Vascular Complications in Human Disease: Mechanisms and Consequences

Vascular Complications in Human Disease David Abraham, Clive Handler, Michael Dashwood, and Gerry Coghlan (Eds.) Vas...

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Vascular Complications in Human Disease

David Abraham, Clive Handler, Michael Dashwood, and Gerry Coghlan (Eds.)

Vascular Complications in Human Disease Mechanisms and Consequences

David Abraham, PhD Professor of Cellular and Molecular Biology Royal Free and University College Medical School London UK

Clive Handler, BSc, MD, MRCP, FACC, FESC Consultant in Pulmonary Hypertension The National Pulmonary Hypertension Unit Royal Free Hospital London and Honorary Senior Lecturer Department of Medicine Royal Free and University College Hospital Medical School London UK

Michael Dashwood, PhD Principal Research Fellow Clinical Biochemistry Royal Free and University College Hospital Medical School London UK

Gerry Coghlan, MD, FRCP Consultant Cardiologist Royal Free Hospital London UK

British Library Cataloguing in Publication Data Vascular complications in human disease : mechanisms and consequences 1. Blood-vessels – Diseases – Congresses 2. Diseases – Complications – Congresses I. Abraham, David 616.1′3 Library of Congress Control Number: 2007928817 ISBN: 978-1-84628-918-7 e-ISBN: 978-1-84628-919-4 © Springer-Verlag London Limited 2008 Apart from any fair dealing for the purposes of research or private study, or criticism or review, as permitted under the Copyright, Designs and Patents Act 1988, this publication may only be reproduced, stored or transmitted, in any form or by any means, with the prior permission in writing of the publishers, or in the case of reprographic reproduction in accordance with the terms of licences issued by the Copyright Licensing Agency. Enquiries concerning reproduction outside those terms should be sent to the publishers. The use of registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant laws and regulations and therefore free for general use. Product liability: The publisher can give no guarantee for information about drug dosage and application thereof contained in this book. In every individual case the respective user must check its accuracy by consulting other pharmaceutical literature. 9 8 7 6 5 4 3 2 1 Springer Science+Business Media springer.com

Foreword

Vascular disease provides a very clear example of the inherent and practical relationship between basic science and clinical medicine. Basic science underpins the rationale of treatment and potential future therapeutic approaches; problems in clinical management often direct new lines of laboratory research. Progress in treatment cannot continue without this close collaboration. Indeed, our improved understanding of pathobiological mechanisms in vascular disease has resulted in several exciting advances in clinical management, for example, pulmonary arterial hypertension. Endothelin, nitric oxide, and prostanoid biology has led to the development of new treatment options for patients with life-threatening and rare conditions. We hope that this short book conveys the flavor of the meeting and is a useful contribution to the literature. We have attempted to present our current knowledge and way of thinking about vascular complications of disease which will be of relevance and interest to basic scientists, the many and varied clinical specialists who look after patients with vascular disease, as well as those whose responsibility it is to provide treatment for patients with these common, challenging, and complex conditions. Although the full picture remains hazy, this book presents our understanding of some of the key bits of the jigsaw, which we hope will offer our patients improved survival and a better quality of life. Professor Dame Carol Black, DBE, FRCP, FMedSci Chairman, Academy of Medical Royal Colleges, London Emeritus Professor of Rheumatology Royal Free Hospital London, UK Lewis J. Rubin, MD, FRCP (Hon) University of California San Diego School of Medicine La Jolla, CA, USA Jeremy D. Pearson, PhD, FMedSci Professor of Vascular Biology Cardiovascular Division King’s College London London, UK v

Preface

The vascular system in mammals provides a conduit for the transport of oxygen and vital nutrients to all tissues and cells of the body. Vascular disease results in malfunction or death of organs. It has been nearly 400 years since the English physician, William Harvey, described his revolutionary theory on the circulatory system. Interest has evolved from mere anatomical observations to the detailed scrutiny of the cellular and molecular mechanisms that underlie the pathophysiology of human vascular diseases and associated complications. This book is a compilation of 18 papers presented by world experts on important aspects of vascular biology and the maintenance of normal blood vessel integrity and function and tone and common complications of vascular diseases. The papers were presented at a meeting held at the Royal College of Physicians in London on July 7, 2006, chaired by Professor Dame Carol Black. The aim of the meeting was to provide a forum where basic and clinical scientists, clinicians in several different specialties, and vascular surgeons, were able to discuss established and evolving parts of the jigsaw of this fascinating field of medical science. This book is divided into six sections, bringing together key and contemporary areas in the study, pathogenesis, management, and treatment of some of the most common vascular diseases that affect humans. It includes discussions of approaches to gene therapy and aspects of regenerative medicine. Section one starts with a retrospective account and visionary exploration of treatments for pulmonary vascular disease. It also focuses on the major endothelial-derived mediators, including nitric oxide, endothelin, and the TGF-β/ BMP pathways that impact upon cardiovascular physiology, influencing both endothelial cell activity and smooth muscle cells responses, and cardiomyocyte function. Section two concerns the use of model systems to study and repair vascular dysfunction. These studies highlight the importance of the inflammatory response and the soluble and transcription factors that promote and modulate the characteristic vascular remodeling processes that take place following injury. Section three focuses on vascular complications of systemic disease, therapies for vasculopathy in connective tissue diseases, and transplant-related vasculopathy in the kidney. Surgical approaches to the management of vii

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Preface

coronary bypass graft patency and saphenous vein harvesting for coronary artery bypass grafting and the medical treatment for peripheral vascular disease are presented in section four Section five is concerned with the importance of, and evidence for lowering total cholesterol and LDL cholesterol in clinical practice with statins. Novel preclinical gene therapies to modulate lipid composition in vascular diseases are also reviewed. The final section is concerned with genetics, gene therapy, and tissue engineering for vascular diseases. We believe that it is paramount that clinical specialists who look after patients with any form of vascular disease should understand relevant aspects of the basic scientific rationale for disease pathogenesis. We have therefore tried to present a “joined-up” review of our current understanding of what we think are the most important areas of clinical science and medicine in vascular disease and, we hope, identified areas for research in the future. We hope that this book will be a useful reference to basic and clinical scientists—clinical specialists interested in this broad-ranging subject. We are very grateful to our authors who made the meeting very enjoyable and a great success, and who have provided authoritative chapters for this book. William Harvey’s revolutionary theory has withstood the test of time. However, we do not, unfortunately, know which areas of current research that we present here will be similarly enduring. It is this uncertainty that excites those involved in this fascinating area of medical science and practice. David Abraham Clive Handler Michael Dashwood Gerry Coghlan

Contents

Foreword by Dame Carol Black, Lewis J. Rubin, and Jeremy D. Pearson . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

v

Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

vii

Section One Treatments for Pulmonary Vascular Disease 1

Therapy for Pulmonary Vascular Disease: Past, Present, and Future . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Lewis J. Rubin

3

2

Nitric Oxide Axis in Cardiopulmonary Disease . . . . . . . . . . . . . . . . . Patrick Vallance

9

3

Endothelin Signaling in the Cardiomyocyte . . . . . . . . . . . . . . . . . . . . Peter H. Sugden and Angela Clerk

14

4

TGF-β/BMP Signaling in Pulmonary Vascular Disease. . . . . . . . . . . Rachel J. Davies and Nicholas W. Morrell

46

Section Two Model Systems 5

Endothelin System in Chronic Kidney Disease. . . . . . . . . . . . . . . . . . Neeraj Dhaun, David J. Webb, and Jane Goddard

6

Endothelial Activation in Inflammation: Lessons Learned from E-Selectin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dorian O. Haskard

77

Pathogenic Mediators of Vessel Sclerosis: Regulation of Vascular Smooth Muscle Cell Proliferation by Growth Factors, the Extracellular Matrix, and the Endothelium . . . . . . . . . . . . . . . . . Mark Bond, Yih-Jer Wu, Graciela Sala-Newby, and Andrew C. Newby

94

7

8

Control of Interstitial Fluid Homeostasis: Roles of Growth Factors and Integrins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Kristofer Rubin, Åsa Lidén, Tijs van Wieringen, and Rolf K. Reed

63

105

ix

x

Contents

Section Three 9

10

Vascular Complications

Vascular Complications of Systemic Sclerosis: A Molecular Perspective . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Daryll M. Baker and Christopher Denton

119

Therapeutic Options for Preventing Transplant-Related Progressive Renal and Vascular Injury . . . . . . . . . . . . . . . . . . . . . . . . Susanna Tomasoni and Ariela Benigni

128

Section Four Surgical Approaches 11

12

13

Current State of Medical Therapies for Peripheral Vascular Disease. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Janice C.S. Tsui and Daryll M. Baker

139

Advantages of Harvesting the Saphenous Vein for Coronary Artery Bypass Surgery Using the “No-Touch” Technique . . . . . . . . Domingos Sávio Ramos de Souza and Bruno Botelho Pinheiro

150

Toward the Prevention of Vein Graft Failure . . . . . . . . . . . . . . . . . . . Jamie Y. Jeremy, Sarah J. George, Nilima Shukla, Marcella Wyatt, Jonathon Bloor, Andrew C. Newby, and Gianni D. Angelini

158

Section Five Genetics, Gene Therapy, and Tissue Engineering 14

Statins and Cholesterol: How Low Can You Go? . . . . . . . . . . . . . . . . Dimitri P. Mikhailidis

15

Endothelin-1–Promoting Actions in the Growth and Angiogenesis of Solid Cancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Marilena Loizidou

187

Gene Therapy for Apolipoprotein A-I and HDL—The Ultimate Treatment for Atherosclerosis. . . . . . . . . . . . . . . . . . . . . . . . Petra Disterer, Eyman Osman, and James S. Owen

197

ETS Family of Transcription Factors and the Vascular System. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Masaomi Yamasaki

213

16

17

18

179

Aortic Valve: From Function to Tissue Engineering . . . . . . . . . . . . . Adrian H. Chester, Najma Latif, Magdi H. Yacoub, and Patricia M. Taylor

229

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

241

Contributors

Gianni D. Angelini, MD, MCh, FRCS, FETCS Bristol Heart Institute University of Bristol Bristol, UK Daryll M. Baker, PhD, FRCS Vascular Unit Department of Surgery Royal Free and University College Medical School Hampstead, London, UK Ariela Benigni, PhD Department of Molecular Medicine “Mario Negri” Institute for Pharmacological Research Negri Bergamo Laboratories Bergamo, Italy Jonathon Bloor, PhD Bristol Heart Institute University of Bristol Bristol, UK Mark Bond, BSc, PhD University of Bristol Bristol Heart Institute Bristol Royal Infirmary Bristol, UK

Adrian H. Chester, PhD Department of Cardiothoracic Surgery Heart Science Centre Harefield Hospital National Heart and Lung Institute Imperial College of Science Technology and Medicine Harefield, UK Angela Clerk, BSc, PhD National Heart and Lung Institute Faculty of Medicine Imperial College London London, UK Rachel J. Davies, MA, MRCP Department of Respiratory Medicine University of Cambridge Cambridgeshire, UK Christopher Denton, MD, PhD, FRCP Centre for Rheumatology Royal Free and University College Medical School London, UK xi

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Contributors

Neeraj Dhaun, MBChB, MRCP Department of Renal Medicine and Clinical Pharmacology Unit The Queen’s Medical Research Institute University of Edinburgh Edinburgh, UK Petra Disterer, MSc UCL Institute of Hepatology Royal Free and University College Medical School London, UK Sarah J. George, BSc, PhD Bristol Heart Institute University of Bristol Bristol, UK Jane Goddard, MD, PhD, FRCPE Department of Renal Medicine Royal Infirmary of Edinburgh Edinburgh, Lothian, UK Dorian O. Haskard, DM, FRCP, FMedSci BHF Cardiovascular Medicine National Heart and Lung Institute Imperial College London Hammersmith Hospital London, UK Jamie Y. Jeremy, MSc, PhD, FRSH Bristol Heart Institute University of Bristol Bristol, UK Najma Latif, PhD Department of Cardiothoracic Surgery Heart Science Centre Harefield Hospital National Heart and Lung Institute Imperial College of Science Technology and Medicine London, UK

Åsa Lidén, PhD Department of Biomedicine University of Bergen Bergen, Norway Marilena Loizidou, BSc, MSc, PhD Department of Surgery Royal Free and University College Medical School London, UK Dimitri P. Mikhailidis, MD, FFPM, FRCP, FRCPath Department of Clinical Biochemistry Royal Free and University College Medical School London, UK Nicholas W. Morrell, MA, MD, FRCP Department of Respiratory Medicine University of Cambridge Cambridgeshire, UK Andrew C. Newby, MA, PhD University of Bristol Bristol Heart Institute Bristol Royal Infirmary Bristol, UK Eyman Osman, BSc UCL Institute of Hepatology Royal Free and University College Medical School London, UK James S. Owen, BSc, PhD UCL Institute of Hepatology Royal Free and University College Medical School London, UK

Contributors

Bruno Botelho Pinheiro, MD Department of Cardiovascular Surgery Hospital Santa Genoveva-Clinicord Goiânia-Goiás, Brasil

Peter H. Sugden, MA, DPhil National Heart and Lung Institute Faculty of Medicine Imperial College London London, UK

Domingos Sávio Ramos de Souza, MD, PhD Department of Thoracic and Cardiovascular Surgery Örebro University Hospital Sweden

Patricia M. Taylor, PhD Department of Cardiothoracic Surgery Heart Science Centre Harefield Hospital National Heart and Lung Institute Imperial College of Science Technology and Medicine Harefield, UK

Rolf K. Reed, MD, PhD Department of Biomedicine University of Bergen Bergen, Norway Kristofer Rubin, PhD Department of Medical Biochemistry and Microbiology Uppsala University Uppsala, Sweden and Department of Oncology Clinical Sciences Lund Lund University Lund, Sweden

xiii

Susanna Tomasoni, PhD Department of Molecular Medicine “Mario Negri” Institute for Pharmacological Research Negri Bergamo Laboratories Bergamo, Italy Janice C.S. Tsui, MD, MRCS Vascular Unit Department of Surgery Royal Free and University College Medical School Hampstead, London, UK

Lewis J. Rubin, MD, FRCP (Hon) Pulmonary and Critical Care Medicine University of California San Diego School of Medicine La Jolla, CA, USA

Patrick Vallance, MD, PhD, FRCP, FMedSci Department of Medicine University College London London, UK

Graciela Sala-Newby, BSc, MSc, PhD University of Bristol Bristol Heart Institute Bristol Royal Infirmary Bristol, UK

David J. Webb, MD, DSc, FRCP Clinical Pharmacology Unit The Queen’s Medical Research Institute University of Edinburgh Edinburgh, UK

Nilima Shukla, PhD Bristol Heart Institute University of Bristol Bristol, UK

Tijs van Wieringen, MSc IMBIM Uppsala University Uppsala, Sweden

xiv

Contributors

Yih-Jer Wu, MD, PhD Cardiovascular Division and Institute of Traditional Medicine Mackay Memorial Hospital and National Ying-Mang University Taipei, Taiwan Marcella Wyatt, PhD Bristol Heart Institute University of Bristol Bristol, UK

Magdi H. Yacoub, FRS Department of Cardiothoracic Surgery Heart Science Centre Harefield Hospital National Heart and Lung Institute Imperial College of Science Technology and Medicine Harefield, UK Masaomi Yamasaki, MD Division of Rheumatology and Allergy St. Marianna University School of Medicine Kawasaki City, Japan

Section One Treatments for Pulmonary Vascular Disease

1 Therapy for Pulmonary Vascular Disease: Past, Present, and Future Lewis J. Rubin

Introduction Although pulmonary arterial hypertension (PAH) had been comprehensively described both clinically and pathologically by the early 1970s, there were no effective treatments for this condition until 10 years ago. Until then, the approach to treatment was quite simplistic, based on the observation in the early 1950s by Paul Wood that some patients with PAH exhibited a vasodilator response to infused acetylcholine, prompting him to postulate the presence of a “vasoconstrictor factor” in pulmonary hypertension.1 Vasodilators used to treat systemic hypertension were then administered to patients with PAH in the hopes of reversing the vasoconstrictive component and reducing pulmonary artery pressure. Unfortunately, this approach met with very limited success, and PAH remained a devastating condition with a life span of only several years. A greater understanding of the pathogenesis of PAH at the cellular and molecular level was required before treatments could be developed that specifically targeted these pathogenic processes. A fresh and detailed look at the pathology of pulmonary vessels in PAH provided tremendous insight into the processes of injury and repair, demonstrating that extensive vascular remodeling, characterized by growth and proliferation of all the layers of the vessel wall, are characteristic features of PAH.2 The focus shifted, then, from thinking of PAH as a vasoconstrictive disease to an angioproliferative process, with efforts then directed at identifying the mechanisms responsible for this proliferative vascular response (Figure 1-1).

Endothelial Injury as a Central Process in PAH The normal pulmonary vascular endothelium is a rich source of the production and metabolism of a variety of vasoactive mediators, including endothelin, nitric oxide (NO), and prostacyclin (prostaglandin I2). As data emerged showing that endothelial injury results in impaired endothelial function in PAH, a number of potential pathogenic mediators were identified that became targets 3

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FIGURE 1-1. Schematic of the molecular and cellular pathways implicated in the pathogenesis of pulmonary artery hypertension (PAH). (From Yuan XJ, Rubin LJ. Circulation 2005;111:534–538, with permission.)

for therapy. Endothelin, a potent vasoconstrictor and mitogen, is overexpressed by the injured endothelium in PAH.3 This seminal observation led to the development of endothelin receptor antagonist therapy for PAH: by blocking the endothelin receptors, these drugs inhibit the vascular growth induced by the overexpression of endothelin. Bosentan, a dual ETA and ETB receptor antagonist, was the first orally active therapy approved for the treatment of PAH;4,5 selective ETA receptor antagonists have completed clinical trials,6,7 and the first of those, sitaxsentan, has recently received European regulatory approval and is undergoing review in the United States. Prostacyclin (PGI2) is a product of the arachidonic acid cascade that has vasodilatory, platelet antiaggregatory and vascular antiproliferative effects—all of which may play roles in angioprotection under normal conditions. The endothelial injury in PAH results in an underproduction of prostacyclin,8 which may contribute to the angioproliferative process; accordingly, prostacyclin replacement therapy for PAH has an appealing rationale. The first of these,

1. Pulmonary Vascular Disease Therapy

5

intravenous epoprostenol, was approved 10 years ago for the treatment of PAH and remains the most potent but complex therapy for PAH because it requires continuous intravenous infusion through a central venous catheter and portable infusion pump.9 Subsequently, alternative modes of delivery for prostacyclin therapy have emerged, including the inhaled and subcutaneous routes.10,11 These therapies have proved to be highly effective in improving symptoms and survival in this disease. Nitric oxide is an endothelial-derived vasodilator and antiproliferative agent whose production by the injured endothelium in PAH is also diminished.12 Unfortunately, in physiological conditions NO is in a gaseous phase, making it impractical to replace this deficiency in the clinical arena with continuously inhaled NO gas; however, since NO exerts its effects through cGMP, drugs that slow the breakdown of cGMP by inhibiting the enzyme responsible, phosphodiesterase type 5 (PDE5), augment the diminished NO-mediated effects. Sildenafil, a PDE5 inhibitor that is used for male erectile dysfunction (also due in part to a local deficiency of endothelial-derived NO), has now been approved for the treatment of PAH.13 Thus, while we do not have the cure for PAH, and none of these pathways seems to be the exclusive mechanism responsible for the development of this disease, we have now entered into a period of targeted therapy for PAH. Furthermore, treatment strategies that combine medications, thereby targeting multiple pathogenic pathways, have now been demonstrated to be both safe and more effective than monotherapy, and are emerging as the preferred approach to management.

Novel Pathways of Potential Importance in PAH A variety of other substances may play roles as mediators through a final common pathway of pulmonary angiogenesis and may therefore be appealing therapeutic targets for many forms of pulmonary hypertension. These include angiopoeitin,14 platelet-derived growth factor (PDGF),15 and serotonin and its receptors and transporter.16 Abnormalities in the production or activity of these mediators have been described in PAH, and therapies that target these abnormalities are just beginning to be explored. Angiopoeitin is upregulated in the setting of a variety of forms of pulmonary hypertension, both pre- and postcapillary.14 By turning off the production of the bone morphogenetic protein receptor 1A (BMPR1A), the switch for regulating bone morphogenetic protein receptor 2 (BMPR2) production, angiopoeitin upregulation may result in inhibition of apoptosis and thereby contribute to angioproliferation. Since mutations in the BMPR2 gene are associated with familial pulmonary artery hypertension (FPAH), 17 inherited or acquired abnormalities in BMPR2 activity may be a central pathway for the vasculopathy of PAH. Platelet-derived growth factor appears to be a potent mitogenic stimulus in the pulmonary vasculature and is upregulated in several animal models of

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pulmonary hypertension.15 In these models, and more recently in a handful of patients with PAH, imatinib, a PDGF inhibitor that is used to treat acute leukaemia, has been reported to improve pulmonary vascular remodeling and pulmonary hypertension.18 These preliminary observations are encouraging and warrant further investigation in large scale clinical trials. Vasoactive intestinal polypeptide (VIP) is a neurotransmitter with a role in gastrointestinal water and electrolyte balance. Vasoactive intestinal polypeptide is a potent systemic vasodilator, which decreases pulmonary arterial pressure and pulmonary vascular resistance in the monocrotaline model of PAH. It also has effects on platelet activation and vascular smooth muscle cell proliferation. Serum levels of VIP are decreased in patients with PAH compared to normal.19 These observations suggest that VIP may play a role in the pathogenesis of PAH. Indeed, when VIP was administered by inhalation four times daily for 24 weeks to a small series of PAH patients, they experienced improved exercise capacity and pulmonary arterial pressure and vascular resistance.19 While preliminary, these encouraging observations suggest a potential role for VIP replacement therapy in PAH.

Smooth Muscle Dysfunction in PAH Hypoxia induces pulmonary vasoconstriction, which is mediated by a unique pulmonary artery smooth muscle cell membrane-bound voltage-gated potassium channel (Kv). Yuan et al. have demonstrated that this voltage-gated potassium channel, the KV1.5, is not expressed in pulmonary artery smooth muscle cells in patients with Idiopathic PAH.20 Inhibition of this channel’s activity would result in membrane depolarization, opening of voltage gated calcium channels, and resulting in an intracellular influx of calcium; this increased cytosolic calcium would contribute not only to vascular smooth muscle contraction, but to growth and proliferation as well. Thus, altered K+ channel activity, or the resultant increased in cytosolic calcium, may be additional novel targets for therapy of PAH.

Stem Cell Replacement/Transplant Therapy: Rebuilding the Damaged Circulation Endothelial progenitor cells have recently been explored as a potential source for neovascularization of the diseased pulmonary circulation of PAH. A recent study in animals with monocrotaline-induced PAH in whom autologous endothelial progenitor cells were infused demonstrated improvement in pulmonary hemodynamics and reduction in the medial thickness of the small pulmonary arteries.21 Endothelial progenitor cells transfected with nitric oxide synthase appear to augment the therapeutic response. Clinical trials with stem cell therapy are likely to begin soon.

1. Pulmonary Vascular Disease Therapy

7

Summary and Conclusion The management of PAH has evolved dramatically over the past decade—we now have an array of effective treatments that are helping patients with this disease live longer and more comfortable lives. Nevertheless, there is still no cure for PAH, and many patients remain symptomatic despite therapy or are refractory to existing therapies, underscoring the need for additional treatments. As interest in this field increases, new findings regarding the pathogenesis of PAH are emerging and are opening up new opportunities to target these pathways with novel therapies. These approaches, along with exciting technologies such as cellular transplantation, are likely to dramatically change the management algorithm for PAH over the next decade.

References 1. Wood P. Pulmonary hypertension with special reference to the vasoconstrictive factor. Br Heart J 1958;21:557–570. 2. Wagenvoort CA, Wagenvoort H. Primary pulmonary hypertension: a pathologic study of the lung vessels in 156 classically diagnosed cases. Circulation 1970;42: 1163–1184. 3. Giaid A, Yanagisawa M, Langleben D, et al. Expression of endothelin-1 in the lungs of patients with pulmonary hypertension. N Engl J Med 1993;328:1732–1739. 4. Channick RN, Simonneau G, Sitbon O, et al. Effects of the dual endothelin receptor antagonist bosentan in patients with pulmonary hypertension: a randomised placebocontrolled study. Lancet 2001;358:1119–1123. 5. Rubin LJ, Badesch DB, Barst RJ, et al. Bosentan therapy for pulmonary arterial hypertension. N Engl J Med 2002;346:896–903. 6. Barst RJ, Langleben D, Frost A, et al. Sitaxsentan therapy for pulmonary arterial hypertension. Am J Respir Crit Care Med 2004;169:441–447. 7. Galiè N, Badesch D, Oudiz R, et al. Ambrisentan therapy for pulmonary arterial hypertension. J Am Coll Cardiol 2005;46:529–535. 8. Christman BW, McPherson CD, Newman JH, et al. An imbalance between the excretion of thromboxane and prostacyclin metabolites in pulmonary hypertension. N Engl J Med. 1992;327:70–75. 9. Barst RJ, Rubin LJ, Long WA, et al.; and the PPH Study Group. A comparison of continuous intravenous epoprostenol (prostacyclin) with conventional therapy for primary pulmonary hypertension. N Engl J Med 1996;334:296–301. 10. Simonneau G, Barst RJ, Galié N, et al. Continuous subcutaneous infusion of trepostinil, a prostacyclin analogue, in patients with pulmonary arterial hypertension: a doubleblind, randomized, placebo-controlled trial. Am J Respir Crit Care Med 2002;165: 800–804. 11. Olschewski H, Higenbottam TW, Naeije R, et al. Inhaled iloprost for severe pulmonary hypertension. N Engl J Med 2002;347:322–329. 12. Giaid A, Saleh D. Reduced expression of endothelial nitric oxide synthase in the lungs of patients with pulmonary hypertension. N Engl J Med. 1995;333, 214–221. 13. Galiè N, Ghofrani HN, Torbicki A, et al. Sildenafil citrate therapy for pulmonary arterial hypertension. N Engl J Med 2005;353:2148–2157.

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14. Du L, Sullivan CC, Chu D, et al. Signalling molecules in nonfamilial pulmonary hypertension. N Engl J Med 2003;348:500–509. 15. Morrell NW, Yang X, Upton PD, et al. Altered growth responses of pulmonary artery smooth muscle cells from patients with primary pulmonary hypertension to transforming growth factor-β and bone morphogenetic proteins. Circulation. 2001; 104:790–795. 16. Eddahibi S, Humbert M, Fadel E, et al. Serotonin transporter overexpression is responsible for pulmonary artery smooth muscle hyperplasia in primary pulmonary hypertension. J Clin Invest 2001;108:1141–1150. 17. Newman JH, Trembath RC, Morse JA, et al. Genetic basis of pulmonary arterial hypertension. J Am Coll Cardiol 2004;43:33S-39S. 18. Ghofrani HA, Seeger W, Grimminger F. Imatinib in the treatment of pulmonary artery hypertension. N Engl J Med. 2005;353:1412–1413. 19. Petkov V, Mosgoeller W, Ziesch R, et al. Vasoactive intestinal peptide as a new drug for treatment of primary pulmonary hypertension. J Clin Invest 2003;111:1339– 1346. 20. Yuan JX-J, Aldinger AM, Magdalena J, et al. Dysfunctional voltage-gated potassium channels in pulmonary artery smooth muscle cells of patients with primary pulmonary hypertension. Circulation 1998;98:1400–1406. 21. Zhao YD, Courtman DW, Deng Y, et al. Rescue of monocrotaline-induced pulmonary arterial hypertension using bone marrow-derived endothelial-like progenitor cells: efficacy of combined cell and eNOS gene therapy in established disease. Circ Res 2005;96:442–450.

2 Nitric Oxide Axis in Cardiopulmonary Disease Patrick Vallance

Synthesis of Nitric Oxide Nitric oxide is made by a wide variety of cells, but for the purpose of this chapter it is the endothelial cell and underlying smooth muscle that are of particular interest. The nitrogen in nitric oxide comes from the amino acid L-arginine, and the oxygen comes from molecular oxygen; this reaction is catalyzed by nitric oxide synthase. Nitric oxide has a number of effects in target cells, but one of the major effectors in terms of vascular relaxation is activation of guanalyl cyclase. It does this by interacting with the heme moiety of this enzyme; binding of nitric oxide causes a configurational change in the heme, which then activates guanalase cyclase to increase cGMP synthesis, which in turn leads to vascular relaxation.1

Effects of Nitric Oxide One of the reasons that very early on in the discovery of this pathway it was possible to understand something about the implications of this discovery for human biology was the ability to block nitric oxide synthase with the compound NG monomethyl-L-arginine (L-MNMA) (Figure 2-1). Using this compound and others like it, it is possible to elucidate many of the biological effects of nitric oxide. For example, if a nitric oxide synthase inhibitor is infused into healthy volunteers, the tonic vasodilator tone caused by continuous synthesis of nitric oxide is removed, and this causes an increase in blood pressure.2 The increase in blood pressure is due largely to an increase in systemic vascular resistance, indicating an intense vasoconstriction of the resistance vessels. Using inhibitors such as L-NMMA a large number of effects of endothelial drive nitric oxide have been elucidated. Nitric oxide causes vasodilatation; it can inhibit platelet aggregation and adhesion of platelets to the endothelium. It can block white cell adhesion, inhibit vascular smooth muscle cell growth, and affect cell movement. Through effects on mitrochondial cytochrome c oxidase, it can regulate oxygen consumption, and through some or all of these effects, basal nitric oxide 9

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GENERATOR CELL

02

TARGET CELL

L-NMMA L-citrulline

GTP

NO

L-arginine

cGMP

NO-haem GC

NO synthase

?x Nitric oxide is synthesied from L-arginine by the action of nitric oxide synthase. The nitric oxide generated activates guanylyl cyclase by interacting with the haem moiety of the enzyme. Nitric oxide can be destroyed More rapidly or stablilised through interaction with other species (X)

FIGURE 2-1. Schematic of NO synthetic pathway.

generation exerts an antiathrogenic effect. If nitric oxide synthesis is blocked in experimental models, atheroma formation is enhanced (for review, see Ref. 1). A wide variety of vascular effects of nitric oxide have been demonstrated in a large number of models, but I will focus on pulmonary vasculature.

Nitric Oxide in Pulmonary Vasculature The changes in pulmonary vasculature that are consequent upon disrupting nitric oxide synthase are evident from birth, and there may be important gender differences. For example, in genetically modified mice that lack the gene for endothelial nitric oxide synthase, there is evidence of structural changes in pulmonary vasculature from a very early stage.3 The effects seen in the systemic vasculature when nitric oxide synthesis is inhibited are mirrored in changes in the pulmonary vasculature. For example, when a nitric oxide synthase inhibitor is injected into healthy volunteers, pulmonary tone and pressure increase, and when a nitric oxide synthase inhibitor was given to patients with sepsis, pulmonary vascular pressure rose.4 So, just as in the systemic vasculature, there is a tonic release of nitric oxide from the endothelium of the pulmonary vasculature. Of course it is now well known that inhaled nitric oxide can decrease pulmonary vascular pressure, and cyclic GMP phosphodiesterase inhibitors may have therapeutic utility. Drugs such as sildenafil will amplify the nitric oxide pathway by causing an exaggerated increase in cytic GMP generation.

2. Nitric Oxide Axis in Cardiopulmonary Disease

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There is much evidence that the nitric oxide pathway is important in regulating pulmonary vascular tone and some circumstantial evidence that it might be causal in disease. However it is very difficult to infer causality just because pressure decreases with an agent like inhaled nitric oxide or with a cGMP phosphodiesterase inhibitor. What I want to focus on is evidence that this pathway may be causal in certain types of pulmonary hypertension.

Probing Causality One of the ways to assess this is to determine what happens if the pathway is disrupted; does that lead to a phenotype of pulmonary hypertension? If a nitric oxide synthase inhibitor is administered to an animal or human, pulmonary pressure increases.4 This clearly demonstrates that there is a tonic release of nitric oxide maintaining low pulmonary vascular pressures. Knockout mice that lack the gene for endothelial nitric oxide synthase also demonstrate the phenotype of pulmonary hypertension,5 although the picture is not consistent and there are contradictory publications in this field, possibly related to gender differences, background strain differences, and the age at which mice are studied.

Asymmetrical Dimethylarginine One way in which nitric oxide pathways may be regulated is through the control of endogenous inhibitors that are structurally very similar to L-NMMA. The key endogenous inhibitor seems to be asymmetrical dimethylarginine (ADMA) (Figure 2-2). There are several papers suggesting that ADMA levels are elevated

ARGININE

L-N MONOMETHYL ARGININE (L-NMMA)

ASYMMETRIC DIMETHYLARGININE (ADMA)

SYMMETRIC DIMETHYLARGININE (SDMA)

FIGURE 2-2. Arginine (substrate) and methylated analogues that inhibit NO synthesis. Note that symmetric dimethylarginine is not an inhibitor whereas asymmetric dimethylarginine (ADMA) and L-NMMA are.

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Vallance

in patients with pulmonary hypertension,6–8 but again, there is not an entirely consistent story. However, a recent paper showed that ADMA levels are indeed increased in some patients with pulmonary hypertension and that there appears to be a relationship between the level of ADMA and both the hemodynamic changes and overall survival.8 The question again is whether any of this is causal or whether this is just an epi-phenomenon. ADMA is eliminated from the body by renal excretion and by metabolism by dimethylarginine dimethylaminohydrolase (DDAH). It has become clear that metabolism by DDAH is the major route of elimination of ADMA.9 DDAH expression is decreased in models of pulmonary hypertension, although it is currently unclear which of the two isoforms of DDAH changes most.10,11 Our hypothesis was that if DDAH activity is reduced, ADMA would accumulate, which should inhibit nitric oxide synthesis and, if the pathway is important in disease, should produce a phenotype of pulmonary hypertension. We reduced DDAH activity in two ways—we deleted the gene for DDAH in a mouse and developed small molecule inhibitors of DDAH activity. We found that when the gene is deleted or the enzyme is inhibited chemically, this disrupts endothelial function in the pulmonary vasculature and impairs the ability of the blood vessel to relax to endothelium-dependent dilators. These changes in vascular reactivity are also associated with an increased thickness of the muscular wall of small pulmonary arteries and a rise in right ventricular pressure. The phenotype of the mice is consistent with mild pulmonary hypertension and suggests a causal link between an increase in ADMA and the development of pulmonary hypertension. Similar changes are produced when DDAH is inhibited chemically. Of course vasoconstriction is only part of the story, and it is important to note that there is vascular remodeling in the DDAH knockout animals and in the endothelial nitric oxide synthase knockout mice. The mechanisms underlying these changes are not yet clear, but preliminary data suggests that alterations of cell mobility and migration may be important.

Pulmonary Phenotype as a Consequence of Disrupted Nitric Oxide Signaling So two different approaches—direct loss of nitric oxide synthase and inhibition by endogenous ADMA—cause pulmonary hypertension. The third approach we took was to ask what happens if you interfere with a cofactor for nitric oxide synthase? We looked at mice that are unable to generate tetrohydrobiopterin, a cofactor for nitric oxide synthase. These animals also develop pulmonary hypertension.12,13 Tetrathydrobiopterin-deficient mice have increased right ventricular weight and elevated right ventricular pressures compared to the wild-type mice. Thus, is seems that whichever way the nitric oxide pathway is disrupted, the endpoint is a phenotype of raised pulmonary pressure and pulmonary vascular

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remodeling. The really exciting challenge in this pathway now is to translate these observations into clinical investigation and potential new therapies.

Acknowledgments. The work described in the lecture was undertaken while I was at UCL. It was funded by grants from the BHF and Wellcome Trust.

References 1. Vallance P, Leiper J. Blocking NO synthesis: How, where and why? Nat Rev Drug Discov 2002;1:939. 2. Achan V, Broadhead M, Malaki M, Whitley G, Leiper J, MacAllister R, Vallance P. Asymmetric dimethylarginine causes hypertension and cardiac dysfunction in humans and is actively metabolized by dimethylarginine dimethylaminohydrolase. Arterioscler Thromb Vasc Biol 2003;23:1455–1459. 3. Miller AA, Hislop AA, Vallance PJ, Haworth SG. Deletion of the eNOS gene has a greater impact on the pulmonary circulation of male than female mice. Am J Physiol 2005;289:L299-L306. 4. Petros A, Leone A, Moncada S, Bennett D, Vallance P. Effects of a nitric oxidesynthase inhibitor in humans with septic shock. Cardiovasc Res 1994;28:34–39. 5. Fagan KA, McMurty I, Rodman DM. Nitric oxide synthase in pulmonary hypertension: lessons from knockout mice. Physiol Res 2000;49:539–548. 6. Gorenflo M, Zheng C, Werle E, Fiehn W, Ulmer HE. Plasma levels of ADMA in patients with congential heart disease and hypertension. J Cardiovasc Pharmacol 2001;37:489–492. 7. Pullamsetti S, et al. Increased levels and reduced catabolism of asymmetric and symmetric dimethylarginines in pulmonary hypertension. FASEB J 2005;19:1175– 1177. 8. Kielstein JT, et al. Asymmetric dimethylarginine in idiopathic pulmonary hypertension. Aterioscler Thromb Vasc Biol 2005;25:1414–1418. 9. Achan V, Broadhead M, Malaki M, et al. Asymmetric dimethylarginine causes hypertension and cardiac dysfunction in humans and is actively metabolized by dimethylarginine dimethylaminohydrolase. Arterioscler Thromb Vasc Biol 2003;23: 1455–1459. 10. Millat LJ, Whitley GS, Li D, et al. Evidence for dysregulation of dimethylarginine dimethylaminohydrolase I in chronic hypoxia-induced pulmonary hypertension. Circulation 2003;108:1493–1498. 11. Arrigoni F, Vallance P, Haworth S, Leiper J. Metabolism of asymmetric dimethylarginines is regulated in the lung developmentally and with pulmonary hypertension induced by hypobaric hypoxia. Circulation 2003;107:1195–1201. 12. Nandi M, Miller A, Stidwell R, et al. Pulmonary hypertension in a GTP-cyclohydrolase 1 deficient mouse. Circulation 2005;111:2086–2090. 13. Khoo JP, Zhao L, Alp NJ, et al. Pivotal role for endothelial tertahydrobiopterin in pulmonary hypertension. Circulation 2005;111:2126–2133.

3 Endothelin Signaling in the Cardiomyocyte Peter H. Sugden and Angela Clerk

Introduction Ventricular and atrial cardiomyocytes are the cells responsible for the contractile activity of the heart. This chapter will relate mainly to the ventricular cardiomyocyte. When we discuss the atrial cardiomyocyte, this will be clearly indicated. The majority view is that postnatal mammalian ventricular cardiomyocytes, which constitute the bulk of the cardiac cell mass, are terminally differentiated cells incapable of undergoing complete cycles of cell division (i.e., both karyokinesis and cytokinesis). This has important consequences. Thus, cardiomyocytes respond to a requirement for increased power (such as when the hemodynamic load on the heart is increased) by enlarging and increasing their myofibrillar content in the absence of cytokinesis, a process known as hypertrophy. This response represents growth that is superimposed on normal cardiac growth in developing animals or on the steady-state heart in nongrowing animals. Cardiomyocyte hypertrophy contributes to the clinical condition of cardiac hypertrophy seen in vivo. Equally, the terminally differentiated nature of the cardiomyocyte renders the heart susceptible to myocyte loss such as may occur after myocardial infarction or in heart failure. In this situation, factors that increase cardiomyocyte survival may be crucial. However, the terminally differentiated nature of the cardiomyocyte means that it is essentially resistant to malignant transformation. Any potentially oncogenic mutation would affect only a single cell and the likelihood of clonal division of transformed cells is minimized. The endothelin (ET) isopeptides, of which ET-1 is the most extensively studied, have three major effects on cardiomyocytes.1 They promote hypertrophy (Figure 3-1),2 they increase survival,3 and they affect the contractile properties in a positively inotropic manner.4 The hypertrophic effect is dependent on the ability of ET-1 to promote transcription of genes and translation of mRNA species.5,6 The pro-survival effect may be related to inhibition of apoptosis.3 The basis for the positive inotropic effect is poorly understood and may be speciesspecific but presumably involves changes in the movement of Ca2+ and/or increases in the sensitivity of the myofilaments to Ca2+ concentrations.7 The 14

3. Endothelin Signaling in the Cardiomyocyte

B

A

CONTROL

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C

ENDOTHELIN-1

FIGURE 3-1. The induction of hypertrophy in cardiomyocytes by ET-1. Cardiomyocytes from neonatal rats were either maintained under serum-free conditions (A) or exposed to 100 nM ET-1 (B) for 24 hours. They were immunostained for β-myosin heavy chain and viewed by fluorescence microscopy. Note that myocytes exposed to ET-1 show increased sarcomerogenesis and myofibrillar organization, are larger and more regularly shaped, and form cell–cell contacts. Expression of β-myosin heavy chain, an “index gene” of the hypertrophic response, is also increased. Because they are removed from the constraints of the extracellular matrix and surrounding myocytes, the morphology of the neonatal cells does not resemble that of the adult (panel C). The cells stained for the Z-disc protein, α-actinin, and viewed by confocal microscopy. (From Severs NJ. The cardiac muscle cell. BioEssays 2000;22:188–199, Copyright © 2000; reprinted with permission from Wiley-Liss, Inc., a subsidiary of John Wiley & Sons, Inc.)

inotropic effect may augment the hypertrophic response by subjecting the cardiomyocyte to the allegedly prohypertrophic strain.8 The effects of ET peptides on the growth and survival of the cardiomyocyte depend on their ability to modulate the activities of intracellular signaling pathways,9 and a description of the regulation of these by ET-1 is the major topic of this review. We will not discuss the involvement of the various signaling pathways in the overall regulation of gross cardiomyocyte biology (hypertrophy, survival, and contractile activity) in any detail as these are likely to involve the complex integration of diverse signaling events. A common feature of many signaling pathways is posttranslational, reversible covalent modification of proteins such that the biological activity of the modified protein is altered. Phosphorylation (catalyzed by protein kinases) and dephosphorylation (catalyzed by phosphoprotein phosphatases) are probably the most common forms of modification encountered in eukaryotes (Figure 3-2). The amino acid residues in proteins that are susceptible to phosphorylation are Ser-, Thr-, and Tyr-. Protein kinases and phosphoprotein phosphatases each fall into three groups: Ser-/Thr-specific, Tyr-specific and “dual-specificity” (i.e., they potentially modify Tyr- and Ser-/Thr-residues). There about 400–450 apparently bona fide encoded by the human10or mouse11 genome. The majority are Ser-/Thr-specific or “dual-specificity” (about 350). About 80 are Tyr-specific, and an additional 100 are classed as pseudogenes. Protein kinases often participate in “cascades” in which a tier of protein kinases is organized in a sequential, serial hierarchy, an arrangement which, because of the catalytic nature of the phosphorylation modification, allows signal amplification and, because of the opportunity for inputs at multiple levels, also allows signal integration. In addition to the

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ADP Protein kinase PSer/PThr

Ser/Thr

Phosphoprotein

Protein Tyr

Phosphoprotein phosphatase

PTyr

Pi

FIGURE 3-2. Reversible protein phosphorylation and dephosphorylation. Proteins are phosphorylated by protein kinases (ATP as phosphate donor), which are either Ser-/Thr-specific, Tyr-specific, or dualspecificity (i.e., phosphorylate both Ser-/Thr-residues and Tyr-residues within the same protein). In regulatory protein phosphorylation/dephosphorylation, the dephosphorylated protein and the phosphoprotein possess different biological activities (e.g., catalytic activity, transcriptional transactivating activity, etc.). Phosphoproteins are dephosphorylated by phosphoprotein phosphatases, which are specific for phosphorylated Ser-/ Thr-residues or phosphorylated Tyr-residues or are dual-specificity, thus reversing any activity changes.

multitude of protein kinases, there are about 100 phosphoprotein phosphatases (Tyr-specific and “dual-specificity” phosphatases number about 40 each, and there are about 20 Ser-/Thr-specific enzymes).12,13

The Extracellular Signal–Regulated Kinase 1/2 (ERK1/ERK2) Cascade One cardiomyocyte signaling pathway that is particularly responsive to ET-1 is the extracellular signal–regulated 1/2 (ERK1/ERK2) cascade [also known as the p42/p44 mitogen-activated protein kinase (MAPK) cascade].14 This three-tiered protein kinase “cassette” consists of Raf family (c-Raf, A-Raf or B-Raf) initiator MAPK kinase kinases, an intermediate MAPK kinase step (MKK1/MKK2, also known as MAPK or ERK kinases 1 and 2, MEK1 and 2), and then finally ERK1/ ERK2 (Figure 3-3). One or more of the Raf family activate MKK1 and MKK2 by phosphorylating two Ser-residues (Ser217/Ser221 in human MKK1, Ser222/Ser226 in human MKK2). MKK1/MKK2, which are dual-specificity kinases, then activate ERK1 and ERK2 by phosphorylating a Tyr- and a Thr-residue in the ERK1(Thr202Glu-Tyr204)/ERK2(Thr185-Glu-Tyr187) activation loops (numbering for human proteins). Subsequently, ERK1 and ERK2 phosphorylate myriad other proteins, probably, in many cases, modifying their biological activities.15 These proteins include transcription factors, protein kinases, and phosphoprotein phosphatases (and other signaling proteins), cytoskeletal proteins, proteins involved in the regulation of apoptosis, etc. While attention needs to be paid not only to the specificity of small molecule inhibitors,16,17 but also to the concentrations at


17

which they are used (see, e.g., Ref. 18), there are two commercially available and widely used ERK1/ERK2 cascade inhibitors which are reasonably reliable in terms of their specificity, namely PD98059 and U0126.19–21 Although these have actions that are independent of their ability to inhibit the ERK1/ERK2 cascade,6 they are useful in implicating the cascade in biological processes. In terms of biological responses, the activation of the ERK1/ERK2 cascade is generally thought to be anabolic and, in cells capable of division, it promotes this process. In the heart, our view has been that the ERK1/ERK2 cascade stimulates growth to promote an adaptive hypertrophy,22 although this is still debated. In dividing cells, growth of individual cells can be considered to be part of the

ET-1, ETA receptor, Gq, Phospholipase Cb, PtdInsP2 hydrolysis

Diacylglycerols

Protein kinase C Ras.GTP c-Raf MKK1/MKK2 ERK1/ERK2 (Thr-Glu-Tyr)

PD98059, U0126

ERK1/ERK2 (PThr-Glu-PTyr)

DuSPs, (PSer/PThrPases, PTyrPases)

FIGURE 3-3. Activation of the ERK1/ERK2 cascade by ET-1. The ERK1/ERK2 cascade is strongly activated by ET-1 in cardiomyocytes. The first stages occur in the plane of the plasma membrane. Binding of ET-1 to the ETA receptor (the predominant isoform in cardiomyocytes) activates heterotrimeric Gq proteins ([αq. GDP].βγ → [αq.GTP] + βγ) by GDP/GTP exchange. αq.GTP and possibly βγ activate phospholipase Cβ which hydrolyzes phosphatidylinositol 4,5-bisphosphate (PtdInsP2) to diacylglycerols, which are retained in the plane of the membrane, and soluble inositol 1,4,5-trisphosphate. As described in the text, this initiates the cascade of reactions that result in activated of ERK1 and ERK2. Activation is reversed by dualspecifity phosphoprotein phosphatases (DuSPs), and, because phosphorylation of both a Thr- and a Tyr-residue is necessary for ERK1/ERK2 activation, Ser-/Thr- and Tyr-phosphoprotein phosphatases may also participate. Two widely used and reliable inhibitors (PD98059 and U0126) prevent activation or the cascade at the MKK step.

