell proliferation index by BRDU immunostaining. Induces EGF receptor expression by Western blot eNOS protein expression is temporally upregulated (peak day 7) in wildtype (WT) pneumonectomy. eNOS / mice had severely inhibited postpneumonectomy response by weight and volume. BRDU immunostaining remained at control values. Similar results obtained by treating WT group with eNOS inhibitor Lung tissue and plasma HGF elevated after pneumonectomy 27

30

Transcription factor involved in lung morphogenesis (7)

13

77

Gene involved in angiogenesis is also vital for postpneumonectomy lung growth Acts on endothelial component of lung (7,70)

Transcription factor important in lung morphogenesis (7,91) acting on epithelial component of lung Transcription factor important in lung morphogenesis (7) also may act through EGF pathway

Molecular Response to Pneumonectomy 473

Effect on compensatory lung growth Anti-HGF blocks cell proliferation and lung growth, while exogenous HGF augments the early growth response Total lung PDGF-BB mRNA and protein increased proportional to compensatory lung growth Truncated soluble receptor attenuated DNA synthesis and total lung DNA content on day 3 postpneumonectomy

Experimental model/intervention

Rat, left pneumonectomy/ Intraperitoneal soluble truncated PDGF-h receptor

Table 3 Continued

Compensatory lung growth permissive for PDGF

Comment

84

Reference

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content (84). These findings imply that PDGF increases in concert with lung growth and is likely to be what is termed a permissive factor: it is not upregulated in response to growth stimuli yet is clearly required for compensatory growth to occur. It is possible that other factors act in this permissive fashion and would not be detected as upregulated by the experimental techniques described above. To support this concept of permissive, but not necessarily upregulated, genes in compensatory lung growth, Landesberg et al. (85) studied the mRNA expression of vascular endothelial growth factor (VEGF) and its receptor flk1/KDR in adult Sprague-Dawley rats following left pneumonectomy. Angiogenesis is a complex biological process by which endothelial cells are stimulated to proliferate, migrate, and form new blood vessels (86). Any compensatory lung growth must include expansion of the vascular endothelial surface area to allow for proper ventilation–perfusion matching and gas exchange. VEGF and its receptor family are likely to be important in pulmonary angiogenesis (87,88). Landesberg et al. (85) found that despite compensatory lung growth over the expected time course, VEGF and its flk-1/ KDR receptor are expressed, but not up- or downregulated, at the mRNA level. Therefore, it is possible that the VEGF system also performs in a permissive role. Future studies should test this hypothesis. VI. Future Directions The study of postpneumonectomy lung growth has gone through stages as experimental technology and investigative techniques have developed to allow for the testing of hypotheses. First, the morphological and biochemical responses were documented to show that compensatory lung growth is indeed a hyperplastic response. Second, molecular techniques were developed so that the mechanisms responsible for the observed morphological changes could be elucidated. Currently, those same techniques are being combined with the newest technologies including microarray analysis and the use of recombinant protein and monoclonal antibody strategies to attempt to augment or inhibit the response. Future studies will likely continue to utilize these techniques as more pathways involved in postpneumonectomy compensation are studied. VII. Conclusion In animal models, postpneumonectomy compensatory lung growth has been extensively studied. However, many of the molecular mechanisms responsible for initiating and ultimately concluding the response remain unknown. It represents a highly coordinated, hyperplastic cellular response leading to the

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restoration of a normal histological appearance and gas exchange. Based on the available data, a preliminary sketch can begin to be made that combines the known morphological, biochemical, and molecular events of the overall compensatory response (Fig. 1). The molecular mechanisms are becoming elucidated through the use of modern techniques including application of knockout models and recombinant protein and antibody strategies. Future studies are likely to begin to complete the picture. Our understanding of this model may eventually have an impact on our ability to treat human suffering from lung diseases in which regrowth of normal lung would have an impact on patients survival and symptoms. References 1. 2. 3.

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14. Kaza AK, Kron IL, Kern JA, Long SM, Fiser SM, Nguyen RP, Tribble CG, Laubach VE. Retinoic acid enhances lung growth after pneumonectomy. Ann Thorac Surg 2001; 71:1645–1650. 15. Kaza AK, Cope JT, Fiser SM, Long SM, Kern JA, Tribble CG, Kron IL, Laubach VE. Contrasting natures of lung growth after transplantation and lobectomy. J Thorac Cardiovasc Surg 2002; 123:288–294. 16. Mueller MP, Thet LA. Changes in lung glycosaminoglycans during postresectional lung growth. J Appl Physiol 1987; 63:1033–1039. 17. Tatar-Kiss S, Bardocz S, Kertai P. Changes in L-ornithine decarboxylase activity in regenerating lung lobes. FEBS Lett 1984; 175:131–134. 18. Wandel G, Berger LC, Burri PH. Morphometric analysis of adult rat lung after bilobectomy. Am Rev Respir Dis 1983; 128:968–972. 19. Boatman ES. A morphometric and morphological study of the lungs of rabbits after unilateral pneumonectomy. Thorax 1977; 32:406–417. 20. Brody JS, Burki R, Kaplan N. Deoxyribonucleic acid synthesis in lung cells during compensatory lung growth after pneumonectomy. Am Rev Respir Dis 1978; 117:307–316. 21. Cagle PT, Thurlbeck WM. Postpneumonectomy compensatory lung growth. Am Rev Respir Dis 1988; 138:1314–1326. 22. Cagle PT, Langston C, Thurlbeck WM. The effect of age on postpneumonectomy growth in rabbits. Pediatr Pulmonol 1988; 5:92–95. 23. Cowan MJ, Crystal RG. Lung growth after unilateral pneumonectomy: quantitation of collagen synthesis and content. Am Rev Respir Dis 1975; 111:267–277. 24. Das RM, Thurlbeck WM. The events in the contralateral lung following pneumonectomy in the rabbit. Lung 1979; 156:165–172. 25. Landesberg LJ, Ramalingam R, Lee K, Rosengart TK, Crystal RG. Upregulation of transcription factors in lung in the early phase of postpneumonectomy lung growth. Am J Physiol Lung Cell Mol Physiol 2001; 281:L1138– L1149. 26. Langston C, Sachdeva P, Cowan MJ, Haines J, Crystal RG, Thurlbeck WM. Alveolar multiplication in the contralateral lung after unilateral pneumonectomy in the rabbit. Am Rev Respir Dis 1977; 115:7–13. 27. Leuwerke SM, Kaza AK, Tribble CG, Kron IL, Laubach VE. Inhibition of compensatory lung growth in endothelial nitric oxide synthase-deficient mice. Am J Physiol Lung Cell Mol Physiol 2002; 282:L1272–L1278. 28. McBride JT. Postpneumonectomy airway growth in the ferret. J Appl Physiol 1985; 58:1010–1014. 29. Nobuhara KK, Di Fiore JW, Ibla JC, Siddiqui AM, Ferretti ML, Fauza DO, Schnitzer JJ, Wilson JM. Insulin-like growth factor-I gene expression in three models of accelerated lung growth. J Pediatr Surg 1998; 33:1057–1060. 30. Sakamaki Y, Matsumoto K, Mizuno S, Miyoshi S, Matsuda H, Nakamura T. Hepatocyte growth factor stimulates proliferation of respiratory epithelial cells during postpneumonectomy compensatory lung growth in mice. Am J Respir Cell Mol Biol 2002; 26:525–533. 31. Sery Z, Keprt E, Obrucnik M. Morphometric analysis of late adaptation of the

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49. Karl HW, Wolpert EB, Rannels DE. Minimizing perioperative hypoxemia does not affect postpneumonectomy lung growth. Am J Physiol 1988; 255:E65–E69. 50. Gashler A, Sukhatme VP. Early growth response protein 1 (Egr-1): prototype of a zinc-finger family of transcription factors. Prog Nucleic Acid Res Mol Biol 1995; 50:191–224. 51. Romanova LK, Leikina EM, Antipova KK, Sokolova TN. The role of function in the restoration of damaged viscera. Sov J Dev Biol 1970; 1–2:384–390. 52. Tartterr PI, Goss RJ. Compensatory pulmonary hypertrophy after incapacitation of one lung in the rat. J Thorac Cardiovasc Surg 1973; 66:147–152. 53. McBride JT, Kirchner KK, Russ G, Finkelstein J. Role of pulmonary blood flow in postpneumonectomy lung growth. J Appl Physiol 1992; 73:2448–2451. 54. Inselman LS, Mellins RB, Brasel JA. Effect of lung collapse on compensatory lung growth. J Appl Physiol 1977; 43:27–31. 55. Berg JT, Fu Z, Breen EC, Tran HC, Mathieu-Costello O, West JB. High lung inflation increases mRNA levels of ECM components and growth factors in lung parenchyma. J Appl Physiol 1997; 83:120–128. 56. Gilbert KA, Rannels DE. Increased lung inflation induces gene expression after pneumonectomy. Am J Physiol 1998; 275:L21–L29. 57. Rannels DE, White DM, Watkins CA. Rapidity of compensatory lung growth following pneumonectomy in adult rats. J Appl Physiol 1979; 46:326–333. 58. Romanova LK, Leikina EM, Antipova KK. [Nucleic acid synthesis and mitotic activity during the development of compensatory pulmonary hypertrophy in rats] [Ob osobennostiakh sinteza nukleinovykh kislot i mitoticheskoi aktivnosti v protsesse razvitiia kompensatornoi gipertrofii legkogo krys.]. Biull Eksp Biol Med 1967; 63:96–100. 59. Koh DW, Roby JD, Starcher B, Senior RM, Pierce RA. Postpneumonectomy lung growth: a model of reinitiation of tropoelastin and type I collagen production in a normal pattern in adult rat lung. Am J Respir Cell Mol Biol 1996; 15: 611–623. 60. Rannels DE, Burkhart LR, Watkins CA. Effect of age on the accumulation of lung protein following unilateral pneumonectomy in rats. Growth 1984; 48:297– 308. 61. Thet LA, Law DJ. Changes in cell number and lung morphology during early postpneumonectomy lung growth. J Appl Physiol 1984; 56:975–978. 62. Columbano A, Shinozuka H. Liver regeneration versus direct hyperplasia. FASEB J 1996; 10:1118–1128. 63. Lai WS, Carballo E, Strum JR, Kennington EA, Phillips RS, Blackshear PJ. Evidence that tristetraprolin binds to AU-rich elements and promotes the deadenylation and destabilization of tumor necrosis factor alpha mRNA. Mol Cell Biol 1999; 19:4311–4323. 64. Dubaybo BA, Bayasi G, Rubeiz GJ. Changes in tumor necrosis factor in postpneumonectomy lung growth. J Thorac Cardiovasc Surg 1995; 110:396– 404. 65. Dovat S, Gilbert KA, Petrovic-Dovat L, Rannels DE. Targeted identification of zinc finger genes expressed in rat lungs. Am J Physiol 1998; 275:L30–L37.

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19 Pulmonary Limitations to Exercise Performance: The Effects of Healthy Ageing and COPD

JORDAN D. MILLER and JEROME A. DEMPSEY University of Wisconsin–Madison Madison, Wisconsin, U.S.A.

Physiological limitation to exercise performance in health and disease is a complex phenomenon. We simplify our approach to this problem by first
defining exercise capacity objectively as the VO2max, or that level of oxygen consumption obtained as work rate is gradually incremented to the point
where VO2 no longer increases linearly with further increases in work rate.
V O2max often correlates quite well with endurance exercise performance. However, there are exceptions to this tight correspondence; for example, with physical training in which endurance performance times may be dissociated
from corresponding changes in VO2max. Generally speaking, this quantity is determined according to the Fick principle:
VO2MAX ¼ Cardiac outputmax  ½arterial O2 content  venous O2 contentmax In turn, COmax is determined by maximum heart rate and stroke volume, CaO2 by the gas exchange and ventilatory functions of the lung and chest wall, as well as the blood’s O2-carrying capacity, and the a-v O2 difference by the metabolic capacity and diffusion capability of the working skeletal muscles. 483

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We discuss the role of each of these components of the Fick equation in
limiting V O2MAX. The important lesson is that the relative vulnerability to failure of each of these functions is not a fixed one, but will change both within and among subjects depending upon the relative adaptability of each of these steps of O2 transport to such situations as physical training, with healthy aging, and with specific organ system diseases such as chronic obstructive pulmonary disease (COPD). I. Limitations in the Young Healthy Adult A. Importance of O2 Delivery and Blood Flow

In healthy young adult humans, several studies point strongly to systemic O2 delivery, or the product of arterial O2 content and blood flow, as the key
determinant of VO2MAX. Cross-sectional studies show a strong correlation

of VO2MAX with CaO2
Q among subjects varying widely in VO2MAX. In both
acute and chronic experiments, VO2MAX has been shown to be increased in proportion to CaO2 when either O2-carrying capacity is augmented via added red cells (i.e., blood doping) or inspiring very high concentrations of O2 (1); or when COmax is increased by adding total circulating blood volume (2) or surgically removing the pericardium to reduce cardiac constraint (3). Only very rarely, as in markedly deconditioned sedentary humans or ani
mals, does a change in O2 delivery not influence VO2MAX (4), showing that the oxidative capacity of the locomotor muscles in such subjects is likely the
major weak link limiting VO2. Of the two major determinants of O2 delivery, maximal cardiac output (and locomotor muscle blood flow) is the overwhelming consensus candidate
for the major limiting factor to VO2MAX in our young adult reference man. This choice clearly makes sense in healthy, active young subjects for two reasons. First, blood flow is a major determinant of O2 delivery and alterations in maximum stroke volume and cardiac output induced experimentally
or via physical training have a major effect on VO2MAX. Second, the healthy respiratory system is considered to be substantially overbuilt and therefore capable of economically providing adequate O2 and CO2 exchange, even in the face of the demands imposed by either maximum or high-intensity endurance exercise. The following types of evidence obtained at peak exercise in the healthy, young adult, normally active male and female subjects support this generalization (5). Arterial PO2 is maintained near resting levels, although the alveolar-toarterial PO2 (A–aDO2) difference does increase 2–2.5 times resting levels.

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The tidal flow/volume loop remains well within the maximal (volitional) flow/volume envelope. VD/VT is markedly reduced below resting values and alveolar ventilation increases out of proportion to CO2 production as PaCO2 is reduced 7–10 mmHg below resting levels at maximum exercise. Airway resistance and lung compliance are maintained near resting levels, the pressures developed by the inspiratory muscles approximate 40–60% of their dynamic capacity and the oxygen cost of
breathing approximates 8–10% of the total boy VO2MAX. B. The Respiratory System as a Weak Link in Young, Physically Trained Adults

The respiratory system does become an important limitation to exercise performance in a significant number of highly trained, young adult, healthy subjects as shown by the following evidence obtained during maximum
exercise at this abnormally high VO2MAX (6,7): Exercise-induced arterial hypoxemia (EIAH) occurs due to an abnormal widening of the alveolar to arterial O2 difference and a limited hyperventilatory response. Athletes’ high ventilatory demands mean that they use most of their reserve for flow rate and volume within their maximum flow/volume envelope, leading to significant expiratory flow limitation. Inspiratory muscles must develop large pressure swings that exceed 90% of dynamic capacity and require an O2 cost of breathing that
approximates 15% of VO2MAX (and 15% of max cardiac output). Why do these respiratory system limitations occur in many highly fit, young, healthy subjects? In health, EIAH occurs almost exclusively in habit
ually active, fit male and female humans ( VO2max 55–85 ml/kg) and in
thoroughbred horses ( VO2max 150–170 ml/kg) (6). In conceptual terms, there
are two reasons. First, trained individuals increased their VO2MAX because of large structural and functional changes in their heart and systemic circulation, including upregulation of endothelial arteriolar responsiveness, all of which augment maximum cardiac output and locomotor muscle blood flow (8,9). Even larger relative changes occur with training in skeletal muscle mitochondrial volume and oxidative capacity (9). However, the lung diffusion surface, airways, and pulmonary vasculature show virtually no adaptation to chronic physical training (10). Recent in vitro studies in a porcine model showed a short-term training-induced increase in endothelial nitric oxide synthase (eNOS) protein and endothelium-dependent relaxation in conduit pulmonary

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arteries similar to that observed in skeletal muscle arteries (11). However, these changes did not persist with longer training periods; nor is their evidence in humans that pulmonary vascular pressures are reduced at any given cardiac output in the trained individual. Even in the highly trained athlete with a 150–
200% > predicted VO2MAX, lung diffusing capacity for oxygen (DLCO) and the maximum volitional flow/volume loop are not substantially different than in their sedentary counterparts of similar body dimensions and age (7). Swimmers appear to be different than runners in this regard as their lung volumes are substantially greater than normal for a given height and age and their diffusion capacities are often substantially higher than normal. It is not known whether swimmers bring these extraordinary respiratory structures to their sport or whether it occurs in part as a result of swim training (12,13). The raised demand for systemic O2 transport therefore exceeds the lungs and airways structural capability for providing the necessary ventilation and diffusion capacity, and arterial hypoxemia ensues. Selected cross-species com
parisons of similarly sized animals with up to threefold differences in VO2MAX, (i.e., cow vs. horse or goat vs. dog), also show that skeletal muscle mitochon
drial volumes are upregulated to a similar relative degree to VO2MAX in the athletic species, but that the lung’s alveolar–capillary surface area is only 20– 30% larger in the athletic compared with the sedentary animal (9). The respiratory muscles do show an adaptive response in their oxidative capacity in response to intense, prolonged, whole body physical training in rats, but not to the same extent as in the trained locomotor muscles (5,14). The result is that both relatively sedentary and highly trained subjects do show exercise-induced diaphragm fatigue resulting from exhaustive whole-body exercise; however, it takes a great deal more work by the diaphragm to cause its fatigue in the trained subject (15). Although the lung and airways show virtually no adaptation to the
chronic stimulus of physical training and the increase in VO2max associated with it, this does not mean that the lung is not adaptable to other stressors. To the contrary, remarkable increases in the alveolar–capillary surface area and DLCO occur in animals and humans born and raised in the hypoxic environments of high altitude (16). Adaptive structural responses in alveolar septation and alveolar surface area also occur in humans and/or rats in response to pneumonectomy and caloric restriction and refeeding (17). Furthermore, in some athletic, aerobic animals, such as the prong-horned antelope, the
alveolar–capillary surface area is enlarged in proportion to their V O2MAX (>300 ml/kg) (18); this proportionality in adaptation prevents EIAH. A second and less certain reason for this failure in gas exchange in the highly trained human is that habitual, daily, hard physical training itself, especially in combination with specific environmental insults such as cold dry air or urban pollution, may actually have detrimental effects on lung structure

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and function. Two types of evidence support this idea. First, biopsy studies show that structural changes occur in the bronchiolar airway walls of crosscountry skiers. Perhaps release of inflammatory mediators may be in part responsible for this increased collagen deposition and remodeling effect on the airways basement membrane in these athletes (19,20). The high prevalence of asthmalike symptoms among populations of elite athletes may be attributed, in part, to this so-called training effect. On the pulmonary vascular side, a thinning or even fracture of the alveolar–capillary interface has been shown to occur because of extremely high levels of pulmonary capillary pressure, secondary to large increases in cardiac output. This type of injury has been documented via direct evidence in the thoroughbred horse who undergoes pulmonary hemorrhage in heavy exercise. In human athletes there is indirect evidence of some form of leakage across the alveolar–capillary membrane from the presence of increased protein in lavage fluid following maximal exercise (21,22). A second line of evidence shows that athletic subjects who experience
EIAH at maximal exercise and who have an abnormally high VO2max also begin to show significant abnormalities in gas exchange (increased A–aDO2, increased PaCO2, and decreased PaO2), even at submaximal steady-state exercise intensities (23,24). Thus this EIAH is not explained solely by the concept of an increased maximal demand compared with normal structural capacity (as outlined above). Instead, this observation suggests that with physical training not only does the lung lag behind the adaptations made by the cardiovascular system and locomotor muscles, but key elements of lung structure may even be damaged to the point where this interferes significantly with normal gas exchange, even at equal submaximal requirements of O2 transport. Confirmation of this postulate requires much more detailed longitudinal study of the lung and airways throughout training in a variety of endurance athletes. The evidence that these respiratory system abnormalities do indeed effect exercise performance is shown experimentally by preventing these prob
lems. First, if EIAH is prevented by supplementing FIO2, then V O2MAX is shown to increase with a consistent, significant effect beginning when SaO2 drops more than 3% below resting values (25,26). Second, if mechanical ventilation is used to lower the work of breathing by 50–60% during heavy exercise, then endurance exercise performance increases significantly and the rate of rise of perceptions of effort (limb discomfort and dyspnea) throughout the exercise period are reduced substantially (27). This positive effect of decreasing the work of breathing on endurance exercise performance also had the effect of preventing diaphragmatic fatigue (28) and of causing local vasodilation and an increased blood flow to the working limbs (29) (Fig. 1).

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Figure 1 Relative effects of changing the work of breathing on limb blood flow (A:


Qlegs) and limb oxygen consumption (B: VO2legs) at VO2MAX (29). The observation that the changes in limb blood flow are so closely related to changes in the work of breathing supports the hypothesis that as the work of breathing increases, more blood is diverted to the respiratory muscles (i.e., blood is stolen from the limb). The
changes in Q legs also closely relate to limb vascular resistance and norepinephrine spillover across the leg (29), which suggests that the changes in limb blood flow are sympathetically mediated.

II. Limitations in Healthy Ageing A. Exercise Performance in Healthy Ageing

The primary effects of healthy ageing (i.e., independent of secondary aging
effects from disease development) have substantial influences on V O2max.
Primarily cross-sectional data show a 10% per decade decline in V O2max beginning in the third decade and roughly about half that effect on endurance performance, although the evidence on endurance performance is less well documented (30,31). These effects of aging are highly variable among the healthy population. There is a consensus in both human and animal studies that major mechanisms of these aging effects on performance must include the following: The age-dependent reduction in fat-free muscle mass and myofibrillar protein in muscle. According to this concept, muscle strength decreases at a rate of about 7% per decade after the third decade,

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with precipitous falls after 50 years of age. In the human, the decline in limb muscle cross-sectional area is due to loss of muscle fibers (32). The cardiovascular system is also compromised significantly via
primary aging effects. Most relevant to the reduction in V O2MAX are reductions in maximum cardiac output due primarily to a reduced maximum exercise heart rate, secondary to a falling intrinsic heart rate plus a reduced responsiveness to beta-adrenergic stimulation. A reduced maximum stroke volume also contributes, due in part to a reduced total circulating blood volume (33). Furthermore, during exercise, locomotor muscle vasoconstriction is enhanced and limb blood flow is reduced in the old compared with the young for any given cardiac output (34). B. Exercise Training in the Elderly

Contrary to some popular concepts, evidence over the past decade shows that in healthy people in their sixth and seventh decades, endurance training can
have markedly beneficial effects on VO2MAX (+20–25%). Training-induced cardiovascular changes are similar in most, but not in all, respects to those in younger subjects. These effects include an increased maximum cardiac output (due to an increased stroke volume with no change in maximum heart rate), along with a widened maximum a–vO2 difference, increased capillary density, and oxidative capacity of skeletal muscle, and increased vasodilatory responsiveness of skeletal muscle arterioles (33,35). Furthermore, the ability of skeletal muscle to adapt to weight training with increased protein synthesis, hypertrophy, and strength is maintained well into the eighth and even into the ninth decade of life (36). Exercise training alone also offers a significant protection against some of the secondary effects of aging. For example, as a result of low physical activity and a relatively high caloric intake, (i.e., positive energy balance), the abdominal obesity syndrome has become a major cause of secondary aging in our society and specifically in the development of type II diabetes and ischemic heart disease. Exercise training in middle age and older (even in the face of only modest reductions in body weight) can reduce abdominal obesity and markedly increase glucose tolerance (37,30). C. Healthy Ageing (and Training) Effects on Pulmonary Structure and Function

Healthy ageing has substantial effects on the structure and function of the lung parenchyma, airways, vasculature, and chest wall. In the lung, the two major structural changes that occur during healthy aging (even in never

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smokers) are loss of elastic recoil, most likely due to a gradual change in the spatial arrangement and cross-linking of the lung’s elastic and collagen fiber network. Additionally increases in alveolar size combined with reduction in the number of alveoli results in a decrease in the total gas exchange surface area with age (38,39). The loss of elastic recoil, combined with a reduced number of attachments of supporting alveoli to airways, means that airways narrow excessively during a forced expiration. Longitudinal population studies show that the forced expiratory volume in one second (FEV1) drops gradually, beginning at about age 25 years with disproportionate reductions in the fifth and sixth decades of life (40). These normal age-dependent reductions in FEV1 occur over a lifetime and rival those attributable only to habitual smoking (41). The increased tendency for small-airway closure also means that functional residual capacity (FRC) increases with age as does the airway closing volume (i.e., the lung volume at which small airways close). The closing volume at age 70 resides very close to FRC during eupneic breathing at rest (see Fig. 2); distribution of inspired air therefore becomes less uniform with ageing. The loss of alveolar surface area translates into an age-dependent
fall in DLCO (DL/VA) beginning in the third decade. VA/Q distribution is also less uniform with aging, although the A–aDO2 at rest is increased only about 10 mmHg by age 70. The equivalent fall of PaO2 is of negligible consequence to

Figure 2 Maximum flow/volume loops at rest, residual volume, closing volume, and the isovolume pressure/flow relationships in 30-year-old and in 70-year-old men (47,48). Note the scooping in the flow/volume loop, the higher closing volume (denoted by the vertical dashed lines) and end-expiratory lung volume, and the lower critical closing pressure for expiratory flow in the 70-year-old subjects. These changes result from the reduced lung elastic recoil in the older subject, causing reduced maximal expiratory flow rates and lower expiratory pressures and higher lung volumes at which airways are dynamically compressed and flow rate becomes independent of effort.

