Endothelial Dysfunction and Inflammation (Progress in Inflammation Research)

Progress in Inflammation Research

Series Editor Prof. Michael J. Parnham, PhD Director of Science MediMlijeko d.o.o. 10000 Zagreb Croatia Advisory Board G. Z. Feuerstein (Wyeth Research, Collegeville, PA, USA) M. Pairet (Boehringer Ingelheim Pharma KG, Biberach a. d. Riss, Germany) W. van Eden (Universiteit Utrecht, Utrecht, The Netherlands)

Forthcoming titles: Muscle, Fat and Inflammation: Sustaining a Healthy Body Composition, S.A. Stimpson, B. Han, A.N. Billin (Editors), 2011 The Inflammasomes, I. Couillin, V. Petrilli, F. Martinon (Editors), 2011 Proteases and their Receptors in Inflammation, N. Vergnolle, M. Chignard (Editors), 2011 Antimicrobial Peptides and Innate Immunity, P.S. Hiemstra, S.A.J. Zaat (Editors), 2011 (Already published titles see last page.)

Endothelial Dysfunction and Inflammation

Shauna M. Dauphinee Aly Karsan Editors

Birkhäuser

Editors Shauna M. Dauphinee Aly Karsan BC Cancer Research Centre 675 West 10th Avenue Vancouver BC, V5Z 1L3 Canada

Library of Congress Control Number: 2010928044

Bibliographic information published by Die Deutsche Bibliothek Die Deutsche Bibliothek lists this publication in the Deutsche Nationalbibliografie; detailed bibliographic data is available in the internet at http://dnb.ddb.de

ISBN 978-3-0346-0167-2 The publisher and editor can give no guarantee for the information on drug dosage and administration contained in this publication. The respective user must check its accuracy by consulting other sources of reference in each individual case. The use of registered names, trademarks etc. in this publication, even if not identified as such, does not imply that they are exempt from the relevant protective laws and regulations or free for general use. This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, re-use of illustrations, recitation, broadcasting, reproduction on microfilms or in other ways, and storage in data banks. For any kind of use, permission of the copyright owner must be obtained. © 2010 Springer Basel AG P.O. Box 133, CH-4010 Basel, Switzerland Part of Springer Science+Business Media Printed on acid-free paper produced from chlorine-free pulp. TCF ∞ Cover design: Markus Etterich, Basel Cover illustration: Early atherosclerotic lesion in LDL receptor deficient mice. With friendly permission by Myron Cybulsky. Printed in Germany ISBN 978-3-0346-0167-2 e-ISBN 978-3-0346-0168-9 9 8 7 6 5 4 3 2 1 www.birkhauser.ch

Contents

List of contributors Preface

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John M. Harlan Endothelial activation and dysfunction in sepsis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1

Grietje Molema Heterogeneity in responses of microvascular endothelial cells during inflammation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 Stephanie T. de Dios, Christopher G. Sobey and Grant R. Drummond Oxidative stress and endothelial dysfunction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37 Fanny Desjardins and Jean-Philippe Gratton Post-translational regulation of eNOS activity in inflammation . . . . . . . . . . . . . . . . . . . 65 Dominique Yelle, Lakshmi Kugathasan, Robin E. MacLaren and Duncan J.Stewart Endothelial dysfunction in pulmonary hypertension . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81 Carol Yu, Arpeeta Sharma, Andy Trane and Pascal Bernatchez Endothelial dysfunction in systemic hypertension. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103 Bodo Levkau Sphingosine-1-phosphate as a mediator of endothelial dysfunction during inflammation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 119 Wang Min, Ting Wan and Yan Luo Dissecting TNF-TNFR1/TNFR2 signaling pathways in vasculature. . . . . . . . . . . . . . . . 137

Contents

Anna M.D. Watson, Aino Soro-Paavonen and Karin A. Jandeleit-Dahm AGE-RAGE signalling in endothelial dysfunction and atherosclerosis in diabetes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161 Jenny Jongstra-Bilen and Myron I. Cybulsky Regional predisposition to atherosclerosis – An interplay between local hemodynamics, endothelial cells and resident intimal dendritic cells. . . . . . . . . . . . . . 175 Mary Y.K. Lee and Paul M .Vanhoutte Inflammation and endothelial dysfunction with aging. . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189 Elizabeth A. Ellins and Julian P. Halcox Clinical approaches to assess endothelial function in vivo. . . . . . . . . . . . . . . . . . . . . . . . . 201 John H. Boyd and Keith R. Walley Therapeutic approaches towards targeting endothelial dysfunction Index

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Pascal Bernatchez, Providence Heart and Lung Institute at St. Paul’s Hospital, James Hogg Research Centre, 1081 Burrard St, room 166, Vancouver (BC) Canada, V6Z 1Y6; e-mail: [email protected] John H. Boyd, Critical Care Research Laboratories, 1081 Burrard Street, Vancouver, BC Canada V6Z 1Y6; e-mail: [email protected] Myron I. Cybulsky, Toronto General Hospital, 200 Elizabeth Street, Max Bell Research Centre, 2-402 R9, Toronto, ON M5G 2C4, Canada; e-mail: myron.cybulsky @utoronto.ca Fanny Desjardins, Institut de recherches cliniques de Montréal (IRCM), 110 des Pins Ave West, Montreal, QC, H2W 1R7, Canada; e-mail: fanny.desjardins@ircm. qc.ca Stephanie T. de Dios, Monash University, Department of Pharmacology, Building 13E, Wellington Road, Clayton Campus, Clayton, Victoria 3800, Australia; e-mail: [email protected] Grant R. Drummond, Monash University, Department of Pharmacology, Building 13E, Wellington Road, Clayton Campus, Clayton, Victoria 3800, Australia; e-mail: [email protected] Elizabeth A. Ellins, Cardiff University, Wales Heart Research Institute, Heath Park, Cardiff CF14 4XN, UK; e-mail: [email protected] Jean-Philippe Gratton, Institut de recherches cliniques de Montréal (IRCM), 110 des Pins Ave West, Montreal, QC, H2W 1R7, Canada; e-mail: jean-philippe. [email protected]

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Julian P. Halcox, Cardiff University, Wales Heart Research Institute, Heath Park, Cardiff CF14 4XN, UK; e-mail: [email protected] John M. Harlan, Harborview Medical Center, Mailstop 359756, 300 Ninth Ave, Seattle WA 98115, USA; e-mail: [email protected] Karin A. Jandeleit-Dahm, Baker IDI Heart and Diabetes Institute, PO Box 6492, St Kilda Road Central, Melbourne, Victoria 8008, Australia; e-mail: [email protected] Jenny Jongstra-Bilen, Toronto General Hospital, 200 Elizabeth Street, Max Bell Research Centre, 2-402 R9, Toronto, ON M5G 2C4, Canada; e-mail: jbilen@ uhnres.utoronto.ca Lakshmi Kugathasan, Terrence Donnelly Vascular Biology Laboratories, St Michael’s Hospital, Toronto, ON, M5B 1W8, Canada; e-mail: lakshmi.kugathasan@utoronto. ca Mary Y.K. Lee, Department of Pharmacology and Pharmacy, Li Ka Shing Faculty of Medicine, L2-48, Laboratory Block, 21 Sassoon Road, Pokfulam, Hong Kong; e-mail: [email protected] Bodo Levkau, Institut für Pathophysiologie, Zentrum für Innere Medizin, Universitätsklinikum Essen, Hufelandstr. 55, 45122 Essen, Germany; e-mail: bodo. [email protected] Yan Luo, State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou, China; e-mail: [email protected] Robin E. MacLaren, Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, ON, K1H 8L6, Canada; e-mail: [email protected] Wang Min, Interdepartmental Program in Vascular Biology and Therapeutics, Department of Pathology, Yale University School of Medicine, 10 Amistad St, 401B, New Haven, CT 06520, USA; e-mail: [email protected] Grietje Molema, University Medical Center Groningen, Dept. Pathology & Medical Biology, IPC EA11, Hanzeplein 1, 9713 GZ Groningen, The Netherlands; e-mail: [email protected]

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Arpeeta Sharma, Providence Heart and Lung Institute at St. Paul’s Hospital, James Hogg Research Centre, 1081 Burrard St, room 166, Vancouver (BC) Canada, V6Z 1Y6 Christopher G. Sobey, Monash University, Department of Pharmacology, Building 13E, Wellington Road, Clayton Campus, Clayton, Victoria 3800, Australia Aino Soro-Paavonen, Division of Nephrology, Department of Medicine, Helsinki University Central Hospital, Helsinki, Finland Duncan J. Stewart, Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, KIH 8M5, Canada; e-mail: [email protected] Andy Trane, Providence Heart and Lung Institute at St. Paul’s Hospital, James Hogg Research Centre, 1081 Burrard St, room 166, Vancouver (BC) Canada, V6Z 1Y6 Paul M. Vanhoutte, Department of Pharmacology and Pharmacy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hongkong; e-mail: [email protected] Keith R. Walley, Critical Care Research Laboratories, 1081 Burrard Street, Vancouver, BC Canada V6Z 1Y6; e-mail: [email protected] Ting Wan, Eye Center, Affiliated Second Hospital, College of Medicine, Zhejiang University, Hangzhou, China; e-mail: [email protected] Anna M.D. Watson, Baker IDI Heart and Diabetes Institute, PO Box 6492, St Kilda Road Central, Melbourne, Victoria 8008, Australia; e-mail: anna.watson@bakeridi. edu.au Dominique Yelle, Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, ON, KIH 8M5, Canada; e-mail: [email protected] Carol Yu, Providence Heart and Lung Institute at St. Paul’s Hospital, James Hogg Research Centre, 1081 Burrard St, room 166, Vancouver (BC) Canada, V6Z 1Y6

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Preface

The vasculature is an extensive network of blood vessels that distributes oxygen and nutrient-rich blood throughout the body. The endothelium, which represents the inner cell lining of blood vessels, exhibits complex spatial and functional heterogeneity that can be disturbed by prolonged cellular activation. Endothelial dysfunction is a general term used to describe a diminished capacity to produce vasodilators and a tendency toward a prothrombotic and proinflammatory state. The pathological changes associated with endothelial dysfunction include a loss of vascular integrity and permeability functioning, thrombosis, leukocyte infiltration into the surrounding tissue and increased cytokine production. This endothelial activation and dysfunction culminates in the clinical manifestations of various inflammationassociated disease states, such as hypertension, diabetes, atherosclerosis and sepsis. This volume was designed to be a source of information for academic scientists, clinicians and trainees interested in endothelial biology and inflammation. Although the fields of vascular biology and inflammation are vast, this book is intended to provide an overview of endothelial dysfunction and the associated consequences of vascular injury in inflammatory disease. We hope that a single volume including a range of topics on the molecular basis of endothelial dysfunction to the therapeutic approaches used to target this dysfunction will assist the reader in deriving connections in the field that are imperative in translational research. We have organized the material to provide an introduction to endothelial activation and dysfunction, specifically as it relates to bacterial infection. This is followed by a description of the heterogeneous responses of the endothelium in different vascular beds. Since endothelial dysfunction is often characterized by a reduction in nitric oxide (NO), we have included several chapters discussing various aspects of NO biology, including the contribution of oxidative stress to reduced NO bioavailability, post-translational regulation of endothelial NO synthase (eNOS), and the relevance of NO to pulmonary and systemic hypertension. These chapters are followed by a look at the role of sphingosine-1-phosphate as a mediator of inflammation and cardiovascular function. This is succeeded by a detailed review of tumor necrosis factor receptor (TNF-R) signaling and the distinct pathways

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and endothelial functions elicited by different receptors for TNF. Next are several chapters on disease pathologies associated with inflammation, including diabetes and atherosclerosis, as well as a chapter on the association between aging, chronic inflammation and endothelial dysfunction. Finally, the book concludes on a clinical note, with a discussion of the methods used to assess endothelial function and current therapeutic approaches. We are grateful to the many expert scientists that have contributed to this volume and assisted greatly in its preparation with insightful reviews of their respective fields. April 2010

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Shauna M. Dauphinee Aly Karsan

Endothelial activation and dysfunction in sepsis John M. Harlan  Harborview Medical Center, Mailstop 359756, 300 Ninth Ave, Seattle WA 98115, USA

Abstract Severe sepsis is a major cause of morbidity and mortality worldwide. Recent advances in our understanding of the pathophysiology of sepsis have emphasized the pivotal role of the innate immune system in the development of a deleterious host response to bacterial infection. It is now recognized that the endothelium is an important effector cell of the innate immune system. This review examines evidence for endothelial dysfunction in experimental sepsis as manifested by activation of coagulation and fibrinolytic systems, alterations in vasomotor tone, increased permeability, augmented leukocyte adhesion, and enhanced apoptosis. As discussed here, many of these perturbations are observed in patients with severe sepsis, suggesting that endothelial dysfunction is also important clinically and may be an important target for new therapeutic approaches.

Introduction Severe sepsis and its major sequelae of multiple organ dysfunction and shock are major causes of morbidity and mortality worldwide, affecting millions each year and increasing in incidence [1]. Although there have been substantial improvements in antimicrobial therapy and supportive critical care, severe sepsis remains the leading cause of death among hospitalized patients in non-coronary intensive care units. Unfortunately, decades of clinical trials with a variety of drug candidates have largely failed to reduce mortality [2]. Consequently, there is an urgent need to gain a better understanding of the pathophysiology of severe sepsis and to identify new therapeutic targets. This chapter examines selected aspects of how endothelial cells respond to and contribute to severe sepsis, emphasizing clinical correlations.

The sepsis continuum Sepsis is defined as infection plus systemic manifestations of infection as evidenced by the presence of two or more systemic inflammatory response syndrome (SIRS) Endothelial Dysfunction and Inflammation, edited by Shauna M. Dauphinee and Aly Karsan © 2010 Springer Basel

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criteria (e.g., fever, tachycardia, leukocytosis) and has a mortality of 5–10% in hospitalized patients. Severe sepsis is defined as sepsis plus sepsis-induced organ dysfunction and mortality rates are about ~30%. Septic shock is defined as sepsisinduced hypotension persisting despite adequate fluid resuscitation and mortality rates are over 50%. The sepsis continuum, as defined by sepsis, severe sepsis, and septic shock, reflects increasing clinical-pathological severity in the host response to microbial infection [1, 2]. Although infection is the inciting event, uncontrolled infection is rarely the dominant factor clinically. Rather, it is the response of the host innate immune system, which evolved to contain and eliminate the infection that drives sepsis pathophysiology [3, 4]. The septic response is highly complex and involves multiple cell types, amongst which the endothelial cell is pivotal [5–8].

Activation and dysfunction The endothelium is a dynamic and heterogeneous organ at the interface between blood and tissue. Under quiescent conditions it promotes homeostasis by sensing and transducing signals between blood and tissue, regulating blood flow and the basal trafficking of hematopoietic cells, and maintaining a non-thrombogenic surface permitting the flow of blood. When perturbed, the endothelium is capable of responding rapidly to diverse stimuli such as microbial components, shear stress, coagulation proteins, cytokines, and growth factors. These ‘activation’ responses evolved for host defense against microorganisms and for repair of tissue injury, and are generally localized and beneficial. However, under some circumstances endothelial responses are detrimental to the host, i.e., there is endothelial ‘dysfunction’. This is the case in severe sepsis in which endothelial activation becomes exaggerated, sustained, systemic, and ultimately deleterious [5].

Endothelial responses in sepsis Endothelial cells express most of the key pattern-recognition receptors of the innate immune system involved in sensing and responding to bacterial infections, such as membrane toll-like receptors (TLRs) and intracellular nucleotide-binding domain, leucine-rich repeat receptors (NLRs) [3, 4, 8]. Activation of endothelial cells by mediators of sepsis, such as lipopolysaccharide (LPS) in gram-negative or lipoteichoic acid in gram-positive infections, or cytokines, such as interleukin-1-beta (IL1b) or tumor necrosis factor-a (TNF-a), results in a complex pro-inflammatory and prothrombotic phenotype and induction of certain cytoprotective genes. Multiple transcription factors, particularly nuclear factor (NF)-kB and early growth response gene-1, regulate these responses [9]. Compelling evidence for the contribution of

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endothelial activation via NF-kB to deleterious changes in experimental sepsis comes from the recent study by Ye et al. [10] in which a degradation-resistant mutant of the NF-kB inhibitor IkBa was overexpressed using an inducible, endothelialrestricted promoter. Remarkably, transgenic mice in which NF-kB activation was blocked only in endothelial cells showed improved survival, decreased permeability edema and organ infiltration by leukocytes, decreased coagulation activation, and decreased hypotension. Importantly, the transgenic mice had comparable ability to eradicate pathogenic bacteria, indicating that NF-kB-dependent endothelial cell activation played little role in host defense.

Hemostasis Normal unperturbed endothelium presents a non-thrombogenic surface to the circulation by inhibiting platelet aggregation, preventing the activation and propagation of coagulation, and enhancing fibrinolysis. Both passive and active processes regulate these activities. Direct injury to the vessel with loss of endothelial cells exposes underlying matrix, which is procoagulant by virtue of its binding to and activation of platelets. Further, endothelial cells undergoing apoptosis in response to various septic stimuli expose procoagulant phosphatidylserine on their surface, down-regulate their intrinsic anticoagulant properties, and show increased adhesion to unactivated platelets [11, 12]. Even without overt vascular injury or apoptosis, when activated by inflammatory mediators, as occurs in sepsis, the endothelium may become procoagulant (or less anticoagulant). Endothelial perturbation may contribute to the activation of a coagulation cascade that is uniformly observed in sepsis as well as to the occurrence of overt disseminated intravascular coagulation (DIC), seen in 25% of patients with severe sepsis [13]. Importantly, the development of DIC has been shown to be an adverse prognostic factor in sepsis. In DIC there is intravascular generation of thrombin, which is postulated to lead to the diffuse deposition of fibrin, thereby obstructing microvascular beds and provoking organ dysfunction [14]. Consequently, the role of the endothelium in controlling thrombin generation and fibrinolysis is of considerable importance. There are three main pathways utilized by endothelial cells to inhibit thrombin generation and limit coagulation: the antithrombin system, the protein C system, and tissue factor pathway inhibitor (TFPI) [15, 16]. Impairment of one or more of these endothelial mechanisms in sepsis could result in increased activation of the coagulation system [17]. Notably, there have been clinical trials in human sepsis targeting each of these procoagulant pathways [18–20]. The antithrombin system involves heparan sulfate proteoglycans on the luminal surface of endothelial cells. These are capable of binding and activating antithrombin III (ATIII), thereby accelerating the inactivation of several procoagulant serine

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proteases including thrombin, activated factor X (Xa), and factor IXa. Binding of ATIII to the transmembrane proteoglycan, syndecan-4, may also transduce antiinflammatory signals [21]. A trial of high-dose ATIII in patients with severe sepsis failed to show any reduction in mortality and noted an increase in bleeding in patients receiving heparin in addition to ATIII [18]. The protein C pathway involves two endothelial receptors, thrombomodulin (TM) and the endothelial protein C receptor (EPCR) [22, 23]. Protein C is cleaved and activated by thrombin bound to TM. Activated protein C (APC) then binds to EPCR, which further stimulates activation of protein C. APC is a potent endogenous anticoagulant that degrades activated coagulation factors Va and VIIIa, limiting further thrombin generation. APC bound to EPCR also activates protease-activated receptor-1 (PAR-1), and cooperative signaling by EPCR and PAR-1 can elicit cytoprotective and anti-inflammatory responses [22, 23]. TNF-a, IL-1b, and LPS have been shown to down-regulate TM, which would reduce generation of APC and tip the balance toward activation of coagulation. Administration of recombinant human APC (drotrecogin a) showed a modest survival benefit in patients with severe sepsis [19, 24]. However, its benefit was restricted to patients at high risk of death, whereas it increased bleeding risk without survival benefit in patients with low risk of death [as defined by an Acute Physiology and Chronic Health Evaluation (APACHE) score < 25 or single-organ failure] [25]. Whether the beneficial effects of APC in the subset of patients with severe sepsis are primarily related to its anticoagulant and profibrinolytic properties or also to potential endothelial protective and anti-inflammatory activities is uncertain [26]. TFPI is a Kunitz-type serine protease inhibitor, which modulates tissue factor (TF)-initiated coagulation [27]. Endothelial cells have been shown to express TF when stimulated by TNF-a, IL-1b or LPS in vitro, although it is controversial whether endothelial cells express luminal TF in vivo [28]. TFPI directly inhibits Xa and produces feedback inhibition of the factor VIIa/TF catalytic complex [27]. It is mainly produced by and bound to endothelial cells, likely to surface glycosaminoglycans. Notably, a trial of recombinant TFPI in sepsis showed no survival benefit but increased the risk of bleeding [20]. If coagulation occurs despite the many anticoagulant mechanisms, endothelial cells also provide proteins to promote fibrinolysis [15]. The endothelium is a major source of tissue-type plasminogen activator (tPA). About 40% of tPA is bound to its inhibitor, plasminogen activator inhibitor-1 (PAI-1), which is also secreted by endothelial cells in response to diverse stimuli. PAI-1 levels are often strikingly elevated in sepsis and portend a poor prognosis [29]. However, other cell types also produce PAI-1 so the endothelial origin of elevated levels seen in sepsis is unproven. Moreover, in a murine model of gram-negative pneumonia PAI-1 was found to be protective by enhancing host defense [30]. More recently, circulating microparticles generated by leukocytes and vascular cells have been shown to be a source of blood-borne TF and to contribute to

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coagulation [31]. While most microparticles are probably derived from platelets and monocytes, under conditions of drastic activation, such as occurs in severe sepsis, endothelial-derived microparticles may be an important source of circulating TF [32].

Vasomotor The endothelium plays a major role in the regulation of blood flow through the production of vasoactive mediators, including the vasodilators nitric oxide (NO) and prostacyclin, and the vasoconstrictor endothelin [33]. In particular, NO is thought to contribute to vasomotor instability in sepsis [34]. Basal production of NO occurs predominantly by conversion of l-arginine by constitutive endothelial NO synthase (eNOS: NOS1) [35, 36]. In response to inflammatory stimuli, inducible NO synthase (iNOS; NOS2) generates large amounts of NO, which contributes to the cardiac depression, vasodilation/impaired vasoconstriction, and hypotension characteristic of sepsis [34, 37]. Several small trials of NO inhibitors in sepsis showed improvement in blood pressure and vaso-responsiveness [2]. However, a Phase III trial of an NOS inhibitor was terminated early because of increased mortality in the treated patients, despite improvements in blood pressure and vascular reactivity [38]. The reasons for the increased mortality are unclear, but may reflect deleterious effects of NO blockade on the microcirculation as well as loss of the cytoprotective and anti-inflammatory properties of NO [7, 34].

Permeability The endothelium constitutes a semi-selective barrier that maintains the fluid balance between intravascular and extravascular compartments, while allowing certain antibodies, hormones, cytokines, and other molecules to access the interstitial space during inflammation, immune response, and wound repair. The loss of the selective permeability barrier results in pathological vascular leak, a hallmark of severe sepsis. Clinically, this increase in vascular permeability is most apparent in the pulmonary microcirculation, resulting in acute lung injury (ALI)/adult respiratory distress syndrome (ARDS), a devastating complication that occurs in up to 40% of patients with severe sepsis [39]. Although there are transcellular pathways of permeability, the vascular leak in sepsis likely occurs by a paracellular pathway involving retraction of the endothelium. Many inflammatory mediators (e.g., thrombin, histamine, and growth factors) increase permeability by a process involving phosphorylation of cytoskeletal and junctional proteins [40]. Phosphorylation of non-muscle myosin light chains by myosin light chain kinase triggers the contractile apparatus and cell retraction. Phosphorylation of junctional proteins (VE-cadherin and/or catenins)

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promotes their internalization or degradation, reducing interactions with cytoskeletal anchors and thereby weakening cell-cell adhesion [40]. Endothelial activation by LPS triggers a variety of signaling cascades [41], including the activation of pro-apoptotic caspases [42]. In an in vitro model of permeability edema, LPS was found to induce caspase-mediated cleavage of adherens junction proteins, thereby increasing albumin flux across the monolayer [43]. Vascular endothelial growth factor (VEGF-A) is a potent vascular permeability factor that also has pro-inflammatory activities. VEGF-A binds to two transmembrane receptors on endothelium, fms-related tyrosine kinase 1 (Flt-1; VEGFR1) and fetal liver kinase 1 (Flk-1)/kinase insert domain receptor (KDR; VEGFR2) [44]. Flt-1 is also produced as a soluble receptor (sFlt-1) via alternative splicing of the precursor mRNA [44]. Soluble Flt-1 can function as a decoy molecule by competing with membrane-bound Flt-1 for binding to VEGF [45]. In two murine sepsis models, blockade of VEGF signaling in endothelial cells by administration of exogenous sFlt-1 or antibodies to Flk-1 reduced permeability edema and mortality [46]. In patients with suspected sepsis, plasma levels of VEGF at initial presentation in the emergency department and 24 hours later correlated with the presence of septic shock at 24 hours [45]. Initial and 24-hour levels of soluble Flt-1 correlated with APACHE and Sepsis-related Organ Failure Assessment scores determined initially and after 24 hours [45]. This observational study supports the experimental data, suggesting that VEGF is an important mediator of sepsis. The angiopoietin (Ang-1)/tyrosine kinase with immunoglobulin-like and epidermal growth factor-like domains 2 (Tie-2) system is also an important regulator of endothelial permeability that has been implicated in vascular leak in sepsis, particularly in ALI/ARDS [47]. Ang-1 and Ang-2 are peptides that act via the Tie-2 receptor on endothelial cells. Ang-1 is an agonist of Tie-2, whereas Ang-2 acts predominantly as an antagonist through inhibition of Ang-1 action. Ang-1/Tie-2 signaling promotes vessel stability and at supra-physiological levels Ang-1 acts as an anti-permeability agent, whereas high levels of Ang-2 in patients with sepsis may contribute to hyper-permeability [48]. In animal models, increasing the Ang-1 levels protects against endotoxic shock [49] or LPS-induced lung injury [50]. Consistent with the experimental data, Ang-2 levels are increased in patients with severe sepsis [48, 51, 52].

Leukocyte adhesion The endothelium regulates leukocyte trafficking via expression of adhesion molecules and chemokines/cytokines [53, 54]. Many of the mediators generated in sepsis (e.g., thrombin, TNF-a, IL-1b, and LPS) induce expression or up-regulation of adhesion receptors for leukocytes on endothelial cells, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), P-selectin,

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E-selectin, and junctional adhesion molecules (JAMs) [55]. The interaction of leukocyte and endothelial adhesion receptors, in concert with chemoattractants (e.g., chemokines, bioactive lipids, bacterial products) in adjacent tissue or bound to the surface of endothelial cells, mediates the recruitment of leukocytes at sites of infection or inflammation. This occurs in a multi-step adhesion cascade, involving initial tethering/rolling supported by endothelial P-selectin, subsequent firm adhesion to endothelial ICAM-1 or VCAM-1, and finally diapedesis via endothelial JAMs [53]. Leukocytes, particularly neutrophils, are crucial for host defense against bacterial infections [56], and profound neutropenia induced by chemotherapy is well recognized as a major risk factor for the development of sepsis. Similarly, impaired neutrophil adhesion to the endothelium increases the risk of infection by preventing neutrophil accumulation at the site of infection. This is evidenced by the occurrence of severe infections in individuals with genetic defects that reduce the expression or function of leukocyte or endothelial adhesion molecules [57]. Although critical for host defense, under some circumstances unregulated or exaggerated leukocyte adhesion may contribute to tissue injury and inflammation in sepsis. For example, in a rabbit model of peritoneal sepsis, blockade of neutrophil integrin CD11/CD18, the counter-receptor for endothelial ICAM-1, after the induction of sepsis decreased mortality [58]. Similarly, gene-targeted mice deficient in endothelial selectins were resistant to lethality in a septic peritonitis model [59]. Notably, in patients with sepsis elevated plasma levels of E-selectin, a marker of endothelial activation, correlate with severity and a poor prognosis [60, 61].

Apoptosis Apoptosis is a fundamental process that is essential for development and homeostasis [62, 63], but also contributes to diverse pathological processes [64]. In contrast to rapidly proliferating epithelial cells in the gut, quiescent endothelium exhibits very low levels of apoptosis (< 0.1%) [65]. However, situated at the interface between blood and tissue, the endothelium is often exposed to pro-apoptotic stimuli. Vascular homeostasis requires robust endogenous endothelial mechanisms to prevent apoptosis. An imbalance between pro-apoptotic stimuli and endogenous anti-apoptotic defenses could contribute to vascular injury in diverse diseases, including sepsis [66]. In necrotic cell death the cell swells, the plasma membrane is disrupted, and intracellular constituents are released extracellularly, eliciting an inflammatory response. In contrast, during apoptotic cell death the plasma membrane remains intact as the cell shrinks. Additionally, there is expression of membrane molecules that trigger rapid engulfment by phagocytic cells [67]. Consequently, there is little inflammation during apoptosis unless secondary necrosis occurs. However, there are

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reasons why endothelial cell apoptosis might not necessarily be ‘quiescent’ in vivo. As discussed previously, activation of caspases during apoptosis triggers degradation of endothelial cell adherens junction proteins with disruption of barrier function in vitro [43]. If this occurred in vivo, it would provoke vascular leak [66]. Furthermore, since endothelial cells are exposed to flowing blood, apoptotic endothelial cells could detach prior to engulfment by phagocytes or adjacent cells. Detachment of apoptotic endothelial cells could lead to vascular leak and expose subendothelial matrix that triggers thrombosis. Finally, since the apoptotic endothelial cells become proadhesive for platelets [12] and procoagulant [11], they could promote coagulation in situ prior to detachment. In vitro studies of endothelial apoptosis in sepsis have focused on apoptotic signaling by the LPS/Toll-like receptor-4 (TLR4) pathway [41]. Although signaling induced by ligation of TLR4 with LPS is mostly pro-inflammatory, it can also trigger apoptosis. The LPS/TLR4-induced pathway leading to apoptosis involves the death receptor adapter Fas-associated death domain (FADD) as well as Toll receptor-IL-1 receptor domain-containing adapter protein (TIRAP), myeloid differentiation factor 88 (MyD88), IL-1 receptor-associated kinase-1 (IRAK-1), TNF- receptor-associated factor-6 (TRAF-6), and c-Jun N-terminal kinase (JNK) [42]. Endothelial cell apoptosis may be difficult to observe in vivo since apoptotic cells are rapidly phagocytosed and apoptotic endothelial cells may readily detach from the vessel wall into the circulation. For these reasons it may be difficult to document endothelial cell apoptosis in situ. Consequently, detection of circulating apoptotic endothelial cells or endothelial microparticles has been proposed as an alternative technique to evaluate endothelial cell dysfunction, including apoptosis, in vivo [68, 69]. Nevertheless, in several animal models of sepsis, significant endothelial cell apoptosis has been identified [70]. Circulating endothelial cells have also been detected in patients suffering from severe sepsis, consistent with vascular injury [71]. In contrast, using electron microscopy Hotchkiss and colleagues did not identify significant endothelial cell apoptosis in rat cecal ligation and puncture and murine pneumonia models [72] or in patients who died with sepsis [73]. However, as they note, conclusions from these studies are limited by the focal nature of the electron microscopic examination and the possibility that apoptotic endothelial cells were rapidly cleared. Intriguing studies in vitro have suggested that the beneficial effect of APC in the treatment of severe sepsis [19] may relate in large part to anti-apoptotic effects on endothelial cells [74, 75].

Conclusion There is compelling evidence that endothelial cells are both important target and effector cells in sepsis and that endothelial dysfunction contributes significantly to the pathophysiology of sepsis. Clinical, as well as experimental, studies in sepsis

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Endothelial activation and dysfunction in sepsis

demonstrate that changes in hemostasis, vasomotor tone, permeability, and leukocyte trafficking result in large part from perturbations of endothelial function. Although administration of activated protein C has shown some efficacy in severe sepsis, other attempts to correct endothelial dysfunction (i.e., antithrombin III, tissue factor pathway inhibitor, nitric oxide synthase inhibitor) have not resulted in significant clinical benefit. Nevertheless, given its pivotal role, further studies of endothelial-directed therapies are warranted.

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Heterogeneity in responses of microvascular endothelial cells during inflammation Grietje Molema Department of Pathology and Medical Biology, Medical Biology section, Laboratory for Endothelial Biomedicine and Vascular Drug Targeting research, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands

Abstract Each segment of the vascular system has its own function, based on its location in the body. The endothelial cells lining these vascular segments are an intrinsic part of this segmental variation, and it is not surprising that throughout the vascular bed they have a highly heterogenic appearance. This chapter starts with a description of the heterogenic phenotype of endothelial cells in the body in quiescent conditions. Focus is next on endothelial engagement in inflammatory processes and how the microvascular endothelial cells in the different organs and within an organ respond to tumor necrosis factor α, interleukin-1, and lipopolysaccharide as inflammatory stimuli. Studies on endothelial responsiveness both in vitro in culture systems and in vivo in the intact body are discussed. From a pharmacological point of view, knowledge of the molecular basis of heterogeneity in endothelial cell behavior will be critical for successful drug development to counteract endothelial cell engagement in disease. This chapter therefore concludes with a short overview of technological advancements that may assist in unveiling the mechanisms responsible. When applied to address microvascular endothelial heterogeneity, major steps forward in endothelial biomedicine are anticipated that will assist in defining the right molecular targets for the microvascular segments involved in the pathology under study.

Introduction Throughout the circulatory system, endothelial cells lining the blood vessel wall are exposed to different microenvironmental conditions. As a consequence they intrinsically behave differently [1, 2]. Mechanical stress induced by blood flow, interactions with cellular components in the blood, and their location within the organ, all contribute to the nature and extent of endothelial heterogeneity in gene expression and consequent performance. The features that all endothelial cells share include their direct interaction with the blood due to their location at the inner lining of the blood circulatory circuit and their anti-thrombotic properties under normal, quiescent conditions. Furthermore, they share the expression of endothelial restricted genes such as CD31 (or platelet/endothelial cell adhesion molecule-1, PECAM-1) and vasEndothelial Dysfunction and Inflammation, edited by Shauna M. Dauphinee and Aly Karsan © 2010 Springer Basel

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cular endothelial (VE)-cadherin [3]. These molecules are, however, not exclusively expressed by the endothelial cells of the vascular wall. Moreover, the observed differences in levels of expression of these molecules in the various (micro)vascular segments in the body imply heterogenic functionality. The aim of this chapter is to give an overview of endothelial responses to an inflammatory insult with focus on the differences in reaction between endothelial cells from different segments of the vasculature, or endothelial subsets. Information obtained from endothelial cell culture systems is discussed first, followed by studies reporting on endothelial heterogeneity in reaction to inflammatory stimuli in in vivo models. These observations are put into a pharmacological perspective, as many of the investigations on endothelial biology converge at the mutual aim to pharmacologically address dysfunctional microvascular endothelium as a treatment modality for inflammatory diseases.

Endothelial engagement in inflammation The possibility to culture endothelial cells in vitro, reported first in 1973 [4–6], created a wealth of opportunities to study in greater detail the endothelial membrane [7], interactions of endothelial cells with platelets and leukocytes [8], and endothelial responses to proinflammatory cytokines such as interleukin (IL)-1 [9] and changes in fluid shear stress [10]. Shortly after, antibodies against a variety of (inducible) endothelial antigens were raised to study antigen expression and function, both in animal and human tissues and cell preparations. This eventually led to the definition of the ‘three step area code model’ of leukocyte recruitment to peripheral tissues in an inflammatory process as proposed by Springer in 1994 [11]. This model has been fine tuned in recent years to a more complex model in which organ specificity, and vascular segment specificity of the molecular processes are taken into account [12]. Also in the absence of an inflammatory stimulus, leukocyte subpopulations exhibit highly selective migration patterns to peripheral sites of the body, as was, for example, recently reported for T memory cell recruitment [13]. However, this chapter focuses on endothelial responses to inflammatory conditions, and specifically on how endothelial cells located in different sites in the body engage in an inflammatory reaction. The vasculature consists of branches with different architecture and function. Besides conduit vessels with large diameter that transport the blood through the body, the smaller arteries and arterioles actively engage in blood pressure control. The capillaries that reside inside the organs exhibit organ-specific microvascular functions, including maintenance of a blood-brain barrier in the brain, clearance function of sinusoidal endothelial cells in the liver, and filtration of the blood by the glomerular vascular segment in the kidney. Leukocyte recruitment represents a process that needs to be executed in any organ upon demand. According to our current view, leukocyte migration into

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Heterogeneity in responses of microvascular endothelial cells during inflammation

inflamed tissues takes place in the first segments of the post-capillary venules [12]. This vascular bed is not primarily committed to essential organ functions, so engaging in other processes would initially not disturb organ microvascular function. Moreover, the membrane-membrane interaction between leukocytes and endothelial cells is highly efficient in this particular microvascular segment, as the diameter of the vessel is of a similar order of magnitude as that of the leukocytes, facilitating extensive contact between leukocyte and endothelium. This paradigm of preferential leukocyte recruitment in the postcapillary venules is, however, not observed in all organs. For example, in lungs the principal site of leukocyte migration is the capillary bed [14]. In a study by Aoki et al. [15], leukocytes activated with the IL-8 homologue cytokine-induced neutrophil chemoattractant (CINC)/gro were mainly sequestered in the lung capillary vessels. In contrast to leukocyte sequestering in the mesentery microvasculature, hardly any interaction with pulmonary postcapillary venules was observed. A vessel wall architecture-driven effect on location of leukocyte transmigration has been proposed. This is based on the observation that in cytokine-activated cremaster venules migration of neutrophils mainly takes place at sites where the key vascular basement membrane constituents (laminin 10, collagen IV, and nidogen-2) are expressed to a considerably lower extent [16]. A similar, basement membrane related fine tuning of leukocyte recruitment was observed in the brain, in which encephalitogenic T lymphocytes extravasated by virtue of the fact that they carry their own address code to do so [17]. Upon an inflammatory insult, one of the first reactions of the endothelial cells is to exocytose the stored, ready-to-release contents of Weibel-Palade bodies, including the blood coagulation factor von Willebrand Factor (vWF) and the adhesion molecule P-selectin [18]. By this means, a rapid interaction between stressed endothelium, platelets and neutrophils is created to facilitate leukocyte rolling [19]. Directly afterwards, the endothelium starts to produce E-selectin, capable of interacting with the tetrasaccharide sialyl-LewisX expressed on monocytes and activated lymphocytes, leading to a further rolling adherence of leukocytes to the endothelium. E-selectin is also implicated in the generation of activated integrin microdomains at the leading edge of neutrophils via E-selectin ligand-1, as a consequence of which erythrocytes and platelets can be captured to create additional inflammatory damage [20]. Firm arrest of leukocytes to the endothelium is next facilitated by immunoglobulin superfamily members vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1), and local production and display of chemokines and cytokines. The latter molecules rapidly activate integrins on leukocytes to bind with high affinity to G protein-coupled receptors. The last step is leukocyte transendothelial migration, which is triggered by endothelial–integrin interaction and clustering, and affects many downstream effector molecules as reviewed recently in detail by Wittchen [21]. The above paradigm of consecutive steps leading to efficient leukocyte recruitment to sites of inflammation is a simplified model. For example, in skeletal

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muscle, heart, kidney, and skin, selectins are key mediators of leukocyte rolling, yet leukocyte recruitment to the liver is largely selectin independent [22]. In addition, activation of both the endothelial cells and leukocytes is fine tuned by a multitude of factors. Vascular endothelial growth factor (VEGF), for example, is important in vascular permeability control during an inflammatory insult. It can, however, also enhance tumor necrosis factor (TNF-) α-induced monocyte recruitment. This may be explained by its effect on adhesion molecule expression [23, 24], or on a more broad effect on inflammatory gene expression, as it was recently shown that VEGFA can induce a gene expression pattern in human umbilical vein endothelial cells (HUVEC), which overlapped to 60% with the genes induced by IL-1 [25]. Interestingly, TNF-α-induced polymorphonuclear neutrophil (PMN) recruitment was not affected by additional VEGF treatment [23], which underscores the fact that selectivity of leukocyte recruitment is controlled at both the leukocyte and endothelial cell level. All vascular beds are under the influence of the VEGF/VEGFR system as the cells of the vascular walls all express these genes. Yet, the expression levels of VEGF and its receptors can vary, between organs as well as between vascular bed segments within organs. For example, in kidneys different microvascular segments can be discriminated, i.e., afferent and efferent arterioles, glomeruli, descending and ascending peritubular vessels, and postcapillary venules. In mice, especially the glomerular microvascular segments code for VEGF and VEGFR-2 expression (Fig. 1) [26, 27]. It is highly likely that heterogenic expression of VEGF, VEGFRs and VEGFR-2 dephosphorylating phosphatase (VE-PTP) [28] in endothelial subsets in different vascular segments can affect VEGFR-2 signaling capacity, and thus sensitizes responsiveness to inflammatory stimuli. Another exquisite example of molecular cross-talk between endothelially restricted genes that may be fine tuned in a vascular segment-specific way was provided by Weis and colleagues some years ago [29]. In an in vivo setting, they showed that VEGFR-2 and VE-cadherin communicate in the heart via the adapter protein Src to regulate endothelial permeability in response to VEGF. Local differences in expression levels of VEGFR-2 and VE-cadherin in the different microvascular segments throughout the body will likely affect the nature and extent of these molecular interactions, and thus their functional consequences, including inflammatory reactivity. In addition to VEGF and its receptors, many other molecules, including members of the Angiopoietin/ Tie2 system [30], and Robo4 and its ligands [31], can directly or indirectly affect each other in controlling endothelial responses to inflammatory stimuli. From the examples discussed above one can start to draw a picture in which the complexity of (microvascular) endothelial signaling in response to an inflammatory stimulus becomes clear. The challenge for successful therapeutic intervention in inflammatory disease aimed at the microvascular segments engaging in the inflammatory process is to understand the involvement of the different molecules, in health (to determine whether unwanted toxicity is expected from a therapeutic intervention) and disease (to choose the right drug for the right disease).

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Heterogeneity in responses of microvascular endothelial cells during inflammation

Figure 1. Microvascular segment specific expression of genes underlies microvascular endothelial heterogeneity in vivo. Within a C57BL/6 mouse kidney, the expression of VEGF and one of its receptors, VEGFR2, is mainly localized in the glomerular compartment. The graph represents relative mRNA levels of both genes in arteriolar, glomerular and venular vasculatures obtained from snapfrozen tissue sections by laser microdissection prior to quantitative RT-PCR (from [26], supplemental data set). Immunohistochemical studies demonstrated VEGF expression within the glomeruli to be mainly podocyte restricted [27], while VEGFR-2 was mainly endothelially expressed [34].