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overall process of cell division because cells have to grow before they divide. In the cardiomyocyte, hypertrophy could therefore be viewed as an unsuccessful attempt to divide. It is important now to consider the mechanisms by which the Raf family is activated.

Small Guanine Nucleotide-Binding Proteins Small guanine nucleotide-binding (small G) proteins are important in intracellular signaling by virtue of their ability to act as molecular switches or timers.23 More than 100 have been identified in Homo sapiens, and these have been divided into five subfamilies. Bioinformatics approaches have suggested that there may be double this number.24 In their inactive state, small G proteins are noncovalently ligated to GDP. Activation involves exchange of GDP for GTP, a process that is enhanced by guanine nucleotide exchange factor (GEF) proteins. The activation process for Ras is shown in Figure 3-4. Exchange of GDP for GTP causes a conformational change in the small G protein such that its biological activity is altered. Small G proteins are also GTPases, and this activity causes their reversion to the GDP-ligated, inactive conformation. Although the GTPase activities of some small G proteins are relatively low, they can be enhanced by GTPase activating proteins (GAPs). Ha-Ras, Ki-Ras (two alternatively spliced transcripts of Ki-Ras are translated in Homo sapiens) and N-Ras are archetypical members of the Ras subfamily of small G proteins.23,25 We will use the term Ras here to refer collectively to these four isoforms. Plasma membrane localization of Ras is essential for many of its biological activities since it places it in proximity to the transmembrane receptors participating in its activation and also localizes its effectors to the plane of the plasma membrane, a necessity for onward signal transmission. However, there is evidence that additional membrane compartments such as the Golgi may also be involved in Ras signaling.26 Membrane localization is at least partly

GTP

GDP GEF (e.g. Sos)

Ras.GDP

Ras.GTP

GAP

Pi

Raf activation H2 O

FIGURE 3-4. Activation of the small G protein Ras. As described in the text, activation of biologically inactive Ras.GDP involves exchange of GDP for GTP. The cycle is reversed by hydrolysis of GTP to GDP and inorganic phosphate.


19

attributable to covalent irreversible C-terminal posttranslational modification of the immature 189-amino-acid residue Ras precursors.26 This involves (probably irreversible) farnesylation of Cys186, cleavage of the C-terminal tripeptide, and (probably irreversible) carboxyl methylation of the now C-terminal Cys186residue. In the case of Ha-Ras and N-Ras, there is also reversible palmitoylation of C-terminal region Cys-residues. The net result of these modifications is to increase the hydrophobicity of their C-terminus. A number of mutants of wild-type Ras have been identified, and some of these are important in cell transformation and malignancies. In v-Ras, Gly12 of wild-type Ha-Ras is mutated to Val, and this was one of the first viral oncogenes to be identified. This mutation is a common feature in a number of human carcinomas. Analogous mutations in other small G proteins are also activating. V12-Ras has a lowered GTPase activity, and the affinity for GAP binding is diminished.27 The result is a more prolonged activation of Ras, and this explains its oncogenicity, although as mentioned V12-Ras should not be oncogenic in cardiomyocytes, but would be hypertrophic as is indeed the case.28 Other mutations produce as “dominant-negative” species, which interfere with endogenous Ras signaling and can be of use in demonstrating the dependence of biological processes on Ras. Thus, mutation of Ser17 to Asn (N17-Ras) produces a species with lower affinity for GDP/GTP and a higher affinity for GEFs.29 This leads to sequestration of GEFs, thus preventing them from activating endogenous Ras.

Activation of Raf One of the essential stages of activation of the Raf family members (best characterized for c-Raf) is their interaction with the activated forms of Ha-Ras, KiRas, or N-Ras. c-Raf binds to activated Ras (Ras.GTP) at the plasma membrane through the c-Raf N-terminal regulatory domain, thus translocating c-Raf from the cytoplasm to the membrane (Figure 3-3).30 Although binding of c-Raf to Ras.GTP probably does induce a certain level of c-Raf activation, the situation is far more complex and involves numerous, temporarily-distinct, directly- or indirectly-activating or inhibitory phosphorylations of c-Raf along with other modifications,30–32 which are outside the scope of this chapter. Two modifications that appear to be particularly important in the activation of c-Raf are the phosphorylation of Ser338 and Tyr341 by poorly characterized protein kinases. These phosphorylations probably occur in the plane of the membrane, which is why c-Raf is placed in this location through its association with Ras.GTP. Following its activation, Raf is able to phosphorylate and activate MKK1/MKK2.

Activation of the ERK1/ERK2 Cascade in Cardiomyocytes In 1993, we suggested that activation of the ERK1/ERK2 cascade could be important in hypertrophic signaling in the cardiomyocyte.33 This hypothesis was based on the initial demonstration that ERK1 and ERK2 were rapidly

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activated (maximum activation at 3–5 minutes) by ET-1 [and by other hypertrophic agonists such as phorbol 12-myristate 13-acetate (PMA)] in the context of what was known of the growth-promoting effects of ERK1/ERK2 activation in noncardiomyocytic cell lines. One feature of the activation of ERK1/ERK2 by ET-1 and other hypertrophic agonists was its transience with activity stimulated about 3.5-fold at 5 minutes returning to baseline by 60 minutes.34 This is a matter to which we will return when considering the time course of the signaling response. With PMA as agonist, phosphorylated ERK1/ERK2 initially appears in the cytoplasm of the cardiomyocyte and subsequently appears in the nucleus,35 thus appropriately localizing it to influence transcription. Furthermore, we demonstrated that MKK1/MKK2 and c-Raf and A-Raf are powerfully and rapidly (within 3–5 minutes) activated by ET-1 in cardiomyocytes.34,36 In addition, ET-1 rapidly activates Ras, promotes association of Ras with c-Raf and stimulates a Ras-associated MKK1-activating activity (to which c-Raf presumably contributes).35 More detailed examination has shown that all three isoforms of Ras are activated by ET-1 in cardiomyocytes (A. Clerk and P.H. Sugden, unpublished observations) and that it is possible to inhibit phosphorylation of c-Raf(Ser338) without affecting c-Raf activity, suggesting that this phosphorylation is not limiting for c-Raf activation by ET-1.37 [We were unable to examine phosphorylation of c-Raf(Tyr341) because a suitable antibody was not available.] There is good evidence that the non-RPTK and protooncoprotein c-Src and/ or Src family proteins are involved in the activation of c-Raf and possibly A-Raf potentially by phosphorylation of Tyr341 in c-Raf and Tyr302 in A-Raf.30,38–40 There is also evidence that ET-1 activates c-Src ond Src family non-RPTKs in cardiomyocytes.41 Activation of c-Src involves dephosphorylation of an inhibitory phospho-Tyr-residue and an activating autophosphorylation of a catalytic domain Tyr-residue, in addition to less well-characterized phosphorylation and dephosphorylation events.42 However, the mechanisms whereby ET-1 regulates c-Src and Src family non-RPTKs are not well understood.

Upstream from Ras and the ERK1/ERK2 Cascade: Phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] Hydrolysis and Protein Kinase C As reviewed in more detail elsewhere,1 the initial stages of ET-1 signaling in the cardiomyocyte involve proteins and phospholipids associated with the plasma membrane (Figure 3-3). The ETA receptor, which is the predominant ET-1 receptor in rat cardiomyocytes,43,44 is a member of the heptahelical or 7transmembrane span G protein-coupled receptor (GPCR) superfamily. The specific subfamily to which the ETA receptor belongs is the Gq/11PCR subfamily. Activation of Gq/11PCRs generally leads to dissociation of downstream heterotrimeric Gq/11 proteins, activation of phosphoinositide-specific phospholipase Cβ isoforms and hydrolysis of the membrane phospholipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] to produce two “second messengers”:


21

hydrophobic diacylglycerol (DAG), which remains in the plane of the membrane, and soluble inositol 1,4,5-trisphosphate [Ins(1,4,5)P3], which diffuses into the cytoplasm. ETA receptor–stimulated formation of Ins(1,4,5)P3 (and, by implication, DAG) is detectable within seconds of exposure of myocytes to ET1.45 Although Ins(1,4,5)P3 is extremely important in regulating Ca2+ movements through the Ins(1,4,5)P3 receptor Ca2+ channels from stores in the endoplasmic reticulum in nonmyocytic cells, it is not clear whether it fulfills any analogous function in myocytes. In terms of the overall Ca2+ movements associated with the contractile cycle, the general view has been that Ins(1,4,5)P3 receptor– regulated Ca2+ movements play at best a minor role in the ventricular cardiomyocyte, although they may be more significant in the atrial cardiomyocyte.46 Equally, more specialized roles cannot be excluded. Thus, the predominant Ins(1,4,5)P3 receptor (which is effectively a Ca2+ channel) in both ventricular and atrial cardiomyocytes is the type 2 receptor,46–48 and this is associated with the nuclear envelope in the ventricular cardiomyocyte.49,50 Recent evidence suggests that ET-1 stimulates the production of Ins(1,4,5)P3 in the region of the nuclear membrane (Figure 3-5).50 This nuclear system, which is distinct Excitationcontraction coupling

SR Ca2+i mobilisation

MEMBRANE ET-1, ETA receptor, Gq, Phospholipase Cb, PtdInsP2 hydrolysis

Increased Ca2+i CYTOPLASM

Ins(1,4,5)P3 (+ DAG)

Calmodulin (CaM) Calcineurin 2+ Ca i/CaM (CaN)

PhosphoHDAC5?

Ins(1,4,5)P3 receptor

Increased Ca2+i Calmodulin (CaM)

FK506, CsA

PhosphoNFAT

NFAT

HDAC5? NUCLEUS MEF2C

CaMKII GATA4

Transcription MEF2C 0

NFAT

0

Transcription

FIGURE 3-5. Ca2+ signaling to transcription by calcineurin and the nuclear membrane Ins(1,4,5)P3 receptor. As described in the text, formation of inositol 1,4,5 trisphosphate [Ins(1,4,5)P3] stimulates uptake of Ca2+ into the nucleus. By binding to calmodulin (CaM), this increases the activity of Ca2+/CaM kinase II (CaMKII) to phosphorylate histone deacetylase 5 (HDAC5), promoting its nuclear export and derepressing MEF2Cactivated transcription. Alternatively, increased cytoplasmic Ca2+ may lead to FK506/cylcosporin A (CsA)sensitive activation of calcineurin (CaN) and dephosphorylation and nuclear import of NFATs. Again, this may activate transcription, possibly in association with the cardiac-restricted transcription factor GATA4. SR, sarcoplasmic reticulum; DAG, diacylglycerol.

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from sarcoplasmic reticulum-based control of Ca2+ movements involved in excitation–contraction coupling, may control nuclear Ca2+ movements via the Ins(1,4,5)P3 receptor.48 Ins(1,4,5)P3/Ins(1,4,5)P3 receptor-mediated uptake of Ca2+ into the nucleus leads to binding of Ca2+ to calmodulin, which then activates a Ca2+/calmodulin-activated protein kinase (CaMKII). This, in turn, phosphorylates the MEF2C transcription factor–associated transcriptional repressor histone deacetylase 5.50 Phosphorylation of histone deacetylase 5 promotes its export from the nucleus and this allows histone acetylation and derepression of MEF2C-dependent transcription. In contrast to the novel role proposed for the Ins(1,4,5)P3/Ins(1,4,5)P3 receptor, it has been known for many years that DAG exerts a major influence on cardiomyocyte biology. It is a physiological ligand and a major regulator of the majority of the isoforms of the phosphatidylserine-dependent protein kinase, protein kinase C (PKC),51 namely those belonging to the classical subfamily (cPKCs, which are regulated additionally by Ca2+) and the novel subfamily (nPKCs, which are not regulated by Ca2+). As DAG concentrations increase in the membrane, DAG-binding PKC isoforms present in the soluble (cytoplasmic) fraction of the cell translocate to the particulate (membrane) fraction. In contrast to direct assay of the enzymic activities of PKC isoforms, this event is easy to detect by Western blotting and is taken to be indicative of PKC activation. In cardiomyocytes, the soluble pools of nPKCδ and nPKCε translocate to the membrane in their entirety in response to ET-1 within 15–30 seconds.52,53 A gradual retrotranslocation back in to the soluble fraction follows over the next 5–15 minutes.52 Interestingly, the EC50 of ET-1 for translocation of nPKCδ (10 nM) is about 10-fold greater than that for nPKCε,52 meaning that, at low (physiological?) concentrations of ET-1, nPKCε should be preferentially translocated. Although cPKCα is a DAG-binding PKC present in myocytes, it does not detectably translocate in response to ET-1.52 However, since the molar abundance of cPKCα is about 10-fold greater than nPKCε,54 it is feasible that even the translocation of a minor proportion of the cPKCα pool could result in significant changes in PKC activity. Indeed, there is evidence that cPKCα plays important roles in cardiac biochemistry including the development of hypertrophy55 and contractility.56 The molecular events associated with the translocation of PKCs in cardiomyocytes are becoming clearer. PKCs are known to be phosphoproteins, and phosphorylation influences their activities.57 For two agonists of nPKCδ— PMA and H2O2—the translocation events are associated with phosphorylation of the enzyme.58,59 Furthermore, the nPKCδ that retrotranslocates from the particulate to the soluble fraction is a “lipid-independent” form of nPKCδ, i.e., one that does not require exogenous phosphatidylserine and PMA/DAG for activity (although it may contain bound lipids).58,59 We have observed similar responses with ET-1 as an agonist.60 Membrane-localized phosphorylation of PKCs and their concurrent conversion to “lipid-independent” species may be a general scheme activation of DAG-sensitive PKCs in primary cells.


23

Even although an older report suggests that PKC activates Raf directly by phosphorylation,61 it seems highly likely that in cardiomyocytes, PKC is involved in Ras activation (although the mechanism is unclear),35 and this leads to Raf activation. In view of the rapidity of the response, the likeliest candidate mechanism would involve the activation of a GEF (see below). Other PKCindependent mechanisms of Ras activation through DAG-activated GEFs exist in other cells,62 although even here such GEFs may also be regulated by PKCdependent phosphorylation.63

Upstream from Ras and the ERK1/ERK2 Cascade: Other Mechanisms Transactivation of the Epidermal Growth Factor Receptor by GPCRs Activation of the ERK1/ERK2 cascade by transmembrane receptor tyrosine protein kinases (RPTKs) and their ligands is an important facet of normal cell growth and division, and its derangement is important in oncogenesis. As their name implies, they bind extracellular ligands and possess Tyr-kinase activities. Their general mechanisms of activation were elucidated in the 1990s,64 and the following is a simplification (Figure 3-6). The EGF receptor (EGFR or ErbB1)

ET-1

HB-EGF

EXTRACELLULAR

Pro-HB-EGF

ADAM 12

MEMBRANE P

P

P

P

PKC (PKCd?)

REACTIVE OXYGEN SPECIES

EGF Receptor Protein Tyrosine Phosphatases

Sos/GRB2, Ras, ERK1/ERK2 cascade

INTRACELLULAR

FIGURE 3-6. Transactivation of the EGF receptor by ET-1: possible mechanisms. As described in the text, ET-1 activates protein kinase C (PKC) and increases the production of reactive oxygen species. PKC (possibly PKCδ) is suggested to stimulate the activity of the membrane metalloproteinase ADAM12, which cleaves membrane bound pro-heparin-binding EGF-like growth factor (pro-HB-EGF) to HB-EGF. HB-EGF binds to the extracellular domain of the EGF receptor, inducing its dimerization and transautophosphorylation of tyrosine residues in the intracellular domain. This then initiates activation of the ERK1/ERK2 cascade through activation of Ras. Alternatively, reactive oxygen species may inhibit protein tyrosine phosphatases, and tonic autokinase activity of the EGF receptor may also lead to its activation.

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is the archetypical RPTK. Three EGFR orthologues—ErbB2, ErbB3, and ErbB4—have been identified, although of these, only ErbB4 possesses both the ligand-binding and Tyr-kinase activities. ErbB2 (no known ligand) and ErbB3 (no Tyr-kinase activity) are heterodimerization partners either of ErbB1 and ErbB4 or one another. Binding of the ligand (e.g., EGF) to the extracellular domain of an RPTK (e.g., EGFR) causes a conformational change and receptor dimerization, followed by activation of the RPTK cytoplasmic domain Tyrkinase activity. This leads to autophosphorylation of Tyr-residues in the cytoplasmic domains and is followed by specific binding of docking proteins (e.g., Grb2) and/or adaptor proteins (e.g., Shc) to the RPTK phospho-Tyr sequences through their specific recognition domains (SH2 or PTB domains). Grb2 also recognizes Pro-rich sequences in the Ras.GEF Sos through the Grb2 SH3 domains (of which it has two), and its binding to an RPTK places Sos at the membrane where it activates Ras. The ERK1/ERK2 cascade is then activated as described above. In cardiomyocytes too, EGF activates the ERK1/ERK2 pathway through Ras and Raf.65 Adaptor proteins become Tyr-phosphorylated by RPTKs following their binding and their multiple phospho-Tyr sequences are then recognized by docking proteins with resulting signal amplification, leading to Ras activation. In noncardiomyocytes, transactivation of RPTKs (particularly the EGFR) by GPCRs has been suggested as a mechanism whereby GPCR agonists stimulate the signaling pathways that are intimately linked to RPTK activation,66–69 henceforth to be referred to as the “indirect pathway.” The GPCR-stimulated phosphorylation of the EGFR is induced by cleavage of pro-heparin binding EGF-like growth factor (HB-EGF), which is anchored to the external face of the cell surface. This cleavage is effected by a member of the ADAM (a disintegrin and metalloproteinase) family of matrix metalloproteinases (MMPs), followed by release of HB-EGF, which then binds to the EGFR and induces EGFR phosphorylation.67,68,70 A similar scheme has been proposed in cardiomyocytes (Figure 3-6). Here, the overall proposal is that the EGFR becomes Tyr-phosphorylated on exposure to ET-1 following a MMP (ADAM12)-induced cleavage of pro-heparin-binding EGF-like growth factor (HB-EGF).71–73 This leads to activation of the ERK1/ERK2 cascade by the classical RPTK pathway described above.71 EGFR transactivation has thus been proposed to be responsible for GqPCR-mediated hypertrophy, and MMP inhibition has been proposed as a means of its reversal.71 A Role for Oxidative Stress? A normal feature of cellular metabolism is the production of reactive oxygen species (ROS; e.g., superoxide anion radicals, hydroxyl radicals, H2O2) and reactive nitrogen species, and there is a view that ROS function as signaling intermediates or second messengers in their own right.74,75 However, in our view, there are general problems with the concept that ROS are bona fide physiological second messengers. First, unlike other signaling intermediates (e.g., second


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messengers such as cyclic AMP), ROS would seem inherently unlikely to possess the high level of specificity necessary for a signaling molecule. There are no known defined specific receptors for ROS analogous, for example, to cyclic AMP-dependent protein kinases. Second, at higher concentrations, they are cytotoxic. Be that as it may, a number of investigators have shown that ET-1 increases the production of ROS in cardiomyocytes,76–80 possibly by activation of NAD(P)H oxidase77,80 or by increasing their production in the mitochondria.81 The suggested means through which ROS might act as second messengers is by eliciting the reversible oxidation of cysteinyl sulfydryl groups in proteins either to their sulfenic (—SOH) or sulfinic (—SO2H) acid derivatives,75,82 or by conjugation of signaling proteins with small molecules containing —SH groups such as glutathione or Cys itself to form disulfide bonds.83 It has been known for many years that certain phosphatases contain sulfydryl groups at their active sites, and their activities are thus very sensitive to inhibition by oxidation. Phosphatases affected in this way include protein Tyr-phosphatases such as SHP2, “dual specificity” phosphatases such as those that dephosphorylate MAPKs, and the lipid phosphatase PTEN, which dephosphorylates the second messenger PtdIns(3,4,5)P3 (see below) to PtdIns(4,5)P2.84 Thus, one means by which ROS could activate Ras and the ERK1/ERK2 cascade would be through the tonic, basal activity of an RPTK such as the EGFR in combination with inhibition of a protein Tyr-phosphatase (Figure 3-6). Indeed, in cardiac fibroblasts, evidence has been presented that ET-1 stimulates the production of ROS through activation of NAD(P)H oxidase, and the ROS then transiently inhibit SHP2 to cause Tyr-phosphorylation of the EGFR.85 Comments on ET-1–Stimulated EGFR Transactivation and Production of ROS in Cardiomyocytes In our view, there are some problems with the hypothesis that GPCRs utilize the indirect pathway in cardiomyocytes through ADAMs, HB-EGF shedding, and EGFR phosphorylation. For example, the mechanism of GPCR-induced activation of ADAMs is unclear. Furthermore, this proposed pathway of activation of Ras and the ERK1/ERK2 cascade by GqPCR agonists would be very indirect (GPCR activation, ADAM activation, HB-EGF cleavage, EGFR activation, then Ras activation) and involves alternating steps at extracellular and membrane/intracellular locations. This is difficult to rationalize given the rapidity of Ras activation by ET-1 in cardiomyocytes.35 Furthermore, although EGF and ET-1 both activate the ERK1/ERK2 cascade to a similar extent in cardiomyocytes, only EGF activates protein kinase B/Akt,65 implying that there is a basic difference between EGF and ET-1 with respect to their signaling rather than integration at the EGFR level. The indirect pathway was proposed largely on the basis of experiments with inhibitors, particularly with inhibitors of the Tyr-kinase activity of the EGFR.66 As reviewed,86 it assumes a linear sequence of events, but an alternative expla-

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nation is that there are parallel pathways (one from basal EGFR activity, one from GPCR and/or PKC signaling), which come together at the level of Ras.87,88 The EGFR-dependent activation of Ras is mediated by Sos, and the PKCdependent activation of Ras results from activation of a Ras.GEF (possibly Sos). It is clear that GPCR signaling or PMA leads to Tyr-phosphorylation of the EGFR.87,88 Rubio et al.88 propose that this results from a PKC- mediated increase in the production of ROS, followed by HB-EGF shedding and reversible inhibition of protein Tyr-phosphatases (see above). How activation of PKC could promote formation of ROS remains obscure, although, in relation to the increased ROS produced in diabetes, there are reports indicating that PKC activates NAD(P)H oxidase (Nox) in noncardiomyocytes (e.g., Ref. 89). An alternative pathway could involve the established37 ET-1–mediated activation of the small G protein Rac (see below) since Rac.GTP is an activator of the Nox2 isoform,90 one of the isoforms expressed in cardiomyocytes.91

Other Signaling Pathways Modulated by ET-1 Activation of Small Guanine Nucleotide-Binding Proteins of the Rho Family by ET-1 There are about 20 further members of the Ras family in addition to H-Ras, KRas, and N-Ras (the regulation of which by ET-1 has already been described), as well as small G proteins belonging to the Rho, Rab, Ran, and ARF/SAR1 families.23 The best characterized members of the Rho family (RhoA, Rac1, and Cdc42) are principally involved in the regulation of cell shape and migration. In cardiomyocytes, ET-1 rapidly activates RhoA and Rac1,37 but the mechanisms are unclear. Activation of Rho leads to actin polymerization and, with angiotensin II as agonist, leads to premyofibril formation in cardiomyocytes.92 Rho signaling may also exert transcriptional effects through actin polymerization and the nuclear import of the myocardin family transcriptional coactivators,93,94 although the effects of ET-1 on the myocardin family have not yet been reported for cardiomyocytes. Whether the remaining small G proteins are activated in cardiomyocytes is not known, and even their expression characteristics have not been investigated systematically. However, it should be borne in mind that the effects of V12-Rac1, wild-type or (activated) V14-RhoA, or wild-type Rab have been studied in the myocardium of transgenic mice although the resulting phenotypes are not simple and seem predominantly to lead to various cardiomyopathies.95–97

Activation of Stress-Activated Protein Kinase Cascades After identification of ERK1 /ERK2 signaling cascade, the existence of related protein kinase cascades was recognized: the c-Jun N terminal kinase (JNK) cascade and the p38-MAPK cascade.98 These exhibit the same three-tiered orga-

3. Endothelin Signaling in the Cardiomyocyte Cellular stresses

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ET-1

SAPK cascade MKKKs, e.g. ASK1 MKK4/MKK7

MKK3/MKK6

JNKs JNKs (Thr-Pro-Tyr) (PThr-Pro-PTyr)

p38-MAPKs p38-MAPKs (Thr-Gly-Tyr) (PThr-Gly-PTyr)



SB203580 SB202190

FIGURE 3-7. Activation of the SAPK cascades. As described in the text, the SAPK cascades are analogous to the ERK1/ERK2 cascade. However, the processes upstream from the MKK steps are not as well understood. Providing they are used at carefully considered concentrations, SB203580 and SB202190 directly inhibit the activated (phosphorylated) α and β isoforms of p38-MAPK, rather than inhibiting upstream.

nization as the ERK1/ERK2 cascade, and the JNKs and p38-MAPKs are also activated by the dual phosphorylation of an Thr-Xaa-Tyr motif in the activation loop (Figure 3-7). In the case of the JNKs, the phosphorylation motif is ThrPro-Tyr, whereas it is Thr-Gly-Tyr for the p38-MAPKs. Because of general similarities with the ERK1/ERK2 cascade, these cascades were originally classified as MAPK cascades. However, they are most strongly activated by cytotoxic stresses (e.g., hyperosmotic shock, ROS, inhibitors of protein synthesis, arsenite) and are therefore known collectively and more accurately as stressactivated protein kinase (SAPK) cascades. As in the ERK1/ERK2 cascade, the SAPKs are activated by MKKs (or better, SAPK kinases), with MKK3 and MKK6 being primarily responsible for activation of the p38-MAPKs, and MKK4 and MKK7 being responsible for activation of the JNKs. Numerous protein kinases analogous to the Raf MAPK kinase kinases lie upstream.98 The most reliable small molecule inhibitors (SB203580 and SB202190) inhibit p38-MAPKα/β.99 Although a JNK inhibitor (SP600125) has been described,100 there are indications that its use requires careful consideration of its specificity.17 ET-1 activates the JNK and p38-MAPK cascades in cardiomyocytes, although the activation is not as great as that by our “standard” cytotoxic stress of hyperosmotic shock (0.5 M sorbitol).101,102 It is unlikely that activation of JNKs or p38-MAPKs is an epiphenomenon resulting from the use of high concentrations of ET-1.102 How ET-1 signaling leads to activation of SAPKs is unclear. Direct acute activation of Gαq using Pasteurella multocida toxin leads to stimulation of JNK and p38-MAPK (and ERK1/ERK2) phosphorylation, although this is relatively modest for the SAPKs compared with that by hyperosmotic shock.103 This suggests that a pathway does indeed exist from GqPCRs to SAPKs. In

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transgenic mice cardiospecifically overexpressing Gαq or in adenovirally transduced cardiomyocytes expressing mutated, constitutively-activated Gαq, SAPKs are activated (although in these longer-term experiments, it is difficult to know whether this is direct or simply in response to preceding changes).104,105 As mentioned earlier, ET-1 activates nPKCδ and nPKCε in cardiomyocytes.52 Experiments with activated nPKCδ suggest that this PKC isoform may be involved in the activation of SAPKs.106 This contrasts with analogous experiments with nPKCε where only ERK1/ERK2 were strongly activated.106 In addition, in other cell types, activation of SAPKs may involve members of the Rho family of small G proteins such as Rac and Cdc42,98 and, as described above, ET-1 activates Rac in cardiomyocytes.37 Equally, ROS stimulate SAPKs in cardiomyocytes,107,108 and thus, given the observation that ET-1 increases ROS production in these cells,76–80 this may represent an alternative route. However, a detailed description of GqPCR signaling to SAPKs has yet to be forthcoming.

Calcineurin The Ca2+/calmodulin-activated protein Ser-/Thr-phosphatase calcineurin (also known as protein phosphatase 2B) represents an important signaling pathway in the development of cardiac hypertrophy and heart failure.9,109 Unlike the MAPKs/SAPKs (see above) and protein kinase B(PKB)/Akt (see below), its in situ activity can be assessed only indirectly because the noncovalent interactions responsible for its activation are disturbed on cell lysis. Its participation in biological responses can be inferred by its inhibition with the immunosuppressants cyclosporin A or FK506. A major molecular role of calcineurin in T lymphocytes involves the dephosphorylation of NFAT (nuclear factor of activated T cells) transcription factors. In their phosphorylated form, these transcription factors are retained in the cytoplasm, but, on dephosphorylation, they migrate to the nucleus where they affect transcriptional activity. A similar scenario has been proposed for the cardiomyocyte (Figure 3-5). The attraction in this proposal is that it represents a mechanism whereby the force of contraction (itself largely dependent on cytoplasmic Ca2+ concentrations) can affect growth of the cardiomyocyte. ET-1 causes an apparent dephosphorylation of NFATc (NFATc1), and its translocation into the nucleus in cardiomyocytes and translocation was prevented by cyclosporin A.110 Calcineurin-dependent effects of ET-1 on gene transcription have also been identified.94,111 The overall conclusion is that ET-1 is able to stimulate calcineurin activity in the cardiomyocyte presumably by increasing the binding of Ca2+ to calmodulin.

Phosphoinositide 3-Kinase and Protein Kinase B/Akt Phosphoinositide 3-kinase (PI3K), which phosphorylates PtdIns(4,5)P2 to PtdIns(3,4,5)P3, and PKB/Akt represent a second membrane-based signaling pathway that promotes cell growth and survival. Lipid phosphatases such as PTEN and SHIP hydrolyze PtdIns(3,4,5)P3 to bisphosphates [PtdIns(4,5)P2 in


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the case of PTEN, PtdIns(3,4)P2 in the case of SHIPs). A detailed description of the signaling (see Figure 3-8) is beyond the scope of this article but can be found elsewhere.112–115 Experiments with transgenic mice show that activation of the PKB/Akt pathway promotes myocardial growth.116 In cardiomyocytes (as in other cells), the pathway is strongly activated by insulin and peptide growth factors.65,117 However, we have never been able to detect a similar level of activation (as assessed by the occurrence of the activating phosphorylation of PKB/ Akt or by PKB/Akt activity) by ET-1 or other GqPCR agonists,65,117 and we believe that PKB/Akt activation is not an important facet of ET-1 signaling. This does not necessarily preclude the possibility that PtdIns(3,4,5)P3 may possess signaling functions independent of PKB/Akt, although, equally, we are not aware of any reports that have demonstrated direct stimulation of PtdIns(3,4,5)P3 production by ET-1 in cardiomyocytes. One protein kinase whose activity is known to be influenced by PKB/Akt is glycogen synthase kinase 3 (GSK3), of which there are two isoforms, GSK3α and GSK3β.118–120 In its dephosphorylated state, GSK3 is active and it phosphorylates and inhibits glycogen synthase, the enzyme that extends the hexose chains of glycogen through the formation of α(1,4) glycosidic links by the transfer of hexose moieties from UDP-glucose, thus promoting glycogen synthesis. On activation, PKB/Akt phosphorylates GSK3α(Ser21) and GSK3β(Ser9) to inhibit their activities (Figure 3-8), and this is one mechanism that accounts

ETA receptor? PtdIns(4,5)P2 Phosphoinositide 3-kinase (PI3K)

PTEN PtdIns(3,4,5)P3 PtdIns(3)P-dependent kinase 1 (PDK1) PKB/Akt activation GSK3 inhibition

Dephosphorylation and nuclear translocation of NFAT?

Cardiomyocyte hypertrophy?

FIGURE 3-8. Activation of the PI3K/PKB(Akt) pathway. As described in the text, activation of PI3K induces protein kinase B/Akt activation. This inhibits GSK3 and induces dephosphorylation and nuclear translocation of NFAT transcription factors, resulting in cardiomyocyte hypertrophy.

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for the ability of insulin to stimulate glycogen synthesis. It is now recognized that GSK3 fulfills multiple roles in addition to its effects on glycogen synthesis. One finding is that GSK3 phosphorylates the regulatory domain of NFATc (NFATc1), and this causes its cytoplasmic retention.121 In the cardiomyocyte, ET-1 has been reported to result in the phosphorylation and inhibition of GSK3 by a mechanism dependent on activation of PI3K (and presumably of PKB/ Akt).122 This finding does not coincide with our failure to detect significant phophorylation of PKB/Akt by ET-1,65,117 although equally PKB/Akt is not the only kinase able to phosphorylate Ser21/Ser9 in GSK3α/β. For example, 90 kDa ribosomal protein S6 kinase (RSK or MAPKAPK1)123 also phosphorylates GSK3,119 and activation of the ERK1/ERK2 cascade leads to phosphorylation and activation of RSK.123 This suggests that there may be a PKB/Akt-independent route through to GSK3 phosphorylation in cardiomyocytes. In addition to these effects on GSK3 phosphorylation, ET-1 also promotes nuclear accumulation of NFAT (probably NFATc1) in a manner that is dependent on inhibition of GSK3.122 Although incorporating NFAT translocation, this pathway is clearly mechanistically different from the calcineurin-dependent pathway described earlier. The signaling pathway has subsequently been extended to encompass a second substrate of GSK3, namely the transcriptional regulator, β-catenin.124,125

Phospholipase D The phospholipase D (PLD) signaling pathway is activated by ET-1 in cardiomyocytes in a PKC-dependent manner.45 Phospholipase D hydrolyzes phosphatidylcholine to phosphatidate and choline with essentially instantaneous hydrolysis of phosphatidate to DAG. While early studies suggested that PLD could produce DAG for activation of PKCs, its biological function may be to produce phosphatidate rather than DAG, with phosphatidate acting as a signaling intermediate in its own right.126–128 Phosphatidate is also formed by the action of DAG kinase, which terminates the signaling function of DAG formed by phosphoinositide-specific phospholipase Cβ. Because of the differences in degree of saturation of the acyl side chains of PtdIns(4,5)P2 and phosphatidylcholine, DAG and phosphatidate formed by the phosphoinositide-specific phospholipase Cβ/DAG kinase pathway differ from PLD-derived phosphatidate and DAG.126 However, although present, the biological role(s) of the PLD pathway in cardiomyocytes, as in other cells, is obscure.

What Happens After the ERK1/ERK2 and SAPK Cascades Are Activated? The events lying downstream from activation of ERKs and SAPKs by ET-1 are still incompletely characterized in cardiomyocytes (and other cells). These


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protein kinases are proline-directed and phosphorylate Ser-/Thr-residues that lie N-terminal to Pro-residues, although it is necessary for the potential substrate to possess an appropriate docking site for phosphorylation to occur.129 Potential targets for ERK1/ERK2 have been described earlier.15 Activation of SAPKs leads to the activation of MAPK-activated protein kinases 2 and 3 in the case of p38-MAPKs, and phosphorylation of c-Jun in the case of JNKs.98 Some of these events have been shown to occur with ET-1 in myocardial preparations. Thus, 90 kDa ribosomal protein S6 kinases are phosphorylated through the ERK1/ERK2 cascade,130,131 and MAPK-activated protein kinase 2 is activated via the p38-MAPK cascade leading to phosphorylation of the small heat shock protein, Hsp25.102 In some cases, ERK1/ERK2 and SAPKs may “collaborate” in signaling events even though in isolation their biological effects may be very different. This is true in the case of c-jun gene and c-Jun protein expression, as is discussed subsequently. This putative collaboration between MAPKs and SAPKs has yet to be explored in thoroughly either in isolated cells or in vivo.

Changes in Gene Expression Induced by ET-1 ET-1 causes changes in gene expression in cardiomyocytes, and some of these are used as defining criteria of the hypertrophic response. ET-1 induces expression of transcription factor–encoding immediate early genes (c-fos, c-jun, egr1),2,132 re-expression of genes normally expressed only in the foetal cardiomyocyte (e.g., natriuretic peptide genes,2,133 and changes in the expression of genes encoding myofibrillar proteins (e.g., myosin light chain-22) or involved in excitation–contraction coupling (e.g., SERCA2134). In some cases there has been relatively detailed analysis of the signaling pathways regulating expression of specific genes/proteins. Two types of experimental approach have been used. One involves the use of transfected reporter genes [e.g., luciferase (LUX)] under the control of appropriate regulatory promoter regions to examine promoter activity, with extrapolation to regulation of endogenous genes. There are advantages in this approach including its relative ease and speed and site-directed mutagenesis of the promoter is a simple way of examining the involvement of specific nucleotide sequences in expression. The disadvantages include the fact that it is an epigenetic expression of a foreign DNA that is being studied and the fact that, although such an approach is often said to assess transcription, the assessment also involves translation of the encoded reporter. The second involves the study of endogenous gene and protein expression, which we feel is the more likely to produce results of direct relevance to the in vivo situation.

Regulation of Natriuretic Peptide Expression by ET-1 By way of example of the transfection approach, the induction of ANF or BNP reporter genes by ET-1 has been studied. The principal findings of Nemoto et

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al.135 related to the effects of manipulating the activities of the JNK and p38MAPK cascades on the expression of a LUX reporter construct regulated by an extended ANF promoter (-3003ANF-LUX) rather than to the effects of ET-1. However, this work suggests that a small molecule inhibitor of p38-MAPKα/β (SB202190) prevents stimulation of -3003ANF-LUX expression by ET-1. Transfection of wild-type p38-MAPK decreased the stimulation of -3003ANF-LUX expression by ET-1, whereas a nonactivatable JNK mutant increased the stimulation by ET-1, presumably by acting as a dominant-negative inhibitor. The conclusion is that ET-1 activates -3003ANF-LUX expression through the p38-MAPKα/β cascade and this is inhibited by the JNK cascade. The effects of inhibition of the ERK1/ERK2 cascade were not reported. In contrast, direct measurement of the stimulation of endogenous ANF mRNA expression by ET-1 using Northern analysis demonstrated that the SAPK cascades (probably the JNK cascade) stimulated ANF expression.136 [The reason for the equivocation with respect to the SAPK cascades is that the inhibitor used (a dominantnegative MKK4/SEK1 in an adenoviral vector) may inhibit both the JNK and p38-MAPKα/β cascades as the wild-type SEK1/MKK4 has been reported to activate both JNKs and p38-MAPK.137 However, whether SEK1/MKK4 activates p38-MAPK in vivo is controversial.98] In contrast, other work examining the effects of U0126 on the stimulation of endogenous ANF mRNA expression (assessed by Northern analysis) by ET-1 has suggested that activation of the ERK1/ERK2 cascade was responsible.138 The effects of inhibition of the SAPK cascades on the ET-1-induced increase in ANF mRNA abundance was not reported. There have been investigations into the role of specific transcription factors in ET-1–regulated cardiomyocyte gene expression. Cardiomyocyte-restricted members of the GATA transcription factor family are important in the regulation of ANF and BNP expression,139 and their transactivating activity is increased by phosphorylation, although any of a number of protein kinases may be responsible.140 The proximal rat brain BNP promoter region contains two (A/T)GATA(A/G) consensus binding sequences for GATA factors with a further GATA core sequence at the TATA box.141 With specific reference to ET-1, Kerkelä et al.142 showed that the increased binding of GATA-4 to an oligonucleotide probe from the BNP promoter sequence containing the two (A/T)GATA(A/ G) motifs was stimulated by ET-1, and this was inhibited by SB203580 (albeit at a relatively high concentration) but not by (albeit a relatively low concentration of) PD98059, thus implicating p38-MAPKα/β rather than the ERK1/ERK2 cascade in the regulation of GATA-4 binding. Further analysis of the regulation of the BNP promoter by ET-1 by the same group of workers using a LUX reporter regulated by the proximal 534 bp BNP promoter (-534BNP-LUX), which contains all three GATA sequences, showed that stimulation of LUX expression by ET-1 was specifically prevented when an Ets transcription factor binding sequence was mutated.143 Furthermore, electrophoretic mobility shift assays showed that ET-1 increased the binding of an Ets transcription factor (Elk-1) to an Ets sequence oligonucleotide probe. Ets transcription factors are


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regulated by its phosphorylation by ERKs (especially in the case of Elk-1144,145) or SAPKs,146 and here the p38-MAPK cascade was identified as being responsible for the ET-1–induced increase in -534BNP-LUX expression by using transfected constructs encoding dominant-negative components of the ERK1/ERK2 or SAPK cascades. However, when the Ets-binding sequence was mutated, expression became ERK1/ERK2-dependent, the suggestion being that p38MAPK signaling somehow represses ERK1/ERK2-dependent signaling. Overall, apart from being able to state that it would appear that some of the effects of ET-1 on ANF or BNP expression could involve ERK1/ERK2 or SAPKs, very little further can be concluded.