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arterial HbO2 saturation and O2 content. Significant gender differences exist in these ageing effects because the rate of decline in FEV1.1 and DLCO is 15–30% less in any decade in women than in men. After the onset of menopause, the rates of decline in lung function are similar in men and women. The influence of habitual physical training on these ageing effects has received some attention. Cross-sectional comparisons of habitually active
subjects (20+ years of daily vigorous exercise) with a VO2MAX 1.5–2 fold higher than normal with sedentary subjects in their sixth and seventh decade tend to show significantly higher expiratory flow rates (FEV1.0 and maximal expiratory flow at 50% of the forced vital capacity volume, or MEF50) and DLCO, when compared to predicted values (33,42). Do these comparisons demonstrate a true positive influence of habitual training on the loss of lung elastic recoil and diffusion surface with healthy aging, an effect of training that is not evident in young, highly trained adults? To the contrary, a 7 year longitudinal study (subjects aged 66–73 years) in highly fit, habitually active, competitive endurance runners showed that their age-dependent reduction in MEF50, and increases in residual volume (RV) and closing capacity were nearly identical to what would be predicted from longitudinal data obtained in the general population (43). Apparently the enhanced lung function as determined in cross-sectional comparisons in the highly fit septagenarian (see above) is most likely brought to, rather than resulting from, their active lifestyle. These differences between the cross-sectional and longitudinal findings likely reflect an effect of preselection, whereby subjects with inherent more age-resistant lung structure are able to stay active longer. Other relevant age-dependent changes in pulmonary system structure include a reduced chest wall compliance secondary to costal cartilage calcification, a narrowing of intervertebral distances, and an increase in the anterior–posterior diameter of the chest (39), and a reduced compliance of pulmonary arterioles and an increased pulmonary vascular resistance (44). In the diaphragm muscle, ageing rats show cellular hypertrophy and a 25% increase in fiber diameter (45). This contrasts with the reduction in fiber diameter in most limb muscles with aging (see above). In healthy humans, cross-sectional population studies show a substantial reduction in maximal inspiratory pressure (MIP) with aging (46). Interpretation of this finding is confounded by the dependence of the MIP test performance on volitional effort, and the accompanying increases in lung volume (FRC and RV) with normal ageing (see above), which place the diaphragm and other inspiratory muscles on a less optimal point in their length–tension relationship. D. Ageing and the Pulmonary System Response to Exercise

Major changes in the healthy lung and chest wall with ageing have significant implications for both the magnitude and the efficiency of the respiratory

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system response to exercise. A few studies in the past 10 years have examined several aspects of the mechanics of breathing and of gas exchange during exercise in the healthy elderly (47–49,33). In contrast to the fairly uniform respiratory response to exercise in young adults (see above), there is marked interindividual variability in the effects of aging on organ system function. It is therefore not surprising that the response to exercise also has wide interindividual variability. Generalizations can nevertheless be drawn regarding healthy ageing effects on the acute response to exercise. Increased Exercise Hyperpnea

The overall ventilatory response to any given exercise work rate is increased in the aged because dead space ventilation is increased, both at rest and during exercise (see Fig. 3). This high VD/VT likely reflects heterogenous airway narrowing leading to a maldistribution of ventilation in the elderly (see above), both at rest and during exercise. A raised frequency and reduced
VT for any given VE reflects the reduced vital capacity with aging and con-

Figure 3 Ventilatory responses to steady-state exercise in the average healthy 70year-old (—) untrained 30-year-old ( : : : : ), and highly trained 30-year-old (  ). Note the higher ratios of dead space to tidal volume in the elderly, their higher
overall ventilatory response (VE) to a given exercise intensity, and their comparable
levels of alveolar ventilation (VA) (47,48). PaCO2 was likewise similar between young and old over all exercise intensities.

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tribute to the increase in anatomical dead space ventilation (Vd). The result is
that alveolar ventilation ( VA) is adequate to maintain PaCO2 throughout all

exercise intensities, but the overall ventilatory response (VA + Vd) in the aged is high and therefore inefficient. Exercise-Induced Expiratory Flow Limitation

The mean values for flow/volume relationship at rest and exercise are shown for healthy 30-year-olds and fit and healthy 70-year-olds in Fig. 4. The most striking contrast is that the elderly begin to reach significant expiratory flow limitation during submaximal exercise at relatively low minute ventilations
(60–70 L/min). This flow limitation worsens as exercise intensity and V E increase further in fit elderly subjects. The response is in sharp contrast to that
of the 30-year-old who does not begin to show any flow limitation until VE exceeds 120–130 L/min. Recent data obtained in 80–90-year-old active adults (44) show that significant expiratory flow limitation in exercise occurs at even
lower levels of exercise intensity and VE than in 70-year-olds (49). Thus the
exercise-induced flow limitation at low VE in the elderly results from a coin-

Figure 4 Maximum flow/volume and tidal flow/volume loops generated during exercise in a typical healthy 30-year-old and a 69-year-old (48). The tidal flow/ volume loops for the young are shown over a wide range of exercise intensities and
for a range of VO2MAX values of 40–80 mk/kg/min. Expiratory flow limitation in the
young only begins to occur at VE f110–120 L/min and reaches complete flow
limitation at VE > 160–180 L/min. In the older subject, significant expiratory flow
limitation begins at much lower VE and increases in severity with increasing exercise intensity. Note in the older subject the increase in end-expiratory lung volume back to and even in excess of resting levels as expiratory flow limitation increases with increasing exercise. RV, residual volume; TLC, total lung capacity. (From Ref. 48.)

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cidence of two factors: a reduced lung elastic recoil and therefore a reduced maximum flow/volume envelope, combined with an augmented hyperpneic response to exercise at any given work rate or metabolic rate. This combination varies widely with ageing, primarily because of the wide variations in aging-induced losses in lung elastic recoil (see Fig. 4). However, the great majority of healthy fit subjects tested in the 60–80-year-age range showed significant intersection of their tidal and maximum flow/volume loops at moderate exercise intensities. At maximal exercise in the very fit older
subjects, VE achieves truly maximal levels, as suggested by the failure of
added inspired CO2 to increase VE further (47). Mechanical and Metabolic Consequences of Expiratory Flow Limitation and Hyperinflation in Exercise

The work of breathing is increased at any given ventilation in the older, exercising subject. Expiratory flow limitation accounts for much of this increase because it not only increases expiratory resistance, but more importantly causes hyperinflation and therefore increased elastic inspiratory work. End-expiratory lung volume (EELV) reaches and even exceeds resting (relaxation) levels, and end-inspiratory lung volume (EILV) can exceed 90% of total lung capacity (TLC) during heavy exercise (see Fig. 4). This
contrasts with findings in the young subject at similar levels of exercise VE who, in the absence of expiratory flow limitation, drives his or her EELV 300– 900 ml below resting levels. The consequence to the older flow-limited fit subject is that he or she must breathe at the upper stiffer portion of the pressure/volume relationship of the lung, where dynamic compliance is reduced. Thus, what is initiated during exercise as a flow resistance problem on expiration leads to an elevated elastic load on inspiration. The lower compliance of the older subject’s chest wall also contributes to increased ventilatory work in exercise. The relative hyperinflation also means that the diaphragm and inspiratory muscles will be shortened and therefore have a reduced force-generating capability. During exercise, the inspiratory muscles are therefore working in excess of 85–95% of their dynamic capacity, in contrast with the younger

subject at comparable VO2MAX and VEmax who operates at 40–60% of maximum dynamic capacity pressure generation (7). In theory, then, the inspiratory and expiratory muscles in the older fit subjects are operating at fatiguing levels (15), although whether exercise-induced diaphragmatic fatigue actually does occur during less intense exercise in the elderly than in the young has not been directly tested. The oxygen consumption of the respiratory muscles during exercise in the fit elderly was estimated conservatively using pressure/volume loop measures of the work of breathing and applying these to the oxygen costs

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Figure 5 Oxygen cost and the work of breathing with increasing exercise ventilation in young untrained subjects, young highly trained subjects, and in older healthy fit subjects. (From Ref. 33.)

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of breathing as determined in young adults (see Fig. 5). These comparisons
reveal that the O2 cost of breathing was f10% of the total VO2 during
moderate intensity exercise and was 15–20% of VO2MAX at maximal exercise in the aged. These metabolic costs of breathing are substantially above those
in the young adult at comparable VO2max (8–10% and 12–15%, respectively). Gas Exchange During Exercise

In theory, several changes in the ageing lung would predispose the subject to

VA/Q mismatch and diffusion limitation leading to exercise-induced arterial hypoxemia (EIAH). On the ventilation distribution side, these mechanisms would include maldistribution of airway resistance and mechanical time constants associated with a high airway closing volume, as well as the restriction in volume expansion imposed by hyperinflation during exercise. A substantial reduction in the alveolar/capillary diffusion surface area is another key factor, especially during exercise in which the compromised ability to increase pulmonary capillary blood volume in the face of increasing cardiac output would greatly shorten red cell transit time in the pulmonary capillary and lessen the opportunity for diffusion equilibrium. The pulmonary vasculature, like the systemic vasculature and chest wall, has reduced compliance with ageing: during exercise at any given cardiac output, pulmonary vascular resistance and pulmonary capillary pressures are significantly elevated (44). These elevations in pulmonary vascular pressures may be sufficient to cause excessive leakage of plasma water and its accumulation in interstitial fluid spaces as shown in some young athletes. The limited measurements of arterial blood gases during exercise in healthy aging subjects show that EIAH does not occur in those relatively
sedentary subjects with normal age-predicted VO2MAX, but that it does occur
with significant prevalence in habitually active fit subjects with VO2MAX 150– 200% of age-predicted (see Fig. 6) (33,50). Hypoxemia occurs in the aged athlete for similar reasons as in their younger counterparts: an excessively widened A–aDO2 and limited hyperventilation during heavy exercise, the latter reflecting mechanical constraints by the limits of the age-compromised flow: volume loop. Prefaut et al. (50) also report absolute CO2 retention (i.e., greater than resting PaCO2) during moderate to heavy exercise in older endurance athletes. However, in our laboratory we have never observed this absolute CO2 retention, even in highly fit healthy subjects, young or old, and not even in those who ventilate up to the very limits of their flow: volume loop in maximum exercise (see Fig. 7). Like the young, therefore, the healthy elderly do not normally experi
ence EIAH exercising at their normal age-predicted VO2MAX. EIAH only
occurs in a fraction of highly trained subjects at elevated VO2MAX. The only

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Figure 6 Comparisons of arterial PO2 and the alveolar to arterial PO2 difference at rest and at maximum exercise in highly trained 70-year-olds compared with
untrained 30-year-old subjects, both with similar VO2MAX. Mean values (FSD) are shown for young subjects (closed circle) and both individual and mean values (straight bar) are shown for the older subjects. At rest, the arterial PO2 is about 5 mmHg lower and the A–aDO2 5 mmHg wider in the older subjects. At comparable
levels of VO2MAX, the older subject shows an 8 mmHg wider average A–aDO2 and a 10 mmHg lower PaO2 than in the young. Six of these 20 older fit subjects showed significant exercise-induced arterial hypoxemia (i.e., SaO2 87–94%), whereas none of the 50 untrained younger subjects tested showed significant arterial hypoxemia while
exercising at the same VO2MAX. (From Ref. 33.)

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Figure 7 Interindividual variability in flow limitation during exercise. Subject A, shown at rest and during progressive exercise to maximum, demonstrates the usual age-related decline in lung function (FEF50 = 100% predicted, DLCO = 190%
predicted). With progressive exercise, mechanical limits to VE are reached during both heavy and maximal exercise. Maximal effective expiratory pressures were exceeded (also see Fig. 1) and the capacity for inspiratory pressure development (PCAPI) was reached at max exercise. The oxygen cost of breathing approached an
estimated 23% of total VO2MAX. PaO2 fell to 59 mmHg from a normal resting level and PaCO2 rose through the final workload. In the bottom panel, subject B of similar
age but a substantially lower VO2MAX and reduced maximum flow/volume envelope (FEF50 = 50% predicted). This subject also reaches flow limitation and achieves maximum inspiratory and expiratory pressures, but at a substantially reduced ventilation and metabolic demand during exercise compared to subject A. (From Refs. 47, 48.)

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clear, significant effect of aging on gas exchange is that the EIAH in the
trained elderly occurs at a much lower VO2MAX (35–59 ml/kg) and in a range
of VO2MAX where it is never observed in young adult men (33) and only rarely in young active women (51). Thus, the fit elderly are clearly more susceptible to EIAH than the young. However, these failures in exercise gas exchange do not occur with the prevalence or severity one might predict from the aging effects on airway closure, alveolar–capillary surface area, and pulmonary vascular resistance, as outlined above. Perhaps, during exercise, these aging effects are overridden by the homogeneous ventilation distribution promoted by inspiratory flow rates that are 8–10 times the resting level, and by the fact that the great majority of the augmented tidal inspiration still occurs above airway closing volume on the linear portion of the pressure/volume relationship, where most of these airways are likely to open. Furthermore, at high exercise intensities expiratory flow limitation caused most of these subjects to raise their EELV, moving their tidal breath not only away from flow limitation but also well above the their closing capacity. A critical factor here may also be the high



overall VA/Qachieved in heavy exercise by most fit elderly (VA/Qf 3–4 vs.< 1 at rest); this ensures adequate oxygenation of end-capillary pulmonary blood,

even in the face of a moderate worsening of VA/Q uniformity during exercise (52). Finally, although the ageing effect on diffusion surface area is certainly significant (a 30% decrement in DLCO from age 30 to 70), the available reserve for diffusion in the healthy elderly is apparently sufficient to meet the reduced maximum demands for oxygen transport imposed by their coincident
age-dependent fall in VO2MAX. Taken collectively, these observations suggest that exercise-induced arterial hypoxemia in the aged, as in the young, most
commonly occurs under conditions in which the VO2MAX is high and the ventilatory response reaches maximal or near-maximal levels (see example in Fig. 7: subject A). E.

Pulmonary Limitations to Exercise in Healthy Ageing

First, it is important to clarify that the specific role of the respiratory system as a limiting factor to exercise with aging will depend critically on the relative age-related deterioration in lung, airway, and chest wall structure and
function on the one hand and the age-related change in VO2MAX on the other. For example, the evidence is clear that the structural limits of the flow/ volume loop, the respiratory muscles, and the lung’s diffusion surface area available to a healthy nonsmoking 70-year-old are clearly not sufficient to
support the ventilatory and gas transport needs of a VO2MAX in excess of 65–70 ml/kg/min, (i.e., that of a well-trained 30-year-old) (53). However, the
VO2MAX will always be reduced with aging to some significant extent below

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these high levels achieved in the young adult, and the rate of normal aging effects on the lung is highly variable from person to person. Therefore the degree to which age-dependent changes in maximum capacity of the lung, airway, and chest wall match (or fail to match) those of a coincidently changing maximal metabolic requirement will determine whether the respiratory system is a weak link in determining exercise performance in the healthy elderly. It is reasonable to speculate from available data that in the untrained sedentary elderly the respiratory system does not play any substantially greater role in limiting peak or endurance exercise performance than it does in their younger sedentary counterparts. Even though the lung and airways age substantially, and most noticeably, throughout the sixth through eighth
decades, V O2MAX is also markedly reduced and nearly in parallel to lung
function. The concomitant fall in VO2MAX therefore reduces the demand on the lung and chest wall for O2 transport. It is most likely that the maximum cardiac output along with reduced blood flow distribution to the limbs (at any
given cardiac output) remain the dominant limitations to VO2MAX in most untrained older subjects. The structural and functional cardiovascular deter

minants of VO2MAX that were so important as limiting factors to VO2MAX in young adults (see above) will decline with age and thus remain major
determinants of a declining VO2MAX. On the other hand, the relatively overbuilt respiratory system in the young also undergoes about equal decrements in capacity (to their cardiovascular counterparts) with normal ageing, and its capacity continues to exceed the declining maximal metabolic demand. Exceptions to this generalization will occur with the extremes of age
dependent loss of elastic recoil, where even relatively modest increases in VE during exercise will encounter flow limitation and its sequelae (see Fig. 7: subject B). We emphasize that it is also likely that a reduced metabolic capacity of locomotor muscles, as opposed to oxygen delivery, would become even more important in the untrained, very sedentary elderly. We believe that it is only the habitually active elderly who retain a
relatively high VO2MAX with a commensurate high demand on the ageing respiratory system for ventilation and gas exchange who experience a significant prevalence of respiratory system limitations to exercise performance. The importance of the respiratory system as a limiting factor, especially in the active, fit, elderly subject, is exacerbated by the ineffectiveness of physical training in altering the aging effect on respiratory system and structure as opposed to the highly significant positive effects of training on cardiovascular system and locomotor muscle structure and function (33). In a significant but fairly small proportion of fit elderly subjects who experience EIAH and a reduced CaO2, it is expected that they would, like their younger counterparts, limit their maximum a-v O2 content difference and therefore

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their VO2MAX (25,26). Expiratory flow limitation leading to increased elastic and flow-resistive work of breathing in heavy exercise likely occurs more consistently than does EIAH in the fit elderly. As shown by respiratory muscle unloading studies in the young (27), the increased loads on the respiratory muscles in the elderly subject would therefore enhance the probability of occurrence of exercise-induced diaphragm fatigue and redistribution of blood flow from the working locomotor muscles, along with enhanced perceptions of breathlessness and limb discomfort. It is conceivable that these changes would limit endurance performance, but these experiments have not yet been applied to the fit elderly. Finally, there is the truly exceptional extremely fit elderly athlete (as shown in Fig. 7: subject A) whose respiratory system has undergone near-normal aging effects and
whose VO2MAX is more than twice the age-predicted normal. Both EIAH (due to a widened A–aDO2 and a mechanically limited ventilation) and excessive respiratory muscle work will prevail in this subject, resulting most
certainly in a primary respiratory system limitation to both VO2MAX and endurance exercise performance.

III. Exercise Limitation in COPD The long-standing incorrect intuition that dyspnea is the sole determinant of exercise capacity in patients with COPD has avoided confrontation for many years. However, a growing number of reports with large numbers of patients suggest that leg discomfort, by itself or in addition to dyspnea, contributes significantly to the termination of exercise in patients with COPD (54). Alterations in respiratory and skeletal muscle histology, metabolism, and blood flow refute the notion that COPD is purely a disease of the lungs, and only a multifactorial, integrative viewpoint will truly bring us closer to understanding exercise limitation in this population. We again utilize the Fick equation to examine the physiological basis for exercise limitation in patients with COPD, beginning with the lung and its ability to oxygenate the blood adequately, followed by the potential cardiovascular limitations to O2 delivery, and ending with the ability of the muscle to extract O2 from the blood. A. Lung Function and Arterial Oxygen Content Limitations in Patients with COPD Mechanical Constraints to Ventilation: Maintaining Alveolar Gas Concentrations

As discussed in the previous two sections, the maximal capacity of the pulmonary system is rarely approached in the normal, healthy adult. The large

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force reserves of the inspiratory muscles allow for adequate increases in inspiratory flow rates as inspiratory time decreases commensurate with increases in breathing frequency, ensuring an adequate increase in inspired volume as exercise intensity increases. Equally important is that the normal alveolar elastic properties and airway tethering allow healthy individuals to dictate EELV by increasing or decreasing expiratory muscle force, even during the highest intensities of exercise in most individuals. In patients with COPD, however, the voluntary control of EELV is a luxury long lost. The obliteration of alveolar walls along with the degradation of alveolar and parenchymal elastin in COPD cause marked reductions in elastic recoil and airway tethering, both of which predispose the airway to collapse when intrathoracic pressure is increased during active expiration (55). To minimize the work of expiration and maintain EELV, patients with COPD typically exhibit a prolonged expiratory time (TE) (56). However, the tachypnea (and shortened TE) of even mild exercise forces EELV upward due to the premature collapse of the airways, despite substantial increases in expiratory pressure (Fig. 8). Such marked dynamic hyperinflation lies at the core of the mechanical ventilatory constraints observed in patients with severe COPD. It also results in substantial increases in the work of breathing due to the mechanical inefficiency of the respiratory muscles and the increased elastic load presented by the lungs and chest wall at higher thoracic volumes. As EELV rises, the respiratory muscles are placed in a mechanically compromised position. In particular, the diaphragm loses its ability to produce an effective force due to two primary factors: Its progressive shortening as lung volumes increase, which places the diaphragm at a less efficient point on its length–tension relationship. The diaphragm becomes less dome-shaped (i.e., becomes flattened), such that a change in sarcomere length will result in a smaller downward displacement of the diaphragm. Thus, as lung volume increases, a given change in diaphragm sarcomere length translates into a smaller downward displacement of the diaphragm and, in turn, a smaller change in intrathoracic pressure. As a consequence, much greater increases in diaphragm muscle fiber force production (and central motor output) must take place in order for the same tidal volume excursion. As a result of this mechanical inefficiency of the diaphragm, the accessory inspiratory muscles (intercostal muscles, sternocleidomastoids, scalenes, etc.) are frequently recruited in patients with COPD. As lung volumes are forced upward by dynamic hyperinflation, the work of breathing is increased not only because of the geometric disadvantage of the respiratory muscles but also due to the mechanical properties of the lung and chest wall. The increased inward recoil in combination with dynamic

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Figure 8 Spontaneous tidal flow/volume loops at rest (solid line) and during exercise (dotted line) plotted within a maximal loop performed prior to exercise in a patient with COPD (134). Note that this patient exhibits marked flow limitation during spontaneous breathing even at rest, as demonstrated by the intersection of the tidal flow-volume loop (FVL) with the maximal FVL over most of the breath. With the onset of exercise, EELV is driven upward due to the inability to empty the lung in the expiratory time allowed. Even though EELV shifts upward by nearly 0.33L during exercise, this subject is still flow limited over 90% of his expiration.

airway collapse often results in a positive alveolar pressure at end expiration (i.e., air trapping), and is commonly referred to as intrinsic positive endexpiratory pressure (PEEPi). When PEEPi is present, the amount of pressure required for the generation of inspiratory flow is elevated, and a significant amount of work may be required simply to overcome this threshold for inspiratory flow. During exercise in patients with COPD, PEEPi may comprise up to 50% of the work of breathing (57), and forms the physiological basis for the common complaint of what is termed an unsatisfied inspiratory effort (i.e., respiratory muscle contraction/effort with little or no inspiratory flow) (58). The outward recoil of the chest wall is reduced at higher lung volumes (and actually shifts to inward recoil at lung volumes above 70% TLC), placing a

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greater load on the accessory inspiratory muscles for any given change in lung volume (59). The mechanically disadvantaged respiratory muscles can usually overcome the elevated workload placed on them at rest, and many subjects maintain normal arterial O2 and CO2 levels during the early stages of disease progression. However, as the disease progresses, the ventilatory demands and expiratory pressures generated during even modest physical activity result in premature airway closure and dynamic hyperinflation. The combination of mechanical inefficiency and respiratory muscle load result in an inability to achieve an adequate tidal volume at elevated breathing frequencies (see Fig. 9), where inspiratory and expiratory times are greatly reduced, and alveolar ventilation is compromised substantially. The ensuing hypercapnia and hypoxemia result in a decreased systemic O2 delivery and reduced pH, both of which compromise peripheral skeletal muscle function and stimulate ventilatory drive and the sensation of dyspnea.