Microvascular endothelial heterogeneity in normal conditions One of the main functions of endothelial cells in the microvascular segments of an organ is to direct inflammatory cells into the tissues during an inflammatory insult. They do so by regulation – induction or repression – of the expression of a variety of molecules in a spatiotemporally highly controlled fashion. Interestingly, although not surprisingly, microvascular endothelial heterogeneity can already be observed in quiescent conditions. Heterogeneity in basal adhesion molecule expression has been well documented. For example, using radiolabeled monoclonal antibodies, Eppihimer and colleagues showed in vivo that E-selectin protein expression was completely absent throughout the vasculature in healthy, cytokine-naive mice [32], which was corroborated at the mRNA level [33]. P-selectin was at the same time expressed to a significant extent in the lungs, mesentery, small intestine and pancreas [32]. Northern blot analysis furthermore extended inflammatory gene expression analysis to VCAM-1 and ICAM-1, and demonstrated that both adhesion molecules were constitutively expressed especially in the lungs [33]. Using real-time RT-PCR technology to quantitatively analyze mRNA levels of genes with a relatively low

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detection limit, a more detailed view on the inter-organ microvascular differences in adhesion molecule expression was obtained (Tab. 1). Detailed immunohistochemical analysis of protein expression levels revealed an even more complex situation (Fig. 2). E-selectin is absent, or at least below detection limit in all vascular beds in the five major organs. VCAM-1, on the other hand, is highly expressed in arteriolar endothelium of kidney, brain and lungs, absent on almost all capillary segments except for some expression on a subset of peritubular endothelial cells in the kidney, and highly expressed in the postcapillary venule endothelium of only the lungs. ICAM-1 is more equally expressed by all vascular segments, with some exceptions. The functional meaning of these differences remains to be established. Table 1. Gene expression levels of endothelial adhesion molecules in main mouse organs The values represent real-time RT-PCR analysis threshold (Ct) values of the molecules relative to the house keeping gene GAPDH, as determined in whole organ RNA isolates. The higher the value, the lower the mRNA expression level of the gene (n = 3 ± SEM; Kuldo and Molema, unpublished)

Kidney

P-selectin

E-selectin

VCAM-1

ICAM-1

16.3 ± 1.1

14.4 ± 0.4

10.3 ± 0.2

9.2 ± 0.1

Brain

16.8 ± 0.3

17.9 ± 0.7

9.4 ± 0.3

12.3 ± 0.2

Heart

14.7 ± 0.2

13.8 ± 0.2

10.6 ± 0.2

10.8 ± 0.2

Liver

12.6 ± 0.3

16.5 ± 1.7

8.8 ± 0.9

7.9 ± 0.6

Lung

8.2 ± 0.2

12.6 ± 1.2

5.6 ± 1.2

2.0 ± 0.9

Other endothelial restricted genes have also been reported to be heterogenically expressed in the vascular tree under quiescent conditions. These include VEGF and its receptors, as already briefly mentioned above. Using VEGF-LacZ mice, it has also been shown that vascular-associated VEGF expression coincided with VEGFR2 phosphorylation [27]. Co-expression of VEGFR-1, -2, and -3 was reported for human and monkey microvessels adjacent to epithelia in the eye, gastrointestinal mucosa, liver, kidney, and blood vessels and sinusoids of lymphoid tissues. In contrast, in the microvascular endothelial cells of the brain and retina only VEGFR-1 could be detected [34]. Using a pharmacological approach, it was demonstrated that especially the fenestrated capillaries – of the pancreatic islets, thyroid, adrenal cortex and epididyma adipose tissue – in the adult mouse are VEGF dependent. This dependency was associated with high levels of VEGFR-2 and -3 expression in these vascular beds [35]. Vascular heterogeneity in gene expression was also reported for members of the connexin family. Connexins Cx37, Cx40, and Cx43 are components of gap junc-

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Heterogeneity in responses of microvascular endothelial cells during inflammation

Figure 2. Heterogeneity in endothelial adhesion molecule expression in microvascular segments of naive 10-week-old C75BL/6 mouse organs. Simplified scheme of protein expression levels of the three major inflammation associated adhesion molecules E-selectin, VCAM-1, and ICAM-1, as semi-quantitatively assessed by immunohistochemistry. White: not detectable; gray: expressed at intermediate levels; black: highly expressed; P: patchy (not all endothelial cells were positively stained/those cells expressing the adhesion molecule did not do so evenly distributed over their cell membrane). Figure adapted from [39].

tions, clusters of transmembrane protein channels used for intercellular communication. While rat aorta and coronary artery endothelial cells extensively express Cx40, Cx37 protein is heterogeneously distributed in both segments. Also, while aorta endothelium abundantly expresses Cx43, this connexin is absent in the coronary artery [36]. A similar segmental variation in Cx expression patterns was reported

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for three different vein segments, i.e., the rat cranial vena cava, thoracic section of the caudal vena cava, and the abdominal section of the caudal vena cava [37]. Another reflection of vascular endothelial heterogeneity in quiescent microvessels is the molecular difference between arterial and venous endothelial cells [38]. Transmembrane molecules shown to be mainly expressed in venule endothelium include endomucin [38, 39] and the transcription factor COUP-TFII. The latter molecule inhibits Notch signal transduction, as a consequence of which a venous identity is maintained [40]. Arterial markers include Neuropilin-1 [41], a receptor for the class 3 semaphorin family as well as for VEGF164/165, Delta-like ligand 4 (DLL4) [42], and various Notch pathway genes [43]. EphrinB2 and EphB4 were initially identified as markers of arterial and venous identity during embryogenesis, respectively [44]. Recent evidence suggests, however, that, at least in mesenteric tissue, EphB4 expression is not restricted to the vein segments in the mature vasculature [45]. These examples emphasize the importance of judging each segment of the vascular tree of the circulatory system on its own merits, including the underlying mechanisms of participation in physiological and pathological regulatory processes, instead of adopting observations from one segment to another without any further consideration.

Endothelial origin affects response to an inflammatory stimulus TNF-α and IL-1β are prominently produced at sites of inflammation and are cytokines that have been extensively studied in in vitro cell culture systems for endothelial cell activation responses. TNF-α mainly signals via TNFR1, leading to intracellular signaling cascades involving the activation of the NF-κB and p38 MAPK pathways [46]. Similar to TNF-α, IL-1 is an early inflammatory cytokine. It exists in two isoforms, IL-1α and IL-1β, the latter being the primary isoform circulating in humans. Both isoforms bind to IL-1 receptor type 1 (IL-1R1), leading to activation of p38 MAPK and NF-κB and AP-1 transcription factors [47]. Both cytokines induce a plethora of pro-inflammatory genes in various cells, including endothelial cells. One of the first studies on endothelial subset differences in reaction to TNF-α dealt with the exposure of human endothelial cells derived from glomeruli (GEC), dermal microvasculature (MvEC), and umbilical vein (HUVEC) to different concentrations of TNF-α. Using ELISA technology, it was shown that all three cell subsets exhibited reduced expression of CD31 and induced expression of E-selectin protein, while VCAM-1 was only up-regulated in GEC and HUVEC [48]. Using a microarray approach, Viemann and colleagues compared TNF-α responsiveness between HUVEC and the dermal microvascular endothelial cell line HMEC-1 [49]. Of the 93 genes that were up-regulated by HMEC-1, 47 were shared with HUVEC, while 46 were only up-regulated in HMEC-1. Conversely, 76 genes were up-regulated in

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HUVEC, 33 of which were non-responder genes in HMEC-1. In HUVEC, differentially up-regulated genes mainly included those encoding for adhesion molecules, cytokines and chemokines, while in HMEC-1 they represented mainly signaling, transcription factor, apoptosis, and proliferation associated genes. This latter outcome is likely associated with the fact that HMEC-1 is an immortalized cell that may in life have lost and gained various functions and associated gene repertoires [50]. Organ-encoded differences in extent and nature of responses to inflammatory stimuli are already visible in endothelial cells derived from fetal tissues [51]. Under the influence of TNF-α, fetal heart, kidney and aorta endothelium derived from 12-week-old human embryos highly expressed E-selectin, while IL-1β treatment only induced this selectin in fetal kidney and aorta endothelium. ICAM-1 expression was mainly induced in fetal heart, liver, and lung endothelium when exposed to IL-1β, and at an early time point also in fetal kidney and aorta endothelium, and less in lung endothelium when exposed to TNF-α. The induction of ICAM-1 in fetal liver and lung endothelial cells increased in time only upon exposure to TNF-α. VCAM-1 induction was lacking in fetal brain-derived endothelial cells, irrespective of the stimulus, while it was a late event in all except fetal heart-derived endothelium upon TNF-α exposure. In contrast, IL-1β only induced VCAM-1 in fetal heart endothelial cells at the later time point, with concurrent minor responses in the other cell subsets [51]. Other inflammation-related endothelial activators have been studied with regard to endothelial subset-specific responsiveness to stress. Direct protein kinase C activation for example activated HUVEC to express E-selectin and VCAM-1, while dermal microvascular endothelial cells were fully devoid of such a response [52]. Nguyen and colleagues [53] exposed primary bovine mesenteric artery and primary bovine vein endothelial cells to Angiopoietin (Ang)-1 and Ang-2. Basal expression levels of Angiopoietin receptor Tie-2 were higher in the artery endothelial subset, yet in both cell types Tie-2 phosphorylation was induced to a similar extent upon Ang-1 and Ang-2 treatment. ERK1/2 phosphorylation was mainly induced by Ang-1 exposure, and did not differ between the two subsets. Methe et al. [54] studied the influence of flow on vascular bed-specific adhesion molecule expression induced by cytokines. In this study, human saphenous vein endothelial cells (HSVEC) and human coronary artery endothelial cells (HCAEC) were exposed to venous and coronary artery flow patterns at, respectively, 2.2 dyn/ cm2 (static) and 17  dyn/cm2 (at 1  Hz). Under static conditions, only the venous endothelial cells exerted an induced expression of VCAM-1 in response to TNF-α treatment. This difference was not observed for the other two adhesion molecules E-selectin and ICAM-1. Exposure to both types of flow conditions increased TNFα-induced E-selectin and ICAM-1 expression by the saphenous vein endothelium, while only coronary artery flow increased VCAM-1 expression. Interestingly, venous and coronary artery flow exerted different effects on the coronary artery

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endothelium. In these cells, flow attenuated TNF-α-induced E-selectin and VCAM1 expression. In addition, TNF-α-induced ICAM-1 expression was only increased further by coronary artery flow, not venous flow. Both flow patterns induced the expression of Kruppel-like factor (KLF)-2 and KLF-4 in the endothelial subsets, while exogenously overexpressed KLF-4 significantly affected TNF-α-induced E-selectin and VCAM-1, not ICAM-1 expression [54]. Not only differences in gene expression induction, but also differences in processes downstream of transcription control have been reported to exist in endothelial cells from different origins. For example, kinetics of E-selectin down-regulation was reported to be different between human dermal microvascular endothelial cells (HDMEC) and HUVEC. In E-selectin cDNA-transduced HDMEC, the rate of E-selectin internalization was independent of transmembrane expression level, exerting a prolonged half-life of more than 4 hours. In HUVEC, on the other hand, the rate of E-selectin internalization was dependent on the level of surface expression and showed a persistent internalization half-life of less than 2 hours. E-selectin internalization in microvascular endothelial cells from lung and subcutaneous fat followed HDMEC kinetics, whereas internalization in large vessel endothelium from saphenous vein and aorta was rapid, like HUVEC [55, 56]. In response to nucleotides released by damaged cells and platelets, endothelial cells lose their purinergic P2Y G protein-coupled receptor expression. Both bovine retina endothelial cells, as a model for microvascular endothelium, and HUVEC, as a model for macrovascular endothelium, down-regulated the receptor when exposed to nucleotide UTP, but did so at a different rate, extent and UTP sensitivity [57].

In vivo microvascular heterogeneity in reaction to inflammatory stress The in vitro examples of endothelial origin-related responsiveness to inflammatory stimuli, such as inflammatory cytokines, growth factors, and nucleotides, have urged researchers to address the question of how the microvascular endothelial subsets behave when challenged in vivo. The easiest model to study in this respect is systemic injection of pro-inflammatory cytokines. Tamaru and colleagues [33] used Northern blot analysis to show that adhesion molecule induction after IL-1β administration was mainly visible in liver, kidney, heart and lungs, and less prominent in brain and skin. Variations in reactivity to IL-1β and TNF-α between the different microvasculatures in the organs were also shown to exist using in situ hybridization. Using real time RT-PCR technology, we quantitated the in vivo microvascular endothelial cell responses to TNF-α and IL-1β administration (Fig. 3). In all organs, the microvasculature responded by the induction of expression of the adhesion molecules P-and E-selectin, VCAM-1 and ICAM-1, although the fold induction differed per organ. Based on these data one can conclude that the microvasculature throughout the body responds in a highly complex manner even when challenged

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Figure 3. Variation in in vivo endothelial responsiveness to TNF-a or IL-1b between mouse organs. Using real-time RT-PCR, mRNA expression levels relative to house keeping gene GAPDH were determined in mouse organs 2 hours after i.v. administration of 200 ng mouse TNF-a (black bars) or IL-1b (white bars) per mouse. Values represent n = 3 animals per group ± SEM. Linear mixed effects models and analysis to account for duplicate PCR analysis values were used to address the significance of observed effects [81]. Multiple testing was controlled by the False Discovery Rate-controlling procedure of Storey and Tibshirani [82] at the level of q < 0.05. All analyses were performed using R [83]. * Significant difference in gene expression induction between TNF-a and IL-1b for the particular organ, q < 0.05. (Kuldo and Molema, unpublished data).

by a well-defined stressor. Similar to their behavior in quiescent conditions, each microvascular segment should therefore be judged on its own merits with regard to the underlying mechanisms of activation, in addition to the consequences for engagement in inflammatory leukocyte recruitment.

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A more complicated model of endothelial cell activation is the induction of sepsis by bolus administration of lipopolysaccharide (LPS), which consists of an LPS effect and secondary cytokine effect. Direct LPS signaling in endothelial cells mainly takes place via Toll-like receptor (TLR)-4 and indirectly via TNF-α released by liver macrophages. A well-know response to LPS is the occurrence of hypotension, which is located in the macrovessels and consists of a blunted vasoconstrictor response to norepinephrine, and an impaired endothelium-dependent vasodilator response to acetylcholine, both of which are mainly NF-κB controlled [58]. Moreover, in the microvasculature of main organs such as the heart, lung, brain and liver, the expression of E- and P-selectin and ICAM-1 were all significantly induced in response to systemic LPS treatment. For VCAM-1 the situation was different, and represents an example of organ-related microvascular heterogeneity in reaction to LPS, in which brain and liver endothelium did not express VCAM-1 upon LPS exposure [59, 60]. Interestingly, the skin microvasculature showed a rather aberrant gene induction profile compared to the other organs [60]. This expands the examples of heterogenic responsiveness of organ-restricted microvasculatures and at the same time questions the validity of using skin biopsies as surrogate read-out of endothelial activation. In addition to induction of cell adhesion molecules, LPS can enhance microvascular thrombosis. In mouse cremaster venules, this enhanced thrombosis was mediated by vWF, not by P-selectin which is also stored in Weibel Palade bodies [61]. As vWF is highly heterogenically expressed throughout the vascular bed [62], it is possible that LPS induced enhancement of thrombotic events mediated by vWF presents as a vascular segment-restricted feature. Other systemic activation models have revealed intra-organ microvascular segment specific reactivity to a stimulus. For example, we showed that induction of hemorrhagic shock, by withdrawal of blood to 30 mm Hg and maintenance thereof for 90 minutes, led to expression of E-selectin in renal glomerular endothelial cells, while it was much less pronounced or even absent in the endothelium of afferent and efferent arterioles, and peritubular and postcapillary venular segments. In contrast, VCAM-1 expression was induced in all vascular segments except in glomerular endothelium [63]. A similar observation of intra-organ segment-restricted endothelial responses was reported by Tamaru et al. [33], who used in situ hybridization to show that systemic IL-1β administration mainly induced VCAM-1 mRNA in the larger vessels of the lungs and the heart, not in the small capillaries. The above examples cover a selection of studies reporting on how the location of the endothelial cells within the vascular system encrypts basic and inflammatory stress-induced gene expression patterns. The question that remains is what the molecular basis of this heterogenic behavior is. The number of studies reporting on the molecules underlying variation in endothelial responsiveness to inflammatory stimuli is limited. In recent years, however, new techniques have been developed that can have a crucial contribution to the field of endothelial biomedicine in the coming years.

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Studying the molecular basis of endothelial heterogeneity – Some technological considerations The ideal way to answer the question of how the molecular basis of endothelial heterogeneity is encoded in the in vivo environment would be to isolate the different endothelial cell subsets from an organ and to either directly map the molecular basis of in vivo differential behavior or to study molecular responses to stress in culture. By this means, sufficient protein, mRNA, microRNA and/or DNA sample can be generated as input for the vast array of (bio)chemical and molecular analyses that are currently available. Using fluorescence activated cell sorting it is now possible to isolate peritubular endothelial cells from human kidneys by virtue of the fact that this endothelial subset selectively expresses MHC class II as positive sorting antigen [64]. This is, however, a rare example of an endothelial subset-specific molecule, as knowledge on the existence and identity of these kinds of antigens is fairly limited. Another complicating factor in using endothelial cell cultures as a source of material to study the molecular basis for responsiveness is the loss of nurture-driven endothelial cell performance when the cells are taken into culture. For example, glomerular endothelial cells are almost devoid of vWF expression in their natural context [65], yet cultured glomerular endothelial cells express high levels [66]. Molecular changes upon isolation from their natural habitat can occur rapidly, as was recently shown with endothelial cells isolated from human umbilical cord veins [67]. In situ they responded to TNF-α exposure with the expression of E-selectin and VCAM-1, but this response was lost within 72 h of culturing the cells. This particular change in E-selectin responsiveness was associated with loss of NF-kB/p65, ATF-2 and p300 transcription factor binding to the E-selectin promoter with concomitant enhanced binding of c-jun upon transition to the in vitro culture conditions [67]. Animal models overcome these endothelial cell culture-related artifacts, and several transgene mouse models have been used in recent years that have an overexpressed, knocked out, or reporter gene in the endothelial cell compartment [27, 62, 68, 69]. Application of such models to address heterogenic endothelial responsiveness to an inflammatory stimulus recently revealed that the Down syndrome critical region gene 1, short variant (DSCR-1s) plays a role in this process [70]. DSCR-1s promoter-lacZ mice showed enhanced promoter activity in brain, lung and kidney, but not in spleen, liver and thymus, after LPS challenge. In lung, both macro- and microvascular endothelial cells exerted enhanced promoter activity, while in kidney non-endothelial cells in the glomeruli were responsive. LPS challenge studies next showed that Dscr-1–/– mice experienced superinduction of E-selectin and ICAM-1 mRNA in the heart and of VCAM-1 mRNA in the lung vasculature. This clearly indicates a role for DSCR-1s in a negative feedback loop of endothelial cell activation that is differentially regulated throughout the vascular system. Laser microdissection (LMD) of endothelial cells from the microvascular segments in tissues prior to analysis of gene and protein expression does not depend on

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advanced transgene animal models and can be applied to both animal and human tissues. It has the advantage of maintaining the RNA, protein and DNA status as they were at the time of harvesting the tissues. Moreover, it allows the enrichment of endothelial cells from predestined (micro)vascular segments and, as such, can become an important tool for studying endothelial heterogeneity issues. Although LMD downstream analyses in theory could consist of any biochemical read-out technique, the yield of material is often highly limited. Therefore, quantitative RTPCR has been most frequently used as an LMD downstream read-out application, although microarray studies with linearly amplified RNA probes have been reported [71]. By this means, it is possible to study endothelial segment-restricted basal gene expression [26, 72] (see also Fig. 1), as well as local responsiveness to an inflammatory stimulus and local consequences of drug treatment [73, 74]. Kinases are considered to be the most druggable target for treatment of disease. They are instrumental in relaying signals from outside the cell into the nucleus, and a vast array of kinase inhibitors have been developed as potential drugs in the last two decades [75–77]. For pharmacological purposes, knowledge about the kinase activity status in the tissues is of crucial importance. To study the activation status of endothelial cells by analyzing kinase activity, phosphoprotein immunofluorescence detection on tissue sections is a feasible approach [78]. By this means, Liu and colleagues [67] demonstrated that in arterial and venous endothelial cells in the human umbilical cord in situ no difference in phospho-p65 and phospho-jun existed after TNF-α administration, while phospho-ATF2 was slightly higher in endothelium in the venous segment. In human glomerulonephritis, p38 MAPK in endothelial cells, as well as in other glomerular cells, was shown to be prominently activated using this approach [79]. Also, the consequences of drug treatment on kinase activity of the endothelium can be studied [80]. Novel, superior performance phosphoproteinspecific antibodies are awaited to expand the number of kinases and transcription factors to be analyzed this way. It will provide us with a wealth of new information about activation of endothelial cells in the different vascular segments and will be instrumental in creating a view on the full extent of the diversity of endothelial cells in vivo.

Concluding remarks A generic endothelial cell does not exist. Depending on the location in the body, endothelial cells have their own molecular make up that drives basic behavior, as well as responses to inciting stimuli. Loss of microenvironment-driven control when using cultured endothelial cells is a major hurdle in obtaining a detailed picture of the molecular basis of heterogeneity that drives endothelial performance in vivo. Application of new technologies may assist in overcoming some of these hurdles, as may well-designed so-called in vivo-in vitro-in vivo experimental schemes in which

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in vivo observations are modeled in vitro to obtain a more detailed understanding of the molecular identity of processes, which are next immediately validated in the in vivo environment. Although mouse models are a powerful tool to study endothelial engagement in inflammatory diseases, from a pharmacological and drug development point of view, knowledge of their role and responses to drug treatment in patients is crucial. For this we need to identify systemic biomarkers of activation of the endothelial cells in the specific vascular segments that are involved in the disease. We should carefully monitor the endothelial compartment in animal models for genes that engage in disease initiation and/or progress, that are mainly endothelial cell driven, and that can be monitored in serum by virtue of the fact that the proteins they encode are shed from the cell membrane. Validation of their potential as real biomarkers in patients will not be easy, but should be pursued. Together with our ongoing quest to unravel the molecular and functional meaning of microvascular endothelial heterogeneity, this will eventually enable us to develop pharmacologically effective drugs targeted at those endothelial compartments that engage in disease while bypassing the ones that are not involved.

Acknowledgements Dr. Joanna M. Kuldo is acknowledged for skillful execution of experiments that led to the data in Table 1, Figures 2 and 3, and for assistance in making Figures 2 and 3. Mr. Jan Schouten is acknowledged for statistical analysis of the data sets of Figure 3.

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HLA-DR-expressing renal microvascular endothelial cells: Characterization, isolation, and regulation of MHC class II expression. J Am Soc Nephrol 14: 1336–1348 Pusztaszeri MP, Seelentag W, Bosman FT (2006) Immunohistochemical expression of endothelial markers CD31, CD34, von Willebrand factor, and Fli-1 in normal human tissues. J Histochem Cytochem 54: 385–395 Nolasco LH, Turner NA, Bernardo A, Tao Z, Cleary TG, Dong JF, Moake JL (2005) Hemolytic uremic syndrome-associated Shiga toxins promote endothelial-cell secretion and impair ADAMTS13 cleavage of unusually large von Willebrand factor multimers. Blood 106: 4199–4209 Liu M, Kluger MS, D’Alessio A, Garcia-Cardena G, Pober JS (2008) Regulation of arterial-venous differences in tumor necrosis factor responsiveness of endothelial cells by anatomic context. Am J Pathol 172: 1088–1099 Monvoisin A, Alva JA, Hofmann JJ, Zovein AC, Lane TF, Iruela-Arispe ML (2006) VE-cadherin-CreERT2 transgenic mouse: A model for inducible recombination in the endothelium. Dev Dyn 235: 3413–3422 Gareus R, Kotsaki E, Xanthoulea S, van der Made I, Gijbels MJ, Kardakaris R, Polykratis A, Kollias G, de Winther MP, Pasparakis M (2008) Endothelial cell-specific NFkappaB inhibition protects mice from atherosclerosis. Cell Metab 8: 372–383 Minami T, Yano K, Miura M, Kobayashi M, Suehiro J, Reid PC, Hamakubo T, Ryeom S, Aird WC, Kodama T (2009) The Down syndrome critical region gene 1 short variant promoters direct vascular bed-specific gene expression during inflammation in mice. J Clin Invest 119: 2257–2270 Buckanovich RJ, Sasaroli D, O’Brien-Jenkins A, Botbyl J, Hammond R, Katsaros D, Sandaltzopoulos R, Liotta LA, Gimotty PA, Coukos G (2007) Tumor vascular proteins as biomarkers in ovarian cancer. J Clin Oncol 25: 852–861 Asgeirsdottir SA, Werner N, Harms G, van den Berg A, Molema G (2002) Analysis of in vivo endothelial cell activation applying RT-PCR following endothelial cell isolation by laser dissection microscopy. Ann NY Acad Sci 973: 586–589 Zhang L, Zhang ZG, Liu XS, Hozeska-Solgot A, Chopp M (2007) The PI3K/Akt pathway mediates the neuroprotective effect of atorvastatin in extending thrombolytic therapy after embolic stroke in the rat. Arterioscler Thromb Vasc Biol 27: 2470–2475 Asgeirsdottir SA, Kamps JAAM, Bakker HI, Zwiers PJ, Heeringa P, van der Weide K, Van Goor H, Petersen AH, Morselt HW, Moorlag HE et al (2007) Site-specific inhibition of glomerulonephritis progression by targeted delivery of dexamethasone to glomerular endothelium. Mol Pharmacol 72: 121–131 Fabian MA, Biggs WH III, Treiber DK, Atteridge CE, Azimioara MD, Benedetti MG, Carter TA, Ciceri P, Edeen PT, Floyd M et al (2005) A small molecule-kinase interaction map for clinical kinase inhibitors. Nat Biotechnol 23: 329–336 Keri G, Orfi L, Eros D, Hegymegi-Barakonyi B, Szantai-Kis C, Horvath Z, Waczek F, Marosfalvi J, Szabadkai I, Pato J et al (2006) Signal transduction therapy with rationally designed kinase inhibitors. Curr Signal Transduct Ther 1: 67–95 Fedorov O, Marsden B, Pogacic V, Rellos P, Muller S, Bullock AN, Schwaller J,

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Oxidative stress and endothelial dysfunction Stephanie T. de Dios, Christopher G. Sobey and Grant R. Drummond Department of Pharmacology, Monash University, Clayton Campus, Clayton, Victoria 3800, Australia

Abstract Oxidative stress is a hallmark of all cardiovascular risk states (e.g. hypertension, diabetes, hypercholesterolemia, cigarette smoking) and a major underlying cause of endothelial dysfunction, vascular inflammation and blood vessel pathology. Under physiological conditions, cells of the vessel wall produce reactive oxygen species (ROS) such as superoxide (O2• –) and hydrogen peroxide (H2O2) in a deliberate and tightly regulated manner for use as second messengers in redox signalling pathways. However, in vascular pathophysiology, the production of ROS in vascular cells is elevated such that these molecules escape detoxification by cellular antioxidant pathways. When present at higher concentrations, ROS may undergo direct chemical interactions with other biomolecules. Of particular importance are the reactions between O2• – and nitric oxide (NO), which give rise to peroxynitrite (ONOO–), and the iron-catalysed Haber-Weiss reaction between O2• – and H2O2, which gives rise to hydroxyl radicals (OH•). Peroxynitrite and OH• are extremely powerful oxidising species and, along with O2• – and H2O2, cause endothelial dysfunction through direct oxidative damage to cellular macromolecules, impairment of the NO signalling pathway, and activation of pro-inflammatory signalling cascades. Recent evidence suggests that the elevated ROS production in vascular pathophysiology is the result of a complex feed-forward mechanism whereby a primary source of ROS (NADPH oxidases) leads to dysfunction of endothelial nitric oxide synthase, xanthine oxidase and the mitochondrial electron transport chain, so that these enzymes become secondary sources of ROS and major contributors to vascular oxidative stress.

Introduction Oxidative stress may be defined as an imbalance in the amounts of oxidants and antioxidants within a biological system that favours the former. The direct consequence of oxidative stress is that the normal redox state of the biological compartment (e.g. intra-mitochondrial, cytosolic or extracellular space) is shifted towards one that is more oxidising and which may potentially lead to oxidative damage to macromolecules such as proteins, lipids, nucleotides and carbohydrates. Over the past decade it has become apparent that oxidative stress is a hallmark of most, if not all, cardiovascular risk conditions including hypertension, diabetes, hypercholesterolemia and cigarette smoking. Moreover, there is now a large body of evidence Endothelial Dysfunction and Inflammation, edited by Shauna M. Dauphinee and Aly Karsan © 2010 Springer Basel

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to suggest that, in addition to causing gross chemical modifications to cellular macromolecules, oxidative stress may activate signalling cascades that lead directly to inactivation of nitric oxide (NO), vascular remodelling and inflammation. As such, oxidative stress is emerging as a critical early trigger of atherosclerosis. Oxidative stress is caused by an increase in the generation of pro-oxidant molecules in a given cellular compartment and/or by down-regulation of the antioxidant systems that normally remove these molecules. Reactive oxygen species (ROS) and reactive nitrogen species (RNS), which include superoxide anions (O2• –), hydrogen peroxide (H2O2), hydroxyl radicals (OH•), NO and peroxynitrite (ONOO–), are especially important regulators of cellular redox status. ROS and RNS are metabolites of oxygen and/or nitrogen that can either oxidise (abstract electrons), reduce (donate electrons), or form an adduct with (react with and become part of) other molecules [1, 2]. Until relatively recently, ROS and RNS were considered only as damaging by-products of cellular metabolism or as environmental pollutants, respectively. However, it is now recognised that both families of molecules are generated in a highly controlled and deliberate fashion by cells and serve critical second messenger roles in signalling processes that are essential for their function and progression through the cell cycle. It is also emerging that these same pathways responsible for ROS and RNS production for use in normal physiological processes may become ‘over-activated’ in the setting of chronic disease states. In this chapter, we describe the chemical reactions that lead to the formation and subsequent breakdown of ROS and RNS in vascular cells under physiological conditions. We also describe some of the key chemical reactions that ROS and RNS participate in when their formation in cells increase to levels that allow them to escape detoxification by endogenous antioxidants systems. Further, we explain how elevated ROS levels lead to endothelial dysfunction through interfering with the NO signalling pathway and activation of pro-inflammatory signalling cascades. Finally we outline a working hypothesis to explain how elevated ROS production by one enzymatic source, namely NADPH oxidase, may give rise to ROS production by other, normally dormant sources of ROS, setting up a vicious cycle of ROS production that ultimately leads to cellular oxidative stress.

Formation and breakdown of ROS and RNS under physiological conditions Reactive oxygen species The formation of ROS within cells usually begins with the enzyme-catalysed, oneelectron reduction of molecular oxygen giving rise to superoxide anions (O2• –) (Eq. 1). For many years it was recognised that O2• – is produced at complexes I and III of the mitochondrial respiratory chain as a by-product of aerobic metabolism. However, more recently, several extramitochondrial enzymes including NADPH

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Figure 1. Major pathways for ROS formation and breakdown during normal cell physiology.

oxidases, xanthine oxidase and uncoupled endothelial nitric oxide synthase (eNOS) have emerged as potential sources of O2• – in vascular physiology and/or pathophysiology (Fig. 1). O2 + e– → O2• –

Eq. (1)

Although O2• – is a free radical, it normally does not pose a major threat to cells. Firstly, O2• – is a relatively weak oxidising agent and, in some instances, may even act as a reducing agent. Secondly, being a charged molecule, O2• – does not freely cross biological membranes and thus remains in the biological compartment within which it is formed. Finally, O2• – rarely gets the chance to interact with other endogenous molecules due to its propensity to react with itself. This process, called dismutation,

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gives rise to peroxide [hydrogen peroxide (H2O2) in aqueous solutions] and oxygen, and occurs either spontaneously within cellular compartments or via the catalytic actions of a family of enzymes known as superoxide dismutases (SOD) (Eq.  2). SOD-catalysed dismutation is favoured when the concentration of O2• – is low and the concentration of SOD is high, as occurs under physiological conditions. 2 O2– + 2 H+ → H2O2 + O2

Eq. (2)

H2O2, the product of superoxide dismutation, is a more stable molecule than O2• – (half-life in biological systems of 1 ms compared to 1 μs for O2• –) and is arguably a more important ROS in terms of redox signalling [3]. Unlike superoxide, H2O2 is uncharged and can diffuse freely across cell membranes. Furthermore, although H2O2 is a mild oxidant and relatively inert with most biomolecules, it readily and reversibly oxidises cysteine thiol groups (R-SH) within proteins, thereby modulating their structure and function [4, 5]. These properties allow H2O2 to act as an important autocrine and paracrine signalling molecule in vascular and non-vascular cells alike. Nevertheless, H2O2 is a precursor for more powerful oxidising species such as OH• (see ‘Elevated ROS production in vascular disease leads to the formation of powerful oxidising species’ below), and thus it is important that its concentration is maintained within ‘safe’ limits in cells. This is largely achieved by the actions of the family of selenium-containing enzymes known as glutathione peroxidases, which utilise glutathione to metabolise H2O2 into water and oxygen (Eq. 3). In addition, catalase, a haem-containing enzyme largely confined to peroxisomes, also plays a significant role in the detoxification of H2O2. 2 H2O2 → 2 H2O + O2

Eq. (3)

Reactive nitrogen species RNS formation generally begins with the production of NO by a family of enzymes called nitric oxide synthases (NOS). Three members of the NOS family have been identified including NOS1, a constitutive isoform expressed primarily in neurons; NOS2, an inducible isoform expressed mainly in leukocytes; and NOS3, also known as endothelial NOS (eNOS) due to its constitutive expression in this cell type [6, 7]. eNOS catalyses the conversion of l-arginine and molecular oxygen into l-citrulline and NO, in a complex reaction that consumes five electrons and is dependent on NADPH as an electron donor as well as several other co-factors including haem, flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and (6R-)5,6,7,8tetrahydrobiopterin (BH4) [8]. The biochemistry of eNOS catalysed NO formation is discussed further in the section ‘The enzymatic sources of ROS production in physiology and pathophysiology’ below, but can be summarised as follows (Eq. 4):

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l-Arginine + O2 → l-Citrulline + NO

Eq. (4)

NO is one of the most important vasoprotective molecules yet identified, mediating a host of athero‑protective effects including inhibition of vascular smooth muscle cell tone, proliferation and migration, and suppression of the expression of inflammatory molecules on the endothelium. Most of these protective actions of NO are mediated by activation of the ‘NO receptor’, soluble guanylyl cyclase (sGC), which, upon binding its ligand at a haem site, catalyses the conversion of GTP into the second messenger cyclic GMP [7, 9]. Within cells, NO can exist in at least two distinct redox states. During normal physiology, NO is primarily produced by eNOS in its radical form, NO•. NO• is the redox variant of NO that binds to the ferrous (Fe2+) haem of sGC, which is the predominant oxidation state that exists during physiological conditions. As such, NO• is responsible for most of the biological actions of NO. However, under certain conditions, NO• may be reduced to its nitroxyl anion (NO–). The reversible reduction of NO• to NO– has been shown to be catalysed by mitochondrial cytochrome C, xanthine oxidase, haemoglobin and manganese SOD. Furthermore, NO– may also be formed directly by eNOS when BH4 bioavailability is low [10–12]. At physiological pH, NO– is protonated (i.e. HNO) and thus, like NO•, it is uncharged and relatively free to move across cellular membranes into different compartments to act as a paracrine signalling molecule. HNO appears to have many biological properties that are distinct from NO•. Notably, HNO can regulate the activity of many proteins including glyceraldehyde 3-phosphate dehydrogenase (GAPDH), the N-methyl-d-aspartate receptor, as well as cardiac (RyR2) and skeletal (RyR1) sarcoplasmic ryanodine receptors by interacting directly with key cysteine thiol groups [13]. Interestingly, HNO preferentially binds to ferric (Fe3+) over ferrous (Fe2+) haem proteins. Recent evidence suggests that in vascular pathophysiology, when the redox state of the endothelial cytosol is elevated, a pool of sGC becomes oxidised such that its haem group is present in the ferric (Fe3+) form [14]. Thus, it is conceivable that increased HNO formation in vascular pathology represents an adaptive response of endothelial cells to ensure that sGC activity is not compromised despite oxidation of its haem group. Interestingly, HNO is also resistant to degradation by superoxide, which is another reason why its preferential formation over NO• during times of vascular disease is likely to be advantageous. The vast majority of NO formed in the endothelium is lost to the circulation. This is because the haemoglobin of circulating erythrocytes represents a significant sink for NO. Indeed, NO undergoes multiple reactions with haemoglobin that lead to a reduction in its bioavailability in the vascular wall. These include the reaction of NO with (i) oxyhaemoglobin to form nitrate; (ii) haemoglobin to form nitrosylhaemoglobin; or (iii) with the Cys93 residue of the haemoglobin β-subunit to form nitrosohaemoglobin. Due to this massive loss of NO to the circulation, it has been estimated that endothelial cells need to produce 10–40 times more NO than they would actually need to activate sGC in the underlying smooth muscle. NO also

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undergoes chemical reactions in the aqueous phase that are important for maintaining its levels within the physiological range. The most important of these reactions in the context of ‘detoxification’ of NO into a relatively inert product is the reaction with oxygen resulting in the formation of nitrite (Eq. 5). 4 NO + O2 + 2 H2O → 4 NO2− + 4 H+

Eq. (5)

Elevated ROS production in vascular disease leads to the formation of powerful oxidising species In the previous section we discussed the key chemical reactions involved in production and metabolism of ROS and RNS under physiological conditions. However, many cardiovascular risk states are associated with a marked and chronic increase in the production of ROS in vascular cells that overwhelms endogenous antioxidant defence mechanisms and allows ROS to interact with other biomolecules. In this section, we describe some of the key chemical reactions that arise when ROS are present in excess within cells. These reactions not only result in the depletion of important signalling (e.g. NO•, tetrahydrobiopterin) and cell defence molecules (glutathione, ascorbic acid), but also lead to the formation of powerful oxidising species with the potential to inflict oxidative damage to cellular macromolecules such as lipids, proteins and nucleotides. Arguably, the most important chemical reaction that arises when ROS production exceeds the antioxidant capacity of the vessel wall is the bi-radical reaction between O2• – and NO• (Eq. 6), first described by Beckman et al. [15]. This reaction occurs at a rate of 6.7 × 109 M–1.s–1, which is at least twice the speed of the reaction of O2• – with SOD [16]. In fact, the speed of the reaction between O2• – and NO• means that it is likely to be limited only by diffusion. This reaction has two very important consequences. First, it results in depletion of NO•, and thus a loss of its important anti-inflammatory, vasodilatory and anti-thrombotic actions. In addition, the product of the reaction between O2• – and NO• is the powerful oxidising species peroxynitrite. At physiological pH, peroxynitrite exists predominantly in its anionic form (ONOO–). However, a significant proportion of peroxynitrite (~20%) is protonated (HONOO). Peroxynitrite is likely to play a key role in the depletion of small molecule antioxidants such as glutathione, ascorbate and urate, thereby increasing the vulnerability of the cell to damage by further oxidative insults. Furthermore, peroxynitrite is particularly efficient at oxidising the eNOS cofactor BH4 (discussed in ‘Secondary sources of ROS in vascular disease’ below) and is therefore a key player in a series of events that leads to the propagation of ROS production by oxidases that are normally dormant under physiological conditions [17]. Finally, peroxynitrite is a powerful oxidiser of cellular proteins, lipids and nucleotides. These effects are mediated both directly by peroxynitrite-mediated nitration of

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protein thiol groups, and indirectly following the decomposition of HONOO in the presence of CO2 into radical species such as NO2• –, CO3• – and OH•, all of which are highly effective oxidisers of cellular macromolecules [18]. NO + O2• – → ONOO–

Eq. (6)

Another type of chemical reaction that is likely to contribute to the formation of powerful oxidising species in the vessel wall during vascular disease is the HaberWeiss/Fenton reaction. This reaction generates OH• from H2O2 and O2•– and requires free iron as a catalyst. The reaction occurs in two steps as follows: O2• – + Fe3+ → Fe2+ + O2

Eq. (7a)

H2O2 + Fe2+ → Fe3+ OH• + OH–

Eq. (7b)

O2• – + H2O2 → OH• + OH– + O2

Eq. (7; summary)

Unlike O2• –, H2O2 and NO•, which are relatively weak oxidants and display a certain degree of selectivity in terms of their targets for oxidation, OH• are extremely powerful oxidants and will react with virtually any molecule they come into contact with, including proteins, lipids, sugars and nucleotides. One might ask the question, if cells are constantly producing O2• – and H2O2 under physiological conditions, why are OH• radicals not also formed continuously? The answer to this question is that the concentration of free iron in cells is normally maintained at extremely low levels. Iron homeostasis in cells consists of (i) uptake of iron by the transferrin receptor or divalent metal transporter-1; (ii) utilisation of iron for the synthesis of iron-containing proteins such as haemoproteins and 4Fe-4S cluster proteins; and (iii) storage of any excess iron in the iron-sequestering protein, ferritin [19]. While bound in proteins, iron is said to be catalytically inactive. However, when present at excess levels, ROS, such as superoxide and H2O2, can liberate iron from these stores [18, 20]. For example, O2• –, H2O2 and even NO• (either directly or indirectly via ONOO–) can react with the [4Fe‑4S] clusters of proteins such as aconitase, dihydroxy acid dehydratase and fumarases A and B, converting them into their inactive 3Fe-4S forms and liberating iron in the process [21].

ROS-mediated damage to cellular macromolecules Within the vessel wall, the major cellular targets of ROS are membrane lipids, proteins, nucleic acids, and carbohydrates. At higher concentrations, ROS, and in particular OH•, initiates lipid peroxidation via the one-electron oxidation of lipids

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that are present in increased levels within the vessel wall in diseased states such as atherosclerosis. Lipid oxidation leads to the formation of a carbon-centred radical, which is further attacked by O2, forming an unsaturated conjugate peroxide that undergoes further spontaneous cleavage, releasing aldehydes and hydrocarbons [22, 23]. Lipid peroxidation is particularly harmful because the damage induced can be amplified by the additional release of reactive substances, radicals or fatty aldehydes providing an opening for further modification of cellular structures. For instance, once fatty acids have been oxidised, they can activate cell-signalling pathways via (i) covalent pathways leading to direct protein modification; (ii) the activation of pathways leading to calcium influx and intracellular ROS/RNS formation; or (iii) non-covalent mechanisms involving binding to a protein receptor [22]. Therefore, the end products of lipid peroxidation can cause secondary damage or direct protein attack by ROS/RNS, resulting in oxidative protein damage [24]. Radical-mediated damage to proteins, such as protein oxidation, may also be initiated by electron leakage and metal-ion-dependent reactions. Various RNS, such as ONOO–, can attack tyrosine, as well as initiate the nitration of phenylalanine and tryptophan. While ROS attack of proteins by either OH• or singlet O2, can generate multiple end products, there is little or no effect of H2O2 or O2•– on proteins at physiological conditions unless there is an easily‑oxidisable and accessible thiol (‑SH) group in the structure [24]. The side chains of amino acid residues of proteins, such as cysteine and methionine, are also particularly susceptible to oxidation via ROS/RNS attack, leading to the reversible formation of mixed disulphides between protein thiol groups and low molecular weight thiols [25, 26]. The oxidative damage to proteins cascades to the damage of receptors, enzymes, signal transduction pathways, as well as transport proteins and contributes to secondary damage to other biomolecules [24]. Oxidised proteins are often functionally inactive and their unfolding is associated with enhanced susceptibility to proteinases and can generally be removed by proteolysis from cells [27]. ROS is also an important mediator of damage to cellular structures and nucleic acids. At physiological conditions ROS/RNS such as superoxide, nitric oxide or H2O2 do not appear to significantly react with any DNA or RNA bases or sugars [24]. However, the highly reactive OH• attacks all components of the DNA molecule to produce damage involving single- or double-strand DNA breaks, damage to both the purine and pyrimidine bases, DNA cross‑links and deoxyribose modifications to the DNA backbone, with permanent modification of genetic material initiating mutagenesis, carcinogenesis and aging [24, 28]. It is understood that metal‑induced generation of ROS results in an attack not only on DNA, but also on other cellular components such as polyunsaturated fatty acid residues of phospholipids forming peroxyl radicals (ROO•) [26]. Nuclear proteins can also be attacked by ROS resulting in protein-driven radicals that are able to cross-link to base-derived radicals if the two meet in the chromatin to produce DNA–protein cross-links that interfere with chromatin unfolding, DNA repair, replication and transcription [24].

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How does oxidative stress lead to endothelial dysfunction? The previous section focussed on the direct oxidising and damaging effects of higher concentrations of ROS and RNS on cellular macromolecules. However, as with any chronic disease process, oxidative stress is likely to be a gradual process beginning with moderate elevations in ROS production and modest changes in the cellular redox state. While at these levels, ROS are unlikely to cause oxidative damage to cellular constituents, they can mediate more subtle, albeit pathologically relevant, effects at the level of cell signalling. Since the early 1980s, the endothelium has become recognised as far more than a mere barrier between the intravascular and interstitial compartments. Indeed, the endothelium is now known to mediate a diverse range of functions including regulation of vascular tone, vascular remodelling, anti-inflammatory, and anti-thrombogenic processes. Thus, in a broad sense, endothelial dysfunction can be defined as a disturbance in any or all of these normal functions and there is evidence that elevated ROS and oxidative stress play roles in impairing many of these processes.

Impaired NO signalling The most prominent feature of endothelial dysfunction is a reduction in the bioavailability and efficacy of the potent vasodilator, anti-thrombotic and anti-inflammatory molecule, endothelium-derived NO. Reduced NO function in vascular disease is mediated at multiple steps along the NO signalling pathway. As discussed above, elevated superoxide production is likely to be a major cause of NO breakdown in vascular disease. However, the generation of NO is also impaired in vascular disease states due to a reduction in the normal catalytic activity of eNOS. This is brought about in part by an increase in the concentration of an endogenous antagonist of NOS, asymmetric dimethyl arginine (ADMA) [29, 30]. ADMA is released in cells when methylated proteins are degraded into their amino acid constituents as part of hydrolytic protein turnover. Within endothelial cells, ADMA is normally rapidly converted into dimethylamine and l-citrulline by dimethylarginine dimethylaminohydrolase-2 (DDAH-2). However, increased ROS production following exposure of endothelial cells to pathological stimuli, such as tumour necrosis factor-α, oxidised LDL and lysophosphatidylcholine, causes inactivation of DDAH-2 and accumulation of ADMA. ROS also affects eNOS activity by oxidising the critical co-factor BH4. As discussed in detail below (see ‘Secondary sources of ROS in vascular disease’), a loss of BH4 causes eNOS uncoupling, switching the enzyme from NO to superoxide generation. Finally, the ferrous haem group of sGC, which acts as the receptor for NO, is susceptible to oxidation into the ferric form, which is less responsive to NO. The mechanisms by which ROS and oxidative stress impair NO signalling are summarised in Figure 2.

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Figure 2. Mechanisms of NO signalling impairment.

Activation of pro-inflammatory signalling cascades As mentioned above, cells generate ROS, especially O2• – and H2O2, in a deliberate and tightly controlled fashion and use these molecules as second messengers in a wide variety of signalling pathways. The number of signalling elements that are now known to be redox-sensitive are extensive and include receptor and non-receptor tyrosine kinases, protein tyrosine phosphatases, serine threonine kinases and nuclear transcription factors (e.g. AP-1, NF-κB, p53, NFAT, HIF-1). To attempt to describe the downstream consequences of redox activation of these signalling elements is beyond the scope of the present discussion; suffice to say that many of them could be described as ‘pro-inflammatory’. Thus, it stands to reason that even modest elevations in the concentrations of O2• – and H2O2, as are likely to occur in the early stages of vessel disease, will, at the very least, lead to prolonged and/or over-activation of many of these pro-inflammatory signalling pathways.