Regulation of Expression of the c-jun Gene and c-Jun Protein by ET-1 We have examined the role of ERK1/ERK2 and the SAPKs in the regulation the expression of the c-jun gene and the c-Jun transcription factor protein by ET-1 in cardiomyocytes.132 The c-jun gene is normally expressed at relatively low abundance, but c-jun mRNA and c-Jun protein are rapidly and transiently induced by ET-1, and this may important in the development of the hypertrophic phenotype. c-Jun binds as a heterodimer to the closely related activator protein-1 (AP-1) or cyclic AMP responsive element (CRE) oligodeoxynucleotide consensus sequences found in the promoter regions of many genes. Preferred partners in the heterodimers are the c-Fos (in the case of AP-1) or ATF2 (in the case of CRE) transcription factors. The transactivating activity of c-Jun is stimulated by phosphorylation of two Ser-residues (Ser63 and Ser73) in its Nterminal transactivation domain.147,148 There are two further phosphorylation sites in this region (Thr91 and Thr93) whose function is less clear,148,149 and there are also sites in the C-terminal DNA-binding domain whose dephosphorylation increases the DNA-binding activity of c-Jun.148,150 The relative biological significance of the individual sites is somewhat unclear. As shown by inhibitor studies, ERK1/ERK2 are involved in the regulation increase in c-jun mRNA and c-Jun protein expression by ET-1 in cardiomyocytes.132 Presumably, the ERK1/ERK2-dependent increase in c-Jun protein is at least in part a reflection of the ET-1–induced increase in c-jun mRNA.132 The ability of the ERK1/ERK2 cascade to stimulate expression of c-jun mRNA is likely to be related to the phosphorylation of transcription factors that transactivate at the c-jun promoter region. There are several different consensus sequences in this region (including two c-Jun-binding AP-1/CRE-like sites, jun1 and jun-2), and thus c-Jun itself could potentially upregulate expression of the c-jun gene. Ser63 and Ser73 of c-Jun can be phosphorylated by ERK1/ERK2 (indeed, these kinases were initially thought to be responsible), and, even recently, it has been suggested that ERK1/ERK2 are important in nonmyocytes when the agonist is PMA.148 However, the kinases now thought to be primarily responsible are the JNKs.151 It is thus possible that JNKs, by phosphorylating

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c-Jun, could upregulate c-jun transcription. Although ET-1 increases c-Jun phosphorylation and although inhibition of JNKs reduces this stimulation, inhibition of JNKs (unlike inhibition of the ERK1/ERK2 cascade) does not reduce stimulation of c-jun transcription by ET-1 in cardiomyocytes.132 By implication, it would appear that the stimulation of c-jun transcription by the ERK1/ERK2 cascade involves transcription factors other than c-Jun or that pathways other than modulation of transcription factor transactivating activities (e.g., mRNA stabilisation) are involved. In addition to binding c-Jun/ATF2 heterodimers, the CRE consensus sequence also binds cyclic AMP–responsive element–binding protein (CREB)/ATF1 heterodimers. Phosphorylation of CREB by a variety of protein kinases on Ser133 increases its transactivating activity. Although there is no change in CREB protein expression (contrast with c-Jun), ET-1 increases phosphorylation of CREB in cardiomyocytes in an ERK1/ ERK2-dependent manner,152 although it is likely that protein kinases lying downstream of ERKs are directly responsible.153–155 The jun-1 core sequence (TGACATCA) more closely resembles a CRE consensus sequence (TGACGTCA) than an AP-1 (TGACTCA) consensus sequence. However, although electrophoretic mobility shift/supershift assays with a jun-1 probe show that there are two protein–probe complexes (one containing CREB, the other containing c-Jun) formed under basal conditions with nuclear extracts from cardiomyocytes, there is no increased binding with ET-1 treatment.152 (Incidentally, mobility shift also show two bands with an AP-1 consensus sequence, although the proteins present were not characterized. ET-1 treatment did not increase in band intensity.152) The overall conclusion is that ERK1/ERK2-mediated phosphorylation of CREB may influence ET-1–regulated c-jun transcription.152 The situation becomes more complicated when the abundance of c-Jun protein is considered. Thus, inhibition of JNKs does reduce the ET-1–induced stimulation of expression of c-Jun protein, and the simplest explanation, for which there is other experimental evidence,156 is that the phosphorylation of cJun by JNKs may increase its stability by reducing its rate of degradation.132 Because c-Jun is induced in cardiomyocytes by ET-1 (i.e., the abundance of cJun is not in a steady state), the overall regulation of c-Jun abundance and transactivating activity by MAPK cascades is clearly a complex matter. This is likely to be true for many transcription factors, particularly those whose abundance changes significantly with time.

Regulation of Overall Gene Expression by ET-1: Microarray Data Until recently, analysis of the regulation of gene expression by ET-1 has focused on only a few genes because techniques for examining a more complete spectrum were unavailable. This has changed with the advent of sequence analysis of expressed transcripts and microarray technology. Using the relatively restricted U34A microarrays, we showed that short-term (2- or 4-hour) exposure of cardiomyocytes to ET-1 increased expression of 72 genes by more than twofold or decreased expression of 6 genes by more than 50%. Using U0126 (a


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relatively reliable inhibitor of activation of the ERK1/ERK2 cascade), we also examined the involvement of the ERK1/ERK2 cascade in these effects. U0126 alone upregulated the expression of 6 genes and downregulated the expression of nine genes, indicating that U0126 alone had effects of gene expression that were independent of the ERK1/ERK2 cascade or that the inhibition of any tonic activity of the ERK1/ERK2 cascade was involved. Four of the genes downregulated by U0126 were also upregulated by ET-1, suggesting that the tonic activity of the cascade was important. Four of the genes that were upregulated by U0126 alone are known to be regulated in noncardiac tissues by the xenobiotic response elements in their promoters and the aryl hydrocarbon receptor and are important in xenobiotic metabolism. The genes whose expression was altered by ET-1 could be clustered into (1) those increased at 2 hours significantly more than at 4 hours with partial inhibition by U0126 (20 genes), (2) those significantly increased at 4 hours significantly more than at 2 hours with partial inhibition by U0126 (27 genes), (3) those increased at both 2 and 4 hours to about the same extent with partial inhibition by U0126 (11 genes), (4) those decreased by ET-1 with partial reversal by U0126 (6 genes), and (5) those increased at 2 or 4 hours which were unaffected by U0126 (14 genes). The general conclusions are that, at the time points chosen, (1) the ERK1/ERK2 cascade regulates, either directly or indirectly, about 80% of the genes whose expression is altered by ET-1 and (2) gene expression even in the relatively short term is distinctly phasic. Given that activation of the ERK1/ ERK2 cascade by ET-1 is transient (returning to baseline activity at 1–2 hours), the latter probably means that an initiating signal from the ERK1/ERK2 cascade is being transduced sequentially from one effector to the next, thus producing a phasic pattern of gene expression. In other words, the effects of ET-1 on gene expression does not simply involve a single step-up or step-down effect, but rather involves a diversification of the initial signal from ERK1/ERK2 that is mediated in a temporal fashion by a relay of signaling molecules. The genes themselves can be divided into (1) genes that encode proteins involved in adhesion, the extracellular matrix, or the cytoskelton, (2) genes that encode growth factors, receptors, or other cell signaling proteins, (3) genes that encode transcriptional regulators, (4) a miscellaneous group of genes, and (5) an unidentified group of expressed RNAs. In view of what has been said before with respect to receptor transactivation, it is of interest that there is upregulation of the genes encoding the ErbB ligands amphiregulin (7.1-fold at 2 hours) and HB-EGF (4.4-fold at 2 hours) and the ERBB feedback inhibitor 1 (3.6-fold at 2 hours) by ET-1 through the ERK1/ERK2 cascade. Also upregulated by ET-1 with inhibition by U0126 are three dual-specificity protein phosphatases (Dusp1, 5, and 6) and three Krüppel-like transcription factors (Klf4, 6, and 10). As described above, the Dusps inactivate ERKs and SAPKs, and their induction through the ERK1/ERK2 cascade represents a feedback mechanism which is responsible for the transience of ERK1/ERK2 activation.157 The possible involvement of Klfs in ET-1 signaling in the cardiomyocyte has not previously been recognized.

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Conclusion In cardiomyocytes, ET-1 is a powerful activator of the ERK1/ERK2 cascade, and this is mediated through Ras and (probably) PKC. The majority of early (2- and 4-hour) transcriptional effects are affected (usually inhibited) by U0126. The ET-1–mediated development of the hypertrophic phenotype is also susceptible to inhibition of the ERK1/ERK2 cascade. The ERK1/ERK2 cascade therefore acts as an important conduit from the ETA receptor GqPCR to intracellular locations (particularly the nucleus) in these cells. ET-1 also activates SAPKs, but the mechanisms and consequences are less clear than for ERK1/ERK2. The activity of PKB/Akt is scarcely affected by ET-1.

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137. Dérijard B, Raingeaud J, Barrett T, et al. Independent human MAP kinase signal transduction pathways defined by MEK and MKK isoforms. Science 1995;267:682– 685. 138. Yue TL, Gu J-L, Wang C, et al. Extracellular signal-regulated kinase plays an essential role in hypertrophic agonists, endothelin-1 and phenylephrine-induced cardiomyocyte hypertrophy. J Biol Chem 2000;275:37895–37901. 139. Temsah R, Nemer M. GATA factors and transcriptional regulation of cardiac natriuretic peptide genes. Regul Pept 2005;128:177–185. 140. Liang Q, Molkentin JD. Divergent signaling pathways converge on GATA4 to regulate cardiac hypertrophic gene expression. J Mol Cell Cardiol 2002;34:611–616. 141. Thuerauf DJ, Hanford DS, Glembotski CC. Regulation of rat brain natriuretic peptide transcription. A potential role for GATA-related transcription factors in myocardial cell gene expression. J Biol Chem 1994;269:17772–17775. 142. Kerkelä R, Pikkarainen S, Majalahti-Palviainen T, Tokola H, Ruskoaho H. Distinct roles of mitogen-activated protein kinases pathways in GATA-4 transcription factor-mediated regulation of B-type natriuretic peptide gene. J Biol Chem 2002; 277:13752–13760. 143. Pikkarainen S, Tokola H, Kerkelä R, Majalahti-Palviainen T, Vuolteenaho O, Ruskoaho H. Endothelin-1-specific activation of B-type natriuretic factor gene via p38 mitogen-activated protein kinase and nuclear ETS factors. J Biol Chem 2003; 278:3969–3975. 144. Marais R, Wynne J, Treisman R. The SRF accessory protein Elk-1 contains a growth factor-regulated transcriptional activation domain. Cell 1993;73:381–393. 145. Hill CS, Marais R, John S, Wynne J, Dalton S, Treisman R. Functional analysis of a growth factor-responsive transcription factor complex. Cell 1993;73:395–406. 146. Sharrocks AD. The ETS-domain transcription factor family. Nat Rev Mol Cell Biol 2001;2:827–837. 147. Smeal T, Binetruy B, Mercola D, et al. Oncoprotein-mediated signaling cascade stimulates c-Jun activity by phosphorylation of serines 63 and 73. Mol Cell Biol 1992;12:3507–3513. 148. Morton S, Davis RJ, McLaren A, Cohen P. A reinvestigation of the multisite phosphorylation of the transcription factor c-Jun. EMBO J 2003;22:3876–3886. 149. Papavassiliou AG, Treier M, Bohmann D. Intramolecular signal transduction in cJun. EMBO J 1995;14:2014–2019. 150. Boyle WJ, Smeal T, Defize LHK, et al. Activation of protein kinase C decreases phosphorylation of c-Jun at sites that negatively regulate its DNA binding activity. Cell 1991;64:573–584. 151. Weston CR, Davis RJ. The JNK signal transduction pathway. Curr Opin Genet Dev 2002;12:14–21. 152. Harrison JG, Sugden PH, Clerk A. Endothelin-1 promotes phosphorylation of CREB transcription factor in primary cultures of neonatal rat cardiac myocytes: implications for the regulation of c-jun expression. Biochim Biophys Acta 2004; 1644:17–25. 153. Xing J, Ginty DD, Greenberg ME. Coupling of the RAS-MAPK pathway to gene activation by RSK2, a growth factor-regulated CREB kinase. Science 1996;273: 959–963. 154. Deak M, Clifton AD, Lucocq JM, Alessi DR. Mitogen- and stress-activated protein kinase-1 (MSK1) is directly activated by MAPK and SAPK2/p38, and may mediate activation of CREB. EMBO J 1998;17:4426–4441.


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155. Shaywitz AJ, Greenberg ME. CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals. Annu Rev Biochem 1999;68: 821–861. 156. Musti AM, Treier M, Bohmann D. Reduced ubiquitin-dependent degradation of c-Jun after phosphorylation by MAP kinases. Science 1997;275:400–402. 157. Farooq A, Zhou MM. Structure and regulation of MAPK phosphatases. Cell Signal 2004;16:769–779.

4 TGF-b/BMP Signaling in Pulmonary Vascular Disease Rachel J. Davies and Nicholas W. Morrell

Introduction The transforming growth factor β (TGF-β) superfamily is a group of multifunctional proteins with over 35 distinct members, including TGF-β, activins, bone morphogenetic proteins (BMPs), and growth differentiation factors.1 They all have profound effects on developmental processes ranging from soft tissue and skeletal development to vasculogenesis.2 The effects are not limited to embryogenesis alone as these molecules are also known to play significant roles in the maintenance and control of adult tissues.2–4 Although TGF-β is the prototypic member of the family, the largest group of cytokines within the TGF-β superfamily comprises the BMPs. They were originally identified as molecules regulating growth and differentiation of bone and cartilage. However, BMPs are now known to regulate growth, differentiation, and apoptosis in a diverse number of cells lines, including mesenchymal and epithelial cells.2–4 There has recently been an increasing body of evidence that abnormalities in TGF-β/BMP signaling results in pulmonary vascular pathologies. Mutations in the bone morphogenetic type II receptor (BMPR-II) have recently been found to account for up to 70%5–7 of cases of familial pulmonary hypertension as well as approximately 25% of sporadic cases.8 Mutations in the TGF type I receptor, ALK1,9 and in endoglin,10 an accessory receptor, have been linked to the development of pulmonary vascular abnormalities seen in hereditary hemorrhagic telangectasia. This chapter explores this evidence further, examining the roles of receptor mutations as well as abnormalities of downstream signaling, which lead to pulmonary vascular pathologies.

Normal TGF-b/BMP Signaling Although the TGF-β superfamily is a large and complex group of ligands and receptors, signaling occurs in a similar way throughout the group via complexes of respective type I and type II receptors. In humans there are seven type I receptors, which are also known as the activin receptor-like kinases (ALK 1–7), and five type II receptors (BMPR-II, ActR-II, ActR-IIB, TGF-βR-II, and TGF46

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β-RIIB). The processes of ligand binding, receptor complex formation, and downstream signaling differ between the TGF/activin receptors and the BMP receptors. BMPR-II, which is a constitutively active serine/threonine kinase, exists in preformed heterodimers with type I receptors, or dimerizes with type I receptors following ligand stimulation.10 Upon complex formation, the type I receptor is phosphorylated by the type II receptor, leading to a conformational change. This in turn leads to phosphorylation of downstream signaling molecules, the main group of which are the small mothers against decapentaplegic (Smad) molecules.11 BMPs signal via a restricted set of receptor-mediated Smads (R-Smads), 1, 5, 8, and 9, which upon phosphorylation form a complex with the common Smad 4 to be translocated to the nucleus. TGF-β and the activin molecules signal via a different set of R-Smads, namely Smads 2 and 3. Subsequent target gene transcription is regulated by a variety of mechanisms, including direct binding of the Smad complex to DNA, and by interaction with other DNA proteins, such as FoxO, E2F4/5, ATF3, and C/EBPb.11 The composition of the resulting complexes dictates whether Smads act as activators or repressors of transcription. Switching off Smad signaling in the cell is achieved via Smad ubiquitination and the regulatory factors, Smurfs12 and Smad phosphatases13 (Figure 4-1).

FIGURE 4-1. Potential interaction between BMP and TGF-β signaling in pulmonary artery smooth muscle cells. Activation of the Smad 1/5 second messengers may inhibit Smad 2/3 phosphorylation, leading to moderated TGF-β signaling. The figure indicates potential sites of therapeutic intervention, including the site of action of the c-ab1 kinase imatinib.

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Although Smad signaling has been recognized as the canonical BMP signaling pathway, there is mounting evidence that the mitogen-activated protein kinases (MAPKs), including p38MAPK, p42/44MAPK(ERK1/2), and JNK/SAPK, are regulated by BMPs and TGF-βs in certain cell types.14,15 MAPK signaling has been reported to positively and negatively regulate Smad signaling depending on the cell type and system studied. The specific pathway activated may depend on which type I/type II heterodimers are stimulated by ligand. For example, the pathway activated by BMPs may depend on whether the preformed type I/type II heterodimers are stimulated by ligand (Smad-dependent pathway) or whether ligand leads to recruitment of type I and type II receptors to the signaling complex (MAPK-dependent pathway).16 It is likely that preformed complexes are heterodimers containing one type I and one type II receptor, while ligandrecruited complexes consist of homo-oligomerized type I receptors and one type II receptor. Thus, structural differences in the receptor complex may account for the selection of distinct signaling pathways.

BMPR-II Mutations in Familial Pulmonary Arterial Hypertension Familial pulmonary hypertension accounts for up to 10% of cases of pulmonary arterial hypertension (PAH),17 and it has been known for some time to be inherited in an autosomal dominant fashion with markedly reduced disease gene penetrance.18 However, it was not until relatively recently, in 2000, that two independent groups identified heterozygous germline mutations in BMPRII in several affected families.19,20 Since this discovery some 144 distinct mutations have been identified in 210 independent patients with familial PAH.21 Subsequent research examining patients with sporadic PAH has also identified germline mutations in BMPR-II in at least 26% of cases.22 Approximately 30% of these are missense mutations occurring in highly conserved amino acids with predictable effects on receptor function, for example, affecting the structure of the extracellular ligand-binding domain. The remaining 70% are frame-shift and non-sense mutations, many of which would be expected to produce transcripts susceptible to non-sense–mediated mRNA decay (NMD). Thus, haploinsufficiency for BMPR-II, resulting in reduced receptor expression, is thought to be the predominant molecular mechanism underlying the inherited predisposition to familial PAH.

The Consequences of BMPR-II Mutation for BMP/TGF-b Signaling Two recent studies have shown that the mechanism by which BMPR-II mutants disrupt BMP/Smad signaling is heterogeneous and mutation specific.23,24 Of the missense mutations, substitution of cysteine residues within the ligand binding or kinase domain of BMPR-II leads to reduced trafficking of the mutant protein


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to the cell surface, a process that may also interfere with BMP type I receptor trafficking. In contrast, BMPR-II molecules with noncysteine mutations within the kinase domain reach the cell surface but fail to activate Smad-responsive luciferase reporter genes due to an inability to phosphorylate BMP type I receptors. Mutations in the ligand-binding and kinase domains exhibit a dominant negative effect on wild-type receptor function in terms of Smad signaling. Interestingly, BMPR-II mutants with missense mutations involving the cytoplasmic tail are able to traffic to the cell surface and are capable of activating Smad-responsive luciferase reporter genes to some extent, but are almost certainly relatively deficient in their ability to transduce signals via Smads. In addition, pulmonary artery smooth muscle cells from mice heterozygous for a null mutation in the BMPR2 gene are also deficient in Smad signaling.25,26 Thus haploinsufficiency or missense mutation seems to lead to a loss of signaling via the Smad1/5 pathway. One study has reported that marked siRNA knockdown of BMPR-II leads to increased Smad signaling in response to some ligands, for example BMP7.25 The significance of this observation remains to be determined. Another potential gain of function as a consequence of BMPR-II mutation was reported in a mouse epithelial cell line. In these cells, transfection with BMPR-II mutant constructs led to ligand-independent activation of p38MAPK.33 Furthermore, these cells showed enhanced serum-induced proliferation as compared to wild-type transfected cells; this abnormal proliferation was completely abolished by treatment with a selective p38MAPK inhibitor, SB203580. Subsequently, however, we were unable to find evidence for constitutive activation of MAPK pathways in pulmonary artery smooth muscle cells (PASMCs) isolated from patients with familial PAH.27 or in cells from heterozygous BMPR-II knockout mice.26 The constitutive activation of MAPK pathways by mutant BMPR-II may be cell type specific. Further detailed studies are needed to elucidate how BMPR-II mutation impacts on MAPK signaling. Our group has previously shown that PASMCs isolated from idiopathic or familial PAH patients exhibit an exaggerated growth response to TGF-β1.28 TGF-β1 is not a ligand for the BMP receptors. In addition, the abnormal response to TGF-β does not seem to be due to alterations in the expression of TGF-β type I, II, or III receptors.28 Of note, mutations in the type I TGF-β receptor, ALK-1, have been observed in patients with severe PAH occurring in families with hereditary hemorrhagic telangiectasia.29 ALK-1 is unusual among the TGF-β receptors in that it signals via Smad1/5, rather than Smad 2/3. This again highlights the potential importance of a loss of Smad1/5 signaling in the vasculature as a cause of pulmonary vascular remodeling. However, a further important functional consequence of loss of Smad1/5 signaling may be a gain of TGF-β signaling via Smad 2/3. Support for this concept comes from experiments in endothelial cells designed to elucidate the diverse responses of cells to TGF-β.30–32 In endothelial cells Smad1/5 functionally antagonizes Smad2/3 signaling. This may be because Smad1/5 signaling competes for availability of the co-Smad, Smad4. In addition, Smad1 may physically interact with Smad3

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and lead to degradation or prevent phosphorylation. This antagonism between TGF-β and BMP signaling pathways provides a mechanism for their often observed functional antagonism in diverse settings. For example, BMP7 can antagonize the epithelial–mesenchymal cell transition induced by TGF-β.33;34 BMP7 inhibits TGF-β–dependent renal fibrosis in animal models.35 In cultured cells BMPs can antagonize TGF-β–induced COX-2 expression.36 and TGF-β– dependent myofibroblast transformation.37 Thus, failure of BMP signaling via Smad1/5 can increase TGF-β/ALK-5/Smad2/3 signaling, which may be part of the molecular switch that determines the altered responsiveness to TGF-β. The potential importance of this is that strategies aimed at reducing TGF-β signaling become rational and realistic therapeutic goals in the treatment of familial PAH.

Studies in Cells and Tissues from PAH Patients BMPR-II expression is widely expressed in normal tissues and cells.38 In the lung BMPR-II is highly expressed on the vascular endothelium of the pulmonary arteries.39 The receptor is also expressed, albeit at a lower level in PASMCs and fibroblasts. We have demonstrated that expression of BMPR-II is markedly reduced in the pulmonary vasculature of patients with mutations in the BMPRII gene.39 Notably, BMPR-II expression was also significantly reduced in the pulmonary vasculature of patients with idiopathic PAH in whom no mutation in the BMPR2 gene was identified. These studies suggest that a critical reduction in the expression of BMPR-II may be important to the pathogenesis of PAH, whether or not there is a mutation in the gene. In addition, since the level of BMPR-II expression in familial cases was considerably lower than predicted from the state of haploinsufficiency, this suggests that some additional environmental or genetic factor may be necessary to further reduce BMPR-II expression below the threshold, which triggers profound vascular remodeling. In further studies we provided evidence that phosphorylation of Smad1/5 was reduced in the pulmonary arterial wall of patients with underlying BMPR-II mutations, and also patients with idiopathic PAH with no identifiable mutation.27 Thus, not only is BMPR-II expression reduced, but activation of the main downstream signaling pathway in patients with familial and idiopathic PAH is also decreased.Other investigators have demonstrated that expression of the type I receptor, BMPRIA, is reduced in PAH due to diverse underlying causes.40 Animal models of pulmonary hypertension, such as that induced by high flow in pigs41 or chronic hypoxia in rats,42 have now also demonstrated reduced BMPR-II expression. Whether these changes in BMPR-II expression observed in animal models are a cause or the consequence of pulmonary vascular remodeling remains to be determined. One study has demonstrated increased expression of phospho-Smad2 in small pulmonary arteries of patients with idiopathic PAH, lending support for the concept that a reduction in BMPR-II/Smad1/5 signaling can lead to increased signaling via TGF-β/ALK-5/Smad2/3.43 Earlier


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studies in idiopathic PAH lung had confirmed increased expression of TGF-β isoforms in remodeling arteries.44

The Effects of BMP Stimulation on PASMCs Several groups have begun to explore the response of pulmonary vascular cells from patients and controls in vitro. The response of PASMCs to BMP ligands depends to some extent on the anatomical origin of cells. The serum-stimulated proliferation of cells harvested from the main or lobar pulmonary arteries tend to be inhibited by TGF-β1 and BMPs 2, 4, and 7.27 Indeed, BMPs may induce apoptosis in these cells.45 Using a dominant negative Smad1 construct, the growth inhibitory effects of BMPs have been shown to be Smad1 dependent.27 In contrast, in PASMCs isolated from pulmonary arteries 1–2 mm in diameter, BMPs 2 and 4 stimulate proliferation.27 This pro-proliferative effect of BMPs in peripheral cells is dependent on the activation of ERK1/2 and p38MAPK. Both Smad and MAPK pathways are activated to a similar extent in cells from both locations, but the integration of these signals by the cell seems to differ (Figure 4-2). Pulmonary artery smooth muscle cells from patients with familial PAH and mutations in BMPR-II can be shown to have a reduced capacity to activate Smad1/5.27 This is coupled to a reduced ability to suppress proliferation of PASMCs isolated from lobar pulmonary arteries. In PASMCs from peripheral small arteries, which proliferate in response to BMPs in a MAPKdependent manner, the prediction would be that in the absence of a counteractive Smad pathway, that MAPK dependent proliferation would go unchecked.

PAEC

BMPR2-wt TGF-β1

PASMC

Serum factors

BMPR2-mutant

Apoptosis of PAECs

TGF-β1

Proliferation and survival of PASMCs

FIGURE 4-2. Model of the contrasting effects of BMP signaling in the endothelium and smooth muscle layers of the pulmonary artery. Endothelial cells harboring mutations in BMPR-II are prone to apoptosis, leading to the release of TGF-β, which acts as a pro-proliferative stimulus to the underlying BMPR-II mutant smooth muscle cells.

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The Effects of BMP Stimulation on Vascular Endothelial Cells The response of vascular endothelial cells to BMPs in vitro is in complete contrast to PASMCs. Endothelial cells proliferate, migrate, and form tubular structures in response to BMP4.46 The proliferation is driven via Smad1/5 activation and is dependent on the induction of the inhibitors of DNA binding (ID) family of transcription factors. In addition, BMPs protect endothelial cells from apoptosis.47 Knockdown of BMPR-II with siRNA increases the susceptibility of pulmonary artery endothelial cells to apoptosis. The contrasting effects of BMPs in the pulmonary vascular endothelium and the underlying PASMCs provide a compelling model for pulmonary vascular damage and remodeling in familial PAH. A critical reduction in BMPR-II function in the endothelium may promote increased endothelial apoptosis, which compromises the integrity of the endothelial barrier and contribute to endothelial dysfunction. This would allow ingress of serum factors to the underlying intima and stimulate activation of vascular elastases. In addition, apoptosis and engulfment of apoptotic cells is known to be accompanied by the robust release of TGF-β.48 In the underlying media, PASMCs already compromised in their ability to respond to the growth suppressive effects of BMPs are exposed to TGF-β, . . . , which, because of a deficient Smad1/5 pathway, causes an exaggerated growth response, as described above. This emerging hypothesis is now open to direct testing both in vitro and in vivo.

BMP Studies in Transgenic and Knockout Mice Studies of knockout mice reveal the critical role of the BMP pathway in early embryogenesis and vascular development. Homozygosity for a null mutation in BMPR2 is lethal prior to gastrulation.49 Mice deficient in Smad-5, one of the BMP-restricted Smads, die due to defects in angiogenesis, specifically failure to recruit vascular smooth muscle to endothelial structures.50 Heterozygous BMPR-II+/− mice survive to adulthood and breed normally with no readily discernable phenotype. This mouse, at least at the genetic level, might mimic the state of haploinsufficiency underlying the majority of families with PAH. In general, the mouse pulmonary vascular bed seems resistant to extensive and severe vascular remodeling seen in human disease. Heterozygous BMP-II2+/− mice have been shown to have no,26 or little,51 resting elevation of pulmonary arterial pressure under normal conditions. However, when heterozygotes are exposed to lung overexpression of IL-1β52 or chronically infused with serotonin,26 they develop a greater elevation of pulmonary artery pressure compared with wild-type littermate controls. These observations support the hypothesis that BMPR-II dysfunction increases the susceptibility to pulmonary hypertension when exposed to another environmental stimulus. However, this response depends on the stimulus because chronic hypoxia, a commonly used


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animal model, did not increase susceptibility to pulmonary hypertension in BMP-II2+/− mice.26 The relatively low penetrance of the PAH within families supports a “two-hit” hypothesis in which the vascular abnormalities characteristic of idiopathic PAH are triggered by accumulation of genetic and/or environmental insults in a susceptible individual. For example, a germline BMPR-II mutation in combination with a somatic mutation in the BMP pathway or one of the related pathways regulating cell growth and apoptosis may be sufficient to generate clinical disease. We recently excluded somatic mutations in BMPRII in microdissected lesions of pulmonary vascular lesions of familial PAH.53 Environmental injury, such as the ingestion of appetite suppressants resulting in an increase in serotonin signaling, may impose an additional burden predisposing to disease. Acquired somatic mutations in the TGF-β type II receptor and Smad-4 are well-recognized associations with certain gastrointestinal cancers,54 a disease process in which such a two-hit paradigm is well recognized. There is some evidence that increasing the level of BMPR-II dysfunction will cause pulmonary hypertension in mice. Thus, transgenic overexpression of a dominant negative kinase domain mutant BMPR-II in vascular smooth muscle causes increased pulmonary vascular remodeling and pulmonary hypertension.55 Interestingly, transgenic mice expressing a hypomorphic BMPR-II survive gastrulation but die at midgestation with cardiovascular and skeletal defects, including defects in the outflow tract of the heart.56 This study demonstrates the importance of gene doseage in BMP signaling. In addition, cardiac defects have been recognized in some individuals with BMPR2 mutations.57 Further studies are needed with conditional knockout mice to overcome the essential requirement of BMPR-II during early embryogenesis and to examine the importance of endothelial versus smooth muscle expression of mutant BMPR-II. If a more robust model of PAH could be established, this would clearly benefit the search for targeted therapies and in addition would provide a means of searching for genetic modifiers of disease expression.

The Role of the TGF-b Superfamily in Hereditary Hemorrhagic Telangectasia Hereditary hemorrhagic telangectasia (HHT) is an autosomal dominant disorder of vasculogensis characterized by small, localized vascular malformations called telangiectasia.58 Patients are diagnosed with HHT according to the presence of three out of four diagnostic criteria established by the Scientific Board of the HHT International Foundation in 2000.59 These include spontaneous recurrent epistaxis, cutaneous telangiectases, internal organ involvement with lung, central nervous system or liver arteriovenous malformations, and a family history of the disease. Two types of HHT have been described—namely, HHT1, where patients exhibit a higher incidence of pulmonary arteriovenous

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malformations, whereas patients with HHT2 have a later age of onset along with a tendency to developing pulmonary arterial hypertension that is clinically and histologically indistinguishable from IPAH. The two syndromes have been found to be linked to different mutations in TGF-β receptors, with HHT1 associated with mutations in the endgolin accessory receptor,60 whereas positional cloning has mapped the locus of HHT2 to the ALK1 receptor.61 The majority of mutations associated with HHT1 correspond to the extracellular domain of the endoglin receptor, in the first 12 exons, with no mutations as yet identified in nucleotides encoding the transmembrane or cytoplasmic domains.62–64 In contrast, more than 100 mutations have been described for ALK1, all of which encode for the intracellular kinase domain, exons 7 and 8.65 A third group of patients, however, display the clinical characteristics of HHT but do not have mutations in either endoglin or ALK1, and in 2005 a locus was found on chromosome 5, leading to the postulation that there might be a third sydrome, HHT3.66

The Consequences of Mutations in Endoglin and ALK1 Two distinct phases bring about successful angiogenesis. In the first phase, or activation phase, the endothelium is quiescent while mural cells undergo stabilization. In the second activation phase, smooth muscle cells detach from the vessel wall and endothelial cells start to proliferate and migrate to form a tube or primitive vessel.67 These two phases are thought to be mediated by TGF-β through ALK5 and ALK1 receptors.68 ALK5 is ubiquitously expressed on cells and signals through Smad2/3 phosphorylation., whereas ALK1 is mainly expressed on endothelial cells signaling via Smad1/5. Although TGF-β is a ligand for both receptors, when present in low concentrations it binds to ALK1 and promotes endothelial cell migration and proliferation by inducing the expression of proangiogenic genes (Id1, endoglin, and IL1RL1). However, when present in high concentrations, TGF-β binds to ALK5 to inhibit cell proliferation but promote extracellular matrix deposition through phosphorylated Smad2/3 activating the expression of maturation-specific genes (connexin 37, βIG-H3, and plasminogen-activator inhibitor-1). These findings have led to the concept that successful angiogensis occurs as a result of an appropriate balance between ALK1 and ALK5 signaling in the vessel wall and that disruption of this balance by mutations in the endoglin and ALK1 receptors, as occurs in HHT, interferes with this process, leading to abnormal vessel formation.69,70 The interaction between the ALK1 and ALK5 pathways appears not to be exclusively regulated by levels of TGF-β present in the cells, but there may be a direct interaction between the two receptors. In 2002, Goumans et al found that on stimulation of ALK5 by TGF-β ALK1 is recruited into the receptor complex and phosphorylated by the ALK5 receptor kinase. Phophorylated Smad1/5 then antagonizes the activated ALK5-Smad 2/3 pathway downstream


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to shift gene expression from a cell maturation profile to a proangiogenic one.71 From recent evidence the TGF-β superfamily clearly plays an important role in pulmonary vascular remodeling and the pulmonary pathology associated with this process.However, further work is still needed to elucidate how the interaction between the TGF-β and BMP pathways, both intracellularly and between different types of cells, regulates homeostasis of the vascular wall.

References 1. Chang H, Brown CW, Matzuk MM. Genetic analysis of the mammalian transforming growth factor-β superfamily. Endocr Rev 2002;23:787–823. 2. Miyazono K, Maeda S, Imamua T. BMP receptor signaling: Transcriptional targets, regulation of signals, and signaling cross-talk. Cytokine Growth Factor Rev. 2005;16:251–263. 3. Kawabata M, Imamura T, Miyazano K. Signal transduction by bone morphogenetic proteins. Cytokine Growth Factor Rev 1998;9:49–61. 4. Massague J, Chen Y-G. Controlling TGF-β signaling. Genes Dev 2000;14:627–644. 5. The International PPH Consortium, Lane KB, Machado RD, Pauciulo MW, et al. Heterozygous germ-line mutations in BMPR2, encoding a TGF-β receptor, cause familial primary pulmonary hypertension. Nat Genetics 2000;26:81–84. 6. Deng Z, Morse JH, Slager SL, et al. Familial primary pulmonary hypertension (Gene PPH1) is caused by mutations in the bone morphogenetic protein receptor-II gene. Am J Hum Genet 2000;67:737–744. 7. Machado RD, Aldred MA, James V, et al. Mutations of the TGF-β type II receptor BMPR2 in pulmonary arterial hypertension. Hum Mut 2006;27:121–132. 8. Thomson JR, Machado RD, Pauciulo MW, et al. Sporadic primary pulmonary hypertension is associated with germline mutations of the gene encoding BMPR-II, a receptor member of the TGF-β family. J Med Genet 2000;37:741–745. 9. Johnson DW, Berg JN, Baldwin MA, et al. Mutations in the activin receptor-like kinase 1 gene in hereditary haemorrhagic telangiectasia type 2. Nat Genet 1996;13: 189–195. 10. Attisano L, Wrana JL. Signal transduction by the TGF-beta superfamily. Science 2002;296:1646–1647. 11. Massague J, Seoane J, Wotton D. Smad transcription factors. Genes Dev 2005;19: 2783–2810. 12. Shi W, Chen H, Sun J, et al. Overexpression of Smurf1 negatively regulates mouse embryonic lung branching morphogenesis by specifically reducing Smad1 and Smad5 proteins. Am J Physiol Lung Cell Mol Physiol. 2004;286:L293-L300. 13. Chen HB, Shen J, Ip YT, Xu L. Identification of phosphatases for Smad in the BMP/ DPP pathway. Genes Dev 2006;20:648–653. 14. Nohe A, Keating E, Knaus P, Petersen NO. Signal transduction of bone morphogenetic protein receptors. Cell Signal 2004;16:291–299. 15. Massague J. Integration of Smad and MAPK pathways: a link and a linker revisited. Genes Dev 2003;17:2993–2997. 16. Nohe A, Hassel S, Ehrlich M, et al. The mode of bone morphogenetic protein (BMP) receptor oligomerization determines different BMP-2 signaling pathways. J Biol Chem 2002;277:5330–5338.

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growth factor {beta} and bone morphogenetic protein. Mol Cell Biol 2004;24: 4241–4254. Zeisberg M, Hanai Ji, Sugimoto H, et al. BMP-7 counteracts TGF-β1-induced epithelial-to-mesenchymal transition and reverses chronic renal injury. Nat Med 2003;9:964–968. Zeisberg M, Shah AA, Kalluri R. Bone morphogenic protein-7 induces mesenchymal to epithelial transition in adult renal fibroblasts and facilitates regeneration of injured kidney. J Biol Chem 2005;280:8094–8100. Sheares KKK, Jeffery TK, Long L, Yang X, Morrell NW. Differential effects of TGF-β1 and BMP-4 on the hypoxic induction of cyclooxygenase-2 in human pulmonary artery smooth muscle cells. Am J Physiol Lung Cell Mol Physiol 2004;287: L919-L927. Jeffery TK, Upton PD, Trembath RC, Morrell NW. BMP4 inhibits proliferation and promotes myocyte differentiation of lung fibroblasts via Smad1 and JNK pathways. Am J Physiol Lung Cell Mol Physiol 2005;288:L370-L378. Rosenzweig BL, Imamura T, Okadome T, et al. Cloning and characterization of a human type II receptor for bone morphogenetic proteins. Proc Natl Acad Sci USA 1995;92:7632–7636. Atkinson C, Stewart S, Upton PD, et al. Primary pulmonary hypertension is associated with reduced pulmonary vascular expression of type II bone morphogenetic protein receptor. Circulation 2002;105:1672–1678. Du L, Sullivan CC, Chu D, et al. Signaling molecules in nonfamilial pulmonary hypertension. N Engl J Med 2003;348:500–509. Rondelet B, Kerbaul F, Van Beneden R, et al. Prevention of pulmonary vascular remodeling and of decreased BMPR-2 expression by losartan therapy in shuntinduced pulmonary hypertension. Am J Physiol 2005;289:H2319-H2324. Takahashi H, Goto N, Kojima Y, et al. Downregulation of type II bone morphogenetic protein receptor in hypoxic pulmonary hypertension. Am J Physiol Lung Cell Mol Physiol 2006;290:L450-L458. Richter A, Yeager ME, Zaiman A, Cool CD, Voelkel NF, Tuder RM. Impaired transforming growth factor-β signaling in idiopathic pulmonary arterial hypertension. Am J Respir Crit Care Med 2004;170:1340–1348. Botney MD, Bahadori L, Gold LI. Vascular remodelling in primary pulmonary hypertension: potential role for transforming growth factor-beta. Am J Pathol 1994;144:286–295. Zhang S, Fantozzi I, Tigno DD, et al. Bone morphogenetic proteins induce apoptosis in human pulmonary vascular smooth muscle cells. Am J Physiol Lung Cell Mol Physiol. 2003;285:L740-L754. Valdimarsdottir G, Goumans MJ, Rosendahl A, et al. Stimulation of Id1 expression by bone morphogenetic proetin is sufficient and necessary for bone morphogenetic protein-induced activation of endothelial cells. Circulation 2002;106:2263–2270. Teichert-Kuliszewska K, Kutryk MJB, Kuliszewski MA, et al. Bone morphogenetic protein receptor-2 signaling promotes pulmonary arterial endothelial cell survival: implications for loss-of-function mutations in the pathogenesis of pulmonary hypertension. Circ Res 2006;98:209–217. McDonald PP, Fadok VA, Bratton D, Henson PM. Transcriptional and translational regulation of inflammatory mediator production by endogenous TGF-β in macrophages that have ingested apoptotic cells. J Immunol 1999;163:6164– 6172.

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49. Beppu H, Kawabata M, Hamamoto T, et al. BMP type II receptor is required for gastrulation and early development of mouse embryos. Dev Biol 2000;221:249– 258. 50. Yang X, Castilla LH, Xin X, Li C, Gotay J, Wienstein M, Liu PP, Deng CX. Angiogenesis defects and mesenchymal apoptosis in mice lacking smad5. Development 1999;126:1571–1580. 51. Beppu H, Ichinose F, Kawai N, et al. BMPR-II heterozygous mice have mild pulmonary hypertension and an impaired pulmonary vascular remodeling response to prolonged hypoxia. Am J Physiol Lung Cell Mol Physiol 2004;287:L1241-L1247. 52. Song Y, Jones JE, Beppu H, Keaney JF, Jr., Loscalzo J, Zhang YY. Increased susceptibility to pulmonary hypertension in heterozygous BMPR2-mutant mice. Circulation 2005;112:553–562. 53. Machado RD, James V, Southwood M, et al. Investigation of second genetic hits at the BMPR2 locus as a modulator of disease progression in familial pulmonary arterial hypertension. Circulation 2005;111:607–613. 54. Miyaki M, Iijima T, Konishi M, et al. Higher frequency of Smad4 gene mutation in human colorectal cancer with distant metastasis. Oncogene 1999;18:3098–3103. 55. West J, Fagan K, Steudel W, et al. Pulmonary hypertension in transgenic mice expressing a dominant-negative BMPR-II gene in smooth muscle. Circ Res 2004; 94:1109–1114. 56. Delot EC, Bahamonde ME, Zhao M, Lyons KM. BMP signaling is required for septation of the outflow tract of the mammalian heart. Development 2003;130:209– 220. 57. Roberts KE, McElroy JJ, Wong WPK, et al. BMPR2 mutations in pulmonary arterial hypertension with congenital heart disease. Eur Respir J 2004;24:371–374. 58. Shovlin CL, Letarte M. Hereditary haemorrhagic telangiectasia and pulmonary arteriovenous malformations: issues in clinical management and review of pathogenic mechanisms. Thorax 1999;54:714–729. 59. Shovlin CL, Guttmacher AE, Buscarini E, et al. Diagnostic criteria for hereditary hemorrhagic telangiectasia (Rendu-Osler-Weber syndrome). Am J Med Genet 2000; 91:66–67. 60. McAllister KA, Grogg KM, Johnson DW, et al. Endoglin, a TGF-beta binding protein of endothelial cells, is the gene for hereditary haemorrhagic telangiectasia type 1. Nat Genet 1994;8:345–351. 61. Johnson DW, Berg JN, Baldwin MA, et al. Mutations in the activin receptor-like kinase 1 gene in hereditary haemorrhagic telangiectasia type 2. Nat Genet 1996; 13:189–195. 62. Pastella P, Sabba C, Lenato GM, et al. Endoglin gene mutations and polymorphisms in Italian patients with hereditary haemorrhagic telangiectasia. Clin Genet 2003; 63:536–540. 63. Lesca G, Plauchu H, Coulet F, et al; French Rendu-Osler Network. Molecular screening of ALK1/ACVRL1 and ENG genes in hereditary hemorrhagic telangiectasia in France. Hum Mutat 2004;23:289–299. 64. Cymerman U, Vera S, Karabegovic A, Abdalla S, Letarte M. Characterization of 17 novel endoglin mutations associated with hereditary hemorrhagic telangiectasia. Hum Mutat 2003;21:482–492. 65. Abdalla SA, Cymerman U, Johnson RM, Deber CM, Letarte M. Disease-associated mutations in conserved residues of ALK-1 kinase domain. Eur J Hum Genet 2003;11:279–287.


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66. Cole SG, Begbie ME, Wallace GM, Shovlin CL. A new locus for hereditary haemorrhagic telangiectasia (HHT3) maps to chromosome 5. J Med Genet 2005;42:577– 582. 67. Carmaliet P. Mechanisms of angiogenesis and arteriogenesis. Nat Med 2000;6:389– 395. 68. Pepper MS, Vassalli JD, Orci L, Montesano R. Biphasic effects of transforming growth factor-beta 1 on in vitro angiogenesis. Exp Cell Res 1993;204;356– 363. 69. Goumans MJ, Valdimarsdottir G, Itoh S, et al. Activin receptor-like kinase (ALK)1 is an antagonistic mediator of lateral TGF-β/ALK5 signaling. Mol Cell 2004;12: 817–828. 70. Ota T, Fujii M, Sugizaki T, et al. Targets of transcriptional regulation by two distinct type 1 receptors for transforming growth factor-β in human umbilical vein endothelial cells. J Cell Physiol 2002;193:299–318. 71. Goumans MJ, Valdimarsdottir G, Itoh S, et al. Balancing the activation state of the endothelium via two distinct TGF-β type 1 receptors. EMBO J 2002;1743–1753.