Figure 9 Changes in operating lung volumes during exercise in normal subjects, patients with COPD who do not retain CO2 during exercise (NR), and patients with COPD who do retain CO2 during exercise (R). Note that in the normal subject, endexpiratory lung volume is well maintained over all levels of ventilation shown. However, in both groups of patients with COPD, EELV is markedly increased at baseline and increases further with increases in minute ventilation. Such a marked elevation in EELV is an excellent indicator of expiratory flow limitation, dynamic hyperinflation, and mechanical constraint. Thus, one mechanism thought to contribute to the retention of CO2 in many patients with COPD is the inability to increase ventilation adequately due to the elevated levels of mechanical constraint (note that EELV/mechanical constraint is elevated over all levels of ventilation in

the R group when compared to the NR group), combined with underlying VA/Q inequality (84).

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Respiratory Muscle Adaptations to a Chronically Increased Load

The previous section detailed the mechanical constraints to increases in ventilation that are commonly observed in patients with COPD, most of which place a chronically elevated load on the respiratory muscles. It is well documented and widely accepted that peripheral skeletal muscle adapts to chronic physical stress by changing its phenotype to a more oxidative, fatigueresistant phenotype, but can the same be said for the respiratory muscles? The diaphragm is generally the first muscle to experience the chronic loading conditions imposed by COPD, as it is the predominant muscle used for inspiration at rest. As a consequence, the diaphragm has been examined in both animal models of COPD and humans with COPD, and remarkable adaptations have been documented. The diaphragm exhibits plasticity during the progression of COPD by developing a more oxidative, fatigue-resistant phenotype and reducing the number of sarcomeres in series. The remarkable changes in diaphragm oxidative capacity in response to COPD have been examined in hamsters with emphysema induced via intratracheal elastase instillation. Significant increases in the cross-sectional area of type I (slow twitch-oxidative) and significant decreases in type IIa (fast twitch-oxidative) fibers in the diaphragm have been reported (60). These changes were also accompanied by increases in both succinate dehydrogenase and citrate synthase activity (markers of oxidative capacity) and decreases in phosphofructokinase activity (a marker of glycolytic capacity) (61). Similar findings have been reported following the analysis of diaphragm biopsies from patients with COPD, with increases in the oxidative capacity of both type I and II fibers (62), decreases in glycolytic activity (63), and shifts in myosin heavy chain isoforms to a more oxidative phenotype (i.e., from type IIa/IIb to type I) (64). However, it is important to note that while elastaseinduced emphysema results in little or no change in the proportion of fiber types (e.g., nonspecific fiber atrophy), the limited observations in humans suggest that there are significant increases in the proportion of type I fibers and decreases in the proportion of type IIax fibers (i.e., specific atrophy of fast twitch glycolytic fibers) (63–65). It is also of interest to note that these biochemical and histological changes are only observed in patients with severe COPD. Patients with mild–moderate COPD do not show increases in oxidative capacity or phenotype (66–68). However, both groups exhibit varying degrees of nonspecific diaphragm muscle fiber atrophy (63,65–67). It is a common observation that diaphragmatic force production is reduced during acute hyperinflation. However, during states of chronic hyperinflation, there appears to be a leftward shift in the length–tension curve of the diaphragm: i.e., the optimal length for force production (LO) is shorter (68). Farkas and Roussos, and others, have shown in the emphysematous hamster that this is largely due to a reduction in the number of

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sarcomeres in series in the diaphragm (60,64,69,70). This adaptation can be reversed following lung resection (i.e., sarcomeres are added back in series), demonstrating the extraordinary malleability of the diaphragm (71). Indeed, such reductions in LO preserve sarcomere force production in the diaphragm during an acute episode of lung hyperinflation, which frequently occurs during exercise (64,69,70). The accessory muscles of inspiration are also frequently recruited during exercise in patients with COPD, and are even recruited at rest with severe disease progression. There is likewise a growing body of evidence that the accessory muscles of inspiration also undergo a marked shift to a more oxidative phenotype during the development of COPD. Such a phenomenon has been demonstrated in hamsters with emphysema, in which the scalene (72) and intercostal (61) muscles possess a much higher oxidative capacity than animals that do not have COPD. Can Exercise Cause Respiratory Muscle Fatigue in Patients with COPD?

It is unclear whether the respiratory muscles fatigue during symptom-limited exercise in patients with COPD. The work of breathing is undoubtedly substantially higher in patients with COPD at any given level of ventilation compared to normal individuals (57,73,74). However, when diaphragm fatigue is quantified using bilateral phrenic nerve stimulation (BPNS) following symptom-limited maximal exercise in patients with COPD, few or no changes are observed in transdiaphragmatic twitch amplitude (75,76). When diaphragm fatigue is examined using the maximal relaxation rate (MRR) of the diaphragm following a sniff maneuver, significant decreases in MRR are observed that are thought to represent the earliest stages of fatigue (77,78). The notion that the reductions in MRR are due to fatigue is supported by the observation that unloading the respiratory muscles during exercise using pressure support ventilation eliminates the reductions in MRR (79). Although mechanical factors undoubtedly constrain the maximal alveolar ventilation in patients with COPD, the fact that minute ventilation at any given workload may be higher in patients with COPD (80) suggests that mechanical factors alone do not limit arterial O2 content. As will noted in the following section, changes in lung morphology result not only in significant changes in the diffusion of oxygen across the alveolar membrane but also the distribution of both pulmonary ventilation and blood flow. Diffusion of O2 into the Blood: Alveolar and Circulatory Constraints

The maintenance of arterial oxygen content is determined by the diffusion capacity of the lung, the ability of the lung to match alveolar ventilation to the

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appropriate metabolic rate, and the ability to direct pulmonary blood flow to alveoli that are adequately ventilated. As COPD progresses, significant derangements in all three of these components contribute significantly to the hypoxemia and hypercapnia observed during exercise. Changes in the diffusion capacity of the lung (DL) are classically estimated using the diffusion of carbon monoxide across the alveolar membrane (DLCO). The measurement of DLCO has two components: the diffusion capacity of the membrane (DM), and the pulmonary capillary blood volume (Vc). The progressive destruction of alveolar walls decreases the surface area for gas exchange in COPD, and chronic inflammation frequently results in increases in alveolar thickness, both of which are major determinants of DM. A significant number of pulmonary capillaries are also destroyed during the progression of COPD, resulting in significant reductions in Vc. It is not surprising, therefore, that the severity of emphysema (as assessed by high resolution computed tomography) correlates well with DLCO (81). Taken collectively, these changes suggest that as pulmonary capillary transit time decreases concomitant with the increases in pulmonary blood flow during exercise, the impaired DL has been postulated to play a significant role in EIAH in patients with COPD (82,83). However, the difficulties in interpreting DL measurements under conditions of uneven alveolar ventilation distribution in combination with difficulties in measuring the individual components of DL during exercise preclude a quantitative analysis of its contribution to EIAH in patients with COPD (83).

The measurement of ventilation–perfusion (VA/Q ) relationships has provided a significant amount of insight into the causes of arterial hypoxemia in patients with COPD both at rest and during exercise. At rest, patients with

emphysema typically exhibit areas with low VA/Q (due to high blood flow in

poorly ventilated alveoli), some areas with near normal (VA/Q = 1.0), and areas with abnormally high VA/Q ratios (due to the excessive ventilation of poorly perfused alveoli) (83). Patients with chronic bronchitis have much

more variable VA/Q ratios, with many patients showing only regions of low



and normal VA/Q, with areas of high VA/Q virtually absent, and other patients

showing areas of high and low VA/Q (83). Unlike normal, healthy subjects,

whose mean V A/Q increases in response to exercise (sometime greater than four times resting levels), patients with severe COPD rarely show any

significant change in VA/Q with increasing exercise intensities (83), although

patients with less severe COPD may show improvements in VA/Q mismatch

ing during exercise (82). Both direct (83) and indirect (84) estimates of VA/Q maldistribution accurately predict changes in both PaO2 and PaCO2 during

exercise (i.e., the more deranged the VA/Q regions, the more severe the hypoxemia and hypercapnia observed during exercise), with the magnitude of these changes determined in part by the amount of ventilatory constraint during

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exercise (i.e., a function of dynamic hyperinflation) (84). Because as there are typically not substantial amounts of intrapulmonary shunting in patients with COPD, increasing the fraction of inspired O2 is frequently and effectively used to increase the arterial O2 content, which dramatically improves exercise performance and reduces exertional dyspnea (see later sections). These

observations as a whole support the hypothesis that the severity of V A/Q maldistribution during exercise is a major determinant of exercise-induced arterial hypoxemia and hypercapnia in patients with COPD, both of which contribute substantially to exercise limitation in this patient population. B. Cardiac Function in Patients with COPD Effects of Intrathoracic Pressure on Cardiac Function

As discussed previously, patients with COPD generate excessive intrathoracic pressure swings during exercise, which are primarily a consequence of altered pulmonary mechanics and the resultant dynamic hyperinflation. How do these pathological breathing mechanics affect cardiac function? Negative swings in intrathoracic pressure have long been known to increase ventricular afterload. When these intrathoracic pressure swings become excessive, substantial decrements in stroke volume have been observed, although the healthy heart can usually catch up in the ensuing beats as a result of increases in cardiac preload (85). However, given the cause of most cases of COPD (i.e., smoking), the vast majority of patients suffer some sort of comorbidity, with the primary comorbidity being heart disease. A growing body of evidence suggests that the compromised ventricle is much more sensitive to changes in intrathoracic pressure and may not be able to compensate completely by increases in cardiac preload, resulting in a reduced cardiac output during the steady state (86,87). Although the excessive negative intrathoracic pressure swings observed in patients with COPD may contribute to impaired cardiac function, the excessive positive pressures produced during expiration are likely to play a greater role due to the marked TE prolongation (TE/TTOT increases by up to 20% or more) (56). Intermittent increases in intrathoracic pressure synchronized with cardiac contraction have been shown to improve cardiac function (88,89), but constant increases in positive pressure applied over the course of several cardiac cycles have been shown to reduce ventricular output via reductions in cardiac preload (90,91). A reduction in steady-state cardiac output has been thought to be mediated by a transient increase in ventricular output followed by insufficient ventricular filling (due to compression of the right atrium and a decrease in the pressure gradient for venous return), which results in a net translocation of blood out of the thorax (92–96). Whether the large negative pressures produced during inspiration can cause compensatory

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increases in venous return is unclear, since collapse of the inferior vena cava has been shown during inspiration in these patients at rest with intrathoracic pressure excursions as small as 10 cmH2O (97). Effects of Hypoxemia on Cardiac Function in Patients with COPD

The marked reductions in blood oxygen content have a significant impact on cardiac function in patients with COPD. Although mild hypoxia may reduce left ventricular afterload (via peripheral vasodilation), right ventricular afterload is markedly increased due to hypoxic pulmonary vasoconstriction. The right ventricle typically responds to the chronic increase in afterload via hypertrophy (98), although the increase in ventricular mass is rarely great enough to produce the pressures required to maintain a normal maximal cardiac output in the face of elevated pulmonary vascular resistances (98,99). Supporting the notion that right ventricular function is a limiting factor in peak cardiac output, there is a strong negative relationship between peak pulmonary vascular pressure (an index of right ventricular afterload) and VO2peak. Acutely reducing pulmonary vascular resistance increases exercise capacity (98) with little effect on arterial oxygen content in mild COPD (82). As the RV function deteriorates and its end-diastolic volume rises (as is the case in cor pulmonale), LV function may also be compromised by reductions in preload due to reduced pulmonary venous return and ventricular interdependence (i.e., compression of the LV due to the limited amount of space in the pericardium and cardiac fossa) (100–103). In addition to its effects on ventricular afterload, hypoxia directly hinders the response of the ventricles to any given stress by blunting the inotropic and chronotropic effects of catecholamines (104–107), which inevitably limits peak cardiac output. Such a phenomenon appears to be present in COPD, where cardiac h-adrenergic receptor density and function are abnormal (108). Following the induction of experimental emphysema, the heart does attempt to improve myocardial oxygen delivery by significantly increasing myocardial capillarity (109,110). However, as the duration and severity of emphysema increase, the excessive afterload placed on the right and left ventricles appears to outweigh its capacity for angiogenesis, and capillary density decreases as a result of myocyte hypertrophy (110). Is Total Cardiac Output Reduced in Patients with COPD?

There are limited data on changes in peak cardiac output in patients with COPD during high-intensity exercise. This stems from the fact that the most reliable techniques used to measure cardiac output require a steady-state VO2, which is not easily accomplished by most patients with COPD during highintensity exercise. The available data do suggest that the relationship between

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cardiac output and VO2 is relatively preserved in COPD (i.e., cardiac output at any given VO2 is unchanged), but peak cardiac output has been shown to be limited by as much as 60% in these patients (111,112). It is not clear whether this limitation in peak cardiac output is due to the effects of hypoxemia on cardiac function or the mechanical interactions between the cardiovascular and pulmonary systems. Reductions in peak cardiac output are likely to play a major role in the limitation of exercise capacity, but of equal importance is the way in which the limited cardiac output is distributed to the metabolically active tissues during exercise.

C. Distribution of Blood Flow in COPD Demand vs. Supply: The Metabolic Consequences of COPD

As mentioned above, the respiratory muscles are exposed to a much greater workload in patients with COPD compared to normal individuals at any given level of oxygen consumption. Thus, the oxygen demand within the respiratory muscles at any given external workload is exaggerated in COPD, resulting in a greater demand for blood flow at any given external workload. Such a phenomenon has been shown to occur in the emphysematous hamster, in which blood flow to the diaphragm was increased by 137% during brisk walking, whereas diaphragm blood flow was increased by only 40% in control animals (113). Furthermore, intercostal muscle blood flow in the emphysematous hamsters was significantly greater than that of the control animals (113). How does this relate to exercise intolerance in humans with COPD? As discussed earlier, data from the healthy, exercising human suggest that the blood flow demand by the respiratory muscles can substantially compromise leg blood flow during maximal (29), but not submaximal (114), exercise. The emphysematous hamsters examined by Sexton and Poole did not show a decrease in hindlimb blood flow during brisk walking, demonstrating that the increases in diaphragm blood flow did not compromise blood flow to the locomotor muscles (113). This observation is similar to that observed in healthy humans (114), but in sharp contrast to the observations made on rats with congestive heart failure (CHF), in which diaphragm blood flow is increased (115) and leg blood flow is compromised (116) during submaximal exercise. It is important to note that these animal experiments were carried out after a fairly short duration of emphysema (e.g., 16–20 weeks), and do not address the changes in circulatory and metabolic control following years of COPD in human subjects. There are limited data on the distribution of blood flow in humans with COPD. However, the few studies available provide excellent examples of the

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heterogeneous nature of this patient population. In particular, there appear to be two distinct patterns of leg blood flow in patients with severe COPD: Patients whose leg blood flow increases commensurate with increases in workload Patients whose leg blood flow plateaus early on in an incremental exercise test The latter group is perhaps the most interesting. Figure 10 shows that leg VO2 plateaus with leg blood flow as early on in the exercise test as 25% of the subjects’ peak workload (117). Of particular interest is the observation

Figure 10 Changes in limb blood flow during an incremental exercise test in
patients with COPD who demonstrate plateaus in limb blood flow and limb VO2 (A,
C) and those who do not plateau (B, D) (117). Note that limb VO2 follows the same pattern as Qlegs, similar to the data presented in young, healthy subjects (see Fig. 1),
but whole-body VO2 does not plateau. Of particular interest was the observation that the plateau group had significantly higher levels of ventilation at any given workload (117), which is supportive of the notion that a greater proportion of the available cardiac output was being diverted to the respiratory muscles due to an increased work of breathing.

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that those subjects who showed a plateau in leg blood flow also had significantly higher levels of ventilation and dyspnea during the exercise test, suggesting that blood flow may in fact be redirected to the respiratory muscles in these patients (118). Indirectly supporting this mechanism is the observation that reducing the competition for a limited O2 supply, by increasing CaO2 with hyperoxia, significantly increases maximal leg blood flow in patients with COPD (117). The fact that both leg blood flow and VO2 appear to be blunted in a specific subpopulation of patients with COPD raises an even more intriguing question: If oxygen delivery to the lower limb was increased in these patients, would the muscle be able to utilize it? The fact that the a–vO2 difference across the exercising limb plateaus as exercise intensity increases suggests that the limb has exhausted its capability to extract O2 (118). However, two experimental studies have shown otherwise. Several research groups have demonstrated that increasing oxygen delivery to the exercising limb via increasing FIO2 increases both peak work rate and peak VO2 (117,119). In patients with COPD, the increase in peak limb muscle VO2 is in direct proportion to the increases in oxygen delivery to the limb (117). The suggestion that O2 supply limits peak limb VO2 is further supported by the observation that single-leg knee extension elicits a much higher muscle-mass-specific VO2peak than twolegged cycling (119). This is very similar to findings in patients with CHF, in whom optimal muscular perfusion is maintained only when a small muscle mass is exercised (i.e., one-legged exercise), and both muscular blood flow and VO2peak decrease with the recruitment of a larger muscle mass (e.g., twolegged exercise) (120). Limits of O2 Utilization in Patients with COPD: Histological and Biochemical Changes in the Peripheral Musculature with COPD

The changes in the peripheral musculature that occur in COPD have been well documented and reviewed in great detail elsewhere (121). However, a brief overview of these changes and their potential physiological ramifications provide insights into the maximal capability of the peripheral muscles to extract O2. Perhaps the most widely documented changes in limb skeletal muscle in patients with severe COPD are those reported for the quadriceps femoris muscle, where there is a shift from type I fibers (e.g., slow twitch, fatigue-resistant, oxidative phenotype) to type IIa fibers (fast twitch, non-fatigue-resistant, glycolytic phenotype). The biochemical alterations in the quadriceps femoris parallel the histological changes listed above, which include reductions in oxidative enzyme activities (e.g., citrate synthase, h-hydroxyacyl-CoA dehydrogenase, hexokinase, and succinate dehydrogenase), and increases in the

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concentrations of glycolytic enzymes (e.g., phosphofructokinase and lactate dehydrogenase) (122,123). It is unclear whether these changes are elicited by generalized deconditioning, corticosteroid therapy, chronic hypoxemia, or a specific aspect of the disease per se (124). However, the fact that the activity of citrate synthase is significantly higher in the deltoid muscle of patients with COPD than in controls (125), in addition to a preservation of handgrip strength (125) and arm ergometry exercise capacity (126), suggests that generalized deconditioning of the locomotor muscles as a result of physical inactivity plays a prominent role in the changes observed in the quadriceps femoris muscle. Also, the observation that patients with CHF show nearly identical changes in peripheral muscle phenotypes despite a different disease pathogenesis supports the generalized deconditioning hypothesis (127,128).




D. Is VO2MAX Ever Reached in Patients with COPD? Role of Symptom Limitation

Although the data presented above do provide a compelling physiological basis for the reductions in exercise capacity observed in patients with COPD, are the true physiological limits of any one component (e.g., O2 delivery or extraction) ever reached? Indeed, confounding our analysis of the physiologic limitations to exercise in this patient population is the fact that many patients discontinue exercise due to what are termed intolerable symptoms of discomfort (a subjective endpoint that often leaves the true limits of the system open to speculation). In the following sections, we discuss two of the most common causes of exercise termination in patients with COPD: dyspnea and leg fatigue. Dyspnea

Dyspnea, or breathlessness, is one of the most common complaints of subjects with COPD during exercise (129). Despite the recognition of its prevalence in this population for many years, the sources and mechanisms of dyspnea remain poorly understood. From an integrative standpoint, one can view dyspnea as a mismatch between respiratory motor output and the physiological/mechanical response, a process frequently referred to as neuromechanical uncoupling (130). Perhaps not surprisingly, the sensation of respiratory effort is closely related to the level of diaphragm activation (131). Such uncoupling between respiratory effort and response can occur as a result of the mechanical properties of the respiratory system at elevated lung volumes, where lung compliance is decreased and large changes in intrathoracic pressure translate into relatively smaller changes in lung volume

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(compared to lower operating lung volumes). The descriptive correlate of this phenomenon is frequently referred to as an ‘‘unsatisfied inspiratory effort’’ (58). Several studies have shown strong relationships between the magnitude of dynamic hyperinflation and the intensity of dyspnea during exercise (132–135). What are the events leading to the intolerable sensation of dyspnea during exercise in patients with COPD? As we have discussed in the previous

sections, both V A/Q mismatching and mechanical limitations to increasing minute ventilation predispose patients to the development of hypoxemia and hypercapnia (83,84,133), both of which are powerful stimulants of the drive to breathe. As ventilatory drive increases, so do expiratory pressures, resulting in premature airway closure that drives EELV upward and places the subject on the poorly compliant region of the lung’s pressure–volume relationship. When the lung is operating at such elevated volumes, neuromechanical uncoupling is worsened and the sensation of dyspnea is intensified (135).
Furthermore, VA is compromised due to the reductions in tidal volume, which worsen hypoxemia and hypercapnia, and the vicious cycle continues until inevitable subject intolerance is reached. Dyspnea is frequently reduced significantly by the administration of supplemental oxygen, which improves exercise tolerance not only by improving oxygen delivery to the exercising muscles (as discussed earlier) but also by reducing the hypoxic ventilatory drive. The reduction of hypoxic ventilatory drive reduces respiratory muscle pressure production as a result of reductions in motor output, which in turn reduces dynamic hyperinflation and improves neuromechanical coupling by placing the lung on a more compliant region of its pressure–volume relationship. The hypothesis that dynamic hyperinflation and the associated unsatisfied inspiratory efforts limit exercise performance are supported by the observation that supplemental oxygen (which lowers EELV) or the unloading of the respiratory muscles using mechanical ventilation (which improves neuromechanical coupling) results in dramatic reductions in dyspnea at any given workload and improvements in exercise tolerance (56,84,134,136). However, confounding the interpretation of the effects of alleviating dyspnea with hyperoxia on exercise performance is the fact that CaO2 is also significantly increased, which increases O2 delivery to the locomotor muscles. Dyspnea is reduced following exercise training, and has often been attributed to reductions in peripheral muscle lactate production, which decrease ventilatory drive and allow for better maintenance of EELV at any given external workload (137). However, it is important to note that reductions in ventilation and increases in exercise tolerance have been observed in the absence of changes in lactate production (138); these changes appear to be a result of improved ventilatory mechanics (e.g., reduced dead space ventilation) in such cases.

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Leg Fatigue

As many as 80% of patients with COPD complain of severe leg discomfort and fatigue during exercise, and up to 50% state that leg discomfort is the main factor limiting their exercise performance (54). This observation is not surprising given the marked changes in the peripheral musculature in these patients. Of great interest is the observation that the magnitude of increase in exercise capacity following exercise training is related not only to the severity of ventilatory impairment at baseline but also to the degree of peripheral muscle weakness and intensity of leg discomfort at maximal exercise (139).




Is V O2MAX Ever Reached?


VO2MAX is strictly defined as an increase in the external workload without an additional increase in oxygen consumption. Those familiar with exercise testing know that such criteria are rarely met in the normal, healthy population, since a high intensity of exercise requires the tolerance of a great deal of discomfort. Can we expect such an effort from patients with COPD in the presence of severe hypoxemia, hypercapnia, and neuromechanical uncoupling? In all likelihood, no. Even when plateaus in limb VO2 are observed with increasing workloads, whole-body VO2 continues to rise and does not plateau (118). Although the physiological mechanisms discussed herein undoubtedly contribute to exercise intolerance, our understanding of the limits of physical capacity in vivo will ultimately be limited by interindividual variability in the tolerance of dyspnea and discomfort inherent in volitional exercise testing. References 1. 2.