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Enzymatic sources of ROS production in physiology and pathophysiology In the previous section we discussed the consequences of elevated vascular ROS levels in terms of their effects on NO and pro-inflammatory signalling cascades and as mediators of oxidative damage to cellular macromolecules. In this section we discuss which enzymes are responsible for ROS production in vascular physiology and which factors cause these and other enzymes to become over-activated or dysfunctional, such that they generate inappropriate amounts of ROS in the setting of vascular disease.

NADPH oxidases are primary sources of ROS in the vessel wall NADPH oxidases are a family of multi-subunit enzyme complexes that are unique in being the only enzymes whose primary function appears to be the generation of ROS. This sets NADPH oxidases apart from all other ‘oxidases’ that produce ROS either as a by-product of another catalytic function or, alternatively, when the enzyme is rendered dysfunctional, such as by oxidative modification. To date, seven members of the NADPH oxidase family have been described on the basis of the catalytic subunit they utilise (either Nox or Duox) to generate ROS. These include Nox1-5 and Duox1-2. Furthermore, NADPH oxidases may require up to five additional protein subunits for activity including: a small, membrane-bound protein (p22phox), which forms a heterodimer with certain Nox proteins to stabilise their expression within biological membranes; cytosolic ‘organiser’ (p47phox or Noxo1) and activator (p67phox or Noxa1) proteins; a third cytosolic protein (p40phox) that is known to associate with the Nox2-containing enzyme complex but whose function remains to be defined; and, finally, a small GTPase (Rac1 or 2) [31–35]. The Nox2 (formerly known as gp91phox) -containing isoform was the first member of the NADPH oxidase family to be identified on the basis of its function in generating the ‘respiratory burst’ of phagocytic leukocytes. However, it is now apparent that this ‘classical’ isoform, along with the six remaining members of the NADPH oxidase family, are also expressed in non-phagocytic cells and tissues [31]. With regards to vascular (patho)physiology, four NADPH oxidase isoforms – Nox1, 2, 4 and 5 – have been identified in vascular cells [33, 36]. The molecular structures of each of the vascular isoforms of NADPH oxidase are depicted in Figure 3. The individual Nox proteins utilised by each of the vascular isoforms of NADPH oxidase represent separate gene products and display a wide range of variation in their primary amino acid sequences. Thus, whereas Nox1 and Nox2 exhibit a high degree of homology, Nox4 and Nox5 only display 27% and 37% homology, respectively, to Nox2 [37, 38]. Nevertheless, topology modelling of the different vascular Nox proteins predicts that, irrespective of their primary amino acid sequences, each of them are built on the same general molecular plan. This includes a cystolic

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Figure 3. Vascular isoforms of Nox.

C-terminal tail containing NADPH and flavin adenine dinucleotide (FAD) -binding regions, as well as six transmembrane-spanning α-helices containing histidine residues for the binding of two prosthetic haem groups (Fig. 4) [37]. An understanding of the membrane topology of the vascular Nox proteins is critical for making predictions about which cellular compartments they are likely to generate ROS in. Studies examining the subcellular distribution of Nox2, Nox4 and Nox5 in endothelial cells indicate that the proteins are localised to varying extents on plasma, endoplasmic reticulum and nuclear membranes [39–43]. If one accepts that the NADPH-binding C terminus protrudes into the cytosol irrespective of the organelle

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Figure 4. Topology of Nox catalytic subunit of the NADPH oxidases.

that the Nox protein is expressed on, then it follows that electron flow through the proteins will always result in ROS generation either directly to the outside of the plasma membrane, or within the luminal compartments of subcellular organelles (Fig. 3). When O2• – is the end product of NADPH oxidase activity (which is likely to be the case for at least Nox1 and Nox2) it would not be expected to affect the redox environment in the cytosolic space unless its passage across biological membranes was facilitated by anion channels or by dismutation to membrane-permeable H2O2. Either way, one would predict that NADPH oxidase-derived O2• – is likely to be present at highest concentrations within the luminal compartments of intracellular organelles or outside the cell. In this latter location, O2• – would be strategically placed to interact with endothelium-derived NO• as it transverses the interstitial space between endothelial and smooth muscle cells. Since the seminal observation that angiotensin II stimulates NADPH oxidase activity in rat cultured vascular smooth muscle cells [44], a wealth of evidence has been generated demonstrating that expression of at least two isoforms of NADPH

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oxidase – Nox1 and Nox2 – are up-regulated in cultured vascular cells (endothelial cells, vascular smooth muscle cells and adventitial fibroblasts) following stimulation with proatherogenic stimuli such as angiotensin II, platelet-derived growth factor, thrombin and oscillatory shear stress [33, 45]. Further, these same isoforms of NADPH oxidase are markedly up-regulated in the vessel wall in vivo in the settings of experimental hypertension [46], hypercholesterolemia ([47], and Judkins et al., 2009, unpublished) and diabetes mellitus [48, 49], as well as in patients with established atherosclerosis [50, 51]. By contrast, the vascular expression of Nox4 either remains unchanged or is down-regulated in these vascular pathophysiological states. There is currently very little information on regulation of Nox5 in the vasculature due mainly to the fact that this Nox homolog is not expressed in rodents [34], which are the focus of most experimental studies into vascular disease mechanisms. As for the cellular localisation of the pathologically relevant vascular isoforms of NADPH oxidase, cell culture studies and immunohistochemical analysis of transverse sections of blood vessels identify endothelial cells, adventitial fibroblasts and leukocytes residing within atherosclerotic plaques as major sources of Nox2NADPH oxidase activity, while medial smooth muscle cells appear to be the primary site of Nox1-NADPH oxidase activity (Fig. 5).

Figure 5. Cellular distribution of Nox isoforms in the vascular wall.

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In addition to the studies outlined above that demonstrate an association between elevated Nox1 and Nox2 expression and vascular disease, several reports have provided direct evidence for a cause-and-effect relationship between NADPH oxidase activity and vascular oxidative stress and disease. Such studies have utilised genetically modified mice that are deficient in either Nox1 [52–54] or Nox2 [55–58], or in the NADPH oxidase organiser subunit p47phox [59]. For example, the induction of hypertension in wild-type mice by treatment with angiotensin II or deoxycorticosterone acetate (DOCA) salt, or following renal artery clipping is associated with marked elevations in vascular O2• – production and markers of oxidative stress (e.g. 3-nitrotyrosine) and reductions in NO bioavailability. However, while the above hypertensive stimuli still caused elevations in blood pressure in mice lacking Nox1, Nox2 or p47phox, the associated changes in vascular ROS and NO levels were not observed. A major limitation with current hypertension models in mice is that while they are associated with medial vascular hypertrophy, they do not lead to the development of true atherosclerotic plaques. Thus, in all of the above studies it remains unclear whether the reductions in vascular ROS levels and improvements in endothelial function would have actually translated to a reduction in atherogenesis. Thus, we and others have focussed our attention on apolipoprotein E-deficient mice. These animals are spontaneously hypercholesterolemic and develop fatty streaks and fibro-fatty plaques along their aorta and major arterial branches thereof. Several studies have shown that the absence of either Nox2 or p47phox in novel strains of Nox2–/–/ApoE–/– and p47phox–/–/ApoE–/– markedly reduced the development of atherosclerotic plaques along the length of the aorta [60–62]. Moreover, we have recently shown that similar atheroprotection is afforded to ApoE–/– mice when they are crossed with Nox1–/– animals (unpublished data). Thus, collectively, the above findings in cultured vascular cells and experimental models of hypertension, diabetes and hypercholesterolemia provide strong evidence for a role for NADPH oxidases, and in particular the Nox1- and Nox2-containing isoforms of the enzyme, as primary sources of ROS in the vessel wall in vascular disease states. However, as discussed below, NADPH oxidases are unlikely to be the only source of ROS in vascular disease. In fact, there is emerging evidence that NADPH oxidase activity results in only a modest, albeit pathologically relevant, increase in vascular ROS production. This ultimately leads to activation of dormant sources of ROS such as uncoupled eNOS, xanthine oxidase and the mitochondrial electron transport chain, each of which may actually be quantitatively greater sources of ROS in the vessel wall, especially at later stages of disease. This notion of ROS production by one enzyme source begetting further ROS production by other enzymes was first proposed by Prof. David Harrison (Emory University, Atlanta, USA) who subsequently termed it as the ‘Kindling-bonfire hypothesis of vascular oxidative stress’.

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Secondary sources of ROS in vascular disease – Uncoupled eNOS, xanthine oxidase and the mitochondrial electron transport chain eNOS is arguably the most important athero-protective enzyme in the blood vessel wall. It is responsible for the production of endothelium-derived NO, which plays a critical role in the maintenance of a healthy vasculature through its powerful vasorelaxant, anti-inflammatory and anti-clotting actions. However, there is a growing body of evidence to suggest that eNOS may also have a dark side – as a significant source of intracellular ROS and thus as a major contributor to vascular oxidative stress and endothelial dysfunction in cardiovascular risk states, such as hypercholesterolemia, hypertension and diabetes. eNOS is a cytosolic enzyme that is associated with caveolae via myristolation and protein-protein interactions with caveolin-1. In its normal functional state, eNOS exists as a homodimer in which the C-terminal reductase domain of one monomer is functionally ‘coupled’ to the N-terminal oxygenase domain of the other monomer [63–65]. Electrons enter and are transported through the reductase domain via NADPH, FAD and FMN. They are then transferred to the prosthetic haem group in the oxygenase domain of the second monomer, which is the site where l-arginine is ultimately oxidised to l-citrulline and NO. Studies on purified enzyme preparations demonstrate that the transfer of electrons from one eNOS monomer to the other is critically dependent on the cofactor, BH4. In the absence of BH4, electron flow from the FMN group of the reductase domain to the ferrous-dioxygen haem complex on the adjacent oxygenase domain is disturbed such that electrons are diverted to molecular oxygen leading to superoxide production (Fig. 6) [66, 67]. Thus, as BH4 levels progressively diminish, eNOS becomes increasingly enzymatically ‘uncoupled’, thereby progressively switching from an enzyme that almost exclusively generates NO, to one that generates superoxide. A further implication of this progressive uncoupling is that at intermediate stages of the process, eNOS produces both NO• and O2• – thereby effectively becoming a peroxynitrite-generating enzyme [68]. Peroxynitrite generation by eNOS is likely to have major implications both as a significant contributor to vascular oxidative stress per se, and also as a mediator of further BH4 depletion and eNOS uncoupling. It is now well established that vascular disease states are associated with marked reductions in endothelial BH4 availability. A major factor that contributes to this reduction appears to be a diminished capacity of endothelial cells to recycle BH4 from its oxidation product 7,8-dihydrobiopterin (BH2) [69–72]. Recycling of BH4 is normally facilitated by the enzyme dihydrofolate reductase (DHFR). Recent studies demonstrate that pro-atherogenic stimuli such as angiotensin II, down-regulate expression of DHFR in cultured endothelial cells resulting in depletion of BH4 and an increase in superoxide production from eNOS [70]. Importantly, this process of DHFR down-regulation and eNOS uncoupling was inhibited by a scavenger of

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Figure 6. eNOS uncoupling.

H2O2 and an antagonist of NADPH oxidase, suggesting a critical role for NADPH oxidase-derived H2O2 as the mediator of DHFR down-regulation. The concept of elevated NADPH oxidase being the upstream mediator of BH4 depletion and eNOS uncoupling is also supported by in vivo observations [73]. For example, in wild-type

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mice, DOCA salt-induced hypertension was associated with a reduction in the ratio of BH4:BH2 and an increase in the amount of O2• – detected in freshly isolated aortic segments. Importantly, the elevated O2• – levels detected in these isolated rings could be partially attenuated by an NOS inhibitor, providing evidence for eNOS uncoupling in this experimental model of hypertension. By contrast, in p47phox–/– mice, DOCA salt treatment had little effect on aortic BH4 levels, nor was it associated with an increase in superoxide production. In addition to evidence for impaired BH4 recycling in vascular disease states, there is also strong evidence for a role for direct oxidation of endothelial BH4 by peroxynitrite [74]. As mentioned above (see ‘NADPH oxidases are primary sources of ROS in the vessel wall’), O2• – generated as a direct result of elevated Nox1 and Nox2 NADPH oxidase activity could potentially interact with NO• as it traverses the interstitial space between the endothelium and underlying vascular smooth muscle, thereby giving rise to peroxynitrite. However, this interaction would result in peroxynitrite generation in the extracellular compartment where it is unlikely to play a significant role in oxidising intracellular (cytosolic) BH4. Thus, it is more likely that the source of peroxynitrite responsible for BH4 oxidation in vascular disease is a cytosolic oxidase such as uncoupled eNOS. Therefore, we propose that the role of NADPH oxidase in eNOS uncoupling is not as the direct oxidiser of BH4 but rather as the source of H2O2 that leads to down-regulation of DHFR expression and impaired recycling of BH4. This likely leads to partial eNOS uncoupling, such that the enzyme simultaneously generates O2• – and NO•, which rapidly combine to form peroxynitrite. The eNOS-derived peroxynitrite, which is generated in the cytosol, can then directly oxidise BH4 leading to further eNOS uncoupling.

Xanthine oxidoreductase Xanthine oxidoreductase (XOR) is a widely expressed homodimeric enzyme that plays a critical role in purine degradation by acting as the rate-limiting enzyme for the conversion of hypoxanthine into xanthine and xanthine into urate. XOR actually exists in two distinct and interconvertible forms, i.e. xanthine dehydrogenase (XDH) and xanthine oxidase (XO) [75, 76]. Under physiological conditions in vivo, XOR is mainly expressed in its dehydrogenase form. In this form, the enzyme has a high affinity for NAD+, which it utilises as the main electron acceptor in the catalytic conversion of (hypo)‑xanthine to urate, with NADH being formed as a by-product. However, XDH can be readily converted into its oxidase form, either reversibly by thiol oxidation of critical cysteine residues involved in disulphide bridge formation, or irreversibly by proteolysis. In it oxidase form, XOR is still capable of metabolising (hypo)‑xanthine into urate; however, it has a markedly reduced binding affinity for NAD+. Instead it uses molecular oxygen as an electron acceptor and, in doing so, generates equimolar amounts of superoxide and H2O2, as opposed to NADH. 54

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Experimental and clinical studies demonstrate that many cardiovascular risk states are associated with a marked increase in plasma XOR activity and that XOR activity is a strong predictor of clinical outcomes. Furthermore, allopurinol, a selective inhibitor of XOR that has been in clinical use for over 40 years for the treatment of gout, improves endothelial function and reduces vascular oxidative stress in animals and patients with chronic heart failure, coronary artery disease, hypertension, diabetes, sleep apnoea, etc. [77–82]. XOR is normally expressed at very low levels in the blood vessel wall. However, in patients with coronary artery disease, XO activity in coronary arteries, as measured by xanthine-mediated or allopurinol-sensitive O2• – production, was increased approximately twofold compared to control patients [51, 83]. This increase in XO activity is largely the result of an increase in binding of circulating XOR to aminoglycans on the surface of the endothelium, although, recent cell culture studies indicate that endothelial cells are also capable of de novo synthesising their own supply of XOR following stimulation with angiotensin II [84]. As stated above, XOR is synthesised in its dehydrogenase form and thus would normally not be expected to be a major source of ROS in the vasculature [85]. In contrast, XDH may actually play an important antioxidant role through the generation of uric acid, which is a powerful scavenger of peroxynitrite. However, when the prevailing redox environment is oxidising in nature, such as would be expected in the vessel wall when the activity of other oxidases (NADPH oxidase and uncoupled eNOS) is elevated, XDH is likely to be converted to XO, thereby becoming yet another contributor to vascular oxidative stress [86]. Analogous to the situation with eNOS, the above discussion indicates that XOR is only likely to become a contributor to vascular oxidative stress following its dysregulation by an external source of ROS. Moreover, recent studies support the notion that NADPH oxidase is the enzyme system that provides this external source of ROS. Exposure of endothelial cells to oscillatory shear stress resulted in an increase in the ratio of XO to XDH expression [87]. Oscillatory shear stress also caused elevations in O2• – and H2O2 generation that were sensitive to inhibition by oxypurinol [87]. Importantly, oscillatory shear stress failed to increase XO expression and oxypurinol-sensitive ROS production in endothelial cells with impaired NADPH oxidase activity either through a lack of p47phox expression or as a result of chronic exposure to apocynin [87]. Thus, like uncoupled eNOS, XO is most likely secondary to NADPH oxidase as a source of ROS production in vascular pathophysiology.

Mitochondrial respiratory chain During normal mitochondrial respiration, the leakage of electrons to molecular O2 at complexes I (NADH dehydrogenase), II (succinate dehydrogenase) and III 55

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(ubiquinone-cytochrome bc1) of the electron-transport chain results in the generation of O2• – towards the mitochondrial matrix. Despite estimates that as much as 1–2% of total O2 that enters the electron transport chain is converted to O2• –, an abundance of antioxidant defence systems within the organelle ensure that this ROS is normally rapidly detoxified. Manganese SOD, which is expressed at high levels in the matrix, rapidly converts mitochondrial O2• – into H2O2. The net amount of H2O2 left available for diffusion out of the mitochondria and into the cytosol (potentially for use in redox signalling) is governed by the actions of several antioxidant enzymes such as catalase, glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase and thioredoxin 2, as well as small molecule antioxidants such as glutathione. Importantly, the mitochondrial matrix also expresses several enzymes involved in the regeneration of oxidised antioxidants including glutathione reductase, thioredoxin reductase, glutaredoxin reductase and peroxiredoxin. Numerous stimuli that are known to induce endothelial dysfunction have been shown to increase mitochondrial ROS production. These include angiotensin II, the adipokine leptin, CD40, hypoxia, cyclic strain, oxidised LDL, electrophilic lipid oxidation products and high glucose [88]. Importantly, excessive ROS generation by mitochondria appears to be a direct contributor to endothelial dysfunction and vessel disease. For example, whereas genetic deletion of manganese SOD in ApoE–/– mice reduced NO bioavailability and accelerated atherosclerotic lesion development [89], endothelial-specific overexpression of the mitochondrial thioredoxin 2 gene in the same strain of mice preserved endothelial function and afforded protection against atherosclerosis [90]. Of particular interest in the context of the over-arching theme of this discussion that ROS from one source may stimulate further ROS production by other enzymes, is the observation that ROS, such as H2O2 and ONOO–, can themselves directly increase mitochondrial ROS production. Furthermore, Doughan et al. [91] provided evidence that, similar to uncoupled eNOS and XOD, excessive ROS generation by mitochondria may occur downstream of elevated NADPH oxidase activity. Specifically, these authors demonstrated that mitochondria isolated from bovine aortic endothelial cells that had undergone prior treatment with angiotensin II, generated substantially more ROS and contained less glutathione than those from untreated cells. Importantly, this effect of angiotensin II on mitochondrial ROS production was prevented by treatment of the endothelial cells with an NADPH oxidase inhibitor, apocynin, or by siRNA-mediated knockdown of p22phox [91]. Of further interest was the observation that angiotensin II‑induced mitochondrial ROS production was also negated by the NOS inhibitor, L‑NAME, and the peroxynitrite scavenger, urate, raising the tantalising possibility that mitochondria may be a tertiary source of ROS production in the setting of vascular disease, with NADPH oxidase and eNOS acting as primary and secondary sources of ROS, respectively. Figure 7 provides a summary of the ‘Kindling-bonfire hypothesis of vascular oxidative stress’ whereby NADPH oxidase acts as the primary source of O2• – and H2O2 in the 56

Oxidative stress and endothelial dysfunction

Figure 7. Kindling-bonfire hypothesis of oxidative stress.

v­ essel wall. NADPH oxidase-derived ROS then act as ‘kindling’ to cause activation of secondary (uncoupled eNOS, XO) and tertiary (mitochondria) sources of ROS, which contribute to the ‘bonfire’ of radicals and oxidative stress seen in later stages of disease.

Conclusion This chapter details the complexity of the enzymatic pathways and redox chemistry that underlie the elevated ROS production and oxidative stress in the blood vessel wall during pathophysiological conditions such as hypertension, diabetes hypercholesterolemia, and cigarette smoking. In doing so, it also highlights the inherent difficulties that scientists and clinicians are likely to face in attempting to devise therapeutic strategies to reduce vascular oxidative stress and its downstream sequelae of endothelial dysfunction and atherogenesis. Indeed, several large-scale clinical trials investigating the therapeutic potential of antioxidant supplementation

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(primarily with vitamin E) in patients with cardiovascular disease, have failed to demonstrate any benefit of such a strategy. We would argue that this is not surprising and likely due to a combination of factors, including: (i) that the reaction kinetics of vitamin E with its target ROS is relatively slow compared to the rate of the reaction of these same ROS with endogenous biomolecules (e.g. NO•); and (ii) that even if vitamin E does react with endogenous ROS to an appreciable degree, it is likely to initiate the formation of downstream ROS and radicals, such as H2O2 and tocopheroxyl radicals. Thus, we would suggest that future strategies for ameliorating vascular oxidative stress may involve the use of compounds that react and form stable adducts with ROS, thereby obviating the formation of downstream ROS and radicals. Such compounds must also possess reaction kinetics that allow them to compete favourably with endogenous molecules (e.g. NO) for ROS. Alternatively, future therapies might involve direct inhibition or restoration of normal catalytic function of the enzymes responsible for ROS production in vascular disease, thereby blocking ROS production and all of the downstream redox chemistry and signalling from the outset. With regards to this latter strategy, the kindling-bonfire hypothesis of vascular oxidative stress may provide a framework for future diagnostic applications focussed on assessing patients’ oxidative status, and for potential therapeutic strategies for reducing individual patient oxidative stress.

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Post-translational regulation of eNOS activity in inflammation Fanny Desjardins and Jean-Philippe Gratton Laboratory of Endothelial Cell Biology, Institut de recherches cliniques de Montréal (IRCM), Université de Montréal, Montreal, Quebec, Canada

Abstract Endothelial nitric oxide synthase (eNOS) produces low basal amounts of nitric oxide (NO), which is responsible for overall vascular homeostasis. However, in some conditions, such as during inflammation, local agonists that act on endothelial cells cause a series of post-translational modifications that influence eNOS activity. Furthermore, the balance in the influence of positive and negative post-translational regulators of eNOS activity will contribute to its activation by autacoids, growth factors or shear stress. Here, we highlight the molecular mechanisms that regulate eNOS activity at the post-translational level, such as the cellular localization, protein-protein interactions, and phosphorylation. The impact of post-translational regulation on eNOS-derived NO in inflammation is also discussed.

Introduction Nitric oxide (NO) produced by endothelial cells plays an essential role in the regulation of the normal physiological function of the vascular wall and it is recognized as a key regulator of blood vessel integrity and cardiovascular homeostasis. NO is produced in a variety of tissues by one or several of the three isoforms of nitric oxide synthase (NOS); the endothelial isoform (eNOS; NOS3), expressed in endothelial cells and cardiomyocytes; the neuronal isoform (nNOS; NOS1), mainly expressed in neurons but also found in heart, skeletal muscle and vascular smooth muscle cells; and the inducible isoform (iNOS; NOS2), the expression of which is controlled by inflammatory mediators and cytokines [1]. For a long time, the role of NO in inflammation was thought to be only the result of iNOS activation, but the involvement of eNOS in inflammatory processes is now recognized. Indeed, in different models of acute and chronic inflammation, endothelial NO production has been implicated in modulating vascular permeability, edema formation, as well as angiogenesis induced by inflammatory mediators [2]. Compared to iNOS, which produces large amounts of NO in virtually all cell types following its induction by bacterial lipopolysaccharides or by other inflammatory cytokines, eNOS activity is Endothelial Dysfunction and Inflammation, edited by Shauna M. Dauphinee and Aly Karsan © 2010 Springer Basel

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tightly regulated at the post-translational level and produces a constant basal level of NO that contributes to the normal physiological function of the vascular wall [3]. Upon stimulation of endothelial cells with proper extracellular stimuli, eNOS activity is increased following a coordinated sequence of phosphorylation events and association of proteins that positively or negatively influence eNOS activity. These events act together with the modulation of intracellular calcium levels to induce NO release from endothelial cells. Described below are some of the important posttranslational regulators of eNOS activity that have putative roles in the regulation of eNOS-derived NO in inflammatory conditions.

Influence of cellular localization on eNOS activity An important consideration regarding regulation of eNOS activity is its localization within cells. The hypothesis that eNOS had to be localized to proper intracellular membranes to be near other regulatory proteins (scaffolds, chaperones, kinases) provided the rationale for the discovery of additional protein regulators of eNOS function. Indeed, it was shown that expression of eNOS in various cellular compartments influenced the efficiency of NO release [4–7]. It has been demonstrated that eNOS is anchored to cellular membranes via co-translational N-myristoylation (at Gly2) and post-translational palmitoylation (at Cys15 and Cys26) [6, 8] that direct the protein to discrete pools of cellular membranes. In endothelial cells, eNOS is found both at the plasma membrane and on the cytoplasmic face of the Golgi apparatus. Mutation of the myristic acid acceptor site on eNOS results in the cellular mislocalization of the protein into the cytoplasm and in reduced stimulated NO production from cells [8]. In addition to myristoylation, eNOS is dually palmitoylated on Cys15 and Cys26, which directs eNOS localization to the plasmalemmal caveolae [9, 10]. The palmitoyl transferase DHHC-21 is thought to be responsible for the palmitoylation of eNOS in the Golgi [11]. Caveolae are invaginations of the plasmalemmal membrane that are enriched in cholesterol and sphingolipids. They are created by the membrane association and homo- and hetero-oligomerization of caveolins, which also serve as scaffolds for the assembly of multiproteins signaling complexes, including eNOS, at the plasma membrane. Again, mutation of the acceptor cysteines of eNOS, Cys15 and Cys26, results in reduced cellular NO production without affecting the enzymatic activity per se, suggesting that eNOS localization to proper membrane microdomains provides some of the regulatory proteins essential for NO production [7]. The thiopalmitoyl bonds on Cys15 and Cys26 can be disrupted by acyl-protein thioesterase 1 (APT-1) [12], indicating that the targeting of eNOS to caveolae is dynamically regulated, and upon specific stimuli, the enzyme can shuttle to intracellular compartments. Indeed, intracellular calcium increases translocation of eNOS from the detergent-insoluble fraction (caveolae) to the detergent-soluble fraction (Golgi complex and cytoskeleton) [9, 13]. Dissocia-

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tion from caveolin interaction is also a critical early step in eNOS activation, as detailed below. While the importance of proper intracellular localization of eNOS is well established, the specific cellular pool of eNOS responsible for NO production in cells has been the subject of much speculation. Recent evidence suggests that both Golgi and plasmalemmal eNOS participate in overall NO production. However, the relative contribution of both sites is affected by the type of stimulus used and is mostly defined by phosphorylation levels of the enzyme [4, 14]. The intracellular distribution of eNOS is also regulated by the expression in endothelial cells of NOSTRIN and NOSIP. These two proteins have been identified as novel eNOS interacting proteins; however, their inhibitory effects on eNOSmediated NO release are most likely due to their influence on eNOS cellular trafficking. NOSIP, a 34-kDa protein, was initially identified as an eNOS C-terminal domain-binding protein [13]. NOSIP overexpression results in reduced NO release from intact cells due to redistribution of eNOS from the plasma membrane to the intracellular compartment. Interestingly, stimulation of cells with the calcium ionophore A23187 does not affect the association of eNOS with NOSIP [13]. Similarly, NOSTRIN, a 58-kDa protein that binds the eNOS oxygenase domain through a SH3 type sequence was discovered by yeast two-hybrid [15]. Overexpression of NOSTRIN results in a profound relocation of eNOS from plasma and Golgi membranes to vesicle-like structures within the cytosol. Thus, inhibition of NO production in NOSTRIN-overexpressing cells may be the result of this cellular redistribution of eNOS [15–17]. The identification of NOSIP and NOSTRIN as modulators of eNOS cellular localization demonstrates that membrane compartmentalization of the enzyme is necessary for optimal activity and NO release. Inflammatory stimuli, such as LPS treatment of liver sinusoidal endothelial cells, affect the ability of endothelin-1 to reduce the translocation of eNOS from the perinuclear/Golgi region to the plasma membrane associated with eNOS activation, suggesting that LPS induces an uncoupling of endothelin receptor activation and eNOS translocation, explaining in part the hyperconstrictive effects of endothelin-1 during inflammatory conditions [18]. Interestingly, increased gene and protein expression of NOSTRIN has been observed in liver samples of alcoholic hepatitis patients, which may contribute to the reduced eNOS activity and increased intrahepatic resistance in these chronically inflamed cirrhotic livers [19].

Modulation of eNOS activity by regulatory proteins eNOS activity is positively or negatively regulated by several associated proteins, all of which have been extensively studied in different experimental settings. Among these protein-protein interactions that regulate eNOS, caveolin-1 and Hsp90 are discussed here further given their implication in normal physiology and in the modulation of eNOS activity in inflammation. eNOS is a bi-domain protein, in which

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the oxygenase (N-terminal) and reductase (C-terminal) domains are separated by a calmodulin (CaM) binding site. At resting intracellular calcium (Ca2+) concentration, eNOS is inactive and its activation is largely dependent on Ca2+/CaM association. Furthermore, the targeted localization of eNOS to the caveolae in endothelial cells has prompted the investigation of its association to caveolin, the major coat protein of caveolae. This association has been reported to reduce enzyme activity [20–22]. When Ca2+ increases in cells in response to agonist stimulation, the Ca2+/CaM complex leads to the dissociation of caveolin-1 from eNOS, the enzyme is activated and produces NO [23]. Thus, the interaction of CaM and/or caveolin-1 with eNOS appears to be mutually exclusive, suggesting dynamic regulation of the interaction by intracellular Ca2+ concentration [24, 25]. Association of eNOS with caveolin results in direct steric inhibition of CaM binding to eNOS, which can be reversed by addition of exogenous CaM, supporting a reciprocal regulation of the enzyme by inhibitory caveolin versus activating Ca2+/CaM. Caveolin-1 has the capacity to directly interact with other intracellular signaling proteins, such as c-Src and H-Ras, through amino acids 82–101, the putative caveolin scaffolding domain (CSD) [26]. eNOS was shown to directly interact with caveolin-1 [20, 22, 27] through a primary binding region of caveolin-1 within amino acids 60–101 [21, 22] and the association of caveolin and eNOS is disrupted in the presence of peptides coding for the CSD (amino acids 82–101) [25, 28]. Furthermore, eNOS contains a putative caveolinbinding motif [29] located within amino acids 350–358 (FSAAPFSGW), which is thought to be responsible for its association to caveolin-1 [21]. The consequence on eNOS activity of caveolin-1 association has been best demonstrated by the incubation of pure eNOS with CSD peptide or glutathione S-transferase (GST)-caveolin fusion protein, which results in inhibition of eNOS activity in vitro [21]. In addition, cotransfection experiments showed that caveolin-1 overexpression resulted in reduced eNOS activity [24] and in reduced NO release [21]. The reduction of eNOS activity by caveolin peptides, or overexpressed caveolin, is reversed by exogenous addition of CaM, confirming a reciprocal regulation of eNOS by the two proteins [25]. Evidence for a physiological relevance of the negative regulation of eNOS by caveolin initially came from work using the CSD as a surrogate for caveolin. Interestingly, delivery of CSD to permeabilized cells [30] or a cell-permeable peptide containing the CSD sequence as a C-terminal fusion protein with the homeodomain of Antanapedia, a transcription factor of the Drosophilia, namely AP-Cav (or cavtratin [31, 32]), can block NO release, inhibit endothelium-dependent responses of isolated blood vessels, inflammation, vascular permeability and edema, angiogenesis and tumor growth in vivo [31, 32]. Moreover, mice genetically deficient in caveolin-1 exhibit a hyporesponsiveness to constrictor agonists and enhanced vasorelaxation ex vivo [33, 34], as well as decreased blood pressure variability in vivo [35]. These results suggest that the absence of caveolin-1 and caveolae in endothelial cells leads to increased eNOS activity and thus NO release resulting in reduced vascular tone.

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The 90-kDa heat shock protein (Hsp90) belongs to a family of highly expressed molecular chaperones involved in maintaining appropriate folding and conformational maturation of proteins [36]. In addition to this chaperoning function, Hsp90 can also act as an integral part of numerous signal transduction cascades. Hsp90 can associate with eNOS in resting endothelial cells, and stimulation of endothelial cells with vascular endothelial growth factor (VEGF), histamine, fluid shear stress, and estrogen all enhance the interaction between Hsp90 and eNOS, at the same time as increasing NO production [37, 38]. The rapid stimulus-dependent formation of the Hsp90-eNOS hetero-complexes suggests that it occurs simultaneously with other signaling events, such as the mobilization of intracellular calcium and/or protein phosphorylation. Hsp90 was shown to directly activate eNOS in vitro [37], and coexpression of eNOS with Hsp90 in COS cells increased NOS activity in broken cell lysates. These results suggested that Hsp90 may act as an allosteric modulator of eNOS by inducing a conformational change in the enzyme that results in increased activity [39]. However, it has been shown that Hsp90 facilitates the CaM-induced displacement of caveolin from eNOS [28]. Moreover, the complexity of Hsp90mediated activation of eNOS has been further revealed. Upon the characterization of the domains of Hsp90 that are required for binding eNOS, it was revealed that Akt, a kinase involved in eNOS phosphorylation (see later sections), was recruited to an adjacent region on the middle domain of Hsp90, which facilitates eNOS phosphorylation and enzyme activation [40]. Lastly, Hsp90 was shown to function as a scaffold for eNOS and its downstream target, the soluble guanylate cyclase, suggesting that Hsp90 also participates in the actions of NO through its effectors [41].

Protein-protein interactions in inflammation Studies demonstrating that altering specifically some of the post-translational regulators of eNOS support the hypothesis that eNOS-derived NO is involved in changes in vascular permeability during acute inflammatory processes. Recent findings show that AP-Cav, which binds to and inhibits eNOS, and geldanamycin, an inhibitor of Hsp90, inhibit increases in vascular permeability and edema in vivo and support the contribution of eNOS-derived NO to acute inflammation [2, 32, 42, 43]. In mouse models of inflammation, mice treated with AP-Cav, the cell permeable CSD fusion peptide, exhibited less interstitial edema and microvascular leakage by reducing eNOS-dependent blood flow and permeability changes without any changes in hemodynamic profile. This underscores the importance of eNOS activity in vascular permeability during inflammation [32]. Administration of AP-Cav also blocked the microvascular hyperpermeability responses to platelet-activating factor (PAF) [44]. Recent studies using cell permeable caveolin-1 peptides have dissected the CSD sequence important for eNOS inhibition and NO-dependent inflammation and showed that amino acids 89–95 of the CSD (82–101) are important for eNOS

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inhibition by AP-Cav and caveolin-1 [45]. Vessels hyperpermeability is also a hallmark of tumor vasculature [46], and it has been demonstrated that acute administration of AP-Cav decreases eNOS activity as well as tumor vascular permeability and tumor progression in vivo [31]. Moreover, acute administration of AP-Cav can normalize the hyperpermeable state of the tumor microvasculature observed in caveolin-1-deficient mice [47]. VEGF is of particular interest in the context of inflammation because it increases vascular permeability and is a potent angiogenic factor. Thus, VEGF induces these important components of the inflammatory response, which are both dependent on NO derived from eNOS [48–50]. Since eNOS activity can be modulated by caveolin-1, its regulatory roles in vascular permeability and angiogenesis have been investigated. Indeed, overexpression or down-regulation of caveolin-1 in cells leads to, respectively, enhanced or reduced capillary-like tube formation of endothelial cells [51]. However, others have shown that caveolin-1 overexpression in endothelial cells leads to a reduced VEGF-induced migration and prevents endothelial tube formation when cultured on matrigel [52]. Similarly, in vivo studies showed that NO-dependent angiogenesis through the VEGF receptor-2 (VEGFR-2), as well as basic fibroblast growth factor (bFGF)-induced angiogenesis in matrigel plugs, are markedly impaired in caveolin-1 null mice, confirming an important regulatory role for caveolin-1 in this process [53, 54]. Finally, basal NO release, responses of aortas to acetylcholine, as well as VEGF-mediated angiogenesis and increase in vascular permeability, were reduced in a mouse model of endothelial specific overexpression of caveolin-1 [55]. The regulatory role of the molecular chaperone Hsp90 on eNOS activity in inflammation has also been studied. Hsp90 inhibitors, such as geldanamycin, which binds specifically to the ATP binding pocket of Hsp90, have been shown to interfere with eNOS activation and to inhibit endothelium-dependent relaxation and NO release from endothelial cells [28, 37]. Hsp90 inhibition has also been shown to dose-dependently inhibit edema formation through inhibition of eNOS activity in a mouse model of inflammation induced by carrageenan [42]. Notably, pharmacological inhibition of Hsp90 and overexpression of a dominant-negative Hsp90 construct have been linked to reduced VEGF-stimulated NO release and vascular permeability, tubule formation of human umbilical vein endothelial cells (HUVEC) and angiogenesis [56, 57]. Interestingly, blockade or genetic inactivation of eNOS mostly affects vascular leakage in models of acute inflammation; however, geldanamycin treatment also inhibits neutrophil emigration and activation, indicating that Hsp90 inhibition also produces NO-independent anti-inflammatory effects [42, 43].

Regulation of eNOS activity by phosphorylation eNOS activity is also modified by changes in its phosphorylation state, which is one of the major mechanisms of signal integration in eukaryotic cells. Indeed, eNOS

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protein is phosphorylated on several serine residues, i.e., Ser1177 (for human and Ser1179 for bovine eNOS), Ser116, Ser614, and Ser633. Shear stress, VEGF, insulin and bradykinin, induce Ser1177 phosphorylation and activation of eNOS through PI3K-dependent activation of Akt (protein kinase B) [58]. Studies using mutated forms of the enzyme that mimic the phosphorylated state of eNOS (mutations of Ser1177 to aspartate; S1177D) showed that this mutated form of eNOS retains enzymatic activity despite low levels of calcium both in vitro and in cells, which results in increased basal and stimulated NO production [59]. From these experiments, it was also reasoned that phosphorylation on Ser1177 enhances NO production due to a configuration change in the C-terminal autoinhibitory loop of the enzyme that increases the rate of electron transfer from the reductase to the oxygenase domain of eNOS [60]. Notably, Ser1177 phosphorylation requires efficient membrane targeting of eNOS, since myristoyl- or palmitoyl-deficient eNOS failed to be phosphorylated following stimulation of cells with agonists [5]. Several other proteins kinases were proposed as being involved in the phosphorylation at this site: AMP kinase [61], protein kinase A (PKA) [62], protein kinase G [62] and calmodulin-dependent kinase II (CaMKII) [63]. However, the choice of the kinase depends largely on the stimulus applied. For example, estrogen, VEGF or shear stress elicit the phosphorylation of Ser1177 through Akt, while histamine and thrombin stimulations need AMP kinase activation [64]. Bradykinin-mediated phosphorylation occurs through CaMKII, although in this case Akt activation has also been implicated [63]. Finally, the phosphatase PP2A appears to be involved in dephosphorylation of Ser1177 [65, 66]. On the other hand, phosphorylation of eNOS at Ser116 inhibits enzyme activity, and it has been shown that VEGF-dependent dephosphorylation of this site leads to enzyme activation [67]. Ser614 is localized in the oxygenase domain of eNOS and it does not seem to be crucial for eNOS activity, but appears to be phosphorylated in the initial stage of eNOS activation, rendering eNOS significantly more susceptible to activation by Ca2+/CaM [59, 68]. Subsequently, Ser633, which is located within the first autoinhibitory loop, which is thought to physically impede the access of CaM to the binding domain of the Ca2+-dependent NOS isoforms, is phosphorylated, increasing eNOS maximal activity to an extent equal to that produced by phosphorylation of Ser1177 [68]. Conversely, the Thr495 residue is constitutively phosphorylated by protein kinase C (PKC) in endothelial cells and this is known to maintain eNOS in a less active state [63, 66, 69]. This residue is located within the CaM-binding domain and it was shown that upon stimulation with selected agonists (bradykinin, VEGF, histamine, and Ca2+ ionophore), eNOS is dephosphorylated on Thr495 and activated. The phosphatases PP1 and PP2A were shown to dephosphorylate Thr495 [66, 70]. The increase in the enzymatic activity observed with the non-phosphorylable mutant eNOS-T497A confirmed that this site is a negative regulator of the enzyme [71] and if phosphorylated, it causes a reduction in the affinity of eNOS for Ca2+/

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CaM [63]. Recently, it has been proposed that this site could also act as an intrinsic switch, determining the generation of NO versus superoxide [71]. Recently, Srcmediated phosphorylation of Tyr83 was shown to contribute to eNOS activation, but it is not clear at the moment if this phosphorylation functions as a docking site for Src homology-containing adaptor molecules [72].

Phosphorylation of eNOS in inflammation Among the numerous potential eNOS phosphorylation sites, Ser 1177 in the autoinhibitory loop of the reductase domain and Thr495 within the CaM-binding domain are the most studied in eNOS regulation, perhaps due to their well-established roles in the regulation of enzyme activity. The importance of signaling pathways that lead to eNOS phosphorylation in models of inflammation, as well as in the response of endothelial cells to inflammatory stimuli, has been demonstrated. Firstly, in HUVEC, LPS treatment leads to the activation of eNOS through a PI3-kinase- and Akt-dependent phosphorylation of eNOS on Ser1177, suggesting that the pathogenesis of sepsis is characterized by an initial activation of eNOS [73]. Interestingly, inhibition of PI3-kinase using LY294002 reduces carrageenan-stimulated edema formation and protein leakage, but not leukocyte influx in mice treated with zymosan similar to that observed in eNOS–/– mice [43]. Furthermore, the direct role of the Akt/eNOS pathway in acute inflammation was studied in vivo in mice deficient in either Akt1 or Akt2 [74]. The loss of Akt1, but not Akt2, suppresses microvascular permeability in acute inflammation and in response to histamine and bradykinin. This effect was proposed to result from Akt1-mediated activation and phosphorylation of eNOS on Ser1177, which promotes junctional permeability [74]. The reciprocal phosphorylation of Ser1177 and dephosphorylation of Thr495 is essential for eNOS activation by many agonists: bradykinin, VEGF, ATP, and insulin [63, 66, 69, 75]. Altering the balance between these two phosphorylation events results in eNOS inhibition and reduced NO production. As mentioned, the Thr495 residue is phosphorylated by PKC isoforms and dephosphorylated by the PP1 phosphatase [63, 66]. Phosphorylation on Thr495 by several PKC isoforms results in eNOS inhibition [76–78]. Angiopoietin-1 is an angiogenic growth factor, which prevents VEGF-stimulated increase in vascular permeability and displays anti-inflammatory effects [79–83]. Many mechanisms have been proposed to explain the inhibition of VEGF-induced endothelial permeability by angiopoietin-1, e.g., junctional protein stabilization, cytoskeletal rearrangements, and more recently, inhibition of VEGF-stimulated NO release [84–87]. It has been shown that angiopoietin-1 induces a PKCzeta-dependent phosphorylation of eNOS on Thr495, which inhibits eNOS, NO production, and endothelial permeability stimulated by VEGF [87]. These results suggest that eNOS inhibition mediates, at least in part, the anti-inflammatory effects of angiopoietin-1.

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Phosphorylation events are central to the regulation of eNOS activity and pharmacological modification of protein phosphorylation provides an interesting and powerful means to modulate eNOS function during inflammation. A model for eNOS activation is shown in Figure 1.

Conclusion It is well established that eNOS has a key role in regulating the homeostasis of the cardiovascular system. The contribution of eNOS-derived NO to inflammation, especially acute inflammation, is gaining credence and warrants further investigation. eNOS can be activated post-translationally and modulation of these regulatory

Figure 1. A Model for endothelial nitric oxide synthase (eNOS) activation. Under basal condition (less active state), membrane-bound eNOS is associated with the negative regulatory proteins caveolin, NOS-interacting protein (NOSIP), NOS trafficker (NOSTRIN) and is basally phosphorylated on Thr495. After endothelial cell stimulation by various stimuli and elevation of intracellular Ca2+ levels, eNOS associates with calcium-activated calmodulin (CaM), dissociates from caveolin and is transiently dephosphorytated on Thr495. Increase in eNOS activity and NO release are associated with increased interactions with the positive regulatory proteins heat shock protein 90 (Hsp90) and phosphorylation by Akt on Ser1177.

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events alters eNOS activity and NO release. The accumulation of evidence showing that interfering with eNOS phosphorylation or with the association of regulatory proteins to eNOS, such as caveolin and Hsp90, inhibits acute inflammation in vivo, suggests that post-translational regulators of eNOS activity may provide new pharmacological targets in inflammation.

Acknowledgements F.D. holds a post-doctoral fellowship from the Fonds de la recherche en santé du Québec (FRSQ). J.P.G. is recipient of a Tier II Canada Research Chair. Work performed in J.P.G. laboratory is supported by grants from the Canadian Institutes of Health Research (CIHR).