Section Two Model Systems

5 Endothelin System in Chronic Kidney Disease Neeraj Dhaun, David J. Webb, and Jane Goddard

Introduction Since its discovery in 1988,1 endothelin (ET) has been widely implicated in the pathophysiology of renal disease. Endothelins are a family of three 21amino-acid peptides, each with distinct genes and tissue distributions, with powerful vasoconstrictor and pressor properties.1–3 Of the three peptides, ET-1 is the major endothelial isoform and, in the human kidney, the only one so far shown to be expressed at the protein level.4 Its main site of vascular production is the endothelial cell, but it is also produced by other cell types, including vascular smooth muscle cells and epicardial cells.5 Within the renal system, it is produced by glomerular epithelial and mesangial cells and renal tubular and medullary collecting duct cells.6 Furthermore, the renal medulla is not only an important site of ET-1 generation, but also contains among the highest concentrations of immunoreactive ET-1 of any organ.7 Regulation of ET synthesis occurs at the level of gene transcription, with the gene product being the 212-amino-acid pre-pro-ET-1. Enhanced gene transcription occurs with a wide range of stimuli.8,9 Those pertinent to chronic kidney disease (CKD) include other vasoactive hormones, such as angiotensin and vasopressin (AVP), the cytokine interleukin-1, oxidized low-density lipoprotein (LDL), reduced extracellular pH, and cyclosporin A (CyA). In contrast, prostacyclin, nitric oxide (NO), and the natriuretic peptides all inhibit gene transcription. Pre-pro-ET-1 is cleaved to big ET-1 (38 amino acids), which is largely biologically inactive.10 Endothelin-converting enzyme (ECE) then splits big ET-1 to the biologically active ET-1 and C-terminal fragment. Once synthesized, the secretion of mature ET-1 from endothelial cells is largely abluminal,11 toward the adjacent vascular smooth muscle, suggesting an autocrine or paracrine mechanism of action. ET-1 acts by binding to two distinct receptors, the ETA and ETB receptors.12,13 Within blood vessels, both receptors are found on smooth muscle cells and their activation results in vasoconstriction. ETB receptors are, however, predominantly found on the vascular endothelium, where their activation results in vasodilatation via prostacyclin and NO.14 Because most ET-1 is released 63

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abluminally, plasma concentrations of ET-1 do not accurately reflect ET-1 production. However, some is released into the circulation, and the ETB receptor also acts as a clearance receptor for this circulating ET-1. The half-life of ET-1 in the healthy circulation is approximately 1 minute,15 with removal through receptor- and non–receptor-mediated mechanisms. ET-1 binds to ETB receptors, with subsequent ligand–receptor complex internalization and intracellular degradation accounting for the majority of clearance, particularly in the pulmonary circulation,16 although the splanchnic and renal circulations also contribute.8 Therefore, reductions in ETB numbers, or ETB receptor blockade, may reduce ET-1 clearance, increasing plasma concentrations without altering production. ET receptors are widely distributed within the human kidney, with the ETA subtype localized to vascular smooth muscle, notably in the glomeruli, vasa recta, and arcuate arteries, whereas ETB receptors are more numerous (ETB-toETA ratio 2 : 1), and more widespread, with a high concentration in the collecting system.4,17

The Role of Endothelin in Renal Physiology Evidence exists for renal and vascular ET-1 acting as two independent systems.18 After systemic infusion of radiolabeled ET-1, labeled compound makes up less than 1% of total urinary ET-1.19 Therefore, neither glomerular filtration nor tubular secretion of plasma ET-1 accounts for urinary ET-1, which is therefore assumed to be primarily of renal origin. Urinary excretion of ET-1 is thus thought to reflect renal ET-1 production. Renal ET-1 is thought to have a role in the paracrine/autocrine regulation of renal and intrarenal blood flow, glomerular hemodynamics, sodium and water homeostasis,20 and acid–base balance.21 ET-1 is a potent vasoconstrictor in vitro and pressor in whole animals.22 With respect to the kidney, exogenous ET-1 causes renal vasoconstriction and an overall reduction in renal blood flow (RBF),23 effects mediated via the ETA receptor.24,25 Indeed, the renal vasculature is more sensitive to the vasoconstricting effects of ET-1 than other vascular beds.26 Although exogenous ET-1 reduces total RBF, a regional difference has been observed, with cortical vasoconstriction that is ETA receptor mediated27–29 and ETB and NO dependent medullary vasodilatation.27 Furthermore, in vitro studies have shown that combined ETA/B receptor antagonism is required to fully abolish the vasoconstricting effects of exogenous ET-1 on the afferent arteriole, suggesting that both ETA and ETB receptors are involved. At the efferent arteriole the effect of ET-1 is blocked by ETA receptor antagonism alone, and enhanced by ETB receptor blockade, suggesting that ET-1 can modulate efferent arteriolar tone via the ETA receptor and that the balance of ETB receptor effects here is to produce vasodilation.30 By this action on efferent and afferent arterioles, ET has the ability to regulate glomerular capillary pressure and so glomerular filtration. Additionally, ET-1 has been shown to reduce filtration coefficient by mesangial cell contraction31 (Figure 5-1A).

5. Endothelin System in Chronic Kidney Disease

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Vasoconstriction Efferent arteriole

Vasodilatation ETA ETB

ET-1 (vascular/renal origin)

Δ Glomerular haemodynamics Mesangial cell contraction/proliferation Glomerulosclerosis

ETB ETA Afferent arteriole Vasoconstriction

A FIGURE 5-1. Schematic of renal ET-1 effects (data derived from in vitro and animal studies). (A) Role of ET-1 in the regulation of glomerular hemodynamics.

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Fenestrated endothelium

Glomeru lar basement membrane

ET-1

Podocyte

Podocyte de-differentiation

Further podocyte de-differentiation

↑ Podocyte ET-1 production

Protein

Breakdown of glomenular filtration barrier

Δ Glomerular haemodynamics Mesangial cell contraction/proliferation Glomerulosclerosis

B Proximal Tubule

Fibroblast proliferation

ETA

ET-1

Increased ECM

ET-1

Protein Tubular lumen

IL-β TGFβ

Fibrosis (glomeruloscierosis and interstitial fibrosis)

Interstitium TNFα IL-1 Vasoconstriction

C

ETA

Macrophage/ monocyte activation

Inflammatory reaction

Pentubular blood vessel

FIGURE 5-1. Continued. (B) role of ET-1 in podocyte dedifferentiation and proteinuria; (C) role of ET-1 in interstitial fibrosis; (D) role of collecting duct cell ETB receptor in natriuresis and diuresis.


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Urine flow ET-1 H2O AVP ETB NO/ PGE2

Na+ ETB

Natriuresis/ Diuresis ET-1

Endothelial cell of pentubular blood vessel

Tubular lumen Collecting duct cell

D FIGURE 5-1. Continued.

In humans, a similar vasoconstrictor10 and pressor response has been demonstrated,32 as well as renal vasoconstriction, a fall in total RBF (with a consequent reduction in glomerular filtration rate), and increase in filtration fraction.33 As yet, there are no studies of the effects of ET-1 on intrarenal distribution of blood flow in humans. Also, there are few studies using ET receptor antagonists. One has demonstrated an increase in RBF after combined ETA/B receptor blockade.34 Most, however, do not demonstrate an effect of selective ETA receptor blockade,35–39 or combined ETA/B receptor blockade,38 on basal renal hemodynamics, suggesting that ET-1 acting via the ETA receptor does not contribute to the maintenance of renal vascular tone in health. Selective and unopposed ETB receptor antagonism can, however, produce profound renal vasoconstriction, suggesting that ET-1–mediated tonic renal vasodilatation via the ETB receptor is important.38 With respect to renal tubular functions, there is now a substantial body of evidence supporting a role for ET-1 in the regulation of volume homeostasis. ET-1 is produced by inner medullary collecting duct cells (IMCD), where it inhibits the AVP-stimulated retention of water,40 and extracellular sodium concentrations may regulate IMCD ET-1 production.18,41 Studies have suggested a natriuretic role for the tubular ETB receptor, which is linked to NO generation. A potent inhibitory action of NO on tubular sodium reabsorption is well described.42 ET-1, acting via ETB and NO, can inhibit chloride transport in the medullary thick ascending limb of Henle, thus promoting natriuresis.43,44 Furthermore, picomolar concentrations of ET-1, binding to ETB receptors, activate

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amiloride-sensitive sodium channels in distal tubular cells in vitro, although higher, nanomolar doses inhibit this channel by a non-ETB receptor–dependent mechanism.45 This has been supported by in vivo experiments in rats demonstrating natriuresis due to reduced sodium transport in the proximal and distal nephron segments in response to low-dose exogenous ET-1, with higher doses resulting in sodium retention due to glomerular vasoconstriction46 (Figure 5-1D). Elegant knockout animal models have further advanced our understanding of the role ET-1 plays in renal tubular function. A rat model deficient in renal ETB receptors displays a salt-sensitive hypertension, with restoration of normal blood pressure by amiloride, suggesting that the ETB receptor regulates sodium excretion at the epithelial sodium channel in collecting duct (CD) cells.47 More recently, Kohan et al. have successfully created a tissue-specific knockout of the renal ET system. Mice lacking CD expression of the ET-1 gene are hypertensive and have an impaired ability to excrete a sodium load.48 Interestingly, these knockout mice excrete acute water loads less well than wild-type mice and have a heightened physiological response to AVP, consistent with an intrarenal role for ET-1 in blunting the response to AVP.49 Antagonist studies have also proved helpful. ETB antagonist-treated rats develop a sodium-dependent hypertension.50 Additionally, in the face of acute ETB receptor blockade, pressure– natriuresis curves are shifted to the right such that a greater renal perfusion pressure is needed to excrete the same amount of sodium.51 Finally, administration of exogenous low-dose ET-1 to dogs in the presence of high-grade selective ETA receptor blockade results in renal vasodilatation and natriuresis, presumably by unmasking an ETB receptor–mediated effect.52 Dissecting the different actions of the intrarenal ET system has, however, proved difficult, in part from an inability to discriminate between effects of ET-1 in vivo on the nephron and vasculature. To date, ET-1–associated natriuresis and diuresis have not been demonstrated in humans.

The Role of Endothelin in Renal Pathophysiology: Glomerulopathies, Proteinuric Nephropathies, and Chronic Kidney Disease Glomerular injury is frequently associated with progressive CKD. This process typically involves glomerular sclerosis and interstitial fibrosis and occurs regardless of the nature of the initial renal insult. The mechanisms responsible for this continued renal deterioration are not fully understood, but likely involve a number of common pathways and may be distinct from those responsible for the original injury. Glomerular hypertension, glomerular cell hypertrophy, and extracellular matrix accumulation are all likely involved. Significant proteinuria, a marker of CKD, has emerged as a powerful predictor of renal disease progression53,54 regardless of underlying diagnosis, and proteinuria


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reduction is an important strategy to retard or prevent loss of renal function.55 Furthermore, reduction of proteinuria confers cardiovascular protection.56 Once filtered through the glomerulus, excess protein is tubulo-toxic and excess protein reabsorption in the tubules can lead to an activation of tubulardependent pathways of interstitial inflammation and fibrosis, with progressive renal scarring.57 The ET system has been implicated in these processes.6 In the remnant kidney model renal ET-1 gene expression and urinary ET-1 excretion correlate with the degree of proteinuria and extent of renal damage.58 Also, transgenic animal studies, where renal ET pathways are upregulated, display glomerulosclerosis and renal tubulointerstitial lesions independent of changes in blood pressure that are usually characteristic of such models.59 These blood pressure–independent effects of ET are supported by antagonist studies, where ET receptor antagonists led to a slowing of progressive renal damage, even in the absence of blood pressure modification.60 Activation of the ET system exacerbates proteinuria. Through its vasoconstrictive properties, ET-1 leads to glomerular hypertension, increasing glomerular capillary permeability and resulting in excessive protein filtration. The development of proteinuria is also associated with damage to the renal podocyte, the highly specialized glomerular epithelial cell that maintains the integrity of the glomerular filtration barrier. Podocytes possess a contractile structure that responds to vasoactive hormones to control glomerular capillary surface area and, in turn, ultrafiltration coefficient. Recent in vitro studies suggest that podocytes undergo phenotypic changes as a result of exposure to large amounts of protein that resemble dedifferentiation.61 In parallel with these changes there is increased ET-1 production by the podocyte, which is, at least partly, dependent on the cytoskeletal rearrangements brought about by excess protein exposure. In the same model administration of exogenous ET-1 brings about similar podocyte cytoskeletal changes as protein loading. Thus, one can infer podocytederived ET-1 acting in an autocrine and paracrine manner to promote further podocyte ultrastructural degeneration and hence its own production, with both of these contributing to a further breakdown in the glomerular filtration barrier (Figure 5-1B). These data are supported by in vivo evidence in a murine model of protein overload that displays increased renal ET-1 production alongside development of podocyte structural damage.62 Podocyte-derived ET-1 may of course act on other glomerular cells, increasing the tone of the glomerular capillary, enhancing vascular permeability, and stimulating mesangial cell contraction by virtue of its vasoconstrictor effects, all effects leading to glomerular hypertension and a further decline in functional renal mass. Thus, a role for ET system antagonism appears logical in preventing, or at the very least slowing, the early pathological effects of protein overload on glomerular structure and function. At present there are only a few studies that support this hypothesis. In a rat model of age-related glomerulosclerosis and proteinuria, there is a significant reduction in both these parameters following selective ETA receptor blockade.63 This effect was independent of changes in blood pressure. Although

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these changes did not translate to beneficial effects on glomerular filtration rate or tubulointerstitial injury, ETA receptor blockade did result in a reduction in the gene expression of matrix metalloproteinase-9, an enzyme involved in collagen breakdown and so glomerular matrix turnover, and an associated normalization of glomerular basement membrane hypertrophy. This is supportive of an antifibrotic role for ET system antagonism, certainly with respect to the glomerulus, in conditions associated with proteinuria. Studies also suggest a link between upregulation of the ET system and tubular protein reabsorption. Exposure of proximal tubular cells in vitro to a protein load leads to a dose-dependent increase in ET-1 production.64 This phenomenon is not exclusively associated with albumin but may be seen with other proteins such as IgG and transferrin.64 As the majority of ET-1 is secreted abluminally, one can infer that in vivo there is a build-up of ET-1 in the renal interstitium in proteinuric nephropathies. Here ET-1 may bind to interstitial fibroblasts and promote their proliferation and generation of extracellular matrix, 65 which in turn is capable of further inducing ET-1 synthesis. Furthermore, ET-1 is chemotactic for blood monocytes66 and leads them to secrete proinflammatory cytokines and growth factors, events that would contribute to interstitial remodelling and scarring (Figure 5-1C). A number of clinically relevant animal models also link the ET system to glomerular damage. In a mouse model of IgA nephropathy, the most common glomerulonephritis worldwide, both ET-1 and ET receptor message increased alongside progression of the nephritis, and treatment with a selective ETA receptor antagonist ameliorated the histopathological lesions and proteinuria observed in this model.67 In a rat model of proliferative nephritis, upregulation of ET-1 protein and receptors has been shown at the level of the glomerulus,68 and in this case non-selective ET receptor blockade ameliorated both the mesangial expansion and proteinuria characteristic of this model. Similarly, in a murine model of lupus nephritis, again defined by abnormal glomerular proliferation, selective blockade of the ETA receptor improved glomerular structure and function translating to an overall benefit in renal function.69 Finally, in rats with accelerated passive Heymann nephritis, a model of membranous glomerulonephritis, combined blockade of the ET and angiotensin systems showed greater renoprotective benefit than either treatment alone.70 Animal models of diabetic nephropathy, now the most common cause of end-stage renal disease in the developed world, and in which proteinuria is a hallmark feature, are also consistent with an etiological involvement of ET-1. Similar to reduced renal mass models, ET-1 activity appears to be increased in the diabetic kidney. Renal ET-1 mRNA levels are elevated in rats with streptozocin-induced diabetes,71 while glomerular ET receptor expression is upregulated in a diabetic rabbit model.72 Alongside these observations, urinary ET-1 excretion, a marker of intrarenal ET-1 production, is increased in diabetic patients with microalbuminuria.73 Inhibition of ET-1 production in diabetic rats leads to reduced interstitial matrix turnover and proteinuria, effects similar to those seen with angiotensin-converting enzyme (ACE) inhibition. With


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regards to antagonist studies, both selective ETA and combined ETA/B receptor antagonism have shown beneficial effects in animal models of diabetic nephropathy.74 Diabetic rats treated with bosentan for 1 month failed to show a rise in either urinary protein excretion or the profibrotic factors collagen I and transforming growth factor β compared to controls.75 Importantly, one chronic dosing study with bosentan showed that in a rat model of type 1 diabetes the early hyperfiltration was prevented, and the subsequent proteinuria and renal structural changes diminished with treatment, effects seen on top of ACE inhibition, the current treatment of choice in diabetic nephropathy.

Patient Studies Studies in patients with CKD are limited. In respect to systemic vascular tone, as with healthy subjects, acute ETB receptor antagonism produces systemic vasoconstriction while ETA or combined ETA/B receptor antagonism produce systemic vasodilatation similar in degree to that seen in healthy controls.38 Blood pressure falls to a greater degree, however, after ETA receptor antagonism in CKD patients due to lesser increases in cardiac index in response to this vasodilatation. The mechanism behind this observation is not, as yet, clear. In the renal circulation, in contrast to healthy controls, acute selective ETA but not combined ETA/B receptor antagonism produces a sustained increase in renal blood flow and fall in renal vascular resistance, suggesting that ET-1 is important in maintaining renal vascular tone (which is about four times higher at baseline than in healthy controls) via the ETA receptor. Because glomerular filtration rate does not significantly alter, this is accompanied by a reduction in filtration fraction that, in the absence of changes in filtration coefficient, may indicate efferent arteriolar dilatation, consistent with animal data suggesting a role for ET-1 in maintaining efferent arteriolar tone via the ETA receptor.38 The attenuation of the renal vasodilatory effect of ETA receptor antagonism by concomitant ETB receptor antagonism suggests that the ETB receptor is important in maintaining renal vasodilatation in CKD and that the renal vasoconstriction seen after selective ETB receptor blockade alone is not simply due to reduced clearance and displacement of ET-1 onto the unblocked ETA receptor, but also due to a specific role for ET-1–mediated tonic renal vasodilatation via the ETB receptor.38 Studies with healthy subjects have suggested that this observation in CKD patients may be accounted for at least in part by concomitant administration of ACE inhibitors, as these are synergistic with ETA receptor antagonism via an NO-mediated, ETB receptor–dependent mechanism.39

Conclusion ET has been widely implicated in renal physiology and pathophysiology. Studies suggest that ET receptor antagonism could be of therapeutic importance in CKD. Studies in humans show that ET receptor antagonsim can sucessfully

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reduce blood pressure, endothelial dysfunction, and arterial stiffness as well as renal vascular tone and proteinuria. Animal studies suggest that there is further potential benefit in terms of prevention of proliferation and fibrosis and also atherosclerosis, an important comorbidity in CKD. To date, two longer-term studies in CKD have been reported, both focusing on proteinuria as a renal outcome. These studies suggest that ET receptor antagonism will be renoprotective. Honing et al. demonstrated that 6 weeks of the selective ETA receptor antagonist ABT 627 reduced proteinuria in 10 patients with diabetic nephropathy.76 More recently, Wenzel et al. have studied the effects of 12 weeks of treatment with the selective ETA receptor antagonist SPP301 on top of standard treatment (97.6% on ACE inhibitors or angiotensin receptor blockers or both) in 256 patients with diabetic nephropathy and demonstrated a reduction in proteinuria with all doses studied.77 A phase III trial recruiting approximately 2500 patients with long-term follow-up is now in progress. Hopefully this and other such trials will report on the ability of this class of drugs to delay the progression of CKD and its cardiovascular complications.

References 1. Yanagisawa M, Kurihara H, Kimura S, et al. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988;332:411–415. 2. Inoue A, Yanagisawa M, Kimura S, et al. The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc Natl Acad Sci USA 1989;86:2863–2867. 3. Arinami T, Ishikawa M, Inoue A, et al. Chromosomal assignments of the human endothelin family genes: The endothelin-1 gene (EDN1) to 6p23-p24, the endothelin-2 gene (EDN2) to 1p34, and the endothelin-3 gene (EDN3) to 20q13.2-q13.3. Am J Hum Genet 1991;48:990–996. 4. Karet FE, Davenport AP. Localization of endothelin peptides in human kidney. Kidney Int 1996;49:382–387. 5. Eid H, de Bold ML, Chen JH, et al. Epicardial mesothelial cells synthesize and release endothelin. J Cardiovasc Pharmacol 1994;24:715–720. 6. Kohan DE. Endothelins in the normal and diseased kidney. Am J Kidney Dis 1997;29:2–26. 7. Morita S, Kitamura K, Yamamoto Y, et al. Immunoreactive endothelin in human kidney. Ann Clin Biochem 1991;28:267–271. 8. Attina T, Camidge R, Newby DE, et al. Endothelin antagonism in pulmonary hypertension, heart failure, and beyond. Heart 2005;91:825–831. 9. Wesson DE, Simoni J, Green DF. Reduced extracellular pH increases endothelin-1 secretion by human renal microvascular endothelial cells. J Clin Invest 1998;101: 578–583. 10. Haynes WG, Webb DJ. Contribution of endogenous generation of endothelin-1 to basal vascular tone. Lancet 1994;344:852–854. 11. Yoshimoto S, Ishizaki Y, Sasaki T, et al. Effect of carbon dioxide and oxygen on endothelin production by cultured porcine cerebral endothelial cells. Stroke 1991;22:378–383.


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12. Arai H, Hori S, Aramori I, et al. Cloning and expression of a cDNA encoding an endothelin receptor. Nature 1990;348:730–732. 13. Sakurai T, Yanagisawa M, Takuwa Y, et al. Cloning of a cDNA encoding a nonisopeptide selective subtype of the endothelin receptor. Nature 1990;348:732–735. 14. DeNucci G, Thomas R, D’Orleans-Juste P, et al. Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endothelium-derived relaxing factor. Proc Natl Acad Sci USA 1988;85:9797–9800. 15. Gasic S, Wagner OF, Vierhapper H, et al. Regional haemodynamic effects and clearance of endothelin-1 in humans: renal and peripheral tissues may contribute to overall disposal of the peptide. J Cardiovasc Pharmacol 1992;19:176–180. 16. Dupuis J, Stewart DJ, Cernacek P, et al. Human pulmonary circulation is an important site for both clearance and production of endothelin-1. Circulation 1996;94:1278–1284. 17. Kuc R, Davenport AP. Comparison of endothelin-A and endothelin-B receptor distribution visualized by radioligand binding versus immunocytochemical localization using subtype selective antisera. J Cardiovasc Pharmacol 2004;44(suppl 1): S224–226. 18. Serneri GGN, Modesti PA, Cecioni I, et al. Plasma endothelin and renal endothelin are two distinct systems involved in volume homeostasis. Am J Physiol 1995;268: H1829–1837. 19. Benigni A, Perico N, Gaspari F, et al. Increased renal endothelin production in rats with reduced renal mass. Am J Physiol 1991;260:F331–339. 20. Kohan DE, Padilla E. Osmolar regulation of endothelin-1 production by rat inner medullary collecting duct. J Clin Invest 1993;91:1235–1240. 21. Wesson DE. Endogenous endothelins mediate increased acidification in remnant kidneys. J Am Soc Nephrol 2001;12:1826–1835. 22. Yanagisawa M, Inoue A, Ishikawa T, et al. Primary structure, synthesis, and biological activity of rat endothelin, an endothelium-derived vasoconstrictor peptide. Proc Natl Acad Sci USA 1988;85:6964–6967. 23. Chou SY, Porush JG. Renal actions of endothelin-1 and endothelin-3: Interactions with the prostaglandin system and nitric oxide. Am J Kid Dis 1995;26:116–123. 24. Evans RG, Madden AC, Oliver JJ, et al. Effects of ET(A)- and ET(B)-receptor antagonists on regional kidney blood flow, and responses to intravenous endothelin-1, in anaesthetized rabbits. J Hypertens 2001;19:1789–1799. 25. Abassi Z, Francis B, Wessale J, et al. Effects of endothelin receptors ET(A) and ET(B) blockade on renal haemodynamics in normal rats and in rats with experimental congestive heart failure. Clin Sci (Lond) 2002;103(suppl 48):245S–248S. 26. Pernow J, Franco-Cereceda A, Matran R, et al. Effect of endothelin-1 on regional vascular resistance in the pig. J Cardiovasc Pharmacol 1989;13(suppl 16):S205– 206. 27. Rubinstein I, Gurbanov K, Hoffman A, et al. Differential effect of endothelin-1 on renal regional blood flow: role of nitric oxide. J Cardiovasc Pharmacol 1995;26: S208–210. 28. Gurbanov K, Rubinstein I, Hoffman A, et al. Differential regulation of renal regional blood flow by endothelin-1. Am J Physiol 1996;271:F1166–1172. 29. Denton KM, Shweta A, Finkelstein L, et al. Effect of endothelin-1 on regional kidney blood flow and renal arteriole calibre in rabbits. Clin Exp Pharmacol Physiol 2004;31:494–501.

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30. Inscho EW, Imig JD, Cook AK, Pollock DM. ETA and ETB receptors differentially modulate afferent and efferent arteriolar responses to endothelin. Br J Pharmacol 2005;146:1019–1026. 31. Sorokin A, Kohan DE. Physiology and pathology of endothelin-1 in renal mesangium. Am J Physiol 2003;285:F579–589. 32. Sorensen SS, Madsen JK, Pedersen EB. Systemic and renal effects of intravenous infusion of endothelin-1 in healthy human volunteers. Am J Physiol 1994;266: F411–418. 33. Rabelink TJ, Kaasjager KAH, Boer P, et al. Effects of endothelin-1 on renal function in humans. Implications for physiology and pathophysiology. Kidney Int 1994; 46:376–381. 34. Freed MI, Wilson DE, Thompson KA, et al. Pharmacokinetics and pharmacodynamics of SB 209670, an endothelin receptor antagonist: Effects on the regulation of renal vascular tone. Clin Pharmacol Therap 1999;65:473–482. 35. Schmetterer L, Dallinger S, Bobr B, et al. Systemic and renal effects of an ETA receptor subtype-specific antagonist in healthy subjects. Br J Pharmacol 1998;124:930–934. 36. Montanari A, Biggi A, Carra N, et al. Endothelin-A blockade attenuates systemic and renal hemodynamic effects of L-NAME in humans. Hypertension 2000;35:518–523. 37. Montanari A, Carra N, Perinotto P, et al. Renal hemodynamic control by endothelin and nitric oxide under angiotensin II blockade in man. Hypertension 2002;39: 715–720. 38. Goddard J, Johnston NR, Hand MF, et al. Endothelin-A receptor antagonism reduces blood pressure and increases renal blood flow in hypertensive patients with chronic renal failure: a comparison of selective and combined endothelin receptor blockade. Circulation 2004;109:1186–1193. 39. Goddard J, Eckhart C, Johnston NR, et al. Endothelin A receptor antagonism and angiotensin-converting enzyme inhibition are synergistic via an endothelin B receptor-mediated and nitric oxide-dependent mechanism. J Am Soc Nephrol 2004;15:2601–2610. 40. Kohan DE. Autocrine role of endothelin in rat inner medullary collecting duct: inhibition of AVP-induced cAMP accumulation. J Cardiovasc Pharmacol 1993;22 (suppl 8):S174–179. 41. Yang T, Terada Y, Nonoguchi H, et al. Effect of hyperosmolality on production and mRNA expression of ET-1 in inner medullary collecting duct. Am J Physiol 1993;264: F684–689. 42. Wilcox SC. L-Arginine-nitric oxide pathway In: Seldin DW, Giebisch G, eds. The Kidney, Vol. 1, Philadelphia: Lippincott, Williams and Wilkins; 2000:849–871. 43. Plato CF, Pollock DM, Garvin JL. Endothelin inhibits thick ascending limb chloride flux via ET(B) receptor-mediated NO release. Am J Physiol Renal Physiol 2000;279: F326–333. 44. Herrera M, Garvin JL. Endothelin stimulates endothelial nitric oxide synthase expression in the thick ascending limb. Am J Physiol Renal Physiol 2004;287: F231–235. 45. Gallego MS, Ling BN. Regulation of amiloride-sensitive Na+ channels by endothelin1 in distal nephron cells. Am J Physiol 1996;271:F451–460. 46. Harris PJ, Zhuo J, Mendelsohn FA, et al. Haemodynamic and renal tubular effects of low doses of endothelin in anaesthetized rats. J Physiol 1991;433:25–39. 47. Gariepy CE, Ohuchi T, Williams SC, et al. Salt-sensitive hypertension in endothelinB receptor-deficient rats. J Clin Invest 2000;105:925–933.


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48. Ahn D, Ge Y, Stricklett PK, et al. Collecting duct-specific knockout of endothelin-1 causes hypertension and sodium retention. J Clin Invest 2004;114:504–511. 49. Ge Y, Ahn D, Stricklett PK, et al. Collecting duct-specific knockout of endothelin-1 alters vasopressin regulation of urine osmolality. Am J Physiol Renal Physiol 2005; 288:F912–920. 50. Webb DJ, Monge JC, Rabelink TJ, et al. Endothelin: new discoveries and rapid progress in the clinic. Trend Pharmacol Sci 1998;19:5–8. 51. Vassileva I, Mountain C, Pollock DM. Functional role of ETB receptors in the renal medulla. Hypertension 2003;41:1359–1363. 52. Brooks DP, DePalma PD, Pullen M, et al. SB 234551, a novel endothelin-A receptor antagonist, unmasks endothelin-induced renal vasodilatation in the dog. J Cardiovasc Pharmacol 1998;31(suppl 1):S339–341. 53. Mallick NP, Short CD, Hunt LP. How far since Ellis? The Manchester Study of glomerular disease. Nephron 1987;46:113–124. 54. Cameron JS, Turner DR, Ogg CS, et al. The long-term prognosis of patients with focal segmental glomerulosclerosis. Clin Nephrol 1978;10:213–218. 55. Ruggenenti P, Perna A, Remuzzi G. Retarding progression of chronic renal disease: the neglected issue of residual proteinuria. Kidney Int 2003;63:2254–2261. 56. Ibsen H, Olsen MH, Wachtell K, et al. Reduction in albuminuria translates to reduction in cardiovascular events in hypertensive patients: losartan intervention for endpoint reduction in hypertension study. Hypertension 2005;45:198– 202. 57. Remuzzi G, Bertani T. Pathophysiology of progressive nephropathies. N Engl J Med 1998;339:1448–1456. 58. Orisio S, Benigni A, Bruzzi I, et al. Renal endothelin gene expression is increased in remnant kidney and correlates with disease progression. Kidney Int 1993;43: 354–358. 59. Hocher B, Thone-Reineke C, Rohmeiss P, et al. Endothelin-1 transgenic mice develop glomerulosclerosis, interstitial fibrosis, and renal cysts but not hypertension. J Clin Invest 1997;99:1380–1389. 60. Benigni A, Remuzzi G. Endothelin antagonists. Lancet 1999;353:133–138. 61. Morigi M, Buelli S, Angioletti S, et al. In response to protein load podocytes reorganize cytoskeleton and modulate endothelin-1 gene: implication for permselective dysfunction of chronic nephropathies. Am J Pathol 2005;166:1309–1320. 62. Benigni A, Corna D, Zoja C, et al. Targeted deletion of angiotensin II type 1A receptor does not protect mice from progressive nephropathy of overload proteinuria. J Am Soc Nephrol 2004;15:2666–2674. 63. Ortmann J, Amann K, Brandes RP, et al. Role of podocytes for reversal of glomerulosclerosis and proteinuria in the aging kidney after endothelin inhibition. Hypertension 2004;44:974–981. 64. Zoja C, Morigi M, Figliuzzi M, et al. Proximal tubular cell synthesis and secretion of endothelin-1 on challenge with albumin and other proteins. Am J Kidney Dis 1995;26:934–941. 65. Ong AC, Jowett TP, Firth JD, et al. Human tubular-derived endothelin in the paracrine regulation of renal interstitial fibroblast function. Exp Nephrol 1994; 2:134. 66. Achmad TH, Rao GS. Chemotaxis of human blood monocytes toward endothelin-1 and the influence of calcium channel blockers. Biochem Biophys Res Commun 1992;189:994–1000.

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67. Nakamura T, Ebihara I, Fukui M, et al. Effect of a specific endothelin receptor A antagonist on glomerulonephritis of ddY mice with IgA nephropathy. Nephron 1996;72:454–460. 68. Yoshimura A, Iwasaki S, Inui K, et al. Endothelin-1 and endothelin B type receptor are induced in mesangial proliferative nephritis in the rat. Kidney Int 1995;48: 1290–1297. 69. Nakamura T, Ebihara I, Tomino Y, et al. Effect of a specific endothelin A receptor antagonist on murine lupus nephritis. Kidney Int 1995;47:481–489. 70. Benigni A, Corna D, Maffi R, et al. Renoprotective effect of contemporary blocking of angiotensin II and endothelin-1 in rats with membranous nephropathy. Kidney Int 1998;54:353–359. 71. Benigni A, Colosio V, Brena C, et al. Unselective inhibition of endothelin receptors reduces renal dysfunction in experimental diabetes. Diabetes 1998;47:450–456. 72. Khan MA, Dashwood MR, Thompson CS, et al. Up-regulation of endothelin (ET(A) and ET(B)) receptors and down-regulation of nitric oxide synthase in the detrusor of a rabbit model of partial bladder outlet obstruction. Urol Res 1999;27:445–453. 73. Lee YJ, Shin SJ, Tsai JH. Increased urinary endothelin-1-like immunoreactivity excretion in NIDDM patients with albuminuria. Diabetes Care 1994;17:263–266. 74. Hocher B, Schwarz A, Reinbacher D, et al. Effects of endothelin receptor antagonists on the progression of diabetic nephropathy. Nephron 2001;87:161–169. 75. Cosenzi A, Bernobich E, Trevisan R, et al. Nephroprotective effect of bosentan in diabetic rats. J Cardiovasc Pharmacol 2003;42:752–756. 76. Honing MLH, Bouter PK, Ballard DE, et al. ABT-627, a selective ETA-receptor antagonist, reduces proteinuria in patients with diabetes mellitus. In: Regulation of Vascular Tone in Humans by Endothelium-Derived Mediators. Utrecht, the Netherlands: 2000:89–102. Thesis 77. Wenzel RR, Mann J, Jürgens C, et al. The ETA-selective antagonist SPP301 on top of standard treatment reduces urinary albumin excretion rate in patients with diabetic nephropathy. Abstract Am Soc Nephrol 2005: F-FC093.

6 Endothelial Activation in Inflammation: Lessons Learned from E-Selectin Dorian O. Haskard

Research over the last two decades has produced major insights into the role of vascular endothelium in the orchestration of inflammatory responses. Endothelial cells play an active part in a variety of inflammatory and thrombotic processes, not least in the recruitment of leukocytes from the blood into surrounding tissues.1 This chapter focuses on the role in inflammation of a particular endothelial surface glycoprotein, E-selectin, which acts as an inducible adhesion molecule for leukocytes and as an excellent reporter of endothelial cell (EC) activation. Following the advent of techniques to isolate and culture ECs,2 a number of groups performed experiments investigating how ECs respond to stimulation by a variety of proinflammatory agonists. By the mid-1980s, it was clear that activation of ECs with pro-inflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1α, or interferon (IFN)-γ or with bacterial lipopolysaccharaide (LPS) led to the induction of a prothrombotic and proadhesive state.3 The mechanism(s) for this change in EC phenotype involved gene transcription and de novo protein, with altered function developing over the course of 3–4 hours. Recent work using microarray technology has shown that approximately 140 genes are upregulated in ECs by stimulation with IL-1β.4 The conclusion to be drawn from these studies was that cytokine activation of EC was likely to lead to a change in surface glycoprotein expression, and that some of these putative inducible glycoproteins might act as adhesion substrates for leukocyte recruitment. This led on to attempts to define the molecules by generating monoclonal antibodies (mAbs) in mice immunized with activated EC. Pober and colleagues raised an antibody, H4/18, which bound an approximately 115 kDa antigen expressed minimally by resting ECs but inducible by IL-1α, TNF-α or LPS, but not directly by IFN-γ. This antigen was initially designated dndothelial leukocyte adhesion molecule (ELAM)-1, on the basis of mAb-mediated inhibition of neutrophil adhesion to IL-1α– or TNF-α–activated ECs under static conditions.5 Subsequent cDNA cloning of ELAM-1 showed that the molecule was structurally similar to two other C-type animal lectins involved in leukocyte–EC adhesion, which at that time were designated LECAM-1 and GPM-140/PADGEM.6–8 The three glycoproteins were 77

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subsequently renamed E-selectin, L-selectin, and P-selectin, respectively, with the prefix chosen to reflect the initial discovery of the molecules on ECs (E-), leukocytes (L-), and platelets (P-). The genes encoding the three selectins are derived by replication of a single ancestral gene and lie in tandem on the long arm of chromosome 1.9,10 The reader is recommended to several excellent reviews on the structure and function of the family (e.g., Refs. 11–13).

Structure of E-Selectin Each of the three selectins consists of an N-terminal C-type lectin domain, an epidermal growth factor (EGF) domain, a series of complement regulatory protein-type short concensus repeats, a transmembrane domain, and a cytoplasmic tail (Figure 6-1). E-selectin is intermediate in size between P-selectin and L-selectin and in humans has six short consensus repeats. This is in contrast to eight to nine short consensus repeats in P-selectin (depending upon alternative splicing) and two in L-selectin. Apart from the cytoplasmic tails, there is considerable conservation between the three selectins in the structure of the separate domains. E-selectin is highly conserved across species, the most obvious species difference between species being the variation in number of short-consensus repeats. Analysis of crystal structure shows little contact between the lectin and EGF domains of E-selectin, suggesting a rod-like conformation.14

Human L-selectin Human P-selectin E-selectin Human Pig Rabbit Rat Mouse

Cytoplasmic tail

complement regulatory protein-type short concensus repeats

EGF domain

Lectin domain

FIGURE 6-1. Domain structures of E-, L-, and P-selectins: The structures of three selectins are highly conserved, although there are differences between species in the number of short-consensus repeats.

6. Endothelial Activation in Inflammation

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E-Selectin as an Adhesion Molecule The primary function of selectins is to facilitate the capture of leukocytes from flowing blood to enable their trafficking into tissues. Adhesive bonds between selectins and their glycoprotein or glycolipid ligands have greater affinity under flow than under static conditions, but have fast on–off kinetics and last only a second or less.15–17 On their own, therefore, selectins do not normally immobilize leukocytes within blood vessels. Instead, leukocyte capture leads on to loose rolling interactions on endothelium, the velocity of which is largely independent of local hemodynamics.18,19 During rolling, the leukocyte becomes available for stimulation by local activating factors (e.g., chemokines) expressed on the endothelial surface.20 Subsequent activation of leukocyte β2-integrin function results in a reduction in rolling velocity, followed by leukocyte arrest and transmigration through endothelium into the perivascular space. This series of molecular interactions by which leukocytes are guided from the blood into inflamed tissue has been termed the adhesion cascade.21 As might be expected from their N-terminal lectin domains, carbohydrate determinants on selectin ligands are critical for the formation of adhesion bonds.22 The best characterized selectin-binding carbohydrate is the sialylated, fucosylated terminal tetrasaccharide sialyl-3-fucosyl-N-acetyllactosamine (sialyl Lewis x), which can bind each of the selectins. However, high-affinity selectin interactions depend not only on the carbohydrate but also its presentation by specific proteins, particularly for P- and L-selectin. The principal counterreceptor for P-selectin is P-selectin glycoprotein ligand-1 (PSGL-1), which is expressed by most myeloid and lymphoid cells. However, not all lymphocytes express PSGL-1 in a posttranslationally modified state capable of binding selectins. While E-selectin binds PSGL-1, it also binds a number of additional counterstructures if appropriately posttranslationally modified. These may include L-selectin, β2-integrins, CD44, and, in mouse, a 150 kDa (reduced) glycoprotein designated E-selectin ligand-1 (ESL-1).23–26 However, these different ligands may be utilized at different stages of the adhesion cascade. Thus, PSGL-1 and L-selectin, which are expressed on the tips of microvilli, may be particularly important for initial capture of leukocytes by E-selectin under flow.27–29 In contrast, CD44, which is primarily expressed on the cell body, may make more of a contribution to slow rolling following leukocyte capture.25 The greater diversity of E-selectin ligands may contribute to the slower and less variable rolling velocity of cells rolling on E-selectin compared to P-selectin.30 E-selectin ligands are expressed by all myeloid cells. Although initially lymphocytes were thought not to bind E-selectin, Graber et al. showed that anti-Eselectin mAbs could inhibit T cells from binding to cytokine-activated ECs.31 T cells that bind E-selectin were then found to be predominantly memory CD45RO+, which express cutaneous lymphocyte antigen (CLA), a carbohydrate variant of sLex that decorates PSGL-1 and acts as an E-selectin ligand.32–34 The population of T cells that binds E-selectin falls within the population that binds P-selectin. Thus, whereas CLA-bearing T cells bind E-selectin and also

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P-selectin, CLA negative cells may bind P-selectin but not E-selectin.34 Whether or not a T cell binds just P-selectin or both E- and P-selectin depends on the level of expression of fucosyl transferase VII.35,36 Only about 15–30% of T cells express CLA, and these cells are thought to particularly migrate to skin.32,37 This may be due to expression by these cells of CCR4 and hence responsiveness to CCL17 (TARC).38 αβ and γδ T cells have similar expression of CLA and ability to adhere to E-selectin.39 In the mouse, T cells that bind E-selectin are predominantly Th1 cells.40,41

Regulation of E-Selectin Expression In the absence of inflammation, leukocytes do not adhere well to endothelium in large part due to the low expression of E- and P-selectins and other adhesion molecules. During inflammation, however, adhesion molecules and chemoattractants become expressed in a kinetically orchestrated fashion, providing the means for the delivery of leukocytes into the tissues in a quantitatively and qualitatively appropriate fashion. Adhesion molecule expression is tightly controlled by a number of intracellular checks and balances, restricting the extent/ duration of an acute inflammatory response and thereby limiting bystander tissue injury. The first wave of endothelial activation during an acute inflammatory response occurs within seconds of EC stimulation by agonists such as thrombin, histamine, or complement C5a and is due to the translocation of WeibelPalade bodies from the cytoplasm to the plasma membrane, leading to surface expression of P-selectin and release of IL-8.42–44 However this rapid response is short-lived, as the newly surface expressed P-selectin is internalised within minutes into endosomes, from where it can be transported to lysosomes for degradation or shuttled back into Weibel-Palade bodies.45,46 More delayed changes in adhesion molecule expression come from transcriptional activation of adhesion molecule gene expression and de novo protein synthesis. The initial transcriptional response is epitomized by expression of E-selectin, which becomes expressed in response to cytokines such as TNF-α or IL-1α/β, along with a number of other adhesion molecules and chemokines, including ICAM-1, VCAM-1, IL-8, MCP-1, and fractalkine.4,47 E-selectin expression becomes maximal after about 4–6 hours of stimulation in vitro.48 However, it is important to note that the kinetics of expression of these cytokine-driven genes differ significantly. Thus, while E-selectin expression tends to be transient in vitro, expression of ICAM-1 and VCAM-1 is more sustained.49 This is consistent with E-selectin acting primarily as an amplifier of leukocyte– EC interactions in relatively acute and acute-on-chronic inflammation (see below). Restriction of E-selectin expression to ECs is likely to be attributable at least in part to silencing of the gene in other tissues by DNA methylation of the


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promoter.50 The mechanisms responsible for the induction in ECs of the otherwise transcriptionally silent E-selectin gene by proinflammatory cytokines have undergone intense investigation. Reporter gene experiments using deletion mutants of the E-selectin promoter have shown that the region between −170 and −85 in relation to the transcription start site is critical for TNF-α responsiveness.51 E-selectin is one of many genes that are activated in EC by nuclear factor (NF)κB.47 p65, p50, and RelB subunits have each been shown to bind the Eselectin promoter.52 There are three NFκB-binding sites—between −129 and −117, between −116 and −108, and between −94 and −85—which cooperate in inducing E-selectin expression.51,53,54 The sites starting at −116 and −94 bind either p50 homodimers or p50/p65 dimers with high affinity, whereas the site starting at −129 is bound by NFκB with lower affinity and is preferentially recognized by p65 homodimers.51 There are also sites (−140 to −135, −125 to −121) that bind the chromatin architectural protein high-mobility group protein I(Y) (HMG-I(Y)), which is essential for optimal NFκB binding.52,54 Transcriptional activation of E-selectin requires the subsequent assembly of an enhanceosome complex of coactivating proteins, including CREB-binding protein (CBP) and p300, related histone acetyltransfereases, which modify histones, steroid receptor coactivator-1 (SRC-1), and p300/CBP-associated factor. Together, these integrate interactions between NFκB and the transcriptional apparatus.55– 57 Whereas p65 can recruit coactivators into the complex, p50 does not, providing a mechanism for inhibition by p50/p50 homodimers.56 Activation of c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (MAPK) pathways is needed in addition to NFκB for activation of the E-selectin gene.58 There is an element (TGACATCA) at positions −154 to −147, which varies at one site (underlined) from the consensus cAMP response element/activating transcription factors (CRE/ATF) sequence TGACGTCA, and which mediates the effects of JNK and p38 pathway activation. Proteins binding this element (e.g., ATF-2 and c-Jun) contribute to the formation of the enhanseosome complex with p65/p50 by creating the optimal conformation of the promoter.54,59–62 There is evidence that c-Jun/ATF-2 heterodimers are the preferred complexes that bind the CRE/ATF element and that both components become transiently phosphorylated upon TNF-α stimulation.63 Expression of E-selectin is under very tight control. This tight restraint comes partly through regulation of transcription and partly through the intrinsic instability of E-selectin mRNA. Furthermore, the protein is rapidly removed from the cell surface following expression by internalisation into endosomes, a property that favours the use of E-selectin for targeting (see below).46,64–66 E-selectin transcriptional repression occurs at a number of levels, including inhibition of NFκB by synthesis of IκBα and A20.67,68 However, EC exposed to continuous TNF-α or IL-1 stimulation do not show a shut-off of p65/p50 NFκB nuclear localization in parallel with the drop in E-selectin gene transcription,64 probably in part due to the persistent downregulation of IκBβ.69 This may

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explain the continued expression of some NKκB-regulated genes, such as ICAM-1 and VCAM-1, beyond the time when E-selectin gene transcription has fallen. The early observation that suppression of E-selectin expression in the continuous presence of IL-1 can be overridden by TNF-α, and vice versa, suggests that specific suppression of early signal transduction is also involved.48 Differences also exist between ECs from different vascular beds in the duration with which E-selectin is expressed in cells under continuous stimulation. Thus, although E-selectin expression by human unbilical vein endothelial cells (HUVECs) returns to near baseline by 16–24 hours, expression is more prolonged in dermal EC.70 This is consistent with the sustained expression of Eselectin that is seen on dermal venules in many chronic skin diseases.71 These differences in duration of expression have been attributed to slower rates of protein endocytosis and degradation in microvascular compared to large vessel ECs.72,73

Effects of Hemodynamic Forces on E-Selectin Expression Leukocyte emigration in many inflamed tissues occurs through postcapillary venules under hemodynamic conditions in which the shear force (mechanical drag) of blood flowing over endothelium is relatively low. In arteries, adhesion molecule expression is suppressed by the biomechanical effects on EC function of high shear laminar blood flow. ECs exposed to high shear laminar flow (>10 dynes/cm2) for durations of 16 hours or more show an upregulation of genes with protective functions (e.g., eNOS, hemoxygenase-1, MnSOD, Bcl-2, A1).74,75 In contrast, ECs preconditioned by high shear laminar flow show reduced proinflammatory potential with suppression of E-selectin, VCAM-1, and many other TNF-α– or IL-1–regulated proinflammatory genes.76 The molecular mechanisms underlying the inhibitory effects of flow on expression of E-selectin and other proinflammatory genes are currently under investigation in a number of laboratories and appear to include suppression of JNK activation.77 This adaptation of ECs to high shear laminar flow is no doubt of great importance in protecting arteries from atherosclerosis. Conversely, ECs exposed to complex nonlaminar flow patterns are less protected, providing an explanation for the susceptibility of bifurcations and curvatures to atherosclerosis.78

Expression of E-Selectin in Models of Inflammation Early studies using immunocytochemistry showed that expression of E-selectin in most normal human tissues is very low. Moreover, examination of biopsies of skin from human volunteers confirmed that E-selectin shows the reversible expression during the course of inflammatory responses that was predicted


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from in vitro studies.79,80 In an attempt to obtain more quantitative information than is usually possible with immunocytochemistry, our group devised an approach for assessing luminal expression of E-selectin, measuring the localization of intravenously injected radiolabeled anti-E-selectin mAbs. Initial experiments in the pig demonstrated the feasibility of this technique and showed that the initial expression of E-selectin occurs in vivo between 30 and 45 minutes after EC stimulation, with peak expression at around 2 hours. The expression of E-selectin is therefore significantly faster in vivo than seen on cultured ECs stimulated in vitro.81,82 Using this approach to analyze E-selectin expression in detail in models of delayed hypersensitivity, we were able to show that expression of E-selectin is closely regulated and fluctuates over the course of inflammatory responses.83–85 In both pigs and mice, two phases of endothelial activation could be detected during a delayed hypersensitivity response: an initial immunologically nonspecific phase that occurs in naïve as well as in sensitized animals, followed by an immunologically specific phase seen in sensitized animals only.85 Presumably the second phase is stimulated by cytokines released locally following contact with antigen by T cells recruited earlier in the response. Harari et al.85 also showed the similarity in expression between E- and P-selectin in the mouse. This is distinct from the situation in humans and due to differences in the mouse and human P-selectin promoters.86 Thus, although human P-selectin can be regulated by cytokines (e.g., IL-4 or oncostatin-M), it is not responsive to p50/p65 NFkB and is not unpregulated by IL-1 or TNF-α.86,87 We have used the same approach to study E-selectin expression during the course of a chronic inflammatory disease, using murine lupus as a model. In these experiments, we compared E-selectin with that of ICAM-1 and VCAM-1 over the course of 20 weeks, during which the MRL/lpr strain develops a lupuslike illness. Endothelial ICAM-1 and VCAM-1 expression increased significantly with disease evolution in kidney, heart, and brain, correlating with increasing circulating levels of TNF-α, IL-1α and IL-1ß.88 Moreover, antisera against these cytokines inhibited ICAM-1 and VCAM-1 expression, providing a link between circulating cytokines and chronic EC activation. A subsequent study using intravital microscopy of leukocyte–EC interactions in cremaster muscle showed that the increase in ICAM-1 expression was functionally relevant. Thus, compared with MRL/++ mice, MRL/lpr mice showed an increase in leukocyte recruitment and transendothelial migration in response to TNF-α, and these were inhibited by anti-ICAM-1 mAbs.89 However, although endothelium appears to be chronically activated in MRL/lpr mice, there was little evidence for chronic upregulation of E-selectin, apart from slight increase in E-selectin in the kidneys.90 E-selectin was, however, induced normally in MRL/ lpr mice by injection of LPS. Extrapolating to human systemic lupus erythematosus, one can postulate that episodic expression of E-selectin on a background of enhanced ICAM-1 and VCAM-1 expression may contribute to exaggerated leukocyte–EC interactions, and this may be a mechanism underlying the triggering of lupus disease flares by infections.