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Wagner PD. New ideas on limitations to VO2MAX. Exerc Sport Sci Rev 2000; 28:10–14. Warburton DE, Gledhill N, Quinney HA. Blood volume, aerobic power, and endurance performance: potential ergogenic effect of volume loading. Clin J Sport Med 2000; 10:59–66. Stray-Gunderson J, Musch TI, Haidet GC, Swain DP, Ordway GA, Mitchell JH. The effect of pericardectomy on maximal oxygen consumption and maximal cardiac output in untrained dogs. Circ Res 1986; 58:523–530. Roca J, Agusti AGN, Alonso A, Poole DC, Viegas C, Barbera JA, RodriguezRoisin R, Ferrer A, Wagner PD. Effects of physical training on muscle O2
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Johnson BD, Saupe KW, Dempsey JA. Mechanical constraints on exercise hyperpnea in endurance athletes. J Appl Physiol 1992; 73:874–886. McAllister RM, Laughlin MH. Short-term exercise training alters responses of porcine femoral and brachial arteries. J Appl Physiol 1997; 82:1438–1444. Weibel ER, Taylor CR, Hoppeler H. Variations in function and design: testing symmorphosis in the respiratory system. Respir Physiol 1992; 87:325–348. Ross KA, Thurlbeck WM. Lung growth in newborn guinea pigs: effects of endurance exercise. Respir Physiol 1992; 89:353–364. Johnson LR, Rush JW, Turk JR, Price EM, Laughlin MH. Short-term exercise training increases ACh-induced relaxation and eNOS protein in porcine pulmonary arteries. J Appl Physiol 2001; 90:1102–1110. Clanton TL, Dixon FG, Drake J, Gadek JE. Effects of swim training on lung volumes and inspiratory muscle conditioning. J Appl Physiol 1987; 62:39–46. Shephard RJ, Godin G, Campbell R. Characteristics of sprint, medium and long-distance swimmers. Eur J Appl Physiol 1974; 32:99–116. Halseth AE, Fogt DL, Fregosi RF, Henriksen EJ. Metabolic responses of rat respiratory muscles to voluntary exercise training. J Appl Physiol 1995; 79:902–907. Babcock MA, Pegelow DF, Johnson BD, Dempsey JA. Aerobic Fitness effects on exercise-induced low-frequency diaphragm fatigue. J Appl Physiol 1996; 81:2156–2216. Cerny FJ, Dempsey JA, Reddan WG. Pulmonary gas exchange in non-native residents of high altitude. J Clin Invest 1973; 52:2993–2999. Massaro D, Massaro GD. Pre- and postnatal lung development, maturation, and plasticity (Invited Review: Pulmonary alveoli: formation, the ‘‘call for oxygen,’’ and other regulators). Am J Physiol Lung Cell Mol Physiol 2002; 282:L345–L358. Lindstedt SL, Hokanson JF, Wells DJ, Swain SD, Hoppeler H, Navarro V. Running energetics in the pronghorn antelope. Nature 1991; 353:748–750. Karjalainen E-M, Laitinen A, Sue-Chu M, Altraja A, Bjermer L. Evidence of airway inflammation and remodeling in ski athletes with and without bronchial hyperresponsiveness to methacholine. Am J Respir Crit Care Med 2000; 161:2086–2091. Anderson SD, Holzer K. Exercise-induced asthma: is it the right diagnosis in elite athletes? J Alergy Clin Immunol 2000; 106:419–428. Hopkins SR, Schoene RB, Henderson WR, Spragg RG, Martin TR, West JB. Intense exercise impairs the integrity of the pulmonary blood-gas barrier in elite athletes. Am J Respir Crit Care Med 1997; 155:1090–1094. West JB. Cellular responses to mechanical stress: pulmonary capillary stress failure. J Appl Physiol 2000; 89:2483–2489. Dempsey JA, Hanson PG, Henderson K. Exercise-induced arterial hypoxemia in healthy humans at sea level. J Physiol (London) 1984; 355:161–175. Harms CA, McClaran SR, Pegelow DF, Nickele GA, Nelson WB, Dempsey JA. Exercise-induced arterial hypoxemia in healthy young women. J Physiol 1998; 507:619–628.

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20 Pulmonary Adaptation to Sustained Changes in Metabolic Rate

JACOPO P. MORTOLA McGill University Montreal, Quebec, Canada

I. Introduction In mammals, the respiratory apparatus participates in gas exchange through two physical mechanisms: convection and diffusion. Gas convection is repre. sented by the regulation of pulmonary ventilation (VE), of which the larger . fraction reaches the gas exchange region as alveolar ventilation (VA). Then the flow of O2 from the alveoli to the blood in the pulmonary capillaries, which in . steady state corresponds to the consumption of O2 at the tissue level (VO2), is controlled by the O2 pressure gradient (yPO2) between alveoli and pulmonary capillaries, and the diffusion conductance of the gas exchange barrier V
O ¼ yPO
diffusion conductance ð1Þ 2

2

Note that the conductance is proportional to the diffusion coefficient and the surface area, and inversely proportional to the length of the diffusion pathway. In the lung, pulmonary conductance is equivalent to the diffusing capacity, DL (mlgas/min/mmHg). The pulmonary diffusing capacity, DL, is largely determined by the surface area of all the alveoli participating to gas exchange, referred to as pulmonary surface area (SAlung). 525

526

Mortola

With sudden changes in metabolic demands, such as muscle exercise or
exposure to cold, increases in VO2 are accompanied by quasiproportional
changes in VE, with only small deviations in the normal values of the alveolar and arterial PO2 and PCO2 (1,2). PAO2 ¼ Pb
½inspired O2 concentration  ðV
O2 =V
AÞ

ð2Þ

PACO2 ¼ Pb
ðV
CO2 =V
AÞ;

ð3Þ

and

where Pb is dry barometric pressure, PAO2 and PACO2 the alveolar O2 and CO2 partial pressures. The diffusion properties of the lungs change only to the extent that lung volume increases and new alveoli are functionally recruited; in essence, the widening of yPO2 and the increase of blood flow are sufficient . . to accommodate sudden increases of VO2. An increase in VO2 is accommodated by an increase in cardiac output and a decrease in mixed venous O2; this latter means a drop of the PO2 in the arterial side of the pulmonary capillaries, and a widening of the alveolar–capillary PO2 gradient. The question addressed in this chapter is whether increases in metabolic . rate sustained for weeks or months modify the convection (namely, V E control) and diffusion characteristics (notably, SAlung) of the respiratory apparatus. There are several reasons for asking this question. The development of neural functions is influenced by the nature of information received . by the nervous system. With respect to the control of VE, this concept of neural plasticity was shown by altering the afferent inputs from the chemo. receptors, which resulted in long-term effects on VE chemosensitivity (3–8). Hence, the possibility exists that with the persistent hyperpnea accompanying . the rise in VO2, sustained differences in the afferent activity from the chest wall . and vagal airway receptors may have a long-term impact on VE control. With respect to pulmonary diffusion, an increase in DL through a restructuring of . the SAlung would accommodate the higher values of VO2 with a lower yPO2 (see Eq. 1). Therefore, the advantage of such a restructuring would be that of . . expanding the metabolic scope (ratio between VO2max and VO2resting), lowering the chances that the lung may impose a limit on the maximal values . of VO2. We know that the lungs have enormous capabilities for structural adjustments, as it happens, for example, in the process of recovery after correction of diaphragmatic hernia (9), which stunted lung growth, or after tissue loss, as with pneumonectomy, even at an age when the normal process of lung growth has long ceased (10–14, also for additional references). The approach to be followed in attempting to answer the question ‘‘Do sustained changes in metabolic rate modify the lung convection and diffusion properties?’’ will be twofold. First, we will consider how the respiratory diffusion (Sect. III) and convection properties (Sect. IV) vary among species

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with huge differences in metabolic rates. This approach stems from the belief that the current design of the respiratory system, and its structural and functional variations among species, represent the optimal solutions to differences in metabolic needs. Then in Section V attention will be paid to the respiratory adaptations of animals, which, for various periods of time, have experienced prolonged changes in metabolic rate. The response to sustained changes in oxygenation (Sect. VI) will also be considered, for a comparison to what emerged from the previous analysis, and as a necessary premise to the interpretations and conclusions of sections VII and VIII. II. Glossary of Terms and Definitions The following terms and definitions are used in this chapter: a, A, v CaO2, CvO2 CO2, O2 DL F, P HVR PCO2, PO2 SAlung Tb . . VA, VE . VT . . VCO2,VO2 W

arterial, alveolar, venous arterial, venous oxygen content carbon dioxide, oxygen pulmonary diffusing capacity force, pressure hypoxic ventilatory response partial pressure of O2, CO2 pulmonary surface area body temperature alveolar, pulmonary, ventilation tidal volume carbon dioxide production, oxygen consumption body weight . . Gaseous metabolism (VO2 and VCO2) and metabolic rate are used interchangeably. Hypoventilation and hyperventilation designate, respectively, a de. crease or increase in VE relative to metabolic demands (i.e., respectively, an . increase or decrease in PACO2), irrespective of the absolute value of VE. Hypopnea and hyperpnea are, respectively, a decrease or increase in the . absolute level of VE, relative to normoxia. III. Interspecies Comparisons: Pulmonary Diffusion Volumetric, surface, and linear parameters that change among species according to strict geometrical rules scale with body weight (W), respectively, as ~W1, ~W2/3, and ~W1/3. It has long been known (15,16) that small species, per unit W, have higher values of metabolic rate than larger species. In . particular, the exponent of the allometric relationship scaling VO2 (Y axis) to

528

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W (X axis) has been consistently found to be 0.75, or very close to this value. . In other words, VO2 does not scale in direct proportion to body mass (~W1), nor in proportion to body surface (~W0.66), as one may have expected if heat production was adjusted to compensate for heat loss. Animated discussions on the interpretation of the 0.75 exponent have been frequent in the last 50 . years. The relationship VO2~W0.75 does not apply only to mammals but also to the other classes of vertebrates, invertebrates, and even unicellular orga. nisms (17). Some exceptions do occur, such as the scaling of VO2 in newborn . mammals (VO2~W0.92) (18). The generality of the metabolic relationship, and the factors responsible for it, have been the object of debate and speculations, but the matter remains unresolved. For the purpose of the present discussion, . the large range in W and metabolic needs and the fact that VO2/W changes among animals offer excellent opportunities to examine the extent of the correlation between metabolic requirements and the diffusive and conductive characteristics of the respiratory apparatus. Close correlations would not prove, but would at least agree with, the general hypothesis that metabolic rate represents an internal force, through selective evolution, in the development of the respiratory apparatus as we currently know it. This information may also tell us to what extent the lung is amenable to adaptive changes to new behavioral or environmental conditions. A. Adult and Newborn Mammals

Because the primary function of the lung is that of exchanging O2 and CO2, if the pulmonary diffusing capacity (DL) was designed to meet, but not exceed, the organism metabolic requirements, it should vary among species not . according to geometric scaling, but according to VO2. Taylor and Weibel (19) used the term symmorphosis to describe the principle that structures are designed to meet, but not exceed, the maximal requirements. In other words, DL should scale to W0.75 rather than W2/3. DL can be measured functionally, or it can be estimated from morphometric measurements. Although the two approaches do not give the same absolute numbers, their differences would be by a constant factor (14,20). Morphometric estimates can be represented by the SAlung. In fact, the alveolar–capillary distance is almost constant among species (21), and the pulmonary capillaries provide a dense covering of the alveolar surface, such that their total surface area is considered to be proportional to the alveolar area (22). In a pioneering study on mammalian species ranging in size from a few grams to more than a ton, Tenney and Remmers (23) set out to measure some parameters related to lung size and function. They found lung volume (volume of air within the lung at a fixed transpulmonary pressure) to be proportional to the first power of W (~W1), as expected by the rules of

Lung Adaptation to Metabolic Change

529

geometric similarity. The direct proportionality of lung volume (and lung weight) to W has been a consistent finding ever since (e.g., 22,24,25), and it applies also to neonatal mammals (18). Alveolar linear dimensions were neither constant among species nor proportional to W1/3; rather, they scaled to W0.14, indicating that interspecies differences in lung volumes were accommodated by changes in both the size and the number of alveoli. The SAlung, calculated from the dimensions of a sample of subpleural alveoli by light microscopy, scaled to W0.75, a result that would seem to satisfy the expectations of the lungs designed optimally to meet the functional needs of the organism. Whether the presumptive selective pressure of metabolic rate . would be better represented by the maximal value (VO2max), than the resting . or basal values of VO2 may not be a crucial question to answer because the . . ratio between VO2max and VO2resting (or what is termed metabolic scope) is, in first approximation, an interspecies constant (25,26), although some important interspecies differences are known (27). Weibel (22) performed electron microscopic morphometry on the lungs of six mammalian species ranging in size from 2.5 g to 25 kg; his results disagreed with those of Tenney and Remmers, because Weibel’s data indicated . that SAlung was directly proportional to W, and not to VO2. The discrepancy was not due to the different techniques. In fact, Weibel’s conclusion could have been obtained also by Tenney and Remmers had these authors limited their observations to land mammals and excluding the marine species. The rattling observation that the choice of the species covered by the study can modify the allometric relationship, and the conclusions that could be drawn from it, is not a rare occurrence in comparative physiology. One could argue that the inclusion of some large marine mammals with unusual low metabolic rate, such as the dugong and the manatee, is the appropriate decision to determine better the assumed correlation between SAlung and the . species’ VO2. On the other hand, the life history of marine mammals is so different from that of land species that their inclusion could artificially distort the general pattern. Further measurements by Weibel and collaborators (21), on a larger number of wild and laboratory land species, again confirmed the earlier findings, whether based on data of SAlung (~W0.95) or on morphometric estimates of DL (DL ~ W0.99); in fact, both parameters scaled to W with . . exponents significantly higher than VO2 or VO2max (Fig. 1). Lechner (25) reviewed data pertinent to only small-size mammals, between 2 and 3700 g, . and found that the allometric exponent of VO2 was 0.73 while that of SAlung . was 0.89. Hence, also his set of data confirmed that the SAlung-VO2 ratio was not an interspecies constant. A comparative analysis based on physiological measurements of DL in land mammals found DL to be more closely propor. tional to W than to VO2 (28).

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. Figure 1 Allometric relationships of maximal oxygen consumption (VO2max, left Y axis) and pulmonary diffusing capacity (DL, right Y axis). DL, like pulmonary surface area, . scales to body weight with an exponent higher than that of resting or maximal VO2. Both axes are represented in log-scale. (Slightly modified from Ref. 73.)

In newborn mammals the only data of SAlung would seem to be the ones collected by Bartlett and Areson (29) on 11 species. They found a significant . difference between the allometric relationship of SAlung and that of VO2, and . the exponent relating SAlung to VO2 was 1.2, which is significantly higher than unity. They commented that in neonates of the large species, which are usually born more developed, the relatively greater SAlung may meet the additional O2 demands for locomotion and heat balance; these needs are less compelling in the newborns of smaller species, which have limited activity and maternal heat protection. Also, the lungs of some rodents and other small species at birth are often characterized by sacs with few alveoli (30), and this reduced compartmentalization decreases the total SAlung. Whatever the reason, their data . agree with the interspecies differences in DL/VO2 among adult mammals, presumably indicating that this pattern is programmed very early in life, possibly on a genetic basis. . Going back to Eq. 1 above, the finding of a larger DL/VO2 in the larger species implies that the yPO2 of these species is less than in smaller species.

Lung Adaptation to Metabolic Change

531

Because yPO2 is the O2 pressure difference between alveoli and capillaries, the values of either of these two parameters should change systematically with body size. Alveolar size tends to increase with animal size (21,23,31), and in larger species the longer path length for diffusion and gas stratification could reduce the mean alveolar PO2 (32). However, it would be unrealistic to think that changes in alveolar PO2 alone could account for the interspecies differ. . ences in DL/VO2. In a 70 kg man alveolar PO2 is f100 mmHg. Because VO2/ SAlung scales to W0.22 (21), if differences in alveolar PO2 had to fully account . for the interspecies differences in VO2/SAlung, in a 25 g mouse alveolar PO2 should exceed 400 mmHg! In addition, the tight direct proportionality . . between VE and V O2 (see Sect. IV) also suggests that alveolar PO2 and PCO2 cannot vary much among species. The blood of smaller species has characteristically low O2 affinity (33– 35), and Weibel (22) suggested that this could be an additional factor favoring the higher yPO2 in small species. Lindstedt (36), argued that the interspecies differences in cardiac output, relative to blood volume, must cause differences in the transit time of the blood in the pulmonary capillaries; specifically, transit time is ~W0.21. In the small species, the short transit time could pose a limit to the full arterialization of the blood in the lungs, especially during exercise, widening the yPO2. Therefore, in the small species, on the one hand . VO2max may be close to the limits imposed by the time course of O2 binding to hemoglobin. On the other hand, the larger yPO2 permits achieving the same gas exchange as larger species. Finally, one should not lose sight of the possibility that factors other than gas exchange may play a role in dictating the size of the SAlung. Heat and water conservation, even in mammals, can occasionally take priority over blood gas homeostasis (37). These issues are bound to be more important in small-size species, and could set a limit to the dimensions of the SAlung, which is a large source of water and heat loss. B. Sedentary and Active Species

. The large range in body size and VO2 among mammals permits interspecies . analysis based on VO2 rather than W. In other words, it is possible to compare species, which, although similar in W, differ in metabolic needs because of their average activity levels. With this type of approach, it appears that those species with higher metabolic needs also have higher SAlung. . For example, a horse could have a VO2 almost three times higher than a cow of similar W, and a correspondingly higher DL (38), and domestic bovids . show a tendency for lower values of VO2max as well as DL, compared to wild species (21). In fact, since one of his earlier comparative studies, Weibel (22) noticed that the data of some species, mice, rats, and rabbits, could be separated from those of dogs, monkey, and shrews. He pointed out that the former animals were raised under the sedentary conditions imposed by

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generations in captivity. The latter were wild, or free-living, specimens, which presented relatively high SAlung. From a series of studies specifically designed to compare dogs with goats, and ponies with calves (39) (i.e., pairs of species with similar W but . different athletic characteristics and VO2max), it emerged that the differences . in VO2 were contributed to by differences in both DL and yPO2 (cfr. Eq. 1). The data also indicated that the blood transit time in the pulmonary capillaries was amply in excess of the time needed for O2 exchange, even in dogs and . ponies at VO2max (40). Two main points can be made in summarizing the results of these studies . on the interspecies relationships between DL and V O2. First, the lungs’ structures appear to be in excess of that needed for gas exchange, and more so in larger species. This pattern, which is already apparent at birth, differs . from the proportional correlations with VO2 found for other steps of the oxygen cascade, from the cardiovascular system to cellular respiration (41). Second, species with higher metabolic requirements do have lung structures larger than expected from their W. Hence, DL correlates both with the size and the metabolic needs of the species. The latter information confirms, after all, what Tenney and Remmers (23) proposed 40 years ago, from their comparative study that included marine species and animals with unusually . . low VO2/kg, like the sloth and the dugong. The fact that, in addition to VO2, W . per se influences the size of DL tells us that factors other than VO2 are important in the design of the lung. As mentioned earlier, water loss by evaporation through the airways could become an important issue in small species, limiting lung size. Also, if the SAlung of a very small species retained . the same proportionality to V O2 as in a large species, because surface properties pose a lower limit to the dimensions of alveoli, the lungs would need to be disproportionately big. [The collapsing pressure (P) generated at the alveolar air-liquid interface depends on surface tension g and the radius r of the alveolus (P ~ g/r; Young–Laplace relationship)]. For example, assuming that a change in lung mass was the only mechanism to change SAlung, with no changes in alveolar size, it can be calculated that in a 25 g mouse lung mass would need to be 2% of W instead of the usual 0.3%. Lung volume (at functional residual capacity) would occupy 20% of the body, instead of 3%; at total lung capacity, more than half of the body would be occupied by lung volume! C. Nonmammals

Morphometric studies of the gas exchange area and of its possible correlation with metabolic rate have been performed in different classes of animals (42– 45). However, the data accumulated are not as abundant as in mammals, limiting the value of the correlation analysis. In addition, in cold-blooded

Lung Adaptation to Metabolic Change

533

. animals values of resting VO2 can be more difficult to obtain than in mammals, as they vary drastically with temperature and oxygenation, in addition to the diurnal and seasonal changes in activity patterns. In reptiles, SAlung scales close to W0.75, whereas in amphibia the allometric exponent would be much higher (42). In amphibians, the contribution of extrapulmonary structures (e.g., skin and gills) to gas exchange is not necessarily the same for O2 and CO2, nor it is a constant fraction of the total gas exchange. Data on the surface area of fish gills are more numerous than in other classes; Schmidt-Nielsen (46), after differentiating species according to their activity behavior, concluded that ‘‘the surface area of the fish gill is related to the requirements of oxygen supply and that the surface area is scaled to the body size with a relationship similar to that for oxygen consumption.’’ Interesting observations were conducted also in eggs of different species. Toward the end of incubation, the values of PO2 and PCO2 in the egg’s air cell, before the onset of pulmonary ventilation, depend on the embryo’s metabolic rate and the diffusion conductance of the egg shell and membranes, as expressed by Eq. 1. Hence, the experimental finding that the air cell values of PO2 and PCO2 are interspecies constants indicates the existence of a close proportionality between the conductance of the structures responsible for gas diffusion and the embryos’ metabolic rate (47,48). Unusual forms of adaptation are known to occur for species laying eggs in wet and dry environments, or in the hypoxic conditions of high altitudes. Up to moderate altitudes (f2800 m) changes in egg-shell conductance favor the conservation of water, but at higher altitudes gas permeability increases and water conservation is sacrificed at the advantage of O2 exchange (48,49). IV. Interspecies Comparisons: Pulmonary Convection in Normoxia and Hypoxia The force F generated by activation of the respiratory muscles is proportional to the muscle cross-section, and, applied to the surface of the thoracic wall (S), produces the pressure (P = F/S) responsible for lung inflation. If we assume that, as a first approximation, mammals were built according to the principle of geometric similarity, among species of different body weight (W), F and S should change in proportion to each other, resulting in nearly similar P values. Because the respiratory system compliance is directly proportional to W (24,50), a constant P implies that also tidal volume (VT) is directly propor. tional to W, and this, indeed, has been experimentally confirmed (24) (VT = Crs
P, where Crs is the compliance of the respiratory system, and P represents the elastic P component, which, during resting breathing, is the largest fraction of the total P required to inhale).

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Differently from VT, breathing frequency decreases with body size (f ~ . W0.25); hence, VE scales to W10.25, or W0.75, an exponent identical to that of . . metabolic rate. In newborn species, in which VO2 scales to W0.92, VE is proportional to W0.91 (18) (VT is directly proportional to W in both newborns and adults, whereas f decreases with W, being ~W0.25 in adults, and ~W0.09 in newborn mammals) (18). Hence, in both newborns and adult . . mammals, VE scales in proportion not to W, but to the VO2 of the species. It . follows that, on average, the ventilatory equivalent, (i.e. the ratio between VE . and VO2) has similar values among species (18), and it is probably rather constant also during growth (51). . . If VE retained the same proportionality to VA at all ages, the interspecies . . constancy in VE/VO2 could imply (from Eq. 2 and 3) that the values of blood gases are all within a narrow range. In adults, the PCO2 values, obtained from the arterial blood or from tissue air pockets do not show any systematic variation with the species’ W (35,52). Probably the same conclusion can be drawn for the newborn species, although data at this age are far more limited (18). In growing rats, values of arterial PCO2 and pH remained stable, . because the decline in VCO2 with age is accompanied by a proportional . decrease in VA (53); PaO2, however, gradually dropped, presumably because . of the age-related worsening of the ratio between V E and pulmonary perfusion. The same pattern, with nearly constant PaCO2 and drop in PaO2, occurs in aging humans (54), whereas in dogs PaCO2 shows a small (f2 mmHg), yet significant, rise with age (55). The considerations made above regarding the P generation among . animals with geometrically similar structures can be extended to the V E response to hypoxia (HVR). Hence, one could anticipate that among animals of different W, the magnitude of the hypoxic hyperpnea should be a constant . proportion of the normoxic VE value. Attempts to verify this prediction experimentally, by comparing the HVR of various species at the same inspired O2 level, have led to discordant conclusions (56,57). Indeed, there is a large interspecies variability in the HVR, a variability manifest also among subjects of the same species and even within the same individual when measured on different occasions, and caused by numerous factors (58). However, if the . HVR was examined not as function of W but as function of VO2, the results . . are less scattered, and the hypoxic V O2–V E relationship falls essentially parallel and above the normoxic function (Fig. 2). The reason for a clearer pattern when metabolic rate is taken as the independent variable is that in mammals, not dissimilarly from other vertebrates, hypoxia often causes a . drop in VO2, which is especially pronounced in small species (57) and in . . newborns (59). Because the lowering in VO2 during hypoxia reduces VE, and because the degree of hypometabolism depends on numerous conditions (58), the HVR can be quite variable. Indeed, in some cases, the hypometabolism is

Lung Adaptation to Metabolic Change

535

. Figure 2 Average values of pulmonary ventilation (VE) plotted against the . corresponding values of oxygen consumption (V O2) for newborn (circles) and adult mammals (triangles) during normoxia (open symbols) and hypoxia (filled symbols). Both axes are represented in log-scale. In all cases hypoxia consisted of an inspired oxygen concentration of 10%, for a duration varying between 15 and 60 min. (Data from Refs. 18 and 57, and additional sources.)