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synthase to endothelial cell caveolae via palmitoylation: Implications for nitric oxide signaling. Proc Natl Acad Sci USA 93: 6448–6453 Shaul PW, Smart EJ, Robinson LJ, German Z, Yuhanna IS, Ying Y, Anderson RG, Michel T (1996) Acylation targets endothelial nitric-oxide synthase to plasmalemmal caveolae. J Biol Chem 271: 6518–6522 Fernandez-Hernando C, Fukata M, Bernatchez PN, Fukata Y, Lin MI, Bredt DS, Sessa WC (2006) Identification of Golgi-localized acyl transferases that palmitoylate and regulate endothelial nitric oxide synthase. J Cell Biol 174: 369–377 Yeh DC, Duncan JA, Yamashita S, Michel T (1999) Depalmitoylation of endothelial nitric-oxide synthase by acyl-protein thioesterase 1 is potentiated by Ca(2+)-calmodulin. J Biol Chem 274: 33148–33154 Dedio J, Konig P, Wohlfart P, Schroeder C, Kummer W, Muller-Esterl W (2001) NOSIP, a novel modulator of endothelial nitric oxide synthase activity. FASEB J 15: 79–89 Govers R, Oess S (2004) To NO or not to NO: ‘where?’ is the question. Histol Histopathol 19: 585–605 Zimmermann K, Opitz N, Dedio J, Renne C, Muller-Esterl W, Oess S (2002) NOSTRIN: A protein modulating nitric oxide release and subcellular distribution of endothelial nitric oxide synthase. Proc Natl Acad Sci USA 99: 17167–17172 Icking A, Matt S, Opitz N, Wiesenthal A, Muller-Esterl W, Schilling K (2005) NOSTRIN functions as a homotrimeric adaptor protein facilitating internalization of eNOS. J Cell Sci 118: 5059–5069 Schilling K, Opitz N, Wiesenthal A, Oess S, Tikkanen R, Muller-Esterl W, Icking A (2006) Translocation of endothelial nitric-oxide synthase involves a ternary complex with caveolin-1 and NOSTRIN. Mol Biol Cell 17: 3870–3880 Kamoun WS, Karaa A, Kresge N, Merkel SM, Korneszczuk K, Clemens MG (2006) LPS inhibits endothelin-1–induced endothelial NOS activation in hepatic sinusoidal cells through a negative feedback involving caveolin-1. Hepatology 43: 182–190 Mookerjee RP, Wiesenthal A, Icking A, Hodges SJ, Davies NA, Schilling K, Sen S, Williams R, Novelli M, Muller-Esterl W, Jalan R (2007) Increased gene and protein expression of the novel eNOS regulatory protein NOSTRIN and a variant in alcoholic hepatitis. Gastroenterology 132: 2533–2541 Feron O, Belhassen L, Kobzik L, Smith TW, Kelly RA, Michel T (1996) Endothelial nitric oxide synthase targeting to caveolae. Specific interactions with caveolin isoforms in cardiac myocytes and endothelial cells. J Biol Chem 271: 22810–22814 Garcia-Cardena G, Martasek P, Masters BS, Skidd PM, Couet J, Li S, Lisanti MP, Sessa WC (1997) Dissecting the interaction between nitric oxide synthase (NOS) and caveolin. Functional significance of the NOS caveolin binding domain in vivo. J Biol Chem 272: 25437–25440 Ju H, Zou R, Venema VJ, Venema RC (1997) Direct interaction of endothelial nitricoxide synthase and caveolin-1 inhibits synthase activity. J Biol Chem 272: 18522– 18525

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Endothelial dysfunction in pulmonary hypertension Dominique Yelle1,2, Lakshmi Kugathasan 3, Robin E. MacLaren 2 and Duncan J. Stewart 1,2 1 

Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Canada Regenerative Medicine Program, Ottawa Hospital Research Institute, Ottawa, Canada 3  Terrence Donnelly Vascular Biology Laboratories, St Michael’s Hospital, Toronto, Canada 2 

Abstract Pulmonary arterial hypertension (PAH) is a rare disease caused by functional and structural abnormalities in distal pulmonary arterioles that result in progressive increases in pulmonary vascular resistance, often leading to right heart failure and death. Endothelial dysfunction, defined as a shift in the balance of production of endothelial vasodilator factors (i.e., nitric oxide and prostacyclin), and vasoconstrictor and proliferative factors (i.e., endothelin-1 and thromboxane A2), has been strongly implicated in PAH. Here we review the evidence supporting a central role for endothelial dysfunction in the pathogenesis of PAH as a result of genetic and environmental influences, and extend this concept to include the critical balance between pathways for endothelial growth and survival. In addition, we present support for the hypothesis that the initial loss of endothelial cells, largely by apoptosis, an extreme form of endothelial dysfunction, triggers a cascade of events that ultimately result in the typical, complex constellation of functional and structural lung vascular abnormalities, including formation of the proliferative intimal and plexiform lesions. This novel paradigm may help in the design of strategies that better address the root cause of PAH and may possibly lead to more effective treatments.

Pulmonary arterial hypertension Pulmonary arterial hypertension (PAH) is an often fatal disease caused by functional or structural abnormalities involving the precapillary pulmonary arteriolar bed that result in progressive increases in pulmonary vascular resistance [1]. PAH has recently been subclassified in three main groups: (1) idiopathic (IPAH, previously known as primary pulmonary hypertension), which occurs in the absence of any known contributing disorders; (2) heritable or familial PAH (FPAH); and (3) associated PAH (APAH, previously termed secondary PH), which is associated with other diseases such as collagen vascular diseases (i.e., scleroderma), HIV, and systemic to pulmonary shunt (i.e., congenital heart disease) [2]. Clinically, the pathological features of advanced PAH are characterized by medial hypertrophy, adventitial thickening, intimal fibrosis and obliteration of small pulmonary ­arteries and arterioles, and often Endothelial Dysfunction and Inflammation, edited by Shauna M. Dauphinee and Aly Karsan © 2010 Springer Basel

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include the presence of plexiform lesions, which are thought to result from dysregulated endothelial cell (EC) proliferation [1, 3, 4]. Other characteristic pathological findings include in situ thrombosis and varying degrees of inflammation [1, 4]. The narrowing of the lumen diameter and functional pruning of pulmonary arterioles results in increased pulmonary vascular resistance and pulmonary arterial pressure, leading to right ventricular hypertrophy and ultimately to right heart failure [5–8]. Unfortunately, despite recent advances in therapeutic approaches, the prognosis for patients with PAH remains poor, as many succumb to the disease within 3–5 years of diagnosis [9].

Endothelium and endothelial dysfunction in PAH The pulmonary bed is quite distinct from systemic arterial beds in that it is required to accept the entire cardiac output, and functions normally at very low basal levels of vascular resistance. This is accomplished in large part due to the large crosssectional area of the normal pulmonary vascular bed, of which only a fraction is required at rest; thus, increases in cardiac output associated with exertion can be accommodated by progressive recruitment of this excess capacity with little to no impact on resistance and pressures. The hallmark of PAH is massive “pruning” of this distal arteriolar microcirculation, either due to functional or structural abnormalities, resulting in an inadequate number of vascular channels to accommodate the cardiac output, even at rest. However, as in systemic vascular beds, the pulmonary vascular endothelium not only acts as a structural barrier to the passage of fluids and proteins into the interstitium, but also actively regulates the tone, growth and differentiation of vascular smooth muscle cells (SMCs), and exerts potent antithrombotic and anti-inflammatory influences by preventing the adhesion and activation of platelets and leukocytes under physiological conditions [10]. These effects are mediated largely through the release of vasodilator substances such as the endothelium-derived relaxing factors nitric oxide (NO) and prostacyclin, as well as constricting factors, including endothelin-1 (ET-1) and thromboxane A2 [10–13]. These mediators are also capable of modulating migration and proliferation of vascular SMCs, and thus likely play an important role in mediating pulmonary arterial structural changes in PAH [10]. In healthy individuals, production of endothelial vasodilator, antiproliferative and antithrombotic factors, such as NO and prostacyclin, predominates, and these factors contribute to the maintenance of pulmonary vascular structure and function [10]. However, as in the systemic circulation, the balance of production of endothelial vasoactive factors can shift to favor the release of vasoconstrictor and proliferative factors, and this is classically referred to as endothelial dysfunction [10]. Consistent with endothelial dysfunction, PAH patients demonstrate an increase in ET-1 [14] and thromboxane A2 [15] production across the pulmonary bed, as well as reduced prostacyclin and NO bioactivity

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[16, 17]. The initiating mechanism leading to endothelial dysfunction in this disease is unknown, but may include hypoxia, shear stress, inflammation or response to drugs or toxins on a background of genetic susceptibility [4, 18]. Current evidence strongly suggests that endothelial dysfunction plays an integral role in mediating the structural changes in the pulmonary vasculature observed in PAH. Here, we briefly review the evidence supporting a central role for endothelial dysfunction in the pathogenesis of IPAH, and extend this concept to include the critical balance between endothelial growth and survival pathways and the genetic and environmental influences that predispose to EC damage and apoptosis. The loss of endothelial integrity by programmed cell death can be viewed as an extreme form of endothelial dysfunction, which is likely the trigger for a cascade of events that results in the constellation of functional and structural abnormalities in the lung vasculature that characterizes the clinical condition of PAH, including arterial remodeling, and intimal and angioproliferative plexiform lesions.

Endothelium-derived vasoactive factors in PAH As in the systemic vessels, ECs play a vital role in regulating the function and structure of pulmonary arteries and arterioles, which is mainly mediated by the release of endothelial factors.

Nitric oxide NO plays an important role in modulating pulmonary vascular tone. It is a potent endogenous endothelium-derived vasodilator that directly relaxes vascular smooth muscle [10, 19]. In addition to its vasorelaxation effect, NO also exerts potent antiplatelet, antithrombotic and antimitogenic effects, thereby preventing platelet aggregation, cell adhesion and SMC proliferation [3, 10]. NO signals through its cytoplasmic receptor, the soluble guanylyl cyclase (sGC), which results in the generation of the second messenger cGMP that mediates most of the physiological effects of NO [20]. NO production is catalyzed by endothelial NO synthase (eNOS) [21]. This complex enzyme has both reductase and oxygenase domains and shuttles electrons from free oxygen to its substrate, l-arginine, resulting in the production of NO and l-citrulline [21]. eNOS also requires several cofactors, notably tetrahydrobiopterin, to maintain its active state as a homodimer, with electrons passing from one subunit to the other [21]. In its monomeric state, eNOS is unable to reduce the guanido terminal of l-arginine, and electrons are passed to molecular oxygen, resulting in the production of superoxide anion [21]. NO is a freely diffusible and highly unstable free radical that reacts extremely rapidly with superoxide to produce peroxynitrite, a highly reactive radical that can act in a manner similar to the highly toxic hydroxyl radical [21]. Thus, the biological activity of NO depends not only

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on the level of its production, but also on the microenvironment in which it is produced. Under physiological conditions, even small amounts of NO can have powerful vasodilatory effects; however, in the presence of oxidative stress, much larger levels of NO release may have little effect on vascular tone, and may even contribute to oxidative damage. Thus, the bioactivity of NO is a more important parameter than simply the level of eNOS expression or NO release. Lungs from PAH patients have been reported to demonstrate reduced eNOS expression [16], although this has not been confirmed by other groups [22]. A contributory role for deficient NO production in the pathogenesis of PAH is further supported by transgenic animal models, in which overexpression of eNOS prevented hypoxia-induced PAH [23], while exposure to mild hypoxia resulted in more severe PAH in eNOS-deficient mice [24]. However, again there are conflicting findings, and some groups have reported that eNOS-knockout mice do not exhibit any increase in hypoxia-induced PAH, and may even show less arterial remodeling in response to chronic hypoxia than wild-type (WT) mice [25]. More recently, it has been recognized that in addition to its classical vascular regulatory effects, NO plays a key role in controlling EC growth and angiogenesis [26], and is a critical downstream mediator of the effects of vascular endothelial growth factor (VEGF) and basic fibroblast growth factor [27]. Interestingly, congenital cardiac defects including bicuspid aortic, atrial and ventricular septal defects were recently reported in the offspring of eNOS-deficient mice [28, 29], associated with a high rate of neonatal mortality of up to 75%, clearly out of proportion to the severity of the cardiac abnormalities in these animals. Given the importance of the pulmonary vasculature and the dramatic changes it undergoes immediately after birth, Han et al. [30] carefully examined lung vascular development in these animals. They reported a severe defect in maturation of the blood-air barriers at late stages of development, with a paucity of septal capillaries and marked thickening of the septae. These are the hallmark features of alveolar capillary dysplasia (ACD), a universally lethal form of persistent pulmonary hypertension of the newborn believed to occur due to lack of arteriolar ingrowth into the developing saccular and alveolar structures.

Prostacyclin Prostacyclin is another important endothelium-derived vasodilator factor and has also been strongly implicated in pulmonary vascular homeostasis. In addition to promoting vasodilation in the pulmonary circulation, like NO, it is a potent endogenous inhibitor of platelet aggregation [4, 10]; however, unlike NO, it does not prevent platelet adhesion to the endothelial surface. The effects of NO and prostacyclin in the pulmonary vasculature are thus highly complementary. Prostacyclin also protects against pulmonary remodeling by inhibiting the growth of SMCs [19, 31]. The production of prostacyclin from prostaglandin H2 is catalyzed by prostacyclin­

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synthase [32]. Prostacyclin binds to a G protein-coupled receptor, which activates adenylate cyclase, resulting in increased production of cAMP [33]. This second messenger has diverse intracellular effects including the activation of protein kinase A and myosin light-chain kinase within SMCs, leading to vasodilation [34]. Deficiency in prostacyclin production has been strongly implicated in PAH. Prostacyclin synthase expression is decreased in lungs from patients with severe PAH [17] and prostacyclin receptor-deficient mice develop severe PAH in response to chronic hypoxia [35]. In addition, overexpression of prostacyclin synthase protects mice from chronic hypoxia-induced PAH [36]. Therefore, it is widely accepted that decreased prostacyclin levels in PAH may account for pulmonary vasoconstriction, SMC proliferation and the pro-thrombotic state observed in this disease.

Endothelin-1 ET-1 is secreted by the endothelium and is one of the most potent endogenous vasoconstrictor peptides known [3, 10]. In addition to its vasoactive properties, it also has platelet-aggregating properties as well as a growth-promoting effects on SMCs and other cell types [10, 31]. The effects of ET-1 are mediated through the ETA and ETB receptors [19]. Activation of ETA receptors, located mainly on vascular SMCs, causes sustained vasoconstriction and proliferation of vascular SMCs [11, 19]. In contrast, ETB receptors, which are found mainly on ECs, mediate pulmonary endothelin clearance and induce the production of NO and prostacyclin by ECs [11, 19]. There is strong evidence that ET-1 is a major player in the vasoconstriction and remodeling in PAH. Our group was the first to demonstrate that levels of circulating ET-1 are increased in patients with PAH [14, 37], and that this was associated with massive up-regulation of endothelial ET-1 expression within the pulmonary vasculature of patients with either IPAH or APAH [37]. Furthermore, in a number of experimental models, PAH is associated with selective increases in ET-1 and ETA receptor expression in the pulmonary vasculature [38]. It has been suggested that a selective increase in the synthesis and release of ET-1 from pulmonary ECs accompanied by a paracrine effect mediated by ETA receptors on underlying pulmonary SMCs could contribute to both the vasoconstriction and vascular remodeling seen in PAH [10].

Thromboxane A2

Thromboxane A2 is another potent vasoconstrictor, as well as a smooth muscle mitogen and platelet agonist [15, 31], in many ways counteracting the effects of prostacyclin. Indeed, in endothelial dysfunction, the balance of vasodilator and vasoconstrictor prostaglandin production is shifted, and an increase in the production of thromboxane A2 together with a decrease in prostacyclin have been reported in patients with PAH [15, 31]. The resulting imbalance between thromboxane A2 and prostacyclin may thus contribute to the platelet activation and abnormal 85

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response of the pulmonary vascular endothelium in this disease. Whether the imbalance in the release of these mediators is a cause or a result of PAH is unknown, but it likely plays an important role in the progression of the disease.

Endothelial-derived hyperpolarizing factor Over the years, there has been increasing evidence suggesting the existence of an endothelial-derived relaxing factor distinct from NO that mediates endothelialdependent hyperpolarization, causing the relaxation of vascular SMCs. This factor was termed endothelial-derived hyperpolarizing factor (EDHF) and, although its chemical identity remains uncertain, it appears to act on K+ channels in vascular SMCs to increase membrane potential and thus reduce contractility [39, 40]. It has been suggested that EDHF acts in parallel with NO, and represents a compensatory mechanism in conditions associated with reductions in NO release, therefore having an important role in maintaining normal vascular tone in disease states [39]. Indeed, while NO-mediated vasodilation was impaired in rats with monocrotaline (MCT)induced PAH, EDHF function was reported to be preserved [41] and even increased in this model [42]. However, histamine-induced relaxation of the pulmonary artery in both normotensive and hypertensive rats was shown to be mediated mainly by NO, whereas EDHF seemed to play a minor role [43]. Therefore, the relative contribution of EDHF in regulating pulmonary vascular tone in PAH remains less well established than for other endothelial factors.

Summary In summary, endothelial dysfunction in PAH is manifested by a reduction in the release of endothelial vasodilator, antiproliferative and antithrombotic factors, such as NO and prostacyclin, together with an increase in the production of factors that promote vasoconstriction, vascular remodeling and thrombosis, including endothelin and thromboxane A2. Although it remains unknown whether this is causal in the pathogenesis of PAH or merely a result of other more fundamental mechanisms, current evidence points towards an important contribution of dysregulated endothelial vasoactive factors in the progression of this disease.

Current PAH therapies and endothelial dysfunction Although the existence of a vasoconstrictor factor has long been suspected in PAH [44], only a small minority of IPAH patients (< 13%) show evidence of an acute vasodilator response at their initial hemodynamic assessments [45]. Moreover, only about half of these (~7% of all IPAH patients) will respond to long-term treatment with high-dose calcium channel blockers, and they likely represent a distinct sub-

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group of PAH patients with a favorable prognosis [45]. Until relatively recently, there was no specific treatment for the vast majority of patients suffering from this disease. Over the last two decades, the development of new therapies has focused on restoring the balance between vasoconstrictor and vasodilator pathways, which represent logical pharmacological targets, although they may also impact on the process of vascular remodeling [46]. The first such therapy to be introduced in the “modern era” of PAH management was systemic prostanoid infusion. The administration of prostacyclin by continuous intravenous infusion was pioneered by Higenbottam and his group at Papworth Hospital in the early 1980s [47], and later developed as the gold standard for treatment of severe PAH. Parenteral delivery of Flolan® via an indwelling central catheter has been shown to improve function, hemodynamics and survival in WHO class III or IV patients [48, 49]. However, this is a cumbersome and expensive treatment that is increasingly being reserved for the most difficult patients to manage, in favor of more convenient prostaglandin delivery systems including subcutaneous (treprostinil) or inhaled (iloprost) prostanoids [13, 19], although these are supported by less definitive evidence of efficacy. The next major advance came from the introduction of endothelin receptor antagonists (ERAs), which provided the first effective oral agents for patients with PAH. Bosentan is a dual (ETA and ETB) ERA and was the first shown to be effective in large-scale clinical trials [50], and is now widely used as a first line treatment for PAH [19, 31]. More recently, a number of other ERAs have been introduced and there is an ongoing debate as to the benefits of dual versus ETA-selective ERAs for this disease. ETA-selective receptor blockers have the theoretical advantage of sparing the endothelial ETB receptors, which mediate ET-1 clearance and endothelium-dependent vasodilation [19]. Sitaxsentan and ambrisentan are two such ETAselective receptor antagonists that have been approved in some jurisdictions for the treatment of PAH. However, to date, there is no clear evidence from clinical studies to establish the superiority of an ETA-selective strategy. The understanding of the importance of the NO pathway has also led to the development of inhaled NO as a therapeutic strategy [51], and although useful for the acute management of PAH in a critical care setting, such as in the neonatal treatment of persistent pulmonary hypertension of the newborn, it has proven to be difficult to deliver over the long term for the treatment of ambulatory patients. Oral phosphodiesterase type V inhibitors such as sildenafil, which increase the levels of the second messenger cGMP, have been shown to induce acute pulmonary vasodilation during short-term administration and improve exercise capacity and pulmonary hemodynamics in patients with PAH during long-term oral therapy [19, 52]. Sildenafil is now widely used in the treatment of this disease, and other agents such as tadalafil, which has a longer half-life, are being introduced [53]. However, despite the proliferation of new drug therapies, nearly all patients will progress and ultimately fail medical management. For this reason, the use of combi-

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nation therapy with agents acting via different mechanisms is becoming increasingly prevalent to maximize the clinical benefit [19]. Nonetheless, the impact of medical management on the overall prognosis of PAH has been limited. A recent systematic review of modern PAH therapies did not support any meaningful benefit on survival in these patients [54], highlighting the need for more effective treatment strategies.

Endothelial dysfunction and thrombosis in PAH In situ pulmonary artery thrombosis, which is commonly seen in PAH, may be initiated or aggravated by endothelial dysfunction and contribute to the progression of arteriopathy [4]. Indeed, there is evidence that procoagulant activity is increased and fibrinolytic function of the pulmonary endothelium is reduced in PAH [4]. As discussed above, endothelial dysfunction may be an important contributor to these abnormalities by virtue of the loss of antithrombotic activity (i.e., prostacyclin and NO), and an excess of prothrombotic factors such as ET-1 and thromboxane A2 [11, 31]. Imbalances in the production of other endothelial proteins are also likely to be involved, including fibrinopeptide, von Willebrand factor, tissue plasminogen activator, plasminogen activator inhibitor-1, factor X, and tissue factor (TF) [55–57]. TF in particular is interesting as a candidate mediator of coagulation and thrombosis. TF is a cell surface glycoprotein that is rarely expressed in healthy pulmonary ECs but is highly expressed in pulmonary ECs in experimental models of PAH [58, 59], as well as in lungs of PAH patients, in particular those with plexiform lesions [58]. In a rat model of severe PAH induced by MCT combined with unilateral pneumonectomy, TF expression was low at 1 week when the lung morphology was relatively normal, but increased markedly by 2 weeks, correlating with the appearance of vascular remodeling and characteristic pathological changes (i.e., plexiform-like lesions) [58]. In addition to thrombosis, TF may contribute directly to vascular remodeling in PAH by virtue of its roles in angiogenesis and SMC proliferation [60–62]. Furthermore, it has been proposed that TF activation may be an important link between inflammation and thrombosis [63]. TF is induced in vitro in ECs by inflammatory factors such as TNF-α, IFN‑γ and oxidized low-density lipoprotein [62, 64]. In addition, TF is the receptor for factor VIIa, which can signal intracellularly via protease-activated receptor (PAR), activating factor Xa and thrombin [65, 66]. Given its role in thrombosis and inflammation, TF is therefore an interesting new candidate in the pathogenesis of PAH.

Endothelial dysfunction and inflammation in PAH Inflammation is strongly associated with PAH, although its contribution to the pathogenesis of this disease remains poorly understood. Lung sections from patients

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with IPAH often show extensive inflammatory infiltrates [67, 68], and the most common causes of APAH are autoimmune disorders, such as scleroderma and systemic lupus erythematosus [69]. However, inflammation is often overlooked as a key component in the pathogenesis of PAH. Endothelial dysfunction predisposes to vascular inflammation by resulting in the up-regulation of surface adhesion molecules involved in the trafficking of leukocytes as well as the release of cytokines and chemokines that recruit circulating inflammatory cells [70]. Immunological disturbances have been reported in patients with IPAH, such as increased circulating levels of the pro-inflammatory cytokines IL-1, IL-6 and TNF-α [4, 11]. IL-1 has been shown to stimulate ET-1 production and may act as a link between endothelial dysfunction and inflammation [11]. Similarly, IL-1 and IL-6 promote thrombosis and are potent SMC mitogens [12]. ET-1 itself can act as a cytokine by priming neutrophils, activating mast cells and stimulating monocytes to produce a variety of cytokines such as IL-1, IL-6, IL-8, TNF-α and TGF-β [71]. Of note, macrophages and T lymphocytes are found in plexiform lesions and small arterioles in severe PAH [4]. Furthermore, pulmonary ECs from patients with PAH have been reported to markedly enhance monocyte migration compared to controls [18]. A contributory role for IL-6 in the pathogenesis of PAH has also been demonstrated experimentally. Delivery of recombinant IL-6 protein in rats resulted in PAH associated with luminal occlusion of the small pulmonary arteries, formation of microvascular thrombi and localized hemorrhage [72]. In another study, mice treated with recombinant IL-6 demonstrated increased right ventricular systolic pressure (RVSP) and right ventricular hypertrophy, which was further augmented upon hypoxic exposure [73]. Moreover, IL-6-knockout mice demonstrated decreased hypoxia-induced PAH, which was associated with attenuated macrophage recruitment within the lungs [74], whereas IL-6-overexpressing mice developed spontaneous PAH associated with the formation of neointimal occlusive lesions and inflammatory cell infiltration within the peri-arteriolar vasculature [75]. It is also of interest to note that this genetic model represents the only model that recapitulated the pathophysiological changes observed in IPAH patients in the absence of an exogenous trigger. This model may be particularly useful in further elucidating the role of chronic inflammation in PAH progression.

EC apoptosis and the genetics of PAH It has been recognized for many years that injury to the endothelium is likely a critical initial step in the pathogenesis of PAH. Indeed, in the rat MCT model, which is one of the most widely used experimental models of this disease, PAH is thought be caused by direct injury to the endothelium, leading to apoptosis. A central role for EC apoptosis was further supported by the seminal study of Taraseviciene-Stewart et al. [76] in which hypoxic rats treated with a VEGF receptor 2 antagonist dem-

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onstrated worsening of hypoxia-induced PAH associated with marked potentiation of arterial remodeling. In particular, the authors described an early increase in EC apoptosis followed by the appearance of proliferative intimal lesions, which in some cases obliterated small pulmonary arterioles [76]. Moreover, treatment with a pancaspase inhibitor prevented the development of severe PAH, as well as the remodeling changes induced by VEGF blockade in the chronic hypoxia model [76]. The authors suggested that widespread EC apoptosis triggered by withdrawal of VEGF survival signaling together with chronic hypoxia led to the emergence of abnormal apoptosis-resistant and hyperproliferative ECs, leading to the development of the characteristic intimal lesions [76]. A protective role for VEGF was further supported by gene transfer experiments in both the chronic hypoxia and MCT-induced models of PAH [77, 78]. These findings suggest that VEGF survival signaling acts as a protective mechanism in PAH and support a significant role for EC apoptosis in the pathogenesis of this disease.

Bone morphogenetic protein receptor 2 mutations Arguably, the most significant advance in understanding the pathogenesis of PAH has been the recent identification of the ‘PAH gene’. Genetic studies have revealed that heterozygous mutations in the bone morphogenetic protein receptor 2 (BMPR2), a ubiquitously expressed member of the TGF-b superfamily of receptors, are found in up to 70% of patients with FPAH and up to 40% of patients with IPAH [5]. However, the penetrance of the PAH phenotype is variable and the likelihood that carriers of BMPR2 mutations develop clinical PAH is less than 20% [12]. Thus, it has been suggested that “multiple-hits” are required for the development of overt PAH, whereby a susceptible person with genetic predisposition may need to be exposed to additional environmental triggers before the disease is manifested [3, 46]. Over 160 different mutations have been reported involving many different regions of the BMPR2 gene, but all of the mutations identified to date are likely to produce loss of function of BMPR2 [79]. The function of BMPR2 is quite complex. This ubiquitously expressed receptor has control over a wide variety of developmental processes such as embryogenesis, organogenesis, bone and cartilage morphogenesis, vasculogenesis, angiogenesis and hematopoiesis, in addition to regulating cell proliferation and apoptosis [7, 8]. Signaling through BMPR2 results in pleiotropic effects depending on the cell type, the specific ligand and the environmental context [79]. Based on recent loss- and gain-of-function studies in vascular cells in vitro, opposing roles for BMPR2 and its ligands, the BMPs, in vascular homeostasis have been reported. BMPs were shown to have an antiproliferative effect on pulmonary arterial SMCs, which were partly mediated through induction of apoptosis [8]. In contrast, BMPR2 signaling promotes pulmonary arterial EC survival [79]. Consequently, this paradigm in BMPR2

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function suggests that the BMPR2 pathway may play a critical role in preventing EC apoptosis and maintaining the integrity of the lung microvasculature. Loss-offunction mutations in BMPR2 are thus thought to enhance pulmonary remodeling and play a significant role in the early endothelial dysfunction in patients with PAH. In addition, Hagen et al. [80] recently demonstrated a negative feedback loop between IL-6 and BMPR2, suggesting a possible link between inflammation and EC vulnerability in PAH patients with BMPR2 mutations. The precise mechanism by which reduced BMPR2 signaling is linked to the functional and morphological abnormalities of PAH remains uncertain, and in vivo models have not provided a clear mechanistic explanation. For example, under unstressed conditions, BMPR2+/– mice failed to develop overt PAH [81] and even transgenic mice expressing only 5–10% of BMPR2 compared to WT mice did not spontaneously develop PAH [82]. These data indicate that reduced BMPR2 gene dosage alone is insufficient to cause PAH in mouse models. In contrast, transgenic mice overexpressing a “dominant negative” mutant BMPR2 gene targeted to SMCs exhibited significant elevation in RVSP compared to WT controls under basal conditions, but this occurred in the absence of significant arterial medial remodeling of the pulmonary microvasculature [83]. A more recent study from the same group has demonstrated the presence of obliterative luminal lesions in a small proportion of mice overexpressing the BMPR2R899X deletional mutation in SMCs [84]. To explain the increased penetrance of the phenotype in these mice, the authors have suggested that loss of the BMPR2 C-terminal cytoplasmic tail results in a stronger phenotype when the more proximal kinase domain function is preserved. Consistent with the “multiple-hit” hypothesis, serotonin exposure alone and together with hypoxic exposure has been reported to result in exaggerated PAH and arterial remodeling in BMPR2+/– mice compared to WT littermates [81]. Furthermore, adenoviral overexpression of 5-lipoxygenase, a pro-inflammatory gene, alone [85] and in combination with MCT [86] resulted in more severe PAH in BMPR2-deficient compared to WT littermate mice. Thus, defects in the BMPR2 signaling pathway appear to increase the susceptibility of mice to PAH, but by themselves do not produce overt PAH.

Mutations in Alk-1 and other members of the TGF-β superfamily The absence of BMPR2 mutations in some families and in a large proportion of patients with IPAH and APAH suggests that there may be additional genes involved in the pathology of this disease. Indeed, mutations in two other members of the TGF-b superfamily, activin-like kinase-type 1 (Alk-1) and endoglin, have also been identified in patients with PAH [4, 46, 87], although overall these mutations account for a relatively small number of cases. Relatively few studies have been reported to date that identify the direct role of these proteins on endothelial function, and even fewer are reported looking at functional consequences of the muta-

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tions; preliminary data from EC lines, however, do suggest that signaling via these receptors plays a role in regulating proliferation [88] and apoptosis [89]. Further work is needed to clarify the exact role of these proteins and their mutations in ECs, as well as loss- and gain-of-function models to clarify their physiological significance. However, with the evidence to date, it is becoming increasingly apparent that alterations in TGF-β signaling pathways may play a central role in initiating PAH or causing disease progression, thereby moving away from the traditional view of endothelial vasomotor dysfunction as the key player and pointing towards cell growth and survival as the most likely molecular mechanism underlying the pathogenesis of this disease.

Ang1-Tie2 pathway Another pathway that has been implicated in PAH is the Ang1-Tie2 pathway. Ang1, an angiogenic and EC survival factor, activates the Tie2 receptor that is selectively expressed on ECs, and is thought to promote postnatal vascular homeostasis by protecting against EC apoptosis and inflammation [90, 91]. Despite substantial evidence supporting a protective role for the Ang1-Tie2 pathway in the systemic vasculature, the contribution of this pathway in the pulmonary vasculature has been fraught with controversy, largely due to a series of studies emerging from one group suggesting that Ang1 contributes to the pathogenesis of PAH [92–95]. Thistlethwaite and colleagues implicated Ang1 as playing a causal role in the development of PAH based on their initial observations that Ang1 expression in the lung was negligible in healthy individuals, but significantly up-regulated in PAH patients [92, 93]. Moreover, expression levels of Ang1 protein and Tie2 activity were found to strongly correlate with increased pulmonary vascular resistance. However, some of this work has since been challenged, and there is now emerging consensus in the field that Ang1 is highly expressed in normal human lung and its expression is not changed in the lungs of PAH patients [96, 97]. More recently, we have observed that Tie2-deficient mice develop significant elevation in RVSP following exposure to the relevant triggers of PAH, serotonin or IL-6, and exhibit significantly higher rates of apoptosis in the periphery of the lung compared to WT mice [98]. Since Tie2 is known to play an important role in EC survival signaling, this is consistent with the concept that predisposition to endothelial apoptosis is paramount in the pathogenesis of this disease. Moreover, inhibition of apoptosis using Z-VAD, a pan-caspase inhibitor, rescued the PAH phenotype in Tie2-deficient mice. Lung Tie2 expression and activation are also significantly down-regulated in pulmonary hypertensive rats after exposure to MCT or hypoxia [97, 99–101]. In addition, it was demonstrated that enhanced Ang1 expression through cell-based gene transfer can protect against the development of MCT or hypoxia-induced PAH, potentially through the prevention of early EC death [97, 99]. Thus, these findings highlight the role of EC

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survival-signaling via the Ang1-Tie2 pathway as a protective mechanism in PAH and further emphasize the importance of EC apoptosis in the onset of this disease.

Mechanistic link between EC apoptosis and PAH As discussed above, several lines of evidence point to endothelial damage and apoptosis as a central trigger for the development of PAH. However, the mechanisms by which EC apoptosis can lead to a full PAH phenotype, including the typical pathological vascular lesions characterized by abnormal EC proliferation, remain unclear. Here, we propose a unifying hypothesis to bring together many of the seemingly divergent ideas surrounding the role of EC apoptosis in PAH (Fig. 1). Firstly, this paradigm suggests that EC apoptosis could contribute directly to ­classical

Figure 1. Proposed role of endothelial cell apoptosis in the pathogenesis of pulmonary arterial hypertension.

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endothelial dysfunction, since it has been previously demonstrated that regenerated endothelium following denudation-induced injury exhibits reduced vasodilator function, with the attendant consequences of vasoconstriction and arterial remodeling [102]. In addition, these changes could potentiate the loss of vascular SMC growth inhibition that may arise as a consequence of mutations in the TGF-b receptor superfamily of genes. Secondly, loss (i.e., dropout) of ECs, particularly at the level of the fragile precapillary arteriole, could lead directly to the degeneration of these fragile EC structures, which normally have little or no mural supporting cells. A progressive loss of functional continuity at the precapillary arteriolar level would result in loss of efficient (i.e., low pressure) perfusion of the distal lung microvasculature, and thus reduced effective cross-sectional vascular area. In turn, this would compromise the ability of the pulmonary bed to accommodate the cardiac output, particularly during exercise, and result in elevated pulmonary vascular resistance. Finally, widespread and recurrent pulmonary vascular EC apoptosis could also set up the conditions that favor the selection of apoptosis resistant and hyperproliferative ECs, as suggested by Tuder’s group [76], which could contribute to the intimal and plexiform lesions found in many PAH patients. Indeed, it was shown in vitro that after initial apoptosis of ECs induced by VEGFR blockade, there is increased proliferation of apoptosis-resistant ECs [103]. Furthermore, ECs cultured from the pulmonary arteries of PAH patients show evidence of increased proliferation and decreased apoptosis compared to those isolated from controls [104]. At present, it is not clear which of these potential consequences of EC apoptosis may be most important in the pathogenesis of PAH, and this will need to be elucidated in future studies. However, the evidence supporting a central role for EC apoptosis is becoming increasingly compelling and this may suggest novel strategies to detect, prevent and treat PAH. To the extent that apoptosis represents the initiating mechanism underlying the pathogenesis of PAH, strategies designed to enhance vascular repair may represent an attractive therapeutic approach. Indeed, alternative approaches to pharmacological treatment of PAH are currently being explored, including cell therapy using endothelial progenitor cells (EPCs). Given the accumulating evidence supporting a role for circulating EPCs in repairing and regenerating damaged blood vessels throughout the body, exploiting the potential of these progenitor cells alone or in combination with genetic engineering represents a prospective future in PAH therapy. A number of clinical trials using progenitor cell therapy for PAH have been registered in clinicaltrials.gov, including the PHACeT trial (NCT00469027), which is currently enrolling patients. A recent pilot randomized trial has been reported showing that transplantation of autologous EPCs into patients with IPHA is safe, and the authors noted that the patients receiving EPC therapy had significant improvements in both primary and secondary endpoints of 6-min walk test and mean pulmonary arterial pressure [105]. Therefore, EPC therapy may be a promising avenue for the treatment of PAH.

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Conclusions PAH is a complex disease and abnormalities in endothelial biology likely play a determining role in its pathobiology. Insights into the mechanisms underlying PAH over the last several decades, particularly with respect to endothelial dysfunction and abnormalities in the control of vascular tone, have resulted in the development of a number of effective pharmacological therapies. However, these have only limited impact in forestalling the progression of this debilitating disease. It is hoped that recent advances in our understanding of novel pathways in this condition will lead to the development of novel therapeutic strategies. Indeed, the elucidation of the central role of EC apoptosis in PAH has resulted in a shift in emphasis from therapies focusing on pulmonary vasomotor tone to the exploration of modalities to influence survival and growth of vascular cells, so as to inhibit or reverse vascular remodeling and the development of angioproliferative lesions. These strategies may better address the root cause of PAH and possibly represent a more effective treatment approach that in the future may have a greater impact on the long-term prognosis of the disease.

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Endothelial dysfunction in systemic hypertension Carol Yu, Arpeeta Sharma, Andy Trane and Pascal Bernatchez Providence Heart and Lung Institute at St. Paul’s Hospital, The James Hogg Research Centre, Department of Anesthesiology, Pharmacology and Therapeutics, University of British Columbia, Vancouver, BC, Canada

Abstract Hypertension, defined as a rise in arterial blood pressure in the absence of a specific cause, leads to a myriad of cardiovascular complications that account for a high number of deaths globally. Progress in understanding hypertension comes from pioneering studies of the vasculature in both in vivo and in vitro settings. A common factor in hypertension and cardiovascular diseases is a decrease in the bioavailability of nitric oxide (NO), a potent endogenous vasodilator and a direct marker of endothelial function. This phenomenon, called endothelial dysfunction (ED), has increasingly gained importance in cardiovascular pathogenesis. Indeed, clinical evidence supports strong associations between ED and cardiovascular disease arising from common risk factors such as smoking, diet, lack of exercise, aging and genetic determinants. Besides NO, other factors that are produced in the endothelium, such as prostacyclin and endothelium-derived hyperpolarizing factor, can also be involved with, or act synergistically in, ED. This chapter focuses on the importance of NO in settings of ED and hypertension, and discusses the arguments around ED as a cause or consequence of hypertension by providing experimental and clinical evidence. We also aim to highlight current therapeutic strategies to improve vascular health by targeting levels of NO synthesis and raise questions emphasizing the need to further our knowledge in the molecular determinants in ED and hypertension.

General considerations Systemic (primary or essential) hypertension can be defined as a rise in arterial blood pressure in the absence of a specific cause. Considered a ‘silent killer’, as it does not cause symptoms unless severely high [1], extensive epidemiological evidence suggests that hypertension leads to atherosclerosis [2], strokes [3], myocardial infarction [4] and chronic renal failure [5]. Lifestyle factors, such as physical inactivity and salt- and fat-rich diets, are at the heart of this increased disease burden. Combined with the fact that the average population in developed countries is aging and that hypertension is spreading at an alarming rate in developing Endothelial Dysfunction and Inflammation, edited by Shauna M. Dauphinee and Aly Karsan © 2010 Springer Basel

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nations, the future consequences of systemic hypertension are likely to confirm the classic ‘tip of the iceberg’ idiom. In 2000, the estimated global number of adults with hypertension was 972 million, and is expected to increase to 1.56 billion by 2025 [6].

Blood pressure regulation and endothelial dysfunction Recent advances in hypertension research, spearheaded by the development of isolated vessel preparation and in vitro culture of endothelial cells (EC) and smooth muscle cells (SMC) led to the current explosion of information regarding the dynamic nature of the vasculature in health and disease. A salient example of this was the discovery describing the profound importance of the vascular endothelium in regulating the contractile activity of vascular SMC and therefore vascular tone, which was suggested by the pioneering studies of Furchgott and Zawadzki [7]. This led to the identification of an elusive but potent endothelium-derived relaxing factor, which was ultimately identified as nitric oxide (NO), which plays a central role in vascular tone regulation because of its capacity to directly induce SMC vasorelaxation independently from upstream vasoconstrictors [8]. A plethora of clinical data demonstrates deficient NO-dependent vascular tone regulation in patients with cardiovascular disease, with the most common condition being hypertension [9–11]. Indeed, “all known cardiovascular disease risk factors primarily induce functional alterations in EC characterized by a lowered bioavailability of NO” [12, 13]. This condition, called endothelial dysfunction (ED), is observed in almost all patients with cardiovascular diseases and is directly linked to aging [14]. The attenuation of the expected blood flow response to acetylcholine (Ach), an endothelium-dependent vasodilator, is a classic observation in patients with ED [15]. However, in virtually all of these cardiovascular pathologies, the vasodilatory responses to endothelium-independent exogenous NO donors are preserved. ED appears to occur in large or conduit (thoracic aorta), but not small or resistance arteries (mesenteric arteries), in human and animal models of hypertension. Endothelium-dependent relaxations are unimpaired in resistance arteries of transgenic hypertensive rats, whereas they are attenuated in conduit arteries. A likely explanation for such disparity is the known difference in the relative contributions of NO and endothelium-derived hyperpolarizing factor (EDHF) in these vascular beds. Inhibition or gene disruption of endothelial NO synthase (eNOS), the enzyme that is constitutively expressed by the endothelium and synthesizes NO, leads to a nearly complete loss of Ach-induced vasorelaxation in aortic vessels, whereas they have a lesser effect on relaxation of mesenteric vessels. Impaired NO release occurs in most human and animal models of hypertension, contributing to increased peripheral resistance and, most likely, pathogenesis of cardiovascular disease.

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Endothelial regulators of vascular tone and ED Nitric oxide NO is a potent vasodilator that significantly contributes to resting vascular tone and participates in maintaining the non-thrombogenic surface of the vascular endothelium. eNOS is the NOS isoform constitutively expressed by EC that synthesizes NO under basal conditions and agonist challenge, and does so via a 5-electron oxidation of l-arginine [16]. Targeted disruption of the eNOS locus in mice, or chronic NOS inhibition, have notable effects on cardiovascular homeostasis, causing increases in heart rate, blood pressure, renin activity [17–19] and atherosclerosis progression [20, 21], which illustrates the broad protective aspects of NO and endothelial function as a whole in the vasculature. The central role of eNOS-derived NO to vascular tone and blood pressure regulation is likely attributable to its capacity to directly induce vascular SMC relaxation independently of upstream vasoconstrictors. NO-induced activation of soluble guanylate cyclase (sGC) in SMC [22] results in decreased calcium-dependent activation of myosin light chain kinase along with increased myosin phosphatase activity. This leads to decreased myosin light chain phosphorylation and SMC relaxation [23]. Since NO is generally considered as the most potent endogenous vasodilator, any kind of decrease in NO activity would likely lead to hypertension and increased ED.

NO bioavailability and inactivation Although the underlying cause for reduced eNOS-derived NO synthesis and downstream NO ‘bioavailability’ in ED is still poorly understood, a likely candidate for the rationalization of some of these observations is oxidative stress and its main mediator, superoxide anion (O2–). It is clear that hypertension increases vascular production of O2–, which can directly react with labile NO to form peroxynitrite (ONOO–), thereby scavenging NO [24]. Both O2– and ONOO– are powerful oxidants that cause structural damages to DNA [25], as well as protein and lipid oxidation, which are key steps in atherosclerosis progression [26]. However, many investigations on the effect of radical scavengers, such as vitamin E, on mortality and incidence of vascular diseases have been disappointing [27, 28], again suggesting that our knowledge of the causes of ED and decreased NO bioavailability is insufficient. From a different perspective, the contemporaneous identification of Angiotensin II (AngII), a well-defined hypertensor, as a highly potent inducer of O2– generation has provided insights into the effect of hypertension in the pathogenesis of ED. AngII stimulates the activity of NADP(H) and xanthine oxidases, which are the major sources of oxygen free radicals within the vascular wall [29]. On the other hand, others have shown that AngII-induced hypertension increases superoxide dismutase (SOD) expression, which is obviously considered an endogenous com-

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pensatory mechanism that impairs the hypertensive response, prevents endothelial damage and helps restore endothelial function [30].

NO resistance A provocative concept closely linked to decreased endothelial activity comes from the possibility that subphenotypes of ED, characterized by NO resistance, may exist. Indeed, a recent report [31] provides evidence that clinical states of ED can be associated with a pool of deficient sGC, the enzyme that is activated by NO and responsible for the downstream activation of cGMP-dependent protein kinase. Such sGC deficiency is believed to be a direct consequence of the oxidative environment associated with ED, leading to oxidized and heme-free sGC that cannot be activated by NO. If true, this unexpected form of NO resistance might not only further complicate the treatment of ED in the clinic, but also complicate the accurate assessment of ED, defined by the maximal dilatation of a vessel in response to exogenous NO donors, which is artificially abrogated in these patients.