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Imaging E-Selectin At present, techniques for assessing the state of endothelial activation in patients are very limited. E-selectin represents a potentially useful target for imaging endothelial cell activation in inflammation, since as a lumenally expressed molecule it is readily accessible. Furthermore, since E-selectin is internalized following expression, agents conjugated to anti-E-selectin antibodies are taken with E-selectin into the cell, allowing accumulation of targeting agent and increased imaging signal (Figure 6-2). Having shown the feasibility of quantifying endothelial activation in tissue samples using anti-E-selectin mAbs, our group established proof-of-principle for the use of E-selectin as a target for clinical imaging. In initial studies, we studied porcine monoarthritis induced by injection of phaetohemagglutinin or monosodium urate (MSU) crystals.91–93 In this model, intravenous injection of 111In-labeled anti-E-selectin F(ab)2 fragments led to focal gamma camera imaging of synovitis, and also provided images of the activated regional lymph nodes. Interestingly, images of the skin were also obtained, a finding explicable by the relatively high constitutive expression of E-selectin in pig skin compared to human skin. This is presumably an adaptation that gives the pig innate immune system an advantage in responding to cutaneous abrasions. An experiment using anti-TNF-α in pigs with MSU crystal induced arthritis demon-

FIGURE 6-2. Imaging of E-selectin in rheumatoid arthritis. Images obtained 4 hours after injection of 99mTc1.2B6-Fab (anti-E-selectin) (left) and 99mTc-oxidronate (HDP) (right) in two patients with rheumatoid arthritis. The images on the top correlate well; the bottom images show discordance between the lack of uptake of the mAb fragment and diffuse bony uptake of HDP. (From Ref. 96.)


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strated the potential of anti-E-selectin imaging for quantifying endothelial responses to treatment.94 We have developed the concept of E-selectin imaging of arthritis further in groups of patients with rheumatoid arthritis. Compared with 111In-labeled human immunoglobulin (HIG), 111In-labeled anti-E-selectin F(ab)2 fragments provided superior images in terms of sensitivity and image intensity. Furthermore, the distribution of uptake in inflamed joints was different for the two tracers, with 111In–anti-E-selectin showing a more focal localization in synovium. Whereas 111In–HIG only imaged joints that were obviously inflamed clinically, 111 In–anti-E-selectin identified joints with subclinical inflammation.95 Further studies have demonstrated the superiority of 99mTc over 111In as a radiolabel for E-selectin imaging and have shown the superiority of anti-E-selectin imaging over bone scanning for detecting active synovitis.96 We have also shown the potential of this approach for identifying bone or joint infections.97 Thus, 111In-labeled mAb 1.2B6 F(ab)2 compared favorably with simultaneously injected 99mTc-labeled leukocytes, particularly for identifying infections involving bone marrow, which takes up labeled leukocytes nonspecifically. As shown in Figure 6-3, anti-E-selectin imaging can also be used to detect active inflammation in inflammatory bowel disease.98 Colonic biopsies showed that expression of E-selectin was enhanced in patients with active inflammation, with weak or absent expression in inactive disease, and healthy controls. 111 In-labeled mAb 1.2B6 F(ab)2 scans were compared with 9mTc-labeled leukocyte scans performed 24 hours earlier. Anti-E-selectin imaging identified areas of inflammation in both Crohn’s disease and ulcerative colitis and may therefore be useful for identifying the site and extent of disease. Magnetic resonance imaging (MRI) affords noninvasive high-resolution multiparameter analysis of tissues and has the advantage over other imaging modalities of not involving ionizing radiation. We have recently explored the possibility of E-selectin–targeted MRI, using anti-E-selectin–conjugated iron oxide particles. These were shown to be taken up specifically by activated endothelium at sites of contact hypersensitivity in the mouse and provided T2 images detectable by MRI.99

Therapeutic Possibilities Although a number of preclinical studies have shown anti-inflammatory effects of inhibiting E-selectin with monoclonal antibodies, a clinical trial of anti-Eselectin mAbs in patients with chronic plaque psoriasis showed no benefit.100 Whether or not inhibiting E-selectin functions in other clinical situations remains unknown. Besides blocking E-selectin, another option is to use the molecule as a target for delivery of therapeutics.101–103 This is an attractive strategy, given the internalization of surface-expressed E-selectin discussed above.

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A

B

C

D

FIGURE 6-3. Comparison of 99mTc-labeled leukocyte and 111In-labeled anti-E-selectin scanning in Crohn’s disease. (A,B) 99mTc-labeled leukocyte images in a patient with Crohn’s disease. (A) One-hour image localizes inflammation in terminal ileum and cecum. (B) At 4 hours, leukocytes have transmigrated (large arrow) into the bowel lumen, making it difficult to estimate the true extent of disease. (C,D) 111In-labeled anti-E-selectin images in the same patient. Both 4-hour (C) and 24-hour (D) images localize inflammation in the same area. Arrows indicate areas of inflammation. (From Ref. 98.)

Conclusion The last 25 years has seen an enormous increase in our understanding of the role that endothelium plays in inflammatory processes. Studies focused on Eselectin have been particularly revealing, both with respect to understanding how leukocytes interact with endothelium during their recruitment into


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inflamed tissues as well as with respect to determining how endothelial activation is regulated during the evolution of simple and more complex inflammatory responses. For clinical purposes, E-selectin offers an attractive target for the molecular imaging of inflammation and possibly also the delivery of therapeutics to activated endothelium.

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95. Chapman PT, Jamar F, Keelan ETM, Peters AM, Haskard DO. Use of a radiolabeled monoclonal antibody against E-selectin for imaging endothelial activation in rheumatoid arthritis. Arthritis Rheum 1996;39:1371–1375. 96. Jamar F, Houssiau FA, Devogelaer JP, et al. Scintigraphy using a technetium 99mlabeled anti-E-selectin Fab fragment in rheumatoid arthritis. Rheumatology (Oxford) 2002;41(1):53–61. 97. Malik IS, Al-Nahhas A, Peters AM, Haskard DO. Imaging musculoskeletal infection with 111indium labeled anti-E-selectin monoclonal antibody 1.2B6 as an alternative to 111indium labeled leukocyte scintigraphy. Arthritis Rheum 2004;50:S263 (abstr 588). 98. Bhatti M, Chapman P, Peters AM, Haskard DO, Hodgson H. Visualizing E-selectin in the detection and evaluation of inflammatory bowel disease. Gut 1998;43: 40–47. 99. Reynolds PR, Larkman DJ, Haskard DO, Hajnal JV, George AJT, Edwards AD. Detection of vascular expression of E-selectin in vivo by MR imaging. Radiology 2006;241:469–476. 100. Bhushan M, Bleiker TO, Ballsdon AE, et al. Anti-E-selectin is ineffective in the treatment of psoriasis: a randomized trial. Br J Dermatol 2002;146(5):824–831. 101. Spragg DD, Alford DR, Greferath R, et al. Immunotargeting of liposomes to activated vascular endothelial cells: a strategy for site-selective delivery in the cardiovascular system. Proc Natl Acad Sci USA 1997;94:8795–8800. 102. Harari OA, Wickham TJ, Stocker CJ, et al. Targeting an adenoviral gene vector to cytokine-activated vascular endothelium via E-selectin. Gene Ther 1999;6(5):801– 807. 103. Everts M, Kok RJ, Asgeirsdottir SA, et al. Selective intracellular delivery of dexamethasone into activated endothelial cells using an E-selectin-directed immunoconjugate. J Immunol 2002;168(2):883–889.

7 Pathogenic Mediators of Vessel Sclerosis: Regulation of Vascular Smooth Muscle Cell Proliferation by Growth Factors, the Extracellular Matrix, and the Endothelium Mark Bond, Yih-Jer Wu, Graciela Sala-Newby, and Andrew C. Newby

Role of Vascular Smooth Muscle Cell Proliferation in Vessel Wall Sclerosis Arterial sclerosis is by definition an expansion of its connective tissue component. Normal arterial connective tissue comprises multiple layers of vascular smooth muscle cells (VSMCs), each surrounded by a basement membrane (BM) composed of type IV collagen, laminin, and heparan sulfate proteoglycans (Figure 7-1A). An interstitial extracellular matrix (ECM), consisting of fibrillar types I and III collagen, glycoproteins such as fibronectin, and dermatan sulfate proteoglycans such as versican, further surrounds the VSMCs.1 Separated from the media by the internal elastic lamina is the intima, often a simple monolayer of endothelial cells and their basement membrane. During atherosclerosis (Figure 7-1B) VSMCs and newly formed ECM thicken the intima together with inflammatory cells and sometimes microvascular endothelial cells (not shown). VSMCs predominate in the intimal thickenings in pulmonary hypertension,2 in veins used as arteriovenous fistulas,3 in coronary or peripheral vein grafts4,5 and after balloon angioplasty with or without stent implantation.6,7 Although adventitial fibroblasts8 and various types of progenitor cells9 could contribute, most intimal VSMCs probably originate from the media. In the rat carotid artery after balloon injury, VSMCs can be observed crossing the internal elastic lamina.10,11 Furthermore, wasting of the media is often observed at the base of spontaneous human and experimental animal atherosclerotic plaques, consistent with the idea that fibrous cap VSMCs derive ultimately from medial cells. VSMCs do not proliferate in normal arteries and do so only rarely in atherosclerotic plaques.12 Nevertheless, VSMC numbers double in the human aorta during age-related intimal thickening and double again in atherosclerotic plaques.13 By contrast, rapid VSMC proliferation is observed in the media of rat 94

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FIGURE 7-1. Normal vessel wall structure and intimal thickening by atherosclerosis. (A) Normal artery: The media comprises an interstitial extracellular matrix with multiple layers of embedded smooth muscle cells (SMCs) between the internal elastic lamina (IEL) and external elastic lamina (IEL). A basement membrane underlies the endothelial cells (EC) and surrounds each SMC. (B) Atherosclerotic artery: The intima is thickened by SMCs that have migrated from the media under the influence of growth factors and chemoattractants produced from ECs, SMCs, platelets (P), and foam-cell macrophages (FCs) that may be activated by the soluble cytokines interleukin-1 (IL-1) and tumor necrosis factor (TNF) or by contact with T lymphocytes (T) through the CD40/CD40 ligand (CD40L) system.

or pig arteries 48 hours after balloon injury10 and in the intima after 7–10 days. VSMC proliferation is also observed in experimental models of stent implantation,14 and coating stents with antiproliferative agents prevents intima formation in experimental models and reduces restenosis clinically.15–19 Similarly, VSMC numbers increase progressively in pig and mouse vein grafts,20,21 and antiproliferative agents decrease intimal thickening in experimental and clinical vein grafts.22–24

Growth Factors and the Cell Cycle in VSMCs Isolated VSMCs in culture appear to respond to growth factor stimulation in a manner typical of other cells (Figure 7-2A). There is immediate activation of signal transduction pathways, leading ultimately to phosphorylation of the

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FIGURE 7-2. Regulation of the cell cycle in smooth muscle cells (SMCs). (A) Comparison between aorta and collagenase-isolated SMC: Addition of serum leads to activation of aortas and isolated SMCs that are initially in the quiescent Gap0 (G0) state. In both aorta and isolated SMCs, typical Gap1 (G1) signal transduction pathways are triggered, and this causes assembly of cyclin-dependent protein kinases (CDKs). However, the decline in cyclin-dependent kinase inhibitors (CKIs) only occurs in isolated SMCs, not aortas, and hence only isolated SMCs can pass the restriction point (R), hyperphosphorylate retinoblastoma protein (Rb), and therefore progress into DNA synthesis phase (S). Once in S-phase, SMCs must complete the cell cycle by passing through Gap2 (G2) and entering mitosis (M). (B) A variety of growth factors can start G1 phase progression and cause assembly of CDKs. However, activation of focal adhesion kinase (FAK), as a result of extracellular matrix remodeling, must occur so that S-phase kinase-associated protein2 (Skp-2) can be upregulated. Skp-2 recruits CKIs to a ubiquitin ligase, which targets CKIs for proteasomal degradation; this allows SMCs to pass the restriction point and enter the S-phase. Upregulation of Skp-2 is blocked by cAMP elevation either through blocking FAK or more directly. Skp-2 is therefore the gatekeeper of the cell cycle in SMCs.


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extracellular receptor-related kinases ERK1/ERK2.25,26 Progression through the G1 phase of the cell cycle is controlled by cyclins D and E, which associate and activate their catalytic partners, the cyclin-dependent kinases (CDK4 and CDK2, respectively).27 Active CDKs hyperphosphorylate retinoblastoma protein (Rb), which triggers release of sequestered E2F transcription factor.27 In turn, this initiates S-phase–specific gene expression and progression through the G1 restriction point, beyond which proliferation becomes mitogen independent. The activity of the cyclin–CDK complexes is negatively regulated by the cyclindependent kinase inhibitors (CKIs).28,29 Important growth factors for VSMCs include members of the platelet-derived growth factor (PDGF) and fibroblast growth factor (FGF) families, which act through receptors with intrinsic tyrosine kinases.30,31 Insulin-like growth factor1 (IGF-1)32 and heparin-binding epidermal growth factor (HB-EGF)33 are also potent growth and survival factors that act through similar receptors. In addition, peptides that act through G-protein–coupled receptors, including thrombin, angiotensin II, and endothelin, may stimulate proliferation directly or via transactivation of EGF receptors.34–37 However, VSMCs in intact arteries are refractory to growth factor stimulation unless there is concomitant cell injury (e.g., after angioplasty) or inflammation (e.g., in atherosclerosis). Two principal mechanisms are thought to contribute to maintaining the quiescence of normal arterial smooth muscle cells, namely, interactions with the ECM and mediators derived from the endothelium. Heparin-like components of the ECM can sequester endogenous growth factors and also directly inhibit VSMC proliferation.38–40 Interactions between VSMCs and other components of the basement membrane, in particular polymerized collagen and laminin, also suppress or at least do not support VSMC proliferation.25,41,42 By contrast, components of the remodeling ECM, including monomeric collagen and fibronectin, promote VSMC proliferation.40 Like most nontransformed cells, VSMCs deprived of cell matrix attachments by culture in suspension fail to proliferate. Hence, attachment to permissive ECM components appears essential for VSMC proliferation, and it is possible that the ECM in uninjured arteries simply prevents these interactions. Another relevant issue is VSMC phenotype.43 VSMCs in normal arteries are specialized for contraction and relaxation. To participate in repair they need to alter their genetic program, and this phenotypic change is dependent on interactions with the remodeling ECM.44 Heparan sulfate proteoglycans secreted by the endothelium suppress VSMC proliferation.39,45,46 as do the endothelium-derived mediators prostacyclin and nitric oxide.47 Prostacyclin acts through adenylyl cyclase to elevate cAMP levels in VSMCs, while nitric oxide acts through guanylyl cyclase to increase cGMP levels. Analogues of cAMP and cGMP, activators of the cyclases (forskolin and organic nitrates, respectively), and phosphodiesterase inhibitors are all pharmacological inhibitors of VSMC proliferation.47

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Coordination of Action of Growth Factors and ECM at the Level of the Cell Cycle in VSMCs To ask this question we developed a very simple model system where we compared VSMC proliferation in segments of de-endothelialized rat aorta with cells isolated by collagenase digestion for the same tissue. First we noted that VSMC proliferation in response to serum, PDGF, or FGF-2 is almost totally suppressed in aorta even in the absence of endothelium.26 More importantly, we found that activation of the ERK1/2 growth factor signaling pathway and induction of CDK subunits was virtually identical in aorta and isolated VSMCs. Even so, CDK activity failed to occur in aortas and this was associated with constitutive high levels of CDK inhibitors, p16INK4 and p27Kip126. Since p27Kip1 apparently plays a more important role in VSMC proliferation in vivo, we concentrated on this CKI. We found that the decline in p27Kip1 levels late in G1 was mediated by proteasomal degradation and therefore hypothesized that ubiquitination of p27Kip1 might be a key difference between aortas and isolated VSMCs.48 Moreover, since the E3 ubiquitin ligase subunit S-phase kinase-associated protein-2 (Skp-2) was believed to be essential for recruitment of p27Kip1 to the ligase,49,50 we wondered whether its expression was altered in aorta compared to cells. This proved to be the case because serum upregulated Skp-2 in cells but not aorta.48 Moreover, if we prevented Skp-2 upregulation with small interfering RNA or prevented its function with a dominant negative Skp-2 mutant protein, p27Kip1 levels failed to fall and the cells did not progress into the S-phase.48,51 Skp-2 levels increased in isolated VSMCs after approximately 12 hours, just when p27Kip1 levels declined. Even more important, however, the time course and location of Skp-2 levels in the rat carotid balloon injury model exactly paralleled proliferation measured by PCNA staining.48,51 All this data demonstrates that Skp-2 is necessary for VSMC proliferation in vitro and in vivo, but is it sufficient? Amazingly, when we overexpressed Skp-2 at high levels in the VSMCs of intact rat aortas in culture48 or after gentle denudation of rat carotid arteries in vivo (Y.-J. Wu, M. Bond, and A.C. Newby, unpublished observations), VSMC proliferation was dramatically increased. This showed that Skp-2 is not only necessary, but when overexpressed in the presence of growth factors, it can be sufficient to drive cells into the S-phase. Regulation of Skp-2 levels proved to depend on interactions with the ECM. VSMCs in suspension culture or attached to fibrillar collagen or laminin, matrices that support proliferation poorly, had low levels of Skp-2. Conversely, cells grown on plastic or fibronectin proliferated well and had high levels of Skp-2.48 Not surprisingly, these ECM effects were mediated through integrin binding and activation of focal adhesions.52 We showed, for example, that blocking activation of focal adhesion kinase (FAK) using a dominant negative mutant prevented Skp-2 accumulation and inhibited VSMC proliferation. Hence, in conclusion, growth factors were needed to promote assembly of G1 cyclins. However, activation of FAK by ECM components was also necessary for Skp-2 upregulation, degradation of p27Kip1, and therefore activity of the CDKs, which


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finally promoted S-phase entry (Figure 7-2B). In fact, the situation is slightly more complex since Skp-2 is also essential for downregulation of another CKI, p21Cip1, and both of these events have to occur for VSMC proliferation53 (Figure 7-2B). Downregulation of p16INK4 also occurs in late G1, but we do not yet understand its basis. Elevation of p16INK4 alone does not seem enough to block VSMC proliferation after injury54 but does appear to be responsible for the inhibition of SMC proliferation by PPARα agonists.55

Involvement of Skp-2 in Inhibition of VSMC Proliferation by cAMP Since FAK is known to be inhibited by cAMP,56 we questioned whether FAK inhibition might underlie suppression of SMC proliferation by endotheliumderived mediators. We first confirmed that cAMP inhibited FAK phosphorylation in VSMCs and found that cGMP caused a contrary small activation.51 Furthermore, addition of cAMP analogues or elevation of endogenous cAMP dramatically inhibited Skp-2 protein levels, while cGMP had no effect. Intriguingly, the effect of cAMP on Skp-2 was only partially rescued by a constitutively active FAK mutant, which suggests there may also be other, perhaps more potent upstream regulators of Skp-2. More importantly, however, overexpressing Skp-2 substantially reversed the inhibitory effect of cAMP on VSMC proliferation, which showed that Skp-2 inhibition is an important mediator of the effect of cAMP. Inhibitory effects of cGMP, observed only in VSMCs under low serum conditions, were also mediated through downregulation of Skp-2.51 Skp2 did not completely reverse the antiproliferative effects of cyclic nucleotides and hence previously described mechanisms, including downregulation of cyclinD1 expression, c-myc expression, and activity of ERK1/2, may also contribute.57–59 Neverthless, Skp-2 appears to be a gatekeeper of the cell cycle in VSMCs, balancing the positive effects of the ECM and the negative effects of endothelium (Figure 7-2B).

Conclusion: Integrating Skp-2 into the Cycle of Vessel Wall Sclerosis In the normal vessel wall VSMCs are kept quiescent by the combined effects of the basement membrane and endothelium-derived mediators (Figure 7-3). The initial response to injury may include death and release of endothelial cell and SMC cellular proteins, including FGF-2 and lysosomal proteases, accompanied by adhesion of blood platelets to surface denuded of endothelial cells. Oxidative stress and the induction of NFκB-dependent genes, including cytokines, growth factors, and metalloproteinases,60 are initiated. In some cases this encourages the ingress of inflammatory cells, which amplify the production of growth

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FIGURE 7-3. The cycle of vessel wall sclerosis. Smooth muscle cells (SMCs) are maintained quiescent by nitric oxide (NO) and prostacyclin (PGI2) from the uninjured endothelium. Injury causes platelet (P) adhesion and upregulates production of platelet-derived growth factor (PDGF) and interleukin-1 production, which leads to an inflammatory state. Ingress of foam cells (FC) and T lymphocytes (T) amplifies the production of growth factor cytokines and extracellular proteases that include urokinase type palsminogen activator (uPA) and metalloproteinases (MMPs). Matrix remodeling ensues, including loss of basement membranes and upregulation of integrins and fibronectin. Hence, all the conditions are in place for phenotypic modulation, activation of growth signal transduction pathways, FAK activation, and hence upregulation of S-phase kinase-associated protein-2 (Skp-2), which removes the last barrier to cell proliferation. Upregulation of PGI2 and heparan sulfate proteoglycan production mediates the return to quiescence, together with the production of the fibrogenic cytokine transforming growth factor-β (TGF-β).

factors, cytokines, and extracellular proteases. ECM remodeling, including loss of basement membranes and synthesis of new ECM components such as fibronectin, ensues, accompanied by changes in cell surface integrin expression. Hence microenvironments are created around individual VSMCs, where all the conditions are in place for phenotypic modulation, activation of growth signal transduction pathways, and assembly of G1 cyclins. FAK activation then signals upregulation of Skp-2, which mediates destruction of p21Cip1 and p27Kip1 CKIs removing the last barrier to cell proliferation. Clearly, mechanisms must also exist to restore quiescence. Upregulation of PGI2 and heparan sulfate proteoglyan production in the regrown, inflamed endothelium must be a possibility together with production of the fibrogenic cytokine, TGF-β, from VSMCs. Interestingly TGF-β also downregulates Skp-2 in fibroblasts and so probably plays an important part in this aspect of VSMC quiescence, too. Finally, the VSMCs stop proliferating, reverse their phenotype, and the cycle is complete (Figure 7-3).

Acknowledgment. The author’s work is supported by the British Heart Foundation and The Mackay Memorial Hospital, Taipei.


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36. Dzau VJ, Gibbons GH, Pratt RE. Molecular mechanisms of vascular reninangiotensin system in myointimal hyperplasia. Hypertension 1991;18(suppl 2): II-100-II-105. 37. Eguchi S, Dempsey PJ, Frank GD, Motley ED, Inagami T. Activation of MAP kinases by angiotensin II in vascular smooth muscle cells: metalloprotease-dependent EGF receptor activation is required for activation of ERK and p38 MAP kinase, but not for JNK. J Biol Chem 2000;276:7957–7962. 38. Clowes AW, Clowes MM. Kinetics of cellular proliferation after arterial injury. IV. Heparin inhibits rat smooth muscle mitogenesis and migration. Circ Res 1986;58: 839–845. 39. Segev A, Nili N, Strauss BH. The role of perlecan in arterial injury and angiogenesis. Cardiovasc Res 2004;63:603–610. 40. Hedin U, Roy J, Tran PK. Control of smooth muscle cell proliferation in vascular disease. Curr Opin Lipidol 2004;15:559–565. 41. Assoian RK, Marcantonio EE. The extracellular matrix as a cell cycle control element in atherosclerosis and restenosis. J Clin Invest 1996;98:2436–2439. 42. Morla A, Mogford J. Control of smooth muscle cell proliferation and phenotype by integrin signaling through focal adhesion kinase. Biochem Biophys Res Comm 2000;272:298–302. 43. Owens GK, Kumar MS, Wamhoff BR. Molecular regulation of vascular smooth muscle cell differentiation in development and disease. Physiol Rev 2004;84:767– 801. 44. Hedin U, Bottger BA, Forsberg E, Johansson S, Thyberg J. Diverse effects of fibronectin and laminin on phenotypic properties of cultured arterial smooth muscle cells. J Cell Biol 1988;107:307–319. 45. Garl P, Wenzlau J, Walker H, Whitelock J, Costell M, Weiser-Evans M. Perlecaninduced suppression of smooth muscle cell proliferation is mediated through increased activity of the tumor suppressor PTEN. Circ Res 2004;94:175– 183. 46. Walker H, Whitelock J, Gark P, Nemenoff R, Stenmak K, Weiser-Evans M. Perlecan up-regulation of FRNK suppresses smooth muscle cell proliferation via inhibition of FAK signalling. Mol Biol Cell 2003;14:1941–1952. 47. Jeremy JY, Rowe D, Emsley AM, Newby AC. Nitric oxide and the proliferation of vascular smooth muscle cells. Cardiovasc Res 1999;43:580–594. 48. Bond M, Sala-Newby GB, Newby AC. Focal adhesion kinase (FAK)-dependent regulation S-phase kinase associated protein-2 (Skp-2) stability: a novel mechanism regulating smooth muscle cell proliferation. J Biol Chem 2004;279:37304– 37310. 49. Carrano A, Eytan E, Hershko A, Pagano M. SKP2 is required for ubiquitin-mediated degradation of the CDK inhibitor p27. Nat Cell Biol 1999;1:193–199. 50. Sutterluty H, Chatelain E, Marti A, et al. p45skp2 promotes p27kip1 degradation and induces S phase in quiescent cells. Nat Cell Biol 1999;1:207–214. 51. Wu Y-J, Bond M, Sala-Newby GB, Newby AC. Altered S-phase kinase-associated protein-2 levels are a major mediator of cyclic nucleotide-induced inhibition of vascular smooth muscle cell proliferation. Circ Res 2006;98:1141–1150. 52. Giancotti FG, Ruoslahti E. Integrin signaling. Science 1999;285:1028–1032. 53. Bond M, Sala-Newby GB, Wu Y-J, Newby AC. Biphasic effect of p21Cip1 on smooth muscle cell proliferation: role of PI 3-kinase and Skp2-mediated degradation. Cardiovasc Res 2006;69:198–206.

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8 Control of Interstitial Fluid Homeostasis: Roles of Growth Factors and Integrins Kristofer Rubin, Åsa Lidén, Tijs van Wieringen, and Rolf K. Reed

Fluid Homeostasis: Role of Loose Connective Tissues Surrounding the Vessels Fluid and solutes are constantly filtered from the blood stream to the surrounding loose interstitial connective tissue. This flow of fluid has importance for, e.g., cellular metabolism and immunosurveillance. The filtered fluid is moved through the tissues into the lymphatic vessels, which eventually return fluid and solutes back into the blood circulation. The driving force for the filtration results from differences between fluid pressures in the blood vessel and loose connective tissue. The Starling equation, JV = ΔPK, describes fluid filtration (JV) across a capillary wall (for a review, see Ref.1). K is a constant expressing capillary area and permeability. ΔP is the differences in the colloid osmotic pressures in plasma (COPc) and interstitial fluid (COPif), and between capillary hydrostatic pressure (Pc) and interstitial fluid pressure (Pif) according to: ΔP = (Pc − Pif) − σ (COPc − COPif), where σ is the plasma protein reflection coefficient. Normally, σ is close to 1, reflecting the low leakage of plasma proteins from normal blood vessels. The higher concentration of diffusible proteins in plasma compared to interstitial fluid together with the properties of the capillary wall causes a higher COPc than COPif, generating a pressure difference that tends to keep fluid within the vessels. Inflammatory processes result in an increased vascular permeability for plasma proteins with a lowering of σ that may cause edema formation since σ (COPc − COPif) is lowered. Vascular permeability factor (VPF), also known as vascular endothelial growth factor A (VEGF-A), is a major determinant for the increased microvascular permeability of proteins in carcinoma and chronic inflammatory conditions (for recent reviews, see Refs. 2–4). The VEGF family of proteins includes several members and splice variants of these. The VEGFs signal through cell surface receptors (VEGFR1–3) equipped with intracellular tyrosine kinase domains. These receptors are chiefly expressed by endothelial cells but also by other cell types, including vascular smooth muscle cells, myofibroblasts, and macrophages. Ligand-stimulated receptors elicit specific signaling, 105

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which in cultured endothelial cells stimulates cell-survival, proliferation, migration, and cytoskeletal rearrangements. The VEGF family is of fundamental importance for vasculogenesis during development and for angiogenesis in the adult. Also, it has an important role in modulating vascular permeability and thereby in fluid homeostasis. Pif in normal loose interstitial connective tissues is close to zero or slightly negative.1 However, during anaphylaxis and burn injuries the loose connective tissue surrounding the blood vessels changes its properties in a manner that is reflected in a rapid lowering of Pif. This lowering of Pif raises fluid filtration into the interstitium and thereby increases the interstitial fluid volume that will dissipate the lowered Pif. Therefore, the lowering of Pif has been studied under conditions where fluid filtration is abolished, i.e., after circulatory arrest. We have proposed a model for how loose connective tissue cells control Pif and thereby fluid homeostasis.5–7 This model is based on data suggesting that connective tissue cells, i.e., fibroblasts and/or pericytes, exert a tension on the collagen/microfibril network. The collagen/microfibril network in turn restrains the intrinsic swelling pressure of the hyaluronan/ proteoglycan ground substance of the ECM.8 The swelling pressure of the hyaluronan/proteoglycan ground substance is due to a relative underhydration of the tissues. The peri- and/or extravascular cells of the loose connective tissues are able to actively modulate Pif by regulating the tension they apply on the ECM.

Interstitial Fluid Pressure Is Modulated by Prostaglandins, Cytokines, and Growth Factors Several cytokines and inflammatory mediators can modulate Pif. Local injections in rat dermis of the pro-inflammatory cytokines interleukin-1 (IL-1), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) lower Pif within 20 minutes.9 Prostaglandin E1 (PGE1) and the prostacyclin analogue carbacyclin similarly lower Pif and generate edema in rat dermis.10 The latter agents increase the concentration of intracellular cAMP, and the synthetic cell-permeable cAMP analogue dibutyryl-cAMP also increased the negativity of Pif in normal rat dermis.11 IL-1 induces increases in cAMP in a number of cultured cell types, including murine fibroblasts,12 but it is not known to what extent the effects of IL-1, IL-6, or TNF-α on dermal Pif depends on increases in intracellular cAMP. Pif lowered after anaphylaxis is normalized by local injection of PDGF-BB but not PDGF-AA or fibroblast growth factor (FGF).13,14 The PDGF family of dimeric growth factors encompasses, in addition to the classical isoforms PDGF-AA, -AB, and -BB, the recently discovered PDGF-CC and PDGF-DD (for a review, see Ref. 15). The receptor-binding domains of the PDGF chains share structural

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characteristics with the VEGF family of proteins. The classical PDGFs are secreted in active forms, whereas both PDGF-CC and PDGF-DD have an additional domain that blocks the receptor-binding domain and has to be removed proteolytically in order for the growth factor to become active. The different PDGF dimers bind and stimulate cognate receptors that contain intrinsic tyrosine kinase domains, which are phosphorylated and activated as a result of ligand binding. The PDGF receptors are homo- or heterodimers of two different gene products: the PDGF receptor α- and β-chains. PDGF-AA and PDGF-CC specifically bind and activate the PDGF receptor-αα, PDGF-BB binds all three possible PDGF receptor dimers, i.e., -αα, -αβ, and -ββ, whereas PDGF-DD binds the PDGF-ββ receptor. The finding that PDGF-BB but not -AA stimulated a normalization of Pif that had been lowered as a result of anaphylaxis suggests that the PDGF receptor-ββ is of particular importance for this in vivo function of PDGF. In addition to PDGF-BB, insulin16 and a prostaglandin F2α analog (Latanoprost)10 also normalize Pif in rat dermis lowered by inflammatory challenge. The ability of PDGF-BB to normalize dermal Pif requires that the PDGF receptors be able to activate phosphatidylinositol-3-kinase (PI3K).14 Furthermore, the Pif-normalizing activity of insulin is abolished by the selective PI3K inhibitor wortmannin. Local injection of wortmannin in naive rat dermis also lowers Pif, suggesting that PI3K activity is of importance for maintaining Pif in naive rat skin.17 Finally, disruption of contractile apparatus of connective tissue cells by local injection of cytochalasin D lowers dermal Pif. The available data suggest that increases of intracellular cAMP in connective tissue cells relax these cells, which in turn leads to a lowering of Pif. Stimulation of PI3K activity in the connective tissue cells counteracts such lowering of Pif. The findings described above suggest an intricate interplay between growth factors, cytokines, and prostaglandins in the control of Pif.

Interstitial Fluid Pressure and Integrins The tension generated in connective tissue cells is conveyed from the intracellular cytoskeletal machinery to the ECM via cell surface integrins.6,7,18 Integrins are transmembrane heterodimeric glycoproteins built from noncovalently associated α- and β-subunits that mediate both intercellular and cell-ECM adhesion.19,20 The eight β-, and 18 α-integrin subunits are combined into the 24 integrin heterodimers identified to date. Several integrins recognize the tripeptide Arg-Gly-Asp in ECM ligands, e.g., the fibronectin-binding integrins α5β1 and αVβ3.19,21 Four integrins bind triple helical interstitial collagens in a non–RGD-dependent fashion, namely, the integrins α1β1, α2β1, α10β1, and α11β1.19,22 Cell surface integrins are concentrated at focal adhesion sites in cells cultured on ECM substrates. These sites contain accumulations of cytoskeletal molecules and several signaling proteins, including ligand-activated growth factor receptors.20 The focal adhesions have been described as “signaling

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organelles,” in which signals guiding proliferation, migration, and survival are integrated. Clustering of integrins in the plasma membrane by antiintegin antibodies also activate intracellular signal pathways.20,23 Integrins function in mechanoreception that sense the tension exerted on cells by ECM structures.20 Injection of specific anti-β1 integrin IgG, or anti-α2β1 IgG but not anti-α1β1, lowers Pif in normal rat dermis.13,18 These findings point to a particular importance of the collagen-binding integrin α2β1 in maintaining Pif. Pif lowered as a result of blockade of the α2β1 integrin is normalized by local injections of PDGF-BB.13 Furthermore, PDGF-BB–elicited normalization of Pif after anaphylaxis induced by mast cell degranulation depends on the αVβ3 integrin.24 This normalizing effect is abolished by specific anti-β3 integrin IgG, but not by anti-β1 integrin IgG. Also, PDGF-BB had no effect on Pif in β3-integrin–deficient mice. Injection of anti-β3 integrin IgG had, however, no effect on Pif in naive mouse dermis, and dermal Pif in β3-integrin–deficient mice was similar to that recorded in wild-type mice. Thus, it seems likely that whereas the β1-integrin participates in fluid homeostasis, the β3-integrin participates in Pif recovery after inflammation-induced lowering of dermal Pif.

Cell-Mediated Contraction of Collagen Gels as an In Vitro Model for Control of Interstitial Fluid Pressure In Vivo Fibroblasts cultured in a three-dimensional collagen lattice contract the lattice typically within 24 hours (for a review, see Ref. 25). This process is often referred to as cell-mediated collagen gel contraction and shares many characteristics with connective tissue cell-directed control of Pif in vivo. The nature of the force generator in cells contracting a collagen gel is not fully elucidated but relates to the traction forces that characteristically are generated by cultured cells on the culture support.26,27 Newly formed integrin focal adhesion complexes at the cell periphery are constantly transported toward the cell center in stationary cells.28 In collagen gels fibroblasts form discernable contact sites resembling FAs where they presumably attach to the collagen fibers.29,30 It can be hypothesized that collagen gel contraction involves such contact sites being constantly transported towards the cell center. Fibroblast-mediated collagen gel contraction depends on β1 integrins; available data show that α1β1, α2β1, and α11β1 can mediate collagen gel contraction in different cell types (for a review, see Ref. 22). In addition, the Arg-Gly-Asp–directed integrin αVβ3 is able to mediate collagen gel contraction by cells, particularly when the collagenbinding β1-integrins are either absent, perturbed, or deficient in signaling properties.24,31,32 PDGF stimulates fibroblast-mediated collagen gel contraction,29,33 and this effect depends on the αVβ3 integrin.24,31,32 Dibutyryl-cAMP and proinflammatory agents, such as IL-1 and PGE1, inhibit fibroblast-mediated


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collagen contraction. These inhibitory activities of IL-1 and PGE1 can be at least partly overcome by PDGF-BB.10,34 Furthermore, dibytyryl cAMP inhibits contraction. These and other examples of similar characteristics of fibroblast-mediated collagen gel contraction in vitro and control of Pif in vivo provide further support for the notion that Pif is modulated by contractile activity of connective tissue cells. The fact that PDGF-BB can stimulate collagen gel contraction through engaging the αVβ3 integrin and also overcome the inhibitory activities of pro-inflammatory agents is of relevance in the further delineation of cellular and molecular mechanisms involved in edema control.