. so pronounced that the HVR has a negative value, meaning that VE in hy. . poxia drops below the normoxic level, even though the increase in VE/VO2 and the drop in PaCO2 reveal the occurrence of normal hypoxic hyperventilation (60). In summary, among adult and neonatal mammals, lung mass, lung volume, and VT are directly proportional to W, as one may expect on the basis of structural and geometric similarities. This design of the respiratory pump, coupled with the interspecies differences in breathing frequency, allow . . VE to be directly proportional to the animal’s resting VO2. The similarity . . between the slopes of the normoxic and hypoxic V O2–V E relationships indicate a general uniformity in the magnitude of the hypoxic hyperventilatory response. The fact that this happens despite the large interspecies variability in HVR and in hypoxic hypometabolism, supports the concept . . that VO2 plays a crucial role in setting the VE level. V. Pulmonary Diffusion and Convection with Sustained Changes in Metabolic Rate This section will review what is known about the convection and diffusion properties of the lungs in human subjects or animals that, for whatever

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reason, had protracted modifications in metabolic rate. As it will be apparent, the experimental observations are usually limited to a few standard laboratory species, often mice and rats, and very few data have been gathered on the . . control of VE. Because changes in VO2 sustained for long periods of time, especially in young animals, often alter body growth, an important analytical and conceptual issue is related to the type of normalization of the data. As an example, let us consider the case of an experimental intervention . causing a sustained increase in VO2 and greater body growth. In this case, the finding of a larger SAlung could be considered of interest per se, since it implies that the alveolar region has been under a process of restructure and expansion. In interpreting this result, however, it could be argued that the larger SAlung is the unavoidable process accompanying the increased body growth, and that only an increase in the specific value (SAlung/W), rather than the absolute value, would truly indicate an effect of the intervention on the SAlung. If one accepts that the data need to be normalized, it is not immediately obvious whether lung weight, volume, or W should be the normalizing parameter. The latter is the most commonly adopted, but it is clear that it includes the weight of organs and tissues (limbs, head, subcutaneous fat) that have little to do with the lungs. Furthermore, lung mass and volumes during growth are not linearly related to W; rather, their specific values decrease with age (18), which further complicates the comparisons between experimental and control animals with different W. In the context of the present chapter, . VO2 would be the most relevant normalizing parameter, given the hypothesis that metabolic rate may be the controlling factor of SAlung. Hence, with respect to the experimental interventions to be considered, emphasis will be placed on studies that provided both metabolic and morphometric measurements. There are few such studies, and even fewer combine measures of . metabolic rate with those pertinent to the control of VE. The main findings of some of the studies here discussed are summarized in Tables 1–4. A. Level of Activity Muscle Exercise

A regimen of controlled exercise can improve aerobic fitness in various groups of patients with respiratory disorders (e.g., 61,62), and steady activity can limit the normal age-related deterioration of respiratory function (63). Nevertheless, whether physical training can specifically modify the structure of the respiratory system, and improve pulmonary gas diffusion, is not a question that is easily answered. In humans, studies are almost invariably crosssectional in design. It has been often reported that athletes have better pulmonary function test results, including a larger DL, than the general sedentary population, a difference implicitly attributed to their enhanced

=

=

na

na

na

=

=

#

#

#

na

na

na

=

=

=

/W

#

=

=

na

na

=

(abs.)

=

z

z

na

na

=

/W

Lung air vol.

=

z

na

z

z

=

Alveolar density

#

=

z

na

na

=

(abs.)

=

z

z

na

na

=

/W

SAlung

=

z

z

=

z

=

/Vol.

=

=

z

na

na

na

(abs.)

. VO2

z

z

z

na

na

na

/W

3 wk exercise (71) 4 wk exercise (72) 8 wk exercise (72) IDPN (76, 77) genetic alteration(74) genetic alteration (75)

Notes

b

Compared to stock mice Compared to phenotypically normal littermates. When not specifically indicated at the source, values . have been calculated from the data reported. W, body weight. SAlung, pulmonary surface area. VO2, oxygen consumption. abs., absolute value. na, data not provided. Alveolar density, either reported or calculated from lung volume and alveolar dimensions.

a

Mice, 3 weeks old Waltzing micea Waltzing miceb

=

=

Rats, 1 month old Rats, 1 month old 1 month old

(abs.)

W

Species

Lung weight

Table 1 Sustained Increases in Muscle Activity

Lung Adaptation to Metabolic Change 537

W

na z

na z

z z

# =

z

# = =

= z

z

=

(abs.)

z

z na =

z

z

= na =

=

/W

=

(abs.)

=

#

= = =

= z

z

=

/W

Lung air vol.

=

=

= # =

z z

z

=

Alveolar density

z

z

# # =

z z

z

=

(abs.)

=

#

= # =

z z

z

=

/W

SAlung

=

#

= # =

z =

z

=

/Vol.

na

na

# na #

na z

z

z

(abs.)

. VO2

na

na

# na #

na z

z

z

/W

104

102

71 83 82

83 82

80,81

71

Reference

When not specifically indicated at the source, values . have been calculated from the data reported. W, body weight. SAlung, pulmonary surface area. VO2, oxygen consumption. abs., absolute value. na, data not provided. Alveolar density, either reported or calculated from lung volume and alveolar dimensions.

Increased growth hormone Rat, 3 month old z female Rat, adult female z

Decreased thyroid hormones Rat, 1 month old # Rat, newborns = Hamster, 1.5 = months old

Increased thyroid hormones Rat, 1 month = old Rat, adult = female Rat, newborns = Hamster, 1.5 = mo old

Species

Lung weight

Table 2 Changes in Thyroid Activity or Growth Hormone

538 Mortola

# # # # # # # # # # #

Guinea pig day 0a dd 1–21 dd 21–40 Mice, adults Hamsters, adults Rats, adults Rats, adults Rats, adults Rats, adults Rats, neonates Rats, neonates

# na na na # # # # # # na

(abs.)

z na na na z = z = z z na

/W # # = = z # na = # # #

(abs.) = = z z z = na z = z z

/W

Lung air vol.

= = = # # = # # = # =

Alveolar density # = # # # #b # # # # #

(abs.) = fz = z z = = # = z z

/W

SAlung

= fz # # # # na # = # =

/Vol. na na na na na na na na na na na

(abs.)

. VO2

na na na na na na na na na na na

/W

113, 113, 113, 122 115 118 116 124, 143 117 121

126

133, 139 139 139

Reference

b

After caloric restriction of the dam during gestation. In this study, not SAlung but DLCO, by the single-breath method, was measured. When not specifically indicated at the source, values . have been calculated from the data reported. W, body weight. SAlung, pulmonary surface area. VO2, oxygen consumption. abs., absolute value. na, data not provided. Alveolar density, either reported or calculated from lung volume and alveolar dimensions.

a

W

Species

Lung weight

Table 3 Reduced Caloric Intake

Lung Adaptation to Metabolic Change 539

z

na

=

=

na

z

na

/W

z

z

z

(abs.)

z

z

z

/W

Lung air vol.

z

z

=

Alveolar density

z

z

z

(abs.)

z

z

z

/W

SAlung

=

=

=

/Vol.

na

z

z

(abs.)

. VO2

na

z

z

/W

146

82

145

Reference

When not specifically indicated at the source, values . have been calculated from the data reported. W, body weight. SAlung, pulmonary surface area. VO2, oxygen consumption. abs., absolute value. na, data not available. Alveolar density, either reported or calculated from lung volume and alveolar dimensions.

na

=

Rats, 1 month old Hamsters, 1.5 months old Guinea pigs, 3 weeks old

(abs.)

W

Species

Lung weight

Table 4 Prolonged Exposure to Cold

540 Mortola

Lung Adaptation to Metabolic Change

541

. physical activity and higher maximal VO2 (64–67). Better support for the possibility of a cause–effect relation between a sustained increase in activity . (and VO2) and an increase in DL would emerge from longitudinal studies, but these are few and their results are variable. Five months of vigorous physical training in university students did not produce any difference in DL (68). No effects were likewise seen in 11–13-year-old boys after 16 weeks of physical training (69). On the other hand, in a preliminary study in teenaged girls training with swimming lung volumes were large in comparison to a population of sedentary controls and increased disproportionally more when studied longitudinally over a few years (70). The variability among the findings of these and other studies may be attributed to the differences in protocols, since duration and intensity of the physical regimen, and the age at its onset, are likely to be important factors. These issues are also important in experimental studies on animals. Rats at about 1 month of age were made to run on a sloping treadmill, daily, for 20–30 min (71). After 3 weeks, none of the variables measured (W, lung weight and volume, alveolar diameter and number, and SAlung) differed from the nonexercising group. A positive result was obtained on 1-month-old rats forced to swim daily for 4 or 8 weeks (72); this exercise did not modify the normal progression of body growth but increased the alveolar density and the SAlung–lung volume ratio. Because these positive results were seen after 4, but not 8, weeks of exercise the author concluded that in the rat there is a critical period before the third month for lung proliferation. Weibel (73) mentioned data reported by H. Gehrig in a dissertation thesis of 1951, indicating that rats . forced to swim increased the SAlung in proportion to VO2. Geelhaar and Weibel (74) performed morphometric measurements on the Japanese waltzing mice, a variety of mice with a genetical vestibular defect that force them into almost constant motion. When compared to standard laboratory mice, which had larger W, the waltzers presented larger lung volumes and SAlung once these parameters were normalized by W. A later study compared these waltzing mice with littermates phenotypically normal because heterozygous for the waltzing trait and of almost similar W (75). The . waltzers had indeed higher activity and VO2/kg than non-waltzing littermates, but not larger lung dimensions, alveolar size, or SAlung, whether in absolute values or after normalization by W. Hence, the high SAlung of the waltzing mice would seem to be a genetic characteristic, just like their small W and waltzing habits, and not an acquired adaptation to their life-long activity. (Table 1). Drug-Induced Muscle Activity

An abnormally high physical activity has also been generated in mice by use of imino-h,hV- diproprionitrile (IDPN), a drug that induces hyperkinesia and

542

Mortola

choreatype movements of the head (76,77). The treated mice grew less than . controls, with higher VO2 both in absolute values and per W. The weights of several organs changed in proportion to W, but lung volume/W was increased. The internal compartmentalization of the lung was also increased, leading to higher values of the SAlung and of the morphometrically estimated DL. In the past, these results had been taken as an additional indication that . increased metabolic requirements (and VO2) can be a driving force shaping the alveolar structure. One should nevertheless be cautious about the effects of a drug such as IDPN, which has very profound and general effects on tissue metabolism and growth of individual organs, to the point that it may not be compatible with life. In addition, the blunting in body growth greatly complicates the interpretation of the results. In fact, the higher values of . VO2 and DL, after normalization by W, in the IDPN-injected mice than in the untreated controls is, at least qualitatively, what one would expect when comparing small and large animals (see Sect. III). B. Hormone-Mediated Changes in Metabolic Rate and Body Growth

Many hormones and drugs have an important impact on the lung tissue and its structural development. Some of these substances, such as corticosteroids, can modify the SAlung (78). In this section, however, attention will be paid only to thyroid and growth hormones, because of their well-documented . effects on whole body VO2 and growth. Thyroid Hormones

The effects of the thyroid on growth and development are well established, as . is its importance in controlling the VO2 level (79). Hence, chronic treatments enhancing or depressing thyroid activity have been used as an experimental approach to study the pulmonary adaptation to chronic changes in metabolic level. Bartlett (71) treated 1-month-old rats with thiouracil or thyroxine, which, respectively, depresses and enhances the activity of the thyroid. After . about 3 weeks, the VO2 of rats with hyperthyroidism was 35% higher than in controls, and 80% higher than in rats with hypothyroidism. Morphometric measurements were performed after 3 weeks of treatment; alveolar dimensions, lung volume, and SAlung did not differ significantly among groups. Later studies in which the treatment was maintained for longer periods gave different results. Desiccated thyroid supplementation of the food of rats . for 8 weeks increased VO2 and slightly decreased body growth; the lungs had larger volumes and SAlung, with more alveoli of smaller size (80,81). In hamsters (82), daily subcutaneous injection of 3,5-triiodothyronine for 4 . weeks resulted in an increase in V O2 of f37%. This treatment did not

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significantly alter W nor modify the individual alveolar size, but increased the SAlung because of a greater number of alveoli and a larger lung volume. Higher dosages of thyroid hormones (from 5 to 100 Ag) induced larger . increases in VO2, but did not increase SAlung beyond the value already attained with 5 Ag, as if this dosage was sufficient for the maximal effect on SAlung. Chronic treatment with methimazole, a depressor of thyroid activity, reduced . VO2 to 68% of the control value but had no effect on W, nor on lung morphometry (82). In neonatal rats, daily treatments to enhance or suppress thyroid activity resulted in, respectively, an increase and decrease of SAlung (83). Hence, from these data taken globally, it seems that the thyroid has an important impact on the pulmonary structures involved with gas diffusion; in particular, hyperthyroidism increases alveolar number, lung volume, and SAlung, as long as the treatment is sufficiently prolonged. Humans with reduced thyroid function, as in myxedema, often present hypoxemia, which is believed to be contributed to in part by a low DL (84). The opposite condition, that of an increased DL in hyperthyroidism that regresses with therapy, has also been reported (85). It should be noted, however, that the changes in cardiac output and pulmonary perfusion as well as in hemoglobin content with hyper- or hypo-thyroidism could explain the differences in DL, with no need for structural changes of the SAlung. Animal experimental observations on what thyroid-related changes in . VO2, may do to pulmonary gas convection are lacking. In humans, chronic hyperthyroidism is associated with increased HVR (86). The opposite condition, myxedema, is accompanied by a reduced HVR, which returned to normal after administration of thyroid hormone (87). Growth Hormone

An excess in growth hormone (or somatotropin) secretion is the cause of acromegaly or gigantism, depending upon whether the disease started, respectively, in adulthood, or in young individuals before closure of the epiphyses of the long bones. One may expect an increase in metabolic rate associated with the growth stimulation, but only few measurements are . available. In adult humans with acromegaly an increase in VO2 may occur because of the associated hyperthyroidism or a generalized hyperactivity of the pituitary gland. In adult horses chronically treated with equine somato. . tropin, resting VO2 did not change (88), whereas VO2 did increase when a similar treatment was delivered to growing pigs (89). Pulmonary function tests in male patients who have acromegaly have revealed large lung volumes and normal diffusing capacity. These results that have been interpreted as indicating that the enlargement of pre-existing alveoli, rather than an increase in their number, was the basis for the larger lung volume (90). In one patient of the same study with pituitary gigantism,

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lung volume was increased in proportion to body size. Subsequent studies in acromegalic patients have confirmed the occurrence of large lung volumes, either with normal or (less commonly) a slightly increased DL (91–96). Patients with adult onset of hypopituitarism, or with deficiency of only the growth hormone, were originally reported to have low lung volumes and a high pulmonary recoil pressure (97). Later studies on hypopituitarism in children or adults have not given uniform results with respect to the changes in lung volumes (93,98,99); DL was consistently found to be within the normal range (100,101). The fact that female acromegalic patients had far fewer pulmonary alterations than male patients (90–92) indicates that sexual hormones also play an important role in the process of lung restructuring. Adult rats with an implanted noninvasive tumor secreting not only growth hormones but also ACTH and prolactin for 3–6 weeks showed increased body weight, length, and lung weight (102). Lung volume and alveolar size also increased, the latter fully explaining the increase in volume. These effects were attributed to the increased levels of growth hormone, because qualitatively similar effects were seen in animals without adrenal glands, and, although to a lesser extent, in two rats with daily injections of . . only growth hormone for 3 weeks. VO2 and VE were increased in the tumorimplanted group, and, respectively, unchanged and increased in those with growth-hormone injections; however, these measurements were performed under barbiturate anesthesia and their meaning is therefore very questionable. In another experiment (103), injections of homogenates from a growthhormone-secreting tumor accelerated the postpneumonectomy lung growth response, and this accelerated response was prevented after hypophysectomy. Hence, these experiments, in combination with the clinical data on humans, were interpreted as suggestive of a somatotropin-mediated stimulation of lung growth. . What remains unclear is whether VO2 can be even considered a potential stimulus for the lung growth that occurs with treatment with growth . hormone, because the measurements of VO2 are too few and not convincing. In addition, it is not clear to what extent the stimulation is specific for the lung, or is part of the general process of increased body growth. This latter possibility emerges from two studies conducted in rats and guinea pigs. In one (104), adult female rats were injected daily with growth hormone for 3 weeks. (It is of interest that these and other experiments on rats had to be conducted on females; several authors have pointed out that male rats seem to be quite insensitive to growth hormone.) Treated rats had larger lung volume, alveolar number, and SAlung than controls, but the difference was what had to be expected from the differences in W. In fact, when control rats were allowed to grow until they reached the same W of the somatotropin-treated group, none of the pulmonary morphological differences was significant. Alveolar

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linear dimensions were the same for all groups, indicating that changes in lung volume, whether during normal growth or because of the growth hormone, were contributed by differences in the number of alveoli. The author pointed out that the results in the rat resembled more closely what is observed in human gigantism than acromegaly. Rats, quite differently from humans, continue to grow through their life time because the epiphyses of their long bones never close. Hence, the rat may not be the ideal animal model for human acromegaly. Essentially the same results were found in 2–4-week-old guinea pigs treated daily for 2 weeks (105). Also in this case the treated animals presented increased lung weight, volumes, and compliance, all in proportion with body weight. . Data on pulmonary convection and VE chemosensitivity in individuals . with acromegaly are almost inexistent. In acromegalic patients, the V E response to hypoxia or hypercapnia was, on average, as in controls (95) and no metabolic data were provided. C. Caloric Intake

Many studies over the last 50 years have addressed the issue of the restriction in caloric intake, or of specific nutrients, on lung growth, development and function, and several review articles have summarized this information (e.g., 106–108). Here, it will be considered only the material strictly pertinent to the theme of this chapter. In experimental animals, food restriction lowers metabolic rate, and . refeeding re-establishes the normal metabolic level (109). Also the VO2 of slices of lung tissues from food-deprived rats or rabbits was found to be decreased (110). Rat pups of small body size caused by raising them in large litters have a . lower VO2 (both in absolute values and after normalization by W) than larger pups growing in smaller litters (111). In humans, caloric restriction main. tained for several days reduces VO2; these metabolic changes regress once the normal caloric intake is re-established (112). Morphological, morphometric, and biochemical measurements of the lungs in food-restricted animals have been performed by many authors in numerous species, including the guinea pig (113,114), hamster (115), rat (116–121), and mouse (122). The findings of a decrease in the absolute values of lung weight, volume, DNA, and protein contents are not surprising, given the drop in body weight and the stunting in body growth. However, once the data are normalized (by W, or by wet or dry lung weight), the results among studies are no longer consistent. Some of the variability is introduced by differences in protocols, especially in the duration of the intervention, the timing of the measurements after termination of the intervention (e.g., 117), and the age of the animals. For example, the lung volume or lung weight of

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food-restricted animals always decreased when considered in absolute values. However, after normalization by W, lung volume could be increased when the intervention was performed on adult animals, not changed or even decreased when the intervention was performed in juvenile animals. It seems that organ hypoplasia, including the lungs, is quite marked in pups starved when young or in newborns after food limitation of the pregnant dam (e.g., 113,117,123). A consistent finding, especially in animals treated at a young age, is the increased size of the most peripheral airways, possibly with destruction of the alveolar walls. An exception seems to be offered by the guinea pig, an animal born at a very early stage of development, and with a large number of alveoli, by comparison with other rodents. Even in this species, however, SAlung, or DL estimated morphologically have been found decreased after starvation (Table 3). The increased size of the peripheral airways, and some other biochemical modifications (124,125), have led some authors to liken the lungs of food-restricted animals to those typically observed in human, or experimental, emphysema. The functional data, however, do not convey the same impression. In fact, the reduced lung recoil pressure and increased compliance characteristic of emphysema have been observed in some cases (116,126,127), but not in others (115,118,121,128), or depending upon the age of the caloric restriction (119). This variability in the mechanical characteristics is probably contributed to by the fact that starvation not only alters the connective tissue elements, in a way that may decrease lung recoil pressure and increase lung compliance, but also decreases the lung surfactant and, possibly, modifies its composition, which cause the opposite effects on lung mechanics (106,124,126,129–131). Morphological alterations qualitatively similar to those mentioned above have been observed also in the lungs of newborn mammals after prenatal starvation, obtained by limiting food intake of the dam during gestation. As the postnatal studies, these studies have demonstrated, in addition to a decrease in tissue mass and volume, protein, and DNA content, a retarded process of alveolarization that contributes to the reduced SAlung and DL (114,132,133). The situation opposite to caloric restriction, (i.e., overfeeding and obesity) in humans compromises the lung diffusive (DL) and convective . properties (VE and HVR), probably because of the mechanical load that the body fat imposes on the chest wall (134). In animals, overgrowth favored by a small litter size or artificial overfeeding seems to stimulate lung growth by increasing the rate of cell division (135,136). However, the high-fat diet of some experimental interventions greatly alters the lung biochemical composition and complicates the interpretation of the results. Rats raised in small . litters had elevated VO2 and lung mass compared to small pups raised in large

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litters (111). By 8 weeks of age, the pups with high-caloric intake presented large lung volumes, even after normalization by W, large alveolar size, and increased SAlung (137). Differently from the morphological data, measurements pertinent to . aspects of VE control following a regimen of altered caloric intake are limited. . In humans, sustained increases or decreases in VO2 with changes in the caloric . value of the diet resulted in corresponding changes in resting VE; the hyperventilatory response to hypoxia was maintained stable because the absolute . value of the HVR changed in proportion to the normoxic VO2 (112,138). In 1month-old rats that were calorie restricted during the first 2 postnatal weeks, . . resting VO2 and VE were both below the values of rats with high caloric intake, in proportion to each other. As in humans, in rats with high or low caloric . intake the HVR was adjusted to the normoxic VO2 such that the degree of hypoxic hyperventilation did not differ (111). Newborn guinea pigs, after prenatal starvation, seemed to have a poor HVR (139). On the other hand, in examining the results of that study, which did not report the metabolic responses, what seems striking is that the majority of the experimental . animals had values of VE and HVR remarkably similar to controls, despite a 20% reduction in W. In conclusion, with differences in caloric intake, both gas convection and pulmonary diffusion change, at least qualitatively, as expected from the . changes in VO2. It would be of interest, with temporally serial measurements, . to observe the time-course of the changes in VO2 and in SAlung, or DL, during fasting and recovery. In fact, despite the often proposed correlation, I am not aware of any study that performed both sets of measurements. The finding of a good temporal correlation would give some additional strength to the possibility of a mechanistic link between diet-induced changes in metabolic . rate and lung restructuring. A reduction in the size of SAlung when VO2 drops would not be an unusual phenomenon in the general physiological focus on saving energy whenever possible; it could be likened to the recovery of calcium from the bones in iguana or the reduction of the intestinal organs of reptiles and amphibia during fasting (140–142). However, the fact that the pulmonary structural response is just about the same when starvation occurs prenatally, (i.e., at a time when the lungs do not contribute to the oxygen requirements of . the fetus) suggests that VO2 is only one of the numerous factors involved in the structural response of the lungs to food limitation. D. Cold-Induced Thermogenesis

In adult mammals cold exposure stimulates shivering and nonshivering . thermogenesis, and substantially increases VO2. Newborn mammals respond . to cold with an increase in VO2, but their thermogenic effort is often short-

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lasting, and in small species it is not sufficient to prevent hypothermia. When . this occurs, it will lower VO2 because of the Arrhenius effect (or Q10, which expresses the change in reaction velocity for a 10jC change in temperature) . (18). The stimulatory effect of cold on the VO2 of some tissues is probably mediated by the increase in thyroid hormones, and hypothyroidism may significantly reduce the response to cold (79,144). Hence, prolonged cold exposure offers an excellent opportunity to study the adaptation of the lungs to sustained metabolic demands. This is also true because a mild cold stimulus has modest or no effect on body growth, making the results more readily interpretable than with other experimental approaches. In 1-month-old rats (145) cold exposure for 3 weeks did not significantly alter W, and increased lung volume and SAlung. Essentially the same results were obtained in hamsters (82) and guinea pigs (146). These authors maintained the cold exposure for variable periods of time between 2 and 18 weeks. The results were unequivocal in indicating that, at any W, lung volume and SAlung were larger in the exposed animals (Fig. 3). The difference between the two groups decreased with age, and in animals of W > 700 g the values were no longer statistically separable. Similar results were obtained in another series of experiments in which cold was coupled to hypoxia: the increased O2 demands were accompanied by a reduced O2 availability (147). Hence, cold exposure appeared to stimulate the lungs to grow toward their

Figure 3 Lung volume (V, left panel) and pulmonary surface area (SAlung, right panel) in guinea pigs-cold acclimated (filled circles) and controls (open circles). Acclimated animals had higher V and SAlung, although the difference was progressively less marked as body weight increased. Both axes are represented in log-scale. (Modified from Ref. 146.)