Prostacyclin Prostacyclin (PGI2; epoprostenol) is an eicosanoid synthesized mainly by endothelial cyclooxygenase (COX) enzymes and is considered a powerful vasodilator and inhibitor of platelet aggregation [32]. However, endogenous PGI2 synthesis and vasodilation play a lesser role than NO and EDHF in the regulation of measurable blood pressure in conduit arteries and vascular tone in resistance arteries, respectively, which led many to believe that PGI2 synthesis and responses are not altered in ED. For instance, in contrast to NO, the PGI2 system was characterized as intact in the coronaries of atherosclerotic mice [33]. However, since ED is mainly characterized by decreased EC-dependent vasorelaxation, one might assume that PGI2 responses should also be abrogated in ED/hypertension. Emerging evidence supports this concept since PGI2 vasorelaxation was reported as impaired when aldosterone was used to induce ED in normotensive and hypertensive animals [34]. From a patient’s perspective, however, an interesting but unexpected piece of information on the role of PGI2 in hypertension and ED comes from the Vioxx studies and withdrawal. The adverse cardiovascular effects of Vioxx were believed to be partly attributable to its inhibitory effect on protective PGI2 synthesis (along with thromboxane up-regulation), leading to increased thrombosis and heart attacks. Although general ED was not cited as a likely cause of these cardiovascular events, analysis of multiple studies show that COX-2 inhibitors cause an inconsistent increase in blood pressure, likely through PGI2 inhibition [35]. On the other hand, it remains to be confirmed that the effect of ED on PGI2 release is comparable to the direct inhibition of PGI2synthesizing enzymes by Vioxx. Presently, it might be too early to consider PGI2 as 106

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a primary cause of ED and essential hypertension, and more studies are needed to fully elucidate its importance in vascular disease.

EDHF EDHF is best characterized as a group of substances and/or electrical signals that originates from the endothelium and causes hyperpolarization of SMC, leading to vasorelaxation. Because EDHF is the main vasodilator in resistance arteries, which mostly determines an individual’s blood pressure, minor changes in the balance of EDHF might result in profound changes in tension. However, the role of EDHF in hypertension/ED remains controversial. For instance, EDHF was reported to not only be up-regulated in a salt-induced model of animal hypertension [36] and diabetes [37, 38], presumably in a compensatory way, but was also shown to be up-regulated by angiotensin converting enzyme inhibitor (ACEi) in ED settings [39], which controversially suggests that both pathogenesis of ED and anti-ED/hypertension therapy enhance EDHF activity. In general, most of the literature seems to suggest that EDHF release is not impaired in ED but rather slightly up-regulated, which is in drastic contrast to NO, and this is likely attributable to its resistance to oxidative stress. Finally, it is relatively safe to say that the elusive nature of EDHF is the main factor impeding the study of this endothelial mediator in health and disease.

ED vs hypertension: Which comes first? A mechanistic evaluation Despite clear evidence that hypertension and ED are closely linked, it remains enigmatic whether ED is a primary cause or a consequence of elevated blood pressure, as there is convincing evidence to support both theories. Initial experiments, as well as most animal models of genetic hypertension and data in patients, support the concept that direct damage to EC occurs as a result of long-term increases in hemodynamic load and mechanical stress [40–44]. One might also speculate that an increase in physical pressure on EC is likely to interfere with the physiological integration of extracellular forces, such as sheer stress by the mechanosensory machinery, termed mechanotransduction, which has become a fertile ground for discoveries in recent years. As such, an increasing number of endothelial signaling molecules that mediate the transmission of extracellular forces to the intracellular compartment have been proposed. For instance, vascular endothelial (VE)-cadherin, platelet-endothelial cellular adhesion molecule (PECAM)-1 and focal adhesion kinase (FAK) were suggested to constitute mechanosensory/transducing complexes in cultured EC that sense the mechanical flow upstream of integrins; intracellular signal amplification for this complex is presumably relayed to the src/PI3K and nuclear factor-κB pathways [45], a known

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stress mediator involved in the pathogenesis of many diseases. Although the role of PECAM-1-dependent signaling complexes in flow sensing and flow-dependent NO activation [46] remains controversial [47–50], caveolin-1 knockout vessels, which lack plasma membrane caveolae [51], show aberrant mechanotransduction [52]. Since several of the above-mentioned signaling molecules are caveolae-resident proteins, it is possible that these delicate caveolar macrostructures are a direct target of deleterious physical stress caused by hypertension on the vascular wall. However, arguments against hypertension triggering the onset of ED cannot be ignored. Studies have shown that potential endothelial damage caused by hypertension can be reversed by some, but not all, anti-hypertensive therapies, arguing against the role of hypertension as a cause of ED. This puzzling topic is further discussed in the following section of this chapter. It has been shown that treatment of hypertension does not consistently improve endothelial function [53], and that ED appears to precede insulin resistance-induced hypertension [54]. Taken together with a recent study that shows that acute smoking induces ED within minutes [55], these findings cast doubts about the role of hypertension in inducing EC damage in ED. From a purely physical perspective, it remains to be seen if decreasing blood pressure by 20–30  mmHg, which is within the average limit of anti-hypertensive therapy in patients, prevents direct endothelial damage to a significant extent. In fact, non-conduit artery EC, such as endocardial EC and myocardial vasa vasorum EC are known to cope with far greater pressure and physical stress during cardiac systole than conduit EC. To err on the side of caution, it would be reasonable to conclude that ED can lead to the pathogenesis of hypertension and vice versa, but that setting-specific factors, such as genetics, may dictate the initial prevalence of one versus the other (Fig. 1). Key variables are likely to include smoking, diet, exercise, aging and genetic determinants.

Hitting two birds with one stone: Improving endothelial function and blood pressure control Inhibition of AngII Angiotensin converting enzyme inhibitors (ACEi) are believed to increase endothelial activity by decreasing the effect and synthesis of AngII, leading to decreased oxidative stress and increased NO bioavailability. ACEi also increases blood levels of bradykinin via inhibition of its degradation, causing increased NO release. ACEi have been shown to improve endothelial function in vivo and in vitro [56], and the therapeutic effects of quinopril and lisinopril have been confirmed in patients with atheroscleorosis [57, 58]. Insights into the pathogenesis of ED come from studies that report improvement in endothelial function with type 1 angiotensin II receptor (AT1R)-selective antagonists [59]. Indeed, experiments in animal models suggest

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Figure 1. It remains enigmatic whether hypertension leads to ED, or vice versa. It is, however, safe to assume that other factors, such as aging and genetic predisposition, are likely influences. On the other hand high salt diets accelerate the pathogenesis of hypertension, whereas smoking and high-fat diets cause ED by activating inflammatory pathways in EC and decreasing NO bioavailability. Both ED and hypertension lead to atherosclerosis, arteriosclerosis, cardiac insufficiency, myocardial infarction, strokes and kidney injury.

that type 2 angiotensin II receptor (AT2R) activation is linked to a mild but significant hypotension, lending credence to the assumption that selective inhibition of AT1R combined with AT2R activation might act in a beneficial fashion, although this raises questions about the importance of the dynamics between bradykinin accumulation and AT2R stimulation when a therapeutic regimen includes ACEi and/or AT1R antagonists. On the other hand, it is of interest to note that diuretics, which are often used in combination with ACEi, appear to have a much more moderate effect, if any, on restoring endothelial function [60]. Considering that decreased blood volume should lead to reduced direct endothelial damage, this supports the hypothesis that hypertension is a consequence of ED rather than a cause.

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Statins An interesting but unexpected link between cardiovascular risk factor management and blood pressure comes from the observations that statins have been shown to decrease systolic, diastolic and mean arterial blood pressure in various settings, including normotensive and hypercholesterolemic men [58, 61, 62], amongst others [63, 64]. However, clinical and basic science reports provide evidence that decrease in blood pressure induced by statin therapy is achieved in a manner that is independent of their lipid lowering effect, further adding to their impressive list of pleiotropic effects; meta-analysis of past clinical data showed lower systolic blood pressure in patients treated with statins versus placebo or even comparative lipid-lowering drug [65]. Increased NO bioavailability, through decreases in AT1R expression [66] and activation of Krupple-like factor-2 [67], an athero-protective transcription factor capable of promoting eNOS expression, provides interesting means of rationalizing the vasculoprotective effects of statins and lends credence to new guidelines that are supportive of broadening the use of statins for managing blood pressure in patients at high risk of cardiovascular disease [68–70].

Tetrahydrobiopterin (BH4) supplementation In the context of ED and O2– release attributable to eNOS uncoupling, recent reports suggest oral BH4 supplementation as a possible mean to improve endothelial function by increasing NO bioavailability. The rationale behind these investigations is that BH4 is essential to optimal eNOS coupling and that low BH4 contributes to a decrease in NO bioavailability by causing increased eNOS O2– release. The first study used serotonin (EC-dependent vasodilator), nitroprusside (EC-independent vasodilator) and NG-monomethyl l-arginine (NOS inhibitor), and reported that infusion of BH4 in the brachial artery of hypercholesterolemic patients improved endothelial function, without showing a significant effect in control patients [71]. Similar conclusions were drawn in coronary vessels [72], and in settings of angina [73], smoking [74], and aging [75], amongst others. In settings of hypertension, BH4 was shown to prevent ED and decrease blood pressure. Since BH4 is a critical mediator of endothelial function, these studies are a salient illustration of ED as a cause, rather than a consequence, of hypertension. l-Arginine

supplementation

Similar to BH4, l-arginine supplementation is believed to increase endothelial function at the level of the eNOS enzyme by improving substrate availability and downstream NO release. However, considering that intracellular levels of l-arginine are

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30–800-fold higher than the Km of eNOS [76], caution must be taken when speculating on the underlying mechanism of action of oral l-arginine supplements. Despite this caveat, the fact that this natural amino acid is well tolerated and considered as semi-essential led to its rapid use in animal and clinical trials. Indeed, l-arginine was shown to augment endothelium-dependent vasodilatation in hypercholesterolemic humans and animals [77–79]. Most interestingly, a 20 mmHg decrease in systolic blood pressure was observed in l-arginine treated patients with hypertension (166 vs 146 mmHg) and it was found that endothelial function and quality of life was improved in the treated group [80]. These data were corroborated by a recent study reporting that oral l-arginine supplementation at a dose of 7 g, three times a day was shown to improve flow-mediated dilatation, a marker of endothelial function, as well as aortic stiffness following acute smoking-induced injury [81]. On the other hand, larger multi-center studies are still required to determine if l-arginine supplements promote long-term reductions in cardiovascular disease risks and events. A possible explanation for lack of large clinical trials for l-arginine-based therapies is due to insufficient financial incentives linked to this natural product.

Conclusion Despite being identified more than a century ago, hypertension and ED have been, and continue to be, fertile grounds for discoveries as well as interesting controversies. New therapeutic avenues aimed at efficiently preventing both pathologies are clearly warranted. As such, these two related but separate concepts will likely require more definitive molecular approaches.

Acknowledgements This work is supported by grants from the Canadian Institutes for Health Research, Michael Smith Foundation for Health Research, Canadian Foundation for Innovation and the British Columbia Proteomics Network. C.Y, A.S. and P.B. are supported by salary awards from the Canadian Institutes of Health Research and Michael Smith Foundation for Health Research.

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Sphingosine-1-phosphate as a mediator of endothelial dysfunction during inflammation Bodo Levkau Institute of Pathophysiology, Universitätsklinikum Essen, Germany

Abstract Sphingosine-1-phosphate (S1P) is a biologically active sphingolipid that regulates immunity, inflammation, and cardiovascular function. S1P gradients between plasma and the interstitium, and dynamically regulated S1P receptors in hematopoietic and endothelial cells, orchestrate immune and inflammatory cell trafficking. S1P generating enzymes are activated in inflammation, and high local S1P levels boost inflammatory responses by inducing adhesion molecules, recruiting and retaining inflammatory cells and lymphocytes, and activating lymphatic dendritic cells. At the same time, S1P has clear anti-inflammatory effects: it strengthens endothelial cell-cell contacts, decreases permeability, and inhibits cytokine-induced leukocyte adhesion. Thus, S1P activates both positive and negative feedback mechanisms in inflammation and determines not only the build-up, magnitude, and duration of inflammation but also its restraint and resolution. This review elucidates these mechanisms and the important issue of S1P bioavailability and delivery by high-density lipoproteins as its major plasma carrier. Finally, the potential application of S1P receptor analogues as new treatment strategies in inflammation is discussed.

Sphingosine-1-phosphate: Sources, metabolism and carriers In 1884, the German physician J. L. W. Tudichum first discovered a new class of biochemical substances by fractional crystallization from human brain. Utterly fascinated by its enigmatic behavior, he named it “sphingolipids” after the Sphinx from Greek mythology. Because of their presence in virtually all types of biological membranes, the main role of sphingolipids has long been perceived as a solely structural one. It is only over the last two decades that sphingolipids, such as sphingosine and sphingosine-1-phosphate (S1P), have been shown to exert a variety of biological effects [1]. It has now become evident that this biologically extremely versatile molecular class, with S1P as its main representative, controls physiological processes as fundamental as immunity, inflammation, and cardiovascular function [2]. S1P is an integral membrane component, especially in lipid rafts, and is synthesized intracellularly from sphingomyelin via phosphorylation of sphingosine on its primary hydroxyl group by the obligatory action of two distinct sphingosine Endothelial Dysfunction and Inflammation, edited by Shauna M. Dauphinee and Aly Karsan © 2010 Springer Basel

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kinases: Sphk1 and Sphk2. The major source of S1P in plasma is hematopoietic cells (mainly erythrocytes, but also platelets and leukocytes), but vascular and lymphatic endothelial cells have also been shown to synthesize and release S1P [3, 4]. Inside the cell S1P moves freely between membranes but requires specific transport mechanisms to translocate to the outer leaflet of the cytoplasmic membrane due to the low propensity for spontaneous flip-flop [1, 5]. In the majority of cell types, these transport mechanisms have remained uncharacterized, but in some cells, such as platelets and mast cells [6, 7], ABC-type transporters have been implicated in S1P export. However, further mechanisms must exist as, for example, plasma S1P levels are completely unaltered in ABCA1-, ABCA7-, or ABCC1-knockout mice [8]. In plasma, S1P occurs in concentrations of 200–1000 nM [9, 10] and is contained mainly in the fraction of high-density lipoproteins (HDL) (~50–70%) followed by binding to albumin (~30%), and low-density lipoproteins (LDL) and very-lowdensity lipoproteins (< 10%) [11]. S1P binds with extremely high affinity to HDL, making these lipoproteins the primary acceptors and carriers of plasma S1P [12]. Accordingly, plasma S1P levels positively correlate with HDL-cholesterol (HDL-C), and apolipoprotein AI and AII levels [10]. Intriguingly, total plasma S1P levels are 100-fold higher than the Kd value of its receptors [11, 13], but only ~2% of it is biologically active. It has thus been suggested that HDL functions not only as a carrier of S1P in plasma but also as a buffer of free S1P [14]. The degradation of S1P occurs either via dephosphorylation to sphingosine, through the action of specific intracellular S1P phosphatases, or through irreversible cleavage by the S1P lyase to ethanolamine phosphate and hexadecenal [1, 15].

S1P and its receptors S1P specifically binds to and activates five cognate G protein-coupled cell surface receptors designated S1P1–5 with a Kd of 8–20 nM [16]. The molecular details on receptor binding and activation are complex. Individual S1P receptors couple to one or more G  protein subunits, with considerable overlap for some but not all receptors: S1P1 is coupled to Gi/o, preferentially Gia1 and Gia3; S1P2 is associated to Gi/o,G12/13 and Gq; S1P3 activates either Gi/o, Gq or G12/13 proteins, and S1P4 and S1P5 signal through Gi/o or G12/13 and Gi/o or G12 subunits, respectively [1, 16]. The complexity of S1P coupling to G proteins drives the multiplicity of downstream signaling pathways: phospholipase C activation and Ca2+ mobilization (via Gq); ERK and PI3-kinase activation, and inhibition of adenylate cyclase (via Gi); and activation of Rho and actin cytoskeleton reassembly (via G12/13). In this manner, the relative expression levels of the different S1P receptors on the cell surface determine the net cellular response to S1P [2]. In addition, S1P receptors have been shown to transactivate tyrosine kinase receptors such as vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and epidermal growth factor

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(EGF) [17–20], as well as other G  protein-coupled receptors such as the CXCR4 [21–23], and to cross-activate the transforming growth factor-b (TGF-b) signaling pathway by phosphorylating Smad proteins [24]. S1P receptor signaling occurs at the full spectrum of the receptor occupancy curve: extremely low occupancy leads to transactivation of the PDGF receptor, occupancy at the steep slope of the receptor binding curve activates a toggle switch that turns off lymphocyte recirculation, and finally, high receptor occupancy (concentrations 50–100-fold higher than the Kd) leads to receptor desensitization and degradation [25].

S1P and lymphocyte trafficking Much of the knowledge on the biological effects of S1P stems from the unique role it plays in the homeostasis of the immune system: it is instrumental for the exit of T and B lymphocytes from the thymus and secondary lymphoid organs (spleen and lymph nodes) during lymphocyte recirculation, a process that is indispensable for immunosurveillance [26]. To exit the lymph nodes, lymphocytes employ a simple mechanism of active migration along a S1P concentration gradient that exists between lymph node (< pM), lymph (pM) and plasma (mM). Ablation of this gradient by genetic or pharmacological manipulation leads to a complete loss of lymphocytes from the peripheral blood through their “trapping” in secondary lymphoid organs [27]. In systemic inflammatory response syndromes, such as bacterial sepsis and viral hemorrhagic fevers, S1P was also shown to couple coagulation with inflammation. There, S1P promotes the dissemination of inflammation by participating in the coagulation-induced activation and trafficking of dendritic cells in the lymphatics [28]. Substantial evidence has accumulated on the specific role of S1P receptors, and specifically S1P1, in lymphocyte biology: genetic deletion of S1P1 on lymphocytes or the administration of S1P1 agonists/antagonists have revealed a S1P1-dependent mechanism of lymphocyte egress. Remarkably, only S1P1 receptor agonists, but not antagonists, induce lymphopenia, indicating that initial activation with subsequent down-regulation, rather than inhibition of S1P1, impedes lymphocyte egress [26, 27]. This has made the pharmacological development of synthetic S1P agonists a novel approach to immunomodulatory therapy, for example in transplantation medicine and in the treatment of multiple sclerosis [29]. Thus, S1P causally influences the efficiency and duration of the immune response and has a major impact on the regulation of inflammation.

S1P in endothelial cell migration, angiogenesis and morphogenesis The S1P receptors most abundantly expressed in endothelial cells are S1P1, S1P2 and S1P3 with S1P1 > S1P2 ≈ S1P3 [30]. Although not systematically analyzed, endothelial 121

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cells of different origin – arteries, capillaries, veins, lymphatics – appear to express different levels of these particular receptors, and, to add a further level of complexity, differences may exist in S1P receptor expression between endothelial cells from different arteries, for example among aortic, cerebral, coronary, renal, and mesenteric arteries. S1P exerts a profound variety of effects, via its receptors, on the endothelium, such as proliferation, migration, angiogenesis, survival, permeability control and adhesion molecule expression [31, 32]. In vitro, S1P induces endothelial cell proliferation and serves as a potent chemoattractant for endothelial cells [33]. It further promotes directed migration, vascular differentiation, and formation of capillary networks on complex extracellular matrices [34, 35]. S1P receptor-mediated activation of small GTPases thereby plays an important role: coupling of S1P1 to Rac1 induces focal contact assembly, membrane ruffling, and cortical actin formation via Gi, while RhoA activation by S1P3 promotes stress fiber assembly via Gq. In contrast, S1P2 signaling appears to have anti-migratory and anti-angiogenic effects: pharmacological S1P2 blockade enhances the pro-migratory response of S1P elicited by S1P1 in endothelial cells, while overexpression of S1P2 inhibits the pro-migratory response to S1P by suppressing Rac1 activity [36]. In vivo, S1P plays an important role in vascular morphogenesis during development: the S1P1 receptor is particularly involved as both general and endothelial-specific S1P1-knockout mice die in utero due to vessel rupture and hemorrhage caused by severe vascular maturation defects [35]. Interestingly, this phenomenon is not due to any apparent defects of endothelial proliferation, migration or tube formation, but instead to the inability of pericytes and smooth muscle cells to sheath and stabilize the nascent vessel. This process seems to be controlled by the endothelial and not the smooth muscle S1P1 because both the global and endothelial-specific S1P1-knockouts exhibit exactly the same vascular hemorrhage phenotype [37]. The identical phenotype is also observed in mice lacking S1P itself caused by a genetic deletion of both S1P-synthesizing enzymes Sphk1 and Sphk2 [38]. The two other S1P receptors, S1P2 and S1P3, also appear to play a role in vascular development, which is evident from the additional defects in vasculogenesis that become apparent especially in the double and triple (together with S1P1) knockouts [39]. While loss of S1P1 signaling impairs vascular development, the loss of S1P2 renders mice resistant to the exuberant pathological angiogenesis triggered by hypoxia, implying that S1P2 (a hypoxia-regulated gene) is a causal factor in ischemia-driven retinopathy [40]. S1P has also been shown to promote tumor-associated angiogenesis. In vivo, evidence has come from studies using a blocking anti-S1P monoclonal antibody that binds to and neutralizes bioactive S1P: in several xenograft and allograft tumor models in the mouse, the anti-S1P antibody reduced tumor progression, and even eliminated measurable tumors in some cases. This has been attributed both to inhibition of tumor angiogenesis as well as an impact on tumor cells themselves [41]. In vitro, the same anti-S1P antibody inhibited endothelial cell migration and

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capillary formation as well as VEGF- and bFGF-induced vessel formation. In vivo, the humanized antibody has been shown to inhibit retinal and choroidal neovascularization in oxygen-induced ischemic retinopathy in mice [42].

S1P and the maintenance of the endothelial barrier S1P effectively increases endothelial barrier function and profoundly decreases permeability in a variety of different endothelial cell types. As a direct measure of permeability in vitro, S1P increases the transmonolayer electrical resistance of microvascular endothelial cells. Again, this appears to be S1P receptor subtype specific, as S1P1 and S1P3 strengthen the formation of endothelial cell junctions [43–45], while S1P2 weakens them [46, 47]. The barrier-enhancing effect of S1P is mediated by several signaling events and cytoskeletal molecules, which all act in promoting adherens junction assembly; these include Ca2+, PI3-kinase, Tiam1/Rac1, VE-cadherin and β-catenin, a-actinin and ZO-1 [34, 45, 48]. Several studies have also linked the transient S1P-evoked intracellular Ca2+ increases to stabilization of the endothelial barrier as well as endothelial nitric oxide synthase (eNOS) activation [49–51]. In fact, these two processes are closely interrelated as both the sealing of the endothelial barrier and the prevention of microvascular leakage is inherent to nitric oxide (NO). While S1P1 seals the endothelial barrier, the S1P2 receptor acts yet again in an opposite manner – its engagement promotes vascular permeability via disruption of adherens junctions, which is dependent on its downstream effectors Rho-ROCK and PTEN in vitro [52], and contributes to the hypoxia-triggered pathological retinal angiogenesis in vivo [40]. Virtually nothing is known on the effects of S1P3 on endothelial permeability in vivo. The only study suggesting a role for S1P3 in promoting permeability has been retracted [53]. The S1P source required for the maintenance of endothelium-dependent vascular integrity is found in the plasma compartment rather than in the endothelium itself. Mice engineered to selectively lack S1P in plasma (“pS1Pless” mice) display vascular leakage and impaired survival after anaphylaxia, PAF and histamine administration, and related inflammatory challenges [54]. Transfusion of erythrocytes as the major source of plasma S1P restores S1P levels in the circulation and reverses the endothelial leak phenotype, as does administration of S1P1 receptor agonists [54]. Although these experiments show that S1P stemming from erythrocytes is crucial for preventing endothelial leakage, they do not exclude that S1P produced by endothelial cells themselves can also contribute to vascular sealing. In fact, endothelial cells exposed to physiological laminar shear stress produce and secrete S1P [4]. Furthermore, the global knockout of Sphk1, which abolishes its function in endothelial cells but does not suffice to decrease plasma S1P levels, also exhibits an endothelial leak phenotype that resembles that of the “pS1Pless” mice, albeit with a much less severe presentation [55]. In fact, Sphk1 activity has been shown to maintain basal endothelial

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barrier function in these same mice [54], and to mediate the anti-permeability effect of angiopoietin-1, although the latter appears to occur independently of S1P receptors [55]. Convincing evidence for a role of S1P1 in the homeostasis of endothelial permeability in the adult organism comes from application of pharmacological S1P1 antagonists. These have been reported to induce capillary leakage in the lung, kidney, skin and intestine in vivo [25, 56, 57]. Vice versa, S1P agonists inhibit the vascular leakage induced by VEGF from skin capillaries [56, 58]. Thus, the expression of different S1P receptor subtypes in different vascular beds, their regulation under different pathophysiological conditions, and the levels of S1P acting on them determine the net effect of S1P modulation of vascular permeability in any particular disease process [59].

S1P and adhesion of inflammatory cells to activated endothelium Leukocyte-endothelial interactions occur in a complex multi-step process of rolling, adhesion and transmigration. S1P has been shown to inhibit TNF-α-induced expression of endothelial adhesion molecules in vitro [60], and stable long-term knockdown of S1P1 in endothelial cells decreased the expression of PECAM-1 and VE-cadherin and abolished the induction of E-selectin after stimulation with TNF-a or LPS [61]. In agreement with an inhibitory function of S1P on adhesion molecule expression, another study showed that S1P suppressed the adherence of inflammatory cells to TNF-a-activated aortic endothelium in vivo [62]. However, this was not accompanied by the inhibition of adhesion molecule up-regulation (VCAM-1 and ICAM-1). Instead, S1P reduced the concomitant production of the pro-inflammatory chemokines IL-8 and MCP-1, which the authors could attribute to S1P1 in vitro using siRNA knockdown [62]. Thrombin can induce MCP-1 during inflammation in a reactive oxygen species (ROS)-dependent manner, which S1P was shown to abrogate by inhibiting the cytoplasmic NAD(P)H oxidase [63]. A study performed in the aortic endothelium of diabetic NOD mice showed that S1P and S1P1 agonists inhibited VCAM-1 expression but had no effect on ICAM-1 or chemokine production [64]. In that study, S1P also abrogated monocytic cell adhesion to the diabetic endothelium, which the authors attributed to inhibition of VCAM-1 by an NO-dependent mechanism, as L-NAME blocked the inhibitory effect of S1P by half [64]. They also suggested a general anti-inflammatory effect for S1P because it potently inhibited the concomitant activation of NF-kB. In contrast, there has also been evidence arguing in favor of a pro-inflammatory effect of S1P in endothelial cells: TNF-α-mediated induction of adhesion molecules has been shown to depend on the generation of S1P by activated Sphk1, and S1P alone, in the absence of TNF-α, has been shown to stimulate the expression of VCAM-1 and E-selectin via the transcriptional activation of NF-kB [60, 65–67]. However, this has been challenged by a report showing that TNF-a-induced adhe-

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sion molecule expression is independent of S1P [68]. Again, in support of a proinflammatory role of S1P, chronic overexpression of Sphk1 has been shown to lead to a pro-inflammatory phenotype in cultured endothelial cells that is represented by higher constitutive expression of VCAM-1, an augmented VCAM-1 and E-selectin response to TNF-a, and enhanced neutrophil binding [69]. In addition, Sphk1 has been implicated in mediating the induction of cyclooxygenase (COX)-2 by TNF-a and the subsequent production of inflammatory prostaglandins such as PGE2 [70]. In agreement, knockdown of the S1P-degrading enzymes S1P phosphatase and S1P lyase have been shown to augment prostaglandin production and raise S1P levels in parallel [70], suggesting that S1P is mediating the COX-2-dependent pro-inflammatory effects of cytokines. Currently, several explanations have been put forward to reconcile these opposing views. The first postulates that the pro-inflammatory role of S1P is due to the action of an intracellular S1P pool that is generated by Sphk1 following TNF-α stimulation, although its molecular targets remain unknown [766]. The second explanation suggests that S1P may be simultaneously exerting two opposing effects on adhesion molecule expression through its different receptors. Accordingly, S1P1 acts as the mediator of the inhibitory effect of S1P on TNF-α-induced adhesion molecules, while S1P3 mediates its stimulatory effect on adhesion molecule expression [71]. In this scenario, G12/13 proteins have been suggested to provide the molecular bias towards a pro-adhesive role of S1P because only S1P3, and not S1P1, activates them [71]. This view is supported by data showing that, in S1P3-deficient mice rendered diabetic by streptozotocin, S1P and S1P1 agonists were still effective in inhibiting monocytic cell adhesion [64], and that TNF-a-stimulated monocyte adhesion was similarly inhibited by S1P in S1P3-deficient and wild-type endothelial cells [64]. In contrast, in an experimental model of myocardial ischemia/reperfusion in mice in vivo (in which recruitment of neutrophils caused reperfusion damage), it was the S1P3 receptor that mediated the inhibitory effect of S1P on the recruitment of neutrophils in a NO-dependent manner [72]. Therefore, differences in endothelial and inflammatory cell origin, S1P receptor occupancy and stimuli that regulate S1P receptor expression (e.g., hypoxia versus cytokines) may account for such discrepancies. Finally, one hallmark of S1P receptor signaling that may also reconcile different experimental findings is its concentration dependency. Super-physiological levels of S1P often inhibit the very same processes that lower concentrations activate [16]. Indeed, studies that have reported that S1P induces endothelial VCAM-1 and E-selectin expression [65, 66] have used rather high concentrations of S1P (5–20 mmol/L), while studies describing VCAM-1 inhibition have used nanomolar concentrations of S1P [64]. Remarkably, when the same authors [64] looked at S1P concentrations greater than 5  mmol/L, they also noticed increased monocyte adhesion [64]. At the same time, the effects of S1P on inflammatory cell adhesion in vivo under physiological conditions have been quite straightforward throughout

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all studies:­ S1P has been shown to inhibit TNF-α-mediated monocytic cell adhesion in large vessels after TNF-α stimulation [62, 64], and to attenuate neutrophil recruitment to the coronary microcirculation during the post-ischemic inflammation caused by myocardial reperfusion injury [72].

S1P as a dynamic modulator of inflammation Increased levels of S1P have been shown to exist at inflammation sites, and are generated there through local activation of Sphk1 by inflammatory cytokines such as TNF-α and IL-1b, as well as LPS and thrombin [73, 74]. Such high S1P concentrations at the site of inflammation may boost local inflammatory responses by augmenting PGE2, inducing adhesion molecules, recruiting inflammatory cells (S1P is pro-migratory for neutrophils [75, 76] and monocyte/macrophages [77]), retaining T cells at sites of inflammation [78] and promoting activation of dendritic cells in the lymphatics [28]. At the same time, S1P may be acting through a negative feedback mechanism to limit the local inflammatory response by decreasing the abnormal permeability inherent to inflamed endothelium, by enhancing the endothelial barrier function, and by inhibiting leukocyte adhesion. Therefore, locally produced S1P appears to be an important determinant of the build-up, magnitude, and duration of the inflammatory response, but may also contribute to its restraint and resolution. This is nicely exemplified by models of inflammatory lung injury. There, the initial increase in vascular permeability induced by LPS or PAR-1 activation is gradually counteracted and ultimately reversed by a concomitant activation of Sphk1, which seals the endothelial cell barrier through S1P production and S1P1 activation [74, 79]. Vice versa, the barrier-enhancing functions of agents protective against lung injury such as activated protein C (APC), a serine protease critically involved in the regulation of coagulation and inflammatory processes, can be partially attributed to their activation of Sphk1, production of S1P and engagement of S1P1 [80]. The APC receptor, EPCR, has even been described to transactivate S1P1 directly [81]. Such protective, endothelium-sealing effects of S1P in the course of inflammation are not restricted to the local vascular beds of particular organs but can be observed in the general circulation under conditions of generalized inflammation: administration of S1P1 agonists have been shown to protect mice devoid of plasma S1P from the generalized vascular leak and death induced by PAF [54].

Interplay of S1P with HDL as its major carrier, buffer and distributor An important issue in all biological effects exerted by S1P is the regulation of its bioavailability. Total S1P levels in plasma are 20–100-fold higher than the Kd value 126

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of its receptors [11, 13]. However, the biologically active fraction is only 1–2% [11], suggesting that a large portion of the total plasma S1P is either buffered or held in a tightly “packaged” compartment, or both. These roles can be easily assumed by HDL as the major carriers of S1P in plasma. In fact, the capacity of HDL to take up S1P from exogenous sources is enormous (up to 10-fold more than its original content per milligram protein; unpublished observations), so that they could easily remove excess S1P produced at sites of inflammation, buffer it and even carry it away, thus helping in the resolution of inflammation. Indeed, HDL and other lipoproteins are present in the interstitial space in amounts that correspond to ~25% of their plasma concentration (in the case of HDL) [44, 45], circulate with the lymph fluid [43], and increase several-fold in inflammatory exudates [46]. On the other hand, HDL-bound S1P itself has been shown to be biologically active because several effects of HDL can be partially or entirely attributed to their S1P content; these are NO-dependent vasodilation, angiogenesis, and several aspects of the their anti-oxidative, anti-apoptotic and anti-inflammatory actions [14]. One plausible explanation may be that the amount of S1P carried by HDL is packaged in such a way that only part of it is accessible to S1P receptors, allowing HDL to act both as a “sink” and a “master” of S1P presentation. This presentation may, in fact, be highly selective, as HDL need to bind to their own receptor, Srb1, before their S1P content is able to engage its receptors [63, 82–84]. In this way, a confined activation of S1P receptors by the S1P cargo of HDL is not only dependent on their distribution and expression, but also on that of their HDL co-receptors, and, of course, on the amount of S1P carried within the HDL particle, its presentation, and the HDL levels in plasma. Clearly, this hypothesis has yet to be proven by experimental S1P distribution and functional studies under normal and pathological conditions. One such proof-of-principle study has observed that loading of HDL with exogenous S1P increased their ability to inhibit oxidized LDL-induced apoptosis in endothelial cells [12].

S1P agonists and antagonists as possible therapeutics in endothelial inflammation Most of the experimental and clinical experience with S1P analogs has come from studies with FTY720, an immunosuppressant that engages four out of the five S1P receptors (with S1P2 as the only exception). FTY720 acts both as agonist and functional antagonist of S1P1 (through initial receptor activation followed by its internalization and proteasomal degradation). FTY720 has been considered a potential candidate for immunosuppressive therapy in the prevention of renal allograft rejection, and has completed Phase III clinical studies. However, it did not prove superior to standard treatments and exhibited more adverse effects. Much in contrast, FTY720 has shown extremely promising results in the proof-of-concept

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trials for treatment of multiple sclerosis [85], although the underlying beneficial mechanisms have not yet been identified. Nevertheless, FTY720 has clear effects on the endothelium that may be highly beneficial in an inflammatory scenario: it confers protection against ischemia/reperfusion injury [86, 87], decreases endothelial leakage [58], inhibits ROS generation by the NAD(P)H oxidase and production of chemokines (MCP-1) [63], promotes NO-dependent vasodilation in the endothelium [88], and attenuates the development of atherosclerosis [89, 90]. Unfortunately, its major drawback (together with that of more selective S1P1 agonists) for potential endothelium-targeted therapies remains its immunosuppressive effect mediated exclusively by S1P1. Another way of targeting the S1P-S1P receptor axis for therapeutic purposes is modulation of circulating S1P levels. While drugs inhibiting the degradation of S1P are also immunosuppressive [91], they may add to the repertoire of S1P-targeted strategies, especially as a careful evaluation of risk and benefit of S1P-based therapies must be assessed on an individual basis. Intriguingly, circulating amounts of S1P are highly associated with histamine levels and the anaphylactic response following passive systemic challenge, while the resistance to anaphylaxis in Sphk1-deficient mice has been attributed to their lower plasma S1P level [92]. This also suggests that circulating S1P may be a factor that elicits an overly extensive degranulation of mast cells that is thought to cause the anaphylactic response [92]. Thus, the Sphkspecific inhibitors that are currently under development may be a further way of modulating the S1P-S1P receptor system by altering the systemic S1P levels. As S1P has also clear effects on vascular tone and tissue perfusion, including that of the kidney [93], a further level of complexity but also one of therapeutic opportunity is revealed when aiming at complex disease syndromes, in which micro- and macrocirculation are simultaneously affected. There, agonists and antagonists of S1P2 and S1P3 (the two S1P receptors that regulate vascular tone) may come into play together with S1P1-targeted strategies, allowing their fine-tuning and compensation of side effects. In summary, combining the use of S1P agonists and antagonists for individual S1P receptors may be a promising concept to custom-tailor therapies that modulate the S1P-S1P receptor axis to treat different aspects of endothelial dysfunction in inflammation.

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Dissecting TNF-TNFR1/TNFR2 signaling pathways in vasculature Wang Min1,2, Ting Wan 1 and Yan Luo 2 1

Interdepartmental Program in Vascular Biology and Therapeutics, Yale University School of Medicine, 10 Amistad St, New Haven, CT 06520, USA 2  State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou, China

Abstract Inflammation is a process essential for host defense and tissue repair. However, excessive inflammation (excessive and/or prolonged production and effects of pro-inflammatory cytokines) may cause pathogenic changes leading to vascular diseases such as rheumatoid arthritis and atherosclerosis. Since inflammatory mediators primarily target vascular endothelial cells (EC), one therapeutic approach to control inflammation is to block specific cytokine signaling in EC. This strategy works in some disease settings but has failed in others. Therefore, anti-TNF therapy targeting both TNFR1 and TNFR2 has been successful in treating rheumatoid arthritis but has failed in the treatment of the cardiovascular diseases. This is because TNF utilizes TNFR1 and TNFR2 to induce different signaling pathways and different EC functions. TNFR1 activates multiple signaling pathways including NF-kB, ASK1-JNK and apoptosis, which can be also activated by other inflammatory stimuli such as endoplasmic reticulum stress. These pathways have been linked to inflammation and EC dysfunction. While NF-kB activates cell survival, ASK1-JNK induces apoptosis. Therefore, inhibition of ASK1-JNK without disruption of cell survival signaling may provide a new strategy for anti-inflammatory therapy. Studies suggest that activation of ASK1-JNK signaling is mediated by a unique AIP1 complex, which is structurally and functionally distinct from the NF-kB-activating complex I. Moreover, AIP1 inhibits the formation of the NF-kB-activating complex I while facilitating the formation of the ASK1-JNK-activating signalosome. Therefore, inhibition of the AIP1-ASK1-JNK pathway may provide a potential anti-inflammatory strategy. In contrast to TNFR1 signaling, much less is known for TNFR2-mediated signaling events. Our data suggest that TNFR2 signaling plays a beneficial role in the cardiovascular system, in part, by activating Bmx and TRAF2-dependent angiogenic and survival pathways. Therefore, specific inhibition of TNFR1 pathways without disruption of TNFR2 signaling may provide new therapeutic opportunities for the treatment of cardiovascular diseases.

Introduction The cell type that normally limits the inflammatory process is the vascular endothelial cell (EC) that lines blood microvessels. Therefore, inflammation can be analyzed as a response to changes in the EC. Under normal conditions, the endothelium Endothelial Dysfunction and Inflammation, edited by Shauna M. Dauphinee and Aly Karsan © 2010 Springer Basel

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maintains a vasodilator, antithrombotic and anti-inflammatory state [1]. Acute inflammation results from EC activation in response to ligands of heterotrimeric G protein-coupled receptors such as histamine, or inflammatory cytokines such as tumor necrosis factor-a (TNF) or interleukin-1 (IL‑1). Time-dependent changes in molecules expressed by cytokine-activated endothelial cells underlie the change in inflammation from neutrophil-dominated to mononuclear cell-dominated responses or T  cell-dominated adaptive immunity. Chronic inflammation involves distinct endothelial cell responses such as neovascularization (angiogenesis) and formation of an inflammatory neo-tissue (e.g., the pannus in a rheumatoid joint and plaque in atherosclerosis) [2]. Therefore, inflammation is a process essential for host defense and tissue repair. However, exuberant defense may cause pathogenic changes leading to vascular diseases such as rheumatoid arthritis and atherosclerosis. EC dysfunction, as determined by vasomotor dysfunction, may occur well before the structural manifestation of overt vascular disease, such as atherosclerosis, and thus represents the first step and an independent predictor of potential cardiovascular events. One approach to control harmful inflammation has been to block specific cytokine, e.g., TNF or IL-1. This strategy works in some disease settings but has failed in others. This is because multiple cytokines are involved in the process of inflammation with each cytokine activating multiple pathways. For example, TNF utilizes two receptors (TNFR1 and TNFR2) to induce different signaling pathways and different EC functions. TNFR1-mediated signal events use both NF-kB and c-Jun N-terminal kinase (JNK) to promote inflammation; while NF-kB activates cell survival, JNK induces apoptosis. TNFR2 is proposed to attenuate or enhance TNFR1-dependent signaling. However, data from our and other labs indicate that TNFR2 induces additional signaling that plays a beneficial role in the cardiovascular system [3–6]. Therefore, dissecting signaling pathways by cytokines may provide therapeutic strategies for the treatment of inflammatory diseases. In this chapter, we focus on TNF signaling to elucidate the following aspects: how a single cytokine triggers multiple signaling pathways, inducing multiple effects on EC; how endogenous inhibitors regulate TNF signaling and whether these endogenous inhibitors could provide novel therapeutic tools to treat inflammatory diseases; how the second receptor TNFR2 mediates a protective effect on EC, and whether specific inhibition of TNFR1 or activation of TNFR2 signaling in EC may be a novel target for the treatment of vascular diseases.

TNFR1 signaling and inflammation Components in TNFR1 signaling Vascular EC are among the principal physiological targets of prototypic inflammatory cytokine TNF [7–9]. In EC, as in other cell types, TNF elicits a broad spectrum

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of biological effects including proliferation, differentiation and apoptosis [10–12]. Specifically, TNF induces activation of EC, leading to increased vascular leakage of plasma proteins, and increased leukocyte recruitment at the site of inflammation. In combination with other cytokines such as interferon-g (IFN-g), TNF may induce EC injury or cell death. In chronic inflammatory settings, TNF may induce angiogenesis. The role of TNF in regulation of EC inflammation and the TNFR1-mediated apoptotic signaling pathways have been reviewed in depth elsewhere [7, 13]. The best-characterized TNF signaling is initiated by TNFR1 [14], and mediated by several different signaling proteins interacting with TNFR1 [15–17]. Cytokines of TNF superfamily utilize receptors that are devoid of intrinsic catalytic activity [10, 18]. The intracellular part of TNFR1 can be subdivided into a membrane proximal and a membrane distal part. The latter contains a “death domain”, which can also be found in other receptors involved in cell death (e.g., Fas), and is critical for TNF-induced cell death, activation of NF-κB and the MAPK cascades [18]. A current model postulates that TNF binding triggers trimerization of TNFR1, which recruits adaptor proteins and signaling molecules through their intracellular domains to form a receptor-signaling complex [19, 20]. Alternatively, the ligand causes a conformational change in pre-associated receptors. Many proteins have been shown to be recruited by TNFR1 into a signaling transducing complex. The first to be recruited is the TNF receptor-associated death domain protein (TRADD) [21, 22]. TRADD functions as a platform adaptor that recruits TNF receptorassociated factor 2 (TRAF2), RIP, and Fas-associated death-domain protein (FADD) to form the TNFR1 signaling complex and activate several distinct signaling cascades [23–25]. TRAF2 and receptor-interacting protein kinase 1 (RIPK1) act as an assembly platform for the recruitment of the members of MAP kinase kinase kinase (MAP3K) family, leading to activation of the JNK pathway. TRAF2 and RIPK1 also recruits IkB-kinase (IKK) complex leading to activation of the NF-kB pathway [20, 26] (Fig. 1). Specifically, IKK is composed of two catalytic subunits IKKa and IKKb and an essential regulatory subunit NEMO (NF-kB essential molecule; also known as IKKg). IKK phosphorylates the NF-kB inhibitor IkBa and targets this inhibitor for degradation by the classic K48-conjugated ubiquitination-proteasome pathway [27]. The structure and function of TRAF2 and RIPK1 have been extensively investigated. TRAF2 contains an N-terminal RING domain commonly found in ubiquitin ligase (E3). Indeed, recent studies have shown that TRAF2 catalyzes the synthesis of non-classic K63-linked polyubiquitination chains. Several targets of polyubiquitination in the TNF-induced NF-kB pathway have been identified, and these include RIPK1, NEMO and TRAF2 itself [28]. Polyubiquitination of these proteins has been proposed to mediate protein-protein interactions between signaling molecules including RIPK1 and NEMO. RIPK1 contains an N-terminal kinase domain, an intermediate domain and a C-terminal death domain. The intermediate domain, but not the kinase domain of RIPK1, mediates IKK-NF-kB activation [29]. Recently,

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Figure 1. A model for the disassembly of complex I and formation of the AIP1 complex: (1) a rapid (2–5 min) formation of a membrane-bound complex I, which is specific for activation of the IKK-NF-kB pathway; (2) endocytosis of TNFR1 concomitant with a dissociation of complex I (and release of TRADD-TRAF2-RIPK1 from TNFR1) at 5–15 min; and (3) formation of AIP1 complex (TRADD-TRAF2-RIPK1-AIP1 devoid of complex I-component TNFR1), which specifically activates ASK1-JNK but inhibits IKK-NF-kB signaling.

it has been shown that TNF induces polyubiquitination of RIP1 at K377 located within the intermediate domain, and that this polyubiquitination is required for the binding of NEMO and the recruitment of the IKK complex to RIPK1/TRAF2, leading to activation of the IKK complex [30]. In contrast, the role of RIPK1 in JNK activation has not been exclusively established [29, 31]. Our recent work reveals that the kinase activity of RIPK1 is critical for TNF-induced JNK activation by phosphorylating JNK upstream activator AIP1 [apoptosis signal-regulating kinase 1 (ASK1)-interacting protein-1 (also named DAB2IP)] [32].