Interstitial Fluid Pressure and Capillary-to-Interstitium Transport in Carcinoma Carcinomas consist, in addition to the malignant cells of a proliferative stroma, which is a prerequisite for tumor growth. The size of the stroma compartment varies between different types of carcinomas. In some highly malignant carcinomas, e.g., scirrhous ventricular, pancreas, and anaplastic thyroid carcinomas, the stroma compartment is particularly prominent. The tumor stroma corresponds to the loose connective tissue embedding blood vessels in normal tissues and shares many characteristics with inflamed connective tissue, hence the notion that tumors are “wounds that do not heal.”35 The generation and growth of the stroma is due to malignant cell-driven activation of the normal, i.e., nontransformed host cells, such as macrophages, vascular cells, and fibroblasts. At the same time the latter cells promote survival and proliferation of the malignant cells.36,37 There is a marked upregulation of cytokines and growth factors and their receptors in wound healing, inflamed tissue, and carcinomas. Furthermore, inflamed tissues and tumor stroma, as opposed to normal loose interstitial connective tissues, maintain a high synthesis of ECM components, such as collagen type I. Inflammatory cells infiltrate carcinoma, and their role in tumor progression is complex and debated.36,38,39 On the one hand, macrophages and macrophage products promote tumor growth in some experimental systems.36,40 This can be exemplified by the finding that experimental tumors grown in mice deficient in either IL-1α or IL-1β display reduced angiogenesis and invasiveness.40 On the other hand, tumor-associated lymphocytes have been implicated in immune reactions that inhibit tumor progression.38 Activated macrophages produce a number of growth factors, cytokines, and other inflammatory mediators that potentially influence tumor progression such as prostaglandins, VEGF, IL-1, PDGF, tumor necrosis factor-α (TNF-α), and TGF-β. Due to the resemblance between tumor stroma and chronically inflamed loose connective tissue, it seems plausible that the study of carcinoma physiology should also provide insights into the physiology of chronic inflammatory lesions. The physiological properties of carcinomas are determined by the stroma. Both human and experimental carcinomas characteristically have a

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pathologically elevated Pif, with recorded values as high as 30–40 mmHg depending on tumor type and size.41,42 Fibrosis and abnormal activation of loose interstitial connective tissue cells in the tumor stroma, as well as deranged vascular structures, high capillary permeability for plasma proteins, and impaired lymphatic drainage, have all been suggested to be important factors for the high tumor Pif.43–46 Successful pharmacological treatment of carcinoma requires that the anticancer agents reach the malignant cells. They must cross capillary barriers and diffuse through the ECM of the tumor stroma. Inadequate transport of anticancer drugs into the tumor tissue may explain in part why chemotherapy for solid malignancies often proves less effective.47 The high tumor Pif forms or reflects a barrier to penetration of anticancer drugs into tumors, thus limiting drug efficacy.41A number of agents lower tumor Pif in experimental cancers including prostaglandin E1 (PGE1),48 a bradykinin agonist,49 inhibitors of PDGF or PDGF receptors,50 an inhibitor of TGF-β1 and -β3,51 antivascular endothelial growth factor (VEGF) IgG,52 and dexamethasone.53 In several studies a parallel increase in transport of low molecular weight components into the tumor tissue and/or increased efficacy of chemotherapeutics and lowering of Pif has been observed.48,49,54–57 An emerging picture of the mechanisms by which some of these agents lower Pif involves a dampening of macrophage activity in the carcinoma.57 This lowering of macrophage activity results in reduced leakage of plasma proteins and changes in the ECM. Thus, a reduction of the inflammatory characteristics of carcinoma may be a rewarding avenue to in part normalize the pathophysiology of carcinomas to enable more effective chemotherapy regimes. A hypothetical model for how agents lower Pif in carcinoma function is schematically illustrated in Figure 8-1.

Conclusion In the present chapter we have discussed how loose connective tissue cells play an important role in the control of Pif and fluid transport in tissues by their ability to alter the physical properties of the tissue within a few minutes. This activity of loose connective tissue cells relies on the tension the cells exert on the surrounding ECM via integrins. Inflammatory mediators, cytokines, and chemokines have been demonstrated to modulate the tension that connective tissue cells exert on the ECM as well as the metabolism of ECM constituents. Although an acute effect on the loose connective tissues has been demonstrated for many inflammatory mediators, cytokines, and chemokines, our data suggest an intricate in vivo interplay between these factors. The details of this interplay are currently far from fully understood—neither their relative importance nor the time at which they may operate following a specific inflammatory stimulus. For example, although direct evidence for a role of endogenous PDGF to counteract excessive edema formation during inflammatory processes is presently lacking, this seems to be a viable possibility that calls for further investigations.


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FIGURE 8-1. (A) A schematic representation of characteristics that determine the pathophysiology of a carcinoma stroma. Tumor blood vessels have an increased permeability for plasma proteins (red dots) creating a high fluid pressure in the constrained stroma. Extracellular matrix (ECM) fibers in stroma are abundant and thick, possibly due to the stress resulting from the increased fluid pressure. Furthermore, the stroma contains activated macrophages (cyan) and connective tissue cells (yellow). The exchange of fluid from the tumor vessels to the stroma is ineffective (blue arrow). (B) Effects of specific agents that lower the interstitial fluid pressure (Pif) in experimental carcinoma. Thus, in addition to a lowering of Pif, plasma protein leakage and numbers of activated macrophages are reduced after treatment with, e.g., an inhibitor of TGF-β1 and -β3. It can be hypothesized that the connective tissue cells are relaxed and that the density of the ECM is reduced. Furthermore, available data suggest that the capillary-to-interstitium transport of fluid and solutes increases.

This is also the case for the role of endogenous insulin and prostaglandin F2α under similar conditions. The dynamic relationship between connective tissues cells and the ECM seems to be of interest and relevance for treatment of carcinomas. In at least

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some respects carcinomas may be regarded as a model for chronic inflammatory lesions. Specific interferences with receptors for specific growth factors, cytokines, and prostaglandins result in changes in the relationship between the connective tissue cells and the interstitial matrix. Several agents that lower Pif and increase efficacy of chemotherapy in experimental carcinomas also reduce inflammatory parameters in the stroma. These findings point to the idea that anti-inflammatory treatments can provide an interesting avenue to improve existing chemotherapy. Furthermore, the data demonstrate the importance of inflammation-induced changes in the loose connective tissues for tissue physiology.

Acknowledgments. Original work from the laboratories of the authors was supported by grants from the Swedish Cancer Foundation (to K.R), the Swedish Science Council (to K.R.), the Gustaf V:s 80-årsfond (to K.R.), and the Research Council of Norway (to R.K.R.). Ann-Marie Gustafson and Gerd Salvesen are gratefully acknowledged for technical assistance.

References 1. Aukland K, Reed RK. Interstitial-lymphatic mechanisms in the control of extracellular fluid volume. Physiol Rev 1993;73:1–78. 2. Dvorak HF. Discovery of vascular permeability factor (VPF). Exp Cell Res 2006; 312:522–526. 3. Tammela T, Enholm B, Alitalo K, Paavonen K. The biology of vascular endothelial growth factors. Cardiovasc Res 2005;65:550–563. 4. Weis SM, Cheresh DA. Pathophysiological consequences of VEGF-induced vascular permeability. Nature 2005;437:497–504. 5. Rubin K, Gullberg D, Tomasini-Johansson B, Reed RK, Rydén C, Borg TK. Molecular recognition of the extracellular matrix by cell surface receptors. In: Comper WD, ed. Extracellular Matrix, Vol. 2, Molecular Components and Interactions. Reading, UK: Harwood Academic Publishers, 1996:262–309. 6. Reed RK, Berg A, Gjerde EA, Rubin K. Control of interstitial fluid pressure: role of β1-integrins. Semin Nephrol 2001; 21:222–230. 7. Wiig H, Rubin K, Reed RK. New and active role of the interstitium in control of interstitial fluid pressure: potential therapeutic consequences. Acta Anaesthesiol Scand 2003;47:111–121. 8. Meyer FA. Macromolecular basis of globular protein exclusion and of swelling pressure in loose connective tissue (umbilical cord). Biochem Biophys Acta 1983;755:388–399. 9. Nedrebø T, Berg A, Reed RK. Effect of tumor necrosis factor-α, IL-1β, and IL-6 on interstitial fluid pressure in rat skin. Am J Physiol 1999;277:H1857– 1862. 10. Berg A, Ekwall, AK, Rubin K, Stjernschantz J, Reed RK. Effect of PGE1, PGI2, and PGF2α analogs on collagen gel compaction in vitro and interstitial pressure in vivo. Am J Physiol 1998;274:H663–671.


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11. Rodt SÅ, Reed RK, Ljungström M, Gustafsson TO, Rubin K. The anti-inflammatory agent a-trinositol exerts its edema-preventing effects through modulation of β1 integrin function. Circ Res 1994;75:942–948. 12. Shirakawa F, Yamashita U, Chedid M, Mizel SB. Cyclic AMP—an intracellular second messenger for interleukin 1. Proc Natl Acad Sci USA 1988;85:8201–8205. 13. Rodt SÅ, Åhlen K, Berg A, Rubin K, Reed RK. A novel physiological function for platelet-derived growth factor-BB in rat dermis. J Physiol (London) 1996;495:193– 200. 14. Heuchel R, Berg A, Tallquist M, et al. Platelet-derived growth factor beta receptor regulates interstitial fluid homeostasis through phosphatidylinositol-3′ kinase signaling. Proc Natl Acad Sci USA 1999;96:11410–11415. 15. Fredriksson L, Li H, Eriksson U. The PDGF family: four gene products form five dimeric isoforms. Cytokine Growth Factor Rev 2004;15:197–204. 16. Nedrebø T, Karlsen TV, Salvesen GS, Reed RK. A novel function of insulin in rat dermis. J Physiol 2004;559:583–591. 17. Åhlen K, Berg A, Stiger F, et al. Cell interactions with collagen matrices in vivo and in vitro depend on phosphatidylinositol 3-kinase and free cytoplasmic calcium. Cell Adhes Commun 1998;5:461–473. 18. Reed RK, Rubin K, Wiig H, Rodt SÅ. Blockade of β1-integrins in skin causes edema through lowering of interstitial fluid pressure. Circ Res 1992;71:978–983. 19. Hynes RO. Integrins: bidirectional, allosteric signaling machines. Cell 2002;110:673– 687. 20. Miranti CK, Brugge JS. Sensing the environment: a historical perspective on integrin signal transduction. Nat Cell Biol 2002;4:E83–90. 21. Ruoslahti E, Reed JC. Anchorage dependence, integrins, and apoptosis. Cell 1994; 77:477–478. 22. Gullberg DE, Lundgren-Åkerlund E. Collagen-binding I domain integrins—what do they do? Prog Histochem Cytochem 2002;37:3–54. 23. Schwartz MA, Ginsberg MH. Networks and crosstalk: integrin signalling spreads. Nat Cell Biol 2002;4:E65–68. 24. Liden Å, Berg A, Nedrebø T, Reed RK, Rubin K. Platelet-derived growth factor BB-mediated normalization of dermal interstitial fluid pressure after mast cell degranulation depends on β3 but not β1 integrins. Circ Res 2006;98:635–641. 25. Grinnell F. Fibroblast biology in three-dimensional collagen matrices. Trends Cell Biol 2003;13:264–269. 26. Harris AK, Stopak D, Wild P. Fibroblast traction as a mechanism for collagen morphogenesis. Nature 1981; 290:249–251. 27. Balaban NQ, Schwarz US, Riveline D, et al. Force and focal adhesion assembly: a close relationship studied using elastic micropatterned substrates. Nat Cell Biol 2001;3:466–472. 28. Smilenov LB, Mikhailov A, Pelham RJ, Marcantonio EE, Gundersen GG. Focal adhesion motility revealed in stationary fibroblasts. Science 1999;286:1172– 1174. 29. Gullberg D, Tingström A, Thuresson AC, et al. β1 Integrin-mediated collagen gel contraction is stimulated by PDGF. Exp Cell Res 1990;186:264–272. 30. Cukierman E, Pankov R, Stevens DR, Yamada KM. Taking cell-matrix adhesions to the third dimension. Science 2001;294:1708–1712. 31. Cooke ME, Sakai T, Mosher DF. Contraction of collagen matrices mediated by α2β1A and αVβ3 integrins. J Cell Sci 2000;113:2375–2383.

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32. Grundström G, Mosher DF, Sakai T, Rubin K. Integrin aVb3 mediates plateletderived growth factor-BB-stimulated collagen gel contraction in cells expressing signaling deficient integrin α2β1. Exp Cell Res 2003;291:463–473. 33. Clark RA, Folkvord JM, Hart CE, Murray MJ, McPherson JM. Platelet isoforms of platelet-derived growth factor stimulate fibroblasts to contract collagen matrices. J Clin Invest 1989;84:1036–1040. 34. Tingström A, Heldin C-H, Rubin K. Regulation of fibroblast-mediated collagen gel contraction by platelet-derived growth factor, interleukin-1α and transforming growth factor-β1. J Cell Sci 1992;102:315–322. 35. Dvorak HF. Tumors: wounds that do not heal. Similarities between tumor stroma generation and wound healing. N Engl J Med 1986;315:1650–1659. 36. Pollard JW. Tumour-educated macrophages promote tumour progression and metastasis. Nat Rev Cancer 2004;4:71–78. 37. Bhowmick NA, Neilson EG, Moses HL. Stromal fibroblasts in cancer initiation and progression. Nature 2004;432:332–337. 38. Jakobisiak M, Lasek W, Golab J. Natural mechanisms protecting against cancer. Immunol Lett 2003;90:103–122. 39. Bingle L, Brown NJ, Lewis CE. The role of tumour-associated macrophages in tumour progression: implications for new anticancer therapies. J Pathol 2002;196: 254–265. 40. Voronov E, Shouval DS, Krelin Y, et al. IL-1 is required for tumor invasiveness and angiogenesis. Proc Natl Acad Sci USA 2003;100:2645–2650. 41. Jain RK. Barriers to drug delivery in solid tumors. Sci Am 1994;271:58–65. 42. Gutmann R, Leunig M, Feyh J, et al. Interstitial hypertension in head and neck tumors in patients: correlation with tumor size. Cancer Res 1992;52:1993– 1995. 43. Brekken C, Hjelstuen MH, Bruland OS, de Lange Davies C. Hyaluronidaseinduced periodic modulation of the interstitial fluid pressure increases selective antibody uptake in human osteosarcoma xenografts. Anticancer Res 2000;20:3513– 3519. 44. Jain RK. Transport of molecules in the tumor interstitium: a review. Cancer Res 1987;47:3039–3051. 45. Leu AJ, Berk DA, Lymboussaki A, Alitalo K, Jain RK. Absence of functional lymphatics within a murine sarcoma: a molecular and functional evaluation. Cancer Res 2000;60:4324–4327. 46. Carmeliet P, Jain RK. Angiogenesis in cancer and other diseases. Nature 2000; 407:249–257. 47. Tannock IF. Tumor physiology and drug resistance. Cancer Metastasis Rev 2001; 20:123–132. 48. Rubin K, Sjöquist M, Gustafsson AM, Isaksson B, Salvessen G, Reed RK. Lowering of tumoral interstitial fluid pressure by prostaglandin E1 is paralleled by an increased uptake of 51Cr-EDTA. Int J Cancer 2000;86:636–643. 49. Emerich, DF, Snodgrass P, Dean RL, et al. Bradykinin modulation of tumor vasculature: I. Activation of B2 receptors increases delivery of chemotherapeutic agents into solid peripheral tumors, enhancing their efficacy. J Pharmacol Exp Ther 2001;296:623–631. 50. Pietras K, Östman A, Sjöquist M, et al. Inhibition of platelet-derived growth factor receptors reduces interstitial hypertension and increases transcapillary transport in tumors. Cancer Res 2001;61:2929–2934.


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51. Lammerts E, Roswall P, Sundberg C, et al. Interference with TGF-β1 and -b3 in tumor stroma lowers tumor interstitial fluid pressure independently of growth in experimental carcinoma. Int J Cancer 2002;102:453–462. 52. Lee CG, Heijn M, di Tomaso E, et al. Anti-vascular endothelial growth factor treatment augments tumor radiation response under normoxic or hypoxic conditions. Cancer Res 2000;60: 5565–5570. 53. Kristjansen PE, Boucher Y, Jain RK. Dexamethasone reduces the interstitial fluid pressure in a human colon adenocarcinoma xenograft. Cancer Res 1993;53:4764– 4766. 54. Salnikov AV, Iversen VV, Koisti M, et al. Lowering of tumor interstitial fluid pressure specifically augments efficacy of chemotherapy. FASEB J 2003;17:1756–1758. 55. Pietras K, Rubin K, Sjöblom T, et al. Inhibition of PDGF receptor signaling in tumor stroma enhances antitumor effect of chemotherapy. Cancer Res 2002;62:5476– 5484. 56. Willett CG. Boucher Y, Di Tomaso E, et al. Direct evidence that the VEGF-specific antibody bevacizumab has antivascular effects in human rectal cancer. Nat Med 2004;10:145–147. 57. Salnikov AV, Roswall P, Sundberg C, Gardner H, Heldin N-E, Rubin K. Inhibition of TGF-β modulates macrophages and vessel maturation in parallel to a lowering of interstitial fluid pressure in experimental carcinoma. Lab Invest 2005;85:512– 521.

Section Three Vascular Complications

9 Vascular Complications of Systemic Sclerosis: A Molecular Perspective Daryll M. Baker and Christopher Denton

Introduction Striking vascular abnormalities are a hallmark feature of all types of systemic sclerosis (SSc). Individuals with the limited cutaneous (lcSSc) subset typically manifest Raynaud’s phenomenon many years before onset of frank scleroderma symptoms. Repeated and prolonged peripheral vasospasm frequently leads to painful digital ischemia, ulceration, and gangrene, which often requires surgery and amputation.1 But vascular disease is known to extend beyond the peripheral circulation, and while its role in overall disease evolution is yet to be fully defined, it is likely that some internal organ complications of SSc may be the result of end-organ vascular injury.2 Indeed, scleroderma renal crisis, often an early feature of diffuse (dc) SSc and frequently a cause of mortality in this group, is thought to have an underlying vascular pathophysiology.3–5 And there is evidence that dysregulated vasomotor tone in the pulmonary circulation causes pulmonary artery hypertension secondary to lcSSc.6–8 The nature of SSc vascular dysfunction was long thought to be essentially a heightened vasoconstricting potential, particularly affecting the microcirculation. This belief was largely based on observations of patients with Raynaud’s phenomenon secondary to SSc as well as on empirical data from patients treated with vasodilating agents.9–11 It is now increasingly clear that vascular pathophysiology in SSc involves several mechanisms, which together have the potential to affect all parts of the systemic vascular tree.12–16 First, there is evidence of abnormalities of multiple components of the vessel wall, including endothelial cells,13,14,17,18 smooth muscle cells,19 and extracellular matrix (ECM)20,21 (Figure 9-1). Second, these changes are not likely to be confined to the microcirculation as previously thought, but are probably a feature of vessels of various sizes, including medium-sized muscular arteries22 and large elastic arteries.23,24 Third, significant changes occur within the vessel lumen, probably secondary to endothelial dysfunction, and resulting in a heightened tendancy to coagulate.25,26 All this equates to a disease state in which there is abnormal vasoconstriction, reduced arterial elasticity, and prolonged and repetitive microvascular occlusions. This leads to significant hemodynamic 119

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Baker and Denton Tunica Intima Consists primarily of a layer of endothelial cells. Endothelial dysfunction impairs release of vasoactive mediators such NO and ET-1. This increases the vasoconstricting potential and causes clotting abnormalities. Tunica Media Consists of smooth muscle cells, collagen and elastin fibres. Marked fibrosis causes alterations in vessel elasticity. Endothelial dysfunction impairs regulation of smooth muscle tone. Tunica Adventitia Consists of collagen and elastin fibres. Marked fibrosis contributes to alterations in vessel elasticity Lumen Platelet dysfunction renders blood hypercoagulable

FIGURE 9-1. Diagram summarizing the effect of SSc on a typical artery.

disturbances and ischemic injury, both of which are major factors in disease progression. This chapter seeks to shed light on the mechanisms of vascular injury implicated in SSc. These mechanisms are complex and highly intertwined, and the evidence is occasionally conflicting. But they can be broadly divided into those that involve injury to vascular endothelial cells and those that promote fibrotic vascular disease, and these are outlined here (and summarized in Table 9-1). TABLE 9-1. Mechanisms of vascular injury in systemic sclerosis Site

Etiology

Effect

1. Endothelial dysfunction

Injury mechanism

Endothelial cells of the tunica intima Principally affecting the microcirculation Possibly affecting medium-sized arteries (e.g., brachial)

?AECA ?Viral infection

2. Fibrotic vascular disease

ECM and smooth muscle cells of the tunica media and tunica adventitia Known to affect large elastic arteries such as the carotid Possibly affects medium-sized arteries (e.g., brachial)

?Fibrillin gene defects ?Secondary to endothelial cell dysfunction

Dysregulated vasomotor tone Heightened constricting potential Altered clotting status Repeated microvascular occlusions Dysregulated ECM and SMC interactions Increased arterial stiffness

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Mechanisms of Vascular Injury in SSc Endothelial Dysfunction The vascular endothelium is a cellular monolayer that makes up the tunica intima and is present on the luminal surface of vessels of all sizes throughout the systemic vascular tree. In essence, it is a highly active paracrine organ that secretes a variety of vasoactive mediators, such as nitric oxide and endothelin-1, that work in harmony with each other, and with the sympathetic nervous system, to maintain normal vasomotor tone and coagulation status.27 Endothelial dysfunction is known to be a precursor to most cardiovascular diseases, including coronary artery disease and atherosclerotic peripheral vascular disease. Although yet to be conclusively demonstrated in vivo, endothelial dysfunction is highly likely to be a crucial component in the vascular pathogenesis of SSc.13,14,28–33 The triggering event for endothelial injury is not known, although several theories have emerged. It has been postulated that a class of autoantibody, distinct from ANA, Scl-70, and other rheumatic autoantibodies, may specifically target vascular endothelial cells in autoimmune vascular disorders; these have been dubbed antiendoethelial cell autoantibodies (AECAs).34–36 Carvalho et al., in a study where they treated cultured human endothelial cells with AECA-positive sera drawn from SSc patients, have demonstrated that AECAs cause endothelial cell activation and release of inflammatory mediators, in particular interleukin (IL)-1, with subsequent lymphocyte infiltration.37 Another putative effect of AECAs may be to induce spontaneous endothelial cell apoptosis by binding to cell surface receptors. Worda et al. have demonstrated, in an avian model of SSc, that endothelial cells undergo apoptosis in the presence of AECAs.38 It is likely that AECAs are damaging to endothelial cells, but it is not yet clear whether they cause primary endothelial injury or secondary injury in response to other immunological stimuli, such as viral infection. Certainly, there is evidence to support a viral etiology for SSc, possibly involving latent cytomegalovirus (CMV) infection of endothelial cells.39–43 Lunardi et al. have shown that antibodies with CMV affinity have the ability to induce apoptosis in endothelial cells cultured from SSc subjects.44 No studies have yet been conducted to identify any homology between AECAs and these “anti-CMV” antibodies. A possible scenario is that CMV infection, together with an environmental or genetic factor (possibly a mutation of the fibrillin-1 gene), triggers the initial immune reponse, with AECAs produced as a by-product. In this scenario AECA functions as a potent endothelial activator, and the inflammatory stimulus is propelled forward towards endothelial injury. Whatever the precise etiology of endothelial injury, once established, it sets in motion a vicious cycle that is critical to the evolution of SSc vascular disease. Dysregulated release of vasoactive mediators leads to the heightened

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vasoconstricting potential seen universally in these patients. There is activation of leukocytes in end organs and in the vessel walls themselves, with the release of pro-inflammatory cytokines and growth factors.42,45,46 There is a surge in fibroblast activity, causing excessive collagen synthesis and ECM deposition.47–50 Furthermore, endothelial injury dysregulates platelet function and causes abnormal release of clotting factors, leading to a hypercoagulable state and repeated microvascular occlusions.51 The resulting ischemia/reperfusion episodes generate reactive oxygen species, exacerbating endothelial cell injury.52 As the disease progresses, the repeated inflammatory stimuli cause arterial smooth muscle proliferation and intercellular scarring, resulting in an abnormal thickening of the tunica media.13 This increases arterial stiffness, adds to the vasoconstricting potential, and further contributes to endothelial injury.

Fibrotic Vascular Disease The tunica media is composed of a circumferential arrangement of smooth muscle cells embedded in a collagen and proteoglycan rich ECM. This layer, assisted by the thin outermost tunica adventitia, is responsible for sustaining a semi-rigid arterial structure. The processes that trigger abnormal stiffening usually fall into broad and identifiable categories such as mechanical stress, inflammation, genetic factors, nicotine toxicity, and hormonal influences. However, ultimately any factor that affects arterial stiffness must impact directly on the relationship between arterial ECM and medial smooth muscle cells.53 Under normal circumstances, the collagenous ECM of the tunica media maintains smooth muscle cell alignment and integrity, while providing a suitable framework in which smooth muscle cells can contract and relax—an essential requirement for the maintenance of normal vessel tone.54 Smooth muscle cells are linked to elastic lamellae of the ECM by microfibrils composed of fibrillin, and also by various collagen types: I, III, IV, and VI.55 Collagen, in particular types I and III, has a high tensile strength and elastic modulus, but has a low strain. Other molecules that have important roles in this interaction include elastin, glycoprotein, and proteoglycans.56 The ECM provides a scaffold for translating the force generated by individual smooth muscle cells into organized arterial wall contraction during systole, while also accounting for passive stiffness during diastole.57 Any disease process that interferes with this interaction, for example, by means of excessive collagen deposition or by affecting linking molecules such as fibrillin, will result in abnormalities of arterial stiffness. These effects can be demonstrated in arteries of various sizes, including large vessels such as the aorta58–60 and medium-sized muscular arteries such as the coronary artery.61 Indeed, increased arterial stiffness is a well-recognized feature of cardiovascular disease in general, and it is known to occur secondary to essential hypertension, advancing age, presence of atherosclerosis, and menopause.62–64 Increased


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stiffness is also classically found in genetic connective tissue disorders such as Marfan’s syndrome, where it often causes aortic dilatation.65 Systemic sclerosis is primarily a disease of excessive fibrosis and uncontrolled collagen deposition, caused by biosynthetically activated fibroblasts. These changes are known to occur in the skin and internal organs of patients,66 but are also likely to occur in arterial walls. Preliminary evidence for this is provided by Cheng et al., who have demonstrated reduced compliance and increased stiffness in the carotid arteries of SSc patients67,68. But given the structural importance of collagenous ECM in arteries of various sizes, it is probable that the arterial elasticity changes observed in SSc are not confined to large vessels such as the common carotid, but are in fact present throughout the vascular tree including medium-sized muscular arteries such as the brachial artery.

Discussion Since we presently await the emergence of a definitive triggering agent for SSc, this review is essentially an analysis of the downstream cellular mechanisms that ultimately disrupt normal arterial function and lead to a typical SSc morphology (digital ischemia, gangrene, and internal organ failure). The mechanisms that are implicated in SSc vascular injury are highly complex and involve several closely related but distinct components of arterial function. Given our current level of knowledge of normal arterial function, making sense of these wide-ranging mechansims and the way in which they interact is a challenge. One convenient way in which these processes can be categorized is into those that involve primary injury to vascular endothelium and those that involve primary fibrotic vascular injury. It is highly likely that endothelial dysfunction is a critical step in SSc vascular pathogenesis, although this has yet to be conclusively demonstrated in vivo. The triggering event is unknown (although latent CMV virus infection and AECAs are thought to play a role), but the downstream effects, caused by dysregulated release of edNO and ET-1, are unmistakable. There results a heightened vasoconstricting potential, and a hypercoagulable state that causes repeated microvascular occlusions, both of which are typical features of SSc. Since SSc is essentially a disease of widespread excessive fibrotic activity, it is likely that this fibrosing process also contributes to SSc vascular disease. Indeed, at least one group have already demonstrated increased carotid arterial stiffness in vivo in SSc.69 The underlying cause is thought to be one or more mutations of the gene that encodes fibrillin-1, an essential arterial ECM component. This leads to microfibrillar instability and degradation, with inappropriate release of TGF-β and continued fibroblast activation. The result is uncontrolled collagen synthesis and eventual disruption of normal smooth muscle cell–ECM interactions, which is manifest as increased arterial stiffness.

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An interesting issue is the relationship between primary fibrotic vascular disease and fibrotic vascular disease secondary to endothelial dysfunction. Dysregulated release of edNO and ET-1 by endothelial cells results in medial fibroblast activation and smooth muscle cell proliferation. Studies into atherosclerotic vascular disease provide evidence that prolonged endothelial dysfunction leads to structural changes within the arterial wall that eventually manifests itself as increased arterial stiffness. As yet it is not clear to what extent fibrosis secondary to endothelial dysfunction contributes to SSc vascular disease.

References 1. Clements PJ. Systemic sclerosis (scleroderma) and related disorders: clinical aspects. Baillieres Best Pract Res Clin Rheumatol 2000;14:1–16. 2. Stratton RJ, Coghlan JG, Pearson JD, et al. Different patterns of endothelial cell activation in renal and pulmonary vascular disease in scleroderma. QJM 1998; 91:561–566. 3. Stratton RJ, Coghlan JG, Pearson JD, et al. Different patterns of endothelial cell activation in renal and pulmonary vascular disease in scleroderma. QJM 1998; 91:561–566. 4. Steen VD, Mayes MD, Merkel PA. Assessment of kidney involvement. Clin Exp Rheumatol 2003;21:S29–S31. 5. Steen VD. Scleroderma renal crisis. Rheum Dis Clin North Am 2003;29:315–333. 6. Stratton RJ, Coghlan JG, Pearson JD, et al. Different patterns of endothelial cell activation in renal and pulmonary vascular disease in scleroderma. QJM 1998; 91:561–566. 7. Orfanos SE, Psevdi E, Stratigis N, et al. Pulmonary capillary endothelial dysfunction in early systemic sclerosis. Arthritis Rheum 2001;44:902–911. 8. Kawut SM, Taichman DB, Archer-Chicko CL, Palevsky HI, Kimmel SE. Hemodynamics and survival in patients with pulmonary arterial hypertension related to systemic sclerosis. Chest 2003;123:344–350. 9. Clements PJ. Systemic sclerosis (scleroderma) and related disorders: clinical aspects. Baillieres Best Pract Res Clin Rheumatol 2000;14:1–16. 10. Leighton C. Drug treatment of scleroderma. Drugs 2001;61:419–427. 11. Pope J, Fenlon D, Thompson A, Shea B, Furst D, Wells G, Silman A. Iloprost and cisaprost for Raynaud’s phenomenon in progressive systemic sclerosis. Cochrane Database Syst Rev 2000;CD000953. 12. Cheng KS, Tiwari A, Boutin A, et al. Carotid and femoral arterial wall mechanics in scleroderma. Rheumatology (Oxford) 2003. 13. Herrick AL. Vascular function in systemic sclerosis. Curr Opin Rheumatol 2003;12: 527–533. 14. Kahaleh BM. Endothelin an endothelium dependent in scleroderma. Enhanced production and profibrotic action. Arthritis Rheum1991;34:978–983. 15. Livi R, Teghini L, Generini S, Matucci-Cerinic M. The loss of endotheliumdependent vascular tone control in systemic sclerosis. Chest 2001;119:672–673. 16. Schachna L, Wigley FM. Targeting mediators of vascular injury in scleroderma. Curr Opin Rheumatol 2002;14:686–693. 17. Livi R, Teghini L, Generini S, Matucci-Cerinic M. The loss of endotheliumdependent vascular tone control in systemic sclerosis. Chest 2001;119:672–673.


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18. Schachna L, Wigley FM. Targeting mediators of vascular injury in scleroderma. Curr Opin Rheumatol 2002;14:686–693. 19. Andersen GN, Mincheva-Nilsson L, Kazzam E, et al. Assessment of vascular function in systemic sclerosis: indications of the development of nitrate tolerance as a result of enhanced endothelial nitric oxide production. Arthritis Rheum 2002;46:1324– 1332. 20. Cheng KS, Tiwari A, Boutin A, et al. Carotid and femoral arterial wall mechanics in scleroderma. Rheumatology (Oxford) 2003;42:1299–1305. 21. Cheng KS, Tiwari A, Boutin A, et al. Differentiation of primary and secondary Raynaud’s disease by carotid arterial stiffness. Eur J Vasc Endovasc Surg 2003;25: 336–341. 22. Andersen GN, Mincheva-Nilsson L, Kazzam E, et al. Assessment of vascular function in systemic sclerosis: indications of the development of nitrate tolerance as a result of enhanced endothelial nitric oxide production. Arthritis Rheum 2002;46:1324– 1332. 23. Cheng KS, Tiwari A, Boutin A, et al. Carotid and femoral arterial wall mechanics in scleroderma. Rheumatology (Oxford) 2003;42:1299–1305. 24. Cheng KS, Tiwari A, Boutin A, et al. Differentiation of primary and secondary Raynaud’s disease by carotid arterial stiffness. Eur J Vasc Endovasc Surg 2003; 25:336–341. 25. Cerinic MM, Valentini G, Sorano GG,et al. Blood coagulation, fibrinolysis, and markers of endothelial dysfunction in systemic sclerosis. Semin Arthritis Rheum 2003;32:285–295. 26. Ames PR, Lupoli S, Alves J, et al. The coagulation/fibrinolysis balance in systemic sclerosis: evidence for a haematological stress syndrome. Br J Rheumatol 1997;36: 1045–1050. 27. Vallance P, Chan N. Endothelial function and nitric oxide: clinical relevance. Heart 2001;85:342–350. 28. Livi R, Teghini L, Generini S, Matucci-Cerinic M. The loss of endotheliumdependent vascular tone control in systemic sclerosis. Chest 2001;119:672–673. 29. Schachna L, Wigley FM. Targeting mediators of vascular injury in scleroderma. Curr Opin Rheumatol 2002;14:686–693. 30. Sgonc R, Gruschwitz MS, Dietrich H, Recheis H, Gershwin ME, Wick G. Endothelial cell apoptosis is a primary pathogenetic event underlying skin lesions in avian and human scleroderma. J Clin Invest 1996;98:785–792. 31. Sgonc R, Gruschwitz MS, Boeck G, Sepp N, Gruber J, Wick G. Endothelial cell apoptosis in systemic sclerosis is induced by antibody-dependent cell-mediated cytotoxicity via CD95. Arthritis Rheum 2000;43:2550–2562. 32. Sud A, Khullar M, Wanchu A, Bambery P. Increased nitric oxide production in patients with systemic sclerosis. Nitric Oxide 2000;4:615–619. 33. Mayes MD. Endothelin and endothelin receptor antagonists in systemic rheumatic disease. Arthritis Rheum 2003;48:1190–1199. 34. Carvalho D, Savage CO, Black CM, Pearson JD. IgG antiendothelial cell autoantibodies from scleroderma patients induce leukocyte adhesion to human vascular endothelial cells in vitro. Induction of adhesion molecule expression and involvement of endothelium-derived cytokines. J Clin Invest 1996;97:111–119. 35. Sgonc R, Gruschwitz MS, Boeck G, Sepp N, Gruber J, Wick G. Endothelial cell apoptosis in systemic sclerosis is induced by antibody-dependent cell-mediated cytotoxicity via CD95. Arthritis Rheum 2000;43:2550–2562.

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36. Worda M, Sgonc R, Dietrich H, et al. In vivo analysis of the apoptosis-inducing effect of anti-endothelial cell antibodies in systemic sclerosis by the chorionallantoic membrane assay. Arthritis Rheum 2003;48:2605–2614. 37. Carvalho D, Savage CO, Black CM, Pearson JD. IgG antiendothelial cell autoantibodies from scleroderma patients induce leukocyte adhesion to human vascular endothelial cells in vitro. Induction of adhesion molecule expression and involvement of endothelium-derived cytokines. J Clin Invest 1996;97:111– 119. 38. Worda M, Sgonc R, Dietrich H, et al. In vivo analysis of the apoptosis-inducing effect of anti-endothelial cell antibodies in systemic sclerosis by the chorionallantoic membrane assay. Arthritis Rheum 2003;48:2605–2614. 39. Hamamdzic D, Kasman LM, LeRoy EC. The role of infectious agents in the pathogenesis of systemic sclerosis. Curr Opin Rheumatol 2002;14:694–698. 40. Hamamdzic D, Harley RA, Hazen-Martin D, LeRoy EC. MCMV induces neointima in IFN-gammaR-/- mice: intimal cell apoptosis and persistent proliferation of myofibroblasts. BMC Musculoskelet Disord 2001;2:3. 41. Lunardi C, Bason C, Navone R, et al. Systemic sclerosis immunoglobulin G autoantibodies bind the human cytomegalovirus late protein UL94 and induce apoptosis in human endothelial cells. Nat Med 2000;6:1183–1186. 42. Kahaleh MB, LeRoy EC. Autoimmunity and vascular involvement in systemic sclerosis (SSc). Autoimmunity 1999;31:195–214. 43. Neidhart M, Kuchen S, Distler O, et al. Increased serum levels of antibodies against human cytomegalovirus and prevalence of autoantibodies in systemic sclerosis. Arthritis Rheum 1999;42:389–392. 44. Lunardi C, Bason C, Navone R, et al. Systemic sclerosis immunoglobulin G autoantibodies bind the human cytomegalovirus late protein UL94 and induce apoptosis in human endothelial cells. Nat Med 2000;6:1183–1186. 45. Matucci Cerinic M, Kahaleh BM, LeRoy EC. The vascular involvement in systemic sclerosis. In:Furst D, Clements PJ, eds. Systemic Sclerosis. Baltimore:Lea and Febiger; 1995:153–174. 46. Sgonc R, Gruschwitz MS, Boeck G, Sepp N, Gruber J, Wick G. Endothelial cell apoptosis in systemic sclerosis is induced by antibody-dependent cell-mediated cytotoxicity via CD95. Arthritis Rheum 2000;43:2550–2562. 47. Shi-Wen X, Denton CP, Dashwood MR, et al. Fibroblast matrix gene expression and connective tissue remodeling: role of endothelin-1. J Invest Dermatol 2001;116:417– 425. 48. Xu S, Denton CP, Holmes A, Dashwood MR, Abraham DJ, Black CM. Endothelins: effect on matrix biosynthesis and proliferation in normal and scleroderma fibroblasts. J Cardiovasc Pharmacol 1998;31(suppl. 1):S360–S363. 49. Xu SW, Denton CP, Dashwood MR, Abraham DJ, Black CM. Endothelin-1 regulation of intercellular adhesion molecule-1 expression in normal and sclerodermal fibroblasts. J Cardiovasc Pharmacol 1998;31(suppl. 1):S545–S547. 50. Schachna L, Wigley FM. Targeting mediators of vascular injury in scleroderma. Curr Opin Rheumatol 2002;14:686–693. 51. Ames PR, Lupoli S, Alves J, et al. The coagulation/fibrinolysis balance in systemic sclerosis: evidence for a haematological stress syndrome. Br J Rheumatol 1997;36:1045– 1050. 52. Herrick AL, Matucci CM. The emerging problem of oxidative stress and the role of antioxidants in systemic sclerosis. Clin Exp Rheumatol 2001;19:4–8.


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53. Kingwell BA, Medley TL, Waddell TK, Cole TJ, Dart AM, Jennings GL. Large artery stiffness: structural and genetic aspects. Clin Exp Pharmacol Physiol 2001;28:1040– 1043. 54. Gospodarowicz D, Vlodavsky I, Savion N. The extracellular matrix and the control of proliferation of vascular endothelial and vascular smooth muscle cells. J Supramol Struct 1980;13:339–372. 55. Dingemans KP, Teeling P, Lagendijk JH, Becker AE. Extracellular matrix of the human aortic media: an ultrastructural histochemical and immunohistochemical study of the adult aortic media. Anat Rec 2000;258:1–14. 56. Dingemans KP, Teeling P, Lagendijk JH, Becker AE. Extracellular matrix of the human aortic media: an ultrastructural histochemical and immunohistochemical study of the adult aortic media. Anat Rec 2000;258:1–14. 57. Kingwell BA, Medley TL, Waddell TK, Cole TJ, Dart AM, Jennings GL. Large artery stiffness: structural and genetic aspects. Clin Exp Pharmacol Physiol 2001;28:1040– 1043. 58. Berry KL, Cameron JD, Dart AM, Dewar EM, Gatzka CD, Jennings GL, Liang YL, Reid CM, Kingwell BA. Large-artery stiffness contributes to the greater prevalence of systolic hypertension in elderly women. J Am Geriatr Soc 2004;52:368–373. 59. Ahimastos AA, Formosa M, Dart AM, Kingwell BA. Gender differences in large artery stiffness pre- and post puberty. J Clin Endocrinol Metab 2003;88:5375–5380. 60. Medley TL, Cole TJ, Gatzka CD, Wang WY, Dart AM, Kingwell BA. Fibrillin-1 genotype is associated with aortic stiffness and disease severity in patients with coronary artery disease. Circulation 2002;105:810–815. 61. Shaw JA, Kingwell BA, Walton AS,et al. Determinants of coronary artery compliance in subjects with and without angiographic coronary artery disease. J Am Coll Cardiol 2002;39:1637–1643. 62. Berry KL, Cameron JD, Dart AM, et al. Large-artery stiffness contributes to the greater prevalence of systolic hypertension in elderly women. J Am Geriatr Soc 2004; 52:368–373. 63. Ahimastos AA, Formosa M, Dart AM, Kingwell BA. Gender differences in large artery stiffness pre- and post puberty. J Clin Endocrinol Metab. 2003;88:5375– 5380. 64. Kingwell BA, Gatzka CD. Arterial stiffness and prediction of cardiovascular risk. J Hypertension 2002;20:2337–2340. 65. Arteaga-Solis E, Gayraud B, Ramirez F. Elastic and collagenous networks in vascular diseases. Cell Struct Funct 2000;25:69–72. 66. Clements PJ. Systemic sclerosis (scleroderma) and related disorders: clinical aspects. Baillieres Best Pract Res Clin Rheumatol 2000;14:1–16. 67. Cheng KS, Tiwari A, Boutin A, et al. Carotid and femoral arterial wall mechanics in scleroderma. Rheumatology (Oxford) 2003;42:1299–1305. 68. Cheng KS, Tiwari A, Boutin A, et al. Differentiation of primary and secondary Raynaud’s disease by carotid arterial stiffness. Eur J Vasc Endovasc Surg 2003;25: 336–341. 69. Cheng KS, Tiwari A, Boutin A, et al. Carotid and femoral arterial wall mechanics in scleroderma. Rheumatology (Oxford) 2003;42:1299–1305.

10 Therapeutic Options for Preventing Transplant-Related Progressive Renal and Vascular Injury Susanna Tomasoni and Ariela Benigni

Introduction The first organ transplantation occurred in 1954 in Boston under the direction of Joseph Murray: a kidney removed from a healthy donor and transplanted into his identical twin promptly started to function, and the recipient survived for 9 years. Since then, attempts to suppress the recipient’s immune system were pursued with the aim to extend the possibility of transplantation beyond involving identical twins.1 The first approach to suppress the rejection process employing the use of sublethal total-body irradiation combined with cortisone had a poor outcome, however. The rate of successful transplantation of kidneys from cadaveric donors and familial human leukocyte antigen (HLA)-matched living donors slowly increased during the 1960s and early 1970s following the introduction of azathioprine with corticosteroids.1 But a prolonged use of corticosteroids was the cause of high mortality due to excessive immunosuppression. Only the introduction of cyclosporine in 1980 really improved the rate of 1-year graft survival from 70 to more than 80%.2 Thanks to the availability of novel immunosuppressants, short-term results of renal transplantation have improved considerably in the past 20 years, but similar amelioration in long-term outcome has not been achieved. Even in the case of grafts with good function in the early years after transplantation, progressive tissue damage and impairment in graft function could develop, often associated to considerable vasculopathy.3 This chronic allograft nephropathy, also termed chronic rejection, represents the primary cause of late graft loss after the first years after transplantation. Chronic rejection is defined as a relentlessly progressive form of dysfunction spanning years to decades and characterized morphologically by obliterative vasculopathy, interstitial fibrosis with variable degree of mononuclear cell infiltration, and, in the case of kidney, glomerulosclerosis.4 Neither its etiology nor its pathophysiology is clearly understood, although it is well known that both alloantigen-dependent and alloantigen-independent factors are implicated. Antigen-independent factors involve initial insult due to ischemia and reperfusion of the graft and, at least for the kidney, calcineurin inhibitor nephrotoxicity and the low-nephron 128

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mass.5–7 Loss of renal mass, responsible for glomerular hypertension and proteinuria, plays a key role in the progression of renal damage to end-stage renal failure. Alloantigen-dependent events include histocompatibility differences between donor and host, chronic immune stimulation involving the donorderived endothelium with vascular and inflammatory cell activation, and the appearance of allospecific antibodies.5,8,9 The most important predictor of chronic rejection is a previous episode of acute rejection, especially when followed by partial loss of graft function.10 No effective therapy exists for chronic allograft dysfunction. Thus, the definition of strategies that may improve longterm outcome has become a priority in transplantation. Targeting both alloantigen-independent and -dependent factors may have implication for preventing progressive deterioration of graft function. In this chapter the focus will be on kidney transplant, although chronic lesions similarly occur in other grafted organs.