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adult dimensions, but not to cause a permanent restructuring of their dimensions. Mammals living in arctic conditions (e.g., reindeer, polar fox, lemming) probably have larger metabolic demands to protect body temperature. Although no data on lung volumes or SAlung of these species are . available, several morphological peculiarities that could improve VE distribution and gas exchange, such as larger or more numerous interalveolar pores and pulmonary capillaries and a thinner air–blood barrier, have been described (148). . Cold exposure raises the VO2 of the pregnant animal, but, like other forms of stress during gestation, also retards fetal development. In rats, cold exposure of the dam during gestation resulted in small neonatal pups, with . . low lung mass and volumes (149). Their VO2/W and VE/W were normal. Specific metabolic and ventilatory rate were also normal in 1-month-old rats exposed to cold for the first 3 postnatal weeks, although their breathing . pattern was slower and deeper than in control rats; in addition, their VE and metabolic responses to hypoxia and hypercapnia were within the normal range (150). In lambs previously reared in the cold, resting breathing rate was . as in those reared in warm conditions (151). Hence, the prolonged rise of VO2 . and VE that accompanies the sustained cold exposure would seem to have minimal, if any, consequences on the development of the mechanisms that regulate these variables. E. Gender and Gestation

. Men usually have higher resting VO2 than women because of their larger fatfree mass and aerobic fitness. It is of interest, however, that, in a large population spanning from 18 to 80 years of age, a small but detectable difference between men and women remains even after accounting for differences in body composition, cardiovascular fitness, menopausal status, or age . (152). To what extent this difference in VO2 is coupled with differences in the lung diffusion and convection properties is unclear, and the information is rather fragmentary. Any gender-related-difference, if present, is likely to be small and probably demonstrable only with a large and carefully controlled sample size. The issue of normalization is particularly tricky because lung dimensions change both with body size and age; yet, the two cannot be simultaneously controlled in comparing men and women. Lung volumes are smaller in girls and women than in boys and men of the same height (153). DL for carbon monoxide is less in girls and in women than in men and boys after adjustments for age, height, and hemoglobin concentration (154,155). These differences could suggest that, in humans, . SAlung differs between men and women in the same direction as resting VO2.

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. However, studies that compared men and women by measuring VO2 and parameters related to lung size (volume, DL, etc.) in the same individuals have not been performed. . In adult rats, females have slightly lower VO2 than males of the same W, . but, of course, the males are younger, a factor that itself raises their VO2. It is interesting, however, that the usual drops in W-specific lung volume and SAlung with growth are blunted in females at a time corresponding to sexual maturity (Fig. 4). The effect of this change is that at this age female rats and . mice have smaller alveoli and a larger (15–20% more) SAlung–VO2 ratio than males (156). These differences do not result from hormonal inhibition on the male but from the stimulatory effect of estrogen on the female SAlung (157). One interpretation of this sexual dimorphism is that the extra SAlung of the females is in preparation for the larger metabolic demands of gestation and lactation, which occupy these species for a large proportion of their adult life. . The 1.6–2-fold increase in V O2 eventually occurring with pregnancy or lactation is not accompanied by a further increase in SAlung. It would be informative to extend these measurements to other species with high or low reproductive burden. The restructuring of an organ in anticipation of later demands would be one example of design not dictated by immediate needs but by an evolutionary acquired process of life-time energy optimization. Faridy and co-workers (158) reported that, toward the end of gestation, pregnant rats with litters of 15–18 pups had lungs of greater mass, volumes, and DNA content than the lungs of dams of similar body weight with only 1–3

Figure 4 Body weight (W, dashed lines, left Y axis) and pulmonary surface area per unit of body weight (SAlung/W, continuous line, right Y axis) in male and female rats at different postnatal ages. After puberty, SAlung/W of females exceeds that of males. (From the data Ref. in 156.)

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pups per litter. Such a relationship could not be demonstrated for the heart, kidney, and liver. Surgical reduction of the litter, especially if performed early in gestation, hindered the enlargement of the maternal lung (159). Most intriguing was the finding that the lungs of the fetuses also changed with these interventions, approximately in proportion to the changes in the maternal . lungs. Further increases in maternal VO2 by cold exposure during gestation stimulated the growth of the lung in the dam, as expected (see Sect. V.D) but had inconsistent effects on the lungs of the fetuses (160). These results, despite the lack of morphometric data, indicate that in the pregnant rat the lungs do show some signs of growth in response to the demands of gestation. Although . not refuting the possibility of the increased maternal VO2 as a stimulus for lung growth, the results also suggest other interpretations. For example, the parallel responses of the fetal and maternal lungs during gestation to the size of the litter could suggest the role of bloodborne factors targeting the lung tissues. These could originate from the placenta, the total mass of which is proportional to litter size, or from the lungs themselves. When pneumonectomy is performed during gestation, compensatory lung growth in the dam is also accompanied by stimulation of the mitotic index and DNA in the lungs of the fetus (161,162). During the follicular phase of the cycle, PaCO2 in women is within the . range measured in men, indicating that resting V E matches the gender . difference in V O2 (163,164). Because progesterone stimulates breathing, PaCO2 decreases in the luteal phase of the cycle and even more so during pregnancy (165). Studies addressing the possibility of a gender-based-difference in HVR gave mixed results. In the only two studies comparing males . and females as function of resting VO2, the HVR was found either similar (164) or larger in women (163). In humans, small differences are difficult to demonstrate given the large variability of the HVR. In rats, the HVR is higher . in females over a wide spectrum of VO2s (166,167). The greater hyperventilation in females is confirmed by the slightly yet significantly larger drop in PaCO2 (see Eq. 3). The mechanisms for the gender-based difference in hypoxic hyperventilation are still unclear. Sex hormones, although wellknown to influence breathing, are probably not the full explanation, at least in rats, because the difference between males and females was also present before puberty, and ovariectomy did not greatly alter the females’ hypoxic response (166).

VI. Alveolar–Capillary O2 Pressure Gradient The previous sections have reviewed the experimental evidence for change in . diffusion conductance with sustained changes in VO2. In this section, Eq. 1 is

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. applied from the other angle: examining, the possibility that, for a given VO2, an inverse proportionality may exist between diffusion conductance and the O2 pressure gradient (yPO2). A. Lower yy PO2: Hypoxia

Very few studies have examined the morphological and morphometric characteristics of the lungs in species permanently living at high altitude, and the results are mixed. The comparison of populations of guinea pigs, sheep, or gophers living at sea level or at high altitudes did not reveal major differences in lung volume or SAlung (168,169). The alveolar dimension and SAlung (normalized by lung volume) of one high-altitude llama were not out . of the range expected from land mammals of comparable W and VO2 (168). A variety of mice living at high altitude presented heavier and larger lungs, and greater SAlung than their same-W counterpart living at sea level (170), Obervations on humans, natives or low-landers, living at high altitude, led to the impression that the lung adapted to sustained hypoxia by increasing the volume and, possibly, DL. Monge and Leon-Velarde (49), after reviewing the literature, concluded that ‘‘although the thoracic shape and dimensions are probably subject to genetic influences, the respiratory functions seem to be under the influence of developmental adaptations to the hypoxic environment.’’ In animals, most of the studies addressing the issue of lung growth in hypoxia were performed by exposure to normobaric or hypobaric hypoxia, for various periods of time, and the large majority of studies are on laboratory rodents. In interpreting the results, one has to take into account that body growth is usually blunted, and that the incidence of pulmonary hypertension can drastically modify the structural development of the lung. When studying the prenatal effects of hypoxia or hypoxia in the neonatal period, one also needs to consider that the maternal reaction to hypoxia, with loss in lactation and reduced care for the litter, can aggravate or modify, even qualitatively, the effects of hypoxia on the fetus and the newborn. Although in prolonged hypoxia stunted body growth is the rule, the lung is among the protected organs. To what extent lung mass, DNA, and protein contents can actually increase depends probably on the duration and intensity of hypoxia. The presence of some edema, which increases the wet, but not the dry, weight of the lung can be a confounding issue (18). Some of the studies reporting on lung volumes and SAlung are summarized in Table 5. . In these studies, measurements of VO2 have rarely been performed. However, . it is well known that in the first days of hypoxia VO2 drops, especially in small . species and almost invariably in young animals (1); then, in a few days, VO2 usually rises back to, or almost to, the control values (171,172).

z

f=

na

z

z = z # # na

na

na

na

na

#

f#

#

# # # # # #

na

=

=

=

Rats, 1 month old Rats, 3 weeks old Rats, 1 month old 1 month old Rats, newborn 9 weeks old Rats, newborn newborn Rats, 4 weeks old Rats, 3 weeks old Guinea pigs, 2.5 weeks old Dogs 2.5 months old 16 months old =

z

z

z

z z z = = =

z

=

#

(abs.)

=

z

z

na

z z z z z z

z

z

=

/W

Lung air vol.

na

na

z z =

zb =b

na

z z z z z z

z

z

=

/W

z

z

# =

z z z = = =,z

z

fz

#

(abs.)

z fz fz f= f= =,#

z

=

=

Alveolar density

SAlung

=

=

=

=

= = = # # z

=

=

=

/Vol.

na

na

na

na

na na na # # na

na

na

na

(abs.)

. VO2

na

na

na

na

na na na # = na

na

na

na

/W

33 mo @ 3100 m (177)

14 mo @ 3100 m (177)

2.5 wk @ 10.5% (176)

3 wk @ 12% (174) 3 wk @ 12.5% (184) 3 wk @ 12.5% (184) 6 dd @ 10.5% (175) 12 dd @ 10.5% (175) 3 wk @ 10.9% a (185,186) 3 wk @ 13% (187)

1 wk @ 12% (174)

3 wk @ 13.5% (182)

15 dd @ 10% (173)

Exposure time and Reference

When not specifically indicated at the source, values have been calculated from the data reported. When hypoxic exposure was in hypobaria, the corresponding fractional O2 concentration at 1 atm. is what here indicated. W, body weight. SAlung, pulmonary surface area. VO2, oxygen consumption. abs., absolute value. na, data not available. Alveolar density, either reported or calculated from lung volume and alveolar dimensions. a Hypoxia was either in normabaric or hypobaric conditions. b In this study, not SALung but DLCO, by the single-breath method, was measusred

na

na

na

na

z z z z z na

z

na

/W

(abs.)

W

Species

Lung weight

Table 5 Sustained Hypoxia

Lung Adaptation to Metabolic Change 553

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There is a good agreement among studies regarding the increase in W-specific lung weight and volume with sustained hypoxia. The negative results by Bartlett (173, Table 5) in which the animals were exposed to 10% O2 for 2 weeks were not confirmed by a subsequent study from the same laboratory using 12% O2 for 3 weeks (174). In discussing the discrepancy, these authors suggested that hypoxia, especially if severe, at first causes a substantial edema with no tissue proliferation. Later, the edema subsides and tissue growth accelerates. They also quoted some of their own unpublished observations of striking increases in lung volume and alveolar number in adult rats maintained in hypoxia for 3 months. In agreement with this interpretation, a more recent study with neonatal rats exposed for 1 or 2 weeks to 10% failed to show an increase in lung volume and SAlung (175), whereas volume expansion and tissue proliferation were found in all studies with milder hypoxic exposures (Table 5). Hence, the experimental conditions can qualitatively affect the results. In young guinea pigs exposed to 10.5% for up to 3 months, W-specific lung volumes and surface area increased; however, the difference from controls decreased with the age of the animals, and eventually, the lungs no longer differed significantly between the two groups (176). These results could signify that the hypoxia-induced acceleration in lung growth has a limit, possibly determined by the size of the thorax itself. Also, the process of acclimatization, which includes increases in the blood O2 capacity and in . VE, should reduce the yPO2, and, with it, one of the putative stimuli to the lung tissue (see below). Few studies have considered very long periods of hypoxia. In beagles maintained at 3100 m for more than 1 year, lung volume and DL increased as long as the high-altitude exposure was initiated when the animals were young puppies (177). In this respect, it is interesting that some of the pulmonary changes introduced by hypoxia can still be observed long after return to normoxia, as long as the exposure occurred in the very young animal (178). As mentioned above, however, measurements of lung morphometry in animals permanently living at high altitude have given conflicting results.

B. Higher yy PO2: Hyperoxia

Oxygen at high concentration has severe effects on the lung, and is eventually lethal. Mild hyperoxia has probably no important effects on body growth. Postnatal growth of rats in hyperoxia stopped the process of alveolarization, lowering the DNA content, mass, volume, and SA lung –volume ratio (179,180); these effects regressed only partially upon return to air (181). Hyperoxic exposures of older animals resulted in a decrease in lung volume and SAlung (173,182,183).

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VII. Interpretations The observations reviewed in the previous sections can be summarized as follows: . 1. Among species, both VO2 and SAlung increase with W, but not with the same proportionality. In fact, small species have smaller SAlung/ . VO2 than larger species. Comparisons of species with similar size and . . different VO2 reveals that SAlung depends both on W and VO2. . 2. Of the conditions known to be associated with an increase in VO2, some (increased thyroid or growth hormones, cold exposure, pregnancy) give clear indications of increased SAlung. Observations after increased muscle activity gave mixed results. . 3. A decrease in VO2 with hypothyroidism or reduced caloric intake is consistently accompanied by a drop in SAlung. 4. The increase in SAlung with chronic hypoxia is a consistent finding when the hypoxia is prolonged and not too severe. 5. The interspecies analysis indicate a remarkable close link between . . normoxic VO2 and VE and its response to hypoxia. Whether this link is maintained when an animal is subjected to sustained changes in . VO2 it is too premature to conclude, because the data are too few and scattered. Hence, whether interspecies or intraspecies, and despite the fact that each condition or experimental situation has its own peculiarities, a pattern emerges for the diffusion properties of the lung. This is structurally repre. sented by the SAlung, having some proportionality with VO2 and an inverse proportionality with the level of oxygenation. The existence of these relationships is functionally and teleologically satisfactory. However, if we could identify some potential mechanisms, we could convince ourselves that these are more than simple correlations. Many potential mechanistic pathways can . be proposed, and each condition or intervention altering VO2 or oxygenation may act in its own specific way on lung structure. The alternative possibility is that of a common mechanism responsible for triggering a chain of events that eventually leads to an alteration in lung structure. This latter eventuality is considered here, and, based on this assumption, numerous potential mechanisms can be identified and discussed. A. Whole Body or Lung Metabolism

. The VE level is often determined by the metabolic rate, both in normoxia and in hypoxia (1). Could one hypothesize that metabolic rate, or any of the variables associated with it including its gaseous components, may represent the signal for changes in lung structures? The answer must be negative if one

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takes into account that, in hypoxia or hyperoxia, metabolic rate either does not change or, often, respectively decreases and increases (1). Hence, with changes in oxygenation, the metabolic changes are often opposite to what one would need to entertain the hypothesis that changes in whole body metabolism govern the processes of lung restructuring. Metabolic changes of the lung tissue have not been extensively tested but, based on what we know, they do not occur consistently among interventions. . For example, with caloric restriction, the VO2 of lung slices drops, but in hypothyroidism, which has qualitatively similar effects on SAlung, lung tissue . VO2 does not change (144). B. Mechanical Stretch

Some in vitro experiments have indicated that pulmonary cell growth is favored by mechanical stretch (188,189), and diaphragmatic paralysis blunts . lung growth (190). Because, with an increase in VO2 as well as during hypoxia, . VE rises, one could hypothesize that the hyperpnea mechanically stimulates further lung growth. Several considerations argue against this possibility, however. Hyperoxia would seem to reduce SAlung, whereas sustained hyper. . oxia increases VE and, in young animals, also VO2 (1). Other conditions . . causing a sustained increase in VT or VE, such as vagotomy (183,191) or chronic hypercapnia (174,192–194), do not result in lung tissue growth. Hence, what is termed mechanical stretch is an unlikely explanation for the . lung growth during hypoxia, or with increases in VO2. The chorioallantoic membrane of chick embryos (195), the gills of some lower vertebrates (196,197), the tracheal system of arthropods (198), and the placentas of pregnant animals (49) are other examples of gas exchange organs that overgrow during chronic hypoxia, with no need for enhanced mechanical stimuli. C. Tissue Inhibitors

. Increases in V O2 are accompanied by increases in cardiac output and pulmonary blood flow. Hence, the hypothesis could be proposed that the increased blood flow through the lungs removes tissue-specific inhibitors of cell division, or chalons (199), favoring lung growth by a mechanism similar to that proposed for lung regeneration (200,201). This hypothesis would account for the large majority of the experimental observations reviewed in this article. However, it would not account for the fact that, during hypoxia, . some young animals do not increase VO2 nor cardiac output (202–207). On the other hand, sustained hypercapnia increases cardiac output and catecholamine concentration (208), as probably happens with other forms of stress, but does not modify the lung weight or the alveolar structure (174,193–195).

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D. Alveolar and Blood Gases

. . The general equation relating VO2 to lung diffusion (VO2 = yPO2
DL, Eq. 1) can be rewritten as ½Cardiac output
ðCaO2  CvO2 Þ ¼ ½ðPAO2  PvO2 Þ
DL;

ð1bÞ

where CaO2 and CvO2 represent the O2 content of, respectively, arterial and venous blood, and PvO2 is the partial pressure of O2 in the mixed venous blood of the pulmonary artery. Most of these variables have qualitatively . different changes in conditions of increased VO2 or in hypoxia; hence, none of them can be taken as a putative factor that may play a common role in the response of SAlung to changes in oxygenation or metabolic rate. For instance, both alveolar and arterial PO2 drop in hypoxia, whereas they hardly change with moderate increases in metabolic rates. The arterial–venous O2 difference . (whether content or pressure) widens with an increase in VO2, but remains constant (O2 content) or drops (O2 pressure) when the inspired O2 drops. The same can be said for the PAO2–PvO2 difference. From the viewpoint of CO2 transport, PaCO2 drops in hypoxia, because of the hyperventilation whereas, typically, it does not change in conditions such as cold exposure or moderate muscle exercise, because the hyperpnea matches the rise in metabolic demands. Venous CO2 increases with metabolic rate, whereas it drops in hypoxia, especially if this is accompanied by hypometabolism. A special case is that of the venous PO2 (PvO2), which drops both with a . rise in VO2 because of the increased tissue O2 extraction, and in hypoxia because of the low arterial O2 content. In a tissue capillary, PvO2 is a closer reflection of the driving pressure for O2 than the arterial value is. Because O2 is mainly transported by the Hb, as O2 is unloaded from the arterial to the venous end of a capillary, PO2 drops drastically at first, and less thereafter. Hence, the average value of PO2 in a tissue capillary must be closer to the venous value than to the arterial–venous average. Therefore, changes in PvO2 are a good reflection of changes in the PO2 gradient between the capillary and the mitochondria. Cellular hypoxia elevates many gene products, including growth factors within the lung tissue (209). Conversely, hyperoxia blunts the expression of pulmonary growth factors and their receptors (210,211). Because many enzymes (e.g., oxidases and oxygenases) in the cytosol have a high Michaelis constant (Km: concentration that halves the maximal rate of the reaction) for O2, within the 20–60 mmHg range, any of these would be well suited to signal even minute variations in PO2. If PvO2 was indeed a good indicator of the stimulus promoting the lung structural response, it should be possible to anticipate the results of some experimental interventions. For example, one would expect that the stimulatory effects of cold exposure on SAlung (Sect. V.D) should be reduced or offset by an elevation of the inspired O2, which raises PvO2. The same effect could

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possibly be reached by a previous adaptation to hypoxia, because, the hypoxia-induced polycythemia, for a given metabolic rate and CaO2–CvO2 difference, raises the PvO2. A reduction in the blood O2 capacity, as with CO poisoning or anemia (other factors being constant) should lower PvO2 and stimulate lung growth. This latter expectation would seem to contradict what is experimentally observed in 3–4-week-old rats chronically exposed to carbon monoxide, which showed no significant changes in lung structure (212). However, because in young animals these interventions are accompanied by . a drop in VO2, PvO2 may not have decreased. In normoxia, for the same . increase in VO2 (and in CaO2–CvO2 difference), the drop in PvO2 is larger in animals with low Hb-O2 affinity, which are the smaller-species (33–35). Hence, one would expect the structural response of the lung to increases in . VO2 to be more easily detectable in small than in large species. By a similar . argument, the pulmonary responses to increases in V O2 should be less apparent in species resident at high altitude, which have typically a high Hb–O2 affinity (47). Also the Hb of the fetus has a very high O2 affinity. However, because of the gas exchange characteristics of the placenta, the umbilical venous PO2 of the fetus (which is its most oxygenated blood) is a linear function of the uterine venous blood (213,214). Because of the steep portion of the Hb–O2 curve, even small changes in uterine PvO2 will affect the fetal oxygenation. Hence, a large uterine O2 consumption, as occurs with large litters, could lower both the maternal and fetal PvO2, possibly contributing to the parallel maternal–fetal pulmonary responses of some experiments reviewed above (Sect. V.E). VIII. Concluding Remarks The majority of observations indicate that the diffusion properties of the . lungs vary with VO2, both among species and within animals. The same cannot be concluded, as yet, for the convection mechanisms, but what is available, . mostly from interspecies comparison, suggests that also VE, and possibly the . gain of the HVR, change in parallel with normoxic VO2. The mechanistic interpretation of these correlations, of course, is not proved but their mere existence adds fuel to the view that the animal’s metabolic rate has been the important selective force in shaping the pulmonary gas exchange structures, and continues to do so as the animal’s needs change. The next important step would be to understand the mechanism(s) by which metabolism informs the lungs, triggering the responses that will optimize pulmonary convection and diffusion to the new needs. As proposed above, these changes, whether on a short time scale in a single individual or on an evolutionary scale among species, could occur via changes in PvO2.

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Comparative observations on the responses of animals that, because of habitats, age, or maturity at birth, have large differences in lung development and Hb–O2 affinity, could offer significant insights. In addition, several experiments can be designed to explore further the potential role of PvO2, keeping in mind that any intervention is bound to have its own peculiarities and aspects to be accounted for. Prominent among all is the fact that chronic . interventions causing sustained changes in VO2 most often also alter body growth, which is by itself a modifier of the lung properties. Same-W controls, in addition to the more customary same-age controls, could offer some insight into the tricky issue of normalization. Better yet, given the hypothesis of a causative link between metabolic requirements and lung structures, . would be to consider V O2 or other indexes of metabolic rate as the independent variable against which to refer the measures of lung convection and diffusion. References 1.