Divergence of TNF-TNFR1-mediated NF-kB and JNK signaling pathways An important question in TNF signaling field is how TNF-induced NF-kB and JNK pathways are differentially regulated. Tschopp’s group has dissected TNFR1 signaling complexes and has proposed a sequential signaling complex model: the initial plasma membrane-bound complex (complex I, comprising of TNFR1, TRADD, RIPK1 and TRAF2) for NF-kB activation, and a cytoplasmic complex (complex II) in which the

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internalized TRADD, TRAF2 and RIPK1 recruits FADD and pro-caspase-8 for apoptotic signaling [33]. Complex II formation is detected at 2–8 h after treatment with TNF in cells overexpressing the NF-kB inhibitor IkBa. NF-kB-induced protein synthesis of FLIP normally prevents the recruitment of pro-caspase-8/10 to FADD, and, therefore, the formation of complex II. A recent study from Schutze’s lab obtained a similar pattern of complex I formation, but different results on the formation of the apoptotic complex [34]. However, it has not been determined whether complex I or complex II is active in the initiation of JNK signaling. Since TRAF2 is critical for JNK activation, it seemed reasonable to assume that complex I, which contains TRAF2, would recruit one or more MAP3Ks to induce JNK activation [33, 35]. However, our recent data support the existence of a unique complex, which lacks TNFR1, as being responsible for TRAF2-mediated ASK1-JNK activation. First, AIP1 forms a preexisting complex with TNFR1 in EC. However, in response to TNF, AIP1 is dissociated from TNFR1 and in turn forms a complex with TRADD, TRAF2, RIPK1 and ASK1 (the so-called AIP1 complex) (Fig. 1). Thus, the AIP1 complex is structurally different from complex I. Second, the AIP1 complex is formed after 15  min in response to TNF and is dissociated by 60  min. We could not detect an association of AIP1 with FADD and pro-caspase-8 in response to TNF in EC. These data suggest that the AIP1 complex is also structurally different from complex II or the death-inducing signaling complex (DISC). Most significantly, AIP1 overexpression enhances, whereas knockdown of AIP1 inhibits, TNF-induced ASK1-JNK activation. However, AIP1 has opposite effects on the TNF-induced IKK-NF-kB pathway. These data suggest that the AIP1 complex is functionally different from the complex I. In some cell types, including EC, ASK1-JNK activation induces apoptotic signaling, which is dependent on intrinsic (mitochondria-dependent) but not extrinsic (caspase-8-dependent) pathways. We have recently shown that AIP1 enhances ASK1-mediated JNK activation and EC apoptosis [32, 36, 37]. Thus, the AIP complex is functionally different from complex II, as a complex mediating an extrinsic apoptosis pathway.

Endocytosis and TNF-R1 signaling Another relevant question is how TNFR1 is induced by TNF to undergo endocytosis and the effects of TNFR1 internalization on NF-kB and JNK signaling. Previous results suggest that clathrin-mediated endocytosis plays a role in TNFR1 internalization and this internalization is required for termination of IKK-NF-kB signaling [34, 38]. A TRID domain comprising a YxxW internalization motif in the cytoplasmic tail of TNFR1 has been identified and implicated in mediating endocytosis of TNFR1, presumably through binding to the clathrin adaptor protein-2 (AP2). AP2 is a classical major adaptor protein in clathrin-mediated endocytosis. It binds to the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) enriched at the plasma membrane and recruits clathrin from cytosol to form a clathrin-coated pit. AP2 also

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functions as a prominent adaptor protein to recruit endocytic cargo proteins to the clathrin-coated pits through recognizing endocytic motifs including YxxY (hydrophobic) and di-leucine motifs that are commonly present in a number of receptors, such as transferrin receptor, low-density lipoprotein receptor, epithelial cell growth factor receptor and TNFR1 [38, 39]. However, the detailed mechanism by which clathrin/AP2 mediates TNFR endocytosis has not been determined. Many questions remain to be addressed, e.g., does TNFR1 directly bind to endocytic adaptor protein AP2 via its YxxW motif (the TNFR1 internalization domain, TRID); how is TNFR1 recruited to endocytic machinery in response to TNF; and do other proteins also participate in this process? Addressing these questions may help to elucidate how TNF-activated NF-kB and JNK signaling pathways diverge (Fig. 1). Given the distinct function of the NF-kB and JNK signaling in EC, it is conceivable that inhibition of the JNK inflammatory and apoptotic pathway without disruption of cell survival signaling (NF-kB) may provide a new strategy for anti-inflammatory therapy. Therefore, identifying novel mediators/processes specifically involved in TNFR1-induced JNK signaling will provide potential targets for this strategy.

Mechanism for ASK1 activation ASK1 activation by TNF Apoptosis signal-regulating kianse-1 (ASK1) is an MAP3K that is critical for TNFinduced JNK signaling [40, 41]. Studies on the EC suggest that ASK1 is activated not only by TNF, but also by other pro-inflammatory stimuli, such as IL-1 and lipopolysaccharide (LPS), as well as by oxidative and endoplasmic reticulum (ER) stress [40, 41]. The mechanism for ASK1 activation is not fully understood. The identification of proteins associated with ASK1 has provided some insights. ASK1 is a 170-kDa protein that functionally is composed of an inhibitory N-terminal domain, an internal kinase domain, and a C-terminal regulatory domain [42]. ASK1 activity appears to be enhanced by its binding to a number of important regulatory proteins such as TRAFs, AIP1, Daxx and JSAP/JIP3 [37, 43–46]. On the other hand, several cellular factors, including thioredoxin, glutaredoxin, Hsp72, Hsp90, Raf-1 and 14-3-3, have been reported to interact with different ASK1 domains and inhibit ASK1 activity [44, 47–51]. Consistently, a recent report suggests that ASK1 constitutively forms a high molecular mass complex (ASK1 signalosome), and that H2O2 induces alterations of ASK1 signalosome components [52]. However, the underlying mechanism for regulating the ASK1 signalosome is not understood. Associations of ASK1 with 14-3-3 have been extensively investigated in our and other labs. 14-3-3 is a phosphoserinebinding protein, and binds to pSer967 located at the C-terminal domain of ASK1. ASK1 is phosphorylated at Ser967 under basal conditions, and TNF/reactive oxygen species (ROS) induce dephosphorylation of pSer967, leading to a release of 14-3-3.

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We have recently shown that AIP1, a novel Ras-GTPase-activating protein (GAP) protein that forms a complex with TRAF2-ASK1 and recruits a phosphatase PP2A, facilitates the dissociation of 14-3-3 from ASK1 [36, 37, 53–55]. Thus, release of inhibitors from ASK1 is a critical event in ASK1-JNK signaling (Fig. 2).

Figure 2. ASK1 plays a central role in stress-induced EC dysfunction. In resting EC, the intracellular protein thioredoxin in a reduced form and the phosphoserine-binding protein 14-3-3 associate with ASK1, maintaining ASK1 in an inactive state. Stress signals, such as TNF and ER stress, induce ASK1 activation by dissociating ASK1 from its inhibitors thioredoxin and 14-3-3. Several proteins are involved in the dissociation of ASK1-Trx/14-3-3 complexes in a stress-specific manner. TRAF2-RIPK1-AIP1 and IRE1-AIP1, form a complex with ASK1 in response to TNF and ER stress, respectively, to dissociate Trx and 14-3-3 from ASK1, leading to activation of ASK1-JNK signaling. This pathway may also activate p38 mitogen-activated protein kinase (p38 MAPK).

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ASK1 activation by ER stress Recent studies suggest that ASK1-JNK signaling pathway is also activated by ER stress. Accumulation of misfolded protein in the ER (called ER stress) under pathological conditions triggers an adaptive stress response, the unfolded protein response (UPR) [56, 57]. It has been recently recognized that many atherosclerotic risk factors such hyperlipidemia (causing lipid loading), hyperglycemia (high glucose), oxidative stress and inflammation induce ER stress [56, 57]. For experimental purposes, chemicals such as thapsigargin (depleting Ca2+ from ER), tunicamycin (inhibiting protein N-linked glycosylation) and dithiothreitol (DTT, disrupting protein disulfide bond in ER) are used to induce ER stress in cultured cells or animals. There are three distinct signaling pathways that are triggered in response to ER stress, mediated by PRK-like ER kinase (PERK), activating transcription factor-6 (ATF6) and inositol-requiring enzyme (IRE) 1 [56, 57]. PERK is an ER-resident serine/threonine protein kinase that phosphorylates eIF2a (the subunit of translational initiation factor 2), attenuating protein synthesis to prevent protein loading into the ER. ATF6 is cleaved to release a cytosolic fragment that translocates to the nucleus to induce gene expression of chaperones for protein folding. IRE1 (a transmembrane kinase and endonuclease), through its endoribonuclease activity, cleaves XBP-1 mRNA and converts it into potent transcriptional activator, which in turn induces gene expression of proteins involved in protein degradation [58, 59]. David Ron’s lab first demonstrated that IRE1, through its kinase activity, couples ER stress to activation of JNK [60]. Subsequently, studies from several labs suggested that TNFR1 signaling components including TNFR1, RIP1, TRAF2 and ASK1 are involved in the activation of JNK, as ER stress-induced JNK activation is reduced or diminished in MEFs with deficiency of each individual component [41, 43, 61–63]. Consistent the role of AIP1 in TNF signaling, our recent results suggest that ER stress-induced IRE1 activation and its downstream ASK1-JNK signaling are blunted in AIP1-knockout (KO) EC. Moreover, AIP1 associates with IRE1 in response to ER stress [64] (Fig. 2).

ASK1 activation by oxidative stress ASK1 is also activated by oxidative stress. ROS activation of ASK1 involves several parallel actions. First, ROS may decrease the quantity of reduced glutathione or of reduced thioredoxin in the cell; these proteins normally bind to ASK1 and keep it in an inactive state [65, 66]. Second, ROS may induce the release of ASK1 from its cellular inhibitors such as protein 14-3-3, which in unactivated ECs binds to pSer967 on ASK1 [49]; phosphoprotein 2A may be the ROS-activated enzyme that is responsible for ASK1 dephosphorylation at pSer967 in ECs [55]. Third, ROS may activate protein kinase D (PKD). H2O2 induces phosphorylation of PKD, translocation of PKD from the EC plasma membrane to the cytoplasm, and association of PKD with

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ASK1, which facilitates ASK1 oligomerization and autophosphorylation at Thr845, leading to ASK1 activation. At the same time, H2O2 induces 14-3-3-binding (likely via phosphorylation of PKD at Ser205/208 and Ser219/223), which is critical for PKD-mediated ASK1-JNK signaling. Inhibition of PKD by pharmacological inhibitors or siRNA blocks H2O2-induced ASK1-JNK activation and EC apoptosis [13, 54].

Inhibiting ASK1-JNK signaling is an anti-inflammatory strategy Anti-inflammation strategies Both pharmacological and biological agents have been used for the treatment of inflammatory diseases. Biological agents that inhibit the pro-inflammatory activities include cytokine receptor antagonists, anti-cytokine monoclonal antibodies, and fusion molecules consisting of soluble cytokine receptors combined with human fusion protein constructs or polyethylene glycol [67]. The best-known example is Etanercept, which is a soluble TNF receptor decoy that binds circulating TNF and renders it inactive. In addition, chimeric monoclonal antibodies to TNF include infliximab, adalimumab, certolizumab pegol. Another anti-inflammatory approach is to block the cytokine-induced signaling pathways. For example, endotheliumrestricted inhibition of NF-κB activation, achieved by expression of IκBα, an endogenous NF-kB inhibitor specifically in ECs, results in reduced atherosclerotic plaque formation in ApoE−/− mice fed with a cholesterol-rich diet. Also, inhibition of NF-κB abrogates adhesion molecule induction in ECs, and reduces macrophage recruitment to atherosclerotic plaques and expression of cytokines and chemokines in the aorta [68]. As stated earlier, complete inhibition of NF-kB will cause EC apoptosis. One approach is to retain EC survival while inhibiting NF-kB signaling. The importance of NF-kB-induced protective proteins is demonstrated by the potentiation of TNF-mediated EC death when TNF is provided in combination with a global inhibitor of new gene transcription (such as actinomycin D), an inhibitor of protein synthesis (such as cycloheximide CHX or Shiga-like toxin), or a selective inhibitor of NF-kB activation (such as pyrrolidine dithiocarbamate) or overexpression of a nonphosphorylatable mutant of IkB. TNF-mediated EC death often involves a DISC that forms about 6  h after TNF binding to TNFR1 [33]. Before the DISC is assembled, a different signaling complex composed of TRADD, RIP1, and TRAF2 functions to activate IKK and to generate functional NF-kB. In human EC, the predominant form of NF-kB activated by TNF is a heterodimer of NF-kB1 (also known as p50) and of phosphorylated Rel A (also known as phospho-p65). Activated NF-kB induces transcription of specific gene products that inhibit cell death by both the caspase and the JNK pathways. A key example is the induction of cFLIP, which competes with pro-caspase 8 for binding to FADD and prevents the

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autocatalytic activation of caspases 8 (and 10) by the TRADD/RIP1/TRAF2/FADD complex [69]. Therefore, expression of cFLIP while inhibiting NF-kB may be a better anti-inflammatory approach.

Linkage of ASK1-JNK to inflammation Almost all inflammatory stimuli such as TNF, IL-1 and ROS activate ASK1. Interestingly, anti-inflammatory factors such as laminar shear stress and anti-oxidants inhibit ASK1. These in vitro studies on EC have demonstrated that ASK1 is an effector of inflammation in EC [36, 37, 50, 53, 54, 66, 70–72]. Studies using ASK1-deficient mice have also linked ASK1 to cardiovascular pathogenesis. ASK1 deletion in mice attenuated angiogiotensin II-induced cardiac hypertrophy and remodeling. Neointimal formation due to proliferation of smooth muscle cells in a cuff injury model is also attenuated by ASK1 deletion in mice [73, 74]. Similarly, the linkage of the ASK1-downstream target JNK to inflammation is also very strong. First, JNK is activated by almost all inflammatory mediators such as TNF, IL-1, LPS and oxidative stress [71, 75, 76]. Second, JNK activation is essential for expression of many pro-inflammatory molecules such as E-selectin, RANTES, IL-12, IL-6 and IL-8 by activating transcription factor c-Jun and ATF-2 [77–79]. This has been demonstrated by both dominant negative and antisense approaches. For example, overexpression of dnJNK or antisense RNA to JNK can block IL-1induced IL-6 and IL-8 expression; overexpression of dnJNK can block EC surface expression in human EC (HUVEC). Third, JNK antagonizes anti-inflammatory molecules. It has been shown that JNK phosphorylates glucocorticoid receptor, a hormone-dependent transcription factor that suppresses inflammation [80, 81]. Fourth, blockage of the JNK pathway by either genetically knocking out JNK or specific JNK inhibitors can prevent inflammatory responses and atherosclerosis progression in mouse models [82–84]. Conversely, deletion of JNK negative regulator MAPK phosphatase-1 (MKP-1) enhances inflammatory responses and progression of atherosclerosis in several models [85]. Moreover, known antiinflammatory drugs such as glucocorticoids have recently been shown to target JNK, in part, by inducing gene expression of MKP-1 [86–88]. Thus, ASK1-JNK signaling may provide a novel target for prevention of vascular disease and atherosclerosis.

Inhibiting ASK1-JNK signaling may be a better anti-inflammatory strategy Here we discuss more specific endogenous inhibitors involved in ASK1-JNK signaling, and application of these proteins may provide better strategies to treat inflammatory diseases. ASK1 is retained in an inactive state by association with multiple

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cellular factors. The best-characterized inhibitors are redox protein thioredoxin (Trx) and phosphoserine-binding protein 14-3-3 [44, 47, 49–51]. It is worth noting that ASK1 can be detected in both cytosol and mitochondria, where ASK1 is bound and inhibited by Trx1 and Trx2, respectively [51, 66]. In addition to Trx and 14-3-3, other cellular factors including glutaredoxin, Hsp72 and glutathione S-transferase have also found to negatively regulate ASK1-JNK signaling [48, 89, 90]. We have shown that the athero-protective factor laminar flow inhibits TNF signaling by preventing release of thioredoxin and 14-3-3, highlighting the significance of physiological blood flow [50]. The Hsp90-Akt complex (in which Hsp90 may hold Akt and ASK1 in a close proximity) has been shown to phosphorylate endothelial nitric oxide synthase (eNOS), leading to increased eNOS activity and EC function [91, 92]. Interestingly, the Hsp90-Akt complex also phosphorylates ASK1 at Ser83. In contrast to the effect of eNOS phosphorylation, phosphorylation of ASK1 by Hsp9-Akt leads to an inhibition of ASK1 activity [93]. Suppressor of cytokine signaling (SOCS) proteins have also been shown to inhibit ASK1-JNK activities. Eight SOCS family members (CIS, SOCS1–7) have been identified and are defined by a characteristic structure composed of a highly variable N-terminal region, a central SH2 domain, and a highly conserved 40–50-amino acid motif (called SOCS-box) at the C  terminus [94, 95]. The SOCS family of proteins can inhibit signaling activated by a variety of cytokines. In resting or IFN-g-treated EC, ASK1 is phosphorylated at Y718, and SOCS1 binds to pY718 to induce ASK1 degradation [96].

Beneficial side of cytokine signaling: Lessons from TNFR2 studies Role of TNF-R2 in inflammation TNF is a major mediator of inflammation and inflammatory diseases such as rheumatoid arthritis (RA), Crohn’s disease, inflammatory bowel disease (IBD) and multiple sclerosis. TNF has also been implicated in several cardiovascular diseases. In humans, there is a direct correlation between cardiac functional capacity, survival and circulating levels of TNF [97]. While pharmacological inhibition of TNF has yielded encouraging results in RA and IBD, the use of anti-TNF has not proven efficacious in patients with chronic heart failure despite the evidence supporting TNF as a causative agent in animal models [97]. The exact reason for this is unclear. Recent data support the idea that TNFR1 mediates the pro-apoptotic function of TNF, whereas TNFR2 can enhance TNFR1-induced cell death or promote cell activation, migration, growth or proliferation [98–106]. Therefore, inhibition of TNF may eliminate the TNFR2-mediated protective effects. The relative contribution of TNFR2 in TNF function and signaling has been underestimated for several reasons. First, most studies have used soluble TNF as an

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agonist, and soluble TNF acts predominantly on TNFR1 and is a poor activator of TNFR2. In vivo, membrane-bound TNF is a better ligand for TNFR2 and binds to TNFR through cell-cell contact. Second, TNFR2 expression is limited to specific cell types including EC, cardiac myocytes and hematopoietic cells. Moreover, TNFR2 is often inducible in response to certain stimuli. For example, stimulation of macrophages with LPS, or T lymphocytes with phytohemagglutinin, results in a marked increase in TNFR2 levels. TNFR2 is also induced by TNF in several cell lines [107]. As we recently demonstrated, TNFR2 is highly induced in vascular EC in hindlimb ischemia [5]. In recent years, the development of TNF muteins with preferential binding to either TNFR1 (R1-TNF-, e.g., R32W) or TNFR2 (R2-TNF, e.g., D143F), as well as the development of TNFR-specific antibodies that function as agonists or antagonists, has circumvented the problems associated with the use of soluble TNF [108, 109]. Furthermore, the availability of TNFR1- and TNFR2-deficient mice, and cells isolated from these mice, has provided the field with critical molecular tools for examining the functions of TNFR2 in vitro and in vivo. For example, it has been shown that TNF mutein R2-TNF up-regulates TNFR2, suggesting a feedback loop. Conversely, engagement of TNFR1 using R1-TNF down-regulates expression of TNFR2 in human kidney organ culture [110]. Using TNFR2-specifc antibody (as an agonist) and TNFR2-null EC isolated from TNFR2-KO mice, we have recently identified Bmx, a non-receptor tyrosine kinase implicated in cell migration, as the first TNFR2-specific tyrosine kinase. Furthermore, we have shown that Bmx binds to the C-terminal 16-amino acid sequence of TNFR2 to mediate TNFR2-induced EC migration and angiogenesis [111]. Both TNFR1-KO and TNFR2-KO mice are viable and do not show any overt phenotypic abnormalities. Data from studies using the TNFR1-KO and TNFR2-KO mice suggest that TNFR1 is primarily responsible for TNF-mediated host defense and inflammatory responses. TNFR2-KO mice are only partially resistance to the systemic actions of TNF and LPS. However, TNFR2-KO mice exhibit reduced sensitivity to TNF-induced skin necrosis, and have decreased Langerhans cell migration [107]. The role of TNFR2 in inflammation has been examined using TNFR2transgenic mice [112]. Constitutive expression of TNFR2 in mice (comparable to disease-relevant levels of human TNFR2), promotes a severe, lethal, multi-organ inflammatory syndrome with elevated NF-kB activation. Interestingly, this process appears to be independent of the presence of TNF, lymphotoxin (another ligand binding to TNFR) or TNFR1. A transgenic line expressing a non-cleavable transmembrane form of TNF, a preferable ligand for TNFR2, using Tie-2 EC-specific promoter has also been generated (tmTNF-TG) [113]. These mice develop chronic inflammatory pathologies in the kidney and liver, characterized by perivascular infiltration of mononuclear cells into these organs. A recent study using these transgenic mice indicates that tmTNF induced continuous EC activation and angiogenesis in vitro and in vivo [114].

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Function of TNFR2 signaling in inflammatory angiogenesis The hindlimb ischemia model is a well-characterized model of inflammatory arteriogenesis and angiogenesis [115, 116]. Briefly, the proximal end of the femoral artery and the distal portion of saphenous artery are ligated. All branches between these two sites are ligated or cauterized, and an arteriectomy is performed. This leads to tissue ischemia, thereby creating a regional mismatch of oxygen supply versus demand in skeletal myocytes. The reduction in tissue perfusion correlates with activation of vascular endothelial cells and the recruitment of inflammatory cells, in particular macrophages. Activated macrophages then secrete chemokines and cytokines [e.g., vascular endothelial growth factor (VEGF), TNF] that may participate in arterializing pre-formed collaterals (termed arteriogenesis) to provide a stable conduit for blood flow to the distal limb. Contemporaneously, redistribution of blood flow and the attendant changes in shear stress may collaborate with local cytokines to arterialize immature collaterals and perhaps induce angiogenesis. Once a stable, collateral circulation has been established, the improvement in distal blood flow and shear stress triggers an increase in capillary angiogenesis, increasing capillary to fiber ratios and oxygen delivery to the dependent portions of the lower limb. Therefore, proportionally regulated arteriogenesis and angiogenesis are necessary to improve nutritive blood flow to tissue and promote functional limb salvage. Thus, the hindlimb ischemia model is a very useful tool to study the function of genes critical for inflammatory arteriogenesis/angiogenesis. Studies from our own lab demonstrate that TNFR2 signaling promotes ischemia-mediated arteriogenesis and angiogenesis. This is based on the functional analyses of mice genetically deficient in TNFR2 using a femoral artery ligation model, and mechanistic studies in mouse EC isolated from these mice [5]. Specifically, TNFR1-KO mice had enhanced, whereas TNFR2-KO had reduced, capacity in clinical recovery, limb perfusion and ischemic reserve capacity compared to the wild-type mice. Interestingly, TNFR2 protein is highly up-regulated in vascular endothelium in response to ischemia. Recently, we have generated EC-specific TNFR2 transgenic mice (TNFR2-TG) and demonstrated that EC-specific TNFR2 is sufficient to mediate ischemia-induced arteriogenesis and angiogenesis [117]. Moreover, we have identified two downstream effectors TRAF2 and Bmx in TNFR2-mediated adaptive responses. Therefore, TNFR2 proteins, TNFR2-TRAF2 complex formation and Bmx/Etk activation are highly up-regulated in vascular endothelium in vivo in response to ischemia in the hindlimb ischemia model [5, 116]. Six members of the TRAF family of proteins have been described. The JNK and NF-kB pathways can be activated by overexpression of TRAF2, 5 and 6, but not by overexpression of TRAF1, 3 and 4, suggesting that different TRAFs, in spite of their structural homology, might perform very different functions. TRAF2-deficient (TRAF2-KO) mice appear normal at birth but become progressively runted and die

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prematurely [118]. Atrophy of the thymus and spleen and depletion of B cell precursors were also observed. Thymocytes and other hematopoietic progenitors were highly sensitive to TNF-induced cell death, and serum TNF levels were elevated in these TRAF2-KO animals [118]. In TNFR1 signaling, TRAF2 is absolutely required for activation of JNK, although it may be redundant in some cell types for NF-kB activation due to presence of TRAF5. However, the role of TRAF2 in TNFR2 signaling is not clear. Data from previous reports [23, 119, 120] and our recent studies suggest that TRAF2 may be critical for TNFR2-transduced NF-kB activation and NF-kB-dependent survival [5]. Bmx is highly expressed in cells with great migratory potential including metastatic tumor cells and EC [121–123]. Bmx can be activated by various angiogenic stimuli such as integrin engagement via FAK [121], VEGF via VEGFR2 [124], and TNF via TNFR2 [111] and/or via transactivation by VEGFR2 [3]. However, the mechanism by which Bmx mediates EC migration has not been determined. Several downstream effectors of Bmx involved in cell migration have been reported. Bmx through its pleckstrin homology (PH) domain directly binds to and activates Rho A (but not Rac1 and Cdc42) [125]. Similarly, Bmx through its PH domain binds to and activates PAK1 [123], a 65-kDa serine/threonine kinase implicated in integrininduced EC migration and angiogenesis by modulating EC contraction [126]. We have shown that Bmx mediates the TNF-induced PI3K-Akt angiogenic pathway [3], which has been well documented in growth factor-stimulated cell migration [127–130]. PI3K-Akt may induce angiogenesis by multiple downstream effectors including the Rho family of small GTPase, PAK1 and eNOS [131]. Both Bmx/Etk-dependent EC migratory and TRAF2-dependent NF-kB survival pathways are critical for TNFR2-mediated angiogenesis. In contrast, activation of TNFR1 signaling caused inhibition of EC migration and EC apoptosis [5]. These studies suggest that specific inhibition of TNFR1 or activation of TNFR2 signaling in EC may be novel targets for the treatment of vascular diseases such as coronary artery and peripheral arterial diseases.

Disclosure statement The authors are not aware of any biases that might be perceived as affecting the objectivity of this review.

Acknowledgements W.M. is supported by grants from the National Institutes of Health.

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AGE-RAGE signalling in endothelial dysfunction and atherosclerosis in diabetes Anna M. D. Watson1, Aino Soro-Paavonen 2 and Karin A. Jandeleit-Dahm1 1 

Baker IDI Heart and Diabetes Institute, Melbourne, Victoria 3004, Australia Division of Nephrology, Department of Medicine, Helsinki University Central Hospital, Helsinki, Finland

2 

Abstract Advanced glycation end products (AGEs) are elevated in diabetes and contribute to the endothelial dysfunction that occurs in this condition by binding to the receptor for AGEs, RAGE. AGE-RAGE binding results in increased generation of reactive oxygen species, decreased nitric oxide bioavailability and increases in pro-sclerotic and pro-fibrotic growth factors. The development of diabetesassociated atherosclerosis in the RAGE/apoE double-knockout mouse is significantly attenuated when compared to that in the diabetic apoE single-knockout mouse. This anti-atherosclerotic effect is accompanied by reduced macrophage infiltration and decreased expression of inflammatory markers. The literature to date points to AGE-RAGE interactions being an important mediator of inflammation in diabetes, and thus the AGE-RAGE signalling pathway presents a promising target for novel therapies that aim to reduce endothelial dysfunction, a precursor of the development of diabetes-associated atherosclerosis development.

Endothelial dysfunction in diabetes and advanced glycation end products It is well documented that hyperglycaemia leads to endothelial dysfunction and accelerated formation of atherosclerosis [1–3]. These changes in endothelial function include reduced vasodilatory responses [including a reduction in nitric oxide (NO)], enhanced inflammatory responses and increases in the deposition of matrix proteins [4–6]. Endothelial dysfunction is also a well-known precursor for the development of atherosclerotic plaques, especially in the diabetic setting [7]. In this chapter we briefly outline how advanced glycation end products (AGEs) significantly contribute to endothelial dysfunction and contribute toward the development of diabetes-associated atherosclerosis. AGEs are the result of the Maillard reaction, a non-enzymatic irreversible process whereby glucose binds to proteins and lipids resulting in changes in protein structure and function (for review see [8, 9]). Accumulation of AGEs over time occurs in long-lived proteins as a natural part of the aging process, with AGE formation­in Endothelial Dysfunction and Inflammation, edited by Shauna M. Dauphinee and Aly Karsan © 2010 Springer Basel

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skin collagen, for example, resulting in excessive cross-linking between proteins and a loss of elasticity, whereas AGE formation in lens crystalline protein may decrease lens function [10–12]. The hyperglycaemic milieu that occurs in diabetes leads to an increase in the formation of AGEs, far in excess of that seen during the normal aging process [10, 13]. AGEs can affect endothelial function via receptor signalling (see below). However, in the vasculature, AGE formation can lead to the entrapment of low-density lipoprotein (LDL) and IgG and alter structure and function of intact vessels in diabetes [14], as well as impairing the turnover of LDL [15] and proteins [16]. AGE formation also enhances the production of extracellular matrix proteins and stiffening of the aorta [17], as well as disrupting the structural integrity of extracellular matrix components causing the cross-linking of matrix molecules and the disruption of matrix-matrix and matrix-cell interactions. AGE-related collagen cross-links diminish arterial and myocardial compliance and increase vascular stiffness [18]. In addition, AGEs quench NO and generate reactive oxygen species (ROS) in a receptor independent manner by stimulating NAPDH oxidase activity [19–22]. AGEs also change the structural integrity of proteins, disturbing their cellular function, and thus have severe consequences on the affected organs [23]. Use of compounds such as alagebrium (also known as ALT-711) and aminoguanidine can reduce AGEs in tissue in vivo [24] and reduce diabetes-associated atherosclerosis in the hyperlipidaemic apolipoprotein E knockout (apoE KO; also see below) mouse [25]. In hypertensive patients, alagebrium treatment was found to improve arterial compliance [26].

AGE binding proteins AGEs can also effect endothelial function and repair by receptor-mediated binding (Tab. 1). Of the many AGE binding proteins, two in particular have been linked to atherosclerosis and inflammation. AGE-R3 is found in both apoE KO mouse and human atherosclerotic plaque [27, 28] and apoE KO mice lacking AGE-R3 have reduced aortic atherosclerosis on a standard chow diet [28]. AGE-R3 is also known to promote LDL uptake [29] and to be up-regulated during monocyte-macrophage differentiation [30]. In addition, it promotes macrophage chemotaxis [31], and potentates IL-1 generation in macrophages [32]. However, AGE-R3-deficient mice fed a high-fat diet for 8 months showed increased atherosclerosis in the aortic sinus [33]; therefore, the role of AGE-R3 in atherogenesis remains controversial. It should also be noted that AGE-R3 forms complexes with two other AGE-binding proteins, AGE-R1 and AGE-R2 [34, 35]; however, the physiological conditions under which any complex forms, and the functionality of such complexes, have not been investigated, thus additional research is needed to determine the role of this receptor in the pathogenesis of atherosclerosis.

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Table 1. Receptors which are known to bind advanced glycation end products (AGEs). Receptor/binding protein

Alternate name(s)

References

AGE-R1

p60, oligosaccharyl transferase-48

[88, 89]

AGE-R2

p90, 80K-H

[88, 89]

AGE-R3

Galectin-3, Mac-2, CBP35, L-34 or L-29

[35]

CD36

[90, 91]

Lysozyme

[92]

Lectin-like, oxidised low-density lipoprotein receptor-1 (LOX-1)

[93]

Fasciclin EGF-like, laminin-type EGF-like, and link domain-containing scavenger receptor 1 and 2 (FEEL-1 and FEEL-2)

[94]

Scavenger receptors: class A scavenger receptor (SR-A)

[95]

Class B scavenger receptor type I (SR-BI)

[95]

Macrophage scavenger receptor type II (Scr-II) Receptor for AGEs (RAGE)

[96] Amphoterin-binding protein

[36, 37]

Most literature points to AGE receptor-mediated vascular inflammation as being caused by the receptor for AGEs (RAGE; see below). RAGE was characterised as a multi-ligand member of the immunoglobulin superfamily [36, 37] and is a pattern recognition receptor and, as such, binds many other ligands including S100/calgranulins, high-mobility group box 1 (amphoterin) [38, 39], EN-RAGE (extracellular newly identified RAGE-binding protein [40]; also known as S100A12), β-amyloid peptides [41], and amyloid A [42]. However, for the purposes of this chapter we only review the endothelial dysfunction associated with AGE-RAGE signalling. In the non-diabetic state, RAGE probably plays a role in the amplification of immune and inflammatory responses, similar to Toll-like receptors [43, 44]. The extracellular domain of the RAGE protein includes two C- and one V-type immunoglobulin-like domains [36, 37], as well as a single transmembrane domain and cytosolic tail. The V domain is responsible for ligand binding (including ligands other than AGEs) and the cytosolic tail for intracellular signalling [45]. However, RAGE can undergo post-translational cleavage, which leads to various truncated isoforms of the receptor. Cleavage of the cytosolic tail leads to a membrane-bound dominant-negative form of RAGE. Cleavage of both the transmembrane domain

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and cytosolic tail by the membrane protease ADAM10 results in the extracellular ligand–binding domain of RAGE being shed to form soluble RAGE (sRAGE) [46, 47]. sRAGE is thought to act as an AGE decoy receptor as it binds AGEs competitively and may aid in their secretion, probably via the kidney [48], which is the major site of clearance of AGEs from the body [49]. A recent study found an association between sRAGE levels and the prevalence of cardiovascular disease in type I diabetic patients [50], although whether this association is linked to causation is unclear. Thus, the exact role of sRAGE in diabetes and endothelial dysfunction has yet to be determined. Endogenous secretory RAGE (esRAGE) is an endogenous splice variant of the RAGE gene (rather than a product of proteolytic cleavage, as with sRAGE), which lacks the C’ transmembrane domain of the receptor [51]. Decreases in esRAGE have recently been associated with the severity and progression of atherosclerosis in diabetic patients [52, 53].

Vascular AGE-RAGE signalling Excess formation and/or decreased clearance of AGEs from the body, such as occurs in diabetes, leads to increases in AGEs binding with full-length RAGE and thus contributes to endothelial dysfunction and atherosclerosis formation. In post-mortem coronary atherosclerotic plaque, RAGE was found in macrophages, smooth muscle cells, and endothelial cells with diabetic subjects having more intense RAGE staining than non-diabetics [54]. AGE-RAGE binding has been shown to increase JNK signalling [55] (Fig.  1), and in addition to receptor-independent ROS generation by AGEs, AGE-RAGE binding also causes ROS generation [56–59], probably via activation of NADPH oxidase [58], but also reduces eNOS levels and NO bioavailability [22, 60, 61]. RAGE signalling causes translocation of nuclear transcription factor-kB (NFkB), resulting in pathological changes in gene expression [62]. RAGE was found to be up regulated in carotid plaques taken from type 2 diabetic patients, with RAGE correlating to increases in NF-κB activation, as well as levels of cyclooxygenase-2 and prostaglandin synthase-1 [63]. NF-kB activation up-regulates NADPH oxidase and enhances the activation of secondary messenger pathways such as the mitogenactivated protein kinases (MAPKs), p21ras, extracellular signal-regulated kinase (ERKs) p38, and protein kinase C (PKC) [64, 65]. Subsequently, the production of several growth factors and cytokines is up-regulated, including VCAM-1 [66], intercellular adhesion molecule-1 and E-selectin [66, 67], as well as transforming growth factor-β1 (TGF-β1), connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF), tumour-necrosis factor-α (TNF-α), interleukin-1 (IL-1), and IL-6 [68–72]. This signalling ultimately leads to inflammation and tissue damage. In vivo, Gao et al. recently showed that the AGE/RAGE signalling induced TNF-α

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Figure 1. Although advanced glycation end products (AGEs) can cause receptor mediated effects via other receptors (including, amongst others, CD36 and AGE-receptors 1–3), the main pathogenic receptor is though to be the receptor for AGEs (RAGE). AGE-RAGE binding in the vascular endothelial cell results in increases in JNK signalling, decreases in nitric oxide (NO) and increases in reactive oxygen species (ROS), increases in mitogen-activated protein (MAP) kinase-extracellular signal-regulated kinase (ERK) signalling. This leads to increases in expression of pro-sclerotic and pro-fibrotic factors including transforming growth factor b (TGF-b), connective tissue growth factor (CTGF), platelet-derived growth factor (PDGF), macrophage chemoattractant protein-1 (MCP-1), tumour necrosis factor a (TNF-a), interleukins (IL)-1 and -6, and vascular cell adhesion molecule-1 (VCAM-1).

activation and stimulation of NADPH oxidase-dependent ROS production, which is central to the endothelial dysfunction in diabetes [73].

Diabetes-associated atherosclerosis and RAGE blockade RAGE blockade in mice by antibodies to RAGE [anti-RAGE F(ab′)2] or by sRAGE (that competes for ligand binding) has been shown to suppress the NF-kB activa-

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tion and the expression of pro-inflammatory cytokines [40]. sRAGE treatment also decreases the formation of new atheroma [74, 75] and arrests the progression of already established atheroma [76]. Preventing RAGE activation via sRAGE treatment results in reduced inflammation, reduced activity of matrix-degrading proteinases and increased levels of interstitial collagen, potentially increasing plaque stability [76, 77]. In parallel, treatment with either sRAGE or anti-RAGE F(ab′)2 decreases the arterial neointimal expansion in both diabetic and control animals [78, 79]. RAGE knockout (RAGE–/–) mice are protected from arterial and myocardial thickening as well as diabetes-associated albuminuria and glomerulosclerosis [79–82]. The apoE KO mouse is a well-characterised mouse model of atherosclerosis, and when kept under standard conditions mice develop endothelial dysfunction and atherosclerotic lesions [83, 84]. When fed a high-fat diet, or rendered diabetic with injections of streptozotocin, animals develop more advanced and complex atherosclerosis [25, 76, 85, 86]. The atherosclerotic plaques in the diabetic apoE KO resemble the complex morphology seen in patients with diabetes, including enhanced accumulation of macrophages, foam cells and cholesterol clefts within the fatty atheroma and increased expression of inflammatory markers, pro-inflammatory cytokines and chemokines. Importantly, RAGE-dependent pathways play a central role in promoting vascular lesions in these mice [55, 87]. Diabetic apoE mice have significantly increased vascular RAGE protein and mRNA expression and signs of inflammatory process on the vascular endothelium [77, 87]. Recently, we examined the development of diabetesassociated atherosclerosis in apoE KO mice, which lack the RAGE receptor (RAGE–/–/ apoE–/– double-KO mice). Absence of RAGE in these mice caused a significant attenuation of atherosclerotic plaque accumulation [87]. The reduction in atherogenesis was accompanied by reduction in the inflammatory response, as reflected by decreased accumulation of macrophages and T lymphocytes and reduced aortic expression of inflammatory cytokines and adhesion molecules, including the NF-kB subunit p65, VCAM-1, MCP-1 and NADPH oxidase subunits gp91phox, p47phox and rac-1. In addition, even in the non-diabetic context, control RAGE–/–/apoE–/– have reduced plaque accumulation and improved endothelium-dependent vasodilatation to acetylcholine [55]. These data imply that RAGE antagonism or treatment with sRAGE could become an effective tool in combating diabetic vascular disease and endothelial dysfunction.

Conclusion AGE-RAGE signalling plays a significant role in the development of endothelial dysfunction and the development of diabetes-associated atherosclerosis. The identification of the key role of the AGE-RAGE axis in the development of vascular lesions

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provides a strong rationale for novel therapies that target AGE-RAGE interaction in the prevention of endothelial dysfunction and management of diabetes-accelerated atherosclerosis.

Acknowledgements A.S.-P. was supported by Finnish Cultural Foundation, Maud Kuistila Foundation and The Finnish Diabetes Association. A.M.D.W. is supported by a National Health and Medical Research Council of Australia (NHMRC) Australian Biomedical Fellowship (472698). K.A.M.J.-D. is supported by a NHMRC Senior Research Fellowship.

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Regional predisposition to atherosclerosis – An interplay between local hemodynamics, endothelial cells and resident intimal dendritic cells Jenny Jongstra-Bilen 1,2,3 and Myron I. Cybulsky 1,3 1 

Toronto General Research Institute, Cell and Molecular Biology Division, University Health Network, Toronto, Ontario, M5G 2C4, Canada 2  Department of Immunology, University of Toronto, Toronto, Canada 3  Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada

Abstract Atherosclerosis develops in regions of the arterial tree with disturbed hemodynamics, but the underlying mechanisms of regional susceptibility to atherosclerosis are not fully understood. In this review, we summarize studies on intimal gene expression and cellular composition in atherosclerosis-susceptible regions of the normal mouse aorta, and discuss the implications to atherogenesis. Low-grade inflammation and accumulation of bone marrow-derived subendothelial resident intimal dendritic cells (RIDC) are unique features of atherosclerosis-susceptible regions. Upon induction of hypercholesterolemia, the RIDC rapidly accumulate intracellular lipid and become the initial foam cells of nascent atherosclerotic lesions, a step that precedes accelerated monocyte recruitment. Thus, unique regional intimal microenvironments established in the normal aorta play key roles in atherogenesis.

Introduction Atherosclerosis remains prevalent in industrialized nations despite therapies and lifestyle modification to reduce known risk factors, including hyperlipidemia, hypertension and diabetes. The recent rise in obesity and diabetes especially in youth forecasts an increased incidence of atherosclerosis and related complications, such as myocardial infarctions and strokes. Inflammation, which involves the recruitment of leukocytes from the blood into tissues, is an important factor in atherogenesis, and endothelial cells are critical regulators of inflammation. Endothelial cells form the interface between the blood and tissues, and, in addition to controlling key steps of leukocyte recruitment, they have important homeostatic functions that include regulation of the antithrombotic properties of the vascular surface and the permeability of plasma proteins and lipoproteins [1]. Thus, it is important to understand endothelial cell biology and functions both in the normal and the diseased artery. Endothelial Dysfunction and Inflammation, edited by Shauna M. Dauphinee and Aly Karsan © 2010 Springer Basel

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Endothelial cells respond to biochemical and mechanical stimuli derived from the blood as well as the artery wall. Biochemical stimuli include inflammatory cytokines and modified lipids released from oxidized lipoproteins trapped below the endothelium. These stimuli can drastically change the endothelial cell phenotype, a process that is referred to as endothelial cell activation (reviewed by Pober and Cotran [2]). For example, exposure of endothelial cells to interleukin-1 (IL-1) or tumor necrosis factor-a (TNF-a) induces signaling and transcription of leukocyte adhesion molecules E-selectin, VCAM-1 and ICAM-1, which mediate inflammation. In most tissues, leukocyte recruitment during an inflammatory response occurs in postcapillary venules and small veins, and leukocytes usually migrate through the venular wall with little or relatively mild and reversible vascular injury. In arteries, leukocytes accumulate in the intima, the inner layer of the artery wall, and this can lead to its expansion and remodeling by accumulated cells and deposited matrix. There is abundant evidence for endothelial cell activation in atherosclerotic lesions, consistent with the endothelium regulating the adherence and recruitment of blood monocytes to the intima, which is one of the earliest events observed in atherogenesis. The first report was published in the early 1990s and described selective expression of VCAM-1 by endothelium overlying early atherosclerotic lesions in the rabbit aorta, but not in regions that were not involved by lesions [3]. Subsequent observations in rabbit and mouse models, as well as in human tissues, documented increased expression of VCAM-1 and ICAM-1 in endothelium overlying lesions (reviewed in [4]). Endothelial cells sense fluid frictional forces and modulate their phenotype in response to different hemodynamic environments [5]. When exposed to uniform laminar shear stress, endothelial cells assume an elongated morphology and align parallel to the direction of flow. They also express a subset of genes, such as endothelial nitric oxide synthase (eNOS), that reduce inflammation and protect the artery wall from atherosclerosis. When exposed to disturbed flow, endothelial cells assume a polygonal morphology and alter their gene expression pattern so that their phenotype can be characterized as pro-atherogenic. The geometry of arteries greatly influences local hemodynamics, and this may be important in shaping regional predisposition to atherosclerosis. Straight segments of arteries experience uniform laminar flow and higher levels of mean shear stress and are relatively protected from atherosclerosis. In contrast, disturbed laminar blood flow typically is found at bifurcations and curvatures. Its features include boundary layer separation, flow reversal, secondary flows, and shifting stagnation points during each cardiac cycle. As a result, endothelial cells are chronically exposed to shear forces with cyclical variation in direction and relatively low mean shear stress. Consistent with preferential pro-inflammatory gene expression in atherosclerosis-susceptible regions is the accumulation of leukocytes, particularly dendritic cells (DC), in the sub-endothelial intima. Our recent studies have focused on the phenotypic characterization of the resident DC and the mechanisms of their accumulation in the intima to reconcile the specific endothelial gene expression patterns in these

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regions with atherosclerosis susceptibility. In this chapter, we review these studies and those of others on endothelial and DC biology in atherosclerosis-susceptible regions of the normal aorta and a potential cross-talk between the two cell types. In addition, we describe an important role that intimal DC play in the initiation of atherosclerosis.