Features of Chronic Graft Rejection The structural and functional changes of chronic renal allograft rejection share similarities with those observed in other forms of chronic progressive renal disease in which inadequate functioning nephron mass has been considered the key event.7,11 In experimental animals, an insufficient number of nephrons, due to a reduction in the renal mass, is the trigger of a self-perpetuating cycle of events, the morphological hallmarks of which are interstitial inflammation and fibrosis, tubular atrophy, glomerular sclerosis, and obliterative intimal fibrosis of arteries. A key functional counterpart is an excessive urinary protein excretion.12 Hemodynamic determinants of renal injury in this setting are enhanced intraglomerular pressure and flow, closely involved in the development of renal structural damage.13 Glomerular hypertension enhances filtration of macromolecules across the capillary barrier, proteins that are then largely reabsorbed by proximal tubuli.12,14 This tubular cell activation induces upregulation of gene encoding inflammatory and vasoactive proteins that also contribute to renal scarring.12,15

Alloantigen-Independent Factors The Role of the Renin–Angiotensin System Of the various mechanisms that contribute to renal function deterioration in proteinuric chronic renal disease, activation of the renin–angiotensin system (RAS) certainly plays a critical role. It is known from several experimental and human studies that blockade of RAS reduced the urinary protein excretion and protected against renal structural injury better than conventional antihypertensive therapy in nontransplant models of chronic renal disease

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caused by lower than normal nephron numbers16 as well as in humans with proteinuric renal disease.5 In patients with chronic nephropathies and proteinuria, ramipril, a long-acting angiotensin-converting enzyme (ACE) inhibitor, slowed the rate at which renal function was lost, safely reduced proteinuria and the rate of glomerular filtration rate (GFR) decline (REIN study). Similarly, RAS inhibitors that were given very early in the posttransplant course have been successful in preventing chronic graft injury in animal model,17–19 and more importantly, a recent study has demonstrated that late treatment with the ACE inhibitor trandolapril fully restored normal graft function, limited progressive proteinuria, and stabilized glomerulosclerosis changes in a rat kidney transplant model with chronic renal dysfunction and overt proteinuria.20 This latter study represents the proof of concept that chronic rejection is not an inexorable process but can be interrupted by the RAS blockade.

The Role of the Endothelin System Since its introduction into clinical practice, cyclosporin A (CsA) has contributed to ameliorate graft survival of various organs despite a variety of side effects, the most relevant of which is the renal toxicity possibly due to vasoconstriction and vascular injury.2,21,22 CsA renal toxicity can manifest as an acute but rapidly reversible decrease in renal function23 or as a chronic form of renal damage, particularly in patients treated for more than 1 year.21 These clinical syndromes share features of vascular damage, functional in the former and structural with histological evidence of obliterative arteriolopathy in the latter.24 It is likely that in CsA-treated patients the cause of renal vasculopathy is an intense vasocostriction. This CsA-induced renal vasoconstriction may be mediated by endothelin-1 (ET-1).25 Endothelin is a 21-amino-acid endogenous peptide with mitogenic and vasoconstrictive properties. It exists in three isoforms, with ET-1 as the predominantly produced isoform responsible for most of the physiological and physiopathological changes seen in humans. Endothelin acts in an auto/paracrine fashion by high-affinity binding to specific cell surface receptors, ETA and ETB. ETA receptor binds ET-1 with a higher affinity than the other isoforms, whereas ETB receptor has similar affinity to all three isoforms.25 Evidence that ET-1 is released in excessive amounts in the supernatant medium when cultured endothelial cells were exposed to CsA has been documented.26 A direct toxic effect of CsA on cultured endothelial cells has been also described.27 Similarly, in an experimental model in vivo, a direct infusion of CsA into the isolated renal artery in dogs induced a significant increase of ET-1 in the renal circulation, indicating a direct effect of CsA on ET-1 production that could be partly responsible for the increase in the vascular resistance.28 In rats chronically treated with CsA, a significant increase in the urinary excretion of both big-ET-1 and ET-1 was observed.24 In CsA-treated renal transplant patients, a chronic administration of CsA was associated with higher blood pressure and increased plasma ET-1 levels.29 Kidney-ransplanted patients on


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CsA trough level, after each administration of CsA developed an acute and transient form of renal hypoperfusion that could be due to an excessive renal synthesis of ET-1.30 Despite the ET-1 involvement in chronic rejection, treatment with ET-1 receptor antagonists gave conflicting results. After 1-week administration of CsA, healthy subjects showed changes of renal hemodynamics with a fall of renal plasma flow, increased blood pressure, and decreased glomerular filtration rate. Co-administration of CsA with bosentan, a mixed ETA/B receptor antagonist, significantly attenuated the CsA renal toxicity by markedly blunting the renal hypoperfusion effect of CsA.31 Conflicting results have been obtained in experimental models. In a rat model of chronic renal allograft rejection, treatment with LU224332, a mixed ETA/B receptor antagonist, did not prevent the progression of chronic renal allograft rejection or prolong survival.32 By contrast, the use of LU135252, a selective ETA receptor antagonist, while not affecting blood pressure and proteinuria, significantly reduced renal function impairment and structural changes and improved survival in chronic allograft rejection in the rat.33 These data would suggest that, as for the prevention of chronic nephropathies, selective inhibition of ETA receptor seems to offer advantages over nonselective antagonists on progressive renal injury underlying renal allograft rejection.

Alloantigen-Dependent Factors T-cell recognition of alloantigens plays a key role in initiating mechanisms critical for chronic rejection, including activation of CD4+ and CD8+ T cells, macrophages, and B cells. T cells require two distinct signals for full and complete activation: the first signal is delivered by the T-cell receptor (TCR) after interaction with antigens presented on MHC molecules of antigen-presenting cells (APCs), and the second is defined as a costimulatory signal, the most characterized of which is the binding between CD28 (on T cells) and B7 (on APCs). Once activated, T cells produce different sets of cytokines that identify two types of CD4+ T-helper cells. Th1 cells produce interleukin-2 (IL-2) and interferon-γ, which promote macrophage activation and delayed-type hypersensitivity responses. Th2 cells produce IL-4, IL-6, IL-10, and IL-13, which differentiate B cells into antibody-producing cells. Cytokines secreted by activated T cells and macrophages in the graft in turn activate endothelial cells to express cell surface adhesion molecules and class I and class II MHC molecules, which mediate further recruitment and activation of lymphocytes.

Optimizing Chronic Immunosuppressive Strategies A combined strategy able to target both the antigen-dependent and independent determinants of the chronic rejection might represent a step forward over the use of single therapies. Mycophenolate mofetil (MMF), a pro-drug of

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mycophenolic acid (MPA), has several immunosuppressant actions specifically inhibiting the proliferative responses of both T and B lymphocytes and blocking humoral responses in vitro and in vivo.34 Originally developed for the prevention and treatment of acute graft rejection, it would also affect processes thought to be involved in chronic graft rejection. Indeed, MMF exerts anti-inflammatory activity, which could attenuate both acute and chronic rejection.35 MMF may also have a synergistic activity with other agents, such as ACE inhibitors or AII receptor antagonist. This concept has been demonstrated in a recent study in which administration of MMF alone to transplanted Fisher-to-Lewis rats only partially protected animals from developing chronic rejection.36 MMF significantly limited intragraft cell infiltration but only partially decreased the amount of proteinuria and preserved the glomerular and tubular graft structure. Moreover, it displayed only a minimal impact on graft survival in respect to untreated transplanted control rats. This partial effect of MMF may occurr as a consequence of nonimmunological factors leading to the nephron loss. In the same model, treatment with losartan improved graft survival but only partially prevented the development of proteinuria and glomerular and tubuointerstitial injury. By contrast, the combined treatment of MMF and losartan completely prevented the development of proteinuria and protected renal graft structure to the extent that graft function was well preserved and all animals survived at the end of the study period. The simultaneous blockade of alloantigen-dependent and independent mechanisms could result in downregulation of cytokine and growth factor gene expression into the graft that ultimately prevented both cellular and molecular events leading to development of chronic rejection.

Blocking the Costimulatory Pathway Using a Gene Therapy Approach Blocking the costimulatory signal represents a strategy to impede fully activation and proliferation of T cells responsible for graft rejection. The cytotoxic T-lymphocyte antigen-4 (CTLA4) is a molecule highly homologous to CD28, expressed on activated T cells, that binds B7 with high affinity. It has been shown that CTLA4Ig was effective in preventing acute rejection of kidney allografts. Direct injection of a recombinant adenovirus encoding for CTLA4Ig into the renal artery of the rat donor kidney before transplantation significantly prolonged allograft survival, although not indefinitely, without the need of systemic immunosuppression.37 Despite the mild infiltration of mononuclear cells observed in the transfected organs, renal function was well preserved in the longlasting animals that showed donor-specific unresponsiveness. Next we tested whether a similar approach could be usefully applied to prevent chronic rejection. Long-term immunomodulation via gene therapy could be ideally achieved by a vector able to drive high-level expression of the therapeutic gene into the graft for a prolonged period of time without exerting any cytotoxic


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effect or eliciting any inflammatory or immune response. Adenoviral vectors provide effective means of gene delivery in several organs, including the kidney, since they do not require cell replication for transduction. However, a major disadvantage is that they trigger host immune response and inflammation, which eventually extinguish transgene expression over time.38 This is why adenovirus cannot be the answer for chronic rejection. Vectors based on the nonpathogenic, single-stranded DNA adeno-associated virus (AAV) have the peculiar advantage over adenovirus of efficiently transducing different tissues persisting for months or years in vivo either after random integration into the host cell genome or in an episomal form.39 The whole viral genome is deleted in recombinant AAV with the exception of two short-inverted terminal repeats, which are required for packaging, integration, and replication of the viral genome. No viral proteins are expressed in recombinant AAV avoiding inflammation or immune response, rendering it an ideal tool to target chronic rejection via gene therapy. We assessed whether chronic allograft rejection could be effectively prevented by local delivery of recombinant adeno-associated virus (rAAV) vectors encoding the CTLA4Ig immunosuppressant protein to the donor kidney before transplantation.40 We chose a fully MHC-mismatched rat strain combination Wistar Furth (WF, RT1u)-to-Lewis (LW, RT1l) renal allograft recipient as a model of chronic rejection. After transplantation, animals received CsA (5 mg/kg) for 10 days to control acute rejection. AAVCTLA4Ig prevented progressive proteinuria and protected transplant kidneys from renal structural injury, completely halting the progression of glomerular lesions and reducing the severity of tubulointerstitial injury. Moreover, AAVCTLA4Ig caused a marked and significant reduction in macrophage infiltration. Analysis of graft-infiltrating lymphocytes showed an increased percentage of CD4+CD25+ T cells in AAVCTLA4Ig-treated grafts compared to untransduced allografts (43% vs. 2.3%, respectively). These cells were in a state of anergy as evidenced by reduced expression of both Th1 and Th2 cytokines. Lymph node T cells isolated from AAVCTLA4Ig-treated animals showed hyporesponse to the donor but not to third-party stimulators, which was reversed by the addition of rIL-2. These T cells significantly inhibited alloreactivity of naïve T lymphocytes to donor WF alloantigens but not to third-party antigen, indicating the generation of T cells with regulatory properties eventually responsible for the induction of tolerance. These data indicate that AAV-mediated CTLA4Ig gene transfer to donor graft represents a promising tool to prevent the onset of the chronic rejection, circumventing the unwanted systemic side effects of the administration of immunomodulatory protein.

Conclusion In the past two decades, long-term results of renal transplantation have not paralleled the progress shown in short-term results because of the inability to prevent progressive chronic allograft dysfunction, which represents the major

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hurdle in transplant medicine. Besides previous acute rejection episodes, HLA matching, pretransplantation injury, and the immunosuppression regimen represent risk factors that could influence the outcome of the graft in the long term. Approaches to protect the allograft kidney in the long term have been designed and tested to control both nonimmune and immune events. These included inhibitors of angiotensin and endotelin receptor antagonist combined with low-dose immunosuppressants. However, the most promising and innovative results for treating chronic allograft rejection derive from the use of a gene therapy approach in a rat model, confining the administration of immunosuppressive therapy at a very early stage after transplantation and at low dose. Additional studies in large animals are now mandatory to prove the concept that a similar approach could be applied to humans in the next future.

References 1. Sayegh MH, Carpenter CB. Transplantation 50 years later—progress, challenges, and promises. N Engl J Med 2004;351:2761–2766. 2. Kahan BD. Cyclosporine. N Engl J Med 1989;321:1725–1738. 3. Pascual M, Theruvath T, Kawai T, Tolkoff-Rubin N, Cosimi AB. Strategies to improve long-term outcomes after renal transplantation. N Engl J Med 2002;346:580– 590. 4. Tilney NL, Whitley WD, Diamond JR, Kupiec-Weglinski JW, Adams DH. Chronic rejection—an undefined conundrum. Transplantation 1991;52:389–398. 5. Remuzzi G, Perico N. Protecting single-kidney allografts from long-term functional deterioration. J Am Soc Nephrol 1998;9:1321–1332. 6. Tullius SG, Hancock WW, Heemann U, Azuma H, Tilney NL. Reversibility of chronic renal allograft rejection. Critical effect of time after transplantation suggests both host immune dependent and independent phases of progressive injury. Transplantation 1994;58:93–99. 7. Mackenzie HS, Brenner BM. Antigen-independent determinants of late renal allograft outcome: the role of renal mass. Curr Opin Nephrol Hypertens 1996;5: 289–296. 8. Paul LC, Fellstrom B. Chronic vascular rejection of the heart and the kidney—have rational treatment options emerged? Transplantation 1992;53:1169–1179. 9. Suciu-Foca N, Reed E, Marboe C, et al. The role of anti-HLA antibodies in heart transplantation. Transplantation 1991;51:716–724. 10. Hariharan S, Johnson CP, Bresnahan BA, Taranto SE, McIntosh MJ, Stablein D. Improved graft survival after renal transplantation in the United States, 1988 to 1996. N Engl J Med 2000;342:605–612. 11. Brenner BM, Milford EL. Nephron underdosing: a programmed cause of chronic renal allograft failure. Am J Kidney Dis 1993;21:66–72. 12. Remuzzi G, Bertani T. Pathophysiology of progressive nephropathies. N Engl J Med 1998;339:1448–1456. 13. Brenner BM. Hemodynamically mediated glomerular injury and the progressive nature of kidney disease. Kidney Int 1983;23:647–655.


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14. Remuzzi G. Abnormal protein traffic through the glomerular barrier induces proximal tubular cell dysfunction and causes renal injury. Curr Opin Nephrol Hypertens 1995;4:339–342. 15. Benigni A, Zoja C, Remuzzi G. The renal toxicity of sustained glomerular protein traffic. Lab Invest 1995;73:461–468. 16. Taal MW, Brenner BM. Renoprotective benefits of RAS inhibition: from ACEI to angiotensin II antagonists. Kidney Int 2000;57:1803–1817. 17. Mackenzie HS, Ziai F, Nagano H, et al. Candesartan cilexetil reduces chronic renal allograft injury in Fisher—>Lewis rats. J Hypertens Suppl 1997;15:S21–S25. 18. Amuchastegui SC, Azzollini N, Mister M, Pezzotta A, Perico N, Remuzzi G. Chronic allograft nephropathy in the rat is improved by angiotensin II receptor blockade but not by calcium channel antagonism. J Am Soc Nephrol 1998;9:1948–1955. 19. Ziai F, Nagano H, Kusaka M, et al. Renal allograft protection with losartan in Fisher—>Lewis rats: hemodynamics, macrophages, and cytokines. Kidney Int 2000;57:2618–2625. 20. Noris M, Mister M, Pezzotta A, et al. ACE inhibition limits chronic injury of kidney transplant even with treatment started when lesions are established. Kidney Int 2003;64:2253–2261. 21. Myers BD. Cyclosporine nephrotoxicity. Kidney Int 1986;30:964–74. 22. Williams D, Haragsim L. Calcineurin nephrotoxicity. Adv Chronic Kidney Dis 2006;13:47–55. 23. Remuzzi G, Bertani T. Renal vascular and thrombotic effects of cyclosporine. Am J Kidney Dis 1989;13:261–272. 24. Benigni A, Perico N, Ladny JR, Imberti O, Bellizzi L, Remuzzi G. Increased urinary excretion of endothelin-1 and its precursor, Big-endothelin-1, in rats chronically treated with cyclosporine. Transplantation 1991;52:175–177. 25. Benigni A. Endothelin antagonists in renal disease. Kidney Int 2000;57:1778– 17794. 26. Bunchman TE, Brookshire CA. Cyclosporine-induced synthesis of endothelin by cultured human endothelial cells. J Clin Invest 1991;88:310–314. 27. Zoja C, Furci L, Ghilardi F, Zilio P, Benigni A, Remuzzi G. Cyclosporin-induced endothelial cell injury. Lab Invest 1986;55:455–462. 28. Carrier M, Tronc F, Stewart D, Pelletier LC. Dose-dependent effect of cyclosporin on renal arterial resistance in dogs. Am J Physiol 1991;261:H1791–1796. 29. Cauduro RL, Costa C, Lhulier F, et al. Endothelin-1 plasma levels and hypertension in cyclosporine-treated renal transplant patients. Clin Transplant 2005;19:470–474. 30. Perico N, Ruggenenti P, Gaspari F, et al. Daily renal hypoperfusion induced by cyclosporine in patients with renal transplantation. Transplantation 1992;54: 56–60. 31. Binet I, Wallnofer A, Weber C, Jones R, Thiel G. Renal hemodynamics and pharmacokinetics of bosentan with and without cyclosporine A. Kidney Int 2000; 57:224–231. 32. Braun C, Conzelmann T, Vetter S, et al. Treatment with a combined endothelin A/Breceptor antagonist does not prevent chronic renal allograft rejection in rats. J Cardiovasc Pharmacol 2000;36:428–437. 33. Braun C, Conzelmann T, Vetter S, et al. Prevention of chronic renal allograft rejection in rats with an oral endothelin A receptor antagonist. Transplantation 1999;68: 739–746. 34. Lipsky JJ. Mycophenolate mofetil. Lancet 1996;348:1357–1359.

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35. Allison AC, Eugui EM. Mechanisms of action of mycophenolate mofetil in preventing acute and chronic allograft rejection. Transplantation 2005;80:S181–S190. 36. Noris M, Azzollini N, Pezzotta A, et al. Combined treatment with mycophenolate mofetil and an angiotensin II receptor antagonist fully protects from chronic rejection in a rat model of renal allograft. J Am Soc Nephrol 2001;12:1937–1946. 37. Tomasoni S, Azzollini N, Casiraghi F, Capogrossi MC, Remuzzi G, Benigni A. CTLA4Ig gene transfer prolongs survival and induces donor-specific tolerance in a rat renal allograft. J Am Soc Nephrol 2000;11:747–752. 38. Yang Y, Li Q, Ertl HC, Wilson JM. Cellular and humoral immune responses to viral antigens create barriers to lung-directed gene therapy with recombinant adenoviruses. J Virol 1995;69:2004–2015. 39. Conlon TJ, Flotte TR. Recombinant adeno-associated virus vectors for gene therapy. Expert Opin Biol Ther 2004;4:1093–10101. 40. Benigni A, Tomasoni S, Turka LA, et al. Adeno-Associated Virus-Mediated CTLA4Ig Gene Transfer Protects MHC-Mismatched Renal Allografts from Chronic Rejection. J Am Soc Nephrol 2006;17:1665–1672.

Section Four Surgical Approaches

11 Current State of Medical Therapies for Peripheral Vascular Disease Janice C.S. Tsui and Daryll M. Baker

Introduction Atherosclerotic narrowing of the lower limb arteries is the most common cause of peripheral vascular disease (PVD), affecting 27 million people in Europe and North America.1 The prevalence of PVD increases with age, and with an aging population it is becoming an increasing burden in the West. Patients may suffer from intermittent claudication (IC)—muscular pain in one or both legs on walking, or the limb may become critically ischemic with pain at rest, gangrene, and ulceration (Figure 11-1). The prevalence of IC varies with age, sex, and geographic location of the population, but rates of up to 14.4% have been reported.2 Five to 10% of patients with claudication will develop critical limb ischemia (CLI) and 2% will eventually require an amputation. CLI affects 20,000 people in the United Kingdom, with a prevalence of 1 in 2500. Over 20% of these patients will require a major amputation.3 Patients with PVD are also at an increased risk of death from cardiovascular causes, mainly from myocardial infarctions and fatal strokes.4 Management options for patients with PVD include conservative measures such as pharmacotherapy, exercise therapy, and cardiovascular risk factor modification, as well as revascularization using either endovascular techniques or open surgery. The choice of treatment depends on the patients’ symptoms, quality of life, disease severity and distribution, and comorbidities. This chapter will focus on pharmacotherapy available for the treatment for PVD.

Intermittent Claudication In patients with IC, the main aims of treatment are to improve walking capacity and quality of life and to reduce the risk of coronary and cerebrovascular events by modification of cardiovascular risk factors. While the risk of complications during revascularization procedures is low,5 it may still outweigh the benefits 139

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FIGURE 11-1. Critically ischemic foot with gangrene of the second toe and ulceration of the big toe.

since IC is a relatively “benign” disease. Conservative measures are therefore preferred for the majority of these patients.

Risk Factor Management Sound evidence exists to support active risk factor intervention in PVD patients to reduce cardiovascular mortality.6 Briefly, patients should be encouraged to stop smoking; hypertension, hypercholesterolemia, and diabetes should be aggressively controlled; and an antiplatelet agent should be prescribed. In addition to reducing cardiovascular deaths, there is also evidence that some of these measures may directly affect PVD symptoms or progression. Smoking is the single most important risk factor for PVD, associated with a threefold increase in risk of developing the disease.7 Persistent smoking contributes to disease progression and poorer outcome following intervention, with higher amputation rates.8 Several observational studies have found an improvement in walking distance and less risk of amputation in patients with claudication who gave up smoking.9 The benefit of statins in primary and secondary prevention of coronary events is well recognized.10 Specific to PVD, the Heart Protection Study11 showed that statin therapy reduced the risk of cardiovascular events by at least a third in patients with PVD. In the Scandinavian Simvastatin Survival Study, the incidence of new-onset or worsening IC was found to be reduced by 38% in patients on statin therapy versus placebo after 3 years.12 Statins have also been shown to improve walking distance in patients with IC.13 This may be attributed to their beneficial effects on endothelial function independent of their cholesterollowering properties.

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Similarly there is clear evidence that antiplatelet therapy confers significant benefit in the secondary prevention of vascular events.14 In addition, the Physicians’ Health Study, a primary prevention trial, found that aspirin reduced the subsequent need for peripheral arterial surgery.15 In patients with PVD who have had revascularization surgery, aspirin may also reduce graft occlusion rates.16 Subgroup analysis of the Heart Outcomes Prevention Evaluation trial showed that ramipril, an angiotensin-converting enzyme inhibitor, reduced major cardiovascular events in PVD patients.17 Recent evidence from a small randomized trial showed that ramipril also improved symptoms in patients with IC.18 Despite the evidence however, it has been shown that cardiovascular risk factors are not always well managed in this group of patients in both primary and secondary care settings.19,20

Pharmacotherapy Various drugs have been used to improve walking distance in claudicant patients for almost 50 years. However, there is little evidence of their efficacy. Many studies are poorly designed, and the use of different protocols makes data comparison difficult. Most studies show a strong placebo effect, which may be due to improved education and awareness and increased exercise in patients enrolled in studies. There is also likely to be significant publication bias. Drugs that have been used to improve walking distance include cilostazol, pentoxifylline, naftidrofuryl, buflomedil, prostanoids, cinnarizine, inosital nicotinate, and levocarnitine.

Cilostazol Cilostazol is a phosphodiesterase type III inhibitor, which increases cellular levels of cyclic adenosine monophosphate, resulting in vasodilation, inhibition of platelet aggregation and thrombus formation, and reduction of vascular smooth muscle cell proliferation.21 It was approved by the U.S. Food and Drug Administration for use in IC in 1999. A meta-analysis of eight randomized, double-blind, placebo-controlled trials involving 2702 patients comparing cilostazol with placebo or pentoxifylline showed that cilostozol increased pain-free walking distance (PFWD) by 67% and maximal walking distance (MWD) by 50% from baseline. These changes were significantly greater than the increases in MWD observed with pentoxifylline and placebo.22 Benefits were seen as early as 4 weeks into treatment.23,24 Another meta-analysis of six randomized, doubleblind, placebo-controlled trials including 1751 patients assessing the effect of cilostazol on community-based walking ability and quality of life also showed an improvement in these outcome measures.25 Reported side effects included headaches, diarrhea, dizziness, and palpitations.

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Pentoxifylline Pentoxifylline (also known as oxypentifylline) is a methylxanthine derivative that has the ability to reduce blood viscosity, increase erythrocyte deformability, and increase microcirculatory flow.26 A meta-analysis of 17 randomized double-blind controlled trials including 1041 patients showed significant improvements in PFWD and MWD in the treatment group. Additional PFWD and MWD of 138.4 m (95% confidence interval [CI] 12.7–264.0) and 356.9 m (95% CI 208.0–505.8), respectively, were found at 24 weeks.27

Naftidrofuryl Naftidrofuryl is thought to act at the tissue level, improving oxygenation, increasing ATP levels, and reducing lactic acid. It may also act as a 5-hydroxytryptamine antagonist.28 A meta-analysis including 901 patients with moderate claudication in eight randomized, placebo-controlled, double-blind clinical trials found modest treatment effects with naftidrofuryl.27 Pooled estimates of PFWD were significantly greater at 24 weeks, reaching 101.5 m (95% CI 12.7–264.0); MWD improved by 31.8 m (95% CI −26.5–90.2) at 12 weeks, but was not significantly increased at 24 weeks. An earlier meta-analysis of five trials showed that the treatment group also had significantly fewer cardiovascular events during the treatment period.29 Longer-term effects are uncertain.

Buflomedil Buflomedil has vasodilatory properties via its anti-α1- and -α2-adrenergic and mild calcium antagonist effects. It also inhibits platelet aggregation and may improve erythrocyte deformability and reduce plasma viscosity.30 A Cochrane review identified two eligible randomized controlled trials.31 The first is a multicenter trial of 113 patients, which showed a significant increase in PFWD and MWD in the buflomedil group versus placebo during a 12-week intervention period.32 The second study of 40 diabetic claudicant patients found a statistically significant increase in MWD at 6 months in the treatment group (191.9 m vs. 20.5 m for placebo) but not in PFWD.33 No attempt was made to pool the results, and the authors concluded that current evidence for the efficacy of buflomedil was poor.

Prostanoids Prostaglandin E1 (PGE1) and other prostanoids have vasodilatory, antiplatelet, and anti-inflammatory activities.34 While there is a role for prostanoids in the management of CLI, their use in IC is less clear. A recent Cochrane review identified 18 randomized clinical trials looking at this area.35 Heterogeneity between the studies prevented pooling of data by meta-analysis. Five studies


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comparing the effects of PGE1 versus placebo showed significant increases in PFWD and MWD with intravenous or intra-arterial PGE1. Intravenous prostacyclin (PGI2), venous iloprost, and oral berapost did not result in any improvement in walking distances. Side effects occurred in 23.6% of patients treated with PGI2 and 13.7% of those treated with PGE1. The authors concluded that PGE1 may have a role in the treatment of IC, but its side effects and the invasive route of administration need to be considered.

Other Agents Less commonly used agents include inosital nicotinate, a derivative of nicotinic acid thought to have vasodilatory effects; cinnarizine, which has unproven effects on reducing blood viscosity, and carnitine supplements, which attempt to improve oxidative phosphorylation in ischemic muscle. Evidence for these agents in the form of recent clinical trials is lacking.

Critical Limb Ischemia The aims of treatment in CLI are to reduce pain, control infection, and promote ulcer healing and prevent limb loss. Risk factor management remains important but is a lesser priority in these patients. Revascularization is the preferred option for limb salvage, with pharmacological agents reserved for patients with nonreconstructable disease (Figure 11-2).

Prostanoids Prostacyclin (PGI2) and its analogs such as iloprost and ciprostene have been used in the treatment of CLI to alleviate pain and avoid amputation. Various reports have shown their benefits in ulcer healing and pain relief. Most of these were small studies with variable treatment periods, and the endpoints of ulcer healing or pain relief were difficult to assess. Seven randomized placebo- or reference-controlled studies on the efficacy of PGEI in CLI involving 643 patients were studied in a meta-analysis.36 Using endpoints of complete or partial ulcer healing or complete pain relief in patients with rest pain but no ulceration, 60.2% of patients in the pooled treatment group showed response to treatment compared to 25.2% in the placebo group. When the three placebo-controlled trials were analyzed together (n = 254), a significantly better response was found in the PGE1 group, with a pooled rate difference in response of 19.9%. A significant difference in the combined endpoint of “major amputation or death” at 6 months was also found: 22.6% in the PGE1 group versus 36.2% in the placebo group (p = 0.029). Side effects occurred at 39.6% in the treatment group compared to 15.4% in the placebo group. These results support the use of PGE1 in patients with CLI not suitable for surgery.

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Tsui and Baker FIGURE 11-2. Magnetic resonance angiography (MRA) of the lower limb arteries of a 73-year-old female with ischemic rest pain of the right foot. The arteries are diseased at multiple levels. On the right are atheromatous stenoses and occlusions of the superficial femoral and popliteal arteries. The distal runoff vessels in the calf are also diseased. These lesions are not suitable for angioplasty, and the diffuse disease and lack of patent run-off vessel render bypass surgery difficult.

Other Agents Vasoactive agents used in the treatment of intermittent claudication have also been assessed for CLI. Parenteral pentoxifylline has been shown to reduce rest pain,37,38 and intravenous naftidrofuryl showed marginal benefits but was withdrawn for treatment of PVD due to reported side effects.39

Potential Strategies L-Arginine

The nitric oxide (NO) pathway plays an important role in vascular endothelial function. L-Arginine is the substrate for NO formation, and its potential role in the treatment of IC and CLI has been shown in early clinical studies using


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intravenous infusions of L-arginine.40,41 The exact mechanism of its efficacy is unclear and implicates the presence of endogenous NO synthase inhibitors.42,43 Further understanding of the complex NO pathway and larger clinical studies of l-arginine therapy may lead to the development of useful agents in the management of PVD.

Endothelin Antagonists Endothelin (ET)-1 is an important endothelial-derived vasoconstrictor and has been implicated in the pathophysiology of PVD.44 The orally active ET antagonist, bosentan, has been shown to reduce recurrence of digital ulcers in patients with systemic sclerosis.45,46 ET-1 antagonism may also benefit PVD patients, for example, in aiding ulcer healing in CLI. The use of ET-receptor–selective antagonists may inhibit the pathological effects of ET-1 in PVD while minimizing side effects.

Therapeutic Angiogenesis Initial studies of therapeutic angiogenesis in PVD were carried out in patients with CLI who were either unsuitable for surgical revascularization or in whom other options had failed. Vascular endothelial growth factor (VEGF) gene transfer showed promise in early studies, with improved ankle-brachial index, enhanced collateral vessel growth, improved ulcer healing, and successful limb salvage reported.47 A larger randomized controlled trial of VEGF gene therapy, however, failed to show significant improvement in amputation rates, ulcer healing, or severity of rest pain.48 More recently, autologous implantation of bone marrow mononuclear cells directly into critically ischemic limbs has been found to improve rest pain and ulcer healing.49 The use of therapeutic angiogenesis in patients with IC has also been investigated. The TRAFFIC study assessed intra-arterial delivery of recombinant basic fibroblast growth factor (bFGF) and found a significant improvement in maximal walking time.50 The RAVE trial investigated intramuscular injection of replication-deficient adenovirus encoding the 121-amino-acid isoform of VEGF and found no siginificant difference in maximal walking time, anklebrachial index, or quality of life.51 Results from some of these early successes have been encouraging, but further research is required to select the appropriate angiogenic agents and effective delivery modalities and to identify potentially serious side effects.

Conclusion The treatment of PVD remains difficult. In terms of pharmacotherapy, cilostazol and pentoxifylline may be useful in patients with intermittent claudication, while prostanoids are the only effective pharmacological agents for CLI

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TABLE 11-1. Summary of pharmacotherapy for peripheral vascular disease Drug

Symptoms

Efficacy

Level of evidence

Ref.

Cilostazol Pentoxifylline Naftidrofuryl Buflomedil PGE1

IC IC IC IC IC

I (MT) I (MT) I (MT) II (2 small, acceptable RCTs) I (MT)

21,24 26 26 31,32 34

PGE1

CLI

Beneficial Some benefit Unlikely beneficial No proven benefit Beneficial but requires iv/ia administration Beneficial

I (MT)

35

IC: intermittent claudication; MT: meta-analysis; RCT: randomized controlled trial; iv: intravenous; ia: intra-arterial; CLI: critical limb ischemia.

(Table 11-1). Better understanding of vascular biology and subsequent development of new strategies, combined with advances in endovascular and surgical techniques, are required to improve the outcome of these patients.

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12. Pedersen TR, Kjekshus J, Pyorala K, et al. Effect of simvastatin on ischemic signs and symptoms in the Scandinavian simvastatin survival study (4S). Am J Cardiol 1998;81:333–335. 13. Mohler ER 3rd, Hiatt WR, Creager MA. Cholesterol reduction with atorvastatin improves walking distance in patients with peripheral arterial disease. Circulation 2003;108:1481–1486. 14. Collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high risk patients. BMJ 2002;324: 71–86. 15. Goldhaber SZ, Manson JE, Stampfer MJ, et al. Low-dose aspirin and subsequent peripheral arterial surgery in the Physicians’ Health Study. Lancet 1992;340:143– 145. 16. Antiplatelet Trialists’ Collaboration. Collaborative overview of randomised trials of antiplatelet therapy II: Maintenance of vascular graft or arterial patency by antiplatelet therapy. BMJ 1994;308:159–168. 17. Ostergren J, Sleight P, Dagenais G, et al. Impact of ramipril in patients with evidence of clinical or subclinical peripheral arterial disease. Eur Heart J 2004;25:17–24. 18. Ahimastos AA, Lawler A, Reid CM, et al. Brief communication: ramipril markedly improves walking ability in patients with peripheral arterial disease: a randomized trial. Ann Intern Med 2006;144:660–664. 19. Hirsch AT, Criqui MH, Treat-Jacobson D, et al. Peripheral arterial disease detection, awareness, and treatment in primary care. JAMA 2001;286:1317–1324. 20. Rehring TF, Sandhoff BG, Stolcpart RS, et al. Atherosclerotic risk factor control in patients with peripheral arterial disease. J Vasc Surg 2005;41:816–822. 21. Sun B, Le SN, Lin S, et al. New mechanism of action for cilostazol: interplay between adenosine and cilostazol in inhibiting platelet activation. J Cardiovasc Pharmacol 2002;40:577–585. 22. Thompson PD, Zimet R, Forbes WP, et al. Meta-analysis of results from eight randomized, placebo-controlled trials on the effect of cilostazol on patients with intermittent claudication. Am J Cardiol 2002;90:1314–1319. 23. Dawson DL, Cutler BS, Hiatt WR, et al. A comparison of cilostazol and pentoxifylline for treating intermittent claudication. Am J Med 2000;109:523–530. 24. Beebe HG, Dawson DL, Cutler BS, et al. A new pharmacological treatment for intermittent claudication: results of a randomized, multicenter trial. Arch Intern Med 1999;159:2041–2050. 25. Regensteiner JG, Ware JE, Jr., McCarthy WJ, et al. Effect of cilostazol on treadmill walking, community-based walking ability, and health-related quality of life in patients with intermittent claudication due to peripheral arterial disease: metaanalysis of six randomized controlled trials. J Am Geriatr Soc 2002;50:1939– 1946. 26. Dettelbach HR, Aviado DM. Clinical pharmacology of pentoxifylline with special reference to its hemorrheologic effect for the treatment of intermittent claudication. J Clin Pharmacol 1985;25:8–26. 27. Moher D, Pham B, Ausejo M, et al. Pharmacological management of intermittent claudication: a meta-analysis of randomised trials. Drugs 2000;59:1057–1070. 28. Lehert P, Riphagen FE, Gamand S. The effect of naftidrofuryl on intermittent claudication: a meta-analysis. J Cardiovasc Pharmacol 1990;16(Suppl. 3):S81–S86. 29. Lehert P, Comte S, Gamand S, et al. Naftidrofuryl in intermittent claudication: a retrospective analysis. J Cardiovasc Pharmacol 1994;23(Suppl. 3):S48–S52.

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30. Diamantopoulos EJ, Grigoriadou M, Ifanti G, Raptis SA. Clinical and hemorheological effects of buflomedil in diabetic subjects with intermittent claudication. Int Angiol 2001;20(4):337–344. 31. De Backer TLM, Vander Stichele RH, Bogaert MG. Buflomedil for intermittent claudication. The Cochrane Database of Syst Rev 2000:CD000988. 32. Trubestein G, Balzer K, Bisler H, et al. Buflomedil in arterial occlusive disease: results of a controlled multicenter study. Angiology 1984;35(8):500–505. 33. Diamantopoulos EJ, Grigoriadou M, Ifanti G, Raptis SA. Clinical and hemorheological effects of buflomedil in diabetic subjects with intermittent claudication. Int Angiol 2001;20(4):337–344. 34. Grant SM, Goa KL. Iloprost. A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic potential in peripheral vascular disease, myocardial ischemia and extracorporeal circulation procedures. Drugs 1992;43(6):889–924. 35. Reiter M, Bucek RA, Stumpflen A, Minar E. Prostanoids for intermittent claudication. Cochrane Database Syst Rev 2004:CD000986. 36. Creutzig A, Lehmacher W, Elze M. Meta-analysis of randomised controlled prostaglandin E1 studies in peripheral arterial occlusive disease stages III and IV. Vasa 2004;33:137–144. 37. Intravenous pentoxifylline for the treatment of chronic critical limb ischemia. The European Study Group. Eur J Vasc Endovasc Surg 1995;9:426–436. 38. Efficacy and clinical tolerance of parenteral pentoxifylline in the treatment of critical lower limb ischemia. A placebo controlled multicenter study. Norwegian Pentoxifylline Multicenter Trial Group. Int Angiol 1996;15:75–80. 39. Smith FB, Bradbury AW, Fowkes FGR. Intravenous naftidrofuryl for critical limb ischemia. Cochrane Database Syst Rev 2000:CD002070. 40. Boger RH, Bode-Boger SM, Thiele W, et al. Restoring vascular nitric oxide formation by L-arginine improves the symptoms of intermittent claudication in patients with peripheral arterial occlusive disease. J Am Coll Cardiol 1998;32:1336– 1344. 41. Bode-Boger SM, Boger RH, Alfke H, et al. L-Arginine induces nitric oxide-dependent vasodilation in patients with critical limb ischemia. A randomized, controlled study. Circulation 1996;93:85–90. 42. Boger RH, Bode-Boger SM, Thiele W, et al. Biochemical evidence for impaired nitric oxide synthesis in patients with peripheral arterial occlusive disease. Circulation 1997;95:2068–2074. 43. Tsikas D, Boger RH, Sandmann J, et al. Endogenous nitric oxide synthase inhibitors are responsible for the L-arginine paradox. FEBS Lett 2000;478:1–3. 44. Tsui JC, Baker DM, Biecker E, et al. Evidence for the involvement of endothelin-1 but not urotensin-II in chronic lower limb ischemia in man. Eur J Vasc Endovasc Surg 2003;25:443–450. 45. Korn JH, Mayes M, Matucci CM, et al. Digital ulcers in systemic sclerosis: prevention by treatment with bosentan, an oral endothelin receptor antagonist. Arthritis Rheum 2004;50:3985–3993. 46. Humbert M, Cabane J. Successful treatment of systemic sclerosis digital ulcers and pulmonary arterial hypertension with endothelin receptor antagonist bosentan. Rheumatology (Oxford) 2003;42:191–193. 47. Baumgartner I, Pieczek A, Manor O, et al. Constitutive expression of phVEGF165 after intramuscular gene transfer promotes collateral vessel development in patients with critical limb ischemia. Circulation 1998;97:1114–1123.


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48. Makinen K, Manninen H, Hedman M, et al. Increased vascularity detected by digital subtraction angiography after VEGF gene transfer to human lower limb artery: a randomized, placebo-controlled, double-blinded phase II study. Mol Ther 2002; 6:127–133. 49. Tateishi-Yuyama E, Matsubara H, Murohara T, et al. Therapeutic angiogenesis for patients with limb ischemia by autologous transplantation of bone-marrow cells: a pilot study and a randomised controlled trial. Lancet 2002;360:427–435. 50. Lederman RJ, Mendelsohn FO, Anderson RD, et al. Therapeutic angiogenesis with recombinant fibroblast growth factor-2 for intermittent claudication (the TRAFFIC study): a randomised trial. Lancet 2002;359:2053–2058. 51. Rajagopalan S, Mohler ER, III, Lederman RJ, et al. Regional angiogenesis with vascular endothelial growth factor in peripheral arterial disease: a phase II randomized, double-blind, controlled study of adenoviral delivery of vascular endothelial growth factor 121 in patients with disabling intermittent claudication. Circulation 2003;108: 1933–1938.

12 Advantages of Harvesting the Saphenous Vein for Coronary Artery Bypass Surgery Using the “No-Touch” Technique Domingos Sávio Ramos de Souza and Bruno Botelho Pinheiro

Introduction Surgery for direct myocardial revascularization was introduced at the end of the 1960s and has dramatically changed the management of patients with ischemic heart disease. In 1967 Favaloro successfully reconstructed the right coronary artery by interposing a segment of saphenous vein (SV) and, later, using the SV graft directly from the aorta to the coronary arteries, established the concept of SV grafting for coronary artery bypass surgery (CABG).1 Since then, the SV graft has been the most commonly used conduit. In the 1980s several studies published by Campeau,2,3 Bourassa,4 and Grondin5 demonstrated that severe atherosclerotic deterioration of SV grafts occurs between 6 and 11 years after implantation. At the same time Loop6 showed a clear advantage of using the internal thoracic artery (ITA) over the SV. Due to the excellent results of the ITA graft, several other arterial grafts have been utilized to substitute the SV graft. In 1987, Pym,7 Suma,8 and Attum9 reported the first studies on the use of the gastroepiploic artery for direct myocardial revascularization. In 1990, Puig10 introduced the use of the inferior epigastric artery, and today the radial artery is also used.11 Despite the widespread use of the ITA and other arterial conduits, the SV continues to be the most commonly used conduit for CABG. However, vein graft failure is associated with recurrence of angina3 and is one of the primary reasons for reoperation. Early vein graft failure occurs in approximately 18% of SV grafts within the first month of implantation, and approximately 30% of SV grafts occlude within the first year.12 At 10 years, graft occlusion rates are more than 50% and the grafts that remain patent frequently show angiographic evidence of luminal narrowing. Every effort should be made to improve the patency rates of SV grafts. Preparation of the graft is important since there is direct evidence that injury during surgery causes severe intimal loss as well as biochemical and functional changes to the graft.13 During harvesting of the SV, a pronounced and protracted spasm occurs that is triggered when removing the perivascular tissue adherent to the vein. Various pharmacological agents have been used to relax the vein,14 but 150

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high-pressure distension of the SV graft is often required to overcome the spasm and establish a lumen adequate for grafting. It therefore seems reasonable to assume that dissection, manipulation, and distension of the SV induces damage to the media and intima of the graft that impacts on its performance. These factors led us to develop a novel “no-touch” technique of harvesting the SV, in which the cushion of tissue surrounding the vein is preserved. The underlying hypothesis was that this cushion of tissue would prevent the vein going into spasm, thus avoiding the need for manual distension and its deleterious effects. Furthermore, the adventitial vasa vasorum may be involved by acting as an important source of nitric oxide to the media.15 We have previously shown that veins harvested with the “no-touch” technique possess a better preserved endothelium16,17 and maintained nitric oxide synthase,15,18 suggesting the potential to improve graft patency. Subsequent prospective randomized studies showed a significant improvement in shortterm19 and long-term20 patency rates for veins harvested with the “no-touch” technique when compared with conventionally prepared SV grafts, supporting our hypothesis.

Surgical Technique Noninvasive preoperative vein mapping of the greater SV using B-mode ultrasonography is crucial to predict the location of the vein, (Figure 12-1). A longitudinal incision is made through the skin over the vein and the subdermal vessels ligated to avoid overuse use of diathermy. The edge of the wound is elevated using forceps, and with the subcutaneous tissue under tension a plane is created around the vein using scissors, the vein being exposed without damage to its perivascular tissue (Figure 12-2). The vein is protected by a thin layer of adherent tissue anteriorly and posteriorly and by a 0.5-cm pedicle of fat on either side. The SV, together with its perivascular tissue, is separated from its bed using scissors and diathermy, and all branches are dissected, ligated, and divided (Figure 12-3). This continuous cushion of perivascular tissue protects the vein from direct handling by surgical

FIGURE 12-1. Vein mapping of the saphenous vein after using ultrasound sonography.