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Index

Abboud, RT, 166 Aber, V, 6–7 Accutane, 156 Acinar arrest, 8–9 Acinus, 68 Acromegaly, 544–545 Acupuncture, 136 Addis, T, 459 Adenoviruses antimicrobials and, 129–132 latent gene expression of, 89–90 Adventitia, 76–78 Affymetrix microarrays, 198–200 advantages of, 199, 208–209 as standard, 199–200 Age alveoli and, 440–442 exercise and, 484–501 FEV1 and, 440–442, 489–491

[Age] in postpneumonectomy compensatory lung growth, 456–457 pulmonary diffusion and, 528–531 pulmonary response to exercise and, 491–499 pulmonary structure/function and, 489–491 Agency for Health Care Policy and Research, 134 AHR. See airway hyperreactivity (AHR) Airflow obstruction, FEV1 measure of, 56–57, 58–59 Air sacs, avian, 338–339 Airspace enlargement, 418–419 loss-of-function models for, 419–428

573

574 Airway hyperreactivity (AHR), 116–119 pathological mechanism in, 117 prognostic importance of, 116–117 Airway obstruction, site of, 70–71 Airway reactivity, 116–117 Aldh-1, 365 All-trans retinoic acid (ATRA). See also retinoids in alveolus formation, 437–439 in alveolus regeneration, 439–440 in branching morphogenesis, 358 promyelocytic leukemia and, 153 results with, 171–175 side effects of, 154–155 as treatment, 150–152 Alpha-1 antitrypsin (A1AT), 124–125 Alsberg, E, 247–274 Alveogenesis, 418 Alveolar degeneration apoptosis and, 399–401 expression profiling and, 203–204 Alveolar development in bronchopulmonary dysplasia, 1–19 glucocorticosteroids and, 436–437 in period of septation, 435–436 retinoids in, 437–440 Alveolar ducts anatomy of, 68, 70 Alveolar gases, 557–558 Alveolarization, 364–367 in alveolar stage, 295–303 capillary network folding and, 295–298 completion of, 300–302 glucocorticoids in, 303 retinoids in, 153, 303 Alveolar macrophages (AM), 83 Aveolar sacs, 435–436 anatomy of, 68, 70

Index Alveolar stage, 4, 275, 276, 295–303 alveolarization in, 295, 300–302 capillary growth in, 298–300 capillary network folding in, 295–298 septation in, 295, 300 Alveolar ventilation, 525 Alveoli, 433–454 age and, 440–442 age/gender and, 440–442 anatomy of, 68, 70 attachment of, 103 destruction and regeneration of, 444–445 diffusing capacity of, 441 elastic recoil in, 440–441 expansion and septation of, 300 formation of, 435–436 gender and, 440–444 inflammation of, 99–114, 103–105 nitrogen washout in, 441 numbers of, 12, 442 oxygen and surface area of, 433 postnatal formation of, 435–436 quantitating, 434 regeneration of, 439–440, 444–445 size of, 435 timed expiratory volume of, 441 turnover in, 433–454 Alveolization in BPD, 3–12 BPD and, 9–11, 12–14 glucocorticoids and, 436–437 postnatal, 12–14 regeneration of, 52 retinoids and, 437–438 American Academy of Pediatrics, 36 American Thoracic Society, 53 Aminophylline, 119 Amoxicillin, 131 Amphibian lungs, 324–331 Angiogenesis, 298–300, 367. See also vascularization BPD and, 31 oxygen and, 31

Index Angiopoietin, 367–368 Animal models baboon, 11, 15 BPD and, 11, 24–26 cigarette smoke exposure in, 412–413 of COPD, 412–429 dog pneumonectomy model, 370–371 of emphysema, 416–418 guinea pig, 129–130 lung development in, 275–317 mouse, 416–428 nonmammalian, 319–354 in postpneumonectomy compensatory lung growth, 456–457, 457–481 in pulmonary convection, 533–535 in pulmonary diffusion, 527–533 retinoid treatment and, 151–152 Ansai, TW, 74 Anthonisen, NR, 131 Antibiotics, 131–132 Anticholinergic medications, 116–117. See also bronchodilators Antimicrobials, 129–132 Antioxidant enzymes, 24 Aoshiba, K, 401 Apoptosis, 307–308, 395–409 alveolar septal cell, 399–401 caspase-dependent/-independent, 396–397 CD8 cells and, 105 emphysema induced by, 414–416 expression profiling and, 203–204 inflammation and, 396–398 lung structure maintenance and, 398–399 Areson, JG, 530 Array Express, 207, 208 Astrocytes, 225 ATRA. See all-trans retinoic acid (ATRA) Attenuation statistics, 164–165

575 Avian lungs, 337–347 Azurophilic granule release, 78 Bacterial infections antimicrobials and, 129–132 COPD exacerbations and, 106–108 Bae, KT, 167 Bailey-Healy, I, 150–151 BALT, 87 Baraldo, S, 107–108 Barnes, NC, 74 Bartlett, D Jr, 530, 554 Basement membrane (BM) characterization of, 285 development of, 249 proteins, 285–287 terminal bud, 282, 284 Bates, DV, 52 Bcl-2, 397–398 Beck Depression Inventory, 160 Behavioral therapy, smoking cessation and, 134 Beghe, B, 107–108 Behzad, AR, 80 Belloni, PN, 150–151 Bensch, K, 3–4 Beta-2 agonists, 117, 118. See also bronchodilators h-agonists, inflammation reversibility and, 108–109 Beta-aminopropionitrile (BAPN), 412 h1-antitrypsin, 402 Beta-galactosidase (h-gal), 228 Biopsies, 162 Birds, lungs of, 337–347 Bland, RD, 21–49 BLAST searches, 186 Blau, HM, 217–245 Blood–gas barrier in amphibians, 328 avian, 341, 345, 347 formation of, 290–292 in reptiles, 336 Blood gases, 557–558 Blue bloaters, 54–55

576 B lymphocytes, 75 BMDCs. See bone-marrow-derived stem cells (BMDCs) BMP-4, 250–251 Bone marrow, leukocyte traffic and, 75–82 Bone-marrow-derived stem cells (BMDCs) in adult tissues in vivo, 229–235 in cardiac muscle, 232–233 cell fusion in, 235–236 in the central nervous system, 231 classic stem cell concepts and, 236–238 de novo cell fate changes in, 235–236 in the epithelium, 234 establishing cell fate changes in, 227–229 in the liver, 233–234 marrow stromal cells, 217, 234–235 plasticity of adult, 217–245 in skeletal muscle, 230–231 transplantation protocol for, 230 Bone morphogenetic protein-4 (BMP-4), 250–251 Bonikos, D, 3–4 Boschetto, P, 107–108 Botallo’s duct, 280–281 Boukedes, SS, 445 Boyden, E, 12–13 Branching morphogenesis, 355–393. See also lung development alveolar development and, 364–367 epithelial differentiation in, 362–363 epithelial–mesenchymal interactions in, 359–362 regeneration and, 370–376 vascular development and, 367–369 Brazelton, TR, 217–245 Breen, EC, 401 Brody, JS, 437 Broman, I, 12 Bronchial-associated lymphoid tissue (BALT), 87

Index Bronchioles anatomy of, 68, 70 inflammation of, 99–114 postnatal production of, 12–13 Bronchiolitis obliterans, 11 Bronchodilators airway hyperreactivity and, 116–119 BPD and, 34 classes of, 117 nebulized, 117 Bronchopulmonary dysplasia, 1–49 alveolization in, 3–12 animals models of, 24–26 arrested alveolar development in, 1–19 bronchodilators and, 34 characteristic features of, 21 definition of, 22 diuretics and, 32–33 evolution of, 1–3 fluid/salt intake and, 31–32 glucocorticoid treatment and, 28, 35–36 incidence of, 22 long-term outcomes in, 14 maternal factors in, 15, 24, 37 new alveoli and, 12–14 old vs. new, 3–12, 21, 22–24 oxygen and, 30–31 retinol treatment and, 33–34 surfactant replacement and, 26–28 survivors of, 11–12 treatment and prevention of, 14–15 ventilation and, 1, 7–9, 11, 15, 22, 28–30 Bronchospasm, 155 Brown, C, 115–147 Bruce, M, 8 Bucher, S, 550–551 Buproprion, 135 Burri, P, 13, 275–317, 288, 308, 435 Butylated hydroxytoluene (BHT), 368–369

Index Cadmium chloride, 413 Cagle, PT, 456 Calcitonin gene-related peptide (cGRP), 374 Calmodulin, 468 Calorie reduction, 414, 539 alveolar destruction and, 444–445 metabolic rate and, 545–547 regeneration and, 444–445 Campbell, EJ, 445 Campbell, MA, 445 Canadian Paediatric Society, 36 Canalicular stage, 4, 275– 277, 290–292 BPD and, 2, 3 preterm infants and, 12 Canaliculi, 290 Cantor, J, 412 Carbon dioxide BPD and, 29 in exercise, 496–499 Cardiomyocytes, 232–233 Cardoso, WV, 375–376 Casoni, G, 72 Caspases apoptosis and, 396–398 emphysema induced by, 401 Cathepsin G, 124, 125 CD3 cells, 74 CD8 cells parenchymal destruction and, 105 in peripheral airway inflammation, 102–103 CD25 cells, 74 Cell proliferation, 247–248 alveolar stage, 302–303 apoptosis and, 307–308 in postneumonectomy, 468–470 Center for Epidemiological Studies Depression Scale, 160 Central nervous system (CNS) BMDCs in, 231, 236 neural stem cells and, 225 nicotine’s effects on, 133–134 Centriacinar emphysema, 104–105

577 Centrilobular emphysema, 52 Chambers, H, 8 Cherukupalli, K, 11–12 Cho, Y-J, 185–196 Christie, RV, 52 Chronic airflow limitation (CAL). See chronic obstructive pulmonary disease (COPD) Chronic airflow obstruction (CAO). See chronic obstructive pulmonary disease (COPD) Chronic bronchitis definition of, 53 pathology of, 74 small airway obstruction in, 72–75 V/Q inequality and, 119–120 Chronic obstructive airway disease (COAD). See chronic obstructive pulmonary disease (COPD) Chronic obstructive lung disease (COLD). See chronic obstructive pulmonary disease (COPD) Chronic obstructive pulmonary disease (COPD) alveolar/brochiolar inflammation in, 99–114 animal models of, 412–429 blood flow and, 510–513 cardiac function and, 508–510 chemical/particulate exposure and, 413–414 cigarette exposure and, 412–413 clinical spectrum of, 54–55 definition of, 99 diagnosis of, 52–55, 60–62 Dutch hypothesis on, 51–52 dyspnea in, 513–514 early diagnosis of, 59 early history of, 52–54 exacerbations in, 106–108, 127–129 exercise and, 501–515 factors in development of, 57–58

578 [Chronic obstructive pulmonary disease (COPD)] FEV1 and, 58–59, 70–71 genetics in, 62 imaging modalities and, 60–61, 62–63 inflammation in, 62 leg fatigue in, 515 lung regeneration and, 62 LVR and, 63 outcomes for, 59 oxygen content limitations in, 501–508 as pandemic, 63 physiology of, 56–57 prevalence of, 59–60, 99 problems in defining, 51–52 risk factors for, 75 small airways disease in, 67–97 subclinical course of, 70–71 susceptibility to, 99–100, 404 tobacco and, 56 treatment of, 115–147 Chronic Respiratory Disease Questionnaire, 161 Chu, F, 80 Ciaccia, A, 72 CIBA symposium, 72 13-cis retinoic acid. See also retinoids side effects of, 154–155 treatment with, 153–157 Clara cells, regeneration and, 374–375 Clara-cell-secreted protein (CCSP),359 Clausen, JC, 119–120 Clerch, L, 197–216, 433–454 Clinical trials, 157–174 goals/outcome measure for, 159–171 inclusion/exclusion criteria for, 157–159 lung remodeling biomarkers and, 168–171 physiological response monitoring in, 161–162 quality of life measures in, 160–161

Index [Clinical trials] radiographic imaging and, 162–168 results from, 171–175 toxicity monitoring in, 159–160 Clinidine, 135 Cloning, 232 Coalson, JJ, 1–19, 25 Cochrane Collaboration, 115, 136 Cochrane Database, 130 Cold exposure, 540, 547–549 Collagenase (MMP-1), 126. See also matrix metalloproteinases (MMPs) Collagens ECM turnover and, 252–253 in lung development, 250–251, 286–287 Comorbid conditions, 152 Computed tomography (CT), 60–62 clinical trial protocols, 162–164 emphysema diagnosis with, 157–158 high-resolution, 162 QIA and, 162, 164–168 Continuous positive airway pressure (CPAP), 28–29 Cooper, JD, 167 COPD. See chronic obstructive pulmonary disease (COPD) Corbino, L, 72 Corticosteroids antibiotics and, 131–132 clinical efficacy of, 127–128 inflammation reversibility and, 109, 124–129 inhaled, 127–128 oral, 128–129 retinoids and, 159 Cosio, M, 72 Costarangos, C, 123 Cosxson, HO, 74–75 Coxson, H, 80 Crypts of Lieberku¨hn, 218 Crystal, RG, 467

Index CT. See computed tomography (CT) Culpitt, SV, 127 CXCL10, 102 CXCR3, 102 Cytodifferentiation, 251 Cytokines bronchiolar inflammation and, 101 in ECM turnover, 252–253 in immune response, 83–84, 86 leukocyte traffic and, 75–82 lung branching morphogenesis and, 252, 253–254 Cytoskeleton (CSK) cell shape and, 256–260 ECM and, 248 tension in lung development, 261–262 Dahms, B, 8 Dantzker, DR, 119–120 Data visualization, 213–215 Data warehouse design, 206–209 DD. See differential display (DD) del Riccio, V, 355–393 Dempsey, JA, 483–524 Denaturing polyacrylamide gel electrophoresis, 191–192 Density mask analysis, 166–168 Depression, 155, 160 Desai, R, 6–7 Desmosines, 170 Destructive index (DI), parenchymal, 104–105 Dexamethasone, 35–36, 303 Diaphragm, 505–506 Differential display (DD), 185–196 development of, 186 factors affecting, 194–195 fluorescent, 186, 191–193 isotope-labeled, 190–192 materials in, 188 methods in, 189–194 Northern blot analysis of, 188, 194

579 [Differential display (DD)] polymerase chain reaction for, 190–191 principles of, 186–188 Differential screening, 185 Diffusing capacity (DL), 441 COPD and, 507 Diffusion conductance, 525 Dipnoi, 321–324 Diuretics, 32–33 DNA (dioxyribonucleic acid) expression profiling, 197–215 in postneumatic lung growth, 460 reamplification of cDNA bands, 192, 194 spotted vs. Affymetrix microarrays of, 198–199 Doerschuk, CM, 80 Double capillary arrangement, 323–324, 336 Downey, GP, 80 Doxycycline, 131 Dry-powder inhalers (DPIs), 117–118 Ductus arteriosus, 280–281 BPD and, 15, 28 Dueck, R, 119–120 Dunnill, MS, 71 Dutch hypothesis, 51–52 DyBuncio, A, 166 Dyspnea, 513–514 ECM. See extracellular matrix (ECM) Edema BPD and, 31–33 diuretics and, 32–33 Edwards, D, 3–4 EELV. See end-expiratory lung volume (EELV) Egr-1, 467–468 EILV. See end-inspiratory lung volume (EILV) Elastase -induced emphysema, 151–152, 411–412 porcine pancreatic, 412

580 Elastic recoil, 440–441 Elastin alpha-1 antitrypsin and, 124–125 in alveolarization, 365 BPD and, 24, 26, 31, 33–34 extracellular matrix turnover and, 170–171 retinol and, 33–34 Elliott, M, 74–75 Elliott, WM, 107–108 Embryonic stage, 4, 277–281 esophagus/trachea development in, 279–280 lung anlage in, 277–279 pleura and lobe formation in, 280 pulmonary vasculaturization in, 280–281 Emphysema animal models of, 412–429 apoptosis and, 395–409 apoptosis-induced, 414–416 in BPD, 1 captase-induced, 401 centriacinar, 104–105 centrilobular, 52 in COPD, 103–105 definition of, 395 diagnosis of, 54, 157–158 elastase-induced vs. human, 151–152 FEV1 and, 56–59 genetic analysis of, 412–429 inflammatory cells in, 74–75 irregular, 52 mouse models of, 416–418 oxidative stress and, 402 panacinar, 104–105 panlobular, 52 pathology of, 52 protease-antiprotease hypothesis on, 395–396 QIA monitoring for, 166–168 quantification of, 60–62 retinoid treatment of, 152–153 reversibility of, 150–151 steroid hormone-induced, 402–404

Index [Emphysema] susceptibility to, 404 types of, 52, 103–104 as vascular disease, 398 V/Q inequality and, 119–120 End-expiratory lung volume (EELV), 494, 496 in COPD, 502–503 End-inspiratory lung volume (EILV), 494, 496 Endostatin, 286–287 Endothelial nitric oxide synthase (eNOS), 371 BPD and, 25–26 in postpneumonectomy compensatory lung growth, 469–470 ENOS. See endothelial nitric oxide synthase (eNOS) Eosinophils in chronic bronchitis, 74 COPD exacerbations and, 107 Ephrins, 367 Epidermal growth factor (EGF), 366–367 in lung regrowth, 471 Epithelial growth factor (EGF), 284–285 Epithelial–mesenchymal induction, 282–284 Epithelial–mesenchymal interactions, in branching morphogenesis, 359–362 Epithelium apoptosis and, 307–308 blood–gas barrier formation, 290–292 BMDCs in, 234 cell differentiation in, 287–288, 290–292 differentiation in, 362–363 embryonic development of, 248–249 evaluation of, 171 in lung development, 251–253

Index [Epithelium] mesenchymal interactions with, 251–253 in reptiles, 335–336 Epstein, RH, 456 Erythrocytes, traffic in, 79–80 Escobedo, MB, 25 E-selectin, 101 Esophagus development, 279–280 Estrera, AS, 456 Estrogen, 443–444 Exercise COPD and, 501–515 dyspnea in, 513–514 gas exchange in, 496–499 healthy ageing and, 488–501 hyperpnea and, 492–493 hypoxemia with, 120–121, 123 -induced arterial hypoxemia, 484–487 leg fatigue in, 515 limb blood flow and, 487–488 metabolic rate and, 536–542 pulmonary limitations in, 483–524 smoking cessation and, 136 in young healthy adults, 484–488 Exogenous surfactant therapy, 2, 8–9. See also surfactants Experimental design, 200–214 data visualization in, 213–214 data warehouse design, 206–209 noise in, 209–213 statistical analysis in, 214–215 supervised training/test studies, 201–202 time series studies, 202–206 variables in, 200–201, 209–213 Expiratory flow limitation, in exercise, 493–496 Expression profiling, 197–216, 404 data visualization in, 213–215 data warehouse design in, 206–209 experimental design in, 200–213 noise in, 209–213 spotted vs. Affymetrix arrays in, 198–200

581 [Expression profiling] statistical analysis in, 214–215 supervised training/test study in, 201–202 time series study in, 202–206 unsupervised hierarchical clustering in, 210–211, 212 variables in, 200–201, 209–213 Extracellular matrix (ECM) cell traction and, 254–256 integrins and, 252, 253–254 lung branching morphogenesis and, 252, 253–254 in lung development, 247, 285–287 micromechanics of, 254–256 turnover in, 170–171, 251–253 Fabbri, LM, 72, 107–108 Facchini, FM, 72 Faridy, EE, 550–551 Feasibility of Retinoid Therapy for Emphysema (FORTE), 174–175 Fetal period, 281–294 FEV1. See forced expiratory volume (FEV1) FGFs. See fibroblast growth factors (FGFs) Fiberoptic bronchoscopy, 74 Fibrillins, 287 Fibroblast growth factors (FGFs), 284–285 10, 250–251, 284–287, 357 airspace enlargement and, 419, 425 in branching morphogenesis, 357, 359–361 in lung development, 250–251 Fibroblasts, 80 Fibronectin, 252–253 Fibrosis alveolar attachment and, 103 BPD and, 1, 8, 15 Fine, A, 375–376 Fletcher, JG, 123 Fletcher, RV, 123

582 Fluorescent-activated cell sorting (FACS), 222–223, 228–229 Fluorescent differential display (FDD), 186, 191, 192, 193 F-met-leu-phe (FMLP), 78 FMLP. See f-met-leu-phe (FMLP) Forced expiratory volume (FEV1), 56–57, 58–59 age and, 440–442, 489–491 CD-8 cells and, 89 as diagnostic standard, 70–71 as early sign, 61–62 rate of decline in, 105–106 small airway obstruction and, 72 smoking cessation and, 132 as standard in COPD diagnosis, 56–57, 58–59 Forced vital capacity (FVC), 58–59 Formichi, B, 107–108 Formoterol, 118 FORTE. See Feasibility of Retinoid Therapy for Emphysema (FORTE) Foxf1, 368–369 Functional residual capacity (FRC), 70–71 age and, 489–491 Furosemide, 32–33 Gain-of-function models, 418–419 Garvin, L, 150–151 Gelatinase B, 125, 126 Genbank, 186 Gender alveoli and, 440–444 lung development and, 499–501, 549–551 Gene expression, 185–196 Genetic engineering, 416–428 Genetics, 52 differential gene expression, 185–196 expression profiling, 197–216 latent adenoviral gene expression, 89–90 Gestation, 550–551

Index Ghezzo, M, 72 Gierada, FD, 167 Gigantism, 543–545 Gilbert, KA, 463, 467 Gills, 325 Global Initiative for Chronic Obstructive Lung Disease (GOLD), 54, 58–59, 63, 115 Glucocorticoids in the alveolar stage, 303 in alveolus formation, 436–437 BPD and, 28, 35–36 GM6001, 402 Goblet cells bronchiolar inflammation and, 101–102 differentiation of, 287–288 metaplasia of, 74, 124–125 GOLD. See Global Initiative for Chronic Obstructive Lung Disease (GOLD) Goldin, JG, 149–184, 166 Gorecka, D, 122–123 Gorzelak, K, 122–123 Green fluorescent protein (GFP), 228, 376 Gross, P, 412 Growth factors in lung development, 250–251, 284–285 soluble, 250–251 Growth hormone, 543–545 H. influenzae, 130 Harari, S, 107–108 Harlan, JM, 80 Harris, TA, 127 Harvey, CS, 437 Hayashi, S, 74–75 Hematopoietic stem cells (HSCs), 217, 218. See also bone-marrowderived stem cells (BMDCs) differentiation pathways in, 219–220 identification of, 222–223 isolation of, 224–225

Index [Hematopoietic stem cells (HSCs)] long-term reconstituting capacity, 219 short-term reconstituting capacity, 219 Henson, PM, 80 Hepatic stem cells, 225–226 Hepatocyte growth factor/scatter factor (Hgf), 361–362 in lung regrowth, 469 Hes-1, 362–363 Heterokaryons, 221–222 Hepatocyte nuclear factor 3h (Hnf3h), 356–357 High-resolution chest CT (HRCT), 162 Hislop, A, 6–7, 12 Histamine responsiveness, 116–117 Histodifferentiation, 251 Hoffman, EP, 197–216 Hogg, JC, 57–58, 67–97, 72 Homeobox gene, 222 HSCs. See hematopoietic stem cells (HSCs) Hsia, CC, 456, 462–463 Huang, S, 247–274 Human leukocyte antigen (HLA), 87 Human umbilical vein endothelial cells (HUVECs), 401 Husain, A, 8–9 HU-time curve, 168 HUVECs. See human umbilical vein endothelial cells (HUVECs) Hyaline membrane disease (HMD), 1 Hyaluronidase, 412 Hyde, DM, 80 Hypercapnia, 322, 504 BPD and, 29 Hypercholestolemia, 154 Hyperinflation, 494, 496 Hyperlipidemia, 154, 159 Hyperoxia, 554 BPD and, 11, 30–31 Hyperpnea, 492–493 Hyperthyroidism, 542–543 Hypertriglyceridemia, 154

583 Hypnotherapy, 136 Hypocapnia, 29 Hypopituitarism, 543–545 Hypoxemia cardiac function and, 509 in COPD, 507–508 in exercise, 496–499, 507–508 exercise-related, 120–121, 123, 484–487, 504 oxygen supplementation and, 119–123 pathophysiology of, 120–121 in sleep, 121, 123 Hypoxia environmental, 322 gene products in, 557–558 lung growth in, 552–554 pulmonary convection and, 533–535 Hypoxia-inducible transcription factors, 369 IgA, 87–88 IgM, 87–88 IL-ih. See interleukin 1 beta (IL-1h) Imaging modalities, 60–62 Immune response, 82–89 adaptive, 83, 85–88 innate, 82–84 Inducible nitric oxide synthase (iNOS), 25 Inflammation alveolar, 103–105 apoptosis and, 396–398 BPD and, 15, 24, 26 bronchiolar, 100–103 changes in severe COPD, 105–108 in COPD, 62 corticosteroid therapy and, 124–129 induced sputum and, 74–75 inflammatory cells in, 107–108 innate/adaptive immune response and, 82–88 leukocyte traffic and, 75–82 PMNs in, 75–82