Endothelial cell phenotype in arterial regions predisposed to atherosclerosis In humans and experimental animals, hemodynamic factors may precondition endothelial cells for atherogenesis even in the absence of systemic risk factors. Our laboratory has explored this hypothesis. Although we acknowledge that many important insights about endothelial cell biology have been gleaned from studies with cultured cells, we think that it is impossible to fully reproduce distinct arterial regional microenvironment in vitro. Thus, we selected two regions in the ascending mouse aorta – the lesser or inner curvature, with an extremely high (virtually 100%) predisposition for atherosclerosis in hypercholesterolemic Ldlr–/– and Apoe–/– mice, and the greater or outer curvature, with a very low probability for atherosclerosis [6]. We compared various aspects of the endothelial cell phenotype of these regions in C57BL/6 mice with normal circulating lipoprotein levels using two main experimental approaches: en face immunoconfocal microscopy and reverse transcriptase real-time PCR of RNA isolated from microdissected intimal cells. Our initial experiments compared endothelial cells in the lesser (LC) and greater curvatures (GC) of the normal mouse aorta and found higher VCAM-1 expression preferentially in the LC region (high predisposition for atherosclerosis) [6]. The VCAM-1 promoter contains two adjacent consensus elements for binding of nuclear factor kappa B (NF-kB) transcription factor family members [7, 8]. The functional significance of these cis elements was revealed in studies demonstrating that a 258-bp region of human Vcam1 5’ flanking sequence was capable of directing full cytokine-induced expression of a reporter in endothelial cells. Subsequent studies revealed that the integrity of both NF-kB cis elements was necessary but not sufficient for full cytokine-mediated transcription activation [9, 10]. NF-kB/Rel is a family of transcription factors, and prototypic NF-kB is the p65/ p50 heterodimer (reviewed in [11]). In quiescent cells, the majority of NF-kB is complexed to an inhibitor (IkB) that retains NF-kB in the cytoplasm. Inflammatory cytokines (IL-1, TNF-a) activate the canonical NF-kB signaling pathway, which involves activation of IkB kinases (IKKs) that phosphorylate IkBs and target them for ubiquitinylation and proteasomal degradation. This exposes NF-kB nuclear localization sequences required for transport to the nucleus where NF-kB transactivates gene expression. Since NF-kB regulates the expression of cytokine-inducible adhesion molecules, chemokines and other molecules implicated in atherogenesis, we investigated the

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expression of NF-kB components and NF-kB activation in endothelial cells in normal mice. We discovered that endothelial cell NF-kB signal transduction was primed for activation in regions predisposed to atherosclerosis [12]. In wild-type C57BL/6 mice, expression of p65 (RelA) and IkBs was significantly higher in the cytoplasm of endothelial cells in atherosclerosis-predisposed regions, but NF-kB activation (determined by nuclear translocation of p65) was present in only a minority of cells. When mice were exposed to systemic endotoxin or hypercholesterolemia, NF-kB activation and up-regulated expression of NF-kB-responsive genes such as VCAM1 and E-selectin was found preferentially in this region. These data illustrate how a topographic difference in endothelial signaling mediates accentuated regional target gene expression in response to systemic risk factors for atherosclerosis. Regional activation of endothelial NF-kB by systemic stimuli, such as hypercholesterolemia, may contribute to the localization of atherosclerotic lesions at sites with high steady-state expression levels of NF-kB/IkB components. In contrast to NF-kB, the expression of eNOS, a potential athero-protective gene, was lower in regions of the normal mouse aorta that are predisposed to atherosclerosis [13]. Experiments using eNOS promoter-b-galactosidase reporter transgenic mice, suggested that the rate of transcription contributes to regional eNOS expression levels. To ascertain the role of hemodynamics in NF-kB and eNOS gene expression, endothelial cells were cultured in parallel plate/step flow chambers that model regions of disturbed and uniform laminar flow found at arterial branches. After 2 or 3 days, the morphology of endothelial cells as well as their expression patterns of eNOS and p65 were similar to those in vivo. Heterogeneous nuclear RNA expression and RNA polymerase II chromosome immunoprecipitation (ChIP) studies demonstrated that increased eNOS transcription contributed to up-regulated eNOS mRNA and protein levels in response to uniform laminar flow. This is consistent with up-regulation of transcription factor krupel-like factor 2 (KLF2) by uniform laminar shear stress [14]. Collectively, our studies suggest that disturbed hemodynamic patterns found at arterial branches and curvatures uniquely modulate endothelial cell gene expression by transcriptional regulation, and this may explain why these regions preferentially develop atherosclerosis when risk factors such as hypercholesterolemia are introduced.

Abundance of resident intimal DC correlates with regional and genetic susceptibility to atherosclerosis Studies described above have implicated that disturbed hemodynamic forces contribute to regional differences in atherosclerosis susceptibility. In this context, the up-regulation of pro-inflammatory adhesion molecules and chemokines such as VCAM-1 (see above) and MCP-1 [15] in the regions of aorta with disturbed blood

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flow suggest that these endothelial gene products promote the recruitment of leukocytes, which contribute to atherosclerosis susceptibility. Several studies showed that DC are abundant in atherosclerosis-susceptible regions of human, rabbit and mouse arteries [16, 17]. Carotid arteries from healthy children and young adults displayed enrichment of CD1a+S100+HLA-DR+ DC in the intima where occasional T  cells and macrophages were also found. [18, 19]. We assessed the abundance of intimal leukocytes in normocholesterolemic C57BL/6 mice in the entire ascending aortic arch and compared the atherosclerosis-prone LC to the atherosclerosis-resistant GC using en face confocal microscopy and immunostaining for markers of myeloid cells (CD68), DC (CD11c, MHC class II and 33D1) and T cells (CD3). We found abundant CD68+ myeloid cells co-expressing the DC markers with typical dendrites only in the LC intima. In contrast, very few T  cells were present in the LC or GC regions [17, 20, 21]. Leukocytes were also present in the arterial adventitia (the outer portion of the artery wall) in both the LC and GC regions. Most expressed CD68 and MOMA-2, which are macrophage markers, and occasional CD3+ T cells, but relatively few DC were seen [17]. The quantification of cell marker gene expression by real-time PCR in the intima of the LC versus GC confirmed a selective enrichment of CD11c+ DC in the LC of the ascending arch, while the expression levels of endothelial cell markers, ICAM-2 and CD31, were indistinguishable between the two regions [17, 20]. Other investigators also reported enrichment of DC in atherosclerosis-predisposed regions of the mouse aorta [22–24]. Choi et al. [24] showed that DC are particularly enriched in the cardiac valves and the aortic sinus of normal C57BL/6 mice. Throughout this review, we refer to DC located in the arterial intima as resident intimal DC (RIDC). We also found a correlation between the abundance of RIDC and the genetic susceptibility of mice to atherosclerosis. Hypercholesterolemic Apoe–/– mice on C3H/ HeJ or BALB/c backgrounds develop significantly smaller lesions than those on a C57BL/6 background [25, 26]. A contributing factor may be that endothelial cells in the LC regions of the resistant strains have attenuated production of reactive oxygen species in response to local hemodynamic forces [27, 28]. We found significantly lower numbers of RIDC in the aorta of C3H/HeJ and BALB/c strains relative to the C57BL/6 strain [17]. Collectively, these findings support the possibility that RIDC abundance in the healthy aorta correlates with the extent of endothelial cell oxidative stress and pro-inflammatory gene expression in response to disturbed hemodynamics, which may be a determinant for susceptibility to atherosclerosis.

Cellular and molecular mechanisms underlying the accumulation of RIDC in the atherosclerosis-prone areas of the aorta Since the abundance of RIDC correlates with susceptibility to atherosclerosis, elucidation of underlying mechanisms for RIDC accumulation may provide new

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strategies to prevent or delay the onset of atherosclerosis, especially in individuals with risk factors. The abundance of RIDC may be dependent on local proliferation, recruitment of bone marrow-derived cells from the circulation, and survival of these cells. We adapted an in vivo BrdU pulse labeling approach to quantify the contribution of cell proliferation and monocyte recruitment in both normal and hypercholesterolemic mice [17, 21]. BrdU has a short half-life and selectively labels cells that are in the DNA synthesis phase of cell cycle, which includes monocyte precursors in the bone marrow. In the blood, BrdU+ cells do not appear in the circulation until 4–6  hours after labeling; thus, providing a window of opportunity to determine the number of proliferating cells in the aorta within 2–3 hours after BrdU-labeling. Using this approach, we found that intimal cell proliferation was very low in the LC region of C57BL/6 mice fed a standard chow diet. In contrast, about a 20-fold increase in the number of BrdU+ cells was detected after 24 hours of labeling, when only BrdU-labeled CD11b+Gr-1+ monocytes, and not granulocytes or T cells, were found in the blood. Collectively, these findings suggest that RIDC are derived from monocytes that are produced in the bone marrow and recruited from the blood. The bone marrow origin of RIDC is supported by experiments in which green fluorescent protein (GFP) bone marrow was transplanted into Ldlr–/– mice, and after engraftment, GFP+ cells with dendrites that also expressed CD68 accumulated in the aortic LC intima prior to the initiation of hypercholesterolemia [23]. Circulating mouse monocytes express the receptor for macrophage colony-stimulating factor (M-CSF, CD115) and consist of at least two major subpopulations with distinct functions and expression levels of Ly6C and chemokine receptors, CCR2 and CX3CR1. Mouse Ly6ChighCCR2highCX3CR1low monocytes (CD14hiCD16– subset in humans) are implicated in inflammatory activities and are recruited preferentially to atherosclerotic lesions, while Ly6ClowCCR2lowCX3CR1high­ monocytes (CD14lowCD16+ subset in humans) exhibit patrolling behavior in the microvasculature and promote healing and angiogenesis [29–35]. Our studies using BrdU labeling demonstrated a relatively high proliferation rate of Ly6Chigh monocyte precursors in the bone marrow and recruitment of Ly6Chigh monocytes into the normal LC intima [21]. However, we cannot rule out the possibility that circulating Ly6Clow monocytes or even DC precursors that are not labeled by BrdU within the time frame of our experiment may also be recruited to the LC and differentiate into RIDC. Endothelial cells in the lesion-prone regions of the aorta that accumulate RIDC express VCAM-1 [6, 12, 17]. A critical role for this adhesion molecule in atherogenesis was described using Vcam1D4D/D4D mice, where the fourth immunoglobulin-like domain of VCAM-1 was targeted by homologous recombination [36, 37]. En face confocal microscopy of the ascending aorta in normocholesterolemic Vcam1D4D/D4D mice (C57BL/6 background) revealed significantly lower number of RIDC in the LC relative to age-matched Vcam1+/+ controls. Since the number of circulating monocytes are comparable in Vcam1D4D/D4D and Vcam1+/+ mice [36], these findings suggest that VCAM-1 participates in the steady-state recruitment of

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circulating monocytes into the mouse aorta. The fact that RIDC were not absent in Vcam1D4D/D4D mice suggests that residual low-level expression of the VCAM-1 protein in the aorta (< 10% of the wild type) or other adhesion molecules contribute to monocyte recruitment. In addition to VCAM-1, endothelial cells in atherosclerosis-susceptible regions of the aorta also express higher levels of ICAM-1 [6, 38]. ICAM-1 binds b2 (CD18) integrins: CD11a/CD18 (LFA-1), CD11b/CD18 (Mac-1), CD11c/CD18 (p150/95) and CD11d/CD18, which are expressed by leukocytes. Cd18–/– mice are resistant to atherosclerosis [39]; however, it is not known whether CD18 deficiency affects the abundance of RIDC. A recent study described that CD11c expressed on blood monocytes during severe hypercholesterolemia contributes to atherogenesis [40], and in vitro experiments showed that CD11c contributes to monocyte arrest on endothelial cells by cooperating with CD49d binding to VCAM-1 [41]. These findings raise the possibility that CD11c/CD18 expressed on monocytes could contribute to RIDC accumulation. CX3CL1 (fractalkine) is a hybrid chemokine-transmembrane protein expressed by activated endothelial cells and leukocytes. As a membrane protein, it can function as an adhesion molecule, and when severed by membrane proteases the secreted form is a chemokine [42, 43]. Its receptor CX3CR1 is expressed by monocytes, with higher levels on the Ly6Clow (CD16+) subset [30], and by RIDC [22]. CX3CR1 deficiency significantly reduced the accumulation of RIDC [22] and the number of Ly6Clow monocytes in the circulation, which was attributed to the function of CX3CR1 and CX3CL1 in the survival of this monocyte subset [44]. In vitro experiments identified CX3CL1 as a mediator of CD16+ monocyte arrest and migration on endothelial cells [45], suggesting that the role of CX3CL1/CX3CR1 in RIDC accumulation may stem from support of Ly6Clow monocyte recruitment and/or survival. The preferential expression of CX3CR1 and CD11c by Ly6Clow (CD16+) monocytes [30, 34, 45, 46] and reduced RIDC in the setting of CX3CR1 deficiency implicates the Ly6Clow monocyte subset in RIDC homeostasis in normal aorta. The importance of this monocyte subset relative to Ly6Chigh monocytes, which we have shown to be recruited to the normal LC intima [17, 21], remains to be determined.

Potential functions of RIDC in the normal aorta RIDC in humans and mice have an immature phenotype. Flow cytometry analysis of CD11c+ RIDC isolated from aortic tissue of normal mice revealed little if any cell surface expression of the activation markers CD86, CD80 or CD40 [24] that are critical for the initiation of the adaptive immune response [47]. Expression of CD83 or CD86 was not detected by en face confocal analysis in mouse RIDC [22] and intimal CD1a+S100+ DC from healthy children [18], while CD83+ cells were found in human atherosclerotic plaques [48]. RIDC isolated from the aorta and

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valves of wild-type mice have the capacity to cross-present antigen to CD8+ T cells in vitro [24], indicating that RIDC are functionally competent DC with antigenpresentation capacity. However, given the scarcity of T  cells in the aortic intima [17], it is unlikely that antigen presentation is their primary function. Alternatively, it is conceivable that RIDC tolerize T cells that enter the artery wall against arterial antigens [49, 50]. RIDC may also survey the arterial intima for invading pathogens, and migrate through the arterial wall to regional lymph nodes or via the blood to the spleen. Recently, a subpopulation of resident tissue DC that expresses the aEb7integrin (CD103) has been described in the gut [46, 51]. These cells are derived from a common macrophage and DC precursor (MDP), have constitutive CCR7-dependent migratory capacity, and are found in the mesenteric lymph nodes both under steadystate conditions and after bacterial infection [46]. A CD103+CD11c+MHC+ subpopulation of DC was also found in the lung, residing in airway mucosa and in the perivascular region under the vascular endothelial cells [52]. Whether aortic RIDC express CD103 and possess similar migratory properties remains to be determined. In addition to antigen presentation, DC can produce pro- and anti-inflammatory cytokines [51, 53–55]. Consistent with this, we detected expression of pro-inflammatory cytokine IL-1b and IL-6 mRNA in the normal LC intima, and demonstrated that expression was significantly reduced following conditional depletion of RIDC [20]. This experiment was carried out in CD11c-diphteria toxin receptor (CD11cDTR) transgenic mice, with a simian DTR under the transcriptional control of the CD11c promoter. The CD11c promoter is constitutively active in DC [56]; thus, DC in CD11c-DTR mice selectively express the simian DTR, and a single injection of DT induces apoptosis and depletes virtually all DC. The endogenous murine DTR binds DT with a relatively low affinity [57], which is why cells with low or no expression of CD11c are relatively resistant to DT. Our data using this model suggest that RIDC produce or promote the local production of pro-inflammatory cytokines in the LC of the ascending arch, which may activate endothelial cells to express leukocyte adhesion molecules and chemokines. However, depletion of RIDC did not reduce monocyte recruitment to the LC intima [20]. Imaging by electron microscopy and confocal microscopy coupled to three-dimensional analysis [19, 24] revealed a close physical contact between the endothelium and RIDC in human and mouse aorta, which may facilitate cross-talk between the two cell types. Further studies will be required to elucidate the nature of communication between cells, and the role of cytokines and cell surface molecules.

RIDC participate in the initiation of atherosclerosis Initiation of hypercholesterolemia in Ldlr–/– mice by feeding a cholesterol-enriched diet revealed that within the first 5  days RIDC engulf and accumulate lipid and

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transform into foam cells [20]. During this initial phase of atherogenesis, the extent of monocyte recruitment and cell proliferation in the intima is not increased and is comparable to normocholesterolemic mice [21], which is consistent with the initial foam cells in lesions derived from RIDC rather than newly recruited monocytes. In support of this, foam cells in nascent lesions co-expressed DC markers, including CD11c, MHC class II and 33D1, and at the periphery of early lesions foci of lipid accumulation were observed in dendrites and the cell body of RIDC [20]. The role of RIDC in the formation of nascent atherosclerotic lesions was assessed by their conditional deletion in CD11c-DTR transgenic mice bred onto the Ldlr–/– background. When RIDC were deleted immediately prior to induction of hypercholesterolemia, a paucity of foam cells and a significant reduction of intimal lipid accumulation was observed after 5 days. Furthermore, the majority of lipid was trapped in the intimal extracellular matrix in RIDC-depleted mice, in contrast to mice with intact RIDC, in which the majority of intimal lipid was accumulated in foam cells [20]. These data reveal an important role for RIDC in the initiation of atherosclerosis prior to increased recruitment of blood monocytes. Monocyte recruitment is also critical to atherosclerotic lesion formation, but at a subsequent stage. Abundant literature supports the role of monocytes in atherogenesis (reviewed in [58, 59]), including a relatively recent study that demonstrated reduced lesion burden following repeated depletion of CD11b+ monocytes in Apoe–/– mice bearing a CD11b-DTR transgene [60]. Lipid uptake by RIDC is likely mediated by scavenger receptors. Consistent with the lipid retention hypothesis [61], low and intermediate density lipoprotein particles are retained in the intima through binding of apolipoprotein B100 to matrix proteoglycans [62], which leads to oxidation and aggregation of particles. Oxidized lipoproteins are recognized by scavenger receptors expressed by myeloid cells [63], and also likely RIDC, and are internalized by receptor-mediated endocytosis. In the gut and the respiratory mucosa DC can extend dendrites through the epithelium into lumina of these organs [64–66], and similarly it was shown that intravenously injected antigens rapidly accumulate in RIDC [24], suggesting that RIDC may extend dendrites through the endothelial monolayer into the artery lumen. Based on these findings, it is possible that RIDC can capture lipoproteins directly from the circulation. Future studies will evaluate if the accumulation of lipid by RIDC and their transformation into foam cells promotes inflammation and accelerates atherosclerosis, or protects the artery wall from potentially harmful effects of oxidized lipids.

Significance Hemodynamic forces in arterial regions predisposed to atherosclerosis contribute to a unique endothelial cell phenotype that promotes RIDC accumulation and predis-

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poses these regions to atherosclerosis. Identification of signaling pathways induced by hemodynamics in atherosclerosis-predisposed arterial regions may provide therapeutic targets for reducing atherosclerosis in individuals at risk.

Acknowledgements We acknowledge support for our research from the Heart and Stroke Foundation of Ontario (HSFO) and the Canadian Institutes of Health Research (CIHR). M.I.C. is a HSFO Career Investigator.

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Inflammation and endothelial dysfunction with aging Mary Y. K. Lee and Paul M. Vanhoutte Department of Pharmacology and Pharmacy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, Hong Kong

Abstract Aging is an independent risk factor for cardiovascular disease that is associated with chronic inflammation. The turnover of the endothelial layer is accelerated in aged subjects and endothelial dysfunction is observed after regeneration. Such dysfunction is the first stage of vascular disease. It is characterized by a reduced availability of nitric oxide (NO, an important mediator that inhibits vasomotor tone), thrombosis and vascular inflammation. The reduced availability of NO associated with aging not only results in blunted endothelium-dependent vasodilatations, but also leads to sequential oxidative and pro-inflammatory events that ultimately facilitate the occurrence of atherosclerosis. Increased intracellular oxidative stress is due not only to the augmented expression of oxidant-generating enzymes, such as NADPH oxidase, xanthine oxidase and uncoupled endothelial NO synthase, but also to the down-regulation of endothelial anti-oxidative enzymes, in particular mitochondrial superoxide dismutases. Consequently, the accumulated oxidative stress causes modifications of key proteins (nitrosylation by peroxynitrite), facilitation of the production of endothelium-dependent vasoconstrictor prostanoids and activation of stress-responsive mechanisms (transcription factors in particular nuclear factor kB). These events initiate a proinflammatory response, a key initial event in the genesis of the atherosclerotic plaque.

Introduction Endothelial dysfunction occurs with aging even when the other traditional risk factors such as hypertension, hyperlipidemia and diabetes are adequately controlled. This dysfunction is characterized by reduced endothelium-dependent relaxations, while endothelium-dependent contractions become prominent. The aging endothelium also exhibits a number of phenotypic changes including increased uptake of modified low-density lipoprotein (LDL), production of reactive oxygen species (ROS), accelerated senescence and apoptosis. The present brief review summarizes experiments performed to gain an understanding of the possible molecular mechanisms underlying endothelial dysfunction during aging.

Endothelial Dysfunction and Inflammation, edited by Shauna M. Dauphinee and Aly Karsan © 2010 Springer Basel

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Endothelium-dependent relaxing factors and aging Nitric oxide The release of nitric oxide (NO) plays a major role in mediating endotheliumdependent relaxations in large conduit arteries, including the coronary arteries. This release can be stimulated by various physiological factors including shear stress, circulating hormones (catecholamines, vasopressin, aldosterone), platelet products [serotonin, adenosine diphosphate (ADP)], thrombin, autacoids (histamine, bradykinin) and certain prostaglandins [1]. In the endothelium, the activation of the constitutive form of NO synthase (eNOS) by these stimuli converts l-arginine into NO with the stoichiometric formation of l-citrulline. This reaction is calcium dependent and is catalyzed by the presence of cofactors such as reduced nicotinamide adenine dinucleotide phosphate (NADP), flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN), heme and tetrahydrobiopterin (BH4). NO then diffuses to the underlying smooth muscle to activate soluble guanylyl cyclase, which in turn produces cyclic GMP, causing relaxation [1]. Nevertheless, uncoupled eNOS, possibly due to oxidized/inadequate BH4 in pathological conditions, such as aging, stimulates the production of superoxide anions [2–4]. Two major G protein families are coupled to eNOS, one of which is pertussis toxin sensitive (Gi  proteins) and the others not (Gq proteins). Pertussis toxin (PTx) is the toxin that causes strong ADP-riboxylation at one of the Gi protein subunits and inhibits the subsequent signaling events in the endothelium. This blocker is usually used to differentiate other endothelial receptor-linked Gq-protein-dependent pathway. The former mediate the response to alpha 2-adrenergic agonists, serotonin (5-hydroxytryptamine, 5-HT) and thrombin, and the latter that to bradykinin, histamine and ADP (Fig. 1) [1, 5, 6]. The normal physiological function of eNOS, and hence the production of NO in healthy arteries, permits the maintenance of a balanced vasomotor tone. In addition, the anti-inflammatory and anti-thrombotic properties of NO in synergy with prostacyclin provide the vascular system with an athero-protective mechanism [5–8]. Endothelial dysfunction is characterized by reduced endothelium-dependent relaxations [1, 5, 6]. The reduced production of NO shifts the equilibrium from vasodilatation towards the production of oxygen-derived free radicals and cyclooxygenase-derived prostanoids [9, 10], thus favoring the occurrence of endotheliumdependent constrictions. Like hyperlipidemia, hypertension and diabetes, aging is an independent risk factor for the development of atherosclerosis [11, 12]. The endothelial dysfunction with aging is exemplified by the reduced production of endothelium-derived NO [13–17] in both aged adults and animals, leading to reduced flow-dependent dilatations. This is likely due to an impairment of the phosphorylation of vascular endothelial growth factor receptor 2 and the subsequent diminished phosphorylation of phosphatidylinositol 3-kinase/protein kinase B (Akt) that activates eNOS [16]. The reduced bioavailability of NO with aging

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Figure 1. Schematic diagram postulating possible pathophysiological events leading to the activation of inflammatory responses in endothelial cells by aging. Aging is a risk factor that accelerates the turnover of endothelial cells and the regeneration of endothelial cells, which results in altered phenotypic changes including dysfunctional Gi protein and endothelial nitric oxide synthase (eNOS), disregulated activation of cyclooxygenase (COX) to produce endotheliumdependent vasoconstrictor prostanoids (EDCF) and, reactive oxygen species (ROS). The accumulated oxidative modification of mitochondrial proteins increases cellular oxidative stress. Superoxide anions combine with nitric oxide (NO) to form peroxynitrite, which further reduces the NO availability. Activation of nuclear factor (NF)-kB leads to the transcription of various inflammatory genes. Gq, pertussis toxin-insensitive protein; Gi, pertussis toxinsensitive protein; ox-LDL, oxidized low-density lipoprotein; PLA2, phospholipase A2; AA, arachidonic acid; ACh, acetylcholine; SOD, superoxide dismutase; XO, xanthine oxidase

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can be accompanied by either a reduced [18, 19] or increased expression of eNOS [20]. One explanation for this reduced bioavailability is that with aging, increased levels of ROS contribute to endothelial dysfunction. In particular, the increased interaction of superoxide anions with NO reduces the bioavailability of the latter, impairing endothelium-dependent relaxations, and leading to augmented levels of peroxynitrite (augmenting nitrosylation of key proteins) [15, 21, 22]. In addition to reducing the NO bioavailability, aging affects a diverse range of physiological/ pathological events ultimately leading to atherosclerosis [6, 15, 22–24].

Prostacyclin Prostacyclin is a vasodilator prostaglandin produced by the activation of the constitutive form of cyclooxygenase (COX) in the endothelium. The activation of phospholipase A2 following stimulation of the endothelial cell membrane by agonists leads to the generation of arachidonic acid. COX converts arachidonic acid into prostaglandin endoperoxides, which, in turn, are transformed into prostacyclin by prostacyclin synthase in the endothelial cells [5, 7]. The prostanoid inhibits platelet aggregation, an effect which synergizes with that of NO [7, 25]. Prostacyclin also stimulates adenylyl cyclase in the vascular smooth muscle and thus the production of cyclic adenosine monophosphate, acting on inositol phosphate receptors to induce hyperpolarization and relaxation of certain, but not all, vascular smooth muscle cells [26, 27]. If present, the vasodilator response to prostacyclin is reduced in aged subjects [23].

Endothelium-dependent hyperpolarizations Endothelium-dependent relaxations can result from hyperpolarization of vascular smooth muscle triggered by the endothelial cells and thus not due to either NO or prostacyclin. Such responses have been attributed to endothelium-derived hyperpolarizing factor (EDHF) [28–31]. EDHF requires an increase in potassium (K+) conductance, as the response can be abolished by inhibitors of small conductance calcium-activated potassium channels (SKCa) and/or specific blockers of intermediate-conductance calcium-activated potassium channels (IKCa) [31]. Activation of endothelial IKCa and SKCa leads to opening of myo-endothelial gap junctions and provides a continuous uniform membrane potential among coupled cells. Other possible mechanisms leading to smooth muscle hyperpolarization include activation of K+ conductance and/or Na+/K+-ATPase in smooth muscle cells [31], epoxyeicosatrienoic acids (EETs) from cytochrome P450 2C or 2J epoxygenase ([30, 32]; see also [31]), and hydrogen peroxide (H2O2) formed by anti-oxidative enzymes (mainly superoxide dismutases, which dismutate superoxide anions into

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the peroxide­) [27–31]. The contribution of EDHF-mediated responses to vasomotor control differs depending on the size of the arteries and becomes more prominent as the blood vessel diameter decreases [27, 33]. In a number of arteries, EDHF-mediated responses are reduced with aging. In small resistance arteries, this is probably due to a reduced expression of endothelial IKCa and SKCa [13, 31]. When the NO component of endothelium-dependent relaxations is reduced, an increased contribution of EDHF can compensate for that reduction [31]. For example, in porcine coronary arteries covered with regenerated endothelial cells, in which NO production is lower, the compensatory EDHF response to bradykinin helps to maintain a near normal relaxation after angioplasty [34].

Endothelium-depending contracting factors The endothelium can induce contraction of the underlying vascular smooth muscle. In the presence of normal amounts of endothelium-derived NO and if endotheliumdependent hyperpolarization mechanisms function normally, the release of endothelium-derived constrictor factors is minimal [35–37]. Endothelin-1 (ET-1) is a potent vasoconstrictor produced by endothelial cells [38]. In the healthy endothelium, the production and the bioactivity of ET-1 is counteracted by NO such that the endothelium-dependent relaxation is preserved. The protein expression of ET-1 and hence its production and bioactivity is increased in cultured endothelial cells derived from older adults compared to those from younger subjects, whereas that of eNOS is comparable. Antagonism of dual ETA/ETB receptor could significantly inhibit the vascular remodeling as well as the progression of hypertension in aged spontaneously hypertensive rats compared to the young [39]. These results imply that an increase in ET-1 bioavailability may explain increased vasoconstrictions with aging [40]. Other endothelium-derived contracting factors (EDCF) are produced, mainly by COX. When endothelial cells are activated, cytosolic phospholipase A2 is stimulated, which makes arachidonic acid available to COX for endoperoxide synthesis [36]. Removal of the endothelial layer or blockade of the COX activity eliminates the vasoconstrictor response elicited by the endothelial cells. This emphasizes that COX activity in the endothelium (but not at the underlying smooth muscle level) is the major source of EDCF. Endoperoxides and prostacyclin are the two major mediators of the resulting endothelium-dependent vasoconstriction. Indeed, these two endothelium-derived prostanoids diffuse to the underlying smooth muscle where they activate thromboxane-prostanoid (TP) receptors to induce contraction [29, 39]. Endothelium-dependent vasoconstrictor responses increase with aging [24, 41]. When NO is diminished during aging, EDCF will become significant and shift the balance to increase the vasomotor tone. This augmentation probably reflects the

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reduced release of NO with aging. Indeed, the presence of NO exerts a long-term inhibition on the production of EDCF; conversely, inhibitors of eNOS augment endothelium-dependent contractions [42]. With aging, the expression of COX and prostacyclin synthase may increase [43]. This leads to the elevated production of endoperoxides and prostacyclin in the endothelial cells, which in turn elicit stronger vasoconstrictions through the activation of TP receptors of the underlying vascular smooth muscle [36]. Thus, endothelial dysfunction is exacerbated if endotheliumdependent vasoconstriction becomes prominent when the availability of NO is reduced by aging [10, 14, 24, 44].

Oxidative stress and endothelial dysfunction ROS further amplify the EDCF-mediated responses [42]. In particular, in aged animals, increased oxidative stress impairs endothelial function due to inactivation of mitochondrial proteins, such as manganese superoxide dismutase (MnSOD) [19, 45, 46] and extracellular superoxide dismutase (ecSOD) [47, 48], important antioxidant enzymes catalyzing the conversion of superoxide anions into H2O2 [19]. The importance of this mechanism is illustrated by the observation that supplementation with vitamin C significantly improves flow-mediated, endothelium-dependent vasodilatation in the brachial artery of middle-aged and older adult males and females [49]. Another possible mechanism explaining enhanced cellular oxidative stress with aging is the generation of free radicals by NADPH oxidase and xanthine oxidase in the mitochondria, causing oxidative DNA damage to the cells [3, 50]. The production of superoxide anions in response to changes in intravascular pressure is increased in arteries from aged when compared to young animals [51], and an increased activity of NADPH oxidase and xanthine oxidase is involved in this pathological response [17, 50, 51], although the latter enzyme may not be a key player [52]. The resulting increased production of superoxide anions and the accumulated oxidative modification of various cellular macromolecules impair endothelial function, cause irreversible vascular damage [53], and eventually explain the deleterious effect of aging on endothelium-dependent vasodilatation.

Oxidative stress and inflammation in aged animals In addition to dysregulating the local control of vasomotor tone, the increased oxidative stress of aging also activates various inflammatory signaling pathways. There is an age-related association between increased level of oxidative stress, arterial inflammation and atherosclerosis [12, 54]. Nuclear factor (NF)-kB plays a role in this inflammatory response. NF-kB is an oxidative stress-responsive transcription factor that is expressed abundantly in endothelial cells and can translate the

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signaling­mechanism regulating pro-inflammatory (IL-1b, inducible NOS, cell adhesion molecules, matrix metalloproteinases) and anti-apoptotic responses, and the subsequent increased monocyte adhesion [55–57]. Activated NF-kB phosphorylates IkB-a (an inhibitory protein in complex with NF-kB in the unstimulated situation) and causes subsequent degradation of the NF-kB-IkB complex in the cytoplasm. This results in the activation of p50 and p65 subunits of NF-kB to form p50-p65 dimers. These active dimers translocate and accumulate in the nucleus, bind to their recognition DNA elements and cause activation of transcription. As a result, a large range of genes implicated in endothelial inflammation (cytokines, antimicrobial peptides, chemokines, adhesion molecules, stress-response proteins and anti-apoptotic proteins) are expressed [53]. Chronic activation of NF-kB predisposes to endothelial dysfunction in older humans [49]. Inhibition of NF-kB activation prevents the phosphorylation of IkB and thus the translocation of the p50/p65 subunit of NF-kB to the nucleus for pro-inflammatory gene transcription. Such inhibition improves the flow-mediated, endothelium-dependent vasodilatation in the brachial artery of middle-aged and older adults with low-grade inflammation [49]. On the other hand, inhibition of NF-kB leads to reduced expression of the p47phox subunit of the oxidant-producing enzyme NADPH oxidase and decreases the level of nitrotyrosine (a marker for oxidative modification of proteins) [49]. This suggests a possible role played by oxidative stress induced by chronic inflammation (activation of NF-kB) in endothelial dysfunction. The sequential inflammatory and oxidative responses associated with endothelial dysfunction leads to a pro-atherosclerotic phenotype in the endothelium [49, 53, 58–60].

Regenerated endothelium Endothelial cells possess limited proliferative capacity. The turnover of endothelial cells in vivo, especially in areas prone to disturbed blood flow, is accelerated in older people [61]. This process involves apoptotic death, desquamation and regeneration of endothelial cells [1, 5]. This layer of regenerated endothelium, despite the complete relining of the damaged area, produces less NO and exhibits characteristics of endothelial dysfunction and senescence. In particular, endothelium-dependent relaxations evoked by aggregating platelets, serotonin or thrombin (Gi protein dependent) are reduced, while to those of bradykinin, ADP and A23187 (Gq protein dependent) are initially not affected. This is likely due to the selective dysfunction of the Gi protein-dependent pathway in the regenerated endothelium. The increased generation of oxidative stress [62–64] and hence the increased presence of oxidized forms of LDL in regenerated endothelium probably underlies this endothelial dysfunction. The reduced level of NO facilitates the inflammatory responses in regenerated endothelial cells and thus the occurrence of atherosclerosis [6, 24].

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Conclusion The initial step in atherogenesis is the abnormal functioning of the intimal layer of the vasculature, the endothelium. Endothelial regeneration, which is a natural repair process in response to vascular injury results in altered endothelial phenotypes, including reduced ability to evoke endothelium-dependent relaxations in terms of NO production and increased presence of the oxidized form of LDL. This process becomes critical due to the accelerated turnover of the endothelium in aged subjects. With aging, endothelial dysfunction appears to alter the vasomotor balance towards an augmented production of the endothelium-dependent vasoconstrictor prostanoids, an increased production of intracellular oxygen-derived free radicals and an enhanced activation of the stress-responsive NF-kB transcription factor. These changes initiate a pro-inflammatory response, which is a key event permitting the occurrence of atherosclerosis (Fig. 1).

Acknowledgements This work was supported by grants from the Research Grant Council of Hong Kong (HKU7490/06M) and the Research Centre of Heart, Brain, Hormone & Healthy Aging of The University of Hong Kong.

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Clinical approaches to assess endothelial function in vivo Elizabeth A. Ellins and Julian P. Halcox Department of Cardiology, Wales Heart Research Institute, Cardiff University, Cardiff, UK

Abstract Atherosclerosis begins early in life. Inflammation has a major role in the disease process. One of the earliest signs of disease is endothelial dysfunction. There are a number of tests that have been developed to assess endothelial function, allowing a valuable insight into the function of the endothelium in both health and disease. This chapter reviews the techniques that are currently used and their utility in assessing the impact of inflammation on vascular pathophysiology.

Introduction Atherosclerosis is an inflammatory disease process that often begins very early in life [1]. Endothelial dysfunction is a consequence of endothelial activation associated with increased expression of leucocyte adhesion molecules, reduced anticoagulant activity and the release of growth factors, inflammatory mediators and cytokines. Thus, endothelial dysfunction is an important indicator of the changes in the arterial wall that signal an increased risk of atherosclerotic disease progression, which can be detected by clinical testing even during the very earliest stages of the disease process. Continuous cycles of inflammation and arterial damage lead eventually to the development of atherosclerotic plaques, which can lead to luminal obstruction and ischaemia, or destabilisation, resulting in acute events such as myocardial infarction and stroke. A hallmark of endothelial dysfunction is reduction of nitric oxide (NO) bioavailability and up-regulation of endothelium-generated vasoconstrictors such as endothelin-1, angiotensin-II and vasoconstrictor prostanoids. Our increasing understanding of the vascular biology of the endothelium and its interactions with the rest of the arterial wall has led to the development of a number of tests that are able to evaluate the functional properties of the normal and activated endothelium [2]. No single method is able to fully meet the requirements of an ideal test, i.e. non-invasive, reproducible, repeatable, cheap, safe, standardisable between laboratories, and able to reflect the biology of the endothelium. For a comprehensive evaluation of the many relevant facets of endothelial biology a combination of tests may be required. Endothelial Dysfunction and Inflammation, edited by Shauna M. Dauphinee and Aly Karsan © 2010 Springer Basel

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Clinical assessment The most widely used clinical means of assessing the endothelium is measuring endothelium-dependent vasomotion. Changes in the arterial diameter and blood flow are relatively easy to measure both invasively and non-invasively. Testing primarily involves pharmacological and/or physiological stimulation of the endothelium, which leads to release of NO and other vasoactive compounds. In normal endothelium, these cause vasodilatation of the artery. Both the local bioavailability of NO and responsiveness of the vascular smooth muscle contribute to this vasodilator response. If a defective vasodilator response is identified, it is important to assess the response to an endothelium-independent dilator for comparison to determine whether this abnormal response is a consequence of endothelial dysfunction or defective smooth muscle relaxation.

Invasive assessment of endothelial function Furchgott and Zawadzki’s work looking at the relaxation of rabbit aorta with intact endothelium after local infusion of acetylcholine (ACh) was the basis for the first clinical studies that were performed in the coronary circulation [3]. These studies involve the evaluation of epicardial and microvascular responses to the infusion of endothelium-dependent pharmacological probes. Assessment requires cardiac catheterisation with the use of quantitative coronary angiography and Doppler flow wire techniques. ACh releases NO from vessels with intact healthy endothelium, leading to vasodilation. In subjects with endothelial dysfunction and reduced NO bioavailability, decreased dilatation and even vasoconstriction results as a consequence of direct ACh stimulation of the smooth muscle muscarinic receptors, overwhelming a diminished or absent dilator effect of endothelium-derived NO [4, 5]. Infusion of N-monomethyl-l-arginine (L-NMMA) inhibits endothelial NO synthase (eNOS), the enzyme responsible for generation of NO, which causes an increase in coronary vascular resistance and constriction of epicardial coronary arteries in subjects free from vascular risk factors, confirming the importance of basal NO in the maintenance of coronary vasodilator tone [4]. In those with risk factors of atherosclerosis, this effect is reduced; suggesting diminished NO bioavailability in these individuals. Responses to a wide range of endothelial agonists including substance P, adenosine and bradykinin can also be assessed as measures of endothelial function. The invasive nature of this technique clearly limits its wider applicability and is only relevant for study of small, high-risk populations. Another technique based on similar methodology can evaluate the forearm microcirculation by measuring forearm blood flow (FBF) responses to these agents using venous occlusion strain-gauge plethysmography [6]. This method utilises the contralateral arm as a control, allowing adjustment for minor systemic fluctuations

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in basal blood flow and pressure that may occur during the study measured in this non-infused limb. Thus, the majority of studies measure percentage differences in FBF and vascular resistance between experimental and control arms following administration of endothelium-dependent and -independent agonists. Evaluation of the contribution of NO to vasomotor regulation can be made using eNOS antagonists such as L-NMMA. Although this technique enables the study of endothelial pathophysiology during the preclinical stages of disease, its invasive nature limits its repeatability and also prohibits use in larger cohort studies. Additionally, some have questioned the clinical relevance to atherosclerosis, as microvascular pathophysiology does not necessarily reflect changes in the conduit arteries in which atherosclerotic disease develops.

Non-invasive methods The endothelium responds to physical and chemical stimuli to maintain healthy blood vessels by regulating tone and adjusting regional blood flow in response to changes in the local environment. Conduit arteries dilate in response to local increases in flow and shear stress. This phenomenon is known as flow-mediated dilatation (FMD), which, in conduit arteries, is predominantly mediated by endothelium-derived NO [7–9]. Exploiting these principles, Celermajer and colleagues developed a non-invasive technique for the assessment of FMD [10]. This entails using ultrasound to measure the change in diameter of the brachial artery (or other conduit arteries) in response to increased blood flow, typically induced by a period of forearm ischaemia. Doppler is used concurrently to quantify blood flow and the magnitude of the hyperaemic response. High levels of reproducibility are attainable when strict attention is paid to methodology [11, 12]. FMD of the brachial artery has been shown to be progressively depressed in individuals with increasing cardiovascular risk factor burden and atherosclerosis (Fig. 1) [9, 13]. Additionally, it correlates with coronary vascular endothelial vasodilator function and circulating markers of endothelial activation, as well as predicting long-term cardiovascular outcome [14, 15].

Clinical evaluation of FMD Assessment of FMD is carried out using a high-resolution ultrasound machine with a high frequency (5–13 MHz) linear array probe. Ideally, this is held in a stereotactic clamp with micrometers for fine image adjustment; the participants lie supine on a bed. To induce the reactive hyperaemia, a sphygmomanometer cuff is positioned around the forearm. A rapid blood pressure cuff inflation system maintains a stan-

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Figure 1. An illustration of the inverse relationship between FMD and coronary risk factor burden in 500 asymptomatic subjects. Adapted with permission from [13].

dardised cuff pressure of either 50 mmHg above systolic pressure, or to 300 mmHg for adults and 200 mmHg for children, for the 5-minute occlusion period. The brachial artery is imaged in B-mode with Doppler so that simultaneous diameter and flow measurements can be recorded both before and after cuff occlusion. Images are acquired on a computer with specialist analysis software for diameter and flow measurements. The brachial artery response to shear stress can additionally be contrasted to the endothelium-independent response to sublingual glyceryl trinitrate. To ensure reduced variability between subjects it is necessary to minimise the impact of environmental and physiological factors that can affect the FMD response. Endothelial function studies should be carried out in a darkened, temperature controlled room (22–26°C). Furthermore, allowing the participants to rest for 10 minutes before the study permits haemodynamic stabilisation and thus more accurate baseline readings. Subjects should ideally refrain from alcohol and strenuous exercise for 24  hours prior to the study and abstain from smoking, caffeine and high-fat food for at least 2 hours, as these can all affect FMD. Additionally, for female subjects, the stage in their menstrual cycle should be noted. Vasoactive medicine can confound assessment by attenuating both basal and stimulated responses.

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Thus, such agents should not be taken on the day of the study if possible and should be preferably withheld for at least five half-lives prior to the vascular assessment. This may not always be practically feasible in patient groups, but is likely to make an important contribution when interpreting data.

Analysis and interpretation of FMD studies Analysis of the study involves measuring the change in diameter of the brachial artery from baseline to peak dilatation in response to the hyperaemic stimulation. It is also essential to record the Doppler trace to measure the change in blood flow, which serves as the shear stress stimulus. These measurements allow quantification of the magnitude of the stimulus and the subsequent vasomotor response profile of the conduit vessel.

Assessment of diameter change Brachial artery diameter is assessed at the intima-lumen interface. The baseline diameter is measured as an average of ten frames and the peak as the mean of three consecutive images measured at the time of the maximal increase in diameter after cuff release. FMD is represented as the maximum change in diameter from baseline, expressed most commonly as a percentage, but can also be reported as an absolute change (Fig. 2).

Figure 2. Ultrasound acquisition of brachial artery endothelial function data: Left panel: B-mode image of the brachial artery demonstrating the selected region of interest and identification of the lumen-intima interface by edge detection software. Right panel: A typical profile of the changes in brachial artery diameter observed during a standard FMD protocol in a healthy individual.