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FIGURE 12-2. Saphenous vein exposure by longitudinal incision.

instruments, dramatically reducing the incidence of venous spasm (Figure 12-4). The vein is then kept in situ covered with saline-soaked swabs until heparinization. After excision the vein is stored in blood obtained from the aortic cannula. While performing the anastomosis, the perivascular tissue is used to grasp the vein, thereby avoiding direct contact between the vein and instruments (Figure 12-5). After each distal anastomosis is completed, the graft is perfused with blood directly from the arterial cannula for approximately 10 seconds to detect any

FIGURE 12-3. Ligation of side branches of the vein at the edge of the pedicle.


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FIGURE 12-4. Saphenous vein removed from its bed without any spasm.

bleeding from the anastomoses or missed side branches. This is performed through a three-way stopcock delivery system (Figure 12-6). After removal of the aortic cross-clamp and before suturing the proximal anastomoses, all vein grafts are connected to the arterial line to allow early myocardial reperfusion. This procedure also helps to determine the graft length, recheck and identify any further bleeding, as well as helping to maintain relaxation of the vein (Figure 12-7).

FIGURE 12-5. While performing the anastomoses, the surrounding tissue is grasped to avoid direct contact between the surgical instruments and the vein.

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FIGURE 12-6. The graft is connected to the arterial cannula via a three-way stopcock to check for leakage.

FIGURE 12-7. The vein without spasm being perfused via the three-way stopcock delivery system after removal of arterial cross-clamping. A vasa vasorum can be clearly seen in this vein.


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FIGURE 12-8. Leg wound closed in two layers.

If using off-pump CABG, checking for bleeding from the grafts and anastomoses would be done at the time of graft perfusion. Manual perfusion by syringe should be avoided as high pressure will damage the graft intima and endothelial cell lining. The leg wounds are closed with interrupted 3-0 suture to the subcutaneous tissue and a continuous subcuticular undyed 4-0 absorbable synthetic polyfilament suture to the skin (Figure 12-8).

Special Considerations Ultrasonography Preoperative vein mapping is fundamental to optimize the “no-touch” technique. The course of the vein should be marked on the overlying skin (Figure 12-1). This facilitates rapid and accurate location of the SV at operation, thus reducing soft tissue injury and the creation of tissue flaps that could lead to wound complications. This is of particular importance in obese patients. In addition, information is obtained to aid selection of the best vein segments for grafting.

Venous Spasm Most veins harvested with the “no-touch” technique relax spontaneously as the graft is perfused. If necessary, spasm can be overcome using local papaverine solution application to the vein. The vein should never be distended manually.

Handling of the Graft The graft should be manipulated using the perivascular tissue as direct handling of the graft by forceps causes adventitial, medial, and endothelial damage.

Incisions at the Knee Level Incisions at the knee level should be avoided as this region of the SV has a larger number of branches and the vein at this level is often of inferior quality. Futher-

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more, it is more comfortable for the patient if an incision crossing the knee is avoided. We recommend performing separate incisions at the calf and thigh (Figure 12-8).

Choice of Graft The side branches of the distal portion of the SV, at the calf, are more easily identified and ligated than the side branches of the proximal SV in the thigh. The vein from the thigh should be used to bypass the left anterior descending coronary artery, the right coronary artery, or their branches since, should leakage occur, it is easier to control bleeding from the graft when it is located anteriorly or on the right side of the heart.

Conclusion Vascular damage caused to the SV during conventional harvesting contributes to the high failure rate associated with this conduit. The patency of the SV is dramatically improved if damage is reduced using the “no-touch” technique.

Acknowledgments. The authors thank medical photographer Stefan B. Larsson, Örebro University Hospital, for his valuable assistance and copyright of the figures. Our studies were supported by the financial fundings from The Swedish Heart Lung foundation and the joint Örebro-Värmland Research grants.

References 1. Favaloro RG. Surgical Treatment of Coronary Arteriosclerosis. Baltimore: Williams & Wilkins; 1970. 2. Campeau L, Enjalbert M, Lesperance J, et al. The relation of risk factors to the development of atherosclerosis in saphenous-vein bypass grafts and the progression of disease in the native circulation. A study 10 years after aortocoronary bypass surgery. N Engl J Med 1984;311:1329–1332. 3. Campeau L, Enjalbert M, Lesperance J, et al. Atheroscloris and late closure of aortacoronary saphenous vein grafts: sequential angiographic studies at 2 weeks, 1 year, 5 to 7 years, and 10 to 12 years after surgery. Circulation 1983;68:1–7. 4 Bourassa MG, Enjalbert M, Campeau L, et al. Progression of atherosclerosis in coronary arteries and bypass grafts: ten years later. Am J Cardiol 1984;53:102C–107C. 5. Grondin MG, Campeau L, Lesperance J, et al. Comparison of late changes in internal mammary artery and saphenous vein grafts in two consecutive series of patients 10 years after operation. Circulation 1984;70:208–212. 6. Loop FD, Lytle BW, Cosgrove DM, et al. Influence of the internal-mammary-artery graft on 10-year survival and other cardiac events. N Engl J Med 1986;314:1–6. 7. Pym J, Brown PM, Charrette EJ, et al. Gastroepiploic-coronary anastomosis. A viable alternative bypass graft. J Thorac Cardiovasc Surg 94:256–259.


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8. Suma H, Fukumoto H, Takeuchi A. Coronary artery bypass grafting by utilizing in situ right gastroepiploic artery: basic study and clinical application. Ann Thorac Surg 1987;44:394–397. 9. Attum AA. The use of gastroepiploic artery for coronary artery bypass graft: another alternative. Tex Heart Inst J 1987;14:289–292. 10. Puig LB, Ciongolli W, Cividanes GV. Inferior epigastric artery as a free graft for myocardial revascularization. J Thorac Cardiovasc Surg 1990;99:251–255. 11. Acar C, Jebara VA, Portoghese M, et al. Comparative anatomy and histology of the radial artery and the internal thoracic artery. Implication for coronary artery bypass. Surg Radiol Anat 1991;13:283–288. 12. Mehta D, Izzat MB, Bryan AJ, et al. Towards the prevention of vein graft failure. Int J Cardiol 1997;62(Suppl. 1):S55–S63. 13. Mills NL, Everson CT. Vein graft failure. Curr Opin Cardiol 1995;10:562–568. 14. He NL, Rosenfeldt FL, Angus JA. Pharmacological relaxation of the saphenous vein during harvesting for coronary artery bypass grafting. Ann Thorac Surg 1993; 55:1210–1217. 15. Tsui JC, Souza DS, Filbey D, et al. Localization of nitric oxide synthase in saphenous vein grafts harvested with a novel “no-touch” technique: potential role of nitric oxide contribution to improved early graft patency rates. J Vasc Surg 2002; 35(2):356–362. 16. Ahmed SR, Johansson BL, Karlsson MG, et al. Human saphenous vein and coronary bypass surgery: ultrastructural aspects of conventional and “no-touch” vein graft preparations. Histol Histopathol 2004;19:421–433. 17. Souza DS, Christoffersson RH, Bomfim V, et al. “No-touch” technique using saphenous vein harvested with its surrounding tissue for coronary artery bypass grafting maintains an intact endothelium. Scand Cardiovasc J 1999;33(6):323–329. 18. Tsui JC, Souza DS, Filbey D, et al. Preserved endothelial integrity and nitric oxide synthase in saphenous vein grafts harvested by a “no-touch” technique. Br J Surg 2001;88(9):1209–1215. 19. Souza DS, Dashwood MR, Tsui JC, et al. Improved patency in vein grafts harvested with surrounding tissue: results of a randomized study using three harvesting techniques. Ann Thorac Surg 2002;73(4):1189–1195. 20. Souza DS, Johansson B, Bojö L, et al. Harvesting the saphenous vein with surrounding tissue for CABG provides long-term graft patency comparable to the left internal mammary artery: results of a randomized longitudinal trial. J Thorac Cardiovasc Surg 2006;132:373–378.

13 Toward the Prevention of Vein Graft Failure Jamie Y. Jeremy, Sarah J. George, Nilima Shukla, Marcella Wyatt, Jonathon Bloor, Andrew C. Newby, and Gianni D. Angelini

Introduction Autologous saphenous vein is used as a conduit to bypass atherosclerotic lesions in both the coronary artery (coronary artery bypass graft surgery [CABG]) and in femoral arteries (infrainguinal bypass graft surgery [IIBS])1–4 (Figure 13-1). Although arteries (e.g., internal mammary and radial) are widely used as alternative conduits in CABG and have proven less susceptible to complications and failure, the saphenous vein will still be used in CABG in the foreseeable future. In IIBS, autologous saphenous vein is still the treatment of choice for treating critical limb ischemia and disabling claudication. Despite the undoubted success and benefits of the procedures, early graft failure due to thrombosis occurs in as many as 18% of cases within the first week after surgery.1–4 Intermediate graft failure (30 days to 2 years after surgery) and late graft failure (>2 years after surgery) occurs in 20–50% of cases at 5 years. A principal cause of intermediate-to-late vein graft failure following bypass is intimal and medial hyperplasia, particularly at the proximal and distal anastamoses (Figures 13-1 and 13-2).5 Apart from lipid-lowering therapy,6 no intervention has hitherto proved clinically effective in preventing late vein graft failure.7 This clearly constitutes a major clinical and economic problem that needs to be urgently resolved. In this chapter, therefore, we will summarize the pathophysiology of vein graft disease and then consider interventional approaches to prevent late vein graft failure, which include conventional pharmacology, external sheaths, cytostatic drugs, and gene transfer.

Mechanisms Underlying Neointima Formation, Graft Thickening, and Atherogenesis The pathophysiology of vein graft failure is complex, involving disparate factors that include adhesion of platelets and leukocytes, rheological forces, metalloproteinase (MMP) expression, proliferation and migration of vascular smooth 158

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Infrainguinal bypass grafting surgery (IIBS) Common femoral Deep femoral

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Popliteal internal mammary artery diverted saphenous vein graft

Reversed saphenous vein graft Posterior tibial

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1–10 years: atherogenesis

FIGURE 13-1. Saphenous vein graft disease following either coronary artery bypass graft (CABG) or infrainguinal bypass graft surgery (IIBS). Within 1 month after implantation, the saphenous vein graft has thickened markedly and a new layer of cells, the neointima, has formed. This process involves the proliferation and migration of vascular smooth muscle cells, the expression of peptide growth factors, remodeling by metalloproteinases, and deposition of matrix proteins. The neointima renders the graft susceptible to atherogenesis, macrophages infiltrating this layer to develop into the foam cell, and then a plaque, such that as many as 50% of grafts will occlude within 10 years after the procedure (see Figure 13-2).

muscle cells (VSMCs), neointima formation, and superimposed atherogenesis. Medial thickening and neointima (NI) formation are mediated by the proliferation and migration of VSMCs (Figure 13-2). Superimposed on NI formation is atherogenesis, which ultimately leads to plaque rupture and graft occlusion.1–5

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Surgical preparation of saphenous veins for bypass surgery results in removal of the endothelium and immediate adhesion of platelets and leukocytes, which not only precipitateS acute thrombosis but also triggers NI formation8 (Figure 13-3). Endothelial removal also results in the loss of vasculoprotective systems that prevent inflammation and thrombosis, these being principally nitric oxide (NO) and prostacyclin (PGI2).9,10 NO and PGI2 inhibit blood cell adhesion VSMC proliferation and migration, MMP expression, proteoglycan synthesis, tissue plasminogen activator release, and cholesterol metabolism.9,10 The NO-cGMP and PGI2-cAMP axes at the medial level are also impaired in vein grafts.11,12 Endothelial cells proliferate and migrate to “reline” vein grafts, complete coverage occurring within 1–2 weeks.13 Although it has been suggested that the regrown endothelium is dysfunctional, accelerated re-endothelialization in vein grafts reduced both thrombogenicity and NI formation.14 The adhesion of leukocytes to vascular cells, each other, and platelets is mediated principally by the selectins, intracellular adhesion molecule (ICAM) and vascular endothelial cell adhesion molecule (VECAM).8 In turn, a causal link between adhesion molecule expression and NI formation has been demonstrated.15 Vein graft surgery is associated with an increased expression of adhesion molecules.15 Monocyte adhesion is another early event.15 Monocytes then infiltrate the neointima and become resident macrophages, which then become foam cells, the epicenter of an atherosclerotic plaque, a process that results ultimately in vein graft failure.15 Following implantation, the vein graft is also immediately subjected to arterial pressure, increased wall tension, shear stress, and pulsatile blood flow.16 These are all associated with an increase in the expression of growth factors, adhesion molecule expression, and proliferation. The process of vein graft remodeling also intrinsically alters intragraft hemodynamics. The assymetrical hyperplasia of the graft may also promote chaotic blood flow patterns, which in turn promote platelet and leukocyte adhesion, thrombosis, and graft hyperplasia.15 (Figure 13-3). Peptide growth factors are expressed in vein grafts and include endothelin-1 (ET-1), platelet-derived growth factor (PDGF), and fibroblast growth factor (FGF), all of which promote the proliferation and migration of VSMCs15 and are induced in vein grafts by hemodynamic forces, platelet, and leukocyte release substances.15 In situ, VSMCs are surrounded and embedded in the extracellular matrix (ECM) proteins, such as collagens and elastin, which act as a scaffold for cell and tissue architecture17 and exert an inhibitory influence on VSMC proliferation in situ.17 In vein grafts, the ECM is “dissolved” by MMPs, which allows VSMCs to migrate to the intima to form the NI.17 Upregulation and activation of certain MMPs has been implicated in negative vein graft remodeling.17 Superoxide (O2•−) derived from NADPH oxidase is a component of vein graft pathobiology.18,19 O2•− promotes VSMC proliferation and migration and upregulates MMPs.18,19 O2•− also reacts with NO, reducing NO bioavailability, which is

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FIGURE 13-3. Representative photomicrographs demonstrating morphological changes at the anastomoses of end-to-side saphenous vein into carotid artery grafts 1 month after implantation in the pig (A–D). Please note the aggressive neointimal hyperplasia and thickening at these sites: (A, C) sections taken directly at the anastomotic interface; (B, D) sections directly adjacent to these sites. Note the complete lack of a neointima in the ungrafted saphenous vein and host carotid artery


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itself associated with vein graft disease.18,19 NADPH oxidase is rapidly expressed in vein grafts, which in turn is upregulated by platelet and leukocyte release substances.20–22 Hypoxia appears to play a key role in mediating vein graft disease. Surgical removal of the saphenous vein ipso facto results in a loss of continuity of the vasa vasorum, a microvessel complex that infiltrates and oxygenates large blood vessels, which in turn would result in hypoxia of the tissue.23–25 Since the vein graft thickens rapidly, the graft is probably subject to an increase in oxygen demand, which may also increase hypoxia. Hypoxia promotes O2•− formation via activation of NADPH oxidase, xanthine oxidase, and mitochondrial respiratory chain19,20 and rapidly induces the expression of NADPH oxidase and an increase O2•− formation in arteries.26

Surgical Technique Surgical technique is a major determinant of the success rate of bypass graft surgery. Furthermore, recent studies based on a no-touch or rather “minimal touch” approach have generated promising data in patients undergoing CABG.27–29 In this approach, care is taken when removing the saphenous vein from the leg not only to avoid endothelial damage but also to avoid removing the adventitia, the tissue that surrounds conduit vessels and that houses the vasa vasorum and the vasa nervorum. Follow-up studies have indicated that early patency rate of saphenous veins harvested with surrounding tissue is high, even in saphenous vein grafts (SVGs) demonstrating low blood flow.27–29 Preservation of graft endothelium may explain this success, since the endothelium protects against thrombosis and also generates NO and PGI2. It was demonstrated that NO release in vivo is eliminated during conventional saphenous vein harvesting, which was preserved after no-touch harvesting,28,29 suggesting the potential to improve coronary artery bypass graft patency. The same could be the case for IIBS. These authors also suggested that the integrity of the vasa vasorum may play a role in mediating improved patency rates. As mentioned above, saphenous vein preparation effectively removes the vasa vasorum, which may result in hypoxic stress, which may promote vein graft pathology.

Conventional Pharmacology Many pharmacological agents have proven effective in reducing neointima formation and vein graft thickening in preclinical studies using animal models.30 Remarkably few clinical trials in bypass graft patients have been carried out with drugs, however. Aspirin (acetylsalicylic acid, ASA) has been the most widely investigated drug in patients who have undergone CABG and IIBS.31 Although ASA reduces the

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incidence of vein graft thrombosis in both the short and long term, it has no effect on late vein graft failure.31 Because of its beneficial effects, however, ASA is routinely administered to patients following CABG and IIBS and continued indefinitely thereafter.31 The relative lack of effect on eventual outcome has been ascribed to a lack of effects on VSMC proliferation and minimal effects on the adhesion and release reactions of platelets and leukocytes.8,32 Another major drawback of ASA is the promotion of gastric erosion and ulceration over the long term.33 In contrast to ASA, NO is a potent inhibitor of platelet and leukocyte adhesion and the release of mitogens, dilates saphenous vein, and inhibits neointima formation.10 It was suggested, therefore, that the co-administration of a NO donor may compensate for the limitations of ASA.5 A novel drug type that intrinsically fulfills these pharmacological criteria are the NO-releasing aspirins (NO-ASA),34 which in vitro inhibit the proliferation of VSMCs and relax isolated human saphenous vein.34 In vivo, NO-donating aspirin (4016) inhibits neointima formation in the porcine model.35 Endothelin-1 and angiotensin II (ANG II) are involved in every facet of vein graft disease since they are potent vasoconstrictors and are mitogenic and chemotactic.36–38 Specific ETA antagonist is a potent inhibitor of neointima formation and vein graft thickening in an experimental model and blocks each of the above-listed events.39 Other potential benefits of ETA blockade in patients following cardiac surgery include a reduction in early thrombosis due the relief of vein graft spasm, improved compliance in distal runoff coronary arteries, improved functional recovery of hibernating myocardium, a reduction in postoperative arrhythmias, and attenuation of renal dysfunction.40 Oral administration of ANG II–converting enzyme (ACE) inhibitors (which prevent the formation of ANG II from ANG I) reduces vein graft thickening.41 Perivascular application of veins with the chymase inhibitor Suc-Val-Pro-Phe-(OPh)2 for 20 minutes inhibits neointimal formation and total ANG II–forming activity when assessed at 3 months after operation in dogs.42,43 Oral administration of the ANG II receptor antagonist L-158,809 and local treatment of rabbit vein grafts with L-158809 reduced neointimal thickness.44 MMPs play a key role in modulating SVG remodeling. In a cultured human saphenous vein model the broad-spectrum MMP inhibitor marimastat reduced neointimal hyperplasia (NIH) and reduced MMP-2 and MMP-9.45 Doxycycline (10 μg/mL) also led to a reduction of NIH that was associated with a reduced MMP-9 but with no changes in tissue inhibitor of metalloproteinases (TIMP)-1 and TIMP-2 between doxycycline-treated veins and controls.46 In vivo administration of the MMP inhibitor BB2983 (a broad-spectrum MMP inhibitor) reduces intimal hyperplasia in a porcine arteriovenous graft model 1 month after implantation.47 Calcium mediates VSMC replication and migration48 and as such would be expected to exert an effect on SVG thickening. Verapamil, a calcium channel blocker applied in a periadventitial matrix, reduced NIH49 and when administered systemically reduced NIH in vein grafts of rabbits.50 It has also been


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advocated that local administration of thapsigarin, which depletes intracellular calcium pools, may be effective in reducing NIH. As has been emphasized in this chapter, oxidative stress, in particular, the upregulation of NADPH oxidase and superoxide formation, appears to play a key role in promoting vein graft disease.16,17 Furthermore, those risk factors associated with increased incidence of graft failure (diabetes mellitus, hyperhomocysteinemia, hypercholesterolemia, and smoking) all promote intravascular oxidative stress.16,17 It would seem logical, therefore, that the administration of antioxidants may be effective in preventing vein graft disease. Perivascular application of vein grafts with polyethylene glycolated superoxide dismutase reduced NIH at 4 weeks postoperatively.51 Oral administration of the antioxidant methylaminochroman reduced NIH compared with controls.52 Other antioxidants that have proven effective in preventing NIH include penicillamine and desferrioxamine.16,17 In recent studies we also found that a number of important drugs are potent inhibitors of NADPH oxidase expression. These include NO donors and nitroaspirin, iloprost, a prostacylin analogue, and sildenafil, a type 5 phosphodiesterase inhibitor.16,17,20–22 These drugs may therefore be effective in preventing vein graft failure through suppression of oxidant stress.

External Sheaths In a porcine model, placement of a porous, nonrestrictive, external, polyester Dacron® stent around saphenous vein–carotid interposition grafts significantly reduced neointima formation and total wall thickness at both 1 and 6 months after implantation53 (Figure 13-4). In order for the external polyester stent to prevent neointimal thickening, it had to be loose-fitting54,55 and macro-porous.55 Tight-fitting external stents may even exacerbate neointima formation or graft thickening.56 With the loose-fitting polyester stent, the space between the graft and the stent becomes organized into a cell-rich “neoadventitia,” abundant with microvessels following entrapment of a fibrin-rich exudates (Figure 13-5). Loose-fitting PTFE (micro-porous) stents not only promote neointimal and medial thickening, but also prevent microvessel formation.55 Porosity may be crucial since it allows the microvessels that form in the neoadventitia to connect with the vasculature outside the stent, allowing a fully integrated blood flow to the graft, thereby obviating hypoxia. Since the external Dacron stent blocks neointima formation in vein grafts within the first month after implantation, long-term support with a prosthetic sheath may not be necessary. We therefore studied an external polyglactin (Vicryl®), which matched the structure and dimension of the original polyester Dacron external stent. Vicryl is hydrolyzed by macrophages between 60 and 90 days.56 The polyglactin sheath reduced porcine vein graft thickening at 1 month,57 which persisted for 6 months.58 One clear feature of the vein grafts fitted with the vicryl sheath was the presence of a microvessel-rich adventitia,

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IEL media

NI lumen EEL

stented

FIGURE 13-4. Effect of the external Dacron stent. In these studies, a loose-fitting Dacron sheath or stent (upper left panel) was placed around a saphenous vein into carotid artery interposition graft (upper right panel). After 1 month, the graft was excised and studied histologically. As can be seen (lower left panel), there is a marked increase in graft size and neointima (NI) formation (the layer between the internal elastic lamina [IEL] and the lumen) compared to the original ungrafted saphenous vein (inset). It is this thickening that is the basis of vein graft failure. The graft fitted with the external stent, however, shows a profound reduction of graft thickening (small arrow IEL and large arrow external elastic lamina [EEL]) and a complete inhibition of the neointima.

indicating that the main mode of action of the sheath is to promote angiogenesis and therefore prevention of graft hypoxia. We therefore concluded that external vicryl sheaths sheath allows for adaptation of the vein graft to arterial conditions while at the same time preventing the “overshoot” of intragraft VSMC proliferation and migration. The long-term presence of the sheath may not be necessary to prevent graft thickening and failure over the long term. This may constitute a distinct advantage over the Dacron stent, especially in patients undergoing IIBS. Another facet of the external stents or sheaths is that they impose symmetry on the graft when it thickens in response to arterial hemodynamics. As mentioned earlier, vein grafts thickening is markedly asymmetrical. Aggressive


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FIGURE 13-5. Time course of events that occur with loose-fitting external Dacron stent on porcine vein grafts: (A) 1 day after, (B) 1 week after, and (C) 1 month after implantation. From (A) it can be seen that the stent is loosely fitting. By 1 week after implantation, however, the space between the graft and the stent has filled with a fibrin-rich exudates (B). By 1 month this area has organized into a “neoadventitia” (C) that is rich in microvessels. We suggest that the fibrin-rich exudate that forms in the space between the graft and the stent (B) promotes the formation of new microvessels, which in turn oxygenates the graft, thereby preventing hypoxia-induced pathogenesis, including cell proliferation. (D) Unstented grafts (trichromes). Note relative lack of microvessels.

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thickening is also apparent at the anastomoses. In turn, these elicit marked alterations of blood flow through the graft, which are associated with blood stasis, turbulent blood flow, which can elicit thrombosis, and hyperplasia. The stent, in effect, keeps the graft contained in what one could describe as a “jellymold” effect (Figure 13-6). This, in turn, prevents trubulent blood flow and imposes laminar and symmetrical flow. The aforementioned studies were carried out in an end-to-end interposition model of vein into carotid artery grafting, whereas in practice end-to-side anastomosis is used to implant SVGs in CABG and IIBS. Hyperplasia is more aggressive in SVGs at these anastomotic sites.3,4 We therefore set up a novel end-to-side model in the pig and examined the effect of a Dacron cuff placed specifically at the anastomoses (Figure 13-3), which we found to inhibit hyperplasia at these sites. We intend to determine whether placement of a vicryl sheath at the anastomoses inhibits hyperplasia over the long term.

Cytostatic Drugs Because VSMC replication and migration are early events following SVG implantation, incubating saphenous veins with drugs that these process prior to implantation may reduce neointima formation. We found that thapsigargin, in vitro, inhibits VSMC replication for up to 14 days.59–61 We then found that preincubation with thapsigargin (TG) prevented neointima thickening when assessed at 1 month but has no effect at 6 months after implantation (Jeremy et al., unpublished). Similarly, pretreatment of SVGs with rapamycin elicited an inhibitory effect at 1 month but not at 3 months (Newby et al., unpublished observations). We also found that perivascular fibrin glue elicited a similar pattern.62 Schachner et al. also found that rapamycin promotes inhibition of NIH.63,64 These effects are possibly elicited by an initial induction of apoptosis, which, although reducing graft thickening in the short term, would elicit thickening in the longer term through a “rebound” mechanism.

Gene Therapy The saphenous vein lends itself to impregnation with oligonucleotides or gene therapy prior to implantation in the same way as it does for cytostatic drugs.65–69 Consequently, a number of molecular strategies using antisense oligonucleotides and gene therapy approaches to overexpress specific genes that target smooth muscle cell proliferation, migration and survival, inflammation, or endothelial function appear to be effective in experimental models. Molecular strategies have included local treatment of porcine veins with antisense oligonucleotides complementary to the messenger ribonucleic acid of c-myc, which significantly reduced neointimal thickness 3 months


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FIGURE 13-6. Vein graft thickening elicits marked alterations of hemodynamic blood flow, which in turn can promote thrombosis and hyperplasia at both the neointimal and medial levels. By contrast, the external sheath or stent imposes symmetry (cylindrical) on the vein graft as it thickens, such that normal laminar blood flow is retained.

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postoperatively.67 Similarly, local treatment of rat veins with c-fos and c-jun antisense oligodeoxynucleotide (ODN) (in pluronic gel) decreased NIH. Furthermore, both transcription factors nuclear factor(NF)-κB and E2F were successfully targeted by decoy ODNs to prevent vein graft NIH.66 However, the translation of molecular therapy from experimental models to clinical application has been limited. The PREVENT (Project of Ex-Vivo Vein Graft Engineering via Transfection) studies provide extensive experience of molecular therapy in venous bypass grafts to date. Based on promising experimental in a rabbit vein graft model,67 the E2F decoy oligonucleotides were delivered with pressure intraoperatively to coronary and peripheral vein grafts. Phase I/II trial data were encouraging from PREVENT I and II studies.70 However, the large-scale multicenter PREVENT IV study found no benefit of E2F decoy therapy in more than 3000 patients undergoing CABG with at least two saphenous vein grafts.71 Similarly, numerous gene therapy approaches have been performed in animal models. Since cell migration is critical to intimal thickening and requires remodeling of the matrix by proteolytic enzymes, such as matrix-degrading MMPs, they have been investigated as a target for gene therapy. Local overexpression of TIMPs reduced intimal thickening in a human in vitro model of vein graft failure and ex vivo adenoviral73–75 or ultrasound-mediated72 delivery of TIMP-3 gene attenuated intimal thickening 1 month after grafting in a porcine vein graft model, highlighting the potential use of TIMP gene therapy. Inhibition of extracellular matrix deposition has also been investigated as a potential strategy. A recent study investigated the effect of antisense to transforming growth factor-β1 (TGF-β1), which affects inflammatory cell chemotaxis and extracellular matrix synthesis in epigastric vein to common femoral artery vein grafts in rats.77 However, a decrease in neointimal area was detected, but monocyte/macrophage infiltration was unaffected, implying that reduced ECM synthesis was primarily responsible for the attenuation of intima thickening after treatment with antisense to TGF-β1. Several studies that increase expression of nitric oxide synthase (NOS) have previously been reported as beneficial for reducing vein graft intimal hyperplasia.76–78 Interestingly, only in the latter study was a reduction in inflammation observed. This suggests that NO is affecting other key processes such as smooth muscle cell proliferation and thrombosis. In addition, together with the study of Wolff et al. described above,76 these studies highlight that attenuation of inflammation is not essential for the reduction of intimal hyperplasia. In fact, the observation that overexpression of 35K, a CC-chemokine inactivator, significantly reduced intimal thickening and macrophage accumulation in rabbit vein grafts at 2 weeks,79 but the benefit was lost by 4 weeks, indicating that a purely anti-inflammatory strategy is ineffective at reducing vein graft hyperplasia. In contrast, demonstration that overexpression of 35K reduced atherosclerosis in carotid-caval vein grafts in apolipoprotein E–deficient mice80 highlights its potential for improving vein graft patency by attenuating superimposed atherosclerosis.


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Two studies have recently examined the potential of combination gene therapy strategies for the reduction of vein graft stenosis using a rabbit jugular vein graft model.81,82 Combination of TIMP-1 and vascular endothelial growth factor A and C overexpression had no additional benefit to TIMP-1 alone.81 In contrast, the second study revealed additional benefit of combining extracellular superoxide dismutase (EC-SOD) and TIMP-1 overexpression on intimal thickening at 4 weeks after grafting.82 Interestingly, combination of the antiinflammatory strategy of overexpressing 35K with TIMP-1 overexpression only reduced intimal thickening at 2 weeks after grafting, and the effect was not retained at 4 weeks. This illustrates not only that combination gene therapy can be more effective than treatments based on single genes, but also that inhibition of migration as well as oxidative stress and inflammation with EC-SOD is more effective than inhibition of migration alone or combined inhibition of inflammation and oxidative stress. It also supports the previous suggestion that purely anti-inflammatory strategies may not be beneficial for the reduction of intimal hyperplasia. Pro-apoptotic strategies have also been considered. Delivery of TIMP-3, which in addition to inhibiting MMP activity and smooth muscle cell migration promotes smooth muscle cell apoptosis significantly reduced intimal thickening in a porcine vein graft model.72,74 Furthermore, adenoviral delivery of wild-type p53, which promotes smooth muscle cell apoptosis reduced intimal hyperplasia in porcine vein grafts 3 months after grafting.83 Despite initial concerns, neither pro-apototic strategy led to loss of smooth muscle cell density or thinning of the graft wall which could lead to aneurysm formation. However, despite promising results from these numerous animal experimental models, to date there have been no published clinical trials utilizing gene therapy for vein grafting.

Conclusion Several diverse strategies have proven effective in preventing neointima formation and potentially late vein graft failure in preclinical animal models. These include surgical techniques (no-touch), conventional pharmacological approach, the placement of external stents or sheaths, gene transfer, and cytostatic drugs. However, it is becoming apparent that acute exposure of saphenous veins prior to implantation to oligonucleotides, adenoviruses, and cytostatic drugs, although initially effective, may not be effective in the longer term. Thus, surgical technique, prosthetic sheaths, and conventional pharmacology may be the more effective approaches. However, lamentably few clinical trials have been undertaken to explore these possible strategies. It is important, therefore, to endeavor to perform clinical trials with these approaches in order to solve a hitherto intractable clinical problem, which has enormous economic and human consequences.

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38. Dashwood MR, Tsui JC. Endothelin-1 and atherosclerosis: potential complications associated with endothelin-receptor blockade. Atherosclerosis 2002;160:297–304. 39. Wan S, Yim A, Shukla N, et al. The endothelin-1A receptor antagonist, BSF 302146, is a potent inhibitor of neointimal and medial thickening in porcine saphenous vein-carotid artery interposition grafts. J Thorac Cardiovasc Surg 2004;127:1317– 1322. 40. Jeremy JY, Shukla N, Wan S, et al. Are ETA receptor antagonists the solution to preventing vein graft failure? Curr Vascr Pharmacol 2005;3:315–323. 41. Yuda A, Takai S, Jin D, Sawada Y, et al. Angiotensin II receptor antagonist, L-158,809, prevents intimal hyperplasia in dog grafted veins. Life Sci 2000;68:41– 48. 42. O’Donohoe MK, Schwartz LB, Radic ZS, et al. Chronic ACE inhibition reduces intimal hyperplasia in experimental vein grafts. Ann Surg 1991;214:727–732. 43. Tsunemi K, Takai S, Nishimoto M, et al. Lengthy suppression of vascular proliferation by a chymase inhibitor in dog grafted veins. J Thorac Cardiovasc Surg 2002; 124:621–625. 44. Fulton GJ, Davies MG, Barber L, et al. Localized versus systemic angiotensin II receptor inhibition of intimal hyperplasia in experimental vein grafts by the specific angiotensin II receptor inhibitor L158,809. Surgery 1998;123:218–227. 45. Porter KE, Loftus IM, Peterson M, et al. Marimastat inhibits neointimal thickening in a model of human vein graft stenosis. Br J Surg 1998;85:1373–1377. 46. Porter KE, Thompson MM, Loftus IM, et al. Production and inhibition of the gelatinolytic matrix metalloproteinases in a human model of vein graft stenosis. Eur J Vasc Endovasc Surg 1999;17:404–412. 47. Rotmans JI, Velema E, Verhagen HJ, et al. Matrix metalloproteinase inhibition reduces intimal hyperplasia in a porcine arteriovenous-graft model. J Vasc Surg 2004;39:432–439. 48. Shukla N, Rowe D, Hinton J, Angelini GD, Jeremy JY. Calcium and the replication of human vascular smooth muscle cells: studies on the translocation of extracellular signal regulated kinase (ERK) and cyclin D1 expression. Eur J Pharmacol 2005;509: 21–30. 49. Brauner R, Laks H, Drinkwater DC, et al. Controlled periadventitial administration of verapamil inhibits neointimal smooth muscle cell proliferation and ameliorates vasomotor abnormalities in experimental vein bypass grafts. J Thorac Cardiovasc Surg 1997;114:53–63. 50. El-Sanadiki N, Cross KS, Murray JJ et al. Reduction of intimal hyperplasia and enhanced reactivity of experimental vein bypass grafts with verapamil treatment. Ann Surg 1990;212:87–96. 51. Huynh TTT, Davies MG, Trovato MJ, et al. Reduction of lipid peroxidation with intraoperative superoxide dismutase treatment decreases intimal hyperplasia in experimental vein grafts. J Surg Res 1999;84:223–232. 52. Davies MG, Dalen H, Barber L, et al. Lazaroid therapy (methylaminochroman: U83836E) reduces vein graft intimal hyperplasia. J Surg Res 1996;63:128–136. 53. Mehta D, George SJ, Jeremy JY, et al. External stenting reduces long-term medial and neointimal thickening and platelet derived growth factor expression in a pig model of arteriovenous bypass grafting. Nature Med 1998;4:235–239. 54. Izzat MB, Mehta D, Bryan AJ, et al. The influence of external stent size on early medial and neointimal thickening in a pig model of saphenous vein bypass grafting. Circulation 1996;94:1741–1745.


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55. George SJ, Izzat MB, Gadsdon P, et al. Macro-porosity is necessary for the reduction of neointimal and medial thickening by external stenting of porcine saphenous vein bypass grafts. Atherosclerosis 2001;155:329–336. 56. Vijayan V, Smith FC, Angelini GD, et al. External supports and the prevention of neointima formation in vein grafts. Eur J Vasc Endovasc Surg 2002;24:13–22. 57. Jeremy JY, Bulbulia R, Vijayan V, et al. A bioabsorbable (polyglactin) external sheath inhibits porcine saphenous vein graft thickening. J Thoracic Cardiovasc Surg 2004; 27:1766–1772. 58. Vijayan V, Shukla N, Smith FCT, et al. A polyglactin biodegradable external stent prevents medial and intimal thickening but promotes marked neo vasa vasorum formation in porcine saphenous vein grafts in both the short and long term. J Vasc Surg 2004;40:1011–1019. 59. George S, Johnson JL, Angelini GD, Jeremy JY. Thapsigargin inhibits smooth muscle cell proliferation and intima formation. Arterioscler Thromb Vasc Biol 1997;17: 250–256. 60. Birkett S, Jeremy JY, Angelini GD, McArdle C. Time-dependent inhibition of intracellular calcium mobilisation by low concentrations of thapsigargin in human vascular smooth muscle cells. J Cardiovasc Pharmacol 1999;33:204–211. 61. Shukla N, Rowe D, Hinton J, Angelini GD, Jeremy JY. Calcium and the replication of human vascular smooth muscle cells: studies on the translocation of extracellular signal regulated kinase (ERK) and cyclin D1 expression. Eur J Pharmacol 2005; 509:21–30. 62. Wan S, Arifi AA, Chan MCW, et al. Perivenous application of fibrin glue in porcine saphenous vein-carotid artery interposition grafts: impact on medial thickening. Eur J Cardiothorac Surg 2006:Mar 30;[E pub ahead of print]. 63. Schachner T, Zou Y, Oberhuber A, et al. Local application of rapamycin inhibits neointimal hyperplasia in experimental vein grafts. Ann Thorac Surg 2004;77:1580– 1585. 64. Schachner T, Oberhuber A, Zou Y, et al. Rapamycin treatment is associated with an increased apoptosis rate in experimental vein grafts. Eur J Cardiothorac Surg 2005;27:302–306. 65. Yamashita A, Hanna AK, Hirata S, et al. Antisense basic fibroblast growth factor alters the time course of mitogen-activated protein kinase in arterialized vein graft remodeling. J Vasc Surg 2003;37:866–873. 66. Suggs WD, Olson SC, Madnani D, et al. Antisense oligonucleotides to c-fos and c-jun inhibit intimal thickening in a rat vein graft model. Surgery 1999;126:443– 449. 67. Mannion JD, Ormont ML, Magno MG, et al. Sustained reduction of neointima with c-myc antisense oligonucleotides in saphenous vein grafts. Ann Thorac Surg 1998; 66:1948–1952. 68. Shintani T, Sawa T, Takahashi T, et al. Intraoperative transfection of vein grafts with the NFkappaB decoy in a canine aortocoronary bypass model a strategy to attenuate intimal hyperplasia. Ann Thorac Surg 2002;74:1132–1138. 69. Ehsan A, Mann MJ, Dell’Acqua G, Dzau VJ. Long-term stabilization of vein graft wall architecture and prolonged resistance to experimental atherosclerosis after E2F decoy oligonucleotide gene therapy. J Thorac Cardiovasc Surg 2001;121:714– 722. 70. Mann M, Gibbons G, Kernoff R, et al. Genetic engineering of vein grafts resistant to atherosclerosis. Proc Natl Acad Sci USA 1995;92:4502–4506.

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71. Alexander J, Hafley G, Harrington RA. Efficacy and safety of edifoligide, an E2F transcription factor decoy, for prevention of vein graft failure following coronary artery bypass graft surgery. PREVENT IV: a randomized controlled trial. JAMA 2005;294:2446–2454. 72. Akowuah E, Lawrie CG, Sheridan PJ, et al. Ultrasound-mediated delivery of TIMP-3 plasmid DNA into saphenous vein leads to increased lumen size in a porcine interposition graft model. Gene Ther 2005;12:1154–1157. 73. George S, Johnson J, Angelini G, et al. Adenovirus-mediated gene transfer of the human TIMP-1 gene inhibits smooth muscle cell migration and neointimal formation in human saphenous vein. Hum Gene Ther 1998;9:867–877. 74. George S, Lloyd C, Angelini G, Newby A, Baker AH. Inhibition of late bein graft neointima formation in human and porcine models by adenovirus-mediated overexpression of tissue inhibitor of metalloproteinase-3. Circulation 2000;101: 296–304. 75. George SJ, Baker AH, Angelini GD, Newby AC. Gene transfer of tissue inhibitor of metalloproteinase-2 inhibits metalloproteinase activity and neointima formation in human saphenous veins. Gene Ther 1998, 5:1552–1560. 76. Wolff R, Ryomoto M, Stark E, et al. Antisense to transforming growth factor-ß1 messenger RNA reduces vein graft intimal hyperplasis and moncyte chemotactic protein 1. J Vasc Surg 2005;41:498–508. 77. Matsumoto T, Komori K, Yonemitsu Y, et al. Hemagglutinating virus of Japanliposome-mdiated gene transfer of endothelial cell nitric oxide synthase inhibits intimal hyperplasia of canine grafts under conditions of poor runoff. J Vasc Surg 1998;27:135–144. 78. Kibbe MR, Tzeng E, Gleixner S, et al. Adenovirus-meidated gene transfer of human inducible nitric oxide synthase in porcine vein grafts inhibits intimal hyperplasia. J Vasc Surg 2001;34:156–165. 79. Puhakka H, Turunen P, Gruchala M, et al. Effects of vaccinia virus antiinflammatory protein 35K and TIMP-1 gene transfers on vein graft stenosis in rabbits. In Vivo 2005;19:515–521. 80. Ali Z, Alp N, Tatham A, et al. Gene transfer of a broad specturm CC-chemokine inhibitor reduces vein graft atherosclerosis in apolipoprotein E-knockout mice. Circulation 2005;112:U500-U500 2130 Suppl. S. 81. Puhakka H, Turunen P, Juha R, et al. Tissue inhibitor of metalloproteinase 1 adenoviral gene therapy alone is equally effective in reducing restenosis as combination gene therapy in a rabbit restenosis model. J Vasc Res 2005;42:361–367. 82. Turunen P, Puhakka H, Heikura T, et al. Extracellular superoxide dismutase with vaccinia virus anti-inflammatory protein 35K or tissue inhibitor of metalloproteinase-1: combination gene therapy in the treatment of vein graft stenosis in rabbits. Hum Gene Ther 2006;17:1–10. 83. Wan S, George SJ, Nicklin SA, et al. Overexpression of p53 increases lumen size and blocks neointima formation in porcine interposition vein grafts. Mol Ther 2004; 9:689–698.

Section Five Genetics, Gene Therapy, and Tissue Engineering

14 Statins and Cholesterol: How Low Can You Go? Dimitri P. Mikhailidis

Introduction Current treatment guidelines1–3 recommend targets for serum/plasma lowdensity lipoprotein cholesterol (LDL-C) that range from 1.8 to 2.0 mmol/l. The 1.8 mmol/l goal is included in the National Cholesterol Educational Program (NCEP) Adult Treatment Panel (ATP) III guidelines (USA)1,2 as an option for very high-risk patients. The same guidelines recommend 2.6 mmol/l for highrisk patients. The December 2005 Joint British Societies’ 2 (JBS2) guidelines3 on prevention of cardiovascular disease in clinical practice recommend an LDL-C of 2.0 mmol/l for high-risk patients. This brief review considers the evidence that supports the guideline goals outlined above. The treatment options available to reach these targets will also be considered.

Key Trials That Support the “Lower Is Better” Concept Several trials support the concept that “lower is better” for cholesterol as well as the target values included in the NCEP ATP III and JBS2 guidelines.1–3 A selection of the key trials is presented below.

Heart Protection Study (HPS)4 HPS compared simvastatin 40 mg with placebo in 20,536 stable high-risk patients over an average 5-year period. Treatment was beneficial despite a 17% ”drop-in” treatment (mainly with statins) in the placebo group. Patients with a baseline LDL-C of 2.6 mmol/l had as much risk reduction after treatment with simvastatin as those with higher LDL-C values. This finding suggests that there is benefit to be gained in lowering LDL-C beyond 2.6 mmol/l, the target value in the NCEP ATP III guidelines.1,2 This benefit may be attributed to LDL-Cdependent and/or -independent effects. 179

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Pravastatin or Atorvastatin Evaluation and Infection Therapy (PROVE-IT)5 This trial enrolled 4162 patients who had been hospitalized for an acute coronary syndrome (ACS) within the preceding 10 days and compared 40 mg of pravastatin daily with 80 mg of atorvastatin daily. The primary endpoint was a composite of death from any cause, myocardial infarction (MI), documented unstable angina requiring rehospitalization, revascularization, and stroke. Follow-up lasted a mean of 24 months. The median LDL-C level achieved during treatment was 2.46 mmol/l in the pravastatin group and 1.60 mmol/l in the atorvastatin group (p < 0.001). The primary endpoint showed a 16% reduction in the hazard ratio (HR) in favor of atorvastatin (p = 0.005; 95% confidence interval [CI] 5–26%). Several secondary endpoints also favored atorvastatin.

Treating to New Targets (TNT)6 Patients (n = 10,001) with clinically evident coronary heart disease (CHD) and LDL-C levels of

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