584 [Inflammation] protease-antiprotease hypothesis and, 395–396 reversibility of, 108–109 smoking and, 107–108, 428–429 vicious circle hypothesis on, 130 Influenza vaccination, 130 Ingber, DE, 247–274 Inorganic dusts, 414 iNOS, 25 Integrins, 252, 253–254 basement membrane and, 285–287 Interalveolar pores, 306, 308 Intercellular adhesion molecule 1 (ICAM-1), 101 Interferon (IFN), 83, 102 Interferon (IFNg), 102 Interleukin 1 beta (IL-1h), 83 Interleukin-10 (IL-10), 101 International Classification of Diseases, 54 Intersaccular septa, 292 Intra-acinar airways, 68, 70 Intrathoracic pressure, 508–509 Intrinsic posititive end-expiratory pressure (iPEEP), 503–504 Intussusceptive growth, 298–300, 302 Iptratropium bromide (IB), 118 Irregular emphysema, 52 Islets of Langerhans, 226, 227 Isotope-labeled differential display, 186, 190–192 Jackson, SK, 437 Jeffrey, PK, 74 Jobe, AH, 26 Kaltenborn, WT, 166–167 Kang, K, 401 Kaplan, NB, 437 Keratinocytes, 227 Kinsella, M, 166 Klienerman, J, 100 Knudson, RF, 166–167 Kotton, DN, 375–376

Index Kruppel-like factor 2, 203 Kuehl, TJ, 25 Kutka, N, 123 LaBarge, MA, 231, 235–236 Lagasse, E, 233–234 Lamina propria, 76–78 Laminins, basement membrane and, 285–286 Landesberg, LJ, 467 Lange, P, 72, 73 Langston, C, 13, 456 Larson, J, 11–12 Laser capture microscopy (LCM), 211 Law, DJ, 460, 462 Leaffer, D, 150–151 Lee, K, 467 Lefrak, SS, 167 Leg fatigue, 515 Leukemia inhibitory factor (LIF), 83 Leukocyte protease inhibitor, 24 Leukocytes, traffic of in the lungs, 75–82 Liang, P, 185–196 Liebow, A, 398 Life-style, 442 Lindstedt, SL, 531 Lineage depletion panels, 223 Lipid interstitial cells (LICs), 437, 438 Liver BMDCs in, 233–234 hepatic stem cells, 225–226 Llewellyn-Jones, CG, 127 Logvinoff, MM, 4 Long-term reconsistituting capacity (LT-HSC), 219, 223, 224 L-ornithine decarboxylase (ODC), 468 Loss-of-function models, 419–428 Lower respiratory tract infections (LRTIs), 75 antimicrobials and, 129–132 Luce, RE, 72 Luckett, RA, 123 Lumen occlusion, 72–73

Index Lung anlage, 277, 279–280 Lung development, 247–274 alveolar stage, 4, 275, 276, 295–303 BPD and, 2, 3 branching morphogenesis in, 355–393, 356–362 calorie intake and, 545–547 canalicular stage in, 4, 276, 290–292 cell shape and cytoskeleton in, 256–260 cold exposure and, 540, 547–549 cytoskeletal tension in, 261–262 differentiation in, 362–363 early, 356–359 embryonic, 248–260, 277–281 epithelial–mesenchymal interactions in, 251–253 extracellular matrix in, 253–256 gender and, 549–551 in hypoxia, 552–554 integrins in, 253–254 mammalian, 275–317 mechanochemical model of, 260–262 point of birth and, 277, 294 prenatal, 277–281 pseudoglandular stage in, 4, 276, 282–290 regeneration and, 355–393 saccular stage of, 4, 275–277, 292–294 soluble growth factors in, 250–251 stages of, 4, 275–277, 355–356 zones of prenatal, 288–290 Lungfish, 321–324, 326, 327 Lung Health Study, 56, 127–128, 132 Lungs age/gender and, 440–442, 499–501 amphibian, 324–331 avian, 337–347 body mass and, 443–444 common structures in, 320–321 dimensions of human, 278 evolution of, 320–321 growth of, 308–310 lungfish, 321–324

585 [Lungs] multichambered, 335 nonmammalian vertebrate, 319–354 nonrespiratory roles of, 321 postpneumonectomy compensatory growth of, 455–481 reptilian, 331–337 single chambered, 335 Lung transplantation, 149–150 Lung volume reduction (LVR) surgery, 63 concerns about, 150 lung pathology studies in, 107–108 QIA and, 167 Lymphocytes B, 89 bronchiolar inflammation and, 100, 102–103 immune response and, 82–85 migration of, 85 parenchymal destruction and, 104–105 T, 89 traffic of, 81–82 Lysyl oxidase, 170–171 Ma, BY, 375–376 Macklem, PT, 57–58 Macrophage inhibitory protein (MIP), 83 Macrophages in BPD, 24 bronchiolar inflammation and, 100, 102–103 COPD origins and, 125–126 corticosteroids and, 127–128 in lung extracellular matrix turnover, 170–171 Maestrelli, P, 72, 107–108 Maina, JN, 319–354 Major histocompatibility complex (MHC), 83 Manfreda, J, 131 Mao, CP, 150–151 Mao, JT, 149–184

586 Mapp, CE, 72 Margraf, L, 8 Marijuana, 168–169 Marrow stromal cells (MSCs), 217, 234–235. See also bonemarrow-derived stem cells (BMDCs) Mash-1, 362 Massaro, D, 150, 153, 197–216, 300–301, 433–454 Massaro, GD, 150, 153, 197–216, 300–301, 433–454 Maternal chorioamnionitis, 24, 37 Matrix metalloproteinases (MMPs) airspace enlargement and, 418–419 cell traction and, 255–256 corticosteroids and, 125–126 in lung branching morphogenesis, 252 in lung extracellular matrix turnover, 170–171, 251–253 MMP9, 125, 126 MMP12, 125–126 small airways disease and, 89 steroid hormone-induced emphysema and, 402 Matsuba, K, 71, 89 Maximal inspiratory pressure (MIP), 491 Maximal relaxation rate (MRR), 506 McGowan, SE, 437 Mclean, K, 52 Mechanotransduction, 254 Medical Outcomes Study Short Form-36, 160 Medical Research Council, 121 Mercer, RR, 300 Meshi, B, 74–75, 89, 107–108 MessageClean Kit, 189–190 Metabolic rate, 525–571 alveolar-capillary oxygen pressure gradient and, 551–554 cold exposure and, 540, 547–549 hormone-mediated changes in, 542–545

Index [Metabolic rate] interspecies comparisons in, 527–559 in nonmammals, 532–533 pulmonary convection and, 533–535 pulmonary diffusion and, 527–533 in sedentary/active species, 531–532 sustained changes in, 535–551 terms/definitions in, 527 whole body vs. lung, 555–556 Metered-dose inhalers (MDIs), 117–118 Methylprednisolone, 402–404 Methylxanthines, 34, 117–119. See also bronchodilators Mice expression profiling and, 211–212 ROSA, 228 Microarrays, 197–216 data format for, 207–209 quality control and, 209, 213 spotted vs. Affymetrix, 198–200 Microfabrication technology, 259 Microvascular beds, 76–78 Miller, JD, 483–524 Miller, T, 123 Minimum information about a microassay experiment (MIAME), 207, 208 Mitotic index, 460 MMP-1. See collagenase (MMP-1) MMP9. See gelatinase B; matrix metalloproteinases (MMPs) MMPs. See matrix metalloproteinases (MMPs) Mononucleate proliferative myoblasts, 237 Monti, S, 107–108 Moore, K, 247–274 Morphogenesis. See lung development Morrison, NJ, 166 Mortola, JP, 525–571 Moschopulos, M, 288 Msx-1, 222 Mucosal-associated lymphoid tissue (MALT), 87

Index Mucosal lymphoid follicles, 88 Mucus gland hyperplasia, 61–62 Mucus hypersecretion, 72–73 bronchiolar inflammation and, 101–102 Mullen, JBM, 72 Muller, NL, 166 Multinucleate myotubes, 237 Multipotent progenitor cells, 219–220 Murphy, TF, 130 MyoD, 200 Myoseverin, 222 Nagai, A, 401 Nagay, A, 108 Naphthalene, 374 Nasal cannula systems, 121, 122 National Cancer Institute Clinical Toxicity Criteria, 159–160 National Emphysema Treatment Trial, 63 National Health Lung Education Program (NHLEP), 59, 63 National Heart, Lung, and Blood Institute (NHLBI), 115 FORTE study, 174–175 National Institutes of Health, 22 National Lung Health Education Program, 54 Natural killer (NK) cells, 83 NCBI GEO, 207–208 Necrosis, 397. See also apoptosis Nestin-positive cells, 227 Neural stem cells, 225 Neuroendocrine bodies (NEBs), 374 Neurological development, 35–36 Neurons neural stem cells and, 225 Purkinje, 231, 236 Neutrophils BPD and, 24 bronchiolar inflammation and, 100–103 in chronic bronchitis, 74 COPD exacerbations and, 106–107

587 [Neutrophils] elastase and, 124–125 in vitro vs. in vivo response of, 78–79 Nicotine, effects of, 132–134. See also smoking Nicotine replacement therapy, 63, 135. See also smoking Nidogen-1 basement membrane and, 286 Niewoehner, DE, 100 Nitric oxide (iNO), 34 in lung regrowth, 469–470 Nitrogen washout, 441 Nocturnal Oxygen Therapy Trial (NOTT), 121 Nonmammalian vertebrate lungs, 319– 354 Northern Blot analysis, 188, 194 Northway, W, 1–4 Nortriptyline, 135 Obesity, 546–547 Oligodendrocyte precursor cells (OPCs), 237 Oligodendrocytes, 225 Organogenesis, 275. See also lung development Orlic, D, 232 O’Shaughenessey, TC, 74, 89 Overfeeding, 546–547 Owen, CA, 445 Oxidant-mediated injury, 414 Oxidative stress, 402 Oxygen alveolar/circulatory constraints in diffusion of, 506–508 BPD and, 30–31 exercise and, 484–485, 494–496 hypoxemia and, 119–123 hypoxia and lung growth, 553–554 indications for, 122 nocturnal, 121, 123 value of, 122–123 Oxygenation, 1–19, 22, 23, 30–31. See also ventilation

588 Panacinar emphysema, 104–105 Pancreatic stem cells, 226–227 Panlobular emphysema, 52 Papi, A, 107–108 Parabronchi, avian, 342–344 Pare, PD, 72, 74–75 Parenchyma avian, 338, 341 destruction of, 104–105 postpneumonectomy compensatory growth of, 455–481 Paul, JL, 108 Perikarya, 324 Peripheral airways alveolar inflammation and, 103–105 bronchial inflammation and, 100–103 emphysema and, 104–105 lesions in, 102 Peripheral musculature, 512–513 Phosphorylglyceral kinase (PGK), 417 Pink puffers, 54–55 Platelet-derived growth factors (Pdgf), 364–367 in lung regrowth, 471, 475 overexpression of, 418–419 Pleura, development of, 280 PMNs. See polymorphonuclear leukocytes (PMNs) Pneumococcal vaccination, 130–131 Pneumonectomy, 370–371, 455–481 compensatory lung growth after, 455–481 Pollution, 76 Polte, T, 247–274 Polymerase chain reaction (PCR), 186 differential display and, 190–191 Polymorphonuclear leukocytes (PMNs), 74, 75, 413 activation of, 78 deformability of, 79–80 leukocyte traffic and, 75–82 margination of, 78 migration of, 80–82 smoking amount and, 75

Index Porcine pancreatic elastase (PPE), 412 Pores of Kohn, 306, 308 Porter, D, 1, 2 Posititive end-expiratory pressure (PEEP), 459 intrinsic, 503–504 Post, M, 355–393 Postpneumonectomy compensatory lung growth, 455–481 age and size in, 456–457 arterial blood flow in, 458 biochemical/morphological analysis of, 459–463 cell proliferative phase in, 469–470 early phase in, 463, 467–469 factors modulating, 457–458 late phase in, 470 mechanisms for inducing, 456–459 models to augment or inhibit, 470–475 molecular responses in, 463–470 phases in, 460, 462, 463, 467–470 tissue distortion/stretch in, 458–459 Premature infants, BPD in, 1–49 Prescott, E, 72, 73 Priming, 78 Promyelocytic leukemia, 153, 155 Protease/antiprosease imbalance, 124–129, 149 Protease-antiprotease hypothesis, 395–396 Protein expression, 228 Proteoglycans, 252–253 Proteolytic enzymes, ECM turnover and, 252–253 Pseudoglandular stage, 4, 275, 276, 282–290 Pseydotumor cerebri, 154 Pulmonary diffusion, 527–533 Pulmonary endocrine cells (PNECs), differentiation of, 362–363 Pulmonary function tests (PFTs), 161–162 Pulmonary microvascular endothelial cells (PMVCs), 438

Index Pulmonary surface area, 525–526 Pulmonary vascularization, 280–281 Pulmonary ventilation, 525 Purkinje neurons, 231, 236 Quality control, 209, 213 Quality of life, 160–161 ATRA and, 172 Quantitative computed tomographic morphometry (CTM), 61–62 Quantitative image analysis (QIA), 162, 164–166 emphysema monitoring with, 166–168 Quokka wallaby, 300 Radiographic imaging, 162–168 clinical trial protocols, 162–164 Raldh-2, 365 Ramalingam, R, 467 Randell, SH, 300 Rannels, DE, 463, 467 RANTES. See regulated upon activation normal T-cell expressed and secreted (RANTES) RARs. See retinoic acid receptors (RARs) Ratemales, I, 74–75 Rea, F, 107–108 Regeneration. See also postpneumonectomy compensatory lung growth alveolar, 444–445 ATRA in alveolus, 439–440 biomarkers of, 168–171 BPD and, 12 branching morphogenesis and, 370–376 calorie reduction and, 444–445 Clara cells in, 374–375 COPD and, 62

589 [Regeneration] dog pneumonectomy model for, 370–371 FGFs in, 360–361 muscle, 204 potential morhogenetic factors for, 372–373 RA in, 365–366 stem cell plasticity and, 222 stem cells and, 371–376 time series studies of, 203–206 vascularization and, 367–369 Regulated upon activation normal T-cell expressed and secreted (RANTES), 107 Reid, L, 72 Remmers, JE, 443, 528–529, 532 Reptilian lungs, 331–337 Respiratory distress syndrome (RDS), 23, 26–27 Respiratory Function in Disease (Bates, Christie), 52 Retamales, I, 107–108 Retinoic acid (RA), in lung regrowth, 471 Retinoic acid receptors (RARs), 152 in alveolus formation, 438 as biomarkers of lung activation, 169–170 Retinoic X receptors (RXRs), 438 Retinoids accumulation of in the lung, 152–153 in alveolar development, 437–440 in the alveolar stage, 303 approved for human use, 153 in branching morphogenesis, 358 clinical availability of, 153–155 clinical trial goals/outcome measures for, 159–171 clinical trial inclusion/exclusion criteria for, 157–159 clinical trial results with, 171–175 emphysema treatment with, 152–153 evaluating as treatment, 149–184

590 [Retinoids] evaluating derivatives of, 155–157 inflammation reversibility and, 109 inhaled, 156–157 lung remodeling biomarkers and, 168–171 nonselective, 156 physiological responses and, 161–162 quality of life and, 160–161 radiographic imaging and, 162–168 receptor expression as biomarker, 169–170 receptor-selective, 156–157 side effects of, 154–155 toxicity of, 159–160 Retinoid X receptors (RXRs), 152 Retinol, 33–34 Rho, 261–262 Rice, D, 100 Rinopathy, 30–31 RNAimage Kit, 190–191 RNA (ribonucleic acid) DNase treatment of, 189–190 expression profiling, 197–216 isolation of from cell cultures, 189 in postneumonectomy lung regrowth, 467 reverse transcription of mRNA, 190 RNAspectra Red Kit, 191 ROCK inhibitors, 261–262 Rogers, R, 74–75 Rosan, R, 1, 2 Rosengart, TK, 467 Roth, MD, 149–184 Rotschild, A, 11–12 S. pneumoniae, 131 Saccular stage, 4, 275, 276, 277, 292–294 Saetta, M, 72, 107–108 Salamanders, 330 Salmeterol, 118 Salt intake, 31–32 Sanderson, EA, 375–376 Sanii, MR, 550–551

Index Satellite cells, 218, 226 Schittny, JC, 275–317 Sciurba, F, 74–75 Secreted protein, acidic and rich in cysteine (SPARC), 286 Septa in amphibians, 328 apoptosis and, 399–401 cell death in, 414–416 corticosteroids and formation of, 436–437 critical period for, 436–437 development of, 292, 295, 300, 302–303 immature/primitive, 296 lungfish, 323–324, 326, 327 maturation of, 304, 305 period of septation, 435–436 secondary, 298, 299 Serial analysis of gene expression (SAGE), 207 Sethi, S, 130 Shh. See sonic hendgehog (Shh) Short,RHD, 300 Short-term reconsistituting capacity (LT-HSC), 219, 224 Siddiqui, N, 8–9 Single Gene Query Tool, 203–206 Skeletal muscle stem cells, 226–227 Skin as respiratory pathway, 326, 330 stem cells, 227 Sleep, hypoxemia with, 121, 123 Sliwinski, P, 122–123 Small airways anatomy of, 67–70 BPD and, 11 chronic bronchitis and, 72–75 in COPD, 67–97 hyperresponsiveness of, 13–14 immune response and, 82–88 leukocyte traffic and, 75–82 pathology of disease in, 71–72

Index [Small airways] site of obstruction in, 70–71 structural changes in, 100–101 Smoking animal models in, 412–413 anti-smoking campaign and, 60 bronchitis and, 58–59 clinical trial inclusion and, 158–159 in COPD, 56 Dutch hypothesis on, 51–52 inflammation and, 107–108, 428–429 mouse models of, 426–428 nicotine replacement therapy, 63 peripheral airway structural changes and, 100–101 retinoid treatment during, 154–155 as risk factor, 99–100 Smoking cessation, 132–136 nicotine replacement therapy in, 63, 135 personal/social factors in, 134 quit rate, 132–134 therapies, 132–136 Smooth muscles in amphibians, 331 cell differentiation in, 287–288 hypertrophy of, 102, 103 in reptiles, 336–337 Sobonya, RE, 4, 11 Somatotropin, 543–545 Sonic hendgehog (Shh), 284–285 in branching morphogenesis, 357–358 in epithelial differentiation, 363 in lung development, 250–251 SPARC. See secreted protein, acidic and rich in cysteine (SPARC) Spirometric gating, 163 Spirometry in clinical trials, 161–162 early diagnosis with, 59, 157 FEV1 and, 58–59 SPORE, 58–59 Squamous metaplasia, 1, 8, 74

591 St. George’s Respiratory Questionnaire, 160–161 Standard operating procedures (SOPs), 209 Standen, JR, 166–167 Starvation, 414 Statistical analysis, 214–215 Stem cells. See also bone-marrowderived stem cells (BMDCs) adult issues in vivo and, 229–235 airway regeneration and, 371–376 in the alveolar stage, 302–303 bone-marrow-derived, 236–238 cloning, 221 concepts in, 218–220 definition of, 236–238 detecting, 220 differentiation pathways in, 218–220 establishing cell fate changes in, 227–229 fusion of in vitro, 221–222 hematopoietic, 222–225 hepatic, 225–226 identifying, 222–223 neural, 225 pancreatic, 226–227 plasticity mechanisms of, 235–236 regeneration and, 222 skeletal muscle, 226 skin, 227 tissue-associated adult, 224–227 tissue-specific nature of, 218 transdifferentiation of, 221 types of, 222–223 undifferentiated, 218, 220 Steroids in BPD, 2, 23 inflammation reversibility and, 109 Stocker, J, 8–9 Stockley, RA, 127 Stone, RM, 167 Stratum basalis keratinocytes, 218 SU5416, 414–416 Substractive hybridization, 185 Summer, RS, 375–376

592 Superoxide production, 78 Surfactants airway epithelium and, 171 blood–gas barrier and, 291–292 in BPD, 2, 23, 26–29 exogenous surfactant therapy, 2 in mouse models, 425–426 Takeda, S, 456 Taraseviciene-Stewart, L, 395–409 Taussig, LM, 4 Tenascin-C (TN-C), 286 Tenney, SM, 443, 528–529, 532 TGF-h. See transforming growth factor-h (TGF-h) Theophylline, 118–119 Theriault, A, 4 Thet, LA, 460, 462 Thiazides, 32–33 Thiazidespironolactone, 32–33 Thiouracil, 542–543 Thurlbeck, W, 11–13, 52, 57–58, 71, 89, 108, 456 Thyroid activity, 538, 542–543 Thyroid transcription factor-1 (Ttf-1), 358–359, 366, 425 Thyroxine, 542–543 Time series studies, 202–206 sampling issues in, 205–206 TIMPs. See tissue inhibitor of metalloproteinases (TIMPs) Tiotropium, 118 Tissue distortion/stretch, 458–459, 556 Tissue heterogeneity, 211 Tissue inhibitor of metalloproteinases (TIMPs), 251–253, 428 Tissue inhibitors, 556 TNFa. See tumor neurons factor alpha (TNFa) Tobiasz, M, 122–123 Tomashefski, J, 8 Total lung capacity (TLC), 494, 496 Toxicity monitoring, 159–160 Trachea, development of, 279–280

Index Transcription factors, 356–359 in postpneumonectomy lung regrowth, 467–469 Transdifferentiation, 232, 235 Transforming growth factor-h (TGF-h), 284–285 cell traction and, 255–256 in lung development, 361, 366–367 Transforming growth factor-h1 (TGF-h1) in lung development, 250–251 Transgenic models, 418–419 Trans-retinoic acid all-, 150–152 BPD and, 33–34 Tree frogs, 326, 330 Trimethoprim-sulfamethoxazole, 131 Tristetraproline (TTP), 467–468 Ttf-1. See thyroid transcription factor-1 (Ttf-1) Tuberculosis, 52 Tuder, RM, 395–409 Tumor necrosis factor, 252 Tumor neurons factor alpha (TNFa), 83, 84, 87–88 Turato, G, 72 Two-dimensional protein electrophoresis, 185 UCLA ATRA Emphysema Study, 171–174 University of Pittsburgh Spore Lung Cancer Screening program, 58–59 Unsupervised hierarchical clustering, 210–212 Vaccaro, C, 437 Van Tuyl, M, 355–393 Van Velzen, D, 8 Vascular endothelial growth factor (VEGF) apoptosis-induced emphysema and, 414–416 BPD and, 25, 31, 33

Index [Vascular endothelial growth factor (VEGF)] in lung development, 284–285, 367–368 lung structure maintenance and, 398–404 parenchymal destruction and, 105 in postpneumonectomy compensatory lung growth, 470, 475 retinol and, 33–34 Vascularization, 367–369 artery/vein development, 288, 294 capillary fusion in, 304, 306–307 capillary growth, 298–300 capillary network folding, 295–298 capillary network formation, 292 epithelial differentiation and, 290–292 leukocyte traffic and, 76–78 microvascular maturation, 303–308 pulmonary, 280–281 Vasculogenesis, 367 VEGF. See vascular endothelial growth factor (VEGF) Ventilation. See also oxygen BPD and, 7–9, 11, 15, 22 continuous positive-pressure, 28–29 high-frequency mechanical, 29–30 Ventilation/perfusion (V/Q) inequality, 119–120 Venturi masks, 122 Vestbo, J, 72, 73 Vicious circle hypothesis, 130 Viral infections antimicrobials and, 129–132 COPD exacerbations and, 106–108 parenchymal destruction and, 105

593 VISDA, 213–215 Visual quantitation scoring systems, 164–165 VLA-1 positive cells, 74 Voelkel, NF, 395–409 Wagner, PD, 120, 401 Walker, DC, 80 Wang, Y, 197–216 Warren, CPW, 131 Weibel, ER, 529 Weiss, M, 308 West, JB, 119–120 West, WW, 108 Wigglesworth, J, 6–7 Wiggs, B, 72 Williams, MC, 375–376 Win, RK, 80 Wise, RA, 115–147 Wnt signaling, 286–287 World Health Organization, 63, 99, 115 Worthen, GS, 80 Wright, JL, 72 Wu, EY, 456 Y27632, 261–262 Yokohori, N, 401 Young, SL, 300 Yusen, RD, 167 Zeltner, T, 13 Zielinski, J, 122–123 Zinc-finger transcription factors, 467–469 Zonal development, 288–290