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In the earlier studies, diameter measurements were made using the calipers on the ultrasound machine. However, this is a (very) time consuming method that can be adversely influenced by operator subjectivity. Semiautomatic analysis technology, such as wall-tracking and edge-detection software, are now routinely used. These are faster and provide a more objective and accurate measurement of arterial diameter, together with improved reproducibility. However, it is still necessary to be able to review and edit the analysis manually where appropriate. There are differences between the available methods. A-mode wall tracking measurements are made at one point across the artery requiring a very stable image. With this technique, the occluding cuff position has to be altered slightly, being placed lower down the forearm to reduce movement on inflation/deflation to aid image stability, but typically results in reduced FMD due to decreased hyperaemia. Additionally, measurements must be taken at set time points, which can result in failing to capture the maximal diameter change, also resulting in a lower FMD measurement. In contrast, the use of edge detection software enables data that encompass the whole time course of the study to be easily acquired and analysed. This helps to ensure that the true peak of the response is assessed and gives greater scope for the measurement of additional parameters such as time to peak and area under the curve (AUC). Some groups do still only make maximal diameter measurements at preselected time points, most commonly at 60 seconds after cuff release. Hence, in a significant proportion of studies the maximal FMD values are likely to have been underestimated [16]. Of the available measures FMDmax (the maximum flow-mediated dilatation percentage change from baseline) has been shown to be the most reproducible, and the use of edge detection software has been found to be less variable [17].

Assessment of flow Attention has more recently focused on evaluation of not just the magnitude of the flow response, but also a more detailed appraisal of shear forces created by the increase in blood flow. This is directly related to the velocity and viscosity of the blood, and inversely correlated to the vessel diameter. Laminar shear stress is the major factor responsible for basal maintenance and stimulation of NO release. As the hyperaemic stimulus has itself been shown to be related to risk factors, it is essential to measure basal and stimulated blood flow in the brachial artery to allow adjustment for its influence on the observed vasodilator responses [18, 19]. Release of the occluding cuff results in a sudden increase in blood flow through the artery (Fig. 3). Peak flow is normally reached within 15 seconds of cuff deflation. The peak reactive hyperaemic response is the most common measure used for evaluating the magnitude of the flow stimulus, defined as the maximal increase in blood flow from baseline between 5 and 15 seconds after cuff release. This can be presented either as a percentage or absolute increase.

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Figure 3. Doppler trace from the brachial artery demonstrating baseline flow (upper panel) and reactive hyperaemia shortly (< 15 s) after release of the occluding cuff (lower panel).

There is growing interest in obtaining a more detailed picture of the shear forces acting on the endothelium during the period of reactive hyperaemia. Shear rate is commonly used as a surrogate measure of shear stress as it does not require blood viscosity measurements. There are a few methods that can be used for evaluation of these measures, which has caused some debate within the literature on which is the most effective and accurate method for expressing the information. Both peak shear rate and the shear rate AUC have been advocated as suitable methods and it has been proposed that the FMD response should be normalised for the shear “burden”. For example, a study of repeat assessment within ten subjects suggested that the change in brachial diameter should be corrected for shear rate AUC as this was responsible for 56% of the variation in FMD response [20]. However, further studies have shown that the relationship between shear rate AUC and FMD is complex and that the variance in FMD explained by this shear parameter may be negligible after appropriate adjustment [21, 22]. This remains a very relevant field of study and emerging data will undoubtedly determine how to deal with this most appropriately.

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Impact of different hyperaemic stimuli on FMD The stimulus for endothelium-dependent vasodilatation can be altered by modifying the technique used to generate the hyperaemic response, for example, by changing cuff location, duration of cuff occlusion or use of alternative hyperaemic stimuli. These factors should be taken into account when comparing FMD data from different studies, as the mechanisms responsible for vasodilatation appear to differ according to the nature of the stimuli. Cuff position influences the response to the occlusion depending on its location. The greatest hyperaemic response occurs when the cuff is placed proximal to the arterial segment being imaged, as the entire arm is rendered ischaemic [7, 23]. Although this results in greater dilatation of the artery, which may be advantageous for discriminating between groups in some circumstances [24], this response is only partially mediated by NO and may not allow discrimination of specific endothelial responses as when the occluding cuff is placed distally, below the elbow [7, 25]. Duration of occlusion also influences the vessel magnitude of dilatation, presumably through modifying the shear stress. A 4.5-minute cuff inflation was found to give an almost maximal dilatation in comparison with a range of occlusion periods up to 8  minutes, with periods of 3.5  minutes resulting in a smaller response [26]. Of note, much longer occlusion periods increase the response in comparison to 5 minutes. Importantly, mechanisms other than NO play a more important role in the FMD response following a 15-minute occlusion [9]. Similarly, isometric exercise during the cuff occlusion period increases the magnitude of the hyperaemic response, and consequent brachial artery vasodilatation, which is also only partially dependent on NO [23, 27]. More sustained hyperaemia, such as by hand warming, mediates a larger FMD response despite inducing a lower peak flow velocity than reactive hyperaemia [9].

Assessment of endothelium-independent vasodilatation Glyceryl trinitrate (GTN)-mediated brachial artery dilatation is calculated as the maximum change from baseline as expressed as a percentage. Many studies use a dose of 200–400 mg GTN. However, the much lower dose of 25 mg produces a response of a similar magnitude to FMD in healthy controls. As the artery returns to baseline diameter more rapidly, typically within 20 minutes, this reduces the delay between serial measures facilitating its use in acute intervention studies. Side effects of GTN are negligible with this smaller dose.

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Emerging methods Pulse wave velocity Measuring the speed of transit of the arterial pulse-pressure waveform through an artery provides a measure of its stiffness, which can be harnessed to assess endothelial function. Following a similar protocol to that of FMD a reactive hyperaemic stimulus can be used to mediate release of NO which reduces arterial tone and stiffness. This can be measured as a reduction in pulse wave velocity (PWV). The extent of this slowing gives an indication of endothelial NO release. This method has been shown to discriminate between healthy subjects and those with heart failure, but further validation studies are required (Fig. 4) [28].

Figure 4. Flow-mediated and GTN-mediated deceleration of pulse wave velocity (PWV) in upper and lower limb arteries of healthy subjects. Upper (triangles) and lower (circles) limb PWV responses to hand and foot hyperaemia in healthy subjects (n = 17) (mean ± SD, † p < 0.05 at that time point compared with baseline, * = p < 0.05 cf. baseline for PWV averaged over 1–10 min during hyperaemia). Printed with permission from [28].

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Peripheral waveform analysis Methods utilising analysis of changes in the peripheral waveform in response to endothelium-dependent and -independent stimuli have been developed and validated. These involve delivery of the b2 agonist salbutamol, either via an inhaler or parenterally, which stimulates endothelial NO release and reduction of arterial tone and stiffness. Importantly, the administration of salbutamol at standard clinical doses produces a measurable effect on arterial wave reflection without significantly reducing blood pressure [29–31]. This response can be characterised using both pulse wave analysis (PWA) by radial artery tonometry and pulse contour analysis (PCA) by digital plethysmography. A transfer function, which is validated in adults, can also be used to measure changes in the central aortic waveform derived from radial PWA data. Alternatively, changes in augmentation index and reflection index can be measured from the peripheral arterial waveform (Fig. 5) [29–31]. Although technically and practically appealing, we have found these methods to be somewhat less reproducible than FMD especially in children and with PCA.

Figure 5. An example of the effect of salbutamol and glyceryl trinitrate (GTN) on the radial arterial pressure waveform in a single individual. It can be seen that the second systolic peak, obvious at baseline, is diminished by salbutamol and almost completely abolished following GTN. The changes in the waveform are quantified using the augmentation index (AIx), calculated as the ratio of the pulse pressure at the second systolic peak to that at the first systolic peak. BP, blood pressure. Printed with permission from [30].

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Interestingly, little correlation was observed between FMD and results with these techniques, implicating distinct pathophysiological influences at different levels of the vasculature, which require further evaluation [12].

Endo-pulse amplitude tonometry Endothelial function can be assessed by measuring the change in finger tip pulsatile arterial volume using pulse amplitude tonometry (PAT). A finger plethysmography device employs small pressure sensors to detect isolated pulsatile arterial volume changes. These register the signal electronically and are analysed by an automated computer algorithm. Measurements are made at rest, before inflation of a forearm cuff and during reactive hyperaemia (RH). This technique provides its own internal control as both the left and right index fingers are studied (reactive hyperaemia being induced in the non-dominant arm alone). Results are presented as the “RH-PAT index” which is calculated as the ratio of the average pulse amplitude in the hyperaemic phase divided by the average baseline amplitude, with normalisation to the signal in the control arm to compensate for any systemic influences on the digital pulse amplitude. Importantly, the RH-PAT signal is decreased with risk factor expression and correlates well with cardiovascular risk factor burden. It can also help identify those with coronary vascular dysfunction and is licensed for this indication in the USA [32–34]. However, this method has been shown to be only partially mediated by NO, and other important extravascular influences are likely to be involved, including the autonomic nervous system [35]. Nonetheless, RH-PAT appears to be a valuable and practical method for identifying those with endothelial dysfunction despite the potential confounding factors.

Practical application of endothelial function testing for assessment of the vascular effects of inflammation A number of different experimental clinical models have been developed and validated to explore the functional reserve of the endothelium. Acute systemic inflammation can be induced by administration of the typhoid vaccine (Salmonella typhi) or lipopolysaccharide (Escherichia coli endotoxin). This causes transient conduit and microvascular endothelial dysfunction evidenced by diminished microvascular responses to agonist such as bradykinin or ACh, reduced FMD and effect of Salbutamol on the peripheral augmentation index, and an increase in PWV [36–39]. These models are useful for studies exploring underlying mechanisms of inflammationinduced vascular dysfunction [38, 40] or the effect of therapeutic interventions, for example vitamin C, aspirin and statins [41–44]. Use of FMD or plethysmography

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enables these studies to be carried out in relatively small study populations. Doubleblind, randomised crossover studies can be carried out using lipopolysaccharide as this can be given to subjects more than once, unlike the typhoid vaccine. C-reactive protein (CRP) is a useful measure of inflammation for use in clinical studies. It is relatively easy to measure and lends itself well to larger cohorts, including children. Associations have been found between endothelial function and CRP in studies of coronary blood flow, forearm blood flow and FMD in healthy and disease groups [45–47]. It also has utility for the evaluation of the effect of interventions on inflammation and vascular dysfunction, for example intensive periodontal therapy or bariatric surgery in morbidly obese subjects [48, 49]. Some caution is required when interpreting CRP data as not all studies are consistent. For example, no incremental association was found between inflammation and endothelial function in a large healthy population study once the effect of standard risk factors had been accounted for [50]. Furthermore, following typhoid vaccination, the measurable rise in CRP lags behind the deterioration of FMD and is discordant with FMD recovery [37]. Thus, it is most appropriate to consider CRP as a biomarker of the extent of underlying inflammation rather than a directly causal agent when interpreting vascular data. A number of other provocative models that involve an inflammatory component have been developed and validated that test the functional reserve of the endothelium. These include mental stress testing and ischaemia-reperfusion [51, 52]. Psychophysiological stress, for example a pressurised mathematical or public speaking task, has been shown to induce acute inflammation and also endothelial dysfunction in healthy adults [51, 53–56]. Endothelin-A receptor activation is mechanistically implicated, as FMD is not affected by mental stress during administration of the selective antagonist BQ-123 [54]. In contrast, a similar level of psychophysiological stress does not further exacerbate the significant endothelial dysfunction observed in diabetics and is therefore a less useful research tool in these subjects. The magnitude of the acute inflammatory response to psychophysiological stress, as defined by the increase in fibrinogen and TNF-a levels, has recently been shown to predict increasing arterial stiffness [56]. However, there are limited studies looking at the effect of mental stress testing on both inflammatory markers and endothelial function, but we believe this is an important research area given the important influence of psychosocial factors on cardiovascular events [57, 58]. Ischaemia-reperfusion of the arm causes damage to the endothelium. The resultant transient endothelial dysfunction can therefore be exploited to test interventions such as the protective effects of pre-conditioning or inorganic nitrogen oxides [52, 59, 60].

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Conclusion Assessment of endothelial function provides important complementary insights into the vascular consequences of both acute and chronic inflammatory stimuli. It is essential that all tests of dynamic vascular physiology are conducted in a rigorous and consistent fashion and that the selected methodology is capable of addressing the specific hypothesis. There are clear roles for both invasive and non-invasive methods with the former more suited to smaller studies in which a detailed evaluation of specific vasomotor pathways is required, and the latter for evaluation of larger cohorts and longer-term interventions. Emerging non-invasive methodology and vascular functional “stress testing” are expanding the range of questions that can be answered but, as always, more work is required to realise their potential.

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Therapeutic approaches towards targeting endothelial dysfunction John H. Boyd and Keith R. Walley Critical Care Research Laboratories, Heart and Lung Institute, St. Paul’s Hospital, University of British Columbia, Vancouver, BC, Canada

Abstract Endothelial dysfunction is a major cause of organ dysfunction in diseases involving systemic inflammation. The disease with the highest attributable mortality and morbidity due to explosive systemic inflammation is septic shock. In this chapter we use septic shock to illustrate how traditional approaches to therapy have fallen short, and how targeting the microcirculation might be of benefit. Specific therapies are discussed, divided into sections according to their molecular target and mode of action.

Microcirculatory failure mediates organ dysfunction in inflammatory disorders such as sepsis Severe sepsis or septic shock arguably represents the most serious acute inflammatory disorder, with the resultant derangement in physiology serving as an extreme phenotype of inflammatory endothelial dysfunction. The classic presentation of the patient in septic shock has traditionally been described as low blood pressure in conjunction with warm extremities and bounding pulses (reflecting a very low diastolic blood pressure due to dilated arterioles), even after initial resuscitation efforts. The initial clinical presentation in these patients often includes signs of circulatory failure such as cool mottled extremities, multiple organ failure (most notably pulmonary, renal and coagulation) and a low mixed venous oxygen saturation, reflecting inadequate oxygen delivery. Some of these cases of acute circulatory failure are due to concurrent cardiac impairment, as 50–100% of patients in septic shock have demonstrable reductions in cardiac ejection fraction [1–5]. This cardiac impairment has been found to be related to myocardial microvascular dysfunction and impaired oxygen extraction in the heart [6]. However, the clinical picture of circulatory failure can be present even with normal or supranormal cardiac output and global oxygen delivery. All microcirculatory beds are involved and despite the variability of clinical presentations, the outcome of septic shock is effected in large part through endothelial dysfunction leading to microcirculatory failure. Endothelial Dysfunction and Inflammation, edited by Shauna M. Dauphinee and Aly Karsan © 2010 Springer Basel

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Not well understood until recent advances in assessment of the microcirculation is the patient who presents clinically with preserved cardiac function and reversal of hypotension (with or without vasoconstrictor medications), but who has mottled extremities and organ dysfunction. At the level of the large blood vessel there is adequate oxygenated blood flow being delivered to organ systems, but these organs (and the tell-tale mottled extremities) are dysfunctional due to an increased heterogeneity of capillary flow. In those with a low blood pressure as a result of severe sepsis, it has been demonstrated that fluid resuscitation alone results in a small improvement in microvascular permeability [7, 8]; however, it is clear that simply restoring an adequate blood pressure and cardiac output is not enough. This is well illustrated in a study by Ledoux et al. [9], where organ function and tissue oxygenation in patients with septic shock did not increase with increases in blood pressure and cardiac output. Patients in this study received fluids and norepinephrine titrated to mean arterial pressures of 65, 75 and 85  mmHg, with the clinical efficacy of this medication demonstrated by an increased heart rate and cardiac index with increased dosing. However, when these investigators then looked for evidence of changes in organ function and evidence of adequate tissue oxygenation they did not find that increases in cardiac output resulted in improved organ function. In fact, urine output and capillary blood flow tended to be lower in those with a noradrenaline-induced high cardiac output, while increased intramucosal carbon dioxide and decreased oxygen extraction (measured via gastric tonometer) suggested impaired tissue perfusion. These results are compelling evidence in human septic shock that macrovascular resuscitation of mean arterial pressure and cardiac output alone is inadequate, and attention must be paid to the microcirculation. This concept of sepsis-induced microvascular dysfunction has been demonstrated convincingly in skeletal muscle (Fig.  1) with an increased proportion of capillaries having stopped blood flow for substantial periods of time resulting, in those capillaries with normal flow having increased oxygen extraction [10, 11]. It appears that in some cases the septic microcirculation is unable to regulate or redistribute oxygenated red blood cell flow to tissue regions of high oxygen demand resulting in local regions of tissue hypoxia [12]. The clinical applications involving microvascular dysfunction are in their infancy, but some clinician-investigators are actively pursuing how best to incorporate an index of vascular function into protocols for resuscitation.

Measures of success in reversing endothelial dysfunction In septic shock the majority of therapeutic approaches were developed for the classic macrovascular pathophysiology (excessive vasodilation and possible concurrent cardiac dysfunction). In the 2008 Surviving Sepsis Guidelines, patients with a mean arterial pressure under 65  mmHg are given first-line therapy with intravenous fluids, followed by a vasopressor agent with some positive inotropy (dopamine or

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Figure 1. Reconstructed 3D confocal microscopy images of skeletal muscle microcirculation in (A) control mice and (B) in mice 5 hours after an endotoxin infusion. The normal homogenous appearance in control mice is disrupted in endotoxin-treated mice. Reprinted with permission from [43].

noradrenaline) [13]. Measures of success in the early treatment include achieving a central venous oxygen saturation of ≥ 70% and normalizing elevated lactate levels. Both of these measures assess global circulatory status, with a central venous oxygen saturation of ≥ 70% providing some assurance that cardiac output/oxygen delivery is adequate, while a decline in lactate reflects a shift to aerobic metabolism – essentially another measure of adequate oxygen delivery. How are investigators using new insights into microvascular dysfunction to guide therapy? To evaluate the efficacy of resuscitation on heterogeneity of capillary flow in the micro-circulation, a new diagnostic instrument using polarized light microscopy of sublingual microvessels has been developed. This has been used to demonstrate increased heterogeneity of microvascular blood flow in patients having septic shock [14], while those patients who survived demonstrate improved microvascular function over the first several days of hospitalization compared to non-survivors [15]. More recently, this technique has found that improving microcirculatory flow using a protocol based on the 2008 Surviving Sepsis Guidelines was correlated with improved outcomes including reversal of organ function and survival [16]. This has led these investigators to propose using an index of microcirculatory flow to target therapies in resus-

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citation, although this approach has not yet been proven to be clinically superior to standard care. In the following sections we discuss the classes of therapy that have been investigated for the treatment of the severe endothelial dysfunction associated with sepsis. In all cases, without a direct measure of endothelial function/microcirculatory flow, the relatively crude measure of mortality and global organ function have served as surrogates.

Specific therapies We have chosen to group therapies targeting microvascular dysfunction into those which target circulating pathogen-associated molecular patterns (PAMPs) and their receptors; those which target circulating inflammatory cytokines and their receptors; the large scale removal of mediators; modulation of the coagulation pathway; and finally modulation of the intracellular inflammatory signaling (Fig. 2).

Figure 2. Schematic diagram of a blood vessel and endothelial cell. Therapies aimed at inhibiting the interaction between circulating mediators of endothelial dysfunction and their receptors include anti-TNF-a antibody, intravenous immune globulin (IVIG), anti-TLR2 antibody, anti-TLR4 antibody and the HA-1A antibody. High-flow dialysis aims to clear the blood of all circulating mediators, while intracellular inhibitors of the inflammatory cascade include ibuprofen, steroids and the NO synthase inhibitor, L-NAME. The sole agonist therapy is activated protein C (APC), which suppresses inflammation when recognized by the endothelial protein C receptor (EPCR). * denotes therapies that have shown promise for the treatment of septic shock.

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Therapies targeting circulating PAMPs and their receptors Exposure of the endothelium to circulating PAMPs derived from cellular components of pathogens results in the early phase of septic shock (a profound drop in blood pressure within 5 minutes followed by transient improvement), and is felt to contribute to the excessive systemic inflammation. Therapies aimed at neutralizing PAMPS therefore have great intuitive appeal. Therapy aimed at non-specifically “shielding” the PAMP from the host has centered on pooled intravenously delivered immune globulin (IVIG). Multiple small studies examining IVIG treatment in septic shock resulted in the suggestion that treatment may improve mortality [17]. However, a large multicenter trial was unable to show any mortality benefit in those randomized to receive IVIG(G) [18]. Despite this result, there remains debate about the utility of IVIG, as it appears that studies in which IVIG is enriched in IgA or IgM may yield superior results. As the predominant organisms responsible for severe sepsis through the 1990s, gram-negative bacteria [19, 20] have been the target of most anti-PAMP therapies. The first line of anti-PAMP therapy to be tested clinically was using an antibody, named HA-1A, directed against endotoxin [21]. Studies in the early 1980s yielded promising results, with antiserum generated through injection of volunteers with heat-killed E. coli improving mortality in a single-center study of patients with septic shock [21]. Unfortunately, the subsequent multicentered randomized control trial (RCT) of HA-1A in septic shock failed to demonstrate any reduction of mortality in this group of very sick patients (mortality rate of 33%) [22]. A similarly powered study in 2000 using a murine anti-endotoxin antibody confirmed the lack of benefit of early administration in patients with septic shock [23]. In an attempt to target the predominant organism responsible for gram-negative sepsis (enterbacteriaciae), an antibody was developed against the enterbacteriaciae common antigen, and this was tested in a multicentered RCT of patients with septic shock [24]. Again, no improvement in mortality was observed in those patients randomized to the antibody treatment. As circulating PAMPs exert their effects mainly via activation of innate immune receptors, this has led to the development of an antibody directed against Toll-like receptor (TLR) 4, whose cognate ligand is lipopolysaccharide. A phase II clinical trial with an anti-TLR4 antibody, eritoran tetrasodium, is currently underway (clinicaltrial.gov # NCT00334828).

Therapies targeting inflammatory cytokines and their receptors Following recognition of PAMPs by innate immune receptors expressed on white blood cells, endothelium and other tissues, there is an explosive production of proinflammatory cytokines. Of these, TNF-a has received the majority of attention as increasing levels correlate with worse outcome, and exogenous administration can

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reproduce many of the physiological derangements seen in sepsis. These observations led to numerous trials using neutralizing antibodies to TNF-α and its receptor [25–29]. None of these studies convincingly demonstrated a survival benefit with therapy. However, in subgroup analysis it appears that patients demonstrating the most profound inflammation (those in shock and/or with IL-6 levels > 1000 pg/mL) had a more rapid improvement in shock and showed trend towards lower mortality when treated with anti-TNF-α agents. It has been suggested that this therapy holds promise if one could identify those experiencing profound ongoing inflammation through a combination of clinical scoring and biomarkers such as IL-6. More recently, circulating levels of macrophage migration inhibition factor (MIF) has, like TNF-α, been shown to correlate with outcome, and in an animal model of peritonitis a neutralizing antibody to MIF was able to reverse shock and prevent death even if given up to 8 hours after injury [30]. This efficacy, despite being given following the injury, is unlike any of the other anti-cytokine therapies, and may relate to the ability of MIF to restore the sensitivity of tissues to the anti-inflammatory effects of glucocorticoids.

Removal of circulating inflammatory mediators by hemofiltration and/or dialysis There is intuitive appeal to the removal of plasma containing pro-inflammatory mediators through hemofiltration or hemofiltration combined with dialysis. Although most clinicians find large volume hemofiltration (35  L over 4  hours) impractical given the need to deal with massive amounts of effluent and replacement fluids, this approach has provided the best results in patients with septic shock who have refractory hypotension. In a series of 20 patients with septic shock, over 50% had major reductions in vasopressor requirements combined with improved cardiac output and measures of tissue perfusion [31]. When a more practical application of high-dose (35  mL/kg/h) hemofiltration was compared to lower dose (20  mL/ kg/h) in those with renal failure due to systemic inflammation, there was no benefit with higher dose treatment [32]. However, the volume of plasma filtered was approximately 2 L per hour in the “high-dose” group [32] compared to over 9 L per hour in the large volume hemofiltration study. Thus, the concept of high volume hemofiltration to remove circulation mediators may have merit, but due to practical considerations, has not been studied in a systematic fashion.

Modulation of the coagulation-inflammatory pathway It has long been recognized that in individuals with severe sepsis the coagulation system is often deranged. Of clinical importance is that there is a profound decrease

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in circulating protein C and antithrombin levels, while there is a significant increase in activity of the tissue factor (thromboplastin) pathway. Prothrombin binds thrombomodulin on the endothelial surface, while tissue factor mediates thrombin generation from prothrombin. The thrombin-thrombomodulin complex cleaves protein C to produce activated protein C, which then binds the endothelial protein C receptor (EPCR). Binding of EPCR by activated protein C initiates signaling through the thrombin receptor PAR-1, and modulates NF-kB activity with a resultant downregulation of inflammatory cytokines and apoptotic pathways. A number of therapies targeting coagulation have been devised. Neither antithrombin [33] nor recombinant human tissue factor [34] were able to improve mortality in patients with septic shock. However, targeting the role of the endothelium in both inflammation and coagulation has resulted in the first successful phase III randomized controlled trial in a severe sepsis patient population [35]. Through specific anti-inflammatory effects or as the result of its combined actions at the endothelial cell luminal surface, activated protein C results in decreased organ dysfunction and improved survival in patients with severe sepsis [35]. This effect was most pronounced in patients with high severity of illness and early treatment was most effective.

Therapies targeting intracellular inflammatory signaling PAMP signaling via innate immune receptors leads to secondarily increased cytokines and causes the endothelial cell to shift to a pro-inflammatory phenotype. This phenotype features dramatically increased nitric oxide (NO), which is responsible for the loss of blood vessel tone and the failure of organs to regulate blood flow. Investigators have aimed directly at one of the final mediators of vascular dysfunction (NO) through the use of an NO synthase inhibitor N-mono-methyl-l-arginine (L-NMMA). Although this agent had been shown in animals and small numbers of humans to improve sepsis-induced hypotension, a large RCT was stopped early due to excess mortality. The increased mortality may have been a result of pulmonary hypertension [36], but the microcirculation was not assessed so that adverse microvascular effects have not been ruled out. Indeed, infusion of the NO donor nitroglycerin is reported to improve microvascular flow. Ibuprofen is in standard clinical use as an anti-inflammatory agent through inhibition of prostacyclin and thromboxane, and was studied in patients with septic shock. Although efficacious at inhibiting the products of prostacyclin and thromboxane and successful at reducing fever and lactate levels, there was no improvement in organ function or survival in a large RCT [37].

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Steroids Steroids are the cornerstone of therapy for endothelial dysfunction mediated through classical immune mechanisms, such as small vessel vasculitis (Wegener’s disease or polyangiitis), and are extremely effective. These disorders are characterized by activated T lymphocyte infiltration into the capillary wall, and corticosteroids effectively suppress this immune cell-based endothelial damage. However, in the endothelial dysfunction induced through the systemic inflammation of septic shock the use of steroids remains controversial. In the 1980s, three RCTs were performed examining the use of high-dose (30  mg/kg methylprenisolone) steroids in patients with septic shock [38–40]. These failed to show any benefit for those randomized to receive intravenous steroids, demonstrating a trend to faster resolution of shock at the cost of increased secondary infections. More recent well-powered studies have used lower doses of steroids, and have conflicting results as to benefits associated with steroid therapy [41, 42]. Taken together, it appears that steroids may be beneficial in those with the most severe inflammation, as characterized by the most profound drop in blood pressure. However, no definitive conclusion can be made as to their benefit with regards to mortality or preservation of organ function. While the potential clinical benefits of steroids occur early, their adverse consequences appear later. It is possible that using anti-inflammatory steroids during the later compensatory anti-inflammatory syndrome (CARS) phase of sepsis may be detrimental.

Future directions in the treatment of endothelial dysfunction As with any disease, the first and most important step towards designing treatment is the development of the tools through which one can monitor the response to treatment. The technique of directly visualizing the microvasculature may be the first step towards rational therapy of endothelial dysfunction rather than the use of relatively crude endpoints such as global organ dysfunction and mortality (both of which are essentially composite endpoints of the initial insult combined with the patient’s response).

Acknowledgements Keith R. Walley is a Michael Smith Foundation for Health Research (MSFHR) Distinguished Scholar. John H Boyd is a Providence Health Research Institute Physician Scholar.

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signs of systemic sepsis. The Veterans Administration Systemic Sepsis Cooperative Study Group. N Engl J Med 317: 659–665 Bone RC, Fisher CJ, Jr., Clemmer TP, Slotman GJ, Metz CA, Balk RA (1987) A controlled clinical trial of high-dose methylprednisolone in the treatment of severe sepsis and septic shock. N Engl J Med 317: 653–658 Annane D, Sebille V, Charpentier C, Bollaert PE, Francois B, Korach JM, Capellier G, Cohen Y, Azoulay E, Troche G et al (2002) Effect of treatment with low doses of hydrocortisone and fludrocortisone on mortality in patients with septic shock. JAMA 288: 862–871 Sprung CL, Annane D, Keh D, Moreno R, Singer M, Freivogel K, Weiss YG, Benbenishty J, Kalenka A, Forst H et al (2008) Hydrocortisone therapy for patients with septic shock. N Engl J Med 358: 111–124 Bateman RM, Walley KR (2005) Microvascular resuscitation as a therapeutic goal in severe sepsis. Crit Care 9 (Suppl 4): S27–32

Index

acetyl choline  202

apoptosis, endothelial  7, 8

activated protein C (APC)  4

apoptosis signal-regulating kinase 1 (ASK1) signalosome  142

activin-like kinase-type 1 (Alk-1)  91 acute lung injury (ALI)  5

artery/arteriole, role in blood pressure control 16, 26

acute physiology and chronic health evaluation (APACHE)  4, 6

asymmetric dimethyl arginine (ADMA)  45

adult respiratory distress syndrome (ARDS)  5

atherosclerosis  176–183, 201

advanced glycation end product (AGE) 



initiation  182

161–163



nascent lesion  183

AGE-R3  162



predisposition  176–179

AIP1 complex  137, 140, 141



progression  201

Akt (protein kinase B)  71

augmentation index  210

allopurinol  55 AMP kinase  71

bariatric surgery  212

angiogenesis  68, 149, 150

basal and stimulated blood flow  206

angiopoietin-1 (Ang-1)  6, 72, 92, 93

blood pressure  104

Ang1-Tie2 pathway  92, 93

bone morphogenetic protein receptor 2

angiopoietin-2 (Ang-2)  6

(BMPR2)  90, 91

angiotensin  49, 105

bradykinin  71, 202

angiotensin converting enzyme inhibitor

bromodeoxyuridine (BrdU)  180

(ACEi)  108 type 1 angiotensin II receptor (AT1R)  108, 109

calmodulin (CaM)  68 calmodulin-dependent kinase II (CaMKII)  71

antioxidant supplementation  57

capillaries, role in leukocyte recruitment  17

antithrombin III (ATIII)  3, 4

caspases, pro-apoptotic  6, 8

aorta, lesser (LC) and greater curvature

caveolin  67, 68

(GC)  177

CD11c-diphteria toxin receptor (DTR)  182

apocynin  56

cell adhesion molecule  17, 19, 21–27

apolipoprotein E knockout (apoE KO) mouse

clinical testing, atherosclerotic disease

162, 166

progression  201

231

Index

coagulation  224

endothelin-A receptor  212

connexin  20

endothelium, regenerated  195

coronary vascular dysfunction  211

endothelium, role in inflammation  16, 17

C-reactive protein (CRP)  212

endothelium-dependent relaxing factor  190

CX3CL1 (fraktalkine)  181

endothelium-derived hyperpolarizing factor (EDHF)  86, 104

cyclooxygenase (COX)  106, 192–194

endothelium-derived relaxing factor  104 dendritic cell, resident intimal (RIDC)  179–183

endothelium-independent vasodilatation  208

diabetes  166

epicardial and microvascular function  202

digital plethysmography  210

ER stress  143, 144

CD11c-diphteria toxin receptor (DTR)  182

E-selectin  7, 17, 19, 24 extracallular matrix  162

dismutation  39 disseminated intravascular coagulation (DIC)  drotrecogin a  4

3 fas-associated death domain (FADD)  8 flavin adenine dinucleotide (FAD)  48

early growth response gene-1 (EGR-1)  2

flow-mediated dilatation (FMD)  203–208

edge-detection  206

fluid shear stress  69

endogenous secretory RAGE (esRAGE)  164

fms-related tyrosine kinase 1 (Flt-1,

endoglin  91 endothelial activation  201

VEGFR2)  6 fraktalkine  181

endothelial cell, signal transduction  22, 27, 28 endothelial cell apoptosis  89, 90

geldanamycin  69

endothelial cell activation  22, 24, 176

gene expression, NF-kB priming in endothelial



in vitro  22



in vivo  24

cell  178 genetically modified mouse  51, 162, 166

endothelial dysfunction  45

Gi protein-dependent pathway  195

endothelial heterogeneity  15, 19, 22–28

glomerular endothelial cell  19, 22, 26–28



in quiescent condition  19, 22

glyceryl trinitrate (GTN)  208



in response to inflammation  22–24, 26, 27

guanylate cyclase  69



in response to shear stress  23



molecular basis  27, 28

endothelial nitric oxide synthase (eNOS)  41,

Haber-Weiss/Fenton reaction  43 90-kDa heat shock protein (Hsp90)  67, 69

45, 52, 53, 57, 65–74, 83, 178

hemofiltration  224



expression  178

high-density lipoprotein (HDL)  126



interacting protein  67

high-resolution ultrasound  203



phosphorylation  70–73

histamine  69



uncoupled   52, 53, 57

hydrogen peroxide (H2O2) 

43, 46, 53

endothelial permeability  5

hydroxyl radical (OH•)  43

endothelial pharmacology  18, 29

hyperpolarization, endothelium-dependent

endothelial protein C receptor (EPCR)  4

192, 193

endothelin receptor antagonist (ERA)  87

hypertension, systemic  103–111

endothelin-1 (ET-1)  82, 85

hypertension model in mouse  51

232

Index

inflammation induced vascular dysfunction  211



inflammatory arteriogenesis  149

nitric oxide synthase interacting protein (NOSIP)  67

intercellular adhesion molecule-1 (ICAM-1)  6, 7, 17, 19, 24 interleukin-1 (IL-1), endothelial activation  2, 4, 22, 24 intermediate-conductance calcium-activated potassium channel (IKCa)  192 intravenously delivered immune globulin (IVIG)  223 ischaemia-reperfusion  212

endothelial, see endothelial nitric oxide synthase (eNOS)

inflammatory angiogenesis  149, 150

nitric oxide synthase trafficker (NOSTRIN)  67 nitroxyl (HNO)  41 nitroxyl anion (NO–)  41 N-myristoylation  66 Nox protein  47, 48 nuclear factor (NF)-kB  2, 3, 194

expression  178



in endothelial activation  2, 3

junction protein  6, 7

occlusion, cuff position and duration of  208

junctional adhesion molecule (JAM)  7

oxidative stress  37, 144–146, 194, 195

kinase, as pharmacological target  28

palmitoylation  66

kindling-bonfire hypothesis  51, 57

pathogen-associated molecular pattern

l-arginine  110

periodontal therapy  212

leukocyte adhesion  6, 124

peroxyl radical (ROO•)  44

leukocyte recruitment  16–18

peroxynitrite  42, 105

leukocyte trafficking  6

pertussis toxin sensitive  190, 195

lipid oxidation/peroxidation  44

phosphatidylserine  3

lipopolysaccharide (LPS)  2, 4, 26

plasminogen activator inhibitor-1 (PAI-1)  4

low-density lipoprotein (LDL)  195

plexiform lesion  94

(PAMP)  222, 223

postcapillary venule, role in leukocyte manganese superoxide dismutase  56, 194

recruitment  17

microcirculation  219

progenitor cell therapy  94

microenvironment, endothelial  15, 27

prostacyclin  82, 84, 106, 192

microparticle, endothelial, in sepsis  4, 5

protease-activated receptor-1 (PAR-1)  4

mitochondrial matrix  56

protein kinase A, B and G  71

mitochondrial respiratory chain  55

protein oxidation  44

monocyte subpopulation  180

P-selectin  6, 7, 17, 19

N-myristoylation  66

psychophysiological mental stress testing  212 pulmonary arterial hypertension (PAH) 

NADPH oxidase  47–53, 194 nitric oxide (NO)  5, 38–42, 82, 83, 104, 162, 164, 190, 193, 195

81, 82 pulmonary vascular endothelium  82 pulse amplitude tonometry (PAT)  211

NO inhibitor, therapy  5

pulse contour analysis (PCA)  210

nitric oxide radical (NO•)  41, 42

pulse wave analysis (PWA)  210

nitric oxide synthase (NOS)  5, 40

pulse wave velocity  209

233

Index

radial artery tonometry  210

superoxide anion  38, 39, 42, 43, 46, 49, 105

reactive hyperaemia  203, 211

superoxide dismutase (SOD)  40, 105

reactive hyperaemia pulse amplitude tonometry

systemic inflammatory shock syndrome (SIRS)  1

(RH-PAT)  211 reactive nitrogen species (RNS)  40 reactive oxygen species (ROS)  38, 46, 162, 164, 165 receptor for advanced glycation end products (RAGE)  163–166

tetrahydrobiopterin (BH4) 

45, 52, 54, 110

thrombomodulin (TM)  4 thromboxane A2  82, 85 thromboxane-prostanoid (TP) receptor  193

RAGE, endogenous secretory (esRAGE)  164

tissue factor  4, 88

RAGE, soluble (sRAGE)  164, 165

tissue factor pathway inhibitor (TFPI)  3, 4

reflection index  210

tissue-type plasminogen activator (tPA)  4

resident intimal dendritic cell (RIDC)  179–183

tocopheroxyl radical  58

resuscitation  220

toll-like receptor (TLR)  2 tumor microvasculature  70

salbutamol  210

tumor necrosis factor-a (TNF-a)  2, 4, 22, 24

P-selectin  6, 7, 17, 19

tumor necrosis factor receptor 1 (TNFR1)

sepsis, severe  1–5, 219

definition  1

signaling  138–142 tumor necrosis factor receptor 2 (TNFR2)

septic shock  2, 219

definition  2

signaling  137, 138, 149, 150 type 1 angiotensin II receptor (AT1R)  108, 109

shear rate  207 shear stress  107, 206

urate  54

signal transduction in endothelial cell  22, 28 small conductance calcium-activated potassium

vascular cell adhesion molecule-1 (VCAM-1) 

channel (SKCa)  192

6, 7, 17, 19, 24, 176, 177, 180

smooth muscle cell (SMC)  104, 105, 107

VCAM-1 expression  176, 177



vascular dysfunction, inflammation induced  211

hyperpolarization  107

soluble guanylate cyclase (sGC)  105

vascular endothelial growth factor (VEGF)  6, 18–20, 69, 70

soluble RAGE (sRAGE)  164, 165 sphingosine-1-phosphate  121–127



heterogeneity in expression  18-20



angiogenesis  121



role in leukocyte recruitment  18



endothelium  121

VEGF receptor-2 (VEGFR-2)  6, 70



inflammation  126

vascular permeability  65



leukocyte adhesion  124

vascular segment specificity  17



permeability  123

vasomotor tone, in sepsis  5

sphingosine-1-phosphate agonist  127

vessel wall, role in leukocyte recruitment  17

statin  110 steroid  226

wall-tracking  206

strain-gauge pletysmography  202 substance P  202

234

xanthine oxidase (XO)  54, 57, 194

The PIR-Series Progress in Inflammation Research Homepage: www.birkhauser.ch Up-to-date information on the latest developments in the pathology, mechanisms and therapy of inflammatory disease are provided in this monograph series. Areas covered include vascular responses, skin inflammation, pain, neuroinflammation, arthritis cartilage and bone, airways inflammation and asthma, allergy, cytokines and inflammatory mediators, cell signalling, and recent advances in drug therapy. Each volume is edited by acknowledged experts providing succinct overviews on specific topics intended to inform and explain. The series is of interest to academic and industrial biomedical researchers, drug development personnel and rheumatologists, allergists, pathologists, dermatologists and other clinicians requiring regular scientific updates.

Available volumes: T Cells in Arthritis, P. Miossec, W. van den Berg, G. Firestein (Editors), 1998 Medicinal Fatty Acids, J. Kremer (Editor), 1998 Cytokines in Severe Sepsis and Septic Shock, H. Redl, G. Schlag (Editors), 1999 Cytokines and Pain, L. Watkins, S. Maier (Editors), 1999 Pain and Neurogenic Inflammation, S.D. Brain, P. Moore (Editors), 1999 Apoptosis and Inflammation, J.D. Winkler (Editor), 1999 Novel Inhibitors of Leukotrienes, G. Folco, B. Samuelsson, R.C. Murphy (Editors), 1999 Metalloproteinases as Targets for Anti-Inflammatory Drugs, K.M.K. Bottomley, D. Bradshaw, J.S. Nixon (Editors), 1999 Gene Therapy in Inflammatory Diseases, C.H. Evans, P. Robbins (Editors), 2000 Cellular Mechanisms in Airways Inflammation, C. Page, K. Banner, D. Spina (Editors), 2000 Inflammatory and Infectious Basis of Atherosclerosis, J.L. Mehta (Editor), 2001 Neuroinflammatory Mechanisms in Alzheimer’s Disease. Basic and Clinical Research, J. Rogers (Editor), 2001 Inflammation and Stroke, G.Z. Feuerstein (Editor), 2001 NMDA Antagonists as Potential Analgesic Drugs, D.J.S. Sirinathsinghji, R.G. Hill (Editors), 2002 Mechanisms and Mediators of Neuropathic pain, A.B. Malmberg, S.R. Chaplan (Editors), 2002 Bone Morphogenetic Proteins. From Laboratory to Clinical Practice, S. Vukicevic, K.T. Sampath (Editors), 2002 The Hereditary Basis of Allergic Diseases, J. Holloway, S. Holgate (Editors), 2002 Inflammation and Cardiac Diseases, G.Z. Feuerstein, P. Libby, D.L. Mann (Editors), 2003 Mind over Matter – Regulation of Peripheral Inflammation by the CNS, M. Schäfer, C. Stein (Editors), 2003 Heat Shock Proteins and Inflammation, W. van Eden (Editor), 2003 Pharmacotherapy of Gastrointestinal Inflammation, A. Guglietta (Editor), 2004 Arachidonate Remodeling and Inflammation, A.N. Fonteh, R.L. Wykle (Editors), 2004 Recent Advances in Pathophysiology of COPD, P.J. Barnes, T.T. Hansel (Editors), 2004 Cytokines and Joint Injury, W.B. van den Berg, P. Miossec (Editors), 2004

Cancer and Inflammation, D.W. Morgan, U. Forssmann, M.T. Nakada (Editors), 2004 Bone Morphogenetic Proteins: Bone Regeneration and Beyond, S. Vukicevic, K.T. Sampath (Editors), 2004 Antibiotics as Anti-Inflammatory and Immunomodulatory Agents, B.K. Rubin, J. Tamaoki (Editors), 2005 Antirheumatic Therapy: Actions and Outcomes, R.O. Day, D.E. Furst, P.L.C.M. van Riel, B. Bresnihan (Editors), 2005 Regulatory T-Cells in Inflammation, L. Taams, A.N. Akbar, M.H.M Wauben (Editors), 2005 Sodium Channels, Pain, and Analgesia, K. Coward, M. Baker (Editors), 2005 Turning up the Heat on Pain: TRPV1 Receptors in Pain and Inflammation, A.B Malmberg, K.R. Bley (Editors), 2005 The NPY Family of Peptides in Immune Disorders, Inflammation, Angiogenesis and Cancer, Z. Zukowska, G.Z. Feuerstein (Editors), 2005 Toll-like Receptors in Inflammation, L.A.J. O’Neill, E. Brint (Editors), 2005 Complement and Kidney Disease, P.F. Zipfel (Editor), 2006 Chemokine Biology – Basic Research and Clinical Application, Volume 1: Immunobiology of Chemokines, B. Moser, G.L. Letts, K. Neote (Editors), 2006 The Hereditary Basis of Rheumatic Diseases, R. Holmdahl (Editor), 2006 Lymphocyte Trafficking in Health and Disease, R. Badolato, S. Sozzani (Editors), 2006 In Vivo Models of Inflammation, 2nd Edition, Volume I, C.S. Stevenson, L.A. Marshall, D.W. Morgan (Editors), 2006 In Vivo Models of Inflammation, 2nd Edition, Volume II, C.S. Stevenson, L.A. Marshall, D.W. Morgan (Editors), 2006 Chemokine Biology – Basic Research and Clinical Application. Volume II: Pathophysiology of Chemokines, K. Neote, G.L. Letts, B. Moser (Editors), 2007 Adhesion Molecules: Function and Inhibition, K. Ley (Editor), 2007 The Immune Synapse as a Novel Target for Therapy, L. Graca (Editor), 2008 The Resolution of Inflammation, A.G. Rossi, D.A. Sawatzky (Editors), 2008 Bone Morphogenetic Proteins: From Local to Systemic Therapeutics, S. Vukicevic, K.T. Sampath (Editors), 2008 Angiogenesis in Inflammation: Mechanisms and Clinical Correlates, M.P. Seed, D.A. Walsh (Editors), 2008 Matrix Metalloproteinases in Tissue Remodelling and Inflammation, V. Lagente, E. Boichot (Editors), 2008 Microarrays in Inflammation, A. Bosio, B. Gerstmayer (Editors), 2009 Th 17 Cells: Role in Inflammation and Autoimmune Disease, B. Ryffel, F. Di Padova (Editors), 2009 New Therapeutic Targets in Rheumatoid Arthritis, P.P. Tak (Editor), 2009 The Hygiene Hypothesis and Darwinian Medicine, G.A.W. Rook (Editor), 2009 Occupational Asthma, T. Sigsgaard, D. Heederik (Editors), 2010 Inflammatory Cardiomyopathy (DCMi) – Pathogenesis and Therapy, H.P. Schultheiss, M. Noutsias (Editors), 2010